JOURNAL OF BACTERIOLOGY, JoLIIC 1967, ). 1888-1896Copyright it 1967 American Society ft r Microbiology

Vol. 93, N,o. 6Pritnted in U.S.A.

Application of the Fluorescent-Antibody Techlniqueto an Ecological Study of Bacteria in Soil

1. R. HILL AND T. R. G. GRAY

HalltleY Botanicao Loaorotories, The Unir.ersitY of Lirvrpoo/, Livr'pool, En,gloatdtl

Received for publicationi 17 March 1967

The fluorescent-antibody technique was used to identify cells and spores ofBaicilluis slubtilis and cells of B. circul/aIs from soil. From cells grown in three brothmedia of ditferenet nutrient status, i.e., a cold extracted soil mediumii (CSE), Canunamended autoclaved soil extract (HSE), and nutrient broth (NB), antisera were

produced with both quantitative and qualitative differences in antibody content.The specificities of antisera to two strains of each of the Bacillus species were de-termined. Antisera for B. slubtilis 0 antigens were species-specific and showed nocross-reactions, whereas those for the B. ciruelli/lis 0 antigens were strain-specific andin some cases showed cross-reactions with B. /lvei. This cross-r-eaction was removedby absorptioni of the antiserum with B. al/ei 0 antigen. Fluorescein isothiocyan'ate

-y-globulin conjugates prepared from these antiser-aL showed the saLmae specificityreactions. A method for staining bacteria on soil particles was developed, by use

of small staining troughs. By mounting stained soil particles on slides and irradiat-ing them with transmitted and incident ultraviolet blue light, bacteria on bothmineral and organic particles, taken directly fiom soil, could be observed. Fluo-rescent antibodies against cells grown in CSE gave brighter- fluorescence of stainedbacteria on soil particles than did fluorescent antibodies against cells grown in eitherHSE or NB. Colonies of both Bacillus species were genercally small and localized.Spore antisera, though not rigorously tested for specificity, were used to identifyspores of B. slubtilis on soil particles. The uses and implications of the techniquein soil bacteriology are discussed.

A major difficulty in interpreting experimentswith soil bacteria in laboratory culture is knowingwhether the conclusions driawn are applicable tobacteria in situ in the soil. Even though it may beshown that a particular species exists in a soil,cultural experiments cannot prove whether theorganism is vegetative or dormant, whether itoccurs as single cells or in colony form, or whetherit is associated with any one type of particle, ormicrohabitat, in the soil. Direct observationtechniques have provided useful evidence on thesepoints, but until recently it has not been possibleto identify the organisms observed. The ecologi-cal value of being able to do this has been stressedby Schmidt and Bankole (17), who used the fluo-rescent-antibody technique to identify the hyphaeand spores of Asspergill/usfluavus in soil (17, 18, 19).They used A. flavus to inoculate sterile soil andsoil preinoculated with known species of fungi,and buried slides in the soils. After incubation,the slides were removed and stained with a specificfluorescent antibody known to react with A.flavus. Hyphae of A. flavu'is stained well whereasmost other fungi examined were either nonfluo-

rescent or showed weak autofluor-escence; only afew strains showed strong autofluorescence. Erenand Pramner (3), also working with artificically,inoculated soils, prepcared fluorescent antisera forArth/robotrvs co7iiclees and used them to study thisnematode-traLpping fungus.

Others have used the fluorescent-Lntibodytechnique to identify colonies of soil bacteria ondilution plates. Unger and Wagner (21) showedthat it was possible to prepaire replicas of coloniesfrom dilution plates, on circular glass slides, andto stain these replicas with fluorescent antisera.When the slides were irr-aidiated with ultravioletlight, the complete colonies of the orgcanism beingstudied fluoresced whereas other- colonies did not.They used antisera for Eschericlia co/i, Bacilluscerelus var. mlVcoi(des, and Sarcina fiava in theirexperiments, but did not attempt to observe oridentify bacteria directly on soil particles or oncontact slides. Except for a photograph, taken byPaton (16), of an unnaLmed species of Pseutldono-)1Cls in the rhizosplhere, no attempts have beenmade to identify soil bacteria in situ.The aiims of this paper are: (i) to describe al

1888

I MMUNOFLUORESCENCE AND SOIL BACTERIA

technique for the application of the fluorescent-antibody technique to the identification of B.subtilis and B. circulaCns; (ii) to describe a methodfor observing the colonization of soil particlesby these organisms; (iii) to present some pre-liminary data on the occurence of the above Btacil-lis spp. in a pine forest soil, developing on a sanddune system at Freshfield, Lancashire (5a).

