SEROLOGICAL GROUPING OF ACTINOMYCES FLUORESCENT ANTIBODIES

Transcription

1 SEROLOGICAL GROUPING OF ACTINOMYCES FLUORESCENT ANTIBODIES BY MEANS OF JOHN M. SLACK, ANN WINGER, AND DANE W. MOORE, JR. Department of AMicrobiology, Medical Center, West Virginia University, Morgantown, West Virginia- ABSTRACT SLACK, JOHN AI. (West Virginia University, Morgantown), ANN WINGER, AND DANE W. MOORE, JR. Serological grouping of actinomyces by means of fluorescent antibodies. J. Bacteriol. 82: Serological groups A, B, C and D of actinomyces were established using fluorescent antibody techniques. One hundred and thirty-eight cultures were included in the study. Eighty-nine were classed in group A, 15 in B, 13 in C, and 21 in D. The isolates were from patients and animals with actinomycosis and from healthy human beings. There was no correlation between source of the isolate and serological group. Furthermore, no one species could be placed exclusively in one group although the majority of those designated as Actinomyces bovis were in group A. Seventeen anaerobic diphtheroids and seven Corynebacterium acnes isolates were placed in group A. One diphtheroid was in each of groups B and D. On this basis it is suggested that these organisms be included in the genus Actinomyces. Additional species of Corynebacterium as well as Lactobacillus Propionibacterium, Streptomyces, and Nocardia did not fluoresce with any of the group antisera. The conjugation of antibodies with fluorescein was successfully accomplished by Coons, Creech, Jones, and Berliner (1942) and Coons and Kaplan (1950). They also demonstrated the specificity of the reaction between conjugated antibodies and the antigen. This procedure has been applied to an antigenic analysis of Salmonella typhosa by Thomason, Cherry, and Moody (1957). Conjugated antibodies have been used for the grouping or typing of bacteria including diplococci (Coons et al., 1942), streptococci (Moody, Ellis, and Updyke, 1958), shigellae (LaBrec, Formal, and Schneider, 1959), salmonellae (Thomason, Cherry, and Received for publication January 3, Edwards, 1959), vibrio (Finkelstein and LaBrec, 1959), and neisseria (Deacon et al., 1959). Serological studies with the actinomyces have been reviewed by Slack et al. (1951) and cn the basis of agglutination tests, serological groups A and B were established (Slack et al., 1955). A third group, C, has also been established but not reported. By the use of fluorescent antibodies the existence of these three serological groups was further confirmed (Slack and Moore, 1960). The present paper extends this work and reports a fourth serological group, group D. MATERIALS AND METHODS Immunizations. Antiserum was prepared by immunizing three rabbits each with group A (culture- A-1), group B (culture A-5), group C (culture D-19), and Actinomyces bovis (ATCC 10048). The organisms were grown in nonantigenic peptone dialyzate, thioglycolate broth; the antigens were prepared and rabbits immunized as described by Slack et al. (1955). All titers were 1:1280 or greater. Labeling of globulins. Globulin fractions of the sera were obtained by three precipitations with ammonium sulfate at 50% saturation and then dialyzed against saline. The nitrogen content of the globulin solution was determined by micro- Kjeldahl and the milligrams of protein per ml calculated. The conjugation procedure with fluorescein isothiocyanate was that of Riggs et al. (1958). Staining. Smears of the organisms were air dried, gently heat fixed, and flooded with the labeled globulin. A wide range of staining times. and temperatures was tried, with 15 min at room temperature proving optimal. The smears were then washed 10 min with saline and 5 min witlh distilled water, air dried, and mounted with a cover slip in buffered glycerine. In all instances,. control smears were made using corynebacteria by either mixing the two organisms together in one smear or placing individual smears on the same

