Testing: Evaluation of Overnight Agar Cultures and the Rapid Inoculum Standardization System

JOURNAL OF CLINICAL MICROBIOLOGY, Mar. 1983, p. 45-457 95-1137/83/345-8$2./ Copyright 1983, American Society for Microbiology Vol. 17, No. 3 Inoculum Standardization in Antimicrobial Susceptibility Testing: Evaluation of Overnight Agar Cultures and the Rapid Inoculum Standardization System CAROLYN N. BAKER,'* CLYDE THORNSBERRY,1 AND RONALD W. HAWKINSON2 Hospital Infections Program, Center for Infectious Diseases, Centers for Disease Control, Atlanta, Georgia 3O333,1 and Riker Laboratories, Inc., 3M Center, St. Paul, Minnesota 551442 Received 26 August 1982/Accepted 26 November 1982 Using the Rapid Inoculum Standardization System [RISS; Minnesota Mining & Manufacturing Co. (3M Co.)], we investigated the use of overnight agar cultures in the preparation of inocula for disk diffusion and microdilution susceptibility tests and compared our results with susceptibility results obtained with the standard methods. The 3M system consists of an inoculation wand, with which a reproducible number of bacteria can be removed from an agar plate, and a diluent in which to suspend the organisms. We used 25 gram-positive cocci, 75 enteric and nonfermentive gram-negative bacilli, and 4 more fastidious bacteria (Haemophilus, Neisseria, and pneumococci) for the evaluation. The geometric mean inoculum size for all organisms tested was 9.7 17 CFU/ml by the standard method and 1.1 18 CFU/ml for the RISS. The categories of susceptibility obtained by both methods in the disk diffusion tests were comparable, as were the minimal inhibitory concentrations. We recommend that the use of overnight agar cultures and the use of the RISS to prepare inocula for susceptibility tests be acceptable alternative procedures in the standard methods for susceptibility tests. Antimicrobial susceptibility testing, whether done by disk diffusion or by dilution methods, has historically been performed by using a culture in the early stage of logarithmic growth in broth (3, 9). This phase of growth is obtained by touching four to five similar colonies from primary agar plates, inoculating them into Mueller- Hinton or Trypticase soy broth (BBL Microbiology Systems), and incubating them for 2 to 5 h at 35 C to obtain cultures containing approimately 18 CFU/ml. If necessary, the inoculum is then adjusted to the proper turbidity (approimately 18 CFU/ml), and susceptibility tests are performed. This process of obtaining inocula for antimicrobial susceptibility tests is time consuming and more epensive than preparing a suspension directly from an agar plate. A Rapid Inoculum Standardization System (RISS) has been developed by the Minnesota Mining & Manufacturing Co. (3M Co.) and features a device that removes a predictable number of organisms from colonies on a primary agar plate, which, when placed into the diluent that is provided, yields a suspension containing approimately 18 CFU/ml (J. H. Wicks, R. L. Nelson, and G. E. Krejcarek, submitted for publication). The purposes of this study were twofold:(i) to determine whether susceptibility results obtained with inocula prepared by placing the organisms from overnight colonies in a diluent and adjusting to the final desired concentration are comparable to antimicrobial susceptibility results obtained with inocula prepared in the manner described in the standard methods (3, 9), and (ii) to determine whether the RISS reproducibly delivers the required number of organisms to the inoculum. MATERIALS AND METHODS Bacteria. One hundred-forty recent clinical isolates were tested. They consisted of 75 gram-negative bacilli, 25 gram-positive cocci, and 4 "fastidious" isolates (Haemophilus, Neisseria, and Streptococcus species). These bacterial isolates, which had been preserved by freezing at -6 C in whole defibrinated rabbit blood, were removed from the freezer and thawed. The gramnegative isolates, ecept for the fastidious organisms, were subcultured onto MacConkey agar, and grampositive isolates, ecept for the fastidious ones, were subcultured onto Trypticase soy agar (BBL) supplemented with 5% sheep blood. The fastidious organisms were subcultured as follows: Neisseria gonorrhoeae onto GC base agar with 1%o GCHI (Carr- Scarborough defined supplement), N. meningitidis onto Mueller-Hinton agar, Streptococcus pneumoniae onto Mueller-Hinton agar with 5% sheep blood, and Haemophilius influenzae onto Mueller-Hinton agar with 1% hemoglobin (Difco Laboratories) and 1% GCHI. All gram-positive and gram-negative isolates were incubated for 18 to 2 h at 35 C. All fastidious 45

