APPLIED MICROBIOLOGY, Feb. 1972, p. 268-275Copyright 0 1972 American Society for Microbiology

Vol. 23, No. 2Printed in U.S.A.

Standardized Antimicrobial Disc SusceptibilityTesting of Anaerobic Bacteria

I. Susceptibility of Bacteroides fragilis to TetracyclineVERA L. SUTTER, YUNG-YUAN KWOK, AND SYDNEY M. FINEGOLD

Anaerobic Bacteriology Laboratory, and Medical Service, Veterans Administration, (Wadsworth) LosAngeles, California 90073, and Department of Medicine, UCLA School of Medicine, Los Angeles, California

90024

Received for publication 4 October 1971

A modified Bauer-Kirby-Sherris-Turck method for disc susceptibility testingof anaerobic bacteria is presented. When tetracycline was used against 100strains of Bacteroides fragilis as a model, reasonably reproducible results were

obtained after overnight incubation in both the GasPak atmosphere and an

atmosphere achieved by adding 10% CO2 to a mixture of 10% H2 and 90% N2.The minimal inhibitory concentration for the strains determined by the agar

dilution technique correlated well with the results of disc tests performed inthe GasPak atmosphere with 30-,ug tetracycline discs. Among 63 strains iso-lated from 1970 to the present, only 24 (38.1%) were found to be susceptible totetracycline.

With the increasing awareness of the impor-tance of anaerobic bacteria in infectious proc-esses, the development of a simple, rapid andreproducible method for determining the sus-ceptibility of these organisms to antimicrobialagents becomes important. Although manyanaerobic bacteria have predictable patterns ofsusceptibility to these agents and clinicianshave relied on these as a guide to therapy (8),there are often exceptions to the general pat-terns. As examples, penicillin G is consideredthe drug of choice for infections due to Clos-tridium species, but occasional strains are re-sistant to penicillin G; tetracycline has beenconsidered a drug of choice for infections dueto Bacteroides fragilis, but resistant strains ofBacteroides species and B. fragilis are now be-ing found (3, 11). Determination of the suscep-tibility of individual isolates to various antimi-crobial agents would provide more specificinformation to the clinician for guidance oftherapy.There is a great degree of variability in

methods now in use for determining antimi-crobial susceptibility of anaerobic bacteria.Investigators have used a variety of broth oragar dilution and paper disc techniques (2, 6,9, 10). Results vary considerably among thevarious studies, with variability probably dueto differences in media used, age and size ofinoculum, and method and length of anaerobic

incubation. Little has been done to stand-ardize methodology and to correlate results ofpaper disc tests with those of broth or agardilution tests. A number of laboratories use themethod and standards of Bauer et al. (1), al-though this has not been standardized for an-aerobic conditions and erroneous interpreta-tions can be made.The method of Bauer et al. (1) has been

found acceptable for aerobic and facultativebacteria and has recently been recommendedby the Food and Drug Administration foradoption as the single recognized method forantibiotic disc susceptibility testing in thiscountry (5). The Food and Drug Administra-tion recommended that susceptibility of Bac-teroides species and other anaerobes be deter-mined by broth or agar dilution methods butdid not specify methods or conditions to beused.We have believed for some time that a

standardized disc test for anaerobic bacteriawith interpretive standards similar to those ofBauer et al. (1) would be of great value to diag-nostic microbiology laboratories, and we havebeen directing efforts toward developing sucha method. Our primary aims have been to se-lect parameters of media, age and size of inoc-ulum, and time of incubation which give satis-factory disc test results after overnight incuba-tion with rapidly growing strains, and to corre-

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ANTIMICROBIAL SUSCEPTIBILITY OF ANAEROBES

late these results with those obtained by a di-lution method with similarly selected parame-ters. Since B. fragilis is one of the most fre-quent anaerobic pathogens and often the mostdifficult to treat, we considered it an impor-tant organism with which to initiate ourstudies. Additionally, tetracycline was selectedfor the initial studies because resistance of B.fragilis to this drug is being reported.The purpose of this report is to present the

methods selected and to analyze the resultsobtained by use of tetracycline against 100strains of B. fragilis as a preliminary model forfurther work toward standardization of suscep-tibility testing of anaerobes. Subsequentstudies will include work with other rapid-growing and slow-growing anaerobes as well.

