AwNrIcRoBuL AIGENTS AND CHUMOT R"PY, Mar. 1973, p. 35O-356Copyright © 1973 American Society for Microbiology

Vol. S, No. 3Prind in U.S.A.

Modified Broth-Disk Method for Testing theAntibiotic Susceptibility of Anaerobic Bacteria

TRACY D. WILKINS AND TERESA THIELAnaerobe Laboratory, Division of Basic &iences, College of Agriculture, Virginia Polytechnic

Institute and State University, Blackburg, Virginia 24061

Received for publication 2 October 1972

The most commonly used method for testing the antibiotic susceptibility ofaerobic and facultative bacteria is the disk diffusion method. However, someanaerobic bacteria do not grow well enough in anaerobic jars for performance ofdisk diffusion tests. A modification of the broth-disk method of Schneierson al-lowed us to determine antibiotic susceptibility in a completely anaerobic environ-ment. Commercial antibiotic disks were added anaerobically to tubes of prereducedbrain heart infusion broth to achieve a concentration of each antibiotic approxi-mating that attainable in blood. The tubes were then inoculated and incubated for18 h. Resistance or susceptibility to each antibiotic was determined according to theamount of growth in each tube as compared with a control culture without theantibiotic. There was good correlation between results obtained by this broth-diskmethod and minimal inhibitory concentrations.

Anaerobic bacteria have been implicated as thecausative agents in several types of tissue infec-tions, and clinical laboratories are often re-quested to process samples for anaerobes. Diskdiffusion methods for testing the antibiotic sus-ceptibility of anaerobic bacteria have beendescribed (1, 8); however, until recently, nostandardized methods based on interpretativezone diameters were available. Two disk diffusionmethods have been standardized for severalantibiotics by the use of regression plots ofininimal inhibitory concentrations (MIC) versuszone diameters (10, 11). However, these tests arelimited to use with common, rapidly growinganaerobic isolates which are not extremelyoxygen-sensitive. During a small-scale clinicaltrial of the disk diffusion method of Wilkinset al. (11), we recognized that a simple methodwas needed which could be used to determine thesusceptibility of both slow-growing and veryoxygen-sensitive organisms that could not betested by this disk diffusion technique.The principles of Schneierson's method (9) for

use with facultative bacteria have recentlyreceived increased usage. In this method, whichwe have termed "broth-disk," a concentration ofan antibiotic approximately equal to thatachievable in blood is put into a tube of brothmedium with commercial filter-paper disks asthe carrier of the antibiotic. Growth of theorganism at that concentration is considered toconstitute resistance to clinically useful levels ofthe antibiotic. This method simply designates

whether the MIC is above or below concentra-tions normally attained in the blood. Abramsonand Smibert (Brit. J. Vener. Dis., in press)recently reported the use of a modified version ofthe broth-disk method for assaying the antibioticsusceptibility of spirochetes, which cannot betested by any disk diffusion method. The needfor automation in antibiotic susceptibility testingalso has resulted in revival of this idea. Isenbergand colleagues have described, in a series ofpublications (5-7), an automated machine fortesting the antibiotic susceptibility of facultativebacteria. This instrument uses antibiotic disks tocarry a single concentration of an antibiotic intoa vial of broth which is then inoculated with astandard inoculum. The results obtained fromthis instrument have reportedly been as accurateas results achieved by the standard Kirby-Bauer(6) disk diffusion method. These authors haveshown that the antibiotic completely diffuses fromthe disk in liquid media within 1 min (5, 7).The present paper describes the use of Schneier-

son's original idea as a simple system for testingthe antibiotic susceptibility of anaerobic bacteria.The results obtained with clinically importantanaerobic bacteria are compared with resultsof MIC determinations and with results ob-tained by the disk diffusion method of Wilkinset al. (11).

MATERIALS AND METHODSBacterial strains. The strains of anaerobes

used in this study were from the culture collection350

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of the Anaerobe Laboratory. The majority wereclinical isolates sent to this laboratory for identifi-cation. Organisms were identified by methodspreviously described (3).Media. Prereduced media were prepared ac-

cording to published methods and formulas (3)and contained 0.05% cysteine. The medium usedfor antibiotic susceptibility testing was pre-reduced BBL brain-heart infusion broth, supple-mented with 0.0005% hemin, 0.002% menadione,and 0.5% yeast extract (BHI-S). Inocula weregrown in prereduced chopped meat (CM), chop-ped-meat carbohydrate (CMC), peptone-yeastextract-glucose (PYG), BHI-S, or Thioglycollate(Difco) broth.Antibiotics. Sources for antibiotic powders

