microdilution antibiotic susceptibility examination of ... · mhbroth, and 46.2 tliters of ts...

APPLIED MICROBIOLOGY, Nov. 1973, p. 658-665Copyright 0 1973 American Society for Microbiology

Vol. 26, No. 5Printed in U.S.A.

Microdilution Antibiotic Susceptibility Test:Examination of Certain Variables

R. C. TILTON, L. LIEBERMAN, AND E. H. GERLACHUniversity of Connecticut Health Center, Farmington, Connecticut 06032 and St. Francis Hospital,

Wichita, Kansas 76307

Received for publication 22 June 1973

A semiautomated microdilution susceptibility test is described. The effect ofcertain parameters such as inoculum size, growth media, incubation conditions,and inoculum dispensing systems was studied. Both medium type and inoculumsize caused significant variations in the minimum inhibitory concentrations(MIC) of certain antibiotic-organism combinations. No effect on MIC was ob-served as a function of incubator type. Efforts to read a reproducible MIC valuein less than 12 h failed. A commercially available wire pronged inoculator was

determined to be inaccurate and unsafe. Disposable dropper pipettes proved tobe economical, accurate, and precise. Although a standard method for microdilu-tion antibiotic susceptibility testing is not proposed, data are presented whichshow that future attempts at standardized procedures are mandatory if inter-and intralaboratory reliability is desired.

Antibiotic dilution techniques are the mostreliable means to determine antimicrobial sus-ceptibility in vitro. Serial dilution techniqueswere revolutionized when Takatsy (18) devel-oped a spiral loop capable of adding, mixing,and removing a small quantity of reagent from atube. This microtitration procedure was firstused in serology and virology but has recentlybeen applied to clinical bacteriology. Marymontand Wentz (15) developed a microtitrationsystem for the determination of minimum in-hibitory concentrations (MIC) of antibiotics anddemonstrated 94% agreement between the tubedilution and microtitration methods. Harwick,Weiss, and Feckety (10) described a multipleloop holder which simplified the microdilutionprocedure and facilitated the detection of mini-mum bactericidal end points (MBC). Theircomparisons between the micromethod and themacromethod were reported as being 92.5% inagreement for MIC and 85.5% agreement forMBC. Gavan and Butler (5) compared thereproducibility of an automated microdilutionMIC method and the manual microtiter MICmethod with eight antibiotics against Staphylo-coccus aureus and Escherichia coli. Abso-lute MIC values obtained by both methods wereidentical for 7 of 13 drugs tested. No deteriora-tion of drugs could be detected when drugs weredispensed in multiwell trays and stored at- 26 C for 2 weeks.

Gavan and Butler (6), Gerlach (8), and Tiltonand Newberg (19) in a collaborative effort havedescribed the detailed procedure and qualitycontrol of the microdilution antibiotic suscepti-bility test.

In this investigation the effects of the follow-ing parameters on MIC were evaluated: inocu-lum concentration, methods of inoculation,growth media, incubation time, and incubationtemperature.

MATERIALS AND METHODSControl organisms. The following organisms were

designated as control cultures for their correspondingantibiotic(s): E. coli WHO-5 (ampicillin, cephalothin,chloramphenicol, colistin, and kanamycin); E. coliWHO-16 (tetracycline); E. coli 4883 (gentamicin);Pseudomonas aeruginosa HC-2 (kanamycin, gen-tamicin, carbenicillin, colistin); S. aureus WHO-4(ampicillin, cephalothin, penicillin); S. aureusWHO-6 (chloramphenicol, erythromycin); S. aureusWHO-4 (clindamycin, tetracycline); S. aureus 2834(methicillin). The organisms were maintained onTrypticase soy agar slants at 4 C and transferredbiweekly. Broth cultures for testing were preparedfrom these stock slants.Growth media. Mueller-Hinton broth (MH)

