RAPI9 FLOW Cl'TOMl:TRIC DETKTION AND IDEN'IDICATION OF MICROBIAL PARTICLES

Hazel M. Davey ad Douglas B. Kell Institute of Biological Sciencet, Untverllly ofWale11, Abay1twyth, Dyfed, \Vales, U.K.

ABSTRACT There iA a cominuina aeed to bt. able to dect •e lipid eltimatiOll of the microbial lo1d in A

sample. IOIMtime1 merely in termJ ofdae totAI munbera of cell• ("dececcima•) but oft• allo with rcipoct to difl°•em c:ell type• ('1idlllti&cation"). 1'lail need ii puticuJarly acute wit• the pretence of pathogemc orpnilw is 1W1pected, ad the b1ckgrmmd coDRiat1 or complex, oAen non .. biolosical puticulatd mad odter, DOD·palhogenic biomateriah. Jt is lhown b«ein that the tect.nique of flow cytoma.ey •ogr..her widl cue.811 eeJectioa of tluorncru stain• may help to meet th.ii need. Optical brighteners repm.mt fiu.on:ICCDt ttain1 with many demable properties.

Dfl'll0DVC110N CytOllUIUy refer110 1he m•mremmt of the phymical ad chemical cbuactailtica of celll. By

m.a-., low C)4IOIDICry nflrl to ~e teadqM wlaen melt mee_....t• ue made 11 the cclla plll ia a 8uicl ltrlllll tbroup I mu.mas poiat IUJJO\mdtd by D any of dlttctorsl ,

In a 1...Ulecl lent.· eyt.oAJtt• l,2, tnclMdual paniclel , .. daroup a ...... pomt where chey ..._ 1 •11111 olllPt tom a II• or IDll'CQI)' uc lamp. Tbe umple la typically coaltniud to tl• omsre of 1 mach ••• 8owlq ••fluid by a procaa bowa u hyctrod)ulr4c fooa•a. aDowtaa rdlMe dellvny otth perdcln to dae Mitre of tlae Dm•i•lrion iou at a rate otsome 1000..3000 eelll.rl. Appropriate dttlCf.Ol'I, "ticb may be gated electromcally. meamre die mapihule of a pulle npre..m1 the cxtaat of Habt IQlttered H dte padde P••• fllouP die m, ••• doa "'*· TU~ olda• , ...... IOlted eJec:troalcdy ilto "Ml11" or •c1wmeJ.e", po1mi1tiq tlat dUplay o£2J>. or ~D-biltopam1 '>f a11mber of cella vi ehftul lnllllber (w cllmmel number tor 1 11eond or ftlnber puunerer) . . Upt acmerecl m tle tonwrd clitlCdma prvvJde1 Wbnuticm 11»0.it the Ut of the ecau.ma panJclc Mio Hpt aftetc4 It~ •r cllp•cla to I WICI exlmt cm~. iatenW lbUcture of th~ coD3. Ill acldldoa, celuler eatoluorwelce may be explokect4 laowev•, ti 11 by the 1ddiltom of tppropriett laoropbor• prior 10 aalylil tUt tilt AaD potmdaJ of fll>w C)'IOlllltfY- Y realile .

A wide variety of fkaort1C411t •ala• have been uted In low cyton•llc n.U..5 and mcJude tho• wlneh bild co moleeulet tuda 11 DNA, RNA or protelD, luoro1.UC mblb'llt* ft>r

593

IDlllUlina the acdYiry of• liv• mzyme, a\cticatora of iDtn1ceD11ltr pH, &aorelCCllt .... which are takm up or txeladecl by cells ill a wcr whidl reftectl the ilatacmeu of their membrane1, and, illcreaiasl>', llll'boctie1 (or olipmadeoddea) taged with a fluoretceDt probe.