were as follows: penicillin G, ampicillin, andtetracycline (E. R. Squibb and Sons, Inc., NewBrunswick, N.J.); clindamycin (The Upjohn Co.,Kalamazoo, Mich.); erythromycin (Abbott Lab-oratories, Chicago, Ill.); and chloramphenicol(Parke, Davis & Co., Detroit, Mich.). Erythromy-cin and chloramphenicol were dissolved in ethanol;ampicillin was dissolved in sterile, pH 8.0 phos-phate buffer (0.1 M). All other antibiotics weredissolved in sterile distilled water.Determination of MIC. All MIC determina-

tions were made by the broth dilution methodpreviously published (11). The MIC determina-tions with erythromycin were performed underoxygen-free nitrogen instead of carbon dioxide,which was used for the other antimicrobial agents.Disk diffusion method. Results of a single

determination by the disk diffusion method (11)were used for calculations of correlation with MICvalues. For purposes of these tests, intermediatezone diameters were reported as resistant.Broth-disk susceptibility determinations.

Rubber-stoppered tubes (18 by 142 mm), each con-taining 5 ml of BHI-S medium, were openedanaerobically under 0-free CO2 or N2, and theappropriate number (Table 1) of antibiotic diskswere added aseptically from a commercial cart-ridge (Difco). The tubes were simultaneously ino-culated from a Pasteur pipette with one drop ofan 18- to 24-h culture of the test organism in CMbroth. A control culture containing no antibioticswas included in each set. Incubation was at 37 Cfor 18 to 24 h. Susceptibility to the test antibioticwas defined as either absence of turbidity or lessthan 50% of the turbidity of the control culture.In some cases, the inoculum was obtained from24-h cultures in PYG, BHI-S, CMC, or Thiogly-collate broth. Results of a single determinationwere used for calculations of the correlation of themethod with MICs.

Antibiotic disks. All antibiotic disks used forthe broth-disk and disk diffusion tests were pur-chased from Difco and were the standard high-concentration disks. Disks were stored refrig-erated in bottles containing desiccant. In oneexperiment, antibiotic disks obtained from PfizerCo., Inc., and BBL were used.

RESULTS AND DISCUSSIONInoculum size and source. The same size

and source of inocula were used for both thebroth-disk and MIC methods. One drop from aPasteur pipette of an 18- to 24-h culture of thetest organism was added to each tube containing5 ml of BHI-S broth to give approximately a1:100 dilution of the culture. This was an inoc-ulum of 106 to 107 bacteria per ml, which is 10-fold higher than the inoculum usually used forbroth MIC determinations with facultativebacteria (2). We chose to use the higher inoculumbecause some anaerobic bacteria did not grow aswell from smaller inocula and sometimes requiredmore than 24 h of incubation to reach maximalturbidity in the BHI-S broth.As a source of inoculum, we routinely used an

18- to 24-h culture in CM broth because, in ourexperience, this medium gave very reproduciblegrowth of anaerobic bacteria. CM broth is alsomore difficult to oxidize than other anaerobicmedia. We found that other media could be usedas sources of inocula without significantly chang-ing the results of either the broth-disk methodor the MIC determinations. When 10 strains ofBacteroidesfragilis, 5 strains of Peptostreptococcusanaerobius, and 5 strains of Fusobacterium variumwere tested by the broth-disk method for sus-ceptibility to five antibiotics with the use ofinocula grown in PYG, BHI-S, CMC, and Thio-glycollate broth, the results obtained with the foursources of inocula were the same as the CMresults in 390 of the total 400 tubes. MIC deter-minations with penicillin gave the same resultswith all five sources of inocula when two strains ofPeptococcus magnus and one strain each ofBacteroides fragilis, Peptostreptococcus anaerobius,Fusobacterium necrophorum, Clostridium per-fringens, Peptostreptococcus intermedius, andBacterioides clostridiiformis were tested. A lightbackground turbidity was present in many of theantibiotic tubes that were inoculated with CMCbroth because of the higher inocula obtained fromthis carbohydrate-containing medium. This couldbe eliminated by the use of a 1 :10 dilution of theCMC broth culture as an inoculum.Reading results. With the CM inoculum,

interpretation of results in approximately 95%of the tests involved merely a judgment of growthversus no growth. Organisms that were resistantto an antibiotic normally grew to almost the sameturbidity as the control culture, whereas sus-ceptible organisms did not grow in the tubescontaining that antibiotic. With a few organisms,the number of cells in the inoculum was highenough so that the initial generations that oc-curred prior to inhibition by antibiotics resulted