(BBL), brain heart infusion broth (BHI) (BBL),Oxoid sensitivity test medium (OST) (Colab), andTrypticase soy broth (TS) (BBL) were freshly pre-pared prior to their use in an experiment.Standard antibiotic solutions. Table 1 lists the

antibiotics tested and pertinent information on their

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MICRODILUTION ANTIBIOTIC TEST

preparation. Stock solutions containing 256 gg ofantibiotic per ml were prepared as previously de-scribed (6, 8, 19) and stored in sterile test tubes (13 by100 mm) at -20 C until used. Carbenicillin wasprepared at an initial concentration of 2,048 Ag/ml.

Preparation of trays containing diluted anti-biotics. Preparation of the antibiotic-containing traysusing a Canalco Autotiter II has been previouslydescribed (6, 8, 19).

Quality control procedures. These procedureshave previously been described in detail (8).Inoculum standardization. The bacterial inocu-

lum was suspended in MH broth. Turbidity of theinoculum was measured by using a nephelometer(Hach Chemical Co.). Standard curves of colony-forming units (CFU) per ml, as a function of turbidityunits, were constructed by diluting an overnight cul-ture of S. aureus WHO-6 and E. coli WHO-5 from 1-2to 1-16. The turbidity of each of these dilutions wasdetermined nephelometrically for each organism anda triplicate standard plate count was performed oneach dilution. This procedure was repeated four timesand standard curves plotted.

Inoculation methods. Two methods of addingorganisms to prepared trays were tested for accuracyand precision. (i) A multi-pronged wire inoculator(Canalco) was flame sterilized and dipped into adisposable styrofoam tray containing 100 ml of a testinoculum (10' CFU/ml, E. coli). The inoculator wasthen used to seed simultaneously each of the 120autotray wells. (ii) Using disposable 50-Mgliter drop-ping pipettes (Cooke Engineering), 50 ;sliters of thesame inoculum was added to each of the wells in asecond tray. Final volume was 100 Mliters ofMH brothper well. A 10-Mliter amount of broth was immediatelyremoved from each well of both trays and a standardplate count performed.A similar experiment was performed in which the

inoculum consisted of E. coli (106 CFU per ml)uniformly labeled with 2H-adenine (New EnglandNuclear Corp.). A 10-Mliter amount of broth wasremoved from all wells after inoculation and placed ina vial containing 5.0 ml of scintillation fluor (NewEngland Nuclear Corp., NEF 934). The vials werecounted on a Beckman scintillation counter modelLS-100 and results were reported as disintegrationsper minute (DPM) per 100 Mliters of inoculated broth.The accuracy and precision of the 50-uliter disposa-

ble pipette were further evaluated. Random samplesfrom a box of pipettes were used to deliver 20 dropseach of 0.85% saline, BHI broth, MH broth, and TSbroth into tared plastic beakers. Each experiment wasperformed 10 times and an average drop size for 200drops was calculated gravimetrically. The meanweight per drop dispensed by the pipette was calcu-lated by weighing each of 40 drops of MH broth on ananalytical balance.

Incubation temperature. The time required forthe liquid inside of a sealed Autotray well to come toincubation temperature and temperature fluctuationsover a 24-h period were determined with three condi-tions of incubation: (i) a forced air incubator, (ii) astatic air incubator, (iii) a water bath in which the

TABLE 1. Antibiotics tested

Antibiotic Solubility

Ampicillin ........... AqueousCarbenicillin ......... AqueousCephalothin .......... AqueousChloramphenicol ..... AqueousClindamycin ......... AqueousColistin ........... AqueousErythromycin ........ Dissolve in 1.0 ml of acetone,

q.s. with waterGentamicin .......... Aqueous (adjust to pH 7.5)Kanamycin .......... AqueousMethicillin .......... AqueousPenicillin .......... AqueousTetracycline ......... Aqueous

sealed plate was floated on the surface of the waterbath. Temperature was monitored by embedding athermistor connected to a digital thermometer in theliquid contained in the well of a sealed tray. Tempera-ture was recorded at 30-s intervals for 10 min and athourly intervals for the remainder of the 24 h.

