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W. etu 1t5n1 • P5M8L2v 0 f Fre5hwat 5urey, ,y901 cE Leathrd • F0 d ,a1r6muC hcrae5 R KU ,n01ta1c05 A ,daehr t L KU J0NA7H N P0R7ER JAME5 R. ,f 1draC C. ment ,y901 cE ,a1r6muC ,5ecn 1 50 8f 1atnem 0r1vnE RAYNE • 0f 1ac1901018 6LEN D0NAt.0 KU ,yt15rev1nUf 1draC 0 f Fre5hwat R08ER750N 570 Dt.EY 0f 1ac1901018 PE7 R 0f 1ac1901018 • y901 r1V and ,5ecn 1c5 ra1uce10M D0na 1a160rc1M ,5e1r0ta 6 L y901 cE -rev1nU 6r0up, NERC ,y901 60rc1M etu 1t5n1 0xfrd, • 1ac 901 8f01 hc5 L0 prev1L 6r0up, Departmn KU KU 1atnem 0r1vnE UP70N KU hcrae5 R 0 f Exetr, L0 prev1L 7H0MP50N MA7HEw Ltd.,5urey yt15rev1nU 10 hc5 KU ,5e1r0ta 6 L D0n a y901 60rc1M 0 f L0 prev1L P. ,a1r6muC KU ,y901 nhce7 ,5ecn 1 5 57R1KE • yt15 ,y901 cE L0 prev1L • ARE 1atnem 0r1vnE • KU ,5ecn 1c5 0 f L0 prev1L J. 0 f Exetr, • 1ac 901 8f01 hc5 RnrcmE yt15rev1nU 6r0up, Re5ach Dprt- yt15rev1nU etu 1t5n1 • A. y901 60rc1M ,5ecn 1 5 RH0 E5 NA1 0 f Fre5hwat 5e1aW J0AN C. PAuL etu 1t5n1 10 hc5f draC • RALPH5 • ,5ecn 1 5 0f KU yt15rev1nU 0 f L0 prev1L KU AN0REw 5. 0f y901 r1V Wm7Et.EY • and 1atnem 0r1vnE ra1uce10M 1a160rc1M ,y901 60rc1M y901 cE 6r0up, NERC 0xfrd, etu 1t5n1 KU M oinc rt f gPa bslme 1 1 Some Problems Posed by Natural Environments for Monitoring Microorganisms Clive Edwards 1. Introduction 1. Tradit on l M cr bio gy The history and em rg nc of microb l gy as a scient f disc pl ne are int ma ely linked with dev lopm nts of methods for isolat n, enrichm t, g r o w t h a , n m d i e c o f r g a n i s m t h l e b o r a py s u c l t e b e n h a s p r o c T i l g y e n t . b as u d h q m c o e x t r m s lu yc f oharen i p l t x d o a mi fcn r g s in terms of disea contr l and er icat on, dev lopm nt of bi technol g a proces , and the volu i n f sophi t ca ed mol u ar gen tic h ques. It h a l rs e o u t i bn md c s a p l e i n t g u d m o r h a e s , bhs eao mcv pd l rti w , n f h e o s al micro g n s . Example inc ud the wi spr ad u e of (model Gram-negative), coelicol r cer vis a Unfortu a ely, it s often the case tha exp rim ntal prot c ls dev lop for model speci are not ranspo ble to ther bacteri . This gulf is often most a p r e n wt h d u c i o ms a fe r g n i m s o w a p u r ce l t is n h labor t y e as um d to p ly hose c ur ing atur l envi o m ts. Until rec ntly, the pro e ti s of micro gan s in their normal habit s a s t u h m e n p y w ci o d r b a t n , could n t be gr a ly dif e nt rom h se ncou t r d i he labor t y. H wg n e o i v w s t r ,a l c y p h d u e n v i r o m s a t e u l y i l i m t e dn Escheri a ol (Gram-posit ve), Bacil us subtil s (industrial y importan mycelia prokaryotes), (model for yeast), and (1) From: Streptomyces Sac haromyces sp . (widely studie fung s). Aspergil u h s a e b dt i mr o n , q u p c s i l n t h a d Methods in B otechnol gy, Enviro mental Monit r ng of Bacteria Vol. 12: Edite by: C. Edwar s © Human Pres Inc., Tot wa, NJ 1 2 sdrawdE bacteri , in particul , have evol d stra egi for coping with condit s of em rtx n i u . o ta m l e S ,s ic p h u sa detim l c obatem ri pe t oda a evit m rp esno r g ivlo n ta urops ) 2 ( . r s e h i t c Od vm p a l no y g w b to be understo by microb l g st . Ther is no doubt tha the pros ect of rel asing, eith r delib rat y or ac ident l y, gen tical y manipul ted microo r g a n i ( s G m M Oe p t )v o h r n a i su m g de f r a t m i o - e f s c u n l v r g t i a o d A my h e s . nal d con ert d s u ie , a number of pr blematic reas h v be n id t laiborc m lato eh tidua ot ,elbis opm t n fi ,tlucif d si t ah tseg u sehT .d if s m at e c ly o .r b n p h i u T f s ea v r n dt y n ea hm gt l o sc ua tb i yf oh s r e v li ad b o c e rhm t i n yo l b a .ygol ib rc m desab-yrot bal noit dar fo s mg d ehsir c dna leh-gno 2. Culturability i m p Ao n s rf a te u l v h y s of the bac ri p es nt ca b ult red by a it on l me h ds, an t i r ges f r o ma p x0 . 1t % ronme t. This means tha it is dif cult to ga e the to al bacteri l divers ty pres nt wi h a g ven s mpl . Th reason f thi are compl t d an robably mu tiface d, but incl de a l ck of kn wledg con er i g the m abolic requi m nts of many speci tha prohib ts suitable isolat n media to be d e v p i rts h o ; cfn m, a ul y r s htb d ov e p m e o t f a b s l h i u c d w n , e a tb y r o l s n d ; t e r c mMu sal n y d o i f . h t p er cm a n s have b n pro sed f r such pe i s ncludi g ormant, dw rf cel s or, m e o f t e n ,v i a b l u o c t r a b l e( V B N C )c s . o i n g l ed f t o h a sp r v e d c oT nh te r v p s i a dl . t e V B N C o f c u r e n t h a s d i f c o r y dni o s u tb h a c e m r t f sod a v i cn ug l r m e fs y bacteri n labor t y exp rim nts, but i s unlike y tha it s a st ble physiol gic phenoty in atur l enviro m ts; rathe it s a tr nsie pro e ty of How- death. c l o im ta n u r e p l g d h o t ansi ey bact ri ev r, it s an mport hen m o as witne d by h fact some bact e n d u m to b r y c a f i l se u t o p a b r h g n the clas i cult ra techniqu s of microb l gy i m p o r t a nf u d e s i t gh e m n a o dc u r e p fa t h o g ni s the nviro m t as wel for p dict ng he fa d consequ f rel asing GM Os to pen vir m ts. A consequ of no cult rabi y observ d in microb al com unit es in natur l enviro m ts is tha it is dif cult to as e whet r the inab l ty to cult re is becaus large p o rti n of bserva l intac el s are d . The , tah ev a erom sul icaB (3) d e p n i go u t r n a ls u dt y p eo f n v i - (4) . Such observati n are M oinc rt f gPa bslme p r o b l e m af s i n tg r u v a b l oy f c t e r i a p u l o n s rd e a t i g h o c u l t r a b i h y es dn mb a i v e s t g o r R . c n l uay m , b e f viab l ty d es hav be n d v lope and tes o enum rat he pro ti n f live/d a cel s in ma y ecos t m and l borat y model sy t m . Thes ar mainly fluoresc nt dyes and are listed in i n c l u d e me m e b rp ao nt i l ) ; c e u a r o v l f g d yn sej t u l i n g t h er l a s o f u c h r m et a i so n l y e d( a t h r f o s i n ) intracel u arly by live cel s; metabolic activ y-dep n t dyes; exclusion mechanis e n o ly in l ve c l s. They av be n ap lied to many exp rimental sy em ( y t i l e a hb o nr p i t g a f e l b ch rm u t a n o ni dets l yd tnecs roulf remun ht iw gn tse yb d g uj sa ,evil ytil baru c dna ytil ba v fo seidut eh l a morf nward eb nac t h noisulc mrif a s fi t or n e m c l b u v a h ne ty r p , ao l d cn i e b a t m sihT .aidem yrota b l hcir ni nworg serutl c morf tner f id ,yl acigol hpr m netfo l a n o i t d e y r b u l i c s a e m t r o h n l p i s e t y c r ua o b f m sdohtem hcus ot narticl e moceb nosaer mos rof tub yrota b l eht ni sdohtem .smet y oc larut n fo sr gi eht o d s pxe ra y ht ne w R e c B n l t o a y m .f i d oc ur en or dev lopm nt of VBNC sta e . Th y pro se tha e failure to recov l s ubject d o in m al pr ces u h a t rv ion s he r ult of an oxidative-m diated suic de of the cel . It is wel know tha bacteria e x p o s e d to ad pt ions e abl th m osurvi ean o m tal yi posed tr uch h a e s t on cu r k i l m a o n sive reduction of growth rate to near zero and the induct o of hig -af n ty sub tra e uptake p thways. Transfe o such organism to rich ult re m dia l e a d t rs o p i w c h n ag f d l o i m e t b p c a h w y s t n o b e coupled im ately o gr wth. Oxidat on f sub tra e l ds to verp ducs u pot efi r n a x d c b l k h m , s g y . Ther is much evid nc ow av il b e to sup ort his pro sal, ris ng from increas d understa i g of the altern iv pathw ys of gen expr s ion tha exist n m cro ga is . 3 Table 1 . Their modes of action brane poten ial-d ent uptake (dea cel s fail to gen rat a se ref. 5 for evi w) and, o the w ol , have d monstra e 1 elbaT (6) pa h od sv e in x bc l t f r in m cal proces undergo b th biochem al nd morph l gica (7–9) i A m n .p o r t a c s e q u n i - 3. Sensor-Regulated Pathways of Gene Expression In bacteri , many stimul -respon networks have be n ident f d, and thes ar of n s ciated w h p ations ch ge in t x r al physic c h e a m nop i d f r t l b T ws v . k of a large , globa cel u ar regulato y network tha respond to a variety of enviro mental stre s (10) . It has be n found tha ther may be overlaps A. 4 sdrawdE Table 1 Some Fluorescent Dyes That Have Been Used to Assess Microbial Viabilitya Dye Mode f Acti n eni aycobr x ly ehiD Ap licat ons Membran pote ial Flow cyt me ri det c ion f bacteri Rhodamine 123 Membran pote ial Micros p enum ratio of viable ct ria Flow cyt me ri as ment of viab l ty n: 1. A range of G m-p sit ve and eg tiv bac eri ; 2. Micro us l te 3. Staphyloc us a re sib cirut b a ly u id-3,1( acid) pent m hi ox n l (O o ) Fluoresc in d a et (FDA) Membran pote ial Intracel u stera Enumeratio f dea c l s Micros p det c ion f v able cleav g to rel as Gram-posit ve bac ri ; v able fluoresc in wh s retain d w h cel s pos e ing a t c me bran mycoba teri ; v able soi acteria; v ble aqu tic b er a Flow cyt me ri nume atio f metabolic y a t ve m rin microalg e; vi b subtil Carboxyflu esc in As for FD Bacil us Flow cyt me ri nume atio diacet (CFDA) and i e t f ca ion v able comp st ba eri ; se a cirtemo yc wolF fo tnems in )lyhte xobrac-2( siB '7, 2 As 5 c-)6( acetoxym h lest r for FDA xobra ulfy emotyc wolF niecs ro Calcein a cirt of a As for FD Sac h romy es c r vi ae ytil baiv f o tnems range of b ct rial spec Micros p det c ion f v able acetoxym h lest r prot z a y t i l b a v f o t ne m s a c i r t e m o y c w o l F of a r nge of bact ri l spec Fluoresc in d - β -D Intracel u nzymic o wl F c y t m e r i cleav g , dye sl ec yb no it e r enarbm tc ni h w )PGF( edisonaryp tc l g Chemc ro Y As for FGP gnitros l ec d tavi of viable y st and b cteria Flow cyt me ri d t c on f Candi lb cans ytil ba v M oinc rt f gPa bslme 5 Table 1 (contiued) Chemc ro B As for FGP cirtemotyc wolF aremun ytil baiv fo noit of a r nge of bact ri l spec Analysi of v ab lity of gen ical y modif e B. subtil Resu cita on f VBNC in comp st Vibr o vulnif c s 5-cyano 2,3 dit ly Respirato y c iv t Micros p det c ion f active edirolhc mu zarte )CT ( aqu tic b er a Flow cyt me ri d t c on f respi ng M. lute s Analysi of d rmancy i Mithramyc n sl ec d tagnole i atS M. lute s FCM monit r g of viable (viable) ft r p olonged i cubat on in the pr s nc of nalid x c Propid um e Dye xclusion by ve 4',6-diam no 2- Dye xclusion Micros p det c ion f v able cel s prot z a nd ye st Micros p det c ion f phenyli do muvrap id sotpyrC a Dat re k n f om . 5 betw n comp ent works in tha pro eins duce by one str espon may lsobeinduc yothers .Agen ricmod lf sti u -re pons networks i hown i nutrie s, mp ratu e, l ravio et (UV) radi t on, r chemi als nd mutage s are d t c by molecu s with n e c l tha r nsmi the forma i n, s et i navm e s r l d o f n m i t e lr c u g h n os , m t ing the xpr s ion f ec g n s tha e cod f r p teins ha ble t w i ot a rh d s e c x p l n W . - t u de ix a m p ln c s the a -shock respon regulon i dam ge of DNA. Thes types of sen or sy tem are rev sibl in tha when condit s rev t o the orig nal ev s, the n w path ys of gen xpres ion q p cu r ei o lt ah s f n Ty w rc e . d drastic l y when t viro mental s i n operat . Yersin a rucke i Fig. 1 . External f uct ions uch fa tors e ntial E. coli and the SO respon t UV light 4. Altered Physiological and Morphological States Arising From Nutrient Limitation or Starvation O u nr d e s t a i o whfg y n bc t e r a iv u nl r o ments and how they may exhib t gros ly dif er nt physiol g ca sta e has st yco 6 sdrawdE Fig. 1 Sensor gulat sy em . r ec ived a great deal of impetus from studies of the response of ent ric b a c t e r i ta po r l o n g e cd u l t r ie sn t a i o n a r py h a s e E. a r l wy o r ik d e n t i f e td h a bacteria such as periods f tarv tion( ary phase E. coli f i se a d g m f c t o n r a m e d o ir ndt e a u l c p v g eo rfnx s i M . c e t l yh , role and regulato y pro e ti s of this sigma factor have be n more clear y d e f i n a , s o m t h g r u p fe n w s o x r i dn e p t on σ S -direct RNA polymeras transc ip o and the functio s they ncode are giv n Table 2 tionary phase su ments, bacteria wil have undergon a prog am of alter d gen expr s ion tha result in a cel tha s di t nc a d if er nt pro e ti s compared with those ncou ter dincel sgrowni utrien -richlabor t ymedia.Theproteins tha re synthesiz d uring starv ion as result of expr s ion ften ar col e tiv ly ref d to as t rva ion- ducible proteins ( s t ip r o e n s ) ,a dt h e i r x p e s i o na ds y t h e s i a v eb n x t e s i v l y t u d ied n Vibr o sp . are cap ble of prol nged survi al during ext nde se ref. 1 ).Otherwo k nthepro ertiesof ta ion, 13) and Salmonel a typhimuri m σ S t , h a l t e r d h s e p c i f t o Ry N fA E. coli (12 (14 , 15) polymeras . By an logy with es labor t y- sed tu ies on starvi al, it is like y tha in nutrien -lim ted natur l enviro - σ (9 , 16) . cel s identi- S -contr l ed g ne M oinc rt f gPa bslme 7 Table 2 Some σ Regulated Genesa S Gen s Functio E and kat G (cat l se HPI kat Prev ntio f DNA dam ge by H 2 O and HPI ) A (exonucl as I ) A, xth bol Repair of H gen s ( spv 2 Over xp s ion re ults in able sph rical cel s. fic plasmid S almone virulenc g s) fic – O 2 and UV-r iat on d m ge. mutan s re ho t ds. Transc ipt o fr m spv prom te is S σ A (of -dep n t. rpo 10 -fold es virul nt. BA oper n ( s o ible f r ots Osmopr tec i n ( S – cel s mor en it v o rpo synthe i of c mpatible solute r ha os ) osm tic hanges) Unk ow ge s di t nc from σ mediat g ne xpr s ion (heat shock igma f ctor) 32 Thermot l ance, m y also be p rtial y mediat by ots S gl Expres ion f a mily of gen s Glycogen s th i . Anaerobic l y ndu e g s. si o b rean y d cu ni ylg orts yb decu ni yl taredom sl i σ S; include a ytochr me xidas , hydrogenas 1, d aci phos at e B and osm osm Y Membran d cel nv ope functio s. Synthesi a d xcretion f m cro ins. c ito b na editp ,7C nicor M tha in b ts pro ein sy th i a Dat re k n f om Starv ion b logy is now a ctive r s a ch rea, nd the p ysiol g and ecol gy f s ow gr th o d rmant cel s ha be n r vi w d n a thoug -pr K o c h brv ye k i n wg n o f g r w s i t a n p h b y c e w r io s a t u n l d h dit ons, mu a t rose du ing tarv on ( fter 12-d incubat o ) h ad superio su vi al pro e ti s xh bited as the abil ty o utgr w 1-d ol ce s from young cult res m u t ia n o w r s g e p d contr ve sial po bi ty ha mut ions the s arving t e could be ir ct d (18) whic in terms of evoluti nary theory had Lam rckian impl cations. R e c n t l y h s o r v e i sw o l v e db ya m n s t r i o h a t n r y phase mut ions wer not direc at sel c d g nes, but oc r ed th oug the genom with n a subpo lation of stre d cel s via a recombinat o - refs. 14 (17) (13) and 15 . f cu or mt s ph e l A i d a g . n . This mutan phenoty was cal ed GASP and was prorpo tr ha eiw so dk n uT y S . ABCD oper n) spv BA. S – mutan s re 2 . 8 d e p n tr o c s m u t s a b l p e o i n wc xr h -f s d ces such as starvation. This itself has prof und consequences for our understa ing of bacteri l behavior in enviro ments tha regula y impose c r a e b n s i l t p d hm o u I . f y p s e o m r a d i c l s yh u f t e g r n im ca k u p s o te x r n af l c s ,h m tu ta ions can be used in an at emp to ensur survi al of a subpo lati n of stre d ind v uals, and tha such a phenom may help to explain such factors he m g nc of GASP mutan s i o ary-ph se cult r , si b a o d c t h p e nr s i w u g l , survi al of p th gens af r xpo u e t a h s ’ im une d f s . sdrawdE (19) T . h i r s e o l u t d n p s h e m r g c o a yf p - 5. Measurements of InSitu B e c a u s o ft h m b l i c a ys h u td o w n e a h t r o p i ca n d s b o a t c h e lsn r yp i x u v m a t s , r e o f n situ activ y are dif cult, esp cial y for an ind v ual speci . It is proba ly more alist c o me ur wh l p oces tha m y be di t y man differ nt speci (e.g , the nitroge cy le). Howev r, this ap ro ch also pose problems for the investigator because bacterial po ulations tha mediate importan bi geoch m al yc es oft n c mprise oduc rs an co sumer f the end products alongside each other. One ap ro ch for measuring activ es i to c n e tra on pr ces tha ve gas ou products. e n v i r o af m t y s ul b gp n ed j o c r h i t s importan proces , and it is im ed at ly ap rent hat produce s and consumer can o xist, e.g ni r f e s a d nitr f e s. Ev n the s ric ly an e obic methanog s and aerobic methano r p s can oc upy the same layers with n soil (20) . A gen ric sum a y of the f c s o nutrie l m ta ion bacteri l pro e ti s in natur l enviro m ts is given in observati n made on the f cts of tures and reinfo c s the argument tha bacteri in natur l enviro m ts can exhib t markedly if r nt p o e i s. W th resp c to de c i n a d sol ti n, many of the c anges po e chal eng s for m dern m thods. For exampl , the reduction of cel u ar rRNA as a result of a downshift of growth rate makes direct d ection by fluoresc nt whole-c hybrid zat on with fluoresc nt oligonucle tid prob s m e dif cult. The ow m tab lic v t es al o m k situ as e m nt of activ y and viab l ty extr m ly dif cult. Thes problems a r pe t i c u l a r iy m p o r t a n f o dr e t c i o n Gf M O is n a t u r a el n v i r o n m e t s or for exploit ng a GM O for environme tal proces such as biorem d at n. Howev r, as conti ual y stre d in the prec ding section , alter d p ns of ge xpr s ion mea th l bora y ctiv es may not be h ar t se c o g b n i d z l , a t i p s o r Te h b l d u . c Activities in in situ Table 3 Table 4 rpo . This underpi s the S expr s ion sta ion ry-ph se cul- in in situ M oinc rt f gPa bslme 9 Table 3 Microbial Production and Consumption of Gases Gas Produce s Hydrogen Nitrogen f x rs; e m nta io Consumer Het ro phs; met anog s; sulf r ed c s Carbon di x e Carbon m xide noita emr f ;noita pser cibo A Unchar te iz d an erob s Auto r phic ba ter Am onia x d zers; carboxyd t phs Nitrous x de Nitr c ox de Nitr f e s; d nitr f e s Denitr f s Nitr f e s; d nitr f e s Denitr f s; het ro p s and meth o r p s Nitrogen Methan Denitr f s Methanog s Nitrogen f x s Methano r p s Table 4 Some of the Responses of Bacteria to Starvation Respon Examples Reductiv s on—ultramic ob ter a Soil bacter ; m ine bact r ; E. coli snietorp s f i ehtnys—r vo ut nie rP S. typhimur E. coli Reduction al ce u r RNA Long-lived mRNA olecu s DNA lev s r main co st r inc eas Reduc m tabolic v ty Morph l gic an es sp. S14 Vibr o Marine ; numero s xample Vibr o Vibr o S14 Vibr o Vibr o Vibr o sp. ANT 30 sp. S14 sp. S14 Numero s xa ple wen fo t mp lev d—ygo is hp der tlA ; various ex mpl E. coli resi tanc pro e ti s Changed ti n c y— ew surfac Vibr o; Escheri a, S lmone a struc e synth ized elbavit ucno b el aiv moceb ya sl eC Numero s xa ple , r or d mant ucit negativ p hogens ni s o ta uM opr S – evit pmoc er sl c , E. coli for esu cita on d survi al a Dat re k n f om . 5 l bed ao tn sm r i p f v e d ing the g n s for phen l tra sfo m i n u der th con r l f prom te s, uch r p o St ,h a r ec i vo n lu y d s t a r i oc n s are wil requi a ful er understa i g of the prog ams of gen expr s ion under nutrie lim ta on condit s as wel as a bet r ap reci t on of the molecu ar biol gy invol ed. Rec ntly, at emp s have b n made to c r elat s i tn u b i o t r a n s f m p l b i y oc (21) F .u t rw eo i knh s lar y G m- 10 a c t i v oe fl y s ten (a measur of growth rate) to its degra tiv rates for a variety of substra e . The ap ro ch proved usef l for model pure cult res, but would be extr m ly labor inte s v to an lyze unk ow bacteri l po ulati ns in soil and w ter sdrawdE s i nt u (2 ) u s im n gc r o p e t h d l s a r ’Rc No An - . 6. Quorum Sensing and Resuscitation—Signal Molecules Many Gram-neg tiv bacteri l speci are now know to regulat gen expr s ion in respon to po ulati n size. This result in group behavior of bacteri l po ulati ns tha requi s interc l u a com uni at , gen ral y b means of dif usible autoinducing molecu s, now identif ed as hexanoyl) m serin lacto (HSL) r its de va . This proce a b n term d “quor m sen i g,” whic is char te iz d by bacteri synthe iz g a c y s ld i -e m g Hn o S L t u p d a e n O r i g . l y , l u m i b n ta e hc s d r o w pv e f iV s cb hr e o o n l y u m i e s c w h np r ta i g hd e n s ,c l a t o w e rd n s i o t u l n e i O d m g h r t s q . ba o f y c e d furthe s di of th s y em for whic t o gen s ar impo t n : encod s an autoind cer- spo iv transc ip o l activ or, and encod s a protein requi d for autoind cer synthe i . Not surp i ngly other factors al o imp nge o luminesc gen xpres ion. lux R requi s activ tion by c li AMP (c ) and the cAMP rec pto r tein, ro can i flul u e m n i c o f s a x tpF r N R d n l u x s T t h u e d Ri . on V. fischer have r sult d in the discov ry f other qu m sen i g–d p dent ac iv s for whic mol gues f lux R and lux I have b n id t f e or pro sed (23) . o e s t m f hq n u w li r c g a T s globa re u to sy em can r sult i comp ex and sophi t ca ed n r ctio s. More importan , it would se m tha es may not be r st ic ed to he signal producing speci alone. Shaw et al. (24) dev lop a thin-layer chromat graphic method for det c ing and char te iz ng N -acyl hom serin lactone signalmo ecu s.Th yt en dHSLsignalmo ecu s ha L-HS , 3-ox , 3-hydrox , and 3-uns b ti ed derivat s purif ed from a variety of Gram-neg tiv speci n a s ay. This te d the abil ty of HSL and its derivat es to induce gen expr s ion of a gen in Agrobacterium tumefaci ns tha w sregul t dbya oin uct a d h w sfu edto orde to as y gen expr s ion a d autoind c . The invest ga or showed tha signal molecu s from dif er nt bacteri could be as yed in this way, whic means th in the rogen us po lati ns fou d in atur l envi o a c t i f h vs e p -n g l m o H b S c y L u t h f s e i , ties of ther sp ci res nt wi h t e sam enviro m t. A sum ary of the N -3 (ox - whic , R, whic lux lux I, whic N -butanoyl lac Zin M oi nc r t f g a P s b m l e 1 Table 5 Processes reported to Be Dependent on Quorum-Sensing Autoinductiona Bacteri l sp HSL-induce a t v y Pseudom na eruginosa B, whic en od s la t e, m al opr te s las V. fischer Rhizob um leg inosarum E. coli Erwin a c tov ra importan f p thogenic y sen g c senimul tavi c h w fo tcud rp eht ,R xul Regulation f cat l se c iv ty Cel div s on a expr s ion f Producti n f ex rac l u enzym s tha re ul s in fts QA gen s ;selbat g v dn stiurf o gn t r fos ni tarec m us it also c rbep n m a tib o c synthe i Init a o f di er nt a io Ti plasm d conjugal tr sfe Resu cita on d reg owth f d rmant cel s by an Ser ati l quefaci ns A. tumefaci ns M. lute s HSL-like s gnali mo ecul a Dat re k n f om . 23 cedlnsity–pdeancivHotSfyLsraivge n Of particul inter s in the det c ion and monit r g of bacteri in natur l enviro m ts is the pos ib l ty of cros induct o of dif er nt activ es betwe n sp ci , the r la ionsh p betw n au oi d cers u h as HSL with o er g l o b ar e u t syi g n a l c h bacteri n oncult rab e sta e may be po ulati n density dep n t and/or reliant o h pr duction f a ut ind c g mole u ar sign l. 7. Summary Analysi of the microb al divers ty of the biospher by tradi on l cu t ral e n v i t r o u d p m a lhT s z y . n d r m i e c v t n hb o - a g w l p s ods f r an lysi , wh c is mportan f many ke ar s of bi l gy. Micro ganism drive the chemistry of natur l enviro m ts, and withou them life would not be pos ible on this planet c o sm p t el r h u ix n f b a c o s e i n p bc o g ar l w e t u h f v s m d y of c m unit es a d pos ibly the r ac iv t es. Such anges m y be xtr m ly importan f hey ct pivo al s ec u h as t e ni r f s. Inter i gly, the s e b af rotm c h n l d y v i e m t ac r o b l p g y , i u l n afo r c y u t s bp e ihl a on r , g c d t e sa r i n g the pro e ti s of micro gan s infer d from labor t y cult res. As this chapter has hig l hted, bacteria inhab ting their natur l enviro ments, to whic they have ad pted and evol ed over mil ons of years, may exhib t T a b l5 e rpo Sa,ntdhpeo s i b l y art u s c i o nf (25) . The abil ty to monit r ind v ual . 12 sdrawdE to al y dif er nt pro e ti s and interac o s to those se n in labor t y cultures. Th is no d ubt ha p lic t on f ew m thods f r an lysi tha re pres nt d in this bo k herald a rich fut re for recogniz new speci of a n d c o m u i t e s , p l x n t r a c h e y o w u n dm i rc s t a g , how t eir ac v ties can be und rsto and pos ibly man pul ted for envi mental bio ch g al pur ose . Ref r nc s 1. Got schal, J. C. (19 0) Phenotypic respon to enviro m tal changes. Microb l. E 2 . E r ( i 1 n 9 gJ t.3 o ) , morph gen si . 3. Pickup, R. (19 ) Dev lopm nt f e hods f r the d c ion f speci ba teri in the vironme t. 4. Colwe ,R. Brayton,P. Her i gton,D. Tal B ,Huq A. andLevi ,M. ( 1 9 6V )i a b l eun toc r a b l e the uman i tes . 5. Edwar s, C. (19 6) As e m nt of viab l ty of bacteri by flow cytome r , in Flow Cytome r eds.), Marcel D k r, New Yo k, p . 291–3 0 6 . G . P oE aw ne Rdr , B I t h . DC o Ad B , l S Fe m wG f a. i r t d M. (19 8) The viable ut no -cult rab e ph nome explain d? 14 , 1–3. 7. Munro, P. M Flat u, G. N Clem nt, R. L and G utier, M. J (19 5) Influe c of the RpoS (KatF) sigma f ctor n mai te nc of viab l ty nd cult rabi y of Escherichia Microb l. 8. Kaprely nts, A. S and Kel , D. B (19 3) Dormancy i sta on ry phase cult r s of Micro us Ap l. Enviro M c biol. 9. Oliver, J. D (19 5) The viable but no -cult rab e sta e in the uman pathogen Vibr o vulnif c s 10. Doul , J. L and Vi ng, L. C (19 5) Globa physiol g ca ontr ls, in and Biochem stry But erwo h-H inema , Bost n p . 9–63 1. Porte , J., Edwards, C., and Pickup, R. (19 5) Rapid as e sment of physio l g i c a sl t u os f 79, 39 –408. 1 2 . K o l t e r ,R . S i g l e D A . ,a n dT o r m ( 1 9 3 )T h es t a i o n r yp h s e ft b a c terial f cy e. 1 3 . s t a ( Ki 1 G l on 9 A R p S f . Z6h P de ) Mr m , y b g 86, 18 – 4. 1 4 . f a sc it go mr h e l H T ( n 1 g 9 a e 4 d-R) A. r C o i s LP w n , in bacter l g oba re ul tion. FEMS 93–102. 74, r e g xu pl n a oc t f si d B a c is lu b t : 1–3 . 57, Microb l. Rev 10 9– . 137, J. Gen Microb l. Vibr o 0 r 1 e v ct a uo l i b s e t n cholera 28–31. 12, World J. Mic ob l. Bi technol. Ap licat ons in Cel (Al-Rubeai, M. and Emery, A. N , Cult re Microb l gy and coli 61, Salmonel a in seaw ter. typhimurium Ap l. Environ. 1853– . : flow cytome ri an lysi of starv ion a d resu cita on. lute s 59, . FEMS 3187– 96. Microb l. 203– 8. 13 , Let . Gen tics of antib o c , (Vin g L. C and Stu r , C. eds ), producti n Escherich a coli u s i n fg l u o r e s c n pt r o b e s . J A. p l B. a c t e r i o l . 47, An . Rev Microb l. 85 – 74. Cel σ An . Rev Microb l. 48, 53–80. s (KatF) M oi nc r t f g a P s b m l e 13 15. Foster, J. W and Spector, M. P (19 5) How survi e ag inst al the Salmone od s. 49, An . Rev Microb l. 145– 7 . 16. Nystrom, T. Albertson, N. H Flardh, K. and Kjel b rg, S. (19 0) Physiol g cal and molecu ar ad pt ion to starv ion and recov y from starv ion by the marine sp. S14 Vibr o 129– 40. 74, FEMS icrob l. E 1 7 . ( 1 KL 9 o.M A c i 7 h ) , r b p a y l s o e c n g d r f w y t h . Microb l. 305– 18. 61, Mol. Bi Rev. 1 8 . C a i r n s , J . d oF t e r , P . L ( 1 9 ) A d a p t i v e r s o n f a r m e s h i t u a o n i 19. Escheri a Rosenb rg, S. M (19 7) Muta ion for survi al. 829–834. . Gen tics coli 695–701. 128, 7, Cur . Opin. Gen tics Dev. 20. Hales,B.A Edwar s,C. Ritch e,D.A Hal G ,Pickup R.W ,andS u ers, J.R (19 6)Isolati n d e tif ca on metha g n-specif DNA romblanket bog peat using PCR amplif cation and sequ nce an lysi . Microb l. Ap l. Enviro . 61, 6 8– 75. 21. Matin, A. Lit le, C. D , Fraley, C. D , and Keyhan, M. (19 5) Use of starv ion prom ters to lim t growth and sel ct for trichlor ethylen and phenol transforma a trc ie o n v m b y t 3 2 – 8. Escheri a coli . A E p n v l i M. r o c b 61, 2 . Whitel y, A. S O’Don el , A. G MacN ughton, S. J and B re , M. R (19 6) C y t o c h e m i a l - o c i s a t n dq u i t a o n fp h e t y i ca n dg e o t y p i c h a r a c t e r i s on fd v ub a lc t e r i s . 62, A p E ln .v i r oM c b l . 1873– 9. 2 3 . F u q a , C . i Wn a s , S . C , a n d G r e b g , P . R ( 1 9 7 ) C e n s u a d c o n s e u i n bacteri l ecosy t m : the LuxR- I family of quor m sen i g transc ip o al regulato s. 24. Shaw, P. D , Ping, G., Daly, S. L , Cha, C., Crona , J. E , Reinhart, K. L , and Far nd, S. K. (19 7) Det c ing and char te is ng N-acyl hom serin lactone signal molecu s by thin layer chromat g phy. 603 – 41. 25. Pace, N. R (19 6) New p rs ectiv on the natur l microb al wor d: molecu ar microb al e ogy. 72 – 51. 50, An . Rev Microb l. Proc. Natl. Acad. Sci. USA ASM News 62, 463– 70. 94, Sampling ed t an Soil 15 2 Sampling Sediment and Soil Use of C ring D v c s Roger Pickup, Glenn Rhodes, and Grahame Hall 1. Introduction 1. Sampling N tura Envi o me ts I p n oc d wl m i t -a ev s r h u b g q y , erful to s in m crobial eco gy, but o h are lim ted wi h resp ct o relating the pres nc and/or divers ty of micro gan s to their functio /a v y in thaenviro m t overc m . Pa ount i h s re p ct a duce sampl in form ep s nta iv of h t enviro m habit To det rmin the roles played by micro gan s in a particul habit , s o m fe r p c d u h a tsbo e n r k t a i n e p r s t v a m p l e s u wp ho rin ec s t a m v u r e bcn d t s thre op i ns av l b e. First, a s mple can be r mov d fr m an e viro m nt lr ae t b hn ou d f y T s p i . c h m o u with “destruc iv sampling,” whic rend s the sample no rep s ta iv of the enviro m t from whic it is remov d (e.g , grab sample from benthic enviro m t; t oc m p l e r a i o s f u n c t a li e g r y .T h s p a t i c u l r y p a e n t when studying geoch mi al proces tha rely on redox gradients or those d i r e ac ft o n l x gh y b s surem nt made r no l nger pres nta iv of tha enviro m t. Second, a sample can be remov d from the enviro m t while at emp ing to maint “ i ns t u ” During the labor y nal si , the ampl c n be mai t n d s clo e a p s- ( 1 , 2 ) .B yc o m b i n gt h e w a p r o c st h i b a l ec n sampling rocedu s tha pro- in s tu (3) ( 3 , 4 ) T h uae. sr l y 4 ).Theno r p s ta iven ur ofth samplei w ng ref. se (5) c o n d i t s u r g a n p o t i ds u b e q n tl a o r y s i . From: Methods in B otechnol gy, Enviro mental Monit r ng of Bacteria Vol. 12: Edite by: C. Edwar s © Human Pres Inc., Tot wa, NJ 15 C o n s. e q u p rat l mcy , - . 16 Pickup et al. sible to exp rim ntal pur ose . Furthe dev lopm nt of this princ le d to sy tem t w s o i - m p h l a e f r v “ t y i c o m n T dh e s a . ” -ides hguor t-w lf esahp- r t( xelpmoc eht o )ria/l os r ia/ret w kal( smet y ; s m e t yr a w k l / n l aiborc m dn , efs t g p a m vo ,l i rus yd t hc w interac o s ( f m i e dnw al o s t , uh r b c T i . x o f l e m e t h - . g ,a s u r d b c p n a m e t r so f g l i e d a o n y d p t i s , h e a n ef l u x r o m p ds i l strain particul y when the transpo i of delicat equipm nt o remot locati ns requ d. condit s, with one or two par met s being varied for “in situ” se 6 s m e t y l d o i v r p a h c .M ) .fer es 7 ). A third opti n is to perfo m the xperim nts in the ref. .T h e s l i m t a o n r s e w i gt l c a o n - (8) 1.2 Sampling Aqu t c E vironme ts This ect on f us edim nts; how ver, ampling of the wa r c lumn b hr e a sv n iH w y d t sent h mai ypes of dim nt sa pler. (4) ( se a C l h s o pG 3 trc ) e .n b d - srelpmaS b rG .1 2 Ther a considerabl um of grab s mple av i ble. A hav t eir adv nt ges and isa v nt ge and o e suit al enviro m ts m i x e d s o. a b p t r hn c u g sb a m l p i n , a c n o v e r d g sm p 1I 0 l b t , i y h x a n e are of 0.1– 2 m g r a b ,S h i p e k E m a ng r b ,O k e a n dt h S m i - M c I y r eg a b , l of whic an be op rated from a bo t; h wev r, they dif er n size and coms c a o m ne p td l fi u h - r g b w a o p l en x, i t y ments sampler in wh c t e in r as mple siz compr ised by t rela iv y large siz nd cumberso natu (4) 2 (4) (4) . Grab sampler include the Pet rs n grab, van Ve n o R b e s t L i u a n dh m r x g c f . k (4) . srelpmaS eroC .2 1 The most cru ial aspect of core design is the ret n io of the core on s e d p tin a r hmv f o c T u l - . y t ers p n t a i g he s dim nt o dep hs >10–2 cm and those in w ch a es to he d vic o urs at he urf c a te r i val tha are char te is c of al core s are the compres i n of the sedim nt and disturbance of the fin up er lay s. Both are in v table cons que of the coring operati n. The simple t core consi t of a pers x tube (e.g , 5 cm diamet r, 30 c length; s h a iw l n ( t o e r m z d p u ) v bA yf s .o a l (4) se ref. 4 ), tha c n be driv nto sedim by hand . Other impo ant fe ur s . For in s tu Sampling ed t an Soil 17 from sedi nt, h e ds of th ube can s led with bungs, prev ting los t rm a S ce n io ls dv p h g. y f are av il b e for sampling at depth. The gravity core s mis le at ch d o r pe, n t a he s dim nt a he cor is l ect d in the c ntral Persp x core tube. The most no able disa v nt ge of this type of devic stha u e bothdis r ance d ompr s i n fthecor ma i l. The Em ry and Dietz cor , the gravi y co er with ex rnal et i ng dev c s, m e a nS ir vh t o c -l p k f sd ug z ronme t (up to 3 m; f r e s h w d ca i ot m n sedim nt of up to 6 m in le gth from lake-w t r envi o m ts. Operation f the ap r tus is pneumatic ( c y l i n d r a h o etm s b T k . n anchor chamber holds the ap r tus firmly in place while the core tube is driven ow ard int he s dim nt by ea s of c mpres d ai . Once ori g is complet d, the anchor chamber is automatical y fil ed with air, the coring tube is removed from the sediment, and the whole ap ratus is recover d to he surface by uoyancy lift ( by oat.The four c ve d s tion . Frame- ount d core s min ze both disturbance and compres i n. The best xample of this type is the “J nki surface-m d sampler” ( w h i c r e t v 3 s 0 – 4 - c m o r e w i t h v l y n g a e s r bd t o h T . e number of anipul t o s ha c n be p rfo m d n Je ki cor tubes ampl demonstra their versatil y. Extrusion of the core, section g fol wed by c m o a n sit p h rue l f H mg w d v . , mp ir co a e n s d , u p s e t b o vc a w rn l y d i g t e h measur d after incubat g under va ying condit s or after ad it on f substra e , .g acet pling ports tha al ow sub tra e ad it ons or sample removal at a variety of depths (13) the Craib s mpler and simultaneo multicore sampler , both f whic ork on the sam princ le (9) 10 ). The Maker th core ret i v s long intac u n d i s r o t e f m p b v h ca l T . ref. se (1 ) ), wher by hydrosta ic pres u acts on a Fig. 1 Fig. 1 ) and returned to he laboratory e c xio tsr u ld b a n y k h o sr Fig. 2 . Some tub s have n dapte wi h a sp r l of am- (13) . This ver atil y s hared by others in th s group, whic n ludes (4) . 1.3 Sampling o E vir nme ts m e f n h t so v r Di w d l a p g , y l r a u m b te o g i s h p c T n . l d co uw tn a e p v i r l g h s u c o b e t a m r pe n s u r t o a i da in m e t Or bs . u c i oh a en l , b k r s d c fibrous mate i l, n ge ral, c n prev t suc e f l p netra io nd af ect h , akin to smal (5) l e n g v toa hfr s i c m y p u bC e . [12] ), 18 Pickup et al. F Ti M1 hg a. e c k r t o : i e 6v s -a f ml d c n o t r T M h . e k c o r desc n the water column (2) from the surface (1) and emb ds itself irmly in the s e i d nt ha(m x 3 cr ) uo , b l T piet . a ratus e rns to he surface by flota i n us g compres d air (4) and is col e t d by the surfac t and re u to he lab r to y f proces ing (5). i n t e g r yo f h c ea t r m o v l .O n c e d ,t h o r ec a nb s p o r t e d to the labor t y. Mainte c of an erobic ondit s is pos ible using the a p r o i t e a u s n dw i l b e c u s d nt h e x c i o ,w h f u s e s a om np l t ie h g d f c r b t i oH n sw .e v r , the ap r us nd p oce ur a di ectly ap b e to sampling f o types. 1.3 Peat S mpling The fibrous nature of undist rbe surface peat prev nts intac cores from v t e r s p wa b d in mT o h f c l g u . aerobic nd tio s n the surface l y rs and the wa r-log ed an robic h r zons is char te iz d by ste p oxygen con e tra ion gradients g r a d i e n t sp l y m o r a n t l ei h v r c a ld i s t b u o n fm i c r a lp o u lations (15) activ es with n e p at, ny compa ti n of the profil disturbance of the r e d o xc n i t s h o u l db ea v i .M o r e ,t h x p o s u r f m e b l i g a t an erobic a ter o air, e.g th me anog s, ev n for sh t periods, c ul m p e r t o h d a u n fc i e t of the p ula ion re t v o he undist rb condi . Ts wt or a e g ih b v u n dc o m p a t i x y g e n m a tion whe sampling eat cor s. One ap ro ch is to cu the p a , using a long knife (18) tube,whic stheni sert dinto hepr formedspace.Thein tialcut ingof t h pe a t c o u l d i n t r o d u c e o x y g e n t o h e d p a n e r o b i c l a y e r s , a n d t h u s , t h e (14) . To ac ur tely det rmin the vertical distr bu ion of microb al (16 o r t h e rc u t i n gd e v i c e (19) , 17) ,t o h es a p e n d p t ho f es a m p l e a t c h e i r n v d f , o uy . Such Sampling ed t an Soil 19 Fig. 2 The J nki core . This dev c us detach bl ore tub s o ret i v 30-cm sedim nt cores a d v rlying water. s h a p e of t i o n f h se a m p l u b I.t c o n ad p r h ,u t i n dg e v c as h t o st b aho mef p ul T ( c . n d r st ) ho e f a of the p at, cu ing the fibrous peat d posit wh le nc osi g the p at core in s at mu hpb el The cor sampl tube s xcav ted from h peat d osi a t e m r p o s vh d i c tw l yur e osn a m p i g place or, t b s reaking lo ts eng h. o s b u t a c n d i e r f h m p M, l a d v l i o s p rt e xh f n u yb g c . the cu sho ld be ac ur t o av id comp t n f the p a on i ser- (20) t h er p , d o bu yc x i nf g a l m t o . (18) becaus 20 Pickup et al. Many repo ts describ the gas flushing of incubat o chambers to establi h d a e n t m c hr p o i b f u s s o m e a t x y g n p b s m d h a e r tli wp o uy T hd s stage during the s c ion g procedu . More v , many s ple ar slu ied, whic remov s th pa i l re t onship am g dif er nt po ula i ns of rgani T s a h m e . o c p b dt rk f n v w y physiol g ca groups of bacteri sample hou d be maint ed o x ic d a n tz m l u e s ry g h b pf vo i ad n e t ben fit h erp ta ion f c v ty measur nt . . (21, ) , and, if pos ible, the struc e of the (23) . Methods at s mple and section res (24, 5) 2. Materials 2.1 Sampling Freshwat S dimen s relpmaS eroC duM-ecafruS nik eJ eht htiw 1. Jenki surface-m d s pler com te wi h rope. 2. Core tub s. 3. Sedim nt x rude . 4. Boat nd s fe y quipm nt. 2. Sampling d A a ysi of Pe t C r s 1. Acryli o e tub s. 2. Cut er. 3. Ap ro iate s l . 4. Periph als ( se ; Fig. 3 Notes 1 –27 ; ref. 5 ). 3. Methods 3.1 Sampling Freshwat S dimen s with e J nki Surface-M d Cor Sample 1. Posit n b a over s mpling te. 2. Load c re tub in o Je k sample ( 3. Lift su pen io r d at ched o r pe, co k sample ( Note 1 se ). se cat h o e “ n” posit ( ), and push afety Note 2 ). Note 3 se 4. (waterhinoldpsumfweghtTak 5. Rel as f ty ca h. 6. Gently a d smo thly wer th sampler into s d ment ( 7. ( elt s o relpmas ht rof dna et r po msinahce g rps ht of iaW 8. Haul the s mp r, ke ing t ver ical, b k to he surfac . 9. Remov sa ple tub and pl ce i a su t ble ho d r p i to ex rusi n. 3.2 Extrusion f he C r ( se ). es se 1. The m c ani l extrusion t s a ched to w rk su face. 2. Fix core tub firmly to he x rusion t. Note 5 N o t e 7) ). 4Note se e 6t o N ). Sampling ed t an Soil 21 S c h a3 e F n. m ti d g s r o p f b l v c e u d i n a g t o of gas ti h p on a lied ft r ieval of th c re p io t ranspo t lab r to y. 3 . L o c a te x r u s i o n b d t e mx r u s i op nl a t e hc r u b do ip knl a c e . 4. Rel as th ecuring ate o h c re tub o m lid. ( e c a f r u s e r o c e h t e v o b a t s u j o t r e t a w g n i y l r e. v5 o n h p i s d n a d i l p o t e v o m R es 8 etoN .) 2 Pickup et al. 6. Fit he sampl co e ting spou t he op f the ub . 7. Use th andw e l o th ex rusion d t ex rud the cor s diment. 8. Extrude h sired amount f sedim nt ( 9. Remov th sec ion f re into a p r iate v s l ( 10. Rep at ). Note 9 se ). Note 10 se as p ro iate. step 8–10 3. Sampling Pe t Cor s 1. Identify sampling s te for c e xtrac ion d place th special y design ampler into s n ( 2. ( r a b ” y m o t “ / r e s n i C V P g n i t f y l t h g i e h t r e s n I ( pilc e ibuJ egral htiw eruc s dna 3 . a s te phm l b c n d o g s a uv t re h f C c w y i o n l core tub p ight on e p at surf ce. 4. Cut hroug the p at, holding the tom y bar nd move with a rota ing moti n (both cl kwise and cou ter l kwis ), th slig down ar p es u and isolate h cor in the sampl tube ( 5 . R e m p t c o a h vu n r d f i bw g s l angled b at e ch nd ( 6. Trim the bo m f the p a core xt ndi g from the cu r with a s rp knife. 7. Fit he gasti h p ston ( core tub ( 8. Remov th cu er ( 9. Store h c (s) upright d n ra spo t i n he labor t y. 3. 1 Section g h Peat Cor Unde A a robic Te hn qu ( se 12 ). ebut eroc eht fo p t eht o ni ) E3 .giF ( edistuo eht no de it sop ) F3 .giF Fig. 3H ) by insert g hroug the cu t r and up into he Notes 17 and ). Note 15 ). Note 16 se ). ) ( se Fig. 3G se 18 ). ). Note 20 ). Note 2 se se Note 23). se se Note 27 ). When p at cores w expos d t air, the m anoge ic a t v ies w r on [5] ). Even brief exposur result d in a .) A4 .giF ) se 3.4 Fut re D v lopm nts aver g 43% lower (range 12–74%; dna 12 etoN es 3. 2 Incubatio d Me han A lysi ( 1. Al ow the incuba o ves l to and f r 48 h ( 2. Flush t e adsp ce with n roge f 5 min after 24 nd 8 h ( 3. Remov sub ample of the headsp c (us al y 0.5 mL) at regula intervals (at least hr ) fo an lysi of methan co e tra i n ( 4 . A f t e ri n c u b a o ,d r y l t h es c i o n fp a t c s n w e i g h ta 6 0 wt de rminat o ( .) 31 etoN es Note 14 se 19 Note 1. Anaerobic se tion g is perfo m d in a flexib gas ho d flushed with n roge gas ( , e b u t e r o c i l y r c a f o ) m c 5 . 2 1 ( s h t g n e l 2. t r o h s o t n i d e u r t x e y l a c i n h e m s i e r o c t a e p e h T ( eciv d a gnisu rebmahc noitabucni sa evr s o la hci w 3. A new i cubat on chamber is then i s rted in o the up r as embly and p s ed s e c i t for u p nh b a d g . s e t o and Notes 1 se Note 25 and ). Note 24 26 ). ° Cf o rd y Sampling ed t an Soil 23 S F c4i h.g e m a dt sr o w p i ehn a g ct d v i e a c h st n m g p l tube o h incubat o sec i n. d e c l i n o 3f 9 (% – 6 ) t, h u s i g l n t h be f i o cedur s. Fut re d v lopments lie not i the d sign of new procedu s (many i r m et p lh c oa nv g d f , b y u e ) m a n y t o e c h i ql u s , a r f t e x p n d i g v r h w i t c o m b n e d h f a c e tom s i r b l g w ynh i ca t v f du i oa nr e l t b d versity and po ul ti /c m n y a l se . i sn t u s a m p l i n gr o - 24 Pickup et al. 4. Notes 1. Ref r to he manu l for instruc o n operati n a d mainte c are loc t d in he ap r tus wi h e tub harnes fl g acin w rd. 2. Once the tube is ecur ly at ched to the sampler, the machine is co ked by a down ar p es ur on the radius m cros ba until he radius m i locked in the prim d os t n. 3. The saf ty c h is pu ed to h “ n” posit . 4. T h e s a m p l e r i s l o w e r d s o t h a t h e l e g s a r e i n t h e w a t e r b e f o r e t h e s a f e t y cat h is rel ased. 5. Any unev move nt during lowering wil activ e the spring mechanis of the sampl r. 6. O n c e t h e s a m p l e r c o m e s i n t o c o n t a w i t h t h e s e d i m n t s ( t h e l o w e r i n g r o p e becom s lack), the spring mecha is w l activ e. Th sampler hou d be l ft for a sh t period set l prio t l f ing. 7. Thet x as ume th operat h spurc a edth p ro iate qu pm nta d ha c s to e p r im n g u ah l , tm o seip n r a u h f ,d provides u f l hints o ef ic nt use. 8. Careful siphon g av ids turbing he top s diment. 9. One r voluti n f he andw l has pitch of 2 m . n i 1 0 d. e l i a t d o h s a g e h t g n i s u y l a c i b o r e a n t u o d e i r a c e b n a c e r u d c o r p s i h T 1 . The cor tub dimens o ar 0.5 m in le gth a d 7.0 cm inter al d m ter. 1 2 . s aT mh cpe ol i r n u t s , yh e d r ci u (ta l l a e c nor b gfy t u h i ( m d i a m e t x s r c l h o y f u Pb i V a C,e ( n v d f i t s g h ol vy e c r u b T sh. v ia e n t r l m y c h i n e d b a (t 3 D i t m l c u o bs e h r d a ) T v y . to he u r wal of the c r ube sing adhe v tap . 1 3 . T hien s r ta m o v b lhe r i z n t a“ o m yb” pr s i ntgh o u a dl s hole t a w ir to escap during the coring p es . 1 4 . A n yc o m p a t i n f h e a p r o i l ( e . g ,s n k i o ft h ep a c r ) nb e a d i l y observ d th oug e transp cryli tub ng a d the r j c d ore. 15. The lower horiz ntal part is triangul in cros section and sharpen d on each edg . The sharpen d leading edg cuts throug the peat as the rod is insert d adj cent o h c re tub , o a depth jus below th cu er (p viously marked on the v rtical section f the rod). The rod is rota ed 360 e t d c h g u r p o is m a t b e hc l u f y so w r t uTo bhp ei .sr f a c l n t y w e z ohid s p , af r l quite long. The lower horiz ntal section is then located on the cut er, and the whole c r tub is gently as d from the p a . 16. The piston is made of PVC with two ext rnal distributor seals located in rec s ed gro ves. 17. The cut r an ow be t ach d o n ther co ube. 18. It is not pos ible to exclude oxygen while the piston is insert d, and, ther fore, the stand r prot col f alw ys ampling the p at least 10 cm de p r . Tubes (12) .02 etoN a t ) c oh e d 3F Ai g . F3 iB g . c Tu ht )e w . i ron ,s a l F i3 gC . t h )a Fig. ° , and the s arpen d si e Sampling ed t an Soil 25 than the d pth required is nec s ary. This provides a “buf er” zone tha may b e x p o s e td o a, n cd o s u m e o, x y g e wn h i l te h d p t oh f e a t bo se u a m p l e d is protec d. 19. For an e obic pro edu s, replicat pe cores a section d at 1.0-cm intervals 20 21 from 3.0 to 9.0 cm. Al gas in procedu s use oxygen-fr nitroge tha has b ep a ns h o v d tc r a l y( sB tA SR F3 ,- r 1 e om ) c v n a i t ing traces of xyg n. The flow rat f g s i ap rox 2 L · min . The an robic h od is made from heavy-g polyeth n bags, whic al ows manipul t on of the equipm nt from the outside and ac es to the section g e q u i p m f btrn oh T l us e. d w i t r o ag f p n ceh has be n pr a d fo the surfac e tion be r mov d. . The d vic s operat d by a screw th ad ( locates n h piston ( ( Fig. 3B a. –1 ; Fig. 4D ) used to al he bot m f he p at core ). P r i o t e x r u s i o n t, h e c u b a t i o cn h a m b e r s a l e d t h o wp i t Pah V C c a p( F i g .4 E “O” ring ( tube and the shoulder of the cap. The top cap lso c ntai s a butyl rub e ( mutpes ) with a pitch of 2 m whic Fig. 4B ; Fig. 4C Fig. 3G . )t h a s ni t e r n a l“ O ”r i n gt oh l di t np o s i t na d l r g e ) tha forms a se l betw n the machined flat edg of the Fig. 4F 4GFig. headspc.tofmlingr,vw) b . T h se a m p l t u b n d h ie c a t o n h m b e r c n t bd y h se c i o n g c. d. e. f. g. h. i. j. devic shown in t o g e h( r F i 4g H. the low r as embly ( s h a t r l e p o fi n d T m c g . y dovetail rebat s ( lie adj c nt o ea h t r. The low r as embly is placed ov r the sample tub and fixed n posit n so tha e op dg f the ub o ches t m al p te. T p h l r e a i t s m o v n cd x r u e t s i h l f a c v w the op f the ub . T i h n e c u b a t o m p i l e s r cu h n d a m t b l o y e f the ub is ag n t he m al p te. The as embli are joined toge h r and fixed in posit n o top f the core tube sing th crew ads n bolt ( The incubation chamber is flushed with nitrogen for 5 min before the p l a t e sa r e m o v e d ,a n dt h ec o r ei s x t r u d e b yt h er q u i r e da m o u n t( y p i cal y 1.0 cm). T p h e c ir ab u s ty l n g e T s h . i o l a t c u e i w o n h t incubat o h mber w il c os ng the cu s rfa e o th main pe t cor . er p u e h T - v a e l r i m n o c v u, a e h b t s d w l y itl nho gewa s r m p b y c d l it nhe g a o s f r m p l e . T h ep l a t f r o mt h eu p ra s e m b l yi sr e m o v dw h i l es m u l t a n e o u s l yi n e r t i nag o t h ePrVsC a l i ncg pT.h i s d e n t i c a lohte sp abluwti h o u t e septum vent. . This consi t of two as emblies tha are joined Fig. 4 T )b h .o e t u fp m a s r b ( l y F i 4g I. Fig. 4J Fig. 4L ) are s l d by remova l t p a es ( a t n )ho d ef p Fig. 4K ), and whe t as embli are join d, the plat s Fig. 4H ). ) 26 Pickup et al. 2 23. 2 .4 2 .5 26 2 .7 k. The xtrud peat lug is ther fo is lated in he cubation ch mber. Gastigh seal of the incubat o h mber a nsured th o g u the n ir cub a tp s 1 ie 5 c or 0“ n yu w -G d m” h . g . Al incubat o s re p fo m d at 20 This al ows the gase pres nt in the peat section (eith r dis olve or in gas bu les) to quil brate w h eadsp c . The adsp ce volum is replac d rox 25 times. As ly r i n ga e d u sft oh r m v a b l p f e ti sohn m c u b a ves l ar p eflush d wit n roge . . M withequpdcromags350ElPekinausgyzd ethn aPorpkNclumndfaeioztr.Injc hegasmpli withflusedopgaTv.mc01-Lby atlesfourimvpnjcgh. Methan c um lation s linear th oug incubat o , and r tes ar c l u ated from the sl p and cor e t f dry wt of he p at s c ion . ° C. References 1. Hal , G. H., Jones, J. G., Pickup, R. W., and Simon, B. M. (19 0) Methods to study he bact ri l eco gy f reshwat enviro m ts. 18 –2 0. 2. Pickup, R. W (19 ) Molecu ar m thods f r e d t c ion f speci ba teri n the nviro m t. 3. Pickup, R. W (19 5) Enviro me ts bridge Un v rsity P e , Cambridge, UK p . 298–315 4 . H e r b t R A (, . 1 9 0 M ) e h o d f s r n u m a t i g c r o a n i s m d e t r n i g biomas n tural envi o m ts. 5 . H a l G . ,S i m o n B M a dP i c k u p ,R .W ( 1 9 6 )M e t h a n p r o d u c t i n b l a k e tb o gp a : r c e d u f o s a m p l i n g , e c t o i n ga d c u b t i n gs a m p l e w h i s t maint g a erobic nd t o s. 6 . M o r gW R A a. Jh n, d e G P s i c k u p t a n l e C yS . , d u R r J s (19 2) The ef ct of micro s design on the survi al of recombina t microorganism lake w t r. 7. Trevo s, J. T. (198 ) Use of micro s to study gen tic interac o s betw n micro gan sm . 8. Baker, J. M , Norman, J. M , and Bl , W. L (19 2) Field sca e p licat on f m f e l a u s x c or n b d y i t m a p l n g . 9. Fench l, T. (1967) The col gy f the microbenth s. I Quanti ve importance of cil ates compred with me azo ns i var ous type of sedim nt . 12 – 37. 10. Ho d, M. A (19 2) Experim ntal e hods f r the s udy of ate nd tra spo f micro gans m in aqu tic sy tem , in and Ap licat ons Hil , New York, p . 51 – 24 23, Methods Microb l. 137, J. Gen Microb l. 10 9– . Sampling a d Det c ing Bacteri l Popu ati ns Natur l , Society for Gen ral Microb l gy Symposiu , seri 52, Cam- 28, Soil B . ochem 1, Microb al Re s 5, Microb l. Sci 1–40. 19, Methods icr b ol. 9–15. 15 – 60. 132– 6. A g r i F c o u l e .M s t y 62, Ophelia Microb al Ecol gy: Princ ples, Methods (Levin, M. A , Seidl r, R. M , and Rogul, M., eds.), McGraw 31–52. 4, Sampling ed t an Soil 1 . Macker th, F. J. H. (1958) A portable core sampler for lake deposit . Oceanogr. 12. Ohnstad, F. R. and Jones, J. G. (1982) The Jenki surface mud sampler: user m a n u O cl , s i o P a b c t F1N r5 e. n s h w a B i o l g Ac s i a t To n , u s Wilson Ke da , Cumbri UK, p . 45 1 3 . J o n e s , .G a dS i m o n ,B .M ( 1 9 8 4 ) e a s u r o fm i c b a lt u r n o v e fc a b ni anoxic freshwater sediments: cautionary com ents 47–5 . 1 4 . g a s e o pmf c t ru n i D e ( 1 9 4 ) . L l o y d , a n J . B e s t d , in peat cor s. 15. Wil ams, R. T. and Crawfo d, R. L. (1984) Methan producti n in Min esota peatl nds. 16. B o n e D, R. ( 1 9 E) c o l g oy mf e t h a n o g e n s i , n Consumption of Gre nhouse Gase : Methane, Nitrogen Oxides, and methan s biol gy, Washin to , DC p . 57– 0 17. Fetz r, S., Friedh lm, B. and Conrad, R. (19 3) Sensit v y of methanoge ic bacteri f om pad y soil t oxygen a d esic at on. 107– 5 18. Fre man, C. Lock M A. and Rey ol s, B. (19 3) Fluxes of CO from a Welsh p atl nd fo l wing s mulation f a w ter abl dr w- o n: p te tial fe db ck to lima c- h nge. 1 9 . r h e i ( s g 1 o A 9 lS C 8 u. t R y a) m p n f , c r e 425– 31. 2 0 . W i l a m s ,B .L n dW h e a t l y ,R .E ( 1 9 2 )M i n e r a l t o g nd y a m i c s p o r l y draine bl k t pea . 21. Mo re, T. R. and Knowles, R. (19 0) Methan emis on from fen, bog and swamp e tl nds i Queb c. 2 . P r i e m ,A .( 1 9 4 )P r o d u c t i na e m s i o n f e t h a i b r c k s ha n d f r e water l nd. 23. Jones, . G and Simo , B. M (1985) Interac io f acetog ns a d meth nog s in a erobic f shwater dim nts. 24. Yavit , J. B., Downey, D. M., Lanc ster, E., and Lang, G. E. (19 0) Methan consumpti in decomp sing Sphagnum-deriv peat. 4 1– 7. 2 5 . ( 1M 9e E tp .0hr )aoLG nd s u cgl , Bi Y vJ e . t o n two Ap al chi n peatl ds. 27 Limnol. 3, 18 – 9 . 3, J. Microbi l. Methods 23 – 40 13, FEMS icrob l. Ec 126 – 7 . 47, Ap l. Enviro M c biol. M i c r o b i a Pl r o d u c t i o an d Halo( R o g e r s J ,E .a n W d h i t m B e , s . ) , A m e r i c a Sn o t fy Mr i c o - 12, FEMS icrob l. Ecol. CH 2, 19, Bioge ch m stry 4 and N 51–60. 2 F u n c t i Eo a l . 13, Biol. Fert i y So ls 96–10 . 7–18 26, Soil B . ochem 45–62. 1, Bioge ch m stry 49, Ap l. Enviro M c biol. Soil Biol. Biochem. Bioge ch m stry 10, 81– 04. O 2 94 – 8 2, Sampling W ter Bodi s 29 3 Sampling Water Bodies Filtra on wT ge Roger Pickup, Helen Mallinson, and Glenn Rhodes 1. Introduction m c i o t r ua b sf n l v y d T h e DNA, or by the ap lic t on f luoresc nt olig uc e t d probes y fluorescent situn h y b r u v i n a d f l eot z s c m , g bial d vers ty n abu d ce in ra g of e vi nm ts. Howev r, n f the major lim ta ons to res a ch into microb al com unit es, and consequ tly the d ction f micro gan s i the nviro me t, is an i b l ty o is late and cult re h vas m jority f c organism . Bec u of th n rep s t a n c i u v ol e f r b h q s a m ,p “ lt i o ne c g ” u f y the pref d opti n. Soil is a dif cult mediu to proces and of ers many i m p a e s t d o r l hx f c , n u w v i p e yt wher as t i proba ly the m s a n ble m diu to sa ple nd roc s . The study of micr b al om unity s r c u e q ir s a rep s nta iv s mple o tf h a c m u n i y o pr c e s n ig fa tm h r e o c n a m i t s h wil nterf with e an lysi . Th s ection de ails the con ribut hat ngential f ow tra i n (TF ) ca m ke to his g al. T r a d i t o n l y ,c e t r a i o n fp c l e s( b i o g a n d b i o l g c a )w s car ied by eith r centrifuga o r “dea end” me bran filtra on (stand r noitar l f ;seuq hc lato eh n d c lp stim eh ra uqin c t s h fo ega n vd i su bO . r tl f emulov f elpmas t h nac eb ,d s corp yti a dn t euq sb gam d ot F T .enarbm ht no sl ec f p i r te o hl bc d sn w v a m u g (3) . Particles >0.2 From: )1( rednu m cav hg o t re i 2.0 ro -4.0 µ m erop zis )2( ygetar s vi n l a sref o tah µ m in diamet r a e con e tra d not by ret n io directly Methods in B otechnol gy, Enviro mental Monit r ng of Bacteria Vol. 12: Edite by: C. Edwar s © Human Pres Inc., Tot wa, NJ 29 b y p atsh e 30 Pickup et al. ag inst he f l r, but wi h n e vo d lum f the TF uni , wh c reas p a ir nt c u l o e p mw r a i t s f n T c h o d . l e a t soluti n (term d “ret n a ”) is retain d with n the unit by set ing up a b ck p r e s u O n c l. a t o h d , e f r i u s a tn g c h bo l e tainer.Th p t cula em rf o 10 –2 L lakew t rc nb du e to r e s u c obi p qn t h I d f a m, L . 5 0 r o vx l u m e a as lit e as 10 mL after c n ifugat o . This rep nts a con e of ap rox 10, 0-fold. The cel s are now amenable to direct DNA extract i o n f o l w e d b y p o l y m e r a s e c h a i n r e a c t i o n ( P C R ) a m p l i f c a t i o n . I n a d ition, enrichment cult res can be set up using the con e tra as an in t al inocul m. m i v c a r o f e t g yn uT sh b d i o includ g hematop i c necrosi viruse e n v i r om a t alg e from eshwat r (1 ) , and for the as m nt of r phic sta u of l kes of the ins r o lem nt, IS The ap r tus ed was the Mil pore P l icon cas et sy tem (Mil pure L W t a d c . fo ,U m K rpi ) l s u ( e n T g F t a , f i lr ate n s d ( v o f i l tm re ha bs o nu , c p t r a d l i f e o w , h b y creat d (5) s a m p l e t h f i sr u a c e o, n l y t i f r a c o n h me d i u ( p r ate; fil r d wate ) p s hroug t e m bran d is e th r col ed in th filtra e s voir discar e . Th ret n a (co entra d s mple) i rec ulated throug the ret n a res voi until the sample and then the ret n a res voi are empty. The ret n a is then col e t d from the filtra on cel . The fol wing section describ , as an exampl , the ap lic t on of TF as detail by Hiorns et al. water fo m nia- x d z g bacteri . tra ion factor (4) , pico- and na opl kt in the ( 5 , 6 , 7 ) a m o n i b - c tx e , rd z g (10) , protis from estuarin d marine vironme ts , Giard (8) cyst , (9) and PCR et c ion (12) . 1 (13) f i pt l uh mr c oea s dg T n ) . 1 Fig. . This flow ke ps the particles su pend in the ret n a . As the (8) tha invol ed sampling a defin depth of lake 2. Materials 1. 2. Oxygen/t mp ra u e ofil met r. 3. Sample tubing w h “T”-s aped inl t a d weight nd ( 4. 1-L steril Schot b le. 5. Two 50 -mL steril Schot b les. Mil ipore Pel icon tangential flow sy tems equip ed with a 3X Durapore microp us me bran cas et GVLP0 5 with a r ted pore size of 0.2 and filtra ionare of4.6m Fig. 1 . The sample was cir ulated by means of a Mil pore vari ble-spe d peristal ic t u bf p ( 3 i X 0 n mw 1 G ) g 8. 2 e s d h - c l t o u b ( i n g e r nal di met r). µ m 2 perm brane.Theunitwas etupas howni se Note 1 ). Sampling W ter Bodi s Fig. 1 Configurat of he tang i l f ow iltra on u it d r ng va ious t ge of operati n. res voi , perm at is col e t d; transfe to next sample res voi as requi d. When al sample r se voi have b n proces d, ret n a res voi s reduc . Perm at is u ed to flush t e final co e tra . 31 (A) Init al set up with sample input and ret n a outp into ap ro i te (B) (C) 32 Pickup et al. 6. A 240-V petrol g n at r. 7. Five sampl bot es (25 L). 8. Sampling bo t w h anc or d life jack ts. 3. Methods 3.1 Sample Co cti n 1. Secur sample bo t a s mple it by mo ring by a chor. 2. Car y out oxygen/t mp ra u e depth profile at sampling site and ident fy oxy/ thermoclin . 3. Con ect sampl ube/w ight v a perist l c pum to he TF unit. 4. Con ect pum o 240-V p wer g n ato d switch on. 5. Place s mp ing tube wa r. 6. Fil samp e tub wi h surface w t r and switch of pum ( 7. Lower sampl tube o r qui ed pth and s bil ze ( 8. Pump water nd iscar the void lume. 9. Col ect r qui ed vol m in prewash d 25-L contai ers ( 10. Stow a y s mple tub ( 1 . Return h sample for TF . ). Note 3 ). Note 4 se ). Note 5 se ). Note 2 se se 3.2 Tange ti l F ow tra i n 10. 1. 12. 13. 1. Set up TF sy tem wi h re filt s n he filtra on u it ( 2. Con ect sampling and ret n a tubing to unit and che k tha they are firmly secur d ( se 3 . I n s e r t a m p l u b ei n t o h s a m p l er v o i a n dr e t u b ei n t or a e s ervoi ( Fig. 1A 4. S w i t c h o n p u m , o p e n p e r m a t e v a l e , a n d s t a r p u m i n g s a m p l e i n t o t h e TF unit. 5 . r e t n h a o c l i p r v a e u s i n gb r 2 a d 1b e t w p r no s a uc k C e t tube o r gulate n ate ou p fl w ( 6. Col ect 1 L of perm at in steril bot le and retain for later use, the r main g perm at is d car e ( 7 . M o n i tTuFr a d s f e m ptlu b ao r es v ia q u r(e d Note 1 ). 8. After last ample r se voi has be n proces d, transfe the sample tube to he ret n a servoi and cl se r t n a v l e nd co ti ue p m ng ( 9. O n c et h r e t n a t er s e r v o i r se m p t y ,s t o p h e u m p ,t r a n s f e rt h es a m p l et u b e to the perm ate bot le col ect d earlier, and close the perm ate outp val e ( Fig. 1C ). P l a cre t n u bi5 0 - msLa p l eo t n d r e t a v l ( Slow y pum perm at throug the TF unit and col e t the first 50 mL of ret n a outp ( Col ect a s ond 50 -mL sa ple. Secur s w top n he bot l s and tore ic for ut e p oc s ing. ). Note 6 se ). Note 7 ; se ). Note 8 ; se Fig. 1A ; se Fig. 1A Note 10 Note 9 ). ). se ). Fig. 1B F i g1.C Fig. 1C ). ). Sampling W ter Bodi s 14. Clean TF filter cas unit a e rl st op r unity ( 15. Proces ampl s requi d ( 3 se ). Note 13 se 4. Notes 1. The l ngt of he ub is det rm n by the r qui d epth of sam ling. 2. Fil ng the ub with s rface w t r duces b oyan . 3. Stabil z ng ref s to al owing the sample tube to unfold an reach t e r qui ed d r v a n e g o t i s c u b l h o i ma t e p c u r s d I n h . with move nt f he boat. F r ive sampling, t s po ible t us a tel scopi pole at ched to the weight d sample tube to obtain water from midstrea or beyond. In ad it on, if sample ne d to be taken at depth, then suf ic ent pi e ne ds to x end b yo the maxi u pole ngth. 4. Contai ers w e rinsed with lake w t r o rem v any traces of det rg n from labor t y washing . 5. By rev sing the pum , the void volume wil be exp l d, making the tubing easi r to g he in. 6. Each filter s epar t d f om the uni a d e ch ot er wi h s l cone gask t ( uppm la b in y eu df c t r T g hs )i . e nq o u d t r F al i . g 7. The sample inlet ube con e ts he sample bot le to he unit, he r ten a pi e c o n e t s h u i o er t n a s e r v o i ,a n dt h e u l p i a o w st h ep r m ate o b c l e t d or isca de . 8 . W hr e nt ao u l( pi sw placed into the first sample res rvoir. As the sample res rvoir empties, it b e c o m e st h er t e n a t er s e r v o i r ,a n dt h es a m p l et u b ei st r a n s f e r dt o h en x t sample r s ervoi . 9. T h e T F p r e s u r e ( i n p u t a n d o u t p ) d e p n d s o n t h r e i n t e r l a t e d f e a t u r e s : the con e tra i of particul es, pum s e d, an the back pres u creat d by the val e/c ip on the ret n a pi e, henc ret n a flow rate. To maint the requi d pres u from waters with a hig con e tra ion of particul es (e.g , eutrophic water or alg blo m) wil requi hig er ret n a flow and slower p s u tm ce h I a ri n d . , o t p s u h lb e c a id r n res voi . F r olig tr ph c waters (low particul e onc tra i ), low ret n a flow (virtual y zero) and hig pum s e d wil ma nt i he pr s u e, and both sample nd r te a ubes can located in h same r voi at le s until he first ample r s voi empty. 1 0 . p T e h r m i q f a s u l t o d n g e h a ft l u i o n the proc du e. 1 . T p h r e s u g a o c b l e d n t i u s m o y t r a e n dw h k i p g s u r e m a i n t e d A. y l v a t i o n dr e p s c a bn e o r t d ay j u s i n tg h pe m s p r eo t d n av l H .w e i s m rt op , a n ud bi es t o r n , particul y on the sample input at he sanit ry clip to the unit. Any distor n should be al vi ted by reducing the pum spe d. TF wil stil conti ue f the pres u d op bel w 1 ar. 12. Filters hou d be cl an d i c or an e with manuf c re ’s in t uc o s. se N o9 t e tr )he , n a s dm p lu eb y Note 12 ). 34 Pickup et al. 1 3 . ( C u m b r iw a ,tE es h f o x d zm r n i ap l g u sC eo d t i n UK; [8] ) are as fol ws: sample depth, 8 m (oxycline); sample volume, 80 L; noitcarxAeN;D,1Lmulovtaner tah e r snom d t eic )7( RC;P )8( -ifusalwoctrepvbah.T tnem oriv ht n daerps iw e .p s aripso t N . )8( References 10. 1. 12. 13. 1. Hal , G. H., Jones, J. G., Pickup, R. W., and Simon, B. M. (19 0) Methods to s t u d y h eb a c r i l o g y f r e s h w a t n v i r o m e t s . 18 –2 0. 2 . L u d w i g K, a. n O s h u e y K, (. 1 9 8 T) a n g e t i l - f o w t r a i n : e c h a l revi w. Am. Biotechn l. 3. Pickup, R. W. (19 5) Sampling and det c ing bacteri l po ulati ns in natur l enviro ments. Society for Gen ral bridge Un v rsity P e , Cambridge, UK p . 298–315 4. Bat s, W. N and i to , J. R (198 ) Conce tra i of n ecti us h ma op iet c necrosi v u from wate s mpl by tange i l f ow iltra on d polyeth n glyco pre i ta on. Can. J Fisher Aquatic S . 5 . B S a c G r h . t n , K e i l (d 1 L C 9 J o 8 R g z ) r , t a f i n live pico lankt and na opl kt n by means of tange i l flow filtra on. Plankto Res. 1 , 12 3– . 6 . G i o v a n SJ,. D e l o n gEF c h m i d tTM,. a nP c eNR( 1 9 0T )a n g t i af l o w r t i a n p d e l m r yh o g e n t ia c l y s om f r np ei c l a k t o n . Ap l. Enviro M c biol. 7. S c h m i d t , T . M . , D e l o n g , E . F . , a n d P a c e , N . R . ( 1 9 ) A n a l y s i o f a m a r i n e p i c o l a n k t r m i 1b u 6o Ss y g a c R e l N n A i q d u g . Bacteriol. 173, 4371– 8. 8. Hiorns, W. D., Hasting R. C., Head, I. M., McCarthy, A. J., Saunders, J. R., ( A 1 m 9 p r H l i5 . g b) e f o 6 n G R s S Nca t , d W P . k u p a u ot f r p hm i c n a - o x d i sb nc gt efl r a ok m w d i n t s . biol gy 14 , 2793– 80 . 9 . H a s t i e J, C. K l y P a, n Bd r o w T J. ( 1 9 2 C) o n c e t r a i g water by tange i l flow filtra on compared with centrifuga on. Freshwat Res. 26, 275– 8. laitneg T )59 1( .J G ,strealA dn ,.N A ,nem rB naV ,.G reiloB ,. iksve urt P .eagl r taw hserf ta nec o t doh em a :noit r l f wo l a i r e d t c b u , ms i . f R n o y e r A dS p h q c a ) M 6H 9 P C 1 ( .seicn f htworg si p n tami se rof n itcud rp Morgan, J. A. W. and Pickup, R. W. (19 3) Activ y of microb al pe tidas , o x i v e d a ws rl t y n f k p , g h c u . 39, 796–803. Pickup, R. W., Rhodes, G., and Saunders, J. R. (19 5) Extrac ion of microb al DNA from aqu tic ecosy tems: Freshwater, in (Ak ermans, A. D L and V Elsa , J. D eds.) Kluwer, Th Net rlands, Section 1. 2, p 1– . 2, Methodsic r b ol. 7, 41– . Symposiu seri 52, Cam- Microb l gy 46, 964– 8. J. 56, 257 – . J. Microcystin Giard NZ J. Marine ,92 .seR r taW ,143 aigol b rdyH M i c JrC.oa bn l Molecu ar Microb al Ecol gy .42 1–9 .321– Diel ctroph si 35 4 Dielectrophoresis D. W. Pimbley, P. D. Patel, and C. J. Robertson 1. Introduction 1. Rapid M crob l gi Ana yse The incr as g t end owar qu lity as ur nce p ograms nd haz r n lysi , con umer d an for wide var ty of wh les m fo d , an legis t v pres u (e.g , the U.K Fo d Safety Act) have increas d the n d for m e rapid m crobi l g a n lyse . A thoug az rd n lysi and crit al contr point (HA CP) prog ams h ve r duc the mp asi on e d pro uct es ing, microb l g a n lyse hav particul ro e in e v ronm tal onit r g, valid t on, verif cat on, d ensur complian e t gisla ve p cif at ons (e.g , EC (European Com unity) microbi logical criteria extr m ly sen it v a d not par icul y apit l n e siv ( xcept wh r automation is requi d), the clas i , cult ra -b sed microb l g a techniqu s are time consumi g, labor-inte s v , and give result tha are only of ret ospectiv value. For exampl , a typical pathogen tes (e.g , i n c l u pd re - h m n st , l e c i v r h m n st e, l c i pv a g n od f r m tion ca ke up to 7 d c mplet . Sign ficant prog es has be n made in “rapid” techniques for microbiol g ca enum ratio and det c ion of pathogens tha reduc s the an lysi t i m q e u s g n f c a t l y F . o r h e s i m a nt f v l b e o r a t , h c n i qd t s u h a er c p f l o i n t e c r h ( q D u E F T ) triphos a e (ATP) biolum nesc fluor cyt me micro gan sm , techniqu s based on enzym -li ked im unos rbe t as y (ELISA), DN probes and lym r se chain t o (PCR), latex g u inat i o n ,e l c r a t h n i q u e s( . g ,B a c t o m e r M l h u s ,a n dR A B I T ) m e t a bolic “marke ”-b s d techniqu s are av il b e From: . Although (1) ) can Salmone (2) (3) (5 ) and biosen r (6) , impedanc . For the det c ion of fo d-p is n g (4) (7) Methods in B otechnol gy, Enviro mental Monit r ng of Bacteria Vol. 12: Edite by: C. Edwar s © Human Pres Inc., Tot wa, NJ 35 . Thes altern iv s to the and, more rec ntly, a d e, n o s i 36 Pimbley t a . clas i cult ra methods for the det c ion of micro gan s in fo ds are more apid, but are g neral y lacking in sen it v y, and most echniqu s for pathogens il requ a p riod f cult ra en ichm t of up 48 h. T ey ar also pr ne t i f renc om th p ne s of th ample nd s turp c a r t o hn s fgl ey i , m - bo r d t y get micro ganism , particular y close y phylogen tical y relat d speci s. b q P i u r A o e T la n s c m h g d p i t f a c n g u r o nD c e v t dE a s i h F T l - m f o n b i u a l e r s ; f a l s eir -n p o um ty P C v R h E b L f dI c S ai A sn o t e c h - m o d r n s f o b u t n e a dr l i s p y , T h e r u l t- n . g a i v o d r a e m f b t s i v nc l p h - q o u y d a r t i l o h s p m e wg d c f n o l x n mt e ah d r , i T cf os l . - n e t q a h u elim nat h cul ra st ge . A range of s p rati n ech qu s a be n r po t d ha exploits vary ng degr s, th cel surfa ch teris c ( .g , char e ntig c y, and h roi m u n o a g e t c b s drih lq u n o f gp m , c t y ) particles (8) biphas c y tem N u m e r op st n ia p l c a t i d o nfe s l c t r o p h e s iab v d n c r i b e d , including the s par tion a d manipulation f no biol gical nd biol gical particles. Biol gical p lications i clude th separ tion f viable and onviable y ast cel s ter focuse on the ap lication of diel ctrophoresi to the isolation and con e tra ion of micro ganism from fo d, bev rage, and environme tal samples. ,i o ne x c h a g r s i (9) ,d i f e r n t a lc i f u g t o n . (12) , can er c l s (13) ,a q u e o s (10) , and iel ctroph si (1 ) , and erythrocytes (14) (15) . This chap- 1.2 Princ ples of Di ctr pho esi Diel ctroph si has be n defin as the moti n of a neutral or charged b e i n g o f r s u l t a p o ru in zd e t g h a s c e l )m i r o b a( . g ,p t placed in o u f rm el ct i a f eld field r sult in a no u if rm o ce distr bu on the now p larized particle ( k n o w a s d i p l e ) ,c u n gt h p a r i c l e om v t w a r d h e g i o n f s t field nt s y ( The t ory has be n w l dev op by Pohl diel ctroph i f rce . The no u if rm ty o he l ctri (15) Fig. 1 ). , who as hown t a he (12) F d can be giv s: F d = (particle vo um ) · ariz b l ty ( oc fie d) · l gra nt (1) or F d= α V · ε∆ ·| E loca |· E loca (2) Diel ctroph si 37 Fig. 1 Princ ples of diel ctroph esi (ad pte from P hl, 1978). The ap lic t on of a n u iform el ct i f eld to a su pen io f microb al e s induce polariz t n of the c l s, whic t en mov t ward he gion f h g est fi ld rength. T is movement is det rmin by the di l ctr p o erti s (condu tiv y and permit v y) of the cel s and the usp ndi g me u , and ot simply he r c a g . For a sphe ic l trode g m y: F d= ε · V )/ r 5 (3) and for cylind a geom try: F d= ∞ ( ε · V )/ r 3 (4) 38 wher the volum f the par icl , nd field (sharp) ect od . Pimbley t a . ε d i t f h p e r s m n c b v a w y t di ul e , It can be infer d from thes equations tha the diel ctroph etic force: increas with particle siz , s tronge with cylindr a r the an spherical particles, nd r ase pidly w th s ance. Unlike el ctroph si , in whic move nt is large y det rmin by the o v e r a l c h g nt ep r i c l ,d e t r o p h i cm v e n t saf u i o h e d i e l c tp r o ( sn d u c t i p vae r m y h s o u f cn - ) ld pendi g me u . The di l ctr p o erti s of a m terial e char te iz d by the sp cif l tr ca ondu tiv y (or el ct i a s vity) and he p rmit vc o Tn ihd teu y f . a r s v g mb port mechanis w t he ma ri l nd ca be t rmin d by easuring th cur ent tha flows throug a sample of the materi l at a given voltage. The permit v y of a materi l gives a measur of the abil ty of the posit ve and negativ ch rges with n a m teri l o separ t (i.e , polarize) und r the f ct of an p lied l ctri f eld. The p rmit v y ma be found by measuring the el ctri a c pa it n e of an el ctri a ch mber tha is f l ed with a s mple of the material. The diel ctric pro erties of fluids are strongly temp rature d e c p o n s u mt r a i , v b y f d l as functio emp ratu . s u r t h o e f n l d i g , a p s m r c t o Ie f v y then body wil move t ward he r gions f hig est l c ri f eld int s y (know as posit ve diel ctroph si ). Convers ly, if the permit v y of the f o r c e d b pw ai l t h s u r feo n d , g t h a n l e s pi r t c toward the regions of lowest el ctric field inte sity (know as negative diel ctroph si ). Thus, both p sit ve and egativ d el ctroph si can be used to par e mic ob al e s from d su pen io a d ther m ic s, by m a n i p u l t c h og e d i v s uf y p n m e g d i a t h r q u n o c f y the ap li d ctr field. l a n r o g e u i s yf c t p dm a l e o vA f t h n u g det rmin by the fr qu ncy of the l c ri f eld an co du tiv y of the su p e n d i m g u t h, l e c r i a t s oc phf ae r i l n u c te s d i e l c t r o p h bi e a v o p Fr . t c m l i e g a ho fn - i e l d inte s y, it must exhib t a hig er specif polariz b ty han the su pendi g mediu . In microb al cel s, various cel surface comp ne ts (e.g , proteins, polysac h ride , and teicho acids) and intracel u comp ne ts (e.g , proteins, ugar RNA, nd D ) contribu e th overal p iz t on. S ce th c o n t r i b u h fe s m p o n w tiv la r c y h s o g ,enp d cies, p fic el typ s give a ch r te is c ol e ti n sp c rum ove a r nge of requ nci s V is r is the dis anc of the par icl f om the ig - (15) . Diel ctroph si 39 1.3 Design of lectr pho si C amber 2.1 Two-Dimens al (2D) and Thre -Dim ns o al (3D) ie ctroph i C ambers The simple t form diel ctroph i chamber consi t of a p ir of el ct r s o u d e ph i n E c a l .m yw b r s t u i e h d w i r eo p n l c t d e s (15) raphy ave m d pos ible th constru i of multie n , paired l ctrode c e s r u a ob gm l i t T d h p y . , n are con e t d to a hig -frequ ncy power sup ly to gen rat he no u if rm el ctri field. Plate 1 show the diel ctroph i col e ti n of lute s f r o sam l v u (e 1 0 d i g t a e l c r o d m T i h y . b e at l p s c o y h r d e s in lo g cha s know “pearl ch ins” ( Tois latemicro ganism fro la gervolumesof dhom genat s nd tm oa x i zt ehd l c t r o p h e t ic o l e t i o n s c e a rt yo n s u cm to r e c o m p l f e x w - t h r o u c g a m b e r T fs h l. o w - t r o u d g i h e l c t r o p h e s i y t m used in our laboratory is shown in phoretic chamber con ected to a peristaltic pump (which circulates test sample through the chamber) and a high-frequency power source. In a f l o w - t h r o u g hd i e l c t r o p h o r e t i c h a m b e r ,t h e r a et h r e f a c t o r st h a t f e c t the diel ctrophoretic col ection of microbial cel s: the diel ctrophoretic force pul ing the cel s toward the high-field region of the chamber, the fluid flow rate acting at right angles to the diel ctrophoretic force, and the ionic composit on of the medium. Thus, the diel ctrophoretic field must be suf ic ently strong to at ract and hold microbial cel s within the chamber. In theory, the diel ctrophoretic field decrease as the inverse fi th power of the distance from the el ctrode; more ef ective separ tion of micro rganism is achiev d only with closely spaced el ctrode ar ays. In our laboratory, we have used chambers contain g el ctrodes of a few microns in width and spacing to achieve the high-field strengths. With a nonoptimized version of a flow-through dielectrophoretic chamber, up to 90% of (<20 µ S/cm) aqueous su pension can be col ected in the chamber and eluted into a smal volume of buf er (Pimbley, D. W. and Patel, P. D., u n p u b l i s h e d a t ) .O t h e rf a c t o r st h a tn e dt ob ec o n s i d e r di nt h ed s i g n of diel ctrophoretic chambers include the avoidance of heating, min m i z a t i o n o f dead flow and turbulence, and elim nation of el ctrolytic ef cts. Fin te el ment an lysi can be used to model the field and field diverg nce with n pre ot ype di lectroph retic hambers and to identify the optimu el ctrode configuration. ,b u ta d v n c e si m r o f a b c t i n dp h o l t g - Micro us µ L o) af n q u e s p i o un ag t e r Plate 2 Fig. 2 Escherichia coli ). . It comprise a 3D diel ctro- in low-conductiv ty 40 Pimbley t a . Plate1.Di ctroph e ic ol t n f el ctrod a y t 10 KHz, 9 V ( Higher ap lied voltages al ow a faster flow rate to be used and, henc , enha c diel ctroph i solati n of micr o gan s from aque s pens i o nM . re f c s t p a i o n l b ec h v rdy u i n“ g e as p c ” betw n el ctrod s. The “p nalty” of hig er voltages i tha ey can le d to Micro usl te × 50 magnif c t o ). usinga terdig a Diel ctroph si Plate 2. Diel ctrophoretic col ection of dig ta ed el ctrode ar ay at 10 KHz, 9 V showing ”pearl chain” formations ( × 10 magnif cation). exc s iv heat produc i n the c amb r. Othe fac ors, u h as t e chamb r dimens o , c nstru io materi ls, and the ig condu tiv y of the s u pensio , ca l inf ue c h at produc i n. 41 Microc cus luteus using an inter- 42 42 Pimbley t a . Fig. 2 System for h diel ctroph e ic ol t n f micro gan sm . Diel ctroph si D i e l c t r o p h i l e c t o n mf r g a i s c bn me u r id v a o s ways. One of the simple t is to measur the length of the “pearl chains” of b a m c i t ue r s o n g dp spectro h m e to asur ptic l dens y, ith r w n e chamb r o in the outle tube 8 cfu (>10 measur ch lowe n tra io s f cel 43 (15) A n a o u t. s ph e r i c , but this requi s hig con e tra i s of microb al cel s (16) /mL). More s phi t ca ed im g an lysi tems hav be n us d to (17) . 