2 1961] SEROLOGICAL GROUPING OF ACTINOMYCES 55 FIG. 1. Group B (culture A-5) and group D (Actinomyces bovis strain 10048) stained with group D conjugated antiserum. Note the numerous single group B cells and the vague filamentous D organisms. Darkfield with white light. (1,500X magnification) slide. Subsequently, six smears were routinely made on one slide each located by scoring the underside of the slide with a series of circles using a diamond-point marking pencil. Sorption. Labeled globulins prepared against group C (culture D-19) were sorbed using equal quantities of globulins and live, packed group A (culture A-1) cells. The sorption was carried out at 55 C for 1 hr with vigorous shaking each 15 min, followed by overnight refrigeration. Successful sorption of labeled group B (culture A-5) globulins proved to be more difficult and in this case the sorbing dose of cells was reduced to ml, but the remainder of the procedure was the same. Observation. A Leitz Ortholux microscope, CS150 Phillips mercury vapor lamp, IJG-1 (2 and 4 mm), BG-12 (4 and 8 mm) filters with Euphos or OG-1 eyepiece filters were used. Early attempts at determining the degree of fluorescence were made but abandoned. The results were then recorded as positive or negative. All isolates were observed unstained for autofluorescence but none was observed. For some time there was difficulty in interpreting smears in which vague shadows of the organisms could be seen. It became apparent, however, that with fluorescence in these preparations there was a definite luminescence and only in this instance were they recorded as positive. Photography. Photomicrographs (Fig. 1 to 8) were made with the Leica camera and micro-ipso attachment. Superanscochrome color film was used with an average exposure time of 5 min. RESULTS Grotup A. Table 1 lists the organisms in group A. These all fluoresced with group A antiserum but not with sorbed group B, C, or D antisera. It should be added that conjugated group A antiserum will fluoresce all group B and group C organisms. There was also a cross-reaction with group D but it was usually quite weak and some group D isolates were negative. Several of the isolates from human infections have been maintained in this laboratory for several years although those received from King

3 FIG. 2. Same field as Fig. 1 using darkfield and ultraviolet light with the group D filaments clearly observable but no visible group B cells. (1,50OX magnification) FIG. 3. Corynebacterium pseudodiphtheriticum and group D (Actinomyces bovis strain 10048) stained with group D antiserum. The masses of cells show up as undifferentiated areas of brightness. Filaments may be seen int the center of the field. Darkfield and white light. (1,500X magnification) 56

4 FIG. 4. Same field as in Fig. S using darkfield and ultraviolet light. Only group D organisms can be seen. Note the filament at the left which is entirely obscured in Fig. 3. (1,500X magnification) FIG. 5. Corynebacterium diphtheriae and group D cells stained with group D antiserum. Most cells are clearly differentiated including the long group D filament. Darkfield and white light. (1,500X magnification) 57

5 FIG. 6. Same field as in Fig. 5 with ultraviolet light. The long filament is brightly fluorescent and the curved end is clearly shown whereas it is very vague in Fig. 5. The Corynebacteria appear as vague shadows but do not fluoresce. (1,50OX magnification) FIG. 7. Propionibacterium pentosaceum and group D stained with group D antiserum. Many single cells can be seen. Darkfield and white light. (1,5OOX magnification) 58

6 196.1 SEROLOGICAL GROUPING OF ACTINOMYCES 59 FIG. 8. Same field as Fig. 7 with ultraviolet light. The Propionibacteria are no longer visible but the filament of group D is clearly fluorescent. (1,50OX magnification) and Meyer and Goldsworthy are recent isolates. It will be noted that of these 16 cultures, 10 were designated as Actinomyces bovis, 1 as Actinomcyes israelii, 1 as Actinomyces naeslundii, and 4 were not given a species name. This points up the fact that actinomyces isolates designated as different species can cause infections in man and that on the basis of the fluorescent antibody technique they can be serologically identical. The majority of those isolated from animal infections were from cattle and designated as A. bovis. Serologically, however, there was no difference between these isolates and those in group A obtained from human infections. Human tonsils and dental calculi were cultured by inoculating infusion agar deeps serially with tonsil washings or crushed calculi. Organisms morphologically resembling actinomyces were isolated. As the criteria for designating these by species name are not too definite, the term "microaerophilic actinomycetes" has been applied to these organisms. Seventeen of these organisms were found to be in group A. Howell et al. (1959) reported a streak plate method of isolating actinomyces from material taken from the oral cavity. At intervals these authors have forwarded cultures to this laboratory for fluorescent antibody study. Ten were designated by them as A. naeslundii and seven to which they gave no species designation were placed in group A. King and Meyer (1957) forwarded certain of their anaerobic diphtheroid cultures to us and we have received additional such organisms from other sources. Seventeen were found to be in group A. Different species of Corynebacterium were obtained from the American Type Culture Collection (ATCC) and the Communicable Disease Center (CDC) to be tested with fluorescent antibodies. Of these, all cultures received and designated as Corynebacterium acnes were placed in group A, whereas none of the other species fluoresced with any of the antisera. A total of 89 actinomyces isolated from various sources and having a variety of species designations were placed in group A on the basis of the fluorescent antibody technique.

