SEROLOGICAL GROUPING OF ACTINOMYCES BY MEANS OFFLUORESCENT ANTIBODIES

JOHN M. SLACK, ANN WINGER, AND DANE W. MOORE, JR.Department of AMicrobiology, Medical Center, West Virginia University, Morgantown, West Virginia-

Received for publication January 3, 1961

ABSTRACT

SLACK, JOHN AI. (West Virginia University,Morgantown), ANN WINGER, AND DANE W.MOORE, JR. Serological grouping of actinomycesby means of fluorescent antibodies. J. Bacteriol.82:54-65. 1961.-Serological groups A, B, Cand D of actinomyces were established usingfluorescent antibody techniques. One hundred andthirty-eight cultures were included in the study.Eighty-nine were classed in group A, 15 in B, 13in C, and 21 in D.The isolates were from patients and animals

with actinomycosis and from healthy humanbeings. 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 designatedas Actinomyces bovis were in group A.

Seventeen anaerobic diphtheroids and seven

Corynebacterium acnes isolates were placed ingroup A. One diphtheroid was in each of groups

B and D. On this basis it is suggested that theseorganisms be included in the genus Actinomyces.

Additional species of Corynebacterium as well as

Lactobacillus Propionibacterium, Streptomyces,and Nocardia did not fluoresce with any of thegroup antisera.

The conjugation of antibodies with fluoresceinwas successfully accomplished by Coons, Creech,Jones, and Berliner (1942) and Coons and Kaplan(1950). They also demonstrated the specificity ofthe reaction between conjugated antibodies andthe antigen.

This procedure has been applied to an antigenicanalysis of Salmonella typhosa by Thomason,Cherry, and Moody (1957). Conjugated anti-bodies have been used for the grouping or typingof bacteria including diplococci (Coons et al.,1942), streptococci (Moody, Ellis, and Updyke,1958), shigellae (LaBrec, Formal, and Schneider,1959), salmonellae (Thomason, Cherry, and

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Edwards, 1959), vibrio (Finkelstein and LaBrec,1959), and neisseria (Deacon et al., 1959).

Serological studies with the actinomyces havebeen reviewed by Slack et al. (1951) and cn thebasis of agglutination tests, serological groups Aand B were established (Slack et al., 1955). Athird group, C, has also been established but notreported. By the use of fluorescent antibodies theexistence of these three serological groups wasfurther confirmed (Slack and Moore, 1960). Thepresent paper extends this work and reports afourth serological group, group D.

MATERIALS AND METHODS

Immunizations. Antiserum was prepared by im-munizing three rabbits each with group A (culture-A-1), group B (culture A-5), group C (cultureD-19), and Actinomyces bovis (ATCC 10048). Theorganisms were grown in nonantigenic peptonedialyzate, thioglycolate broth; the antigens wereprepared and rabbits immunized as described bySlack et al. (1955). All titers were 1:1280 orgreater.

Labeling of globulins. Globulin fractions of thesera were obtained by three precipitations withammonium sulfate at 50% saturation and thendialyzed against saline. The nitrogen content ofthe globulin solution was determined by micro-Kjeldahl and the milligrams of protein per mlcalculated. The conjugation procedure withfluorescein isothiocyanate was that of Riggs et al.(1958).

Staining. Smears of the organisms were airdried, gently heat fixed, and flooded with thelabeled globulin. A wide range of staining times.and temperatures was tried, with 15 min at roomtemperature proving optimal. The smears werethen washed 10 min with saline and 5 min witlhdistilled water, air dried, and mounted with acover slip in buffered glycerine. In all instances,.control smears were made using corynebacteria byeither mixing the two organisms together in onesmear or placing individual smears on the same

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FIG. 1. Group B (culture A-5) and group D (Actinomyces bovis strain 10048) stained with group D con-jugated antiserum. Note the numerous single group B cells and the vague filamentous D organisms. Darkfieldwith white light. (1,500X magnification)

slide. Subsequently, six smears were routinelymade on one slide each located by scoring theunderside of the slide with a series of circles usinga diamond-point marking pencil.

Sorption. Labeled globulins prepared againstgroup C (culture D-19) were sorbed using equalquantities of globulins and live, packed group A(culture A-1) cells. The sorption was carried outat 55 C for 1 hr with vigorous shaking each 15min, followed by overnight refrigeration. Success-ful sorption of labeled group B (culture A-5)globulins proved to be more difficult and in thiscase the sorbing dose of cells was reduced to 0.075ml, but the remainder of the procedure was thesame.