1.3 2 Traveling-W D el ctroph i Electrod A ays In a conve ti al diel ctroph i chamber, the sample su pen io conm i c r t o h e l ga n d - y p v, s i m dt ir ea lv c n og ,p- hw I e l u t d . h n a c o l e t d r i s m m i c r o g a n s ep r o l dt h u g as i o n r y , u p t i gf l db ya p ing d f er t phas of hig - requ ncy l tri a s gn l i equ nc to al ern a t ep i r so f l c d e .T h v l o c i t y f ep a r l sd n o t h e i l c r f o r ec x t b hyd l o e ws , i t c unh dr p o f e l i s t r b u o n and the i l c r p o e ti s f he particl s nd he flui . D d cate in rf c el ctroni s a d oftw re hav be n d v lope t drive th s el c rod a ys. Traveling-wave diel ctrophoretic devices have be n used to separ te micropa t les micro nvey b lt sy em fo d an lysi h ot ye b n valu ted. (18) b s h e u a gi n t d , c o f y l r m (19) . Howev r, th po en ial f thes d vic for 1.4 Factors Af e ing D l ctroph esi f M cro ganism The ef ic n y of diel ctroph i col e ti n of micro gan s is influr a t e , f l o ws v m p q i u n gc y d r b e s a cm op nl de u t i (v ry t e m) p, r d a i h u l n c t e r i s m i t c h er xo F ab f dl s p w . u , n Gram-neg tiv (protein a d lipo ysac h ride) and Gram-posit ve (t icho p ea nc tdi o g l y bh x v s ) e p a t r d co fu ria (20) v ai n o db l e c f s separ t d u ing comb ed i l ctroph esi and l ctro a i n The frequ ncy over whic micro gan s can be col e t d by diel ctrophoresi anges from ap rox 1 kHz to 20 MHz. Over this range micro ganism exhib t typical frequ ncy-d p e t col e ti n profiles ( can be us d, to s me xt n , o res lv mixtures of pure c ltures of di er nt c t e y l s p g r ,o a iI u b nm f . h e l s r a o l v e y cr s m p , t u o w i f l n a r s e c h y v T d . maxi u col e t n ra o m l y c urs at f eq ncy arou d 20 kHz, b t this can vary with e type of rganism and cult ra sta e. Diel ctroph i c o l e t i n r a s e w t hi n c gv o l t a e ,b u g sa b o v e2 0V ,h t - hb ae v n Sac h romye svi a (21) Fig. 3 . ), whic 4 Pimbley t a . Fig. 3 requ ncy-d pe nt di lec roph tic ol e ti n prof les r va ious b cteria. i n ge f c t sw h e a m b rc n d u et h o l c i n f e yb c a u s i n g t h e r m a cl u n s , td h e a i g l ro u c e ts h v i a b l oy f e c t md i r o r g a m c n tbl i h u e s w p , d r a co ling b th r Pel i d v c . 1.5 Techniqu s for Red cing th Perm iv ty of Micr b al Suspen io As previously ind cate , the di lectroph i force is trongly influe c d by the ion c streng h (condu tiv y) of the m diu . Highly polar sub tance (ind cate by hig perm t vi y), such a lts, re a ct d o he r gi ns of hig -f eld nt si ya , fthe r p s nta hig co entra i s,p ev nt h bacteria from col ecting at the el ctrode. Since microbi logical an lysi invol es th su pen io a d growth f targe mic o rgan s i h g -condu w a t e dr ) i o n z l u ( pe r. g t , a m n o f s m e d i a , b r o t h v y r ie sq u t od ch l r p o e ti n c f r d o ms l v ea tI n . tes with a typic l no se ctiv broth cul re m diu an two sel ctiv media, t w s found tha 1: 0 dilut on f he brot s wa equir d ( valent to a condu tiv y of <10 bl ed u o c a i r e t c b e h t f o n i t c e l o c i t e r o h p t c e l i d t n a c i f g s m i c r o g a n s f e t d i l o c a pr s hn v e F o t c u r h e, l a i p v m t so h af ey l u p n i m b os wt e h r a tha of e microb al e s (10 – ). This can be hi v d n arious w y , includ g dilut on i deion z water, desalting, deion zat , and, pos µ S and relativ permit v y of <10 ) befor any observ( d ). F i 4g . sibly, Diel ctroph si 45 .giF .4 tcef E o n itul d fo er ubnotp re aw )WPB( ni dez o r taw no dna ,seitr po cirt el d )A( )B( .V 01 ,zHk 0 1 ta iretcab suoirav fo n itcel o citerohp l id 46 Pimbley t a . t h e u s o f r q n c i e s a b o v 1 M H z . w e r , s i n c d l u t o a s r e dh u c a fo ytiv sne ht cuder of eht dluow na sm i gro cim tegra ht fo lev .der f p si hcaorp evitanr l a ,metsy noi cet d sab- i erohp tcel id Wehaveinvestigatedar ngeofdesaltingtechniques,includingdialysi cas et es (Slide-A Lyzer, Pierce & War iner), dialysi chambers (Spin Biodialyser, Sialomed Inc.) and min columns of desalting gels (PD10, Pharm cia). Dialysi cas et s and chambers are simple to use but relat i v e l ys o w ,d e s a l t i n g5 – 1 m Lv o l u m e so f dh o m g e n a t e si n3 – 5hw i t go d recovery of the to al microbial flora (>95%). Columns of desalting gelsaremuchfaster(5–10min),but hepro rtion fto almicrobialf ora eluted in fractions with low conductiv ty is relatively low (<20%). To addres thes problems, a novel, rapid esalting technique based on dialysi c h a m b e wr d s v l o p e cd a, b rol ef d u c i tn hg oe d u c t i v 5 uo - pfy m L v o l u m e sf 15 min, without sign ficant los of micro ganism (Pimbley, D. W. and Patel,P.D ,unp blished at ).Thisproces alsoclarif esthefo dsu pension. The resulting microbial su pension has be n shown to exhib t hig recoveri s when subject d to diel ctroph retic proces , as il ustra ed in the fol wing section. af o ds u p e n s i o n t a r i n gf r o m> 2 0 µ S / c mt o< 2 0 1.6 Ap licat on f D el ctroph si Micro gan sm can be roadly categoriz d as u ef l (e.g , star e cult re b a c t e r i p) , h o g n( c. colif rms). Diel ctroph si has poten ial p licat ons for the s par tion f al four groups from enviro mental and fo d matrices. The adv nt ges of m i n ) , ( < 1 5 r a p d s i t h a s e p dr c o n l q i u t v s and tha it can be used to separ t micro ganism from relativ complex m a t r i c g e o ls v u p n i t a b f l o e r y m s d n t c i o g e s n y d r a i t c l m o pA , h u . b e n s i microb al f ora (includ g pathogens a d spoilage micro gan s ) from the s a m p l e r i o t n y s u g oe tf h m d r n c i o s y t e m ( . g A, T P biolum nesc ). Alterna iv y, ther ap s to be p t n ial for the sp cif d i ep la t u m ch s o rg n , f i antibody-c uple molecu s to modify the diel ctroph i behavior of the micro gan s (Pimbley, D. W and P tel, . D unp blished at ). Dif er nc s in the c l surface har cte is of subpo lati ns of microorganism caused by injury (e.g , by heating, fre zing, or chemi al dam ge) can be exploited in the separ tion of micro rganism using diel ctrophoresi . Ther is also evidenc tha micro ganism can be char cterized ac ording to seit v cudno a Salmone their ns oe cil t f r pa h u eq o t if c d l )2 ( . , Lister a s)p,o i l a g en d c t( r. , µ S / c mi n Diel ctroph si 47 2. Materials 2.1 Separ tion f T al Microb Fl a from ds 1. Diel ctroph e ic l trode chamb r ( 2. F u n c t i o n g e n r a t o r w i t h a f r e q u n c y r a n g e o f 0 - 2 M H z a n d a v o l t a g e r a n g e of 1–20 V (e.g , Thander TG20 1, Thander Electronics Ltd., Hunti gdon, UK) ( se Note 2 3. I m a g e a n l y s i s y t e m , c o m p r i s n g a l i g h t m i c r o s p e ( e . g , O l y m p u s B H - 2 Olympus GmbH, Germany), a video camer (e.g , Hitach KP-C50 , Hitach Denshi, Toky , Jap n), video tex overlay (e.g , Linkham VTO 23 , Linkam, Tadworth, UK) and visu l d p ay unit (e.g , Hitach VM-920K). 4. Conductiv y me r ( .g , Horiba C172, Horiba nst ume , Kyot Jap n). 5. Peristal c pum ca ble of handli g up to 1 mL · in Gilson I c., Mid leton, USA). 6. Magnetic s r e . 7. Sample ho g nizer ( .g , Stomacher 40 , S ward Lt ., ond UK). 8. Steril d u nt (e.g , Maximu recov y diluent, Oxoid L ., Basing toke, UK). 9. System for stima ng u bers of mic rgan sm ( 1 0 . g lC G ao p s I r ( nWe - thf . i , b L m d l Maidstone, UK). 1 . Dialys ca set (Slide-A Lyz r 10K, Pierc and W r ine Ltd., Chest r, UK) , a i b S m C u - hl). d 6 to c e n 0 w I , za r yi o p e B S b ( m s a h c y l r i d .senarbm t no racyl p Note 1 se ). ). –1 se (e.g , Gilson M ipuls, Note 3 ). µ m 2. Diel ctroph si n E v ro me tal M ni or g e t i s a nr op z t e hr Tp -moc t na si ersidnaesod vitcefniwolas hta ,negohtapn muhenrob- etaw s d o h tn e mi c t en d r . u Co i t a n r o h lsc aud tn eo mi c f n i sd ey l un o m -vel wol ta nes rp netfo si msinagro eht esuaceb lbai ernu dna gnimusnoc-emit era ,dipar A . etaw fo semulov egral fo n itar lif eht gni at s ec n , oitan m tnoc f sle - , o d t e u a r tn -o ei f a z ot n m e r s i f hy t d o p r t c e d l t i a m o u a fo st yco deta r nu d a ,devalc t n a c id fes z n ag i o sc e r m u v r a ip d r o p s t y r C debircsed n eb sah retaw morf muvrap .C 1. Diel ctroph e ic amb r ( 2. Micro mpute o s t pul e vo tag nd frequ ncy ap lied to he l ctrodes an S eA t iX k2 (ma o,PE np c - C . du sg h r w l si, Jap n). 3. P u l s e / f u n c t i o n g e n r a t o r ( e . g , H e w l e t P a c k a r d 8 1 6 A , H e w l e t P a c k a r d , Englewo d, CO). 4. Peristal c pum (e.g , Gilson M puls 3). 5. M i c r o s c o p e w i t h f a c i l t y f o r t r a n s m i t e d a n d r e f l e c t e d l i g h t ( e . g , N i k o n Labphot-2, Nikon Corp., Toky , Jap n). 6. Solid-sta e col r ame ( .g , Hitach KP-C50 ). 7. S-VH video cas t reco d ( .g , NEC DS 60 K, Yoky Jap n). se Note 1 )32( ). . 48 Pimbley t a . 8. 0.5-m 9. Stock su pen io c ta ni g p rox 10 M sodium ecyl su phate (SD ) igma-Aldr ch Co. Ltd , P ole UK). 8 –1 o cyst mL C. parvum . 3. Methods 3.1 Separ tion f T al Microb Fl a from ds Agen ricp ot lhasbe nd v lop f rthes a ion ft alm crobi flora m d s outline and et il x . Fig. 5 1. .rezin gom h ralim s ro ehcamots gnisu t e lid rets ni 4:1 elpmas zinegom H 2. U s i n g a s t e r i l e s y r i n g e , p a s 1 0 m L o f h o m g e n a t e h r o u g h a s t e r i l e , c o a r s e (50µ m) glas -fiber filter (e.g , Whatm n GF) to remove particulate mat er. 3. T r a n s f e r a l i q u o t o f t h e f i l t e r d s a m p l e t o a d e s a l t i n g d e v i c e ( e . g , 5 - m L Biod alyser i s chamber o 5- L dialys c et ). 4. D i a l y z ea g i n s t1Lo fd e i o n i z e dw a t e rw i t hs i r i n g( b o t hd e v i c e sh a v em a g nets for use with magnetic stir ers) at 20 10 µ S/cm (2 to 3 h). 5. T r a n s f e r a 5 - m L a l i q u o t o f d e s a l t d h o m g e n a t t o t h e d i e l c t r o p h e s i s y tem r se voir and reci ulate hroug the c amber fo 15 min at flow rate of 0.5 mL/ in with a signal of 20 kHz, 20 V ap lied to the el ctrod s from a functio ge rato ( se 6. Elute h col e t d bacteri by turni g of the l ctri a signal d flushing the chamber with 0.5 mL of steril d uent. 7. Analyze th r sul ing c ar f ed su p n io f m cr o ganism ( m f i l c o r w d- t a eh3b D u U g s n p m ri c previous pr toc l, we hav demonstra he rapid se ar tion f to al microb i a lf o r ms u p e n i o fv a r u s o d .Ap a t e n ,r i d s a l t n g e c h nique >20 betw n 8 and 94% of the otal microb al f ora f desalt hom genat s of chi ken, m c d be f, and skim e lk powder (SMP) was col e t d in the c h a m b eTd r . s l t i n g c r o p h e sa il m v dr t c u a e , leaving c r su pen io f m cr ganis . s m i n a g r o c e l pf t h d T sitcal 057( D2 citerohp l d s bma ,ye iP( .D W dna ,let P . , D dehsilbupn fo ser p ,yl a im S .)zHM 2( cn uq g s reh i )at d ,suebm lp roc M ,mudi na ,zHM 02 fo ycneuq r a t ,re w d l its n 01 i detul ,k im ohw dez ru t .sel cim n a k ht o f r p d iw ° C until conductiv ty fal s below ). Note 2 ). Note 3 se was used to rapidly reduc the condu tiv y of the sample from (24) µ S/cmtobe w n41a d59 µ S/cmwith n15 . show t a Table1 secymor v ulK dna re b gal ytiv cudno -hg ,deta r nu mo f asonigure a om duesP µ 065( retaw l nim d a )mc/S µ cita s n detar s om n b osla h )mc/S muhcirtoeG dna muil c neP -sap morf deta p s n b evah .p s Diel ctroph si 49 Fig. 5 Outline prot c l for the di l ctroph e ic s par tion f to al vi b e microbial f or m f od h genat s 20 kHz and 20 V. 3.2 Prepa tion f U reat d C. parvum 1. m 5.0 fo L 2 ni st yco f n is ep u kcots a f Lm 1 dnepsuS 2. Centrifug o 10 min at 2,0 3. Draw of supernat d resu p nd el t in 4 mL of 0.5 m Oocyst M .noitul s SD g . M SD soluti n. 50 Pimbley t a . Table 1 Diel ctroph S a i n of T t l M cr bia F o from Chicken, M d B f a Skim e M l Powd r (S ) Hom genat s Af r R pid De alt ng Par met s Chicken Star ing co du tiv y >2,0 Minced b f SMP >2,0 >2,0 ( µ S/cm) Final co du tiv y 41 54 59 ( µ S/cm) Total vi b e count 1.3 × 10 4 1.6 × 10 6 4.8 × 10 7 1.5 × 10 3 1.9 × 10 5 3.1 × 10 6 a (cfu/mL) Befor di l ct oph resi Total vi b e count (cfu/mL) After di l c oph resi % depl tion a 8 8 94 cfu = ol ny rmi g ts. 3.2 1 Prepa tion f Au claved Oocyst C. parvum 8 1. Autoclave sm olu e f st ck u pensio f cyst (10 /mL) at 12 10 min. 2. Proce d as for unt ea d o cyst ( ). Subheading 3.2 3.2 Prepa tion f Oz e-Tr at d Oocyst C. parvum 1. Treat 10-mL sa ple of t ck ys u pen io w th 3. mg/L of z ne. 2. Centrifug o 10 min at ,0 3. Draw of supernat d resu p nd el t in 4 mL of 0. 2 m g . M SD soluti n. 3. Diel ctroph i D f er nt a io f Untrea d and Tre t Oocyst ( C. parvum 1. 2. se Transfer tes su pension of o cyst to the diel ctroph resi sy tem sample res rvoir. Pump sample through diel ctrophoretic chamber for 10 s at a flow rate of 1.5 mL/min. 3. Reduc flow rate 0. 5 mL/ in a d p ly signa t frequ ncy of 1 kHz. 4. Remov signal d conti ue p m ng for 5 s. 5. Increas flow r te 1.5 mL/ in for 5 s. 6. Increm t f quency. 7. Rep at step col e ti n sp c ra ( 8. On completi n of exp rim nts, replay video r c ding a using d tal fre z frame cil ty o un cyst ol ec d n a betw n l c rodes. 1–6 at signal frequ ncy betw n 1 kHz and 50 MHz to gen rat se Note 5 ). N o t e 4) ° C for Diel ctroph si 51 3.4 Fut re P osp c In1978Pohl Certainly great stride have be n made in the technol gy; in particul , the h a v e s y t m i g n o p c a l r f db e t i , s v n led to sign f ca t improve nts in the f ic en y of diel ctroph i chambers and et c ion f the s par ted icles. D pite h s adv nces, th ful poten ial of diel ctroph si as a techniqu for separ ting micro gan s from d an e viro m tal s p e h y t o be r aliz d com er al y fo v r a e oi sf m t n y d , l w p hg e a r i t A so c n . -d l i e t m r h a ,p n c o s l i t d n h g e c r o p t i l e c m o f n r o r g a n i s m ef q u n c y , o d t i v a n p e r m t i v y .M o s m c r b i l g a techniqu s vol e su p n io f the s ample in a h g -condu tiv y solus i g n mc f a r w e u h d l o t b ( ) y ,. i n r a n g e M. H z pd 1 oi – s l 0 t c f v r h qb u e an w i o In ad it on, the ighly particul e nature of s me sample can i terf with col e tion by blocking the diel ctroph etic chamber. A novel and simple desalting procedu has be n dev lop d to overc m thes problems Another pos ible soluti n to the problem of hig condu tiv y is the use of n e g a t id v l c r o p h e s ia n , m t o b r h v e g d - c n ductiv y sample , but has no ye b ful y exp oit d. Despite the ap rent lack of com ercial inter st in the ap licat on of d i e l c t nr vo p h m a bd s i g l ro e y , inb omed cal biote hn l g ca p i t ons, ucha edi l troph e ic m a n i p u l s t o b f c r i ( e . v s g l , u k t a h dI y ) n c m o d i - t f e c r h p n q v wu s l oi d g a m , b e r t c f y d t i h n e g l c r o p t e i s m n c f r o g a dw t l ei hs v , s e p a r t i o n l - m h f o r d i e l scyt pmh n- b a o f e t and et c io f m r ganis from d an e viro m tal s p e . 4. Notes 1. Diel ctroph esi c amb rs e not av il b e com r ial y. The c amb rs u ed a Lt e h r Fd o R wA e s i g n ad f b r c t e d Ey R TA h n o l g Ly t d . The Univ rs ty of Y rk (D . W Bet s) and U ivers ty of Wales, B ngor (D . R Pethig) also h ve xp rtise n h co stru i n of d el ctroph e ic sy t m . 2 . T h eo u t p f T h a n d r G 2 0 1f u n c t i o g e r a i s d e q u a t f o rs m l v ume (<0.5 mL) diel ctroph e ic hambers. Large diel ctroph e ic hambers with more ext nsiv el ctrod ar ys may requi a functio gen rato with a hig er pow ut . 3. The majority of bacteri l cel s are rel as d from the el ctrod s as so n as the e l c t r i s a g n e ml o v H d . w t ar h , e i 0 o fT1n/ w% v t- h8 e diluent may i prove th covery f mic o rgan sm . (15) predict ab gh fu re o bi l g ca d e troph si . (24) . 52 Pimbley t a . 4. ,P gT nA i t eu o ac l d p s m n i t b e a g r o s nwcl ip hm x Fe biolum nesc , and el ctri a mped nc , Steps 2–6 are cont l d au om tic l y us ng the micro pute . The dielectrophoretic response of has be n shown to be dose dependent, and consistent with a decrease in internal conductivity predicted by a mathematical model for two-shel spherical particles 5. 6. refs. se 25 , resp ctiv ly. o cysts treated with ozone C. parvum (26) 3 , 4 , and . Acknowledgments The aut ors w ld ike to hank ERA Technol gy f r design a d f bricating he di l ctroph esi c amb rs, D . Patrick Mu phy (Teag sc) nd Dr. Fabrice P lad n (Dano e) for the sup ly of microb al isolate , and the U.K Min stry of Ag icult re, F sh i and Fo f r undi g the work at Le h rhead Fo RA pres nt d h i . References 1. Ano ymous (198 ) Council directive on hygien and health problems af ecting the production and the placing of eg products. OJ No. 32 (L21 ), 7/2 , European Com is on, Luxemborg, p . 87–10 . 2 . ( 1 D r 9 E P e 8 d F . c B T ) v R : a n t lJ s - , L G r P K e o . t i l f , opments for ds an bev rag s, in B e v r a g Pns hd m c e u t i a l s e d S s o ) c A , i f p t r l B y a e o T g c y h n i S B e 2 la r N 5 . k s w c n tif c, Ox ord UK, p . 3 –46 Rapid M crobi l g ca Methods f r Fo ds, ( S t a n r JCd P. , e i BSt a n k e AFr . , 3 . K y r i a k d e sAL ,. nP t l D( 1 9 4L )u m i n e s c t h n i q u ef som r c b i - 4. 5. 6. 7. 8. logica an lysi of fo ds, in (Patel, . D ed ), Chapm n d Hal , Lond UK, p . 196–23 Arnot , M. L (19 3) Impedanc mi rob l gy in fo d quality con r l, i I n d uF so t r y h e fS n m a sd t i o Hein ma , Oxford p . 49 –520 B r a i l s f o r d ,M .A a n dG a t l e y ,S .( 1 9 3 )I n d u s t r i a l p l i c a t i o n s f l o wc y t o m etry for the rapid det c ion of micro ganism , in and Bev rage Microbiol gy Seri s No. 31 (Krol , R. G., Gilmour, A., and Sus man, M., eds.), Blackwel Scientif c, p . 87–10 . Wagner, G. and Guila t, G. G., eds. (19 4) Dek r, N w Yo k. Patel, P. D., ed. (19 4) and H l , Lond UK. Patel, P. D. and Blackburn, C. de W. (19 1) Det ction of fo d poisoni g agents using im unomagnetic particles, in Ap liedtoCel u ar ndMolecularBiol gy Broughton, Gilford, UK, p . 93–105. Rapid Analysi Techniqu s in Fo d Microb l gy InstruB( uK tr e d s . ) w- , E oR g h New Techniques in Fo d , Society for Ap lied Bacteriol gy Technical . Marcel Fo d Biosen r Analysi Chapm n Rapid Analysi Techniqu s in Microb l gy. Magnetic Separ tion Techniques (Kemshead,J.T ,ed.),Cromwel , Diel ctroph si 53 9. Patel, P. D , Wo d, J. M , and Gib s, P. A (1983) Physico- hemical interaction fmicro ganism with ecationexchanger sinB o-Rex70:aprelim nary ap lication f the t chnique to f ods. Rep. No. 437, Leth r ead, UK. 10. Rodrigues-Sz l , U. M , Ventoura, G. Mackey, B. M , and Payne, M. J (19 6) p h yR sa i c od e tm b f n r p l a , i o be f sur ac . 1 . m i c r o d e g f nt a sp , T h i ( o 1 9 4 ) P . R B e t s , Methods and Automa ion in Microbi logy and Im unol gy Wright, E. P and Newsom, S. W B eds.), Ath naeum, p . 107– 2 12. . s l e c f o s i e r o h p t c e l i D ) 1 7 9 ( . S J , e n a r C d n a . A H , l h o P 13. Markx,G.H Tal ry,M.S andPethig,R.(19 4)Separ tion fv able nd o viable y st u ing d el ctroph esi . 14. Beck r, F. F., Wang, X.-B , Huang, Y., Vykou al, J., Gascoyne, P. R. C., and Pethig, R. (19 4) The r moval f hum n e ka i c l s from b d using terdig ta e m cro l t des. 15. )8791( .A H ,lhoP 16. -erusa m of euqinhc t la i po nA )7891( .R ,gihteP dna ,.H P .J ,truB .R A .J ,ecirP -celE no ec r fnoC .t I h 7 .corP 7891 scita or elE .si erohp tc leid c fo tnem .97–5 p ,KU drofxO ,gnihs lbuP OI ,).de L J ,nots rpS( anemo hP cita sor 1 7 . G a s c o y n e P R C, . H u a g Y t h i R , . V y k o u a l J n B d e c k r F (, . 1 9 2 ) Diel ctroph e ic s par t on f ma li n ce s tudi by computeriz d mage an lysi . Measur m nt Sci. Te h 18. Hagedorn, R., Fuhr, G., Mul er, T., and Gimsa, J. (19 2) Travel ing-wave diel ctroph esi f m cropa ti les. d n a o i t a l u 1 9 p. i n a m o r t c e l E ) 6 9 1 ( . R , g i h t e P d n a , . A J , e m a T , H . P J , t r u B , . S M , y r e l a T .sdleif cirtcel gnil evart gnisu l ec fo n itar pes 20. Markx, G. H., Huang, Y., Zhou, X.-F, and Pethig, R. (19 4) Diel ctroph e ic char te is on a d sep r tion f m cro ganism . 21. Huang, Y. Holze, R. Pethig, R. and Wg, X. B (192) Diferncs the ac combinedthrug lsyeano-vibdlfectroynamis studie.lcroanph 2 . Markx,G.H Dyda,P.A nd ethig,R.(19 6)Diel ctroph e ics par t on f bacteri us ng a co du tiv y grad ent. 23. Archer, G. P., Bet s, W. B., and Haigh, T. (19 3) Rapid dif er ntia on of untrea d, utoclaved n oz e-tr a d diel ctroph esi . 24. Patel, P. D and Pimbley, D. W (19 5) Diel ctroph esi . PCT aten Ap lication N . 950346 . Filed by UK MAF , Lond UK. 25. Bacteriol g a Analytic Manu l. ed., AO C Inter a io l. 2 6 . Q u i n ,C .M A r c h e ,G .P B t s W . ,a n dO ’ N e i l ,J .G ( 1 9 6 )D o s e - d p n dent diel ctroph e ic respon of Let . Ap l Microb l Leather ad Fo d Res. As o. Res. . 80(6), J. Ap l Bacteriol 673– 81. Rapid (Spenc r, R. C., . ,1 J .syhpoiB 29–37. 32(1), J. Biotechn l. . 27, J. Phys D: Ap l. Phys .72 –1 7 2659– . .KU ,egdirbmaC s P yti rev nU gd bmaC .si erohp tc l D . 3(5), 439– 5. , 49–5 . 13( –2) Electroph si . ,92 .302 –8912 syhP .lp A :D .syhP .J . 37(), BiolMed.Phys 51(2), J. Biotechn l. 165– 72. (19 5) Fo d and Drug Admin stra o , 8th Cryptos id um . 2 (3), 2 4– 8. 149–57. 175– 80. o cyst u ing Cryptos id um parvum 73, Microb s. 58 – 91. 140, Microb l gy o cyst reat d with oz ne. Flow Cyt me r and C l Sorting 5 5 Flow Cytometry and Cell Sorting Rapid An lys Separ tion f I d v ual B cteri C s from Natu l Envi e s Jonathan Porter 1. Introduction E f e c m t o i n v b a rh g s ed - l t od l gica chal eng . Det c ion and an lysi of whole cel s or marke mole i c n f u sd r a ltm o v b O k . nec s ary to increas th number of cel s pr ent i a s mple using a cult re step, befor at emp ing the an lysi tep. In ecol gi a studie , such ind rect methodsar ilyc t zedb aus th y el c forba t i su ed oth c lture condit s at the exp ns of the majority of bacteri pres nt (althoug s m u e c t h ra o d i bn f l y o g q c u a i t n y r H l ) w . e v , i . eg o ,at m d lh v r n s py c i g e an lysi of bacteri from a given sample withou a cult re step. A furthe p r o lb e m f thestudyofnatural yoc ur ingbacteria sthenumbersofcel s invol ed. B a c t e r p i o l u n sm f e t b a i r d , p g cif el s ag in t background tha m y consi t f b l ions f targe c l s is t me-con u i g a d f cult. One technique tha of ers a solution to thes problems is tha of flow cytome r (FCM) and cel sorting. FCM can ot s lve a the m od l gica problems f enviro m tal b c eriol gy, and its ap l c tion s lim ted n certain situa ons. Howev r, the claims of the manuf ct re s and of the few microb l g a devot s are bsolute y rue. FCM can lyze thousand of bacteri l s, one at im v ry second. It a ge r d ta on mil s f ind v ual cel s, and ev n stand r instrume are s n it ve nough to have l id t f e c un b a y g s rp ioT h l m . e t f c n d From: Methods in B otechnol gy, Enviro mental Monit r ng of Bacteria Vol. 12: Edite by: C. Edwar s © Human Pres Inc., Tot wa, NJ 5 56 c e li sn v a u b , p e c i l w yh t n u ga dv e r - x p n i ga of e l u resc nt marke dyes is brought into play. Final y, the opti n of cel sorting al ows physical separ tion of specif c cel s of inter st, ont solid cult re m e d i a o r, c a t i o m nf s u l r e c ab i o l g t e c h n q u sF .Ci M oa d alone m th d, bu sho ld be thoug f as n a lysi techn qu t a le ds to biol g ca y re v nt da s mple roc sing. Porte d ie nfb t u o p as r l c , h w d i - 1. Princ ples of FCM and el Sorting FCM instrume specif at ons may vary trem ndously but al fol w the same basic princ le. A flow cytome r in ts implest form is an utoma ed micros pe. The cel sorting opti n, av il b e on some machines, takes the p r o c ef su t a hbl y w ip n g cs e a r t o ifl n sT .a d t o n l f l u i d a I f s l pi ed c - m t o nu . , a fe x m i n v to lc r s p y stream w t o fl w ver th su face o th slide a ong fi ed path, n s et m qrf a u l n x y i ,dh o w e rc v li d su a dif er nt cel s would be pos ible. Provide tha each cel ar ived singly and o s tn u ie f m l , ac g h vr w o f i e s tl d p a m n y , w o u lba de t i n h s r e d f o m a t i nb u T .h e xs t p o m e a sl ui rgn ht c d , ae plb y rs o i t l i g h td e c o r s ,a n f e dt h i g a l s n o c m p u t e rw i ha o t es f ware. Ad it onal use of luoresc nt labe s for the c l s expands the range of measur nt . Becaus fl ore c n mis on gen ral y of a l nger wav e x l tc hi m n ga ( s b o u r d ) , e l y . This t e ba ic pr n le of w cyt me ri an lys . The entir setup is achiev d throug the use of sev ral dist nc sy tem with n the instrume . Thes sy tem are describ briefly her , and more detail scu on a be f u d in M lame t . rF eo p d u c i b l s et v , ar my p i c xbul s o h e d a l m i o c g t u f e h n ;s p w y b . Hydro namic fo us ng i ut l zed o achiev t s. Pre u iz d water p s ing t c h o l r n a i m u f g z s ( ew T dh ) p . l , contai g su pend cel s, is introduce into the cent r of the sheat fluid ( F i g1 . ) S . a m p l f e u i v d o c t y n r e a s p i d l yc t o n s h e a f l u i d . This ac el r t on has e f ct o in reas g th di ance b tw l s in the sample. B c us the flow is am n r d the v loci y f the wo fluid stream b e c o m q u a l t , h s p f e o i wc n t r a d h e o t f l u i s r e a m ; i.e , th sample i hydro namic l y fo used. The width of the xcit ng li ht pa must be gr ate han t e width of this fluid stream to ensur uniform il um natio . Sensit ve cytome ry requi s inte s excita on light, and thus light source (us al y mercu y arc lamp or (1) and i Sh p ro (2) . Flow Cyt me r and C l Sorting laser) focused int a sm l rea. Du l- ser in t um s increa th nge of par met rs tha can be an lyzed. Elim nation of background scat er d light by an p rop iate optical design is also crit cal to btain the b st ignal-to n ise ratio (SNR). Background noise is inher nt, but as cel s pas t h r o u g i s t h rla e n o , an optic l sy em. A com nly i p e t d sy m i the j - n air del v y s t m ( n o z t h l r ep a f us g m i d t h a c e r o w f i s h a ds i c u e p r v o s l y t, h u g e x c i n l h bt a m , id w o s ce l lection sy tem as a jet of water withou conta with a surface. Instrume using this type of configurat are produce by sev ral manuf ct re s ( Subheading 2. tioned gather s much lig t as po ible from the ar whe t cel s m t the lig t beam, nd as lit e light as po ible from ther places. For jet-in a r n u m s l e y oa r wp tg i h c(0 . b 6 ), v whic ol ects a ter d light and fluoresc n from a re sonably rge a . l o w a d e f pt h U c s o l r C i . v n gd e t numerical pertu lens acrif es ome pr cis on f r the b nefit of ensuri g tha cel s are alw ys in focus. Howev r, other machines may be configured d i f e r n t l Ty jh. - i a s r t e m q u h i fg l ro aw t e s m n w c a ht snieo rf , bl m g y sa z ie d on t , cause great inher t background light scat er than altern iv , no s rti g opti ns. I de , so much lig t s cat er d by this ar ngem t ha obscuration, r bl cki g ars, e quir d to p ev n backgrou d li ht e c on. Ac o m na l t e r i v s h u eo fad r k - i l m c o s p y e t u ,i nw h c the fluid stream is d rect on a micros pe cov r slip. Instrume of this type often use a mercu y arc-l mp light source, such as the Skatron Argus (now market d s the Bry s tem by Bio-Rad), P rtec o Bruke ( heading 2. maint ed c ntral y, althoug the c l s are confi ed to a more nar ow c re. cL oi lg s h et n r u d ma c p l ( e . 1 g i 3 , ) r o s c p l im ers on bj ctive l ns o the p si de of th c ver slip. Lower sh at fluid velocit s can be used to maint a stable flow. This design al ows a min u of refl cting surface (al of whic are per ndicula to the light path), a low sheat fluid flow rate, low background light scat er, and use of ef ic nt ol ecting le s . Howev r, light as to be col ted from a sm l er are in orde for the sample cel s to remain in focus. A very hig SNR is obtained,andprecisean lysi ofsmal particles( uchasb cteria) sea ily achiev d. A flow chamber of this type is not as menable to s rting as the jet-in air sy tem, although a flow-s itching ap ratus is av ilab e from P a r t e c( s e 57 c o lb y s e i a m t n d / r ), Fig. 1 se ). In such a y tem, ligh s co e t d by j c ive l ns , po i- se ). Hydro namic focusing is til used to ke p the sample stream S u b h e a d i n g2 . ) .J e t - i n a i rs y t e m s a y l s ob em d i f e dt ou i - Sub- 58 Porte Fig. 1 Diagr m t c rep s nta io f the princ les of FCM and cel sorting. A s u p e n i o fc l s e d o w yi n t h ec ro fa p i ds t e mo f h a l u i dp s - Flow Cyt me r and C l Sorting lize oil- m Ste n 59 ersion l ght c e ion. M r details on uch y tems ar giv n (3) C o l e c t di g h p sa o e c t ru h m l i p et ru b( P M T ) p h o e t l d a c S i su n r g . v a l p s e i u v n g h o m ct w f b r , P M T A t h e u b i sa l om t p y h er c i v ds g n a lt op r u c e v a b l t r i c signal. Es ential y cel s are d tec as pulse of cur ent hat greatly exc d b a c k g r n o s T p u hi e l d t. - v ; , a / n c o l g e r t s a n sd m l c p i t o r as ue d p v i “ m o r y f” t h ce p u r L. i n a a n l d o / g r i t h m p c f s a e n d ou l r g i t y from the d t c ors. Incomi g dat re us d by the computer software to pl t various h tograms of light n e sity ag inst umber ( a l ubs eo ct d n r m i p a e t h r s o g a (m tica ed dat an lysi . Rec nt dev lopm nts have trained neural networks to a n l y d - t ui f h o er m s n g, c l b p i u fz e a t o n s si proces . Automa ic sample loading, machine cleani g, and dat storage al ow “h nds- f ” u e o th ins rume t. Cel sorting ca omplet h roces by ph ical sep r ting ubpo lam e ca spluo rfb d V it h . na m p wl e o i s of the noz le throug whic the sample pas e , using a piezo l ctr crystal devic at a precis frequ ncy, cause droplet formati n of the fluid stream. Dropletsbakwyfromhetadfin stceromhligt/fud i n t( e r s c point al ows the mac ine to rack ny particle on it has p ed throug the light beam. W n p rticle as hroug t e li h b am, sc t er d ligh an fluoresc n are measur d, and the sy tem wil deci whet r to sort tha particle bas d on perato -di c e rit a deriv f om light sca er o flu b a s i t h e f o r m s v a l u e m i n d M a xc h urm t e i s .n of a sort windo . The machine wil then deci whet r tha particle (i.e , targe c l ,no targe c l ,o dirtpa cle) sofint r ( .e , ob s rt d).The only conta left with the particles at this point is throug the sheat fluid, whic an be posit vely or negativ ly e ctri al y ch rged ( i n t o e f pc r al b s d y w h m , k - f p ri a en t fcs h d o l m g , ing throu a coni l z e, to achiev ydro namic fo us ng. The str am of cel s pas e throug a beam of light, and scat er d light and emit d fluoresc n e are det c . Vibrat on f the noz l cause dropl ts form, ideal y contai g one c l d r p o e s t cR h l f a n b u i . g point f break-of , i requ d. Charged opl ts (con ai g cel s) ar then d fl cte by charged pl t s in o c l e ti n ub s. . ). Such plots can Fig. 2 F i 2g . 1 Fig. a) l, o w i sn g p h - db r oe pa lk n - s t i f z , M w uo )r .e m n t Fig. 1 ). As a p r- 60 E F x2i a.g m d p o l utfe cs y r w B a . t e r ci wl s b d im unofl resc nt y i sewag f luent. I al the xampl s, two p ulations c be s e Tp nho . u l a t iw e f s o r c n e p s b ta c k g r o um n d e ia l specif labe ing with the antiser . The second, more fluoresc nt po ulati n rep sent target cel s. Discr minat on of background and target cel s was more than suf ic ent to al ow hig ly suc e f l cel sorting of the targe cel s. histogram of relativ fluoresc n (logarithm c scale) ag inst number of cel s; d o i t m - p u f l n r e s c a g n f i o l w t d er p h c - , sent one vent; l i g h st c a e (r sity ag n li ht sca er, in wh c o entric o u s rep nt c l de sity. b r o k e n f T. h c a g i s p l e ud n t h a r i c l e - o n t dg r p l e h a bs o ken aw y. Thus, the droplet remains charged and fal s throug the space betw n o charged fl ction p a es ( i n ts a o e r l c i o t n u b ( e Porte (A) frequ ncy (B) (C) thre -dim ns o al histogram of luoresc n inte s y ( x -axis), y - a x i s ) , n cd e l u m b (r z -axis); (D) Fig. 1 F i g 1. ) T . h uf a sl o c w y t m e r / l s o t h a e c o n t u pr l f o e s c n i t - ). Then, t drople is d flect Flow Cyt me r and C l Sorting 61 ,ylevitag n ,ylevit sop( yaw e rht fo en i s ylan retfa s lcitrap leb ot y il ba fo elpmas dehcirn ylhgi a ecudorp t lai netop h sa dn ) egrahc yl rtuen o g n .i sc tu ”ed l pR o gr a h c e- svf tl no m “ pi d. e cv g r R a t s , u d h e t c l f r a p eo h nd t s ci u e r l gp n o a hd t c ro sl ew yart e i orc m edul ni sec v d noitcel r htO .sleba tros u f gniwol a enihcam evitc f e ,desu edom tros eht no dnep l iw ycnei f E .sedil epocs r im dna ,et rcsid ,etar p s era sl ec h i w n elpmas ecudorp t y il ba eht dna ,putes l its tub ,noi arep l m on rf ylthgi s etar isyl na rewol gnitr S .del ba e l b i s o p t e l d n a hx o r p 0 2s / t n e vg i d url a , cs ten m h o .s/ tnev 0 , 2 fo gnitr s wol a y m sedargpu s e hw As previou ly t ned, h pri c le of FCM is th ga er n of su p de particles singly and separ t ly into a sen i g region, wher they are pas ed through a light beam of uniform wavel ngth and inte sity. Each particle rec iv s a uniform il um nat o f r a short period f time (typical <10 and emits a burst of scat er d light and fluoresc n over al angles. Light d e t c( Pp l oMr T a s n )u i gh s l c i mo a g er t h n f u F y d b z , . l fluores c e n i t e n s i t c y a b n e l c u a t e d , n h i s t o g r a m o ds t - p l o t es f a c h par met r can be produced. FCM can thus measure sev ral par met rs simultaneo y f r s ve al thou nd cel s a h cond. µ s) 2. Materials 2.1 Major FC nufact re s nd Sup liers The fol wing st con ai s m t ajor cyt me r anuf ct re s and ome r cm e oa s j u g p i n l d t A h - b. v , r s vidual cytome r p n ts i g ven Shapiro manuf ct re s also sel cytome ry reag nts. Sources of antibod es are not include , b cause th r a e f w source for antibod es ag inst mo bacteri , and m y res a ch ise t r own. 1 . 1 9 7 0 ( ) + 4 T e l I .3 n A s H t S b r Y P u 2 acm y i U k K , w h 615284; fax. +4 (0) 1970 6154 . (Micro yte dio e las r cytome r, compa t and truly portable instrume , d signe for the d ction f bacteri l s zed-par ticles and lowi g ac ur te o n s.) 2. B e c t o n D i c k n s o I m u n o c y t o m e t r y S y s t e m s , 2 3 5 0 Q u m e D r i v e , S a n J o s e , CA 9513 -1807, USA. Tel. (80 ) 2 3 82 6; fax. (80 ) 2 3 82 6. (Major cytomet r manufacture , with a wide range of sorting and no s rting machines and reag nts.) 3. Bio-Rad SPD .r l , Flow Cytome r Unit, Via Modigl an , 5/7 20 9 Segrat , Milano,It y.Tel 392 160 4 ;fax.392 160 4 .(BryteHSm cury-a lamp cytome r [o ig nal y market d as the Skatron A gus], volumetric sample inject o a d hig prec s on wh a lyzing bacter .) (2) . Many of the ins rum t 62 Porte 4. Bruke Sp ctros in S.A , 34 Rue d l’In ustrie, F-671 0 Wis embourg, F ance. Tel. (8 ) 73 6 0 ; fax (8 ) 73 6 9. (Mercu y-a l mp cyto e r, hig p ec sion f r mal p tic es.) 5 . 1 N 9 o M 4 v a 7 8 . i 0 , s dC uh R n e m - S A l f x F o r a t ( T , 1 c ) e . 1 9( 2 6C f45 0 h a3. e x ;) m Fr Il g o c s wny t a e d r i ated r agents, market d as robust, ea y-to us in trume d signe for industrial m c ob gy.) 6. Coulter Corp ati n, P.O Box 1690 5, Miam , FL 3 1 6-90 5, USA. Tel. 1 3 0 5 / 8 f; a x 31. 0 5 / 8 ( M a j o ir n s t u m e a f c t u r e w, i d a n g of instrume and re g ts.) 7. Cytoma i n, 40 E. Horset h Road, F rt Col ins, CO 8052 , USA. Tel (970) 2 6 2 0 ; fax. 970 2 6 01 7. (Special st in hig -spe d an lysi and sorting opti ns, provid ng up rades to exist ng i strumen , and m ufact re of hig spe d ort s.) 8. Molecu ar Probes, Inc., P.O Box 2 01 , Eugen , OR 97402- 6 , USA. Tel. ( 5 4 1 ) 6 8 3 0 ;f a x .( 5 4 1 )3 6 0 .( M a i ns u p l e ro f u s c e n tp r o b ,d y e s and c librat on e ds for al p icat ons, ech i al s t nce av il b e). 9 . 2 5 3 4 ( 9 ) O t T G M e uo lD r n- . m sH b a P 8 h 1, y 6 2 S c 80 ; fax. (49) 2534 80 9 . (Laser and mercu y arc lamp based instrume , com er ial y produce piezo l ctri fluid c switch ng sy tem for contai ed, aeros l-f ce l sorting.) 10. Polyscien Inc., 40 Val ey Road, War ingto , PA 18976- 0, USA. Tel. (215) 34 6 8 ; fax. (215) 34 0 . (Gen ral gents a d c libr t on eads.) 1. Sigma Aldrich, Sigma Chemical Company, P.O Box 14508, St. Louis, MO 6 3 1 7 8 - 9 U T ( 5 S e f A l a , . 4 0 x G) ; 1 n r g e t s . ) 3. Methods 3.1 Ap lying FCM a d el Sorting Bac eri l Popu at ns FCM is a powerful nd v satile chn qu a d, s uch an be us d to answer many biol g ca question . Howev r, as ind cate in sample r a tion d instrume s tup de rmin the suc of any l s i T h. u t f , e r p n o c d u ti s e a b l h x r c t i mo en d p s a ue rf nlt o i cm g vh s e to bl ck the noz le f the instrume . Labor t y cult res a gen ral y suitable without a cleanup step, although some worke s have filter d cult re media befor us . Al b f ers and l bo t ry eag n s mu t be fil r d at e s once b f r use, and it mus be orn i m nd tha steril o uti n does n t equat o p rticle-f so uti n. I s often pr able to rins gla w re, nd r a p n g Se m y ul s t i . o p r f a e n w s ic tl oh , from n e at l (e.g , an lysi of lake w t r bacteri ) to ex nsiv blendi g/ a m o u nl t r g e c As i f p a o ) (.l n / ,trg e c h m s oa fm p lr e t i n y o d ub c ea s h m p i t f o n l s u p e n G i o r . a q l t d s y m , u p c eg n Subheading 1. , Flow Cyt me r and C l Sorting crude filtering through nylo mesh befor an lysi , resulting in min al change. FCM an lysi of bacteri from more chal engi enviro m ts has b ep nr f o m td uh , g s c e a p n o f d tiven s of c l extra ion. H a v i no gb t c e d ls u p n i ot h aw l b c kr g F eCf M u i d tubing, w l be n c s ary to l be c s to di ngu sh t em fro n cel u ar particles. Invari bly th s wil requ a fluoresc nt labe , thoug s me orphol gica y dist nc el s ( .g , Head, I. M personal c m u i at on) c be dif r nt a ed from backg und u s i n g h e r tl s c a e r h t i s c .H o w e v r ,t h s x c p i o n a r e a n d f l u o r e s c n ed i s c r i m n a t o rl a b e li sr e q u i r e df o rt h em a j o r i t yo fb a c t e ria. Some bacterial populations can be distinguished on the basis of autofl resc n of specif p gments, bu many other p ocedur s equir an e x p r t a i h m s dn . o l f ue b C c h a i t d . Samples y be pro d f a to l bac eri l count, a vi ble or active l count, a specif cel count, or an ind cat o of cel macro le u conte (e.g , DNA R or t al p ein). Som f the dy s pical y used r li t in Tables 1 and Sample labe ing prot c ls are obvi usly dicta e by the fluoresc nt dye being us d. In ome cas , dye bin g s trongly i f uenc d by salt con e tra ion, wh c as u ed probl ms in the s udy of marine b ct ria. P ot c ls often r qui wash ng d resu p n io te s r move unb d ye. If numeration f cel s i mportan i he xp rim ntal s, i may often b r to amend s pl with con e ra d buf e , n /or ch se dy an prot c ls tha do n requi wash ng tep , o av id cel mag nd los . An im e s (and ev r-inc as g) ran e of lu resc nt probes n w exist for bi l g ca res ch. Many of thes prob have b n dev lop f r ma mali n ce l bio gy, but ac eri l ap ic t ons are pidly ncreasi g. A summary of the major dyes can be found i and T a b2l e ( f l u o r e s c v n i t a b p y o e s ) , g t h w i r x a m p l o e f su in flow cyt me ri s ud of bacteri ; d ls of the m d us in each s can be found i the abl ref nc s. The majority f thes ap lic t ons have used dyes for enum ration and viab l ty as e m nt. Specif det c ion is a c h i e v t d r o u g s f a n i dm o l u r e s c n a b l u f h o r e s c p oh i y n t r( (FISH) using ribos mal RNA-direct olig nuc eotid s. Thes olig nuc eo t i da erf s l nb w h d y ea sl ,t o um g rb n i h a el t tives ha b n i vest ga d Instrume s tup is of great importance. Th mac ine ds to be cl aned regula y ndsteril z op ventdir a /o b filmac u t on,a dregular m inte a c is mportan . C librat on is u al y chiev d t rough e us 63 ; Pickup, R. W and Achromatiu oxaliferum 2 . Table 1 t oh r ) uf gl e s c n t T a b1 l e (4) . (gen ral f uoresc nt labe s) s i nt u hybrid zat on 64 Table 1 General Fluorochromes Used to Label Bacteria for FCM Fluor ch me Target mol cu s Fluoresc in a d Gen ral f uo esc nt labe s, vi oth cyan e group; 4 ,1 te ram hyl odamine 64 Phycoer t in Ethid um bro e Propid um I e Mithramyc n Chrom ycin A Hoechst 3 42 or 58 4',6-Diam d no-2 phe ylindo SYBR Gre n I YO -I, PRO-I PicoGre n Ref r nc s e.g, to al ce prot in, a ser olig nuc e t d s Conjugated o pr in, us al y for im un l oresc n 18 Double-strand uclei a d, often wi h m t ra ycin Labeling dou e-strand uclei a d 32 G/C rich DNA, often us d with e d um bro i e G/C rich DNA A/T rich DNA Nuclei a d ye 3 Nuclei a d Cyani e d -bas nuclei a d Double-strand uclei a d 1–3 28 25 14 49 57 58 58 , 12 , 14–25 28 , 39 , 40 , 19 , 25 , 42 , 43 , 45 , 17 , 25 , 42 , 43 , 45 , 47 , 48 , 15 , 34 , 41 , 51– 6 , , 41 , 70 , 46 , 40 , 41 , 4 , 46 , 70 , 47-50 Porte Flow Cyt me r and C l Sorting Table 2 Fluorescent Probes Used to Assess Bacterial Viability by FCM Fluor ch me Cel functio m as r d Rhodamine 123 Dihexylocarb ni e dy s Fluoresc in d a et Carboxyflu esc in d a et Bis-carboxyeth l-carboxyf u esc in Ref r nc s Membran pote ial Membran pote ial (s ver fo ms exi t) Enzyme activ y, me bran i tegr y Enzyme activ y, me bran i tegr y Enzyme activ y, me bran i tegr y 13 31 59 13 13 Enzyme activ y, me bran i tegr y 26 , 26 , 28-31 , 59 , 63 , 41 , 26 , 35 , 25 , 26 , 36 , 35 , 25– 7 , 34 , 64 , 35 , 36 , 65 , 67 , 31– 5 , 31 , 62 , 65– 8 , 59–62 , 70 , 60 65 acetoxym h l est r Enzyme activ y, me bran i tegr y 13 Respirato y c iv t Double-strand uclei a d Double-strand uclei a d Com ercial k t, nuc ei a d Ac um late in d cel m branes Nuclei a d Nuclei a d Nuclei a d 28 32 62 30 27 31 29 30 65 Calcein toxyme h l st r Chemc ro B Cyanodit l e razolium ch r de Ethid um bro ide Propid um e BacLight Oxon l dyes Calcof u r White Po r SYTOX Gre n , 69 , 60 6 Porte of m nodisper fluoresc nt beads, whic are vail b e in a v riety of size . F o br a c t e i sl u d 0, . t5 2o to use for instrume alignme t. Subseq nt dat handli g and an lysi wil dep n o the instrume specif at ons. Howev r, most machines ow save files n a flow cyt me r s and format. Sev l software p ck g s (in luding fre wa x mples) i t ha elp in FCM dat lysi . µ bm e a d s r p o l ty h me s r v a n i z e 3.2 Enviro me tal M ni or g f Bacteri Us ng FCM and Cel Sorting m i c r o b l a p gn y . t s f u d h a v e o r t i n g c l d F C M 1 and 2 givean d ofh wt e c nol gyhasbe u dtom ni rbac e . Sev ral evi ws al o exist a n i troduc n to he ar of FCM in e vironbacteriol gym n considerably in certain situa ons, e.g , discovery of the sign f ca t marine genus Prochl us D Na An l y s ih v e o wt m a i n sfr d t o am le h f s rn y z i g phot syn e ic mar b te ial p nk o bi mas Tables (5–8) help d a r y s nd i crea g s FCM of use Th . . Observation us g phot syn e ic p gments a d (9) . (10) 3.2 1 Specif D t on f Bacteri Us ng FCM I m u en so b w tl a c g h i r v d y c o n i f j ts u e p r a F w l C d M b h n o m , e r a t i and purif cat on procedu s. Problems with producti n, specif ty, epito expr s ion in stre d cel s, and labe ing of background materi l are wel know ; howev r, extrem ly sen it ve and specif c det c ion is pos ible, includ gthe ab lin of tracel u mo es.Sign f ca t mprove n si det c ion,a SNRsh veb nmad yco bin gla e withmon cl a antibod es and pro id um iod e for the det c ion of waters (1 ) improve nts, FCM has proved t be of nly im ted valu when orki g n soil (12 , 13), a c t i v e ds l u g h a b n c e s f u l ya z e d suc e f l im unofl resc t FCM ap lic t ons t enviro m tal b cteriology have used sewag or water sample d e t c ap on l iy b s c oS ni f g rl me a t . d s c i v o n p l a t e d s o r w e v n t i f l s u o h r c at ew y p i v n d , b m f g l s to n specif b nd g of a tib d es FISH methods labe ri os mal RNA (r ) sequ nc i s de ntac el s. Many of thes udi are p fo m d an lyzed on micr s ope lid s. The revi w of Aman et al. F C Mch oy nb dr i t z a s . e g , p or b a ts i m n ld c u e in co ling Legion l a and the det c ion of in soil Flavob cterium (12) . Despit thes althoug er i hly pa t cu e nviro m ts uch a fe s nd .H o w e v r ,t h m s (14– 6) (1 (19) (4) gives a compreh nsiv background to FISH and . Porte et al. , 17– 9) P o s i -e w a g . n d t r l k e i n E cs o lh ie r a . (18) used Flow Cyt me r and C l Sorting 67 an lyse of mixed po ulati ns of cult red cel s have be n perfo m d W a l .e tn r (21) o p t i m ez b afhd c r s F n C M I l S y H i and sub equ ntly used thes to probe the microfl ra of activated sludge directly (16) . Dat such as thes , and those from micros p observati n , have d monstr ha t e fluor sc n e ig al obt ned is pro t nal he ribos me conte of the c l , and henc , in utrien -po enviro m ts, low ribos me c nt ay re d c l s if u t o de c . a m p o t l v i e f O rb dy n h c F I S H g s targe nuclei acid sequ nces ins de whole cel s. This ap ro ch has be n d e v l h o i p s n t a w g y c , h r e s n d i t v yo e c v i r a l n f e c t o s T . h p r a c m b yu e d s t h o f l r a i n c g e s o tain g p r cula gen s. It ha be n pos i l t erfo m h p cedur on ltured bact ri l ce s, to de ct a pl smid enco d gen gen (23) . The lat r s udy l o em nstra d ve transc ip o d am lificat on r m RNA ins de whol c s. (20) (2 ) or the 16S rRNA 3.2 Viab l ty As e m n of Bacteri Us ng FCM Fluoresc nt probes exist for a range of metabolic functio s, tha aim to refl ct viab l ty w hou t e n d for cult e. Bac riol g st have not y u n a m tb hid ge s o v y l r p fluoresc nt viab l y pro es u d ith r sepa ly or in c mb at o s c u l y r e f l c vt i a b y os p e n q u t i H. o w e v r , h u s Ff C M a c o n t r i b u e d to he incr as g confide n the da . Becaus m ny of the fluor scent viabil ty probes ha com n ex ita on/ m s i wavel ngths, ey ar dif cult o use for simultaneo sample b ing. The sp d of FCM has en bl d res a ch to pr ces multip e s b am l of cel s with n a c ept bl ime period (25– 7) n a u ( d ts r i l ey c m o p x g , d u n t r e m pe r o h(tb u dsna i g c l v f ) , o r e i n t r a c e l u d m g b y , o f i n t a c de s ) x o l n 1 2 3 m a d e , l s o w r c u n t T a dl y e s f ) . o (r g ci m n v b t a using micros py and im unofl resc n . The studie show tha great m i cv f ra o T ts u h n F C p bM y . g e d i o arise f om perato r, and o u if rm, day-to instrume op rati n, and emphasiz the ne d for quality contr l. Howev r, the flow cytome ri / fluoresc nt probe viab l ty estima wer more sim lar to the direct viable f t l h c u e ( o s n - a p U ri d b x .) resc nt viab l ty probes i ap lic b e to s udying active c l s, or cel s whose stre pons i clude ma nt ce of m tab lic v ty. v i a b l - M e m s u p r d n h t oc a i y ity nd ca or. The as y work n the basi th live c s maint embran (6 ,2 4) Whet r. t I h n e. s u d i c v, l a b w t sy e h d i l b o . 68 ytirge n d a noitc uf d a e ulcx ht ,eyd w u n a b l et od .T h s , y e n t r h c l a d b e sn u c i a d .W o r ku s n g the Gram-posit ve bacterium respon ap e rs to be on f metabolic shutdown, i.e , dormancy. Dormant Micro us acid ye, and failed to ac um l te h me bran poten ial probe rhodamine 123 (28 , 29) molecu deta icsu r eb dluoc s e tnamrod t h dewo s ) e ciel un ht o ytil baemr p d sae c dewohs yl ait n sl ec ht ,ycnamrod g i dica ,ey d wol f yb noitalum c f enimadohr 321 dna ylet mi u ac b .elbarut c y nO eht su fo MCF delban hcus eta ir n s m u fo siht xelpmoc .no em hp rehtO srotagi evn evah yl ufs ec desu enarbm ytil ba v fo r c dn sa leb dic un a ot y il b emr p x u l y f t i e p mr o ch s a n b v spmu dael ot a esl f gnidroc f .ytil ba v fI ,es ht ro , eht c rid s ohtem niag ,ec tp y h l iw eb hto er m dipa n erom ta uc n h erutl c secruo f yt i av m b l gn o r f seuqi hc t Porte hci dae ro ad maged cel s are has shown tha the stre Micro us lute s m as e nco d u l i f , r t . Howev r, ap ro i te trea m n (e.g , with interc l signal 82( , )92 -kaerb n hW . -seroulF . )53–0 ( )63( 4. Discussion and Future Prospects M C F s a h y d e r l n v o p u f s r ) l a t n e m o i v , y (g b c d w e n eht gnivorpm yl a s c e t d na gis emur cnavd sMiC,F t n e m o r v a l u c p d fh g .A q t yltrap eb n c oi mu s hT . r t a q fo i yl n de us a detamo u rof enituor ,esu dna at d si ylan si erom d taci s hpo na t .rev sm inagro c f t lup b ez d k w N tnecs roulf y a i m x d p v gn eb r at w o f eniram d retawhs f cino k lp e s selcitrap fo seitr p la c eht nimr d ot ,gni rp e f la tc s fo esu ht ylsuoiverp t d ng a c P .ylsuoe t mi h gn vaw f d r t dezir tca h l po .stnir eg f d luP resa c uo yam osla ev h lar es -sid ot si ylan ec s roulf dev s r- mit gn wol a ,y iretcab n s oitac lp g n s . i e d c r l tm o h a O f p u dev lopm nts include the producti n of cytome rs specif al y design to edoi a htiw ,MCF elbatrop ,d e -yr t ab gnidulc ,sm nagro ci t e d laser and fixed optical alignme t (elim nat g machine setup and focusing) esu dl if gn wo a .seidut lac go b r m n v e itu ,y as c )73( )6( a s MCF h ilbatse o p h dlu s e t a hcus e n vdA . 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S c h u t F, . d Ve r i s E J G, o t c h a l J C. R, o b e r t s n B . H, a p e r W P i n s R, . A., and But o , D. K (19 3) Isolati n f ypical m r ne bact ri y d lution c ture: g owth, main e c and h r cte is of is late und r labo t ry c nditions. Ap l. Enviro M c biol. 56. Montford, P. and Baleux, B. (19 2) Comparison of flow cytometry and epifluorescence microscopy for counting bacteria in aquatic ecosy tems. Cytometry 13, 5 7 . M a r i eD ,.P t n s k yF J ,a c q u e tS . nV d l o D(, 1 9 7E )n u m e r a t i o c d l cy le an lysi of natur l po ulati ns of marine p co lankt by flow cytome r u .I ne rG RBYS niats d c iel un ht g is 58. Marie,D. V ulot,D. andP rte sky,.F(19 6)Ap licat on f he v lnuc ei acid dyes Yo -1, Yo-Pr 1, and Picogre n for flow cytome ric an lysi of marine p ok y tes. 59. Diaper, J. P , Tither, K., and E wards, C. (19 2) Rapid as e m nt of bacteri l viab l ty f ow cyt me r . 6 0 . D i a p EPe nJ d.r w , ( C 1s 9 F 4 l c) o y t m e r d i c t v o f an b l e r i from c p st. 61. Kaprely nts, A. S and Kel , D. B (19 2) Rapid s e m nt of bac eri l v ab ty and vitality by Rhodamine 123 and flow cytometry. 410–42 . 6 2 . L o p e z - VA imCa v n RJ r d . s( , 1c F 9y l t o g5 w ) e i s mentof Escheri a ol water using rhodamine Microb l. 61, 6 3 . m e - i n c h a r g ys t l d e C ( 1 9 6 ) B . a l e u x , n d PM o t f r brane poten ial during growth of fluoresc nt dy a flow cyt me r . J Mic ob l. Meth 905– 1 . 59, Ap l. Enviro M c biol. Salmoa sd e t r m i n b yf l o wc t m e r y . 712– 9. 40, C a n .J M i c r o b l . 621– 3 . 40, 70– 6. 5 8– 63. 10, 8 1– 9 . 59, Ap l. Enviro M c biol. 2150– 6 . 59, 18 –192. ,36 .loib rc M .no iv E lp A 62, Ap l. Enviro M c biol. 1649– 5 268– 7 . 38, Ap l. Microb l. Bi technol. 14, FEMS icrob l. E 213– 0. 72, J. Ap l. Bacteriol. and starv ion-surv alinse - Salmone typhimur 123, propid um iodi e and ox nol. Ap l. Environ. 25 1– 6. Escheri a coli .391–68 as det rmin by a cyani e 25, 79–86. Flow Cyt me r and C l Sorting 73 6 4 . K a p r e l y n t sAS,. K d lDB( 1 9 3T )hu eso5 f- c y a n – 2, 3-ditoly e raz lium chloride and flow cytome r for the visual z t on f respi ato y activ y in ind v ual ce s of Micr us l te . 17, J. Microb l. Methods 1 5– 2 . 6 5 . L o p e z - A m r s ,R . M a o n D J , dL l o y D .( 1 9 5 )U s eo ft w x n l sa d fluoresc nt razolium dye to ni r sta v ion f by flow c t me ry. in seaw t r Escheri a ol 2, J. Microb l. Meth 165– 7 . 6 . De r , . Porte , J. Edwar s, C. and Pickup, R. (19 5) Evalu tion f the sui abil ty of bis-(1,3 d butyl ar i c a id) trime h n ox n l for flow cytome ri as e m nt of bac eri l v ab ty. 67. Jepras, R. I , Carte , J. Pearson, S. C , Paul, F. E , and Wilk nso , M. J (19 5) c r y f o t dlD b e u m wv s a i n p g t r bacteri . 68. Mason, D. J , Al man R. Stark, J. M , and Lloy , D. (19 4) Rapid est ma ion f bacteri l n b otic su ep bil ty w h flo cyt me r . 69. Roth,B.LP MYue,S.Tandilr,P.J(197)Bacterilvb tyand antib otic su ceptib l ty tes ing with SYTOX gre n nuclei acid stain. Enviro . M c biol. 7 0 . n o cS ( u 1 lr f9 tv i 3 W ) a . b e P R k u n p d , G h o W . s A M J r g a n , Aerom nas l o ic da n l ke w t r. 130, FEMS icrob l. Let 61, Ap l. Enviro M c biol. 165– 70. 269 – 701. 8–16. 176, J. Micros Ap l. 63, 24 1– 3 . Ap l. Enviro M c biol. 59, 874– 0. Monit r g Bac e i from Natu l Enviro me ts 75 6 Magnetic Particle–Based Separation Techniques for Monitoring Bacteria from Natural Environments Jonathan Porter and Roger Pickup 1. Introduction Physical separ tion f eith r intac rget c l s or specif molecu s from m a n ye v i r o t sc a n e u l i s p n o f r e c t a m i n gp r t c l e s , n o t a r g e c l s n bd i o g c a l h b i t o r s n d g l ey i c h tn a r g e l s or m lecu s of inter s . Th proces d ampl wi be r ady fo the n x part of the ov ral exp rim ntal prot c l; e.g , a cult re st p, or a molecu ar biologica procedu , and great confide c in a suc e ful outc me wil be w h m o e f l t x vr i - a s c d n g A y . e n v i r o m t sS .u c he d a yi o m b t l n s u p e i ot h a r res nta iv of he bulk c po ulati n, r may i to arge sp cif el s. On o c a s n i e t , b r ly u c k s p e n i o r t c f cel xtrac ion. Th s c apter d ls with e s par tion f speci l s, eith r intac or getin a m rk olecu f int res . M hod f r intac el s p rations include flow cytome ri cel sorting manipulation (3) , diel ctrophoresi sedim nta ion field-f ow fractiona ap lic b ty and the degr of sel ctiv y tha can be achiev d for the cel s e l c t i v o a n r w mh e s O pd f . c i a l o r t n h e x f i s p e r n t v osh i f a k m w c d b l , si en tc rlf uo mg v a , y p w h i ce r l t f i l h t aoe mr p s c bi e r v a t o On p s . h c r fv e a s i b l ef r o m n v t sa c h l e n g i f c s ,p l a n t i u e o rs l h of magnetic p r l se a tion ch l gy. The adv nt ge of r d by magnetic b ad cel sorting s the abil ty o sepap o b s u i l f m rak ce t n yd , g From: Methods in B otechnol gy, (1) , optical trap ing , ultrasound sedimenta ion and elutria on (4) (6) Enviro mental Monit r ng of Bacteria Vol. 12: Edite by: C. Edwar s © Human Pres Inc., Tot wa, NJ 75 (2) , micro(5) (7 , 8) . The g neral , 76 Porte and ickup sen it v y and specif ty. Physical separ tion based on cel densit (e.g , ultraso nd se im nta o , elutria on, sedim nta o field- ow fraction ) ds o ip tf e h ac n v r ( . q gy u , m e n t cel numbers, large final sample dilut on, and the nec s ity for special z d e q u i p Mm b cn ao rt f ) . - u h i l p v d e n o g rial eco g st , and re f ctiv a obt in g pure isolat . H wev r, th p ocedur s a e tim -consu g and l ck in t al specif ty. Flow cyt me ri c l e s f o a r n ti c p gw vd u h l , e n i x c a sm ined and a decis on is made whet r or not to sort. The abil ty to examin single c s ha much to f er mic ob l gy, but flow cyt me ri cel sorting requi s exp nsiv , special z d instrume a ion, and is les robust and les rapid than m gnetic separ tion . Magnetic separ tion are durable, r qui e a min u of special t qu pmen , a d r e sily p formed n th b c . 1. Princ ples of Magn tic P r le–Bas d S p r tions M a g n e t pi c r l sv a b i ne to yf p s d z u,h r i n ciple of the c nique s the sam for al types, and is outl ned i magnetic particle is coated with a hapten tha recogniz s a rec pto on the t a r gc e w m l o u Th . p t d ns a g e r l y i b o d e s , a l t h o u g e r n i v s l a c h t b e n i g a ct sA ld u . p e n o l r a e m b t h cw g p i n o x d v s , with e c l s ( reaction w th al t rge c l s with n e su pen io w thou al owing o specif el at chm n (e.g , arly st e of bi lm sta h en ). T su pensionc ta g hec l sandb i the plac dwi n m g et cfi ld,an the magnetic particles (with at ched cel s) are al owed to con e tra ( 1B ). The particles wil remain held in place for the p riod tha the magnetic force is pre nt ( removal of the remaind of the su pen io (contai g no targe cel s and otherpa icl s), ndthea i o f resh,d in buf er( the con rati s ep al ow ce ashing to be p rf m d o as m ny ti es as nec s ary; gen ral y only one or two washe yield su pen io s tha are adequ t ly c ean d. Cel s can the b remov d from the particles for u ther s t u d y ,a l h o g s m e p l i c a t o n s wu e ft h c l s i e t a c h d o the par icl s. The magnetic separ tion step has be n demonstra d to be ap lic b e in hig ly part cu e nviro m ts, includ g soi . The l mit ng s ep i thus gene r a l ty h e a p t un s e d a, n i t rs e a c t i v t ay f e r t a c h m e n t o h be a d s M. g netic particles can be purchased in a variety of forms, from ready-to use kits to uncoated particles ready for user labe ing. Avail b e formats include the fol wing: Fig. 1 . The ). The tim requi d for this sel ct d so a to al w Fig. 1A Fig. Fig. 1C ); thus, a suitably design ap r tus al ows f r the Fig.1D ).Rep ating Monit r g Bac e i from Natu l Enviro me ts 7 Fig. 1 Purif cat on f arget c l s u ing a m etic par le-b s d par tion echnique. in mag etic f ld. fied c l s are usp nde i buf er sol ti n. (A) Cs eu l p n m i w a ox g tb dh e r c l s . (B) (C) Supernat (contai g no targe c l s) i remov d. M i x tp ul ra ec d (D) 7 Puri- 78 Porte and ickup 1. Magnetic p r les coat d wi h polymer tha l ows c va ent li k g of m n clona tib d es (MA ) tha recogniz the arg c l s (prima y l be ing). 2 . M a g n e t i cp r l e s o a t dw i hM A b sf r o ma l c n b o r a t y n i m l s .P a r i n r d c le wo a v m b t h - u f s y i l n a t b e c d s rato y n ibod es rai d n other anim ls ( econdary af in ty labe ing). As an exampl , a user has r ised an MAb in a mouse c l ine tha show the d sire specif ty with the target cel s. Purchase of magnetic particles coated with a n t i m o u s e b d i r a s e n t , b i o rh e w p la s c o n d rl ya b e i g c o Tm rM ph Atb euw si . a d f n g o l b i e s can ot be guar nt d si g th me od, an the sub q nt i d g cap ity of t h ae n i b o d my r e u c T. h s p a r t i o n c e d u r a b p f o m e id n of two ways: eith r the coated beads are labe d with the antibody and then al owed t r ac with e c l s, or the an ibody s al owed t r ac with e c l s a tn hd e c o b ars d te wc i h n b o f d uAy r . t ae l n tive o his ap ro ch is t e u of pr tein A or p tein G coatings provide th link betw h antibody the magn ic p rt le. 3. S t r e p a v i d n - c o a t e d p a r t i c l e s a r e a v i l b e , t h u s a l o w i n g u s e o f b i o t n y l a t e d antibod es n ith r of e alt rn ive us pr viously t ined. 4. Uncoated beads or activ ed beads are av il b e, al owing the user to coat the particles d t y, b a method f c i e. T h er a n g o fm t i cp a r l e s o m c i a y v l b e s o f r t h u e flexib ty. Vir ual y com nly used particles uper a m gn tic; .e , r e s i d cnu o a l t b m f g ap i r ns o he w t c y activ y when r mov d f the magn ic f eld. Com n y used ar polyst ren -bas d,u iform phe s(b ad ),whic onta m ys l agnetiz b particles hom gen ously distr bu ed with n the matrix. The entir bead is m i f c e r w od p a n l t y s v , h g tive groups for labe ing, and lso wering the otal surface r a of the b ad su pen io . C l da p rticles ( .g , 40–1 nm) are lso vai ble w th v ry l a r g es u f c ,b tr e q u i f a s o n g e rm t i cf s o r n e t a i , such as neodymiu magnets Por us glas may also be used as a car ie for the iron oxide, and the pores o n rl ey q u bi m a gd L s t c . n f r e v l y normal magnets, and theor ical y al ow sev ral cel s to bind to each bead. Multip e col ida p rticles b nd to each l for sepa tion. B th ap ro ches c o n e t r a lf s x i n v m eoy w Sa p r . l c t i o s n uf c yh e m ag l i c n h s v ue g t d a lo f r b e am d vys r fl c t a i ve n b l mh y u d, t ap e c ment poi s f the larg be ds to he ma li n ce s an c use probl m if detachm n is requi d at later s age. Most repo ts u ing bacteri l ce sy tems hav found tha cel viab ty s no af ect d by he ads. Howev r, us l a orbfge cd ts i p o rnue ls d t c i f a p m e n (9) or hig - rad ent magnetic technol gy (10) . Monit r g Bac e i from Natu l Enviro me ts of cel s with n pore spaces betw en beads during con e tra ion. Entrapment would be min mal if col idal magnetic particles wer used. Ad itional y,col idalm terialh sle s f ecton heopticalpro ertiesofcel s, d o e s n o t a g r e g a t e or l a t i n vg e r fy l c s ) a, n d h o w m r e p i c l b n d tg h a r e b d s . Howev r, use of large beads requi s far les time for separ tion to oc ur under o mal c ndit o s. As wel as po it ve s l c ion f targe c l s, it also p ib e to nrich fo specif l typesb d ion f therc l s,an p o cht maybe dv ntageous in some itua ons. Ad it onal y, it s often pos ible to de ach bound c e l sf r o mt h a g n i cp r l e s ,a t h o u g p r c e d s ha t p o l y m e r s chainre t o (PCR), rDNAsequ nci g,ar ot f ec d els of th par icles. 1.2 Ap licat on f M g etic Par le–B s d S par tions M a g n e c st i l p r o d b , u a ts n i g h f o r w p d c e u s , and ideal for proces ing large cel numbers and searching for ra e cel s. In ad it on separ i gwholec s,t ap ro chis l amen b to h d ecs p e m c o i l fa t g n u h R . sd m o e t particles of rit cal mpo n e, a d is prob ly the im ng step i h roc e d u tr I h if . s v p , e o c d u w r i s l t v e y m o c il n f s r est for furthe study. Withou the abil ty o provide a sel ctiv link betw n targe c l s and the magn ic p rt le, h procedu an ot w rk. Fo wh lecel xtrac ions, the arg ec ptor mus ther fo b expr s d on the bac rial cel wal under ap ropriate environmental condit ons. Whole-cel extrac ions of bacteri from enviro m tal sample have targe d struc e s u fac l h g e o r, - w m al k e s u c h r o t y p af g e n bi c t r a , or sugar residues on cel -wal proteins. When extraction of specif c molecules i required, the c oice of target can be xtend to include intracellu la r mar k ers, including nuclei acids and proteins. Whatev r the target molecu , specif ty and avid ty of the hapten must be confirmed prio to at emp ing t c separ ion. m a g t n h e i co s u H a tb v r p y n li e g , d particles.A ou n dpreviously,th canbe i v d y rect oa ing f he p a r t i c ol h e u g n d r p t o c e u D i. l a b rn eg q u i t phs a c l e throug ac iev d s Th apten. c wil tha po ymer with coa ed b to the use of wel - stab i hed sy tem for covalent y link g proteins, such as tosyl gr up (86) . Direct lab ng of eads i r l t ve y s raightfo w d, an p r o c e d u s h l b p r o v i d e ty h m a n u f c r e S. o n d a ly b i g e r al y invo es u f com er ial y v b e magn tic p r les tha r co ed a f w i n m ot lh u e y c s g p U r , b A . n 79 set l ( hu avoid ng the c s i y of m x ng whe iso- pers bylow ev- 80 kind of sy tem nabl s u er- ais d nt era o be ad pt o magnetic s par s a m g rte u h n cb i o d f v y s , w h e na t c h e dt o h eb a ds u r f a c e .I ns u c ha s e ,i tm a ys t i l b ep o s i b l et o use t h ea n i b o d y f l w n ga i d r e c tm n i a p u r e o t c l ,i nw h i c n a e t r l h u b s w d o f y , i- a n v g e t ing with af in ty for the antibody. A sim lar option for af in ty labeling invol es u s e t r h c ’ o p i l a d b g n s f y t r o ue m h antiser , .g , strep avid n/b ot . Use of such a sy tem d pen s o the commercial av ilabil ty of biot nylated antisera, or the wil ng es of the user t o b i o t n y l a e s ir - h o u A g. a tn e, l s i v y r g h t f o w a p d cedur (86) . Physical separ tion using magnetic beads requires ef ctive mix ng to enable the lab , the b ads, n the c l s to c me into c a t. Aqua ic env r o n m e t s a l p r d y u e n s ( ti a o h c f r a t e d ) m b a e us g f n o p r t i S c l w . d a g d i s p e r a l o t h c e d u r S. a n p o c e d u r fs a m p l e oy n the us of a stom cher bring cel s into su pe ion. It is po ble t include filters n h omac erb gt mov lar ep tic sand ro u e cl ans s i p n e a g t O l h .o v r m s i n du c , e t al y rely on blendi g/centrifugat on step in particular buf ers for optimal disper al. Soi d sper al quires opt miza n for each soil tex ural type, and proba ly for ind v ual soil . Use of i n-excha g resin has be n shown to s ef ti c l r ou d -a A g n s m , . i e c pl r a y o v ment s p (often achi ved by centrifuga o ) wil he p roduce a cle n r su pensio fr m wh c to ex ra g t cel s or m cules. The apt n h is be ng us d i then r qui ed to reac with e targ molecul s in the su pen io . If the hapten is at ched to the beads, this simply means d i g labe d s to he c l su pen io . H wev r, th ap en m y be al owed t react wi h t e arg t molecu s and the b ads ed to cap ure the apten. In either case, the use of a blocking reag nt o prev nt onspec i f cr e a t i o n sm a yb en c s a r y .B l o c k i n gr e a n t sa r eo f t np r o t e i n s u c h asbovines rumalbumin(BSA),milk-derivedprotein,orgelatin.Samples are incubated to al ow maximu cel bind g with the hapten. Gen ral y, the reac tion time is ap rox 30 min. Increasi g this time may not increas r e c o v i s .H w e r ,d n i t yo f h eb a d s n e m o t r a d b e fs i g nif ca e, nd g eral y quires b ad num ers g atly in exc s of the arg cel numb rs. After incubat o , the beads can be washed by rep at d con e tra i and n od e its u rc a gh pv bMo f nt e ys u r h. i as 0. 5– 1% Twe n-20, as wel as maint g the blocking reag nt, in the wash buf er. The cel s can be at ched firmly to the beads, and may remain Porte and ickup Monit r g Bac e i from Natu l Enviro me ts 81 at ched uring vo tex . How ver, this may v r with e apt n used, an more gentl washing (e.g , aspir t on throug a pi et tip) may be pref d. Washing tep w l r move akly, n specif al y bound ce s a l o ce s P u a src m ei l df gb . n t p o h ri a e d ready for an lysi at his tage, whet r by cult re o therwis . If a cult re step is used, the magnetic bead separ tion may be consider a more rapid altern iv o a pre n ichm t ul re st p. Al ernativ y, for hig ly sen tiv d e t c m p na i rg s o f b , h l d . Target mol cu es h as nuclei a ds houl a s be uf ic ntly ea s to mag- toler able r ctions Ma y pr edu b. l gic mo a w netic particles up to certain lev s. Magnetic sup ort such as por us glas show great h m l stabi y (e.g , durin PCR cy ling) tha some p ly rb a s e d A l. t r n i v e cy , lmo s u rba n v fe dto hm s , e.g , by boil ng in det rg n , or by ad ing a competing agent for the hapten bind g s te. 1.3 Enviro me tal M ni or g f Bacteri Using Ma et c P r i l –Based S p r tions M a pg n sre t i dc l mb o h vq ua s det c ion l m ts for nit g bac eri n ma y situ on . O e f th larges s u f m b o i j a c en rd h t l p g y , o m i n a u t s c g m e r a c v A s i g f k n t p l y d ob . m , n of targe c l overy has b n chiev d by cult re- as d m tho ( or direct det c ion ( Table 2 improved by lowering the numbers of no targe bacteri and other contamina ts. Achiev ng a cl s mp e, nrich d targe b c ia, h s en bl d avings in overal det ction time. The IMS (im uno-magnetic separ tion) procedu has now be suc f l y ap ied to a wi e r ng of sample ty ( Tables 1 and 2 ). The majority f epor s u the raig fo w d ap r ch of mix ng an environme tal cel su pensio with beads ready-coated in the ap ro i te hapten. Removing the supernat gen ral y leav s con e tra d beads tha can be used for normal spread plates or direct det c ion. This a p r o c h sb e n u f li w a t e r bacte ria via antisera (1 – 3 lectins (19 , 8 ) . Howev r, vari t ons on this them do exist. Jones and Van Vu rde (18) captured magnetic beads irectly ont a flat magnetic surface, sub eq ntly rub ing the magnet over the surface of an gar plate o al ow c u l t r (e m a g n t i fc s h n g ) I. d i r e c t a p u r e i,wn h c t a r g e l as r e o w d tr oe a cw i l h b e a dn t i b o y ,e f rb i nc ga p t u r eb dyso f a w h i c react with the primary antibody label, has be n suc es ful y ap lied (12 , 14 , 40) . Table 1 ). In both case , sen it v y has gen ral y be n (1 ) , 87) or targe in ind ge ous po ulations using a n ds o i l (12 , 13) ,t a r g e i n s p c f ) 82 Table 1 Examples of Culture-Based Detection of Bacteria after Magnetic-Particle Separation Target o nism Hapten Enviro me t MAb MAb MAb/P Unfractio ed s rum MAb PAb Lectin, PAb Lectin, MAb Lectin ( o a v lin A) PAb PAb MAb Com ercial PAb 82 Strep omyc s liv dan Strep os angium fr le Thermod sulf t mac u obile Rhizob um Pseudom na p tid Pseudom na stu zeri Staphyloc us a re Lister a mon cyt ge s Indige ous bacteri epmac s no ht aX sub p. sp . serog up C1 pv. pela i nogr atrosep ic Soil Soil 13 15 14 1 40 78 , 79 , 97 78 , 79 , 96 19 , 8 18 29 3 37 62, 69–71 38 42 , 64,2 4– 8 , 05 , 16–25 Seaw t r Cult re Cult re, ak w ter Seaw t r Milk Milk, che s am Soil, r ve wat Leaf sur ce Pota pe l Water, s lmon ice Mixed cult r s Com ercial k t Wide var ty of ds MAb O157 12 Blo d, st l Com ercial k t Human d bovi e fa c s, be f, milk recta sw b , ice r am, body flui s, o pond water, slu y gras Porte and ickup Erwin a c ot v ra Aerom nas l o ic da Vibr o pa h em lyticus Salmone Salmone Escheri a ol stri Ref r nc s , 78 Monit r g Bac e i from Natu l Enviro me ts 83 U mos afe g n t i c - b d l o r hn ag es p c d t a b l e r native o enrichm t cul re in some ap lic t ons b e n f it oh d u s r y ,e c i nt gh2 4 - r m e p i otu d n h 1, r but stil yield ng an isolate for confirmat y pur ose . Magnetic separ tion c o m p w a t e i r l h d n u m t e b f o r a d h ls - p i v e and egativ solati n targe c l s may ti requ a s l ctive nr hm t s ep Magnetic-bead-based det c ion technol gy has be n dev loped further w i t hD N Ab e i n gt h e a r g e tr a h e rt h a n s p e c i f ch o s tc e l .M i l a re t l . dev loped a magnetic sy tem wher by sequ nces of DNA from the flanking regions of specif c insertion el ments or gen s wer at ched to the beads. When mixed with DNA extract, he b ads wer able to capture sp cif c target DNA of quality suitable for PCR. In ad it on, this sy tem removed false posit ves and other PCR artifacts dev loped a magnetic capture hybrid zation-PCR sy tem (MCH-PCR) to det c the lux r e l a s eo dn tb a r l e yo tA .f eh ry b i d z a t i o nfhD eN Ax t r a cw i tb he a d s car yingasinglestrande capturep obe,theb adswer separ tedan this step removed the hybrid DNA from its sur oundi g contamina ts (e.g , humica ids)withadet c ionlim tof40cel s·cm same method logy but combined with rev rse transcriptase-PCR (RTPCR),ar pidan reliableprocedurefordet c ingpoli v rusingroundwater was dev loped tha is readily ad ptable for other viral pathogens In this method, a biot nylated oligonucleotide capture was hybrid zed to poli v rus-RNA in solution. Streptivadin-coated magnetic beads removed theRNA-oligonucleotidehybridfromthesample rio t RT-PCR ad it on to det c ion f single speci s, MCH-PCR has be n used to dif erentia eb twe nstrainsof cif c gen as marke . Howev r, det c ion f very low numbers of (64– ) . (24) (91) . Jacobsen (91) gen from an e gine r d strain of also (92) Pseudom nas fluoresc ns –1 barleyro t.Usingthe . (93) (93) and Bacil uscer us (94) . 1.4 Discu on a d F ture P osp c m as p gi e r n o c tl f h dT w y s a C m o p r l e n t c d . h g yb , v s u f m a r e specif c bacteria via cel antigens. Other investigators have used marke DNA as a prom ter to det c surface expres ion of an epitope introduced into target cel s and have used this epitope as a means to track rel ased cel s using im u suited o IMS, and woul a ow m nitor g f rel as d b cteria, pos ibly for biotechn l g a pur ose . Magnetic separ tion have almost alw ys include use of a par m gnetic particle, r ying o the ap n to pr vide th bridge tw n he c l and the . This may be of great (64) noflu resc n (7 ) . An ap ro ch such as this would be B.thuring ensi usingaspe- .In 84 Table 2 Examples of Dir ct n of Ba eri t M gn c-Par i le S p t on Target o nism a Det c ion m h d Bacteri l ox ns (b tuli s A, cholera ECL Hapten Enviro me t Ref r nc s MAb — 89 staphyloc ent ro xi ) Hepati s A Rotavirus (g o p A) Oral spi ocet RT-PC MAb RT-PC PCR sret yo , aw es dn r viR MAb Cros - eactiv 73, 5 74 20 Fresh and w ter Subgin val p que MAb PCR ECL ELISA PCR sp. a u r e f sc n o m serog up O-6,7 Soil PAb Milk, fo dstu , oil PAb Water. f c s — PCR PCR PCR ELISA ELISA, C PCR, E L carot v Soil PAb PCR sub p. (includ g om Salmone Salmone s rog up D Salmone virchow L. mon cyt ge s — — MAb PAb PAb Fec s Com ercial k t Pota pe l Pota pe l Fo dstuf Com ercial k t Eg s, fre hwat , serum — 71 , 52 , 13 , 23 , 63 67 , 68 , 70 , 71 , 95 wide var ty of dstu , ntige 1) ELISA 43 76 45 28 4 21 2,9 29 39,68 Porte and ickup Bacil us th ring e s Bacil us nthracis Bacil us tearo h m p ilus Helicoba t r pylo i Alterom nas Mycoba terium v E. carotv Erwin a ch ys nt emi Salmone Salmone t ri d s Salmone typhimur mures na h ,ret w s f MAb Fo d PCR Conducta e PCR — Com ercial k t Human fec s Skim l powder MAb Che s 38, 51 25,39 36 78 pv. O:3 PCR/DIAN PAb PAb O157 ECL, Electro h mi u n s ; RT-PC , rev s transc ip e- olym chain reaction; ELISA, enzym -li k d im unos rbe t as y PCR, E L Nas l ph ryngeal spir te 26 MAb Fo dstuf , MAb, P MAb Fresh wat , fo dstu Mixed cult r s s li not , ec f 27 30,72 34, 5 34, 5 wabs Fec s MAb Com ercial k t 23 Leaf xtr c s ELISA PCR a — 94,1 52 ; 85 DIAN , det c ion f m b l zed a p i nuc d. PCR PCR/DIAN PCR citr Monit r g Bac e i from Natu l Enviro me ts Bordet l a p r us i Xanthom s ax nop dis Porphy m nas gi v l s Yersin a teroc lit a Shigel a dys nteria Shigel a f xn ri E. coli 86 Porte and ickup p a r t i c l e T. h s o w p c i f t by u s oe d r n ht a p e s b, u m k p a r tion of ind genous bacterial po ulations dif cult. Such separ tions may have a role to play in e vironme tal bacteriol gy, as means of btain g clean cel su pensions tha are rep senta ive of the natural po ulation. At empts to achiev this using lectins as haptens have met with only limited suc es cel s and the magnetic particle by coating the particles with a sugar, or by util zing bacterial lectins u n l i k e l yt h a ts e l c t i v t yw i l b ea v o i d e .H o w e v r ,Z b o r w s k ie ta l . demonstrated more gen ral labeling and sepa c o a t i n g h e im tn h le a n t h a n i d me t a l h a s a n e x c e p t i o n a l y h i g h m a g n e t i c d i p o l e mo f o mr a n cy e l s u r f a c e s I. n c u b a t i o n cf e l s u p e n s i o n ws i t Eh r C l cient to impart suf ic ent magnetic mo ent to concentrate cel s when pas ed in solution ver neodymiu -iron-bor n magnets. Ap roaches uch as this, or use of magnetic col ids, with the extrem ly powerful neodymiu -iron-bor n magnets may l ow for dev lopment of automated separations, because they avoid problems with set lem nt inher nt with large particles. The s l ction f m ercial y v ble kits for u e with magne c s par tions conti ues to increas , and it is like y tha any latex ag lutin o kits (e.g , Pseudom na p l ei be asily d pte for IMS. An ther a t may be of int r s fo me purp o sc e u lt di zhn r o b a c t e i lm n g of the magnetic separ tion pr ces may en bl the us of m re g n ral ntig efcnoasrp t u l , o w it nh ge r d u c of a ts l e i v u yb sequ ntly, i creas ng th producib l ty and suc e r t of as y b u ing a hig ly enr c d a purif ed c l samp e. (8 ) . It m a y b e p o s i b l e t o r e v r s e t h e b r i d g e b e t w e n t h e , but with either ap roach it is highly (90) (85) ration of bacterial cel s by 3+ erbium,asEr .Thistrivalentcation ent, and a hig af in ty [84] ; Cryptoc us ne f rma [83]) (16 , 80– 2) 2. Materials 2.1 Gen ral Ap tus and Co m bles 1. Magnetic p r le con tra . 2. End-over shaker. 3. Stomacher bl nder. 4. Quanti ve prot in as y (e.g , Bio-Rad Pr tein as y k t ). D 2 i L. r Ma e b c g ot nl Bf d s with a User-P oduc Antib y 1. Sel ct d an ibo y an p ro iate f rm o at chmen to b ads. 2. Magnetic p r les uitably co ed f r labe ing ( wasuf i3 se Note 1 ). T s h p. e d could Monit r g Bac e i from Natu l Enviro me ts 87 3 . L a b e l ui fn p g h or K s: C , Na 0 t . e 2l ( 87 Pid 4pBn HS ) , 1.4 g Na and m ke up to 1 L). Wash buf er: PBS, pH 7.4 contai g 0.1% BSA 4. 2 HPO 4 , 0.24 g KH PO 2 4 dis olve in ap rox 80 mL dH O; adjust pH 2 2.3 Direct Lab l ng of M etic B ads w h Lectins 1. Sel ct d in a p ro iate f rm o at chmen o b ads. 2. Labeling buf er: sodium borate buf r, pH 9.5 (1 05.1 g citr acid, 30.9 g boric acid, 69.0 g NaH 2 PO 4 ; adjust pH wi h con . NaOH) 3. Wash buf er: PBS, pH 7.3 amend with 0.1% BSA 2.4 Pre n ichm t for Ta ge B ct ria Gen ral enrichment mediu (e.g , buf er d pe ton water) or sel ctive a n t i b o c - e r ( b a. on g t hi ,m c d u sup lem nt d richment d u ). 