JOURNAL OF CLINICAL MICROBIOLOGY, Jan. 1975, p. 15-24Copyright © 1975 American Societv for Microbiology

Vol. 1, No. 1Printed in U.S.A.

Comparison of Three Procedures for Biochemical Testing ofAnaerobic Bacteria

H. B. MOORE, V. L. SUTTER, AND S. M. FINEGOLD*San Diego State University, San Diego, California 92115, Wadsworth Hospital Center, Veterans

Administration, Los Angeles, California 90073,* and Department of Medicine, UCLA Medical Center, LosAngeles, California 90024

Received for publication 6 September 1974

The Analytab Products, Inc. (API), anaerobic multitest microsystem (MI-CRO) was compared with the Center for Disease Control conventional (CONV)thioglycolate (supplemented with hemin and vitamin Kl) system and withpre-reduced anaerobically sterilized (PRAS) media as recommended by theVirginia Polytechnic Institute. Growth from a solid medium was suspended toproduce standard inocula. Substrates included 16 carbohydrates, indole, urea,gelatin, and esculin. API strips were inoculated in air and incubated in GasPak(BBL) jars. MICRO tests were read at 1 and 2 days, CONV tests at 1, 2, and 7days, and PRAS tests at 3 weeks. One hundred thirty well-characterized strainsof anaerobes (76 gram-negative rods, 16 cocci, 26 gram-positive nonsporeformingrods, and 12 clostridia), including 48 reference strains, were studied. Of 2,600tests performed, 2,085 (80.2%) showed agreement with all three methods. Therewas 90.9% agreement between the MICRO and CONV, 84.9% between theMICRO and PRAS, and 84.6% between the CONV and PRAS tests. All MICROtests were reliable except for indole, which was not sensitive enough, and gelatin,which was very insensitive. The MICRO system permits performance ofbiochemical tests at the workbench in the average clinical laboratory without theneed for expensive equipment and time-consuming procedures.

Currently there is considerable interest in thedevelopment and use of modified (abbreviated)biochemical test procedures in clinical microbi-ology. Most systems have been developed foridentification of organisms that grow rapidlyand are enzymatically quite active, such asEnterobacteriaceae. It should be noted, how-ever, that buffered (rapid) substrates were em-ployed by Pickett et al. some time ago forspeciation of Brucella (4, 6) and subsequentlyfor relatively rapid identification of nonfermen-tative gram-negative bacteria (5). Several com-mercial systems are available and share thefollowing objectives: simplicity, use of smallvolumes of substrate, more rapid results, andreactivity patterns that are reproducible andcorrelate well with those patterns recognized asessential for identification.

Starr et al. (10) pointed out the need forsimpler, more rapid, and less costly systems foridentification of anaerobes. They compared amicromethod multitest system (API AnaerobeSystem, Analytab Products, Inc.) with the con-ventional test procedures employed at the Cen-ter for Disease Control (CDC). The conven-tional media were maintained in an anaerobicchamber for at least 48 h before use. Both sys-

tems were inoculated and incubated in ananaerobic chamber. With notable exceptions,such as tests for nitrate reduction, H2S produc-tion, and indole production, there was over 90%agreement between the two systems. A similarAPI system has been used effectively for identi-fication of Enterobacteriaceae (9, 13).

Since most clinical laboratories do not haveanaerobic chambers and since there is evidenceto indicate that strains which have been iso-lated from clinical specimens are quite tolerantto exposure to air during subculture, etc. (1, 7;F. P. Tally et al., Abstr. Annu. Meet. Amer.Soc. Microbiol. 1973, M60, p. 83), we evaluatedthe API Anaerobe System (MICRO), inoculat-ing the strips under aerobic conditions andincubating them anaerobically in GasPak jars(BBL). These results were compared with con-ventional (CONV) test results obtained byinoculating steamed and cooled media undernormal atmospheric conditions and with resultsobtained with pre-reduced anaerobically steri-lized (PRAS) media.

MATERIALS AND METHODSBacterial strains. The 130 strains of anaerobic

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TABLE 1. Anaerobic bacteria included in comparisonof three procedures for biochemical testing of

anaerobic bacteria

Microorganism or group

Bacteroides fragilis (2 subsp. distasonis, 21subsp. fragilis, 1 subsp. ovatus, 13 subsp.thetaiotaomicron, 8 subsp. vulgatus, 7"other") ..............................

B. hypermegas ..........................

B. melaninogenicus (2 subsp. asac-

charolyticus, 6 subsp. intermedius, 5subsp. melaninogenicus) ...............