7 60 SLACK, WINGER, AND MOORE [VOL. 82 TABLE 1. Group A cultures Laboratory designation Source Genus and species Human actinomycosis isolates A-1 and A-2 A-14 and A-15 A-16 A-17 A-19 A-21 A Morgan Butler Hunter Animal actinomycosis isolates B-2 B-3, B-4, and B-5 Heilman; Mayo Clinic Novak; Univ. Illinois Kimball; Kansas State 8374 ATCC, Emmons, ATCC, Emmons, 1946 Thompson; Mayo Clinic Appel; Lynn, Mass. King and Meyer; Chicago King and Meyer; Chicago King and Meyer; Chicago Goldsworthy; Australia Goldsworthy; Australia ATCC, Howell, 1955 ATCC, Meyer, 1958 Actinomyces bovis Actinomyces israelii * Actinomyces naeslundii Kimball; Kansas State, bovine Novak; Univ. Illinois, bovine B ATCC, Emmons, 1942, bovine B-7, B-8, and B-9 Thompson; Mayo Clinic, bovine Pine-6 A-12, Pine, Duke Univ., bovine Pine-7 P-1, Pine, Duke Univ., bovine Pine-9 P-2S, Pine, Duke Univ., bovine 387 CDC, bovine 4501 NCTC, Carnes; Australia, bovine E-1 Kimball; Kansas State, equine E-3 Ennever; Ohio State, equine Human tonsil isolates, nonactinomycotic T-5-2, T-5-3, T-12, T-13, T-47, T-66, Slack; W. Va. Microaerophilic actinoand T-97 mycetes Dental calculi isolates, nonactinomycotic D-68, D-76, D-95, D-113, D-132, Slack; W. Va. Microaerophilic actino- D-143, D-167, D-202, D-341, and mycetes D-342 Oral cavity isolates, nonactinomycotic 286, C-294, C-319, C-323, C-334, Howell; NIH Actinosnyces naeslundii C-342, C-389, C-452, CS-1750, and CS-1752 C-447, C-1021, CS-1181, CS-1759, Howell; NIH CS-1771, CS-1788, and CS-1786 Anaerobic diphtheroids, nonactinomycotic AD-3, AD-12, AD-29, AD-30, AD-32, King and Meyer; Chicago, blood AD-35, AD-37, AD-39, and Al)-41 cultures S-1 and S-2 King and Meyer; Chicago, skin Zach Myers Clinic, W. Va., lung A-8, A-9, A-10, A-12, and A-13 Schain; Staten Island, bone marrow Corynebacterium 6911, 6919, 6921, 6922, 6923, 11827, ATCC C. acnes and *-= Unnamed species.