3. Methods 3.1 Direct Lab l ng of Ma etic B ds with a User-P oduc Antibody 1. Quantify he prot in he purif d hapten su ion (a t b dy). 2 . S u s p e n dt h ea n t i b o d yi nl a b e l i n gb u f e rt oaf i n lc o n e t r a i o n f4 0 o f protein. 3 . S u s p e tn mhd a g i c r l te hs o u g a ny ld i q s o f t c e an m u i t o steril m c otube ( 4. Wash t e aliquoted stock beads thre times by con e tra i g them in the magnetic field, removing the supernat while holding the beads in the magnetic f i r e a l n s d u , P p B b S h f g F e i r n . a l c oy s , u p t - d tra ion p x mately doub tha of e st ck in PBS. 5. Mix equal vo mes f th was ed b and the ibody ( 6. Incubate 4 7. Conce tra h p rticles and w h t re im s n PBS/ A wash buf er. 8. Resu p nd i PBS/ A and store 4 µ g/mL se Note 2 ). ). Note 3 se ° C for 18-24 h wit end-ov r shaking. ° C. 3.2 Direct Lab l ng of M etic B ads w h Lectins 1. 0 4 fo n itar ec o lanif ot ref ub gnileba ni tcel ht dnepsuS µ Lm/nietorp f g in borate uf r. 2 . S u s p e tn mhd a g i c r l te hs o u g a ny ld i q s o f t c e an m u i t o steril m c otube ( 3. Wash t e aliquoted stock beads thre times by con e tra i g them in the magnetic field, removing the supernat while holding the beads in the magnetic field,an r su pe dinfr shbo ate uf r.Final y,resu p ndtoac en r tio ap roxim tely doub tha of e st ck in borate uf . 4. Mix equal vo mes f th was ed b an the l c in ( se Note 2 ). se Note 3 ). 8 Porte and ickup 5. Incubate 4 6. Conce tra h p rticles and w h t re im s n PBS/ A. 7. Resu p nd i PBS/ A and store 4 ° C for 18–24 h wit end-ov r shaking. ° C. 3. Prepa ing C l Suspen io Ther are sev ral methods av il b e to pre a the in t al cel su pen io dep n i g o the s urc of the arg c l s ( ). Note 4 se 1. Fromwate h c l su pen io ca b t ined r ctlyf om hes urc withno precon t a i , by centrifuga on f a r nge of water s mpl vo umes (10 mL t 1o L0 ) b, y a n g e t i fl o w r a t i fn o lm g e vr u tp 1o 0 (L ter 3) o by ther m ods 2. From s il ev ral methods are vail b e for dis oc at n of bacteri l ce s from sm toa hi elr x 3. Gen ral iso t n f bacteri f om dstuf , ch as me t nd ch es , invol es removal of a defin weight of sample fol wed by hom genizat o (using a stomacher), and often precult is car ied out in, e.g , buf er d pe ton water (45– 7 , 49 Chap- se (5 , 6) . (12 ; se t h e rg bn y f ics a u l p ( o n , 13 , 19) se ). N5 o t e ). Note 6 3.4 Separ tion f T get Bac ri (Note 7) 3.4 1 Using A t era 1 . w al t ekF r o (1 ) l a 1 k - e m L s w u t b ( r c p; l n h f e i o m ) main s ple and ce in a 5-mL gl s te ub . 2. Ad 10 3. Ad 30 4. 5. 6. 7. µ L of 10X PBS ( µ L of bead su p n io (a t m use i nogl bu i G [Ig ] she p anti- b o d i ec s u p l ta to he pr vious m xt re and i cub te a 20 At rac he b ads n bead-c l omp ex t h side of th es ub y placing t in the mag ic p rt le con tra ( Pipet of h supernat d wash t e b ds y a ing 1 mL of X PBS. Rep at Resu p nd beads and bead-c l complex s in desir final volume of 1X PBS prio t fu her manipul t o ( ) and ge tly mix. Note 8 se 8 – s p e c i f m o n l a t i b o d1 y0 Pp .u t i d a ° C for 15 min. step 5 ). Note 9 se two m re i s. ). Note 10 se 3.4 2 Lectins 1. Lectin-bou d beads (78) ligande to sp cif le t n;ap rox10 at 4 ° C for 3 h wit end-ov r mix ng ( 4. Notes with pro e n A, r with an bodies ag n t im u ogl b ins from t ani ls. µ L of lectin-a v ted beads (tosyl-ac iv ted 8 2. The b ads r con e t a d s in 3. Rel as cel s from beads using competing sugar specif to the lectin in use prio t fu her manipul t o ( 1 . P a r t i c l pm be u s y h a d t i v f le o r b ( n g . t , s a y c l i v e o d r ) t : ad 50 – 1 beadsmL Note 1 se . step 4–6 se Note 12 ). ) to hesampl ndi cubate ) b e a m d·L s – 1 ) Monit r g Bac e i from Natu l Enviro me ts 89 2. The stabil y of the lab d s may v r (dep n i g o the ap n us d), a also the pr s vati e d by the manuf ct re wil be r mov d prio t labe ing. Thus, it is pref abl to labe smal amounts of beads and discar un sed beads after 2 wk. The amount of beads labe d wil ther fo dep n on how m u c hw o r ki sa n t p e do v ra2 - w kp i o d .S m e n v s t i g a o r h v en t d pres nc of large particula es in some col id pre a tions, whic should be remov d by a rief ncubatio n he mag tic f eld prio t labe ing. 3. Check ol id surface nd labe i g r qu em nts. 4. Optim za on exp rim nts wil requi sample to be spiked with targe cel s; thes can b d e to h c n e tra io s equ r d. 5. T h e c e l c o n c e n t r a t i o n c a n b e a d j u s t e d b y d i l u t i o n o r c e n t r i f u g a t i o n a n d resu p n io a defin volume f di nt. 6. typesamlnoucridb hsevPlt thisofdavngeTbcr.plymu comprised.bwltaghfquny 7 . u n i v o e m r fT s t h a l d g n p o i c r t a b O y m l z . tion ca he most ap r i te xp r m ntal co di ns be d t rmin . 8 . N a o f g 1 . 4 K C l , o f g 2 N a C l , o f g 8 0 o cf n s i Pt B S 1 0 X id Lm 0 8 ni s t i Ha w l Cd ( 7 e j p . r ,u h 4 o b y z c 9 . T h em a g n t i cp r l e o n c t r a w s u p l i e db yD n a ( B r o m b u g h ,U K ) ; altern iv y, a st nd r ba m gnet could be s . 1 0 . F u r t h em a n i p l t oc u i dn v l e t u r( c l m o v a h i e bs dy n c a t i o or v texing; [1 ] ) or di ect oun i g s acrid ne o a g 1 . Payne t al. (78) recom nd tha magnetic-b d ligande lectins are the most ef ic nt or sepa ting b c er a f om cult re and fo s. 12. Recovry of cels was found to be specif, with the majority of bacteril cels relasd from beads by incubato with competing sugar beads:numr of cl was found t be imporan f sucel partion 450atopimzedwsn 0.25mg,or1–7 × 10 –1 (8) 10 5 mL cfu asw recovy nd tail, esd not wr desi cl Grat . 5.0 than lower dnsit cel using por beads ruc eovy, whras diton f great numbs of magnetic bds di not mprve cy. Adial, t ws foun ha ig step lyd a separtionmgcfbdWhvy.l by vortexing fresh bu gave por ecvis, wth e majority f the cls by eads th wing Hovr, as. ft he r supna t i fod beng aditon f resh buf and eithr nvso r gentl aspiro thug a 1-mL dispoable t ip mroved cy, et raind specfty a cels wr esugar.competinfhbdyv Acknowledgments This work was sup orted by funding from the Natural Environment Res arch Coun il, Sw do UK. 2 HPO 4 KH of g 2.4 , PO 2 4 g )n . i v a . (1 ) (78) . The numbers of , (8) µ 52withsupenoclL 7 beads)forclupnitg5.0 µ (aproxbedslfL × 10 × 10 3 cfu Lm –1 magnetic fwr o Aditn . 3 –2.0 × 90 Porte and ickup References 1 . P o r t e ,J . E d w a r s ,C . M o g a n J A .W , dP i c k u p R .W ( 1 9 3 ) a p i d , u t o - 10. 1. 12 . 13 . 14 15 . m a t es d p r i o nf e c b a t r if ol m kw ea ns d gbf yl oc w t m etry and cel sorting. Ap l. Enviro M c biol. 2 . M i t c h e GW lJ. , RB r o nMS i e lJaH . , d ( 1 p 9 r A 3 c ) t i a l o p t i c a rlf m n u t i a gsd o l bn c t e r fi a o m p l x c b i a o m munit es. Microb. E l 25, 1 3– 9. 3. Bradfo , D. Hugenholtz, P. Seviour, E. M , Cun i gham, M. A , Stra on, H. S e v i o u r R J , . a n B d l c k L (, . 1 9 6 ) r S i b o s m a R l N A n y i o s f l a t e obtained from G a -neg tiv , f lamentous b c eria m o nipulated from ctivated-slu g . Syst. Ap l Microb l. 19, 3 4– . 4. M a r k x , G . H . , H u a n g , Y . , Z h o u , X . - F . , a n d P e t h i g , R . 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Kuhn,H.-M ei r D t ,U. andM yer,H.(198 )ECA,thecom n terobacteri l n ge . FEMS icrob l. Rev Somp linksy, D. Hertz, J. B , Hoiby, N. Jens , K. Mans , B. and S mra, Z. ‘ c o m i - a s b T l h e t fr 1 w . n g d a c ( o i A m 9 e 8 0 n ) mon a tige ’ from Pseudom na eruginosa S o m p l i n k s y D, . H e r t z J B o i b y N, . e n s K M a B, . P e d r s n V B . , and S mr , Z. (1980) An a tige com n t a wide r ng of bacteri . 2 A biochemi al study of a ‘com n antige ’ from 8 , 253– 60. Cur ie, B. P., Freundich, L. F., Sot , M. A., and Cas dev l , A. (19 3) Falsew i p t a h e n f s o r g c l u y b x ti d v p a n cult re-posi v crypto al meni g t s. S m i t h M ,D . W u e k a n V , . W l s h A L a n P d i t T , .( 1 9 3 L ) a e x g l u tina o est f r id n catio f 374– 5. in fo ds. Salmone 23, Int. J Fo d Microb l. r fa w o m S a l m o ne t r i d s 149– 58. 23, O-3 in fecal s mp e and to sil wabs from 563– 8. 78, J. Ap l Bacteriol. J. Virol. 327– 8. 5, J. Virol Meth ds ). Let . Ap l Microb l. 10 – 24. 24, spore in so l matrices. 62, Ap l. Enviro . Microb l. with an ige c surfa e Pseudom na putid 214– 0. 62, , i l oac h r e s E suer a c o lyhpatS , airets L 37 .25–14 , .loiretcaB .lp A J 276– 83 74, J. Ap l Bacteriol. 195–2 . 54, . APMIS 8, 143– 9. . APMIS Pseudom na aeruginos J. Clin M crob l. Pseudom na pseudom l i. , 2519– . 31 J. Clin Patho . 46 , 96 Porte and ickup 85. Zbor wski, M. alchesky, P. S Jan, T.-F and H l , G. S (19 2) Quanti ve separ tion f bacteri n saline soluti n usi g lanth ide Er(I ) and magnetic field. J. Gen Microb l. 138 , 63– 8. 86 . Harlow, E. and L e, D. (198 ) Cold Spring Antibod es, a L borat y Manu l. Harbo L at ry, Cold Spring Ha bo , NY. 8 7 . E S d a P w A n W o u i . M R HC r c , J t K k b e s p 8. 89. 90 . 91. 92 . 93 . 94 . 95 . 96 97 (19 7) Det c ion, distr bu on a d proba le fate of asympto ic a tle on dairy f m. Porte , J., Robins , J., Pickup, R., and Edwar s, C. (19 8) An evalu tion of lectin-m d a e magnetic bead c l sorting for the arget d separ tion f ent ric bacteri . J. Ap l Microb l. G a t o m e n k i g ,D .L Y u H B r n o ,J .G d e M T , i l r . a n dZ u l i c h , A. W. (19 5) Sensit ve det c ion of biot x ds and bacteri l spore using an im uno ag etic el ctro hemilu nesc sen or. 10 , 501– 7. Lelwa gur e, J., Ascen io, F., Ljungh, A., and Wadstrom, T. (19 3) Rapid o s l f p e c t ah i n r d z o APMIS 10 , 695–702. Mil ar, D. S Withey, S. J Tizard, M. L V , Ford J. G , and Hermo -Tayl , J. (19 5) Solid phase hybrid zation capture of low abundance target DNA f n o i t c e d a n r i h e c s m y l o p nt i a c l p - s e n u q si olucreb ta p dna m u i r e at c v b o y M .03 –52 M ( i 1 c s9 r p d bo we5 S a ) . t J hf l C n - D , N A a magnetic capture-hyb id zat on and PCR amplif c t on as y. Microb l. 61, 3 47– 52. Regan, P. M and rgolin, A. 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H e l i cp oy b a r t muiretcabo yM psbu . . l ma en hA c o i B mucit vl s , 62 Ap l. Enviro . 64 , 65–72. and B a c i l ut sh r n g i e s Bacil us Sys. 60, Infect. Im un. Listeria Ap l. Environ. Staphylococ us aureus 27, 163 – 5. DNA Extrac ion f m Natur l Envi o me ts 97 7 DNA Extraction from Natural Environments Kenneth D. Bruce, Peter Strike, and Donald A. Ritchie 1. Introduction rU en ct m i s l ou dy b , a p r n e v i s l o t m e n t rs l i od c v e n t a l p i c m r o s p b e v a t i o n cd u l a t p r b A d v olhi s e c u m y n g . , h n a u v m e b lo ifr t n Ts h. e c o m l iy t d a so n e fm r t h ef i n d g a t h em j o r i y f c b a l e si n t u r a l v o n m e t sc a be cult red in the laboratory—the phenomeno of no cult rabil ty. The fraction of bacteria in soil, e.g , whic can be cult red forms only ap rox 0.3% of the to al number of cel s tha are observ d micros p al y contras , new r methods based on the use of molecu ar biol gy methods to an lyze to al extrac d DNA from natur l specim n , poten ial y sample the entir po ula n d, thus provide a b t r ep s nta iv p c ure of th al microb al munity. c u l p t r i he v aq o w n m , V f c u l s e r h p h o s f a t l y i c d e m u x n S vr o s b p . acids (2) wher as others a direct a n lyzi g nuclei ac ds w i t h cn e l os fr l o w i n pg r i o n u c l e i ac ed x t r a c t i o n permit a more compreh nsive understandi g of particular environme tal i s u e s .T h i sc a p t e rf o c u s e o nt h e x t r a c t i o n fD N A r o mn a t u r a ls o i a n d s e d i m e n t sam ples, but the question , techniqu s, and form of an lysi also co f t r m h g ape d l n,s i u x y and so . Anyspecif atur lenvi o m tw l us a ycont i var e yofp k o t ea sn du k r y T .h ib s o t c m p n e f r q u t l y p e s n ai g f c t d i v e r s a t o n , i s a n d mep lv i r o t s n m a o f p r t i n c o im np s t ( 3 ) A l t .h g o e u n i rm ap c f v t nu e - s From: Methods in B otechnol gy, . By (1) eith r (1) Enviro mental Monit r ng of Bacteria Vol. 12: Edite by: C. Edwar s © Human Pres Inc., Tot wa, NJ 97 T. h e s t c h n i q u e s in s tu 98 ral ecosy t m pr e s i r cogn zed, a u rst ndi g of he c mpl x o posit n, divers ty, and functio g of thes biot c comp ne ts is nec s ary (4) . With s under ta i g, he l k i o d f pre ict ng h o sequ nc f enviro m tal ch nge is a c d. Ther is an u derlying as umption hat e DNA extrac ion methods u ed p r o d m u c l e t a h s n r co e gi f v m u T h t y . of meth d s oul be ap ro i te f r he biol g ca question b g ad res m p o l ate nhc r u d y z s tha ve b n describ , ther is a div s on betw n hose t a do an those tha do n t separ t cel s of inter s from the nviro me tal sample prio to cel ysi . In the forme ( m e t h o d s u c a r ge d i n ct r f u g a i o n cel s while stil with n the enviro m tal matrix is used ( ap ro ch provides an extrac ontai g DNA from eith r live or dea lysed proka y tic and eukaryotic cel s in ad it on to any extrac bl extrac l u DNA (7) persi t ng he sampl . Inpracti l e ms, h i olatedDNAmus b of ic entqual yfor sein the most deman i g of the antic p ed biol g ca procedu s. For exampl polymerase chain reaction (PCR) amplif cation requires DNA of a hig er purity han tha tha can be used for estrict on e donucleas an lyzes. To optim ze the quality of sub equ nt an lyzes, certain other considerations are im portan .Thes i clud max zingthelys of arg tcel s nd ov ring the max u q nti y of DNA r m the x ac ion m l eu wh n pos ible. More v , th isola ed DNA l y shou d be f igh mol wt, c requi s extrac ion procedu s tha min ze shearing. Other desirabl featur s of a ts e c f hp i n w a r q l u o d m g y t , e p and reag nts as pos ible to reduc the chan e of introduc g contami g materi l. Many DNA extrac ion prot c ls have b n published. This lack of procedu al nif rm tys e inpar f omthev iabl n tureof h sampl to be an lyz d, with so l and se im nt sa ple roving part cul y recal it n owing to enzymatic inh b tors tha coextra with DNA. In this chapter, we d e s m c r tai h b o l p y v sDd uNe A t a PfbmCol Rr i c w a t e r ) n d l f , p ts e c i ( m o n a v l r u h e o f a c r t i o n prove may th pro c ls e t pos ibl wher f nc mak d proces , more ap i t for he xamin t o f par icul s mp e . 2. Materials B e f o r m b a k i n gD N e A x t r c o s , v e a f l t r h o u b dc e n s i r ( se 1Note D om N te A h R f r a d ) s . i n c 2 ), and some informat is provide for the xtrac ion f RNA as wel ( Note 3 ). Bruce t al. (5) ind rect i s o Dlm Nae At h nd O f . ) case, this epar tion ca be car ied out by (6) M. o r ce m n l y , s i o f direct e x t r a c( i o n ). This lat er se Note se DNA Extrac ion f m Natur l Envi o me ts 9 2.1 Sampling 1. Gam -ir d ate plasticw re ap ro i te to the size and comp sit n of the sample ( 2. Sampling equ pm nt rel va to he s udy nviro me t ( se ). Note 4 ). Note 5 se 2. DNA Extrac ion 10. 1. 12. 13. 1. Enviro me tal s p e ( 2. Bal nce. 3. Gam -ir d ate pl s ic entr fug bes (15 and 0 mL). 4. Benchtop rifuge s tabl for 15- and 0 mL tubes. 5. Water b h (cap le of rating o 70 6. Bead-b ting mach e wit glas be d n ati g ves l ( 7. Ultracen ifug and ltr cen ifug t bes. 8. 1-mL ga ir d ate syring a d 1.5-in e dl s. 9. Ultravio e (UV) transil um tor. Ice ma hin . Refrig ato . 1.5-mL icro ent fug bes. Micro ent fug . se ). Note 6 ° C). se ). Note 7 2.3 DNA Visual z t on Agarose; el ctroph esi buf er of choi e— ther Tris-borate-EDTA or Tris-acet EDTA, N stain e.g , thid um bro e; l ading ye. 3. Methods 3.2 DNA Extrac ion 1. -daeb ht o ni )selpma tne id s/l o r f elbatius g 1( elpmas fo tnu ma hgieW ( sdaeb lg d va cotu f smarg 5.0 ni at oc yd erla s v gnitaeb 2. Ad 5 mL of extrac ion buf er 10. 1. 12. 13. pH 8.0) 3. Bead b t (for his mac ne, 30 s o et ing 1) a d l ow t se l for 15 min. 4. Pour c nte s i o a 15-mL centrifug be and h t 70 5. Co l rapid y on ce a d ntrifuge a 280 6. Transfe th up rnate o 50-mL centrifug be and hol ice. 7. Ad 5 mL of resh xt ac ion buf er to h pel t. 8. Vortex su pend th l e and h t 70 9. Co l rapid y on ce a d ntrifuge a 280 Transfe th upernat o he 50-mL c ntrifuge b and hol ice. Rep at Centrifug he po l d su ernat 80 Transfe th clear sup nate o a fresh 50-mL tube and polyeth n glyco 60 (to a final con e tra i of 15%) and NaCl (to 10% of the volume of the supernat ). M ° C for 1 h. g for 10 min at 4 ° C. ° C for 1 h. g for 10 min at 4 step 7–10 ° C. . g for 30 min at 4 ° C. .) 8 etoN es (1 % sodium ecyl su fate in 0.12 Na 2 HPO 4 , 10 Bruce t al. 1 4 . P r o e 4 v c D i N p anAt h g 15. 16. 17. 18. 19. 20. 21. 2. 23. 24. 25. 26. 27. ° c e n p tb ryla i 5 f d 0 u g C o n min at 4 Discard the supernat and resu p nd the pel t in 8 mL of TE buf er (10 m Tris-1 m Ad 10 Transfe th con e s t an ul r ce t ifug be. After s aling d bal nci g the ub s, centrifug o 18 h at 18 rpm in a Beckm Ti75 rot equival nt (Beckma , High Wycombe, UK). Extrac the single DNA band, visual zed on the UV transil um tor, from the gradient us g a teril sy nge a d le, pi rc ng the sid of the ub . S h a k et D N Aw i ha ne q u lv o m fc e s i u h l o r d e - s a t u di o p r a n l . Remov th (pink) layer cont i g e h dium bro e. Rep at Dialyze th sample ov rnight TE buf er. P r e c i p t a D N w A 0 h . v 1 o ls f d i u a m c e t ethanol (10 %) overnight a 4 Pel t h DNA by centrifuga on t 13,0 Remov th supernat d w sh t e DNA p l et wic th 70% e anol. Resu p nd i a p ro iate v lum of water ( .g , 10 g 10for ° C. M M EDTA, pH 8.0) µ L of 10 mg/ L ethid um bro e and 8 g of cesium hlor de. ° C, using 50, step 20 and 21 twice mor . M p ,4 H . 8 a ) n 2 d v 5 o l u m e f (3 ° C. g for 30 min at 4 ° C. µ L) ( ). Note 9 se 3. DNA Visual z t on 1 . P r e p a n g o s e l( 0 . 7 %a g r o s ei nT B E A b u f e r )c o n t a i g2 µ Lo f 10 mg/ L ethid um bro e. 2. Load a porti n (e.g , 10 µ L of sample) and ap ro i te mol wt marke s (e.g , kilobase d r) into he ag r s el. 3 . F o l w i n eg c t r o p h e s i a 1t 0fV o r, n p e c h g l u, s i nUatV r l u minator, f he siz and tegri y of DNA extrac d ( ). Note 10 se 4 . E s t i m a o nc t h e b m a d o ft h eq u n i yo fm a t e r l x a c t e d( ). N o t e1 se 4. Notes 1. Thed signa ex cution f hesamplingre m sof undame t li por ance; c o s n p t f a i h d er u l g m s c n t p eo ensur ta is c l onsi te cy and re uc vari b l ty a so, rem b tha n ural enviro m ts are in flux and tha it may not be pos ible to rep at a sampling regim . 2. Monit r g s importan i w de numb r of envi m ts, wi h d f er nt vir o n m e v a t s y g i u h n ol b c f e r p s t a hd u e enviro m tal rix. a. Sequ nc s of mi r b al o ig n (v ral, ch e ba t ri l, nd/or fu gal) h ve i sf or l Dam Nt Ae p d b n (8–14) , peat bog materi l a qe un v ti r co m s hot spring sedim nt (25) , marine microb al m ts soil a uch t e r sn v i ao l m t (15 , 16) , landfi s e a w t urc h (19 (23) , marine sedim nt , and sub rfaces (17) , 20) (21 (9 , 24) (26) , hydrot e mal vents ; from (18) f r e s h ,w ad ti m n , biof lm microse t n (27) , and hypersalin , 2) , DNA Extrac ion f m Natur l Envi o me ts 10 b. lakes (28) and blo d products (34) i n c l u d m g k: plants (38 gal comp ne t f lichens Methods have be n dev loped for specif c ap lications, e.g , extracting microb al DNA fre om “c nta i g” plant DNA from c p st t uh dose if r n c t h pe i f g r a v ot u y n s pl r e a mt i x e x t D r d N a i A c f g op u n e l s ods have be n dev lop to extrac DNA from many plant, bacteri l, and fungal speci couple th x raction f DNA with o er c l u ar comp ne ts uch as lip d , from the sa oil r sed m nt a ple. ; from (31 includ g: canker d wo c s ha en u d , g , seagr leav s from , the fun- (41) , and liche s and t ir symb ont fo d and; (37) (40) (42) ; from (3 ) (36) , blo d (30) , bronch alve r lav ge fluids , 32) o y s t, e r (35) , 39) includ g: dental plaque (29) clin a sample (43) . or (4 ) (45) (46 , 47) m oe t Yh - r , . . Some th ds, uc a th of Ke rm y t al. , (48) 3 . S t ui dn v e s g ab t o l i c rh e mf u n a w t i o v r m e n t s 4. 5. 6. 7. 8. becoming increas gly com n as the technol gy adv nces and the ne d for enviro m tal oni r g nc eas . Thi ap ro ch als d an importan ew dimens o t s udie of biol g ca flux in m crobial ecol gy. Moran et al. showed tha rRNA could be recov d from bacteri in various enviro m tal sample inc ud gse im nta dso l.Ahy r x ap ti es n-colum eth d as be n shown to be ef ctiv in extrac ing DNA and RNA rapidly from natur l sedim nt (50) c o m p at hr e i nv b d cl oa m u n i st y e g p r a u - d i ge n lt el ctroph esi rof les ribo mal sequ nc Gam -ir ad te , plastic, Universal screw- ap d contai ers and centrifuge t u b e s , c a hF l o t n u b e ( s . g A , l p h L a b s E t l e i g h U , K ) a r u s d o t i n e l y for sampling so l and surface s dim nt of up to 15 g, with gras nd other su face pl nt grow h fi st remov d xpose bar il. The sampling equipm nt used varies ac ording to he nviro me t, he sample s i z e ,a n dt h p o fs a m l et b k n .T h s ec a r g f o m l a e ds p t u , coring devic s (surface soil and sedim nt sampling), boring equipm nt (de p t a n f g i e l o (s ruw m b ) p , q a . c n fd e Inthela r x mple,c on tra i ,e.g usin m crop e mb an sor y c e n t r i f u g a o n , s f t e r q u i d o v e r c m p b l e so ft h wm i c r b a l o mas in tural w e s. Samples hould be proces d as o n as po ible to av id changes during stora W gs h et .o n r i a l m , pf – r eo2 sz0 t n P r o l n g e sd t a o f m p l e s 4t ref. 5 2 e x p s t R r SN o a n A c f bq m h u d ) i l . k y A B. Braun cel hom geniz r is used her with 0.17– 8-m diamet r glas beads l cte for bacte i l ysi . The method described her is modif ed from previous studies ensure tha e m thod pr vi es a pr enta iv s mple of DNA r m the nvironme tal sample—th orig nal reason for taking a mo lecu ar p roach—it s e p c mf o s ti T h b a y n d l - . m e p t o r a n (49) . It has l o be n show t a nuclei a d extrac s n be us d to (51) . ° b ep o l ri s f Cw , . ° cC a rn e s u l it h g dn e c t i o l v ( se (9 , 53 , 54) . To 102 Bruce t al. ( c b s h e a o w m l t d n i S k D xg ) mize lys . s er u qh it nO v ac d be r s o t a n c e ih r o p f s l e dc y l e a fg s mh on u t i d c e m i t os a u pr n e g c i d b f r e z - t h a w i cn yg l (e s t o c l s i n r e a c l y s i . A t h o u g e s i m a t v r y c o n s i d e a b l y , u p t fo 9 6 % yduts eno i d syl ne b vah stnemid ram fo selp a ni t es rp l c 9 . T h i rs e m o v a n y u b s t c e i n h b t o r y e z m us id n o l e c a br o g i cal pro edu s. H mic a ds, foun req tly in sample fro natu l e vironments, are know inh b tors of enzym s such as Comp unds such as polyvin l poly r olid ne, whic reduc s the ef ct of inh b tory su ance by sor ing hum c o p unds trime hyla onium bro ide wh c over m s the f cts of inh b tory chemicals th coextra wi h DNA extrac ion ( si erohp tc l eg sor Elutipdcomns(Shera,Kigt-upTU) chromatgpyexn 10. h g i f o e b t A N D d e t c a r x e h t r o f s i t n e m r i u q e h T moregnatyxsifcatoherdbnastDNAw.molhig chimer s qu n d g PCR a pl f t o resu th sen g suog l moh .ytisrev d lairetcab fo lev ht fo n ita erp tnis m eht ro seic ps tne 1 . The yield enviro m tal sample. Methods are av il b e tha can be used to quantify the extracted nucleic a ids and tha wil also provide an estimate of the purity of the sample. As exampl s, van Elsa et al. extrac d f om ive d f r nt soil , var ed f om 2 t 35 range of b tw en 2.5 a d 6 9 organic arbon, clay onte , and pH dif er nt soil ranged b tw n 6.1 and 54.0 µ g/ of sedim nt has be r cov ed 1 ( s e l p m a i ) 0o g 75 ) and .fer es 6 h a ) v l eb s oi n c r p a t e d i o f r p n t - ref. se g n i t a e b - d s r c ,n i a )5 ( e s. a n i t o Kr P m a e ( )65( . )42( DNA polymeras Taq (38 (6 , 61 , 59 , or hexad cyl- , 60) , have b n recom nd i other DNA , 62) ag- include; s b have wic stp urfaon Ohe pcds. .sfer es 64 , 36 , )46 noitule r c , 2( hguor t e as p , , )56 ,ion (5) (6) capture-hybidzonmg, (67) . and ytirup hgi tob— ytilauq fon itac filpmaoc )86( -tsixeno f noitp rcsed ht o dael nac siht ,smret lacit rp nI . otf aDlNvA r i ews d ayc o r i ntg he x a c i opnr e s tdh found tha yields of t al DNA, (56) µ g/ of s il. S m arly, µ g/ of s il wa found r eight so l f di er nt , and i a sep rat s udy, ields from (57) µ g/ of s il (24) (62) . For sedim nt , 47 . Acknowledgments .gnid uf l cn oC h raes R tn m orivnE la oit N yb detrop us aw kro sihT References 1. Aman , R. I , Ludwig, W. and Schleif r, K. H (19 5) Phylogen tic dentif caa n dt i o Rev. 59, 2. B a r d g e t , R . D , L e m a n s , D . K , C o k , R . a n d H o b s , P . J ( 1 9 7 ) S e a s o n a l i t yo f h es o i lb o t a fg r z e da n u g r a z e dh i l g r a s l a n d s . 29, 1285–1294. . (58) situ n i n d e t m v o wf c r u la h b s i n . Microb. 143– 69. S o i lB o .B i o c h e m . DNA Extrac ion f m Natur l Envi o me ts 10. 1. 12. 13. 14. 15. 16. 103 3. Kil ham, . (19 4) 4. O’Don el , A. G., Go dfel w, M., and Hawks orth, D. L. (19 4) Theor tical p ra n cd t q i u s h eo l f b a t d i v n e r s m o c y g a n i s m . Phil. Trans Roy. S c 5. Lef , . G Dan , J. R McArthu , J. V and Shimkets, L. J (19 5) Comparis n o fm e t h d s D N A x r a c t i o nf ms r e a d i n t s . 61, 1 4 – 3. 6. Tsu hima, S. Haseb , A. Kom t , Y. Carte , J. P , Miyash t , K. Yok yam , K., and Pickup, R. W. (19 5) Det c ion of gen tical y engi r d micro ganism in pad y soil using a simple and rapid nest d Polymeras Chain-Re ct o method. Soil B . ochem 7. Trevo s, J. T (19 6) DNA in so l—ad rption, ge tic- ransfo m ti , molecular evoluti n a d gen tic m rochip. Microb l. 70, 8. Liesack, W. and St ckebrand , E. (19 2) Oc ur en of n vel groups f the dom a b i n c t e r s v la b y d g o ie f sn t m a c r l o t f e Ada u n ms r lian ter s ial env ro m t. 9 . R i t c a h n d e S P, . r M k O A s b o L n , H J . m D a W i o r n s , K B . u c e D. A (19 2) Amplif cat on f DNA from native po ulati ns of s il bacteri by u s i n gt h eP o l y m r a s C h i nR e c t o . Smal , K. Cres w l , N. Mendo cah gler, L. C , Wolters, A. and v Elsa , J. D. (19 3) Rapid DNA extrac ion p t c l from s il f r Po yme as Ch in Reaction med a plif cat on. Silva, M. C., More, M. I., and Bat , C. A. (19 5) Dev lopm nt of a molecu ar det c ion method for napht le degra in pseudom na . 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(19 6) Distr bu on of sulfate-r ducing bacteria in a stra if ed fjord (Mari ger fjord, Denmark) as evalu ted by most-proba le-number counts and enaturing radient gel ectrophoresi of PCR amplif ed ribos mal DNA fragments. Microbi l. 21. Rochel , P. A., Fry, J. C., Parkes, R. J., and Weightman, A. J. (19 2) DNA e x t r a c if1 o 6 n Sb s mRg aNe lA y d t io s r m g ne d cv r s i tn y de p s im nt co u i es. 2 . Chandler, D. P., Fred ickson, J. K., and Brockman, F. J. (19 7) Ef ect of PCR templa con e tra i on the comp sit n and istr bu on of to al com unity 16S rDNA clone ibra s. 2 3 . B a F M r u . S n , s d J E y e R g f ( P 1W i 9 N c 4 m ) a r k b l e arch e l divers ty det c in a Yel owst n Nation l Park hot spring enviro ment. Proc. Natl A d. Sci U A 24. Gray, J. P and Herwig, R. P (19 6) Phylogen tic a lysi of the bac ri l ommunit es marine s d m t . 25. Taylor, D. G., Bre n, A., and Bishop, P. L. (19 7) Det rminat o of phenol degra ist bu on i f lms u ing e prob s. 26. Muyzer, G. Dewa l, E. C , and Uit erl nd , A. G (19 3) Profil ng of c mplex m i c r o b a el d - p n Pg t u y i h f ro s meras Chain Reaction amplif ed gen s coding for 16S ribos mal RNA. Enviro . M c biol. 2 7 . M u y z e r ,G . T s k A ,W i r e n C .O ,a dJ n s c h ,H .W ( 1 9 5 )P h y l o g e n t i c relationships of hydrot e mal vent sample by denaturi g gradient gel- ctroph esi of 16S rDNA f agments. 28. Martinez- u c a, A. J , Acinas, S. G and Ro riguez-Val r , F. (19 5) Evalu t i po rfn k a y d ic v e r s bt y i c do n g e s t 1 r6f DS N iA c al my p fied rom hype salin v ro ments. 2 9 . D e l a m b r i eX ,.Z a n d o t C V i g l ,.B o e tCa nD d m i c oP(,.1 9 2A ) one st p microb al DNA extrac ion method using Chel x–10 suitable for gen amplif c t on. 3 0 . - i r d u n p o a t c x e f h d i .m p ) A a 5 r 9 , g1 E P ( e b n d . h KG D sa i r P cif ation f DNA rom dental p que. Bruce t al. in a groundwater 5271 86–93. 60, Ap l. Enviro M c biol. Ap l. Environ. 3405– 12. 62, in labor t y micro s . Escheri a coli J. Microb l. Meth- Ap l. Environ. 1405– 62, 14 5. 10 , FEMS icrob l. Let 59–6 . 6, 475– 82. Mol. Ec 9 1, 1609– 3. 40 9– 5 . 62, Ap l. Enviro M c biol. 3 1, 1 9– 2 . Water R s. Ap l. 59, 695–70 . Thiom crospira speci s and their identif cation in de p-sea Arch. Mi ob l . 164, 165– 72. FEMS icrob. E l Res. Microb l. 143, 17, 247– 5 . 785– 90. Ap l. Enviro M c biol. 61, 4120– 3. DNA Extrac ion f m Natur l Envi o me ts 105 3 1 . G o l b a n gN ,.B u r i eJP K l a p r E ,. B o s t c kAa nW di l m s o P( ,.1 9 6 ) Sensit v and u iversal method f r mic ob al DNA extrac ion fr m blo d pro ucts. J. Clin Pathol. 861– 3. 49, 3 2 . T a g l i f e r C L o . , b l M a i t P G e , g r a V ni . o d t e ( l 1 P 9 , 5 ) 3. 34. 35. 36. 37. 38. 39. 40. 41. 42. 43. 4. 45. 46. U s oe raf p i d n m l e t h o d x r a c p v i Dl N A tn h e d i f c a t o n HIV–1 by PCR. Jaulh c, B. Nowick , M. Bornstei , N. Meuni r, O. Prevost, G. Piemont, Y. Fleur t , J., and Monteil, H. (19 2) Det c ion of alveo r age fluids by DNA amplif c t on. Candrian, U. (19 5) Polymerase Chain Reaction in fo d microbiol gy. Microb l. Methods Tola, S. Angio , . R c hig an , A. M Idin , G. Manu t , D. Gal eri, . and Leori, G. (19 7) Det c ion f Polymeras Ch in React o . Brauns, L. A., Hudson, M. C., and Oliver, J. D. (19 ) Use of the Polymeras Chain-Re ction in det c ion of cult rab e and no cult rab e cel s. Ap l. Enviro M c biol. Ehrman , M. Ludwig, W. and Schleif r, K. H (19 4) Rev rs dot bl hy ridizat on— usef l method f r the direct ident f ca io f Lacti Acid bacteri n ferm nt d o . A S B uE Pa r d. i C L y, n H tG o ( 1 cp 9 gh e 6 ) and se it v PCR based y for c n u e t d c ion f bacteri us ng common a d h l b ig ts n bea d. Vol s i uk, T. Rob , E. J , and N zar, R. N (19 5) Direct DNA extrac ion f r o r g a nsi ml . f e d y tP C R Brown, A. E., Muth me nakshi, S., Sre nivas pras d, S., Mil s, P. R., and c i, o fe ) t. n 3 T r 9 u A R pb m 1 s C ( P w S det c ion f the pa og n i ap le wo d. Weidn r,S. A old,W. an Puhler,A.(19 6)Divers tyofunc l redmic o ganism ociated w h t e s agr tion Fragment Length Polym rphis an lysi of PCR amplif ed 16S ribos mal RNA gen s. Grube, M., Depri st, P. T., Garg s, A., and Hafel n r, J. (19 5) DNA isolat n from lichen as om t . Armaleo, D. nd Clerc, P. (19 5) A rapid n exp siv method f r e pu if cation f DNA rom lichens a d t ir symb ont . molecuar nd Purifcato (194) A. Ogram, nd C., Petigrw J., Kain M., alik compst.frDNAibalny Okuda, T., Yan gisaw , A., Fujimor , F., Nish zuka, Y., Takeh na, Y., and Sugiyam , M. (19 5) New isolat n methods and Polymeras Chain Reaction s t r a i dn c m a t i o n e c h q u fs o nr a t pl d u c s r e n i pg o a m s . Bot. 73, S 946- 5 . Porte us, L. A., Armst ong, J. L., Seidl r, R. J., and Watrud, L. S. (19 4) An ef ctiv method to extrac DNA from enviro m tal sample for polymeras 30 – 6. 18, Microb l g a sp . in bronch - Legion l a 920– 4. 30, J. Clin M crob l. J. 89–103. 23, in she p milk sa p e by Mycoplasm g lacti e 54, Vet. Microb l. 17–2 . Vibr o vulnif c s 2651– . 57, 143– 50. 1 7, FEMS icrob l. Let 86, Phytopa l gy 361– . 3972– 6. 61, M E i n c v r Ao pb l . fo r amenor t ph ac dnilyC estima d by Restric- Halophi st ulace 76 – 1. 62, Ap l. Enviro M c biol. Mycol. Res 9, 1 7– 20. 108, FEMS icrob l. Let 132 – 4. Lichenol g st Methodsicrbl.J 207– 13. 27, 20, 183–96 C a n J. 106 Bruce t al. chain re ction amplif c t on a d DNA fingerp t an lysi . 301– 7. . 29, Cur . Mic ob l 4 7 . L a r s e n d , S . DJ gk K o A b a h Ol M r e , . Rn u d i F. (19 7) Rapid, universal method to isolate PCR ready DNA using magnetic beads. Biotechn qu s 506– 1 . 2, 48. Kehrm y ,S.R Ap legat ,B.M Pinkart,H.C edrick,D.B Wh te C., 49. 50. 51. 52. 53. 54. 5. 56. 57. 58. 59. 60. and Sayler, G. S. (19 6) Combined Lip d/DNA extrac ion method for enviro mental s p e . Moran, M. A , Torsvik, V. L , Torsvik, T. and Ho s n, R. E (19 3) Direct xt r a c p iu n o d f r t b s e o Rc m N a Al g i - s t u d e . Microb l. ( eR1 xa9 tp B N ri6. d) c Dwn T S l o kM , E m bJ e P y Ku .r d , o fD N Aa n dr i b s o m lR N Af r e d i m n t sb ya o v e lh d r x y a p t i es n - c o l umn ethod. F e l s k A E . n, g B N u b e U al , d c k h ( H 1s . 9 D 6 i ) r e c t b o m s e x l t b asr fo c i m n R Nu a A i l t y s . Enviro . M c biol. J o s e p h o n ,K .L G e r b a ,C .P n d e p r ,I .L ( 1 9 3 )P o l y m e r a s c h i n - r e a c tion det c ion f no viable bacterial pathogens. 351 – 5. Sel nska, S. and Kli gmu er, W. (19 ) DNA recov y and irect d ion f Tn 5 sequ nc from s il. Bruce,K.D Osborn,A.M Pearson,A.J Strike,P. andRitch e,D.A (19 5) Gen tic diversity with n no cultiva ed so l an edim t bac eri . P i c a C o r . n d , s N e gS E t X m ( 1 P 9 D . 2 , ) c i o n and enum ratio of bacteri in soil by direct DNA extrac ion and polymeras chain-re t o . vanEls , J. D., Manty e , V., and Wolters, A. C. (19 7) Soil DNA extrac ion and s e m nt of the fa o dif er nt soil by 16S ri os mal RNA gen s qu ce bas d mo t-pr ba le number PCR and im u ofl resc n . Zhou, J. Z , Bruns M. A , and Tie j , J. M (19 6) DNA recov y from s il of divers comp it n. Teb , C. C. and Vahjen, W. (19 3) Interf enc of humic acids and DNA e x t r a c d i e t l yf r o ms i nd e t c o a d r n s f m a t i o r e c m b n a tD N A from bacte i nd a ye st. Porte us, L. A. and Armst ong, J. L. (19 3) A simple min - ethod to extrac DNA directly from soil for use with Polymeras Chain Reaction amplif c t on. Cur . Mic ob l Berth l , M. Whyte, L. G and Gre , C. W (19 6) Rapid, rect x a ion f DNA from s il for PCR an lysi u ng polyvi pyr olid ne sp columns. FEMS icrob l. Let 153– 6 . 25, J. Microb l. Methods A pE nlv.i r o 915– 8. 59, 3905– 7. 62, Ap l. Enviro M c biol. Ap l. 4162– 7. 62, 59, Ap l. Enviro . Microb l. 13, Let . Ap l Microb l. 21– 4. gen s directly amplif ed from com unit es of mer 4, 605– 12. Mol. Ec 58, Ap l. Enviro M c biol. 271 – . strain PC -1 in Mycoba terium chlor p en icum 62, Ap l. Enviro M c biol. . 27, 1 5– 8. 138, 316– 2 . 59, Ap l. Enviro M c biol. 17–2 . 18 – 95. 24, Biol. Fert Soils 2657– . DNA Extrac ion f m Natur l Envi o me ts 107 6 1 . C h LJ .o He D, Y a K ( n i 1Sd m 9 6 ) r e c x t a - 62. 63. 64. 65. 6. 67. 68. tion f DNA from s il for amplif c t on f 16S ribos mal RNA gen s quenc by Pol meras Ch in-React o . Wikstrom, P., Wiklund, A., Anders o , A. C., and Forsman, M. (19 6) DNA recov y and PCR quantif c o f cate hol ent soil ype . Erb, R. W., and Wagnerdobl , I. (19 3) Det c ion of polych riated biphenyl p o el xy tm a r n Dd c N s A i b u e g r t a d n s i o chain-re t o . Her ick,J.B Madsen,E.L Bat C A., ndGhiorse,W.C (19 3)Polymeras Chain Re ction amplif c t on f napht le cat boli and 16S ribos mal RNA gs e nq u fi cr od m s e u b a nc t r i . 687– 94. Vescio, P. A and Nierzw ck bauer, S. A (19 5) Extrac ion a d purif cat on f P a C m R p l i f b D N er A o a m c u s t i n b r f a s c e d i m n t . ods 2 1, 2 5– 3 . Lovel , C. R. and Piceno, Y. (19 4) Purif cat on of DNA from estuarin sediments. J. Microb l. Methods ( 1M J 9i a S c .5r o ) bC sd e t p w n l , f h i c D rN A a a magnetic capture-hyb id zat on and PCR amplif c t on as y. Microb l. Wang, G. C Y. and W g, Y. (19 6) The fr qu ncy of chimer olecu s a c o n s e P q a C u m R p f l i r 1 c b 6 o S gt s d e n N m A a f l bacteri l sp c e . 34, J. Microb l. 2 9– 35. 2, 3-dioxygenas from di e 107– 2 . 52, J. Biotechn l. 59, Ap l. Enviro M c biol. 4065– 73. 59, A pE n lv .i rM o c b l . MJ i. c r o b l e t h - 16 – 74. 20, Ap l. Enviro . 6 1, 3 47– 52. Microb l gy 142, 1 07– 4. Sequ nci g of DNA 109 8 Automated Sequencing of DNA Retrieved from Environmental Samples Mathew Upton 1. Introduction g n d e i h t z Ts a f r u v o q l ne hd c t o r m ynaM .daerps diw si ygol iborcim latnem orivne ni esu rieht dna ,ygol ce laiborcim e s v m a h t y s e o ca r l f v i db o g r e n c h i t m a g s e v n i d t u f e so c h r lato m rf secn uqes neg fo n itac filpma det grat fo hcaorp a nom c a detpoda dna gni olc yb dewol f )RCP( noitcaer niahc esar mylop eht gnisu AND detcar xe -imod sah ecn uqes neg ANRr S61 fo yduts ehT .sremilp a fo si ylan ecn uqes s e i d u t has n -s ecorpeht cuderyltaerghci w,seuqinhcetgnic euq sdetamo uafoytil ba i va fo rebmun tneic f us a fo si ylan eht wol a dna seluc om AND dev irte fo emit gni .metsy a niht w tnes rp ytisrevid eht fo evita nes rpe erom sgnid f ekam ot senolc .smet ys gnic euq s AND detamo ua fo esu dna selpicn rp eht srevoc retpahc sihT 1 ( , ) 2 e s ud y f a l o cp e hi s b t v. ra d c u S s 1. Princ ples of Aut ma ed S qu nci g of DNA hguo tlA arev s d t mo u AND ecn uq s i yla smet ra yltnes p -erp noitam f h ,) d wS l s U BKL aic mr P ; oC ,.g e( lb v -nikreP ]IBA[( sm t y o d lp h f u ab i re c s t n , e n s om g )u t Ka A i Uc 3 y 7 r b S N D lq W E h .enihcam d su yl o detamo uA ND gnic euq s g tar e a desab no eht niahc o t nimre .la te r gn S yb d vi c o h m .giF 1 . e sA aN rD m y l io p d u t c n ) - g ( fo dne '3 ht o )PTNd( se ahp irt o lcunyx d g a b eluc om t p a remi p sohw tegra si detacol '5 fo eht noig r f AND ot eb .d cn uq s I noit d a etalpm ,AND ,esar mylop f ub ,remi p dna ,sPTN t h e ni wohs l c t rp a e g f dn )3( From: Methods in B otechnol gy, Enviro mental Monit r ng of Bacteria Vol. 12: Edite by: C. Edwar s © Human Pres Inc., Tot wa, NJ 109 10 Upton s c h e rmA a pt i 1 .F D o N n g lh s fye v d sample fro nvi me tal s p e . reaction mixture contai s di eoxynucl tide triphos ate (d NTP), whic wil be randomly incorp ated in o the synthe iz d DNA strand. Si ce DNA p o l y m e r a s q u i f r e 3 'h y d o x lg r u pf t h e n z y m a i cf o r t n a phos die t r bon wi h c m ng dNTPs, i corp at n f d NTP result in cha termin o , a d re ction ubes wil conta mixture of synthe iz d DNA strand of i er nt l g hs. Clas ic y, d NTP molecu s wer labe d with radio s t pe , and sepad G ( T CP , A N f os u r t h e a c f o r e u t i nd s and T P)wer subj ct o le r ph si onadj ce tl sofap y cr l mide gel. The dev lopm nt of fluoresc nt dye labe ing of dNTPs for DNA sequ nci g l a bn e c, u d s oiNh T j P r g pa t e m l c u h i gs t (4 , 5) a s l r i e n o g wv c t d m p h s i Sequ nci g of DNA 1 o u f wn ai s vq e l T hg x bc try . d al ows d uble-stran d DNA (ds ) to be th rmal y denatur nd result in l ear mplif cat on f synthe iz d DNA. This cy le s qu nci g ap ro ch gives r at yield an more ac u t res l than e stand r e ctions w h heat-l bi DNA polymeras . A variation of the dye terminator method detailed previously use fluoresc nt y labe d prime s and unlabe d d NTPs to produce termina d DNA strand . Dye prime s quenci g requi s four eaction ves l , one for e a c hd N T P , l t o u g h ep r d c t sa o m b i n e d u j c t o l e r p h si n a single an of the g l. Cycle s quenci g an be p rfo med using dye prime s and Taq ing s a f vorable p roach w en clo ing w th vectors c ntai g tar e s for l a b e dc u s o n , a d R e v r s , M 1 3 - 2 a s u c h p r i m e d y t a n prime s a co r i l y av b e (ABI). r e q u ti p h a - s d b c y l n r m e o f gt T h i ments a d xperi nc of the p ra o nd the yp of t m la e o b nalyzed. Taq polymeras cy le sequ nci g has the obvi us adv nt ge of increas g p r i mn eo s c f d ut a l v e h a n d y i e l , p r o u c t an ealing and the ef cts of secondary struct re in template molecules. Althoug dye prime sequ nci g requi s a reaction ves l for each d NTP, increas g cost and the risk of operat er o , and secondary struc e can sequ nc DNA of readings (98%) c u t ve ap ro h st , fal e c u up to 50 b . Dye t rmina o cy le s qu nci g w th com nly used technique and is best uited for an lysi of PCR products. Dye terminator sequ nci g has sev ral adv ntages over primer sequ ncing: the reaction can be perfo med in one tube; false stops wil not be det cted by an lysi software; and obtained from st empla s. Owing to he po ularity of the dy termina o ap ro ch, t is ap er focus n thi ec qu . c hu es t m id r , an y F o l pf w v g e sample are loade on single lanes in a polyacr mide denaturi g gel and s l e Da p Nb A trh , d i n 1 v g 2 u j o c e a l p h f r s i fragments ac ording to size. Standard gels contain either 4.75 or 6.0% acryl mide. Gels cast with 4.75% acryl mide are usef l for det rmin g the s e q u n cob fa l st e hp r i m a ,n6 d. 0g % lw s i v eo r u t n b oa fsd ei tp hr l m D u . n g c o p h r e l s a i b , m nc o 2 g t 4 e h i 0 l p m o s a O u rc n . , f g e h f i e l r x t c p m o h s n g u a d - b, i y e t c multip er b . An el ctro i s gnal cor esp ndi g to he yp and mou t f f l u o r e s c d n at h i y o f w r d u e Taq polymeras t increas g l tren h. Dy prime s qu nc- Taq ac ur te (98%) sequ nc dat can be polymeras i perha s t mo polymeras 12 Upton eht dna ,detai n si si ylan detamo ua ,et lpmoc si noitcel oc at d nehW .gnis ecorp - s e r o u l fe h t om a r g t a m o r h cas d e t n i r pd as e l i fa t d e i s ad e r o t s a t l u s e r g d n e i z s M a y u r t .l d h c en f b E su k t a e n d p c e c n u .q s S i h la bm d t r po f C Px w ,yl aicrem o b v s g p l fo en t i d r m b h ac dna sremi p RCP fo g ht , I v hsotnica .seditoelcunogil 2. Materials 2.1 Prepa tion f Templat DNA from PCR 1. Steril , u t ap re dist l wa er (dH 2 . R e a g n ftPosCr : 2 O) and 0.5-mL icrofuge t b s. p o l y m e r a sbn ud f N, T P p r i m e s c ft ao r g Taq gen , a d mi er l o . 3. -ogil r f stnega r o eci h fo re ub gni ur dna )KU ,elo P amgiS ,. e( soragA .)tiK no cet D dna g ileb L GID miehn aM r g i heoB ,.g ( niborp ed t lcun 4 . M a t p e u r f i o D ml N s c A b y h n p o r t e s l i u w g h m e l t p i o an g r ( s . S e , P l a o q u Br F i M p C d c R t o s , k l a n ME) or by MicroSpinS-40 HRColumns,Pharm ciaB otech;QIAquickPCR urif cation Columns, centrifugation (e.g , Centricon-10 Micro-C nce tra or columns; QIAGEN). 2. Alka ine Lys I olati n f Vec or 1. Soluti n I: 50 m glucose, 25 m M Tris-HCl, p 8.0, 1 m M M 2 te ra c i d (EDTA), autocl ve 10 lb/in 2 . S o 0l .u I 2t i : n N 1N f 0a r O o H m , ethyl n diam e- for 15 min. N s o d t 1 i % uc e mk l , y 2f 0a r (v/ ) stock. 3. 5 p o t a s i u m c e t ( p 4H . 8 ) A: d 1 m5 oL gf l a c i e t ad n 2 8 . m5 L 2 O to 60 mL f 5 M of H 4. Soluti ns f 5 5. 95 and 70% ethanol. 6. TE buf er: 10 m 7. Tris- atu ed ph nol. 8. Chlor f m. 9. 3 M potas ium ceta nd mix wel . M NaCl nd 13% PEG 80 M Tris-HCl, p 8.0 1 m steril z d by autocl ving. M EDTA, pH 8.0 M sodium acet solu i n, pH 5.2 2.3 Cycle S qu n i g React ons wi h Dye T rminato s 1. Dye T rminato Re dy action Cy le S qu nci g K t (ABI). 2 . R e a g n ft Pos Cr 0 .: 5 - m iL c f u tg e b s , Taq p o l y m e r a s bn ud f N, T P prime s ( th r pecif to he arg t ne or h v ctor), and mi er l o . 2.4 Electroph si f Samples Using De atur Polyac mide G ls 1. dH 2. leg rof spmalc dn )KO ,asluT .cnI epaT d r ef P ,lecamr P( epat l c mreP 2 O, warm dH 2 O, Alcon x ( o , New Y rk ) and li t-fre pa . plates. Sequ nci g of DNA 13 3. 40% acryl mide so ut n: 19 acryl mide:b s-acryl mide. 4. Mixed-b on excha g resin (S gma, Po le UK). 5. 10% (w/v in dH 6. N, ' 7. 10X TBE buf er: 108 g T is, 5 boric a d, 8.3 g Na tha e pH is 8.3 and pre ag in f the pH is d f er nt. 8 . B l du e x t r a on i b ug f e 1r : mLblueD xtran) d5 ing 10 mL of orma ide with 1 g of mixed bed resin for 15–20 min, filter ng throug pa e nd stori g n al quots –20 2 O) freshly p ared m oniu pers lfat o u i n. -Tetram hyl en diam (TMED, Sigma). EDTA, 1 L dH 2 µ l oL a d i bn ug f (e m5r 0 M O. Check 2 E D T 8pA .H, m30 g / µ Ldeion z f rma ide.Forma idep r bymix° C. 3. Methods DNA of pre a ti n qu d ech i s t a l ec ion f w g Th templa roduce by PCR and the sub q nt a om ed nalysi o n ABI r e a sc t qi uo n . yg l m r d e f o w i Sn yg s t qm u c 3 7 A r e f b d c a n w mh i u s e rl ’, t f do a p b n h v e Tc l s m fd o e tr a i l n s o dc r p t i e f n a o d l y si of DNA templa fro the s urc . 3.1 Prepa tion f Templat DNA from PCR dna ,gnic euq s AND ni ro e fo esuac nom c tsom eht si etalpmet ytilauq ro P - el ci fn oa h rt p d e s u t o a l c i h t d e m w r o f l e u b n p h i s t a r p e -caer gnic euq s fo emoctu eht cef a osla l iw etalpmet fo yti nauq ehT .desu serud ( at d netsi noc f noitcudorp of dezim tpo eb ot sah dna s oit es .) 1 etoN 3.1 Reamplif c t on Templat DNA from Bacte i l Co n s 1. Suspend single col nies aris ng from cloni g exp riments in 10 double- ist d H 2. Heat o 98 3. Use 1–5 µ L steril 2 O (d H 2 O). ° C for 10 min. µ L as templ in PCR us g the prim s and co it ns of he rig nal amplif c t on rea i . 4. Check for p senc of i sert by g l e ctroph esi r ol g nuc e tid prob ng. 5 . R e m o vu n s d z y m eb ,u f rp i sa ,nd N T Pf r ot me p l a D NbgAy el ctroph esi or centrifuga on throug one of the many com er ial y av ilable co umns. 6 . Q u a l i t y n qd o tf h De N cA a bn r s e d gy l c t r o p h e s i w t a mol wt s and r . 3.1 2 Isolati n f Vec or (Alka ine Lys Proc du e) ( se 1. Harvest h cel s from a 50 -mL cult re by centrifuga on t 60 pend th l e in 10 mL of S luti n I co ai ng 5 m / L of lys z me. 2. S t a n d a t r o m t e m p r a t u r e f o r 5 m i n i n a B e c k m a n ( P a l o A l t o , C A ) S W 2 7 polya lomer tube (or equivalent). ) Note 2 g , and resu - 14 Upton 3 . f r e p o sc i h n t l Sv m y ab L u 2I x d 0 A ; g gently a d s n o ice f r 10 min. 4. 10. 1. 12. 13. 5 dloc-e i f Lm 51 d A M .nim 01 rof eci n d ats )8.4 Hp( eta c muis atop 5. Centrifug he t b a 23,0 g for 2 min at 4 6. Transfe qual quanti es of the supernat to each of two 30-mL Corex tubes 5 o 0vf a. d1l n 6 and st o ice f r 20 min. 7 . P e l c tDb hNyn A r i f u g a 1 2 ot, n 0 Note 3 8. Wash t e p l in 70% etha ol r m te p a ur nd y u er vac m. 9. Resu p nd the DNA in 40 Ad an equ l vo me f ph nol; mix and ce trifug o 1 min. Remov the aqueo s phase, ad 40 centrifug o 30 s. Rep at his ep onc ( 04 d A Remov al tr ces of than l d ry un e vac um. Res p nd the DNA p l et in 20 ° C. M e q v P a u o EN n l G f C d m 80 ( S i g w mMe a l )x . g r maot f3i e 0n p u( r se ). µ L of TE bu er. µ L of chlor f m, mix the soluti n, and ). Note 4 se 0 7 – o t l c d n a l o h t e % 5 9 f o L mµ 1 d n a e t c a m u i d o s f L ° .nim 02 rof C µ L of TE bu er. 3.2 Cycle S qu n i g React ons wi h Dye T rminato s 1. Ad 4 Sequ nci g K t (ABI) o steril 0.5-mL icrofuge t b s. µ L of co ktail mix from the Dye Terminator Ready Reaction Cycle 2. Ad 1 µ L of 20 pm l ri e to ach ube ( 3. Ad templa DNA to ind v ual tubes at the fol wing con e tra ions: PCR product, 20– ng; s DNA, 0.25– 4. P l a c e t u b s i n a P C R t h e r m a l c y i n g m a c h i n e a d p e r f o m t h e c y l e s q u e n c ing reactions u der th fol wing co dit ons: 25 cy les of 96 for 15 s, and 60 3. Electroph si f Samples on De aturi g Poly c amide G ls y r a d n o c e St h y t i l a u qd n t a u qf oe l p m A tN D d e s u g n i c e u q s ,snoitcaer ht y ilauq fo eht dimalyrc op eg dna sti luferac noit per si eb ton dluohs etalp s alG .at d etaruc fo n itcudorp tne si oc eht rof tna ropmi d e ro t unl pai f s b , h d e g r coi t sfa u .slangi t ecs roulf noitce d h iw er f tni yam ht re am t luci rap evom r s i a e dt r o D hg pn c u e l t s n di z y l a w . sr t a m o f d e y c b h u t m o a r p g t u f e o h s ac n p i b r d q e S -ruF .selif cneuq s lbitapmoc-CP r -hsotnicaM s dna leg ht fo gni acs re l .9 retpahC ni d uof eb nac si yl na ec uq s fo liated r h 3. 1 Prepa tion d Cas g of P lyacr mide G ls Because the procedure for set ing up and run i g gels on automated m a r c e h f d i o n g , ks v q t u l w y e m should be ma to h rel vant us ’ m al . se µ g; dsDNA, 1.0 Note 5 ). µ g. ° C for 30 s, 50 ° C for 4 min; soak t 4 ° C. ° C Sequ nci g of DNA 15 1. Wash gl p ates, comb and sp cer with Alcon x a d w rm ate , rins w th warm te fol w d by H 2 . A l pi g an wt s e h c Cr l o . d m ng p a e s i c t r with Permac l t pe, xcluding a y r bu les. R p at o se l h ot er sid . 3 . T 2om5dL H of 40% stock a ryl mide soluti n, a d 1 g of mixed-b resin. Heat nd ge tly stir he mixture ntil he ur a c ystal begin to dis olve. R move from the at and co ti ue m x ntil he crystal e compl t y dis olve . 4. Degas th olu i n f r 5 min us g a 0.25. Ad the d gas e oluti n o a 10 -mL easuring cyl der contai g 8 mL of 10X TBE buf er, and m ke th volume p t 80 mL with dH 6. Pour the soluti n to a 150-mL beak r nd mix n 40 persul hat o u i n a d 45 7 . e s b c u a t p ol i r . w , h f n g m 3 – 5 C p ta l or e h f i u s y 8. Lay the plates flat, we th casting comb in 1X TBE buf er, insert b we n the p l a t e c sn ,d io m A l p. tg he w y mr ao if z 2 s t temp ra u , b t do n use a g l fter mo han 18–24 . 9 . R e m o v a l t p n cd m fs r o t h ge l w, a r c e os f y l a m i d e r o t h plates wi h dH 2 2 O, and ry with l n -fre pa . a O d 4 o g u0 f r e 9 , . m 5 ( L 7 g % e l a ; d 1 m 2 f L o 6 r ) µ m vacu filter un ( ). Note 6 se O. 2 µ L of 10% am oniu 2 µ L of TMED, avoid ng r bu les. O, and l ow them air d y. 3. 2 Gel Loading 1. Remov uni corp ated ye t rmina o s by centrifuga on f reaction products thoug spin colum s (e.g , C ntri-Sep, P nc to Separ tions; M cr Spin -40 HR Columns, Phar ci B ote h, Up sal Swed n). 2. When usi g a 24 wel comb, ad 4 to 5 sample; u only 3 3 . B r i e f vl oy t a cnx d r i f u sg e m p l b o hr a t i 9 n 0 g on ice f r no m e than 1 . 4. Careful y flush al wel s with 1X TBE buf er. Load sample in od -number lanes d loa ing buf er in la s 0 nd 25 or 37 ( 5. R u n a l s a m p l e s i n t o t h e g e l b y e l c t r o p h r e s i f o r a p r o x 5 m i n . F l u s h a l wel s with 1X TBE buf er and load ev n number d lanes. Complet the sequ nci g run. 3.4 Concludi g m ents A l t h o u g ea n y s i o fD N A e q u n c si r a l yu e d c t i n v r o mental monit r g, it is an importan precu so to many of the methods inv o l e A d u . t m a D s N q n c i e g h u a wr os d l v y i b n e e d r f a t n v q o c u i m ws lh p o d s I.cn t r a h e d i o n u c l t e s fd o r a b l i n gD N A m e t s clas i DNA sequ nci g ap ro ches, the fluoresc nt y labe d prime s and d NTPs used by autom ed sy tem are no haz rd us and have long shelf µ L blue d xtran lo ding buf er to each µ L of l ading buf er o a 36-wel comb ( se ° m f ai2oC sn r td e se Note 8 ). Note 7 ). 16 Upton l i v e T sm h .a j d o r w b at ch f ki e s n o l t i gh s ye c a l p i t n v e s t m n . H o w e v r i , t a h p d l e y v o p i t n e g c h q u e m s a , c h i n be r s o m i n g a v able that reportedly are able to read lengths of over 120 bp and perform bid rectional sequencing in one run. Such advances wil reduce run ing cost and can only serv to facil ta e a great understandi g of microb al divers ty. ail- 4. Notes 1. Templat DNA for sequ nc an lysi in enviro m tal microb l gy is most com nly obtained fol wing PCR and cloni g, although shotgun cloni g a p r o c h e su i n g a m p l f i e d x t r a c D N Ah v eb nu s d .M i x e D N As p c i ae ms p l f tb Phyd C rR e a u s i n tg d a cr l o i n eg h q u Vs . c tors com nly used for cloni g include pUC BW a lI n u) d e, s c r ( i S p t a g e C n m , b r i sd U K ) T e h v . c t o r n s a i pv f ra i cso tm e l n gy q u d a c T s e i m p . l f o t r sequ nci g reactions can be obtained by amplif c t on of the targe gen from bacteri l co nies u ng specif prime s, or by isolat n of the v ctor f l owed by sequ nci g from p i ng s te in h vector he targ en . 2 . a l T k h B e i p y r o Dt s n f b c d m u cial y vail b e pr a tion methods include QUIAGEN columns a d Promega Magic n pre s (P om ga). 3. Centrifuga on s mple at 4 4. Removal f traces of ph n l is e nt al s i c n af e t dy p rfo mance. 5. Targets for sequ nci g prime s can be in the vector arms, and can be used to s e q u nc l o Dw Ni dAt h p ur k n l e s do gfq ti h c r p , cif hybr dizat on s e ar chosen wit he clon d DNA. 6. Degas in the soluti n for the sam length of time for ev y gel nsure producible r su t . 7 . F o r m a l d i t s e b u n f gD h r N o A m i t s d a c e r bands blue d xtran s i t n sample vi ua z t on. 8. The ABI 37 Sequ nci g System can ru eith r 24 o 36 sample . If using al lanes, care must be taken to avoid overfl w of sample into adj cent wel s. In a d r i et chf o g n s l , y a um ’ t r ni ce k q s s a m p l e o f r i k t h d u c e o a n t h i s , vb T d e m p w lc r . overfl w, el s are loade altern y, od -number first and then ven umber d on s with br ef p iods f el ctroph esi b tw n loadi g. (6) , pGEM (Promega Madison, (7) C o m .e r - ° C may result in p e g of salt . References 1 . B a r n s ,S .M D e l w i c h ,C .F P a l m e r ,J .D n dP a c e ,N .R ( 1 9 6 )P e r s p c t i v e n v i mr fs o d a q t u chp y l . , Proc. Natl A d. Sci U A , . G N , k e n a j m u R , . A 2 J , s u l a P , . M K , n v ia l u S ’ O , . W P , h c o r k S , . J , n a m e r o B J .L ,N i e n h u s J . ,a dT r i p l e t ,E .W ( 1 9 6 )M o l e c u a rm i o b ld v e r s i t yo fa n agricult soil n W sco in. 93, 918 – 3. Janse , Ap l. Enviro M c biol. 62, 19 35 – .3491 Sequ nci g of DNA 17 3. Sanger, F. Nicklen, S. and Couls n, A. R (197 ) DNA sequ nci g w th c aintermina g hib tors. 4. Smith, L. M Sanders, J. Z Kaiser, R. J Hughes, P. Dod , C. on el , C. R ( 1 E 9 HF . a8 ol L n6 u e d) iB , K r C S s t c a u i n o mated DNA s quenc a lysi . 5 . J o n e s , E . H r C Vh a k iJB n, d g m. H e r S F u n g , R . C o e l , B., Mench S., Mordan W., R f M ecknor, M. Smith, L. M Springe , J. Wo , S., and Hunkapil er, M. W. (1987) Automa ed DNA sequ nc an lysi . BioTechn qu s 6 . Y a n i s c h - P e r VCo .n , iMaJ sd ( 1 9 gI8 m5 p) r o Mv h eca3dl n ing vectors and host rains: ucleotid sequ nc of the M13mp 8 and pUC19 vectors. 7. Birnbo m, H. C., and Doly, J. (197 ) A rapid alk ine xtrac ion procedu for scre ni g combina t pl smid DNA. 74, Proc. Natl Ac d. S i U A 321, Nature 5463– 7. 674– 9. 5, 342– 8. Gen 3, 103– 9. Nuclei A ds Re . 7, 15 3. Analysi of DNA Sequ nc s 19 9 Analysis of DNA Sequences Mathew Upton 1. Introduction M o l e c u a br i g ml e t h o d as r n cw l uy e td o c b a r i n d i v e r sn o m ta g ifnrs l e,d m ta n u g s (3) and ma li n niques mo t widely us in det c o m h ds are th polymeras ch in rea tion (PCR) and olig nucleotid probing. PCR exploits prime s targe in a region of ribos mal RNA (rRNA) know to be specif to the organism of inter s (8) , or functio al gen s for metabolic pathw ys exclusiv to certain b cteria, nd facil t es qualit ve or quanti ve d t ct aoi rf g n e s m u vl i r o n e t s c h f e r l m a u iw d o t p n s b molecu s and use to pr be nucl i ac ds extrac d from sa ple giv ng qualita ive or quanti ative information regarding the oc ur enc of target organism . In ad it on, fluorescently label d olignucleotides are increas inglybe us dincomb at wi hflo cyt me r f l counti g os r t i n g ( 1 2 , 1 3 ) a ,nw di tc ho f l s e r a n i m g c o p y erat in s tu dat rev ling c ose pati l s oc ati n f orga ism n e viro mental s p . A l t h o a u n g s D e yN q f i r c d l n tv y o m e tal monit r g, many p roaches r ly on the an lysi of DNA sequ nc dat fo r d e s i g n o f p r i m e r s a n d p r o b e s . T h i s c h a p t e r f o l o w s t h e a n l y s i o f methanogen DNA sequences recovered from peat bog samples as an example of the types of sequ nce an lyse tha are perfo med in environm e n t a lm i c r o b i o l i T ng h fy e . r m a t p s u id b en o dt eh a ofi p l rs n g u m e at d r i vl y h b e ad res giv n. From: (4 , 5) ptloa n (1 , 2) (6 , 7) . The tech- tis ue and fo d or water sample (9) (1 ) and toxins (10) O l i. g o n u c e t b d a s , ag in (4) (2 , 14) Methods in B otechnol gy, Enviro mental Monit r ng of Bacteria Vol. 12: Edite by: C. Edwar s © Human Pres Inc., Tot wa, NJ 19 tg oe n - 120 Upton 2. Materials w o r k - P C p e f a u s nl i d T Mh y w m g c s o I f p n t a mc w e Ak r i - . y a g t enr sd f oi lb w h a r vne dm s ti u f g l t r a n s p f e o g ( l F T m A P ) i u. t r n g q o a d l p i h y logen tic r s and equ c alignme ts. 3. Methods 3.1 Sequ nc Dat Edi ng u s i n g e d t a l i g n b e c a D N A t r g e o f a m sn t q u I c ie d l y , p t a h co e kf n l g i s d be found by fol wing ks at In er t si e uch as the Ribos mal D t b se d o w n l b ea c v d f p ir l e k b ta hg s o M P( rRn Dy j e) c. t over th In e . Tf ho el w ip nr g t c u a s d l y f o e q n r a g m t s of the P C R b y e n vD iN rA co l m dt a f p be n h d mt a o g e n s with prime s ME1 and ME2 ( formed using an Ap lied Biosy tem Inc. (ABI; Perkin-Elm , War ingto , UK) 37 A Sequ nci g System using dye t rmina o chemistry with prime s ME1, 2 4 and 5 ( e n x T o l h i t a s u. v c d r T a1 b l e α -sub nit of the methyl coenzyme M reductase (MCR) gen of Fig. 1 se Note 1 ) (15) . Automa ed sequ nci g was per- ). 1 . 3 DS A f 7 t ae B r h q I o u m n c i y s g f a p r o t m n e d l y s i software as a Macintosh sequ nc file and as a printou of the chromat g produce by the laser scan i g of the gel during el ctroph esi ( Note 2 ). 2. Visual y inspect hromat g s, whic are p int d col r f easy interp ation, f r mistake d by the au om t d base-c l ing software ( 3. Import sequ nc fragments ME1, 2 4 and 5 i to he S qu nc Navig tor package(ABI).Usingthec roma g f r e nc ,d let po r-qualityd from both ends of the fragments (us al y 10–5 base at the 5' end and ap rox 10 –20 base fromthe3'terminus).Overlap lignfragments1with4and2 with 5 to form a pair of ful - ength sequ nces and rev rse complem nt the lat er strand. Perfo m a compar tive overlap of the two strands. Using the “Cre ate Shadows” featur of the pack ge, compare the forwa d and rev s s t r a n d c o e ta n ym i s c h e b yr f n c et o h r l v a n tc o m g r a s . Compute a c ns u from the di s quenc a d xport i a new fold r as a “GC ” format ile. Th s quenc a lso be transl ed to amin c ds in a y of the p s ibl read ng f mes. 4. Copy the x or d file to a PC-c mp tible f op y disk u ng the Ap l fi e convert prog am (unles using a PowerMac, whic an write directly to a PC-for ) ( se Fig. 2 se Note 3 ). Analysi of DNA Sequ nc s Table 1 Some Sources of Software for Analysis of DNA Sequence Data a Facil ty 12 Sequ nc avig tor Factur Oligo MacDNASI Pro OPD GenBa k EMBL D BJ GSDB Laserg n Functio Source/a s inf m t o re c Sequ nc dit g ABI, Perkin-Elm Sequ nc dit g ABI, Perkin-Elm Prime d s gn/a ly is oftware Nation l B scien Hitach Sof ware Engi r Ame ica ht p:/ w .cme su d /OPD ht p:/ w .ncbi lm h.gov/ ht p:/ w .ebi ac uk/ _home. t l ht p:/ w .d bj nig ac. p/ ht p:/ w .ncgr o / sbd ht p:/ w .dnast r com Prime d s gn/a ly is oftware Dat b se of lig nuc eot d pr be s qu nc Search bl d ta se of DNA quenc s Search bl d ta se of DNA quenc s Search bl d ta se of DNA quenc s Search bl d ta se of DNA quenc s Sequ nc dit g, a b se r f nc , prime and probe sign, re t c ion s te a ly is Wiscon Pa k ge Sequ nc dit g, a b se r f nc , prime Univers ty of Wi c ns , Ge tics Compu er G 12 (GC ) and probe sign, re t c ion s te a ly is, phylogen tic a lys SEQN T Ac es to maj r d t b se and th GC ht p:/ w .dl ac uk/SEQN T home. t l edit ng a phylogen tics prog am RDP Search bl d ta se of rRNA sequ nc , ht p:/ rd .life u c d / phylogen tics prog am PAU PHYLI Clusta W Tre Vi w Phylogen tic a lys Tre alignm t View ng f l s rom t e drawing p cka es a , e t u s ; c K ia y U b r J n o B m D I A d N f p L k E e , S t u G a q ecn r f I ynegol hP , ILYH ;krowteN cn uqeS ,TENQ ;ynomisraP gn U si ylanA cite golyhP , UA ;tcejorP sab t D l mos biR 15 ht p:/ evoluti n.ge cs wa hingto .edu/phyli . tm Phylogen tic a lys ht p:/ w .no emb /gro.te phylogen . tm #Clus a W ht p:/ axom ny.z log a. c uk/rod t e vi w.html ygo , De Pd Oi t o l c u n g b r ; s a t D, k B n e G u r y b e h t l a n o i N u s I f h t l a e H , ) I N ( ; A S U , L B M E n a e p o r u l c M i B ; ,PDR .egakc P 12 Upton Fig. 1 Sequ nce fragments of the α sub nit of the m thyl coenzyme M reduc- tase g ne. F i g2S . e c t o ah nf r m g p o d u c ebt yhA B3 I7 S q n c i gy s t e m . T h ep a k s , n dt h eb sc a l df r o mt h e ,c a nb p r i t e d c o l r( A ,g e n ;C b l u G, black; T red) to as i n terp a ion. mat ed isk). Using FTP, transfe the file from the flop y disk into UNIX file space with ac es to the GC (Wiscon Pack ge, Univers ty of Wiscon , Gen tics Compu er G , Madison WI) uite of pr g ams. 5. Perfo m a FAST se rch ag inst e G Bank d EM L DNA sequ nc dat b a sc t eh o ki m l r MthsfCye R q u n c o p r v i u s l y b m t e d to he da b s ( Note 4 se 3.2 Downstream A ly is of DNA Sequ nc Dat 3.2 1 Sequ nci g Pr me D sign s Me o Eq pn 4uc rw ia m d t g f l o cT sw eh i d n u r and w s rep t d u ing seq c ME2 for design pr me ME5. 1 . I nt h eG C f a c i l t y , g ne d i s q u n c e f r o mp i M E 1u s n gt h eP I L E U prog am ( 2 . a r p l e s i gI dtqo n huf . c m V y 3 ' a s l p e t o ft h ea l i g n m t w h c a l s e q u n h a r e o m l g y n dt h ef o l w i n gc r t e ria e s t fi d: se Note 5 ). ). Analysi of DNA Sequ nc s 123 a. Prime s hould be 17–30 nucleotid s in le gth with a G+C conte of 40–6 % and b. Ther should be no regions of self-comp entari y tha result in hairp n lo p formati n. Th s e p cial y mportan he 3' nd of a prime . c . I d e a l p y r , i m s q u e n c h o l d t ma i sn c h w e t r g i o n I s f . is not p ible to arg suitable , d gen rat b se hould be cat d he p s r e i t q m h b u n o c a y f d l 5 - s 'e i ing at he 3' nd. d. Prime stha rge ions f ec daryst uc ein mplat DNAwi notperform pti al yev nwh sy t e iz d otheab v sp cif at ons( 3.2 1 a,b c T m of at le s 45 ° C. Subheading ). 3.2 PCR rime D s gn 1. Import he s qu nc to be scr n d fo PCR prime a s into he GC pack ge at he SEQN T facil ty ( 2 . RtP uh neIp Mr Eo g as mi t e dq u ar n g cs S h . i e d par met s ucha prime nd o uctleng h.T prog am ut ical ys n the pos ible prime pairs and rej cts any tha do n t sa i fy a r nge of requi ments, i clud ng those at form p i e d m rs and those wi d f er nt ues ( se 3. Prime specif ty should be as e d by perfo ming BLAST/F search ag inst rel vant dat b se (e.g , GenBa k, EMBL) or using the Check Probe facil ty n he RDP ( se ). Table 1 T Note 6 m val- ). Note 7 se ). 3.2 Oligonuc e t d Prob Design 1. Import he s qu nc tha e prob is t arge into he GC pack ge run i UNIX workspace. 2. Using the PILEU prog am, align the sequ nc with cor esp ndi g sequ nc from b th close y r at d n ista x ( 3. Visual y inspect h MSF file produce by the pil u and i e t fy a region tha dif er nt a s he qu nc of the arg o nism fro al thers ( 4 . o l iA gn u c e pt sr o h d b l u c i e n f g or a t ( sp er qi um n c g 15 and 30 ucleotid s n le gth ( 5. Input the sequ nc sel ct d for use as a probe into SEQ D with n GC and p e B r L f Aao S T m / F s r gc ah i n e lyt v d b a (s e . Gg n, k / EMBL) to che k at he prob is ec f to arge x ( ). Note 8 se b e ct ow m n l ay r p hs u g ) , S3 u. b2 h e1 a d i n g se Note 10 ). se 3.2 4 Phylogen tic A alys - n o r i v l ae u t m on sr f c e u q A N ioD s y l a en gh t v o s ei d u yt n a M ( segakc p si ylan citen golyhp fo esu kam stnem se ab t d cilbup ot ecn r f yb sniart de utl c ro/dna sec uq denolc e wt b 1 elbaT ( 1 elbaT .1 dna 0 sretpahC ni ev g ra se yl na cite golyhp f sliateD .) Note 9 se spih no taler fni ot ) Note 1 ). ). 124 Upton The ap ro c s u ed an b pted o al w use of th r pack ges, whi r m e o a d b v Ul i s n K t c y h u g -F . a n l y - t h e f o r l s r a n g e f u l p r o Dv ai d e s b u y t S E Q D o a c e s d m , si of DNA equ nc s. e x t v a n r s -di T h p c ko u f g e vn at il y a b fl DoesNr A q u n c y i ms a,to hf e r n d l cy s i b o ve r the Inter . The abil ty o rapidly an lyze s quenc dat and i ent fy probe ap nr di mt e gs c a r w h t im e o l u c g s at l d i e can be p rfo med. At he most ba ic lev , thes ap ro ches an quickly and easily nd c te h pr s nce o ab f organism , cluding s ow- r ing tax nd those ught o be unc ltivab e. If used j ic ously, the c niques canle dto gr a unde st i gof her l sp ay db t rge o anism its na ur l e vi onm t. 4. Notes 1. published rvoy ben hav ME2 and ME1 primes on Studies Frag . (16) ments ME4 and ME5 wer sequ nced from primers designed by ref renc to the sequ nces of ragments ME1 and ME2, resp ctively (Upton, M., unp blished primes, quncg of desi th on ifrma F dt). 2. The s qu nc files an o be c nv rt d o PC-c mpatible formats. 3. Chromat g sc nbeu dsol yf rc n i mat o fsequ nc i format nby se 3.21 Subheading v i s nu a pl e c dr t qo b m f , h nu y a l into edit ng pack ges. Mistake made by the autom ed base-c l ing software include incor e t as ignme t of base obscured by hig background, cal ing a incor e t number of base to a seri of peaks, and as ignme t of base wher ther s ould be pac s in reg o s f c mpres ion f equ c dat . 4 . R e sF uA a lS o tT f r c b h n d i m e s t a q o u f y n c d 5. 6. 7. 8. wil give an ind cation of the novelty of the cloned DNA. In ad it on, the sequ nc most l e yr at d o he st qu nce a b opiedfr mth a base nd u as ref nc strai d n phyloge tic an lys . When using a single c one/s qu c , the alignme t is not p s ible, and prime design can be p rfo med by visual examin t o f the single chromat g or sequ nc file. PCR prime s can be sel ct d by visual y scan i g the sequ nc for suitable regions. PCR prime s are g neral y 18–2 nucleotid s in length and should be design d under the crite a used for sequ nci g prime design ( 3.2 1 C s a t h ) v r o .b ek u ic g l d n m f p t a r b i o n y h e l ab ne dt w p r i m s ch e u l a tni r p o md e f a t i o n . This cre n is not ec s ary when d sign prime s for nwa d sequ nci g of cloned fragments, althoug obvi usly prime s hould have only one site n the targe mol cu . s e g q r f u n R N o1 A m6 c S x a F p l e , a l i g n e dw t h1 6 Sr D N As e q u n c f r o m t h e b r so ft h ek i n g d mA r c h a e Subheading M e t h a n o s br c i k e bw eo u l d Analysi of DNA Sequ nc s 125 (both eurya ch and crena h ) and a rep s nta ive of the Bacteri . This alignme t lows regi n of ter ax v i b l ty o e asily dent fi . 9. Locating su table r as c n be as i ted by using equ nc s from sev ral o g nism of the targe group in the pileu . Target site can also be sel ct d using some f th so ware list d n 10. Entire gen sequ nc can also be used as probes. Such probes can easily be produce by labe ing PCR products using specif prime s with digoxy en labe d (DIG; Boehring Ma heim) dUTP a e to h reaction m x ure. 1 . D a t b s e r i n u g p d a t e o s cr h u l a b i t s e n b l t s o c r e a n probe for mis atch site ev n when using previously published probes. Thes m h a by v e d n s i g f o h r e m l g s u q n f c r e o m t a g n i s m was ubmit ed o h at b se. . Table 1 References 1. Wagner, M., Rath., G., Aman , R., Ko ps, H. P , and Schleif r, K.-H (19 5) In-situ identif cation of am onia oxid sing bacteria. 18, 251–264. 2. Sier ng, P. L. and Ghiorse, W. C. (19 7) Dev lopm nt and ap lic t on of 16S rRNA-ta ge d probes for det c ion f iron- and mang es -oxid z ng sheat d bacteri n v ro mental s p e . 3. L a c o u r t , I . a n d D u n c a n , J . M . ( 1 9 7 ) S p e c i f c d e t c t i o n o f nicot a e sequ nc of its el c n g e ParA1. 4. H o s h i n a , S . , K a h n , S . M . , J i a n g , W . , G r e n , P . H . R . , N e u , H . C . , C h i n , N . , M o r t m i ,.L g e r f oP a nW d i s t e B( ,.1 9 0D )i r cd te oa n m p l i f i c a to n sie . Diagn. M crobi l. Infect Dis. 5. Ho, S. A , Hoyle J. A , Lewis F. A , Seck r A. D , Cros D., Mapstone, N. P , Dixon, M. F Wyat , J. I Tompkins, D. S Taylor, G. R and Quirke, P. (19 ) Direct polymeras chain reaction tes for det c ion of humans d imals. 6. Yam ot , H., Hashimot , Y., and Ezaki, T. (19 3) Comparis n of det c ion methods for Legion l a speci in enviro m tal sample by col ny isolat n, f l u o p r a e n s ty c im h b d g . 37, 617– 2 . 7. Olsen, J. E., Aabo, S., Hil , W., Notermans, S., Werna s, K., Granum, P. E., Pop vic, T., Rasmu en, H. N., and Olsvik, O. (19 5) Probes and polymeras chain reaction for det c ion of fo d-borne bacterial pathogens. Microb l. e l b a i r v e p y h A ) 4 9 1 ( . P , d 8 n. a m r o N d n a , . J , r e y Z , . K , p e Z , . D , n h a H , . W , e g a l r n o H -nu fo itas re c h if ceps rof teg a nit m rcs d a e ivorp n ge ANRr S32 .aikn rF de utl c na derutl c 9. Joshi, B. and Wali , S. (19 6) PCR amplif c t on of cate hol 2,3-dioxygenas gs e nq u f cra ot m l yu h i nd gr o c a be d i n gt rs o al e d Syst. Ap l. Microbi l. 63, Ap l. Enviro M c biol. 64 – 51. Phytopht ora using the polym rase ch in reaction d prime s ba d on the DNA 73–8 . 103, Eur. J Plant ho . r i b o s g 1f me 6 an S l t r i d c o b p - H e l i c o b a pty r i 13, 473– 9. in Helicoba ter pylori 29, J. Clin M crob l. 2543– 9. M i c rI om b u n l . Int. J. Fo d 28, 1–78. .loib rc M .lp A tsyS ,71 .34 – 126 Upton from pet lium hydroca b n tami ed groun wate . 19, 5–1 . FEMS icrob l. Ec 10. Fach, P. Hauser, D. Guil o , J. P , and Pop f , M. R (19 3) Polymeras ch in reaction f r the rapid entif ca o f det c ion f d sample . type A strain d Clostrid um bot linum 75, J. Ap l Bacteriol. 234– 9. 1 . Butendi ck, B. Morales, P. Figueroa, J. C ncha, M. nd Leo , G. (19 5) Specif gen amplif cation as a means to det c Archivos De M dic na Ve t ri a Renibacterium salmoni arum. 27, 47–5 . 12. Ves y,G. Nar i J ,Ashbolt N.,Wi ams K., ndVeal D.(19 4) et c ion f specif m cro ganism enviro m tal s mp e using flow cyt me r . ods Cel Bio . Meth42, 489–52 . 1 3 . T h o m a s ,J .C D e r o i s ,M . S t - P i e r ,Y . L t e P ,B i s a l o n J .G ,B e a u d t R., and Vil emur, R. micro ganism in soil samples using rRNA targe d fluoresc nt probes and ethid um bro ide. (19 7) Quanti ve flow cytome ri det c ion of specif 3, 2 4– 3 . Cytome r 1 4 . A s H m B u . t , z K l P i e r c h A G o L a f R n . w H, d J t m e 15. 16. A. (19 6) In situ loca iz t n of s c a n - o d l i gt p r u b e f R N Aa s cw n , h l y t ni g co f al ser mic o py. Swof rd, D. (19 ) PAU : phylogen tic an lysi using parsimony, version 3.0. Computer program distributed by Il inois Natural History Survey, Champ ign, IL. Hales, B. A., Edwards, C., Ritchie, D. A., Hal , G. H., Pickup, R. W., and Saunders, J. R (19 6) Isolati n d i ent f ca io f methanog -specif DNA from blanket bog peat using PCR amplif cat on and sequ nc an lysi . Enviro . M c biol Azospir l um brasilen Ap l. Enviro M c biol in the rhizosp e of . 61, 10 3– 9. Ap l. . 62, 6 8– 75. PCR/ FL Monit r g f Gen s 127 01 Fluorescent Polymerase Chain Reaction/Restriction Fragment Length Polymorphism Monitoring of Genes Amplified Directly from Bacterial Communities in Soils and Sediments Kenneth D. Bruce and Mark R. Hughes 1. Introduction Ther as b n growi ackn ledgm t of b h t e col gi a nd b oe n v a i t r u C o b m l c hs . p f g a n e the no rep s ta iv nature of tradi on l an lytic methods, as a result of t h e i r q u m n tf o rp i c u l v a t o n ,h el d i t r o u c n fm l e lar biol g ca p roaches to hes are s of study d e m tv o h n l a u p fc b r i s profile th d v rsi y of m c bial sequ nc i v ronme ts. For many of thes mol cu ar p o ches, t firs tep is the amplif c t on b t y h p e o l m e r a s c h i r n e a c t i o ( n P C R s) f p e c i f t a r g e s t q u e n c p s r e n t in nucleic acids extracted from environmental samples. These target sequ ncesaref quentlyregions fribos malgen s,u edb causeoftheir established phylogen tic framewo k. Howev r, other sequ nc s are being increas gly u d owing t he in r st acking m r ed st ain d fol we v o s a l p ng iur Td t h P C c. R m e s can be m d hig ly spec fi or a single t r ( .g , to rack he p ogr s f a specif strain throug an enviro ment) or can ac es the widest range of sequ nc vari nts of a particul gen tha are av il b e (throug the use of c o n s e u r g i o fd a t b s ev r i n ) .A x a m p l e s , c i f r g o n s 1 6 S rRNA gen s have det cted am onia-oxid zing bacteria of the Nitros pira (2) and m a n y s t u d i e s w h i c u s e “ u n i versal” ribos From: Methods in B otechnol gy, . Considerabl ef ort has (1) gen ra po ulations Nitrobacter (3) omal sequ nc . Describ ng the Enviro mental Monit r ng of Bacteria Vol. 12: Edite by: C. Edwar s © Human Pres Inc., Tot wa, NJ 127 , in comparison to the 128 informat contai ed with n the resulting po l of PCR products, howev r, pres nt a dif er nt chal ge. Sev ral t chniques av be n d vise to examin PCR products amplif ed from natur l com unit es. One of the first ap ro ches requi d ind v ual amplif c t on products to be cloned into plasmid vectors to form “libra es” prio to scre ni g by olig nucleotide hybrid zation or direct sequ nci g. Althoug this method provides detailed information, the time-consuming nature and the poten ial for introducing cloni g biase make it gen ral y unsuitable for environmental monitoring. It was ther fore important to dev lop methods tha resolve the diversity of the amplif ed products more rapidly and pref rably in a single el ctroph retic run. A number of such methods ave si (DG E) phism (RFLP) DG E has be n used to study bacteri l com unity divers ty in marine microb al ts nd biof lms r wa te r atmen pl s rial com un ties w h n a microb l at teriz d the gen tic divers ty with n speci or functio al groups of bacteri . F o re x a m p l ,W w n dM u y z e r speci in a natur l microb al mat than those in an exp rim ntal bioreact using DG E of [NiFe] hydrog nase qu nc s. Sim lar DG E-b sed tu i s e d xh s i a u vtm l for n - y b z d ci eg t r h v a nm sl ( 5 ) s u l a f n d t e r - c m w oi b g a a m p o r b fu lt h d e s i D 1 N v n 6 A S c y a m munit es o l Fluoresc nt polymeras ch in reaction/RFLP ( luRFLP) has be n d vise to pr file va nts of peci s quenc amplif ed rom natu l e vironm ts (1 ) . FluR LP, shown diagr m t c l y in s t u d i e o fm x - c u n i t yP C Rp r o d c si nt h a e o l f c u s t h ea n l y s ia n g fl re m c t o , a s h ue l i p f r g m c n oa t s v e i l RFLP. This fragment is gen rat d by digest on of PCR products (amplif ed eith r f om a single cu t r o f m ixed-co un ty DNA) using a pec fi rest ic on endo ucl as . The rest ic on endo ucl as chosen dif er nt a s, tlahe dvoi fs c r m n e q u i tsdh , nv ca er o f g t on the basi of the distance from the first e rict on e d ucl as ite o the star of the PCR product. The dif r nt size var nts ge rat d by the r s iction e d ucl as re id nt f e by the fluor scent lab on the 5' end of ne t h o s e l c nr yp a d , i g tF s u R L P W h e n C . t i n u s e d p r m s i n g l e - t r a d p o u c bs e r i n tg h f l o s c e a b l r d t e u s i n ag o mated DNA sequ nci g technol gy. In this man er, only size vari nts are det c ed. Furthermo e, through the use of computer software orig nal y Bruce and H gh s d b e v n l i o p c u a g t r e n dl i c o p h (4 , 5 , 6) and those based on rest ic on fragment length polym r(7) . , and b cte- (4) (8) (6) . Subseq nt udies hav c r - f o u n dh i g e r v s t yf o Desulfovibr (9) (10) and i hypersal n w t (7) Fig. 1 . , dif ers from other RFLP RFLP . PCR/ FL Monit r g f Gen s 129 Fig. 1 D a r m tic epr s nta io f he FluR LP proces . 