MATERIALS AND METHODS

Prepairationi offflorescent antisera fir B. sibtilis anldB. circclans. Cultures of B. sithtilis (strain NCTC6284 from the National Collection of Type Cultures,London, England, and a strain freslhly isolated fromthe pine forest soil) and B. (irculanuls (strain NCTC7578 and a strain freshly isolated from the pine forestsoil) were growni in an unamended autoclaved soilextract (HSE; 6), nutrient broth (NB), and CSE, asoil extract prepared by ball-milling soil for 1 weekat 2 C. Somatic (0) antigens were prepared from allfour strains and spore antigens were prepared fromthe B. slubtilis forest soil isolate by use of the methodsdescribed by Norris and Wolf (I15). The antigens wereinjected intravenously into rabbits as follows: day 1,0.5 ml; day 4, 1.0 ml; day 7, 2.0 ml; day 10, 3.0 ml;day 13, 3.0 ml; day 16, 3.0 ml; day 19, 3.0 ml; day22, 3.0 ml. On days 32, 33, 36, and 37, the rabbitswere bled from the marginal ear vein. Secondaryinjections and bleedings were carried out after a 1-month rest period.

Antisera were prepared from the blood samplesand were sterilized by membrane filtration. The ag-glutination titers of the antisera to the 0 antigenswere determined by testing them against homologousO antigens and whole-cell antigens, by use of La-manna's (11) modification of Noble's (14) tube ag-glutination test. If these were satisfactory, appro-priate antisera were bulked and their specificities weredetermined.

In the preparation of the --globulin fractions ofthese antisera, equal volumes of antiserum and 3.9 Mammonium sulfate were mixed at 2 C. The resultingprecipitate was centrifuged, washed with 1.95 Mammonium sulfate, and dissolved in a minimal volumeof distilled water. The solution was dialyzed (in916-inch diameter tubing) against 0.85% sodiumchloride, at 2 C, until all the ammonium sulfate hadbeen removed. The protein content of the /-globulinsolution was determined by the Folin-Ciocalteaumethod (12), and was adjusted with 0.85%c sodiumchloride and 0.5 N4 carbonate-bicarbonate buffer(pH 9.0) to give a sample containing 10 mg of proteinper ml and 10%' carbonate-bicarbonate buffer.The sample was kept at 2 C, and crystalline fluo-

rescein isothiocyanate (BBL) was added over a 1-hrperiod, with continuous stirring for 12 hr. Conjugateswere prepared with the minimal dye-protein rationecessary to produce optimal staining of homologousantigens; this ratio varied between 1:40 and 1: 100for different -y-globulin preparations. The conju-gated sample was then passed through a SephadexfG-25 bead form; Pharmacia (G.B.) Ltd., London,England] column (25 by 2.5 cm), equilibrated with

0.01 NI, pH 7.1 phosphate-butfered saLine (4); anid anlhourly flow rate of 4.5 ml/cm was maintained. Theconjugated protein passed rapidly tlhrough the columiin,and was diluted no more than twofold. This prepara-tion was run into Bijou bottles in 5-ml volumes andstored at -20 C.

The specificity of the conijugates was determinledby staining films of 24-hr-old cultures, grown on 1.2%;oagar media of CSE, HSE, and NB, with the use ofcontrols suggested by Nairn ( 13). Fluorescenice oforganisms was assessed as follows: 4+, maximalfluorescence, brilliant yellow-green; 3+, bright yellow-green fluorescence; 2+, less bright but clearly yellow-green fluorescence; I +, dull fluorescence; 0, no fiLlo-rescence. Whenever possible, all-glass apparatus wasused to avoid fluorescence contaminants (10).

Tech/nique for staininlg soil and observatioul J bac-teria ont soil particles. Freshly sampled soil was placcdin a glass cylinder (1.5 by 1.45 cm), fitted with anylon-weave base (20-, twill weave). The conjugated-y-globulin was pipetted in1to a trough into whiclh theglass cylinder was inserted (Fig. 1). For every 0.1 gof soil, 0.06 ml of conjugate was placed in the troughplus an extra 0.05 ml to occupy the dead space be-tween the two vessels.The soil container was lowered into the troLghIland

left there for 40 min at 18 C, and was gently agitatedat 10-min intervals. The container then was removed,allowed to drain, and placed in 0.01 M, pH 7.1 phos-phate-buffered saline (one rinse and three 2-minwashes) in troughs similar to that used for staining.Soil particles were carefully removed from the con-tainers and placed on slides in a very small volume ofwater (to facilitate their distribution on the slide).When observation was delayed, the slides were stored

0c

45cm

Ca7X-t7X~~~~~

1 8cm.-

SOILCONTAINER

SAND DUNESOIL

NYLONt......WEAVE BASE

STAINING- TROUGH

FLUORESCENT-- ANTIBODY

FIG. 1. Apparatius for stthiilinug soil wit/u f1loresenuut-auutihotlj preparaitolos.

