JOURNAL OF CLINICAL MICROBIOLOGY, Nov. 1982, p. 926-934 Vol. 16, No. 50095-1137/82/110926-09$02.00/0Copyright © 1982, American Society for Microbiology

Development and Evaluation of a Biochemical Scheme forIdentification of Endocervical Lactobacilli

JOAN E. FAGNANT, CHRISTINE C. SANDERS,* AND W. EUGENE SANDERS, JR.

Department of Medical Microbiology, Creighton University School of Medicine, Omaha, Nebraska 68178

Received 4 June 1982/Accepted 18 August 1982

A biochemical scheme for the species identification of endocervical lactobacilliwas developed and evaluated with 10 isolates obtained from the American TypeCulture Collection (ATCC) and 106 endocervical isolates obtained from womenreporting to a local venereal disease clinic and a local hospital clinic. The schemeconsisted of two stages. Stage I included six tests and was tested and modifiedwith results obtained with ATCC strains. From the modified stage I, stage II wasdeveloped. Tests to be performed in this stage were determined from expectedcharacteristics of lactobacilli. Stage II was also tested with the ATCC strains. Ofthe 106 endocervical isolates, 78 (74%) were identified with the two-stage schemeas developed with the ATCC strains. Unexpected results were obtained in one orboth stages with the other 28 isolates. For 10 isolates, the final species identifiedwere not previously expected to be recovered. A "best-fit" method was used todetermine the most likely identification of the remaining 18 isolates. In a fewinstances, the use of a third stage was necessary to reach an identification. Thefinal identification scheme, although complicated in appearance, generated aspecies identification with a total of 12 tests with a range of 7 to 10 tests perisolate.

Lactobacilli are ubiquitous microorganismsinhabiting numerous ecological niches. Thereare many reports in the literature on the identifi-cation of lactobacilli from a variety of sources.Briggs (2), in 1953, was the first to attempt tostandardize the classification of lactobacilli byphysiological tests. Serology, colonial morphol-ogy, nutritional requirements, and cultural, cel-lular, and macromolecular characteristics havealso been utilized in attempts to develop anidentification scheme (4, 6, 7, 10, 11, 13, 14, 16,17). However, to date, there is no widely accept-ed scheme for the identification of species withinthe genus Lactobacillus. There are several rea-sons for the lack of a simple, broadly applicableidentification scheme: (i) the particular speciespresent vary widely from niche to niche; (ii)lactobacilli are usually considered to be non-pathogenic; (iii) the lactobacilli are relativelyfastidious organisms.The results of a previous study indicated that

endocervical lactobacilli may play a role inresistance of females to gonorrhea (15). Wewished to pursue these studies further to deter-mine whether any particular species were moreimportant than others in this natural defensemechanism. However, there was no simple iden-tification scheme in the published literature.Therefore, the present study was designed todevelop and evaluate a simple but reliable identi-

fication scheme for the lactobacilli inhabiting thefemale genital tract.

MATERIALS AND METHODSStrains. Stock strains obtained from the American

Type Culture Collection (ATCC), Rockville, Md.,were (i) L. acidophilus ATCC 4356; (ii) L. leichmanniiATCC 4797; (iii) L. lactis ATCC 12315; (iv) L. lactisATCC 8000; (v) L. brevis ATCC 14869; (vi) L. fermen-tum ATCC 14931; (vii) L. salivarius subsp. salivariusATCC 11741; (viii) L. cellobiosus ATCC 11739; (ix) L.cellobiosus ATCC 11740; and (x) L. casei subsp.rhamnosus ATCC 7469. A total of 106 endocervicalisolates were obtained from women reporting to a localvenereal disease clinic and a local hospital clinic.

Endocervical cultures. Endocervical specimens wereobtained with a sterile calcium alginate swab (Calgi-swab, Inolex Corp.). The swab was immediatelyplaced in 1 ml of eugonic broth (Difco Laboratories)and blended in a Vortex mixer for 3 min. The surfacesof a dextrose starch agar (Difco) plate supplementedwith 5% sheep blood (Colorado Serum Co.) and alactobacillus selective agar (LBS, BBL MicrobiologySystems) plate were inoculated with swabs dipped intothe eugonic broth. The plates were streaked for isola-tion in the four-quadrant fashion and incubated in acandle jar for 48 h at 37°C. Thus, only aerobic andfacultatively anaerobic lactobacilli were examined.