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in a light turbidity that was present with all ofthe antibiotics tested. In such cases, this back-ground turbidity was disregarded, and only thosetubes in which the turbidity was 50% or greaterthan the control culture were considered resistant.Incubation time. We recorded both the

broth-disk and MIC results after incubation for18 to 24 h. Resistance to an antibiotic couldsometimes be reported much earlier, however,since both the control cultures and some culturescontaining antibiotics were often very turbidafter only 4 h of incubation. Definite susceptibil-ity could not be reported until after 18 h ofincubation, since growth could occur later in thetubes containing other antibiotics. However,presumptive susceptibility could be reportedvery early and confirmed after 18 h of incubation.Antibiotic concentrations. Opinions of

various investigators differ as to the concentrationof each antibiotic to which an organism must beresistant in order to be termed "clinicallyresistant." Listed in Table 1 are the test con-centrations we chose for this trial of the broth-disk method. Except for chloramphenicol anderythromycin, the test concentrations were thesame as the MIC break-points used in the diskdiffusion method of Wilkins et al. (11). Otherconcentrations could be used by changing eitherthe number of antibiotic disks or the amount ofbroth used per tube, and more than one con-centration could be tested for each antibiotic.

Correlation of the results of the broth-disk, disk diffusion, and MIC methods. Todetermine how well the results of the broth-diskand the disk diffusion methods correlated withMIC values, we compared the three methodsusing the 145 strains of anaerobic bacteria listed inTable 2. As indicated in Table 2, certain of thespecies were chosen for their significance in clinicalinfections, others for their poor growth char-acteristics which would test the methods mostseverely, and some species for both of thesecharacteristics. Two of the more common clinical

TABLE 1. Test concentrations of antibiotics

Labeled No. of CalculatedAntibiotic disk disks test conen

content tuer per ml

Penicillin G.. 10 units 1 2 unitsAmpicillin........ 10 pg 2 4 pgCephalothin .... 30 ,g 1 6 pgTetracycline ...... 30 pg 1 6 pgClindamycin......2 pg 4 1.6 pgChloramphenicol.. 30 jpg 2 12 pgErythromycin..... 15 pg 1 3 ,ug

TABLE 2. Organisms used and reason they werechosen

Charae-No. of Common teristic

Species strains clinical poortested isolates growth

on plates

Bacteroides fragilis ...... 50 +Peptococcus magnus. 50 + +Peptostreptococcusanaerobius ............ 5 +

P. intermedius .......... 5 +Eubacterium lentum ... 6 + +Clostridium perfringens 5 +Bacteroides melanino-

genicus................. 6 +Fusobacterium varium. . . 5 +F. mortiferum ........... 2 +F. russii ................ 2 +Bacteroides ruminicola... 3 +B. putredinis............ 2 +Actinomyces israelii 2 +A. viscosus.............. 1 +A. naeslundii ........... 1 +

isolates, B. fragHls and P. magnus, were chosenfor detailed analysis, and 50 strains of each ofthese species were tested. P. magnus grows onlyto a very low turbidity in broth, and on agarplates the growth is barely visible so that zonesof inhibition are difficult to see. B. fragilis is avery fast-growing organism which reaches highturbidity in both broth and agar media. We alsotested a total of 45 strains belonging to 11 otherspecies of anaerobic bacteria to determinewhether the broth-disk method could be used fora wide range of anaerobes.The susceptibility of each of the 145 strains

to the seven antibiotics listed in Table 1 was de-termined by both the broth-disk and MIC meth-ods. The MICs for the organisms tested werewithin two double dilutions of the broth-diskconcentration in 26% of the tests. Listed inTable 2 are the number of tests in which thebroth-disk results did not correlate with the MICdata and the calculated percentage accuracy ofthe broth-disk method with each of the speciestested. The overall correlation of the broth-diskresults with MIC values was 97% in these 1,015tests.The results of the disk diffusion and MIC

methods were also compared, but susceptibilityto chloramphenicol and erythromycin was nottested by the disk diffusion method since in-terpretative zone diameters for these antibioticsare not available. The number of cases in whichthe results of the disk diffusion assays did not

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TABLE 3. Accuracy of the broth-disk and disk diffusion tests as compared with MIC results

