APPLIED MICROBIOLOGY, Sept., 1965Copyright @ 1965 American Society for Microbiology

Vol. 13, No. 5Printed in U.S.A.

Specificity of Immunofluorescent Staining for Studyof Aspergillus lavus in Soil

E. L. SCHMIDT AND R. 0. BANKOLEDepartment of Microbiology, University of Minnesota, Minneapolis, Minnesota

Received for publication 8 March 1965

ABSTRACTSCHMIDT, E. L. (University of Minnesota, Minneapolis), AND R. 0. BANKOLE. Speci-

ficity of immunofluorescent staining for study of Aspergillis flavus in soil. Appl. Micro-biol. 13:673-679. 1965.-Fluorescein-labeled antiserum prepared with Aspergillus flavusstrain CS was tested for specificity by staining fungi grown in soil in the vicinity ofburied slides. All 14 strains of A . flavus fluoresced as intensely or nearly as intensely asthe antigen control. Among 21 isolates of species of Aspergillus other than A. flavus, 17reacted with moderate to low fluorescence at intensities readily distinguishable fromthat of A. flavus. The fluorescence of the remaining four cultures, and particularly A.sydowi, was indistinguishable from that of A. flavus. Fungi other than aspergilli weregenerally nonreactive. Interfering cross-reactions were encountered for one strain ofSpicaria and one strain of Stemphylium; three isolates could not be evaluated becauseof interfering autofluorescence. An additional 22 isolates were either wholly negativeor had a low order of fluorescence. Agglutination tests between each of the fungi andA. flavus CS serum revealed close agreement between agglutination titer and fluores-cent-staining reaction. Unknown fungi freshly isolated from soil were checked for re-action to the A. flavus labeled antiserum; only one isolate gave a pronounced stainingreaction, and that one proved to be a strain of A. flavus. In a simplified ecologicalmodel, the fluorescent-antibody technique was used to follow the development of A.flavus in mixed culture in soil with five other soil fungi.

Direct microscopic study of microorganismsin soil would be especially informative if it werepossible to recognize the microorganisms ob-served. The fluorescent-antibody technique hasbeen advanced as a possible means for the detec-tion of a particular microorganism in soil preparedfor microscopic examination (Schmidt and Bank-ole, 1962). Results of initial attempts to applyimmunofluorescent-staining techniques to thedetection of Aspergillus flavus in pure culture onsoil slides were encouraging (Schmidt and Bank-ole, 1962, 1963).

Since the effectiveness of the technique de-pends on the selective adsorption of the fluores-cein-labeled antiserum to microorganisms closelyrelated to the one used as the antigen, data onthe specificity of the antiserum used in stainingare important. Results of studies on the specificityof the fluorescein-labeled antibody preparedfrom A. flavu strain CS, and on its use in thedetection of A. flavu in mixed culture in soil,are presented in this report.

MATERIALS AND METHODSMost of the data were derived from study of

fungi obtained from culture collections and iden-

tified at least to genus. The fungi were grown inpure culture in sterile soil moistened with 1 or 2ml of 1% glucose, and with sterile microscopeslides inserted in the soil. After recovery from thesoil and just prior to examination, each slide wastreated with the labeled antiserum. The antiserumhad been prepared in rabbits by injecting A. flavusCS, and was conjugated with fluorescein isothio-cyanate. Slides were examined in detail with flu-orescence microscopy, and cultures were rated forfluorescence as compared with slides bearing A.flavus CS. Techniques used in the preparation ofthe antigen, conjugation of the antiserum, growthof the test cultures, and staining and examinationof contact slides were as reported previously(Schmidt and Bankole, 1962). A typical schedulefor the immunization of rabbits with mycelium ofA. flavus has been presented also (Schmidt andBankole, 1963). Serological reactions between thetest cultures and A. flavus CS immune serum were-tested by standard tube agglutination assay. Eachfungus was grown in glucose (0.2%)-glutamate(0.4%)-mineral salts broth, and was prepared asantigen for agglutination assay as a suspension ofwashed, minced mycelium in saline, adjusted to anoptical density approximately that of A. flavus CSantigen. A constant volume of 1 ml of antigen wasadded to dilutions of serum.