Fusobacterium (1 each necrogenes, necro-

phorum, nucleatum, mortiferum, 6varium).

Peptococcus (1 Peptococcus species, 1 mag-nus, 2 prevotii, 1 saccharolyticus, 2variabilis).

Peptostreptococcus (2 anaerobius, 1 mi-cros).

Acidaminococcus fermentans .............

Veillonella (2 alcalescens, 2 parvula) ......

Megasphaera elsdenii ....................

Eubacterium (1 aerofaciens, 1 alac-tolyticum, 1 lentum, 1 limosum, 1 tor-tuosum, 1 ventriosum) .................

Arachnia propionica .....................

Propionibacterium (2 acnes, 1 avidum, 1granulosum) ..........................

Actinomyces (1 each bovis, israelii, naes-

lundii, viscosus) .......................

Bifidobacterium (1 adolescentis var. A, 1adolescentis var. D, 1 bifidum var. B, 2breve, 1 eriksonii, 1 infantis subsp. infan-tis, 1 infantis subsp. liberorum, 2 longumsubsp. longum) ........................

Lactobacillus catenaforme ...............

Clostridium (4 innocuum, 1 oceanicum, 3sporogenes, 4 ramosum) ................

Total organisms included in study ........

bacteria included in this study are grouped in Table 1.Forty-eight of these are type, neotype, or referencestrains; see Table 2. The remaining 82 strains were

obtained from the Wadsworth Anaerobic BacteriologyLaboratory (WAL) collection and were isolated eitherfrom normal human feces or from clinical specimens.MICRO system. The API Anaerobe System physi-

cally resembled the API system forEnterobacteriaceae and the API System employed byStarr et al. (10) for anaerobes, but differed in some

substrate components. The 20 substrates includedindole, urea, glucose, mannitol, lactose, sucrose, mal-tose, salicin, xylose, arabinose, gelatin, esculin, glyc-erol, cellobiose, mannose, melezitose, raffinose, sor-

bitol, rhamnose, and trehalose.CONV system. Conventional fermentation media,

indole, gelatin, and esculin broth were preparedaccording to Dowell and Hawkins (2), but vitamin K1

and hemin were added to each to give final concentra-tions of 0.1 gg of vitamin K, per ml and 5.0 jg ofhemin per ml. The rapid urease procedure recom-mended by Sutter et al. (11) was used.PRAS media system. PRAS media consisting of

the same 20 substrates as the API System, plusnecessary media for gas-liquid chromatography(GLC), were prepared as directed by Holdeman andMoore (3). During this investigation, vitamin K1 andhemin were not incorporated in the PRAS media.(Subsequently, W. E. C. Moore [personalcommunication] has indicated that vitamin K1 andhemin should be added to all PRAS media.)

Procedure. Stock cultures frozen with skim milkwere removed from the freezer (-70 C) as needed.These were inoculated into tubes of maintenancethioglycolate broth (BBL 135-C) supplemented withNaHCOs, 1 mg/ml, and vitamin K1, 0.1 ug/ml (8), andonto two brucella (Pfizer) blood agar vitamin K1(BAK1) plates. One plate was incubated aerobicallyand one plate anaerobically (GasPak) at 35 C for 48 to72 h to check for purity. After appropriate anaerobicgrowth, colony morphology and Gram stains wereobserved and recorded. Slide catalase and spot indoletests (12) were also performed. A loopful of growth wasused to inoculate a tube containing 0.5 ml of Stuart etal. urea broth (Difco). This test was read after aerobicincubation at 35 C overnight. Sufficient growth (20 ormore colonies) was emulsified in 10 to 12 ml ofthioglycolate medium without dextrose or indicator(Difco 0432), supplemented with 0.1 ug of vitamin K1per ml and 5.0 gg of hemin per ml, to match aMcFarland no. 1 nephelometer standard. This sus-pension was used to inoculate the MICRO, CONV,and PRAS systems.We inoculated the PRAS tubes first, using a

roll-tube inoculator while flushing with oxygen-freeCO2. Tubes were incubated at 35 C for 3 weeks andthen were read according to the Virginia PolytechnicInstitute Anaerobe Laboratory Manual (3). End prod-ucts of metabolism were determined by GLC, whereindicated, by the procedure described by Sutter et al.(11). Next, the standardized suspension was used toinoculate the CONV media, after the tubes had beensteamed for 10 min and cooled, and sterile carbohy-drate solution had been added as needed. Sterilecapillary pipettes were used to inoculate a droplet tothe bottom of each tube. Tubes were incubated at35 C and read at 24 and 48 h; a final reading was madeat 7 days.

