Specificity of Immunofluorescent Staining for Study

Transcription

1 APPLIED MICROBIOLOGY, Sept., American Society for Microbiology Vol. 13, No. 5 Printed in U.S.A. Specificity of Immunofluorescent Staining for Study of Aspergillus lavus in Soil E. L. SCHMIDT AND R. 0. BANKOLE Department of Microbiology, University of Minnesota, Minneapolis, Minnesota Received for publication 8 March 1965 ABSTRACT SCHMIDT, E. L. (University of Minnesota, Minneapolis), AND R. 0. BANKOLE. Specificity of immunofluorescent staining for study of Aspergillis flavus in soil. Appl. Microbiol. 13: Fluorescein-labeled antiserum prepared with Aspergillus flavus strain CS was tested for specificity by staining fungi grown in soil in the vicinity of buried slides. All 14 strains of A. flavus fluoresced as intensely or nearly as intensely as the antigen control. Among 21 isolates of species of Aspergillus other than A. flavus, 17 reacted with moderate to low fluorescence at intensities readily distinguishable from that 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 were generally nonreactive. Interfering cross-reactions were encountered for one strain of Spicaria and one strain of Stemphylium; three isolates could not be evaluated because of interfering autofluorescence. An additional 22 isolates were either wholly negative or had a low order of fluorescence. Agglutination tests between each of the fungi and A. flavus CS serum revealed close agreement between agglutination titer and fluorescent-staining reaction. Unknown fungi freshly isolated from soil were checked for reaction to the A. flavus labeled antiserum; only one isolate gave a pronounced staining reaction, and that one proved to be a strain of A. flavus. In a simplified ecological model, 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 microorganisms in soil would be especially informative if it were possible to recognize the microorganisms observed. The fluorescent-antibody technique has been advanced as a possible means for the detection of a particular microorganism in soil prepared for microscopic examination (Schmidt and Bankole, 1962). Results of initial attempts to apply immunofluorescent-staining techniques to the detection of Aspergillus flavus in pure culture on soil slides were encouraging (Schmidt and Bankole, 1962, 1963). Since the effectiveness of the technique depends on the selective adsorption of the fluorescein-labeled antiserum to microorganisms closely related to the one used as the antigen, data on the specificity of the antiserum used in staining are important. Results of studies on the specificity of the fluorescein-labeled antibody prepared from A. flavu strain CS, and on its use in the detection of A. flavu in mixed culture in soil, are presented in this report. MATERIALS AND METHODS Most of the data were derived from study of fungi obtained from culture collections and identified at least to genus. The fungi were grown in pure culture in sterile soil moistened with 1 or 2 ml of 1% glucose, and with sterile microscope slides inserted in the soil. After recovery from the soil and just prior to examination, each slide was treated with the labeled antiserum. The antiserum had been prepared in rabbits by injecting A. flavus CS, and was conjugated with fluorescein isothiocyanate. Slides were examined in detail with fluorescence microscopy, and cultures were rated for fluorescence as compared with slides bearing A. flavus CS. Techniques used in the preparation of the antigen, conjugation of the antiserum, growth of the test cultures, and staining and examination of contact slides were as reported previously (Schmidt and Bankole, 1962). A typical schedule for the immunization of rabbits with mycelium of A. flavus has been presented also (Schmidt and Bankole, 1963). Serological reactions between the test cultures and A. flavus CS immune serum weretested by standard tube agglutination assay. Each fungus was grown in glucose (0.2%)-glutamate (0.4%)-mineral salts broth, and was prepared as antigen for agglutination assay as a suspension of washed, minced mycelium in saline, adjusted to an optical density approximately that of A. flavus CS antigen. A constant volume of 1 ml of antigen was added to dilutions of serum. In addition to study on the specificity of the A. 673