MATERIALS AND METHODSPreliminary studies. Thirty-five strains of anaer-

obic bacteria representing the genera Bacteroides,Fusobacterium, Vibrio, Veillonella, Peptococcus,Peptostreptococcus, Bifidobacterium, and Clostri-dium were used in tests for selection of media to beused. Commercially available fluid media [fluid thio-glycollate medium (BBL), Schaedler broth (BBL),and thioglycollate medium without indicator-135C(BBL)] with and without additives such as asciticfluid, dithiothreitol, hemin, menadione, and Na-HCO,, alone and in combination, were surveyed.Thioglycollate medium without indicator-135C towhich was added 5 gg hemin per ml prior to auto-claving and 1 mg of NaHCO,/ml plus 0.5 ug ofmenadione/ml (filter-sterilized) after autoclaving, assuggested by Sawyer et al. (12), was chosen as themedium for growing the inoculum. It was found togive good growth of the rapid-growing strains such asBacteroides fragilis, Clostridium perfringens andFusobacterium varium within 4 to 6 hr of incubationand of the slower-growing strains such as B. melani-nogenicus, B. oralis, and Peptostreptococcus speciesafter overnight incubation. This medium is hence-forth referred to as THCM. Fluid thioglycollatemedium with hemin and 25% ascitic fluid wasequally good for promoting growth but has the disad-vantages that ascitic fluid is extremely variable incomposition, expensive, and difficult to obtain, andthat excessive turbidity which does not representviable cells is present after growth of some bacteria.The same strains were tested on several commer-

cially available agar bases [Brucella (Pfizer Co., Inc.)Columbia (BBL), Infusion (BBL), Mueller-Hinton(BBL), Mueller-Hinton (Difco), Schaedler (BBL),and Trypticase soy (BBL) I with and without variousadditives. Brucella agar with 5% sheep blood and 0.5Lg of menadione per ml was selected as the mediumfor the disc diffusion test. This medium supportedthe growth of rapid-growing strains so that testscould be read after overnight incubation. Althoughmany anaerobic bacteria were able to grow on Bru-cella agar without additives, a number of them ei-ther required or were stimulated by hemin or blood,

and one strain of B. melaninogenicus required bothblood and menadione for growth on solid medium.Therefore, we believed that these ingredients shouldbe added to the test agar to provide a medium onwhich most anaerobic bacteria would grow andwhich could be used in future work with anaerobesmore fastidious than B. fragilis. Schaedler and Co-lumbia agars supplemented with blood and mena-dione were equally good for growth of the organisms.Schaedler agar was not selected because of its highglucose content. The choice between Columbia andBrucella agars was arbitrary. Infusion agar withblood and menadione supported growth of all bac-teria tested, but growth was not as good on this me-dium as on the above three media. Mueller-Hintonagar with blood and menadione yielded no growthafter 18 to 24 hr with three of the strains tested, andTrypticase soy agar with blood and menadioneyielded no growth after 18 to 24 hr with two of thestrains tested.

Fifteen strains of B. fragilis were tested for theirsusceptibility to tetracycline by both broth and agardilution methods. Twofold dilutions of tetracyclinewere made in Brucella broth and agar to which men-adione and laked sheep blood were added so thattheir final concentrations in the test were 0.5 ug/mland 5%, respectively. The blood was laked byfreezing and thawing. An inoculum of 0.5 ml of a 6-hr culture in THCM diluted in Brucella broth to justbeyond visible turbidity (105 to 106 viableorganisms/ml) was added to 0.5 ml of the broth con-taining tetracycline. The agar plates were inoculatedwith 104 to 105 viable cells by means of a replicatingdevice (13) with the inoculum in the wells adjustedto the #1 MacFarland nephelometer standard. Bothsets of tests were read after 18 to 24 and 42 to 48 hrof incubation at 37 C in a modified Brewer jar madeanaerobic by evacuating and filling with oxygen-freegas five times. The final atmosphere was achieved byadding 10% CO2 to a mixture of 10% H2 and 90% N2.This atmosphere will be referred to as 10% H2, 90%N2 + 10% CO2. The jar contained palladium-coatedalumina catalyst. The minimal inhibitory concentra-tion (MIC) in each test was recorded as the highestdilution showing no macroscopically visible growth.MIC values with both tests were usually the same,and were not more than one dilution step differentin instances where variation occurred. With bothtests, the MIC was frequently one dilution steplower at 42 to 48 hr. The agar dilution test was easierto read and interpret at both incubation periods.Because it is more economical to perform the agardilution method if one is testing many strains andbecause results are easier to read, the agar dilutionmethod for determining the MIC was chosen for theremainder of this study.