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flavus CS antibody with respect to isolates ob-tained from culture collections, the staining reac-tion of fungi freshly isolated from soil was inves-tigated.A fertile field soil was plated in rose bengalstreptomycin agar and Czapek agar. Colonies offungi that appeared to be different species wereisolated in pure culture, grown individually in thesterile soil system, and examined for fluorescent-staining reaction as before.The usefulness of the fluorescent-antibody tech-

nique for the detection of A. flavus in mixed cul-ture in soil was tested in studies with sterile soil-slide systems inoculated with a controlled mixtureof several of the fungi freshly isolated from thefield soil, with and without A. flavus CS. Slideswere removed carefully at 2, 5, 7, and 14 days, sothat soil particles adhering to the surface were un-

disturbed. The loosely adhering soil on a partic-ular section of the slide was then scraped asepti-cally into sterile diluent and plated on rose bengalagar. The same slide surface which provided thesoil for plating was prepared for immuniofluores-cent staining.With the above approach, the distribution or

absence of A. flavus in a mixture of fungi was de-tected by specific staining soon after the slide was

harvested. Results of immunofluorescent observa-tions were then checked against results obtainedby plating procedures after suitable incubation.The soil isolates used to provide the mixture offungi were selected because they were nonreactiveto the stain, grew at about the same rate in soil inassociation with A . flavus and each other, and were

distinctive in colonial appearance on the rose

bengal plates. The five isolates used in mixed-cul-ture studies were tentatively identified as A. ustus(two strains), A. fumigatus, Mortierella sp., andTrichoderma lignorum.

RESULTSTests with A. flavus, other aspergilli, and various

other genera. All 14 isolates of A. flavus used inthe study grew well in pure culture in sterilesoil, and ample mycelium was evident on eachof the fresh, unstained slides as seen with a lightmicroscope. None of the unstained slides gave

evidence of autofluorescence when viewed witha fluorescence microscope. The- immunofluores-cent reactions that developed for each A. flavusisolate subsequent to staining with labeled A.fiavus CS antiserum are summarized in Table 1.The fluorescence rating of 4+, based on the in-tense yellow-green fluorescence exhibited by con-trol slides of the antigen, was equalled or ap-proached by each strain of A. flavus tested.Immunofluorescent-stained slides of the variousA. flavus strains also resembled those preparedfrom the antigen, in that all hyphal filaments didnot fluoresce with uniform intensity; conidio-phores, vesicles, and some sections of hyphaecommonly fluoresced intensely, whereas otherhyphae, perhaps older, frequently appeared as

faintly fluorescent. The general equivalence inthe staining reactions of culture-collection strainsof A. flavus and the particular strain used for

TABLE 1. Agglutination titers and immunofluorescent-staining reactions of various isolatesof Aspergillus flavus when reacted with antiserum preparations of A. flavus strain CS

A. flavus strain Agglutination Fluorescence Remarkstiter rating*

Minn. CS ................. 1,280 ++++ Control; most mycelium fluorescent, especiallyhyphal tips, conidiophores, and vesicles

Wheat isolate 1 ........... 640 +++ Intense staining restricted to vesicles and hyphalfragments

Wheat isolate 2 ........... 640 +++ Most mycelium and spores uniformly fluorescentWheat isolate 3 ........... 640 +++ Most mycelium highly fluorescent; spores less soWheat isolate 4 ........... 640 +++ No spores seen; vesicles and mycelial fragments

with intense fluorescenceWheat isolate 5 ........... 640 +++ Mycelial fluorescence stronger than that of

sporesMinn. F 921 ............... 1,280 ++++ Extensive, uniformly intense fluorescenceMinn. F 9807 .............. 1,280 ++++ As in F 921Minn. F 708 ............... 640 +++ As in wheat isolate 3Minn. F 301 ............... 640 +++ Scanty mycelium but strongly fluorescent,

especially vesiclesQM 380 ................... 1,280 ++++ Many fragments intensely fluorescent, but much

weakly fluorescent mycelium alsoQM 630 ................... 1,280 +++ As in QM 380, but lower intensityPeoria 453 ................ 1,280 ++++ As in QM 380Minn. F 16-1 .............. 1,280 +++ Uniformly bright but not intense; few sporesUpjohn 9296 .......I 1,280 +++ As in QM 380, but lower intensity

* Overall fluorescence rating between negative (-) and most intense (++++).

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TABLE 2. Agglutination titers and immunofluorescent-staining reactions of aspergilli otherthan Aspergillus flavus when reacted with antiserum preparations of A. flavus CS

Culture Agglutination Fluorescence Remarkstiter* ratingt

A. sydowi ................. 640 +++ Indistinguishable from many A. flavus strains;vesicles and stalks intense, hyphal filamentsless intense

A. candidus ............... 320 +++ Hyphal filaments weakly fluorescent; sporesintense

A. nidulans ............... 320 +++ Fluorescence restricted to relatively few hyphaeA. caespitosus ............. +++ Fluorescence restricted to vesicles, otherwise

weak

A. ochraceous 17 ........... 40 +1A. ochraceous 18 ........... 40 + Very weak fluorescence; readily distinguishableA. ochraceous 23 ............... .40+ from A. flavus

A. fumigatus ...............

A. niger ................... 40 +1A. niger (Mulder) ......... 40 +| Fluorescence blue-white and of low intensity;A. proliferans ............. ? + s

. .A.amtdm.4 +, spores when visible appeared blueA . amstelodfami ................ 40 + I .