2 674 SCHMIDT AND BANKOLE APPL. MICROBIOL. flavus CS antibody with respect to isolates obtained from culture collections, the staining reaction of fungi freshly isolated from soil was investigated. A fertile field soil was plated in rose bengal streptomycin agar and Czapek agar. Colonies of fungi that appeared to be different species were isolated in pure culture, grown individually in the sterile soil system, and examined for fluorescentstaining reaction as before. The usefulness of the fluorescent-antibody technique for the detection of A. flavus in mixed culture in soil was tested in studies with sterile soilslide systems inoculated with a controlled mixture of several of the fungi freshly isolated from the field soil, with and without A. flavus CS. Slides were removed carefully at 2, 5, 7, and 14 days, so that soil particles adhering to the surface were undisturbed. The loosely adhering soil on a particular section of the slide was then scraped aseptically into sterile diluent and plated on rose bengal agar. The same slide surface which provided the soil for plating was prepared for immuniofluorescent staining. With the above approach, the distribution or absence of A. flavus in a mixture of fungi was detected by specific staining soon after the slide was harvested. Results of immunofluorescent observations were then checked against results obtained by plating procedures after suitable incubation. The soil isolates used to provide the mixture of fungi were selected because they were nonreactive to the stain, grew at about the same rate in soil in association with A. flavus and each other, and were distinctive in colonial appearance on the rose bengal plates. The five isolates used in mixed-culture studies were tentatively identified as A. ustus (two strains), A. fumigatus, Mortierella sp., and Trichoderma lignorum. RESULTS Tests with A. flavus, other aspergilli, and various other genera. All 14 isolates of A. flavus used in the study grew well in pure culture in sterile soil, and ample mycelium was evident on each of the fresh, unstained slides as seen with a light microscope. None of the unstained slides gave evidence of autofluorescence when viewed with a fluorescence microscope. The- immunofluorescent reactions that developed for each A. flavus isolate subsequent to staining with labeled A. fiavus CS antiserum are summarized in Table 1. The fluorescence rating of 4+, based on the intense yellow-green fluorescence exhibited by control slides of the antigen, was equalled or approached by each strain of A. flavus tested. Immunofluorescent-stained slides of the various A. flavus strains also resembled those prepared from the antigen, in that all hyphal filaments did not fluoresce with uniform intensity; conidiophores, vesicles, and some sections of hyphae commonly fluoresced intensely, whereas other hyphae, perhaps older, frequently appeared as faintly fluorescent. The general equivalence in the staining reactions of culture-collection strains of A. flavus and the particular strain used for TABLE 1. Agglutination titers and immunofluorescent-staining reactions of various isolates of Aspergillus flavus when reacted with antiserum preparations of A. flavus strain CS A. flavus strain Agglutination Fluorescence Remarks titer rating* Minn. CS... 1, Control; most mycelium fluorescent, especially hyphal tips, conidiophores, and vesicles Wheat isolate Intense staining restricted to vesicles and hyphal fragments Wheat isolate Most mycelium and spores uniformly fluorescent Wheat isolate Most mycelium highly fluorescent; spores less so Wheat isolate No spores seen; vesicles and mycelial fragments with intense fluorescence Wheat isolate Mycelial fluorescence stronger than that of spores Minn. F , Extensive, uniformly intense fluorescence Minn. F , As in F 921 Minn. F As in wheat isolate 3 Minn. F Scanty mycelium but strongly fluorescent, especially vesicles QM , Many fragments intensely fluorescent, but much weakly fluorescent mycelium also QM , As in QM 380, but lower intensity Peoria , As in QM 380 Minn. F , Uniformly bright but not intense; few spores Upjohn I 1, As in QM 380, but lower intensity * Overall fluorescence rating between negative (-) and most intense (++++).