Laked blood had been used in the studies above toallow observation of growth in the broth dilutiontests. To determine how whole blood or laked bloodwould affect results in the agar dilution method, theMIC for 31 strains of B. fragilis was determined asdescribed above with the use of whole or laked bloodin two sets of plates. At 42 to 48 hr, the MIC valuesfor seven of the strains differed by one dilution step,

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SUTTER, KWOK, AND FINEGOLD

with values higher more often with whole blood thanwith laked blood. Results on laked blood-agar wereeasier to read because growth caused a discolorationof the blood underneath the growth and in the areasurrounding the growth, which made the decision ofgrowth or no growth less difficult in several in-stances.

A similar experiment was performed with tetracy-cline discs, 30 ug (BBL), applied to plates swabbedwith 10 strains of B. fragilis grown for 4 to 6 hr inTHCM, diluted to the density of one-half the tur-bidity of the #1 MacFarland standard, as in themethod of Bauer et al. (1). At 18 to 24 and 42 to 48hr, inhibition zone diameters were often the same ornot more than 2 mm larger or smaller for each strainon the two types of blood-agars if the zone of inhibi-tion was measured by reflected light on the surfaceof the plate. Discoloration caused by growth on lakedblood diffused into the zone of inhibition, and ifplates were read by transmitted light an erroneouslysmall diameter would be measured. Accordingly, itwas decided that whole blood would be used for thedisc test.

Antimicrobial susceptibility tests: bacterialstrains. Susceptibility tests were performed on 100strains of B. fragilis. Fifteen strains had been iso-lated from '1954 to 1959, 22 from 1960 to 1969, andthe remainder from 1970 to the present time. Mostof them had been isolated initially on nonselectivemedia; a few had been received from other laborato-ries, and information regarding initial isolation me-dium was not available. Eighty-three were from clin-ical specimens, and 17 were from feces. They wereidentified by methods previously described (14).

Paper-disc susceptibility test. The inoculum wasprepared by heavily inoculating each strain intoTHCM (three to four colonies or a 3-mm loopful).The medium was prepared fresh each week, steamedfor 10 min, and cooled prior to the addition of mena-dione and NaHCO. Cultures were incubated at 37 Cfor 4 to 6 hr and adjusted to one-half the density ofthe #1 MacFarland standard (1). The inoculum wasapplied by swabbing onto duplicate plates of Bru-cella agar containing 5% defibrinated sheep bloodand 0.5 MAg of menadione per ml, freshly preparedand dried at 37 C for 45 to 60 min prior to use orincubated overnight for use the next day. The agarwas 5 to 6 mm in depth and was at pH 7.4 prior touse. Tetracycline discs, 5 and 30 ug, were applied,and one set of plates was incubated under 10% H2,90% N2 + 10% CO2 as described above; the other setwas incubated in a GasPak jar. Catalysts for bothanaerobic systems were reactivated after each use byheating to 160 to 170 C for 1.5 to 2 hr. Except for thetime required for setting up the tests and the timerequired to achieve anaerobiosis, no prediffusion pe-riod was used. Usually only 12 strains were done atone time, and both jars were processed rapidly andplaced in the incubator. Indicators (Disposable An-aerobic Indicator, BBL) used in the jars usuallyshowed that conditions were completely anaerobicafter 2 to 3 hr. Zones of complete inhibition ofgrowth around the discs were measured with calipersafter 18 to 24 and 42 to 48 hr.

One strain (B-1) was included each day that testswere performed. This strain was also used in 50 ad-ditional determinations on 3 different days withboth 5- and 30-,gg discs to determine reproducibilityof results.Agar dilution tests. Agar dilution tests were set

up as described above in preliminary studies. Testswere incubated in an atmosphere of 10% H2, 90% N2+ 10% CO2, or in a GasPak jar. Strain B-1 was in-cluded with each day's tests to assess reproducibilityof results. It was tested six times.