Identification of lactobacilli. Gram-positive, cata-lase-negative rods forming small translucent colonieson LBS were identified presumptively as lactobacilli.To ensure a pure isolate, a single-colony cloningprocedure was employed. This involved aspirating a

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IDENTIFICATION OF ENDOCERVICAL LACTOBACILLI 927

TABLE 1. Stage I identification testsa

Value

Group 1 test assigned for Group 2 testa positivetestb

Acid from glucose 1 Acid from lactoseGas from glucose 2 Acid from mannitolAcid from amygdalin 4 Acid from galactose

a Sum of results from group 1 tests = first digit ofstage I identification value (0 to 7). Sum of results fromgroup 2 tests = second digit of stage I identificationvalue (0 to 7).

b All negative tests were assigned a value of zero.

colony into a sterile Pasteur pipette from the agarsurface with the use of a dissecting microscope (30x).The colony was placed into broth and incubated for 24to 48 h. The broth was subcultured again onto agar.From this subculture, a single colony was selected asdescribed above, and the cloning procedure was re-peated again. Further tests, including some or all ofthe following, were performed on each isolate foridentification.

Tests for the fermentation of glucose, lactose, man-nitol, galactose, cellobiose, salicin, sorbitol, and xy-lose were performed in LBS broth base (14). Thecarbohydrates were added to the LBS broth base in afinal concentration of 2%. For all except glucose,sterile solutions of the carbohydrates were added tothe broth base after autoclaving. The final volume ofthese media was 3 ml. Glucose was added to the basebefore autoclaving, and the media were dispensed in 8-ml aliquots. A Durham tube was inverted into eachglucose-containing medium, and the medium was thensterilized. All tubes in which the Durham tubes con-tained bubbles after sterilizing were discarded. Thefinal pH was 5.9.The medium for amygdalin utilization consisted of

1.5% neopeptone, 0.1% Tween 80, 0.6% yeast extract,0.15% agar, and 0.4% chlorophenol red (19). Amygda-lin was added to the sterile medium in a final concen-tration of 0.2%. The final pH was 7.0 to 7.2.The inoculum was prepared by suspending the

growth (after single-colony cloning) from a 2-day eu-gonic agar or blood agar plate in 9 ml of saline. Thesaline was centrifuged to obtain a pellet. The superna-tant was discarded, and the pellet was resuspended inthe small amount (ca. 0.2 ml) of saline remaining in thetube. Each tube was inoculated with one or two dropsof this heavy cell suspension (ca. 109 colony-formingunits/ml). After inoculation of the glucose medium, 1ml of melted sterile paraffin was layered over the topof the medium. Inoculated tubes were incubated for 14days in 10%o CO2 in air at 37°C and were examined ondays 1, 2, 4, 7, and 14. Most color changes indicating apositive test occurred within 2 days. In the LBS broth,a color change from slightly green to lemon yellowindicated a positive reaction. For the amygdalin test, acolor change from dark maroon to scarlet red with ayellow tinge was a positive reaction.The arginine hydrolysis medium consisted of 0.3%

yeast extract, 0.5% peptone, 1% sodium acetate, 0.3%glucose, 0.1% Tween 80, and 0.5% salt solution (3, 14).The final pH was 7.4. The sterile L-arginine-hydro-chloride was added in a final concentration of 0.3%

after the medium was sterilized. Tubes were inoculat-ed in the same manner as the LBS tubes and were

incubated for 10 days at 37°C in 10o C02 in air. Apositive test consisted of an orange color after Nesslerreagent was added (8).The ability of a strain to grow at 45°C was tested in 5

ml of Eugon broth. Inoculated tubes were incubated ina candle jar at 45°C until growth was apparent. Thepositive tubes were then subcultured to ensure that nocontamination had occurred. All negative tubes were

held for 7 days before being discarded.

RESULTS

A scheme was developed for the identificationof species of lactobacilli reportedly isolated fromhumans (9). It was designed to identify thesespecies with a minimum number of biochemicaltests.