3 VOL. 13, 1965 IMMUNOFLUORESCENT STAINING OF A. FLAVUS 675 TABLE 2. Agglutination titers and immunofluorescent-staining reactions of aspergilli other than Aspergillus flavus when reacted with antiserum preparations of A. flavus CS Culture Agglutination Fluorescence Remarks titer* ratingt A. sydowi Indistinguishable from many A. flavus strains; vesicles and stalks intense, hyphal filaments less intense A. candidus Hyphal filaments weakly fluorescent; spores intense A. nidulans Fluorescence restricted to relatively few hyphae A. caespitosus Fluorescence restricted to vesicles, otherwise weak A. ochraceous A. ochraceous Very weak fluorescence; readily distinguishable A. ochraceous from A. flavus A. fumigatus... A. niger A. niger (Mulder) Fluorescence blue-white and of low intensity; A. proliferans...? +.. s A. A.amtdm.4 amstelodfami I. +, spores when visible appeared blue A. ruber A. terreus A. glaucus. ++ A. nidulans.- ++ Localized areas scantily distributed on hyphal A. tamarii segments gave fair fluorescence, but most A. echinulatus hyphae were barely fluorescent; spores, vesi- A. restrictus cles, and cross walls did not fluoresce; readily Aspergillus sp. (unidenti- distinguished from A. flavus fied) A. versicolor j * Antigen not dispersible in saline (-); end point uncertain (?). t Overall fluorescence rating between negative (-) and most intense (++++). serum preparation holds also in the agglutination titer data of Table 1. All strains either had agglutination titers equal to the antigen at 1,280, or were at the next lower dilution level at 640. The serological relationship within the group of A. flavus isolates was close, and agglutination data and fluorescent microscopy data are in good agreement. Cultures within the genus Aspergillus but representative of species other than A. flavus were examined. As shown in Table 2, the A. ftavus fluorescent antibody did not react with most aspergilli not in the flavus-oryzae group. The antibody stain, however, did react strongly with A. sydowi, so that this culture of A. sydoui could not have been distinguished from A. flavus on the basis of immunofluorescent-staining reaction. Three other aspergilli evidenced a lesser degree of cross-reaction than A. sydowi, with fluorescence of variable intensity and generally limited to certain structures. The three, A. candidus, A. nidulans, and A. caespitosus, were distinguishable from A. flavus in most preparations by means of direct comparisons. Still, the better preparations and better fields of the three were judged about equivalent in staining intensity to the poorer reactions of some A. flavus strains, and could not be distinguished from A. flavus under these conditions. Agglutination titers again correlated closely with microscopic evaluations. A. sydowi strongly resembled A. flavus both in agglutination titer and fluorescentstaining properties. At least two of the three aspergilli which evidenced a lesser, but still significant, degree of cross-reaction in staining also had a moderately high agglutination titer with A. flavus antiserum. A. caespitosus could not be titrated in tube agglutination, as the blended mycelium was not dispersible in saline. The remaining 17 strains of aspergilli tested gave no evidence of interfering cross-reaction on stained preparations or in tube agglutination reactions. Some notable degree of staining reaction was found in 4 of 27 preparations bearing fungi of

4 676 SCHMIDT AND BANKOLE APPL. MICROBIOL. genera other than Aspergillus (Table 3). The color and intensity most nearly like that of A. flavus was observed for the Stemphylium sp. isolate, with a fairly uniform distribution of moderate fluorescence along the hyphal elements. The Spicaria preparation had a slightly lower degree of specific fluorescence, but some hyphal segments appeared similar to stained fields of A. flavus. Helminthosporium sp. and Zygorrhyncus moelleri were rated at 2+ because of local areas of bright, yellow-green fluorescence on the hyphae. Adjacent areas on the hyphae, if at all fluorescent, appeared red and blue, colors not seen in A. flavus preparations. Three of the fungi listed in Table 3 could not be evaluated, because unstained preparations gave interfering autofluorescence. The two strains of Fusarium and the Pullularia pullulans were the only cultures observed in this study to autofluoresce markedly with the filter systems used. Tests with freshly isolated soil fungi. Since the specificity of the A. flavus CS antibody preparation was generally satisfactory in tests with a variety of common fungi maintained in culture collections, it was of interest to check the staining properties of unknown fungi freshly isolated from soil. A silt loam soil which was plated shortly after collection yielded 21 fungus cultures readily distinguished from one another by gross TABLE 3. Agglutination titers and immunofluorescent-staining reactions of genera other than Aspergillus when reacted with antiserum preparations of A. flavus CS Culture Agglutination Fluorescence Remarks titer* 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 Pullularia pullulans T T T I +++I +I +l Filaments generally fluoresced uniformly with moderate intensity; not readily distinguishable from A. flavus in color or intensity, but distinguishable in morphology As in Spicaria Local small areas on hyphae comparable in color and intensity to A. flavus; other portions of hyphae showed blue-green, yellow, and red fluorescence; all spores were red. Similar to Helminthosporium sp.; occasional weak fluorescein-type fluorescence; generally filaments fluoresced with a blue-white color, and spores, when visible, were blue and red No yellow-green fluorescence Unstained preparations gave interferinig yellowgreen autofluorescence * Antigen not dispersible in saline (-); end point uncertain (?); turbidity interfered with agglutination reading (T). t Overall fluorescence rating between negative (-) and most intense (++++). Autofluorescent (A).