N111it %.

i

--

VOL. 93, 1967 1889

HILL AND GRAY

in moist chamlcrs in the dark at 2 C. No fading orother detrimental chaniges occurred during 24 hr ofstorage.

Preparationis were observed withi a monocularLeitz Orthulux microscope equipped withi a lOXeyepiece and dry objectives (X 10, 25, 40. and 70)the objectives were fitted with ultraviolet-absorbingEuphos glass and were corrected for use without acover glass. Two HBO 200 mercury vapor lampswhichI couLld be ulsed simultaneously were attaclhed tothe microscope, onie providing transmitted illumllina-tion, and the other, incidcnt illuminlation. All tranis-mitted light was passed througlh a dry, dark-fieldcondenser (D, 0.80), anid the incidcnt light was passedthroughi a Leitz vertical illulminator. In this way,bacteria on both trallslucellt and opaquC soil particlescoukld be examined. Schott BG 12 (3 mini) anid OG I(2 mini glasses were used as primary anid barrierfilters, respectively. Photographs were taken wvith aLeitz Orthomiiat camera. with Kodak high-speedEktachromie. or Iltord HP 3. film.

RESULTS

Elfect of growtth inediumn o01 anitigenl produtctioni.When fluorescent antisera are to be used to iden-tify organisms growing in soil, it would seemlogical to obtain antisera against antigens derivedfrom organisms grown as nearly as possible undersoil conditions. To test this proposition. 0antigens were prepared from cells grown in CSE,HSE, and NB. Antisera were produced and weretested, by use of the tube agglutination test,against 0 antigens and whole-cell antigens fromorganisms grown in each of these media (Table 1).Higher- titers were obtained when the antiserumand the test antigen were derived f-om culturesgrown in the same medium. Thus, B. slibtilisNCTC 6284 antiserum agglutinated the homol-

ogous 0 antigen at a higher dilution when bothhad originated from cells grown in the same med-um, i.e., both in CSE, HSE, or NB. The samephlenomenon was observed with the antisera fromall four strains tested. These differences in anti-ser-a titers were not entirely quantitative, since theaLgglutinating capaclity of any antiserum couldonly be partly neutralized by absorption withantigens from cells grown in a different medium tothe cells whose antigens hald been used to pr-oducethe antiserum. Observations on cell growtlh in thethree media showed that cells grown in CSE weremor-e gram-variable and often more irregularlyshaped and smaller in size than cells grown inHSE or NB.

SpecificitY of tlhe (antiserac. Antisera to the 0antigens of the four organisms were prepared.The tube agglutination method was used to testtheir specificity Cagainst 0 antigens, whole-cellantigens, and spore antigens prepar-ed fi-om thefollowing organisnms (the number of strains testedfor each species is indicated): B. siubtilis, 37; B.cir cillalls, 39; B. circlulaniis-calv ei intermediates, 3:B. circilulans-br-ev,is-Jlterospovrlos intermediates, 6:B. alvei, 6; B. cer-eits, 7; B. cer-euts var. in?vcoidles.3; B. lichlenifori-i.si, 10; B. miieguteriemti, 7; B.pluinilhs, 8; B. coaguluins, 4; B. firiniis, 1; B. lentiis.3; B. bwliuis, 1; B. /polfllmv.va, 4; B. inucerans, 4:B. brevis, 9; B. /aterosporus, 3; B. splhaericits, 5;B. /)(iitothen1icIus, 5; B. pisteirii, 1. Antisera werealso tested against 0 antigens and whole-celPantigens fiom thr-ee soil isolates of each of thefollowing generca: Psedollnonas, Arthlrobucter.StireJ)tonyces, A1chlronlobacter, and Flaveobaic-ter'iill?l; two soil isolates of the following genera:StaphYlococcius., Micr-ococcuis, and Nocard(ia; and

TABLE 1. Effect of mnedium used for giowthl of calitige,is, o0lagglutinatiolt titers of talntiscrat,