Inoculated trays (S. aureus WHO-6 and E. coliWHO-5) were placed under the same three conditionsof incubation at 35 C for 18 h. At hourly intervals atray was removed and the MIC of the designated eightantibiotics for the gram-positive control organism andfor the gram-negative control organisms were deter-mined by assessing turbidity visually.

Effect of the growth medium on MIC. Fourdifferent nutrient media (TS broth, BHI broth, MHbroth, and OST broth) were evaluated for their effecton MIC. Trays were prepared by using the broth to betested. The inoculum (10w CFU per ml) was similarlydiluted in the test broth.Reading of the trays. The MIC for each antibiotic

was read as the dilution corresponding to the last wellin the twofold dilution series having no visibly detect-able growth. Growth was defined as (i) confluentturbidity, (ii) light but definite turbidity, or (iii)single or multiple clusters of growth = >2 mm indiameter.

Statistical methods. Following a pattern initiatedby Ericsson and Sherris (4), replicate MIC were ex-pressed as the log, of the geometric mean (X) of theMIC + 9. The interconversion of dilution interval,MIC (jug/ml), log, MIC, and log, MIC + 9 can be seenin Table 2. Calculations using the MIC expressedin this manner provide for better indications of re-producibility than would normally be indicated andthe elimination of negative numbers from the calcu-lations.

Tests of significance were performed by using thenonparametric Kruskal-Wallis one-way analysis ofvariance by ranks (17). Due to the geometric nature ofsome of the data, it was necessary to use this test todetermine if differences among samples signifiedgenuine population differences or whether they repre-sented chance variations such as are to be expectedamong several random samples from the same popu-lation.

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TILTON, LIEBERMAN, AND GERLACH

TABLE 2. Conversion units for expressing MIC asdilution interval in gg/ml, log2 or log, + 9

Dilution Antibiotic concn.

interval jsg/ml log, log2 + 9

1 64.0 6 152 32.0 5 143 16.0 4 134 8.0 3 125 4.0 2 116 2.0 1 107 1.0 0 98 0.5 -1 89 0.25 -2 710 0.12 -3 611 0.06 -4 512 0.03 -5 413 0.015 -6 314 0.007 -7 215 0.0035 -8 1

RESULTSFigure 1 shows the relationship between the

number of bacteria in a broth medium and thedegree of light scattering measured in turbidityunits. The slope of the lines for gram-positive(S. aureus WHO-6) and gram-negative bacteria(E. coli Who-5) differed slightly due to the lightscattering properties of rods and cocci. Lightscattering results on other members of thefamily Enterobacteriaceae and the genusPseudomonas were similar to those for E. coli aswere the results with streptococci similar to S.aureus.Both the wire pronged inoculator and the

50-,uliter disposable pipettes were evaluated foraccuracy and precision. The inoculator theoreti-cally delivered 0.001 ml per wire prong. Actualmean delivery was calculated to be 0.0004 mlper prong. By standard bacteriological tech-niques, the mean number of bacteria deliveredto a broth-containing well was determined to be989 CFU (S.D. 670 CFU, C.V. 66%). The rangeof CFU delivered per prong was from 200 to2,580. By radiometric methods the mean DPMdelivered by each prong was 470.2 (S.D. 320.7,C.V. 68%).The 50-Mliter plastic disposable pipette dis-

pensed a per drop mean volume of 49.2 ,liters ofsaline, 47.0 ,liters of BHI broth, 46.0 Mliters ofMH broth, and 46.2 tliters of TS broth. Theaverage weight of one drop was 40.75 gg (S.D.0.679 fig, C.V. 1.66%). Using radiometric tech-niques, the pipettes delivered a per drop meanof 69,270 DPM (S.D. 1,573 DPM, C.V. 7.46%).Tables 3, 4, and 5 show the effect of inoculum