Stage I. Stage I was developed from thosecharacteristics of lactobacilli commonly recov-ered from human sources and listed in Bergey'sManual of Determinative Bacteriology (9). Aminimum of six tests appeared to be sufficient toyield an identification of most species. Thesewere (i) acid from glucose, (ii) gas from glucose,(iii) acid from amygdalin, (iv) acid from lactose,(v) acid from mannitol, and (vi) acid from galac-tose. Each positive test was assigned a numeri-cal value (Table 1). Each negative test wasassigned a value of zero. The sums of the valuesof the first three tests and the second three testswere determined, and these sums provided a

two-digit numeral. The two-digit stage I valuewas used to provide an initial identification(Table 2).The validity of this scheme was determined by

testing 10 reference strains of various species a

TABLE 2. Identification of lactobacilli by usingstage I valuesa

Expected Other possiblestage I Most likely species speciesvalue

74, 75 L. cellobiosus57 L. casei subsp. rhamnosus55 L. acidophilus L. leichmannii54 L.jensenii51 L. leichmannii37 L. brevis36 L. brevis35 L. fermentum L. brevis34 L. brevis33 L. brevis32 L. brevis31 L. brevis30 L. brevis17 L. salivarius15 L. lactis

a Derived from results of stage I tests as predictedby characteristics listed in Bergey's Manual ofDeter-minative Bacteriology (9).

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TABLE 3. Stage I values modified from results ofmultiple tests with nine reference strains to include

inherently variable tests

Stage I Most likely species Other possiblevalue species

74, 75 L. cellobiosus57 L. casei subsp. rhamnosus L. cellobiosus55 L. acidophilus L. leichmannii54 L.jensenii51 L. leichmannii50 L. leichmannii37 L. brevis L. cellobiosus36 L. brevis35 L. fermentum, L. brevis L. cellobiosus34 L. brevis L. cellobiosus33 L. brevis32 L. brevis31 L. brevis30 L. brevis17 L. salivarius15 L. lactis L. acidophilus

minimum of four times. All but three strainsgenerated the stage I value predicted by Ber-gey's Manual ofDeterminative Bacteriology forthe species they represented in the majority (68to 100%) of replicate tests. One strain, L. lactis(ATCC 12315), was highly variable in the stage Ivalues it generated. In 68% of the replicate tests,it generated a value corresponding to L. leich-mannii. It deviated from the expected results infour major tests and was therefore considered tobe inappropriate for use as a control strain forthis species. Neither L. cellobiosus controlstrain generated the stage I value expected.Amygdalin was not utilized by either strain, thusgenerating a stage I value of 35 that corre-sponded to an identification of L. fermentum.According to Bergey's Manual ofDeterminativeBacteriology, an additional distinguishing char-

acteristic between L. cellobiosus and L. fermen-tum is the ability of the former but not the latterto ferment cellobiose. Since both ATCC strainsof L. cellobiosus fermented cellobiose, theywere considered to be valid control strains.Stage I was modified, however, to include L.cellobiosus under "other possible species" forstrains generating a stage I value of 34 or 35.

Further modifications to stage I were neces-sary when it became apparent that variableresults were obtained in certain specific testseven when they were performed in an identicalfashion with the same control strain. The vari-able tests were amygdalin for L. acidophilus,lactose for L. leichmannii, and mannitol and gasfrom glucose for L. cellobiosus. Results of thesetests varied from the expected in over 10% oftests, indicating that the test was inherentlyvariable for the particular species. The interpre-tation of stage I values was modified to includevalues generated due to "inherently variabletests" (Table 3). With the modifications indicat-ed, stage I should be correct at least 90% of thetime.

Stage II. From the modified stage I, a secondstage of tests was developed to distinguish be-tween those species giving the same stage Ivalues and to verify the species suggested bystage I. As before, the tests to be performed instage II were determined from characteristicslisted in Bergey's Manual ofDeterminative Bac-teriology and evaluated with the nine referencestrains. The tests performed in stage II varieddepending upon the stage I value and represent-ed the minimum number of tests required todifferentiate the strain from all species consid-ered capable of generating the stage I value. Thestage II scheme and expected results for eachspecies are shown in Table 4.