Undl reecady low cytometry had been applied maialy to munmatian cella. Mieroorpnicms, wSJa their .,n., Me IDd Nlldvely Jow OOD~I ofDNA ad other celh1•1r conllitueats .. cl proved to be below the reeohatioa of ma• co1111111rcial flow Q)'t0meten6. However, within the Ian 10-1' year1, wilb iaenued in.,namt lftlitMty ad whit die dwelopmei ofbfiahter 8uorncent proba low q1omaay lw bem 1Ucceufillly applied 10 a DamlNr of ariciobiological problemt 7· IS, •d ~ a book dtYoted w die .mbject U. Me publ•tc1l6.

While the majority of8ow cytomelric applicatioat in microbiolo1Y Jaave illvolvecl the~ of ueaic cultuns tom1 work bu -.. done on dae ;.teatificltion of orpni1m1 • mmure.17·20. FJow C)1omeay o&r1 ICVenl advauuaa for this cype of work, (i) c:ella are aalytecl iu.dividuo.lly, rather tlwa 11 populadoe1 allowin1 Che i~ of cli8iaet cell t)'ptl, (ii) typical dlta acquisidon rMes are in exoeu of J.000 cells.r 1, ..Uliaa rtasoubly nre evata tt> be obterVcd, (iii) dependiq oa tile iulnlmeat, typically 3 to ! cliftiret Ju.met ... of oeda cell may be meatwed limdtan•oully, increa••1 the pollibility of ~8 between lbD1ar ora•imli, ud (iv) if dae prnmce of a ur1et or1n1111 ii iadlclted the eortiDs capalrilitiet of tho flow cytomeler c.n be caDecl '&.:pOll to Mpante the A11pect cen from the mixture for further coa8rmatioa of a idmlity.

Oa• lit\altiol whm it would be adv•t1Pou• to dlt..-. wbedler • w1• or1Nlitlra wu pnNDt WOlild be ;4 oate• ..... the releeM of I biological wadare llml Wll IUipCc:ted. Jn the work delc:ribld below Battllw 1lo11t111 (B. nbtt/11 vs "'P') llpOl'el •ve bom \llecl 11 a tinm1aat olB. "'lllrodl21-23, n.. flow "'')'Oll*rio cUractcildct ofdUI or1f4t.mbv• U.. ttuctied a.ma '°'11 liPt ecattmq ••---• lld by 8uomcmoe mee---. following the additicm of8aortlOlat bftPua ... fluoruont brtabt•., r-~ coamaoa 1ddiliv11 to d~mestc walliq powdm-.. daey iacnue ... .,,., ........... of doda• '>' ablO!bing UV·liPt md •••1ia1 ID dae Yilltle pct of die lpecll\lllL 11a• ... are llOl•toxic uul would be apecttd to MDCI to tlae Nrf'ac~ ofbocttrial tporee. dNa overcomiD1 probleml that may be fcnmd wkb cGer ltUil ~ would require ptrm11biliution of •e tpore prior lo aa•m1.

MATEIUALS AND ME'l'dODS flow Cytomltr)'

All low ~0DMC1ic a1lltyt1t Mn prirmed ulliq 1 Coull• Epica Eli&• low eytometcr (Coubr ilec.v.UC. Lad., Luaon, U.K) ecpU,,ICI will& 1Heed1a .. (325 am), a ar1on laACJ (411 Am) and . HeNe Luer (633 am). 1\e low C)'lometn WH let up aa clelCribed m the mn1d'actama nwmal n. mlllh Add waa prepared um1 Miiiipore MWl-Q weter flltnd te> 0.21 J'Dl ad ocmt.med 1!0 mM l«.1 •d 10 naM HEPES. Tla1 pH w11 iCVulled to 6.1 with KOH and the ..... aaW Wll IMs lkertd to 0.1 JUD WI a Wa..uwa WCN Iller. 'l1le forward ecett• lipal l'om die ar1oa la• wu \&led to dhorimiaatt b«weft lb1 ltpal &om the 91poru llld tile •acqrauad aoiM. De waveleaadal OYW wbWi .We IOltt ... ad Aeorttcence wtre ~ '° ... wllla .... plMMoneldpilr vok1111 ........ p.. .. •Ill•• to ae lpre1.