VOL. 17, 1983 isolates ecept for H. influenzae were incubated in CO2 at 35 C for 24 h. Isolates were then resubcultured in triplicate onto their respective media (see section on testing) and incubated as stated previously. The inocula were prepared from these cultures. Inocula. The RISS consists of a tube containing.81 ml of diluent (1; Wicks et al., submitted for publication) and an inoculation wand. At the tip of the wand are cross-hatched grooves designed to hold a specific number of bacterial cells picked from colonies on an isolation plate. The bacteria are suspended in the diluent by agitation with a vorte mier. Five to 1 colonies from each of the gram-positive or gram-negative culture plates were touched with the tip of the RISS wand, placed in the diluent, and agitated with the vorte mier for 1 s. Ten to 3 colonies of each fastidious isolate were touched with the tip of the RISS wand, placed in the diluent, and agitated with the vorte mier for 15 to 3 s. S. pneumoniae isolates were agitated with the vorte mier for at least 3 s because it was difficult to break up the clumps of bacteria to obtain a smooth suspension. Inocula for standard antimicrobial susceptibility tests were prepared by touching four to five similar colonies of gram-positive or gram-negative bacteria with a loop, transferring these colonies to tryptic soy broth, and incubating them at 35 C for 2 to 5 h until the turbidity was equivalent to a.5 BaSO4 standard. Suspensions in tryptic soy broth equivalent to a BaSO4 standard were prepared from a 24-h culture plate of the fastidious organisms. Colony counts were performed on each inoculum by subculturing various dilutions of the inocula in water (or phosphate-buffered saline [ph 7.2] for Neisseria species). A.1-ml volume of each dilution was subcultured onto an appropriate agar plate, spread with a sterile glass rod, incubated for 24 to 48 h at 35 C, and counted with a Fisher bacterial colony counter, model 48. Antimicrobial agents. Disks containing antimicrobial agents were purchased from Becton, Dickinson & Co., ecept for spectinomycin disks, which were prepared in our laboratory. Antimicrobial reference powders were supplied by the appropriate pharmaceutical manufacturer. Gram-negative organisms were tested with amikacin, ampicillin, carbenicillin, cefamandole, cefoitin, cephalothin, chloramphenicol, gentamicin, nitrofurantoin, tetracycline, tobramycin, and sulfamethoazole/trimethoprim. Gram-positive organisms were tested with ampicillin, cephalothin, chloramphenicol, clindamycin, erythromycin, gentamicin, kanamycin, oacillin, penicillin, tetracycline, sulfamethoazole- /trimethoprim, and vancomycin. H. influenzae was tested with ampicillin, cefaclor, cefamandole, chloramphenicol, rifampin, tetracycline, and sulfamethoazole/trimethoprim. N. gonorrhoeae was tested with penicillin, spectinomycin, and tetracycline. N. meningitidis was tested with penicillin, rifampin, and sulfadiazine. S. pneumoniae was tested with penicillin and oacillin. Susceptibility testing. Disk diffusion, broth microdilution, and agar dilution (N. gonorrhoeae only) tests were performed with the inocula prepared by using the RISS and by the standard method. Disk diffusion and broth microdilution tests were performed according to the National Committee for Clinical Laboratory Standards (NCCLS) methods (9, 1). Agar dilution tests for INOCULUM STANDARDIZATION 451 N. gonorrhoeae were performed by using the method of Biddle et al. (5). RESULTS Viable organism counts, disk diffusion, and minimal inhibitory concentration (MIC) results obtained for the quality control organisms used in the study with each system are shown in Table 1. Paired results were similar for the five organisms and generally were within +2 mm for the disk test and ±1 log2 dilution for the MIC