Broth-Disk test Disk diffusion test

Species tested

No. of No. in- Percent No. of No. in- Percenttestsa correctb correct testse correctb correct

Bacteroides fragilis .......... ........... 350 2 99 250 3 99Peptococcus magnus ..................... 350 25 93 235c 12 95Peptostreptococcus anaerobius .......... 35 2 94 25 2 92P. intermedius.......................... 35 0 100 25 3 88Eubacterium lentum..................... 42 2 95 25c 3 88Ciostridium perfringens ................. 35 0 100 25 23 8Bacteroides melaninogenicus. 42 0 100 30 0 100Fusobacterium varium..... 35 0 100 25 7 72F. mortiferum .......................... 14 0 100 10 2 80F. russii ............................... 14 0 100 5c 0 100Bacteroides ruminicola................. 21 1 95 15 0 100B. putredinis .......................... 14 3 79 5c 0 100Actinomyces israelii................... 14 0 100 -c aA. viscosus............................. 7 0 100 5 0 100A. naeslundii........................... 7 0 100 5 0 100

Total of all species .................... 1,015 35 97 685 55 92

a A test was defined as one strain tested for susceptibility to one antibiotic a single time.b Incorrect results were those which did not correlate with the MIC value.c One or more strains could not be tested by the disk diffusion method.d A dash means no data available.

correlate with the MIC results is given in Table3, as is the correlation of the results of the diskdiffusion method as compared with the MICvalues. With these species, the disk diffusionmethod correlated with MIC values in 92% ofthe tests. However, the disk diffusion methodwas only 8% correct in tests with the five strainsof Clostridium perfringens. For this organism, thezone diameters were slightly smaller than thepublished break-points for susceptibility; yet theMIC results indicated that the five strains wereuniformly susceptible to these antibiotics. Thus,it appears that smaller zone diameter break-points should be considered for the disk diffusiontest with this organism.The disk diffusion method could not be used

for testing the antibiotic susceptibility of eightother strains (three of P. magnus, two of Ac-tinomyces israelii, and one each of Eubacteriumlentum, Fusobacterium russii, and Bacteroidesputredinis), since these organisms would notgrow well enough in the agar medium for zonesto be discerned. Many other strains of these samespecies grew poorly on the susceptibility plates,but zones could be measured, albeit with somedifficulty.The percentage of organisms classified as

susceptible to the antibiotics by each of thethree methods is given in Table 4. The majordiscrepancy was that 88% of the strains of P.

magnus were susceptible to erythromycin asdetermined by the MIC method, but only 44%appeared to be susceptible by the broth-diskmethod. Since it has been reported that erythro-mycin is less active at the lower pH producedby a CO2 atmosphere (4) the MIC determina-tions with erythromycin were performed with N2as the oxygen-free gas. The broth-disk determi-nations were routinely performed with CO2since this is the gas most widely used with pre-reduced media. When the broth-disk methodwas repeated with N2, there were no significantdifferences in the results (Table 4). We cannotexplain this discrepancy between MIC andbroth-disk results which occurred only in theerythromycin tests with P. magnus and not withthe other 12 species. However, the overall cor-relation of MIC values with broth-disk tests ofP. magnus with all seven antibiotics was 93%.

Possible sources of error. The most obvioussource of error in the broth-disk method is theactual concentration of antibiotic contained incommercial antibiotic disks. Since the Food andDrug Administration (FDA) has enforcedstandards on antibiotic disk production in theUnited States, the concentration in commercialdisks is now closer to the labeled value. The FDAroutinely determines a mean value for six disksfrom each batch of antibiotic disks manufacturedin the United States. This mean value must be

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TABLE 4. Percentage susceptibility of organisms as determined by three methods

B. fragili, 50 strains P. magnus, 50 strains Other species, 45 strains

Antibiotics DiskMIC Broth- Disk MIC Broth- Disk MIC Broth- diffu-disk diffusion disk diffusion disk

so

Penicillin G................... 0 0 0 100 100 98 89 91 76Ampicillin .............. 0 0 0 100 100 94 91 95 76Cephalothin .............. 0 0 0 100 100 94 82 82 68Clindamycin ................. 100 100 94 98 96 92 89 89 73Tetracycline ....58 58 58 76 76 74 73 82 59Chloramphenicol .............. 100 100 100 100 100 100Erythromycin (CO2).-. 96 44 82 -

Erythromycin (N2)b........... 100 100 88 46 82 82 -

a Tests performed with 02-free C02.b Tests performed with 02-free N2.