flaoraeeat .... tea .. 11lree dililmt luofllClllt brip.teun were lhlctied iD tenm or ~cir usefiaJD• in 1taiDiD1

B. globlfit 1pote1. Tiaopal CBS·X wl T"aopll SWN wen oluined 11 • Pf\ om Ciba~ ad Chemir.a1' Ltd., Macdedeld, U .K. md Calcoluor Wlake M2Jl wat pun:Usecl &om Sipla, Poole, U.K. They were tested It variou coaemtntiou ad tile time-depmd•cy of ••iDinlJ wH

clecflnri:aocl by uting 1be tia parlmlter on the 8ow cytometer.

Sample Preparation B. tlol>llll 1Por• Mr• oh11iaed form the CBDE, PoltOD Down, Salilbwy, U .K. Two

.amples \vete received. • "wet• ...,•lion of IP.Ore. ;and a "dt)"' preparation. Prior to aaa)yt."J the spores we!e w.p•ded in. lbellb Suki to pe a concatrtdoa or approximlte)y Ix l 06 1port,.mt 1. Unaiaainecl umple1 were rn widao\rt .&nthcr prcpartttion. Stained un:iples werc­pnpued •• delCribed ht the Je1m.!1 to the fipret.

u:sm.:rs AND DISCUSSION On ualy1i1 of the lislit acatterins propertin of the •wee" tu.spension of 1por-:r; mppJjed by tae

CBDE il became appulbt. daat ther& were two subpopulatioti pree....41lt (see Fig.are t ), 11 weD u 1 ___________ ..__, _ _.., _____ ------

M r•rw1rll Sn9er ... lltfel..-r ••

' 1 ,, .. " l : ,.. cy&aa\etrtc ...,. .. of UM,~ .... peoperti• c:IU.. •wm" ....,.UDft or &tn/11&• 11'°""" lpDlll mMltd M die...,.., •rU•h•ld tM dAldaet pap ,...,, .4.IJ a...- \tMf ( .. II nm) wm u.tld M tbt ~ 1GU1ct Ind upt a11.t11 ._ eolllcUd In the farwarcl dltK~oo •I ah' • wide ...-. In bo\h e111• the PMT wltlp w •to 400 Ind dw pla w laprhlanUc, wfdt fall 1eale .....-llti1114 dleld11. 0..J) •11be cw otn .., ...... , ... , ploc u 11 pa111Wt eo f'lllOtw 11w.,. ,......_. ftally, ~ dlmoNCrlUq lhl apia-a PJWI! flmu~ Mll)'llJ.

~----------------------------------------------------------

&9&

noticeable dei-.:~ of chunpinp, leading to the 1pparently larg'-l heterogeneity of the spore suspauioa. ID order to d•ell'Uae whether the two lllbpopulation• were real, or whether they were aa artefa" of the different 1Hpm:trt of the spores 11 they p11ted throup the 11t:a1Uring point of the B.ow cytometer we decided to exploh ., Mting capabilities of tile Cou4er Epics Elite to separate them prior to re-maly6is of the eriJmted mnples by !Ww cytomeuy.

A 90l't protocol for die B. g/obigii was created in the Elite softwere, in~g the 8ow cytometer to place events fi'om !legion 1 (JU in Fipre 2A) bto one collflCtfon tube md events from R2 (f'igurt 2A) .into a second tub~. \\'lien enoush sample had been processed to give at leasr 0.5 ml in each collection tube the two 1Ubumples were paued in tum tMough the flow cytomet~r. If the two aubpopulations were m artefact one would expeci to obtain similar. results from the two subsamples; laowever as caa be seen in Figure lB,C the two populatiuns have been

------------·---A B ~ --....... ~--·----

I : ,R1 ~ ..... .. .... :· ····

g '

... IT ~ ! /\, . ; /' . . .

"' i : .