test. The geometric means of the viable organism counts for the inocula prepared for the test isolates are shown in Fig. 1. The paired counts for the two methods were very similar, the greatest disparity occurring with Staphylococcus aureus. The distribution of the paired zone sizes of the test isolates is shown in Table 2. For the gramnegative isolates, 92.9% had a zone size variation within ±2 mm, and 98.8% of these isolates had a zone size variation within ±4 mm. For the gram-positive isolates, 83.7% had zone size variations within ±2 mm, and 95.9% of these isolates had zone size variations within ±4 mm. For the fastidious isolates, 83.7% had zone variation within ±2 mm, and 95.2% had zone variations within ±4 mm. For most species, the average variation in zone diameter was less than 1 mm. Similar data on the distribution of paired MIC differences for these isolates are shown in Table 3. More than 96% of all strains were within ± 1 log2 dilution. A comparison of susceptibility categories, i.e., susceptible, intermediate, and resistant, for the disk diffusion method is shown in Table 4. There were few errors in categorization, as shown by overall agreements of 97, 96.7, and 97.8% in the three groups of organisms. Only.1% of the gram-negative isolates had very major (resistant by the standard method and susceptible by the test method) or major (susceptible by the standard method and resistant by the test method) discrepancies. None of the gram-positive or fastidious organisms had very major or major discrepancies. Table 5 shows the comparison of results obtained with the dilution tests by both methods. The MICs of all isolates were within ±1 log2 dilution in more than 96% of the pairs and within ±2 log2 dilutions for more than 99%. DISCUSSION In the original description of the standardized agar diffusion antimicrobial disk susceptibility test, Bauer et al. reported that the inoculum should be prepared from a logarithmic culture obtained by growing the bacteria in broth just

452 BAKER, THORNSBERRY, AND HAWKINSON TABLE 1. Results with quality control strains, using RISS and standard inocula Staphylococcus aureus ATCC 25923 Staphylococcus aureus ATCC 29213 faecaliso Antibiotic Diska MICb Disk MIC Disk RISS Std RISS Std RISS Std RISS Std RISS Std 2.2 18c 3.2 17 3.4 16d4.1 i3 3.1 18 6.6 17 4.5 X 16 1. 16 2.6 18 1.3 18 Amikacin NDe ND 1..5 ND ND 2. 1. ND ND Ampicillin 3 32 <.25 s.25 19 22 1..5 26 26 Carbenicillin ND ND s4. <4. ND ND s4. s4. ND ND Cefamandole 29 3.5 s.25 26 26.5 s.25 16 15 Cefaclorf NAM NA 2. 2. NA NA 4. 4. NA NA Cefoitin ND ND 2. 2. ND ND 4. 4. ND ND Cephalothin 34 35 s..25 s..25 32 34.<.25..25 16 18 Chloramphenicol 24 25 8. 8. 23 25 8. 8. 22 23 Clindamycin 26 27.12.12 25 26.12.12 6 6 Erythromycin 27 29.25.25 27 28.25.25 19 18 Gentamicin 23 25 s.25 s.25 23 24 s.25 s.25 15 16 Kanamycin 22 24 1. s.5 22 24 2. 1. 11 13 Nitrofurantoin ND ND 16. 16. ND ND 8. 8. ND ND Oacillin 2 21.25.25 19 2.25.25 6 6 Penicillin 32 34 s.3 <.3 18 22 1..5 23 22 Rifampinf 26 27.3.3 27 25.<.15 s..15 18 18 Spectinomycinf 1 12 >32. >32. 12 12 >32. >32. 8 9 Sulfadiazinef 21 2 64. 64. 22 22 128. 64. 6 6 Sulfa/trimethoprim 28 3 1.2/.6.2/.6 29 29 1.2/.6 1.2/.6 28 28 Tetracycline 24 26 1. 1. 25 25 1. 2. 8 8 Tobramycin ND ND..25 s.25 ND ND..25..25 ND ND Vancomycin 17 18 2. 2. 17 18 1. 1. 19 18 a Values indicate average zone size in millimeters, rounded to the nearest millimeter. b MICs are given in micrograms per milliliter. c Viable organism count; epressed as geometric mean. d Viable organism count in 3 ml of water containing.2% Tween 8. e ND, Not done. f Tests were performed on medium used for fastidious organisms. g NA, Not applicable; cephalothin used as class disk. -J E U CD Ul 1 1i9-5 X 19. 1 X 18 5 X 17. < i 19 5 X 18 IOI o o J. CLIN. MICROBIOL. o S. aureus S. faecalis S. durans S. pneumoniae N. meningiditis XI I XO X O S. epidermidis S. faecium S. bovis H. influenzae N. gonorrhoeae o 1 18 5 X 17 X X X X E. coli P. rettgeri P. vulgaris P. stuartii Enterobacter K. pneumoniae M. morganii P. mirabilis S. marcescens Ps. aeruginosa FIG. 1. Geometric mean of viable counts for clinical isolates obtained by employing inoculum prepared by the standard () and RISS (X) techniques.