concentration on the MIC of eight antibiotics

for S. aureus, seven antibiotics for E. coli, andfour antibiotics for P. aeruginosa. Only fourdrugs, clindamycin (S. aureus), methicillin (S.aureus), tetracycline (E. coli), and carbenicillin(P. aeruginosa), failed to show significant inoc-ulum effects. As expected, a greater range ofMIC as a function of inoculum size was ob-served in penicillinase producing aureus grow-ing in the presence of the penicillinase-sus-ceptible antibiotics such as ampicillin (7.6 to11.14, p < 0.05) and penicillin (7.39 to 12.28, p< 0.001). Two other antibiotics showed highlysignificant inoculum effects with both E. coliand P. aeruginosa: gentamicin (p < 0.001 andcolistin (p < 0.001). The other antibioticstested showed no greater than a two-tube varia-tion as a function of inoculum.

It has been recognized that the incubation ofa bacterial inoculum in a multiwelled polysty-rene tray is not functionally identical to theincubation of the same inoculum in a glass tube.When unsealed inoculated trays were stacked inan incubator, there was a measured 25 to 30%loss of volume in the individual wells, resultingin an increased concentration of the antibiotic.Unsealed trays incubated in a humidity-con-trolled incubator showed less dehydration (5 to10% volume loss). Sealed trays showed nodetectable fluid loss.Experiments were performed to determine

the minimum time required to generate a relia-ble MIC. Although after 8 h of incubationturbidity could be seen in the wells, resultsvaried by four to five dilution intervals between8 and 15 h.Heat exchange between the polystyrene tray

and the liquid medium was increased by float-

GRAM POSITIVE COCCI _/CORRELATION COEFFICIENT-

0.991

10/

GRAM NEGATIVE RODSCORRELATION COEFFICIENT-

10

/ /0.9850/

TURBIDITY UNITS

FIG. 1. Standard curves for adjusting the bacterialinoculum by nephelometry.

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TABLE 3. Effect of the inoculum concentration of S. aureus on MIC

Inoculum concn. (CFU)Antibiotic Control organism

104 105 10' 107

Ampicillin S. aureus WHO-4 p < 0.05 7.60a 8.20 9.30 11.14Cephalothin S. aureus WHO-4 p < 0.05 6.29 6.89 6.66 7.49Chioramphenicol S. aureus WHO-6 p < 0.05 10.50 11.00 11.30 12.00Erythromycin S. aureus WHO-6 p < 0.05 6.89 7.19 7.29 8.49Clindamycin S. aureus WHO-4 n.s. 5.59 5.99 6.19 6.39Methicillin S. aureus 2834 n.s. 9.20 8.79 8.49 8.79Penicillin S. aureus WHO-4 p < 0.001 7.39 7.79 9.30 12.28Tetracycline S. aureus WHO-4 p < 0.001 5.19 6.19 7.00 7.19

a Results expressed as log2 X MIC + 9.

TABLE 4. Effect of the inoculum concentration of E. coli on MIC


104 105 106 107

Ampicillin E. coli WHO-5 p < 0.05 8.71a 8.57 8.71 10.00Cephalothin E. coli WHO-5 p < 0.01 10.28 10.28 11.00 12.00Chioramphenicol E. coli WHO-5 p < 0.05 8.76 8.58 8.91 9.54Kanamycin E. coli WHO-5 p < 0.01 8.77 9.25 10.08 10.77Tetracycline E. coli WHO-16 n.s. 7.40 7.50 7.29 7.86Gentamicin E. coli 4883 p < 0.001 6.50 7.67 8.75 10.00Colistin E. coli WHO-5 p < 0.001 6.00 8.50 9.00 9.75

aResults expressed as log2 X MIC + 9.