Observation. A Leitz Ortholux microscope,CS150 Phillips mercury vapor lamp, IJG-1 (2 and4 mm), BG-12 (4 and 8 mm) filters with Euphosor OG-1 eyepiece filters were used. Early attemptsat determining the degree of fluorescence weremade but abandoned. The results were then re-corded as positive or negative. All isolates wereobserved unstained for autofluorescence but none

was observed. For some time there was difficultyin interpreting smears in which vague shadows ofthe organisms could be seen. It became apparent,however, that with fluorescence in these prepara-tions there was a definite luminescence and onlyin this instance were they recorded as positive.

Photography. Photomicrographs (Fig. 1 to 8)were made with the Leica camera and micro-ipsoattachment. Superanscochrome color film wasused 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 butnot with sorbed group B, C, or D antisera. Itshould be added that conjugated group A anti-serum will fluoresce all group B and group Corganisms. There was also a cross-reaction withgroup D but it was usually quite weak and somegroup D isolates were negative.

Several of the isolates from human infectionshave been maintained in this laboratory forseveral years although those received from King

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FIG. 2. Same field as Fig. 1 using darkfield and ultraviolet light with the group D filaments clearly ob-servable but no visible group B cells. (1 ,50OX magnification)

FIG. 3. Corynebacterium pseudodiphtheriticum and group D (Actinomyces bovis strain 10048) stainedwith group D antiserum. The masses of cells show up as undifferentiated areas of brightness. Filaments maybe seen int the center of the field. Darkfield and white light. (1 ,500X magnification)

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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 areclearly differentiated including the long group D filament. Darkfield and white light. (1,500X magnification)

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FIG. 6. Same field as in Fig. 5 with ultraviolet light. The long filament is brightly fluorescent and thecurved end is clearly shown whereas it is very vague in Fig. 5. The Corynebacteria appear as vagueshadows but do not fluoresce. (1 ,50OX magnification)

FIG. 7. Propionibacterium pentosaceum and group D stained with group D antiserum. Many single cellscan be seen. Darkfield and white light. (1 ,5OOX magnification)

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FIG. 8. Same field as Fig. 7 with ultraviolet light. The Propionibacteria are no longer visible but the fila-ment 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 weredesignated as Actinomyces bovis, 1 as Actinomcyesisraelii, 1 as Actinomyces naeslundii, and 4 werenot given a species name. This points up the factthat actinomyces isolates designated as differentspecies can cause infections in man and that onthe basis of the fluorescent antibody techniquethey can be serologically identical.The majority of those isolated from animal in-

fections were from cattle and designated as A.bovis. Serologically, however, there was no differ-ence between these isolates and those in group Aobtained from human infections.Human tonsils and dental calculi were cultured

by inoculating infusion agar deeps serially withtonsil washings or crushed calculi. Organismsmorphologically resembling actinomyces wereisolated. As the criteria for designating these byspecies name are not too definite, the term "micro-aerophilic actinomycetes" has been applied tothese organisms. Seventeen of these organismswere found to be in group A.

Howell et al. (1959) reported a streak plate

method of isolating actinomyces from materialtaken from the oral cavity. At intervals theseauthors have forwarded cultures to this labora-tory for fluorescent antibody study. Ten weredesignated by them as A. naeslundii and seven towhich they gave no species designation wereplaced in group A.King and Meyer (1957) forwarded certain of

their anaerobic diphtheroid cultures to us and wehave received additional such organisms fromother sources. Seventeen were found to be ingroup A.

Different species of Corynebacterium were ob-tained from the American Type Culture Col-lection (ATCC) and the Communicable DiseaseCenter (CDC) to be tested with fluorescent anti-bodies. Of these, all cultures received and desig-nated as Corynebacterium acnes were placed ingroup A, whereas none of the other speciesfluoresced with any of the antisera.A total of 89 actinomyces isolated from various

sources and having a variety of species designa-tions were placed in group A on the basis of thefluorescent antibody technique.

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SLACK, WINGER, AND MOORE

TABLE 1. Group A cultures

Laboratory designation Source Genus and species

Human actinomycosis isolatesA-1 and A-2A-14 and A-15A-16A-17A-19A-21A-220-2Morgan23163ButlerHunter1210413031Animal actinomycosis isolatesB-2B-3, B-4, and B-5

Heilman; Mayo ClinicNovak; Univ. IllinoisKimball; Kansas State8374 ATCC, Emmons, 194210049 ATCC, Emmons, 1946Thompson; Mayo ClinicAppel; Lynn, Mass.King and Meyer; ChicagoKing and Meyer; ChicagoKing and Meyer; ChicagoGoldsworthy; AustraliaGoldsworthy; AustraliaATCC, Howell, 1955ATCC, Meyer, 1958