\ 1)ist-u: nt to it ;mlitiigei

Baoilh1.ss,/ntili.c NCT6284

B .su/hilit soil isolatc

B. circ(ultals NCTC7is

B,. c irc('l(an.s soilI so la

-Aggglutina tion titer of antiserum anll tie Itot1iolloo,lus alititSt)erivedl fron -_____-

it cells* grottit XXWhole cells \\thole cells \\hlole cells (a)ilitiggrit aniti,gri (-rottn in growtn I 1grotVri II grotnt in gron-It nli(CSiE HSi Nil t sF HSE

C'

578

Itc

CSEHSENBCSEHSENBCSEHSENBCSEHSENB

1:6401:8()1:3201:6401:1601:1601:6401:81)1:1601:6401:161)1:80

1:320 1:160 1:5, 1201:640 1:320 1:1,281)1:640 1:640 1:6401:160 1:80 1:5,1201:640 1:320 1:641)1:641) 1:640 1:6401:160 1:80 1:5,1201:640 1:320 1:321)1:640 1:640 1:640)1:161) 1:80 1:10,240/:640 1:320 1:641)1:320) 1:640 1:640

1:1,281)1:5,/201:2, 5601:1, 28)1:5,1201:2, 5601:1,2801:2,5601:5, 1201:1,2801:5, 1201:_, 56t)

) aliltit'I(),row ill

.\'1R

1:1,28)1 :5, 12(11:5, 1201:641)1:2, 560)1: 10,24(1:3201:1 ,28011: 10.24(t1:641)I : 1 2S

1: 5, 120(

a Titers obtainied when antiserum and antitgen were derived from cLIltLires grown in the sam,,e mui:Jiare in italics.

1890 .1 [(IlR0

,9iM\NIUNOFLUORESCENCE ANI) SOIL BACTERIA

one strain of each of the following organisms ob-tained fromii cultur-e collections: Esclheiichia coli,Prwoieus vulgar-is, M vcobacier-itnn p/lei, Azotobac-ter CIhr'OOCOCCilni, Sarcinia Iiitea, Rhijiobitun sp.,Klebsiella (Aer-obacter aegeniesv and Agigobac-ICeriuni7 tuni1ejaiciens.Table 2 records the iresults obtained for the B.

SilbilisaintiseraL. Both antisera were completelyspecific for the 0 and whole-cell aLntigens of thisspecies. There were no cross-reactions witlh aLnyspore antigens prepared fromii B. sutbtilis, or withwhole cells, 0 aLntigens. or spore antigens of anyother Bacilllus species or species of other- generatested. All the B. subli/is strains tested reactedpositively with both Cntisera. Table 3 recor-ds theresults obtained for the B. circul(iIns antisera. Thereactions of the aLntiser-aL to the two strains weretotally different. Althouglh B. circulans NCTC7578 antiserum agglutinated whole-cell and 0aLntigens from most of the 10 B. circiulais strainsCand the 3 B. circalans-alveli intermediate strainisoriginating from culture collections, it aggluti-nated none of the antigens fr-om the 19 B. circuilansstrains or the 6 B. cirlaCntl(l/s-br-ev,is-laterosporutsintermediate strains isolated from the forest soil.Conversely, the antiserum to the B. circiulans soilisolate agglutinated the whole-cell and 0 antigensfr-omii all the 25 soil isolates of B. circulans acind B.cir-c-tilcias-br-evis-lalter-o.svoruiis intermediates, butwould not react with any of the antigens from theculture collection strains. In addition, it cross-realcted with most of the whole-cell and 0 antigens

of B. abei culture collection aLnd soil strlains. Thesecross-reactions were removed by absorption of theantiserum with B. abei 0 antigen, lecaving aln anti-serum thlat was specific for all soil isolates of B.cir-ciilans and B. iciillis-l'S-l(T( )5JN4iiSintermediates examined.

Specificity of the conjiigcitetl y -globulin prel(prc-tioiis. Determination of the specificity of the con-jugated -y-globulins, by use of the sciaile test cul-tures, confirmed the antiserum agglutinationresults. Nevertheless, ainumber of confirma-Itoryinhibition tests were carried out as suggested byNairn ('13). All bacteria thrat fluoresced whenstained with conjugated, ii-immune y-globulin werealso tested with conjugated. nonimmlllaulne y-globu-lin. No reactions were observed. Inrhibitionl testswere performed, the first involving the treatmentof the antigen with unlabeled specific -y-globulinbefore stLining with the conjugaited, immltlune,y-globulin, and the seconid, the simultarneoustreatment of the antigen with both these pirepaLira-tions. Either a reduction or complete inh[ilbition ofstaining occurred in all suchi tests. Antigens werealso stained with an immilune conjugaLte that hadbeen absorbed with its homiiologous Cantigen, re-sulting in complete inhlibnition of staininig for allantigens tested. All the necessary contr-ols wereincluded in these tests. The intensity of the fluo-r-escence of organisms was assessed. Bacterialstained with their homologous fluorescent anti-body showed 4+ or 3+ fluorescence.