TABLE 5. Effect of the inoculum concentration of P. aeruginosa HC-2 on MIC


104 10' 10' 107

Gentamicin P. aeruginosa HC-2 p < 0.001 1.84a 1.84 3.30 7.19Carbenicillin P. aeruginosa HC-2 n.s. 14.60 15.00 14.90 15.90Kanamycin P. aeruginosa HC-2 p < 0.01 14.00 14.20 14.60 15.00Colistin P. aeruginosa HC-2 p < 0.001 8.49 9.60 11.20 13.00

aResults expressed as log2 X MIC + 9.

ing the inoculated tray in a 35 C water bath.Although the tray in the water bath came totemperature faster than a plate in either aforced or static air incubator (10 min versus 31min), all three incubators produced stable tem-peratures in the medium (i0.25 C) for theduration of the 24-h incubation. Inoculatedtrays showed identical MIC in the three incuba-tion modes with no decrease in the time re-quired to achieve a reliable MIC.

Results in Tables 6, 7, and 8 show the effect ofvarious growth media on the MIC. Variations inmedia induced statistically significant differ-ences in the mean MIC for E. coli (Table 7) byusing ampicillin (p < 0.001), chloramphenicol(p < 0.001), kanamycin (p < 0.02), and tetra-cycline (p < 0.001); S. aureus (Table 6) by

using ampicillin (p < 0.001), cephalothin (p <0.02), chloramphenicol (p < 0.001), and eryth-romycin (p < 0.01); and for P. aeruginosa, byusing gentamicin (p < 0.001), colistin (p <0.001), and kanamycin (p < 0.001). However, inonly three instances did the differences in meanMIC between any two media exceed 1.5 dilutionintervals (chloramphenicol, E. coli WHO-5),(gentamicin and colistin, P. aeruginosa).

DISCUSSIONIt is unfortunate that there are currently no

generally accepted standard methods for dilu-tion tests of antibiotic susceptibility. Althoughthe agar diffusion methodology has been widelyadopted by most clinical laboratories, there aresituations when the MIC of an antibiotic must

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be determined by either broth or agar dilution.Broth dilution is a cumbersome, time consum-

ing, and expensive technique. However, thedevelopment of equipment that partially auto-mated the broth dilution method has led torenewed interest in the routine determination ofMIC. The numerical values generated by thesetechniques are not absolute. Rather, they are

the sum of variation induced by factors such asinoculum preparation, standardization, size,growth media, and conditions and time ofincubation. This current investigation does notpropose a standardized method for microdilu-tion susceptibility tests. It attempts only todefine the extent of variation that can beexpected in MIC if the stated parameters are

altered.Various studies attest to the accuracy and

reproducibility of the microdilution antibioticsusceptibility method. MacLowry and Marsh(14) demonstrated that of 88 microdilutionsperformed in parallel with tube dilutions, 68%produced the same end point and 19% varied byone dilution. Of 336 repetitive assays, 86% wereidentical. Gavan and Town (7) reported that99.1% of manual tube dilution replicates fellwithin the ±41 dilution interval of the mean

compared with 94.8% of microdilution repli-cates. Gerlach (9) tested 191 bacterial strains byboth agar dilution and microdilution. He foundthat the MIC obtained on 186 of 191 strains withmicrodilution were within plus or minus one

dilution step of those obtained with agar dilu-tion.

In the present investigation, of 500 consecu-

tive microdilution MIC determinations, 91% of

TABLE 6. Effect of growth media on MIC (S. aureus)

MediaAntibiotic Control organism

OST BHI TS MH

Ampicillin S. aureus WHO-4 p < 0.001 8.10a 9.57 9.00 8.15Cephalothin S. aureus WHO-4 p < 0.02 5.79 5.99 6.69 6.69Chloramphenicol S. aureus WHO-6 p < 0.001 9.60 9.80 10.00 10.84Erythromycin S. aureus WHO-6 p < 0.01 6.19 5.59 6.12 6.92Clindamycin S. aureus WHO-4 n.s. 4.44 4.71 4.48 5.49Methicillin S. aureus 2834 n.s. 9.00 9.57 8.75 9.08Penicillin S. aureus WHO-4 n.s. 7.44 7.69 8.10 7.61

a Results expressed as log2 X MIC + 9.