Kimball; Kansas State, bovineNovak; Univ. Illinois, bovine

Actinomyces bovisA. bovisA. bovisA. bovisA. bovisActinomyces israelii

*

A. bovisA. bovisActinomyces naeslundiiA. bovis

A. bovisB-6 8373 ATCC, Emmons, 1942, bovine A. bovisB-7, B-8, and B-9 Thompson; Mayo Clinic, bovine A. bovisPine-6 A-12, Pine, Duke Univ., bovine A. bovisPine-7 P-1, Pine, Duke Univ., bovine A. bovisPine-9 P-2S, Pine, Duke Univ., bovine A. bovis387 CDC, bovine A. bovis4501 NCTC, Carnes; Australia, bovine A. bovisE-1 Kimball; Kansas State, equineE-3 Ennever; Ohio State, equineHuman tonsil isolates, nonactinomycoticT-5-2, T-5-3, T-12, T-13, T-47, T-66, Slack; W. Va. Microaerophilic actino-and T-97 mycetes

Dental calculi isolates, nonactinomycoticD-68, D-76, D-95, D-113, D-132, Slack; W. Va. Microaerophilic actino-

D-143, D-167, D-202, D-341, and mycetesD-342

Oral cavity isolates, nonactinomycotic286, C-294, C-319, C-323, C-334, Howell; NIH Actinosnyces naeslundii

C-342, C-389, C-452, CS-1750, andCS-1752

C-447, C-1021, CS-1181, CS-1759, Howell; NIHCS-1771, CS-1788, and CS-1786

Anaerobic diphtheroids, nonactinomycoticAD-3, AD-12, AD-29, AD-30, AD-32, King and Meyer; Chicago, bloodAD-35, AD-37, AD-39, and Al)-41 cultures

S-1 and S-2 King and Meyer; Chicago, skinZach Myers Clinic, W. Va., lungA-8, A-9, A-10, A-12, and A-13 Schain; Staten Island, bone marrowCorynebacterium6911, 6919, 6921, 6922, 6923, 11827, ATCC C. acnesand 11828

*-= Unnamed species.

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Group B. Group B antisera were sorbed withgroup A cells until all cross-fluorescence was re-

moved. This sorbed antiserum did not cross-reactwith groups A, C, or D. Table 2 lists the 15cultures that fluoresced with this sorbed anti-serum and were thus placed in group B.

It will be noted that there are isolates fromhuman and animal infections as well as from thenoninfected, human oral cavity in this group.

Also it can be seen that different species names

had been applied to the majority of these organ-

isms.Group C. Specific antiserum was prepared for

this group by sorption with group A cells.Thirteen isolates listed in Table 3 were placed inthis group, 2 from human infections and 11 fromeither tonsils or dental calculi.Group D. This antiserum showed the least

amount of cross-reaction in that there was weakfluorescence 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 notfluoresce with group A antiserum which indicatesthe possibility of some antigenic differences withinthis group. It is possible that there was inade-quate concentration of antigen or antibodies toprovide visible fluorescence. For specificity, thisantiserum was sorbed with group A cells.Except for group A the greater number of iso-

lates from human infections were placed in groupD-eight in all. Two isolates were from bovineinfections, ten from the oral cavity of man, andone from a blood culture. These are listed inTable 4. The majority of the cultures received anddesignated as A. israelii were found to be ingroup D, although some isolates designated as A.

TABLE 2. Group B cultures

Laboratory designation Source Genus and species

Human actinomycosis isolatesA-3 and A-4 Heilman; Mayo Clinic Actinomyces bovisA-5 Lawrence; Sterling-Winthrop *CC, King and Meyer; ChicagoAnimal actinomnycosis isolateP-i Kimball; Kansas State, porcineHuman tonsil isolates, nonactinomycoticT-6, T-46, and T-48 Slack; W. Va. Microaerophilic actinomy-

cetesDental calculi isolate, nonactinomycoticM-1 Ennever; Ohio State Actinomyces israeliiOral cavity isolates, nonactinomycoticC-286 and C-454 Howell; NIH Actinomyces naeslundii (?)C-456 and C-457 Howell; NIH A. naeslundiiC-959 Howell; NIH A. israeliiAnaerobic diphtheroid, nonactinomycoticA-ll Schain; Staten Island, bone marrow

= Unnamed species.