Autofluorescence of bacterial cells varied from

TABLE 2. Alggluitiln(aionl rectionis of hle (antisealfo thlie somatic antigeni (0) of Baicillai.s so/hills-

Antigen

B. siibtili.s 0B. salbtilis WC"

IB. slhbtilis sporeB. siabtilis 0B. slibtilis WCB. slbtuilis sporeOther Bacillus spp. 0Other Bacillus spp. WCOther Bicillus spp.

spore

Other genera 0Other genera WC

No. ofstrainstested

212121161616

1191I 19C

2929

No. of strains agglutinate(lwith antiserum to liter- ianige oI reactivc st railns

0) antigen fiom

B. sublilisN (T( 62S4

212101616000

0

00

B. sutblilisa B. sb/(bilissoil isolate NCT1C 6284

21 1:80-1:5,12021 1:40-1:1,280016 1:40 1:2,56016 1:20-1:640O0

0-0-0

B. slb/ili.ssoil isolate

1:40 1:2,5601: It) -1:640

1:80-1: 10,2401:40 1:2,560

00

Origi n

Culture col-lect ions5

Forest soil

Culture col-lectionsand forestsoil

,/ Strains were obtained from the National Collection of Type Cultures, the National Collection ofIndustrial Bacteria (Teddington, England), the National Collection of Plant Pathogenic Bacteria (Har-penden, England), and J. Wolf (Department of Agriculture, Univ. of Leeds. Leeds, England).

'? Whole cell., Two strains of B. ceicei5s showed strong autoagglutination.

1891Voi.. ')3 19'67

HILL AND GRAY

TABlE 3. ,oIgghitilatioI r-etactiotIs of tile anitisera for thle sonatic tattigenI (0) of Bacilhls circulans

t ri£i, Antigen

Culture col- B. circulans 0lections B. circulans WCU

B. cir-cilanls spor(Forest soil B. circulans 0

B. circulans WCB. circulans spor(

Culture col- B. circulans-alveilection s B. cireculain.s-al'vei

B. circulans-alveiForest soil B. circulans-brevii

sportis 0B. circulans-brevii

sporus WCB. circulans-brevi

sSpor-lus sporeCulture col- B. alvei 0

lectionS B. alvei WCB. alvei spore

Forest soil B. alvei 0B. alvei WCB. alvei spore

Culture col- Other Baicillus splections Other Bacillus spjand forest Other Bacillus spsoil

.e

0OiWCispores,%-la tE?1() -

i.s l te(r) -

p-. 0p. WCp. spore

Other geniera 0Other genera WC'

No. ofstrainstested

1010101919193336

6

6

4442

112b

11127)112's

2929

-No. of strains agglutiniate(twith antiserum too antigen frorn

B. cic,ulan.s B. circulantsNCTC 5> soil isolate

9 07 0

0 00 190 190 03 03 0

0 6

(1 3

0 3

0 0

0) 0

I'iter- ranige of reactive strains

B. circulans B. circidalnsNCTC 7578 soil isolate

1:10-1:10,240 -1:101 801 1280

1:80-1:10,240I1: 20 -1: 1 280

1:160-1:1,2 801:40---1:640 -

--- 1:80-1:2,560

1:40-1:640

1:160-1:6401:40-1:80

-- 1:160-1:32()1:40-1:80

(I00

a Whole cell.I Two strains ol B. cereus showed strong autoagglutination.

0 to 2 +, but was clearly distinguishable fr-om thefluorescence of stained cells. A number of fungiand actinomycetes, freshly isolated from theforest soil, were also ol:served for autofluo-rescence to examine the possibility of their inter-ference with the correct identification of thebacilli. Autofluorescence of actinomnycete hyphlaeand spores, as well as fungal spores, never ex-ceeded 2+, and only a very small pr-opor-tion offungal hyphae exceeded 2+, suclh hyplhae beingclearly distinguishable from stained bacterial cellsor spores. Sometimes autofluor-escence color-sother than green were seen, particularly anmongstthe actinomycetes. None of these fungal or acti-nomycete strains showed increased fluorescencewhen stained with the fluorescent antibodies.The possibility that older Bacillus cells might