5 VOL. 13, 1965 IMMUNOFLUORESCENT STAINING OF A. FLAVUS colonial appearance. When each of the 21 fresh isolates was cultured individually in the sterile soil-buried slide system and then examined after application of the immunofluorescent stain, it was found that only one isolate fluoresced with an intensity that approximated that of the control. It is of particular interest that the one isolate which was reactive to the stain proved, on further examination, to be a strain of A. flavus. The likelihood of bias was negligible, since this isolate did not look like the control in superficial 677 colonial appearance. Thus, a strain of A. flavus which occurred as a component of a mixed soil population, but was not readily recognized on the dilution plate, was detected by its characteristic fluorescent-staining reaction. This observation provides further evidence that immunofluorescent staining is sufficiently specific for use as a tool for study of the ecology of A. Jlavus in soil, and lends encouragement to the view that the method may prove amenable to other problems in soil ecology. FIG. 1. Field from contact slide after burial for 5 days in sterile soil inoculated with Aspergillus flavus and five other soil fungi. Hyphal diameter of the larger fungus (probably Mortierella sp.) is approximately u. (a) Bright field with ordinary light after staining with A. flavus CSfluorescent antibody. (b) Same field viewed by dark-field fluorescence microscopy. Finer bright filaments are A. flavus which fluoresced bright yellowgreen; the larger fungus was seen in the background with weak blue-white fluorescence.

6 678 SCHMIDT AND BANKOLE APPL. MICROBIOL. A. flavus detection in a mixed soil culture. A. flavus was traced successfully in a simplified ecological system comprised of the antigen fungus and five freshly isolated soil fungi. The objective of this portion of the study was to evaluate the fluorescent-staining technique, applied previously only to single culture preparations, as a means of following microscopically the progressive development of A. flavus in mixed culture in soil. Conventional microscopic examination prior to fluorescence staining showed the presence of abundant hyphae on all slides; the colonies which developed on culture plates inoculated with scrapings from each slide confirmed the presence of A. flavus, usually with three or four of the other isolates. With immunofluorescent examination after staining, no difficulty was encountered in detecting some hyphal filaments of A. flavus on each slide by virtue of their characteristic bright yellow-green fluorescence. In most of the fields, the fluorescent filaments were seen against a dark background intermingled with the hyphae of one or two of the non-a. flavus isolates, dimly visible due to weak, blue-white autofluorescence. A field typical of some of the better preparations is shown in Fig. 1, but necessarily without benefit of the striking color contrasts seen when actually viewed by fluorescence microscopy. If known to be present, A. flavus (bright, thin filaments of Fig. lb) might, of course, be distinguished from the coarser filaments of the other isolate (probably M1Iortierella sp.) with brightfield microscopy and ordinary light, as in Fig. la. The fluorescent-antibody technique, however, serves, not merely to set off the thin filaments from the coarser filaments and the particulate background, but to identify the thin filaments as A. flavus. Inspection of Fig. lb discloses also that not all of the thin filaments fluoresced uniformly, even though they apparently were hyphae of A. flavus. The presence of nonfluorescing hyphal segments was noted earlier in this study, and has been mentioned before (Schmidt and Bankole, 1962). The abundance of brightly stained hyphae appeared to be associated both with the age of the soil culture and its nutrition. In the incubation series in which the soil was treated with glucose, filaments bright with specific fluorescence were most abundant at the 5th day of incubation, and the hyphae appeared to be relatively wide. By the 14th day, filaments with comparable fluorescence were much less frequent, and were shorter and thinner. Those cultures incubated in soil with no amendment exhibited slower development of A. flavus as indicated by brightly fluorescing filaments. Most extensive specific fluorescence was found in 7-day preparations and only slight diminution in abundance was noted at 14 days. In all nonglucose soils, hyphae of A. flavus were thinner than that seen in the early stages of the series with glucose.. DISCUSSION The promise inherent in immunofluorescent