TABLE 3. Group C cultures

Laboratory designation Source Genus and species

Human actinomycosis isolatesANll Salvin; NIH Actinomyces bovisAN14 Salvin; NIH A. bovisHuman tonsil isolates, nonactinomycoticT-113 Slack; W. Va. Microaerophilic actinomyceteDental calculi isolates, nonactinomycotic1)-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

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TABLE 4. Group D cultures

Laboratorp designation Source Genus and species

Human actinomycosis isolatesHill King and Meyer; Chicago Actinomyces bovisCC2 King and Meyer; Chicago *Boyd Goldsworthy; Australia A. bovisLemke Goldsworthy; Australia A. bovisSlamon Goldsworthy; Australia A. bovis10048 ATCC, Emmons, 1946 A. bovis12102 ATCC, Howell, 1955 Actinomyces israelii12103 ATCC, Howell A. israeliiBovine actinomycosis isolatesPine-i A- Pine, Duke Univ. A. bovisi2836 ATCC Georg, CDC,i957 A. bovisOral cavity isolates, nonactinomycotic295, C-355, C-464, CS-963, CS-996, CS-1038, Howell; NIH A. israelii

CS-1042, CS-1064, and CS-1171CS-55 Howell; NIH Actinomyces naeslundiiAnaerobic diphtheroid, nonactinomycoticCC3 King; Chicago, blood

= Unnamed species.

TABLE 5. American Type Culture Collection strains

No. 'Species { Source isolated Person submitting Year SerologicalNo. Species ~~~~ ~~~~fromProsumtig submitted group

8373 Actinomyces bovis Bovine Emmons; NIH 1942 A8374* A. bovis Human Emmons: NIH 1942 A10048 A. bovis Human Emmons; NIH 1946 1)10049 A. bovis Human Emmons; NIH 1946 A12102 Actinomyces israelii Human Howell; NIH 1955 D12103 A. israelii Human Howell; NIH 1955 1)12104 Actinomyces naeslundii Human Howell; NIH 1955 A12836 A. bovis Bovine Georg; CDC 1957 D13031 A. bovis Bovine Meyer; Ill. 1958 A

* See note p. 975, Hazen and Little (1958).

bovis, A. naeslundii, and anaerobic diphtheroidswere also placed in this group.

Table 5 lists the actinomyces which were ob-tained from ATCC giving the original source andthe year in which they were sent to the collection.Five of these cultures were placed in group A andfour in D. There is no correlation between habitat,species, and the serological group. The first fourcultures listed have been maintained in thislaboratory for ten years and when compared withoriginal lyophilyzed specimens they each re-mained in their same serological group.

Culture 10048 has been received from at leastthree different sources and in each instance hasbeen placed in group D. The original culture of

this organism was very rough but over a period oftime has become quite smooth. Serologically it re-mained in group D.

Control cultures. Table 6 lists Corynebacterium,Propionibacterium, Lactobacillus, Streptomyces,and Nocardia species that were included in thesestudies because questions have been raised abouttheir relationships to the actinomyces. Theseorganisms were first carefully screened for auto-fluorescence and found negative. They were thenset up alone or as mixtures with the actinomycesfrom the various groups and stained with thedifferent antisera. There was no fluorescence withany of the group antisera.

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TABLE 6. Control cultures

Laboratorydesignation Source

Corynebacterium1 and 2 CDC

CDCCDC

_ CDCCDC

7, 26, and CDC27

296 ATCC373 ATCC10700 and ATCC

10701Propionibacterium4867 ATCC4870 ATCC4875 ATCC6207 ATCCLactobacillus

Doetsch;Md.

Williams;Pa.

314 and ATCC332

11146 ATCCStreptomyces11426 and ATCC

11429Barnett:

NocardiaF-8

F-9

W. Va.Barnett;W. Va.

Barnett;W. Va.

Barnett;W. Va.

Vet. Hosp.;Iowa

Vet. Hosp.;Iowa

Species

C. ulceransC. xerosisC. hoffmanniiC. ovisC. renaleC. diphtheriae

C. diphtheriaeC. xerosisC. pseudodiph-

theriticum

P. jensenjiP. freudenreichiiP. pentosaceumP. peterssonii

L. bifidus

L. bifidus var.

pennsylvanicusL. acidophilus

L. bifidus

S. griseus

S. intermedius

S. flavovirens

S. albus

S. scabies

N. asteroides

N. asteroides

* = Unnamed species.

DISCUSSION

The fluorescent antibody technique was used inthis study as a means of serologically grouping 138cultures of actinomyces. The results confirm theexistence of previously reported groups A and Bwhich were established by using the reciprocalagglutinin sorption technique. Two additional

groups have been confirmed and established bythe fluorescent antibody procedure and weredesignated as C and D.Two major difficulties occur in identifying and

classifying these organisms on the basis of sero-logical procedures. They are frequently veryrough or filamentous and thus agglutination testscannot be done, or their growth is so scanty thatinsufficient antigen is available. Both of thesedifficulties may be overcome by using the fluo-rescent antibody techniques. In preliminary testsusing suspensions of colonies taken directly fromstreak plates or from agar deeps the organismscould be readily identified. Also in mixed cultureswith bacteria the actinomyces could be selectivelyidentified by fluorescence.