fluoresce less brightly when stained with fluores-cent antibodies was also tested. Stained cells from21-day-old cultures showed the same, or onlyslightly less, specific fluorescence compared with

cells fr0om 24-hr-old cultures. Maximal fluor-es-cence of pure cultur-e preparations was obtainedby use of a 1:4 dilution of the B. subtilis conju-gates aLnd a 1:2 dilution of the B. circulans conju-gates. The greatest dilution of conjugate withmaximal staining power was obtained when thestained cells were grown in the same mlediumii asthat used to produce the fluorescent antibodies.The specificity of the spore antisera was not

determined. However, the fluorescent antibhodiesfor spores of the B. subtilis forest soil isolatestained spores of six strains of B. subtilis with 4+fluorescence. They did not stain the vegetativecells of any of these strains, or thie vegetative cellsor spores of six strains of B. circulans.

Detecdioni of Baic illus spp. on soil particles. Or-ganic and mineral par-ticles from the Al horizonof the Freshlield soil were stained with fluores-cent antibodies for the 0 antigens of B. slibtilisforest soil isolate, B. subtilis NCTC 6284, B.circulans forest soil isolate, and B. circulans

1 892 J. BACTFRIOL.

IMMUNOFLUORESCENCE AND SOIL BACTERIA

NCTC 7578, and for the spore antigen of B.sUbtilis forest soil isolate. Observation of soilstained with fluorescent antibody for the 0 anti-gen of B. circulucnis NCTC 7578 showed that nocells were stained, confirming the results of aLg-glutination tests with the homologous antiseruim.All of the other three preparations, for 0 anti-gens, produced stained cells, and, of the two B.sbbtilis strains used, the fluorescent antibody forthe soil isolate produced slightly brighter staining.Thus, further staining of soil was with the 0antigen fluorescent antibodies of the B. cir-clal(lnsand B. siubtilis forest soil isolates. The antigens ofthese forest soil isolates were grown in CSE,because the brightest staining of the bacteria onsoil particles was from fluorescent antibodiesprepared from these antigens. A 1:2 dilution ofthe B. sbibtilis preparation and a 1 :1 dilution ofthe B. circulans preparation gave optimal fluores-ence.

Figures 2 and 3 show a quartz mineral particleand a fragment of decomposing root, respectively,stained with fluorescent antibody for B. subtilis 0antigen. Very few B. subtilis cells were observedon mineral particles but, when present, weregenerally regular rods (1.0 to 2.0 ,u by 0.3 to 0.5 ,)and were in colonies of 1 to 14 cells. Colonies onmineral particles were composed of an average oftwo to three cells. These cells were very similar tothose of B. subtilis growing in culture, but weresmaller (culture cells: 2.0 to 4.0 , by 0.6 to 0.8 /).Organic material, on which the majority of B.subtiis cells were observed, was most fiequentlycolonized by large, and often curved, cells (4 to11 ,u by 0.8 to 1.1 ,) in colonies of 1 to 40 cells,with an average of 4 to 5 cells per colony. Thus,the cells on organic particles were larger than cellsof B. suhtilis grown on culture media in the

....:.'.;.!:-. :-~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~...

FIc ' 1 lIO)iZoiI foirst SO;l Ni/ Iia/lI ilt/i Bacil/usSUbtilis (IJorest soil iso/ate) 0 (ilntigenI f/O/rescenttnlltibOd/y, s/iowinig tlhe presenlCe ol Ci COOiIy ojf B. sub-

tilis cell/s on a smCall cryptocrv'ysta/line quartZ pCarticlecoated1 w>Eith organic mlatter.

FiG. 3. A, hior0zoui olrest s(oil stai/ietl lit/i BacillusSUbtilis (JOrest soil isolate) 0 antigen Jlulorescent antlti-body, slowing at firagnent 0o' lde¢CoP/)0oXSilgpilg , I'ootcolonized hb cells of B. subtilis.

laboratory. Colony sizes, however, mLay hlcave lbeenslightly underestimated, since a smnall number ofcells were found, on slide surfaces, detached fromsoil par ticles, and others, again in very smallnumbers, were found in the used stain and wash-ing salines. These cells may have become detachedfrom particles or may halve been originally presentin the soil solution.