8 19611 SEROLOGICAL GROUPING OF ACTINOMYCES 61 Group B. Group B antisera were sorbed with group A cells until all cross-fluorescence was removed. This sorbed antiserum did not cross-react with groups A, C, or D. Table 2 lists the 15 cultures that fluoresced with this sorbed antiserum and were thus placed in group B. It will be noted that there are isolates from human and animal infections as well as from the noninfected, human oral cavity in this group. Also it can be seen that different species names had been applied to the majority of these organisms. Group C. Specific antiserum was prepared for this group by sorption with group A cells. Thirteen isolates listed in Table 3 were placed in this group, 2 from human infections and 11 from either tonsils or dental calculi. Group D. This antiserum showed the least amount of cross-reaction in that there was weak fluorescence with group A (culture A-1) but no fluorescence with cultures of either group B or C. There were also cultures in group D that did not fluoresce with group A antiserum which indicates the possibility of some antigenic differences within this group. It is possible that there was inadequate concentration of antigen or antibodies to provide visible fluorescence. For specificity, this antiserum was sorbed with group A cells. Except for group A the greater number of isolates from human infections were placed in group D-eight in all. Two isolates were from bovine infections, ten from the oral cavity of man, and one from a blood culture. These are listed in Table 4. The majority of the cultures received and designated as A. israelii were found to be in group D, although some isolates designated as A. TABLE 2. Group B cultures Laboratory designation Source Genus and species Human actinomycosis isolates A-3 and A-4 Heilman; Mayo Clinic Actinomyces bovis A-5 Lawrence; Sterling-Winthrop * CC, King and Meyer; Chicago Animal actinomnycosis isolate P-i Kimball; Kansas State, porcine Human tonsil isolates, nonactinomycotic T-6, T-46, and T-48 Slack; W. Va. Microaerophilic actinomycetes Dental calculi isolate, nonactinomycotic M-1 Ennever; Ohio State Actinomyces israelii Oral cavity isolates, nonactinomycotic C-286 and C-454 Howell; NIH Actinomyces naeslundii (?) C-456 and C-457 Howell; NIH A. naeslundii C-959 Howell; NIH A. israelii Anaerobic diphtheroid, nonactinomycotic A-ll Schain; Staten Island, bone marrow = Unnamed species. TABLE 3. Group C cultures Laboratory designation Source Genus and species Human actinomycosis isolates ANll Salvin; NIH Actinomyces bovis AN14 Salvin; NIH Human tonsil isolates, nonactinomycotic T-113 Slack; W. Va. Microaerophilic actinomycete Dental calculi isolates, nonactinomycotic 1)-19, D-28, D-64, D-85, D-86, D-106, Slack; W. Va. Microaerophilic actinomycetes D-114, D-150, D-184, and D-312

9 62 SLACK, WINGER, AND MOORE [VOL. 82 TABLE 4. Group D cultures Laboratorp designation Source Genus and species Human actinomycosis isolates Hill King and Meyer; Chicago Actinomyces bovis CC2 King and Meyer; Chicago * Boyd Goldsworthy; Australia Lemke Goldsworthy; Australia Slamon Goldsworthy; Australia ATCC, Emmons, ATCC, Howell, 1955 Actinomyces israelii ATCC, Howell A. israelii Bovine actinomycosis isolates Pine-i A- Pine, Duke Univ. i2836 ATCC Georg, CDC,i957 Oral cavity isolates, nonactinomycotic 295, C-355, C-464, CS-963, CS-996, CS-1038, Howell; NIH A. israelii CS-1042, CS-1064, and CS-1171 CS-55 Howell; NIH Actinomyces naeslundii Anaerobic diphtheroid, nonactinomycotic CC3 = Unnamed species. TABLE 5. No. 'Species { King; Chicago, blood American Type Culture Collection strains No. Species ~~~~ ~ Source isolated Person Year submitting Serological ~~~from Prosumtig submitted group 8373 Actinomyces bovis Bovine Emmons; NIH 1942 A 8374* Human Emmons: NIH 1942 A Human Emmons; NIH ) Human Emmons; NIH 1946 A Actinomyces israelii Human Howell; NIH 1955 D A. israelii Human Howell; NIH ) Actinomyces naeslundii Human Howell; NIH 1955 A Bovine Georg; CDC 1957 D Bovine Meyer; Ill A * See note p. 975, Hazen and Little (1958). bovis, A. naeslundii, and anaerobic diphtheroids were also placed in this group. Table 5 lists the actinomyces which were obtained from ATCC giving the original source and the year in which they were sent to the collection. Five of these cultures were placed in group A and four in D. There is no correlation between habitat, species, and the serological group. The first four cultures listed have been maintained in this laboratory for ten years and when compared with original lyophilyzed specimens they each remained in their same serological group. Culture has been received from at least three different sources and in each instance has been placed in group D. The original culture of this organism was very rough but over a period of time has become quite smooth. Serologically it remained in group D. Control cultures. Table 6 lists Corynebacterium, Propionibacterium, Lactobacillus, Streptomyces, and Nocardia species that were included in these studies because questions have been raised about their relationships to the actinomyces. These organisms were first carefully screened for autofluorescence and found negative. They were then set up alone or as mixtures with the actinomyces from the various groups and stained with the different antisera. There was no fluorescence with any of the group antisera.