TABLE 7. Effect of growth media on MIC (E. coli)


OST BHI TS MH

Ampicillin E. cili WHO-5 p < 0.001 8.69a 9.33 9.80 8.69Cephalothin E. coli WHO-5 n.s. 10.20 10.50 10.50 10.44Chloramphenicol E. coli WHO-5 p < 0.001 7.82 8.90 9.40 8.44Kanamycin E. coli WHO-5 p < 0.02 8.81 9.90 10.00 9.33Tetracycline E. coli WHO-16 p < 0.001 7.11 7.99 8.56 7.61Gentamicin E. coli 4883 n.s. 7.94 7.99 9.10 8.19Colistin E. coli WHO-5 p < 0.001 7.62 7.34 8.56 8.61

. Results expressed as log2 X MIC + 9.

TABLE 8. Effect of growth media on MIC (P. aeruginosa HC-2)


OST BHI TS MH

Gentamicin P. aeruginosa HC-2 p < 0.001 3.94a 3.94 5.57 1.84Carbenicillin P. aeruginosa HC-2 p < 0.01 14.60 14.30 14.00 15.00Kanamycin P. aeruginosa HC-2 p < 0.001 13.10 13.10 14.00 14.20Colistin P. aeruginosa HC-2 p < 0.001 5.66 8.09 9.80 9.60

. Results expressed as log2 X MIC + 9.



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the values were within the 1 dilution intervalof the mean MIC for the antibiotic and orga-nism used.

Prior to testing the effects of inoculum con-centration on MIC end points, the methods ofdelivery of inoculum were evaluated. The wire-pronged inoculators lacked reproducibility(C.V. 66%) and accuracy (actual delivery was50% of stated delivery). The plastic pipettesproved to be more reproducible and accurate(C.V. 1.66 to 7.46%) than the wire inoculatoralthough more time consuming and tedious touse. Cooper et al. (3) found delivery errors of 7.6to 14.4% (mean 3.5%) in similar nondisposabledropper pipettes based on an expected volumeof 25 uliters. Gavan and Butler (5) reported that50-,gliter nondisposable dropper pipettes deliv-ered a mean of 47.4 + 3.6 pliters of broth tostatic-free wells. When the wells were static-charged, drop size decreased to 39.0 Aliters. Inthe present study, the dropper pipette was held2 cm above the plate surface to minimize staticcharges. The mean drop size of 46.0 gliters ofMH broth compared favorably with the similardata of Gavan and Butler (5).

In numerous studies, inoculum size has beenshown to markedly affect disk diffusion suscep-tibility results. These methods suggested byBauer et al. (1) and Ericsson et al. (4) dictatethat inoculum be standardized by visual com-parison with a barium sulfate turbidity stan-dard. Similarly, it is recognized that inoculumsize influences the end point obtained in dilu-tion tests, particularly with the fl-lactamasesensitive antibiotics. Ericsson and Sherris (4)established an arbitrary inoculum size rangingfrom 105 to 106 CFU per ml for broth dilutiontests, designed to include the range of inoculumsizes reported in previous studies.