I~ . ' ......... : ............... : ..... ........ '..· ................ . . . . . ' . . . . . ~ ..

e ~ . I

0 ... . ' ...... ··:. -.

Forward Scatter Forv.ard Scatter c

...,.----~~-,

: ! /

.R1

?!.11-_____ _.,._ -;....-•v• 11)• 101 tO' fQ•

f~ biter f'nf"'9rd scatter Fipr-. l : Pl~· c:ytometric 1nal,:-'li1 of the FJ. 1lt>bJ1ll spc.res. The t!o" C')'fometer wu set ap ns dtscribed :., the Jepnd to )lipre t . A) Thi "Wit" pr.,..ion of spurn canai'11 ~two 1Ubpopulllion1. Th• .. Mre 1oned by aow eytoanetry t.ld on the fflion• (P.l and JU) shown in tb1 8pr1. 8) and C) art the flow cytOrMtric UICU proctuc.d ~ 1111ntultinaIDftld1Ubmnp1N trocn Rl and R.2 relpletivel)'. D) The "dry" prtparlif:jOf' of 1pore11

iupplied by tk CBDP. haw chlncterilria ver; 1imUar co the nutj« subpopulation (JU) of the "wet" population

-------------------......... .._ __ -----··------ ...

59t;

aiccesddly teparakd. A:W.ysis oftlt.e "dry'' 8. globigtl sample revealed that it contained only one popula~ '..mch wt1 lbnilv iD. 8ow cytometric tenm to tile major population (Ill) in the "wet" llq)le (Fipre 2D).

When one's aim ii to dildnp.ilh between differeot orpnilms the presence of two diac::t mbpopulatiow with.iu one nominaDy homogmous organin ••dud is far hm delinble ad su the "day" prepuation of iportl wu cbo11m for UH with tho Aioreteent briPtmers. Of the three 11uoreteat brishteam dWlied Tinop1l CBS.X wa1 the most effective in aldaing die tpores (Table I). It was found that a coacentra~ of 40 µg.mt-1 (mol weight• '62.5) is illflicieat to ~t nyJin11m .. iaiDg of the spores bDt that at this conceatntion and ia the lbsenC6 of ccageners -20 minutet are required for m1xim1m tt•ining to be acldev~. 1he extent of caining of B. globtgtt 1pons ii illd:rated in Fipre 3A,B.

--F-1~----cen--t...,.. ___ lmu--·c---...--------~Ch~enu~·c-~~S~trud--u-~----------·.,--:sx-.w~n~t~~ !ria.htener Cban9cter StlUWla: ... ~ .......................... +---"'!!!!------~~~~----~~-... .... ------.... -i

TillOpll StraDgly IQ\-~Q ~ +++ ces-x Anioruc '~--CH~-a· ~ -~ I

l(\NI Nt09' '---·--t---------.--..-·-----+-------Tinopzl s·wN ·· Weakly cationic ~¥A

~-------...-1------,- __ .._.,,.......__......,_ ---('.a!~,ftuar A.11.ioaic ¥1'\Pf + '.Vbite Kp;K,Pt

(rinopaJ 1.PW)

~> ~M.,tK,DH .~·

6Npt,OH

Tablf.1 : Cbaracterilti<:t Md WUCCUree of tut tla..-nasctnt bnpttnlr& Ulld. Tinopal CBS-X WU atOlt ffft<11ve •n ltainia& the /J. gfob;gii lpOTH.

---·----··------------------,----------

CONCJ .. USIONS

It is often delirable to detect tuget microorplilma again• a hish biological baokpound, not lea• wbaa the pretnce of a p11hosen it su1pected. Modem flow cytomcten are sufficicmtly .aliiive to deteet aach orasnilml ud at O\ltliQod above oft'er CODlidnrable advutages •JVer odaer techniques. lb~ value of the multiparameter uatwe of flow cytometric data in

597

•

.. • • ~­... """ ,_, ... ,...