A. ruber .................. 40 +

A. terreus ................. 40 ++A. glaucus. ++A. nidulans.- ++ Localized areas scantily distributed on hyphalA. tamarii ................ 40 ++ segments gave fair fluorescence, but mostA. echinulatus ............. 40 ++ hyphae were barely fluorescent; spores, vesi-A. restrictus ............... 40 ++ cles, and cross walls did not fluoresce; readilyAspergillus sp. (unidenti- distinguished from A. flavus

fied) .80 ++A. versicolor ....... 80 ++j

* Antigen not dispersible in saline (-); end point uncertain (?).t Overall fluorescence rating between negative (-) and most intense (++++).

serum preparation holds also in the agglutinationtiter data of Table 1. All strains either had agglu-tination titers equal to the antigen at 1,280, orwere at the next lower dilution level at 640. Theserological relationship within the group of A.flavus isolates was close, and agglutination dataand fluorescent microscopy data are in goodagreement.

Cultures within the genus Aspergillus butrepresentative of species other than A. flavuswere examined. As shown in Table 2, the A.ftavus fluorescent antibody did not react withmost aspergilli not in the flavus-oryzae group.The antibody stain, however, did react stronglywith A. sydowi, so that this culture of A. sydouicould not have been distinguished from A. flavuson the basis of immunofluorescent-staining reac-tion. Three other aspergilli evidenced a lesserdegree of cross-reaction than A. sydowi, withfluorescence of variable intensity and generallylimited to certain structures. The three, A.candidus, A. nidulans, and A. caespitosus, were

distinguishable from A. flavus in most prepara-tions by means of direct comparisons. Still, thebetter preparations and better fields of the threewere judged about equivalent in staining inten-sity to the poorer reactions of some A. flavusstrains, and could not be distinguished from A.flavus under these conditions. Agglutinationtiters again correlated closely with microscopicevaluations. A. sydowi strongly resembled A.flavus both in agglutination titer and fluorescent-staining properties. At least two of the threeaspergilli which evidenced a lesser, but still sig-nificant, degree of cross-reaction in staining alsohad a moderately high agglutination titer withA. flavus antiserum. A. caespitosus could not betitrated in tube agglutination, as the blendedmycelium was not dispersible in saline. The re-maining 17 strains of aspergilli tested gave noevidence of interfering cross-reaction on stainedpreparations or in tube agglutination reactions.Some notable degree of staining reaction was

found in 4 of 27 preparations bearing fungi of

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genera other than Aspergillus (Table 3). Thecolor and intensity most nearly like that of A.flavus was observed for the Stemphylium sp.isolate, with a fairly uniform distribution ofmoderate fluorescence along the hyphal elements.The Spicaria preparation had a slightly lowerdegree of specific fluorescence, but some hyphalsegments appeared similar to stained fields of A.flavus. Helminthosporium sp. and Zygorrhyncusmoelleri were rated at 2+ because of local areasof bright, yellow-green fluorescence on the hy-phae. Adjacent areas on the hyphae, if at allfluorescent, appeared red and blue, colors notseen in A. flavus preparations.Three of the fungi listed in Table 3 could not

be evaluated, because unstained preparationsgave interfering autofluorescence. The twostrains of Fusarium and the Pullularia pullulanswere the only cultures observed in this study toautofluoresce markedly with the filter systemsused.

Tests with freshly isolated soil fungi. Since thespecificity of the A. flavus CS antibody prepara-tion was generally satisfactory in tests with avariety of common fungi maintained in culturecollections, it was of interest to check the stain-ing properties of unknown fungi freshly isolatedfrom soil. A silt loam soil which was platedshortly after collection yielded 21 fungus culturesreadily distinguished from one another by gross

TABLE 3. Agglutination titers and immunofluorescent-staining reactions of genera otherthan Aspergillus when reacted with antiserum preparations of A. flavus CS

Culture Agglutination Fluorescence Remarkstiter* ratingt

Spicaria sp................

Stemphylium sp.Helminthosporium sp..

Zygorrhynchus molleri.Penicillium oxalicum......P. roqueforti..............P. purpurogenum..........P. camemberti.............P. nigricans...............P. expansum..............P. brunneo-rubrum........Paecilomyces sp............