130 Bruce and H gh s design for tudies f m cro atel i s n ukaryotic p ula ons and rel tiv abund ce of a h p k c n be stima d c ur tely. If the PCR product has be n g rat d f om ne s qu c vari nt, s gle t y p e s q u nv ca r i g h o f K n d we l t i g .b f r a m eF nl tu R L P can ther fo be us d to c mpile a list of di er nt size vari nts. Thi can be used sub eq ntly when an lyzi g DNA isolated from mixed com unit es. One of the major adv ntages of FluRFLP lies in the as e sment and monitoring of subtypes of particular gen sequ nces in complex gen tic backg r o u n d s u c ha s ( l o a d gs e n f v o l r a t h e o n l q y u a dt i rf e sc F R L P or pe si t nc ), bu also the q n if cat o he r lativ moun s f particular subtype . The drawb cks of the FluR LP technique are those often ident fied with the use of PCR. Care must be taken in making infer c s on the star ing con e tra ions of gen s deriv d from the final ratio of PCR products o w vi t an rg o p u s t e n i b a l s e . , g r a n e l i n d g , e r a c Gyon C, d ten i prime s. Given the p rsi tenc of DNA in atur l enviro ments, it s p o s i b l et h a s i g n l sc a b eg n r a t e df o m a t e r i lo u t s i d eo fc l s w i ta hlP C R - b a s e dt u i sc ,a rm eu bt k n o i t r d u c“ e o n t a m i t ing” DNA sequ nces during the DNA isolati n or PCR amplif cation step . In ad it on, it s po ible tha c imer c sequ nces an be g n rated from the coamplif cation f hom l gous en s ently “novel” types. A stra egy has be n dev loped to try to obviate this poten ial problem in FluR LP. In this tra egy, two separ te PCR reactions a c r e i o u sd t e n a g c h u r o f me p l a D t N O A n r. e i m t o bh , a r ing the fluoresc nt labe , is com n to both reactions, with the two other p r i m e d s i g n dte o f r e cn ot s ru e g i o n aOs m . p l i f c a t i o n pw, r d u c t so fd i e r n tl e g ha r e n r a t e d .H o w e v r ,b e c a u s t h e y“ s t a r e d ”f r o m the sam posit n, the dig st on f eith r s ould pro uce th same fragment when amplif ed from a single s quenc vari nt. It is hoped tha this hould a s i t n h ed i s c r m i n a t o b e w ni f o r m a t i v e n ds p u r i o sP C Rp r o d u c t s , s i n c ef r a g m n t s e r a t e df o ms p u r i o sp d u c t sw o l db e no l yi e digest on pr file. The pres nt FluR LP ap roach can be ext nde to incorp ate know a s m p o e u c f tn i r g q T h P x C s . R a l i c targe sequ nc can be modif e by altering the posit n of the rest ic on endo ucl as ite w h n t e PCR targe ( ith r by sequ nc modif at n o creat a new rest ic on endo ucl as site or by the insert o /d l i n of 10 n o a b t s u i e - r v F l d p hR z L P y c a o s e ) r e a n l v i o m t T s h .w p l r v i a d b e o u t s c r m i n a b o e t w h r g i nal subc e and the “n wly-creat d” v i nts. The can the b used both (12) , the siz t f h o r u se n q d a i v l y m F u t r s . h e o , (13) (14) (15) , leading to he cr ation f ap r- .A s PCR/ FL Monit r g f Gen s 13 in e x p r i m e n t s t o q u a n t i f y t h e r e l a t i v e a b u n d a c e s a n d t o m o n i t o r p e r s i tenc and spread in atural environme ts and in a wider ange of environmental monit r g studie . This chapter outlines the princ les and use of FluR LP to s udy bacteri l sequ nc divers ty and iscu e its role in e v ronme tal i or ng. 2. Materials B pe f ro P Cm Ri ,n g e di r a c e b d l u o h s k r o w y r a n i m l e r p ( out ). 1 Note se 2.1 Amplif cat on PCR r ducts f om Te Sa pl 1. Reactionmpl 2 . , s e t a l p w - 6 r9 so e b u t g f c i Lm - 5 . y0 l a c i p :t s n e g a Rr C P ,)etairpo sa( remi p ,setahp o ir ed to lcunyx ed ,r f ub noitcaer dn esar m ( retaw d l i s e r ts ,)enihcam RCP no g id ep ( lio aren m 3. Agarose, l ctroph esi buf er o ch i e, DNA stain (e.g , thid um bro ide), loading ye. 4. PCR machine. ivne morf detcar x AND ,.g e ta r o n m e t sa l p ( - y l o Ap N D .) 3 etoN es ). N o 2t e se qaT 2. Restric on E d ucleas Dig t on f PCR roducts 1. PCR products, gen ra d by metho s in 2. Micro n-30 spi columns (A ico , Bev rly MA) ( 3. Restric on e d ucl ase of h ic and re tio buf er ( 4. 1.5-mL icro ent fug bes. 5. Water b h o incubate r s ic on e d ucl ase dig t . Subheading 2.1 ). Note 4 se se Note 5 ). 2.3 Electroph si of Restric n E do u leas –Dig t d PCR ro ucts This met od a b en writ fo he Ap li d B osy tem 37 A autom ed DNA sequ nci g ma h e. 1. Digest d PCR pro ucts. 2. Size stand r . For the 37 A Gen sca -50 or -250 , TAMR ( te ram thyl-6 carboxy h damine) inter al marke s (Ap lied Biosy tems) are ap ro i te ( 3. IM NaOH in eth ol ( 4. Sequag l-6 (Nation l D ag ostic , A lant , GA) for a 6% denaturi g polyacr amide g l ( 5. Am oniu pers lfat . 2.4 Analysi of B nd g Pat erns G e n o t y p r a d 1 . 2 - ( v) s i Go en c f t w a r : Rl y m d software (version 1. ) (PE Biosy tem , Nar ingto , UK) run i g on Power Macintosh mpu er . N , N , N ', N '). Note 6 se se se Note 8 ). Note 7 ). 132 Bruce and H gh s 3. Methods 3.1 Amplif cat on PCR r ducts f om Te Sa pl 1 . P r m e a C o s Rp g f t i u x n l c a t i o n d e s a b l i h d previously. 2. Div de mast r ix n o wel s/0.5-mL tubes and cov r with l f requi d. 3. Raise th mp ratu e of h PCR mac ine to 95 4. Ad templa DNA. 5 . F o l w i n gc y ,e x a m i n t h p r o d u c sg e n r a t db y g o s e l c t r o p h e - ° C. si and co firm by DNA h rid zat on. 6. Store PCR p ducts a 4 ° C until res ction e d ucl ase dig t on. 3.2 Restric on E d ucleas Dig t on f PCR roducts 1 . s e t l h Ar c d i n o u c l ( e t a y s p i 5 2. 3. 4. 5. 6. manuf ct re ) o20 Centrifug this m x for 5 min at ro m te p ra u throug a Micro n-30 spin column at 13,0 g Invert h Mic o n-30 lum into a 1.5-mL icro ent fug be. Spin th s a embly t 650 g in a m cro ent ifug or 30 s at o m e p ratu . M e a s tu mhr o fn i e zd y / b u f r i t an s 4 h o de Digest he amplif ed PCR products with an p ro iate volume of the r tained enzym /buf er and final co entra i of 1X rest ic on buf er sing the r action c d s recom nd by the manuf ct re . 3. Electroph si of Restric n E do u leas Dig t d PCR ro ucts 3. 1 Casting Poly cr amide G ls 1. Wash gl p ates, comb and sp cer in wa m ter. 2. Align spacer and plates nd clamp in posit n (fol wing the instruc o supplied n th user man l ). 3. M i x t h e d e s i r d a m o u n t o f S e q u a g l - 6 w i t h T B E r u n i g b u f e r ( f o l w i n g instruc o ). 4 . 1( o0w A mf%/a d.L v 4 ) n ip ue r s l f g a o t v m 6L 0 y i n . 5. Pour the g l avoid ng the forma i n f r bu les. If bu les do ap r, t he glas p te o r l ase th m. 6. Insert a d cl mp the s ac r to f m a wel . 7. Leav to s f r a min u of 2 h rs at o m e p ratu . 8. Unclamp the g l and w sh l traces of rylamide. 3. 2 Gel Loading Electroph si 1. Remov th spacer nd flush wit 1X TBE buf er. 2. Insert h comb (eith r 24- o 36 wel ). 3. Flush t e w l s ith 1X TBE buf er. µ v a b r c u i t e Lo s , d n g µ Lof1Xrest ic onbuf ers p li dw th e nzym . in a m cro ent ifug . ° C. PCR/ FL Monit r g f Gen s 13 4. Prio t loading s mple , th gel is an lyzed to ensur tha no spuri fluorescent sig al re b ing erat d. 5 . d 2 , a b m o l c e w - 6g a 3n i s u e h W 5.0 dna )laun m smetsy oiB EP ni sa( ref ub dorp 6 ot pu( s c 6. Vortex and h t s mple o 90 7. Store sampl t 4 8 . L o t a h d e - n u m b r l a e s d c t o p h r e s a m l if E5 no . c t r phoresi is car ied out with the voltage lim ted to 1 50 V for 3.5 h on 12-cm wel s to r ad pl tes. 9. Load the v n- umber d lan s complet h ctroph e ic run. g n i d a o g l n i r u t a µ n e d i m a r o e f u l n b a r t x e f dL o RCP dets gid eht ot srekµam ARM T fo L . ) d e r i u q µf A N D e t a i p c r ; d e a o l b n c s t u d o r p R C P f o L ° C for 2 min. ° C. 3.4 Analysi of B nd g Pat erns 1. Ther sultingba d p t ernca b lyzedusingth LocalS uthernM od of size cal ing with n Genotyp r software. Other methods are av il b e in the Genotyp r Ma u l. 2 . c ai ln d b u eo G vp f s r t T h gk y n ware (version 1. ) lane by lane using the calibr t on provide by the TAMR labe d m rk s. 3.5 Specif Example of F uR LP b a c mt w re hig s n u l d -y p o F a R L P t a n c( e mer o )p e r n —m a d s ly t f o e c g is u d to he w l -char te iz d nature of its gen tics and biochem stry regions f conse u from dat b se qu nces, olig nuc e t d prime s w r d a ewmtrhp i l vc f y merRT tory and sport gen of the major subcl e of archetyp l G am-neg tiv mer oper ns (T 501 [19] , Tn DNA sequ nc dat h ve b n us d to gr up hes arc typ l Gram-neg tiv mer oper ns (23) . Furthe an lysi of subcla e of mer could be dif rent a d on the basi of the l ngth from RX (the s ar of merR ) to he firs P r e l i m x p n a d t s yh v o w u l in cult res of mercu y- si tan bacteri gave a single fragment of the same p s r e D q a N d u A i b n c z y t l o mE f h . com unity DNA tes d, to date, has contai ed one or more of the fragment i dse nzt f 2 Fig. d i f s a e mo r p l nu D b t h v . c s locati ns. Figure 3 show the profile obtained for a s mple taken from Fiddlers Fer y, on the river Mers y. Because the subclas type is know from the fragment size, any potential link betwe n gen type and dif er nt physiochemi al enviro ments can be explored rapidly. This is poten ial y importan for mer , becaus only certain subcla e car y the m e rc u i a l y s ) g e n w h i c f t s r e a n c o g m e r u i a l c o p n d s (16 owing , 17) . Using (18) ∆ P r( ea gp i o n x , pKLH2 [20] 21 c o tnr h e a gk iub l) s - 1 mer Fok and pDU1358 [21] ). merR [2 ] sequ nc showed tha six major I rest ic on d u leas it ( ). Fig. 2 mer s p e rtq yu n c merB (organ - 134 Bruce and H gh s Fig. 2 merRT d e r i fv o m the arc yp l Gram-neg tiv m rcu y esi tanc ge s. Dif er nt FluR LP fragment siz gen rat d by ∆ P sequ nc . The den rog am on whic the site have be n superim o d, merR (2 ) I. an d i t o h se z n tha wer p dict on he basi of cur ent DNA da b se ntri . Subseq nt s t u dw ei l r m h n ps oe v q t aw ul m r n c i fied rom Fok s e q u dn a c t p f r o m ref. F i g 2. mer 4. Notes 1 . u i s n v o g l T h e c t a . r d b m u p Fs l e w R o i r L k B P n , g fa y d a t b s e m a n i p u l t o s o f t h e s e q u n c o f i n t e r s , t h e d e s i g n o f o l i g n u c -e gen hom l s. a, d i t o n fl r g m e s i z w d t c e 23 s h mto, a w je u br c l s f I digest on f di er nt PCR/ FL Monit r g f Gen s 135 Fig. 3. merRT Fer y soil w th e Proces d image of FluR LP fragment produce by ∆ P P C p R r o d u c t ( s F l R XP o a ) m p i f e d r o D N A x t a c e f d r o F m i l s fragments how in base . mer o p t r ci a P d n C f m ue R , s h l y c t n i d o u l e T a h s . choice of enzyme det rmines the resulting fragment profile and in turn the value of the information tha can be d rived from the digest . Computer p ograms tha are useful in this proces in the Gen tics gc pack ge (Gen tics Computing Group, Madison, WI) include FAST and MAPSORT. Before incur ing the xpens of luoresc nt oligonucleotide primers, it is advisable to che k tha the target sequ nce can be amplif ed using conve tional PCR p r i m e r as n td co f i r tm h i bs Dy N hA b r i d z a t i o n f h ge n r a t e Pd C pR r o d ucts. Furthermo e, although the automated DNA sequ ncer provides hig ly ac urate siz ng information, it is importan , in practi al terms, to rest ic on endo ucl ase tha al ows >10 base betw n each size vari nt and over a gion tha c be siz d y the au om d DNA sequ nc r oftwa e. Numero s prot c ls exist for DNA isolat n. Her , the method used was as d e s c r i nb cel hom g nizer (B. aun Biotech, G rmany) to e sur ly i of bacteri l s in so l a d e im nts. 2. 3. Prime s u d her w taken from 4. 5. 6. 7. 8. m o l e c u b t w n h ef l u o r s c n tm i e ya d h o l i g n u c e t d usef l to bal nce th ydroph bic ty. One olig nuc e t d prime , FluRX, was label d with the gre n fluorescent label TE (4, 7, 2', 7'-te rachlor -6 carboxyflu esc in, O wel Labs, Univer ty of S u hampton, UK). The us of Micro n-30 columns prev nts he prof und istor n caused by a r c e o st m h p i u n d c l pe oa s g i- yb r l n v e d the orig nal (p es in colum ) a t ed s qu nc r s. The choice of restriction endonuclease is case specif c. Man heim, L w s UK) a used h r to p file th amp if ed b a s e < . 5 0 w r g i o z n t h e s c r u d w eT m A a M R k - 5 sh 0 T h i s o l u t c n a b e rd o m v c u l a t e b d k g r o u n f l e s c f r o m sequ nci g plates, if r qu ed. Other no flu esc nt a ryl mide so ut n ca be us d. I digest on of Fok sel ct a ref. 24 B r b a u e s dtw n i h - g o 3 f 0 p ref. 24 . Incorp ating te ra hyl ne g col (25 , 26) I (Boehringer Fok mer gen s. c a nb e 136 Bruce and H gh s Acknowledgments P o s N t E d R F C c e la n ( r G w T u 5 p b h / y i 9 o 4 s t d L K S D ) B , s a u n p G ig b oR r y N 3 E t/ M f e9 HC d0 8 w 1 . l h k s NERC PhD studen hip. We would ike to acknowledg Donald Ritch e and P e t S r i f k o h l p d u s c i o T n h w . a r l b k e f t du o s m of the SEQN T facil ty D resbu y. References 1. Aman , R. I Ludwig, W. and Schleif r, K. H (19 5) Phylogen tic d f ation and in situ Microb l. Rev 2. Hiorns, W. D., Hasting , R. C., Head, I. M., McCarthy, A. J., Saunders, J. R., ( A 1 m 9 p r H l i5 . g b) e f o 6 n G R s S Nca t , d W P . k u p of auto rophic am onia-oxid zing bacteria demonstrates the ubiquity of Nitros p a in the v ronm t. 3 . D e g r a n V ,. B d i R( 1 9 5D )e t c i oa n d u t gf lations il by PCR. 4. Muyzer,G. D Wa l E C., ndUit erl n,A.G (19 3)Profil ng c mp ex m i c r po b d ua e l n t y g i s c r o p h a ne l s y i -f meras chain reaction— mpl f ed gen s coding for 16S rRNA. Microb l. 59, 5. Muyzer, G. Tesk , A. Wirsen, C. O and J sch, H. W (19 5) Phylogen tic relationships of hydrot e mal vent sample by denaturi g gradient gel el ctroph esi of 16S rDNA f agments. 6. W a w e r , C . a n d M u y z e r , G . ( 1 9 5 ) G e n t i c d i v e r s i t y o f environmental samples an lyzed by Denaturing Gradient Gel Electrophoresi of [NiFe] hydrogenase gen fragments. 2 03–2 10. 7. M a r t i n e z - M u r c i a , A . J . , A c i n a s , S . G . , a n d R o d r i g u e z - V a l e r a , F . ( 1 9 5 ) Evaluation of prokaryotic diversity by restriction digestion of 16S rDNA directly amplif ed from hypersaline environments. 17, 247–256. 8 . F e r JiM .su , y z G a W n ( d 1D 9 6 e ) t u r i G n g a d e E lt c troph esi prof les 16S ribos mal RNA defin po ulati ns habit ng a hot spring m c ob al t com uni y. 9. Tesk , A., Wawer, C., Muyzer G., and Ramsing, N. B. (19 6) Distr bu on of s u l f a t e - r d s bc i n g f (j Moe a r d F D n v m, ls u k - ) m a pobt rsye d n ul c Da t s r Gi n g d E e l t c r o p h e si of PCR-amplif ed ribos mal DNA fragments. 1405– . 10. Porte us, L. A., Armst ong, J. L., Seidl r, R. J., and Watrud, L. S. (19 4) An ef ctiv method to extrac DNA from enviro m tal sample for Polymeras 59, det c ion of ind vidual microbial cel s without cultivation. 143– 69. 2793– 80 . 14 , Microb l gy po u- Nitrobac e 2093– 8. 61, Ap l. Enviro M c biol. Ap l. Enviro . 695–701. Thiom crospira speci s and their identif cation in de p-sea 164, Arch. Mi ob l. 165– 72. sp .in Desulfovibrio 61, Ap l. Environ. Microbiol. FEMS Microbiol. Ecol. 62, Ap l. Enviro M c biol. Ap l. Enviro . Microb l. 340– 6. 62, PCR/ FL Monit r g f Gen s 137 C h aR ie n c t om p l f i a t D onN dA g e r p ia t l y s . 301– 7. Bruce, K. D (19 7) Analysi of munit es in soils and sediments resolved by fluorescent PCR/ restriction fragment length polymorphism profiling. 4914– 91 . 1. 29, C u rM i .c o b l gen sub-clas es within bacterial com- mer 63, Ap l. Environ. Microbiol. 1 2 . L e v KC i .Rt DB s, r M a g J w d u XE c e Ak y . , n Dr s Hudson, J. R. (19 4) Fluoresc n -ba ed resou c for semiauto d genomic an lyse u ing m rcosatel i marke s. 24, Genomics 361– 5. 1 3 . S u z kT Ma i.G n , d o v ( J S1 9 B 6 i )c a s u t b e y dm p l n a i g the amplif cation of mixtures of 16S ribos mal-RNA gen s by PCR. Enviro . M c biol. Ap l. 62, 625– 30. 14. Trevo s, J. T (19 6) DNA in so l-ad rption, ge tic ransfo m ti , molecu ar evoluti n and gen tic micro h p. Microb l. Anto ie van Le uw nhoek Int. J. Gen. Mol. 1– 0. 70, 15. Wang, G. C Y. and W g, Y. (19 6) The fr qu ncy of chimer olecu s a 16. 17. 18. 19. 20. 21. 2. 23. c o n s e P q a C u m R p f l i r 1 c b 6 o S gt s d e n N m A a f l bacteri l sp c e . Bark y, T., Lieb rt, C., and Gil man, M. (198 ). Hybrid sat on f DNA probes with whole-c m unity genom for det c ion of gen s tha encod microb al r e s p o ntl u a s : 5 , 1574– . Rochel , P. A Weth rb M. K , and Olso , B. H (19 ) Distr bu on f DNA sequ nc e oding ar ow- nd broa - spectrum esi tanc mer u y esi tanc . Ap l. Enviro M c biol. Hobman, J. L. and Brown, N. L. (19 6) Bacteri l mercu y resi tanc gen s, in Metal ions Biol g ca System New York, p . 527– 68 Brown, N. L , Ford, S. J , Pridmo e, R. D , and Fritz nge , D. C (1983) Nucleo gft s erh i n dq a m u c cury esi tanc . B a r i n e u ,P . G l b r t ,J a c k s o n W . ,J e s C S u m r s ,A .O a n dW i m e F s t r I h c D q u ( n N o i 1 T A f y 9 S 8 a pd . 4 ) m e , plasmid NR1. K h o G l Y .r L d , M i e m nZ k v s u aO y S . , V., and Nik for v, V. G (19 3) Molecu ar char te is on f an ber ant merrc eu s iy t a n p o s e lb vafm inr t l Plasmid G r i f n H, . F o s t e r T J S, i l v . a n Md s r T, K ( 1 9 8 7 C) l o n i ag Dd N A sequ nc of the m rcu i - and organ me cu i l resi tanc det rmina s of plasmid pDU1358. O s b o r n ,A .M B u c e ,K .D S t r i k e ,P . a n dR i t c h e ,D .A ( 1 9 5 )S e q u n c o servation be w r gulato y mercu si tance g s in bacteri f om ercu y pol uted an prist e nviro me ts. 1 07– 4. 142, Microb l gy 2+ g H ea n d s mer 57, resi tanc . A pE n lv .i rM o c b l . 158 – 9. (Sigel, H. and Sigel, A. eds ), Marcel D k r, t r a n s p oT Pseudom na Biochem stry 2, 501 2, 601– 9. J. Mol Ap . Gen t strain. Acinetoba r 30, e n c o dm i r g- 4089– 5. 30 – 8. 84, Proc. Natl A d. Sci U A Syst. Ap l Microb l. 31 2– 6. 18, 1–6. 138 Bruce and H gh s 2 4 . B r u c e ,K .D O s b o r n ,A .M P e a s o n ,A .J S t r i k e ,P . a n dR t c h i e ,D .A ( 1 9 5 ) Gen tic d versity w hin cultiva ed so l an edim t bac eri . 2 5 . B r o w n ,D .J S a dB r o w n ,T .( 1 9 5 ) mer gen s directly amp if ed rom c unites of n 4, 605– 12. Mol. Ec ( N e w t o n ,C .R d ) P C R :E s e n t i a lD Wiley, N w York p . 57– 0 2 6 . G r z y b o w s MJk c.i P, h l BaFp rn ( od 1Tw 9 5 ) P CE Rs : e n t iD a l ton, C. R ed ), Wil y New York, p . 93– 8 (New- Recov ry and A l si of rRNA Sequ nc s 139 1 Recovery and Analysis of Ribosomal RNA Sequences from the Environment Ian M. Head 1. Introduction 1. Histor cal Dev opm nt f Ribos mal NA n ysi of Micr b al Popu ti ns S i n c te h 1 9 8 0 ts h ue o rf i b s o m a Rl N (A r s) e q u n c e - b a s d n l y c hts aoi r e m iz c r o b p a l u t i (o mn as b l cy t e r a i n d h p o l u lations) has increas d sign f cantly. This ncreas d use is n respon e to he r e c o g n i t o h c a u l t r e - b a s m d t h o g r s m l i y r e p s t nc h o e m p s i t o n m oi fc r b p ao l u t i oahnsec y u tr e inher t in cult re-d p n e t s udies of microb al com unit es, it was uggested that, by extraction of nucleic acids directly from environmental samples, g nes tha wer p sent i al t xa could be isolated n sequ nced (2 , 3) . Compar tive an lysi of sequ nces recover d from environme tal samples with those from cult red isolates would permit phylogen tic relationsh p of the unc ltured tax o be d t rmined tribu ed gen s most com nly used for such an lyse are the rRNA gen s, p a r t i c u l a r yt h o s e n c d i gt h es m a l r i b o s m a l u b n i tR N A s( 1 6 Sa n d 8 rRNA).rRNA :gniwol f eht (1) (2 , 3) . The universal y dis- -dulcni ,se g ta d c r h o ev s gatn d y m ev h s n g 1. They ar c u i l omp ne ts f ribo mes. 2. They pos a c m on, es tial func o i al ce s. 3. Functio al e s itycon ra sthei pr ma y ndseco ary t uc eandh c 4. the d gr of iverg nc dif er nt ax . Their primary structure is a mosaic of conserved and variable tracts of sequ nce. This permits unambiguo s alignme t of hom log us posit ons in From: c Ti or. u m v etbnhi a s Methods in B otechnol gy, Enviro mental Monit r ng of Bacteria Vol. 12: Edite by: C. Edwar s © Human Pres Inc., Tot wa, NJ 139 140 Head an cif sequ nc motifs. rRN A sequ nc and ident f ca io of universal y conserv d and taxon-spe 5. Ther is l t e vid nc of h riz ntal sfer o RNA gen s. 6. Extensiv rRNA ef nc s que dat b se xi t. 7. A “tre of li e” based on rRNA sequ nc provides a fr mewo k with n whic sequ nc r ove d fr m natu l s mp e can b om date . Method l gica nstr mea h tin ls ud e w r im t d oan lysi of bacteri l com unit es of lim ted divers ty using 5S rRNA sequ nc extrac d and purif ed directly from enviro m tal sample suf er om being o ly ap r x 120 nucleotid s n le gth and, e c p rmits only re ativ low-res uti n phyloge tic an lyse . M thod wer th fo e dev lop for the r cove y of large and more informat ve rRNA sequ nc i r n R 2 N 3 aA (S 1 d 6 It was in t al y sug e t d hat s o gun clo ing f DNA, extrac d from environme tal s mp e , in phage rRNA gen s ap rox 0.2– 3% of clones in a lambd libra y rRNA sequ nc si g th s ap ro ch w uld req i scre ni g of ar und 30 – 50 clones. Thu , ev n to de rmin the comp sit n of a microb al c m unity co ain g o m re than few domina t speci would be an extr m ly labori us t k. Howev r, the dev lopment of procedur s to sel ctively recov r rRNA s e q u f n r cv o i m p t l h y Dge a N A s d c chain re t o (PCR) relativ y com npla e. Broadly speaking, ther have be n two main ap ro ches adopte for the study of natur l mic ob al p u tions ba ed on the PCR for ampli c t on f rRNA sequ nc : PCR amplif c t on rRNA gen s fol w d by c oni g a d compar tive an lysi of the cloned rRNA sequ nc , or PCR using specif o l i paug w ns t C h-y c P r e R b d. i m s ing and sequ nci g al ow the microb al po ulati n as a whole or a specif subgro p fthe ula iont bech ra iz d,but h s il re at v y borious. By contras , he us of the PCR util z ng specif pr me s al ow rapid ident f ca o p rticula me b rs of the micr b al o munity. Ref m nt t hb oea fs p i cr ( . g l , d a e ton if u r g lc t r o p h m b [ e D xs G i ca f E d n ] ) l g ta ed mor pi an lys of mu tiple sa . This chapter outlines the basic ap ro ches used in rRNA sequ nc -ba d an lysi of tural mic ob p ulations, d he lim ta ons d p licat ons of rRNA-based nviro me tal n ysi are discu e . DG E and whole-c hybrid zat on p cedur s a p e nt d i Chap ers 12 nd 5. (2 , 3) . 5S rRNA and Bacteri Archae i r n R 2 N 8 aA S 1 d , Eukary λ vectors uld be us d for the r cov y f 16S (2 , 3) . Init al emp s r ult d in recov y f 16S rRNA gen s i (2) (4–6) has m de rRNA nalysi of m cr b al po u ti ns . To obtain a single 16S ). Recov ry and A l si of rRNA Sequ nc s 14 1.2 Princ ples 1.2 Recov ry f RNA Gen s U i g the PCR Since ts v ion the PCR as found p licat on lm st ev ry alm of the biol g ca s en , a d vironme tal crobi gy s no exc pti . The s e n i t v oy f h m e d a ls t io u fe r h d c t i o n bf a e r p s n t in very low numbers in e vironm tal m trices ( .g , char te iz on f PCR-amplif ed rRNA gen s i ncreasi gly the m od f choi e f r d t min g he comp sit n f m crobial mun t es. p o mui-c r b a l f n P CyRs e d o m a j r i t h f o p i nst a r g T h e lations a nuclei a d pre ation ex ract d f om an e viro m ntal s p e. A variety of techniqu s have be n dev lop to do this, and any particul m e t h o wd i l h a v ie n h r e n bt i a s e E. x t r a c t i o n f u c l e i ac d fs r o em n v i r o n mental samples is often problematic. Dif iculties encounter d include c o e x t r a i mnf l s h b i t o r y D eNp A l m a s u itdnhP eC R . In ad it o , an extr c ion e h qu dev lop f r one particul samp e ty may not be dir c ly t ansfer bl to di fer n sampl . A det i scu on f nuclei a d xtr c ion e h qu s i g ven Chapt r 7. Stand r PCR condit s are gen ral y adequ t for the amplif c t on of rRNA gen s thou ec niq s u h a ot s r can redu misp ng a d i cre s p if c ty o amplif c t on. refs. se and touch w PCR (9) 1.2 3 Amplif cat on 16S rRNA Gen s U i g Spec f Prim s T h ep r s n c o fv a i b l er g n s t h R N Ap r i m a y u c t ep r m i s h d e s i g no f l u c e t i d s h a nb eu d i g o s t ch y b r d i z a o np b e s and PCR prime s. Oligonucle tid s targe in hyperva i ble regions of the i d - e t s n u a f l m c o w p b yi e q run R N Ac se or Eukary . Thes primers, in princ ple, al ow amplif - (14) sing relativ y low an e li g temp ra u s in the PCR can refs. C.o n s e q u n t l ys,o - c a l ed o m a i n - 1–3 8 ), and (10) 1.2 Amplif cat on 16S rRNA Gen s Using Broad Specif ty Pr me s T h eu s o fP C Rp r i m t a g e n c o s r v d e g i n o ft h r R N Am l e c u has proba ly e n th most frequ ntly adopte a pro ch t he c ar te iz tion of microb al com unit es (e.g , designed tha al ow the sel ctive amplif cation of rRNA gen s from the Archae , Bacteria, cation of rRNA sequ nces from al me b rs of a particular phylogen tic Domain. In reality ther are few posit ons in the rRNA molecule tha are a b s o l u t e l c y o n s e r v i dc n o m p s i t o n specif c or universal primers can ex rt some sel ctiv ty on the sequ nces they amplify. U reduc this le v ty. 7 and ). Prime s have be n 142 Head snoiger , m ht uF .l v seic p b , a mo n d seic p ht a no f fo eht ANRr iw e om d vr sn c uq ekam ti lb s op ngi ed s borp h t i w , s u n oe g v d b a m .l y p c f r H n i e v o g ngised fo hcu seb rp a ton ,l srevi u d a n emos ga il ,. ( eht -dnuor gnimrof–e ps ) l cab hgi ANRr ecn uq s ytiral m eb d vr s o ne ne wt b yl acip oneh d f - l w seic p secn uq ANRr fo esab t d h fo n is apxe yfitned l acov uq s b m r ngi ed t lcu o a cif eps m inagro ANRr desab- cn uq RCP rof eht noi ac f d o ralucit p x ni larut n .sehcaorp t yn m v g d if s e alb . nI , oit d a h w eht dipar )51( )61( a t h nerap g imoc b s ti . ,s el ht noN eht ytil ba ot ”enut“ eht y ic f eps fo )71( 1.2 4 Rev rs T an c ipt se PCR The most straigh forwa d format for PCR-dep n t char te iza on of laiborc m sn t up i tcer d noi a f lpm ANRr sen g morf ”ci neg“ DNA extrac d from a natur l sample. This provides a snap hot of the to al -if lpma eht yb sr am e ufnoc sla y m tub nes rp i tah no lup aib rc m regnol eb yam t h sm inagro f ecn s rp ht gni ac d ,AND dekan fo n itac tnes rp )81( -moc laib r fo srebm vitca yl ob em ht ,erom uF . -nits d eb o ac sm in gr e ht d a , s r ni t om f sen ht fo era ytinum dehsiug morf t a e vi c n t y a e g mi n su yla de b o d e i f r u. pA N D h T R t n o c a l bs e d u f - r etar h wo g cif ps t l ew a n a h t , A N D d e s R m y z r o bu t i q n a ; h e f l ANR ni eht mnoriv dlu s e f ht b yl aredisnoc t h tah fo . A N D R r e sn odil c a b t m f u p h v .gnilpmas fo e t h u b rc m vi a e t fo l A consequ f the r la iv nst b li y of RNA is tha ex r ction f m natur l samp e is more p bl matic h n DNA isolat n. This particul y t r u eo fs i la n d m e t s o n l yr c e t h a v m o d sb e n v l p df o r purif cat on f RNA from thes nviro me ts in a form a en bl to rev s transc ip o (2 w it a hx o n - s p e c f l i g u o t dhb ea s n r l i v u n s a d( t ie m u f. p r g l o q , y c 26 ), but rev s t an crip o f the RNA and et il char te iz on f the active fraction of the microb al po ulati n has only ra ely be n at emp d ( 2 , 27 ; Misk n, I. P et al , unp b ished at ). 1.3 Analysi of PCR-Ampli ed rRNA S quenc s rRNA sequ nces amplif ed from nucleic acids extracted from natural sample can be an lyzed by a number of techniqu s. Rapid, low-res uti n )12–9 ( , 23) elbats y r di noc sla ANR . . Direct p ob ng f rRNA ext ac d from natu l s mp e se refs. 24– Recov ry and A l si of rRNA Sequ nc s p r o b - y s i l e o r gp a n t mc (u ) f r o s e q n c h a t i z o ing blot ed PCR pr ucts wi h pe f c olig nu e t d prob s, an ver l ind cat o f the div rs ty of the microb al po u ati n ca be achi ved using DG E ( sequ nc separ t single s quenc into div ual c l a o i n e b r T y h . R f d N A a g m e ns c b t q u f r d oa l , s e l c t i o n , f h e s a d t i l p c u r oe f h s q n t y p r e s i a p rticul env ro m t can be hi v d. 143 se Chapter 12). Cloni g of the het rog n us po ulati n of rRNA Escheri a clones coli 1.3 Oligonuc e t d Probing P r o b i n g lf, t e Cd R - a m p i f r gN eA n a m t ws i, ph e c f o l gonucle tid probes can be used to det c the pres nc of rRNA sequ nc c h a r t e i s o cp f u l a m r i o g n s r u p o f g a n i s m e v ronme tal sample (e.g , s c r e b i am n u l p od - f g i s v a t n h g e w ic th a l e n g db f r a m e n tg s R N A a m e F tu hr n o s l y . , r a n god efi p t b sor v i da e c h t r i z a o nfh em p t sequ nc types r p es nt d. Howev r olig nuc e t d probes tha rget particular organism are often design d from compar tive an lysi of rRNA sequences from cultured tax , and the pres nce of target sequences in unchar te iz d, unrelat d tax can ot be discounted. Consequ tly, inferenc s, from such studie , about the pres nc of particul microb al groups i n hc or we a vs d , f e g r T h c a u t i o wn . e d b s h o u l c o ns fp a ie r w d m u t h P C R b s a n i t f l y the ident y of the sequ nc amplif ed is obtained by probing with a third specif ol g nuc e tid R e l q a u t i n pv r o s f c b y e olig nuc e t d probing, alth u is ubject o a number of cav ts rel i n t pg o e a b l s x r t e d y h P C a R m p l i f c t o n d h e u r c f multip e rRNA o ns i me organis ( refs. se (28) 27– 9 ). Oligonuc e tid probing has the . using (30) Subheading 1.4 2 ). 1.3 2 Cloni g a d Sequ nci g p r o d - P C R s e q fl u i g n a c d p t r o b , I ucts with sub eq nt compar tive an lysi to det rmin the relationsh p of enviro m tal sequ nc to cult red isolate can be la ori us. It does, howev r, al ow ident f ca o of novel tax and their phylogen tic placem nt in c ur le si t a qo n d vf h m e t s . DG E also f er th op unity o b a n sequ c dat from n vel tax by excis on f ba d , ol we by r amplif c t on a d irect s qu n i g The DG E ap ro ch, wev r is l m ted in the siz of rRNA gen fra m t tha can be an lyzed, and in complex environme ts in whic DG E can (31 , 32) . 14 gen rate large numbers of bands, it can be problematic to obtain single bands, fre sequ nc d ir tly. Head from c nta i o w th er RNA g n fra me ts, h can be 1.4 Lim ta ons The dev lopm nt of molecu ar biol g ca techniqu s to study microb al p e r nhmc a iu ts l o d - y e p n t s r m i l ci om n s- d e r a b t h A pu g n . y e ms i c r o db a l t h f n a t i o ta ions and biase inher t in cult re-bas d techniqu s are cir umvent d by b i a ns te r . l c md h o v , e t l cs u a r h p o i c t o h rnI s e R N qf Ax u v n o d m i r e s t a l p f , fer ntial ys of micr b al e s during DNA extrac ion p edur s can e ult i nc e l st h a r m o e i s t a n l y b e i go v r l k d nm e c u l a ri v n t o ries of microb al divers ty. This discu e in Chapter 7. Howev r, sev ral ad it on l factors also c nfou d at emp s to infer ac ur tely the divers ty of natur l mic ob a p ul tions. 1.4 Sampling d Coverag in Clo e L bra i s mdb i a cvs re o t fly u q n i o s k e d t h a u A n on the an lysi of rRNA sequ nc is ampling. This in part owing to he e l x a p b no dr t s - h i uC f c m . v ge s ie nq o g du b f t r p a c R m N A l y e s and sequ cing of lar e c on libra es o t in d from each s pl i not rs i n g l e f r o m b t a d S q u e n c i . a d o s t f b a i t h e o n f a s i b lm y c l o n e i b r a s , yt h e i rn a u , p e s n ta h o f ed m i n a t b e r s tm ohi fec r b p a l u o ne s i t c a dm l h b go u t e m sp o ar v il F h n .s r,g cR el N oAi b a s m a yt e l u s i b o tw h a e q u n c y p sm i g h tr e n “ y p i c a l ”b t e r from a p ticul r env o m t. H wev r, th discovery f lated rRNA g ne cluster cov ed in p e tly from di e nt loca i ns by dif er nt g oups using a variety of methods has al owed the ident f ca o of novel bacteri l tax , know ly from RNA sequ nc , tha re ap ntly g oba ly distr buted (3 – 5) The introduc of DG E has permit d vari t on betw n sample to be invest ga d. Comparis n of DG E band pat erns of rRNA gen fragments from replicat sample and sample taken over time are now being used to as e s the temporal and spati l vari t on in microbial com unit es. For insta ce, DG E nalyse h v d monstra e h considerabl t i y ex st d o tm hib ena c p r s u l o g f m it a n e s (36) and i waste r atmen pl ts (Craine, N. G and Curtis, T. P , pers o n a lc m u i t o n ) .T h sw r ki m p l e t a f ws n e dt ob c h a r . Recov ry and A l si of rRNA Sequ nc s teriz d to obtain rep s nta iv informat about the microb al po ulati ns p r e s n T t h . v i o m e s n t a r dl i g h u y s t n f o e r m i r e l a t i v s p h yo b fn m c d a l i t ho en s r t e s m p a u n o d hrlib k I w x y v . t e h n m vw i o sr u l bg n m e t s such as oils and se im nt are xamined with s milar igor. Ther is, ther m s o y r an ef t d , p i c hl n g u a r a c t e r i z mo fn b ca l u i t e Ss . b j c rn g p l i a st e m D Go E a n l y s i t do e r m n h g e o vf a r i b l t y n h me c r o i a pl u t n s m o r e f s a pi lg n t h o f s u b e lc a r t i o n yf w h e d , detail nv s gatio , m y be a s n i l w y to pr ce d. Another importan sampling is ue in an lysi of rRNA sequ nc cloned a e cd fn t i rv hu o m l - s w p e n s n i a t y u r l m p e c i o nd b r a y c o v e r a g i n l b e hs a v n o r w fd sm t u i e o p l a n s f macro g nis 145 (37) (38) . Coverag ( C ) is det rm n usi g a mple qu tion: C = 1 - n( wher n 1 is the number of sequ nce types from a clone libra y tha are e n c o u t r al d y , N t o nh i cue as lm b f r y z H d . , l a t f pr h g ie o s n lc a ni der voc secn uq s euqin t e n o d w u s ac r i v y m T l g p . h f e u t o i n for c ve ag , it has be n sug ted ha sequ nc >97% in s m larity should be consider ntical (37) . This based on the obs rvati n hat org nism with 16S rRNA sequ nce hom l gies below 97% are unlike y to exhib t genomic DNA hom l gy >80% (ind cat ve of a relationsh p at the speci lev ; [39] a n I y f t) h. i u m g d , es r a p t c i v e r n s u m - y c l e a r y > 9 % o fh s me q lu gnr ycR N A a v t o k nr g w i s m f b e dist nc speci based on DNA- reas oci t n exp rim nts and phenotypic da (e.g , se refs. 15 and 40 defin g what cons i ute a single qu nce typ wil ead to an u der stimation f the div rs ty if organism with g rRNA sequ nc hom l gies tha cj ou gpns H er - t h wa i d v . , l y tif eds nc h t roge i y fd rent RNAop swith na gleor ism can be sig f cant (41 , 42) . Calcu ations from published dat ind cate tha in clone libra ies from dif er nt enviro m ts,c verag n f omaslit e 4% (37) . This implies tha in clone libra ies wher coverage is low, considerableundiscover d iversityexist .Withcoveragevaluesa lowas4%,the clone a l n i b e r t y cow z h mu d v s g majority f the div rs ty in a s mple. Cons que tly, in such divers n i o - M e. t h o d s i m a 1 N) / (1) lo in be r a y , 1 ). Adopting a 97% cutof r pe ation l y (43) to>80% /N 146 Head d i e f n b v u“ t w k oc r l m y a ” - h x s d mental y i por nt. 1.4 2 Quanti ve d Qualit ve D scr pan ie in PCR-Ge rat d RNA Clone Libra s The PCR is an m e ly pow rfu techniq . T er a , howev r imp t a n l i m t a i o n st w h a tc nb ea c h i e v du s i n gt h eP C R .I np a r t i c u l a r ,q u a n ti ative infer nces from PCR-amplif ed rRNA sequences derived from enviro m tal s mp e should n t be ac pted uncrit al y. Quanti ve disp cr ai tm e w g o h y n P f C Rs l - d : av a r e c o n s q u ft h ep r o i s f R N A e q u n c st h m e l v ,a n dt h o s tha re b ought a by mechanist f ure o th PCR. 1.4 2 I B NTRI S C IASE Anomalies tha re a consequ of eatur s of rRNA sequ nc include s e l c t ia vm p f s o e n q u c v t rh s r e s n t a i o c l e b r s m; o f i c e n at p l i o n rf R N sA e q u c tha re cluster d on the g nome o r m g u a wp l n ie t R sN h A divers ty owing to het rog n i y in rRNA oper ns with n a single organism (41 , 42) . Furthe mo , qualit ve nd qua ti ve anom li s can be th r sul p r o i fs m e l c t E w v“ nh u . r s p a l i ” o m e t d b , h a low ev of mis atch be w n th prime and t rge s qu nc a result in pref ntial amplif c t on of certain rRNA gen sequ nc . Introduc i n of deg n racy i to pr me s qu nce a min ze this, bu deg n rat p imers, es ntial y m xture of sim lar but no ide cal pr m s, al o h ve t po ntial o cause bi s n PCR amplif c t on. This can result from di fer nc s i the an li g temp ra u s of lig nuc eot d s in a eg r t mix u e. Also, exhaustion of the primer sequ nce cor espondi g to the most abunda t sequ nc types in a sample may result in a skew d istr bu on of sequ nc types r cove d in a clone ibra y ecaus mplif cat on f les abund t sequenc s i favored t w h end of t ampli c t on y le 1.4 2 M Competi on betw en primer an ealing and template rean ealing has r e c n t b l y o g f u i a r z e s h c d p t n b P C lm R i f i c a r t gRo eN nA s it was demon tr ha some pri a s g ve trong c elation b we p r o dP uCf cRi t n a lh e o n s d t ma ir x h e g so f a t i h e This wa not c si ently obs rved with al prime a s u ed. In i sta ce in whic the star ing ratio of rRNA gen s was not refl ct d in the final ratio (4 ) (45) l e a d o itv n g r p - ; over p s nta io f sequ nc from (45) o v e a r n dp s t i ; u m l f o (37) ECHANIST C B . IASE (46) U s di. en fg m x t r u Ro N A p l a e s , (46) . Recov ry and A l si of rRNA Sequ nc s o b t a i n e d h P C p R r u c m t i x e ,w a f s o n d h t r i w a g s e l y i g n e w t d s a h o p .r 1 f : c l I n c r e a s i gt h u m b ro fc y l e si nt h P C Rr a c o e n u g dt h i se f c .A p r e md oi c h t l nT x s a . v e wo p m d k i n l t c r p e a n f l i t gw s c u h e t a i n d c b a s P C R o e r v d t h of the templa DNA ( ratio f products rega dl s of the in t al r tio f gen s pres nt was tha in a mixture of w rRNA gen s with one pr s t in exc s , a the PCR proce ds, the con tra i of the mos abund t empla re ch s a crit l con e tration. Once this con e tra i of templa is at ined, rean li g is favored over p im an e li g d amplif c t on his templa d cre s . Thu , t e orig nal y les domina t template becomes more ef ctively amplif ed in the later cy les of the PCR until it to reaches a con e tra ion at whic template ( an e li g pr m outc s n w i ta hlp r m e u s b d c a w h pe rn i m l f ew di t ho cien y, th cri al once tr i fo templa r n e li g was nev r ach d. Howev r, it was rgued tha the phenom may not be a seriou problem when amplify g rom envi tal DNA bec us it wo ld harb v iety of templa s, l at rel iv y low c n e tra io s. Any si gle t mp a e, th r c o n r we ha i t b u l d k g f o y r e , favored p im r an e l g It has l o be n oted hat cloned PCR products gen rat d using d f er nt prime s ult d in s g f cantly dif er nt comp si n of cl ne ibra s Furthe mo , same b tch of PCR pr duct lone si g ther blun - d or sticky-end clo ing procedu s gave dif rent sul . Howev r, it s not clear how inter al estric on e zym cleav g af ect d he r sult since th clone d t o lh s ie y - c b r w a f n z p d u e s insert DNA he scr n d lo es wa n t r po ed. r C e o p n s D a tN d i cq A u . l gy m , e o er o s oc ur during replicat on of PCR-amplif ed gen s. The frequ ncy of nucleotide mis ncorp ation varies for dif er nt thermostable DNA polymeras u ed in the PCR. Enzymes uch as meras ( Pfu v e rl yo wa t n sfu c i dm e o r p a t i nM . c e m o lu ys d z s u ca h T h e r m ua sq t i c u s n u c l e a s c t i v t ya n dh e c h a v e i g h e r o r a t e s .H o w e v r ,t h ed g r e o f er o resultingfrom is ncorp ation fbase duringthePCRisgen ral y r e l a t i v sy m ( f r a c t i o n s 1 % ) m p r e td i f n c s r R N A e q u b e t w e nb a c t e r i a ls p e c i s( a p r o x2t 3 % alw ys hold, and many wel -defined speci s how much lower dif er nces in their RNA 147 ). The explan tio for a tend cy toward a 1: Fig. 1 1 Fig. oc ur n t di Th s ). . (46) (47) DNA poly- Pyroc us f riosu DNA polymeras ) tha have a 3'–5 pro f eading functio have ( Taq D N p A o l y m e r a s l ) pc a rk o f e a d i n e g x o - [39] sequ nc , lose t h lev s of mi nc rpo at e r d fo ) .N o n e t h l e s ,t h i sd o e n t . 148 148 Templat (A) 2, both amplify at he same rate. (B) Templat 1 has reach d a con e tra i at Head p t r m e i a f x o l P d C S n h Rc . 1 t F e i g 1 is orig nal y more abund t han templa w h t i e c m p l a n f v s o r g e i d m n a l p T f h t . i cg r e o m s a l 1 n d y u T . p t e 2 remains at con e tra i at whic prime an e li g s favored an conti ues to be amplif ed f ic ently. reach s on e tra i wh c templa n e i g s favored n amplif c t on s dim he . (C) Templat 2 Recov ry and A l si of rRNA Sequ nc s some thermos able DNA polymeras sequ nc a lysi to re lv ationsh p t e s ci l ve s im t d un er thes cir um tan es. PCR- using d ver ty m c ob al f n ysi w th a oc ed pr bl m fu th A amplif ed rRNA gen s i the formati n of chimer PCR products meric g nes r ult from the incomplet syn he i of an rRNA gen fragment during amplif cat on. If the incomplet fragment an e ls to a hom l g us r g f R e N n a A m h o t i x d b u lc p e g , . This re ults in a rRNA gen fragment ha s be n r plicated from tw (or d m i o f r et ) c s ph na lu q R , N A does n t exis natur l y in a liv ng or a ism ( meric ol u s anbe t d c by ondu ti gphylo en tica ys on t c r sh e i qm u R n NI Af , o p . s d i t e fg i re nt a cw sb o m h q l u -I d . g t e r n i h s a d m o p , l R - N Af t e r g y n s ecul sho d be i nt cal, or t leas v ry im la . Sev r compute r g ams i d e c bv nh t s lo m f q p yru( w a . g , thes have dif cult es in recogniz chimer molecu s in whic the “pare n st ” q u c h a >v 8 5 o% m l g Ty . e ps r a m h o u l td , e r f n y be used as a guide, and the oc ur en of chimeras hould be confirmed by careful n ysi of ec ndary st uc e in ract o s nd i ep nt hylogenetic an lyse with dif er nt regions of the molecu . The frequ ncy of chimera formati n has be n det rmin to be up to 30% when PCR has be n condu te with mixtures of sim lar templa s sequ nc s in clone libra es from natur l sample has be n repo t d to be slight y lower (e.g , deriv sequ nc for their pos ible chimer nature since they can lead to over stima n of he micr b al d ve sity pr n a p rticul samp e. 149 . Howev r, the abil ty of rRNA (15) (48) ). The oc ur en of chi- Fig. 2 se . Oc ur en of chimer (50) ref. se 37 ). Noneth l s , it is advis ble to tes PCR- 1.5 Quanti o s ng PCR P C R - d e p n m t h o f c s r a e i z m n g c r o b a l u i t de r s n tify ng particul organism pres nt in an enviro m tal sample are invaluan lysi qu t ve o it lends PCR the of s n i v ty ex r m Th able. of specif organism when pres nc /absenc dat are requi d. Obtain g quanti ve dat using the PCR is more problematic. The biase outlined in Subheading 1.4 2 a c u r tqe n i a o mf c r g n i s a t u r eA.l h o g a nst l u i o have b n d lope tha w qu nti a o b sed n th u of in er al st dar s competi v mplates ( .g , t h a l e m p i n sr o g u x t a m e p l i f q d y c e n t l . h o l fd t e mn a y A i s u ,g h and al conf u d at emp s to use the PCR for 1.4 2 ref. se (51 , 52) 51 a s v u lm ip dt o n w y s t h i . Chi- ), they r l on the as ump ion ref. 49 but ), 150 Head Fig. 2 Schemati d gr m of chi er p oduct f rma ion du g the PCR. (48) and thus ab olute q an it o f speci rRNA sequ nc based on the PCR must be consider with care. Howev r, quanti ve competi v PCR using a inter al st ndar th can be shown empir cal y to amplify w th e Recov ry and A l si of rRNA Sequ nc s same f ic en y (or at le s at rep oducibl and me surabl ef ic n y relat i ha voer gs q u n c ) t e h o lp dr s m i Q u .a n t v e , competi v PCR using prime s specif for a particul group of organism has be n d monstra e to w rk el for the quanti o f rRNA sequ nc from icrob al tax recogniz d only from enviro m tal y recov d rRNA sequ nc (52) t e m p l aswh o n i f y c t e r s g m p Hl a o r wt ce s i b v. u n qR h N d , A f a v lue for c numbe s or i a em ns probl atic. Det rmina o f the r lative bunda ce of particul organism u ng hybrid zat on wi h spec fi ol g nuc e tid s o quantify spec i quenc typ s in a PCR- mplif ed xtur is al o fe sibl n some cir u stan e . I stance in wh c t s may be u d vali y nc ude tim -s r dat n ep h distr bup a r to if sc eu nql( y . g , r e l a t i v b u n d c oe paf r t i u l s q e n c t y p a d i f r e n o t s p a c r t i m e h a ct bn de t r m i n e d I. wt o u l bd ie m p o s i b l te co n v e r t h i ms e a u r e to a figure for the pro rtion of the to al microbial po ulation tha this rep sents unles al templates pres nt wer know to amplify with the same f ic ency. Likewise, an bsolute number of cel s can ot be infer d without information on the size of the genome and the rRNA gen copy n u m b e rf o a l ft h eo r g a n i s m a u tp o er c i n hsl f g md , a p r o c h t i s b y a n e d um i c h r los t vg b y c oh fne dm ui t ap l y s br o f i n t y p e h s W in wh c a org nism o t abund ,o e can b gi to f rmula e st gi for is lat ng h organism cult re and et rmin g ts me abolic t v ies of bi ge ch m al r ev nc . Comple ntary to this ap ro ch is the relativ quanti o of rRNA ext r a c e d i l yf r o m n v i e t a ls m p r h e t a n R N Ag s P C R - a s m e i p q n l xu g t f rc d , N A L o k v e a i n t pd nh c vro i a e b s l u m , r g a t o p e w i h s c ln a o f r t R e N A h s i n c b u s r p e o al t c i h g n f d v m y relativ bund ce. In con lusi , it se m wise to consider recov y and an lysi of rRNA s e q u n fc r o vm i e st a d l p f n o i , c v e g x r i s but as the first ep toward i ent fy g relativ y abund t, unc lt red m bers of the microb al p u ation. The clon d sequ nc provide mark s tha d e p t r a b hso m fi ln c u x , v g the unc l red po ulati ns time and sp ce in r lat o changi e v ronmental co di ns ca be studi . They also pr vide th informat equir d 15 c o m p e t ai r f u s cdn ae i t g h l p ob sy w T e i . se ref. 5 3 t sh ie u I a n )o . , sp ur a i tn eT c h d o l . y (30 . , 3 , 53) . (24– 6) (54) can it 152 Head c bu el y t nia vr ho g s m d x f k p o t e n c ia l m y bs zr g t o hn ef i u c a I l s . e n v t i h r o s - q ug a l p c e m f n t ment ca b dis over . 1.6 PCR Amplif cat on rRNA Gen s from Envi e tal DNA Pr c i al Cons der ti g e n o t , h r a y d e g i ms R p N l A u jf n o rc T a h t sub tance y PCR the of in b verc m g s a plnviro me t f coextra d with e nucl i a ds. Details of pr cedu s igned to rem v i np vr o lc e d u tE sh a 7 y. C p e r i n g v a s eu b h ti n c o r y dif er ntial preci ta on of the contami ng sub tances with am oniu acet , physical separ tion using el p rmeation techniqu s, and sorpti n of inh b tory materi ls ont ion-excha g matrices and adsorbent such as p o l y v i n r o s l m iey t pf d Ah au n c . P C R l dilut on of the enviro m tal DNA pre a tion to reduc the lev s of the inh b tory c amin ts o bel w h v at whic e PCR s inh b ted. e h t morf d i up n b va y l q e c s t h gnimu A e m a s a t nle m o r i v wA is at nh py l c o P f Ce di R st, r a b h l v c we od k i a n r s edt u m ( p f o R h ) , .g P aC n d t s c u l be stored remot from templa DNA pre a tions. When amplify ng rRNA sequ nc using universal prime s or prime s targe in very broad phylogen g ebr tao icu l p s R N Ah , -m n e t a i o ous pr blem. Fo xa ple, m ny th r os able DNA p ym rase p tions c o n ts au if p r D h eN d Am c ng y P t Co i lR s u negativ contr ls contai g no ad e DNA. Howev r, t ea m n of enzym pre a tions w h DNase I has be n prov t remov DNA ef ctiv ly from enzym pre a tions h b a c r o t n e - s f i u l m P b ar . q c o w n t i e y r e g u - nh oi p t w v q a f s y c l md D , N n A t i g l a r c y e n d m i t a e c , n r s y u f i e D t N aA o l w m p i f c t o n Pp oCr fR d n uie c g t a v o lb Id sn . i t m , e r a v l c y sn be an importan source of contami g DNA. Al reag nts should be prepared with f l er d, st ile d or dei n z water of hig qual ty. If cont a m i n w o e x h g b ua sc t p r iD N lA e n o b mx ,p s ut r ultraviolet (UV) radi t on eith r on a UV transil um nator or using a UV cros linke w also he p r duc DNA ontami . P r i m es l c t o a n u i s d e r t oa n w i l p d h e r t c u l a a p l i c t o w n h d e r R g N A f si a m n o e ( . g a l , me b rs of the domain A NRr fo RCP eht ,tser tni fo elp . r a w o f tdh g i s e u q s i e n g . Bovine s rum alb in (BSA) in PCR buf ers can (5 ) Bacteri ) or a specif group f organism re b ing Recov ry and A l si of rRNA Sequ nc s 153 targe d. It has l o be n fou d tha some pri a rs m y a plif env ro m e n t a lD N As u c f l y ,w h e r a so t d n her wo ks el for the amplif c t on f bacteri l RNA gen s from envi mental s p . Mo t f he comp n ts f he PCR mixture can b o t i ed e c o - a p mn r v s i uyd l e f , t c oa m r i l y n o m i c sa el r v f t h y n coiu s m - d e g n l i o u c e t pd r i m s . Purchase of hig -qual ty reag nts from reputabl sup lier is recom nd becaus this ensur rep oducib l ty betw n batches of reag nts and also reduc s th risk of c ntami o f reag nts wi h lo ev s of DNA during pre a tion, her by saving t me d f ort. (56) .T h ep r o t c ld s i b e 1.7 Analysi of PCR-Ampli ed rRNA S quenc s: Practi l Cons derati 1.7 Use of Diagn t c Pr me s and Oligo uc e t d Probing P Ca Rn - l rmy pz N et Ai o f s d g h a- n T r e w sequences relies on the use of diagnostic oligonucleotides. The oligonucleotid s are used eith r as hybrid zat on probes or as PCR prime s, and a m p l i f r c to hd eu s n z g t i e no s o r g a n pi f s m t u c l r o e h n d a i f y t v (e.g , se ref. gen fra m t c n be o tai d by pro ing the PCR product in e h r a Sout ern blot r dot-bl format (e.g , o l id g a n u c s et w h r b a l s o c n p r i mu e v s a l and some gr of quanti o s f er d by this ap ro ch bined with DG E, the PCR-probe ap ro ch can provide usef l qualit ve informat he comp sit n f m crobial p u t ons 57 ). Great confide in the source of the amplif ed rRNA ref. se 28 ). PCR products amplif ed using (28 (27) , 30) . When com- (30) . 1.7 2 Cloni g a d Sequ nci g PCR-Ampl fied rRNA S quenc s m wT o ih s de at l y p cr e m i z a on bg p l u t in deta l vo es qu nci g of l ned rRNA g f a ments plif ed rom enviro m tal DNA. It is al o p s ible to s quenc dist rRNA gen fra ments r olved n DG E gels ( ev r, gen ral y facil t es the sequ nci g of large fragments. This permits phylogen tic an lysi at great r resolution and of ers more scope for the d e s i g n of P Cp Rr o d u c t sh av ye p n d i otg l a m e h u ds r t i c k y - e n , blunt-e d, a TA cloni g. 1.7 2 S se Chapter 12). The cloni g ap ro ch, w- diagnost c ligonuc e t d s. The t r main p roaches to cl ni g TICKY -E ND C LONI G Sticky-end lo g rf ced loni gas t ome i st r d equi sth ad it on fres ction es h 5' ndoft eampli c on r me s( .g , se 154 refs. 56 porated in each prime , then double digest on ca be car ied out, prev nti g reci ula z t on of the cloni g vector, henc improv ng cloni g ef ic n y. p r o b H - e w P c d C al uv t R n f s, m i h g e o lematic amplif ed rRNA gen products an result in the r cove y of trunca ed rRNA sequ nc i lo e bra i s. Th can be ov r m t so e x nt, by he us of ra e cut ing r s ct on e d u l as ch Head and 5 8 h It a )sd e v n g i f r e t s c i o n a r e - (59 ). Furthe mo , rest ic on endo ucl as cleav g at site with n 1.7 2 B Not Blunt-e d ligat on procedu s are les ef ic nt than sticky-end ligat on. Noneth l s c oni g f PCR products ing blu t-end clo i g f rRNA f agments amplif ed rom enviro m tal s mple has be n widely adopte ( .g , se refs. 60– 2 PCR products an be cloned. Howev r, thermos abl DNA polymeras tha l a c k 3 ' – 5 p r o f r e a d i n gf u n c t i o n( e . g , nal deoxynucleotide transferase activ ty and ad a template-indep nde t deoxyadenosine residue to the 3' ends of the PCR product stan ial y reduces the ef ic ency of blunt-end ligation procedures. Consequently,thePCRproductsmu tbemodif edtopr duceblunte ds.This normal y achiev d using a DNA polymerase tha has a 3'–5 pro freading function (e.g , T4 DNA polymerase or reported hat gives bet r blunt-end cloni g ef ic ency than does T4 DNA polymerase. Alternatively, PCR products amplif ed with thermostable DNA polym e r a s e w i t ah obviate his trea m n si ce th y do n t produce PCR products wi h a single nucleotid 3' ext nsio dures hav b n de lop . Thes maint h g lev of in ar blu t-end vector by inclus o f the ra -cut ing bl t-end r s ict on e d ucl as in the l ga on mixture. C ND ). LONI G ). Since ther is no ne d for estric on digest on, ful - ength Taq D N Ap o l y m e r a s e )h a v et r m i (63) Pfu . This ub- DNA polymerase). It has be n D N Ap o l y m e r a s e( S t r a g e n ,C a m b r i d g e ,U K )g e n r a l y Pfu p r o f e a xd i n ug c l t ( s v . y , Pfu (61 , 62) p o lD yN mA e r a s ) . Rec ntly improved blunt-e d cloni g proceSfi 1.7 2 3 TA-C A l t h o ub g n - ec d i sm p r o ta bh y 3v 'e f n g ni u cg l e o t bd y p s r f a Di oNn lAg y m e r s t fh a , c xi p o l e d for the f ic ent cloni g of PCR products in “T-vectors”. T-vectors a e plasmids tha w en li r z d have singl d oxyth mi ne r s du at he 3' nds. T h i cs a bn e o v t l ay c h i e v bd a g w i t rh e s c o n d u l e a s tha produce bl nt ds. 3' T overhangs c then b g rated by incu at o with Taq obtained com er ial y (e.g , pGEM-T, Promega, Southamp n, UK). Thes -E LUNT I (50 LONI G DNA polymeras nd T P. Vectors gen rat d in this way c n be I Recov ry and A l si of rRNA Sequ nc s 15 v e c t a o l rs i w k y - n g d p P tC o R f u c e s r a b y d o f i n gt h e r m o s a b l D N Ap y m e r a s w i t h o u en df r s t i c o nd g e . 1.8 Scre ni g RNA Clone Libra s g e r n R N 1 A 6 S c t sl q h o au i f m b d T n e g i d e n - lt o b r a sh c y e i n v lf bo rm a t y p s , tify sim lar or ident cal rRNA sequ nces. This can be done using col ny h y b r i d z a t o np c e u r sw i t ho l g n u c e i d p r o b s f e n dp h y l o g e t i c r e s o l( u t. ig n, s p e c i fa g ol r n t y , q u - c e i s p o b a I f c l . est can be overlo ked. Convers ly, it s pos ible to discount unique clones if they contain the target site for the oligonucleotide probe, but are otherwise quite dif er nt. Isolation of plasmids from indiv dual clones and d i g e s t i o nw t h g r o u p e l a t ds q n c ( e . g , using, e.g , sequ nci g prime s with prim ng site hat flank the insert DNA can be used as a rapid scre ni g procedu to det c cloned PCR products. d i r g a Te w hs t c n o u d l r e as i f q t y n c in the clo ibra y. A l t e r n a i v y s, g l e - a n q u c i (g t - r a k n c) l s bo de t a w hig er soluti n cre g ef ctiv way of ident y g sim lar c one s qu ce withou e r qui ment c af t os r l ue q y n i g o H s w . e v m t r h i , d p a a v i l b r o tfp y u d m a Ds eN Aq n c r , u iol f g es a n single pr m can be r l tiv y cos -ef tiv and pro es uf ic nt ormation f r bas c phylogen tic a lys . se ref. 6 1 c r i t s - u p e o dH b h w ) v f . r c , i t y f r e q u c n t l siy g o d e b u a c ns refs. se 60 and 6 2 ) .A l t e r n a i v y ,c o l P C R . If manu l seq cing s u ed, this can be (64) 1.9 Char cte iz on f U cult red Mi o gan sm c h a r t s m e i q n u p R o N l Az Ty- f bial po ulati ns i extr m ly powerful. In the r lative y short ime since th i n c e p t o f h s e c n i q u ,m h a sb e nd i c o v r a b u t s - y e n c l i vated micro gan s in atur l po ulati ns. Consequ tly, his ap ro ch is becoming routine in many res a ch labor t ies. Althoug the t chnol gy is w e l s t a b i h c d , r t e i z a m o f n c r b p l u a t i o Pn h y Cs e R - c l – s e q a u p nr c iol m t b h - d v e s f u I n g . u n l i k e yt ob s da r u i n e v o m t a l n i r g o .I t s ,h w e v r the m od f choi e f r d taile cu t r -indep t char e iz t on f microbial p u tions a d provi es th framewo k f r sub eq nt s udie s ng methods re am n ble to c mpreh nsiv ampling d rapi n lysi (e.g , PCR and probi g, wh le-c ng ol ev r b a , y - d i s t m uc r eoa bx l in s tu hybrid zat on d DG E an lysi ). Sev(25 , 34 , 52) u n bc eo h v a r d 156 Head t a h bp i y s r ow nl cu d k e v m o t w h n r a id I s e .tion he discov ry f n el phy ot s, cl ned rRNA s vide the information nec s ary to design oligonucleotide primers and probes that have facil ta ed autoecol gical studies of uncultured tax (17 , 30 , 3 , 35) bution and temporal po ulation dynamics of unc ltured micro ganism (30 , 3 , 35) . Sequ nc dat can lso be o tained from DG E gels by excis on f ind vidual bands reamplif cation and direct sequ nci g of the PCR product o b t a h i c T dn w e f , s v . m r p u l x a t i e y z d rounds ev al q i m y t pe s qu nc i gl a of b nds pure o tai g nc of purif cat on and DG E under dif er nt denaturi g condit s, to obtain purif ed PCR p o uct and g qu lity se nc dat . equ nces have pro- U s .it nha g p r o b e c d l m it nh s r - 1. 0 Det c ion f Spe ic Grou s f O gani ms PCR util z ng specif prime s or diagnostic olig nucleotid probing of r s R e N q A u n a c m p l i f b dr s o g e c p i t y m r o s b a f l e h most promise for the dev lopm nt of rapid techniqu s to monit r specif m i c r o p b a u l t e T s h n i . q 7 d0 v l : c o w a m t s cel s/g of s il (equ val nt o les than single c per PCR as y) h ve b n claimed (57 proba ly m re alist c, ev n wh using e t d PCR gonucle tid prob ng t i creas n it v y ronme tal s p wer inoculat d w h kno am u ts f c l red ba t i l cel s, and tec io l m s f r ind ge ous p lati n m y be slight er. c a st p e- i f w bn o h u d l r t s e b n h a p r o Tc i s bolic a t vi es n e vironm tal rices ( .g , ticular groups of indigenous pol utant-degrading micro rganism Bioge ch m al y sign f ca t organism have also be n det c using PCR amplif c t on eith r with or withou ligonuc e t d probing pres nc of ath ge s uc sample using PCR amplif cat on of rRNA gen fragments. In the case of Legion l a cial d gnostic k for envi o m tal monit r g (EnviroAmp™, PE Ap lied Biosy tem , War ingto , UK). The use of diagnost c PCR amplif c t on of a d s e w h ri q o Rt m n u N A v , c p s te rn iv c ol ypa m h t s n i ,o . Iie tsq u a l y doh e c t i p nfa g s d o i l er a n m t s h fo d an gricult e nd s ri (e.g , To date, most ap lic t ons of PCR amplif c t on of rRNA sequ nc to enviro m tal oni r g have b n at he l v of pres nc /ab e t s ing. , 65) 3 . Det c ion l m ts of the ord f 10 4 to 10 cel s/g of s il are or sub eq nt oli- (6 ) . In the ci d xamples, nvi- (67) refs. se 6 and ) and p r- 67 (57) (28 sp . and h a s l ob e nd t c i w a e r (70) Legion l a this a be n d v lope int a com er- L. pneumo hila, se refs. 71 and 72 ). , 68 , 69) . The . Recov ry and A l si of rRNA Sequ nc s 157 This has stem d large y from the dif culties in reliab y quantify ng the e x t r a Dc N mA p d l i s n q u ge t r R N cA al o f m u n t from enviro m tal sample , using the PCR. Howev r, the dev lopm nt of g e n u i ql y a t Pv CesR y p a, r t i c u l w h se n f oi c g u l e t d p r i um a se d d e t s aroi cm b l un h p y g t e oa f lr i n gen usi caref l y ontr ed c mp ti ve PCR. How ver, ithou kn w g u n c a l t o is f h v e z r p g m d b n rRNA gen p ome, it w l no be p s i l to c nver his to cel numb rs or bi mas . Th problem ains key is u n PCR-dep nt qua i on of unc lt red mi ob al t x . (52 h o c l ) d n s i e r a p b f tmu h o - I y . 1. Autoec l gi a Stud es of Unc ltured Ba i s l i ng ahb cte - y m R P o C u qs l n ie bat dyv curate and requi careful valid t on, but robust methods tha al ow relativ quanti o f speci rRNA sequ nc ar v il b e ( Thes a y re suit d o a ec l gi stud e of rganism whet r o n they can be cultiva ed. Useful informat on the r lative abund ce of tax recognized only from enviro mental y isolated rRNA sequ nces has be n obtained using this method. For exampl , the relativ abund ce of a novel bacteri l n g (SAR406) relat d o p e3 r0 i- om d a w v S s t g h e n o f r e S x A h R s i 4 t b 0 ov a r c 6 f d w n g l e chlor phyl s e q u n c w r f o td b me s a u n t p ih we a cr o l u m tn h was just below the de p chlor p yl maxi u r e d Sv i nt A f h Ra 1l m o c gb u s - N m o S s A t b R e 1 q i u n - gp l a c r k w x h m t y e r a b u n d ti e p rw a n do t h e sb i gm r a u n d t o w r s h e u f a c of the wa r column Thistec n qu alo gwith e-c l provide usef l means to monit r specif microb al po ulations in natur l b e pn r h o a c tv d u s l i m f T e p n ,y . explor d( m op na ir t c u l g e q d h s rft mp oi - n vation. Pr to he d v lopment f molecu ar biol g ca monit r g me hods this wa no p sible. 1. 2 Changes i M crob al C m unity S r c e a b n e d h v i t o r u p f c s T m l a de n g distr bu on of discret microb al po ulati ns (eith r specif organism or ). Subheading 1.5 se was inve t g d surface Chlor bium (30 r e al b T uth ni -v ) . a con e tra ion in the same samples. Furthermo e, SAR406 (30 (3 ). ins tu Subheading1.4 ). Sim lar studie have and 1.5 ).Noneth l s , ydopr vi eth m ans hybrid zat on( se Chapter15) 158 groups f elat dorg nism ).It al op s ib et mon rva i t on m cr bial po ulati ns at a more gros lev . Two prima y ap ro ches have be n used to achiev this with amplif ed rRNA sequ nc : DG E, and amplif ed ribos mal DNA re t ic on a lysi (ARD ). DG E al ows r pid com ar s n of micr b al om unit es ( ubj ct o he cave ts above, c o n s m dup i e f r t b a g l , o n s or c upled with e us of ligonuc e t d probing, the dynamics of specif groups f ani ms c be onit r d. c y s a f p n o dr l h i b m -g e w t T z o n turbance has be n studie using DG E of PCR-amplif ed 16S rRNA gen s (73 ). This tudy emonstra d h t rapid ecol nizat of the dis urbe mats oc ur ed, but no al of the orig nal cy noba teri l sequ nc types orig nal y det c wer p sent v after 40 d, an ovel cyanob teri w e r sponsible for much t e in al r co niz t . Lo ger-t m ni or g d cate t h a b e c r i p ol u a t n s e i h m o p r ng v e w t r rathe s bl over a 10-mo peri d an th er was uc es ion f bacteri l types along the rmal g dient from the source po l f the o spring d e a m pn o l D s tG uy h ir E f - x b d c z n g am s e r qw oi u y -nt d l h c v a e r s i nia oxid zers found large y on the seaw rd side of the dunes. In ad it on, dif er nt po ulations of as oci ted w h sample of di r nt pH A furthe c nique tha s be n i troduce ntly o char te iz comp l e xm i c r o b a u n i t e sb a do P C R - m p l i f e dr N Ag n si R D (74 , 75 ). This method is ba ed on vari t ons i the fr quency a d locati n of te ram ic rest ic on enzym recognit site in rRNA gen s. Almost ful length rRNA gen s are amplif ed from enviro m tal y isolated DNA and digest with rest ic on endo ucl as tha recogniz te ram ic sequ nc (e.g , Alu I, Bst gen s produce dif er nt sized rest ic on fragments and henc can be dist nguished on a r s gel Monit r g inocula t and ind ge ous groundwate bacteri in a fluid ze bed reactor trea ing toluen -contamin ted groundwater has be n a usef l A R D . a op f l i c t n and Burkholde ia picket tha can be recogniz d on ag rose gels bacteri l strain to a fluid ze b d reacto , reating f lter-s i z d groundwater con i g t luen , cha g s i t e po ula i n f the r bac i wer monit red using ARD . After an i t al period f 53 d, unfilter d groundw a t e r m n d i h o l u e w a s td f h b i o r e a c I. wt s l fr o m Head ). It permits emporal nd spati l changes in Subheading 1.4 ). (36 -like organism wer , in some case , (32 ). Nitros pira UI, Hae I, I, Mbo Hha (74 , 76 I, Msp I, Rsa a In ,d Taq ID )i .f e r nR tN A ). PaW1, P ps ue t id o am n PKO1 produce dist nc ve ARD band pat erns (74 ). Fol wing inoculat of thes B u cr ek ph ao l id G4, Recov ry and A l si of rRNA Sequ nc s 159 tA hR eDd a p n r l c u , t e - d p n tu m r a i oh fe c l n t bacteri h st ain P W1 outc mpe d h ot er w bact i for luen a d, u l t i m b a e o x rhf c y , C d n . i g wa o t u h ter bac i h d oc ur e by d 38 of the xp rim nt, a d this wa cle r y vid e f n r t c o h m a g i A s R D b p d t e 5 r 8 B n ly . o u a i f i n d g e r o u s bw Pt a l c W vh 1if n d p e , si run egt T a h c ow d . l b u n l i k e yt oc m p w l a g i n s t d e o u r g a n i s m t l z g h es a m c r b o ns u r c e , v i ft h n o c u l a s r i h d n t a l yc o i z e dt h b r a c o . c m o i - fr w nb h g ea l p t s q u d r a p i s A R D munit es. Unlike DG E an lysi , ther is no requi m nt for elativ y complex o yacr mide natur g dient ls. How ver, th l so uti n f ag rose g ls means tha m ny sim lar but no ident cal y sized ban s wil be se n a single ba d, thus nder tima g the ru dive s ty. This exac rbated y h fact digest on w h at le s hr est ic on d ucleas i requi d to give maxi l taxon mic resoluti n using reacto b d flui z a from s ple icat w h ob ned p t r s a t h r e d i f n t s r i c o e n d u l a s h o w e dg a r m n ti h ed v r o b s ie tr yv d most abund me b rs of the p ula ion t be char t ized. Cons que tly, m i c r o sp f t b u d ha yl e n i b g l t o k e s y i n d u o m bfi e r a g t l h sI . d c o n u t e , date, using ARD for whole com unity an lysi , the bands det c d in A R D a n l y s e h v c o r p n d e w l i t h A R D p a e r n os b t i d f r ot mhd e i n a c u l r b m e ot sfh a c r i l m u n t ys o a eb d dilut on enrichm t techniqu s d e c o p t n h g l sr a y m f u i v w , d e u t i l co fy m b An Rg D i t r a c ud l v i to en h q u Ts a . b l i t y to use molecu ar techniqu s to targe isolat n of key speci is sign f ca t. Physiol g ca d t from the isola d t x , whic an be d monstra e by molecu ar methods to be domina t me b rs of the bacteri l po u fo sled m acit meh a gnimrof elbau vni (76 (74 ). Noneth l s , ARD t h ec a il n F o gu w r ks ) . y A m R D e , (74 ). Thes exampl s from biotrea m n , 75 , l n o i e w tb a biotrea m n . 2. Materials 2.1 PCR Amplif cat on 16S rRNA Gen s 1. 10 X PCR buf er: 10 m M MgCl 2. 10 m , 1% (v/ ) Triton X-10 ( 2 M d T P I ) : e a l t h y s o up b dr c a f e m t b s l u p i o e f r c l a b i o l r ge ya n t s. , U l r a p u d eN ™T( P sh r m a cB i o t e SA h l ,.b nU sK ) Dilute aliquots of each dNTP (10 ° C), 50 m Tris-HCl (pH 8. at 25 se M ). Note 1 deoxynucl tide triphos a e (dNTP) soluti n (dATP, dCTP, dGTP, µ L each) with 60 µ L of steril deion z KCl, 15 m M 160 Head µ L vol of this 10 m water. Dispen 10centrifug t bes and store a –20 dNTPs owing t rep a d cy les of r zing a d th wing. 3. Oligonuc e t d prime s: Prime s are dis olve in steril , deion z water and quantif edbytheirUVabsorbanceat260nma d iluted o10 2 ). The diluted oligonucleotide solutions are stored frozen as smal volume aliquots (e.g , 20 r e p a t e f d r z i n a g t d h w i n g M . a p y r i m e r h s a v b e d n s i g n e w d i tr a h n g e of specif cit es for the amplif cation of smal sub nit rRNA gen s. Primers p A ( 5 ' - A G T G A T C G C T A G - 3 ' ) a n d p H r ( 5 ' - A G A G T AC CAG -3') devis by Edwar s c o m gfpre lRn N tAs dure sc ib d. 4 . p ( dr e o .si gy n ,t z a m u D w de o rl i . Mil -Q, Mil pore, Watford, UK) is f lter d throug a steril 0.2a u t o c l v e d I . p f r s i t n D N c A o a m i t np s r o b l e m , h w a t r b y U V ir ad te ( .g , 5 min exposur n a UV tr sil um nator). 5 . Thermostabl DNA polymeras ( fo niarts a morf detalosi emyzan D .)KU ,dleifhc L - u n a me h ty bd r o p s i , y t v c ae s l u n o x g i d a e r f o p' 5 – 3e v a ht o ns d i s r e u t c oa f v h n o i t a er p w c nl h s i m n ar he t s m b o f d i n p u v a h dl .ea st n mpo r i f I v e a n l o i s f e t x 0 1 > n i m s , d e u h t .tcudorp RCP eht fo gni lc-AT wo a t h sgna revo Ad '3 s a o l emyzn M dNTP soluti n into steril micro- ° C. This prev nts xce iv degra tion f the µ M ( se Note µ L) and used as required to prev nt degrad tion owing to . (7 ) et al for the amplif c t on f near- s u b che a d vnp r tofw i l - y Bacteri µ m filter and µ L of Dynaz me (Flowgen, ). 2 U/ Note 3 se hguo tlA . sunaikcorb sumrehT qaT qaT , e sA aN rD dm ny l o p I s e t a l p mAh N Di w r e y l o p 2. Sticky-End Clo i g 1 . V e c t D o N M r A a : n y m c i l v a b p e s m c i l d o n v g t u b rs a e 2. 3. 4. 5. to clone PCR-amplif ed rRNA gen s. The procedur described used pUC18 (BCL, ew s UK). PCR prime s: The prime s are id nt cal to h se d scribe in exc pt ha rest ic on site ( n ital cs) have b n i corp ated the 5' end. pA ( P s t I 5) ' G- T CA G ATC describ n Restric on e donucl ase : The particul enzym s u ed ep nd on the r st iction s te incorp ated in o the prim s. For the prim s de crib her , and Pst I are qui d. 5X Ligation buf er: 250 m adrenosine triphosphate (ATP), 5 m ethylen glycol-80 . a m o l u T i nc h g t e D M s N A . r y p 4 : a v d requi d for the ligat on reaction should be det rmin d with ref nc to the m a n u f c t r e s’ p i f c a t o n Ts h. pi r c w dal e v o p i DTt N4h A g a s e sup lied by L f Technol gi s (Pa ley, UK). Subheading 2.1 CTG A A G T C AG -3'. The PCR is car ied out as A G T G A T C G C T A G - 3 ' p, H (r H I 5) ' - Bam Subheading 3.1 M Bam Tris-HCl (pH 7.6), 50 m M M dith othreitol (DT ), 25% (w/v) poly- MgCl 2 ,5m HI M Recov ry and A l si of rRNA Sequ nc s 16 6. Compet n cel s: compet n cel s ( E. coli ) can be pre a d in the Note 4 se labor t y. Howev r, in my exp rienc , PCR-amplif ed rRNA gen s do not alw ys clone ef ic ently and best results are obtained when com ercial y obtained hig ef ic n y compet n cel s are used (e.g , XL-1 Blue, SURE2, Stra gen ). β -D thiogal c pyr noside (IPTG): 0.1 M i o l f mw La 1-s t T 0Q I e h P r n u . G v g 2 4 i l z e d and store a 4 ° C. 8. -ly odni 3- rolhc 4- morB 5( lag-X gal in N, -dimethyl forma ide. 9. Ampic l n: D s olve 50 mg/ L a pic l n steril d e wat r. 10. Growth mediu for sel ction f recombina ts (LB/Ap IPTG X-gal r): 2.5 g of yeast x ract, 5 g of Tryptone, 2.5 g of NaCl, 7.5 g of Agar e dis olve in s t e r i a l g T N z h w O d H 7 . 5 p j a t ui n o s m L ( w e ) dl 0 r b ya u t o c l v i n g .W h e t a r sc o l e dt 5 0 t i c a l y .0 5m Lo fa p i c l ns o u t i ( 5 0m g / L ) , . 5 o fI P T Gs l u t i o n( 0 . 1 M ) and 0.4 mL of X-gal solution (50 mg/ L in dimethyl forma ide). The m e d is uh o bt l 4 ra for 30–4 min in an incubator set at 37 liqu d from the pla s nd l ows i cret ol ni s t dev lop. 7. Isopr yl- IPTG is pre a d by disM X- mL / g β 0 5 e v l o s i D : ) e d i s o n a r y p t c l a g - D ° Ct h ef o l w i n ga r e d s p - ° au nsw Cdei Tp t hl 3. 0 o br ° C prio to use. This remov s surface 2.3 Blunt-E d C o i g sc tl io n k- y fe rd a c bl o un i t -g e f d r q a m i s l e T h ingexc pt ha res ict on zymeg ratin blu -e d v ctorDNAmus be us d (e.g , Producti n of PCR products with blunt ends requi s polish ng of the PCR products emov 3' rhangs. T e c ary e g nts ar fol ws: I) and prime s contai g rest ic on site ar not requi d. Sma 1. 10XT4DNApolymeras buf :3 0m M sium acet , 10 m 2. Bovine s rum alb in: 1 mg/ L acetyl d BSA. 3. dNTPs: 1 m M dATP, 1 m dCTP, 1 m M) M Tris-HCl (p 8.0), 1 m M M M p r e a f d oc m n e t r a s d o c k l u t i n U sf r a p u ( eP h m c iB a o t e hS , . Albans, UK) dNTPs (10 m 4. T4 DNA polymeras . 5. TE buf er: 10 m Tris-acet (pH7.9),60 m M magnesiu c ta e, 5 m M dGTP, 1 m DT . M dT P. hes ould be . M EDTA. 2.4 TA-Cloni g d e T is A m nc - a r t lf b o q u g 2.3 exc pt he T-v ctor. A number of T-vectors a e v ilab e com r ial y, oab plu e t s r h m n i c T o . d e tl a i bh u ws n c o m e M r p G s (i S E t Pa l u – TU y h K v) g , n av il b e TA-cloni g sy tem also include a contr l insert DNA. It is advisable tha contr l ligat ons be car ied out with this insert since exonucl as a S n u d b 2h .e i g potas- 162 Head p r P o a C vd c R eu t 3 i m ' h f , n g y s ligat on ef ci y. 3. Methods 3.1 PCR Amplif cat on 16SrRNA Gen s It is conve i t o pre a bulk reaction mix for 5–10 PCR reactions f µ d 0i .s t r 2p o e a - h n m c v 5 L u l b 50–1 9 6 - w e lm i c r o t p a ed s i g n f ou r w t h e m ac ly r sT . f o w i n g p r o t c l v i d e s u f c n tr a i o m xf 1 0 X5 - µ Lr e a c t i o n s . 1 . P r e p5a0 2. µ b r u oe l fa k c L t m i n x 5 0 g o fd N T Pm i x( 1 0 p r i m( e1 0 µ M 3 )5 , p o l y m e r U a(/ s2 After the bulk reaction mix has be n pre ared and careful y mixed, aliquots (49 µ L)aredispensedintoindividualreactiontubes.Toeachofthese,ad 1 µ L o f D N A t e m p l a t e( the ub s a neg tiv con r l. µ b P 1u C 0f oR X e L r , M µ L µ Lo f r w a dp i m e ( 1 0 e a c h ) ,1 0 µ s ot fe L r id l w a t e 5 rn ,d µ M ) ,1 0 µ t oh fe L r m s a bD lN A µ L). se µ o f n te L ) s At de ) r . i l w a( 1 N5 o t e 3 . O v e r l a y o tf h c i n ws t fah e d r o p m i n a l ( tf h e r m a cl y is f t ed wi h a e t d li , no is requ d). 4. Subject h sample to he fol wing PCR cy ling pro am: in t al den tura io 95 at an e li g at 5 final ext nsio h ld t e r action 72 plet PCR products ( ° d e c n y o a f t 3 9l u 0 5 m r si b w 4 d C An eali g t mpera u is of v tal impor nce wh amplify ng rRNA en u s e d , a r p i g m n c o f t Iv s e l q u . a then the an e li g temp ra u must be suf ic ently hig to al ow amplif c t a hrs oeg if q u n w c m p l y t h geo rs a q du n c . Convers ly, if un versal p imers a used, lower an li g temp ra u s wil al ow amplif cation of rRNA gen s ev n when ther is some mis atch betw en the prim and t rge si ( t u r foeca hn , w v s u til me p f c a no s e i f P pCc rR d ucts.Thi par cul y ob ematic f h PCR ragmentis ob cl d.The pres nc of multip e PCR products nec s ita gel purif cat on f the PCRfragment o i r s . ° p mr i n ,e 1 f o C ° C for 1 min, a d prime xt nsio at 72 ° C for 1 min. After h ° C for 10 min to ex nd ful y an i comse Note 6 ). ). Lowering th an e li g t mpera- Fig. 3 3.2 Sticky-End Clo i g 1. PCR amplif c t on f rRNA gen s with pr me s contai g rest ic on site can be don usi g the pro c l des rib n 2 . P u r p i a f o m CdT c R hl e s t b : n r c r l e ot m n s x i b p v g a d N IT h fP , . µ Lo fr e v s Subheading 3.1 Recov ry and A l si of rRNA Sequ nc s 163 3. can be done directly from the reaction mix using com er ial y av il b e PCR p r o d u c i t f a ( k e Q . oI g s nA q ,c u p i l m a C r e n w U f K ) y ; , multip e bands re obs ved, th band of the cor t size (ap rox 1.5 kb for the e x gp t u r a l i d n m c f s hb o e ) kit (QIAqu ck gel p rif cat on sy em, Qiag n Cr wley, UK). R e s t r i c t o dn i g e s t i o n D: g e s t h p u r i f e Pd C pR r o d u c at n vd e c t o wr i h and Pst linker ca b moved fr th es rict on d ges u in QIAq ck spin olum s. HI Bam I as recom nd by the nzym sup lier. Th smal fr gment of p ly- 4 . L i g a t o n : i r e a c t o n s i r a gm o f e l t v s c i o n r e t are p d. Molar ti s of 3:1, and 1:3 re us al y dequ t . Th amount o P f C p R r d u c a t n v e o r q u i t gd pv a e r c u l m o a r t i c b n e l u lated using the fol wing equat on. Nanogr ms f i e t v d s r mola ti = [(na ogr m f vect size of v ct r n k l base] × size of n rt k l base)]/ × desir mola t Thus, to b ain 1: molar ti f vec or t inse of a 1.5-kb PCR product an 50 ng of pUC18, use th fol wing equat o : × 1.5)/2 69] [(50 × (1/ )= 27.9 ng i sert qu d) µ d io gf e L s pt U n(C 21 /08 m ) , m i ca r M o n x e t f u 2 g .b 5 T4 DNA ligase buf er, and the ap ro i te volumes of digest PCR product, steril d stil e wat r, nd T4 DNA ligase to give a fin l vo ume f 10 incubate 4 µ 5 Xo f L µ L, and ° C overnight. µ L of 5. Transfo m ti : Thaw e ig - f c en y omp ten c l s o i e. Ad 20 µ Lo f t h ec o m p n t l s oa e r i 1 . 5 - m L c r o e n t i f u g b eo n c .A d 1 ligat on mix to he c l s and t p gently o mix. Do n t vor ex th cel s becaus compet n l sca ber th f agile.Incubat o i ef r30m n.Heatshock c e l sf o r x a t y4 0 i n h o w e rb a t s 4 2 2 min. Ad 80 MgCl 2 , and 20 m 37 ° Cfor1h.Plate u r p icate50-mL liquots nLB/Ap IPTG X-gal r nd incubate ov rnight a 37 Using p C18 and ap ro i te hos train ( sel ction is pos ible. White col nies can be picked and transfer d to a fresh patch plate, and col ny PCR using primers flanki g the insert DNA al ows scre ni g of the puta ive recombina ts for the pres nce of the ap rop iate sized fragment ( sequ nce an lysi . ° C .I n c u b a t e h l so ni c f r µ L of LB broth (sup lem nt d wi h 12.5 m M ° C. se Fig. 4 3. Blunt-E d C o i g 1. Purify the PCR product sing a QIAquick sp n olum (ge p rify equir d). 2. Ad 2 vol f ice- old abs ute hanol t he purif d PCR product an preci ta e –20 MgSO M filter-s ized glucose) to the cel s. Incubate the cel s at ° C. ). Posit ve clones can then be sel cted for sub equ nt Note 4 ), blue/whit col ny 4 , 12.5 m M 164 Head Recov ry and A l si of rRNA Sequ nc s 165 3. Dis olve th DNA p l et in 2 4. Ad 2 5. Ad 2 6. Ad 5 U of T4 DNA polymeras p microg a f PCR product. 7. Make up to 20 8. Incubate 37 9. Inactiv e h T4 DNA polymeras b he ting a 75 1 0 . P r e c v i o a p l b w 2 uDs t dmN f h A n o l v e µ L of 10X T4 DNA polymeras buf . se Subheading2.3 , tem µ LofdNTPs lution( µ L of BSA s lution. ). µ L with s er l di t e wa r. ° C for 5 min. ° C for 10 min. µ L of TE bu er. pel t in 50 1 . Ligat on and transfo m i : ligat on and transfo m i can be car ied out as describ fo sticky-end lo i g ( and Subheading 3.2 , step 4 se 5 ). 3.4 TA-Cloni g 1. Purify CR p oduct sing a QIAqu ck spin olum . Ge p rif cat on he PCR product may be requi d, particul y if the PCR reaction does not produce a single, d stinc DNA band. 2. P r e p a r e l i g a t i o n r e a c t i o n s a d e s c r i b e d f o r s t i c k y - e n d c l o n i g ( ing 3.2 , step 4 control DNA. 3. Ligat on d transfo m i are don as previously de crib ( 3.2 , step 4 Subhead- se ) with a r nge of vector to insert atios with PCR product and Subheading se and 5 ). 