Finally, the standardized suspensions were used toinoculate the MICRO system. Each strip was pre-pared by writing the culture number in the center andplaced in the incubation tray provided, to which 4 mlof water was added. Large capillary pipettes (sterile)and rubber bulbs were used to inoculate the tubes.The tube and cupule of gelatin were filled, but onlythe tube section of the other substrates was filled. Inearly tests, no mineral oil was added to the indole test;later, mineral oil was added to the indole cupule. Aseach strip was filled, it was placed in a metal rackdesigned to hold 12 strips. After the rack was filledwith strips, a catalyst basket was taped to the upperinner surface of a GasPak 100 anaerobic jar which had

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been modified to lie in a horizontal position. Anindicator strip was placed in the jar so that it wouldnot come in contact with the catalyst container. Thetop 2 1/8 inches (5.4 cm) was cut from a GasPakenvelope. An 8-ml amount of water was added to theGasPak envelope, and it was then placed at the side ofthe rack in the jar. The GasPak envelope was tiltedslightly away from the rack and rested on the upperwall of the jar. The lid was clamped on the jar, and thejar was placed horizontally in an incubator at 35 C.The strips were examined through the unopened jar at24 h and the results were recorded. At 48 h, the jar wasopened and the final readings were made. To test forindole, a drop of xylene was added on top of the oiland mixed with a toothpick; then a drop of Ehrlichreagent added. A red color indicated a positive test.Urease activity was indicated by a red color (phenolred). A positive gelatin test was indicated by disper-sion of the carbon particles. Esculin hydrolysis wasindicated by a brownish-black color which was furtherexamined for lack of fluorescence when exposed to a366-nm wavelength ultraviolet light. The carbohy-drate substrates contained bromocresol purple (BCP)indicator; therefore, yellow indicated fermentationand purple indicated lack of fermentation. If theindicator was reduced (colorless), a drop of 0.04%aqueous BCP was added to the tube before thereading was made.

Results of the three procedures were compared withone another and with expected reactions listed byDowell and Hawkins (2), Holdeman and Moore (3),and Sutter et al. (11). With 41 organisms, the MI-CRO, CONV, and PRAS results were recorded onseparate data sheets along with supplementary infor-mation. These 123 data sheets were coded and scram-bled, and identification by the reaction schemes citedabove was attempted.

RESULTS

Table 2 shows the results obtained with the 48type, neotype, and reference strains in all threesystems. The greatest disagreement with ex-pected reactions occurred with gelatin, sinceonly one MICRO gelatin test was positive. Also,the disagreements were greater with organismsthat are slow growers and weak acid producers,such as many of the gram-positive non-sporeforming anaerobic rods.Table 3 summarizes the results obtained with

130 strains, including the 48 type, neotype, andreference strains. The overall agreement of thethree systems was 80.2%. When the very poorresults with gelatin were discounted, the overallcorrelation increased 82.8%. For the 76gram-negative rods, the overall agreement was80.1% (83.3% when gelatin results were omit-ted). For the 16 cocci the overall agreement was94.1% (97.0% without gelatin). Agreement forthe 26 gram-positive nonsporeforming rods was71.3% (71.9% without gelatin). Agreement for

the 12 clostridia was 82.5% (85.1% withoutgelatin).Although indole results showed good overall

agreement, there were nine negative results bythe MICRO system when either the PRAS orCONV or both were positive. Most of thesediscrepancies occurred early in our studies.When the test was repeated on four Bacteroidesfragilis subspecies (three subsp. thetaiotaomi-cron and one subsp. ovatus) with an improvedAPI indole strip, three of the four were positive.There was total agreement with urease tests,

with only 2 positive and 128 negative. The twopositive results were with Peptococcus prevotiiand P. saccharolyticus.The overall agreement for the 16 carbohy-