Mucor sp..................M. circinelloides...........Hormodendrum sp.........H. pedrosoi...............H. resinae.................Neurospora crassa.........Microsporum gypseum.....Trichoderma sp............Alternaria tenuis..........Syncephalastrum sp........Scopulariopsis sp..........Gliocladium sp.............

Fusarium sp...............Fusarium S-14............Pullularia pullulans.......

16080

801020T201010T20

101010101020T2010000

160

+++

++I

+++I+I

++l

Filaments generally fluoresced uniformly withmoderate intensity; not readily distinguish-able from A. flavus in color or intensity, butdistinguishable in morphology

As in SpicariaLocal small areas on hyphae comparable in colorand intensity to A. flavus; other portions ofhyphae showed blue-green, yellow, and redfluorescence; all spores were red.

Similar to Helminthosporium sp.; occasionalweak fluorescein-type fluorescence; generallyfilaments fluoresced with a blue-white color,and spores, when visible, were blue and red

No yellow-green fluorescence

Unstained preparations gave interferinig yellow-green autofluorescence

* Antigen not dispersible in saline (-); end point uncertain (?); turbidity interfered with agglutina-tion reading (T).

t Overall fluorescence rating between negative (-) and most intense (++++). Autofluorescent (A).

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colonial appearance. When each of the 21 freshisolates was cultured individually in the sterilesoil-buried slide system and then examined afterapplication of the immunofluorescent stain, itwas found that only one isolate fluoresced withan intensity that approximated that of the control.

It is of particular interest that the one isolatewhich was reactive to the stain proved, on fur-ther examination, to be a strain of A. flavus.The likelihood of bias was negligible, since thisisolate did not look like the control in superficial

colonial appearance. Thus, a strain of A. flavuswhich occurred as a component of a mixed soilpopulation, but was not readily recognized onthe dilution plate, was detected by its character-istic fluorescent-staining reaction. This observa-tion provides further evidence that immuno-fluorescent staining is sufficiently specific for useas a tool for study of the ecology of A. Jlavus insoil, and lends encouragement to the view thatthe method may prove amenable to other prob-lems in soil ecology.

FIG. 1. Field from contact slide after burial for 5 days in sterile soil inoculated with Aspergillus flavus andfive other soil fungi. Hyphal diameter of the larger fungus (probably Mortierella sp.) is approximately 20 u.(a) Bright field with ordinary light after staining with A. flavus CSfluorescent antibody. (b) Same field viewedby dark-field fluorescence microscopy. Finer bright filaments are A. flavus which fluoresced bright yellow-green; the larger fungus was seen in the background with weak blue-white fluorescence.

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A. flavus detection in a mixed soil culture. A.flavus was traced successfully in a simplifiedecological system comprised of the antigen fungusand five freshly isolated soil fungi. The objectiveof this portion of the study was to evaluate thefluorescent-staining technique, applied previouslyonly to single culture preparations, as a means offollowing microscopically the progressive de-velopment of A. flavus in mixed culture in soil.

Conventional microscopic examination priorto fluorescence staining showed the presence ofabundant hyphae on all slides; the colonies whichdeveloped on culture plates inoculated withscrapings from each slide confirmed the presenceof A. flavus, usually with three or four of theother isolates. With immunofluorescent examina-tion after staining, no difficulty was encounteredin detecting some hyphal filaments of A. flavuson each slide by virtue of their characteristicbright yellow-green fluorescence. In most of thefields, the fluorescent filaments were seen againsta dark background intermingled with the hyphaeof one or two of the non-A. flavus isolates, dimlyvisible due to weak, blue-white autofluorescence.A field typical of some of the better prepara-

tions is shown in Fig. 1, but necessarily withoutbenefit of the striking color contrasts seen whenactually viewed by fluorescence microscopy. Ifknown to be present, A. flavus (bright, thinfilaments of Fig. lb) might, of course, be distin-guished from the coarser filaments of the otherisolate (probably M1Iortierella sp.) with bright-field microscopy and ordinary light, as in Fig. la.The fluorescent-antibody technique, however,serves, not merely to set off the thin filamentsfrom the coarser filaments and the particulatebackground, but to identify the thin filaments asA. flavus.