3.5 Sum ary Rapid, s m le routine a lys are qui d for envi m tal oni r g ap lic t ons. Althoug the PCR can take s veral hours to amplify a specif gen fragment, hig -perfo manc thermal cy lers are now av il b e tha are i d e a l s yu t r o p h g u s tfa m l e the pos ib l ty f run i g 30 cy les of th PCR in u der 15 m if th n-wal ed glas c pi r es a u d, an they r w l suited o r utine a lys of arge n u m b e r os f a p l F. u t h e r m o c n l g hy a bs e d v o p t l w real-tim onit r g of the PCR based on fluoresc n technol gy Fig. 3. r R N Ag e n f r a g m e n t sf r o mg e n o m i cD N Ao fa u t o r p h i ca m o n i a - o x i d z i n gb a c teria using primers Nso190 (5'-CGATC CTGCT TC C-3') and Nso12 5 ( 5 ' - CG AT CG T A-3') ria of the 5 8 ° C .L a n e1 , oD N Ac t r l ; a n e2 , europae N p A V ;l a n e6 , wt marke VI (BCL). The match wi pr me Nso190. T .h e st r m ac ly o f e (78) (79) (previous page) Ef ect of an e li g temp ra u on the amplif c t on of β - Prote bac i . (A) specif or am ni -ox d z ng bacte- (81) ° C. Prime an e li g at 57 0; l N i t r o s p am u l t i f o r s ane 4, Nitros m nas eutropha N. eutropha (B) Prime an e li g at s p .N v 1 4 ;l a n e3 , Nm57; lane 5, Nitros p a Nm5 C-7 Nm57 16S rRNA gen has ingle bas mi - and 1 ;l a n e7 , Nitros m na Nitros pira Nitros p a s p .2 3 1 M ,m o l sp. 16 Head S c F4r i.e p g U noCl t f1 h 8 s R N A b y PCR using pUC/M13 forwa d (5'-GTA C G A T-3') and rev s p imers (5'-CAG TA G C-3'). The PCR product insert was p rox 50 bp a m p l i uf sy e rd t h b c a i s ln o g e r b p 1 2 0 a o x r e d u c Tt hs l n g . r e g i o nf sl a k t gh e rD N AT . s i zv a t o c ns i e w t l h n g v a r i t o n i r n R N g A e f s o d m r e n t a x T . h P C p R o d u c t w s e r l n d i a tg c k y - e n d cloni g procedur . Lanes 1 and 6 show truncated insert tha contai ed inter al rest ic on tes. M, 10 -bp mol wt arke (P om ga). The Int s ba d in the la d r is 50 bp in s ze. instrume tha c n simultaneo y monit r h e fluoresc n chan els are com er ial y av il b e a i m n o p c t l h u ed f s b r y a specif fluoresc nt hybrid zat on probes with n the PCR reaction mix Thus, t e amplif c t on a i tern l s a d n competi r an pote i l y be m asured api ly, and problems uch as pref ntial emp at re n ali g can be id nt f e . How ver, th ins rume ta on is exp n iv a d relativ y r e c d n v t l o p m qi a f h u s y , t P C v R l e c a i o n s has yet o b ful y eva t d. The abil ty o quantify specif groups of rganism , or at least he abundance of specif rRNA gen sequ nc using PCR, is e s ntial if we are to realiz the ful poten ial of rRNA gen s quenc -ba d enviro m tal moni- (80) . With this instrume a on, ther is poten ial to (79) . Recov ry and A l si of rRNA Sequ nc s 167 toring. For insta ce, it may be valu b e to know tha a specif organism cap ble of articul abo ic fun t s pre nt, bu i s m ch ore valuable to b a le to d ermin how large th po ulati n of the organism and, poten ial y, how active it is. It is now ap rent hat reliab PCR-dep n t quantif c o rRNA and ge s i a r l pos ib ty. Poten ial d user fo th ec nol gy i ude th wa r indust y a hose invol ed in cleanup of pol uted land, sedim nts, and water. To be widely - l a r h.o cej A bm i l d , n pr a e h c m , i d s tp e a b ur q n h c e d t p o a n e l d e v r l t o s a p b hc n f i q m u y ek b w i l g e r a p i s d , m l e n y t c f a o r m w iuh bs d e n g t o . s r o e p h a i b n u m t f- g l c d T s q r e s i b l ct o y n d u r g s i e n c t I h . r f o s e n i ta hl w m o c u l artechniquesareus d,suitablem anstodet rmineth qualityofthedat be adopted. Only rec ntly have steps be n taken to det rmine how rep oducible and rep senta ive dat obtained from molecular biol gical techniques are. Such considerations are critical if routine environmental monit r g us n molec ar bi og c l te hniqu s to bec m a r lity. Th s is not say th e larg y adopte mor piec m al p roach t da e is not valid, n it s til nec s ary to car y out baselin tudies o det rmin what microb al divers ty actu l y exist . In the contex of enviro m tal monit ring, howev r a m focused tl k is requ d. 4. Notes 1. Buf ers up lied with particular enzymes are likely to vary in their comp sit i o n T. h be u f e dr s c r i b e d rs c o m e n d f o ur s we i t Dh y n a z y m (e F l o w g e n , Lichfield, UK) DNA polymerase. 2. An 260 µ g / m oL f l i n u c e o t i d a, n h m wl ot f e o i1f es q u v a l n t 2o 0 o l i g n u c e t bd a l u e s i t omn wh f g d l v u a c e o t i d ( s A = 3 0.2, dC = 306.2, dG = 346.2, dT = 321. ) – 79 (98 for the 5' phos ate group, whic s not pre on sy the ic d oxy l g nuc eotid s). 3. Awiderang oftherm s abl DNApo ymeras v ilab ecom r ial yfo use in the PCR, includ g pro f eading e zym s uch as (Stra gen ) d UL modif e version of a b l u n t - y i r a c oe s g n R N A 1 6 S p ro ef a t i n d u c Ps C gR e r a end ligat on. They av lower at of mis ncorp ati n ha enzym s uch as Taq b l d u ig nf re t - p c s o h a y m D N A end ligat on. 4 . T hc e o i f s t r a u n e d hc l o i P gfC R - a m p l i er dR Ng A n o s f considerabl importance. Al organism contai rRNA sequ nc , and a hig degr eofsequ nce onservation sevidentacros awiderangeoftax .Cons e q u n t l y , h pe o t n i a fl o hr m o l g o u rs e c o m b i n a t i o bn e t w e cn l o e rd R N A Pfu DNA polymeras (PE Ap lied B osy tem ) whic s a Tma Thermot ga marit enzym . Thes are often used to DNA polymeras 168 Head sequ nces and the cient E.coli most uc es ful y (e.g , JM109, XL-1 Blue strains). I have found tha SURE2 compet n cel s (Stra gen ) are also go d host for cloned rRNA gen host is sign f cant. Ther fo e, recombinat o -defi E. coli host rainsmu tbeus d.Gen ral y frag- not are cls SURE2 mnt. to respc wih dabl ut mns recA recJ mutan shaveb nused recA and recB tosimlarphenycfuwTg. mutans. recA 5. Theamount fDNAad e p n so a ev r lf cto s,bu ypical 20 ngof templa r eaction w rks el . How ver, smal qu nti es can b used, particular y f dilut on reduc th amoun f i h b tory c n ami ts requi d. Usinglar e qu nti esofDNA,i h g enou p rity,can l owdet c i nof rRNA sequ nc tha re p s nt i low abund ce. 6. This prot c l has be n used with an Omnige thermal cy ler (Hybaid Ltd., A s h f o r Ud atK, m ) p l i y o cs e rt R gN A fn a m (t s p r 1o k.x b5 ) f r w oai m d e n g c u l t r b da e i n v r o m e t as l p u i n gr m e s targe in the distal nd proximal ends of bacteri l 16S rRNA gen s also en b d the amplif c t on f cloned rRNA gen fra ments u i g pUC/M13 prime s to pr a e t mpl s for DNA equ nci g. . It has (7 ) References 1 . B r o c k T, D. 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M a s o l - D e y A W., Rr i o s - H e n a d Lz Z., h o Ju - H i c k e FRy a. , n d f b c iS o ( xu m n 1 e v 9 d a - l r 7 s M g ) t. T J j e , film reactors trea ing arom tic hydroca b ns in groundwater. Microb l. 63, 75. Fries, M. R., Hopkins, G. D., McCarty, P. L., Forney, L. J., and Tiedj , J. M. ( M 1 i 9 c r 7 t s o ) h u eb f v p a l n d i g prima y sub tra e fo Trichlo et n cometab lis . 63, 15 – 2 . 76. Moyer, C. L , Tiedj , J. M , Dob s, F. C , and Karl, D. M (19 6) A computersimulated rest ic on fragment length polym r his an lysi of bacteri l smal sub nit ribos mal RNA gen s: ef ic ency of sel cted te rameric restrict on enzym s for studie of microb al divers ty in ature. 62, 2501– 7. 7 . Edwar s, U. Rogal , T. B öcker, H. Emde M , an Böt ger, E. C (198 ) Isolation a d irect omplet nucleotid et rmina o f entir gen s. Char cte iz g ec t ano i d f 1r 6 Sb s mR N lA . 78. Wit wer, C. T and G rli g, D. J (19 ) Rapid cy le DNA amplif c t on— ime and temp ra u optim za n. 79. Wit wer, C. T., Her man , M. G., Mos , A. A., and Rasmu sen, R. P. (19 7) Continuous fluorescence monitoring of rapid cycle DNA amplification. Biotechn qu s . 18, Clin. I fect Dis 958– 62. cel s Lister a mon cyt ge s 28 7– 31. 58, Ap l. Enviro M c biol. 63, Ap l. Enviro M c biol. 1367– 4. Ap l. Enviro . 270– 6. Ap l. Enviro . M c biol. Ap l. Enviro . Microb l. 17, N u c l eA i R d s . Biotechn qu s 2, 130– 8. 10, 76. 7843– 5 . 174 80. Wit wer, C. T , Rir e, K. M , Andrew, R. V , David, D. A , Gundry, R. A , and Balis, U. J (19 7) The LightCycler™: a microv lu e multisa p e fluorimet with rap d em ratu con r l. 81. Mobar y, B. K Wagner, M. U bain, V. R t man, B. E d Stahl, D. A (19 6) Phylogen tic probes f an lysi g abund ce a sp ti l organ z ti of n tri ying bacter . Head 2, Biotechn qu s Ap l. Enviro M c biol. 62, 2156– . 176– 8 . Ap licat on f DG E t Microb al E gy 175 21 Application of Denaturing Gradient Gel Electrophoresis to Microbial Ecology Richard Hastings 1. Introduction A sign f ca t pro i n f m crobial e gy is now ba ed n th description fc m u tys r c ein atur l yoc ingba ter l s mb age .Th dev lopm nt of m lecu ar biol g ca te hniqu s ha f cil ta ed his ta k, primarily v a the cloni g and sequ nci g of microb al gen s ret i v d from the enviro m t. Howev r, the labor-inte s v nature of a cloni g procedu , as w e l a ts h b i c a n t r o d u e h, v g n r a t d e f o l r n a t i v e l a b o r t ym e h d s a o r c u t e l yd s r i b m c o a l u n i t ys r c t u d r e n ; a g il c o p h ra en s g d l om y p b t c u lar microb al ecol gist to perfo m this functio . Denaturing gradient gel e l c t r o p h ( D s a G im n d E e ) g t r u l c o p h ( e T s G i E ) are t chniques based on the s par tion f polymeras chain reaction (PCR)amplif ed g n fra me ts, no ac rdi g to s ze, but owing vari t on he targe d nucleotid sequ nc s. Nucleotid pair dis oc ati n is mediat in d e n a t c u hg f r o i m ( D b G y l T s E ) d e . denatur s e incorp ated in o the g l in creas g once tra i s o f rm d e t n h a u g r i s o - c A m l . e d t f w Dn a N g h i b e i na g l y z do s th e i c a ly m r st g e h o u l ai n d r t u rg i na e d w m h c l s p t( u r e of the double-stran d struc e of DNA virtual y halts migrat on. Sequ nc s p e c i f d o m ta y n he is v d mu a l t n g p e r s , t h i se n o ,r y m l a - f D z N e A dg nc r t o s m l y a e d i r s resolvab in de tur g ls. TG E use urea nd forma ide in fixed con e tra i s to encourag but not mediat DNA dis oc at n; thus, PCR products are subject d to a linear From: T Methods in B otechnol gy, Enviro mental Monit r ng of Bacteria Vol. 12: Edite by: C. Edwar s © Human Pres Inc., Tot wa, NJ 175 m p o i )n t (1) Los. 176 Hasting i n c t r e m a p s h g u y d o li ne c t r p h s . When a melting domain c e G u a C si - f I r o t l . qh p n 4 c0 x e i d tg s o h a f D t br N h e o A g nc m d i s l y z a ht , - n g t e m p r a u l i d n o g m T h .s f i c a n t l y r e s d h c t ib o a f n pair va i nts over ident cal but no clamped fragments easily be incorp ated into PCR products by the use of one olig nuc e t d prime tha con i s the clamp qu n e at i s 5' nd. q u e s - t o a l p c i rh nd q u g e s , t a c l o n i g G e tions f microb al e gy, are both pr ne to h biase of PCR amplif c t on (4–6) , but he lat r is bel v d to f er c tain dv tages ov r cl ni g techniques. Once optim zed, denaturi g gel el ctroph si is a relativ y quick and easy way of an lyzi g gen tic divers ty with n a microb al com unity. Also, a bro de sp ctrum of enviro m tal gen tic d versity ma be o s rva b l u e s i n d g t r l s b , e c a u t h n y i o ls f b r a c n e dom pr ces influ d by a or t ime and u b r of cl nes av i bl . m u p o s ri b De c t Gf n v El g a y d w h m s r e a d i l y g v c n p r o Ef li e ts h q u m . c a r w i t h b n e d observa l ind cat o s of com unity struc e, but the probing of resolv d bands with ol g nuc e tid s a /or equ nci g of x sed ban r it ons tha c n yield mor ani gful ormati n f crobial d ve s ty. Th re a , howev r, lim ta ons to thes sup lem ntary techniqu s. Bands tha do not hybrid ze an olig nuc e t d probe remain unide t f beyond the lev of e x c yfi rs o dlm qa ut n p e h P f Cri R c y , bands for phyl gen studi lim ted (16S rDNA V3 regions a lyzed t pical y ext nd b w 20 and 5 b se in l gth). DG E was orig nal y used to an lyze g netic muta ion, but since its first microb l g ap lic t on lecu ar mi ob al ec ogist , and the lis of rep ts d ail ng ts u e in umerous and if er t cosy em nti u s o length T m is reach d, dis oc ation oc urs and migrat on (2 , 3) . GC-clamps can (3) , it has becom increas gly po ular with mo. (7–10) 2. Materials 2.1 DG E 1. Ap ro iate g l ctroph esi ap r tus ( 2. Dual-ch mber g adi nt m ker ( 3. G e l r e a g n t s : A c r y l a m i d e s t o c k s o l u t i o n 4 0 % w / v ( 3 7 . 5 : 1 , a c r y l a m i d e : a c r y l m i d e ) ,f o r a m i d e( o n z dw i t hA G5 0 1 - X 8m i x e d b r s i n[ B o - R a d , Hercul s, CA]) 0.5 m M Na u l t r a p u r e a( G i b c o te ram hyl t endiam (TEM D). se se Note 2 Note 1 ). ). - 2 bis Tris-aceta e buf er (0.5X TAE: 20 m EDTA, pH 7.4), molecular biol gy grade sterile dist l ed water, M - BP Ra Li ,s lU m eKn y)do p ur f a t e , Tris, 10 m aceta e, M N, ' - Ap licat on f DG E t Microb al E gy 17 4. DNA stain ( 5. Loading ye 2% (w/v) brom phen l b u . 6. PCR products f r an lysi ( ). Note 3 se ). Note 4 se 2. TG E 1. Ap ro iate g l ctroph esi ap r tus ( 2. Gel reag nts. Acrylamide stock soluti n 40% (37.5 :1, acryl mide: m f i o d r e ) ( 5a , 0 1 x - A X [G w n 8 B st bz o ih d R T a r ] ) - , acet buf er (0.5X TAE: 20 m 7 .m 4o )l ,e c ub i a r g s t yd l e i w a u rt , p ( G eiB bR cL o) glycero , am niu pers lfat , TEM D. 3. DNA stain ( 4. Loading ye: 2% (w/v) brom phen l b u . 5. PCR products f r an lysi ( ). Note 1 se bis Tris, 10 m M acet , 0.5 m M Na 2 EDTA, pH ). Note 3 se M -acryl ). Note 4 se 3. Methods 3.1 Prepa tion f Gels DG E is perfo m d in o e f two ays, the op i n de ing o whet r el ctroph i par met s are to be optim zed or gen tic divers ty is to be a n l y z e d .P r m t o p i z a nr e q u sp d i c u l a rD G Ew h s t b e quent g ic a lys requi pa l e DG E. 3.1 Casting De a uring G d e t l T h i sp r o t c lu e h x a m p o f g e li n c r a t g d e n u r i a d e n t range of 0–1 % (7 M urea, 40% v/ forma ide) t 20-mL volu e. Comp ne t 8% Acrylamide s 50X Tris-acet buf er U rea F orma ide 1. As embl thano -cle d g as pl te . 2. theanigrcm5poxlysetanmkrgdihPlc .pmalc s a htiw ebu wolft s i lae dn evla ht esolC . ta p s lg de bm a 3 . O n tc dhe a u r i g y l m sd oe u t i an pr e d i, a pnt r o v e l h i tc g o en fr u m a d i t s r o m l n u c g x h ea b d b r i e f l yo p nt h v a e , l o w i n gs m ea c r y l i d s o u t n p a s h r o u g t e res voi . W th e val c osed, tran f his olut n back o the mix ng cha d e R t b n h a m r o . ufv s c i l w g y n t h r fresh 8% ac yl mide so ut n. 4. Begin rota i n of the stir e bar at a low rate of revoluti n (but suf ic ent to adequ t ly mix acr l de so uti n whe t val e is op n d). 0% Denaturant olution 10 % 9.8 mL 0.2 mL Denaturan 5.8 mL 0.2 mL — — 4.2 g 4.0 mL t 178 Hasting 5. Pipet an equal volume of the lower con e tra i denaturi g soluti n into the r e s v o A di m f j a . u b t h r e l n y c w t ws o l u i na hv e mo fc r y l a i d t enh x c g a m b w ri lh v e n i n c r e a s b yd t i o hfne r b a T .cp s f o r mtbehyd i fn a compens ti g bar o the s rvoi lut on. 6 . A d t h pe o l y m r i z n ag e t s o a c h m b e r F. o a n c r y l a m i d ve o l u m 1f 0 mL, use 10 nium .yl aun m 7 . r e v l a n p g t d hP s o c u , i f b w l y theclamp, owing ur tobegin.I maybe c s rytoin a e h flowby sucking ol t a ng the ub with a p e ing act o . 8. Insert comb. 9. Once th gel is poured, wash t e gradi nt maker with d s il e wat r o prev nt tube lockag . 1 0 . A l ot whg e s f a r 6m 0i nG . lc s b e da t o r v n i g ha t 4 ° C after s ling w th cling f m. µ L of TEM D and 10 µ L of reshly pre a d 10% (w/v) am o- persulfat . Polymeriz ng agents ad e to the res voi ne d to be mixed 3.1 2 Casting Perp dicula Den t ri g G ad ent l The direct on of el ctroph i migrat on is per ndicula to denatur c o n e t r a i T . h o e n t p f u r i g , h e o m u s b ta r i g h n l e o i n s g e r a t d h o l f xi n e g a c d tA s- m w b l . h ( egnar tneidarg eritn eht s orca AND fo gnidaol gniwol a leg ht o ni es 3.1 Casting P r l e D naturi g G d ent l Becaus the direct on of migrat on is par l e to denatur gradient, the d e tn ha o u f r l w gp m i s e t c n b d l range ( se made since comb te h (and ther fo PCR products when loade ) ext nd a distance o h gradient. Fig. 1B 3.1 4 Casting o a t Den uri g G ad ent l The use of a gradient maker is un ec s ary since gel comp ne t can be mixed n a gl s contai er d pou by ipet ng. A multiwe co b s u ed and can be easily insert d into the gel im ed at ly after pouring withou nec s ita g djus ment o h d atur n ge. 3.2 Run i g a Perp ndicula De t ring G ad e t l 1. Switch on the heating mechanis and al ow the operating temp ra u to read 5 –60 ° C. 2. Posit n the polymeriz d gel into the el ctroph esi ap r tus. This step wil dep n on the make of ap r tus being used. Gel plates may have to be dismantled an remov d (Multiphor I sy tem, Pharm ci B otech, Up sal , Swe- ). An adjustmen to the denatur range may have to be A1 .giF .) Ap licat on f DG E t Microb al E gy 179 179 Fig. 1. g r a d i e n t m s ohg e ar lti n soh g r a d i e n t . is loade t h lower d natu con e tra i nd m grates p l with e ncr asi g d ent of a ur nt. (A) The princ le of per ndicula denaturi g el el ctrophi es . DNA is loade acros the length of the denatur (B) T hp er i n c lo fa ed n t u r i g l e c o p h r s i D .N A 180 Hasting 3. 4. 5. 6. den) or, more us al y, clamped into a sup orting frame of the el ctroph esi ap r tus (Dcode™, Bi -Rad). Clean aw y unpolymeriz d acryl mide soluti n from wel s when the comb is r e m o v w i df t h s a b n u k p e y r t i c g ( o n h s mpeb a yr f d befor th gel is at ched o t lec roph si a p r tus, if more c nv i t). Al ow the g l time o equil brat wi h t e op rating emp ratu of the ating mechanis . Load n ap ro i te volume of DNA and ye in each sample w l (us al y 10 parts PCR poducts:1 par dye). Con ect the power sup ly to the ap r tus el ctrod s and begin cur ent flow. The us al vo tage p lied acros denaturi g acyl mide g ls i 150–2 V. The time of el ctroph esi ne ds to be det rmin for ind v ual PCR fragments ( se Subheading 3. 2 ). 3. Optim za on f DG E Par met s An es tial comp ne t f d a uring el ctroph esi t de rmination f melting behavior f the DNA under invest ga o , whic enabl s the optim za n of el ctroph si par met s. Par met s ap lic b e to DG E include th range of th dena ur t g dien a the dura ion f el ctroph s i .T h e a r d t m i n e p r c a l yb u s ei t m p o r a n s t b l i ho p mal e ctroph si par met s for di e nt PCR products. De rminat o f denatur gradient range is best perfo m d by per ndicula DG E whilst det rmina o f el ctr pho si un t me s p rfo d by par l e DG E. 3. 1 Optim za on f De atur n G die t Rang Itisadv ble o ginw thabr d e u antr ge dobs v th c ara c t m e i r Dg s N A o fl u v n d p h e rt - s a io u g pendicular den turi g adient g l. The r gion f inter s in thes g l is the point f DNA lection a wh c dis ret f agm n s re b i g solved in th gel ( Fig. 2) i g n o r e ad s , u b q gn t l mr e d p a i h rn t og fe s . The d gre of ine-tu g in det rmin g the pr cise gradient range d pen s m o s tq hu lnea y b i r f d oAtx s . e m ,n a u r t gc s be optim zed to a single point (consta denaturi g gel el ctroph si , Subheading 3.1 4 . Denatur con e tra ions flanki g this region can be large y se ). 3. 2 Optim za on f DG E Ru Time The d natur g adients rmined to optim ze el ctroph si run time. Replicat sample of DNA are loade into adj cent lanes of a par l e denaturi g gradient gel with specif time intervals b w en loadi g. M x mu band resoluti n hus ob erv d f m a know ru time. Subheading 3. 1 should be us d Ap licat on f DG E t Microb al E gy 18 Fig. 2 Diagr m t c rep s nta io f per ndicula DG E showing the c ar t e r i “s gc m o d ” h a ei pt n AD N u .f cr o t a i D Nn As , melts on e t ri g he l and migr t on is l m ted. A low denatur con e tra i s, m e l t i n gd o s c u ra n dm i g t o su n i m p e d .P o n t fi l e c o n( s h a d e )i cates op imu denat r g adient whic to res lv ind ual DNA fr gments. 3.4 Casting Temp ratu D n ri g Gel This prot c l des rib a gel comp sit n ha is u t ble for gin the par met o i za n procedu f TG E. As with DG E, e T procedur n eds to be optim zed to res lv band profiles at usable c rity. Gel volume is 40 L. 1. Mix comp ne ts of gel (8% acryl mide soluti n, 1X Tris-acet buf er, 20% deion z f rma ide, 7 2. P o l y m e r i z e g e l b y t h e a d i t o n o f 4 0 pre ared 10% am onium persulfate. Insert comb and al ow gel to set for at least 60 min. 3. Switch on el ctroph esi ap r tus and set to the desir star temp ratu e. Al ow heating m ch s to reach is operat ng mperatu . 4. Posit n he polymeriz d gel into he l ctroph esi ap r tus, and l ow it o reach t opera ing t mpera u . M urea, 2% glycero ). µ L of TEM D and 40 µ L of freshly 182 Hasting 5. Clean aw y unpolymeriz d acryl mide soluti n from wel s when the comb is r e m o v w i df t h s a b n u k p e y r t i c g ( o n h s mpeb a yr f d befor th gel is at ched o t lec roph si a p r tus, if more c nv i t). 6. Load 10 w i t h e a n gr m p t ea df i n l m p e r a t u d m i n e ( 7. Visual ze b nd profiles w th ap ro i te DNA s ain ( µ L of PCR products and 2X loading ye, and el ctroph es at 10 V se Subheading 3.5 ). ). Note 3 se 3.5 Optim za on f TG E Par met s TG E par met s ha ne d to b p im zed nclu the mp ratu e ng o wv he ir lc t p s ri o c e t a u nd Am . b l p r g ae dient o begin w th ould ext n from 30 to 60 da ic vr o s b l e w u g t h e , 1 5 cfe o l nr t i ua psd h i tl se n g h k o w m p e r a t u i n c sA .f t e rl c o p h s ia n dt ing, the region of gel (and ther fo temp ra u gradient) acros whic al melting oc ur ed is term n d. R peat his gel u in the wly defin t mperatu gradient and a justing the ramp rate o give a conve i t run time. Typical , temp ra u gradients of 15 increas p hour gives a n t m of 15 h. ° C. If a r mp ate of 2 ° C/h is ° C are suitable, so a r mp rate of 1 °C 3.6 Electrob ing f Ba d Pro iles 1. Equil brate h g l in e ctroph esi buf r o 15 min. 2. T r a n s f e r b a n d p a t e r n s t o H y b o n d - N suitable el ctrotransfer ap ar tus (Transblot Cel ™, Bio-Rad; Semi-Dry E l e c t r o b ,S c h l e i r& u l ,D a s e G r m n y )b a p l i g c u r e n to f 0.5 mA/c 3. D e n a t u r e t h e t r a n s f e r d D N A b y p l a c i n g t h e m e b r a n e o n a p i e c o f 3 M Whatm n pa er soaked in denaturing solution (0.4 10 min. 4. Neutraliz by two rinse 2.5X S C (0.375 5. Expose for 45 s to ultravio e (UV) light (302 nm) to cros link the DNA fragments o he m bran . 6 . – 2 s 0 t a c o f n l Mr i d e m b g s is delay . 2 + nylon me brane (Amersham) using of gel r 45 min. NaOH, 0.6 NaCl) for M M NaCl, 0. 38 M Na citr e). ° p h r y o bi f e d C z a t n 3.7 Oligonuc e t d Prob Hy id zat on f B d Pro iles Ther are various hybrid zation prot cols av ilable using RNA and DNA probes that have be n radiolabel d or no radiolabel d. Given her is a typical example of probing a denaturing gradient gel band profile 3 2 (P u s i n ag γ A T P ) - l a b e l Dd N oA l i g o n u c l e o t i d (e active m thod). 1. Prehyb id ze blot ed me bran in blocking soluti n (10% Blocking Reag nt [Boehring Man heim], 25% 5X S PE [20X is 3.6 f o nr a d i o - N o t 5e se M NaCl, 0.2 M Na phos- Ap licat on f DG E t Microb al E gy 183 phate, 0. 2 do ecyl su fate [SD ], 20% deion z f rma ide) t 45 2 EDTA, pH 7. ], 0.1% N -lauroylsarcosine, 0. 2% sodium ° C for at le s 60 min. 32 2. Incorp ate Na M P( γ ATP) into he l g nuc eotid (10–2 pmol) using a com er- cial y v ble nuc otide k nas c ording t he manuf ct re ’s in t uc o s. 3. Remov blocking soluti n a d rinse m brane with pre-wa m d hybrid zat on soluti n (25% X S PE, 0.1% forma ide). Im ers m brane i ap rox 20 mL of resh, prewa m d hybridizat on soluti n and ad labe d olig nuc e t d probe. Hybrid ze overnight at the ap ro i te ncuba io temp ra u ( 4 . R eps mro n l vbua t id c f o l u ng yd c e t fh r io m me bran i a seri of washe using fresh, prewa m d hybrid zat on s lution g wes inh t a lkC o . ur e d ni gD Na c tA o h f vs y d w a es xh Rc q pum r o nv b l . i t g a s e y c d soluti n a d wr p me b an i cl g f m. 5. Hybrid zat on signal are det c using autor di g aphy (placing X-ray film P h o s - a p r e o i d n ) t f a x l s w u r me g b t n h a i phor Imager SF sy tem and as oci ted software ac ording to manuf ct re ’s instruc o (M lecu ar Dyn mics, Sev noaks, UK). N -lauroy s c ine, 0. 2% SD , 20% deion z se Note 6 ). 3.8 Excis on f DNA r m Gels 1 . R e m o vt ha r g f c l n i t ghb ea o dfn r sw ti u h a b l e r i t o (e.g , pi t or scalpe b d ), an pl ce i a st r le mic ofug t be. 2 . A d va o l u m e sf t r i d l e w a t r o h u b e , n ad l o tw h D N A i f u s e pas ively from the g l at 4 dent o he v lum of gel r mov d, but smal er vo um s re lt in more c n tra ed mplate for sub eq nt PCR; a ypic l vo ume is 20 3. Remov half t e vo um f water nd us a templ for PCR. 4. Notes 1 . T h v e a r i m o t f ny u c r e h s a t n m l y r k e p a t f du o s n r ing gel el ctroph esi . Considerat should be given for the techniqu to be u ( s i T e . GDd o br E , ) c a pn l t u i s o A v e a . l b ap r tus include th fol wing: Hoef r Scient f (San Fr cis o, CA) SE60 sy tem; Bio-Rad, Protean I and DCode™ sy tem ; Diagen (Düs eldorf, Germ a n T s y G ) S, E t C c e B i ; ( M Df a A y r G sl P h m c i a Biotech, Mul ip or I sy tem. 2. T h e r a r e a v a r i e t y o f m a n u f c t r e s t h a c u r e n t l y m a r k e t g r a d i e n t p o u r i n g devic s, n ludi g G bco and M.S E 3. Ther a e cur ently hre DNA stain g methods ap lic b e to denaturi g els. Choice f a p rticula method s uld be mad fter view of their adv nt ges and is vant ge . a. Ethid um bro ide s quick, nexp siv , and l ows rec v y of bands from the s ain d gel. It has relativ y low sen it v y, can give background fl resc n , a d is h g ly toxic. ° C overnight. (T e quanti y of water d is dep nµ L.) 184 Hasting b. Silver stain g s more xp nsive a d t kes long r t perfo m than e id um a u t ob (me P h y rd B i a c ) s I t 3 b (o . m y pd ie 2c a l h is age n t v d o p y r s ub ca e k g o nt d i Dc b gNa e .A ret i v d from the g l af r its p e a ni g f x t on. c. SYBR Gre n I (Molecu ar P obes, Eug ne, OR) is al o re ativ ly exp nsiv and h s lim ted f once us d. It of ers g d ensit v y, a l ck of t xic y, a n b do c k g r u s t a i n gB . dc s bo e r v u n d Ut V a s i l u m n tion a d rec v from the s ain d gel. 4. The majority of studie into microb al ecol gy b the ap lic t on f DG E or TG E have t rg ed th 16S rRNA gen . With n is r bo mal gen , th variable V3 r gion s exploit d he most becau PCR prime s a re dily av ble for the ubacteri l group of 16S rRNA gen s tha flank this equ nc , and the f r a g m e n t b p y l id a f c ro t s n ( b 2 e z 1 l 6 c -) troph es . Some studie have targe d altern iv vari ble regions of the 16S rDNA gen as probes, PCR prime s u t have th ir optimu an e li g temp ra u det rmined p rical y to pr vide ampl fic t on w h t e d sir pec fi ty (bu i s not wi h n the r mit of this chapter o detail method l gy for det rmina o f this par met of amplif c t on). A touchd wn prot c l of thermal cy ling is given h re whic s uitable for any prime pair used to gen rat GC- clamped PCR products f enaturi g lect oph r si (13). a. Heat he DNA/reaction mix to 94 DNA molecu s mplet y. Co l t 80 b. 01 ot l C c. Heat o 72 d. Denatur94 temp ra u g in for 1 m , and ext prime s at 72 f. Conti ue h rmal cy ing but drop an e li g t mpera u by 3 two cy les unti he op mu an e li g t mpera u is each d. g . p r o d - P at C ne R m h l i c k sgu . y 1e 5 a– n2 Po0 t rh f m uct yield b ctroph esi f 0.1 vol f reaction m x 1.5% ag rose l. , or the , functio al gen s (1 ) (12) . As with ol g nuc eotid s u e ° C for 5 min to denatur double-strand ° C and polymeras . Taq . nim 1 rof dl h na erut ° pm gnilae r mi p u t o ev ba C ° C and hol f r 1.5 min ° f C om 1 ri n a , e l t 0 ° aCb o v ep t i m u r a n e l i g ° C for 1.5 min ° C after v y Prime s uitable for PCR amplif c t on f a 193 nucleotid sequ nc a ros the V3 r gion f Eubacteri 16S DNA ( 4) include th fol wing: ( ' 3 - G A C G A ) d r a w C Go f ( 1 r e m A i G P C A T C - ' 5 )esr v ( 2 emirP ( '3-G TC G C A T -'5 pmalc-CG )853-14 noit s p )715-43 noit s p iloc .E iloc .E G C G C G GC -'5 '3-G C A G 5. Nonis t p c methods f probe la ing have pro d t be po ular owing to he lackofr di t v yand e forsp cial zed ont i m fac lit es.Inad it o , n o r a d i c t pv e o b a r s u l e n g t hr a if o e monly used no radi ct ve labe is the DIG System (Boehring Man heim). m T a h n e u f c t r d l a s e i m o n t v c i m y p a r o h bd f l t e c i v ity. The DIG Syst m a be us d with DNA, R or lig nuc eot d pr bes. 32 ( c d1P oA)4 m . - Ap licat on f DG E t Microb al E gy 185 6. Oligonuc e t d probes ne d to be hybrid ze to im ob l zed targe DNA at a temp ra u at whic one-half of the bound probe is rel as d from the hybrid. This o-cal ed t mpera u of dis oc at n ( grade w sh rie as p viou ly describ T d ) is det rmin by a temp ra u . (15) References 1 . F i s c h e r S,G.a n Ld m ( 1 9 8 3 D) N fA r a g m e n t ds i f r bg y n l e a s p sa ui br t o e n p s a r d it u g n a e clt os r: p n d e wc i t h melting h ory. 1579– 83. 80, Proc. Natl A d. Sci U A 2. Shef i ld, V. C ox, D. R Lerman, . S d Myers, R. (198 ) At achmen o fa4 0 - b s ep i rG + C - c hs e q u n ( G C - c l a m p )t og e n i cD N Af r a g m e n t sb y the polymerase chain reaction results in improved det c ion of single-base changes. Proc. Natl A d. Sci U A 23 – 6. 86, 3. Muyzer,G. D Wa l E C., ndUit erl n,A.G (19 3)Profil ng c mp ex 4. 5. 6. 7. 8. 9. 10. 1. m i c r po b d ua e l n t y g i s c r o p h a ne l s y i -f meras chain reaction-amplif ed gen s coding for 16S rRNA. Microb l. Liesack, W., Weyland, H., and Stackebr nd , E. (19 ) Poten ial risk of gen a m p l i f c t o b Pn y C a dR s e r m i b 1 y 6 S D N a A n l s i mo f x e d - c u l t r o f stric ba oph lic ba ter . S u z k i ,M .T a n dG o v i ,S .J ( 1 9 6 )B i a sc u e db yt m p l a e n i g a P m C p t R l h . b gi e y f r n c N 1 s A 6 S x o u 62, 625– 30. S Wt a i n c B d k . ze ( G , b 1 oD U r V9 g E 7 l F ) m i n a t f o m i c r o db va e l n s t y m a pl ie P ts C f: Ro - b r n aN Ad y si . FEMS icrob l. Rev F e R l hAs W .k oHi, m t S r a n c e b A k L E . d D r ,t m a n s ( 1 9 R7 i) b o s am ne l y ri v p s o m n e a ct i v u o fy l r me d b o f the clas A tinobac er in g as l d oi . M cr b ol gy Fer is, M. J. and Ward, D. M. (19 7) Season l distr bu on of domina t 16S r R N A - d e f i n p o u l a t i n s h o p r gm i c o b a l te x m i n db y a t u r i n g gradient l c roph esi . Ap l Enviro . M c biol. Heu r, H., Krsek, M., Baker, P., Smal , K., and Wel ingto , E. M. H. (19 7) A n a l y s i o f c t n m y e c o u n i t e sb y p c i f a m l c t i o n fg e s c o d ing 16S rRNA and gel- ctroph e ic separ tion denaturi g radients. Ap l. Enviro . M c biol. Ovreas, L., Forney, L., Da e, F. L., and Torsvik, V. (19 7) Distr bution of bacteriopl nk in mero ict lake S lenva t, as det rmin by denaturi g gradient gel el ctroph esi of PCR-amplif ed gen fragments coding for 16S rRNA. p l Enviro . M c biol. N öb e l , U . , E n g e l , B . , F e l s k , A . , S n a i d r , J . , W i e s h u b r , A . , A m a n , R . I . , Ludwig, W. and B ckh us, H. (19 6) Sequ nc h terog n i s of gen coding 16S rRNAs in el ctroph esi . Ap l Enviro . M c biol. Ap l. Enviro . 59, 695–70 . 19 – 8. 21, Microb l. E EA np v Mil r. co b 21, 213– 9. 2983– . 143, 63, 63, 32 – 41. 63, 3 67– . det c by temp ra u gradient g l Paenib c l us polym xa 178, 563 – 4 . 1375– 8 . 186 Hasting 12. Wawer, C. and Muyzer, G. (19 5) Gen tic divers ty of enviro m tal sample an lysed by denaturi g gradient gel el ctroph esi of [NiFe] hydrog nase fragments. 13. Don, R. H , Cox, P. T , Wainwr ght, B. J , Baker, K., and Mat ick, J. S (19 ) ‘Touchd wn’ PCR to cir umvent spurio prim ng during gen amplif c t on. Nuclei A ds Re . 14. Edwar s, U. Rogal , T. B ocker, H. Embe M , and Bot ger, E. C (198 ) IsolaC h a r g ce tn s i. d o u l f m a p e n t i o of a gen codi f r the 16S ibos mal RNA. 1 5 . ( 1 G 9 r A o . 4 u S ) p t D a - n s h R E d l i eM , c L B k m J f y r1 hR6 yNS bA i d s a t p ro n e b c i u oa ml n t e i f h s o g n . Ap l. Enviro M c biol. sp . in Desulphovibr 19, 2 03– 1 . 61, Ap l. Enviro M c biol. 40 8. Nuclei A ds Re . 60, 123 – 40. 17, 7843– 5 Problems f M nitor g c anism 187 31 Reporter Gene Expression for Monitoring Microorganisms in the Environment James R. Firth 1. Introduction 1. Princ ples of R rte G n Expres ion r e t o p Rn g s i a c m h d e u o t b i r s n g - w p x nois ev tac d f h rp o alucit , e f ps n vg r o sen g d t ra yl u , h o f n is erpx t o c lf a r , e t h ni w fo .tser ni oF ,elpmax ht ecn s rp fo hcus a en g yam ek na msi gro dluoc ti r , e pah s n d b c l iw a , o t f ned p h u a s eb tah n g si yl o de rpx nu at c ,s i .g r d ne f segat l n mpo ved of induct o of the SO respon to DNA dam ge gnitcel s yb gnire c t rof de n ht iova desu b nac se g r t op eht raf yb ,rev woH . t p eh sa ti gn su d a msin gro a fo ytrep uqin a noitce d w l a ot lec a otni e g r t op a ecudortni s hcaorp tsen m of a particul organism or to monit r its activ y. The repo t gen can be nac ti l e h w g fo ytil ba s eh rcn d m lp a o et c be incorp ated into the bacteri l chrom s e. Ther are a wide variety of erom ht ka s i p r luc t evah dn , b li s eg r t op ( noitac lp eh g d luf s e ro si m nagro eht ci w o n t em riv h fo si ab eht no dam si en g r t op ot eb ,d sa l r w h n g i ot eb ,d c na hw sr f t gim e .tcudorp nie s / a g h f t w Expres ion f the b s repo t gen s ca be d t c e ph noty ical , thus avoid ng the n d for m lecu ar techniqu s. Mo t repo t gen s code f r an enzym , so tha expr s ion of the gen can be eith r monit red directly or )1( se rt la n m o iv e , 2( , )3 tluser a s riape AND ro , (4) 1 elbaT From: Methods in B otechnol gy, Enviro mental Monit r ng of Bacteria Vol. 12: Edite by: C. Edwar s © Human Pres Inc., Tot wa, NJ 187 ralucit p fo e h T .) . Natur l y-oc ing Method of det c i n Source Report g n Resi tance Mode f det c ion Gen product Various pla mids Antib o c resi tance For exampl , ch oramp enicol gen s, .g amp E Z15 Pseudom nas putida Various pla mids Fluoresc nt mari e b ct ria, β -lact m se antib o c, herbic d or metal 6 5 merA Luciferas lux e.g , V. harveyi, Phot bac erium Scint l a ion 3 , 4 , 9 , 19-26 counter, lumino etry, Vibr o f scheri , sp . C D (charge- 2 Fire ly Click be tl Aequor a victor a Renil a renifo m s luc rluc gfp (jel yfish) (sea p n y) V. fischer Gre n fluoresc nt pro ein (GFP) Yel ow f u resc nt pro ein yfp E. coli, β -Gal ctosida e lacZY, Bga 38 Enzyme as y in 2 , 7 , 8 , 9 35 Catechol 2,3-dioxygenas ap ro iate 31 sub tra e, .g X-Gal, c t hol spectro h metry, visual y 10– 4 Firth β -Gluc ronidase xylE Pseudom nas putida cytome r , conf al pres nc of gusA, id TOL plasmid pW O of 15- 8 3 1 micros py Bacil us tearo h m p ilus E. coli coupled vice) camer , X-ray film, micros py fluoresc nt micros py, flow (YFP) Chrom genic 32 pres nc of Unk ow For exampl , cu i d t se Heavy m t l resi tance, Ref r nc Sel ctiv ult re in acetyl ransfe , catP Herbic d resi tance e.g , Fluoresc n 18 Table 1 Examples of Different Types of Reporter Genes Problems f M nitor g c anism 189 monit red af it on f a sub tr e a nzym ctiv . The sub tra ed normal y p duces ol r d p ucts when r po t gen xpr s ion ha t ken s p eWl ha c r n d t . i fgo m a n s , t h e n ar u m b fo c s t ae k i n o d r t Tn fh. ie s tha er po g n sha tobeu iq h nv ro me ti wh c s obe monit red. Th as to be a m ns of clear s tion f ge xpr s ion tha d i s t n g u h a e r o fi bt s m c k g u n d r o f l T ha me. s t be hig s n t v y of de cti n so ha l rge mp s a not requi d. I al y, det c ion f the r po te g n should be simpl , aking the proc s a quick p o saei n Ab yx l d. v t h uoms w more sample to be an lyzed. The det c ion method for expr s ion of the chosen gen may invol e eith r destruc iv or no destruc iv sampling and wher po ulati n ch ges a d com unity erac ons bei g stud ch aib sn o f l m( Ideal y, the m thods chosen would also al ow actions he actu l nviro me t can b x mi ed. Chaps . 19 and 2 0 )t ,hn eo d s r u c i v p t om nabe y s l . det c ion so tha inter- in situ 1.2 Ap licat ons f Rep rt G n s 1.2 Det c ion f Target O nism e hd tn, a s r i f . T u g o p y w l a r u t n s o e g h a n i t w m o r f l e c t g a h s i u n d o t n m ,c t s o -si er c to b na si yrota b l eh ni metsy r k a desu ylnom c A .noitalup e w d v m o n i s r a g tu l b h c , k ecnatsi r h gn y ac to sm in gr y A .cito b na r lu p a fo ecn s rp ht rek am l iw b nu ot . v s hT er a m i n ecnatsi rl u v h m g o f b e cn as i t yb tuo den rcs l w m i ag ehT .sc to b n f r u a eht vi c l s a p dn uow er f ht kam n i c d o a if ceps m n gro tlucif d on e b s pmi v f l t u ci o b na e s r g .de u -taer ni ytluc f d eh dna s egoht p na i r-yl um t ob snrec ,ylt R -ib tna g s el r fo k p h d y t i a sm n gro cu ne b vah sen g cnatsi er vitanre lA .tnem oriv eht o ni se g cnatsi er cito sedic br h ot na s e g idoc y lpme .sen g hcu fo l t im a r n slatem yv h dn )5( )6( ,niag tub e v in tA a r co ls e ni g r e h t s u c ri enf t o, g pm h c eht ylba orP .l ec ht niht w ruc o t egnahc rol c a esuac ,des rpx nehw , cihw eht no desab i smet y ret op es ht fo ralupo ts m iloc m u i d h en t o lw a y sr b g c di e r p x , h m t s i n a o g r ly odni-3 om rb-4 o lhc-5( laG-X gni at oc .la te foH .ne rg– ulb sraep ynol c eht , myzne ht b deva lc si YZcal edocn se g sehT . fo sen g aihc re sE detr sni ec o ,dna β es mr p esotcal dn esadi otc lagl a G - βX n e h W . ) e d i s o n a r y p t c a l g )7( demialc 190 d n a o h t e ms i g n ul o sf m a r g e p0 1f os i t n e d as l ct e do l b a t d se tm f i o w n a l c v 1 br h p g s e d m e t s ya hn b water sup ly ine by ent ric bacteria problem of the ind ge ous microfl a lso p se ing the pos ib l ty must be taken into considerat befor employing the techniqu and obvi usly the arg o nism u t be Another xample is the plasmid pW 0 of catechol 2,3-dioxygenase (C23O) tha converts catechol, whic is col rles , to hydroxymuconic semialdehyde, whic is yel ow, and, al ows cel s car ying the gen to be dist nguished. The both Gram-positive organisms, e.g., numerous Gram-negative organism the studies on Gram-negative speci s, the broad host range, no c njugative IncQ plasmid pKT230. The gen was expres d from either the under the control of the temperature-sensit ve lambda repres or cI857, whical owsexpres ionat37 s w i t c h e do f u n t i ld e t c i o n sr e q u i r e d ,t h u sr e d u c i n gt h em t a b o l i cb u r d e n caused by the igh lev l of expres ion f used in I cP onjugative plasmids sy tems could be mployed to det c recombina t tions a low as 10 The other g oup f repo t r gen s are th biolum nesc t and fluoresc nt repo t r gen s. Thes include th luciferas nd the gr n a d yel ow fluor e s c n pt r o e i n s G, F P when expr s ed, produce light ways ( se Note 1 (with e xc ption f the po ulations of micro ganism producing the same respon e. Even the s y t eiv m ru ys f i lno a s f dr e h w a t e rs ,i n ct h e o c u n r a s t l m r ay e i o n g s m T r h a. e dn o ig f r e bn it o l u m n e s c t repo t r gen s also produce light of dif er nt wavel ngths, so tha two or more can be us d in co junctio dist nguish betw n dif er nt organism or metabolic proces . One of the major p oblems with using GFP used to be its hig stabil ty, whic meant tha the protein remain d with n the cel long after h gen had be n switched of , a problem wh n trying to investig aw thf e c om ri sg hw t p a cnr i u lp ar o c e s T hp .ir o b l n ea msw be n overc m by the d velopm nt of m re unstable GFPs b de g r a e md o r a s i l by p r o t e a s w i t h n ce l g i v n t h pe r o i han l f Firth )8( s .i h T used to monit r the col nizat on of biof lms in a drink g . Again w th is y tem h r is the (9) gen s, and this lac -minus. lac gen , whic is found atural y on the TOL xylE . The gen codes for the enzyme Pseudom nas putida sy tem has be n used in xylE (10) Streptomyces lividans , 12) (1 gen was cloned into the xylE p or p L prom ter of the lambda bacteriophage R ° Cbutnoa30 ° C.Thisal owstheg ntobe . The sy tem has ince b en xylE (13) . Morgan et al. (14) found tha such at con e tra- P. putida 3 -1 cel s mL of lakewater. a n Yd F P (15– 7) , and as reporte /marke gen . In ( T a b l 1e ) T. h s re p o t e gr n s , (18) tha can then be visual zed in a number of ). Since the source of thes gen s i eukaryotic organism lux sy tem), ther is no pr blem with background lux iw .nh c t V i b r s op (19), whic an Problems f M nitor g c anism 19 e f n io l m n o s i m t l u d a b v r g n f oe s h c mt i u v )2 -noriv e ht ni sm nagro cim gn tce d fo htem l von d a gnitser nA . c i f e p s - i c h t wn o i c u j n is e gr t o p e n c s i m u l o be s t i n m se uriv ecnis , hci w ,en g ret op r eht niat oc ot deif om era s egahp es hT .egahp e h t s c f n i e g a h p t n e h w , r v e o H . d s e r p x t o n s i , e t o r p zi s h t n y o d tsoh l ec dna stre ni st AND otni eht soh ,em r c eht r ope n g si lairetc b fo rebmun a tce d ot desu n b sah i T .pu sthgil ec ht dna es rpx gnidulc sei p utes M re oh t s i n a m f gc u dtil e s o r n k a h b w –02( 32( airets L , )42 , dna .)52( airetc bo yM 1.2 Report G n Fusio t De c G n Expres io Another way in wh c repo t r gen s are us d i n ge fusion whic the repo t r gen is linked to a gen of inter s . Expres ion of the repo t r gen is a umed to refl ct he time of expr s ion f the fused g ne product. This kind of ap licat on is uited to he biolum nesc t repo t r gen s. For example, toluen dioxygenas enzym in to the gen encoding the enzyme, betw n the ime of expr s ion but also betw n the activ y of the nzyme and the amount f biolum nesc e. Tak n furthe , such an p roach n be used to m nitor he con e tra ions f various p l utan s i the nviro ment. H eailt.z r tiga e bioav l bi ty of uel hydroca b ns. Si ce b oluminesc e ap red at he same time as enzym expr s ion, whic , in turn, was pro tional to the con e tra ion f pol utan , biolum nesc e showed a linear co relation with e amount f hydroca b n pol ution soluti n. gen fusion have be n used to monit r the xpres ion of the lux whic (27) ua s e d . Her the (26) P. putida lux not only gen was fu ed al owed a cor elation n fa op rh t lc e n a b i o tv s m - n a h G -f lu s xi o n 1.2 3 Report G n –P om ter Fusi n to M ni r E v o mental C di ons R e c n t l hy r bad s m o v we f u i rn g p t e hos struc al gen s of inter s a d inste fu ing the r po directly o the prom o t e r g i n f h a t e T . i s p r o a c h b e u n iw d v r t y a p o fl i c butn hes I d r . v m o c p r a t ih ne g regulato y gen s ca be ins rted o det c a number of hydroca b n pol utan s The GFP repo t sy tem has be n used to replac the coat protein gen of pota virus X o tha w en th virus nfect d a pot cel , GFP was ynthes i z et doh w c l sab mi en f c t d coat protein tha had be n replac d was es ntial for cel -to move nt. The luxAB yb gnisuf meht o eht r om p f xylS xylR gen s of the TOL plasmid into whic various repo t (28 (30) c y a n o b i t re h m d s u b e n h a v g s IAbsp , a en g th uo eb d cn ulf i yb T .h ia sl o w et d , 29) . 192 Firth smhty r naid cr ) 1 3 ( r- o p e t n c s e i m u l o b t d e i m l t o n e r a s n o i u f r e t o m P . .la te y r h F .sen g r t )23( eht d su retom p fo a en g lbis op er f eht noi cud rp fo nix talf ni suvalf dluow tah sn i c red u g t op eh f n is rpx ot dael sihT . en g c atsi r o b n eh ,yaw r lim s nI . ixot eh f noitcud rp eh s a Ptac fo en g ixot-ahpl e t fo n iger tom p eht o desuf n b sah sneg irf p os tah ecn i r l pm o s y n der f i u - oc nixot eh f cud rp m l w a s Reporte gen s can also be used to demonstra e activ ty of cel s. For example, Mycobacterium tuberculosi l u c i f e r a s g e n h a v eb nu s e dt o v a l u t e h a c t i v e so fan u m b e ro fa n t i microb al comp unds (34) . The more f ectiv the comp und, the l s light i se m t df r o mt h ec l s .T h i sa l ot h eb a s i o ft h ec m r c i a l y v i l a b e Microt x as y (35) . A sim lar p oach s al o be n ap lied but sing the lacZ repo t r sy tem (36) . Report gen s can lso be us d to m nitor he f ect of micro ganism w i t h o u ge n a c t u l by e i n pg r s e n it d e h c l T. pe a t h o g e n i c f e t of Erwin a mylov ra on tobac or pear plants was investiga ed by introducing the luciferas not i o the bacterial ce s but in o the plant cel s T ph ae t o g n ei fc w ta hs me on i t r fbe oyd l w i tn rhg e d u c t i lo fn g h t being mit ed by the plant cel s a they w re kil ed. The tak home s age from al of this tha er is no perf ct eport gen tha c be us d to inve ga l or nism a d l et bo ic pr es in al enviro m ts. Each repo t gen has its own lim ta ons, be they the n a s t p e u r l -o x f c gb h m i v w , d ral y oc ur ing micro gan s car ying the gen , or the ne d for relativ y hig metabol c iv ty o al w det c ion f xpres ion. H w ver, by using a range of the repo t gen s av il b e, we can gather enorm us amounts of informat b u he avior f m c ganis the nviro m t T eh x a m p dl e s c r i b he d u s t p l a m i Ld V 1 0 ( 3 t h se u r v i a ol f P a. e r u g i n o s a i n t r o d u c e i n t so r i l ae n d o s t e r i l a k e w t r micro sm . In this plasmid the expr s ed from the p R prom ter of the bacteriophage lambd . The plasmid a cl os n t ki a m y c s i tn rd- e p o m y c i n - r e s t a gn ec d f o i rs t n a l sel ction method. 2. Materials 2.1 Labeling P. aeruginosa with e 1. 2. 3. P. aeruginos E. coli E. coli recip nt s rai . don r st ai c r y ng the plasmid LV10 3. CA60 car ying the mobil zat n p smid NJ50 . β eht o d suf en g r t op esadin uc lgsul igrep A muid rtsolC )3 ( and M. bovis . expres ing the luc (37) F i 1g . xylE gen is temp rature regulated and xylEReport Gn mt o ) n i r . Problems f M nitor g c anism 193 Fig. 1. Plasmid pLV10 3 car ying the gen s for kan mycin resi tance (Km), streptomycin (Sm), and the enzym s I(xh), Xho 4. Sim ons c tra e g . 5. Kan myci sto k lu i n (50 mg/ L). 2. Inoculati f M cro sm 1. 2. Fresh, no t rile ak w t r. 3. Autoclaved k w t r. 4. Spectro h me r. 5. OD 6. Steril d s e wat r. 7. Desk-top c n rifuge. 2.3 Extrac ion d Det c ion of Marked ic o gan sm fro Mic sm 1. Steril p t. 2. Steril 1 mL Ep endorf tub s. 3. 1% (w/v) cate hol s uti n. 4. Volati z on chamber nd os l. 5. Fume ho d. 6. Glas pre d . reporte gen . xylE I(s), Sma car ying the r po gen . P. aeruginos 50 vs. col ny f rmi g un ts calibr t on cu ve. Hin dI (H), and *Cut ing site for the rest ic on Xba I(Xb) are shown 194 Firth 7. Pot f e hanol. 8. Bunse b r . 3. Methods 3.1 Labeling th Or a ism w th e xylE R porte G n fo lavi rus eht gnirot m sevlo ni er h debircs d tnemir px ehT d eh lt i bw a l iw er h d bircse lo t rp eh fo selpicn r eht yl ai nes tub ,es ri ht fo sliated ( smet y noi cet d sab-erutl c a rof em s ht eb asonigurea .P s r m l e y t n i. w a o g vh p O r Elyx es 1 . G r ou wpv e n i g hc t l u r eo sf c i p n t .) 1 etoN , Ec .o l i Pa .e r u g i n o s don r biot c ( C A 6 0a ,nt dh e strain nutrie broth wi the ap ro i te con tra i of anti- E. coli se 2 . 1 P0 l a c e ). Note 2 µ e o d a r n c f g u h L pt i s m , l e contai g o ntib c and l ow t dry. o 3. Incubate h pl at 30 C overnight. 3.2 Scre ni g of Plasm d Tr n fe 1. water.dislofmL05nghbcpRu 2. Make 10x dilut on seri f the su p n io s g teril d s e wat r. 3 . S p r e a 0 d . m 1o L f c h i l u t n s o e p a r t l o sS f i m n c t r a e g (38) contai g 50 µ g/mL kan yci , ( se o 4. Incubate h pl s at 30 5 . c od lus p a n b yi t e r c o l n 3 ia 0 p s w t x h e S l c t h a p e r s bo u n d e gay r c o l t i n a e Sw m o s g pr l a t e contai g 50 ). Note 3 C overnight. µ g/mL of kan yci d streak ou . o 6. Incubate o f ach dupli te a 30 7. Place th pla esin fum c pboard n sp yev nl witha g covering f 1 c% a t e h o sl u i n I. tf h pe l a s m i d b tn k e u sp c f l by t h re c i p ient those gr wn at 37 o t h ce o l n i gs r w a 3t 0 P a. e r u g i n o s , o o C and o e t 37 C overnight. wC i l r e m a cn i o l r w, h e C wil turn a b ight yel ow. 3. As e m nt of Plasmid St b l y 1 . R e m o sv a i n g l b c t e r a o n f y S m i a s g p r l t e n i d o c u a r p licate n o 10 mL f nutrie b oth. 2. Incubate for 24 h at 30 3 . t r a nh 5s A 0f 2 e 4 grow f a urthe 24 at 30 4. Take a furthe 1 mL sample from each flask and make a 10x dilut on seri of each s mple. 5. P l a t e e a c h d i l u t i o n s e r i e s o n t o n u t r i e n t a g r w i t h a n d w i t h o u t 5 0 kan mycin. 6. 03 ta hginrevo setalp es ht e abucnI 7. e(prviouslydcbahtwS o C. µ f r n i l e c u a s t h o k mb L d ° C. µ L/mL 24 ta h 2 reht uf o a yb dewol f C o 3.2,Subheading (C es 4 etoN step7 .) ). Problems f M nitor g c anism 195 8. Cho se plates with betw n 30 and 30 col nies both with and withou the kan mycin for each replicate and count the number of yel ow col nies and white col nies on each ( ). Note 5 se 3.4 Set ing up of Steril and No -Steril M c o sm 1. mL50inbatches 6xodivanwterlk ofsamp Tke (flaskconi ). 6Note se 2. Autoclave hr of t e lask to eril z th wa er. Take an overnight cult re of the a np de l t hc e l b sy centrifuge. 3. car ying the marke plasmid P. aeruginosa mv 1Lo l u ef m so3ia9 r nt0 centrifug g d iae n s k t o p 4 . s t Pu o h p f e r n a md i L l s 1 c t e h w r . 5 . R e p t ca h n r i f u g t a o n e d s p i t h n r e m o s v a c t y r - e of nutrie s o the micro s . 6. Take a reading of the optical density at 5 0 nm of the cel su pen io using a s p e c t r o h m e a n l d cr u t h s O i D y n g ing U (cfu) alibr t on cu ve ( 7. .ytisned l c riuqe ht vig o n s ep u l c eht iw msoc r i h ae t luconI c v o s l f. n r y m - 50 se ). Note 7 3.5 Extrac ion d Det c ion f xylE Car ing O sm 1 . v r o m e l L A hu f1 t 2 i 4 a c s m e p t i c a l u s y , n g steril p ts ( 2. Make 10x dilut on seri f ach s mple in t r le dis water. 3. P l a t e o u t a n d i n c u b a t e t h e s a m p l e s o n n u t r i e n t a g r c o n t a i n g 5 0 kan myci overn ght a 30 4 . Spray the l s wi the ca ol s uti n a de cr b p viously ( 7item3.2, 5. Cho se plates with ap rox. 30– counts ( se 6t .n e m i r p x e h t f o n i t a r u d e h t r o f s e m i t g n l p m a s d e r i u q e h t a s e c o r p s i h t a e p R se ). Note 8 µ L/m o o C and the for a u the 2 a 4 C. Subheading ). xylE Note 9 posit ve col nies and make ac ur te ). 4. Notes 1 . u w s a ec Ai l n g t b r d h i q u f e l s , o r g c a n h i d m e r p o t gen s ca lso be visual zed n cou ted sing other c nol gies uch as flow c (y t o m e r coupled vice am r s i sn t u dark, becom s expos d by the light being emit d by the c l s. Thes dif er nt visual z t on me h ds av their own adv t ges and i v tages. 2. iloc .E inoragwepLV103,lsmidthcaryngED8654ei 50contaigbrhue may conetri s ad ntiboc The ls. t wihn maed s pli the -irets l f eb dluohs cit b nA .desu gni b era sdim lp hc w no g id ep yrav 5 Chap. ms ic ( a rnl o ef g p y ) , 17 Chap. (2 ) c h a r g e -n d ) . Fluoresc nt repo t gen s ca lso be monit red b uy s i n Xg - r a f l m w, h i c e pn l a od v r x t h se a m p l i n ron d eht dna 0 5JNp dimsal ev t gujnoc eht gniyr ac 06AC iloc .E µ Lm g –1 ensurtoplciakmyf s ,niart 196 Firth o lized and ade to broth or agr after autoclving. Agar should be below 60 befor ading tbocs. An idcato h te agr is col enugh is when t botlecanhdmfryiwusgv. 3. Sim ons citra e ag r 4. 5. 6. 7. 8. 9. source. Since c ao n ,l y also inc rpo ated in o the pla s, only th se up the LV10 3 plasmid houl be pr s nt. Since the expr s ion of the repo t gen plasmid LV103, no exprsi cu whl te organism w at 30 plasmid the cong plas r cute h gn, te xprs o de in Thrfo, 42atincubedlsryg Plasmid tby imporan s the rpo gn must be aind wth e -detcbolsthefrodingeatsubqyinhrdeacl ted. Sabily s xpr the cnag of tl number of cs (th al number of clnies) tha sow exprin of the echol.atwisprydnmv The usef ln of pres nt i the natur l bacteri l po ulati n. It is ther fo es ntial o first pla e u s tn a hi moe pf l c dr s a w on i t m yh c l ther a no tural y oc ur ing A calibr t on cu ve of OD d i l u t o n s fac u r e dt k i n g h O D 10x dilut on seri , whic is spread ont ag r plates and incubated overnight. A f t c e o r u n i g l t hesp o n i bc l r a e t O D h col nies, . cel s in the or ginal s mp e. It may be tha 1 mL of water f om the micro sm ay not contai any of the introduce cel s tha may stil be pres nt but at lower con e tra ions. Under such cir umstances large vols can be extrac ed and con e tra ed up by cent r i f u g a t i o bn e f r s u p e n d i tg h pe l ot cf e l is an m l e vr o u m e wf a t r . Thiscon e tra ionstepmustbe ak ni toc nsiderationwhe quantify ngthe targe organism. One problem often encou t r d when trying to count organism car ying the r e p o t g la n s, p e c i wl hy t n a pr e s i o uw m b r t h , a p l a t e hs k nm i c ur y oU f l g b a w . p e d c n t h y wil help to sel ct ag inst hes background cel s, althoug natur l kan myci resi tanc may stil ead to swamping of the plates. An ad e adv nt ge of the plasmid pLV10 3 is tha it also car ies a gen for strep omycin resi tanc and se x lt ra u pc n i b o w z fh d e , s u cr number of ackg und el s growin the pla s. P. aeruginos s h o a g tp ub r l e n d w S i . k c m y s n a Pe .r u g i n o s Acknowledgment This work was funde by the Natur l Environme t Res arch Council, Swindo , UK. is a mediu tha contai s citra e as the sole carbon (38) can ot u il ze citra e s a c rbon s urce and E.coli cel s tha ve tak n P. aeruginos xylE is temp ra u regulat d in the ° C. ° cathol.wispryng2fC xylE xylE gen. I practie ny cels as a marke gen is dep n t on the gen not being xylE 50 car ying o a sm pre nt. vs. col ny f rmi g un ts i con tru ed by making 50 .E a c hd i l u t o n s e dt m a k 50 n u a tm ob e f r C Problems f M nitor g c anism 197 References 1 . S r i k a n t h T, . K l p a c A L, o r e n z W . T s a i L, K u g h l n L, A. G o r m a A Ja .S n ,o d ( lR D1 9 T 6s hp )e y a sen it v biolum nesc t repo t for dif er nt al gen expr s ion in albic ns. J Bact l u c i f e r as v R e n i lr a f o m s Candi 12 – 9. 178, 2 . 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Infect Dis. 39, 209. Char cte iz ng M ro a ism n the E viro m nt 201 41 Characterizing Microorganisms in the Environment by Fatty Acid Analysis Ian P. Thompson, Mark J. Bailey, and Andrew K. Lilley 1. Introduction 1. Fat y Acid Me h l Est r (FAM ) nalysi as n Ecol gi a T D e t r m i n g h a x o m i c p s t o n ,b i m a dp h y s o l g i c a t u s of micr b al s emblag is t l one f the gr at s chal eng s facing m robial ecol gist . Ther are many reason why as e m nt of microbes in the enviro m t is so deman i g, not least their number, divers ty, and lim ted p e rU u m on k s l a i d z t y h f . c g , n s yields t le or n i format n co erni g the p ylogen tic af li t on r ecol o g ir c f a n e s C m . q u t a l y , h o d i g r e b c s v a t p n vide an indication of biomas , they do not al ow the investigator to distinguish among the many microbial po ulations pres nt in samples. Clas ical ap ro ches tha util ze enrichment methods for the isolati n of micro gan s from the nviro me t c n i ue to pr vide alu b e informat i o n b c h e m a l ,t x o n i c da u t e o l g i c s u d e .T h p r i m a yl ta ions to such ap ro ches are those of no cult rabi y (the active cel u ar c o m p n e t h a bg r o wi n l e a t y r i f c m le d a ) ,nt h c h p a r o f b l t i e d m n z s g y i r c e a l n o u -v m f b t s lates nec s ary to gain ins ght into the po ulati n ecol gy and com unity divers ty of any but the simple t of habit s. Furthe mo , thes ap ro ches ra elyp ovid nf rmat o ic b al s em g l a tr e c h n i q u ms y l tp ro e v m f h s le i t a o n d h e i r p l c a e n m d a v i l “ c s r o tp w h b f , ” and cel u ar locati n and activ y in targe d com unit es From: ins tu .Modernm l cu- (1) Methods in B otechnol gy, Enviro mental Monit r ng of Bacteria Vol. 12: Edite by: C. Edwar s © Human Pres Inc., Tot wa, NJ 201 . Howev r, as 20 t e a w cl ir h b nm qg o u s d y , f a t i n amplif cation of gen tic material, whic impose inevitable sel ction for the sequ nc an lyzed in the col e t d “rep s nta iv ” sample . Althoug this can, o me xt n , b co r l ed, it s far om i ple t rou in ly e at a b u n dt x co e r m il v s t y a c i d n u l e dev lopm nts, uch as den turi g adient g l e ctroph si of p lymeras c o r Dmw (e N h Pa Au C nl Rt i ) s - y p f d c (3) , are b gin to reduc h s if cult es. Cur ent f or s cu ing o the mol cu ar biol gy f nuclei a ds m y, to some ext n , undervalu the enorm us amount of informati n tha can be c o n es tl x h i u a d m r T b f y v g . of thes dat set provides a combined un ersta di g of b th axon my and microb al e gy. Anal si of wh e c l s, .g by mas - pectro y (MS)p y r o l s c i ew a , n t u p sc h i d o g l y a n r , t e i s dp f a m ig -r o uc p s ,b dl t n h e f o r d a tc v m il up b e lies, g n ra peci s, nd ub peci s. A ment of h c l wa omp sit n b a c o t f u e d s r i n g a yc hl t o /s i Gd r m n p e f ’ x e m x f tp i hlcr ao s d n- e w t a i bial t x . One group f cel ar onsti ue , h lip ds, have b n wid ly a ef ctiv ly used to pr vide taxon mic for ati n for ind v ual iso te and whole micr b a s em l g . Th wealt of in rmat lip ds rov e ab ut m i c r o g a n s f , p ht y e l i c d ab s o m f n , t h e toge h r wi t c nol gi adv cem nts i a lyze b s d on ga chr m to raphy (GC) and computeriz d dat handli g, has sign f ca tly improved the understa i g of env r m tal icrob gy. edivorp t ub ,sdica yt af l iborc m fo stcep a l revoc t er h de n t i on si tI gnil forp dica yt f desu ylnom c tsom eht fo wt oh n thgisn lacit rp emos ,)EMAF( sret lyhtem dica yt f dna )AFLP( dica yt f dip loh s p , ehcaorp -noriv e htn oi c ufdnaytisrev dlaiborc mf ydutseh o d ilp ane b v h . t n e mA F L P s a h n e d y blt , i o s wp a x g u r c ie m o f h t u reda eht ,euqinhc t siht fo noitac lp dna seigol d htem ,selpicn r eht fo sliated .la te ihW der f si hcaorp eht gnikam yb stne opm c di a yt f ralu ec fo yduts eh d zino tul ver n . I y t i v s n e f o t l u h i d w e p g s n b m o y rc e s d u n i a t e h o l b a i v fo n itac lp dna esu ht rof sehcaorp deniltuo evah w ,snoitce gniwol f eht treh o i w d e t s a r n o c e v a h , t i r p o a e r h w , d n a l o t c i g l o e n a s E M A F sdohtem f ip l .s y an owT reht s ca p o di l s y an ucof eht roF .e h d r isnoc ylfe b no era hci w ,seno uq di rp na sdip l ra o fo n itac uq d ,noitac f e r x eht rof sd htem no sliated r h uf ralop sdi na dio erp s , n uq eht r da luohs t n c eh sw iv r fo ikuz S .la te Thomps n et al. (2) )4( sah isylan EMAF detamo u i es fo n itcudor ni ehT . )5( ikuz S dna t g moK dna )6( . rN e cv n t h l s ,. Char cte iz ng M ro a ism n the E viro m nt 203 1.2 Isopren id Qu o s Isoprenoid quino es are located in the cytoplasmic me brane of most p r e o l k i a cn t w y h s , v Te r m i a . t a x o n mu is ce f lp r v d t o n g e a l y i s q u p r n o d t i marke s s u br cR (lNf aAo d i t e h mn g s r Prote bac i . (5) I s o p . r e q n u i w bd h a t v xl y s o m c α , β , δ and , 1.3 Lip ds Lip ds are amphi at c molecu s, contai ng both a hydrophil c and a h y d r o p m b i c e t n , l u h d s p o i g a n y c l rd es , t bacteri l me bran s. Vari t on i the polar hydrop bic head h s al o be n used as a taxon mic marke , althoug predomina tly for dist ngu h the actinomycet s a c i d s ,p o l a r ,m y c o l i ca d s ,a n dt h ei s o p r e n o i dq u n o e s .H r ew i l c o n centra e on m y ui t c s h l o e a p f n r d q t i a d f n - g fer nt ax (7 , 8) . Ther are four main clas e of lip d: long-chain fat y the fa y cids an pol r ip ds; other s udie hav confirmed th . (9 , 10) 1.4 Long-Chai F t y Ac ds Carboxylic a ds with long hydroca b n chains re th basic onsti uents of importan lip ds including the glycerid s. Bacterial ip ds range in chain length from simple 2-carbon atom back ones to hose, as in the case of the m y c o a l i t d h s ,n c o a9 vr 0 eb t sT m x . o n i c a l f y t , d s in the range of C d i v e r s a nmogifec r g a n i s m T h. e c l u a snrtd c u r fa l t cy i d rs e dist nc from l wer ight comp unds a ociated with metabolism tha c n also provide taxon mic information, but they are not consider d in this revi w. Most fat y acids are located in the cytoplasmic me brane as consti uents of p lar ip ds an glyco ip ds, in wh c they form the int gral p rt of the lip d layer. In Gram-negative bacteria, fat y acids are also pres nt in the ou r me b anes part of he lipo lysac h ride. Fat y cids are p o erly named by the basic struc e of their carbon skel ton, i.e , the number and posit n of d uble onds, in the carbon chain, d the pr senc of unct i o n ga rl u p s double bonds and sub ti u ed groups as ociated with their hig ly regulated synthesi make them valu b e biomarke s. For example, hydrox lated fat y acids having the –OH group in eith r posit n 2 or 3 can be found in most Gram-negative solates. Fat y cids w th branched alky chains predominate in Gram-posit ve g n ra. 1 (5) D i. v e r s fit any c sd u a h li en g t ph o, s i t hfn e –C 24 provide th great s information a d re p sent i a γ of ) 204 Thomps n et al. 1 . 5 P o l a r iL p d s Amphi at c ol r ip ds con t u e h major p t f bac eri l m b anes. They comprise a hydrop bic head group linked to two hydrop bic fat y a c Vi h d y p rs o t . f m l e n k g b u the fa y cids but, nev r h l s , p ovide g n ral t xo mic nf r at o . 1.6 Nomenclatur Fat y acids are designat ac ording to the number of carbon atoms, the number of d uble onds, and the posit n of the double ond relativ to he methyl r inus ( i (n d c a b t y e branchi g, and br- ind cates an unk own methyl branchi g posit on. For exampl , 10Me ind cates a methyl group on the t n h carbon atom from the carboxyl end of the mol cu and cy- ref s to cy l pr ane f t y acids (e.g , c1 y7 : 0 )T .hp eo s i t a nf d r x yg l o u ps m b e f dr t hc a o x y ln of the fat y acid, with OH as a prefix (e.g , 3-OH17:0; 3-hydroxy-heptadecano t ) ω ) of the m l cu . The onfigurat of he d ubl on is ( ta n d) cis (4 , 1 ) trans i n p d T r ah e c ) f . t x s o - . 1.7 Ap licat ons d E vironme tal M i or ng 1.7 Chemotax n y d Biomas The w alt of in rmat o n the comp sit n of micr b al f t y acids h be n wid ly us b micro l g st o impr ve und sta i g of c m un ty comp sit n a d yn mics the nviro me t. This a be n achi ved us ng two dis nc ve but omple n ary p o ches: a ing com u ty posit on by extrac ion of the to al fat y acid conte of enviro m tal sample andusi g t refa y cids b omarke fsp ci o ulat ns, dculturing d v ual iso te nd char te iz on by the fa cid onte . PLFA an lysi has be n most com nly and ef ctiv ly used to examin microb al m unity s r c u e in whol e vir nm tal s p e , inc d f erent sub of a c m unity have d f r nt PLFA pa erns c h a Pr L o F m Atp e ig s d u bn l v f a c t h e x p l o i s pat erns a d h s be n sculpted in a bro d ange of habit s ( d e t r m ci s on g a u p f d b y e t h r , es ntial to consider the enviro m t from whic the sample was ret i v d (2 , 30) . For insta ce, a sedim nt wo functi al groups (defin by Findla and Dob s tics) an be dist ngu he by PLFA an lysi . F r t, he ukaryotes, c mpo ed of PLFA of both anim l and plant or alg orig n. Thes are dist ngu hable dep n i g o the p si n of the irs un at io be ng th r i e mals) or ω3 . This ap ro c (29) ). When Table 1 as uites of micro gan s sharing biochem al char te is- [2] ω6 (plant) osi f the PLFA. T second fu ti al group c m - (ani- Char cte iz ng M ro a ism n the E viro m nt 205 Table 1 Application of PLFA for Monitoring Microbial Communities in the Environment Microb al m un ty ves iga d Ref r nc s Microb al m s and com u ity n so l Soil bacter spon e t r s nce of r ts Biomas rbu c la soi myc r h zal fungi Biomas nd com u ity s r c u e, biof lms Impact on s il m crob a m unit es of p l uti n s re Impact of l nd ma ge nt prac i es Microb al m s , co unity s r c u e, and physiol g c ta e (5 (57) (58) (59) (1 (1 (68–72) , 56) (73 (16 , 74) , 75 , 76) (13 , 7 , 78) , 56 , 59 , 60– 2) , 59 , 63– 7) in de p-s a diments Biomas nd com u ity s r c u e in rocks Monit r g specif o ulati n (s fate-r ducing ba ter , methano r p ic ba ter ) in so l a d e im nts Quality s rance m thods f ampling d storage m h ds (soil and e im ts) posed of proka y tes can be furthe div e into bacteri util z ng a erobic desatur pathw y, gen ra such as PLFAs. Some ar ven mo sp cif marke s nd hig ly a nostic of specif groups. For insta ce, type I methan -oxid z g bacteri form m o n e u Pwt Lihs F a cA r y p o n l t 1 i 8 sa: u c ,h (27) . Unlike most other bacteri , this group contai more of the 18-carbon 1p 6a n -t o m c (h r e i 8 b : l 0 y ) , f A u g s. t c a les p cif lev , gen ral t ends i PLFA can be o s rved with n specif functio al gr ups. Straigh -c n PLFAs tend o be pr s nt i great qu n it i e s nb a c r t h e1 6 - c a b o nm i e t y( 1 6 : 0 ) ,w h r e a sm i c o u k r y t e sc n tain great amounts of the 18-carbon form. Some dist nc ve groups share sim lar fat y acid consti ue , and, consequ tly, interp a ion of profiles mustbe nd r ak withc e nd habit c r e ist c nm d.For insta ce, rminal y b ched satur PLFAs are com n t Gra -posit ve bacteria but are also pres nt in some Gram-negative an erobic bacteria. Branched mo n ic PLFA are com n i the an robic a b e r c As o t l m i h u .n g f a d e - r b c u t i n g PLFA profiles d not rev al sp cie -l v informat directly, his ap ro ch provides a f ng rp i t of m cr bial d ve s ty pr n . The main p l cation f FAME an lysi ha be n i the id nt f ca o nd elucidat on f he taxon mic relat onship m g cult res of mic rgan s Bacil us Desulfobact r -type Gram-posit ve bacteri and ev n specif , ac ording to the pres nc of specif marke ω 8c Desulfovibr -type 206 Thomps n et al. (17) . Improve nts in GC techniqu s, computeriza on, and more ef ic nt g e n r w t a h i o f xp F A mcM E s e v d rn of instrume -bas d sy tem , for rapid char te iz on of micro gan sm . FAME an lysi of bacteri l u ar f t y acids ext nsiv ly u ed as ither a prima y o an dju ctive m ans for ident f ca o f clin a d phyto a genic bacteri m i c r o g a n p s t x o cm hi a r t e can be lyz d quanti vely o pr ide m o tan x mic nfor at the sp ci and sub peci l ve s the microb al dentif c o sy tem (MIS) has be n com ly ap ied to b cteria, it has also be n used to dif er ntia e yeast ( 3 8 , 3 9 ) s p ,i r o c h e t m o r e t h c w in g u l y b t o p r v g eh da u i n s , r e sd fti a gc - h o n b y l sm i a r - u n cet h o d v ment comparison of to al extrac ed DNA or PCR amplif cation of target r e g i o nM sI .S - F Aa E l y h i b e o p n ts d w c l m u i c o m s p u tf l b ie x a r h rial com un t es In ad it on to provid ng information on com unity comp sit on and d y n a m i c s P, L F A f u l m a n oy t h ce r i q u e td bo f c i v h m a l m a r k e so f i c b a l m s .T h i n c l u d e st r i v a ld s t r b u i o n h e cel u ar comp ne ts of intac el s, but short esid nc time n detri al po ls a f t d e r g I h n . l ,a yx p e s r t d i v c l o y n a e t w i s h c o m u tn hi r y g f weTc l o .P aL n F s A i d r to be f pr d mina tly b c eria o g n: 15 0, i16:0, and 1 . It is wel establi h d tha fat y acid comp sit n of (31– ) ta Fhn dA M E (34) (35 , 36) . Althoug FAME an lysi ut l zing , mycor hizal fungi (37) (40 ai nr d e s c v t u , 41) (42) (43) (45) ( Table 2 soil, m t Iaoh n fes . y u d - mb oa dc net l - (4 ) ). ω 9. 1.7 2 Physiol g ca St us The fat y cid omp ne ts of he ind v ual me br n lip ds are not fixed b uv ta rw yi n h o s lt a u ne d v i r o m t ac l n d i o sB .y u nt gh e changes of ph li d fat y cid prof les, p cial y the pr s nc of ertain PLFA marke s, it s po ible t as e th p ysiol g c ta us of the microb al com unity. For exampl , changes typical found in PLFA profiles when G r a m - n e g b t i c v s r a o ul fd h n e to unsa r ed f t y acids to cis - mon e ic unsat r ed fat y acid. By contras , negli b changes in PLFA profiles are observ d in Gram-posit ve bacteri p r o t h e ia n l d g v s ap te i c orf d s l h v n a g e of polyβ -hydrox alk n ic acid (a storage lip d) in bacteri PLFA, provides a me sur of nutri o al-phys gical st u c h a P v r H n A ui / tl e L b F s o wc k y r a t i o sb e l w1 .B yc n t r a s p o l - (15 , 46 , 47) , and i creas n th ra io f the trans (48) . The observ d relativ to (49) (4) β - h y d r o x a l k n i c dP H A / L F r a t i o s f . Bacteri n - Char cte iz ng M ro a ism n the E viro m nt 207 Table 2. Application of FAME Analysis for Monitoring and Assessing Individual Isolates and Microbial Communities in the Environmenta Microb al m un ty ves iga d Ref r nc s Bacteri l d v s ty inaqu c e osy t m Char cte iz on f xe biot c degra in b cteria Char cte iz on f myc r hizal fung Phytosp er bact i l po u ati n dy m cs Bacteri l om un ty s c e ion ecr ti plan s ue Predict ng b o tr l ac iv ty of en ir m tal b c eri solate (79 (81 (38 (54 (87 (89 , 80) , 82 , 83) , 39) , 84– 6 ) , 8) , 90) on the i d g nous micr b al om unity Impact of gen tical y mod fie bact r um (91) a Since1986th r av b le s 50 tic pub hed nw t MIS asb u ed o e n v i - f r o p m c l u ts y a i n d v e c o l f m p s F t A M E h a r e i z ronme tal s p . 6 or m e hav b n reco d f r bacte i grow n i the nu ri t- ch onditions yp cal of the r iz sp a n c eo mr vt b l i P fs L d F A a the cy lopr PLFA, whic can also provide a usef l ind cator of physiol gica st e s e g n a h C i t A F L P l f o r p a u d i v ns e t w g r l c a e b desu n b vah , c t ixo yl p erus gn w f d c s a e v i rt oc f d n u l p tra ions of organics such as phenol induces a reduction in the pro ti n of mon e ic to satur ed PLFA, and an increas in the pro ti n of detaru sn y f ic o des px air tc b l nu o edarg ht . n im oc ralim S segn hc va ne b d vr s o ni a egn r fo sm i ag rc de px ot s d n ue c h ot- i l r a p v m w g b f sm inahce t r f p d y il ba ni ev ta uq d roce ht guo la ,ecn r t vlos f sm inahce t in fat y acids sug e ts tha me brane repai mechanism , invol ing rapid fat y acid synthesi , are invol ed. Not surp is ngly, since l u ar fat y fo sn it pm c e arb h , d wo g n t ep si m c ,saer hw d tl era c uos tneir a s tna ul op ezi nac t h sl ec air t b .devr sbo a ic yt f u n p h se r , l aci yt 1.7 3 Enviro me tal M ni or g: eth d Ap lica on i n v e t s h a p b r g -I d o c w k i n v t e s preta ion f c m unity fa cid prof les: ir t, exam n io f speci fat y (50) (15) . Starv ion, s t ary-ph se g owt , and . . r o F , e gc n ia st - )15( - trans )35–1 ( fo nw k si elt ,r v H . creas dica 208 acid profiles know or as umed to be uniq e to a given functio al or taxonomic gr up, and seco , ap lic t on f multivar e n lysi to d scrim nate b e t w c o mn p s i r f a tl e h b d s u r a w n iAc e t lyh . d method for monitoring microbial com unit es pres nt in environme tal sample , PLFA and ME lyzes hav d nt ges a lim t ons. c o m - a n db i r s t h l a n y P iL F A o fd vp r tm a g eyT h munity s ruc e an be as d from the sam ple. Th result ob ained integra c os the n ir com unity, a d thus avoid the in v able s ctive p r e s t a h u m o n c l d i g w r yf e t h c o u n l d i t h r e - w s a p k A y o nc ti , . ated with enumerative studies, such as dislodging microbial cel s from sub tra e ( e x t r a c i o n b) v , d e h r f u p c o b l e m s r s e n t a i mv d p l cr g u z a t mTi p eho s n l . c d b to a range of dens and solid sub tra e such as sedim nt , soil , sand, and rock, in whic micros pic methods can be of lim ted value. Relative to t i m e - w f a nh d c ov s r t e n i a p o m c h l u q e r s , out the los of the hig precis on or quality in the dat obtained, althoug time l s and re i bl mo c ng ti u sly are m hod cul s u m i n g t o u n d e r t a k e . F i n a l y , sam ther bioc m al h r cte iza ons FAME an lysi h be n w d ly ac ept in l ca m robi l gy as pr mary or adjunctive means for ident f ca o of medical y importan bacteri (32) H o . w e v F r A ,a M n E l y s i u g t r m e n - b a s y d t m h ,o e n wa is d e l py n t v r , i a b l e n d p m t h fo r c b i a l a c t e r i z n v o m e t ia cl r b o Tg p mhy . w n s t e , capit l cost f equipm nt. Nev rth l s , FAME an lysi compares w l ith other m ods f strain ch ra te iz on such a DNA hybrid zat on r ta ge nuclei acid amplif cation. Inde , when fit ed with an el ctron det c or, femto l (10 ( d n i o r e sfa c m t p l h D N x A / v RF ) i o, n . hig lyspec a z d om unit es cha p yl os her ,FAMEdat ofin vidual pseudom na isolate ligned w l ith genomic studie ance of the reliab lity of FAME dat overcomes the ne d for sup orting char te iz on by tradi on l g stic b o hem al t ods ha re l bo i n t e o r h s g a lv d y b m c i F A e M . aE r - s oI S n ably r pid, eas to p rf m, and equir s l t e p cial z d te hnical r g. Despit h ne d to c si er th con ibu f the ac iv but nc l rab e c o m p n ae i yv t r s lm p c eh , a nr g ift o u s h e d e v l o p m n ta d x i o n fc u r e t h n i q u s a te b l r g n u m e s o sf t r a i n b e p d l y i a b c h r t e i z d A. n a l h o u g e x i s t n d a iden- sp c r ev nti g l m d, b ay str ine v o m l f base Thomps n et al. conple xtrac s n be us d to under ak fu (2) –15 . ) quanti es of at y cids an be d t c e hat m y be mor (54) . As ur- Char cte iz ng M ro a ism n the E viro m nt 209 tif ca on for unk ow isolate , large numbers of enviro m tal isolate are being xam d, whic furt e imp ov s the quali y nd precis o f denti cation. Ther is certa nly ne d for a m n ged at b se, i. a Web sit , for FAME and PLFA profiles tha comp re t h quality of d ab se v lop d for DNA sequ nc i format n FAME and PLFA of nalysi d v t ge . c our , f a e Th r requi s pecial n ytical p r tus ch as gas chrom t g a , in the cas of PLFA, with mas pectrom . S lvents, r agents, d glas w re mu t be scrup lo y c ean d rigo must be o rv d in sample h nd i g a reco d ke ping. Al procedu s hould be stringe and c reful at en io paid to he inclus o f ap ro i te s and r . In ad it on, special z d equipm nt s neces ary nd requi s con iderabl understa i g to p im ze its ap lic t on at t l h i e m s o f n i t v F y u . r t h e m o r i n , t e p r t a i P o L f p n F r A l o i e f ns t xelpmoc dna seriuq a hguor t egd lwonk fo a ylediw der t acs .eruta il , s t d n i a r p e y v l g w cf u F o h .elbis op t n v a u m rc d e fo ,st i l h w Althoug factors for converti g phos li d dat to carbon conte are e s t a b l i h u n d c , r t i e m qs f a u o n y p h g l ti d e oa r n f m s cel numb rs o i v lume. And althoug e fat y cid omp s t n f ma y microb al t x are know , it s dif cult o c nvert PLFA dat for the pr cise descript on f the comp sit n of microb al com unit es. B cause f t y acid c o m p s i t d n r e c l y p og tn w c h d i so ,n eft r g hi t h c dui ba s e n g o l . , d t s c r i p b o e u m n a c h t , b i p e r d t i u af b o l c n x s ,g p e y t i d enviro m tal ch nges ( Fut re d v lopments r qui e nv stme ap ro i te o the ap lic t on f cur ent s a chobje tiv sl nk d oaut m ing dac el r ting h spe dof n o d ei t s a vh b , r l I y wp z m . c resultina om edsy t for ignatu el p dbiomarke n lysi tha w lbe ac omplished n a m t r of h u s, in tead of h cur ent im f a e o d ys. Table 1 ). 2. Materials 1. Four eag nts re qui d to sap nify the c l s, e t rify, ext ac nd base w h f a r t c e A h i l dp g y s . n b o u - w a h r e d , n (light opaque) glas bot les fit ed with volumetric plungers. Extrac ion should only be u d rtak n i gl s tube fi d w th Teflon-c at d s rew-cap d li s. a. 57.3 :)noitac f p s( 1 tnega R b . R e a g n t2( m e h y l a t i o n ) :4 . 7 methanol). c. R e a g e n t 3 ( h e x a n e / m e t h y l - t e r t b u t y l e t h e r [ M T B E ] ) : 2 0 m L h e x a n e , 2 0 m L M T B E. d. Reag nt 4 (b se wa h): 0.3 M M NaOH in de o z ist l ed wa r. M H/lonahtem i HOaN H C li nm e t h a n o l( 3 2 5m L6 MH C l ,2 7 5m L 2 .)emulov yb 05: ( O 210 Thomps n et al. 2 . W a t eb r h s q u i ea1 dt0 ° C8 ,0 ° Ca ,nr do t me p u r Ao . t a i nm g x - ing dev c should be s for te ub s. Al reag nts should be of hig -perfo mance liqu d chromatography an lytical grade. Al procedures hould be undertaken i an p rop iate, ventilated facil ty or chemical fume ho d. Reag nts 1 and 4 are caustic and reag nt is 2 acid c; theyshouldbeonlyhandle byoperatorswearingsafetygo lesandgloves. MTBE and hexane are flam ble; extinguish al flames and sources of heat before use. 3. 