RESULTSInhibition zone diameters around the tetra-

cycline discs were as easily measured at 18 to24 hr as they were at 42 to 48 hr. The diame-ters with the majority of strains varied only 1to 2 mm at the two incubation periods in eachanaerobic system. Exceptions to this were asfollows: in the 10% H2, 90% N2 + 10% CO2atmosphere, there were 28 strains which had 3to 9 mm larger or smaller zones at 42 to 48 hraround the 30-,ug disc and 12 strains with vari-ations of 3 to 8 mm around the 5-,ug disc; inthe GasPak atmosphere, there were 15 strainswith a variation of 3 to 7 mm around the 30-Igdisc and 10 strains with a variation of 3 to 5mm around the 5-,ug disc. Three strainsshowed this variation with discs of bothstrengths in both atmospheres. An unexpectedobservation with regard to variations in zonediameters was that with sensitive strains thezones usually became larger and with resistantstrains they became smaller with longer incu-bation.

In general, the diameters of the zones of in-hibition tended to be slightly larger in the H2,N2 + CO2 atmosphere than in the GasPakatmosphere at both 18 to 24 and 42 to 48 hr.The 5-,ug discs produced zones of inhibitionfrom 5 to 11 mm smaller than did the 30-,ugdiscs tested against the same strains in the H2,N2 + CO2 atmosphere and from 3 to 11 mmsmaller than did the 30-,ug discs in the GasPakatmosphere. The distribution of zone diame-ters with 5- and 30-,ug discs in the two atmos-pheres and for the two periods of incubationfollowed bimodal curves as illustrated in Fig. 1and 2. Most strains appear to fall into either aresistant or susceptible group with a fewstrains in an intermediate group.The diameters of zones of inhibition around

the 5- and 30-,ug discs at both 18 to 24 hr and42 to 48 hr were plotted against a log of theMIC read at the same interval in each anaer-obic system for each of the 100 strains. Regres-sion lines were caluclated by the method ofleast squares (4) and were drawn to demon-

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ANTIMICROBIAL SUSCEPTIBILITY OF ANAEROBES

No. OFSTRAINS

16 _

* * GAS PAKx t10% Ha, 90% Ng., 10% CO,

I0-

6..

4-

1015 20 25 30 35 4ZONE DIAMETER (mm)

FIG. 1. Distribution of inhibition zone diametersaround tetracycline discs, 5 Asg, 18 to 24 hr..No. OFSTRAWNS

16_

14_

12.

10_

8.

4-

2.

,ug/ml, then correlation at 18 to 24 hr is poor.Strains with zone diameters of _11 mm wouldbe considered resistant; 12 to 25 mm, interme-diate; and >25 mm, susceptible. As Fig. 3Aindicates, there were 18 strains with an MIC of;'12.5 jg/ml and 1 strain with an MIC of 0.2jug/ml which had zone diameters in the inter-mediate range and one with an MIC of 3.1jig/ml with a zone diameter in the susceptiblerange. At 48 hr, correlation was better, as indi-cated in Fig. 3B. Zone diameters were inter-

MICjig/ml>25.

25..

12.5_

Log,10 MIC9_ \

8_

3A

7-

6.2. 6.

3.1_- 5.

1.6_ 4.

0.8. 3_

0.4. 2_

0.2_ I_

*- GAS PAKx---_-u 1%Hg.90%Ng. I O%

I I

5 10 15 20 25 30 35

ZONE DIAMETERS (mm)45

3BMIC LogosMug/ml IOMIC>25. 9_

25. a8

12.5.

62._

3.1.

7-

6.

5-

0..I I I I I

0 6 o s 30 35 40 45ZONE DIAMETER (mm)

FIG. 2. Distribution of inhibition zone diametersaround tetracycline discs, 30 jg, 18 to 24 hr.

strate the relationship between zone diameterand MIC data. Figure 3 illustrates the rela-tionship observed between the MIC and zonesof inhibition around the 30-jig disc in theGasPak system at the two time intervals. If wedefine susceptible strains as those having anMIC 1.6 jig/ml, resistant strains as 2_12.5jig/ml and intermediate strains as 3.1 to 6.2

1.6. 4.