Inspection of Fig. lb discloses also that notall of the thin filaments fluoresced uniformly,even though they apparently were hyphae of A.flavus. The presence of nonfluorescing hyphalsegments was noted earlier in this study, andhas been mentioned before (Schmidt and Bankole,1962). The abundance of brightly stained hyphaeappeared to be associated both with the age ofthe soil culture and its nutrition. In the incuba-tion series in which the soil was treated withglucose, filaments bright with specific fluores-cence were most abundant at the 5th day ofincubation, and the hyphae appeared to be rela-tively wide. By the 14th day, filaments withcomparable fluorescence were much less frequent,and were shorter and thinner. Those culturesincubated in soil with no amendment exhibitedslower development of A. flavus as indicated bybrightly fluorescing filaments. Most extensive

specific fluorescence was found in 7-day prepara-tions and only slight diminution in abundancewas noted at 14 days. In all nonglucose soils,hyphae of A. flavus were thinner than that seenin the early stages of the series with glucose..

DISCUSSIONThe promise inherent in immunofluorescent

staining as applied to soil ecology lies in the pos-sibility that a given microorganism may berendered microscopically recognizable, and thuscan be studied directly in soil environments.Soil preparations treated with fluorescent anti-body should, ideally, retain the labeled antiserumat sites which correspond only to the distributionof the soil isolate used to prepare the antiserum.In the case of A. flavus, the only organism usedthus far in attempts to trace the development ofa specific microorganism in soil by fluorescent-antibody techniques, results have at least ap-proached within workable range of the ideal.Nonspecific adsorption by soil materials or auto-fluorescence of soil minerals has not been trouble-some. The preceding data reflect a specificitywhich, although not absolute, appears adequateto justify application of the technique to prob-lems involving the ecology of A. flavus. Therelatively few interfering cross-reactions thatwere encountered among members of the genusAspergillus involved species, which, on the basisof agglutinating antibody, were closely related toA. flavus. Modified methods of antiserum prepa-ration might serve to enhance further the speci-ficity of the labeled antibody with respect toclosely related forms.

Application of fluorescent-antibody staining tothe simple soil ecology problems posed in thestudy was made mainly to explore the techniqueitself, but some of the observations are of furtherinterest to considerations of A. flavus as a soilorganism. A. flavus is known to form nitrate inpure culture (Schmidt, 1954), but its contribu-tion, if any, to nitrification in natural environ-ments is not known, and is difficult to assess.Information on the occurrence of A. flavus insoils is pertinent to evaluation of its importance.When the labeled antiserum was used to stainnumerous fungi isolated at random from a freshsoil, the only isolate to react proved to be astrain of A. flavus. Work is in progress to examinesimilarly many fresh isolates from a wide varietyof soils; this will provide further data both onthe specificity of the stain and on the extent towhich the nitrifying fungus is encountered insoil populations. When A. flavus was inoculatedinto soil with several other soil isolates, its de-velopment could be followed by immunofluores-

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cent examination of slides recovered periodicallyfrom the soil. It was clear that A. flavus grewwell in competition with the other fungi present,was influenced as to size and rate of growth bythe nutritional circumstances of the soil environ-ment, and persisted in an actively growing hyphalform. This ability to compete and persist inmycelial form in soil, rather than merely asspores, is a further essential attribute if the fun-gus is to be seriously considered for its ecologicalsignificance as a nitrifier.

Certain difficulties were encountered in thecourse of the present study. In the A. flavussystem, the limitations merely condition, ratherthan preclude, useful application of the immuno-fluorescent technique. But application to eachnew organism selected for study probably mustbe preceded by rather extensive tests to evaluatethe specificity of the staining reagents. Even inthe A. flavus system where the specificity isgenerally adequate, subjective judgments offluorescence may be uncertain for poorer prepa-rations or for marginal intensities. Referenceslides must be used consistently to aid in judg-ments of fluorescence intensity. When the organ-nism of interest is filamentous, nonfluorescent orweakly fluorescent filaments are likely to occur

in the same field as some with characteristicbright fluorescence; thus, in mixed culture or inunknown preparations, the organism sought mustgo unrecognized save for sites of characteristicreaction. Despite difficulties, the potential of thetechnique may be well worth the effort of ex-ploration and development in connection with avariety of problems in which it is necessary toevaluate the function of a certain microorganismin the soil environment (Schmidt and Bankole,1963).

ACKNOWLEDGMENT

This investigation was supported by NationalScience Foundation Grant G 21009.

LITERATURE CITED

SCHMIDT, E. L. 1954. Nitrate formation by a soilfungus. Science 119:187-189.

SCHMIDT, E. L., AND R. 0. BANKOLE. 1962. Detec-tion of Aspergillus flavus in soil by immunoflu-orescent staining. Science 136:776-777.

SCHMIDT, E. L., AND R. 0. BANKOLE. 1963. The useof fluorescent antibody with the buried slidetechnique, p. 197-204. In J. Doeksen and J. VanDer Drift [ed.], Soil organisms. North HollandPublishing Co., Amsterdam.

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