Colonies of B. circulans detected in the sameway were most frequently composed of 2 to 10cells per colony, on both mineral and organicparticles, thouglh colonies of up to 25 cells wereobserved. The cells were either short rods orcoccoid (see Fig. 4), were most fi-equently re-corded on organic matter, and wer-e quite unlikethe long cells that are characteristic of this specieswhen grown on artificial culture media.The intensity of staining of cells on soil paLrticles

was usually slightly lowei than that obiserved on

FIG. 4. A11 horizon forest soil slaiOlC'd wit/h Bacilluiscirculcals (fo)est Csoil isolate) 0 alligenelfluo)rescenltantibody, sliowinig ai cololiy oj'iiitenisely staitilinig coccoidcells onz the sarfiice ojaC loose acggregate of l/econliposiolgorganic niiatter, boundtl togetlher bY nonfluiorescent lioligalhypliae.

VOL. 9)3, 19t67 1 893

HILL AND GRAY

corresponding pure culture slide preparations.However, B. cir-cuilctis cells on organic particleswere frequently noted as b7eing intensely stainedand halving a diffuse outline (Fig. 4). Biegeleisen(1), obiser-ving tissue imprints of dried m.eatstained witlh B. antlhracis fluorescent antibody,reported cells fluorescing in a very similar mannerand attributed this to the presence of a largequantity of capsular material.

Figure 5 shows part of the surface of a quartzminercll particle stained witlh undiluted fluores-cent anltibody for B. suhbtilis spore antigen. S,ev-eral spores are visible. When soil was stained witha mixture of fluorescent antibodies for both B.suibtilis 0 and spore antigens, free spores appearedto be present within colonies of cells. Both anti-sera, however, were conjugated to fluoresceinisothiocyanate, making a distinction of sporesand cells more difficult.

Autofluorescence and nonspecific staining ofthe soil par-ticles seemed of little consequence inthe soil examined. A few particles (mostly or-ganic autofluoresced bright apple-green or blrightyellow, wher-eas some others took up the conju-gated dye to fluoresce a dull green. However, thegreat major-ity of the particles were nonfluorescentor else autofluor-esced a dull green. Par-ticlesfluorescing white, yellow, red, or blue wererelatively rare, and often these colors were onlypresent as patches or streaks on large particles.Attempts were made to eliminate soil particlefluorescence by use of the following range of po-tential fluorescence quenchers on stained soils (atconcentrations of 1, 0.1, 0.01, aind 0.001'-,):methylene blue, acid fuchsin, congo red, Lugol'siodine, basic fuchsin, phenol, Ziehl's carbolfuclhsin, potassium permanganate, sodium hy-droxide, and sodium pyrophosphate (also at 2(--).

Fic,. 5. "II lhori:oni finrest soil stai,,edl with Bacillussubtilis sp/)or (llntigell fluiorescent antibodv, showintg tilepresence o spores oni tdhe sr/acie oj'c mineral qluarZ't:particle, wit/i o.xidi7ed i-o/I pIresent as ioucliasiotus anld

ti50 ( compon)enlt o/all external coatilng.

In no case did a quenclher- reduce soil palrticlefluorescence, or improve the contrast betweencells and soil paLticles.

Dis(-ussIoN

This investigation hals shown thalt several precautions may le necessary to Laid the successfulapplicaLtion of the fluorescent-antibody techlicqueto the identification of bacteria in soils. It is ev,i-dent from the data obtained on the chanliges inantigen production encountered by growing or-ganisms on difrerent media thalt the medium usedito produce antigens for injection into raLbbitsshould be caL-efully specified. A cold extralctedlsoil medium (CSE) enabled antisera with higlher-titers (and possibly with a more relevant contentof antibodies) to be pr-oduced, when tested againstantigens from cultures grown in CSE, and wheniused als al fluorescent-aLntibody preparation tostain cells on soil par-ticles. High titers halve notalways been regalrded as advantageous when usingfluorescent antisera, since it has been suggestedthat nonspecific reeactions can occur if highlNpotent antiseraL are used. However, witlh the ad-vent of high-purity crystalline dyes, it is possiblethat high-titer antisera conjugated with low dye-protein ratios maLy reduce nonspecific staLiningreactions (2, 5, 7). Also, when antiserum isscarce, high-titer antisera are useful becausethey, can be diluted to give larger quantities ofreactive serum. Although the changes in antigenproduction appeared to be to some extent quanti-tative, qualitative changes did occur, and thesemust always be catrefully considered when prepar-ing antigens for studies on soil organisms. Also,the specificity of antisera aLgaLinst capsulate andnoncapsulate strains might Lbe difTerent. Organ-isms failing to produce a capsule under soil con-ditions might need to be stained withl antiserafrom noncapsular str-ains of organisms, and viceversa, although this might not always be possible.The use of the technique in ecological investi-