VOL. 17, 1983 INOCULUM STANDARDIZATION 453 TABLE 1-Continued ATCC 29212 Escherichia coli ATCC 25922 Pseudomonas aeruginosa ATCC 27853 MIC Disk MIC Disk MIC RISS Std RISS Std RISS Std RISS Std RISS Std 4.1 16 1.5 16 1.2 18 5.7 1' 1.7 16 7.9 1' 2.2 I8 4.8 17 4.4 16 1.1 16 >32 >32 22 22 1. 1. 2 21 8. 4. 1. 1. 18 18 4. 4. 6 6 >32. >32. 64. 32. 25 25 8. 8. 21 22 32. 64. 32. 32. 28 28.5.5 6 6 >32. >32. >32. >32. NA NA ND ND NA NA ND ND >32. >32. 27 27 2. 2. 6 6 >32. >32. 32. 32. 19 19 8. 8. 6 6 >32. >32. 8. 8. 25 24 8. 8. 6 6 >32. >32. >8. >8. 6 6 >8. >8. 6 6 >8. >8. 2. 2. 7 7 >8. >8. 6 6 >8. >8. 8. 16. 22 23 <.25 -.25 17 19 4. 4. 32. 64. 23 22 2. 2. 6 6 >64. >64. 16. 8. 22 23 16. 16. 6 6 >128. >128. >8. >8. 6 6 >8. >8. 6 6 >8. >8. 2. 2. 6 6 >4. >4. 6 6 >4. >4. 1. 1. 9 1 >2. >2. 6 6 >2. >2. >32. >32. ND ND ND ND ND ND ND ND >256 >256 21 21 16. 16. 6 6 >256. >256. 1.2/.6 1.2/.6 26 27 :51.2/.6 s<1.2/.6 6 6 >76/4 >76/4 >16. >16. 21 22 2. 2. 9 12 >16. >16. 8. 16. 22 22.5.5 21 21 1. 1. 4. 4. 6 6 >16. >16. 6 6 >16. >16. TABLE 2. Distribution of paired zone differences for clinical isolates Zone differences (mm) Organism RISS < standard RISS > standard Avg zone difference >-4-3 to -4-1 to -2 1 to2 3to4 >4 Gram-negative Escherichia coli 5 6 169 285 238 15 2.2 Klebsiella pneumoniae 1 9 19 144 9 6 -.1 Providencia rettgeri 1 2 7 37 53 6 2.7 Morganella morganii 1 52 59 15 8 1.2 Proteus vulgaris 1 1 11 58 36 1.3 Proteus mirabilis 5 13 99 148 139 25 3.2 Providencia stuartii 11 36 14 29 -.3 Serratia marcescens 6 18 85 64 7.4 Enterobacter sp. 1 23 11 113 46 4 -.5 Pseudomonas aeruginosa 3 8 22 136 9 2 -.4 Percent.6 2.9 21.5 43.1 28.3 3..6 Gram-positive Staphylococcus aureus 13 24 186 17 95 6 1 -.6 Streptococcus faecalis 5 12 22 56 38 8 3 -.1 Streptococcus faecium 6 24 28 12 2 -.5 Streptococcus durans 1 5 14 21 31.1 Streptococcus bovis 5 16 27 39 11 8 2 -.6 Staphylococcus epidermidis 3 4 22 16 2 7 1.6 Percent 2.7 7.3 3.8 3.3 22.6 4.9 1.4 Fastidious Haemophilus influenzae 2 4 57 85 49 5 2. Neisseria gonorrhoeae 5 11 32 35 22 3 -.7 Neisseria meningitidis 3 4 13 12 16 1 5.5 Streptococcus pneumoniae 2 5 19 19 11 4 1.2 Percent 2.3 4.8 24.6 34.7 24.4 6.7 2.5

454 BAKER, THORNSBERRY, AND HAWKINSON TABLE 3. Distribution of paired MIC differences for clinical isolates No. of pairs showing a well difference of: RISS = higher MIC than RISS = lower MIC than Within ±1 Organism standard standard dilution <-2-2 -1 +1 +2 >+2 Gram-negative E. coli 1 71 512 113 22 1 696 K. pneumoniae 3 75 239 42 1 356 P. rettgeri 1 1 5 74 25 1 1 14 M. morganii 1 11 16 21 4 1 138 P. vulgaris 14 73 18 3 15 P. mirabilis 8 84 282 49 7 2 415 P. stuartii 1 2 133 21 2 3 174 S. marcescens 1 3 133 13 3 176 Enterobacter sp. 2 57 188 4 1 285 P. aeruginosa 1 22 156 1 179 Percent.3.7 14.4 7.2 12.7 1.6.3 97.3 Gram-positive S. aureus 2 9 53 351 14 3 418 S. epidermidis 3 66 3 72 S. faecalis 1 123 1 1 143 S. faecium 2 63 6 1 71 S. durans 6 55 9 1 1 7 S. bot1is 3 92 1 2 1 15 Percent.2 1 8.6 83.3 5.8.6.5 97.6 Fastidious