drates was 81.4%. Among strains failing toferment glucose in the MICRO system were aslow-growing strain of B. fragilis subsp. fragilis,five strains of B. melaninogenicus, three strainsof C. sporogenes, and one each of Peptostrepto-coccus anaerobius, Megasphaera eldenii, Eu-bacterium ventriosum, Arachnia propionica,Actinomyces naeslundii, Fusobacterium mor-tiferum, and F. necrophorum. In looking downthe columns of Table 3, it is apparent thatindividual negative carbohydrate results wererecorded more often with the CONV (66) andPRAS (62) than with the MICRO system (38).Although individual positive carbohydrate re-sults were much higher with PRAS (191) thanwith CONV (19) or MICRO (12), this was largelya result of recording of weak positive results (pH5.6 to 5.9) in the PRAS systems which wererecorded as weak, variable, or negative identifi-cation schemes. Total agreements betweenpairs of procedures were counted so that thedirect comparisons given in Table 4 could bemade.The 41 organisms which were evaluated sepa-

rately by the MICRO, CONV, and PRAS sys-tems consisted of 19 B. fragilis, 1 F. varium, 1Clostridium innocuum, 1 C. ramosum, 1 Acida-minococcus fermentans, 1 M. elsdenii, 1 P.prevotii, 1 P. saccharolyticus, 1 P. variabilis, 1Veillonella parvula, 1 V. alcalescens, 6 Bifido-bacterium species, 5 Eubacterium species, and1 A. propionica. With supplementary informa-tion for preliminary grouping, plus GLC resultswhen needed, 26 were definitively identified bythe MICRO system, 28 by the CONV, and 30 bythe PRAS, but only 19 were correctly identifiedby all three systems. The 22 organisms incor-rectly identified by one or more systems arelisted in Table 5. The first five organisms wereincorrectly identified in all three systems. Thefirst organism listed probably is a B. fragilissubsp. distasonis (and therefore not incorrectly

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MOORE, SUTTER, AND FINEGOLD

TABLE 4. Agreements between pairs ofprocedures forbiochemical testing of 130 strains of anaerobic

bacteria

Tests in agreementTest

No./2,600 Percent

MICRO & CONV ............. 2,364 90.9MICRO & PRAS .............. 2,208 84.9CONV & PRAS ............... 2,200 84.6

identified) since we have never detected indoleby any procedure. E. tortuosum gave negativeor inconsistent biochemical reactions in allthree systems, and the GLC showed only aceticacid. The three Bifidobacterium strains wereplaced in the right genus, but could not becorrectly speciated with the information pro-vided. Organisms 6 through 11 were not cor-rectly identified with two or three systems. Thetwo B. fragilis strains were not correctly sub-speciated because indole was not detected orbecause negative results occurred with key sug-ars. In our tests, V. parvula ATCC 10790 isweakly catalase-positive, and this accounts forthe confusion recorded here. P. saccharolyticusATCC 14953 was called Peptococcus species inthe CONV and PRAS systems because a weakgelatin reaction was reported. Organisms num-bered 12 through 22 were each misidentified inonly one system. The four B. fragilis strainswere incorrectly identified in the MICRO sys-tem because of false-negative indole results. Allfour organisms gave positive spot indole tests,and all four were positive when retested withthe new API indole substrate. A. propionicagave no positive reactions in the MICRO systemand could not be properly identified. Organisms17 through 22 were placed in the proper genus,but could not be correctly speciated or sub-speciated because of discrepant biochemicaltests or GLC results.

DISCUSSIONBecause our overall agreement with the three

procedures (80.2%) was considerably lower thanthe agreement found by Starr et al. (10) be-tween micromethod and conventional tests(91.2%), we made the comparison between pairsof procedures given in Table 4. This shows thatour MICRO and CONV test agreements (90.9%)compare closely with those reported by Starr etal. Also, the MICRO and CONV tests agreemore closely than either of these agrees with thePRAS results; this is largely due to the numer-ous results which were weakly positive only inPRAS tests.

In our early evaluation of the MICRO system,the indole test was inconsistent with knownpositive organisms, and the gelatin test wasalmost totally insensitive. Subsequently, bothsubstrates were improved, and API now recom-mends a sterile mineral oil overlay for the indoletest. When four known indole producers whichwere negative in early MICRO tests were testedagain, the new indole test was positive with allbut one strain of B. fragilis subsp.thetaiotaomicron. Our observation is that thespot indole test is a very reliable screening testand, if positive, further testing is not necessary.If this test is questionable or equivocal, then theMICRO indole test is as reliable as either theCONV or PRAS tests for a secondary test. Alimited evaluation of the new gelatin test indi-cates that it is more sensitive than the earlierone, but further evaluation is necessary. Al-though the urease test has limited applicationat present, the MICRO substrate seems relia-ble. P. prevotii and P. saccharolyticus wereurease-positive in all of the systems, and per-haps further studies will indicate more areas ofusefulness of the urease test for identification ofanaerobes.