TABLE 4. Stage II identification of lactobacilliStage I value Stage II test (result) Identification

74, 75 Cellobiose (+) L. cellobiosus57 Cellobiose (+); sorbitol (+) L. casei subsp. rhamnosus

Cellobiose (+); sorbitol (-) L. cellobiosus55 Cellobiose (+); arginine (+) L. Ieichmannii

Cellobiose (+); arginine (-) L. acidophilus54 Cellobiose (+) L. jensenii51 Cellobiose (+); arginine (+) L. leichmannii50 Cellobiose (+); arginine (+) L. Ieichmannii

37, 34 Cellobiose (+); growth at 45C (-) L. cellobiosusCellobiose (-); growth at 45°C (-) L. brevis

36 Cellobiose (-); growth at 45°C (-) L. brevis35 Cellobiose (-); growth at 45°C (+) L. fermentum

Cellobiose (-); growth at 45°C (-) L. brevisCellobiose (+); growth at 45°C (-) L. cellobiosus

30-33 Cellobiose (-); growth at 45°C (-) L. brevis17 Cellobiose (-); sorbitol (+) L. salivarius15 Cellobiose (-) L. lactis

Cellobiose (+) L. acidophilus

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IDENTIFICATION OF ENDOCERVICAL LACTOBACILLI 929

TABLE 5. Identification of lactobacilli recovered on endocervical cultures

Stage I Stage II

No. ofValue isolates No. of Identification

obtained generating Test (result) isolatesvalue giving result

75 5 Cellobiose (+) 5 L. cellobiosus74 4 Cellobiose (+) 4 L. cellobiosus55 23 Cellobiose (+); arginine (+) 6 L. leichmannii

Cellobiose (+); arginine (-) 17 L. acidophilus54 24 Cellobiose (+) 24 L. jensenji50 11 Cellobiose (+); arginine (+) 11 L. leichmannii35 6 Cellobiose (-); growth at 45°C (+) 2 L. fermentum

Cellobiose (-); growth at 45°C (-) 4 L. brev'is17 1 Cellobiose (-); sorbitol (+) 1 L. salivarius15 4 Cellobiose (+) 4 L. acidophilus

When the control strains were used to assessthe validity of the stage II tests, all strainsproduced the expected results in over 80% ofreplicate runs of each test, with one exception.L. brevis fermented cellobiose in three of fourtests. L. brevis and L. cellobiosus differed intheir ability to ferment cellobiose and amygda-lin. The only situation in which it would benecessary to differentiate between these twoorganisms in stage I is in the instance of a rareamygdalin-negative strain of L. cellobiosus.Therefore, the cellobiose test was left in thescheme with the assumption that the L. breviscontrol strain was a rare cellobiose-positive iso-late.

Use of the scheme for the identification ofendocervical lactobacilli. A total of 106 isolates oflactobacilli were obtained from endocervicalcultures from 72 women. The species of Lacto-bacillus to which a strain belonged could bedetermined for 78 (74%) of the strains (Table 5).The remaining 28 isolates generated either unex-pected stage I values or unexpected stage IIresults or both. Modification and expansion ofthe two-stage scheme was required for identifi-cation of these 28 isolates.Ten isolates gave unexpected stage I values.

A list of species most likely to have given theunexpected stage I values was prepared by (i)assuming that the first digit of the stage I valuewas correct and selecting the most likely speciesfrom Table 3 to generate the first number, (ii)selecting the most likely species from Table 3that could generate the unexpected value ob-served by assuming only one of the six stage Itests was aberrant, and (iii) selecting additionalspecies listed in Bergey's Manual ofDetermina-tive Bacteriology that could have generated theunexpected stage I value. These last specieswere not included in the scheme originally asthere was no indication that they had ever beenrecovered from the human genital tract. Afterthe most likely species had been determined

(from i to iii), stage II tests were devised todifferentiate among them. The isolate was finallyidentified by using a "best-fit" analysis, i.e., foreach species on the list, the number of aberranttest results was determined, and the species withthe fewest aberrant tests was selected. With thissystem, the most likely species for the 10 iso-lates were determined (Table 6).A similar method of "best-fit" analysis was

employed to determine the identification of theremaining 18 isolates that gave unexpected stageII results based on their stage I values. Criteriaused in selecting the most likely species were: (i)an assumption that the stage I value was correctand the aberrant result was in stage II, (ii) anassumption that the stage II result was correctand the aberrant result was in stage I, and (iii)selection of additional species from Bergey'sManual ofDeterminative Bacteriology. In manycases, application of these three criteria generat-ed only one species for which a single character-istic was aberrant (Table 7). Thus, no furthertests were performed as this species was themost likely. In other instances, additional tests(Stage III) were necessary to determine theidentification from among several possible spe-cies.