• 121 rwn • 12&""

.. fleuft 3 : B. 1lob¥ft IPDll' A amtliatd. IDd 9 ltliaed wjtb 40 J.1&,mJ·lrinopll CBS-X. In A there ii little dltlCClble IJuonlC•:e • s" nm and tbt emillicn coUeeced below 44f'> mn ii a ...it f/l lidl alteri.D& and pmlibly •taauon.ceDCt f/ltbe ..-. la••• u.cr.. iD flUOl'llCllnce •bath waveJ_.,. cm be 11111. f1UCNIClnCe below 440 nm w oaUeeted oa PMTl via a 440 didvoic 1 .... while nu~• 525 nrn Wll

collected an PMr3 uaq a "O dichroic lena IDCI a '2' nm brmd pas ftlter. The whip for PMT 1 Wll Mt to 460 and for fMTl a volt.Ip or 610 waa uMd. All pi.al were loprithmic.

~-------------~·~~---------------~--~---dist:inpiQil1 subpopulations in a nomm.Dy laomo1eaou1 llq'le (Figure 1) may ealily be ntauled to dilrinpri"9q bttweco ctiffereot Ol'p'itms m a ll'dttdl'e. Then.ore paramcten one measures thcs more Jikely it ia that o.oe will be able ~hly ~~ •dfy a target microcnpnilm in web a mixture. 'lbe capability of 8ow C)'tometers ·to sort cello 1ccordin1 to their ftow cytometric dwaeteriltica i1 mu.ntecl (fipn 2); this may prove to be very utefilJ in coa&miD1 the ideatity of a ...,~ cqnipn The mn potmtial of .8ow cytometric llll1ysis ia realded 'When fU>re&CCDt suiDs are added to tit cauaple. Pm: die pmpo• of •lining tporcs 111 ideal •am is one which is Aon-to:dc ad bin.dr mrf1ce, avoUling the requiremat for permeebi~ution. It is lbown that the 8uoresccnt bri ilopal CBS.Xis an etrective min fbr B. globigit 1porca (Fipre 3).

ACK.N0'1\i'LEDGEMENTS nae teletll'Qh repoited herein has bem lpODIOred hy the United States Army through ire

Eutopeu llctearcl 08iCC\.

REFEIC.'ENCES

1. Sblpiro, KM. (1988). PrlCtical FlowCylometry (2nd Edirioll). New York: Alan Jl. Lia Inc. 2. Mtltmod, M.ll, Lindmo, T.~ aad McadellOlm, M.L. (1990). Flow Cytommy ad Sortins

(2nd Edition). New York: Wiley·Lilt Inc. 3. Salama, G.C., SiD ... •m, S.B., Jolmrtcm, R.G., ID.d Bohren, C.F. (1990). Ught Scattering and

Cy\OIDC(1y. ID fJgw CY'OPWD' yd Stnia1 (Zad lditin), ed. M.ll Mtlamed, T. LiDdmo, a11d M.L Meacleliolm. 11·107. New York: Wdty·Li&1 Inc.

4. Ominp•m, A (1993). Aaaly• ofMictoaJpe ad Cyanob1cceril by Flow Cytomet1y. ID En C)tomlll)' ilJ6i;r,olrjRJag, ed. D. Lloyd. 131·142. Loadon: Sprlqer Verlaa.

5. Wagnur, A.S. (1990). ftloroteent Probe1 for Cytomeay. In flow Cyrqmrqy m.d Smtiu Qp41:&1~i11), ed. M.ll Melaned., T. Lillclmo, md M.L. MeDclelsolm. 209·225. New York: w0ey .. u.1ao.

598

6. Stem, KB., Sbntad, K., md Boye, E. (J~O} DNA MealW'oments ofBacteria. ln BQw C,ytcunlqy, eel Z. Dlri~ aaJ H.A Crilmwl. 519-5%~. Loadoa: Academic Presa>.

7. Betz., J.W., ~ W., md lland, W. (lgM). :;11 offlow C)'tOmlbyiD ladultri&l Maobialol)' for sum Jmprovemmt J.'latr JIDI, CytOIMtly 5, 14S.150.