Statistically significant differences were ob-served in the MIC end points as a function ofinoculum concentration. When the inoculumwas varied 10,000-fold, MIC differences of fourto five dilution intervals for penicillin andampicillin were noted with the gram-positivecontrols. This marked inoculum effect was notobserved for the other antibiotics tested al-though statistical analysis revealed that in allcases, except methicillin and clindamycin, asignificant difference existed. Although signifi-cant, these differences seen as a function ofinoculum were small, one to two dilution inter-vals over the 10,000-fold range of inoculum size.Data on the gram-negative control organismswere similar. The MIC for tetracycline did notshow significant variation as a function ofinoculum, whereas ampicillin, cephalothin,chloramphenicol, kanamycin, gentamicin, and

colistin did. The most marked inoculum effectwas seen with gentamicin and colistin (3 to 4dilution steps). This result is hard to explain asthere was no evidence that members of the En-terobacteriaceae or P. aeruginosa producedextracellular gentamicin or colistin-inhibitingsubstances. E. coli WHO-5 and P. aeruginosaHC-2 were judged susceptible to gentamicinand colistin by both microdilution and disk dif-fusion methods. The arbitrary choice of inocu-lum size (105 to 106 CFU per ml) in broth dilu-tion tests by Ericsson and Sherris (4) appears tobe satisfactory for the microdilution method. Inonly two cases (S. aureus, ampicillin and penicil-lin) was there greater than one but less than twodilution intervals difference in MIC end pointswhen inoculum was varied between 105 CFUper ml and 106 CFU per ml.Most of the studies reported on the effect of

the growth medium have concerned disk diffu-sion tests. Jay and Sherris (13) reported statisti-cally significant differences in zone size for 10different high-strength antibiotic disks whenfour different nutrient media were tested. Jay,Batjer, and Sherris (11) studied the growthsupporting ability of MH broth and concludedthat it was adequate as a sensitivity test me-dium. The same investigators compared differ-ent batches ofMH broth from two manufactur-ers by both broth and agar dilution methodsand results were essentially identical. Needhamet al. (16) in a similar study observed the effectsof MH and Grove and Randall (GR) media onMIC end points. Results with the two mediawere essentially comparable except that the GRbroth gave a higher MIC for kanamycin againstEnterobacter.

In the present investigation, the MIC of anti-biotics tested against gram-negative organismsgrowing in TS broth were consistently higherthan the other media. This effect of TS brothwas not observed with S. aureus and probablyreflects the reported effect of Mg2+ and Ca2+ onthe antibiotic susceptibility of some gram-nega-tive bacteria (9). The most marked effect ofgrowth media was observed when the MIC ofgentamicin was determined by using P.aeruginosa as a test organism. Predictably, theorganism was more than four times more sus-ceptible to gentamicin when the test was per-formed in MH broth. Other studies (9) reportthat the MIC of gentamicin for P. aeruginosaincreases as the concentration of Mg2+ and Ca2+in the growth medium increases.Growth media should have no more or less

effect in the microdilution procedure than in themanual tube dilution procedure. However, inthe interests of uniformity, one medium should

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be recommended as the one of choice. Thebenefits due to the wide availability of MHmedium, its minimal lot to lot variation, and itsaccepted use for the disk diffusion test appear tooverride the disadvantages of a slightly reducedgrowth rate for the gram-positive cocci and theincreased growth rate for Enterobacteriaceae(11). However, in order to insure reproducibleresults, the concentration of Mg2+ and Ca2+ inthe medium must be standardized.Although variations in inoculum size,

methods of dispensing it, and growth mediahave been shown to effect the microdilution endpoint, certain other variables such as machinefunction, antibiotics, and contamination mustalso be controlled.

In the microdilution susceptibility test cur-rently conceived, the clinical laboratory is boththe producer and the consumer of its ownproduct, i.e., the prepared trays. The dilutorsmust be carefully handled, cleaned regularly,heated to redness, and quenched in sterile waterprior to each use in order to insure dilutionalaccuracy. At regular intervals, trays containinga single antibiotic in all eight rows should beprepared and inoculated with a control orga-nism. Gross machine malfunction resulting in aone-dilution interval or greater error may bespotted in this manner. Dropping needles mustbe calibrated prior to each use. Failure to cleanthe needles after preparation of trays may resultin broth medium drying in the needles, withresultant loss of accuracy. The machine shouldbe visually monitored during operation to insurethat reagents are dispensed and mixed uni-formly.The greatest risk of error may be in the