4. 3. Methods Althoug e princ les of PLFA and FAME an lyzes ar sim lar, the wo methods are gen ral y used for dist nc pur ose . PLFA is most com nly u sa t en o d l y zb h i m c a u n i t o y m p s e f v r n m tal s mple , and FAME to char te iz solate grown defin labor t y media ( se Note 1 ). 3.1 PLFA Extrac ion Enviro me tal s mp e ust be handl with ex r m ca e to lim d sturh am li tc b r yno g- e s v d uT h . bial activ y by rapid fre zing to –20 methods of pres vation should not be used since thes can dvers ly af ect certain lip ds. Gas chromat g p ic an lysi of PLFAs extrac d from envir o n m e t a ls p g n e r a l y q u i sn o m l a r e i t v y .T h sn e c i t a the use of clean glas w re washed in eith r 10% (v/ ) HCl or Decon phosphate-fr det rg n (BDH[Merck] Ltd., Lut erwo h, UK) and baked in an ( 4 5 o0 v e n tion a d stor ge v ntual ex r ction a d lysi . The xtrac ion s u al y undertak ro m te p ra u . Al so vent a d chemi als u ed m st be of an lytic grade. s e t l Px h i c r p o L n a Fd : f A , of phos li d by column chromat g p y, and methyla ion of est rif d fat y acids in the phos lip d fraction. Ther are many vari t ons of the e x t r a pc oi m n d u fhe ,q t r p g oT c d s u . e describ y Wh te al. ° C or by lyophil zation. Alternative ° c s o a l m f p er - t v y o i d b e P m l u a s th ) . c 4 f o r C (4) is um ar zed n xt: 1 . T r a n s h o f u 1 egm – 3 i lc t r f u g e b q s i p w dT t h f l o n - i e d 2. screw ap . E x t r a c is n g l e - p h a s ce l o r f m - e t h a n o ml i x t u r (e 1 : 2 v, / t) h Be l i g a n d Dyer mixture v) c a n a l s o b e u s e d mixture with phosphate or citrate buf er (1:2 0.8, v/ v) to increase PLFA recovery (1 . Alterna ively, a dichlor methan -methanol mixture (1:2 v/ (12) (13) , 14) . . For soils with high clay content, sup lem nt the Char cte iz ng M ro a ism n the E viro m nt 21 3 . C e n t r i f us ga m p 6 l 0 e g m f3ir o0ne , tl vh q u as d w kie l volumes of chlor f m and dist l e water (or buf er) to produce an emulsion tha is l owed t s an over ight. 4 . R e m lto ihv p d - c n a oi r g p h c fs e l, t a onr d i p b y s evapor ti n a 37 5. Dis olve the drie to al lip d extrac in chlor f m, transfe to sil ca acid columns, a d separ t in o eutral, g ycolip d- an polar ip d fractions by elution w i t hs o l v e n t so fi n c r e a s i n gp o l a r i t y pholip dis ubject d otranse t rficationbymilda k linem thanolysi and the capil ry GC. ° C. (15) .T h ep o l a r i p dc o n t a i n gt h ep o s (16) resulting FAME is separ t d, quantif ed, and ten a iv ly ident f by 3 . 2 F a t y cA i d e M h l E s t r Prepa tion f cel u ar f t y acids on ist of hydrol si u ng sodium hydroxi e t form s diu alts, nd the m ylation f the fa y cid est r o make them volati e in the gas chromat g p dures tha ve b n used to b ain the nd pro uct for GC an lysi , and l invol e ac d r b s hy ol i f wed by st rif ca on w th me a ol 2 0 ) .H o w e v r , c n ta d e si h m t o d a v e p i m z dt h r c o v e y f fat y acids tha forme ly wer dif cult o ident fy reliab y. This owing, in M i l e of r t h s p a ,g e dure with NaOH th remov s f e acids n prev ts h tail ng of hydr x l acid pe ks uring GC a lysi . Th dev lopm nt, ge h r wit o her fin p r t o h cm e i n d u s for the p a ion f s mple h24–8forincubated.plsmw)(g50xHv 2. Saponify using a sodium hydroxi e–m thanol s ution f r 30 min at 10 rel as f t y cids from el u ar ip ds. 3. M e t h y l a t e w i t h H C l i n m e t h a n o l a t 8 0 into a 4. Wash t e xtrac in aqueo s NaOH for 5 min. This procedu is mple to perform and up t 120 sam le c n b pro es d in a y. S mples can b r p ed from pu e c lt r s o envir m tal s p e . 5 . i d e t n u o p s F r A f l Ta M y hg E c I b d i e n t f c a o S o f t w a rd e b s( M I D - SN ,e w a r kD l e )H .o w v rd ,a t b s ef o n y p a c r s o t u n i e b g q l d w h m sa gs rp c oe a w nm ti d hf S u . l s r x y a ,i c o e t m l s a m p h l e b d i t o u s c , m r a f e nh l v t o i y microb al m unity. (17) . Ther are various proce(18– ws pai r m d oh e c l v n -g , (21) (18 r e l a t i s v o f m h p yd u - r e c , 19) (2 ) . 1. ° C, to ° C for 10 min and extract the FAME soluti n f hexa nd MTBE for 10 min. 3. FAME nalysi of I ates The fol wing sta d r p ot c l has be n d velop f r cult res p ared after x c ly 24 h of gr wt , a 28 ° C in 85-m Petr dish conta i g 20 mL 21 Thomps n et al. of tryp one s y broth ag r (TSBA) tha is al owed t se and ry at 37 16–20 h befor us . ° C for 1 . sS ip nr ca ge bo l d f t q y u T S v B , A r n plate, s ril ze th lo p and spread second qua r nt f om the dg of the first quadr nt. Rep at this procedur until four reducing densit e of inocula are pres nt i he quadr nts. 2. After xac ly 24 h of incubat o 28 ° C, col e t 50 mg wet of cel s rom the third qua nt si g a lo p nd e sit a he bo t m f a 10 b d enil- o f t h iw ebu gn lio f or many ths). × 1 cm Pyrex glas screw- ap d li s ( ample y b sto 07– ta der 3. 10 at incube ad cels th supend to vrex 1, reagnt of mL 1 Ad 4. 5. 6. 7. 8. 9. °C ° min. 5 for C Vortex h su pen io a d ncub te a 10 Rapidly co l the saponif ed sample to ro m temp ra u by placing tubes in water. Methyla with 2 mL reag nt 2, and i cubate 80 temp ra u . This the most crit al step and should be undertak exactly as describ . r o tm i a np vl 1 b e 0 y f g s x d 3 o , n m t 1 L . A 2 5 at ro m e p atur . Al ow phase to par e, nd col e t and isc r the low r aque s ph . W a s ho r g n i cl a y e w t h3 . 0m Lo fr e a g n t4b ym i x go n h er t a f o 5m i n . Al ow the conte s of the tubes to set l ; ad sev ral drops of satur ed NaCl soluti n a d sep r tion f phase . ° C for a u the 25 min. ° C for 10 min. Co l t ro m 1 0 . g lU p as i c n e o t , w - r h g i p a f n d s tc l e , o r GC-vial, nd se with ample c s. 1 . Store sample at –20 ° C for up to 4 wk and run o the gas chromat g ph ( Note 1 ). 3.4 Sta is c l An y is of Dat m u sl t pbi rjvoa e fc h n d P L F A l t i o s to as e im lar t es b w n PLFA profiles. D ndrog ams f hiera c l cluster an y is re u al y constru ed f om arcsine-t form d PLFA ole perc ntag values, with sim lar t e based on modif e Euclidean distance . T huesotfw - d i m n apl o tgse r fd pam i n c l y s o tn i d e n s t x p r h a f c lP v b L o u F A e profile s m ar ti D a t n l y s ri p e d f c wn t Mh I TS r . e i o n u m s d c t a o l u e q n i v c h t a l g T e q. u i v c n h t a l eg q s u o n u m - s t a r i g oh c f u b m n s d t e y r c a l b i e nu t h y r m p od w f c eu l a p e r c o a n df t l g u i s m y T h e . the to al amount and printed toge h r with the most like y identif cation (4 , 28) . se Char cte iz ng M ro a ism n the E viro m nt 213 ac ording t s m lari y to en s i the da b s d i a n t e , r pf o u s c g n i mt e h lo, d a matching unk ow s with dat b se entri s and resulting in an ident f ca o . Numerical an lysi of fat y acid dat can be ap lied for the constru i of den rog ams. An unweight d pair-m tch ng method can also be ap lied to det rmin the ext n of sim lar ty betw n isolate and the col e ti n to the genus, p ci and sub peci l v . (17 . Multivar e s ti - , 2) 4. Notes 1. Ther av be n two maj r dvances i the 1950s tha ve brought ine f a t y c i d n l s t o h e a b r t o y .T h ef i r s t d e v l o p m n ta di l e m n ta ion f used- il ca pil ary columns ao nt dh bew r i s m c a ,p i l ro y u m n s e wp d c i br l o v e y h oy fd r xa t c i nhbs e l d o y g u is e hv r a lo m f t s c yi d w i t h es a m c r b o n h i l e g t .P a r l e d v o p m n t si h e rm o d s u c h as nuclear magnetic resona c spectrom y, infra ed spectro y, and mas s p e c t r o m hy a v e l s bo wn i d uy e t o n i f y a t c d s major dvance is the d v lopment of micro pute s y tems hat en bl ef icient da proces ing The resulting FAME obtained from the phospholip d as described above, or one of the many vari nts of this procedure, are separ ted on a gas chromatograph equip ed with a flame ion zation det c or and phenylmethyl sil cone capil ary column (14) hydrogen is used as the car ier gas, and injection is made in a split es mode.T nta ive d ntif cation f at yacids basedonret n iontimeonthe c o l u m an s e u r e ad g i n s ct a l i b r a t i o sn t a d r s I. n d i v d u a cl o m p n e t cs a n also be identif ed by mas spectrometric an lysi . With this procedure, the GC condit ons are identical to those used above, but helium is used as the c a r i e rg a s .I d e n t i f c a t i o n fF A M E si b a e do nc m p a r i s o nw i t hs p e c t r a h t are obtained either from stand r s fide ad ucts (27) The fat y acid an lysi of micro ganism is now so c m on tha commercial y av ilab e GLC sy tem is now av ilab e. This ystem was in tial y codev loped by Hewl t -Pack rd and Microbial ID ( the identif cation of aerobic bacteria, but more rec ntly it has be n used for the an lysi of ungi, viruse , spirochet s, and the lip d conte of to al soil extracts. The sy tem consi ts of a gas chromatograph (HP 5890 Seri s I ) e q u i p e wd i tfahl m ie s i l i c a p i l a r y c o l u m n ( 2 5 mX computer and printer. Equipment designed and edicated for the pur ose of microbial identif cation is not a nec s ary requirem nt, but he MIS greatly facil ta estheinterp eta ion fresults mation is ad e by the automatic sampler, whic lets the operator un up to 10 samples without interv ntion. (19 , 23) . In contras to packed columns T. h se c o n d (24) . (25) (14) . In the procedure described by Frostegård et al. or by an lysi of the dimethyl disul- (26) . Newark, o n i z a t i o nd e t c t o r ,5 %m e t h y l p h e n y ls i l c o n ef u s e d 0 . 2m ) ,a u t o s a m p l e r( H P 7 6 3 ) ,i n t e g r a t o r , (17) .Furthermo e,anel mentofauto- Delaw re) for 214 Thomps n et al. References 1. Am an, R. I Ludwig, W. and Schleif r, K.-H (19 5) Phylogen tic den fication and in situ det c ion of ind vidual microbial cel s without cultivation. Microb l. Rev 2 . ( Q 1 u 9 a n C 3 t . ) i d D F e o m s b c v r , p f H - a ln R d . y munit es u ing l p d an lysi , n (Kemp, P. F , Sher , B. F , Sher , E. 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(1986) Dif er nt a io f 32. 3. 34. 35. 36. 37. 38. 39. 40. 41. 42. 43. 4. 45. carot v and Erwin a c ot v ra s p. atrosep ica acid omp s ti n. Can. J Microb l. 32, 796–80 . De s, S. B , Hol is D. G , Weav r R. E , and Mos , C. W (198 ) Cel u ar f t y acids of Brucel a nis and Brucel a si . J. Clin M crobi l. L a m b e AP rM. t , B oC n Ja ( T W1 ds 9 8D 7i )f e r n t a tion f Campylob cter and Campylob cter like organism by cel u ar f t y acid comp sit n. J. Clin M crob l. 25, 706– 13. isolate:nurfdchianlys Ft(1985)G.AO’Donel, posibltefr intus identfcaousgmlvrpeconit, centAdvasiMrob.El (Hatori,T.IshdYMuymR 674–8.pJan,TokyPresScitfd)AUh Mukway , G. M and Welch, D. F 198 Subgro pin of by cel u ar f t y cid omp s ti n. J. Clin M crob l. R a i n e BPy T. , h o m p s I a n d l e r o (JNi 1. , 9 G4 ) n am f e d t cy i an lysi of Pseudom na stu zeri . Int. J Sys Bacteriol. ( c 1 F a o 9 L Mm 8 t i .p 6 d n) ey P r g s , K J o V c C k l j B . e n , a guide for the clas if cation of sel cted gen ra of yeast belongi to the Endomycetal s. J. Gen Microb l. 132, 2397– 40 . Grah m, J. H , odge, N. C , and Mort n, J. B (19 5) Char cte is on f at y acid methyl est r profiles for char cterisat on of Gloma e n fungi and their endomyc r hizae. Ap l. Enviro M c biol. 61, 58–64. B e n t i v g a ,S .P n dM o r t ,J .B ( 1 9 6 )C o n g r u e c f a t y i dm e h l s t r profiles and morphol gical char cters of arbuscular mycor hizal fungi in Gigaspor ce . Proc. Natl A d. Sci U A 93, 56 9– 2. Livesl y, M. A , Thomps n, I. P , Bailey, M. J , and Nut al , P. A (19 3) Comparison fthe a y cidprof les Bor elia , Serpulina Gen. Microb l. 139, 8 9– 5. Livesl y, M. A, Thomps n, I. P , Gern L and Nut al , P. A (19 3) Divers ty in fat y cid prof les Bor elia bu gdorfe i . J. Gen Microb l. W i l T a h n (o d 1m . P 9ps F , I 4 t c r) y i f v l e b a r t i d s cent virus . Arch. Vi ol 140, 975– 81. u n v c e o g Aa (k 1 9d E2 ) . r l Hn , O i e W . L P n lg R t, o diet sh f t e profil human fec l mi rof a: computeris d an lysi of direct sto l amp e g s-liqu d chromat g phy rofiles bacteri l u ar f t y acids. Ap l. Enviro M c biol. 58, 36 0– . C a v i g e l A RM . o , b r t s P aG K n ( d Ju 1 g 9. , F 5 ) t c my i e h s l r (FAME) profiles a measur of s il m crobial com unity s ruct e. 170, 9 –1 3. H a c k ,S .K G r h o w ,H . O d e l s n D A . ,F o r e y L J a n dK l u g ,M .J ( 1 9 4 ) Ac ura y, ep oducib l ty and i erp ta ion f t y acid me h l st r p ofiles model bact ri om un ties. Ap l. Enviro M c biol. s p. Erwin a c ot v ra on the basi of cel u ar f t y 1 , 2. 14, Re- Pseudom na cepa i 2640– . 27, 93–10 . 62, and speci . Leptis ra 139, 1947– 5 . Plant Soil 60, 2483– 9 . J. Char cte iz ng M ro a ism n the E viro m nt 217 4 6 . L i n d ea KO r .l , v ( D J1 9 8 M ) m b r a f n e t c y iv d u l n h e a g i s t vh ie a b l nu o - c t r a b sl e o f 5, . A p El n. v i r o M c b l . V i b r v uo l n f c s 2837– 4 . 47. Rice, S. A and Oliver, J. D (19 2) Starv ion resp of the marin b ophile 48. 49. 50. 51. 52. 53. 54. 5. 56. 57. 58. 59. 60. CNPT–3. Kostiw, L. L., Boylen, C. W., and Tyson, B. J. (1972) Lip d comp sit on of growing and starving cel s of 186 –1874. Nickels, J. S., King, J. D., and White, D. C. 197 . Poly-beta h droxybuta e a c u m l t e i o s n r f b a c g e d o w st f h u a r i n e t m l c o b i a . Ap l. Enviro M c biol. d n, a. Gm h O HRy l d n i, F. S o s O BMr e l x , T. H d i a B A l n u T -yloP dna s ic yt af deknil-r ts dip loh s fo n ita mre D )5891( .C D ,etihW -ozihr e t ni yt v ca dn s amoib la retc b fo n itam se ht rof etar ubyxo d h-ateb tnalp e r ht fo e ps S i k e mJd a. , B M P o A n t l ( 1 9 5e )c h a n i m so f b r e toxic y f h droca b ns. H e i p J Dr. , f e n b a cB h K d w l o( H 1 . ,9 M i ) c r b a e l s p t o r y quino es in the environme t: a sen it ve liqu d chromat graphic method. Microb l. Methods ( 1 e C 9 P n W R . i v o l h 6 a D g ) k f ,t Hd J r p s m c h a n sgioe l v t - r a sn o dl v e t - s i P v e u d o m np a st i r f o l lowing exp sur to O-xylen . Rainey, P. B , Bailey, M. J , and Thomps n, I. P (19 4) Phenotypic and genot d y i p vf el c ru o s n t d m o i s a lf g r b eu w d nt . Microb l gy F r o s t e g B a å n (A d p1. E h9 , u T f 6 ) o l i a t d c n y s o estima b c erial nd fu gal biom s n il. F r o s t e( T B g1 u a AC å9 m E n .h i l , d 6 c ) b o t y struc e during long-term incubat o in two s il exp rim ntal y contami ed with me als. Ols on, P. A., Ba th, E., Jakobsen, I., and Soderst m, B. (19 6) Soil bacteri respond t pres nc of r ts bu not myceliu of arbusc l fungi. Biochem. O l B As a . J P E,o t nkh b S I e d r s p ( 1 o B u9 fT. m h - 5, ) pholi d and neutral lip d fat y acids to estima biomas of arbusc l mycorrhizal fung i so l. W Fh a C i n . D t (d , e 1 H l 9 R B 8 y o ) c m i a r l k f e o s u m n t m e t a - n d s c u or b i y, t l h e n pf r dc a s i o bolic a t v y of micr b al ofi ms. F r a P n D z B N t. m iR , Gv c oM e d h Tw s l ( 1 M 9 i c s6 r h) o a b l u qm w n , f e t i a r o m h d c hydroca b ns: an lysi by phos li d fat y acid conte and comp sit n. Ap l. Bacteriol. 243 – 7. 58, Ap l. Enviro M c biol. . J. Bacteriol. Arth obacter c ystal op iet s 37, 94, 459– 6 . .) L( supan ci rB 59, Microb l. Rev .91 –3 ,31 .loib rc M .J naC 210– . J. 5, 243– 5 . 1 29– 3 . 62, Ap l. Enviro M c biol. 2315– . 140, 2, Biol Fert . Soils 5 –63. 28, Soil B . iochem 59–6 . Soil B o . 28, 463– 70. Mycol. Res 9, 623– 9. Hydrobi l g ca 159, 1 9– 32. J. 80, 617– 25. 218 61. Heip r , H. J , Meul nb d, G. van Oirschot, Q. and eBont, J. A M. (19 6) Ef ecto nvir me talf c ors nthe a s/ci r t o funsa r tedf yaci s n Pseudom na p tid S12. 62. Pen a , T. Frostega d, A. Fritze, H. and B th, E. (19 6) Phosp li d fat y acid omp sit n of s il m crobial c m unit es along tw heavy m t l po uted gradients co ifer us o t . 6 3 . B a t h ,E . F r o s e g å d ,A . P n a e ,T . n dF r i t z e ,H .( 1 9 5 )M i c r o b a l m u nity s ruct e and pH res on i relation soil rganic m t er quality n wo d ash fertil s d, c ear- ut o b rned co if r us o e t s il . 2 9– 40. 6 4 . Z e lR Y a B . L Q c , sk i w C t h z d D e ( n . 1 F , s 9 5 ) r m i natio f phos li d an lipo ysac h ride v d fat y cids a n estima of microb al biomas and com unity struc e in soil . 1 5– 23. 6 5 . J o r d KDa .n e, B m R g f i C Y A Wl ( cd 1N n 9.V o 5 , ) Evalu tion f m crobial methods a p ten ial d c tors f il qua ty in h s or cal gri u t al fie ds. 6. Wander, M. M., Hedrick, D. S., Kaufm n, D., Train , S. J., Stin er, B. R., -orcim eht fo cna i g s lanoitc uf ehT )59 1( .C D ,etihW dna ,.R S rey m h K b i a l o m rs n g c d v e t i o n a l m y g s e d i . 67. Bardget , R. D , Hob s, P. J , and Frostegå d, A. (19 6) Changes i so l fungalbacteri l biomas ratios f l owing reductions i the inte s y of man ge t of an upl d gras n . 68. Rajendr , N. Matsud , O. Ima ur , N. and Urushigaw , Y. (19 2) Vari t on in m crob al i m s and com u ity s r c u e in s d me t of u r phic bays det rmin by phos li d est r linked fat y acids. 58, 562– 71. 69. Parkes, R. J , Dowling, N. J E , White D. C , Herb t, R. A , and Gibso , . R ( 1 9 3C )h a r c t e i s o nfu l p h a t e - r d u c i nb ga t e r p lo u a t i nw s h m r e and estuarine sediments with dif er nt rates of sulphate reduction. Microb l. E 70. Rajendr , N. Matsud , O. Urushigaw , Y. and Sim u, U. (19 4) Char cte im i c r s o a f b ut B O h l n e y , k d m r Jap n, by phos li d fat y acids lip d an lysi . 248– 57. 71. Kieft, T. L , Ringelb r , D. B , and White, D. C (19 4) Changes id est r linked phos li d fat y cid prof les ub rface t ria du ng starv ion d esic at on p rous medi . 72. Guez n , J. and Fi l -Med oni, A. (19 6) Bacteri l abund ce a divers ty in p ah no s l y i . b d e t r mT B c a o s h 19, 83–9 . 73. Amy, P. A Halderm n, D. L Ringelb r , D. and White, D. C (19 4) Changes in bacter l coverabl f om sub rface vol nic r k sample during sto a e 4 ° C. Ap l. Enviro M c biol. Thomps n et al. 27 3– . 62, Ap l. Enviro M c biol. 420– 8. 62, Ap l. Enviro M c biol. 27, Soil B . iochem. 19, Biol. Fertil. Soils 297–30 . 19, Biol. Fert Soils 1 70, P l aS no ti 2, Biol. Fert Soils 87–9 . 261– 4. Ap l. Enviro . Microb l. FEMS 1 02, 235– 0. 60, Ap l. Enviro . Microb l. 329 – . 60, Ap l. Enviro M c biol. E cM oi l .r b F S 60, 2679– 03. Char cte iz ng M ro a ism n the E viro m nt 219 74. Hirsch,P. E k ardt,F.E W andP lmer,R.J (19 5)Methodsf r e tudyof rock-inhab t g micro- an sm : revi w. 143– 67. 23, J. Microb l. Methods 7 5 . B r i n k ,D .E V a c e I , n dW h i t e D .C ( 1 9 4 ) e t c i o n f i no l Desulfobacter f ie n lv d r o m b y-t s a i c p Dr NovAbe s . 42, A pM i lc .r o b B t e h n o l . 469– 75. 76. Sundh, I. Borga, P. Nils on, M. and Sve s on, B. H (19 6) Estima on f cel numbers of methanog ic ba teri n boreal p t ands b ed on a lysi of specif phos li d fat y cids. 7 . Haldem n, D. L., Amy, P. S., Ringelb r , D., White, D. C., Garen R. E., and GhiorseW.C (19 5)Microb alg wth ndresu cita on l erc m unitys r cture af p rtu ba ion. 7 8 . C L R oM e ( iS l. h 1 n w, m 9 W g F a c5 D B r) td b bial com unity lev an lysi ba ed on pat er s of carb n sou ce til za on d phos lip d fat y acid profiles for quality as ur nce of ter s ial sub rface cores. J. Microb l. Methods 7 9 . L e f , .G K r n a R M . , c A r t h u J .V ,a n dS k i m e t s ,L .J( 1 9 5 )I d e n t i f c a tion f aqu tic Burkholde ia (Pseudom na ) cepa i by h brid sat on with species- p f c rRNA gen prob s. 8 0 . B r o w n J,. a L de f G( 1 9 6C )o m p a r i s fn t yc m de h ls a rn y si with use of API 20NE and NFT strip for ident f ca io f aqu tic bacteri . Ap l. Enviro M c biol. 8 1 . M e r g a tJ ,.W b A n d e r s o C ,.W u t A a nS dw i g sJ( ,.1 9 3M ) c r o bial degrad tion of poly-3 hydroxybutyrate. 32 –32 8. 82. Ka, J. O Holben, W. E and Tie j , J. M (19 4) Gen tic a d phenoty ic d versity of 2, 4-dichlor p en xyac ti ac d ( 2, 4-D trea d fi l so . 83. Tons , N. L , Matheson, V. G , and Holben, W. E (19 5) Polyphasi chra te isation f a suite of bacteri l so ate c pable of degra in 30, 3–24. 84 Thomps n, I. P Bailey, M. J El is, R. J & Purdy, K. J (19 3) Sub-gro pin f bacteri l po u ati ns by cel u ar f t y acid omp sit n. FEMS icrob l. Eco 12, 75–84. 8 5 . ( C 1 o 9 m L p 2 . a ) rB iK d tw e n v A ,M c f I W Jl .o a - p y e r tion by fat y- cid an lysi of soil, rhizosp e , and geocarp s h e bacteri of peanut ( Arachis- ypogae 86. Lil ey, A. K., Fry, J. C., Bailey, M. J., and Day, M. J. (19 6) Comparis n of aerobic het ro p ic tax isolated from ur o t d mains of mature s gar be t ( Beta vulg ris 8 7 . F o s t e a rJLMn ,. d g l m C( 1 9 3I )d e n t i f c a o ne d l g b yfa c t r i l com unit es as oci ted with necros of thre ca tus speci . Microb l. 59, 8 . ( B1 a9 F c C o t4. g se) n ur JldM i m , L c t e r c a( g r t i ou fs 103– 2. 18, FEMS icrob l. E 17, FEMS icrob l. E 27–38. 263– 81. 2, 62, 1634– . 61, Ap l. Enviro M c biol. 2183– 5. 59, Ap l. Environ. Microbiol. 2, 4-D) degra in bacteri solated from 60, Ap l. Enviro M c biol. 1 06– 5. 2, L). ). FEMS icrob l. Ec 21, Microb. Ec l 85–90. 139, Plant Soi 4-D. 231– 4 . Ap l. Enviro . 1–6. S t r e n o cg u m s ) . E A n p v M i l r .c o b 60, 619– 25. 20 Thomps n et al. 89. Frachon, E. Hamon, S. Nicolas, L. nd e Barj c, H. (19 ) Cel u ar f t y cid an lysi as a poten ial to l for predict ng mosquitoc dal activ y of sphaericu Bacil us strain . 57, Ap l. Enviro M c biol. 3 94– 8. 9 0 . N d o w r a ,T .C R K i n k e l ,L . J o s R K . ,a n dA e r s o ,N . ( 1 9 5 )F a t y 91. acid n lysi of path genic a d sup re iv strain of Strep omyc s pe i s olated in M sota. Thompson,I.P ,El is,R.J ,andBailey,M.J (19 3)Autecol gyofagen tical y modif ed fluorescent pseudom nad on sugar be t. Ecol. 17, Phytopa l gy 86, 138– 4 . FEMS Microbiol. 1– 4. FISH and A lysi of the S ngl C 21 51 Fluorescent InSitu Hybridization and the Analysis of the Single Cell Anthony G. O’Donnell and Andrew S. Whiteley 1. Introduction 1. Over i w of the M d The discov ry tha prok y tic and euk ryotic el s cou d be mad per able to fluoresc nt y labe d, sequ nc specif olig nuc e t d s makes possible th de rminat v probing f intac m robial ce s t a r g ce l ib s d n f a e u m r t hi nd o g e pu s l a t i o (n r ev n wh pres nt a dosymbi nt of her ganism for di ect sola n d cult re of h ganism of ter s . In microb al e ogy, the prima y t rge s for such pro edu s, ref d to c l e tiv y as fluoresc nt (rRNAs). The rRNAs have pro d exc ptional y go d targe s for det minative probes for sev ral reason . First of al , despit being hig ly conserv d biop lymers wing to he r l in p ote sy h i , t ey also xhib t reg ons c m o n s a e b ri q v kd u f R N t A T h l y , . c o n sh ei rg v l dy f R qa u cb h .e no d s i rg v l y f sequ nc have remain d virtual y uncha ged throug evoluti n and provide al t rge s for s -cal ed universal or c nse u probes and for p obes d i r e c t a h g l v e so ft a x n m i cr k .T h ev a b l r g i o n s , t h e r hand, ve ol d m re apidly n ca be us d to if er nt a mong spes e u v b p o a n r c i t d fh A y . g in hig copy numbers in active cel s (10 – , ribos me per cel ) t hi en src d b ya o f mv g x t i h en r d A o s . s e t q h u a g n r i c D d- N v Ao w f e ing of the rDNA oper ns, prima ly the 16S and 18S rDNA (1.6 and 1.8-kb, . Thus, ind v ual (1) (2) withou e n d r Ri Nb Ao s t mh ae l y n c(d F Iz viS qH u) o s , situ n (3) From: Methods in B otechnol gy, Enviro mental Monit r ng of Bacteria Vol. 12: Edite by: C. Edwar s © Human Pres Inc., Tot wa, NJ 21 , 2 resp ctiv ly), s relativ y s raightfo w d an yields a gre t d al of in rmation f r evalu ting phy o e tic r la onship m g or anis wel a for F I S pH r o b de s i g n . a t o , e q u n c g r a t i o , n hd e c p b s i g n , is ap lic b e to both cult red and “unc lt rab e” tax using stand r gen amplif c t on, l i g a d sequ nci g tra e s. Whole c probes wa first u ed in m crobial eco gy in the la 1980s by De Long a d col eagu s an lysi of microb al com unit es in atur l enviro m ts, and exc l nt revi w of their ap l c tion the d ction a d phylogen tic har te iz on of ind v ual m crobi el s provide by Aman O’Don el a d Whitel y in s tu (1) hybrid zat on wi h fluoresc nt y labe d o ig nucle t d . Since th , FISH procedu s have b com widely us in the et al. . (4) 1.2 FISH: Lim ta ons d P te ial So u ns A l t h o F u I g e S c H n i q r a l s t pv u o bfy c e dn m provide us f l det rmina v formati n o micr gan s i natur l envid e s c r i b , p v o u l y A e x i s t . d o c n r am e hs l g i v o n t , perm abil zation remains an importan lim ta ion and whet r cel s wil t hs a p ob e w r n m di F l c u y z . t o r e , cel s growing in natur l enviro ments such as soil may exhib t dif er nt perm abil t s to olig nuc e t d probes than those grown under labor t y condit s. Macn ughto e al. infect d ro section hat l ough perm abil z t on by mild ac hydrol si n o l t a ws be u c h r f i p F I S H y , g o ne d for sample ocat d the surfac o a growin t. Sim lar f ndi gs wer reported by Hahn et al. perm abil z t on pret a m n used suc e f l y to probe bies i n p u r ec l t w a sn o q u i r e dt p m a b l i z e h s o r g a n mw e it was gro n i ut e -am nd soil. p r o b e t h a g u n o i s t h e r a u d , i s p m e b l z t o nc h u g E v e t o w i n gb e h u s t To c i w rl . e y h b R N d nA tz oa i si t en ab q r uc g o h w f - l somal proteins or to hig ly stable secondary struc e el m nts with n the rRNA (7) . The lat r should be su p cted if a strong hyb id zat on sig al c n be o tained w th a universal p obe tha is know t arget dif er nt, ac ess u c o e r f Rl t Ni a m A - g y h d . n s i t b e l direct FISH is av l b e in a xc l e t r vi w by Aman et l i c s m to ea p n l F I h r u S d H w f q v y f l u o - s i n g e a c r y P oebn vsi m at u. l c r o g n is m d y t roch me wil on y give a strong i al f the c l s are ctiv ly me abo iz ng. This make uch probes id al for studying labor t y cul res, b t for na u l enviro m ts in whic olig tr ph c condit s prevail, altern iv det c ion have s own u i g (5) (6) – Rhod c us fa ciens , who demonstra ed tha the enzymatic Strep omyc s sca- (4) . FISH and A lysi of the S ngl C stra egi may be requi d. A number of ap ro ches have be n pro sed to i sm eptnrh o v f y more than one probe in a single cel al ow f r enzymatic s gn l amp if c t on r enzymatic produ ti n f reaction products ha l ow discr m nat o f probe ind g a inst b ckground fl resc n e r a n t c mv p i h y o g l s u , onlyare tiv smal fr ction he d g ousp lati n,m k g ec ss a r yt o e c hl g n u m b e r so f i c p e l d si no r t c a e h r g t o r g a n i s m R e. l t c a y h, i s m r o c p a n l y st i f r g e o u a n s to organism pres nt at more than 10 overc m by cel sorting using flow cytome r ( form en ich t pr o an lysi . fo melb rp ht ,sm inagro e v d morf secn uq fo ytil ba v re g htiW eborp ytic f s dna h gi e fo c ts n a d b rp i g moce yl n sa ;tlucif d s h a y wl ne b a it op m l eh da rps iw - c l tion of FISH. As for al olig nuc e t d - p techniqu s— o only for rem-81 cif ps a o yt l b r eh —HSIF u n r ae lo 1 : i tg 4 d s m like ho d f n i g18var bleposit n l m ed,an it s or l ke y hat thes Und r po i . base f v or u nly i d f ers p ob th thelikeliho d fencounteringthesame fal s to 1:4 niques can be made more sp cif by using multip e sp cif .eluc om ANRr eht no seti n r f d g ite a dn semorhc ulf tn id h w larev s ni d uof eb yam cn uq s tegr di o lc n e g s a h uo tl ,s T detal r , x eht y il ba orp s hcum rewol tah egr s ti rof la ev s -if c p .sm inagro te p d lcu is y a Brock ac epts on If smal .” o i env r m t s h fo e and sm l i ce “th a this v ew, n to u ders an th ole f micr gan s i the r na u l vir o n m e t s , d o t u ym i c r b a l e s t c a p r o i t e h rs z and to he micr n s they influ c . This dea w r it ed by Brock ve 20 yr later when he ident f tha a major factor lim t ng the use of micros d c ieo t f p h u l n g y m r o a n i s and in using the micros pe to measur cel activ y. In the last 10 yr, the d e v m l o p f c u n i t a r b h e F d Ig Sy o H f n cyto hemi al st n g procedu s hav elp d to res lv th e probl ms. It i exp ct d hat over th next d ca e microb al eco gist w l conti ue o harn e sa d v c im o l u ab r g ynt dos eh v l p m n t s o u d ya , t h ec o r s p a i l c e , m p o r t a n i c b l ym e d a t c o g i p r e s . 23 s i t un h y b r i d z at e c o n q u f sl , dh i g (8 , 9) , the use of det c ion sy tem tha . Another problem with the use of FISH ap ro ches in natur l (1 ) 4 5 –10 mL –1 . Such problems might be se 18 Chapter 5) or by some remi p o b gtarein sequ nceina unrelatedorga av r i b l e v H a o r w i e n . bgt l h , s cir umstances, 5 nism (for ive bas dif er nc s). It has be n pro sed tha FISH techd, es l b o ar p (25) , in his 196 bo k the Princ ples of Microb al Ecol gy , sta ed s i t un 24 O’Don el a d Whitel y 2. Materials 2.1 Gen ral 1. Al reag nts wer obtained from Sigma (Po le, Dorset, UK) unles sta ed otherwise. 2. Fluoresc n mi rosc pe with a rop i te f l r b ocks. 2. Cel Fixat on P a r f o m l d ec i h n y s x a r u t g o f 4%inphosatebufrdlin(PBS):8gNaC1,0.24KH 1.4 g Na made to 10 mL). 2 2 HPO 4 PO 4 ,0.2gKCl dis olve in 80 mL dist l e water, pH 7.4, with HCl and 2.3 Cel P rm abi z t on Cel perm abil z t on was car ied out using a grade seri (in water) of ethanol 50%, 8 and bsolute. 2.4 Cel hybrid zat on ( ref ub noitaz dirbyH es 9.0 :ref ub hsaW .) .1 4 3 gnidaehbuS M m 02 ,lCaN M T r i sH C l ,p 7 . 4 0 1 %v / S D N e g a t i c o n r l s e a t dw i hR N s Ip r o to pr bing. 2.5 Fluoresc nt y Label d O igonucle t d Prob s Oligonucleotide probes wer sup lied 5' label d with fluorescein or rhodamine f GENOSY Biotechn l g s, Cambridge UK. 2.6 Fluoresc n Mi o py Stand r fluo esc n im r o l and m u t . 2.7 Cytochemi al S n g 2,3 5-tri dilute o 1-5 m lium sa t , 15 p -nitrophenyl-2H te razolium chloride (TN C) 15 m M synthe iz d by E. Seidl r Com e cial y v b e t razoM dilute o 1.5 m 3. Methods Fig. 1 provides a gen r l ov iew f the ap ro ch used. Bri fly, ce s ar p m e at fr d i n x b y w l h o d e a t n y r g s m o ca r el itp nh sw , T g f . y for pas ge of the probe throug the c l env lop , and cros link the targe r R N A m, a k i n g t c e s b l po r i n d g O. c fe x , l as r t c h e d o gelatin or 3-aminopr ylt e hox si an (APS) coated micros pe slide and dehy rat p io hybr d zation w h flu resc nt y lab ed o ig nucl t de M M , provide by S gma stock FISH and A lysi of the S ngl C 25 Fig. 1 Schemati d gr m of the basic pro edu s f r FISH p ot c ls . probe (15–2 nucleotid s n le gth). Al ernativ y, f xed c l s an be hy rids u i znp e d t ahm o b l r c s e px ia m natio , or an lyzed by flow cytome ri techniqu s directly from su pen io hybrid zat ons ( se Chapter 5). 3.1 Prepa tion f Gelati -Co d Sli es 1 . W E A RG IL NO Vp r Se , f a ws i o Kn l d h u H tb y g v5 0 95% ethanol (50 mL). 2. Leav slid n the KOH/ anol s uti n for 1 h. 3. Remov th slide an w shi d t l e H 2 O( × 3).Placer konfilterpa nd al ow s ide to a r d y. 4. (SO KCr and w/v) (0.1% gelatin Dsov 70 at bh water in beakr gls a in gelat disov Kep watr. 5. ° (C Put the slides into the gelatin for 1 min lift ng up and down gently to coat them. Remove the slides and al ow them to dry for 5 min. Rep at this four t i m e s . Al ow the slid to air d y, and store h m in the dark in a dust-fre box prio t use. 3.2 Prepa tion f APS-C ated li s Ar Pe Sa cw tifs h y d o xg l u pnea sr f c t h,d l n c o v a l e n t y sdu r f a c e i p o s t v h a r g e y s i o l c ap(Hb t e - 4 ) 2 distle ho f mL 50 in w/v) (0.1% se 1 Note ). 26 O’Don el a d Whitel y rial ce s t nd o be n gativ ly ch rged at his pH). APS trea m n lso eav the surface more hydrop bic than the orig nal glas . Bacteri l cel s can be deposit n he slid u ng e th r ai d y ng or a c t spin ar tus. 1. Clean micros pe lid s by im ers on i aceton , hen dist l e wat r nd blot with s ue. 2 . y a s g L e t, n r h d ) i l k f o ( w m b c u s n o f i t u ) dl v e % / rs 2 a ( p y n h i t , f m d e o l a u c h ( h 42 dna 61 ne wt b rof e utar pm o r ta ev l dna ,e ot ca ni SPA 3 . R e m o tv Ah P S / a c n oe t i s g l u an , md e r ts h l i a cn o f e r 5 min to re v xc s APS. 4. Remov th ac ne di v ual yw sht e lid bys quential y m ersing them in two dis l e wat r she . Not ha c re must be ak n to iden fy the up er (coat d) surf ce ( 5 . 3 t 7 g wip s eb aL l n D ohu r d v y . 6. Store h slide for up t 1 mo in a cle , dry Pet i d sh ( ). Note 3 se .) 2 etoN es ° d r ay i. t o h 1 f r C Note 4 se ). 3. Cel P rm abi z t on d Fixat on 3. 1 Par fo m ldehy Tr atm n r e l i p b a w u t d , o c n g h v y -z s A r e lar y when p m abil z ng Gram- e tiv bac er , is ba ed on th rig nal y p r o s e db yD L n g ta l . spond with e c l u ar RNA conte out n actively growin , log-phase c l ; howev r, this can ot be guar nte d for p bings w th a ur l s emb ag . (1) .G i v e nt h a s i t yo f l u r e c n o , FISH (12) 1. C e n t r i f u gc e l ( s v) Nonidet P-40. Spin down and resu p nd in 0.3 Make up fresh a soluti n of 4% par fo maldehy in PBS. Al ow to c ol and a d0 . 1 µ o t L f h s e l u t i o n h c e l s F . i f x o b r e t w a 3 n( 1 h do 6 v e r n i g h t a ) 4 ° C ( se Note 7 2. Centrifug cel s and resu p nd in Nonidet P-40 (0.1% w/v) so tha ther are 4 –10 ap rox 10 APS-trea d sli e ( 3 . A l o wt d r y , h e n d r a t u s i n ge h a o l : H and 96% for 3 min). Al ow t dry ( mia)n c r o f u ga enr d s u p e ni0d .m 4L , 1( %w / N o t5 e se procedu s a be t c r i d µ L of PBS ( se ). Note 6 5 ). cel s in 5 se Note 8 µ L of soluti n. Spot 5 ). µ L ont a gelatin-sub ed or O( 5 0 %f o r3m i n ,8 2 se Note 9 ). 3. 2 Acid Pret a m n Fixat on us g 4% par fo m ldehy an /or eth l is the com nly used method for perm abil z ng microbial cel s and stabil z ng rRNA prio to f l u o r h y c bw i t d m e z -g a n s erth l s , e failur to perm abil z m ny Gra -posit ve c l s ha be n w l document d (4–6,8 12 5) (6,1 6,17) . At empts have be n made to provide a gen ral Nev- . FISH and A lysi of the S ngl C 27 m e t h o df rp e m a b i l z i n ga l c e lt y p e si n c l u d i n gt h o s ew i t h g l yh d r o phobic el env lopes uch as the actinomycet s . Howev r, dif er nces in cel wal struct re betw en Gram-posit ve and Gram-negative organism make it unlike y tha ny si gle m thod cap ble of perm abil z ng a l org ni s m c a b ne l y h v d T . m e t o s c r i b n d e x tf o M m a u g h n a le .t (17) pwa r n osd tf h e m n tain g c omy et s. s e l c mt y d o a i n - 1. Use xpon tial h se c l for ptimu es lt . 2 . I m g o e b l i c a n t z s - d u h m ye b r i a n t o l (50, 8 and 95%, v/ ) as de crib a ove. 3. A l o w t a i r d y , h e n m r s l i d e n 1 min (for yc li a d cont i g ac nomy et s) ( 4. W a s h t h r e t i m e s i n d i s t l e d w a t e r a n d a i r d r y p r i o t o f i x a t o n i n e t h a n o l / par fo m ldehy ( HClat37 M ° Cforbetw n30a d5 se and Notes 10 ). 1 ). Subheading 3. 1 3.4 Hybrid zat on Once l s hav be n fix d a perm bil z d, they ar d to be hy ridized. Hybrid zat on ne ds to be car ied out in a secur ly seal d chamber to prev nt los of the hybrid zat on buf er throug evapor ti n. If the buf er evapor t s nd he pr a tion s al owed t ry out, hen sig f cant o specif b ndi g w l resu t. We routinely us a sm l airt gh sandwich box as hybrid zat on ch mber, ut a 50-mL poly r en tub ca lso e u d. 3.4 1 Prepa tion f Hybr dizat on Buf er 1 . H y b r i d z a t o n u f e r 1: . m8 L 5 N a C l 0, . m2 oL 1f g / M o f2 5m g / Lp l y v i n r o l d n e ,0 . 4m Lo f5 g / b v i n es r u ma l b o i n , m ( 0 g 1 . o/p 2 A f Ll )y 5 , ( w / v )S D ,0 . 1m Lo f 5 a to l f 10. mL Filter s il ze ach of thes comp ne ts xcept h poly A. Store in 1-mL al quots –20 2. Spot 9 F i c o l 0, . m8 L p h o s b a2 u t0 f e% m L . r 5 , M E D T A ,0 . 7 m Lo f r a i d e ,1 . 9 3m Lo fH M 2 ° C until e d . µ L of hybrid zat on buf er ont he fix d cel pre a tion ( ) A. d 1 N o t 1e 4 se µ oL pf r b se o l u t i o (n 5 0 3. Lineth cuba ion h mber(an i t gh sandwic box) thW a m n 3 M se ) Note 13 se t a k i n cg a r te o v i ad br u l e (s n g / µ Lo fe a c hp r b a e di ns t r l i e dw a t r ) .C e f u l yp a c o v e r slip ont he c l s. ter pa nd wet i h 0.9 equil brat for 30 min. Pour f exc s alt so u i n. Place s id nto chamber. h y ( bL o t re v ia f d 2 w n– z 1 g6 ) m o p e r a t u ap ro i te f r h p obe ( 4. Prio t micros p an lysi , wash lides thor ug ly at he ybrid zat on emperatu using 0.1% SD and 20 m buf er (wash stringe cy can be modif e by lowering the NaCl con e tra i , Of o r M fil° C and NaCl. P ace h mber in a w ter ba h t 37 and Notes 15 M 16 Tris-HCl (pH 7.2) in 0.9 ). M NaCl wash 28 O’Don el a d Whitel y e.g , se 1 8 )T .h i m s o e ta ld y nu i w g s h c a m b ei r s dn ref. a water bath set at he hybrid zat on temp ra u . Cover slip should float of . Remov slide from buf er and w sh in d stil e H a p r o i m n C M ( u e t . d g f Ay l , a ™ o w n p d e r Scient f , Stans ed, UK), and store at 4 epifluor scen mi rosc py ( 2 se Notes 17 ° C in the dark prio to examin t o by and 18 ). 3.5 Micros p Analy is Fol wing hybr d zation e ch sl d i mounted a if ent (C ifluor) and examin d using a epifluor sc n e micros pe fit ed with e r qui ed filter e m iw as n v d ol f g 4 t b 9 h ( 0 .u r e a s, n c i 520 nm wher as hod mine has n absor nce at 5 0 nm a d n emis on at 610 nm). Fluoresc in the mor s n it ve a n d provi es l w r d tec ion lim ts. Howev r, it is also more prone to bleaching and can be dif cult o det c ag inst background fluoresc n e. For thes reason , it s go d practice first to examine samples using phase contrast and to switch to epifluorescence only w micros pyt l a ec sofint re ca l dtoprem u bl aching dto los in fluoresc n inte s y below background lev s. Sim lar precautions should be tak n wh usi g r odam ne. For more detail quanti ve an lysi of a range of fluor ch mes and vari t ons i their nte si y, we r com end using a micros pe with as ocia t i e m d g n l y s c p a b i t er W o .u n la s y v t m e i d c r o p h a provides a t bl p form hysi log c exp r m nts, whic a be oupl d wFe iIx tSp hH r m n s the obj ctive o the d ctor obtain ph se contras , b ight-f eld, or flu esc e i n m p a r g o b ts l h, u i n m w e t a k u l g tiple exposur on conve ti al micros pe . Sev ral sy tem are av il b e for image p c s in , a d et il d scu on f the quipm n , rocedu s and prot c ls an be fou d i Wh tel y a . hen the sample is located. Using epifluoresc n e (19) (20) 3.6 Col ca iz t n of Phe typic and Ge otypic Char e stic in I d v ual B cteri C l s Cytochemical procedures combin g te razolium salts with or without e x o g n u si d z a b l e t r ( s u a e - n h cr d t i o u bs ae )n study he pa t rn of xidat ve m bolis n d vi ual ce s withou e n d to cul re th m bial com unit es in ma y n tural enviro m ts in wh c most f the organism remain largely unc ltured. Howev r, to exploit the poten ial of this cyto hemi al informat and to ap ly it o enviro m tal sample requi s c l e a rd t m i n v e f o r a t i n h e d v u a lc sp r e n t .S u c hi f o r m a - c h l nta ipf eo g r d m s , (21) . This a obvi us mpl cations f r the an lysi of m cr - . O( × 3). Blot s ide on filter FISH and A lysi of the S ngl C 29 tion is now readily av il b e by combin g FISH techniqu s with sub tra eenha c d t r zolium ed ct on si e both me ds hav com n g al: the descript on f a cel u r h acte is h ingle-c v withou e n d for cult re. Thus, the sub tra e- nha ced te razolium reduction is used to as ign a insolub e cel - o a ized preci ta s, wher as the rRNA probing techniqu s (FISH) are us d to c l a e g notyp s prot c l f r this ype of an lysi , ap l c b e to na ur l com nit es o a es respi ato y activ y with n rRNA-p obed c l s, i provide n xt, u il z ng the te razolium s t “TN C,” a ypic l n s i hown m o r ei n - d p t hs g l e c p y s i o g ca n l e su i g y t o c h m a l e n i q u s coupled with FISH s provide n Wh t l y e a . phenoty , since a t v ly respi ng cel s onvert azolium sa t o 1 . A 1d 0 . An over i w f the xp rim ntal (19) . Prot c ls f Fig. 2 . (20) µ m1o 0fL M TN (C se 9t 0o) N o 1t 9e 6 µ co efL l ( as p r 1 0x c e l o n sb / C m f a L i t ) r . y e d o n u g a p t l r iy si f cel on tra i s low. 2. Incubate l s for up t 4 h wi TN C ( Section 3.4 and Notes 20 se ap ro i te f r h condit s from whic t e l s w re d iv . 3. Fix the react d cel s with 3 vol of freshly pre a d 4% par fo m ldehy and proce d as in for FISH p toc ls ( se 21 ) at emp ratu ). Note 2 4. Notes 1 . T hg e l a t i b ns d ohK eC( rS O 4 ) 2 / d i s t l ew a r h t nd i g e soluti n he g latin. 2. We routin ly s ore th undil te APS s ock lution a 4 ° C for a m xi u of 1 mo, becaus w find tha c iv ty de l n s rapidly w th onger st a periods. 3 . M a r kt h ec o ds u r f a c eo t h s l i d f o r u b s e q n ta l y s i ( . e ,t h s i d f a c n g upward in the ac tone/APS soluti n). Marking the op left-hand cor e with an indel b p n serv a go d ref nc . 4. Micros pe slide or cover slip can be coated using the APS method. Cover s l i p t e n db ou f r h y s i l o g c a m e s u r n t o i f m b l z e d a c t r i o n i n v e r t m d c o s p w e h t r v l i o dh af e sn c p l t w y r o u g h whic to view th cel s from bel w hi e soluti n ca be plac d over th cel s a b o v e .A P Sh st a d v n g eo r l a t i nf o h sp r c e d u i nt h a c e l sd on t have to be drie to the slide but can be f ic ently deposit by centrifuga on ont he c v r slip w th n a liqu d me . 5. To facil t e sub q ent micros p examin t o , we us al y tr o ensur tha our sta ing cel once tra i s about 10 by counti g el s in a hemocyt r. 6. Note hat when pre a ing the 4% par fo m ldehy in PBS, the par fo m ldehyde dis olve only at betw n 70 and 80 drops of con e tra d sodium hydroxi e (2M) wil aid the dis olut n of the par fo m ldehy . 8 cel s · mL ° C with stir ng. Ad it on of a few –1 . This can be d t rmine –10 7 230 O’Don el a d Whitel y Fig. 2 Col ca iz t on f TN C with rRNA p obing as p rt of he FISH an lyse a m o n i c- fx ud zte r s . with TN C and view under b ight-f eld micros py. Note h dist nc am oni oxid zer col ni s tiv y same fi ld as top lef but view d th epifluor scen mi rosc py after obing w th e u b a c k t df g E il hr U o B 3 n s 8 - , N p e b . c owing t he d pt of ield an size of th am oni - x d zer col nies, d cating he left) (Top (A) (B) and cel s with an extr m ly hig -resp ato y ctiv y A m o n i a c - r ex u d t z s and the razolium dep s tion d cating s ro e pirato y c(C) . (Top right) The FISH and A lysi of the S ngl C 10. 1. 12. 13. 14. 15. 231 7. Prol nged stora in f xat ve r duc s the quali y nd stre g h of t e luor scent signal. F xed c ls may be tor d f up to 8 wk i h nly s g t de rio at n f signal (2 ) hyde is a po re ch i s n e a hig l ve of aut l resc n a be o s rv d. 8. We routinely spot two sample for probing per conve ti al micros pe slide howev r sp cif sl de ar v ilab e th al ow up to 15 separ t s mple to be proces d n o e slid ( .g , ICN hemicals, OH: Mu ti es l d ). 9 . N o t e h a f rs m c e l ,f i x a t o n 1 %p r a f m l d e h y a n o tb e c s a r y and th cel s an be pr a ed for FISH simply b ethanol dehy ration. I de , B r a u n - H o w l e t. d Micro us l te dehy , r sulting ower The l ngth of trea m n time n 1 o r s gt ah pbne u i c M f y m d l. o r g an ei s -m e r a l y q u i e db t w n3 0a 5 m i h y d r o l s w h e a c l s u h subtil wer nd perm abl in o y 10 min. a c t hi en o mf y Fs r l u d , e ci n t s m h r yea d t h c e l w s r i m e d1 n sion exc d 50 min. A pos ible xp an tio f r the d cr as inte y is tha longer xp su to acid use grad tion f he targ RNA. Thesuc ofthepr c du s e rib d n some ext n , on the chain length of the mycolic acids. Organism such as Tsukam rel from 47– 6 carbon atoms, could not be perm abil z d. Howev r, fortui m (60–9 carbons and 46– 0 carbons, resp ctively), wer moderately perm able, sug esting tha t c w e S l rn os a f. i any of the pr c du s e crib d. am Wh p r y u ev lk t b s n o id f g z a , e r previously the fac o the slid o wh c t e l s w re ad . I bf u l e ds o r wm h an p l y i tg he b r d z a t i o n u f e r , h cy a bn m o v e d by “po ing” w th a fine syr g ne dl . The optimal e p r tu fo hybrid zat on is a functio f he bas comp it n of the probe (dis ociation temperature l a r g ec o n i st b u a c e r i l no g ,a sw e l t h p r n c eo fs m a l r s , o e u b a c t o r i ng l nies. ( B o lt e f m ) a probe specif for am oni oxid zers, as ign thes organism to this specif group (bot mrigh ) w i t c h o l e n p y s , b d i c a t l o n g z r e w d hf ia ts b o c m n ten , or lim ted prob ac es i l ty w hin e d s col ny. . Glutar dehy or formaldehy as a sub ti e for par fo m lde- s u g et h ao mr n i s( (23) S t a p h y l o c u sr e , ) may ev n r spo d a vers ly to fixa on i 1% par fo m lfluoresc n wh probed. in s tu M HCl dif ers ac ording to he c l env lop Bacil us M H C il n c r e a s d , o n l y c r e a s wd h in m r - and Notes10 , Gord na and , Nocardi 1 se m tod p n , lr ea cn hg t m i y s w od Mycoba te- and w,h i cp o s e lso n g ecrh a imny c o l a t e s N o c a r d i as t e r o i d e s chain length alone does not explain the dif er nc s beL a c t o b ipl uns a r m p e r b m c n oa u t li d s z e g T d ) and the complem ntary target c a l n o d s e y i w t ah r m g o n - x i d c z o e l r (D) l caT orh ge (n pi s t wa u br e) q pn t ol i y hd .Note,h w v r t eh og ne usdi tr b on f lu resc n 23 O’Don el a d Whitel y -dnib c f eps on y a ezim n ot yl acir pme d n t eb dluohs i T .ecn uq s ing of the prob wing to misache btwn he prob and its arge squnc forguidehA. (2) T d 2iscaluton ° 4andTorAevyfC ° GevryfoC of temprau h cngi to alerv An oiguctd. he wn bas C or hybridzaton, if the ybridzaon tempu is to hig based on the prob (>5 temprau disocn hybridzaton e° in formade aditon he is C), bufer. Sinc omad s trong deau, i wl dsrupt hige-o c tureswihnRNAmolc(.g,aps)nd b i l t y os e q u n c e sw h i l ea s o l w i n gar e d u c t i o n h e increas probe ac es ihybridzaton equ 0.7 a for cunts mide 1% of atn he grl, I tmpau. ° reduc- C tion in hybrid zat on temp ra u . A compreh nsiv discu on of methods for (195)al.etAmnbyprovidschzg (4) 1 6 . A l t h po ru g c e a d v f i lt s h o b r c ae n i gf u l o t d e 17. p r o b e st,h a f bne sot a i fdr cmo e i aslu p e r a dly b e . To end-lab pro es an mi ohexyl ink r (Am o-Link 2, Ap lied B osy tem , Foster City, CA) is incorp ated into the olig nuc e t d at the 5' end during synthe i .T fluoresc ntdy (formic b alstudie , h s u al yf oresc inisothiocyan te hydrochloride or te ramethyl-6 carboxy-rhodamine) is then r e a c t d w i h t e p mr i cols. Any uni corporated fluor chrome is removed from label d oligonucleotide using an olig nuc e t d s are then purif ed by thin-layer chromat g phy using a Sureo B U(l C i K a ) g m S . b cp n r u P e d f t , Label dprobescanthenb dispens dan storedinsteriled ion zedwaterat –20 ° Cuntilne d .Wehavek pt robespre aredinthiswayforupto2yr. W h e nu s i gF I S Ht e c h n i q u e s ,i t sv i t a l h tp r o e rc o n t r o l sb ei n c l u d e s i n c e n o s p e c i f bc i n d ag n d u t o f l u o r e s c n ae r c o m n W. re o u t i n e l iy n c l u d e , in our an lysi , both a posit ve and a negative control. For the posit ve control, if probing bacteria, we use the eubacterial probe EUB 3 8 (5'GCT AG T- 3') to de rmin whet r c l s a e p rm bil zed p cr a e n l t s i h u y k fo d bW w g . p e r b h w t m y i o l a n cd u z s t i o n s . A sn e g a t i v c o r l s ,w eu ap o b i t h es a m q u n c e st h a r g R N A ( n co m p l e t a r i y , h e f o r n b i d g a)ws e l f i x cd ps r e t a wd i h RNase I (10 µ g · mL cel u ar consti ue . For natur l sample work, counters ai g of the bacteri l cel s with DNA-specif luor ch mes t a h ve mis on wavel gths ou ide t l h a o befs id g n u c l e o t pd r m s v a i cfn e wlg t h f v ai mo ne dl w r , p t cv a ei o hld f u r n t D N A - os fp e c u l r i m b T ah d . o n t s v e for l cating cel s becom s e ntial f olig nuc e t d probing s perfo m d on polycarb n te o c l u se nitra f l ers ary amino group ac ording to he manufacture ’s prot olig nuc e t d purif cat on cart idge. Ful - ength, labe d –1 , 1 h 37 ° C) to as e no p cif robe ind g to her (24) 18. We r com end xamin g pre a tions m ediat ly, but when t is not p ssible d s can be tor d a 4 ° C in the dark fo 4–6 wk. . . FISH and A lysi of the S ngl C 23 19. In our orig nal pa er we used INT ( p -iod n tr e azolium violet)-f rmaz n to measure sub tra e- nha ced reduction. Howev r, INT-formaz n and sev ral other f maz ns, i clud ng CT -formaz n, re xt ac ed by the anol used to dehy rate cel s prio to FISH. This makes col a iz t on more dif cult and f o r im t a eh z sc bn l d oq u y i g r e s s u b a Fe r Iq pt S h H i o .nw y g c l d s e i r o t The use of TN C (whic is not solub e in ethanol) or altern iv perm abild e i h zay n t rs l o c ( p g m ue i bn h y t s trea m n and polyeth n glyco ) could cir umvent his problem when using INT. Cur ently, TN C is not c m er ial y v i able, nd ther fo , its u e i lim ted b caus it m be ch mi al y s nthe iz d. 2 0 . E x o g e n u s b t r a e c n d u r i n gt h e c b a i o nw t hT N C oa s e s u b t r a e n h c d z o l i r u e m c t p I an . l e o , t r w i s n Th l N y C ad it on sh uld a o be pr a d. 2 1 . T hi en c u b a t op r i c ds at loh en y s R . p i d lg r o w nc e m sa y l requi an cub tion per d of the rd of min befor sub tan i l deposit n o b s e m r l Fv g w d . c y ui n t a o s re m , pl c u b a t ei ro n e n t s o a u dh r x b q 4 m v y p e z o l d i u m s t n . The incubation period should be empir cal y det rmined by removing serial sample at dist nc time points and micros pical y che king for te razolium d e p o s i t S u bn . a e x rl c fu o a m d ez p n s i gt o r a l d y c et s lysi ha oc ur ed an th e r action has be n p rfo med for a longer p iod than ec s ry. 2 . Since FISH requi s tha cel s are fixed and perm abil z d prio to probing, it must be p rfo m d a ter h azolium red ct on as y. Acknowledgments We ar g teful o he Natur l Envi o me t R s arch Coun il, the Univ rs w u o p r t k f h . i W a T Sn e g v d N y w u c p , o s f t l e i References 1. De Long, E. F., Wickham, G. S., and Pace, N. R. (198 ) Phylogen tic stain : ribos mal RNA b sed pro f the id n f catio s ngle microb al e s. enc 243, 2 . A m S I a p . R n r , i g L N e u d w G W o D c . H ht , lz ( i 1 f 9 K e r ) e n d bo as ucy tm ri l . f p h g e n a d s i t u I f c a o n 351, 6 – 4. 3. Giovan , S. J , Delong, E. F , Olsen, G. J , and Pace, N. R (198 ) Phylogenetic group-s ecif olig deoxynucleotide probes for identif cation of single microb al- e s. 4 . S L c u H A Wh d I. m l K w a e R i , n g f ( 1 r 9 P 5 y ) o e n i t d c f a i o n and in s tu Rev. 59, Sci1360– . Nature J. Bacteriol. det c ion f d vi ual m crobi el s withou c l ivat on. 143– 69. 170, 720– 6. Microb l. 234 O’Don el a d Whitel y 5 . P h (y 1s 9i - 6 ) G . O ’ DA o n a e dl ,M T E. m b y B o t h J, M a c n S u. g o 10. 1. 12. 13. 14. 15. 16. 17. cal stabil z on a d conf al micros py of bacteri on ro ts u ing 16S rRNA targe d, fluoresc nt-lab ed olig nuc e t d probes. 279– 85. 6. Hahn, D. Aman , R. I Ludwig, W. Ak ermans, A. D L , and Schleif r, K. H ( 1 9D e 2t ) m c i o fr n g a s t le m t a r fg le u o d s c n t y - l a ob e i g d n u c t e s . 7. Frische , M. E., Florian , P. J., and Nierzw ck bauer, S. A. (19 6) Dif er nt al s e n i t v o1 yf6r SR N A - t a g e o dl i n u c e t dp r o b u s ef l r c n e situ hybrid zation is a result of ribos mal higher-order structure. Microb l. 42, 8 . A m a n ,R .I K r u m h o l z ,L . a n dS t h l ,D .A ( 1 9 0 )F l u o r e s c n t l i g o u c e t d d e t rf mo i c pn ha lw y s v g , de t b - u i s in m crob l gy. 9. Le , S. H , Malone, C. and Kemp, P. F (19 3) Use of multip e 16S rRNA-ta get d fluoresc nt probes to increas signal streng h and measur cel u ar RNA from natu l p nkto ic ba er . Schon uber, W., Fuchs, B., Juretschko, S., and Aman , R. I. (19 7) Improved sen it v yofwh le-c ybrid zat onby hec m inat o fh rse adi p rox dase-l b d olig nuc e t d s and tyramide s gnal mp if cat on. Microb l. 63, Aman , R. I , Zard B., Stahl D. A , and Schleif r, K. H (19 2). Identif ca o of ind v ual proka y tic cel s by using enzym -lab ed, rRNA-ta ge d olig nucleotid pr bes. P o u l s e n ,L .K B a l r d ,G . n S t a h l ,D .A ( 1 9 3 )U s eo fr R N A l u e s c n situ hybrid zat on f r measuring the activ y of single c l s in young a d established b of lms. J u r t s h B R.l k ,i cM e JF Ea r oG nu x d t s h ( P1 k.9 , R 2 )p i d situ h y b r i d z t a s e c g R h of m N 1 u n A 6 r S q i tia ng he clos y re at d g m-posit ve rganism lus macer n . Wagner, M. Aman , R. I , Kampfer, P. As mu , B. Hartm n , A. Hutzler, P. Springe , N., and Schleif r, K. H (19 4) Identif ca o and gram negative filamentous bacteria in activated sludge. Microb l. 17, Wal ner, G., Erha t, R., and Aman , R. I (19 5) Flow cytometric an lysi of activated-sludge with rRNA-target d probes. 1859–186 . H a h D nA . ,m I R Z e d y ( J r1 9 W 3 h ) o c l b i d z a t o f n strain w h fluoresc n labe d or ig x en lab d 16S rRNA- ta ge d oligonucle tid probes. Macnaughton, S. J., Odon el , A. G., and Embley, T. M. (19 4) Permeabil zation f mycolic a id contain g actinomycet s for w i t f l u ho r e s c n t l yo - i ag b up r d e s . . 26, J. Microb l. Methods h y b r i d z a tw R o N nhA - s i t nu 879– . 138, M iG ce rn o.J b l in Can. J. 106 – 7 . 762– 0. 172, J. Bacteriol. 193–20 . 10 , Marine Ecol. Pr g Se . Ap l. Enviro . 3268– 7 . 30 7– 1 . 58, Ap l. Enviro M c biol. in 1354– 60. 59, Ap l. Enviro M c biol. in and Bacil us po ym xa 58, Ap l. Enviro M c biol. Bacil- 2571– 8. det c ion f in situ Systematic Ap l. 405– 17. 61, Ap l. Environ. Microbi l. Franki Ap l. Enviro M c biol. 1709– 6. 59, hybrid zation in situ Microb l gy 140, 2859– 6 . FISH and A lysi of the S ngl C 235 1 8 . S n a i d r ,J . A m n R I . ,H u b e r L d w i g ,W . a n S c h l e i f r ,K .H ( 1 9 7 )P h y - 19. 20. 21. 2. 23. 24. 25. logen tic a lysi and Enviro . M c biol. Whitel y, A. S , Odon el , A. G , Macn ughto , S. J , and Bare , M. R (19 6) Cytochemi al co izat on d quanti o f phenoty ic and ge otypic hara c t e r i s n d v b ua c lt e r i - l s . Whitel y, A. S., Hunt, A., Grewal, R., and Bare , M. R. (19 8) A Mn ia cl o ry f s b e Grib on, L. T and Bare , M. R (19 5) Oxidat ve m tabolis in oncult rab e H e l i c o b a t p yr i razolium reduction a d ig tal image-proc s ing. 3 79– 84. Stahl, D. A. and Am an, R. I. (19 ) Dev lopm nt and ap lic t on of nuclei ap b cr io d nt e s y l m a S i q cnu s e , H y b dr g a tT ie oc nh q u s B a c S t y e i s r n W ( Gm l o J d h ,f . Mk ) b E a w n t Chic est r, Engla d. Braun-Howland, E. B., Daniels n, S. A., and Nierzwick bauer, S. A. (19 2) Dev lopm nt of a rapid method for det c ing bacteri l cel s rRNA-ta ge d prob s. Lim, E. Caron, D. A and Delo g, E. F (19 6) Dev lopm nt a d fiel p q u a n t e i x m f c o r h v s p d g a w l tb i e olig nuc e t d probes. Brock, T. D (196 ) Clif s, NJ. ident f ca ion f bacteri n activ ed slu g . in s tu Ap l. 28 4– 96. 63, 1873– 9. 62, A pE n lv .i rM o c b l . Dig tal Image e S d Y c W F a s o h i . N n r u (l , ) k t w y H M and celstudiby rate- nh c dt V i b r v uo l n f c s 61, Ap l. Enviro . Microb l. in situ 13, Biotechn qu s Ap l. Enviro M c biol. Princ ples of Micr b al Eco gy. 928. 62, 14 6– 23. Prentic -Hal , Eng ewo d using 16S Det c ion, As e m t and S i g of Bacteri 237 61 Specific Detection, Viability Assessment, and Macromolecular Staining of Bacteria for Flow Cytometry Jonathan Porter 1. Introduction D i ar neb c lt oy fs um v p n r ie b l t s a D c . e s b t a n c m f hi o r l q x u y p - e s n gist , but is prone to er o , is time-consu g, and can be tedious. In many situa on , the proces can be autom ed using flow cytome r (FCM) FCM can be consider d an alternative and complem ntary technique to micros py, and it c lso ex nd th ra ge nd v lu of micr s p al me s u r e a m t l qb n o c w h i , gf d y v s e at i m e , v r ys c o n d .D a t h u sb eo a i n d m l o s fc e ,w i t hu ful informat acquired for ind v ual cel s. The opti n of cel sorting also a l p ho yw s i ec r t d f n aAl s . p c h u y eo i some and c figur t s,me o n c l a d FCM of basi c l ap lic t ons e vir m ntal b c erio gy a ven i Chapt r 5. Suc es ful ap lic t on of FCM to enviro mental bacteriol gy gen ral y rf el qu o a i d s ct b n v e f( r la c o sk m )g r o u n de v t s( i r o n m e a lp t i c u e s ,m a h n o i e r t a g c l s ) . Dyes or probes of gen ral inter s al ow to al cel enum ratio (DNA stainsi pn e g c ) u , mlf r (a o t b n d c y e s ai v g b) , l t y as e m nt, d physiol g ca es m nt ( ai g cel s for t a nuclei d and protein conte ). The wide range of sample tha may be encou t red a n l ye - c h fp ordvw it b a mn e l u g y s s iH o .w e v tp rh , c d u s l i n e b q u tar o y s nh d p l u s ve a if t d l r p o n g m s a it f e d lc v , b t y a s e m c r n o d t , l u a i P c g w e m. r o s n t d l y From: (1 , 2) . Methods in B otechnol gy, Enviro mental Monit r ng of Bacteria Vol. 12: Edite by: C. Edwar s © Human Pres Inc., Tot wa, NJ 237 238 Porte optim zation for each environment, and can also be asily ad pted (e.g , for dif er nt fluor chromes and for dual- abeling procedures). Ad it onal y, the range of fluorescent probes av ilable continues to expand rapidly (3) .This mproveschoiceofdyeforeachap lication,butmeansthat prot colsmayberapidlyoutdated.Itisworthw iletospendtimed signing the experimental procedure careful y ( g n is le u bq a h c t r o , d e u s n t a h y d m o r c l f being used are suitable for the instrument, the environment from which the cel s orig nate, and to ensure that spectral overlap does not hamper det ction. ), especial y if dual- Fig. 1 2. Materials 2.1 FCM 1. Flow cyt me r wi h ap o ri te f l r b ocks. 2. Filter ap tus wi h 0.1- and 2 3. -1.0 hguor t semi rht sael d r tlif ,d u htae S 4. Filter d buf ers and stock soluti n ( buf ers include filter d dist l e water, phos ate buf er d saline ([PBS]; 8 g NaCl, 0.24 g KH water; pH to 7.4 with HCl and make up to 1 L), Tris buf er (Tris-[hydroxym e t h y l a] m i n o m e t h a n e , . g 1, m0 pH adjusted with and 1 m M EDTA pH 8.0) 5. Enviro me tal c su pen io ( µ m filters o b th large nd smal vo u es. 2 PO 4 , 0.2 g KCl, Na µ ( sretlif m and Notes 2 se 2 HPO M d, i s o l v ie dn s t i l e Hd 2 HClbefor makinguptof alv ume),TE(10 aO n d Tris-HCl M ). Note 4 se 2 .) dis olved in 80 mL dist l ed 4 o 1r 0 m M dna 1 setoN es 3 ): Useful soluti n and 2. Total Ce Enum ratio 1 . 4 ' , 6 - d i a m n o 2 - p h e d i y s l t m H n g ( o/ [ c D kL1A P I ] , 2 ( se Note 5 [dH O 2 ). 2. Filter d fo malin (38–40% formaldehy ) ( ). Note 6 se 2.3 Specif C l Enumeratio 2.3 1 Specif C l Det c ion Us g Fluore c nt In Situ Hybrid zat on (FISH) with Ol gonuc e tid Prob s 1. Label d o ig nucle t d probes ( 2. Freshly p ared, filt red pa form ldehy (4% in PBS) ( 3. H y b r i d z a t o n s o l u t i n : 0 . 9 do ecyl su fate (SD ) or N nidet P-40. se Note 7 ). se M NaCl, 20 m M ). Note 6 Tris-HCl, pH 7.2, 0.1% sodium 2.3 Specif C l Det c ion Us g A tibod es 1. 3% Bovine s rum alb in PBS ( A/ ). 2. A n t i s e r a a g i n s t t a r g e t c e l s : I f p r i m a r y a n t i s e r a a r e n o t l a b e l d , t h e n a fluoresc nt y labe d s con ary tibod s requi d ( se Note 7 ). O]) Det c ion, As e m t and S i g of Bacteri 239 F i g1D . a r s mh o w i n g p e c t sfh x r i m e n t aw lo kh u sbc e n i d r careful y b ore suc f l y ap ing FCM to he s udy of envir m tal b c eri . 2.4 Viab l ty As e m n 2.4 1 Membran Pot i l Us ng Rhodami e 123 or a Cy ni e D 1. Rhodamine 123 (Rh123) (stock 0. 5 mg/mL in PBS or 1X TE buf er) or dihexylo ac rb yani e (DiOC [DMSO] or abs lute hanol). 6 [3]; stock 0.57 mg/ L in dimethyl sulfoxide 240 Porte 2. Valinomyc (st k 1 mg/ L in DMSO or abs lute hanol). 3. Gramic d n S (stock 1 mg/ L in DMSO). 2.4 Membran Pot i l us ng Oxo l 1. B i s - ( 1 , 3 d i b u t y l a r c m i( o e dD x ) h B n l Eugen , OR; stock 0.517 mg/ L in DMSO). 4 2. Heat d con r l e su p n io (80 M o l[ Pe3 rc] u; b a s , ° C for 10 min). 2.4 3 Intracel u Enzyme Activ and Membr Int g i y 1. m 1 kcots ; remosi dexim ,]ADFC[( eta c id niecs roulfyx braC )6( 5 2. C h e m c r o m e B ( b a c t e r i a l v i a b l i t y s u b t r a e f r o m C h e m u n x S A , M a i s o n Alfort, F ance). M .) e n o t c a n i 2.4 Nuclei A d Dy Exclusion A ay 1 . d H i nm g P / r o L1( p 0s t cdu ke 2 4 - [ 3 o mr e t h O y ) l 2 , - d i r o ( b e n z - 1,3-oxazole)-2 methyliden ]-1 (3'-trimethylam onium propyl)-pyrid nium di o e ([PO- R 3]; obtained from M lecu ar P obes a 1 m tion DMSO) or BacLight v ab li y tes ng kit (Molecu ar P obes). 2. Contr l ce su p n io ( M se Note 8 ). 2.5 Cel Macrom ul C nte A alys 2.5 1 Ribos mal NA C nte 1. Propid um e (stock 10 mg/ L in dH 2. RNase A (10 mg/ L stock, b iled an f ter d). 3. Fixat ve (formalin 70% filter d hanol). 2 O). 2.5 Total Ce Prot in C e Fluoresc in th o ya e (FITC) stock 1 mg/ L in aceto ). 3. Methods 3.1 Instrume S p, Calibr t on, a d M itor ng 1. Exact ins ruct o wil vary with eac instrume . Howev r, focusing a d c libration mus be achi v d us ng ta d r p ticles ( 2 . S a cv e l i b r t do an c ih m e s u apt n, od f i r m c s pn eg odical y throug a seri of measur nt . Often this may be achiev d by i n o c u l a t s k m g p w i oe n b f h r a t d s i e , b c l o h p monit r g and cou ti g. 3. Det c ion of bacteri requi s stringe filtra on of buf ers and fluids to ke p background particles to a min u . Al owing growth of bacteri with n the instrume bing w l a so interf with sen iv measur nt . 4. Wash nd flush t e ins rum t fl id sy tem hor ug ly. Rinse thor ug ly with f i l t e r d s i l e w a t r f b l e a c ho rd t g e n c l a i g .H v e s t a n d r c l - se Note 9 ). stock solu- Det c ion, As e m t and S i g of Bacteri 241 i n ag sd h u t o w p r c e d u t h a l s e r m u ft o l w M. n i ur s e t o d n i f y those a d not leav h sy tem cl an. 5. When an lyzi g cel s, it is pref abl to ke p the rate of an lysi at or below 20 cel s/ (althoug some achines may cope with far hig er an lysi rates, esp cial y if upgrade ). Rates uch as this reduc the coin de of two cel s pas ing throu e s n i g re on t g her. 3.2 Total Ce Enum ratio 1. Dilute or c n t a e s mpl unti a co entr i of ap r x 1 6 × 10 achiev d ( se and Notes 10 2. Fix sample f n c s ary ( 3. Ad ye from st ck o a fin l co e tra i n of 0.5 4 . I n c u b a ti dh e ( fr T.1ok cs n i t m ba ey p r o v nd t i m z a - ). Note 6 se cel s/mL i ). 1 µ g/mL ( ). Note 5 se tion d f er nt vi o ments, bu ho ld a w et c ion fr m the i s run.) 5. Analyze b FCM. 6. Contr ls may be pr a ed by inoculat g sample with know numbers of cultured c l s prio t f xa ion. 3. Specif C l Enumeratio 3. 1 Fluoresc nt Hybrid zat on In Situ 1. .SBP ni edyh lamrof p dera ylhserf o l v 3 ni h 61 rof n is ep u l c xiF 2. Wash nd resu p nd cel s in PBS to a fin l co entra i of ap roxim tely 1 10 9 cel s/mL (if pos ble). 3. Ad an equ l vo me f absolute hanol (t e c l s are now ady for FISH, but are st bl and c be stor d a –20 4 . a Ap d 1 r o x water bath (actu l temp ra u wil dep n upon the probe being used; for the gen ral ub cterial p obe, Eu 3 8 hybrid ze at 46 5 . A d b e t w n0 . 1a d 5 g / p e r(f ob m d t i s e p ad 1–5 ng probe/ 6 . W a s hc e l i fn s a r y , n d e u s p i n c e - o l d ,f i t e r b u f .I n ow a s h i g step ar to be p rfo med, a 1 mL of ice- old, fi ter d buf er and hol n ice until a ys . o n u m i 7 f. I l e b a t n e c s r o u l f a s h e b o r p e d i t o l c u n g e h t f i M C F y b e z y l a n A d e r t qb c p i ( uo s n 8. Averyfctionlsubhpm(.g,E38')a used b also my cels targ he to bind to n kow prbes Oth sample. r (usedbalomytrnRNc.igpf × ° C for sev al months). × 10 6 f i c x e 5 l n d0 t s o µ p r o e f w aL h m y b d i z t au o f n e r ° C). µ Lp r o b ea n di c u t f o ra l e s 6hi n w g se 3 Note p o s h t a v y e b r i d w z c l o s nI , f ) . µ L and i cub te for 1 2 h ( se ). Note 12 logic se to g ), 7N o t e S u b h e a3 d. i n g2 se 3. 2 Im unofl resc D t ion 1 . P r e p a c l s u p e n i o B S A / P t oa p r x1 × 10 ing of a t ens c b perfo m d n ixe or l v ce s. 6 c e l s / m L .S u r f a c el b - 13Note ). 24 Porte 2 . a c t i v e o n f r s a g c h i e v st uo p n l h e t o a n i b d y A antibody f betw n 0.1 and 0 tes from1/ 0and , 0 ilut ons.If heprima y nt bod is rectly ab ed with e fluor ch me, it w l be n ces ary to est a r nge of c n e tra io s to find the op imal ev . To much antibody w l give a h b ckground whe as to li e w m an po r det c ion. µ g/mL. If the ac iv once tra ion s u know , 3. Incubate for 30 min o temp ra u . 4 . F odri e c t l ya b dn i o e sw,a hnrd u p e iB S A / P a n d l y zbe F Ct IbMh fa.e c k g r o u n l d e s c h i g r , p t aw e s i n guh o r times un l background is at f c ory. 5 . Is fe c o n d a r y t i o nse q u r dw ,a h e s u p nc d l a te s w i b f o r e a d i n gt h es c o d a r yf l u e s c n ta i b o d y .A g a n , r eo fd i l u t n sw e d to be t s d, but as gen ral u e, try slight y igher dilut ons ha t ose t d for the p imary (e.g , 1/ 0– 5 ). 6. Incubate for 30 min o temp ra u . 7. Wash and resu p nd once in BSA/P and an lyze by FCM. Again, if background fl resc n i h g , perfo m e washing teps. 3.4 Viab l ty As e m n 3.4 1 Membran Pot i l Us ng Rhodami e 123 r a Cy ni e D 1. 2. 3. 4. 5. ( bufer apoit n cels Rupnd , 1 and , 3 Notes se of cel su p n io a p rticle-f 30 mL glas bot e ( Dep ndi g o dye us , ad Rh123 to a fin l co entra i of 5 DiOC ad or stck), 25adsolutin,wrkgpcef Incubate for 15–30 min, w th s aking ( Analyze b FCM ( Contr ls a e pr a ed by incubat g par l e sample with Gramic d n S (ad 5 µ L stock to 5 mL to give a final con e tra i of 0.1 f u r t h c e o n i v s a l f m y c ( n2 d 5 14 mL 5 produce t ) n 50 6of cnetrai fl a to (3) ). µ g/mL (50 Note 2 se µ L M diluton 10 in 1 a (pre µ mL).5tosluinwrkg se ). Note 15 se ). Note 16 µ M active ngredi t). A µ s m t L o5 , c k se ). N o1t7e 3.4 2 Membran Pot i l Us ng Oxo l 1. Prepa 1 mL of cel su p n io 5 m 2. Ad ox n l t a fin l co e tra i n of 1 3. Incubate ro m te p a ur fo 3–5 min. 4. Analyze b FCM. 5. Contr ls fo x n l abe ing may be pr a ed using heat d c l s (e.g , 80 10 min). Other inv s gator h ve us d Gramic n (1 M Tris buf e , pH 7.5 ( µ M (5 se ° C for µ L stock/m ). 3.4 Intracel u Enzyme Activ and Membr Int g i y 1. Resu p nd c l s i ap ro te buf r ( 2. Ad fluor genic sub tra e (10 se µ L stock CFDA/mL or 10 stock/mL ( Note 18). µ L stock). se Note 20 ). Note 19 ). µ L Chemc ro B Det c ion, As e m t and S i g of Bacteri 243 3. Incubate for 10–3 min ( 4. Analyze b FCM. 5. Contr ls may be pr a d using formaldehy -fix d cel s or h ated c l s. se ). Note 21 3.4 Nuclei A d Dy Exclusion A ay 1. Resu p nd c l s i ap ro te buf r ( 2 . f c i o p n r a F l e t 5d 0 y u m f , n se ). Note 2 µ (g2/0m L mL su pen io ). For PO- R 3, ad ye to 3 the BacLig t k , fol w manuf ct re ’s in t uc o s ( µ M se 3. Incubate for 10 min the dark. 4. Analyze b FCM. 5. Contr ls may be pr a ed using formalin- xed c l s, heat d cel s, or includ g octan l (10 µ s t o cLk / final con e tra ion. If using ). Note 23 µ L/m ) in the cubation m x ure. 3.5 Cel Macrom ul C nte 3.5 1 Ribos mal NA C nte 1. Fix cel s n 70% ice- old than ( 2. Wash nd re usp twice n PBS. 3. Ad pro id um o ide t 50 se rega din f x t o in e ha ol). Note 6 µ g/mL (if th s re ults in h g back round stai ng, reduc th pro id um e to 15 4. Incubate i h dark fo 45 min at 4 5. Analyze b FCM. 6. Contr ls for this w l inc ude RNas digest on ( R N bA i d o e c t h rw m f n a l s v ei t g ( se µ g/mL rathe n i troduc ng a w sh tep). ° C. se Note 13 ). Propid um odi e Note 6 regarding fixation ). Note 24 3.5 2 Total Ce Prot in C e 1. F ix cel s in 70% ice-cold, filtered ethanol ( i n ethanol). 2. Wash nd re usp c l twi e n 10 m 3. Ad 2.5 4. Incubate i d rk fo 10 min. 5. Analyze b FCM. 6 . e b n d z ci tyw ag mo u p s l , h f a y n d i o r C c u e tl s se M Tris-HCl, p 9.0 µ L / m s t o c kF I T C l u t i o n . destroy the cel s. Comparis n of sample fluoresc n inte s with cult red c e l s t a n d r m b yp e o s i l n m c a e s T . h i t y p o f a n h g s l b o e ( tne oc i t rp fo sn ita mre d lacim h o b tiw de al r oc 4. Notes 1. It may be h lpfu to aliqu s f ic ent sh a fluid or a y’s work bef autoclaving. A y remain g at he nd of the day c n be discar e after instrume cleani g. The flow cyt me r wil have n i -l e f t r somewh b t e n h sheat fluid nka thes ingr o .Ifit snec arytofi cus m lter,a es 42 etoN .) 24 Porte 2. 3. 4. 5. large f ctive f l ration ea m y be r qui d to main sheat fluid pres u . If the solu i n sed for heat fluid s to be us d for ting, wil ne d to c ntain s lt for the dropl t chargin p oces . Th amount f salt nec s ary fo succes ful sorting may wel be sub tan i l y les than tha recom nde by the manuf ct re . I is mple to ry he quir d b f er in the s rument a d che k forsati c oryd plet f c ion.F al y,it sbe ( fpos ible)t us h ame s u c e a p l t r hw n f oT id b . e col e t d in a separ t tank, to whic it may be pos ible to ad con e tra d dis nfecta o re t bi log ca nd hemical z rds. Routinely filter al buf ers and sheat fluid befor use. Some stock dyes may come in soluti n, or nly a few mil gra s m y be purchased to pre a stock. Since often les than a microl te may be ad e to labe cel s, it is gen ral y un ec s ary to filter such stock (althoug they should be pre a d in filter d buf er). Fo cheap r dy s an l rge vo um s, filtra on f st ck i worth ile. W h f e i n l t r s g o b c u k f e l t i o n p s a , u r q f lt e o h im r s ri untf h oe a lc v gn t i ec ras ,ph v k g o u lad yne , c ts h t i o b nf e l r a d g i n R . e p t h o s c m r e T . u , o l t i n a s r f e t d h times, and th co ainers in ed particle-f so uti n w ce. If absolute c n i g s crit al for the xp rimental objectiv s, t wil be pr f able to lim t cel pel ting/r su pe ion step . Often this may be achiev d by a m e n s d t i h p wg c l o s r e a b t u y w f p c d l i n , g prot c l tha requi s min al washing step (e.g , Hoechst or Chrom ycin l a b e i noD gfN Av , l ta ys e m nu i o gx f lr e n i cs t r F ,I S H using low ev s f olig pr be and lo g hybrid zat on imes, u ng mi al evels of anti er fo ectiv d on, a d s on). Obtain g rep s nta ive s mpl of bacteri l s in uspe ion may be pro lematic for some nviro me ts. In gen ral, marine and freshwat sample are ideal y suited o FCM, althoug crude filtra on (e.g , 50–1 m abp yer f is dno m t u a sT .h e m p l u n toc a i r gp e t c l s tha could g the ins rum t fl id sy tem . Sa ples uch a soil nd se im t p r e b ( a m u . s l yg i d n , / c e t r i u f s h g b aF C M e o , p n ) may risk crit sm over the rep s nta ive s of the su pen io . Howev r, al invest ga or e fac d with e sam proble s f r p e nta iv cel xtra ion, m F i C f c M o r , s l e p yb u a m ot r ni g dc , s f a tory cel su p n io sh uld be pos i l . M s o p r e c i l f a b h m n yu gv s C e d r o A c i n 1 . m2 g / sL t o c ik dn H stock in dH mos e in bacteri after rifamp c n trea m n . Contr l cel s produce in this way (4) sample . Diaper and Edwar s 3 42 fluoresc n with e diph nylam e thod f r biochem al d termination f DNA have great quant m yield (brightnes ). DAPI has often b used at hig er µ m nylo mesh) 3 2 2 O o) Hr e c h s 3t 4 2 / 5 (8 A rT i c Dh N 0; . m5 g / L O). The sp cif ty of thes dy al ows det c ion f discret hro- may help in estima on of DNA conte of cel s from enviro m tal (5) (6) . Thes dyes can be sub ti ed for DAPI in the procedu but cor elat d FCM measur ments of Hoechst ( r DG i N/ c AC h ; Det c ion, As e m t and S i g of Bacteri 245 c o n e t r a i( .2 sg 5 , and stored f ozen. SYBR Gre n I (Molecu ar P obes) al ows pecif measur ments of DNA in marine sample , when the hig ion c streng h soluti n may i n h b t d i n go f h e r y s .D u c ha r i d n eo a g h v r e p u t a i o nf r stain g instrume tubing a d contami g sub eq nt sample . This can be o v e b dar l Dy c t N Asim hnf g w u , is proba ly est o av id ng cr i e o ang . µ g o / ta d s m hl y D Lb i e c ) nq r u . k 6. Optimal f x on c dit s have b n wid ly ebat d; howev r, fixat on by he ad it on f rmalin s qu ck, easy nd oes t prom e c l umping as lcohol fixat n c do. Often sampl h ve to b fixed or st age prio t an lysi . Aldehy fixat on in clean bot les fol wed by storage at 4 adequ t in most i ua ons. I many c se , fixat on is perfo m d at he tim of sampling to al ow an lysi at a later date. Freshly pre a d par fo m ldehy has be n d monstra ed to be an ef ctiv f xative n ma y situa on (e.g , dual labe ingfort alce num ratio c mb nedwithFISH)a fin lco e tra i n ob fe t w a 1n4 d%/ vF . r m l e h y d - b a s f i x t v eh a b s nu g t e do cause no specif bind g of DAPI to cel u ar materi l, but this has not be n repo t d for Hoechst or Chrom ycin dyes. Nuclei acid stain g should stil prove ef ctive if the fixation condit ons are alter d. If alcoh l fixation is requi d, cel clumping can be avoide by inject g the sample g ntly into he centr of ice- old, vortexing ethanol. Cel perm abil z t on after fixat on can often be improved by inclus o of a det rg n in the buf er (e.g , 0.1% SD , Twe n-20, or N nidet P-40). 7. When cho sing a l be for specif det c ion, it s po sible tha dual beling techniqu may be usef l (e.g , to al cel enum ratio , or viab l ty as e m nt). The opti ns av il b e wil dep n o the lig t source f the ins rume t in use. Dual- ser opti ns e abl use of dyes with no spectral overlap. Many viab l ty dyes share the fluoresc in excita on/em s i wavel ngths. Thus, single light source inst ume would req i a phycoer t in labe (or quivalent) for antibody labe ing. Gen ral y, im unofl resc n outp from bacteri is dim (at least compared with ma li n sy tem ), and thus e n c s ary m chine s tt i n m cg a s y u p e r o v l ia I sp t . cb o me n a f t h r i d s , p e n g o tn h se f w a r v i l b e , u t s n i k l y h a t pe r o c d u w i l b aes t r g h forwa d hen p li d to bac eri s t may p e r to b f m clin a ytome r r e s a c hp u b l i t o n s .F I S Hr e q u i sk l e db a c t r i ,w h p e c l u d s a b e l ing w th a vi b l ty d e, althoug s me viab l ty d es ar fix ble n place using an ldehy fixat on/cr s li k ng step. How ver, d t min g to al nd specif cel numbers u ing uclei ac d n olig nuc e t d probing w th a single i ht source inst ume ay stil be pos i le by us of a h pten ( .g , di oxygen or biot n) l ked to h lig nuc eot d pr be inst ad of irectly onjugated fl or o c h m e .T p r o c d u ew l t h nb o y r i d z e nucleotid hapten using (e.g , phycoer t in) im unofl resc n , and det c to al cel s using a 48 -nm excit d nuclei acid ye (e.g , SYBR Gre n 1 from Molecu ar P obes). Autofl res nc from phot syn e ic p gments hould a so ° C should prove i ns t u ,d e t c b o u n l i g - 246 be consid r when d sig the fluor c me o binat s. 8. Contr ls for this type of as y invol e perm abil z ng cel s to al ow fre dye pas ge acros the me bran . This may be achiev d by fixat on in formalin, heating, or d it n of c a l. 9. Clin ca FCM often u il zes chi ken r d blo d cel s a st ndar . Howev r, fo b a c t p e ir so l y f a u btsn oei rl m c e ni t r o s p h ew ,i c a r v e i l s nb o g f z d u r e c n i t s ( e u m b f o l r s cein qu vale tsp rb ad).Usingbead of p r ximatelyb c rial e s z wil help instrum ep. Instrum ha utilze hg-nmrca petu objciv lensforightcayqup lwveris 0.5(eg,badsmlnurt tha cels for alws Thi beads. lrg use to prefabl may it syem, jt-inar focus.inrematghpdlyw 1 0 . a p r nDo i c l ed u t ( s .wf y ga z m k, pn r i l u t e cel s in f lter d ake w t r). Dilut on s ep ar lso an ide l way of altering salt con e tra i s, whic may af ect some dyes bind g to DNA ( such a es, u an p ro iate d lu nt. O her nvi o me ts ay requi sample con e tra i ( 1 . W hc eo n t r a ic e glf s o m n v r e ts a lm p c b yn r i f u g a t o n , fraction f hem ay b dif cult o pe t. If cen ri ugat on is be u d, it may be pr f a le to r m v 90% of the v lum by gentl pi ng from the su fac layer to ry to avoid losing cel s tha have con e tra d near the bot m of the p l s e av t Fro m h g f u ni . , y b l t e flow i tra on (describ n Chapter 3) m y be us d. 12. When pres nt a con e tra i >0.1 notlim g( .e ,th r ismo ep b thanri os me p vid ngthe o al umb r e xa ncp1 do t r l s f µ b ahcg n i / o k um sL t r e) p w d f c n g bai, yu sl e f l r u te o ha c Ds i b v n p . y m d z e a x t i W o r l n s a el t. (7) t i o ns m a e f p w lb r h d o , c e n t r a i o w l s h n g e r h y b r i d z a t o w nm efs b h i g T d a . p r o s t c h n g le y m mend (Porte , J., et al., unp blished dat ). Hybrid zat on stringe cy can be increas d by the a di on f rma ide n th buf er. 1 3 . R d N i a rg s e b o t f n m A c ph l x y , d s e sev ral invest ga or . Howev r, other dat (Porte , J., et al., unp blished dat ) sug e t tha ef ctive and rep oducible digest on requi s large amounts of enzym a d ext n i cubat on peri ds. T eatm n of ixed, wash cel with 1m g / Lo f i n a lc e t r i o n z y m ef rp i o d s 4 – 1 6ha t3 7 u s e (d P o r t J, . a l u n p b i s h e d a t ) S. u c x e n d t r a m dsno t f e c cel integr y, but have on oc asi n be n oted to alter (increas ) cel forwa d light sca er (Po t , J. e al , unp b ished at ). e v i t a g n - m r G s a e h w . S4 B1 P n i y l r o t c a f s i l e b a y r e n g a i t c b e v i s o p - m a r G m 1 gnisu dev hca b n c sihT .pets noi az l b emr p a e iuq r a etc b Porte µ forHwev,suitabl.dm) Note 5 se se Note 1 ). µ g/mL, the amount of ligonuc e tid s × 10 6 pc hro Vi n egb )y . t a ( 2 o 0 n s , r te oc m i n d r e ap t oc b n r a t i o s nh d ey rb i z a - ° Ch a v eb n M ni ATDE ). In Det c ion, As e m t and S i g of Bacteri 247 n o e A s , T a l bt G h E r c. fi) x I D g e ( h u t b m 1.0 ta desu .snoi ep u s o neg r t h of desu b l ohs pet noi az l b emr p a hcus o )8( ,ATDE fo ecn s rp eht ni l ew bal os airetc b vi sop-marG . M 1 5 . C e l a b i wn g e r a l o cy wu i t h m1 5nT . e a d y w os r lk i g h t vR r b t ahm u F 1oc p f s i 2 e 3 d , l n . y g al ow 30 min. For cyani e dy s, al ow 15 min, but increas this o 30 min a protein-c a g su pen io . 