Esculin hydrolysis by the API procedures wasfound reliable when false-positive results, dueto H2S production, were eliminated by readingwith a 366-nm ultraviolet light for nonfluores-cence of hydrolyzed esculin. Table 3 shows thatthe greatest discrepancies with this test weredue to the difference in sensitivity between theCONV and PRAS systems.

Starr et al. (10) found that the micromethodsystem results had to be supplemented withother tests and GLC information in order toidentify more than 66% of the anaerobes theytested. For this reason, we provided supplemen-tary information (GLC data, colony morphol-ogy, Gram reaction, presence of spores, motil-ity, antibiotic susceptibility pattern, and leci-thinase, lipase, and catalase activity) on datasheets for all three procedures when identifica-tion of the 41 coded organisms was beingattempted. The MICRO system performed aswell as the other two procedures in these iden-tifications with two exceptions. False-negativeindole tests resulted in improper subspeciationof four B. fragilis strains, and A. propionicacould not be identified because of biochemicalinactivity. All four of the B. fragilis strains gavepositive spot indole tests, and subsequentlythree of these gave positive MICRO indole testresults (with the improved test). Biochemicalinactivity was noted especially with gram-posi-tive nonsporeforming rods, some cocci, and

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TABLE 5. Comparison of MICRO, CONV, and PRAS results for 22 anaerobes (of 41 tested) incorrectlyidentified by one or more systems

Designation byOrganism Sourcea

MICRO CONV PRAS

1. B. fragilis subsp.thetaiotaomicron

2. E. tortuosum3. B. breve

4. B. infantis subsp. infantis5. B. eriksonii

6. B. fragilis subsp. fragilis7. B. fragilis subsp.

thetaiotaomicron8. V. parvula9. P. saccharolyticus

10. B. adolescentis var. A

11. B. adolescentis var. D

12. B. fragilis subsp.thetaiotaomicron

13. B. fragilis subsp.thetaiotaomicron

14. B. fragilis subsp.thetaiotaomicron

15. B. fragilis subsp. ovatus16. A. propionica

17. E. limosum18. E. alactolyticum19. E. aerofaciens20. B. bifidum var. B21. B. fragilis subsp. fragilis22. B. fragilis subsp. fragilis

NCTC 10582

ATCC 25548ATCC 15700

ATCC 15697ATCC 15423

WALC 2447WAL 2030

ATCC 10790ATCC 14953ATCC 15703

ATCC 15706

WAL 1168

WAL 1423

WAL 2330

ATCC 8483Al'CC 14157

ATCC 8486ATCC 23263ATCC 25986ATCC 15696WAL 2165WAL 2211

distasonis

lentumadolescentis

var. Abrevebreve

fragilisdistasonis

alcalescenssaccharolyticusadolescentis

var. Dspecies

distasonisd

distasonisd

vulgatusd

"other"dPropionibacte-rium orArachnia

limosumalactolyticumaerofaciensbifidum var. Bfragilisfragilis

distasonis

speciesbspecies

speciesadolescentis var.Ddistasonisdistasonis

alcalescensspeciesspecies

species

thetaiotamicron

thetaiotaomicron

thetaiotaomicron

ovatuspropionica

speciesspeciesaerofaciensbifidum var. Bfragilisfragilis

a See Table 2 for source of NCTC and ATCC strains.b Identified as undetermined species of genus in "organism" column.c Wadsworth Anaerobic Bacteriology Laboratory.dCorrectly identified later with improved microsystem.

distasonis

speciesadolescentis var.A

speciesspecies

distasonisthetaiotaomicron

parvulaspeciesadolescentis var.A

adolescentis var.D

thetaiotaomicron

thetaiotaomicron

thetaiotaomicron

ovatuspropionica

limosumalactolyticumspeciesspeciesdistasonisdistasonis

some of the more fastidious gram-negative rodssuch as B. melaninogenicus. Since false-positiveresults with the MICRO system were never aproblem in this study, our limited observationssuggest that two considerations deserve empha-sis. The first is that the inoculum must beheavy, at least matching the density of a no. 1McFarland nephelometer. Concentrationsequivalent to a no. 4 or 5 McFarland nephelom-eter are even better for the poorly growingorganisms discussed above, and this applies forthe CONV and PRAS systems as well. Thesecond consideration is that, with these orga-nisms, sometimes a 3- or 4-day final reading willgive more definitive results. This is in contrastto the rapidly growing, active organisms with

which tests can be read much earlier. Wesuggest that preliminary readings be made at 48h. If the results can be interpreted at that time,then the tests can be discarded. If there areincomplete or questionable results, then thetests should be reincubated and a final readingshould be made in another 48 h.Some of the recommendations of Starr et al.