preparation of antibiotic solutions. Antibioticassay powders should be stored in a dessicatorat a maximum temperature of 4 C. An analyti-cal balance must be used for weighing withquantitative technique used throughout. Fail-ure to thoroughly dissolve the antibiotic in thesolvent will lead to error.Although contamination has not been a prob-

lem, the following elements of the proceduremust be routinely checked for sterility: growthmedium, inoculum dilutors, dispensing needles,dropping pipettes, antibiotic solutions, and au-totrays.The microdilution procedure offers a practi-

cal, cost and time feasible method for perform-ance of broth dilution antibiotic susceptibilitytests. Having defined some of the elements ofvariation in the microdilution test, a collabora-tive study should be convened to standardizeboth agar dilution and microdilution proce-

dures. Such a study should result in (i) uniformmethodology, (ii) availability of quality controlcultures, and (iii) comparative MIC data frommany laboratories indicating both inter- andintralaboratory variability.

ACKNOWLEDGMENTSWe acknowledge the statistical and data processing assist-

ance of Robert Swan of the University of Connecticut HealthCenter, Data Systems Division.

LITERATURE CITED

1. Bauer, A. W., W. M. M. Kirby, J. C. Sherris, and M.Turck. 1966. Antibiotic susceptibility testing by astandardized single disc method. Amer. J. Clin. Pathol.45:493-496.

2. Branch, A., and E. E. Power. 1966. Influence of differentagars on the results of diffusion tests. In Antibioticsensitivity testing: report of an international collabora-tive study. Acta Pathol. Microbiol. Scand. Sect. B.,(Suppl. 21):1-90.

3. Cooper, H. A., E. J. Walter Bowie, and C. A. Owen. 1972.Pitfalls of the microtiter system for serial dilution andstandardization using radio-iodinated albumin. Amer.J. Clin. Pathol. 57:332-335.

4. Ericsson, H. M., and J. C. Sherris. 1971. Antibioticsensitivity testing: report of an international collabora-tive study. Acta Pathol. Microbiol. Scand. Sect. B.,(Suppl. 217):1-90.

5. Gavan, T. L., and D. A. Butler, 1971. Automatedmicrodilution antimicrobial susceptibility testing.Bacteriol. Proc., p. 115.

6. Gavan, T. L., and D. Butler. 1973. Automated microdilu-tion method for antimicrobial susceptibility testing. InCurrent techniques for antibiotic susceptibility testing.Arthur C. Thomas, Inc.

7. Gavan, T. L., and M. A. Town. 1970. A microdilutionmethod for antibiotic susceptibility testing. Amer. J.Clin. Pathol. 53:880-885.

8. Gerlach, E. H. 1973. Microdilution: a comparativestudy. In Current techniques for antibiotic susceptibil-ity testing. Arthur C. Thomas, Inc.

9. Gilbert, D. N., E. Kutscher, P. Ireland, J. A. Barnett, andJ. P. Sanford. 1971. Effect of concentrations of magne-sium and calcium on the in-vitro susceptibility ofPseudomonas aeruginosa to gentamicin. J. Infect. Dis.124 (Suppl.):537-545.

10. Harwick, H. J., P. Weiss, and F. R. Feckety. 1968.Application of microtitration techniques to bacterio-static and bactericidal antibiotic susceptibility testing.J. Lab. Clin. Med. 72:511-516.

11. Jay, V. C., J. D. Batzer, and J. C. Sherris. 1966. Studieson Mueller-Hinton broth. In Antibiotic sensitivitytesting: report of an international collaborative study.Acta Pathol. Microbiol. Scand. Sect. B., (Suppl.21):1-90.

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microdilution antibiotic susceptibility examination of ... · mhbroth, and 46.2 tliters of ts...

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