Final identification scheme. By using results oftests with the 106 isolates, a final identificationscheme was derived (Table 8). The schemegenerated in two or three stages either (i) a"final identification" if the strain possessedcharacteristics expected of a particular species(Table 4), or (ii) a "most likely identification" ifthe strain possessed aberrant characteristics anda "best-fit" analysis was applied. This scheme,although complicated in appearance, requiredthe use of only a total of 12 tests, with a range of7 to 10 tests per isolate.

DISCUSSIONSince Doderlein (5) first described the lactoba-

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cilli in the vagina in 1892, most early investiga-tions assumed that the lactobacilli recoveredfrom the female genital tract were all membersof one species, L. acidophilus. However, it hasbeen shown subsequently that these organismsdo not form a homogeneous group (12). Thus, itwas apparent that any study of the species ofendocervical lactobacilli would require the useof a simple but reliable identification scheme. Asearch of the literature revealed that no suchscheme exists (6, 9, 18). Most identificationschemes involved the use of a multitude of tests,some of which would require expensive equip-ment (9, 12, 14, 16-18). Thus, an identificationscheme was developed in this study that wouldallow maximum identification of lactobacilliwith the use of a minimum number of biochemi-cal tests.With the identification scheme developed for

this study, most isolates (74%) of lactobacillirecovered from the genital tract of women werereadily identified. The remaining 26% were iden-tified by using a best-fit analysis. With somespecies, there were several strains that variedfrom the expected reactions according to Ber-gey's Manual ofDeterminative Bacteriology (9).For example, only 82% of strains of L. acidophi-lus fermented amygdalin. This test was found tobe inherently variable for the species, as theATCC type strain fermented amygdalin only86% of the time. The same was true for thefermentation of lactose by L. leichmannii. Threeof five strains of L. delbrueckii did not fermentglucose. Since we had no ATCC type strain withwhich to compare these lactobacilli, the best-fitanalysis was used to designate these as L. del-brueckii. Of the species of lactobacilli listed inBergey's Manual ofDeterminative Bacteriology(9), L. delbrueckii is the most asaccharolytic.Another aberrant result was found to occur intests with all endocervical isolates of L. caseisubsp. rhamnosus, but in no instance with theATCC type of the species. No endocervicalisolate fermented sorbitol. A stage I value of 57was the only value requiring not only a stage IIbut a stage III determination as well. Stage IIIconsisted of salicin and xylose to distinguishamong three possible species: (i) sorbitol-nega-tive L. casei subsp. rhamnosus, (ii) L. cellobio-sus producing no gas from glucose, or (iii) L.xylosus. Since all strains fermented salicin butnot xylose, the best-fit analysis designated thestrains as sorbitol-negative L. casei subsp.rhamnosus. This designation was also supportedby the VPI identification scheme for the lactoba-cilli, which lists weak acid production fromsorbitol for this species (18). Thus, from thisanalysis, it appears that the scheme developed inthis study permits a relatively reliable identifica-tion of lactobacilli recovered from the endocer-

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IDENTIFICATION OF ENDOCERVICAL LACTOBACILLI 933

vix. Most deviations from Bergey's Manual ofDeterminative Bacteriology were due either toinherent variability in tests or to the isolation oftoo few strains of a particular species for accu-rate analysis.A comparison of results generated by this