1 6 . U s t Lw c h a oi e b p n dg . l v u - 17. fic ty an be confirmed using the contr ls to ensur tha me bran poten ial s being measured. Some previous work has used higher concentrations of rhodamine (9) equil br um. Other repo ts avoid ng wash tep . Valinomyc sel ctively transpo t K poten ial s a functio f the K cel s in low-K ers. Hyp olariz t n ca be hi v d n PBS. and reli d upon the use of ext nsiv wash step to provide dye have advoc ted lower con e tra ions, thus (8 , 10) + + + + /hig Na acros the me bran until me bran gradient. Thus valinomyc wil hyper ola iz + buf ers and epolariz cel s in low Na + /hig -K buf - 1 8 . p er o b m f u L a it s nh y d w l x , v g e m s a t n y d b u r f ie o s c , t n l hy r I b u. oa e fn cs d k r e q u i m n tf o rs a p l e t a m n h sl e dt o u g i n st h a e u p r i o dye for viab l ty s e m nt i some ap lic t ons (1 , 12) . 1 9 . T h i a s p r o vc e y b u s p t r o d e b , u m p r a i l z t o s n e p h u l d c T of w ni(e 0 A a. t mu l1-h s %2 r kd b g ) ef ctiv labe ing of lake w t r bac e i Howev r, a l be ing uf er is p ov de with e Ch mc ro e B kit (Chemun x t a w h S l n o i A s d r p) e T k c . u x a i t y n s b e o d l , s c u e p v t l r (i w h a n 1 /b : m ) o x f y d e t (14) S a .m p h lcbe sn t o d u ry i h ela ,t o u g m p r a ture f cts may be in mal so e nvir me tal s p e a rh e t ( d . g 4 ,0 si toen ur dy et n io (P rte ,J. al unp b ished at ).Thel rg adv nt a g oe f r bd y h s e l u o r g n i c s t e d y h a r ne o - f l u s c e n t i l cleav dins eac l ,thusen ri glowback r undsig al .L be ing f c e y may, howev r a y c ording t he rowt phase of c l s. (13) and m xi zed viable c ounts. (13 , 15) . If sample ° C )s ,u b e q nc th i l o g m eabry q u i d f o ra en l y - 20. Using co ktails of the dy s oe n t i creas vi ble c ounts, g e tin ha h e t b a r i c os n g m l d p u f y e h t 21. Extend incubat o times do not gen ral y improve the viable cel count and may c use no p cif hydrol si f the sub rate. 2 . Often it s pos ible to perfo m thes as y in the orig nal cel su pen io , but some inv t ga ors m y p ef r to ad c n e tra d, efin bu er. 23. It is of en p ibl to sca e d wn m ufact re ’s p ot c l a ow m re s ay from an exp siv k t. 24. FCM measur nt of pr tein have b n cor elat d with b ochemi al det rminatio s (13 (5 , 16) . Thes m t od (6 , 17) requi large numb s of cel s, whic may , 15) . 248 Porte l u etn aod x r p t s i a o m n c le v A d . i t o n a l b y , c k i o gn s p e cif labe ing may prove problematic in some sample , becaus proteinac us blocking a e ts would n t be ap lic e. Acknowledgments c y d f t Fe l o iv m n w a h u s r p prot cols was provide by the Natural Environment Res arch Council, Swindo , UK. References 1. 10. 1. 12. 13. Porte , J., De r , D., Pickup, R., and Edwards, C. (19 6) Fluoresc nt probes a n df l o wc y t o m e t r y :n e wi n s g h t si n t oe n v i r o n m e t a lb c t e r i o l g y . 23, 91– 6. 2. Porte , J. De r , . Hardm n, M. Edwar s, C. and Pickup, R. (19 7) Go with f tl ho c ew y - u s m n iv r e t ac l o b i g y . Ecol., 24, 93–10 . 3. Haugl nd, R. P (19 6) cals , 6th ed. Mol cu ar P obes, Eug n OR. 4. Ste n, H. B , Skarsted, K. and Boye, E. (19 0) DNA measur nt of bacteri . Methods C l Bio . 5. D i a p e r , J . P . a n d E d w a r s , C . ( 1 9 4 ) S u r v i a l o f lakew t r moni ed by flow c t me ry. 6 . H e r b tD ,.P h i l p s J a ,nS dt r g eRE(. 1 9 7 C )h m i c a ln y s om fi c r bial ce s. Methods icr b ol. 7. Wal ner, G. Aman , R. and Beisk r, W. (19 3) Optim z ng fluoresc nt hybrid zat on with rRNA-targe d olig nucleotid probes for flow cytome ric ident f ca io f m cro ganism . 8. Mason, D. J Lopez-Am r s, R. Al man, R. Stark, J. M and Lloy , D. (19 5) The abil ty of me brane poten ial dyes and cal fluor white to dist nguish betw n viable nd o -viable ct ria. 9. Diaper, J. P , Tither, K., and E wards, C. (19 2) Rapid as e m nt of bacteri l viab l ty f ow cyt me r . Kaprelyants, A. S., and Kel , D. B. (19 2) Rapid as es ment of bacterial viability and vitality by Rhodamine 123 and flow cytometry. Bacteriol. 72, Mason, D. J , Al man R. Stark, J. M , and Lloy , D. (19 4) Rapid est ma ion f bacteri l n b otic su ep bil ty w h flo cyt me r . Jepras, R. I , Carte , J. Pearson, S. C , Paul, F. E , and Wilk nso , M. J (19 5) c r y f o t dlD b e u m wv s a i n p g t r bacteri . Ap l. Enviro M c biol. P o r t e J, . D i a p r E d w s C, . a n P i c k u p R ( 1 9 5 D) i r e c mt a s u n o f natural planktonic bacterial com unity viab lity by flow cytometry. Enviro . M c biol. Cytometry FM Ei c Sr o b l . Handbo k f Fluoresc nt Probes and Res arch C emi- 519– 26. 3, in Staphyloc us aure s 35–42. 140, Microb l gy 210–34 . 5B, in s tu 14, Cytome r 136– 4 . 309– 15. 78, J. Ap l Bacteriol. 38, Ap l. Microb l. Bi technol. 268– 7 . J. Ap l. 410–42 . J. Micros 61, 176, 8–16. 269 – 701. Ap l. 61, 2783– 6. Det c ion, As e m t and S i g of Bacteri 249 1 4 . D .e , r s t a rD ( n vi o1 e f9 l d E y c 6 C w ) . sP R , k J u r p t e . J. Fish D . Aerom nas l o ic da 19, 459– 67. 15. Porte , J. Pickup, R. W , and E w r s, C. (19 7) Evalu tion f low cyt me ri methods f r the d t c ion a d viab l ty as e m nt of bacteri n soil. Biochem. 16. Al man, R., Hahn, A. C., Phil ps, A. P., Martin, K. L., and Lloyd, D. (19 0) Growth of cytome ri pa met rs, and ultr s c u e. 17. Lowry, O. H , Rosebr ugh, N. J , Far , A. L , and Rand l , R. J (195 ) Protein measur nt wi h e Folin-phe r ag nt. Soil B o . 29, 91– 0 . Azot bacter vinela di with cor elation of Coulter cel size, flow 1, Cytome r J. Biol Chem. 82 – 31. 193, 265– 7 . CLSM of Envir me tal S p s 251 71 Confocal Laser Scanning Microscopy of Environmental Samples David Lloyd, Anthony J. Hayes, and James R. Ralphs 1. Introduction 1. Conf cal L ser S n i g M cros py f Microb l gy Enorm us techni al adv nces in imag n and dat acquis t on techniqu s, cif eps o gnileba c s rou f epocs d a r ni g u t oc a htiw den bmoc sehcaorp ni tul v b g e ah ,sm in ro v l f t u c ot lacig b .sme rp hW lait no s gr na l rutc s ev dluow evah d m s yletin f h ecnivorp f nortcel yp s im t uj a wef -elpmoc a ylno t ref o nac ypo s r im thg l fo sd htem w n eht won , ga sr ey dna evis o f mr t ni yl a ceps , rom v ih nac tub ,h orp y a nem real-tim measurnt. Confcal laser scanig microspy (CLSM) is the tsom yran i ulove t mp d ni lacitpo y s rcim e n s eht ylrae tn ves htne .yru c mo F laib s’t g ce n op f ,w iv eht y l ba fo eht fo sde n ht yb liram p nev d b sah ci w ,euq nh t w lufre op siht ls ae c in d m o t b f dna ,seluc om r htiw na b e htiw a mu in fo , tabru ep dl c -isyhp la borc m ni sec avd t rg ne s ah yrutnec sap ehT .yl mit ero b ton ygol dna ,yrtsimehco b ylts m htiw sm inagro w g ni o snep u no hcir tah s en r w g i o a er ht yl n c oM .se ar htw g i dna em ecafrus ,htwo g neir u av p d o elitas up n r yc if s e a rom . tnem oriv la u eht o c pser htiw no av luc yr t b fo sed m cit la r lacigo d htem f s a gnirb em t d a c ps ni yt e gor h f yduts eh B .rotcae kn d i s yl uo n t c r ksalf e h t ni der uoc t sm lb rp nI y a ,es c t om f eht sm inagro elb p rof lacimeh g b ,s orp tneir u ,g lcy d a tnem oriv g hc a tey b d i u s n eht -robal atory b tradi onal microb l gical procedur s. CLSM provides the m ans n i s l e oc t h af r , u m ni ut s From: Methods in B otechnol gy, Enviro mental Monit r ng of Bacteria Vol. 12: Edite by: C. Edwar s © Human Pres Inc., Tot wa, NJ 251 25 Lloyd et a . wher by s e l b a n o i s e m d r hng ti a m. Ie c n b r u t s ie dl h wm e t s y o cd l p m a s , rs dh em nt ai w o g p f h r s c n i o m t l a e d h r pc su e . h ) T l a m i, r nt o s p e h c a f r u m ( t s b e uo hc p t rn i w a c o l e h t , . g e (y l v i s n t x ed w i v rn e bs a h u q i n c e t sko b )RO ,en guE( seborP raluce oM yb dehsilbup elba i v ylediw ton l i s t igol ce aiborc m eht o su f noitar pe l a u d i v sn m g r o a c e b d i f t n a r i e h t s v c a d e b o r p n i es )6–4( .sfer t n e l c x r h td n a, ) 3–1 s t .e n b ’ lo c ir a h p T f d y v‘ u m )7( elpmas no noitamr fni ,rev woH . )8( . 1.2 Princ ples a d M tho l gy The first com ercial y produced conf cal laser scan i g microsc pe becam av il b e in 1983. It has revoluti n z d micros py in tha it transf omtrih ces d va p l t rs u c d iw mone to one cap ble of reconst u i g in thre —in real-tim CLSM, ev n in four p r i ns ( c d m l e I t o . ont he sp cimen (us al y b the micros pe bj ctive l ns) a d im ge by the sam l n , o t a pi h le (or a tu ) in fro a ph t mul i er d t ctc o A rm .p l e i a gb s n y d c it oa h fr geb k (in a r ste scan) acros the specim n, so tha the image is built up oint by p o i n tL . g hf r mu - o c p sl a n e b v d ot wh f s ec ai n w i d e l sy p r a t h i n o l e d c i m n a t e d s, o h f c u e id n o r m a tion c r butes v y li t e o h final m ge. A conf al mi rosc pe an thus v se f hraoi y m pt g ,bc n jk B . i h r a o u fg e focal planes, a seri s of optical “sections” through the specimen can be acquired; th se can be us d to rec nst u a thre -dim ns o al o s p e t ld i m c b a - y r f ,h n t T e io s b j . c , n o i r t a f d ec mb w s l v pg h r o t a e d .T h i s t eb a s i cp r o e s fi m a g e c q u i s t o n ,c s t r u c i o n ,a d i s p l at yh im so f t eu n doi r t e n s i a y l s i c no j u c t i ow n s hp e -orp ne b vah stnemur i lacofn emit- r ,yl nec roM .seb p tn c roulf decu )9( .erutc s i a l w o p dn v f y ut es hT . T ph re i n c ol f ma r s c o wp e y t b l i h M d n s k y e r a dl i f n st y e m h a v b u id n c l mg t p e - o i n s c a ug a Nipkow d sc, tage sc n i g, a d be m scan i g b ue mt sa iwo r n g h v c p d m i sa n t h e r o d u c m i dt -h 1e 9 8 n 0 ’ o s a l m o s tf u r e c n , a m t o s u i e d I a l y t w o . u s e t a i r s t i c e a l i n s t r u m e i h rs m a l - c o e d r g n i ,h l u m - e o n rk y p t laser th exci a on s urce. Ap o iate xc ion a d em s ion f lters a used, p n i g o the flu r sc n e ha t ris c of the lu r p o s ed. 1 Fig. p r o js il e cua g t hT d ) . image of (30) cif (10) (2) (1 , 12) p r a i c d -ne m t l h s o , ; u y g . System ha sc n the s e; v - CLSM of Envir me tal S p s Fig. 1. f o c u a s te o d pf o i ct s nuh e a d l r o t i g f c h Ae . n p ( l b a r n o e i k t g h s u )d m , f c p a e n p l e i t a v n h r g o u yc s d , h . i m op fr ve s d l c u tin o m a r s c hl Aeu p y i . n t a o g s r a point bjec , th objec is maged s n Airy d sk of pr g es iv ly ncreasi g nte sity hat sc n acros the d t c or pinhole. the image of a point object in co venti al nd conf al micros pe . (Reproduc with perm s ion f m 253 The layout of a typical conf cal microsc pe. Il uminating light is (A) s p e t c h F i l m u rn o a . f p s g t e d (B) (C) ref. 2 . The r sulting tensi y profile acros b aT sh ie 254 Lloyd et a . 1.3 Ap licat on f he T c niqu Sampling of natur l ecosy t m for CLSM pres nt practi l dif cult es. CA as l d w e t . times al er than we do, an 1 m ours. Henc , lo ki g f r a b cterium on a 1 m single p r o in a 1-km e n v i r o m t as k he l c i o fn m p s g t ae d h i f c u o l nst y very smal p e ( .g (8) h a pv oe i n t udb , c r pa e s ot nlm i 2 2 on tha sc le is equ val nt o 1 km 2 2 on surface i l ke o ing f r a forest. Th marked sp tial he rog n it s of natur l ., 1 m 2 × 0.2 m depth) can b s udie n ta l. Samples of this ze, contai g ind v ual organism , icro l n es, and microb al m unit es a ch d to mineral su f ce in so l , edim nts, or n the surface of plant or anim l tis ue , may be xamined iscret ly. Optical s e c t i o n ag b l u s e fi v c q t od (r . g thicknes w 1T h i c k s e a r m p l u b yt e c i o n f d r s a t , y e c i o n p r g v e i xad cn s l o p t F h r. e m w i s n c a l o t rn es s t r ui cn o f pl am e v t . g r n o is a h , l y Pen tra io f n b dies a of m l cu ar p obes int rga ms nec ita g.e(stn vlo h iwtnem a r s itemo dna it x fro p .)seti no azid rbyh ie t ca r ot sedi lcunogi det gra -l mos b . , s e2 c0 t i .o 5fn µ m inters c o pa ings). ,. -irselban dim rof 2. Materials 2.1 Some up li rs of C n cal L ser S n i g M cros pe 1. Bio-Rad Microscience Ltd., Bio-Rad House, Maryland Avenue, Hemel H e m p s t a ( d r7 , U4 T PK Dl 2 - : 1 3 5 f 4 a 2 x ;- : 1 3 4 ) . 2 . W o 7 Z 8 CLBd eP, at f i. R r x sO l G w H y n A 7 , 1LU (tel: 4 -170 8 2 , fax: 4 -170 8 2 . 3. Olympus Micros pe , 2-8 Honduras Stre , Lond , EC1Y 0TX (tel: 4 -17 250- 179, fax: 4 -17 250 46 8). 4 . N i k oUL nKt d . , H u s e3 8R 0i c h m o n d a K , g s t o nS u r e yK ,T5 2P R (tel: 4 -18 5 4 0, fax: 4 -17 250 46 8). 2. Materi ls R qu ed for Analysi Requir m nts for vital s in g, activ y, and 16S ribos mal RNA (r ) an lysi re d c b lsewh r ( se 3. Methods 3.1 Direct Exam n io f Samples A t r Vi al S n g for in S tu Ac vi es c h a p - r e v i o u s n d t a l i e s c r b d a h i s c u me d t o a n g T h t e r( s se C h a p t e r1 s5n d6 )O . o f m a c t i v en d p o r a - Chapters 1 , 5 and 6). µ m CLSM of Envir me tal S p s 25 tages of the ap lic t on f CLSM in m crobial ec ogy is the imag n of live microbes overal p c s e may v n be pos i l . C n derabl po u ti n he rog i t wy rh e g a d o n m s u r e c t i v (y . g r e s p a t i o n ) v d e c in w e l - m i x a d b o r t c u y l e s h ; ib a n o vm t d y c l e s Flow cyt me r (FCM) p ovides a w rful means o tudy situ.n r e q cl u oat n h T i v d b f s (13) . . (14) In atur l po ati ns her a us l y man dif er nt ypes of rganism crowde t g h r. Using CLSM it s po ible t valu e sp tial d s r bution of activ es w h min al d sturb nce a d th reby p oduc 3D maps of interactions (microbe-microbe, microbe-plant or microbe-animal). Organism f i r a m t l y s cu h o e d ( v r ,m t s c e a l b n directly as mal specim n blocks (1 m tha tend to disag re t (e.g , from lo se y ag re t d sedim nt or soil ) i m p r e ws g a n t h ( T l / d o v ) 2 % . m i b e f s h r u t l d l o w - m e t i np g ( 3 9 smal b ock ef r u th ea m nt wi h fluor p es. O r g a n i l s m q u d p e ( o n. gf rv , a m i ue s tw h c o l umn) pres t ome pr bl ms, ev n if no m tile. Two meth ds of prev nti g Brownia m ve t (as wel im ng ove ts) may be util z d. The i n( m cw t1 e r/ o0 v f % h a) l s y u b i n d , to c mpres h lig t y b w hdra l of exc s luid from n e th cov r slip. A terna iv ly, s ide can be pr coated wi h a thin layer of the polyani poly sine f rm d by ing a 2% (w/v) soluti n. A wide variety ( ind cating fluor ph es are av il b e ( “viab l ty stain g” tha ideal y enabl dif er ntial counti g of “live” and “dea ” organism (e.g , undis closed ye formulation; alternatively, a large number of wel -understo dstain gmethodsare vail b e.Themostfrequ ntlyusedisthedualstain g combination of pro id um iod e (red), and fluoresc in diaceta e ( n o f l u r e s c t T )n h . l a i d e r c t n g o a p l e y r d t m d a oe g r n i s wm h , t a e r u b s l o c a t e i nv z y m s inl vec sbyf uore c inp du t o (yel w-gr n) 6 - c a r b op xe y m l h i g t e o f u s t h e i n c l d p r o a s t h i fluoresc in d acet , Calcein a toxyme h l ster (both fr m Sig a), or the Chemc ro dyes (Chemun x SA, Maison -Alf rt, France) organism h ve no sp cif e lux p m s O n l s y e d o h m a t v i ey f s m a t o l v r g n i s m a t u l p e s be n co firmed by fluoresc n -a tiv ed c l sorting a d sub eq nt cul re (21) . The comple ntary nature of informat obtain le from the CLSM vaire w d etc.) 2 × 0.2 m thick). Natur l sample ° C a ) g r o s e l u t i n a ; f r o d i c t n ,s l e d o se 7 and refs. Bactoli e as wel as Chapter 16) of “activ y”se 15 and ). The pro iety kits for Note 1 from M lecu ar P obes) u e Fungolite (16) .“Improve nt” f (17 (19) tha c n exp l vita dyes , 18) . Some (20) . 256 Lloyd et a . and FCM techniqu s is like y to becom increas gly evid nt in the fut re. Other vital activ y stain include those for transme b el ctro h mi al t a k e nc i v l y d o b t h 1 2 3r, d a m i n e y s c ( t hpeo n i a l e l p c r o t f ba s h i n m y g e u w - p l tive chang o t e u r fac ) nd the io c x n ls, e ud b t se n a “ h a l o . ”C i c e f y a n d i sb e o nh y r p b i c t a n dh e p r m abil ty pro e ti s; DioC usef l cyani e and ox n l dyes, resp ctiv ly s t a i n e db r u o f l m k s c w e i t h a pv b l , s x c i t o n with ndo carb y ni e o x l VI; both emi n red ( 5 - c y a n o 2 , 3C T d i t l s e r z u m h i s t r a n c v y u s e d w i l A t e r a z o l i u mc h d e ) ,w i r a c t s h ed y r o g n a s fe l c t r o a n s portchains duce insol b r df u esc nt ormaz extr m ly bright ma es of bacteri n CLSM ( tron d rs (e.g , or anic ds, H s u b t r a c o e in f w d l v s ut bh r a e . a v l r g i Os o te n h - b f x c u p r s ind cate o n tra io s (e.g , fluor 2 Ca as wel as host f luoresc nt y labe d antibod es for ext ac l u r comp ne ts. Befor m unti g under cov slip , materi l shou d be wash d fre o exc s dyes and su pend in a soluti n of a fre - adic l scavengi agent (e.g , 2.5% DABCO [w/v] (1,4-diaz b cy lo-2, ctane) pro ylga te, or p h e r nd yifvolat mu b ( ) c g 6 (3) and DiBaC 4 (3) ap e r to be the most gen ral y (2 . Provide ap ro i te , 23) . (24) ;itproduces (25) ). Use of di er nt lec- Fig. 2 2 S, H 2 ) can be m asured i ctly b image (7) 2+ t h a m i n yc l u d e ) or pH (BCE F or SNAR ) ). 2 Note se 3.2 Cryosecti n g A ne x c l tb a k g r o u n d h ec y p r a t i o n fb l g cs p e i m n g i t v h e s pao rn dy c f i q Au e v. a n t g cs l p rd o e i n g n i a t e v x r c l u f a e i n d v r o m t s a , n e o c f i v t yz m e s and tige s, and r pi (w th n 0.1– ms) i ob l zat n f org ism . After placing the sample on the microt e chu k, it s placed on a bed of ethanol “slu h” co ed t 7 K in l qu d N liqu d N into7slide at mbient emp ratu . Alterna iv y, the s ction is al owed to fre z dry ove night o the slid . V tal s in g or fixat on s al o perf m d on the m e s i l c ( a n S r E o ut M p b ) d g q y . or mic p be an lysi for e m ntal dis r bu on ( (26) 2 . When froz the sampl y be stor d in 2 , or t ansfe r d to a cryost hamber t 253K, cut wi h a ste l knif µ msection ,a d he l ow t ha f ercol ti n am cros pe se and Notes 3 4 ). 3. Fixed Sampl s Identif ca o m r bes nucleotid s nec sita prio f xation d perm abil z t on i ns t u r e q u i n gt h s o fr R N A - a g e t do l i (27) . A poten ial CLSM of Envir me tal S p s problem is th low c py number of RNA m lecu s pr nt i cel s found i nutrie -l m d enviro m ts, whic an result in low ev s of lu resc n in stained cel s, making their discr m nat o dif cult ( h y d r o l z e p a f r m l d e h y s o u t i n( 4 %w / v ) p h o s a t e - b u f r ds a l i n e m i n a l z p e r s o b m u f t l e s c n F i . x a 4 t h f o3 r ing sco ve tly ar iedou w thag r se- mb d ateri lo w hmaterial at ched to slide . Clean slide (soaked in 10% KOH in ethanol for l h) rinsed tho ug ly with 0.2 with gelatin by dip ng them in a 0.1% gelatin, 0. 1% chromiu potas ium sulfate o i n at 70 Dehydration (3 min each in 50, 8 and 98% [v/ ] ethanol) is fol wed by the in situ s o d i u m e c y l f a t ,2 0m water) can be sup lem nt d with forma ide, or the organism can be subject d o h r pe m abil z t on pr cedu s 257 ). Freshly Note 5 se ° PC r. o c e s - µ m filter d s il e wat r nd i r e a co ted ° C, then al ow d t ir y n a vertic l pos n. hybrid zat on procedu . Hybrid zat on buf er (0.9 M T r i s - H C la tp 7 . 2 n0 M NaCl, 0.1% (8 , 29 , 30) µ mf i l t e r d s . (28) 3.4 Ap licat ons f Me h d CLSM has be n xt sively u d to s udy biof lms growin te h, submerg d st l ruc es, il on rub e cath rs, nd o b u r i ce l pn td v h f a s o, gw y o tf h e i l am n d s u c o f r g a n i s m ,w e l p r o v d i n g f m a t o on pH a d re ox g adi nts her by g n ated. Combin w th microel t d O d e t r om fi ( n a . g , s t a r g e o dl i n u c t p er o b s v i d t a l em po sf c r b i a m u n seit 13( , )23 noitac f r s eht no at d elb u vni s g MSLC tah dnuof evah W . of activ es and i e t s of rganism peat bogs ( rhizosp er organism using rRNA-ta ge d olig nuc eotid probes al ows p l a n t r o e h s i b d gw c - f l a u t y n o s ro ts (34) . Investiga ons of a denitr fy ng sand-filter sludge (36) have also used thes methods. A method for the numeratio f s o i bl a c t e r h n b o a t e d of smal molecu s throug gels have be n det rmin by CLSM activ y and istr bu on f toluen -d gra i evalu t d in a multispec biof lm rev aled by CLSM studies, ev n when their host are very large (e.g , 80 µ m in the case of the rumen entodin om rphid prot zo n multivesc a um) study of pathogenic formis (41) . A negl ct dar ofmi b aleco gy,th p a oc si fba ter yp otoz a (they have a prodig us pro ensity for grazin and can turn over the 2 NO and 3 – . In spe- r R N A – 1 6d Si s t o fh b e u n ) s , , Figs. 3 3 ). Studies of , and of sewag (35) D. i f u s o cn e i t fs o r h me g a i n (37) . The (38) has be n Pseudom na putida . Intracel u bacteri a e spl ndi y (39) Polyp astron . i m tf p e a o cn r hd Tl s q u (40) Legion l ac with n the prot z n Tetrahym n pyri- 258 Lloyd et a . Fig. 2. Fluoresc nt images of plant- s oci ed micro gan sm from a peat core. Thes w r obtained o ptical se tion g f 1-m depths wi n a i t c ore and st i e w h it er azolium (CT ) 3 peat s m le tak n from kn w (A–D) or cya- CLSM of Envir me tal S p s 259 entir bacteri l po ulati n of a sedim nt with n a few days nie tly studie by CLSM. Thus the kinetics of luoresc nt bead uptake by Acanthamoeba c stel ani , t i a t i v e l by F C M a, n td h ce o m p l e m n t a r y e c h n i q u oe Cf L S iM ns e c s a r y to distinguish betwe n ingested and surface-adsorbed particles marine and freshwater an erobic environme ts, it s the major function f large cil ates (e.g ism lack mitoch ndria, nd their hydrogenos mes can be r veal d by the u s oe mf b r a n pe o t n i a l – s e n i t v de y s 2+ as Ca stores, and fluor-3 has be n used to show this in CLSM images. Similar results have be n obtained for the other hydrogenosomes of low e r eukaryotes living in O N e o c a l i m a s t i xf r o n t a l i s , Trichom nas vagin l s vagin ( se Note 6 ), is conve- [42] a com n soil amoeba, c n be m asured quan- . In (43) sp .) to car y out this proces . Thes organ- , Metopus (4 ) 2 T. h e s o r g a n e l s e r v a l s o -deficient or anaerobic environments: ac h y t r i df u n g st h a i n h a b i t s h er u m n (46) (45) ,a n d , a flage t d prot z n par site of the human ). 4. Notes 1 . L i m a vt e d l b c si on g uhf y part m de l c use of usef l ultravio e (UV) excitabl dyes for most com er ial y av il b e c o n f c a l s e r c a n i mg c r o s c p e s H. o w e v r , e a s o n a b l e o n g wr a v e l n g t h alternatives, for the most part, are already in use and this range becomes ext nde daily. 2. In conve tional epifluoresc n micros py, the image fails to rep s nt 3D objects a ur tely. Stray light blurs thi mage, nd becaus the n ire obj ct is i l u m n a t e d f , l o r s c n p t b e m a y c o l h e d I C. n L S M r , j c t i o an f l o u t - f c l si g h t v ear uo p c s l t i ow n ah c k e s l i t a0 . 2 depth resolution is pro rtional to the square of the numerical apertu e Emis onfromafluor phorecanbequantif edforthecal u ation fmolecular con e tra ion. Dig tal imagin of a sequ nce of ptical sections obtained by step ing in the tage of the conf cal method is clearly evident in the images of intracel u ar bacteria with n a rumen prot zo n ( quantif cation of pixel (voxel) image. R pid scan i g of the obj ct y he focus d pot min zes xpo ure t the po n ial hot b eac ing of the lu rop e. 3. The resolving power of the conf al laser scan i g micros pe is margin l y improved (i.e , the min al resolv d istance is 0.7 of tha of the conve ti al n[ iD eO C s w t Sea r c u i m 1n f p o . 2–d l 05 H h ) ( D , ; G C air, wher as tho e from 20 cm wer stained a erobic l y, under N calibr ted n micro et s. 6 (3)] µ m; (2) . z -direction is fol wed by 3D reconstruction. The adv nFig. 3 ). Software is av ilab e for rapid inte s with chosen are s (volumes) of the (E–H) c m ; 3 F ( ) D B s i e , 9a n p fo 0 1 . t h r E l w A g 2 (d, h). Bars e 260 Lloyd et a . .giF 3 a l g ni re o mt u z c b p , m u t a l c i ns oe v pu ym l P s S t 6 dn 1 eo – li b A m N hay Rc r w u p f b e c , n s u fo r i- d l gt a m p E r e v o f l a i r e t c b s e g a m i k , n l tb us o c p m i .noitces lgnis .la te dyo L m rf decu o p R( .airetc b d zilanret d a serut f ca rus woh t ni ut s )B( .noitaz d rbyh )A( l a c o f n r e s g l i a c f o r l in me s c p a )C( yaw tuc dna oita r lanidut retfa detcur sno tub ,)B( ni sa eg mi ]04[ l i g h mt c r o s p e ism n microas e bl g , and ther by valu tion f as oci t n , extrac l u f l u o r e s c dn b a t m i se u o r f b m l t a o i v e p s H r , - [2] ) A. ws e l a n b i g o d e s t r u c i v n m e r a t o f g n - ). CLSM of Envir me tal S p s 261 O ro ,sn i fo stneidarg o sn it f n o i s e r p x d l o t n c - r e m o p i f c e , s . g ( l b i o s pn e r x f gn o i t .)edom gnit uoc n tohp ni xulf nois me tn cse imulo b r nietorp necs roulf ne rg 4. Electron micros py scan i g (SEM) or transmi o (TEM) el ctron microscopy gives much ig er soluti n, but almos ways requi s e of ixed an d e h y r a t m e i lR . p a t e dx m i n o l f vs ea m p bC yL S Ml o wt sh e d i y n c bv a h e mo t s f g r ( p dl i e s c ot n rw ) (hours to months) time scale . Thus, as wel as having great poten ial for the studie of spatio em r l oscil at n (e.g , Ca dev lopm nt of biof lms over ext nd periods of time have also be n document d (38) section g s a extr m ly i e-consum g, operat -in s ve ta k. O her t c niques of scan i g probe micros py (scan i g tun eli g, atomic force, scany h p a r. g o t e c a f r u s n o y l n o i t a m r f e v i g t a h s e b o r p u ) e c n a t d o - i g n An exampl of the compl entari y of s me of thes m ods f r the xamin tion f b ilm has be n pr ted 5. F u r t h e d e v l o p m e n t s i n c l u d e e n h a c e m n t o f a x i l r e s o l u t i n t o b e t r t h a n 0. 5 µ m by standi g-w ve excita on (49) , enabli g more reliab estima on of fluor ph e con e tra i s. A new, Nipkow disc, real-tim conf al micros pe f l u o r e s c b n jm t i A o . p e / m a c r o s n p e b l x a m i s t o fn d large specim ns in a single devic f l u o r p h i ne cs l u d y w i t yo fm e a s u r n t w a v e l g t h s o n e r a c h t e r i s co f h eR a m ns c t tering of water and those com nly encou ter d in autoflu resc n e (e.g , chlor p y ). Fast acquis t on sy tem tha al ow 3D imag n of liv ng microorganism their na u l e vironm ts an obvi us targe 6. H i g h - p o w e r d a r g o n - i l a s e r ( l i n e s b e t w n 3 0 a n d 3 6 4 n m ) h a v e t o b e waterco l d and are very exp nsiv : micros pe lens are not chromati l y H a D oT l A eh t U P cu V I s - , . g x r i d y n b l e DANSYL, fura-2, and indo-l can ot be used with most sy tems, derivatives of luoresc in, rhodamine, BOID PY, Texas red, cyani e, oxazole, thiazole, phena thrid ne, and the phycobil ns are excitable by smal argon-ion laser (48 and 514 m ) or mixed-gas Krypton-a g laser (48 , 56 and 647 nm). New fluoresc nt cal ium ind cators include fluor 3, Calcium gre n, Calcium orange,Calciumcrimson,a dFura ed.pHind catorsu edinco f calscanni gap licationsareBCE FandcarboxySNARF.Newr d yes howgreat promise (53 - a z i l u s v t c e r i D . 2s e i t v c a i l o b a t e m f o s r t a c i d n s a 2+ waves), the stabli hment a d . Thre -dim ns o al p ti reconst u i of TEM images by rial . (47) , and fluoresc n lifet m imag n (48) (50) (51 (53 . The burgeoni g list of av il b e , 52) , 54) show adv nt ges for weakly t ph oa e n i gl rvy a st e n i - (5 ) , 54) . References 1 . F a r k s ,D .L B a x t e r ,G . D B i a s o ,R .L G u g h A ,N e d r l o f M .A ,P a n e D Pane, J. Patek, D. R., Ryan, K. W., and Taylor, D. L. (19 3) Multimode light m i c r o s a tp dn hy e m i oc f ls u e , a t n i d s . 5 , 785– 1 . A n R u e P. v h y s i o l . 26 Lloyd et a . 2. 3. 4. 5. 6. 7. 8. 9. Cox, G. (19 3) Trends i co f al micros py. Boyde, A. (19 0) Conf cal optical m rosc py, in niques and Ap licat ons York, p . 185–204 Wilson, T. and Shep ard, C. (1984) Micros py. ( 1 9e P d a 5.Jw ) l , y Prenum, N w York. Stev ns, J. K Mil s, L. R and Trog is, J. E eds. (19 4) logica Spe m ns. Academi , S n D ego. Haugland, R. P. (19 6) Che micals, The Netherlands. L ( C a 1 Kw n9 R l or Ed . e 2 , DJ b f ) c m i s copy and computer image an lysi in microb al ecol gy, vol. 12, Microb al E gy Xiao, G. Q., Corle, T. R., and Kino, G. S. (198 ) Real-tim conf al scan i g optical m rosc pe. 24, Micron 237– 4 . Modern Mic os p e . T ch(Duke, P. J. and Michel , A. G. eds.), Plenum, New Theory and Practi e of Scan i g Optical Academi , N w York. e d .2 ,n B H i a o nT l dh fbe Cg c k M r s o p y , Investiga on f Bi Handbo k of Fluorescent Probes and Research 6 ed., Molecular Probes, Eugen , OR 97402 and B. V. Leiden, Advances in (Marsh l , K. C ed ), Pl num Lo d , p . 1–68 53, Ap l. Phys Let . 716– 8. 1 0 . c i o n s v f t e m a h M ( 1 r 9l 8 g ) . p k y , Scanni g 10, 28–13 . 1 . White, J. C , Amos, W. B , and Fordham, M. (1987) An evalu tion f conf al versu conve tional imagin of biol gical struct res by fluoresc n e light micros py. 12 . Carls on, K. and Liljeborg, A. (198 ) A conf al aser micros pe scan er for dig tal reco ding f pt cal seri ct ons. 1 3 . m i c r o b f tw ln ea g h s q u , y m F or ( 1 9 3 ) D . L l y d , Cytome r in M c ob l gy 14. Porte , J. De r , . Hardm n, M. Edwar s, C. and Pickup, R. (19 7) Go with the flow—use of flow cytometry in environmental microbiol gy. Microb l. E 15 . Lloyd, D. and Hayes, A. J (19 5) Vigour, vital y and viab l ty of micro ganism . FEMS icrob l. Let 16 . Schup , D. G and Erlandse , S. L (1987) A new m thod det rmin cyst viab l ty: cor elati n of lu resc in d acet and pro id um odi e stain g with an m l i fect v y. 17 . Edwar s, C., Diaper, J., and Porte , J. (19 6) Flow cytome r for the targe d an lysi of structure and function of microbial po ulations, in E n v iA rp o Mt m c ea h b ls g y Elis Harwo d, Chic est r. 1 8 . m ( a iR 1 n c GB9 rp o lS tf3 y. d ) s e g M , u w m c y t o m e r i n, Lond , p . 17 – 80 19. Paulsen, I. T., Brown, M. H., and Skur ay, R. A. (19 6) Prot n-dep nde t multidr g ef lux sy tem . 41– 8. 103, J. Cel Bio . 153, J. Micros 17 – 80. Flow (Lloyd, D. e ) Springe -V la , Lond p . 1– 0 FEMS 24, 93–10 . 13 , 1–7. Giard 53, Ap l. Enviro M c biol. 704– . Molecular e d s . )R, S aJu n rd (RP.i c k p , ( L l o y Dd e., S) p r i n g - V e l a , F l Co yw t m e Mir n c o b l g y Microb l. Rev 60, 57 –608. CLSM of Envir me tal S p s 263 2 0 . M i d g e l y (,.1 9 8 7A )nf us xy t e moc ra i n d y e s l a t c o m p u n di s 4, 125- 7. Escheri a col . Mi r b ol. Sci 21 . Porte ,J. Robins ,J. Pickup,R. andE w r s,C.(19 5)Recov ry fab cte- 2. 23 . 24 . 25. 26. 27. 28. 29. 30. 31 . 32 . 3. rial sub-po lati n from sewag using im unofl resc nt flow cytome r and cel sorting. FEMS icrob l. Let Lloyd, D., Moran, C. A., Sul er, M. T. E., Dinsdale, M. G., and Hayes, A. J. (19 6) Flow cyt me ri on t ri g of rh da ni e 123 and cyani e dy uptake by east. J. Inst Brewi g W i l e t s ,J . S w a r d R ,D i n s a l e M .G ,S u r M .T E ,H i l B a n dL o y ,D . b u t e a n h o - m l di , c r g w y s t e o f V i a l ( 1 t 9 y 7 ) l-o r is butanol. J. 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Singleto , S., Albiston, L., Treloa , R., Mahers, E., Hodgs n, R., Watson, K., S c h i l n g K, . a Ad l i s o n C, (. 1 9 5 O) p t i c a l m g n d h a r c t e i s o n f r a l b i o f l ms t r u c e s i n gv t a l i n s d p e c f i a n t b o d yp r e s ,i n B i oD fe a lt mh H a LGT( n i.W d P pl, b m e y r J -t S c o H., eds ) Biol ne, Cardif p . 3 – 6 Caldwe , D. E , Korbe , D. R , and L wrenc , J. R (19 2) Imagin of bacteri l cel s by fluoresc n exclusion using scan i g conf al laser micros py. Microb l. Methods 15, Zanyk, B. N. Korbe , D. R., Lawrenc , J. R., and Caldwe , D. E. (19 ). 4-D visual z t on f bi lm dev op nt by Schram , A. Larsen, L. H , Revsb ch, N. P , Ramsing, N. B , Aman , R. and Schleif r, K. H. (19 6) Struc e and functio of a nitr fy g biof lm as det rmined by in situ hybrid zat on and the use of microel t d s. Microb l. 62, 46 1– 7. Lloyd, D., Thomas, K. L., Hayes, A., Hil , B., Hales, B. A., Edwar s, C. A., Saunders, J. R., Ritchie, D. A., and Upton, M. (19 7) Micro-ecol gy of methanog s in peat: min al y- v si e an lysi u ng co f al ser can i g 13 , 195– . 103, 79–84. 251– 9. 102, S ep dr ( .i LD)n l ,g o - y V a isnewa t r E s c h e r i ao l 2, 165– 7 . , R oMyiaclr s p S o iMe tcyr s H apn d b o k 172, 762– 0. in situ hy- Microbiol gy T h eL i f a n d J. 249– 61. 3, 24–39. Pseudom na fr gi. B na y Ap l. Enviro . 264 Lloyd et a . microscopy, me brane inlet mas spectrometry and PCR amplif cation of methanog -specif g n sequ c . 179– 8 . 25, FEMS icrob l. E 3 4 . A s H u B m t K . z i , P l r L eac G R h n w o d f J A. (19 5) wheat. loca iz t n of In situ in the rhizosp e of Azospir l um brasilen 10 3– 9. 61, Ap l. Enviro M c biol. 35. Ne f, A. Zagl uer, A. Mei r, H. Aman , R. Lem r, H. and Schleif r, K.-H (19 6) Populati n a lysi n a denitr fy g sand-filter conve ti al nd identif cation of Microb l. in s tu sp . in methanol fed biof lms. Par co us 62, Ap l. Environ. 4329– . 36 . Ros el -M ra, R. A , Wagner, M. 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(Shot n, D., ed.), Wiley-Lis , 5, Monit r g c obial A t v es 267 81 Monitoring Microbial Activities Using Membrane Inlet Mass Spectrometry James R. Firth and Clive Edwards 1. Introduction 1. Princ ples of th T c nique Monit r g m c obial t v es in h v ro ment is d f cult owing he lack of suitable m thods. For a techniqu to be us f l or m nitor g a c t i v e s m, u p o t hf l w i np gr o e t s : i v y ,e l c t i s a c mo b a n i kt l e h r u y , s- bi m n ea v td , sive or p tu bing o he m cr ganis or t he nvi o m t be ng s udi . C u r e m n s t il h d y o c a r n i q m u e c s , o h r i a s c h g rl y o m , t i n p d - e r f o m l a c q h u i d t g r p y , but al h ve t ir l m ta ions d us al y requi s b tan i l d sruption the environme t being studie . The princ ples of me brane inlet mas spectrometry (MI S) have be n described els wher rized n etail to be mad in oth e liqu d an g s ph e , only iqu d phase m ur nts are d t il n h s exampl . Es ential y the method invol es the ion zat of the gas or volati e mole c uf lo s t hp b a wy r d in q e of its mas /ch rge ratio ( s y G dt a ie fm c . o u n v h r s l y b e t i an d cg o a sil con rub e me bran located at the tip of the stainle - dis olve speci rob . Alte na iv m br nes ca b used ch as Teflon, d p i g h p a r s o t b e v f l m i n T g u d h .p e o r t s e x a t n r d c i o l m 0 uf . s 7 b h e w s u c f h o m a w r n i t l d s e y h c r T i m bp o a u g . t s e probes tha c n sample s quential y b prog am in the mas pectrom . in s tu (1 , 2) , and are sum ah A e l r t .a M o I um g S w s n e f r t m/z From: Methods in B otechnol gy, ). Figure 1 show the basic comp ne ts of the Enviro mental Monit r ng of Bacteria Vol. 12: Edite by: C. Edwar s © Human Pres Inc., Tot wa, NJ 267 268 Firth and E w s Fig. 1. A schemati diagr m of the workings of the quadr pole me bran inlet mas pectrom O p e n i vo afg l s wd t c h bn e p r o i as ud nt e m c a l y , and it ther fo requi s a gas cylinder of inert gas, e.g , air or nitroge , to d r a wg n e sp t h oi b O q , c u ) ( . 6 d r e a l ot bh n yi gev c u p r m d a s e t o m r ’ u s b l e c lar pum . On reaching the ion zat chamber, the gas molecu s are bomh i s g to rf- e a n m l b c y d ,i to e i r d fu m l a e n Tt h. ci s o z a t ghfn m e s l c u T v . a m c o n t i d r u a h e l w s q g p o n y z Te chr i. s ft four metal ods, u al y ste or m lybdenu , with a po enti l d f renc a d r a d i of e q u n c y p l i da r o s e c hp i .T n u m b e ro fp s i t v n a g d e f t i l h s co q r au - n b g y , z ing degr s by the magnetic field betw n the rods. The dif er nc s in the d e g r o f l c t i a n d e p o h t m i ca s n d r goe f h , t ih .e r , o n f u r m e ab q d v i l tc D g C s , o f r a n e c t h i u g z chan els b xamined th ons f i ter ocus d nt he c or. e d a A c f s h l i o t m n w e a h g r p s, l i fied, giv n a re dabl cur ent. Since th number of i ns det c is pro c oe t n h p v ir g a s ( l m f m/z n e u a t b r y d o l i s f z h c ,w M n a y . m/ Monit r g c obial A t v es 269 Table 1 An Example of a Mass Spectra Cracking Patterna Gas 2 12 18 2 8 N 30 32 34 40 4 46 64 10 NO 10 2 N O 2 1 NO 3 6 CO 37 1 10 10 1 2 10 O 10 SO 10 2 H 2 S H 2 O 10 4 1 H 10 10 10 Ar 10 a Thes dat r of mpli u es at v o h m j r peak (10 %). ion zat source remains the same), it is ther fo pos ible to quantify the con e tra i of h t gas. Becaus dif rent gas ion zed s multan o y, he ma produc i ns s a tm he o f n ua m b doe ifr g t s T v . e r c o m h pi b l a s , e c t r k ing pat er is u d ( gase of inter s p ent major pe ks at oxide [NO]), 32 (oxygen), and 4 (nitrous oxide [N [CO 2 ]). Ther fo , s me p ak re pot n ial y m de up of c ntribu o s fr m othergase , .g ,chan el4 consi tsofthemajorpeaksofb th[N CO 2 . It is ther fore n ces ary to study ad it onal various minor peaks to establish what contribution each gas makes to a major peak. To dist nguish betw en the two gase , CO m/z c h a n e l1 2 , tw i c h a u s e p kc o r n d i gt o6 % f sc n r i b u t i o a nc h e 4 l( to cal u te what fraction f the c an el 4 peak is owing to CO making up the r maind . The Hal qu dr pole gas n lyzer (Hid n A alytic , War ingto , UK) has the abil ty to measure and reco d up to 16 Becaus of the n d to car y out m ltip e cal u tions, the dat re saved to computer disk by the mas spectrom in the form of a spread h t. The spread h t is then imported into Excel 5.0 (Micros ft), wher a m cro p gram can be design d to perfo m the nec s ary cal u tions. Most of the exp rim nts ca ed out inv l e th s quen ial s of al ur of the dis olv m/z r a tes i.o gm ;, m/z c h a o nm t ey l i s r b u f ). For exampl , wh n studyi g en tr fica o , the Table 1 m/z chan els 28 (nitroge ), 30 (nitr c 2 O] and carbon diox e 2 m/z 2 se T a b l 1e O]and chan els to measure can be m sur d at ) F. r o tm h i s n p e a k , r f o s i b l e 2 m/z chan els simultaneously. with N 2 O 270 Firth and E w s speci rob s, and he c t is m an he d t for al u exp rim nts a e t i a l s o cy n g p e r d h T aot l . y i z s e , f n r c sary to place each dat set into a separ t spread h t cor esp ndi g to the dif er nt exp rim nts. Once the dat has be n proces d each probe is calibrated g ins a k ow c n e tra io f g s t conver h mas pectro units o m lar c n e t io s. 1.2 Ap licat ons f MI S Thisc apterd ib s heu ofMI St s udy eni r f cat o ne vir mental samples. This is an importan res a ch are becaus of the globa importance of the denitr f ca o proces rega din to the gre nhous ef ct and water quality, and the fact hat so many of the comp unds invol ed are O ig .a es , t h ep r o d u c s f n i t a o h sl e dt m n y u i s gM I Sr e p o t i n e a x itdr sh o nbf c enorm us vari t on both ra es nd pro ucts f denitr f ca o betw n differ nt denitr fying isolates under dif er nt condit ons refl ct d in he v ro m nt The t chnique s u ef l or eal-tim onit r g of any microb al proces tha has gaseou or volati e end products. It has be n used to study various m i c r o b pa l e s ti hn b o r a y e d v i n m ra st e w d by Degn t al. (10) One of th irs p oce s tudie w h t e chniqu was n i r t e f l o a xg s h d yw c p in p r o d u c n t i b y g el h a s t d y p b n r u e o , c i is resto d in the pres nc of carbon mon xide, whic inh b ted hydrogen u ph t y b ad e kr o g n s z J m .l t e n i t lcr e o a hv g y s , bf u n d i a c ho yn d re L gta i . s c o r t we h ipu n da k , r e a c h id ys o n g tfl u r h , a o n wd k w h ia c t u l n y b s r o g e a c t i v ( 1 y4 ) , s h no w c lge r a tionsh p be w nitroge as nd hy roge as in troge fixa n. Another microb al sy tem hat s be n studie using MI S is ferm ntaa e br o t s h p i n c w f d m a - o , ta ion of glucose are inh b ted by ethanol and tha hig er alk no s ex rt an increas gly inh b tory af ect on CO i n d u s t r a pl c iMo Ifn S e ms t r i a wnghdB o á ekl . (16) m o d n f e i v t r c a g s w u h ky o n t e p m t h a e yc o u l d i s t n g hc a e si ng o c t r a i n so f< 0 . 2 %v l G a s eous exchang s in the rumen cosy tem both in v tro and i v o have b n examin d ism n a fluct ing e v ro m nt. The pro ti s f MI S make it n exc l- 2 CO , 2 N, 2 N and O, 2 m ao lxn yi gt be s r T d h O . (3–6) d e m h oatT ns r . (7 , 8) . This is also (9) . (1 m o n i t r e d fg x a - (13) 2 (17) g i n vas, c to hm epr l x b it wo e g na - producti n H y d r o. g e n , 12) (15) . Degn (2) pro sed th Monit r g c obial A t v es 271 lent o l for m nitor g pol uti n ported m ans it c be us d to ample nu ro s ite proach, wev r has be n u d by Cristea nd L ger coupled to he wat r plant plants k.From hestalk, xyg n dCO f ur p e w tcg o h a A s d l v . i n t studie using MI S is methanoge si . Benst ad and Lloyd d e c a y r o b il s ,u f t h e i n a t f o u d n c r e s p a t m i nh o g e s o m r e g t a hf xn l i C dc O g Ar te a d p w h s i , cn o b d t p nr se m i a o w d t g r satur ion f he p at, m noge si c uld o r. The adv nt ges of MI S are th i s conti u s, en it v o the l v of parts e il on measur numero s gase and volati es imultaneo s y in eith r the liqu d or gas phase (23) months. Furthermore, response times can be as short as a few seconds dep ndi g on the obtained r lmost in a eous. tm e ho a ub s i ,l n dy z e r M t a I s b i l S T m’ h v y with a gen r to a d g s bot le h ntire sy m can be s tup o in the f ld, next o a river or lake, e.g After the in tial exp nditure on the ap ratus, the cost of the technique is extrem ly cheap, with no conti uo s cost of consumable r q i d. T h pe r i m a ly t o n f h e c i q u s m t bo e h a d c i n g e in gas con e tra i s dep n t on the rat whic t is produce or c nsumed. For xample, wh n gase th oc ur nat l y wi h n a give n ro ment are measured, e.g , N c h a n g e t i r uo m s a cn h e pU , d t f. a o r c t me p a b l n u t r i I e c s l o a - b m . h d w u y ,t n to det c a change in gas con e tra i is unlike y to be a problem becaus proces such as denitr f ca o wil be oc ur ing almost o their maxi u poten ial; he nviro m t, h wev r is ould be pr matic. (18) , and the fact hat i s o easily trans. A more n v l ap- (19) (20) 2 wer monit d,a he f cto (21) 2 (2 ) m e tb hy a n o r p ci e a . , and virtu l y no i vas e. In d it o , can be us d to , and is very stable, requir ng calibration only ev ry few length of the probe being used, meani g tha the result 2 , dif usion out of solution may oc ur at a 2. Materials 2.1 Gen ral Co sum b e for Memb an I l t M s Spec rom t y 1. Membrane inlet mas Hiden Analytical). 2. Mixtures of ga t be m asur d, t know c e tra ions. 3. Acylinderofa tgscpbleofrducingapesrof6btpeah val e sy t m of he as p ctrome . . Her MI S was by insert g he prob into he Elodea c n de si spectromet r (e.g , Hal s eries mas spectrometer, studie 27 Firth and E w s 4. A PC-format ed lop y isk to rec d h at produce . 5. Enviro me tal s p e. 6. In the exampl describ her , the enviro m tal sample was transfe d to a benchtop 2-L ferm nto with stir e , temp ra u , pH, and aer tion contr l in orde t char e iz th deni r f cat on pr es und r co t l ed con it s. 2. Cleani g d Ster liza on f D s lved Sp ci s Probe and Repl c m t of Me bran s 1. 70% (v/ ) Ethanol. 2. m0.63diaetrnlmb,usicoplatn-redRm (Merck agn P rk, Lut e wor h, UK) ( se ). Note 1 3. Scalpe . 4. Chlor f m. 5. Ste l forc ps. 2.3 Calibr t on f he Dis olv d Speci s Prob 1. Steril d st e wa r. 2. Thermo t . 3. Gas mixture of kn w c e tra ions. 4. Gas olubi ty a les. 2.4 Dat Proces ing. 1. A PC cap ble of sup rting a spre d h t pack ge. 2. A spread h t p ckage (Mi ros ft Excel 5.0 ) ( 3. Mas pectral king pat er . ). Note 2 se 3. Methods 3.1 Prog am in the Mas Sp ctrome i n T s h t e r Hu q dam l c p b o e t r sup liedbyH nA al tic (W r ngto ,E la d).Othersy m wil vary d e s pc r oi tb l h f n c i p et s a bl u y , t h e i r o f d a l s t h e i n her wil b the sam for l . 1. Sel ct MID mode t pr g a m the mas pectromet r to record the ap ro m/z c h a n e F l x o s r . m p c h 6 a 4, n 3 0 d 2 1 e 8 l s sel ct d for denitr f ca on. Thes al ow con e tra i s of the gase N NO, C s u c ha y d r o g e ns u l f i d ( H nel 32, wh re oxyg n is mea ur d. priate 2 2 , and O 2 ,N O, 2 to be cal u ted by su trac ing o tr bu i ns from the gas 2 S )a n ds u l f r i o x d e( S O 2 )t h a c o n r i b u t e c h a n - 2 . ( s b e9 t r 0 i p w m lc o f n h a gd S. , se Note 3 fo egnar d t ise droce yln m ts eh 3. Insert a flop y disk an el ct h disk opt n ( ). fo egnar ht in acs et p d ah seru n ihT z/m ,ylsuo nit c sle nah .s 0 9 yrev sl n ahc z/m se Note 4 ). Monit r g c obial A t v es 273 3.2 Steril za on f the Dis olv d Speci s Prob d i s r tc pe o hu q lw - v A n i a f t o s cies prob t e s ril . Th s car ied out s f l w : 1 . I n s e rt hp o bi 1 a0 - mg Ll sp et ov id a m g ,nw r t phe x o s d end i alum n foil. 2. Steril z h probe y aut cl ving at 12 3 . 6 p 0 traOdho v eb y n ° C for 15 min ( se Note 5 ). ° b e fr ho c 2 n ms tap C i r o e . This prev nt mois ure f m nt ri g he mas p ctrome . 4. At ach the probe to the mas spectrom befor insert g into any liqu d, to ensur tha f l v cu m is reach d ( Note 6 se ). 3. Prepa ing th S mple m d e a n s i u t o rb qf h c x y T p l e in r ve wat s mple ak n from the iv M rs y, e id UK. 1 . P l a c et h r i v w s a m p l ei n u t b v s e l , . g af r m e n t o p ,a ds i r t 2. 3. 4. 5. 2r 0ps mu ,f i c ket no h p sl u iba vnto d m gx u c h air nto he s lution. Incubate the sample at a fixed temp ra u ; in this exampl we used 30 increas th e of d nitr ca on. Sup lem nt with 15 m stimula e h d nitr fy g po ulati n. a w sb c v e oh t pm in I r uk l d , g clear of th s ir e baf l s. m S p a t“w f r sn h i o e b c l d ” , y et r o b gin reco d g at . ° C to M of Na O 3 and 50 m M of sodium acet in orde to 3.4 Calibr t on f he Dis olv d Speci s Prob Once th exp rim nt has end , the prob is w tched o “standby” o the manifold rem v f o the riv wa . 1 . I m e r s t h p o b i nd s t l e w a r f t g e n l yw i p ga n yd e b r i st h a may h ve c um lated on i s uter fac . 2. Switch e prob n at he m nifold. 3. Bub le th gase of inter s h oug t e wa r in tu il satur ion s reach d ( se 4. Calcu te h gas olubi ty a know ater mp ature f om dat bles B yd i v n gt h es a u r i o nc e t r a i o n f h eg s ta i v n m p e r a t u b y h a s i g n e b d c o m l t r pa s e h , b r y c o a d i e n g to each m s pectrom unit. Note 7 ). (24) 3.5 Dat Proces ing 1. Removeth diskfromthemas pectromet ronceth exp rimentisf nished and transfer to a PC ( se Note 2 ). The dat are saved by the mas spectrom- . 274 Firth and E w s et r as a text file that can be ac es ed as a spreadshe t using Excel 5.0 (Micros ft). 2 . O p e n i gt h f l e a d rg i v s n f o m a t i b u h es t po f m a s e c t r o m et r, whic is displayed above the reco d dat . The head r information is d e l t a n h c o l u m s fd a t b e l w i h p l a y we id t sh m r c o d e right, .e 12 8, 30 2, 4 0 , 46 and . m/z m/z 3. Using dat from the cra king p t ern ( 4. 5. 6. 7. 8. se by multip y ng the dat from chan el 12 by 16. 7 This is converts the CO reading s reco d t i s ac u l on e tra i . Subtrac thes values from th se in cha nel 4 , wher CO tribu e o th p ak v lue. Th r mainde s owing t N Ap ly thes rinc ples to h r f the da , g in us the cra king p t er o cal u te h valu of e ch gas. Use th calibr t on da convert h s value into gas c entra io s. Recording thes cal u tions as a macro prog am in the Excel pack ge al ows sub eq nt da to be proces d at he push of a but on, as long as the dat re first loca ed in th same pl c in the spr ad et. Plot he dat s a gr ph ( measur d ove 48 h are p s nt d howing t e i al drop in xyge con tration to a low steady sta e as the micro ganism car y out respiration using the ad e suc inate. Subsequ ntly, an increas in N by nitrogen production. This is the result of denitr fication, as the denitr fying organism ing the suc inat pres . c h a n e l u m b r s .D a t ed i - c h a n toe l uf p i g h s n e Table 1 ), the CO value is cal u ted 2 2 2 and N 2 O both c n- 2 O. ). Conce tra ions f nitroge , oxygen, a d N Fig. 2 2 2 O is det c ed fol wed begin to r duce th ad nitra e o gase u form, ag in ut l z- 3.6 Mainte c Wear nd tear m ns tha e sy t m requi s ome l w ev mainte c as de crib h ( se Note 8 ). 3.6 1 Cleani g th Sys em 1. Clean th probe and pl ce th m brane. 2. Im ers th ipof e r b in70%(v/ )ethanol dc tinuo sly amp ewith mt sah p e c r o T sh pi. t e a u d o rfg yh s t ne dm ther fo turns he p ak rofiles t h ir no mal “be ” shap . 3.6 2 Cleani g th D s olved Sp ci s Probe 1. After ext nsiv use, an ac um l tion of dirt may ap e r on the surface of the ste l prob , en ath me bran . 2. Remov the m brane y scoring w th a sc lpe and ge tly p e ing aw y from the prob . 3. Wipe th s l probe c an usi g ethanol-s ked n tis ue ( 4. Ap ly a new m bra to he cl an probe. se Note 9 ). O Monit r g c obial A t v es 275 F i g 2 .t A y p c a r l e s u o t b i n d y s M g I t S m o e a d u r n i t f c a o i n r v e water sup lem nt d with 15 m s h o w na r e i t g n M — ,n i t r o u s x d e nitra e and 50 m M - ,a n do x y g e sodium suc inate. The gase - . 3.6 Membran R pl cem t 1. Soak the m bran i chlor f m 30 s, cau ing t o s f en a d xp . 2 . S l ite dhx p a n m b r o t e h n s u i c rgao t b e l s are p s nt. 3 . R i mtn ehs b r wa l o i t n ez ck s, u r ga h seal i formed at b h ends. 4. Seal th ip of the m brane y gently bu firmly sque zing w th o ste l for(p r ot bh e l fn a g ci q u ot d hs r e p A tigh fit at the top of the me bran is also es ntial to prev nt gase from traveling dow bet n he prob and me bran , whic tends o give ab r nt readings ( 4. Notes 1 . a l s t ir e uT b p n c fo y A , v lrt e h m n- , 2. branes c also be us d ep ndi g on the cir umstan e i vol ed. Th se al ow dif us on f smal , no p lar g s molecu s but are l s perm abl to wa er nd polar m ecu s, whi y sil con rub e was d in th s work. Althoug the descript on given her use a spreadsh et software pack ge to proces th da , this mac ne d others may be us d in co ju ti n w h specif , de cat sof w re p vid w th e mac in . Su h software m y o a not be pr f d. se se Note 1 ). 1 0N o t e ). 276 Firth and E w s 3 . T h pe r i o sd t n ug e h r w a cs o bn e u i pt r o d c e n u g rh a d i ts o give a go d rep s nta ive curve when plot ed as a graph. If the period is to l o n gi ,m p r t a e i o d sfc v tm yab ei s d ,n f o h r t e mousn ber f sult gen ra dm stha longexp rim tsw l producet much dat o fi nt he flop y disk. Such dat wil her fo b l st. 4. Althoug is exampl use dat s ved to a fl p y disk, ome sy t can s ve dat o a sm l inter al h rd ive or linked PC hard ive. Th use of a flop y disk enabl th da o be asily tr n fe d to her PCs. 5. Sil con rub e is r tan o rep at d ry and wet h a steril za on, d henc , gen ral ythep ob scan er p t dlys eri z dw thou ca singd m e.Howev r, heating and co ling may slight y af ect he p rmeabil ty of the sil con to gase , m king cal br tion es al fter ch xperim nt. 6. Water nd other liqu ds po e s a gre t po n ial for expansio when co v rted r e q u it h a d o p f v n m l e s c rT h ai g .o u t vacu m with n e mas p ctrome . 7. -artnec o ni esa rcni ehtruf on ehw d veihca s e ag noitarb l c eht fo n itaru S , s e r u t a p . n m l y i h e T w f dv o t r c b s a p m deir ac eb osla d uohs noitarb l C .der isnoc eb osla tsum eruta pm erof ht dna -percsid, lpmaxeroF. lbis p atnemir px hto ralim s no tid cre nut o a ecnis d ta g si no tul s i arb l c eht ub ci ats elpmas ht fi ruc o na seic .snoit d c ats rednu l i eht dnuora c n oitelp d sag fo en z 8. When me bran integr y is breach d or built- p dirt from the surface of the probe nters the mas pectrom , perfo manc is reduc dram tic l y. This r e d u c p f o r m a n c e i f s t e l a b r n ts p l i e a k r o f l s w i n gt o h e i o n z a t f h ec o n a m i t g e r .O c a s i o n l yt h sd i r m a b e w n t o ms tap h e c r o d l m a i , t nf s c g p r Te ov h . the prob s a e cl n d whe v r discol at n becom s ap rent. 9 . l o n a h t s E i d e u o t n a e l c h s t b o r p n e v t r ag pm w o i f e h n t s y o .sgniraeb pmu raluce om brut eht fo egam d ot dael nac retaw esuac b ,noitcen o r 1 0 . p dn t oi hI s f e m l , b a k r u y o t w n tial moun f liq d to be rawn i to he sy m. 1 . t m I h e f b r a d n o i gp s v t l e , m c b a n contami ed with gase traveling from the a mospher down the l ngth of the probe tw n s e l and me br to he ip. Th s can m ifest lf as un al y stable readings of atmospheric gase at atmospheric lev s. Such readings may lso ind cate l k in the sy m l ewh r . Acknowledgments This work was funde by the Natur l Environme t Res arch Council, Swindo , UK. References 1 . o r c i nms e a dg v l o s i fdt n e m r u s a e tm c r i )D 3 8 9 1 .( I,R t o c dS n .a,D y o l L spectromy.ainlug bial Methodsicrbl.GnJ 127, 31–28. Monit r g c obial A t v es 27 2 . D e g n H (, . 1 9 2 M ) m b r a n i e l t s p c r o m e t i y n u a d p l e m i c r o b ol gy. 185– 97. 15, J. Microb l. Methods 3 . D a v i e sKJP ,. L l o y dD a nB L( ,.1 9 8 E )f e co t x y g d n i r f c a in t o 135, and P a r d ec no i t u s f P s e u ad o rm gn i . M i c rG oe bn . l J 24 5– 1. 4 . V N a i n eJ W ER l. o , b r t s A L C P xa . K n , u d e ( G J 1 9 I 2 ) h i b d e n i o t f r u x yc a g l d z b n i o Ap l. Microb l. T h i o s p a n et r h . G e n .J 5 3– 8. 38, 5 . L l o y d ,D . B L a n dD v i e s ,K .J P ( 1 9 8 7 ) e r s i t n c o fb a e r i ld n t ficat on cap ity under aerobic condit s: the rule rathe than the exc ption. FEMS icrob l. E 185– 90. 45, 6. Lloyd,D.(19 3)Aerobicd n tr fica on s il a d e m nts:fro al ciesto facts. 352– 6. 8, Trends Ecol gy v . 7 . T L h B (l ao . 1 D nm dK , 9y E f s 4 x ) ie tc g p r H 10. 1. 12. 13. 14. 15. 16. 17. c o n e t r a i o dn t r f i c a o bn y 1 8, 18 – 6. 8. Thomsen, J. K , Ge st, T. and Cox, R. P (19 4) Mas pectrom i studie of the ef ct of pH on the ac um lation of interm diates in denitr fication by Par co us denitr f ca s. -9 i. d e s n i r a u t s e n i o t a c i f r n e d f o t n e m r u s a M ) 5 9 1 ( . D , d y o l L n a . L K , s a m o h T .yrtemo c ps am telni arbme gnisu t em L ( a l 1P n o D 9.Cd y R8,e H 5 g x ) t i u m a s r d e o f n t l v gase in biol g ca sy tem with the quadr pole mas spectrom e . Biochem. Anal Jouan e , Y., Kel y, B. C., Berli , Y., Lespinat, P. A., and Vigna s, P. M. (1980) Conti u s monit r g by mas pectrom y f H cling Rhod pseu m na c psul ta. Berli , Y. M and Lespinat, P. A (1980) Mas pectrom i k netic studie of the nitroge as and hydrogenas activ es in in-v o cult res of brasilen Sp.7 J e n s B C . P o a R D, x d ( g H 1 9 8 M ) p s e c t r o m i a s u r e m n t of steady- te kinet cs of cyanob terial nitroge fixat on by monit r g dissolved N 2 in a ope sy t m. Jens , B. B. and Cox, R. P. (198 ) Measur m nt of hydrogen exchang and nitroge up ak by m s pectrom y. C a r l s e NHn D. , g L d o y ( 1 9 E f) e c aot l s h r n p i a tf a ie n or d m s t u e d p n B ia o k f Y r ’ s t . 137, 2879– 3. Bohátka,. S Futó, I. Gál , J. Langer, G. Molnár, J. Paál, A. Pintér, G. S i zMm á.o Jd,n é a k Ge ( l 1 y Q9 g u 3 i ) r smp ao e c trome sy t m for e nta io m t ring. Lloyd, D. El is, J. E , Hil man, K. and Wil ams, A. G (19 2) Membran i let mas pectrom y: probing the rum n ecosy t m. ogy S mp siu lem nt 19 2 speci . Pseudom na F E M S i c r o b l L. e t 536– 41. 60, Ap l. Enviro M c biol. ,61 .locE iborc M S EF .41 –30 Methods 185– 97. 31, producti n a d recy2 628– 3 . 143, J. Bacteriol. Azospir l um 67– 2. 125, Arch. Mi ob l. 37–40. 12, FEMS icrob l. Let 467– . 167, Methods Enzym l. G M e Ji n .c r o b l 4, Vacu m Journal f Ap lied Bact riol73, 15 S– 63 . 6 9– 71. 278 Firth and E w s 18. Virk , . T Ketola, R. A Ojal , M. Kotiah , T. Komp a, V. Grove, A. and 19. 20. 21. 2. 23. 24. O ( F e n 1 a - 9 c v s i 5 S h pr t )m. o b l , y s trome y. Harl nd, B. J. and Nichols n, P. J. (19 3) Conti u s measur nt of volati e organic hem als in tural w e s. Cristea, O. and L ger, G. (19 2) Gas met bolis mea ur nts of aqu tic l vs t ir m nu e gc b p y a l w o ft r ud e i nc o . Sensor a d Actu ors B g a s e o pmf c t ru n i D e ( 1 9 4 ) . L l o y d , a n J . B e s t d , in peat cor s. L a u r Gi ytn R lsd . F e ( 1 S , 9 gO 5 - ) m o n i t b r f l g c e a t i o n s at low parts- e il on ev ls by me ran i let mas pectrom y. Chemistry Lloyd, D. avies, K. J P., and Bo y, L. (1986) Mas pectrom y as n ecof o r tl g i c a Microb l. E Wilhe m, E., Bat ino, R., and Wilco k, R. J. (197 ) Low pres u solubi ty of gase in l qu d water. 142 – 5. 67, Analytic Chem stry 135, Sci. Total Envir . 7, 37–54. 518– 2 . 13, FEMS icrob l. Ec 23 – 40. Analytic 67:14 8– 20. s e yd i tm n g . a o l u vfr e situ n 38, FEMS 1 – 7. Chem. R v 7, 219– 30. Biof lms and E viro me tal P c s e 279 91 Experimental Biofilms and Their Applications in the Study of Environmental Processes Joanna C. Rayner and Hilary M. Lappin-Scott 1. Introduction 1. Why Stud Biof lms? The trend in res a ch in rec nt years has be n to extrapol result from studie of plankto ic bacteri into enviro mental sy tem . This method of s t u p d l y a i n k vg b o c r e importan d in a wide rang of are s; how ver, th examin t o f sev ral enviro m tal h bita s, extr m o therwis , uch as drink g water pi line has rev al d only relativ y low numbers of plankto ic el s. In aqu tic sy tem h biof lm acteri l ount per squa centim r of su ace h s b en estima d to be ap rox 10 -fold hig er than the cor esp ndi g plankto ic count per cubi centim r first ecogn z d as ign f c t as e rly 1943 tion ha we dtos u ymicr gan s ot lya bi f ms utal oin he contex f the biof lm nterac io s w th eir m d ate sur o ndi g a the influe c s they x rt on the nviro me t. The nviro me t has sign f ca t ef ct on the m tabolic a tiv es of bacteri , and studie of biof lm bacteri r e p s b t f h o xn a l m i g r w u p d o e n c i s y tems (3) . The study of biof lms is rel vant to a wide range of are s, and a m u l t i d s c p an m ro h y tei u q f v s w d understa he int rac o s c ur ing ot nly betw n he c l s and the surfaces to whi ey ad r , but e w n h micro l n es tha co xi w th n multispec b ofilms From: c o n u d i ht a s b y e l (1) . Surface ol nizat by micro gan s was (2) (4) Methods in B otechnol gy, . Enviro mental Monit r ng of Bacteria Vol. 12: Edite by: C. Edwar s © Human Pres Inc., Tot wa, NJ 279 , and ther is now a re liz - 280 Rayner d L p in-Scot 1.2 What is B of lm? A microb al iof lm is e ntial y microb al ce s im ob l zed at n i term i c r f oa b wv p e t , l h d xuy s a c i n l T r thy e d . col nizat of surface and sub eq nt growth as a biof lm is the bacteri l s u r v i a le p o n t r m e as l i u c h on w t r e l v s n u r t e h a s ic p o m l g y n air-l qu d o i -s l d nterfac s starved Vibr o g ra con hwd t e i s c sy tem with an increas d dis olve organic carbon conte n u ts r a ib e d c o t an i ls r u e f xo cpv h e a f l s u o cr t b i n m v y m o d e o f g r o w t h i n c l u d e i n c r e a s d p r o t e c i o n a g i n s t a n t i m c r o b i a l a g e nts (10– 3) m ed c hfb a o n isy ’ t — p g o n ai zs d , t so n. I it al y he biof lm was v e d a hom gen us di tr b on f cel s in a conflue t, blanket- i exop lysac h ride matrix ni g laser mic o py (CLSM) has be n t driv ng fo ce b hind alter g ou u n d s p e t r o hc b a iw f T ml g . of no destruc iv visual z t on has al owed the thre -dim ns o al and realtime visualization of hydrated biofilms. Biofilms are now model d as p o r e ws hmi yat d x , n c l eo s m f i u r t n e s o cr h a n e l ts u g o h ne c f l u bt i o m nels facil ta e transport of oxygen and nutrien s to the micro l nies and removal of waste and secondary products, but the biofilm matrix or exop lysac h ride has postula ed role in a tim crob l resi tanc , pos ibly e x cr ih oas n g t c a ot fi m n l e u s res nt a op im zed r ang m t of cel s a i t e m x al nutrie d f sion to enabl the establi hm n of microb al cons rtia al owing metabolic e x c h ar n g d y ls o i f t u r e n s of plasmid enc o g dru an he vy m tal resi nc a result of he cl s p r o x i m tc he f lw y s b n to a prim t ve ukaryotic s ue, with ome sta ic ontr l mechanis and hig lev of physi l g ca o per tiv y 1.3 The Rol f Bi ms in U dersta ing E v ro me tal P c s e u n e d l Ov b r a ip s t o g h f c m y obtained throug the xtrapol i n of dat obtained from labor t y s tem i n v o l gp a k t n i c e l s ;b u t h i n c r e a s g l yi p c a b et or l s y tems (3 , 15) D i o r b e st . c h v an ud f q m g (5) (6 , 7) . Adhesion t a ubmerg d s fac by cel s result d in the cel s regain their normal morph l gy (5) H o w e. av t r , c b h l m s n i d (8) B e n . a t fh o i c s d (9) but conf al scan- (3) (14 (1 ) (16) , 15) i o n h dc ra wfl t e uy s g r a fb ns F e dt oi m u . g c h , lp - f a t c o ni dl , r h e s (17) (17 Tb hi .eo f la ms nk d , 18) (19) . T. h pe o r as n cd - , and hig er a ,n d Biof lms and E viro me tal P c s e 281 chapter have confirmed th domina ce of bi lm bacteri , both numerical y a nm de t b o l i c y n , u r e t - s f i c n v r o m e t s view d as uneq ivocal, owing to its acquis t on using no destruc iv p h e - o f r a n g w i d e p bo s c t frm lanh i q e u d , p l t a hn c eok i du ry f s have norm us p ten ial o be util z d n the bio c n l gy i dustr , beca they x ib t a number of cap bil t es uch as the abil ty o l ca ize specif b i o lr e( gs p c an m t d fb u io) l, s and i cre s d l v of per manc i re to sy m c pared to l nktonic ba er l cu t es t r i c k l n gf e ra dp o u s / n r f l u i d z e b s na u m ro f e s c h as denitr f ca o , xenobi t c detoxif ca n, and heavy metal removal from w a t e r .B i o f l m c t r sh a v eb nd l o p a u t i z e df o r l n g a d t i ro en m, u v af l d h g t yoi r n c a b f w s m e ter sy tem organic comp unds and the transfo m i of inorga c comp unds, sub eq u e n t al cy i m og z bhe u l d p f t a n s human and anim l gastroin e al trac are col nized by bacteri tha form tis ue-pro ctiv b f lms, prev nti g adh s on by f reig act D .a t h c b ne (1 , 15) in situ (20). (20) Wastew r atmen sy com nly uti ze . Biof lms play an importan role in the biodegra tion of M .o s te c i n fh (21) (2 ) 1.4 Detrim n al Ef ects o Bi f lms The unco tr l ed and undesirabl ac um l tion f biof lms in biomed cal a en d g i r s y t e hm a p i r fy e c t s h: i da l m g e ., c o r rosi n and to h decay; reduction in pro e function of the surface, e.g , reduc f i ency of heat xc ngers el ctri p elin s Biof ul ng has be n defin as dam ge to surface or the enviro m t as a direct sul of r ace- s o i t d m cr b al g owth (7) (23) and turbi e pow r l s e in hydro- ; and the cr ation f a res voi f poten ial p thogens. . (23) 1.4 Physical D m ge nd R uctio n S rface E i n y Metal cor si n of ship , pi el n s, and oil rigs is an exp nsiv problem, with b of lm r ation c ur g apidly fo w ng im ers o f the sur ac . Marine macrobi f lms on ship act to increas drag and frict onal forces, c op d i n r sw f e u 1 m 8 % a l - t g ; b i sr o d he f m t p l v a c w n hul s (8) . The p ysical thicknes of the biof lm reduc s the pi e diamet r in industr al heat exchang r sy tem , af ecting flow, and the exchang of heat betw n h liqu d an the co ling surface i r duce estima d cost of £50 mil on an u l y with n e biof lm as depl tion f oxygen oc urs by the a robic m ro ganc o r p i -m a y f g o w t h e ac v n rsm i ub , l t h n p r e s t i m B i o f l .m s (15) (24) (23) . Anaerobic zones are formed , with a sub eq nt . 28 s io r ng a s mub ce h a ts l f e - r d u c ib na g t e r( iS aR B ) c a d l m g o e c u r i n a eg s u l o b t f i m o r a t i o c n l s b e r v i d n the body. Dental caries are an ind rect consequ nce of the formation of a multispeci s oral biof lm (“plaque”) on the enamel surface of the to h. Demin raliz t on f the namel oc urs a result of by-products of he bacterial metabolism, uch as organic ds, whic be om trap ed at he to h surface (19) m e c h a n i s mt oa v i dt h ea n i m c r o b i a l c t i o n fs a l i v r yc o m p n e t s u c ha lysoz me and mechani l removal, but also t facil ta e th optimal util zation f the abund t nu rient sup ly. Rayner d L p in-Scot (23) P h .y s i - . The oral microfl ra util ze biof lm formation not only as a 1.4 2 Creation f Res rvoi f Path gens The prima y con er f the o d, water nd me ical ndustrie to de rmine th po ial f the b o ilm act s p hogen r s v i a d to ev l p ef ctiv on r l st a egi not ac ur tely rep s nt the ext n to whic biof lm formati n is oc ur ing. The contami of d pro ucts may o cur f l wing co ta wi h potential y de rim ntal b c eria s qu ter d wi h n surface- o i t d b ofilms The ac um l tion of colif rm bacteri in biof lms in water distr bu on sy m p t a r h se o c k y i n d f g o s c m r u e a i l n t d e t f r io c ah p m n s be n demonstra to be harbo ed with n biof lms tha would be pres nt in co ling t wers and w ter sy m w e im l an c d t o vr g b h s u p y with a cor esp ndi g creas in os c mial nfectio b r i o a p f l t e h s m g n c, w d i r o v b y e n ut h re i - fb lo dc y a s v u n , t r e i hc s , central venous lines, pacem k rs, heart val es, and prosthe ic hip joints and thes biof lm ay ct s i e for u the dis m nat o f i ect on. Th p h a n g tb o i c y e d m r s f p a n c h r o nd i s p e l t r i n d e v cf s ,t o r m u ag li h e n r s u l t m a y devic -as o ted inf c o s . In most ca e , pl nkto ic el ounts d (6 , 23 , 25) (25 (27) , 26) . L e g i o pn l u ma h has . The r c nt i reas n the us of ind(28) . Extensiv bacte- (7) . (24) 1.4 3 Stra egi s fo B lm C ntro Treatment regimes ag inst biof lm-as ociated infections are normal y dev loped using dat tha measure measures the f ica y of an antim crobial gent ag inst planktonic organism , sub equ ntly resulting inef ct u a le r a d i c a t i o n ft h eb i o f l m t w oa r e s :t h ep r v e n t i o n f i t a lc o n i z a t i o na ds u b e q u n tb i o f u l i n g , and the dev lopment of removal/control stra egi s ag inst he stablished biof lm. . (6) (29–31) .B i o f l mc o n t r o lc a nb ed i v d e i n t o , Biof lms and E viro me tal P c s e 283 1.4 3 P REV NTIO B OF /A IOF UL NG ehT yca if o larev s tn f id g luo a s nit c d a stnel p r iag eniram s l fo b h ne d imax esu hT . fil n ram e to y cix f l ve ht dna sm i o b g yc e f d e ft oa n g r p m i s ch b x u d e eht cn di fo detaic s -r h y n u tcar ;snoi ef t b hn fo -irp yram noisehd t a c-r vl xe s t h a ne b d vr s o i t e h Tn o i t a r p c f s d b u y x ) A P B O ( o - 0 1 , a h n e b w o h s t c u id r l a y v p detarop cni ,ytic bohp rd eg a ,s nh uor ecaf ht fo s eldrag t h antim crob l agents, bacteri wil ev ntual y adher to any surface, and the .gnio s ecafru hd t / l v y pmoc a r f es , tub er h s m b a ecn l wt b )23( )43( /R ANIT Z ON EMOVAL S . , r e v w o tH i s a p TRA EGI S (35) (36) condit s ha led to vel pm nt of del sy t m for he stabli m n . Enviro me tal s u g v i a e r o n tf b p c a i oe ns (36 (38) , 37) sug- (29) (24) t oc r h wu e a n di T s l y. 2. Materials and Equipment for Studying Biofilms 2.1 Model Syst m for E ablish ng Experim tal B ofi ms The complexity of biof lms and the ne d to study them under labor t y . )3 ( 1.4 3 2 S In i dustr al ystem , ch i al b ocides r p ent h prima y str egy for f o r mh sy —p c n d l i - e a t , u n C h l o r i e , c t . b f m rousacid,hyp lor te, ch in d ox e— sth m co nlyusedbiocide for chemi al trea m n of water. Mon chl rami e has be n fou d to be the mos ef ctiv n the inac v t o f bio lm acteri n f u a t c r l s i o e d h , p gy t c e r o f i s b a c t e r ( i . g , o w a h m n d t b l s i c u w ) ,a f e t o h v r p l t i e so f h b l m , u s e q n t ya f c i gb o d e f c i n y .T h e o u i f r m m i r fc e o s l p b a n u t w h v g y m i e trea m n and the pr s nce of dist nc ve bioc d gra ients w hin t e biof lm d l i r o t f eg c h s a w b - n m u rine a d oth r is nfecta . Th fo d in ustry e anit z rs o di nfecta s f o l w i n gd e t r a m e n t ;c o l yu s e d h m i c a n f e t si c l u d chlorine, iod ne, and am oniu -b sed comp unds. Antib o c trea m n of devic -as o ted infect o s i large y dep n t on the organism, or gani s m , n v o l e d H. w r t, h i n e s t a n c oe f h b r i a l o f m s may result in the surgical removal of the infect d devic in orde to dispel c h dr eo vn i - a s c ft e d i o n s d e v l o p m nft h w sai b l e o u g c p t i h fe y organism f ter as biof lm. DHESION 284 Rayner d L p in-Scot Table 1 Experimental Variables and Parameters for the Investigation of Biofilms Using Laboratory Model Systems Vari bles Physical Chemical Biol g ca Par met Temp ratu , s face om pH, sub tra e con tra i , d s olve xyg n co e tra i n Organism type, organism co entra i and stu y of exp rim ntal biof lms. pothes and the extrapol i n of dat under defin , contr l ed condit s. Ther are two main types of exp rim ntal biof lm models whic encompas wide range of c mplex nviro me tal v riables, and i vestiga whic are g n l y simp er and e bl th con r l f a v riety of influe c g a tors. M labor t y s em ar of the la r type and to examin biof lm formati n at solid/ qu interfac s util z ng fixed surface . 1 Table rato y model sy tem , and surem nt of bi lm par et s. Thi section describ only a number of the o t h e r n i f m a uo t h e r s ; i n y t e m s o d l a b r c n m s t sy tem , posit on, surface h arge, su f c ro ghnes Model sy tem nable th esting of hy: replicat v , (39) l e a xub so mi- n g d p c v r t l h e a b s list ome an lytic methods for the m a- Fig. 1 se refs. 4 , 7 , 15 , and 40 . 2.1 The Rob ins D v ce TRhoebidnsvwca lopteUhdnivrsCafylgetxomin biof ul ng in industr al pi el n s e x a m i n l u s od t w f v c g r – , c i a t be d o f l m Ts h. i Re d b n v (c M r aiD s) t g u l P e p x block 4 cm long, 2 cm hig , and 2.5 cm wide, with a 2 m hig by 1 cm c e n t r a l u m s d i e o rf v a b l s t u d p c e o n ig t ls h w c dif er nt su face n be fit d. The sy t m is ter liz d us ng ethyl n oxide gas since hig temp ra u / es r result in warping of the Persp x. This p h s y er a o x l f t i nc mg w b , d par met s on biof lm formati n and an lysi of the respon of biof lms to antib o c d i e tr a m n . in situ (41) . Init al y comp sed of bras or 2.1 Conti u s l re F ow C l m e t a - n wc d p sur h v o l f i T e w s bolic products and the d pletion f oxygen a d nutrie s quently enabl s the contr l of the bacteri l growth condit s dif er nt s g of c nti u s l re f ow c l are u ntly i se, ut l zing and sub e- (42– ) (4 ) . Sev ral Biof lms and E viro me tal P c s e 285 Fig. 1 Experim ntal v ri b es and p r met s for he inv st ga on f exp rim ntal biof ms e tabli h d us ng labor t y m del s t m . r e a l s f t n i o u g mP c v p h d y , x a s t e u r c i h l two, seal d g s cover slip w th sila c rub e t ing and outle s, o the more c pl x. The lim ta ons wi h t e us of l w ce s ar p im ly due to r t e h s i c o u a f n p r t w b ce u h os i n, j d with CLSM, m cros py image n lys , a d met bolic s a n , flow ce s an s t r u cba -i o f lhn m e , g d a l r e t p o v i d ture and f ctio l rgan z tio w h n s gle a d mu tispec b ofilms. 2.1 3 Perfus d Bio lm Fer nt T h pe r f u s bd i o l m e r n t s y wm a d e v l o p t n b de i s c t o n b e t w f g h r a o d n c s l iy o nb a uc sterial po u ati ns ont a cel u os acet m brane; th impregnat d me bran w s remov d and i sert d upsi e down i a conti u s ferm nta io p ar tus m e d i u w a st h np r f e d o mb l wt h r u g ef i l ,w t h en u m b r so f e l u t c d r s a h i n g y t f e p r s — 2 oa x u i n m lt h r of bacteri l su fac n e tio s f ti sue . Th ystem a p lic t ons f r use in xam g the f c s o antib c herapy on s ft i ue n ct o s. (45) . Midexpon t al ph se bact ri l cu t res w filter d (46) 2.1 4 Rot rque Also know as the an ul r eactor, the rot que sy tem is comp sed of two c n e tri cylinders with a number of removabl slide in a conti u s cult re sy tem. Rota i n of the in er cylinder creat s a shear field indep dently of the m diu flow fluid r ct onal resi tanc d is cap ble of varying flu d shear nd stre and (47) . The sy t m i h g ly sen it v o changes i . Fresh 286 Rayner d L p in-Scot resid nc times indep tly. The surface r a of the slide are xposed to uniform shear stre values and complet mix ng of the liqu d in the sy tem enabl s th y is of a r nge biof lm pr ces . 2.1 5 Consta Dep h Film r ent The consta depth film ferm nt is an enclos d ferm nt tha has be n e rxu t iasw o vm c d n y metalworking fluid biof lm fluor ethy n (PTFE) turn able, with a seri of removabl film pans, each contai gs xremovabl p ugs.The iof lm s a nt i ed co stan dep h s c r a b p l ey n d o , t i v s c u w e r my g h a t surem nt , and carbohyd te lev s, is consider to be “quasi steady- ” (40) . Biof lm formation can be control ed, is rep oducible, and is easily sampled un r s cif ed nutr a d g s con it . a nd (48) P s e u d o m an r g i s . It contai s a rota ble ste l or polyte ra- (49) 3. Methods 3.1 Micros py The abil ty o v sua ize th b of lm is p rtan defi g th arc i e u of bi lms and the in ract o s cur ing betw n he c l s and the surfac . Micros py ha be n wid ly use for th di ec v sualiz t on f i al t chment and sub eq nt biof lm formati n fol wing adhes o (4 (15 , 54) and phenoty ic changes , 50– 3) . 3.1 Electron Mi sc py T h em a j o r d v n t g e f l c r o nm i s p y t a b i l or e s v b j c t tha c n ot be s n u i g l ht micros py; the r soluti n of el ctron microscope is ap rox 0.5 nm co pared with e 0.2light absorpti n micros pe tungs e filament gen rat s an el ctron beam tha is focused by a seri of magnetic l s under hig vacu m ont he sp cim n. 3 .1 S U n l i k ce o v t n a bl r i g h - f e d n p a s c o t r m i c o p y S, E dM e s e l c t - h s p b r u y a in f m e S oq c t . g tron beam c use the mis on f secondary el ctrons tha ent r he d tec or and strike a scint l a or, gen rati light flashe , whic are convert d to an el ctri a u ent by he p ot mul i er. Subs q ent amplif c t on a d tr nsm i c s a t h o r n d up eyb li am g e of el ctr ns de ct is dep n t o he surfac top gra hy; t e pr s nc of d e p r s i o cn a u l e t r b os p ad cn, e q u t l y ah r p e s darke compared to raised and ther fo lighter are s. Sample pre a tion µ m resoluti n of di er nt al (5 ) CAN I G E LECTRON M ICROS PY . In el ctron micros pe , the heating of a (SEM) (5 ) Tn hu .em b r Biof lms and E viro me tal P c s e 287 –1 F i g .2 L a m n rf l o wb i m( f l o wr a t e 0 . 