TABLE 5. Information supplied by the Food and Drug Administration on actual contents of batches ofaccepted disks over a 12-month period

Antibiotic disk Labeled concn Mean conce of nRage of all batches StandardManlbacnceo deviation

Ampicillin.................. 10pg 11.7 7.3-14.8 1.93Cephalothin ................ 30gg 33.3 23.2-44.0 5.06Chloramphenicol . ......... 30 g 31.7 29.0-44.0 3.97Clindamycin ............. 2 ;g 2.36 1.79-2.95 0.423Erythromycin............... s15 ;g 19.15 16.3-21.2 1.45PenicillinG. 10 units 10.81 7.8-14.7 1.94Tetracycline ............... 30 ug 35.53 26.7-44.0 4.63

between 67 and 150% of the labeled value or thebatch of disks cannot be sold. Data on all assaysperformed by the FDA on the types of antibioticdisks used in the present study were suppliedto us by Richard Norton and Alfred Giovetti(Table 5). The composite means for all of thebatches of each type of disk were very close tothe labeled values. The maximal variation be-tween batches was 73 to 148% of the labeledvalue. Differences of this magnitude could in-fluence the results of the broth-disk test but,since very few organisms have MICs within atwofold dilution of the break-point, the dis-crepancies are not numerous. In the process ofobtaining the results reported here, severalbatches of antibiotics were used, and there was noevident difference between results obtained withdifferent batches.

Antibiotic disks are a convenient source ofantibiotics for testing purposes, but the disksmust be handled with care to prevent deteriora-tion of the antibiotic. Antibiotic disks are moststable when stored at 4 C, or preferably at -20 C,in sealed bottles containing a desiccant. Theprecautions suggested by the International Col-

laborative Study on Antibiotic SusceptibilityTesting (2) should be followed for both storageand handling of antibiotic disks, and all disksshould be discarded by the expiration date.

Contamination has not been a problem in ourlaboratory in the performance of the broth-disktests; however, this could be a problem if thedisks were heavily contaminated with bacteria.Of several thousand tubes used in our trials ofthe broth-disk test, we have found obvious con-tamination in approximately 10 tubes. Currently,in the United States, disks are manufacturedchiefly by Difco, BBL, and Pfizer Co., Inc. Wetested 100 disks at random from each manu-facturer for levels of contamination that wouldinterfere with the broth-disk test. Each disk wasincubated for 48 h anaerobically in 5 ml ofBHI-S broth and no turbidity developed in anytube. Erroneous results due to introduction ofresistant bacteria on the antibiotic disks wouldthus seem to be very unlikely. However, anyantibiotic tube in which the type of growth ap-pears significantly different from the growth inthe control tube should be stained to test forpossible contamination.

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Organisms for which the MIC is very closeto the test concentration may be reported asresistant in one broth-disk assay and susceptiblein a later assay. This kind of variation is alsoinherent in MIC procedures in which a differenceof a twofold dilution is considered normal vari-ation between assays (2). Organisms with anMIC slightly below the test concentration alsomay appear resistant if the results are not re-corded until well after 24 h of incubation. Pro-longed incubation results in a gradual increasein the MIC of most antibiotics.

Inoculum size does not have a great effect oneither the broth-disk or MIC determinationswith most anaerobic bacteria. We have found thata 10-fold dilution of the inoculum resulted inlowering the MIC by, at most, a twofold dilution.With some anaerobes, such as B. melaninogenicusthe use of dilutions of inocula which are greaterthan 1:10 can result in erratic growth. In ourlaboratory it is much more convenient to uselarger inocula and disregard the infrequent"background" turbidity that sometimes occurs,instead of making an anaerobic dilution to useas an inoculum. Other laboratories may prefer touse a 1:10 dilution in order always to have agrowth versus no growth assay.Advantages of the broth-disk method.

The principal advantage of the broth-disk methodis that almost all clinical isolates can be testedfor antibiotic susceptibility by this one procedure.Although in this trial we reported results for thediffusion test for all except eight of the strainstested, growth was so poor with many of theslow-growing anaerobes that zones of inhibitionon agar plates were very difficult to discern.The results of the broth-disk method were easierto read and there was less variation in the resultsrecorded for the same test by different tech-nicians. The percentage of anaerobic isolateswhich cannot be tested by the disk diffusionmethod would vary among laboratories accord-ing to the types of specimens examined and theisolation techniques used. Of the last 100 an-aerobic bacteria submitted to this laboratory byresearch and clinical laboratories for identifi-cation, 16 did not grow in the pour plates usedfor the disk diffusion test, and an additional 8 didnot grow well enough for accurate measurementsof zone diameters. Of these same 100 organisms,only 3 did not grow well enough in the prereducedbroth for performance of the broth-disk test.Thus, the broth-disk test increased by 21% thenumber of organisms on which we could reportantibiotic susceptibility data.