identification scheme with those from morecomplex schemes used by other investigatorsalso helps to confirm its simplicity and reliabil-ity. In 1955, Davis (3) used an identificationscheme employing over 30 tests to identify 473strains of lactobacilli isolated from the humanmouth. Of these 30 tests, 8 were used in thepresent scheme. A comparison of the results oftests performed by Davis (3) that were includedin the scheme developed in the present studyrevealed excellent agreement between the twostudies. The only disagreement with the charac-teristics listed by Davis was the ability of L.brevis to grow at 45°C. This characteristic wasnot observed in the present study, nor was itexpected to be observed according to Bergey'sManual of Determinative Bacteriology. Also incontrast to Bergey's Manual of DeterminativeBacteriology, but in agreement with this study,Davis found only 80% of 146 strains of L.fermentum capable of fermenting lactose. Thus,the results obtained by Davis support the valid-ity of the identification scheme developed here.There has only been one report in the literatureconcerned solely with the identification of hu-man vaginal lactobacilli. Rogosa and Sharpe(12), in 1960, isolated 35 strains of lactobacillifrom 21 women and studied 21 of the strains indetail. Rogosa and Sharpe found four species oflactobacilli to be present in the vagina: (i) L.acidophilus, 14 strains; (ii) L. casei subsp. rham-nosus, 2 strains; (iii) L. fermentum, 4 strains;and (iv) L. cellobiosus, 1 strain. The reactionsobtained by Rogosa and Sharpe in their identifi-cation scheme were identical to those obtainedin the present investigation, with one exception.Both of their strains of L. casei subsp. rhamno-sus fermented sorbitol. Nineteen tests were per-formed by those investigators, of which 11 wereutilized in the present investigation. Thus, fromthese comparisons, it appears that the newlydevised scheme provides a relatively reliableidentification of endocervical lactobacilli.To establish highly reliable identification

schemes, phenotypes generated with biochemi-cal tests must be considered in combination withgenotypes determined in studies of DNA con-tent and relatedness (1). London has recentlyreviewed the taxonomic status of the lactobacillibased upon these and other diverse characteris-tics (6). However, the purpose of this study wasnot to establish a precise taxonomic descriptionof the lactobacilli. Rather, it was to develop apractical approach to the identification of those

species commonly recovered from the femalegenital tract. To this end, the scheme developedhas the advantage over previous schemes ofusing fewer and relatively simple tests. Thereliability of the identification generated by thisscheme appears to be at least as good as morecomplex schemes (3, 12, 14, 18).

ACKNOWLEDGMENTS

This work was supported by Public Health Service grant 5R01-AI-11584 from the National Institute of Allergy andInfectious Diseases.We thank the staffs of Equilibria Medical Center and

Creighton Health Clinic OB-GYN for their assistance in thisstudy.

LITERATURE CITED

1. Brenner, D. J. 1980. Taxonomy, classification, and no-menclature of bacteria, p. 1-6. In E. H. Lennette, A.Balows, W. J. Hausler, Jr., and J. P. Truant (ed.), Manualof clinical microbiology, 3rd ed. American Society forMicrobiology, Washington, D.C.

2. Briggs, M. 1953. The classification of lactobacilli bymeans of physiological tests. J. Gen. Microbiol. 9:234-248.

3. Davis, G. H. G. 1955. The classification of lactobacillifrom the human mouth. J. Gen. Microbiol. 13:481-493.

4. Davis, G. H. G., K. A. Bisset, and C. M. F. Hall. 1955.Correlation between morphological and physiologicalcharacters in the classification of members of the genusLactobacillus. J. Gen. Microbiol. 13:68-71.

5. Doderlein, A. 1892. Das Scheidensekret und seine Bedeu-tung fur das Puerperalfieber. Leipzig.

6. London, J. 1976. The ecology and taxonomic status of thelactobacilli. Annu. Rev. Microbiol. 30:279-301.

7. McDonald, I. J., and W. C. Frazier. 1951. Variation inmorphology of colonies of lactobacilli. J. Bacteriol.61:627-637.

8. Niven, C. F., Jr., K. L. Smiley, and J. M. Sherman. 1942.The hydrolysis of arginine by streptococci. J. Bacteriol.43:651-660.

9. Rogosa, M. 1974. Genus Lactobacillus Beijerinck 1901, p.576-593. In R. E. Buchanan and N. E. Gibbons (ed.),Bergey's manual of determinative bacteriology, 8th ed.The Williams & Wilkins Co., Baltimore.

10. Rogosa, M., J. G. Franklin, and K. D. Perry. 1961. Corre-lation of the vitamin requirements with cultural andbiochemical characters of Lactobacillus spp. J. Gen.Microbiol. 25:473-482.

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