10 19611 SEROLOGICAL GROUPING OF ACTINOMYCES 63 Laboratory designation TABLE 6. Source Corynebacterium 1 and 2 CDC CDC CDC _ CDC CDC 7, 26, and CDC ATCC 373 ATCC and ATCC Propionibacterium 4867 ATCC 4870 ATCC 4875 ATCC 6207 ATCC Lactobacillus Doetsch; Md. Williams; Pa. 314 and ATCC ATCC Streptomyces and ATCC Nocardia F-8 F-9 Barnett: W. Va. Barnett; W. Va. Barnett; W. Va. Barnett; W. Va. Vet. Hosp.; Iowa Vet. Hosp.; Iowa * = Unnamed species. Control cultures Species C. ulcerans C. xerosis C. hoffmannii C. ovis C. renale C. diphtheriae C. diphtheriae C. xerosis C. pseudodiphtheriticum P. jensenji P. freudenreichii P. pentosaceum P. peterssonii L. bifidus L. bifidus var. pennsylvanicus L. acidophilus L. bifidus S. griseus S. intermedius S. flavovirens S. albus S. scabies DISCUSSION N. asteroides N. asteroides The fluorescent antibody technique was used in this study as a means of serologically grouping 138 cultures of actinomyces. The results confirm the existence of previously reported groups A and B which were established by using the reciprocal agglutinin sorption technique. Two additional groups have been confirmed and established by the fluorescent antibody procedure and were designated as C and D. Two major difficulties occur in identifying and classifying these organisms on the basis of serological procedures. They are frequently very rough or filamentous and thus agglutination tests cannot be done, or their growth is so scanty that insufficient antigen is available. Both of these difficulties may be overcome by using the fluorescent antibody techniques. In preliminary tests using suspensions of colonies taken directly from streak plates or from agar deeps the organisms could be readily identified. Also in mixed cultures with bacteria the actinomyces could be selectively identified by fluorescence. Eighty-nine, or approximately two-thirds of the cultures listed, are in group A indicating that this is the most widely distributed serological group. The isolates in this group are derived from human and animal infections as well as from noninfected human beings. Thus there is no one source of these organisms and their antigenicity is not dictated by their habitat. This same statement holds true for the organisms in group B, C, and D. Thirty-two of the isolates had been identified as, 14 from human sources and 22 from animals. Twenty-two of these were in group A, 2 in B, 2 in C, and 6 in D. Fifteen were designated as A. israelii. One is in group A, 2 are in B, and 12 in D. Sixteen were called A. naeslundii, and of these 11 are in group A, 4 in B, and 1 in D. Thus, of those cultures designated by a species name there was no correlation between the species name and the serological group, although a preponderance of one or more were in a particular group. Twenty-two of the cultures were in group A. However, here again on a serological basis using this procedure no one species is found exclusively in any of these four groups. The term "anaerobic diphtheroid" is widely used to designate organisms having morphological characteristics of a corynebacterium but which cannot be or are not designated by a given species name. Nineteen of these cultures have been included in this study. Seventeen were found to be group A and one each of B and D. Although this study should be extended to include a wider selection of these organisms, this does indicate a serological relationship with the actinomyces and on this basis it could be tentatively proposed that they be included in the genus Actinomyces.