(10) had been incorporated in the API AnaerobeSystem which we used. For example, the car-bohydrates necessary for subspeciation of B.fragilis were included and were reliable. Also,Ehrlich reagent and xylene extraction havereplaced Kovac reagent in the indole test.Although not shown in this report, our resultsconfirm the greater reliability of the Ehrlich

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reagent and xylene extraction. We performedcatalase tests on growth from plates as inocula-tion suspensions were being prepared, but thistest can be performed by adding one to twodrops of 3% hydrogen peroxide to one of the APISystem tubes (not indole) 30 min after thestrips are exposed to air. We agree with Starr etal. (10) that attempts should be made to includein the micromethod system additional supple-mentary tests such as lecithinase, lipase, andgrowth in 20% bile.We emphasize that tests and observations

such as colonial and microscopic morphology,motility, antibiotic susceptibility, lecithinase,lipase, catalase, and GLC are necessary fordefinitive identification of many anaerobic bac-teria. However, pertinent, simplified, rapid bio-chemical test procedures which will permitidentification of most clinically significant iso-lates can be set up at the work bench in theaverage clinical laboratory without cumber-some, expensive equipment and time-consum-ing procedures.

ACKNOWLEDGMENTSWe thank Analytab Products, Inc., for supplying the test

strips used in this investigation. Also, we acknowledge theexcellent technical assistance of Phyllis Stewart and KennethBricknell in certain of the bacteriological and chemicalstudies.

LITERATURE CITED

1. Dowell, V. R., Jr. 1972. Comparisons of techniques forisolation and identification of anaerobic bacteria.Amer. J. Clin. Nutr. 25:1335-1343.

2. Dowell, V. R., Jr., and T. M. Hawkins. 1973. Laboratorymethods in anaerobic bacteriology. CDC laboratorymanual. Center for Disease Control, Atlanta, Ga.

3. Holdeman, L. V., and W. E. C. Moore. 1972. Anaerobelaboratory manual. Virginia Polytechnic Institute andState University, Blacksburg.

4. Pickett, M. J., and E. L. Nelson. 1955. Speciationwithin the genus Brucella. IV. Fermentation of car-bohydrates. J. Bacteriol. 69:333-336.

5. Pickett, M. J., and M. M. Pederson. 1970. Non fermenta-tive bacilli associated with man. II. Detection andidentification. Amer. J. Clin. Pathol. 54:164-177.

6. Pickett, M. J., M. L. Scott, and R. E. Hoyt. 1955.Tableted mediums for biochemical tests in diagnosticbacteriology. Amer. J. Med. Technol. 21:170-174.

7. Rosenblatt, J. E., A. Fallon, and S. M. Finegold. 1973.Comparison of methods for isolation of anaerobicbacteria from clinical specimens. Appl. Microbiol.25:1391-1398.

8. Sawyer, S. J., J. B. Macdonald, and R. J. Gibbons. 1962.Biochemical characteristics of Bacteroidesmelaninogenicus. A study of thirty-one strains. Arch.Oral Biol. 7:685-691.

9. Smith, P. B., K. M. Tomfohrde, D. L. Rhoden and A.Balows. 1972. API System: a multitube micromethodfor identification of Enterobacteriaceae. Appl. Micro-biol. 24:449-452.

10. Starr, S. E., F. S. Thompson, V. R. Dowell, Jr., and A.Balows. 1973. Micromethod system for identification ofanaerobic bacteria. Appl. Microbiol. 25:713-717.

11. Sutter, V. L., H. R. Attebery, J. E. Rosenblatt, K. S.Bricknell, and S. M. Finegold. 1972. Anaerobic bacteri-ology manual. Department of Continuing Education inHealth Sciences, University Extension and School ofMedicine, UCLA, Los Angeles.

12. Sutter, V. L., and W. T. Carter. 1972. Evaluation ofmedia and reagents for indole-spot tests in anaerobicbacteriology. Amer. J. Clin. Pathol. 58:335-338.

13. Washington, J. A., II, P. K. W. Yu, and W. J. Martin.1971. Evaluation of accuracy of multitest micromethodsystem for identification of Enterobacteriaceae. Appl.Microbiol. 22:267-269.

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