staining as applied to soil ecology lies in the possibility that a given microorganism may be rendered microscopically recognizable, and thus can be studied directly in soil environments. Soil preparations treated with fluorescent antibody should, ideally, retain the labeled antiserum at sites which correspond only to the distribution of the soil isolate used to prepare the antiserum. In the case of A. flavus, the only organism used thus far in attempts to trace the development of a specific microorganism in soil by fluorescentantibody techniques, results have at least approached within workable range of the ideal. Nonspecific adsorption by soil materials or autofluorescence of soil minerals has not been troublesome. The preceding data reflect a specificity which, although not absolute, appears adequate to justify application of the technique to problems involving the ecology of A. flavus. The relatively few interfering cross-reactions that were encountered among members of the genus Aspergillus involved species, which, on the basis of agglutinating antibody, were closely related to A. flavus. Modified methods of antiserum preparation might serve to enhance further the specificity of the labeled antibody with respect to closely related forms. Application of fluorescent-antibody staining to the simple soil ecology problems posed in the study was made mainly to explore the technique itself, but some of the observations are of further interest to considerations of A. flavus as a soil organism. A. flavus is known to form nitrate in pure culture (Schmidt, 1954), but its contribution, if any, to nitrification in natural environments is not known, and is difficult to assess. Information on the occurrence of A. flavus in soils is pertinent to evaluation of its importance. When the labeled antiserum was used to stain numerous fungi isolated at random from a fresh soil, the only isolate to react proved to be a strain of A. flavus. Work is in progress to examine similarly many fresh isolates from a wide variety of soils; this will provide further data both on the specificity of the stain and on the extent to which the nitrifying fungus is encountered in soil populations. When A. flavus was inoculated into soil with several other soil isolates, its development could be followed by immunofluores-

7 l QL. 13, 1965 IMMUNOFLUORESCENT STAINING OF A. FLAVUS cent examination of slides recovered periodically from the soil. It was clear that A. flavus grew well in competition with the other fungi present, was influenced as to size and rate of growth by the nutritional circumstances of the soil environment, and persisted in an actively growing hyphal form. This ability to compete and persist in mycelial form in soil, rather than merely as spores, is a further essential attribute if the fungus is to be seriously considered for its ecological significance as a nitrifier. Certain difficulties were encountered in the course of the present study. In the A. flavus system, the limitations merely condition, rather than preclude, useful application of the immunofluorescent technique. But application to each new organism selected for study probably must be preceded by rather extensive tests to evaluate the specificity of the staining reagents. Even in the A. flavus system where the specificity is generally adequate, subjective judgments of fluorescence may be uncertain for poorer preparations or for marginal intensities. Reference slides must be used consistently to aid in judgments of fluorescence intensity. When the organnism of interest is filamentous, nonfluorescent or weakly fluorescent filaments are likely to occur in the same field as some with characteristic bright fluorescence; thus, in mixed culture or in unknown preparations, the organism sought must go unrecognized save for sites of characteristic reaction. Despite difficulties, the potential of the technique may be well worth the effort of exploration and development in connection with a variety of problems in which it is necessary to evaluate the function of a certain microorganism in the soil environment (Schmidt and Bankole, 1963). ACKNOWLEDGMENT This investigation was supported by National Science Foundation Grant G 9. LITERATURE CITED 679 SCHMIDT, E. L Nitrate formation by a soil fungus. Science 119: SCHMIDT, E. L., AND R. 0. BANKOLE Detection of Aspergillus flavus in soil by immunofluorescent staining. Science 136: SCHMIDT, E. L., AND R. 0. BANKOLE The use of fluorescent antibody with the buried slide technique, p In J. Doeksen and J. Van Der Drift [ed.], Soil organisms. North Holland Publishing Co., Amsterdam.