11 64 SLACK, WINGER, AND MOORE [VOL. 82 Seven cultures of C. acnes were obtained from ATCC and all of these were placed in group A. This should be extended to include many more such isolates, nevertheless consideration should be given to placing C. acnes in the genus Actinomyces. Serological relationships between these organisms have also been indicated by Beerens (1953) and Linzenmeier (1957). Moody and Jones (1960) were able to differentiate 26 strains of Corynebacterium diphtheriae from 6 strains of diphtheroids by the fluorescent antibody technique. This further indicates that this procedure can be used to determine antigenic differences between these organisms. Eight other species of Corynebacterium as well as species of Propionibacterium, Lactobacillus, Streptomyces, and Nocardia were fluoresced with each conjugated actinomyces antiserum and were negative. Thus there is no antigenic relationship between these species and the actinomyces as determined by the fluorescent antibody technique. Sharpe (1955) showed definite serological relationship between various species of Lactobacillus although she did not attempt to determine antigenic relationship with other genera. Group A antiserum will fluoresce group A, B, C, and D organisms. However, if the antiserum is sorbed with B, C, and D antigens it will still fluoresce group A cells. When group B and C antiserum is sorbed with A cells, the antiserum no longer cross reacts with other groups but is specific for its respective group. Group D antiserum will only cross-react with group A and sorption with group A cells removes this crossreaction. Thus group A cells contain at least three antigens or haptenes, one or more of which is specific for group A, one in common with both groups B and C and one in common with D. Attempts to chemically isolate these antigens have been initiated. As no correlation has been shown to exist between habitat, species designation, and serological grouping, the question arises as to which species name or names should be applied to these organisms. Before this question can be satisfactorily answered there must be extensive comparative work done including morphological, physiological, and chemical characteristics of these isolates to determine if one or more characteristics will correlate with the serological group. It is also possible that such characteristics as catalase, roughness, and animal pathogenicity will be found to be variable to the degree that they do not support species differentiation. If this should occur and if serology becomes the basis for identification and classification it seems that one species name could be applied to groups A, B, C, D, and others if they are established and then, possibly, types within each group. This would also give rise to controversy as to which species name should be given priority. However, as Erwin (1960) in his extensive study established Actinomyces bovis as the type species, this would have to be given prime consideration. The fluorescent antibody technique is impressive from the standpoint that it provides a method of applying serological procedures to filamentous and other organisms that have defied serological procedures. It is hoped that other workers will attempt to use this as a tool to investigate the intriguing problems of identification and classification. ACKNOWLEDGMENT This work was supported by grant no. E-1801 from the U. S. Public Health Service. LITERATURE CITED BEERENS, H I-tude comparative de six souches de bacteries anaerobies none sporulees. Ann. inst. Pasteur 84: COONs, A. H., AND M. H. KAPLAN Localization of antigen in tissue cells. II. Improvements in a method of detection of antigen by means of fluorescent antibody. J. Exptl. Med. 91:1-13. COONS, A. H., H. J. CREECH, R. N. JONES, AND E. BERLINER The demonstration of pneumococcal antigen in tissues by the use of fluorescent antibody. J. Immunol. 45: DEACON, W. E., W. L. PEACOCK, E. M. FREEMAN, AND A. HARRIS Identification of Neisseria gonorrheae by means of fluorescent antibodies. Proc. Soc. Exptl. Biol. Med. 101: ERWIN L. F The nomenclatural status of the generic names of the Actinomycetales. Intern. Bull. Bacteriol. Nomen. and Taxon. 10 (Suppl.) : FINKELSTEIN, R. A., AND E. H. LABREc Rapid identification of cholera vibrios with fluorescent antibody. J. Bacteriol. 78: HAZEN, E. L., AND G. N. LITTLE Actinomyces bovis and "anaerobic diphtheroids." Pathogenicity for hamsters and some other differ-

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