Eighty-nine, or approximately two-thirds of thecultures listed, are in group A indicating that thisis the most widely distributed serological group.The isolates in this group are derived from humanand animal infections as well as from noninfectedhuman beings. Thus there is no one source ofthese organisms and their antigenicity is notdictated by their habitat. This same statementholds true for the organisms in group B, C, and D.

Thirty-two of the isolates had been identifiedas A. bovis, 14 from human sources and 22 fromanimals. Twenty-two of these were in group A,2 in B, 2 in C, and 6 in D. Fifteen were designatedas A. israelii. One is in group A, 2 are in B, and12 in D. Sixteen were called A. naeslundii, and ofthese 11 are in group A, 4 in B, and 1 in D. Thus,of those cultures designated by a species namethere was no correlation between the species nameand the serological group, although a preponder-ance of one or more were in a particular group.Twenty-two of the A. bovis cultures were in groupA. However, here again on a serological basis usingthis procedure no one species is found exclusivelyin any of these four groups.The term "anaerobic diphtheroid" is widely

used to designate organisms having morphologicalcharacteristics of a corynebacterium but whichcannot be or are not designated by a given speciesname. Nineteen of these cultures have been in-cluded in this study. Seventeen were found to begroup A and one each of B and D. Although thisstudy should be extended to include a widerselection of these organisms, this does indicate aserological relationship with the actinomyces andon this basis it could be tentatively proposed thatthey be included in the genus Actinomyces.

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Seven cultures of C. acnes were obtained fromATCC and all of these were placed in group A.This should be extended to include many moresuch isolates, nevertheless consideration shouldbe given to placing C. acnes in the genus Actino-myces. Serological relationships between theseorganisms have also been indicated by Beerens(1953) and Linzenmeier (1957).Moody and Jones (1960) were able to differ-

entiate 26 strains of Corynebacterium diphtheriaefrom 6 strains of diphtheroids by the fluorescentantibody technique. This further indicates thatthis procedure can be used to determine antigenicdifferences between these organisms.

Eight other species of Corynebacterium as wellas species of Propionibacterium, Lactobacillus,Streptomyces, and Nocardia were fluoresced witheach conjugated actinomyces antiserum and werenegative. Thus there is no antigenic relationshipbetween these species and the actinomyces asdetermined by the fluorescent antibody tech-nique. Sharpe (1955) showed definite serologicalrelationship between various species of Lacto-bacillus although she did not attempt to deter-mine antigenic relationship with other genera.Group A antiserum will fluoresce group A, B,

C, and D organisms. However, if the antiserum issorbed with B, C, and D antigens it will stillfluoresce group A cells. When group B and Cantiserum is sorbed with A cells, the antiserum nolonger cross reacts with other groups but isspecific for its respective group. Group D anti-serum will only cross-react with group A andsorption with group A cells removes this cross-reaction. Thus group A cells contain at least threeantigens or haptenes, one or more of which isspecific for group A, one in common with bothgroups B and C and one in common with D. At-tempts to chemically isolate these antigens havebeen initiated.As no correlation has been shown to exist be-

tween habitat, species designation, and serologicalgrouping, the question arises as to which speciesname or names should be applied to these organ-isms. Before this question can be satisfactorilyanswered there must be extensive comparativework done including morphological, physiological,and chemical characteristics of these isolates todetermine if one or more characteristics will cor-relate with the serological group. It is also possiblethat such characteristics as catalase, roughness,and animal pathogenicity will be found to be

variable to the degree that they do not supportspecies differentiation. If this should occur and ifserology becomes the basis for identification andclassification it seems that one species name couldbe applied to groups A, B, C, D, and others ifthey are established and then, possibly, typeswithin each group. This would also give rise tocontroversy as to which species name should begiven priority. However, as Erwin (1960) in hisextensive study established Actinomyces bovis asthe type species, this would have to be givenprime consideration.The fluorescent antibody technique is impres-

sive from the standpoint that it provides amethod of applying serological procedures tofilamentous and other organisms that have defiedserological procedures. It is hoped that otherworkers will attempt to use this as a tool to in-vestigate the intriguing problems of identificationand classification.

ACKNOWLEDGMENT

This work was supported by grant no. E-1801from the U. S. Public Health Service.

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