0.8. 3.

04.. 2.

II ~~~~~~I5 10 I5 20 25 30 35 40 45

ZONE DIAMETERS (mm)FIG. 3. Relationship of inhibition zone diameters

around 30-jig tetracycline discs and MIC values inthe GasPak atmosphere. (A) Determinations made at18 to 24 hr. Least squares line fory = a + bx; b = -

0.211, a = 9.285. (B) Determinations made at 42 to48 hr. Least squares line for y = a + bx; b = -0.216,a = 10.368.

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SUTTER, KWOK, AND FINEGOLD

preted as follows: 5 16 mm, resistant; 17 to 29mm, intermediate; 230 mm, susceptible. Onlyone strain with intermediate susceptibility bythe agar dilution test fell into the susceptiblecategory by disc test.MIC results were often difficult to interpret

at 18 to 24 hr, and the 42 to 48 hr reading wasconsidered technically better, but there was nodifficulty in measuring zone diameters at 18 to24 hr. The relationship of zone diameters at 18to 24 hr and MIC values at 42 to 48 hr wasthen plotted, and regression lines were calcu-lated for each disc strength and each conditionof incubation. Figure 4 shows the relationshipbetween zone diameters with the 5- and 30-pugdiscs and MIC values in the GasPak atmos-phere. Correlation was slightly better with the5-,pg than with the 30-pug discs. Figure 5 illus-trates the relationship of results of the twotests in the 10% H2, 90% N2 + 10% CO2 at-mosphere. Correlation was about the same fordiscs of both concentrations.

Table 1 indicates tentative interpretation ofzone diameters based on data derived from thedistribution of zone diameters and regressionline analysis of zone diameters measured at 18to 24 hr and MIC values at 42 to 48 hr. Thecriteria for susceptibility are based on tetracy-cline concentrations in the blood achieved withordinary dosage schedules (levels of 1 to 2pig/ml are commonly achieved with oral dosageand of 5 to 10 pg/ml with intravenous adminis-tration).

Results of tests to determine reproducibilityof tetracycline zone diameters with strain B-1are shown in Table 2. In 56 tests with 5-,pg tet-racycline discs performed on 9 different days,the zone diameters in both atmospheres variedover a range of 10 mm. In 56 tests with 30-pAgtetracycline discs performed on 10 differentdays, the zone diameters varied over a range of7 mm in the GasPak atmosphere and 8 mm inthe 10% H2, 90% N2 + 10% C02 atmosphere.Table 3 illustrates an analysis of variance of

results due to disc potency and gaseous atmos-phere at the 95% confidence level. When vari-ances with 5-,pg tetracycline discs were ana-lyzed, there was no significant difference be-tween the two atmospheres. With 30-pug discs,the variance was significantly different. Vari-ances between 5- and 30-pg tetracycline discsin the GasPak atmosphere were significantlydifferent.

In the tests to determine reproducibility ofthe agar dilution method with strain B-1, theMIC was 0.4 pg/ml on all but one occasion.One determination in the 10% H2, 90% N2 +

10% CO2 atmosphere resulted in an MIC of 0.8pg/ml.

DISCUSSIONMost of the recent effort in antimicrobial

susceptibility testing has been directed towardthe development of a standard referencemethod (7) and toward the recommendation ofa single standardized disc method for suscepti-bility testing of rapidly growing aerobic andfacultative pathogens (5). Similar efforts are

MIC Log.,Mg/ml IOMIC

, 9 R̂ q

4A,6 0_ V- \ r -.

25_ 8_ ,I.

12.5_- 7_ "A. .

6.2_ 6..

3.1_- 5-.

1.6. 4_\

0.8. 3_ . ,

0.4 2. . a

0.2_ _ I .

5 10 15 20 25 30 35 40

ZONE DIAMETER (mm)

4BMIC Log.,mg/ml IOMIC

>25_ 9_

25. 8..

12.5_ 7_

6.2. 6.

3.1_ 5_

1.6. 4_-

0.8. 3..

0.4. 2.