8m L· i n f l ao i w n - t h r uc g l Mav Rni eDds zu S ET gMh .e b i o f wl 7am 2s - h d ; c r o b i a l p y d u c e x o l y s a c h r i v d e b la s n k t like ay r(bl ck ow),rev aling E n t e r o c f ua s i m a r = b5 e l c S . s e l a i r t c b d i o c g n i y l r e d u invol es th fixa on the surfac nd t he biof lm us ng tar ldehy or f maldehy , fo l wed by h ration d e th r ai o cr ti al-po n dry ing. The sp cim n s coated wi h a f ne lay r of metal p r ic es, pla d in the micros pe chamber, subject d to a vacu m, and bom arde with el ctrons (56) . Figure 2 3.1 2 E µ m. show a bi f lm on g as vi u l zed by SEM. LECTROS AN SEM ( ) ES M, a modif e form of SEM enabl s imag n of hydrate specim n (57) p lb ay c sti hn e g m r u x c t d o2i 0n r g — h e satur ed p tial res u of water o m te p ra u v i m hs a yu g d p n rl e co f tz w , shrinkage and gen ratio of artif c s compared to conve ti al SEM techniques. Howev r, as i the cas with s and r SEM, the l ctron beam d ages th p cimen a r l tive y shor p i d f t me. 3 .1 3 T (58) RANSMI ON In TEM the l c rons a e c t r d as hey p t rough e sp cim n, the f l u o r a h e s c T Ei n Mm t g . f l o b e y u s c d f o r m e d ng l a s u r f c e E LECTRON M ICROS PY (TEM) . This enabl th 28 Rayner d L p in-Scot -ruc o snoitcaretni eht no dna s enkciht mlifo b no noitamrofni ecudorp ot desu ne b eht fo si ylan deliated selbane tI .mlifo b a fo srebm gnoma lev ralu ec a ta gnir .mlifo b eht niht w nes rp sl ec fo erutc r s alu ec dna stnem gnar lait ps 3.1 4 A TOMIC F ORCE M (AFM) ICROS PE The AFM is a scan i g probe micros pe, in whic vari t ons in voltage astuormf cbey i p A F M t h e a cl o u dr n e f t i w o cg u r (59) . When a sample is can ed in a raste pat ern, vari t ons in the surface top gra hyc use nd latio f hec ntil v r owh c asil n tr de ip s at ched. A laser measur this move nt and fe ds back a signal to the piezoscan r, causing the can ilev r d flection be k pt a consta lev . The voltages p lied to he pi zo scan er can the b convert d o an rtif cial y col red image, whic onsequ tly mi cs the op gra hy of the surface t ons a r te of d l c i n . (57) 3.1 2 Light and P se Co tra Mic s opy Stu d i e s u s i n g b r i g h t - f i e l d a n d p h a s e c o n t r a s t m i c r o s c p y c o u p l e d w i t h i m a ge an lysi have examin d col ny dev lopm nt, ef cts of nutrie concentra io on at chmen , and so on d e m o t n su r b iv a l e m c -o nh f t teria to glas surface t m r i a o c n fh s u e p v l y , r i m t ing their ap lic t on to the study of biof lms on opaque materi ls, exc pt in s i t u a i o n s w h i c s t a i n s u c ha s c r i d n eo r a n g e d5 - c y a n o - 2 ,3 d i t o l y t e r a z o l i u m c h l o r i d e ( C T ) c a n b e u s e d a n d t h e n v i s u a l i z e d u s i n g epifluores nc mi o py. (53 (53) , 60) . Phase contras micros py has . Howev r, most bright-f eld and phase contras 3.1 Dif er nt al I fer nc Co t as (DI M) icros py DICM has a marked lev l of superiority compared to phase contrast micros py, al owing the obs rvati n of bi l g ca s mple withou e g neration f acts. The DICM micros pe a nv tio l gh m cros pe with ultraviolet fluoresc n e, whic has undergon reconfiguration of the epifluor sc n e a d pisco DICM section above th micros pe tag . Thes and ot r p a ions l w the vi ual z on f paque s cim n , a d the light inte s y can be enha c d by mir o s pres nt in the mercu y lamp casing (51) s b u it r p o f h ad e c v lg D m n I C M . y and l ow visua z t on f he bi lm xop ysac h ride (EPS) 3.1 4 Conf cal L ser S n i g M cros py I nC L S M ,p e t r a i o n h c kb f i l m s a d ep o b l w i n gt h eu s of a krypton/a g laser, whic excit s fluor ph e dyes pres nt with n the (57) . Biof lms and E viro me tal P c s e sample. Th r sulting f oresc n i det c by phot mul i er t b s and d i g t a m ol b s e n A d . r t fi o h e c ( a l quent col e ti n of the col e ti n of a seri of ptical se tion hat c n the b computer oc s ed i m a g 3e D c n r u s lo yft iw a e to l for the study of a wide range of biof lm featur s, includ g physiol g c p r o f isaltne d u c h r o g n e (i t y t hael o w i y po Hx f,y g ean u dt r i l m s c o n iu e g r l c t o d e s ; the an lysi of vel city and if us onal proces and number of ther f at u r e sC ,L S M p n t a c h i q uom ef j r p t a n ci hs e u dom yf cal, industr a enviro m tal b fi ms 289 z p l a nd e ) ts hu b x - y plane images (par l e to he surface) nables the (61) se situ n e f ac nt i v s C L S M . a C l h s o p 1 t B 7 e ) r c i. b u f - h m yi ebf dn a u rt sol c , ; . (62) 3.1 5 Metabolic/V Stains Labor t y techniqu s util zed in the enum ratio of plankto ic bacteri , such a vi ble c ounts, po es an i her nt dency to under stima he to al number of viable bacteri pres nt owing to the pres nc of viable but no cult rab e c l s or a biof lm. Direct m rosc pi techniqu s co pled with t h ue s o vf i a l n r e p s mat o c u r e h n i q f o tr u m e a i n 5 - c y a n o s 2 b , M 3 u e t d hv i r l . y c f z a o n te razolium chloride (CT ) and INT (2-[ p h e n y l t r d a m z co u i b s e h nv ) tl - y teriap s n w ter ampl s (64) . In the pres nce of an ctive lectrontransport chain, CT undergoes reduction, resulting in the formation of an crystal tha fluoresc red when excit d with a certain wavel ngth of epifluoresc nt light. CT has the adv nt ge over the relat d comp und INT in tha i al ows the visual z t on f actively r spi ng cel s on me bra filters p l a s t i c n d m e , w o a s u c h r f eo p q t i a l y n d fluor genic comp unds tha have be n used to as e biof lm physiol g ca activ y include rhodamine, whic det rmin s me bran poten ial 4,6-diam no 2 phe yli do (DAPI), whic sta n l vi g d ea c l s. 3.2 Ad it onal Tech iqu s for St dying B of lms 3.2 1 At enua d To l Ref ction F ur e T ansfo m Infra ed Sp ct om ry (ATR/F I ) In the study of biof lms, ATR-I radi t on is d rect hroug an i ternal refl ctan el m nt (germaniu or zinc sel nid crystal ) to whic bacteri are at ched. IR radi t on is absor ed by a molecu when the nergy of the r a d i t e q o s u hn l p mr d c a x i e t v , b o n a l sta e. This ab orpti n o ly c urs at discret f quenci s, and the numb r of p -iod phenyl]-3 [ (63) , on p -nitrophe yl]-5 pi elines, and in dis nfect d biof lms insolub e p r le CT -formaz n Other . (65) (6 ) , and 290 Rayner d L p in-Scot molecu s pres nt is pro ti nal to the amount of radi t on absor ed. This frequ ncy-d p e t absorp i n oduces a uniq e absor nce pat rn of the s p e d c t i f r h b um a n y o l I s . p e c trum is the comp site of the spectral signature of each of the biom lecu s p r e fm s o T l h nw q a t uc .i b y d e r o m n the pr s nce of speci groups f atoms wi h n t e mol cu e. Th group f equency is defin as the ind v ual wave number range at whic a specif group f at ms b or adi t on. D f er c s in molecu ar st e can subsequ ntly be ident f and quantif ed using tables of char te is c frequ ncies to d n fy speci IR absor nce ds (52) . 3.2 Cryoemb d ing C r y o e m b d i a n psg l c t oe f v m a s r i b l h c k n e o r t ment of paque r t nspa e urf c s, and i vol es th fixa on biof lm using a cryoemb d ing comp und tha contai s a number of water-solub polymers to maint the intac biof lm struc e pound is placed ont he biof lm whi e t is t l a ched to he surface. This p r o c aie f s d t n y u hz m l v o, i d n g the forma i n ce rystal . The mb d iof lm s re ov d f m the suro tf pa h n ec d s i m bg r z , o af n dl w i c h e betw n he mb d ing comp und m i cu rs Mo a n g p e l d y . t i c u b e t h n a disrupt on e s tha ind v u l ce s, mi ro l n es, a d w t r ch n els a remain vis ble; physiol g c gradients of metabolic activ y, such as those p r e f s o l n t a w i g b c s m e u r v n a to d ,f i metabolic dy s n fluore c mi os py. 3. Model Syst m and Experim tal B ofi ms Experim ntal biof lms e tablish d using model sy tem r p es nt a usef l to l f r the labor t y- sed tu y of sanit z o and is fect on s rategi , metabolic proces , nutrie util za on, gen transfe , and biodegra t n. d e t r h m iy np ao f s w g e d x a m t i o h n f l T w e y t h p o a if s r n e m l u v c b g a t i o r n , h e c ture, and functio al char te is c . The producti n of rep oducibl biof lms under labor t y condit s rep s nt an importan factor in the study of biof lms, w th par icul e vanc to ir nme tal p oc s e . 3.4 Ap licat ons f Bi lm to S udy In strial Sy ems ae px s l t o ir bcD h mn f d q u y stfe hon u rv d i y m p a l c e s hi r ot bu , d g n z a f l m s are th si e wh r t e majority f enviro m tal proces oc ur, ather an (64) (6 ) . Cros ections f vari ble thicknes . The mbed ing com- Biof lms and E viro me tal P c s e 291 simply a to l to facil t e their study. The study of biof lms encompas a wide range of disc pl ne and has many importan ap lic t ons in furthe ing our understanding of key environmental proces es. The primary use of biof lms, from an industrial point of view, is in the contr l of unwa ted biof lms, e.g in the d v lopm nt f a i oul ng c ati s or u faces th wil reduc o p vent microb al dhesi n. 3.4 1 Influe c o S rfa e Typ on Adhesi a B of lm F r ation SEM ( m e c h a n i s u t l z e db ym a r i n - f o u l gb a c t e r i o l s ,p a t i c n d f o u l p s a u i r n f t e cg d a wide range of surface types in batch or conti u s cult re and is a go d model f r studying flow sy tem biof l s tinc o betw n factors ibuta le o gr wth a e nd those wing to adhesion. Mild-ste l surfaces exhib ted a 10-fold dif er nce in the number of col nized het ro phic bacteria relative to polycarbonate surfaces when examin d us g the an l r cto ( si tenc of li rms n xed-po ulati n b f ms ) has be n used to examine the at chment Subheading 2. 1 . (67) ( MRD The . t o e f s in h a gb l ) S u b h2e.a d i1n g (68) , althoug i does n t al ow dis- Subheading 2. 4 (69) ) in a study of he p r. 3.4 2 Physiol g ca Ef e ts o Bi c de To evalu te a particular antim crobial agent for util za on in trea m nt regim s, it is nec s ary to det rmin the ef cts on the biof lm in terms of alterations to the physiol gy or metabolic activ ty of the bacterial cel s. Biof lms tha form in heat xc ngers, pi l nes, a d rink g water sy m (26 , 38) a r ne o t b l y s i a n co h r we i fs q u n t l y h me a i d s f c t a noc fh i eT . p s v r aq lu t i o n se ;. g I : h r u loai tfn b i t yo f h eb c d p n t r a e h b i o f l m s r u t h ep s n c o fa E P S w i t h c e n l s o f a l d i p r uA s c t e n ? o w i g r m a t x a biof lm equal y af ect d by the trea m n ? The use of chlorine microel trodes an CLSM for the visual z on f chl ri e p n t a o i m xed aeruginos t h pe r s n c o f a t i - d u s o n t e r a c i s w, h e u l t id n m c h o rine pen tra io into the biof lm with n a biof lm o wing b ocide tr a men tha ve b n visual zed using cryoemb d ing a m e an lysi h ve own a u iform l s f re pi atory activ y with n the biof lm visual ze th p ysiol g ca respon f bacteri n of lms t rea n with chlorine (37 b s e o ri la nf g p y d t h v c bi or fa n O l me s . thousand 10, -foldhig erc n t a io s f n m crobial gentsm yb k i r cl e a o q uvf ts n hb g p w d i c P. and r o b t i a gf n l eq wm vu d K l e b s i p n ua m o e (38) (36) , 64) Td.oa t e ,h rs m bn i g lf ea c t o rh b n d i - . The gradients of physiol g c activ y . Cryoemb d ing has also be n used to 29 cel s with rega d to he c l nve op , bind g of antib c mole u s r modif cation f m lecu ar t g s by EPS at dif er n s t wi h n e b ofilm the f c iv n s of a p rticul ea m nt s r egy. Rayner d L p in-Scot (29) I i. pt s o u l a e d h c m b i n t o a f l e r d i b t p c m e a l i y , alter d physiol g ca t us of he c l s (29) (36 , 37) , and growth e al inf ue c (12) 3.4 Bioc r s n a d Pit g The biof lms exist ng i the majority of natur l ecosy t m are p sent as complex i d com unit es, whic pos e comple ntary me bolic fun tions, resulting in the formation of sev ral loca ized microenvironme ts. Biof lms are cogniz d as pl y ng im orta le in b oc r s n, a d thi b i o t f h e l am n u r s b i t o c e d l neity inher t of many biof lms loca ized r s onc l ;a erobicz n s eat dby h u il zat on f xyge tb ahy e r o i fnc d u l t v e a y r o b i c g n s fm a v t hr e o w cd i ity of SRB. Under optimal condit s, he SRB are impo tan co tribu s in cor si n. Enha cem t of their activ y may oc ur as a result of the EPS, w h i c s a p b l o e f t n g m ha s b i d e r t n i o c f r sion pr duct . AFM ( rial biofilm on a cop er surface (previously as umed to be toxic to micro rganism ) and has shown that the organism tested was directly as oci ated with the pit ng cor si n of cop er sample d hy ration a d can provide nformati on the as oci t n betw n the c l s, EPS produce an th surf ce o whi t ey a ch. Posit n g of a micr el t od ip (<10 nelsha b u dtoexamin h pH d s olve xyg n lsi b of m p r e s n ta m l / r i f c a s e w t ri n f a c e s the lev s of dis olve oxygen decr as as the microel t d was moved aw y from the bi l nt rface d p r into he l s a robic ent al zo s of the micr ol ny (70) (47 h e tT r o g .- result in the establi hment of , 71– 3) ) has be n u d to xamine b ct - Subheading 3.1 4 . AFM does not requi (59) µ m) in relat o micr lon es a d w t r ch n(74) (3) .I na P .a e r u g i n o s biof lm, . 3.4 Fluid ow System Labor t y studie invol g exp rim ntal biof lms are rel vant o indust r i a l n d u s y t e m .K n o w l d g c e r n i t h f c so a t r u h s flow rate, hyd o namics, d hear st i ap l c b e not ly the und s i r a b l e o f c m u s i n d g t o fh le r p aw i bn u t s ho e a q u t ib c o f l m r sn k a t - o w i nr g v e s t a m B .i o f l c u a tion in pi el n s can af ect the hydro namics of the sy tem, with consequenc s for heat and mas transfer pro erties. Even under condit ons of turb len f ow, hic are com n i both na ur l nd e gi r d sy tem , a lamin r flow sublayer proba ly exist in the vic n ty of the pi e wal (68) . Biof lms and E viro me tal P c s e Vari t ons he flow rat i l nf ue c di s on rate d u ri nt av l b ity and, sub eq ntly, co izat n lev s. Liqu d flow ve city n model sy tems is an importan factor in predict ng the ef ct of a biof lm on sy tem hydro namics. tions a d v u lize s ng CLSM. The RD as b n u ed i a x m n tio c o n d i t fs l w a m r n d o t bi f l m e w a n s h p o f By tracking fluoresc nt y labe d latex b ads throug the biof lm pres nt o t h es u r f a c o l w u s i n gC L S M , t p o s i b l e n kf o wv c i t y h v a r i o up sh y c l a m e t rs u bh i o f l m c t u r ah e o g n i t y struc al het rog n i y of a biof lm may cor esp nd with het rog n i y in some physiol g c par met s such as dis olve oxygen gradients. Transpo t proces oc ur ing with n biof lms wil nflue c the sup ly of xygen a d nutrie s a d h over l f ic n y o b cides u h a c lorine. 293 show a biof lm dev lop under turb len condi- Figure 3 (68) 3.4 5 Fo d an W ter T a m nt I dus rie -orcim lufmrah yl aitne op r f sriov e sa tc ot laitne op eht s e op smlifo B s me l ib na p g rcfo t e hy a i l u qf o ed h t c su i no r fp e l d b r n a s h i t y g e u m o rc nx h w d s e i g d t a ru s n i o a t ed l z v c r n i f s a e do r h t sm inagro cim noitac f dom ecafrus emylop b ,.g e cn r hda l ir s t n e i gd a r o m x b s e d n y u a r o c p i m g f t w u l respi atory activ y in a mixed cult re biof lm fol wing dis nfection with e n i m a r o l dh c v e a m r o f i n u s l f o y r t a i p s e v c n h t i w e e tn i m agl r o f w h c b n m d e r u ct a o e h f r u so em hl ti fr a bn od i a , k e l c u b f r t n i d e ty cl s fvmg n i a u e r od t l y f ai re h pt s -retn yb tilauq retaw cef a yltcerid yam iretcab mlifo B .ytiv ca ilobatem dna s e n o ufi r t a v d g e y qsk n h p l m u r b )67( airetc b mrofil c , y l t n e u q s lo c i w,h t e r u d c o g np i l m da rs n gt i du e c t lob n i w tceridn ehT .daol iborc m fo smret ni retaw d hsin f eht fo ytilauq e rt h ksam t fo s ce f dna ,rod ro etsa ni segnahc edulcni yam iretcab mlifo b fo ecn serp h ecn seroulF .noitad rgedoib laiborcim ot gniwo retaw dehsin f eht fo n itarol csid a htiw deniats mlifo b retaw gnik rd nworg-y otar b l a fo ypocs r im fo ecn serp eht dewohs eborp esad )52( sa hcus mrofil c fo ytil ba eht gnitar snomed , tner hni esoht ot ralim s noit dnoc ihport gilo t dezitam lc a smrofil C .mlifo b a dlim eht fo n itaz nol c ni lufs ec us eb ot dnuof er w smet ys noitub rtsid retaw ni rotcaer alun a fo secafrus etanobracylop dna le ts y pt os )ca M r C I n D (i m T .h e (71) (75) . sihT )52( ) 6 ( - e t c a bf on i t e v r p h t o fs e i g t a r s e h u fp o l e v d t n a, -anibmoc f esu hT . )57( . ehTts gi ol f ytiv ca )63( )96( sm inagro es ht ecniS .al enoig sa hcus negohtap ro , β -isotcal gl a gni atnoc( iloc .E iloc aihc rehcsE ca )en g ret oper Z niht w detau is emoc b ot )96( ecn ref tni la tner f iD . gnidaehbuS 2. 3 ) combined with fluoresc in . 294 Rayner d L p in-Scot Fig.3 B of lmdev pingu dert b l n f owc dit nso agl c vers ip n a polycarb n te flow chan el at 72 h CLSM in transmit ed light mode. The biof lm was comp sed of Pseudom na fluorensc m s –1 , t h e f l o w d i r c t n s i d c a t e b y h a r o w i n ( A ) . T h e l a r g b c k m a r o n t h e left edg of each panel is a relocati n mark drawn on the outside of the cover slip. Scale (C) = 250 (A) , 98 h (B) , 12 h (C) , and 14 h using (D) , P. aeruginosa , and K. pneumo ia . The aver g flow velocity was 1.8 µ m. (I age sup li d by Paul Sto d ey, Univ rs ty of Exe r.) im unolabe g has demonstra the pres nc of with n a L. pneumo hila secafru l i t m gn b p o w se c lu . )7 ( 3.5 Medical Env ro me ts l a i c ,f r h n et oysd u aib c n l p g m i e y r o A f ehto gniw o tamr f li o br fetisla netop s ne rp , vla tr eh o,tni j ,.g e htiw de aicos m inahce n fed tsoh lamron eht fo ecn sba t mh ue c o s layer techniqu s may result in a los f the prot c ive anti dhes o c atings maintained by h o and the s bli m nt of a p rtunis c 3.5 1 Dev lopm nt f A i oul ng/ dhesio C at ng The d v lopm nt f a i ect v d i s uch a t e rs i d able u to he ig nc de of as ci ted nos mial nfect o a d prim y se t c - (78) . The physical dam ge/ isrupt on of tis ue or organs by invas e noitcef )97( . Biof lms and E viro me tal P c s e 295 mia (80) ef ica y of vari us ncorp ated n im crob al gents minocy l e and rifamp n wer found to be ef ctiv ag inst Gram-posit ve co i, Gram-neg tiv bac l nd yeast uch . The MRD rep s nt a go d in vitro model sy tem for tes ing the (80) . A combinat of . Candi lb c s 3.5 2 Evalu tion f A biot c Sus ep ib l ty Cur ent s for the d rminat o f antib o c sen it v y on whic trea ment stra egi s are based, such as the disk dif us on as y and min um s t ne o i a r t e c n yo t i b h n The rol f bio lms n cli a d se i wel docum nte i n c r e a s t h u o if n d w e l mg c a d v i e s u h t r a n d i f c j i o a l h n b t e v s y d c wr a i n e m o p f l t d devic -as o ted infect o s for more oc ur en s of devic -as o ted infect o than y other micro ganism nizat o with a view to det rmin g ef ctiv con e tra i s of antib o cs, eith r fo trea m n of the inf ct o r as coating the ca ter su face to d e c r ia ns t l h c m e o f s e b ft i h s o u c x a nl m r d p o s i tion (37,52 84) ciprofl xa n into a f r o tm h be u l k i d s r f a c we g n i t l ry e d u c b h p s n oe f the biof lm whic o urs with sof - i ue nf ctio s, and rep s nt a go d m el for examin g the rol f c growth a e in t b o c resi tan by ofilms 03( . s l ce i n o t k a fl es p y hu i v , r , )18 (7 , 28) , and rec t . Coagul se-n tiv staphyloc i ac ount (82) s t c ufmv o d i lhyr e - n g pM R DT a t . s (83) (80) A T R. / F bh s ueaI t o n d y , and lso i med cal ont x i vest ga h pen tra io f biof lm; the pen tra io of the antib o c P. aeruginos . The p rfus d bio lm fer nt close y mi c the si ua on (37) . (12) 3.6 Natur l Sys em s ay q ut p e m r i c w lo v h fd n s B e m and res a ch into his ubiq to s mode f bacteri l growth as ubseq ntly e x p a n d i t o l u r a e n v i o m t s .G r w ha b i o f l me n s x p o i t a tion of the nutrie s whic may be con e tra d at a surface ag inst de ic at on a d changes in the pH, temp ra u , or smolarity of the enviro m t, and y of er inc as d protec i n f m grazin p ed tors uch as moeb nd pr toz a (79) (5 , 9) , protec s (85) 3.6 1 Gen Tra sf /Exch nge The requi m nt to understa gen transfe , as it oc urs in terms of the n a p t r ou b e h x m l g i kD f s N d A , y by con er s about the abil ty of ind ge ous po ulations to uptake gen tic sequ nc from engi r d o ganism . Transfo m ti oc ur ing the riv epil thon (86) h ab se nu g t r do p s e na i b lm c h s w y . , 296 Rayner d L p in-Scot resi tanc gen s could be spr ad throug natur l po ulati ns; the ransfe o m e r c u p o yi -l t f n sh a d recip nts twe n microb al com unit es in aqu tic and ter s ial enviro m ts hould ac ount f r he xist nce of the majority f micro gan s , biof lm c munit es a d xp rimental sy hould be sign ac ord gly. P s e pu d o im t an has be n d mo stra e . Furthe s udi of gen tic ransfe b - (17) 3.6 2 Influe c o Ext rnal F c o s n Mi r b al B ofi m F r at on Enviro me tal f rc s u h as temp r u and utrie con tra i ex t an ef ct on microb al behavior in the natur l enviro m t. Lawrenc and Caldwe o n c s u t m l r a b f e i z v s n h c ar o y t w e f u i m l s ct or me a u Cn i py . - e x h a m t n iou c sb r d p y ds c eu o vr l f a n y p m t m i c r o e n v f t l s a y h u d c e n m i w p ot l a s and to lo k at he f ects of dif er nt con e tra i s of organic nutrie s on bacteri l o n zati (43) d e m - t o c u l r e s i d n o - f wa m c r s p y l i g h t u e d b a c s t e or h f i xv m l n , (43) (43) . (5) 3.7 How Repr s nta iv Are Exp im ntal B of ms? Experim ntal b ofi ms rep nt a compr ise b tw n o extr m yp s of models; holist c, whic invol es a study of the complet sy tems, and r e d u c t i o n f w s m h lp a b , e y r g a c o u n it k e d o s f t e n w h i c b u y s m , t e o f i nl d v us a com unity interac o s studie fal somewh r in the mid le of thes wo clas if t on . The us of h o l i s t c / r e p a i v m o d l sf rp u c i n ge x m t a lb i o f sr e p n t h best y em in terms of their lative s milar ty o enviro m tal y oc ur ing b i o f l m st uv ,h a e r n y d b o c f l g i h m e s a t n y p of m dels can v ry widel the nviro m t, p ducing rep oducibl f ms or the xamin o f eatures com n to dif er nt sy tem . Thes enabl a wider degr of contr l over sp cif ed xp rimental f c ors tha ve a postula ed rol in fluenci g struc al nd fu ctio al pr es Tb hi eo f lp mr s t n v e oi , r b d s lg h u p rep s nt a cons rtia of bacteri , fungi, alg e, and prot z a; biof lms in the b o d a y r c e m p s n fu b o d i e r t a c i s l p e F . o r x a m l i , n thecas ofd n lp queth rima ypl queform sa th l rep oc i, whic are then fol wed by secondary forme s such as fusobacteri m Mon cult re (single speci ) biof lms are widely used in labor t y studie u m s op a r nf e bli t y- cd u o r h s i t . B i o f lcmh a r t e i s c f l t i voe h gr w n i o m e t . Most model sy tem and exp rim ntal biof lm (87) (39) . Reduction s / ve tiga models i p fy (87) . (8 ) (89) a n dr e . Biof lms and E viro me tal P c s e 297 influe c d by nutri o , fluid dynamics, speci comp sit n, and physicoc h e m pi r oa l t T b s . e n vr iy o dm f t g s i c a n hr eol my ext rnal e vironm t i erms of luct a ions nutrie sup ly and em , and growth condit s should ther fo at emp to mi c those observ d in vi o (29) ingthep o y ic har te s ic of he l s consider prio to the extrapol i n of dat from in vitro models to biof lms complex int rac o s uch as predato - y relationsh p , e.g the grazin of prot z a on biof lms c h pa t n i yg e s o m c r a l p e s t u h i f o n v r m e t and the interac o s c ur ing betw n complex microb al com unit es, can l o pa d f rb i t m u e v n c y x F o r p t l . e , the p rfus d bio lm fer nt od l, he nutri co en ati w l be s milar for most of the cel s; howev r, in a natur l y oc ur ing biof lm, nutrie g r a d s i u e bn t q f pc r h yl s i e o a x - tw g n tiald s r bu on fthec l s no dist nc o t be mad betw n featur s oc ur ing as result of adhesion and those wi g ro th a e . The natur of he gr wt -lim ng utrie s mportan i fluenc(90) and, co sequ tly, m be in situ . In the nviro me t, biof lms rep s nt dynamic sy tem , with (39) (85) . Thes relationsh p and others, such as specif .Themodif R b nsdevic (MRD)al ows (85) (40 . , 45) 3.8 Visual z t on f he Bio lm t n e r a p sd o iy m lc , h g M S L C ,secafru iht y v fo setar bu h n c .d imaxe guo tlA fo n itardyhe , m x eb ot secafru o yteirav selban ypoc r im n t el xylaco g detar yh e t s ned oc yler v s noita m xe ot r i p el mas eht g n i y o r t s e d h x l p mu c a f i o b t w ( e h n p c x f - v MES latnem or noitar pe f ,MES gnil mas c f r d st a i e —nom c l yam r ep tn s i a ,t p gn se u wodk rb f ht gni u s -ar b tum, but the pit may actu l y be the conde s residu of the dehy rated xylaco g selpma tn oriv g cse ulf a o eht :MSLC iw segatn vd fo -lausiv n t ecs roulf a htiw no c uj i des n hw m lborp esuac y m gniz stcejbo nih w a ;mlifo b gn w dahs y m se it o ruc gniwo t eh niht ylem r x d a , b es l ht y d ar en p to ah s cejbo f n rp dnif ot lucif d eb yam sne ic p 1 xorp a( 3.9 Sampling Tech qu s Prima y ethods f r the num ratio f bacteri l v ab ty nd activ y re large y d p n t o he abil ty of he bac ri to f m c l nies o ab r t y , )85( ]85[ gnirud tsol e f ra mli o b eht f s n opm c larut S .) )7( emos ra e ht ,smlifo b yduts eh tiw y l baci p st e ip D . )19( µ aer d sopx ht f l y eru cip s ga v dn )m reyal s b eht s nimax yl o RITF/ A . )25( . 298 Rayner d L p in-Scot media. Subletha injury fol wing exposur to antim crob l agents, reduc cult rabi y, and the varied microb al comp sit n of biof lm com unit es s u r f a c e - bo t i m n d h a p e r y cal remov f th ad er n c l s e u t in cha ges t physiol g c ar teris c of the cel s. The spati l distr bu ion of the cel s and intersp cies i n t e m r p a bo f c l u y hd s g e t if a v c o n y biof lm (93) or in det rmin g the f ects of antim crob l agents on biof lm proces (92) dt e h nv c a bl o i p q u s r e f T , h . no destructive an lysi of biof lms, as op sed to destructive sacrif cial procedures such as col ny counts, to al cel counts, and SEM. Metabolic stains such a CT micros p v ualiz t on d use for th activ y (96) . (92) and rho amine 123 (65) (94 in s tu 3.10 Fut re Ap lica ons f r Bi lms in the S udy of Envir me tal P oc s e It is now clear th biof lms rep s nt he prima y to l in the proc s e of gain a clear understa i g of a wide range of enviro m tal proces tha ve pr iously re i d on the x rapol ti n of dat ob ined from planktonic m r b al cu t res a r g fp e ou w i n t s cd ; l b may include th fo wing: 1 . m p e r t o a c sb i l n d u f h T o 2. 3. 4. 5. tha wil enab the d v lopment f a model f struc e/a hit c ure ap lic b e to b h ig - and olig tr ph c nu ie t v ronme ts. The d v lopm nt f sy em to nable h mor ac u te s m nt of he ica y of bi c des an tim crob al gents; i. , les r iance o th us f planktonic el sy tem in the as ment of antib o c ef i acy prio t he tr a men of bi lm-as oci ted nf ctio s r in the d v lopment f sanit z o stra egi in the fo d in ustry. mA o r de t a i l u n r s t a d i og bf ch e l - a n d l i t e r f a c n t i o s enabl the dev lopm nt of antifoul g surface / o tings for use acros a wide range of i dustr e , coupl d with an u derst ing of the m c anis / g alin proces invol ed fin g b o lm struc e/a hit c ure. As t u d yo ft h eu n d e r l y i n g e n t i cp r o c e s t h a i n f l u e n c b i o f l mf o r m a t i o n , such as the expres ion of gen s related to alginate or EPS production; the production of cel signaling factors, and the gen tic char cteristics that ac ountfortheobserv dphysiol gicd f er ncesb twe ntheplanktonicand biof lm cel s. The d t rmina o f the d gra tion rates of p l utan s tha oc ur in the nvironme t by iof lm bacteri h t an owi g t plank o ic el s. For exampl , the adsorpti n of organic pol utan s and surfact n ont sedim nt pres nt in (15) . The study an se of bi lms n e viro m ntal , 95) study of bac eri l m t bo c have b n coupled with Physi- . Biof lms and E viro me tal P c s e 29 s o ri l v e m a t c y u h m e n r t , s u l ai c g e r t b d o g a dation depl tion f he absor d u fact n . (97) 6 . u n d Ae i r s t h a o f c b g uw n e p r i l a t o s pres nt in a multispec biof lm in terms of nutrie exchang and recy ling, u t i l z a o nfx y g e , s du b q ne tf c om s a b l i t v yc ,e ld i s t r b u tion, a d tersp ci o erati n. 3.1 Biof lms and E viro me tal M ni or g m o sdA ye l it n a c r el p o d u s t i m n f c a o of a complex sy tem, whic al ows cal u tions to be made, along with the tes ing of hyp t es and r ict o s. The ic of the sy m o b used f r a n i p l r t ev y o h x s c m b d u f a n of the ultima e end-poi t requi ments: Do we requi qualit ve (SEMs, AFM, light micros py images) or quanti a ve (viable counts, metabolic c o u n t s , a l r b h y d t e v l s ) a A ? r w i n t e bd o l g i c a p , h y s cal, or chemi al par met s? How ac ur te a rep s nta io of the natur l enviro m t d we r qui ? Once d t rmi , we can th b lance th d sirable featur s ag inst the disa v nt ge of the sy tem of inter s . No single m o d e lw i p r u c ab o f i l m p e x a i n g l r e so fi n t m u l t a n e o u s Wl hy . r ct k ai e n m g f l o s u c bi n g f ol rm a s t r u c e o f i n l h o r a e n gt i m s c , b f l t h e i o n , on degra tiv b l y, an w re s of the in r t p oblems c n ted wi h t h e x p r i m n a l o d e s y t m c na l o w h es t i n fa y e mw ho p t i mizedap l c t onsf r hea o int r s be ginv t a ed.Aknowl g f the move nt of particles and fluids, physiol g cal condit ons with n the biof lm, the pr s nce of chemi al nd physical gr dients, h spati l r angeb i t po rw f c a n d l u sh em g , o f m e n t is importan in furthe ing our understa ing of dynamic proces such as n u t r i e a s p o n dh f u i a t m c r o b lg e n t sB .i f ma ru b q s u c h , A e np vr io m . t fa l j y h e o r s i t h e p n a d u i t o s they refo rep s nt a es ntial exp rim ntal o in the qu st o understand ho e vir nm tal sy e nd proc s e f int r s o u . References 1. Coster n, J. W , Nickel, J. 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M eds ), Car if B oL ne, Cardif UK, p . 13– 6 G i l b e r t ,P .a n dA l i s o ,D .G ( 1 9 3 )L a b o r t ym e h o d sf rb i l mp o d u c t i n , in Microb al iof ms: or ati n d contr l Sus man, M. eds ), Blackwe S i nt f c, Ox ord UK, p . 29 R C ( o 1 D B a O b s 9 . r n W t J 8 , y d i e M c) v s in foul g bi f lm or ati n. Schalkows y, S. and Hu t, L. G (19 5) As e m nt of therap u ic poten ial by means of pr ba il ty mode f antim crob al tion. 35, 31–52. L a w r C e n R l d . J c ( 1 E, 9 D B 8 7 h ) a v b i o c f u t r e s l p a m tions with n the hydro namic boundary layers of surface microenv m ts. Microb. E l L(a C 1t S w9 c lnRo E r d .f u e5 i ,J D )h y s f l os w i dc eu t r ,n sy tem , p . 1 7– 38 LeCh val ier, M. W., Cawthon, C. D., and Le , R. G. (198 ) Inactiv on of biof lm acteri . Gilbert, P., Al ison, D. G., Evans, D. J., Handley, P. S., and Brown, M. R. W. (198 ) Growth rate contr l of adher nt bacterial po ulations. Microb l. Gjaltem , A., Arts, P. A. 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W (19 5) Speci s divers ty in dev loping freshwat biof lms, n The Lif and De th of Bi lm D., and L p i -Scot , H. eds ), Biol ne Cardif , UK p . 7 –82 Rayner d L p in-Scot 60(8), 271 – 6. 1 3 – 8. 43, Biotech. ng 173(20), J. Bacteriol. 391– 6. Ap l. Environ. . Burns Staphyloc cus aureus 20(1), ( L a p C i o n s - tdM S e . c r H J , Antim crob. Manu l of I n f e c t i Ao s a w e td h Infectio s As ociated (Wimpen y, J. Nichols, W. Stickler, Biof lms and E viro me tal P c s e 305 8 6 . W i l a G mD H .J s F M , C yr S n t d e w ( 1 9 N 6 a ) u r t l n s f o mation r ve pil thon. 29 4– 8. 62(8), Ap l. Enviro M c biol. 8 7 . W i m p e n y J, . T ( 1 9 5 O) tn h e a u r vd l i t oy mf e s i, n T h Le i f a n d ( W i m p e n y ,J . N i c h o l s ,W . S t i c k l e r ,D . a n dL p i - S c o t ,H . D e a t ho fB i l m eds.), BioL ne Cardif , p . 1–8 8 . Ad y, M. Slayne, M. A and W e, . G (19 2) The formati n d co tr l f dental p que—an ov r iew. 89. Bus cher, H. J and We rkamp, A. H (1987) Specif and on-specif interact i o b n a s c e r dl h i sto n u b r a . 90. Brown, M. W R. and Gilbert, P. (19 5) Some p rs ectiv on pres vation d dis nfect o i he pr s nt-day. 9 1 . L ( C a 1 Kw n9 R l or Ed . e 2 , DJ b f ) c m i s copy and ig tal im ge an lysi n microb al eco gy, in Ecol gy (Marsh l , K. C ed ), Pl num New York, p . 1–67 92. McFet rs, G. A , Yu F. P , yle B. H , and Stewar , P. S (19 5) Physiol g ca methods u y biof lm d s n ectio . 9 3 . ( 1 T C 9 h a E e l . 4 n d )R W D wL o b , M f r J t G s c role f interac o s, e sil growth and utrien amend ts on the d gra tive ef ic n y of a micr b l ons rtium. 94. Ron t, X., Ben l, L., Adolphe, M., and Moun lou, J. (1986) Mitoch ndrial an lysi in liv ng cel s: the use of rhodamine 123 and flow cytometry. Cel 57, 1–8. 95. Matsuy m , T. (1984) Stain g of liv ng bacteri with rhodamine 123. Microb l. Let 96. Pyle, B. H , Broadw y, S. C , and McFet rs, G. A (19 5) Factors af ecting the det rmina o of respi ato y activ y on the basi of Cyanodit l Tetrazolium Chloride r duction with me bran filtra on. 430 – 9. 97. White, G. F Rus el , N. J Marchesi, J. P and House, W. A (19 3) Surfact n adsorpti n, bacteri l at chmen and biodegra t n in rive sedim nt: a thre i n tw ea ry c o , (Wimpen y, J. Nichols, W. Stickler, D. and Lap in-Scot , H. eds.), BioL ne, Cardif , p . 12 – 6 73, J. Ap l Bacteriol. 269– 78. 46, F E M iS c r o b Rl e. v 219– 6. 36( –4), Int. Biode g. 165– 73. Advances in M crobial 15, J. Ind Microb l. 40, Can. J Microb l. 3 – 8. 3 1– 40. Biol. FEMS 153– 7. 21, Ap l. Enviro . Microb l. B a c M t I e C nT di r o h u f s l m y 61( 2), Biof lms U ng the MRD a d Flow Ce s 307 02 Establishment of Experimental Biofilms Using the Modified Robbins Device and Flow Cells Luanne Hall-Stoodley, Joanna C. Rayner, Paul Stoodley, and Hilary M. Lappin-Scott 1. Introduction 1. Prope ti s f B o lms Rec nt s udi have s own tha biof lms (a co plex rganiz t o f bacter i a lc e sp n ta u r f c eo i n t a ,w h c p r o d u e sa l i m - k t r x ) r e p s n t h i c p a lf o r m b t e i a lg r o w h n e v i r o m t s u d i e o date ( 1 ) b Ti ho ef s l m ng . ra c wt du v e T s h . include ext nde protec ion ag inst enviro mental changes, antim crobial a g e ns c tu h m i d l f a n t s i b o c such as meba ents (4) . Biof lms are of inter s in medical, industr al, and atur l enviro mentsfor v al e son .F r xample,th yc na sre voi f mwh c the dis m nat o f pathogens may oc ur. shown t be har o d with n b of lms r ed with n r k g water pi l n s ( 5 ) S. i m l a r y , t ws e b l i h td a o f l m cs n i z e u r o ts y p of medical mp ants fol wing biof lm growth such as reductions in heat- r nsf ef ic n y and flow cap ity. B of uling may lso markedly increas or si n biof lms rep s nt a bacteri l archite u tha may sup ort gen tic transfe , nutrie l zation, d b egra tion (2) , as wel as provid ng increas d ac es to lim ted nutri- (3) has be n Legion l a p eumo hila (6) . In i dustr al sy tem , d trimen al f ects may oc ur (7) . (8) 1.2 Establi h ng Exper m tal Biof ms A major problem as oci ted with the invest ga o of enviro m tal sy tems is the inher t degr of complexity with n a sy tem. To facil t e the b s i t lo u af hd ey n r m , p i o s d y e l t h b a m v n From: ag nr d z ip e t o s Methods in B otechnol gy, Enviro mental Monit r ng of Bacteria Vol. 12: Edite by: C. Edwar s © Human Pres Inc., Tot wa, NJ 307 . Final y, 308 Hal -Sto d ey al. oped tha enabl the growth of biof lms, along with the an lysi of sev ral definedpar met rs,undercondit onstha c nber plicated.Ther a es ve r a ml o d e sl y t e m is cn u r e n ut s (e fied Rob ins device (MRD) and flow cel s have many adv ntages and are r e a d i l ya d p t a b l et oi n d v i d u a le x p r i m e n t a ls y t e m s .T h em a j o r d v a n t g e is tha they trol ed hy namics. C h a p t e 1r 9 ) A. m o n tg h e s t, h me o d i - se al ow the study of biof lms under flowing condit s with con- 1.2 Modif e R b ns Devic i n w a Mt s R T dD h e l v b y u o fp i n t gr a e l s ( 8 ) Ih .tas i n cb e m o d f u s ti n v e g a b o f i l m sr n u e of envir m tal h bi s. The prima y dv nt ge as oci t d w h t e sy m is the numb r of c l nized sampling orts av il b e for an lysi . Th al ows s a t i m k h u pe b ol n r w v f g y single t m poin the d v lopm nt f he biof lm. Quantif c o sev ral p art no d c e il u s , v b o ft cih le m a s p carbohyd te conte is ther fo pos ible. Micros p an lysi is pos ible using co venti al st in g techniqu s of slide-mount sample or el ctron r e a l c s o t m i n v h uM x R p z y fD T d . e It can be us d in both a c (re i culat ng) d flow-thr ug c lture sy t m and c be o n t d a chemost if cl e mon t ri g f owth c ndi s is requ d. Disa v nt ge of h MRD sy tem includ the inab l y to visualize th biof lm a r oh uy nd c m p i s e f b l t y h a n d e v i c , t h o f l e n g t h t h s e a m p l i n g u d F . y t , h M e R i Dp s r o n d a w b c k h r e m y n b i o f l m t h e a n l qy us i t v e F o r b f m . s t u d y h e i n u t l z d s y e m to be car i d out, des r ctiv sampl ng tech iqu s are qui d. Conve ti al techniqu s such as viable cel counts, to al cel counts, and to al protein or carbohyd te c n a lysi u al invo e d sruption f he b ilm. in s tu , the pos ib l ty of nutrie gradients xi t ng alo the 1.2 Flow Ce s The conti u s and no destruc iv monit r g of biof lms is es ntial in understa i g b of lm pr ces cel s uitable for many dif er nt exp rim nts cd t m or h a e f n w y b M F R i D k . s l , o tion of the biofilm in its hydrated form when used in as ociation with c o m p u t e r - n h a c di m g e l y s o rat v i nc m e r a .T h si p t c u l a r y whic des at on, r fix by ofilm the al r tion bec us dv g i m a g u e s n A l v o , d t b r . c f i m e u n k o sh w a v y an lysi , c um lation r es can b lcu ated by comparing tu ed imag s g r o w t h e q u a n f lx i p r c m tg o h , u s e t h a o s e w i t h (9) . Ther a s v l dif er nt yp s of l w (10) . Flow cel s can lso vers i tn u visual z - Biof lms U ng the MRD a d Flow Ce s 309 k i n e t co sfhb l mQ .u a i t v en f o r m a i g d ns u r f a c eo l i z t n is also pos ible. Second, the flat plate reacto (dev lop at the Cent r for Biof lm Eng e ri , Boz man M t ) can om d te various f ce so tha ey ma be compared. Th surface n be r mov d at he nd of the exp rim nt to enabl eith r quantif c o of the biof lm by scraping and/or sonicat of the surfac , s in the MRD, or an lysi u ng sca ing el ctron micros py (SEM). A disa v nt ge of h lat p e r c o is tha constrai ed by h c annel thicknes . This type of flow cel must nec s arily be thin owing to the m i c w t l r o hd e s k b j fn a p g T v u , . a tradeof betw n magnif c t o and hydro namics f l co ew t sh,q u a gr b c t o v, e r m hspi b lf ay c t i n g hig er ma n fic t o he biof lm, a wing the flow c t be vi w d from above, nd th c a el d pth is no re ct d. This flow react s d igne a s m o d e lt u yb i f m o u l i n g d s t r a lp i e n s .M o tf l wr e a c tors tha al ow direct micros p observati n operat at low, lamin r flow rates. How v r, this y em can b op r ted a hig , ful y t rb en flow rat s whic are oft n m re indust al y re v nt. The sy t m can be op rat d using p t a w r o l f c e h s u g i an b td w p e c o l y b e i s o t f ra un cdl m g w o t he n c di , det rmin . The hydro namics of the square tube reacto s have be n wel char cterized using the relationship betw en the frict on factor and the Reynolds umb ra fitwel o s ab i hed qu t ons crib glam n d turb len flow throug a smo th pi e ad pt to particular exp rimental condit ons. Howev r, large bore tubing r t e h gs l m qa i u cb kyn , f N v r o t d h .e p l s - , ing o the xp rim ntal co di ns, ther a s ver l f ow devic s tha permi an lysi of b lms. (1 ) . Another type of . They are also easy to make and (12) 2. Materials 2.1 Modif e R b ns Devic 1. Ethylen -oxide gas steril z d MRD ( whic t e surfac o h ice av b n fit ed. 2. Steril p acem nt s ud . 3. Bacteri l u t re s voir (us al y g s fla k) with an ou fl w c n e tor a d filter d a inlet. 4. Steril mediu res voi with an outfl w con e t r and filter d air inlet ( Note 1 5. Steril f ask or w te c l ion. 6 . S t e r s i l c u o b n f r g em t c h M d o R i D u s n v r , bacteri l u e r s voi , and w ste fla k ( ) fit ed with removable stud to Fig. 1A se ). se Note 2 ). 310 Hal -Sto d ey al. 7. Peristal c pum alibr ted o giv requ d flow rate ( 8. Sampling equipm nt consi t g of steril scalpe blades, buf er soluti n, waste jar contai g bleach or dis nfecta , steril tes tubes contai g diluent (e.g , 0sm.tL9e r i bl u f o t n ) , r c e p s a l b hd oe 7ra0,l% c 5 - m L pi et s, and 5-mL Gilson p et. ). Note 3 se 2. Flow Ce s 2. 1 Flat P e R ctor 1 . S t e c r l i o h s a n d b w t v r m i c 2o 4n s a t f g 2. 3. 4. 5. 6. 7. 8. 9. × 60 m glas cover slip he d n place by ru e gask t nd me al f nge ( Note 4 Steril f ask with outfl w con e t r, filter d air nlet, ubing, flow breaks, and con e t rs fo a t chmen of t l w ce . Steril waste r voi ncludi g nflow c ne tor a d filter d ai outle , bing with flo break, nd co e t rs fo a t chmen o t fl w ce . Peristal c pum alibr ted o sired flow ate. Waterb ho ating rco l units, f ec ary,toke p s cult re a mperatu s o her t an o m e p ratu . Micros pe. Camer ( Computer wi h F amestor b a d ( Image n lysi oftware ( se ). se ). Note 5 Note 6 se se ). ). Note 7 2. Square Gl s T b Reactor 1 . C ( at S mu -g lbs 1 iq r0 n 3 , d e o c f F l s w UK) 3 m wide an 3 m de p an 20 cm long ( Fig. 1C 2. Steril nu e t r s voi . 3 . P e r i s t a l p ud cm v e r w ia t y h f l o ( 4. 5. 6. 7. 8. 9. vane h d pum (Masterfl x, Co e-Parm , Niles IL). F l o w m e t r s (M c i l a n F l o - s e n r m o d e l 1 0 T # 3 7 2 4 a n d 3 8 5 s u p l i e d b y Cole-Parm ). Pres u t ansducer (RS Comp ne ts, Corby N than s, UK model 286- ). Waste r voi . Polycarbonate holder mounted on the stage of an upright microsc pe with epifluoresc n cap bil t es. By posit n g the flow cel s on the holder, the biof lm can be i g d Camer ( se Computer wi h F amestor b a d ( 2.3 Sup liers Al of the described flow devices can be found on the fol wing Web pages: for information on MRDs, contact Environmental Microbiol gy Res arch Group at Exet r University at ht p:\ w .ex.ac.uk/biol gy/ se in s tu Note 5 withou n er pti g flow. ) se Note 6 ). ). [13] N o8 t e c o )n t r l w eia d h Biof lms U ng the MRD a d Flow Ce s 31 Fig. 1 Thre typ s of devic s to udy bi f lms under flow c ndit s. r e s c h . t m l # D r H M L a p i e n f- c lS t o m s , ; BioSurface T hnol gi s C rp. at h :/ w .imt ne /~ bs flowce .htm 3. Methods 3.1 Modif e R b ns D vice 3.1 Prepa tion f he MRD 1. Cut sila c rub e , lack b ing d sc u ing a 0.85-m cork b e . 2. theo r maid nwo k a f ,citsalp ro ,s alg reb u citsal ,.g e s cafru eht ca A black ing d sc u ing a stro dhesiv or wate p of s al nt ( se 3 . W i p te h f sd u r a c e w i t 7h 0 % l o s u t i an ld - f r e t i s u a n d l o w 4. to air d y. Fit hestudsinto heMRDsotha thesurfacesforc lonizationlieflushwith the c ntral umen. 5. Wipe th MRD wi 70% alcoh nd seal i g -perm abl gs. 6. Pack ge 25 replac m nt stud in batches of ap rox 4 stud per bag to prev nt contami during the cours f the xp rim nt. 7. Steril z he MRD using ethyl oxide gas ( 3.1 2 Inoculati f S r aces nd Biof lm Treat n 1. Remov th MRD from the gas-p rm ble ag nd che k for l se u fac . 2. Remov any surf ce , tha ve b com d ta he uring ste l za ion. se Note 10 ). Note 9 ). 312 Hal -Sto d ey al. 3 . C o n e tc Mh R D u l r m e, d i a wn s t e r v o i u wn g d e - b o r , steril ast c rub e t ing ( for exp im ntal se up). Fig. 2 se 4. I noculate the cult re res rvoir to give an ap rop iate planktonic viable cel count, e.g , 3% v/ expone tial bacterial broth cult re and incubate for 18 h or vernight. 5. I n o c u l a t e h e M R D s u r f a c e s . T u r n o t h e p r i s t a l i c p u m a n d e n s u r t h a t h e inoculated cult re is moving throug the MRD sy tem and into he waste jar. After he in t al biof lm has be n formed ( .g , by inoculating the surfaces for 24 h), quickly switch t e sy tem to flowthr ug with only the steril mediu and control agent (antib otic or bioc de), by changi the open and closed clamps. 6. Maint low rate f low during the swi c over t p ven backflow iqu d throug e sy t m. 7 . R e m o av n biy ur l f s t Mhm Re D n i u p g d o aw t l i n g 45 ° angle for a w minutes wh l under th o mal f w condit s. 3.1 Sampling Co zed Surfac s 1 . S w i t c oh f pe u m a r o i a t e m p r o d , n c l a m tp h se i u b c n g at ei h r nd of the MRD. 2 . ( sd te u R a r m n o i v g b l d s e ) p t a c y it w h a steril p acem nt s ud. 3. Hold the remov d stud above a pot contai g dis nfecta or bleach soluti n. Rinse to remov any onadher t bacteri by pi et ng 10 mL of steril buf er soluti n gently ont he side of the stud, so tha the flow is not direct at he im ed at col nized surfac ( 4. Place both e scalpe blade n the scraped surface into a tes ube contai g steril buf er soluti n (for techniqu s for the an lysi of biof lm formati n Subheading 3.2 5. Sonicate h scalpe b ade n surface or ap ox 5 min to disper the biof lm and y clumps of el . 6. Place th us d MRD tu in o he p t of dis n ecta . 7. Rep at for p iate numb r of sa ple . 8. SpraytheMRDwi 70%alcoh ftersampling,w e a dr moveth clamps. 9. Switch the pum back on and turn the MRD upside down to remov any air bu les form d u ing sampl . 3.1 4 Final Procedu f th Experim nt 1. Empty he tubing a d the MRD by tip ng the r s voi wh le maint g he pum rate so h n liqu d s taken up i to he sy m. 2. .DRM eht dna sriov e ht morf tcen o sid na loh c a %07 htiw gn but eh yarpS 3. Seal open ds of tubing w h alumin fo l and utoclave. 4 . S o a t k h M e R i Db n l c d r s f e t a n o r p 1 x 2 – ( h 4i n c r e a s t m o f d i s n f e c t o p h d i n k g e s / v b c i o f l n t ay D r m g k ) e . than 48 . se Note 1 ). se ). Biof lms U ng the MRD a d Flow Ce s 31 Fig. 2. Schematic diagr m of MRD exp rimental setup. clamps: B closed = inoculat of surface , and A closed = flowthr ug with only steril m d u . 5. Con ect o a t p and ri se n cold, run i g water fo 8–12 h to rem v sidual dis nfecta . 6. Soak the used stud in bleach or dis nfecta for ap rox 8 h and then rinse in conti u s r n i g water fo u ther 8-12 . 7. Al ow the MRD and stu o dry p i to reas mbling. 8. Autoclave l mediu , waste, and i ocul m res voi as p ro iate (increas autocl ving me for la g v umes). (A) and (B) ind cate 314 Hal -Sto d ey al. 3.2 Experim ntal M surem nt 3.2 1 Viable C ounts 1 . V o rt c u h eb nx a i s gf c d el b p 1 a5to – r n2 i0 tion ( 2. Serial y d ute in b f r a d pl te ou n a p ro iate s l d growth mediu . 3 . C a l c u t e h n m b r os vf i a l ce p sr q u a ce n t i m r os f u a c e i n tg h fol wing equat o : ). Subheading 3.1 Nu mberofbacterialco nies as umed to c n i 0. 1 wher Note 12 = dilut on factor and Df × 10(biof lmre o × Df × 1/ )Lm isved As 2 = b acteri / m = are of surface in square centim rs ( As se ). 3.2 Scan i g Ele tron Mic s py 1. 4 ta hginrevo h 2 rof e utar pm o r ta ec frus eht xiF 2. Remov from the buf er and use a suc e iv ethanol seri to dehy rat the sample: star with a con e tra i of 30% and work throug 50, 7 and 10 % ethanol f r 3 min. 3. Remov th s lu ionafter3m nd iscar ,then plac with en x soluti n in the s r . 4. Place th s ud ont a SEM mount, coa with s lver using a p l dium cat lys , and view us ng SEM ( ° ( C .3 1 gnidaehbuS se Note 13 .) ). 3.2 Total Ce unt 1 . S ct rh os a nu pe f i g l t r a d e h c y o l b a u t f e ( r 2 . F i a xr t o m e p u r f o h 2 v e n i g a t4 se ). N 1o t e ° C S . a m p l e s y b of r z n and proces d at l er dat . 3. S erial y dilute the sample in buf er solution and filter 3–5 mL ont a black 0.2µ mpolycarb n tem ra .Ap ly ndthe r l as thev cu m. 4 . a c mo r g i / d n 0 L . e 1 c m o b vf r a ( tn e ) u g L h 2 t o 1 w i S h a n soluti n ( phos ate buf r) o 3 min. 5. Reap ly v cu m and while run g destain w h 1.5 mL of is pr yl a coh . 6. Remov filter and ir d y on filter pa , view us ng a under epifluoresc n e microsc py, with a calibrated ey piec graticule, a mercu y lamp nd acrid ne orange filter block (emis on wavel ngth of 48 – 514 nm). 7. Count he fluor scent y ained c l s in ap rox 10 fields o view and ver g to cal u te h o al ce ount. 3. Flat P e R c or 1. Calibr te pum to desir flow rate by volumetric displacem nt prio to autoclaving tubing a d co ne t rs. × 10 oil m ersion lens Biof lms U ng the MRD a d Flow Ce s 315 Fig. 3 Example of at pl e r acto sy em with nu r e s and w te r s voi . 2. Autoclave th flow cel ( 3. 4. 5. 6. 7. 8. ) after it ng with e coup n or tes materi l, Fig. 1B and cover with a ub er g sk t, la cover slip, and met l cov ring. Grow the bact ri l cu t re o the d sire d nsity and t ach o pum , flow ce l, and w ste r voi a steril con rub e t ing ( Init a e flow ( leaks. Tighten s al if nec s ary. M o n i t b h r e f v a l m o u i sp e n d t i o h x ge p r m n t d a l s i g ( se Autoclave h tubing, res voi , and flow ce at h end of th exp rim nt, a d rinse w l run i g water o m ve any biof lm res du . Replac ny tubi g f nec s ary. Clean the flow cel with 70% ethanol t remov any remain g residu , and fit with a ne surf c be o aut cl ving pre a tion f r he xt p riment. Note 15 . Fig. 3) se se ) and monit r flow cel , tubing, and con e t rs for Note 14 ). 3.4 Square Gl s T b Reactor sT qh ue ga lr t b c f o y w ( m se F 4i g . dw ea s ) i h g tn vo lamin r f ow ne l c and turb le f ow in the r. 1 . M e a s u r t h f l o w a e r u g h c o ft e l w s( Q f )u s i n gf l o wm e t r sc n trol edin p e tlyb igh en orl seni gclampsonthei l ub ng.The aver g flow ve city ( u ) is cal u ted from: u = wher CSA is the cros ectional re (in th s ca e 9 number is found r m: Q f /CSA (1) × 10 –6 m 2 ). The R ynolds 316 Hal -Sto d ey al. Fig. 4. Example of biof lm reacto sy tem consi t g of par l e flow cel s in a recy l o p at ched to a mix ng cha ber. Th mix ng chamber w s aer t d an the lev maint ed by ov rfl w t as e. Re = wher for water can be us d), hydraulic met r cal u ted from: uD h /ν (2) ν t k i h s e n m a v c o s t i h e f yd ( l a r w - n u t i e m d v h a l u D is the c ar te is c length, w ich n this ca e is the h D WP is the wet d perim t of the flow cel , 2(width + depth). For thes flow cel s, D m o n l u y s e b d g i r t sc o h a e i z f l o c w n d t i s I . p a r c u l y s e f b e pc ra i u t ds w h l f b m e o rn a u s c d t compar tive am t r fo a divers ang of l w sy tem . =3 h × 10 –3 h = 4CSA/WP (3) m. The R ynolds number is a dimens o l number com- 2. Det rmin the pr s u e drop ( ∆ P ) acros each flow cel using d f er ntial pressure t an d c rs. ∆ P can be us d to fin he Fa ing fr ct o a r ( f ): f =( wher ports b i o f u l n Tg ph. re d i c t the Hag n–Poiseu l q ation) s: ρ (14) w is the d nsity of liqu d me ia nd . f is al o a dimens o l number and can be us d as n i d cator f ∆ P × D h )/2 lpρ w u 2 l p is the distance b twe n pres u f f lo ar m i n t hw s u ag o ( c l e pn i) f r o m (4) Biof lms U ng the MRD a d Flow Ce s 317 f = 16/Re and i the urb l nt egio (5) f is pred ct from he Blasiu form la: 0.25 f = 0. 791/Re 3. 4. 5. 6. The relationsh p betw n Re and transi o betw n lami r nd turb len f ow c ur ed at R = 120 ( cm 3 /s). To increas th ensi v ty of he length d. At he nd of the xp riment, biof lms can be fix d w th 1% par fo m ldehy ( 3 0m i n )a ds t e w i hn u c l a i ds t n u c ha sp r o i d u m e( 0 . 4 % ,a t 25 ° C for 30 min). Biof lms can be i g d mit ed light micros py, or epifluor scent ultravio e micros py, al of whic can be us d in co ju ti n w h mage n lysi . B i o fa c l mu r b t e i on l y ( r .b dg a i , ns u f ce coverag d t ) n relat d o ch nges i pr u e d op. Metabolic activ y of the biof lms can be examin d using the metabolic stain such as 5-cyano 2,3 dit ly te razolium chloride (CT ) in wh c flow cel s are r e m o v df t h er a c o s y t e m n d a i w t hC T ( 0 . 4%w / v )f o r3 0m i n at 25 ° C in a sh ker incubato . (6) f for 20-cm long flow cel s showed tha the Q f ∆ P measur nt, he flow c s an be using co f al ser c n i g m rosc py, t ans- in s tu 4. Notes 1. m e r t d Ah i s n u a o bv f c l y e i n g tes ing u ceptib l y of i lms to an i cr b al gents. 2. Insert m al or thick-wal ed g as tubing to rub e ungs a d pl ce th m into the steril mediu , waste, and inoculat res voi flask . After steril za on, the sila c rub e t ing ca the b a ched to res voi a the ubing. 3 . P r e c a l i b t ph u m r o e q i f ld aw ubt sy n o g e r i l s t w m h water in pl c of the ul r o g wth mediu . 4. Certain d s g al ow desir u face to b fi ed w th various te ma ri ls. 5. We us a COHU 4612-50 charge- oupl d evic (Cohu, San Diego, CA). 6. We us a Scion VG-5 PCI (Scion, Fred ick, MD). 7 . a v H i e l - t h , I o n fN s u a r p m o g 1 N . I 5 H 9 - a t h e u s W able from the Inter by ano ym us FTP from zip y.n mh i gov or flop y disk from the National Techni al Service, Springf eld, VA, part no. PB9550 19 GEI. 8 . v (o lT uh me w a p s r 1 moT7nLxh5u. e t i f l n rt o a (w e a resulting res d c time ( 9. When cut ing surface , nsure tha e diamet r of the surface do s n t exc d tha of the sample port; otherwis , this may interf with removal of the stud from the MRD and the surfac m y be o d tache . 10. Autoclaving and chemi al dis nfecta dam ge the MRD and O-rings used to ensur a tigh seal wh r t e s ud are fit d n o the MRD ( V m ic r txh e a l n yo gbd f p ,u ws ) Q θ = V /Q n n w 4 ma .)Ls 3 / i g n , v ) of 40 min. se Fig.1A ). Check = 3.15 318 Hal -Sto d ey al. 1. 12. 13. 14. 15. for leaks on the MRD and i the ar s wher the ubing s joined to he MRD. Spray with 70% alcoh , wipe, and then seal with a quick-dry ng waterp o f aqu ri m seal nt wh re c s a y. If sampling for SEM, place the rinsed surface in 3–5% glutar dehy buf er ( g S 2 r E 5 a M %l d u e t h y 0 i d . l e 1 n u t c v o i t u e ap f n l br s , h y d t e s c b o a i t lr h f p e n d u m z ( 7 – 1 0 s l be suf ic nt o rem v th ad er ntc l s). The plankto ic v able count i he sy t m can lso be m nitor d e to che k st ril y and cel growth by emoving 0.1-mL sa ple , c r ying out serial dilut ons, a d pl ting ou ap ro i te grow h mediu . e t s h i d a no r c f y l A u p g t e o i v s the sample. Once dehy rat and coated, sample can be stored for 1 to 2 wk until req d. Det rmin plankto ic cel s at the in t a o of flow. A sampling port near the ef lu nt inter up d by a flow break to reduc the pos ib l ty of contami al ows e i r ac s . For example, in tial col nization ev nts may be monitored in the first 24 h, or a biofilm of a certain thicknes may be grown before examination. B i o f i l mt h i c k n e s m a yb em a s u r e dm i c r o s c o p i c a l yb f o c u s i n go t h es u b stratum of the cel cluster and then on the surface of the cel cluster and noting the dif er nce on calibration on the fine focus adjustment important to det rmine the optimal working distance betwe n the microscope objective and the flow cel and to use the ap ropriate objective lens. Once this is established, surface area, as wel as heights and areas of cel clusters can be compared to previous images. The ap ropriate software al ows for images to be linked and provides a virtual record documenting changes over time. Length and width of cel s may be measured, and al the as ays outlined for the MRD are pos ible, but at only one time point. Focusing on a single area of the biofilm enables a cel cluster or groups of clusters to be monitored with time. Such images may be animated to provide a real-time record of cel at achment, ag regation, and sloughing, as wel as the volution of the biofilm with time. The flat plate reactor flow cel is easily exp rim nt and the coup n can be remov d and subject d to the same xperim e n t a l s u r m e n q t d a i s v e m p l u nt M i gh R dDr , a s and so i cation of the coup n resulting in disruption of the se ile organism toyieldviablec l counts( mination of the col point ma in ster l y. References 1. Coster n, J. W , Nickel, J. C , and Lad , T. I (1986) Suitable m thods for the compar tive s udy of re -liv ng a d surf ce-a o i t d bac eri l po u ati ns, M c a o d y l b ut fe s aIrm )p . i n g (12) disa embl d at the end of the se nized surface and SEM is av il b e only at he final time Subheading3.1 )Howev r,viablec l det r- . It is 1ndex a4ue05 A Acanth m0e6a Achr0mat1u en1d rca en150 da 259 ,1 na1 et5ac 36 ,muref1 ax0 0ran9e, (A7P), Ae4u0rea Aer0m na5 A9r06acte1um tumefac1n5, a19e, en1 ak1 A1ter0mna5 tr1eh0xy3-amnp 36, 46, F0rce 1 2, 1 3, n01tce1f R am0n1 am0n1 61 aut0f1re5cn, detam0 u 127, 1am05 61r ,5e1d06 tna 46, 87-89, yt1n f a ,deta1yn 0 6 and d1rect f1u0re5cntday ant19e(5), 83 6 64,ant15er 80, 1 8, 23 951 64, 7 8, 80, 6 0f, 1, 0 f eta1pm 0f, L19ht, 79, DNA, 12, 109- 12 ,de1 6a 65,240, 243,25 84 ,51carhtn 87- 9 ,de1 6a 38 ceru5, 24 ,5u1 hp0mre t a 5 ,51 t6u5 ,51 ne19n ruht 256 78, 41 2, 05 ,5u1 ca8 0f, 1 2, 1 2- 5 8ac detc10n, 238 6, 1 8 80 78, 0 9n1 e6a1 pr1me, term1na0, 5e1p cn1rp 9n1 e6a1 1 ec 109-2 31 24 238, y1tnec5 r0u f ,1an0 c m 158, 901 f0r, ,51 er0hp tc 1e n01tar pe n01tc r 5e (ARD ), 109-7, term1na0, dN7P5 dye dye 165,231, DNA 51 y1an 231,257 ,9n1c eu4 5 021 n1ahc 230 de1f pma 751 63, DNA 6acter1 30, 2975, ,5e1dut 9, 34 ,5e1t num 0c yp0c5 rt e 2 98 0 , 1ac 90 e tua 162 ,5re21d x0 9n12 d1x0 re1 u0F (A7R/F1), 84 24, 26, 29 1nfraed750m ena1 5 (AP5), (AFM), 28, 29, 296 5p ., M1cr05py 70ta1 Aenud 01 ,51 y1 162 291 At0m1c 28 ,ad1c n0m1a5 256, 5p., 1 52 81 ,a1r0tc v 14 ,re5a1 f1avu5, 53 ,ecn 5 1mu 0 6 36 5ytem, 140, 5u1 9rep5A 245 tr1ph05ae and c15ahp 6 Archae , ar90n 28 ,)1(a retc 6 24 140, aut0rph1c, 9 321 84, ,1 4, 5, 132 83, 4 14 ,152 81 23 ~dex ,5m1 f016 ,5e1n0 c yt1num 0c 279 5e1tr p0 e10r 31 ,51 y an 139- 72, 201-2 0 279, 280 9, 201 rRNA 641 ,c15n1rtn1 ,c1t5 nah em 9n1c eu4 5 trea m nt, 29 , 76, 25 0f, 283, 289, 25 , 162 ,9n1 0 c 153, 154, 5erum 290 16 , 3 58 ,51 utrep nemu61a (85A), 832 5 a 9n1kc0 6 293-295 c0n5ta deph m1 f fermnt, 286 5u0 n1tn0c erut1 c w01f , 1 e c 84,2 582 c0ntr1 0f, 28 n015 r c 6 y , 182 cry0em6d1n9 0 f , 290 n01t n1fed 0f, 280 1atnem r d fect5 0f, 2 8 1 - 2 8 3 expr1mnta, 279-319 ta1f 6ed react0r, 14,3 315 w01f 51 ec f0r, 308-31 ,316 fut re 5n01tac 1p a 0f, 298, 29 n 1 ,en1c d m 294, 295 m1cr05py 0f, 2 8 6 - 2 8 9 ,5met y 1 d0 283-6, 290- 4, 296, 7 307- 9 de1f d0M 5n16 0R MRD),( ev1c 284, 30 9, 31 -31 ,317, 318 perfu5 d ferm nter, 85,2 297 (8CE F,56 end 279-319 A7R/F71R ,9n1 u0f 16 245 tnu16 a1 et dr08 en1v06 261,273- 19 ,5n01tac 1p a 162 8 ret5 1yh m x0teca AM), 307, 308 281,28 53 291, 29 ,n015 r c 6 ,)5(m1 f016 tu6e react0, 309, 15 decay, 35, 146- 9 n 1 m1 f016 297, 298 5 a19 t0h ,ecn 5 1mu 0 6 ,n01ta dem r016 ,5r0 ne 16 ,n1t0 6 n1ec5 r0u1fyx06rac-1yhte x06rac-516 35 286 fr0m, 54uare and 297 6eat1n9, 285, 9n1 pma5 84 5ed1c0 6 280, 296 r0t r4ue, 2, 8act0met r, 6ead 61a5e - f0r 307 n 1 nv1r0met5,au 281,295, 127, 39- 201- 20 ,yt15rev d ,c1n0tk a p ,5n1x0t ,yt1 6a1v 0f, a9ent, ,08 ,251 80 a1red 0hk u8 64, PK01, cepa1 1 t ekc1p 851 851 C n1ec a r0u1f c a can er ad1 n C n1ec5 r0u1f-yx06rac ,ret5 1yh m x0 ca 4, 65,2 5 65wh1te, ,51 ec 36 4, 952 eta c 1d 52 ,5nac16 a 4, ,56 cath01, 2 4 0, (CFDA), 81 2,3-d10xy9ena5 1 ec (C230), ,9n1tr05 5 -73, ,5n01tac 1p a ,5e1p cn1rp 18 , 75, 1 9, 62-74, 091 237 25 56- 1 char9e de1pu0c ec1v d (CD) camer, 81 chemr08, chem r0meY, ,5 47,2 650 24, 4 52 xedn1 32 ,5arem1hc ,mu160r 1hC chr0my1n, 64, 2 245 kc1 en01c dur1n9 PCR, 149, 051 cyan1ed5, 24 247, 751 256, cyan06ter1, 162 270 mu1 02arte 1y 0t1d-3,2-0nayc-5 6et1, (C7), 16ray, 143- 5 0frRNA, 147, 109, 61unt 51 1 6, 128, end, tne1dar9 153, 451 51e9 451 mu1d1rt501C mr0f1 c c0mpet n c0ndut1vy, ,5ne9 1rf ep 46, 16 , ,1 0c.E and 361 1 9, 18 , ,51 y1an 291,293,294, 289, n 1 ,y901 6 rc m 5e1p cn1rp 9n15u 280, 285, 8 8, 9 257 ,5et ac meth0d5, 46-48 ,)1PAD( ,5 64, 256, 257 D1AN, c0re(5) extru510n 0f, ,5namr0f en 28 ,5 4 7 - 5 0 , ,muvrap 47-50, 2, 3 29 25 43, 45 35- 3, 57 46-52 50 f0r, 39-43, 47 43, 4 48 43, 4 c1ter0hp tce1 d 52 cham6er(5), 39, n01tce1 0c f0rce, ,n01ta1051 detc10n, 82 enr1chmt, 36 35, 261, 89 37 ,5n01tac1 p a and . C parvum, e4u1pmnt fact0r5 f0r, 0 f ,5m 1na9r0 c m 291,293 meth0d5, 29, 245, pr0et15, 38 71 ,e16arut c 1arut1 c 24 , ,51 er0hp tce1 d 51 10 cry0em6d1n9, 290, 5uc 0 tpyrC mu1d1r1p50tpyrC 0cy5t, 238, 58 ,c1rt e1 d 20-2 81 ,51 type5, 205 e10dn1 ynehp-2 0n1d ma1d-6,4 25 -254 ,5e1pma 238 46-48 cham6er5, 25 -254 f1xed 35- 8, 205 128, 297 and 18 27 -275 270 ,retca60f1u5eD ,01r61v0f1u5eD 515y1a1d 251-265 254-259 f0r m 1 f 0 1 6 180, 270, aer061c, m1cr05py an9 (CL5M), 17 - 80 0f, detc10n mh5, ,5n01tac 1p a c0re(5), 158, 159, ,5re1f rt1ned re5a1 153, ,n01tac f1rt ned 41, 4 7, 48 ,5 1 preat10n, h 156, 50, 51, 84 c0nfa1 ,541- 3 n01ta2 m1tp0 28, 293 37- 9, peat, ,5re1pma 140, 28 291 6acter1, 1e9 51 er0hp0rtce1 (D6 E), 681-57 341 153, 289, 713 9n1rutaned 451 9en5, 28 ,56 3 D 041 153, end, ,5 , 5 6 298, 146, ,9n1 0 c rRNA ykc1t5 7A, ed1r01hc 81 81, 97 931 D16 43, 47, 48, ,5e1 f0rp 39, 36, 8 39, 43, 4 4 , 6 48, 5ytem, 50, 49 184, 245 46, 15 15 423 xedn1 1yxeh d tcer1d 0xacr6yn1e, ecn 5 r0u1f pe (DEF7), 4, 56 eu41nh t retf1 ,1 05 c1f cep5 temp1a vect0r, ,n01ta21 au51v water, 35, 6 tcer1d 9n1 e6a1 e16a v (0f ,)5e1d06 tna 86, c0unt dev105 1d (DVC), 5e1cep5 te1n 27-5 DNA, 67, pr06e, 5 am 78 18 f0r me6ran ,)5M1 ( 64, 6 ,1a retc 6 ,5m1 f0 6 n01ta1c05 1d e16u0d ,51 er0hp tc n015 cxe extrac10n tnec5 r0u1f 9n1c eu4 5 10 , 10 , 10 ,20 , 7d, temprau 10, (d5DNA), 5tmnde 231, 2 ,51e9 0f, 9n1 e6a1 1 0 9 ,2 4 102, 1 2, 381 30, 175, ,n01ta2 d r6yh ,n01ta1051 ,e5a91 98 9n1t em n 1 peat, p01ymera5(), Pfu, 14,709 160, 7a4, ,e16at50mr h pr06e5, 165 rDNA, 185 rDNA, ,9n1 pma5 ,tnem1d 5 e19n15 trande5 (5DNA), 10 , 31 12 , f0r ,5t016 ,9n1tr05 meth0d5, 35, 52 ,51 y an 1, , (7m) 18 281 761 75, 6 38 241,245 dekn1 - my2 e 761 a5y 1mun0r6et (EL15A), 3 6, 84 35 124,176, 184, ,31CrE a1n wrE 01 97-10 58 er61um, 8 12 12 9, 287 faec1um, enumrat10, 35, 28 761 154, 172 ,51 nedan c 5uc 0 retnE 761 149,152, 43 enr1chmt u, 30 81, 6acter1,n 01 154, 60 180-5 n1 D 6 E , aed01E ,n01ta r u1e 061 13 ,132 1 ,176, ,n01ta r ce 147, 59-61 175- 86 241 154, 12 -124 281 268 147, 0f, n 1 1 ec and DNA parmet5, 5e1 f0rp 180, 321 51 y an 16 38, 10 temp ratu e 175,176, 421 1 9- 26, ,9n1t 0 6 r ce1 , n 0 12 t u e r c 1 ,51 er0hp tc 1e 671 9en0m1c, 541 1 9, 123, ,n915ed 97-10 , 41 fra9ment5, 24-6 fun9a1, 761 pum5, ef1x 68 ecn d pm1 a rt 41 0f, 061 701 E 1 ,175 fr0m 12, ,n01ta c 5 er d0t )5(te1p0rd dye(5), 3, 6 7, 68 10 237 13 10 , 50ftware 206 97-10 23 , 5e4unc(), 10 13 PCR, 1 3,1 6,130, 9, ,51 y an pr1me 0f, 109- 17, f0r 1 2, DNA-DNA DNA 83,295 aut0m ed f0r, 152, 36 arche1, 5em0rhc u1f DNAa5e, 5pectr0my 5, 9 2, 30, 5 38, 901 86 ,ar0v 1ym 291 xedn1 325 ,1 car t0v ra, chry5anthem1, 82, 4 510c t rp 84 E5cher1a ,1 5, 6 9, ,1 0c 6, 143, CA60, ED8654, 0157, 168, 18 ,189, 194, 192, ,1 39, w01f 291 mu1d hte EU83 8, Eukary , ra1u 1ecartxe 75 59 f0r , 5 m 1 f 0 1 6 58 01 230, 23, 140, w01f cyt0met r 2 3,259 142 14 n1 ,5m1 f016 29, 8 27, 80 f0r 892 detc10n tnemur 5 1 te5 240, f0r , 9 n 1 r 0 t 1 n 0 m ,51 ec 204-206 207-9 206, 12 1ac 901 5yhp n1ec5 r0u1f 208- 4 4,(FDA)65 etan yc01h 5 (F17C), ,36 18,65 352 16 24 6ead5, dye5, 3 56, 293 24 n1 24, 26 0f, 60, tnec5 r0u1f 542 0f, ,95 56, 7 ,n015 me ,yt15ne 1 m1cr05py, 21-35, 8 140, f0r, 23 , 23, 240, 243 n 1 ut15 , ) n 0 1 t a 7 1 d r 6 y h n01tax f 52 52 21, 213 ,76 3 237-49, eta c 1d ,ecn 5er0u1f 20 -2 0 ,51 y an 2 5, ed150naryp0tca1 9-D 31- d n1ec5er0u1f 81 1 ec n01tp rc5ed 5n01ta 1m 1 n01ta2 1 6aemr p 18, 63, 42 28, n1ec5 r0u1f 207 tnec5 r0u1f( 29, 241, 0 f ,51 ec ,65 76 50rt1n9, 245,261,293 206, 206,5tau 86 cyt0mer1 (F6P), 205, 26 mea5urnt, 67 ,n1ec5 r0u1f 210 21 ,213 ,y1fer H51F yt1 6a1v ,515y1ana 1 ec 204-209 ,9n1 f0rp 1ac1t51ta 5 16 w01f 201-2 0 ,n01tar6 1 c 0f perat10n, 56- 1 and 201-2 0 and 142 5e1p cn1rp 531 1 9, 6, 7 up manufctre5, 208 and 308- 5 -74, 62-74 0f6acter1a, F ,51 y an 5n01tac1 p a n 1 1a retc 6 and ,5 am016 f0r chem0tax n0my, ,de1f r t5e 9n01 032cha1n, methy15r, 20 285, (FCM), ,5n01tac 1p a ed1rahc a5y10p ac1d(5), 284, 31 , 6 6r0m1de, 4 65, FAME- 15, FA57A, fat y 2 4-2 8 6 )5(1 ec cham6er, rate, 5met5y 591 195 82, f0r, F1av06 cter1um, n01ta2 d r6yh 51tu (F15H), F15H 2 1,2 2, 23 f0r, 24, 26 ,51e6a1 de1 6a dN7P5, 56, 3 64 pr1me5, 1 01 5e 326 xedn1 ,5e60rp ,92 ,76 ,36 tnec5 r0u1f ,91 ,25 ,832 952 n01tc r 5e /RCP ht9ne1 PCR/FL), 127-38 ,deta1u9 r- 05n tran5fe, fra9ment m51hpr0 y -u1F( 3 ,61 y1 ac1ten 9 de1f 0m 13 - 5 132, 31 ,5ed1t0e1cun091 0 12 5a pr06e5, 2 4, 23 142 , 3 ,46 ,6 246,3 742 30 cy5t, a1dr 6 e5ad1n0ruc 19-31 60rdna, 9ra6 5mp1e(), 6ram-ne9t1v(5), 43 18 , (9u5A), 291 132 51 190, 203,2 6, 27, 46 95 67, e5tr24 247 and D6 E, 6ram ,)5(ev1t 50p 205, 6 2, 7 246, 7 175- 86 25 43, 68, 190, 203, 295 n1d c mar9 6 ,e5ad150tea1 9-31 5a9 18 , 189, chr0mat 9raphy 18, 20, 6 ,)5(netpah heat retca60c1 eH 51t apeh ,)5(mar90t 1h H0ech5t, 64 2, 45 (6LC), 213,267 (mutan5), 7, 8 )5(1e9 denatur19, 17 , 871 71 denatur19, 178- 0 38 ,ecnat51 r 981 091 ,67 ,97 ,18 0 5h0ck, 3 ,38 ,68 542 ,98 ,5 7 48 ,1r0 yp A, 84 59, 16 162 342, 64, 2 3258, 64, 2 871 9rad1ent, (6FP), H 267 chr0mat 9raphy 5, 240 9renp0t1 f1u0re5cnt 293 (6C), 209, 1 c1t0 61tna ,9n1 0 c exchan9e, ,3 n015 erpx fra9ment5, ,1an0 tc uf 6C-c1amp5, 28, 48 ,mud1 nac 53 ,51 er0hp tc 1e 1e 1ar p denatur19 pc 0 f ,a1retc 6 muhc1rt0e6 de1 6a d1u41 207 charte5 29 123- f0r, ,em0rhc u1f f1u0rcytme, c1ne90r u f f0rma1de ,et1 09nuF 2, ,58 ,c1pyt0ne9 13 0f, y1tnec5 r0u f 9en(5), 5m 1na9r0 c1m (6M 05), ,5n01tac 1p a ,51a ret m 5e1p cn1rp 510c t rp 6A5P 295 1act0ne(5) hmr hum1c n01ta21d1r6yh 56, hydr0nam1c 7fu59, (H5L), ,5d1ca ,01 (f0r 6ufer ,)H51F 27, 82 671 295 e16a1ravrepyh 6 5n019er (0f 561 rRNA), 14 571 1 91 176, 481 e9am1 ,51 y1an 25, 285, 28, 308 1 201 43, 47, 2 8, 251, xedn1 327 1mun0fre5c, 64 7 5cater1n9,6 57, 59, and FCM 6, 2 4 1 , 2 4 5 y1tnec5 r0u f m 1 n1 u6019 num 1 c1ten9am0nu m1 ,de1 6a 60, 6 (196), 79, e1c trap ,)5M1( 36 ,1a retc 6 ,c1 0 ym 8 p01ar, 98 203,204 46, ,a1ret51L 53 ,en1 ayc06r d 1nfraed 5pct0y, 213 n01tre5n1 1N7-f0rma2 n, 1nterc0, d10nerp 51 203 203 )5(n01tar pe5 , 6 381 1mpednc, 16 5d1p1 19 m0ncyt9e5, 256 (15), emnt 30 ,cu1 ,e5ar f1cu ,yrtem0n1 u ,xu1 1uxA8, ,Rxu1 38 23 ,289 021 ,5en0 1u4 203 J 82, 4 81 18 , 091 81 83, 18 , 190, 19 19 01 M Jenk1 c0re , jet ,71 n 1 r1a 20 (and )5(e1uc 0m r a FCM), 57 c0nte, 36 0 f n 1 FCM, 9n1 at5 K a1 e156e1K 5ecym0r v u1K pneum01a, ma9net1c 291,294 ,51tca1 p1antrum, water, 192, 293 6, 8 76, 81, 2 ,6 257, 361 detc0r5, 56 ,n015 me ,n01ta cxe ,yt15ne 1 ,re5a1 67 56, 7 6 95 57, 59, 79-81 75, 6 38 ,a1retca6 ,57 0f, 76-79 ,71 c0re , ,67 81 (m/2), 5 am 86- 9 703 ,ea c 1 en019eL ,n01ta91 th91f 75-96 267-269, 27, 274 53 294, 201 ,)5(n01tar pe5 ,9n1tr05 Maker th ma5/chr9e rat10 57 f10w, 6ead5, e1c trap ,5n01tac 1p a n 1 1 ec f0r 9 n 1 r 0 t 1 n 0 m 5e1p cn1rp 132 18 , 189, 30, PCR 83, c1ten9am 091 ,01 n01ta2 d r6yh (MCH-P R), 48 L ,-ca1 5u1 ca60tcaL ,Y2ca1 eka1 ran1m xeta1 ,)5(n1tce ,a1 hp0muenpa1 en019eL 237 capture 1 ,25 293 28, 156, 5pectr0my 213,269 (M5), ,51 y 0r p-5M ME1, ME2, ME4, ME5, me 6rane 20 120, 120, 421 421 421 421 dye uptake, ,yt1r9e n ,yt1 6aemr p 3 65, 67 67 20 , 209, 81-3 328 xedn1 ,1a tne 0p 65, 9n15u 9n15u 9n15u 67, 68, 239 ,5n01tcare cyan1ed5, 239 24 0xn1, 24 24 rh0dam1ne, 239, 24, 4, ,5 ,1 ,86 ev1t 5ne ,61 7 at0m1c 6r19ht 267-2 8 270, dat 172 ,9n15 ec0rp dark dev105 1d pr06e f0r, 27 -275 267-270 0f, 0f, 27-5 c0ntra5 e1ctr0n, 251,286 230,859 18, 260 ,th91 pha5e c0ntr, 28, 297 9n1 ac5 27 81 28, 296, 29 e1ctr0n 56, 1amp 7 287, 0per n, 13 , 531 ,5d1m a p 68, 285,289 ert1 0 c m n0c1UeP-r0p1 1M 5,m1thraycn 64 de1f d0M 61 6acter1, 205 ,81 5n16 0R 1an01c n0m ,58 172 reducta5, 120, 259 ,5e1t v1tca ,5e1t num 0c ,yt15rev d n01tac f1tned1 21 Muc0r 59 h1t0ram,upe eta1r v t1um Myc06ater1um, ,5c1t5 at5 51, 40 ,51v06 21 ,51 0 ucre6 t , 48 7 21, 213 091 f0rtu1m, (M15),ytem 206 207, (Ma65), 48 481 av1um, 2 (f0r 31 - 8 p1um6e5, 2 2, 29, (MRD) 3 0 98 , 5e1d061tna 421 Met0pu5, 1a 60rc1m ec1v D 284, 31 ,317,318 23,271 ,81 02 C0M 16 291 ,)5m1 f016 2, 287, 305ytem, ca 20 methan0951, (7EM), ,51 ew a5 y, M1cr0tx 9n12 d1x0 pr0duct1n, 314 28 2 f1ux, 297-29 , 286, e1ctr0n 296 5tau, (5EM), n015 1m5nart 26 methan095, ,8 9 Methan05rc1 6arke1, methan0rp5, 8, 9 methy1 75 ,ecn 5er0u1f pe mercu1 dta5, mercu y ,51 y an 251- 28, (D1C) 93 ma1ntec, c1 06atem methane, ,8 1 982 ,d1e f 268 5e1p cn1rp Pr09am1n ,91 28, 5 280, ecn r f t 1 a d 27 0f, 5e1cep5 28 230 ,9n1 nac5 ,562 172 f0r, d1a9rm (AFM), ,d1e f re5a1 c0fn c ,5n01tac 1p a 5e16amu n0c arc mer tna 51 er f0rce 5pectr0my a (M15), 41, 286-289 dye5, 29 te1n ,04 391 m1cr05py, 67 28, 289 ,93 19,23 ,5m 0c r 1 256, me 6rane 61 ,5uet 15uc 0 rc1M 291 132 291 xedn1 923 N 0cy5t, fu510n, nah-1ux 19 c1x d1 an 67 ac1d, na0p1kt, 30 parf0m1dehy 259 fr0nta15, ,)5(e1c trap 561 34, 127, 158, 561 ,51mr0f1t um ,1 97, 102 ,18 ,79 5trande, 0u61 dye(5), 67 8 ,201 ,721 931 56 n015u cxe 240, a5y extrac10n, 97 102, 140, 1 ,n01ta 1m 1 251 2, 3 257, 41, 24- 6, 2, ,5 6, 7 ,81 ,e1 f0rp ,9n1 pma5 ,9n1 0 tce5 312 ,86 ,91 c0re(5), (nmr), re50nc ,)5(d1ca 39, cha1n5, ,81 ,91 2, 4 5p ., 48 37, 8 4, 15 0 f ,)a1retc 6 281,286 ,)5(d1p 0hp5 ,46 ,36 ,21 ,671 ,751 ,351 ,531 ,481 ,deta1yn 0 6 ,c1t50n9a1d y1tnec5er0u1f 209, 1 23 ,14 faty 23 19, ,351 061 RNA-d1rect, 63 15, 157,24 6 257, 204-1, PLFA, 207 51 y an 0 f ,5e1 f0rp 21, 3 ,581 542 1am05 61r ,79 5p ., Ph0t6acer1um ,8 43,1 0 2 19, 184, 3 2 176, 127, 53 1 6, (PLFA), 1ac1t51ta 5 ,12 ,531 821 21,3 d1ca m0ne1c 651 23 ,5rem1 p 207 12 21 153, ,de1 6a1 ,5 1 PLFA, extrac10n,2 38 24, 258 32 dev1c, 1yp0r c ,n01ta2 d r6yh ,)5(e60rp 24- 6, 32- 81 re1t P mu1 1 c1neP ,yt1v mrep phen0ty1c (ar5 0 ,341 91 24 28, 280 ,)5(ed1t0e1cun091 0 43 6095,27 36,4 c1ten9am 46 1raep peat, 132 3, 57 path09en(5), 36, 561 ,5ed10r t a nutr1e 3, 89 env1r0mt, 6 8, 76 mater, 30, 270 eutr0pha, F15H), 87 ,)5(eta1uc r p ,n01tax f n1( 30, 6 38, 51 7, 59 61, 3 75, ,8 9, 1 721 ,e16arut c-n0 ,e16a v-n0 rae1cun c1e1cun n01tax f 26 68 N0card1 47 24,756 P x1rt5am1 ac0eN ne0dym1ua9t5, 78 netw0rk5, ua1 59, 68 ,5re1f rt1n N1tr06ace, n1tr09e N1tr05mna ,ar1p50 t N 0 f .C parvum, 4, ,56 240, ,10n x 260 ph0td1e, 59 re1 p1t1um0t0hp phyc0ert1n, 63, 4 25 c1ten 901yhp P1c09ren,64 e10hn p 81 (PM7), tu6e ,515y1ana n1( CL5M), ,95 ,321 ,421 25, 3 14,2 61,25 30 xedn1 ,)5(d1m a p n915ed ,c1t50n9a d 81 n 1 ,5m1 f016 mercu y pLV10 3, pNJ50 , pUC18, ,yt1 6at5 70L, vect0r5, 280 ,ecnat51 r 591 361 491 190, ,n01ta2 r 10p 01 p v1ru5, 61 61 561 561 561 240, ,)5(1e9 1, 5e4unc19, 0f, 1 4, 1 5, ,)56aP( n1ahc ,48 7 n01tcaer 14 ,146 6 pr0mte5, 9 mu1d1p0rp n1et0rp 1 2- 5 231 5e1d061tna ,ed1d01 20 , n01tac1f1 pma n01tac f1 pma 0fDNA, 13, 02 0frRNA 51 156, 1 rRNA J rRNA L rRNA K rRNA ,)5(rem1 p 15,4302 ,9n1 ae 203 48, 192, 194- 196,28 29, 294, 295 ,5nec r0u1f ,1e am0due5p put1da, 741 83,294 86 82, 158, 19 ,257, 321 18 , 296 28 ,5u 01rt f 741 0 4u0r m 58 481 641 165,203 203 ,1 451 201 ,51 av19n19 203 ,5 a1c6u5 ,5 a1c6u5 5uc 0 ryP P0rphymna5 64 203 ,a50n19ure 260 ,en1d 0r yp1 n vy10p 63, 5an0m due5P ,muta1uc1 5ev1t um 257, ,1 ec ,5 a1c6u5 ,5 a1c6u5 ,1re2tu 5 n0rt5a1py 0P 8 165, 16 5a16 0 f , 741 n 1 etc10n,d 30 n 1 DNA e4unc19,5 1 2- 14 pr1me5, 2 160, 481 pr0duct(5), ,231- 0 147,52 4uant10, 149, 751 rean19, 741 e5r v e5atp1rc5nmt R7-PC),( temp1a DNA, 12, 1 3, 52 201 5e4unc, 146, 149, 78, 9 pr0te1na5K, pr0te6ac1, 206 ,342 ,042 87 1at0 ,041 15-86, ,5 , 6 - 4 6 A, 6, 58 30,(PCR) 35, 10297 142, ,351 ,5uc 0 r 1h P 231 1 2- 5 DNA, 0f 1an01cy10p e5ar my10p 14-8,9en5 ,1a5rev nu 243 ed1ma yrc 10p DNA rRNA 481 38 65, 51 er0hp tce1 9n1da0 14 061 f0r 36, 8 P0-PR0, 14 , 60 751 ,c1f ep5 N5012, 451 124, 061 N5019, 19 128, PCR, 123, 153, 156, d0ma1n pA, pHr, pUC/M13f, pUC/M13r, 296 192- 6 18 , f0r ,9n15 e ,01 R recA mutan5, 861 1 190, xedn1 31 a1 1neR ,51mr0f1ner 81 rep0 ter 9en(5), detc10n ,5en 9 187- 9 5n01tac1 p a ,tnec5 1mu 0 6 chr0m9en1, 0f, 189- 2 ,741 18 , f0r 091 ,5n01ta 1m 1 PCR 591 0 f ,5m 1na9r0 n01tce d examp15, 18 , n 1 9en 5e1p cn1rp n 1 pr0m ter ,E1yx 190, fu510n, 291 ,89 13,2 154, 1en9th m51hpr0 y (RFLP), 451 (R7-PCR), 309, 315, 316 ,mu16021hR 207, ,321 , 4 ,56 261,89 5 5uc 0 d hR 1am05 61r ,86 7-9, 239, 8 24, (rRNA), 256, 4 23, 0 29, 6 752 139- 72, 240 0 f ,5en 9 154, 143, and 5e4unc19 14 , 0f 149, 15 ,153 9en5, D, 5er09up 0-6,7 84 6, 9 84 751 751 9n1 nac5 n0rtce1 256, yp0c5 r 1m 260, 413 309, 9n15 e r05ne , 1 28 84 48 5AR1 , 5AR406, ,16 163-165 5er09up ,mu1rum1hpyt ,w0hcr1v ,10 ,1 4, 43 82, 4 46, 84 5er09up C , 7 , 0 1 ,6 ,041 21-4, 139-72, end 2 1,246 7, 9 35, ,51d1t1retne 67 0f, 9n1 01c 15, ,ea15 v r c 5p ., pr0ject (RDP), ANR1am05 61r 154, 1 a1 en0m1a5 321 mRNA,9 f0r, 561 10 ,139, p01ymera5, 6 2 dat65e 120, 9n1 0 c 0f9en 5 241 892 ,5ne1c5af 51 y an tnu16 041 041 5ac h r0myce5 257 247, 541 5 1 ,er hp5021 en1mad0hr 14 , 2 041 67, ,50pr r0tav1u5,84 28 ,mura50n1mu9e1 9en5, 153, 124, 139, 9n1 0 c RNA num6er, ,91 67, RNAa5e, 14 5d10nyeR 0f rRNA, rRNA, rRNA, rRNA, ,)5(em0 61r PCR 261 16 - 3 16,2 n01tp rc5na t 24, 9n1 0 c 160, 041 rRNA, 821 83- 5, 159, 160, 241 235 285 7A 061 14 , 139- 72 end 561 581 361 761 9en5, 149- 54, 0f, 5 192- 6 ,5et15 0f 154, n01tc r 5e rev5 n01tac f1 pma 187- 9 ,5n01 uf ,n01t5e9 d ,e5a 1cun0d e fra9ment 159, 142, 19 ,192 0f, 15, 8- rec0v ry R7-PCR, ykc1t5 591 156-159 14 - 9 19 - 8 81 f1u0re5cnt, 0 f ,5m 1na9r0 67, 127, 40 -3 146, re910n, deta1u9 r 286, (5EM), 287, 291,7- 16 9en ,n015 erpx ,3 6 32 xedn1 5 ert ruf1 5 ruf1 5 5e4unc() DNA, 109-1 7 ,51 y an , c 91 4r e m h dye fr0m fr0m 901 68 re5p0n, 9n12 d1x0 6acter1 dun9 6acter1, 821 (5R8), 128, 28, 9 e1cy ,5n01tcaer 12, eyd n 0 1 t a n 1 m r e t ,21 ,5n01tcaer 41 rRNA 9en5, a1t r e5 143, 14 , ,5ne1cafeu41 5heat 56-8,243 64, 184, 245 56 7 351 7A 1 ,d1u f 5Y8R6re n, 5Y70X6re n, 41 ,9n1 0 c 153, 1a tne9 at 24 w01f 16 ,165 n01tart1 f ( 7 F ) ,2 9 - 3 4 10 a1 e91h5 ,ea1retn 5yd ,1renx 1f c0n e tra e, ret1 f meth0d5, 32, ret n a e, ,9n1 pma5 58 am915 am915 1an915 58 (t~32), 32 7 5 (05), 6, 7 ,5e1uc 0m rat10 rev1 5 (5NR), ,9n1 at5 256 9n1tr05 and 505, FCM, 59, 16 5 ,5et hc0r1p 5uc 0 1yhpat5 ,1c 0 yhpat5 84, 7a4 206 ,5uer a (571), pr0ten 6 pha5e, t10nry 6, 7 8, 207 ykc1t5 5t0macher, 80 en1d vatper 5 end ,9n1 0 c ,n1t016- 153, 4 160, 80 c0ated ,5e1c trap 78, 261 143, ,e1 9arf 28 154, 7 tne1dar9 061 1e9 51 er0hp0rtce1 175-86 ,51mr0f1ryp 257 ,en1mad0hr-yx 6 ac- 1yhtem 23 230 ,e1 60m ,am1t ram 741 RNA, 240, 243 pr0te1n, 240, e16a v 243 c0unt, 241,289, 9r0up, 49, 50, 63 67, 314 8 1arut c 83, 97 28 761 ,5uc1tau4 238, 091 12 15 ,21 ,201 1y50t 1an01t dart 2 89, 481 18 1 82, ,18 3 120,127, 1 ec 1 ec 1 ec 38 5ecym0tper 5 mu19nar0p5 tpert5 75,8 ,e5aremy10p anemyharte7 tera ,mu1 02arte mu1 cam0t f1u5d0mreh7 a90t mreh7 5umreh7 1at0 5trep0c1, 29 ,r010c1 e0c ,5nad1v1 ,5e16ac5 3 32 (76 E), 5-9 e16 cudn1 30, 1 eruta pm 4 , 132 295 5tarv10n,3 3 3 6acter1, 8 89 ,51 ec DNA, ,5e1uc 0m 5e4unc, 57, 6 481 5NARF, 31, 3 tar9et 01 t 0 e510n 30, ca5et, meth0d5, 152 ,1 3, ,1 xedn1 3 tran5f0m1, 361 n015 m5nart ,yt1 6a1v e1ctr0n (7EM), tre 3, ,5 8 yp0c5 r 1m tnem5 a dye5, 162 0f1e, 041 ,51 an19av 259 6,5p.9 ,42 ,a1 erum k 57 U ,032- 82 32 280 1,8 ,5uc1f n1uv 132 5 ,602 5e ur1v 392 W ,a1retc 60 mart1u derut1 cn 9 ,5m 1na9r0 c1m water e10hw ,751- 289, UV 190, ,01 81 harvey1, (•1N•17C), 25 256 ,1rehc5 f 2,3f1-rp~Hta0um ed1r01hc 237-249, 245,25 , 01r61V 7r1ch0mna5 67, 8 0f, urea n1 D6 E, 259, dye xc1t ,th91 ,r0tan1mu 1 5nart c01umn, 10 , ,51d0p n xa ,51rt epmac 251 28 16 ,18 , xy1E, 0f 561 rRNA ,5e1ra 61 AND ••,7•• 451 e16a1v 6ut e 1 6 a r u t 1 u c n 0 1 ec 0unt,c 4, 36 4, 286, 9 314 19 Y vect0r(5) ,d1m5a1p 192- 6 19 xy15, 481 981 1 8 ,9 0 xy1R, 247 f0r 751 58 ,1a9-X V 5n019er ,5 1 Xanth0m5 5 9en5, 81 X 175- 86 162 ,n1cym0n1 av e16a r v ,61 ut15 ,n01ta21d1r6yh n1 1ec 128, ,5t aey w01 ey a1n 5reY 061 821 (V8NC), ,3 2 ,05 36 ,5 9 247, 4, 5 36, 20 tnec5 r0u1f ,ac1t 0 re n ,1 rekcu n1et0rp Y0 -1, Y0 -PR0-1, (YFP), 58 5 64 64 81