In cases of endocarditis and certain other in-fections, the bactericidal activity of antibioticsis of much more importance than bacteriostatic

effects. Neither disk diffusion tests nor agar di-lution MIC determinations can yield such in-formation. The preferred method is to performa broth dilution MIC determination with sub-sequent subculture of tubes at and above theMIC concentration to test for viable cells. Justas the broth-disk method can be used to approxi-mate whether an organism can grow at concen-trations of antibiotics attained in the blood, tubescontaining antibiotics that inhibit growth inthe broth-disk test can be subcultured to de-termine whether this antibiotic concentrationwas also bactericidal.One disadvantage of the broth-disk method is

that it does not distinguish between degrees ofresistance or susceptibility. Such data theo-retically could be obtained for disk diffusion testsby consulting regression plots of MICs versuszone diameters, but in clinical practice this isnot done routinely. The main disadvantages ofthe broth-disk method are cost of the tubes ofprereduced BHI-S media and the requirementfor a supply of 02-free CO2 or N2.The broth-disk method would seem particu-

larly useful for clinical laboratories which cur-rently identify anaerobes according to methodsdescribed in the Anaerobe Laboratory Manual(3), since susceptibility tests could be performedat the same time as the biochemical tests by thesame technician. However, the method shouldwork the same if performed inside an anaerobicglove box with anaerobic media. The choice ofmethods would depend on the requirements ofindividual laboratories.

ACKNOWLEDGMENTSThis project was supported by Public Health Service

grant 14604 from the National Institute of GeneralMedical Sciences and grant FR07095 from the NationalInstitutes of Health, Biomedical Sciences.

LITERATURE CITED1. B6erens, H., and J. Guillaume. 1954. Technique de

determination de la sensibilit6 des bact6riesana6robies aux antibiotiques par la m6thode desdisques. Ann. Inst. Pasteur (Paris) 86:781-784.

2. Ericsson, H. M., and J. C. Serris. 1971. Antibioticsensitivity testing. Report of an internationalcollaborative study. Acta Pathol. Microbiol.Scand., Sect. B (Suppl. 217): 18.

3. Holdeman, L. V., and W. E. C. Moore (ed.). 1972.Anaerobe laboratory manual. Virginia Poly-technic Institute Anaerobe Laboratory, Blacks-burg.

4. Ingham, J. B., J. B. Selkon, and J. H. Hale. 1970.The effect of carbon dioxide on the sensitivity ofBacteroids fragili to certain antibiotics in vitro.J. Clin. Pathol. 23:254-258.

5. Isenberg, H. D. 1971. Development of an auto-mated method to determine antibiotic suscepti-bility of rapidly growing organisms. Advan.Automated Anal. 1:371-376.

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6. Isenberg, H. D., A. Reichler, and D. Wiseman.1971. Prototype of a fully automated device fordetermination of bacterial antibiotic suscepti-bility in the clinical laboratory. Appl. Microbiol.22:980-986.

7. Isenberg, H. D., and J. Seo. 1971. Experimentalfoundation for the automated antibiotic sus-ceptibility test. Advan. Automated Anal.1:377-380.

8. Merritt, E. S. 1962. A simple method for the deter-mination of the antibiotic-sensitivity of anae-robic organisms. Amer. J. Clin. Pathol. 38:203-206.

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9. Schneiersen, S. S. 1954. A simple rapid disc-tubemethod for determination of bacterial sensitivityto antibiotics. Antibiot. Chemother. 4:125-132.

10. Sutter, V. L., Y. Y. Kwok, and S. M. Finegold.1972. Standardized antimicrobial disc suscepti-bility testing of anaerobic bacteria. I. Suscepti-bility of Bacteroides fragili to tetracycline.Appl. Microbiol. 23:268-275.

11. Wilkins, T. D., L. V. Holdeman, I. J. Abramson,and W. E. C. Moore. 1972. Standardized single-disc method for antibiotic susceptibility testingof anaerobic bacteria. Antimicrob. Ag. Chemo-ther. 1:451-459.

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