0.z.. I.. A

5 10 15 20 25 30 35 40 45

ZONE DIAMETERS (mm)

FIG. 4. Relationship of zone diameters measuredat 18 to 24 hr and MIC values read at 42 to 48 hr inthe GasPak atmosphere. (A) Tetracycline discs, 5 pg.Least squares line for y = a + bx; b = -0.248, a =

9.827. (B) Tetracycline discs, 30 pg. Least squaresline fory = a + bx; b = -0.245, a = 11.101.

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ANTIMICROBIAL SUSCEPTIBILITY OF ANAEROBES

SA TABLE 1. Tentative zone diameter interpretation(18- to 24-hr incubation)

Tetra- Inhibition zone diameter (mm)cyclinedisc po- Atmosphere Resis- Inter- Suscep-tency tanta mediate tibleb(Ag)

5 GasPak s 12 13-24 2255 H2, N2 + CO2 S12 13-24 ' 25

30 GasPak < 17 18-29 23030 H2, N2 + CO2, 22 23-30 231

a Bacteria resistant to 12.5 gg/ml or more.bBacteria susceptible to 1.6 ,g/ml or less.

5 Mi 2 25 3

ZONE DIMETERS

FIG. 5. Relationship of zone diarat 18 to 24 hr and MIC values read10%o H2, 90% N2 + 10% CO2 atmosicycline disc, 5 jg. Least squares libx; b = - 0.245, a = 9.800. (B) Tetrlg. Least squares line for y = a + b= 11.255.

needed for susceptibility testinpathogens.

Development of resistance t(agents has long been recognize(among aerobic and facultativesimilar situation undoubtedlymost anaerobic pathogens. An eis the emerging resistance of B.racycline. In 1966, Keusch andreported only four of nine strroides tested to be susceptible. I

et al. (3) reported that 18 of 38

" \ * , were susceptible. More recently, 45.5% of0 35 4 5 strains of B. fragilis isolated in St. Louis (A. C.

Sonnenwirth, personal communication) and34.5% of strains of B. fragilis isolated at MayoClinic (W. J. Martin, personal communication)have been found susceptible. Among the

5B strains tested in the present study, 14 of 15isolated prior to 1960, 12 of 22 isolated from1960 to 1969, and only 24 of 63 (38.1%) isolatedfrom 1970 to the present were susceptible.Our data with tetracycline against 100

strains of B. fragilis indicate that a modifica-tion of the method of Bauer et al. (1) results ina reasonably reproducible method for disc sus-ceptibility testing of anaerobes. With the vari-ables of gaseous atmosphere, incubation time,and disc potency tested, the system utilizingthe GasPak, overnight incubation, and 5-mgdiscs gave somewhat better correlation be-

.^RA tween MIC at 42 to 48 hr and zone diameterdata. Based on MIC values and regression line

0 36 40 45 analysis, there were no predictions of suscepti-bility or resistance by inhibition zone diame-

,neters measured ters which did not correlate with MIC values.at 42 to 48 hr in With 30-ig discs, under the same conditions,phere. (A) Tetra- three strains resistant by MIC data were pre-ire for y = a + dicted to be intermediate by zone diameters. A-acycline disc, 30 similar analysis of the data obtained in theIx; b = -0.239, a 10% H2, 90% N2 + 10% CO2 atmosphere indi-

cated that, at 18 to 24 hr, zone diameters with5-,gg discs resulted in five predictions which

g of anaerobic did not correlate with MIC data, and zone di-ameters with 30-,gg discs results in two predic-

D antimicrobial tions which did not correlate.d as a problem Tests for reproducibility of zone diametersorganisms. A with strain B-1 showed greater variability withexists among 5-ug discs. An analysis of variance of results

.xample of this with the two disc potencies in the two dif-fragilis to tet- ferent atmospheres indicated the least varia-O'Connell (11) tion with 30-,gg discs in the GasPak atmos-ains of Bacte- phere. Additionally, the GasPak atmosphereIn 1970, Bodner with activated catalyst gave fewer predictions3 strains tested which did not correlate with MIC data in the

MNC Log2

)25_ 9_

25. 8.

12.5. 7.-

6.2. 6.

3.1. 5..

1.6. 4..

0.8. 3.

0.4. 2.

0.2.. I .

SC L",jig/fri lowE>25. 9_-

25. 8.

12.5. 7.

6.2. 6.

3.1. 5.

1.6. 4..

0.8. 3.

0.4. 2..