8. Fnllt, G., Ltplace-Bdle, C., and GnmwlW, D. (1919). Mieiobiel ADalysis by Plow Cytomltly: Pr11ml -1 future. In Emv QrtOAWD! Adyactd..)lmn;l ad gpipel AIJlicationt (Volemc II). ed. A Yen. 256-~ 79. Boca boll FL.: CllC Pie~.

9. BO)e, E. ad i..a--Olelea, A (19'>~). Plow Cytometry: Dlitminltin1 Microbiolol)'. The New Bio/opt 2, 119-125.

10. Sbpiro, KM. ( 1990). FJ.ov. Cyto, ~IY m Labontocy Microbioloay: New Directions. ASM NN$ 56, 584-588.

11. K.B, D.B., llyd~: KM., kqrdyma, AS., ad w·~ RV. (1991). Qumlifyiq !Uterogmdy. l"!Dw C)1omet.iy of~1cterial Culluns. Ant. van UtllWt!n'ltoel fO, 145-1 SS.

12. Kapnlylllta, A.S. •' Kd1, D.B. (1992). Rapid Aa1eem•1 o£Bactmal Vilbility 111d Vitality usiDglUaodamiae 123 ad flow Cytosmtry. J. Appl. Boc:.rtol. 72, 410·422.

13. Dlvey, KM., Dlvey, C.L, ad Kell, D.B. ~1993). OD. the Detennmtioll or~ .. • Sia of Microbial Celli Uling Flow Cytometry. In t..w.w. qytOQMOy ia MigobioloC'., ed. D. i.!~yd. 49· 6,, Lolldon: Spriq•· Verlag.

14. Davey, KM., Klprelyat1, AS., ad Kell, D.B. (1993). Flow Cytometric ADalylil, ulirlr, RhodamiDe 123, of Mlcrococcus Jutr.u at Low Growth Rate in Claemolll1 Culture. bl~ qylQmM':iy ii MkfohloJAI)', ed. D. Uoyd. 83-93. London: Sprinpr-V.Ug.

JS. Fatirllct, P., Jayat, C., Hed1ard, Y., Ratiuud, M.·R, and Frdat, G. (1993). Recent Mvaac• of Flow Cytomeay iD Fundammtal uul Applied MicrobiolOI)'. Btol. Cell. 71, 9S· I~. .

16. Lloyd, D. (1993). Flow Cytomeay ill Microbiolol)'. London: Spriq•·Vedag. 17. Balfoort, RW .• Snoek, J., Smitt, J.llM., Breeclveld. LW., Ho&uut, J.W., and Ringelberg,

I. (1992). Automldc ldmtilcatioa of Alpe: Nemal Network Aul)'lil of Plow Cytometric Dita. J. Planklon &Hlll'Ch 14, 57!1·189.

J 8. Boddy, L. ad Monil, C. W. (1993). Aulylil of flow Cytommy Data • A N1ual Network Approach. Binary 5, 17·22.

19. Boddy, L. ad Monil, C.W. (1993). Neural Network Aulylis ofFlow C)'tOlmby Data. Jn flcnygytgrmgyiaMlcmMolo1Y, ed. D. Lloyd. 159-169. LmadOD: SpriDaer·Verlas.

20. Boddy, L., Morril. C.W., Wi1kiDs, M.F., Tuna, Ci.A, and Burldll, P.K (1994). Neural Network Aulylis of Flow Cytolllltric Dita tor 40 Marine Pbytoplmkton SpeciN. Cytomttry I!, 213-293.

21. Dado, M. (1994). Biological Warfar• in the 21 • Cemmy. London: Bres1ey's. 22.Stevenson1 R. ( 1994 J Chemistry A.icit the Germ War W1cdido11. CMmistry in Britain

693-696 23. CBDE (1994) Prote~-m, by Scimtific Underatmding. London: MoD.

599