gations is governed by the specificity of the anti-sera. Species-specific antisera, e.g., antiserumagainst B. sulhtilis 0 antigen, can be used to studythe distribution of these species in diffeerent soilenvironments. StrLin-specific antisera, e.g., B.cir-culuins 0 aLntigen aLntiserum, cannot be used inthis way but may be vaLluable in studying theestablishment of competing strains in mixed popu-lations, especially if the strains reacting witlh theantiserum haLve key physiological properties.AntiseraL agaLinst difTerent str-ains could be conju-gated witlh different-colored dyes, e.g., fluoresceinisothiocyanate cand r-hodamine isoth iocyainaLte.Finally, antisera that are specific for only onecomponent of the cell or plhaLse of growth can beof considerable importance: e.g., antiseraL aLgainst

1 894 .1J. BAC I i- R{ Rol

IV.6MMUNOF'LUORESCENCE ANI) SOIL BACTERIA

spores of B. suthtilis might Lbe used to study theonset of dormancy in bacterial populations. Un-fortunately, fluorescent antisera will not distin-guish between living and dead cells or, if the cellsaire living, will give no indication of their physio-logical activity. To be certain that stained vegeta-tive cells are active, information on the survivaltime of antigens in dead cells in soil is needed.The method of observing the stained bacteria

is also important. If one wishes to determine thegrowtlh pattern of an organism in soil, especiallymycelial forms which grow from particle to parti-cle, the contact slide method as used by SchmidtLand Bankole (17) is useful. However, one cannotbne sure whether- the forms observed on contactslides are typical of those on soil particle surfaces.The factors limiting growth on the surface of alhumiius paiticle may be quite ditferent from thoseaffecting growth in a water film on a glass slide.For this reason, we have examined the growth ofcells on individual particles taken directly fromthe soil. In the past, examination of soil particleshas been partly impaired by the limitations of theilluminating system. With incident ultravioletlight, however, it is possible to see organisms notonly on mineral particles, but also on opaquepieces of organic matter. This is important, sinceit hals bteen shown that approximcately 60%'C of allbacteria in this soil occur on organic particles,even thouglh these particles pr-ovide only approxi-mately 15', of the observable surface area of thesoil particles (6a). In other soils, e.g., clays, withsmaller- particles, this observational problem maybe less serious; however, difliculty in staining thesoil without washing away the clay fraction maybe encountered. Eren and Pracmer (3) found thatclay particles decreased the intensity of cell fluo-rescence and interfered physically with their ob-servations of Artlhrbotrvs conioicles.

Initial observations of Bacicllls spp. in the soilhave shown that, although B. subtilis retains theapproximate size and shape of cells found in cul-ture, cells of B. circulans beconme more variable,and possibly encapsulate on organic material,suggesting that the ecological roles of the twospecies differ. This conclusion is supported byGoodfellow (Ph.D. Thesis, Liverpool University,Liverpool, England), who studied soil isolates ofthese two species and showed that B. stibtilis wasmluclh mo-e biochemically active than B. c'ir'culcinis,als determined by laboratory tests. These testsshowed that some isolates resembled B. circulans,whereas others were intermediate between it andB. brei.s and B. laICrosporuIs. The B. circulansstr-ains found in this soil are also serologicallyrelated to B. alvlei, further illustrating the difli-culties experienced by Knight and Proom (9),Simitl, Gor-don, and Clark (20). Iyer and Bhalt

(8), and Goodfellow (PhD. Thesis) in delineatingthe soil isolates belonging to morphological group2 of the genus Baicillits (20).We have also shown that cells of B. subtilis are

capcable of forming spores under soil conditions,although the possibility cannot be excluded thatthe spores were formed during the earliei- stagesof preparation of the slides. Althouglh pasteui-iza-tion exper-iments have provided evidence for thepresence of spores, they have not proved it, sinceit is possible that the presence of extraneous or-ganic matter may aftect the survival of the vegeta-tive cells under these conditions. Now thcat theposition of these spores can be determined in soil,more rational experiments on the factors affectingtheir formation and disappearance can be formu-lated. Further data on the occurrence of sporesand vegetative cells of these bacteriaL under difTer-ent environmental conditions will Lbe publishedelsewhere.

ACKNOWLEDGMENTS

This investigation was supported by grants fromthe AgricLiltural Researclh Council of Great Britain.We thank E. L. Schmidt for his criticism of the

manuscript, J. Wolf for providing many of our cul-ture collection strains of Bacilluts spp., and the De-partment of Veterinary Pathology, Liverpool Uni-versity, for providing animtal house facilities.

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1896 J. BACT[l-RIOL.