0.2- I

I5 l 6 20 25 3

ZONE DIAMETERS

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TABLE 2. Variability of zone diameters

Tetracycline Inhibition zone diameters (mm)adisc potency Atmosphere Minimum-

(fig) maximum Range Mean Mode SD

5 GasPak 26-36 10 32 33 2.335 H2, N2 + CO2 27-37 10 33 35 1.97

30 GasPak 35-42 7 38 38 1.4130 H2, N2 + CO2 35-43 8 39 39 2.03

a Based on 56 determinations for each set of conditions.

TABLE 3. Significance of variances betweenmaterials and methods

Tetracycline Meandisc potency Atmosphere squares F ratioa

(Mg) deviation

5 GasPak 5.435 H2, N2 + CO2 3.88 1.39

30 GasPak 1.9930 H2, N2 + CO2 4.12 2.07

5 GasPak 5.4330 GasPak 1.99 2.73

aAt the 95% level of confidence, the confidenceintervals are 0.57 < F < 1.52.

present study, and, because of its simplicity,this atmosphere is probably the one least sub-ject to variation in a diagnostic microbiologylaboratory. Therefore, it has been selected forfuture studies of susceptibility testing in thislaboratory.Further work is in progress with other drugs

against B. fragilis. We also plan to study otheranaerobes and other drugs in this manner.Standards for interpretation of inhibition zonediameters applicable to all anaerobes testedwill then be developed.

Conditions inherent in anaerobic incubation(anaerobiosis and CO2, with consequent low-ering of pH), as well as addition of blood orother substances to culture media, are knownto affect the activity of various antimicrobialagents, but are necessary for the growth ofmany anaerobes. Our approach has been tostandardize conditions as carefully as possibleand to determine the variability of resultsunder these conditions. Once this is known foreach antimicrobial agent to be considered,then the effect of varying conditions or me-dium composition, or both, on the activity ofantimicrobial agents can be determined.

The validity of test results and their inter-pretation can be ascertained only when corre-lative clinical data are available.

ACKNOWLEDGMENTSThis research was supported by Public Health Service

research grant CC00520 from the Center for Disease Control,Atlanta, Ga.We appreciate the kindness of A. C. Sonnenwirth, St.

Louis, Mo., for his contribution of several of the bacterialstrains used in this study.

LITERATURE CITED

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2. Beerens, H., M. M. Castel, and A. Modjadedy. 1962.Recherches sur la determination de la sensibilita desbactaries anaerobies non sporulees a onze antibio-tiques par la methods des disques resultats obtenusavec 180 souches. Ann. Inst. Pasteur Lille 13:105-122.

3. Bodner, S. J., M. G. Koenig, and J. S. Goodman. 1970.Bacteremic Bacteroides infections. Ann. Intern. Med.73:537-544.

4. Brownlee, K. A. 1965. Statistical theory and methodol-ogy, in science and engineering, 2nd ed., chap. 11.John Wiley & Sons, Inc., New York.

5. Edwards, C. C. 1971. Antibiotic susceptibility discs.Drug efficacy study implementation. Fed. Regist. 36:6899-6902.

6. Egerton, J. R., I. M. Parsonson, and N. P. H. Graham.1968. Parenteral chemotherapy of ovine foot-rot. Aust.Vet. J. 44:275-283.

7. Ericsson, H. M., and J. C. Sherris. 1971. Antibiotic sen-sitivity testing. Report of an International Collabora-tive Study. Acta Pathol. Microbiol. Scand. Suppl.217.

8. Finegold, S. M. 1968. Infections due to anaerobes. Med.Times 96:174-187.

9. Finegold, S. M., N. E. Harada, and L. G. Miller. 1967.Antibiotic susceptibility patterns as aids in classifica-tion and characterization of gram-negative anaerobicbacilli. J. Bacteriol. 94:1443-1450.

10. Ingham, H. R., J. B. Selkon, A. C. Codd, and J. H.Hale. 1968. A study in vitro of the sensitivity to anti-biotics of Bacteroides fragilis. J. Clin. Pathol. 21:432-436.

11. Keusch, G. T., and C. J. O'Connell. 1966. The suscepti-bility of Bacteroides to the penicillins and cephalo-thin. Amer. J. Med. Sci. 251:428-432.

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