H. influenzae 7 14 154 25 9 1 193 N. gonorrhoeae 3 113 1 117 N. meningitidis 8 5 5 63 S. pneumoniae 8 46 6 6 Percent 1.6 7.3 8.7 8.2 2.2 96.2 before the testing, and diluting, if necessary, to appropriate turbidity (3). The disk diffusion method approved by the Food and Drug Administration (16) and the approved standard method of NCCLS (9) also reported this method of inoculum preparation. In the agar overlay method of Barry et al., the bacteria are grown in broth to a stationary phase and then are diluted to the appropriate concentration (2). These methods have been proven to be efficacious but have the disadvantages of being time consuming, in that 2 to 6 h is required for incubation, and more epensive, since the broth used to grow the organism is more epensive than a saline diluent. In our laboratory, we have recommended that with certain organisms, e.g., Haemophilus spp., Legionella spp., Neisseria spp., S. pneumoniae, and anaerobes, inocula should be prepared by suspending bacteria from an overnight agar culture, because we have previously demonstrated that we obtained the desired organism concentration more consistently than if we prepared it from a broth culture (5, 6, 8, 9, 12, 13, 15). In this study, we etended the use of such procedures to other bacterial species that are likely to be tested for susceptibility to antimicrobial agents. J. CLIN. MICROBIOL. These data indicate the preparation of inocula by suspending cells from an overnight agar culture yields results, either by disk diffusion or by dilution tests, that are comparable to the results obtained with conventionally prepared inocula. The comparability of data obtained with the different inoculum preparation methods is probably best demonstrated by the data on the ability of the disk diffusion method to predict categories of susceptibility (Table 4). Only three very major or major discrepancies were seen, one each with Providencia rettgeri, Providencia stuartii, and Serratia marcescens, organisms that have been shown to have discrepancies in other studies (11, 14). Most of the discrepancies were minor, indicating that one or the other technique yielded an intermediate result. We were not surprised by some discrepancies, e.g., cephalothin with Escherichia coli; nitrofurantoin with Klebsiella and Enterobacter spp.; enterococci with cephalothin, erythromycin, and kanamycin, because they have been seen in other studies (11, 14) or because of the closeness of the mean zone diameter to a category breakpoint. the E. coli and cephalothin combination is a well-recognized problem because the mean zone diameter is approimately 19 mm and the breakpoint is 18

VOL. 17, 1983 Organism TABLE 4. INOCULUM STANDARDIZATION 455 Comparison of disk diffusion susceptibility categories: RISS versus Std Discrepanciesa Percent agreement Very major and major Minor All gram-negative 97..1% 2.9%o E. coli 97.5 ceph (7/6) K. pneumoniae 96.7 nitr (6/3), tetr (4/3) Enterobacter sp. 92. nitr (9/24), tetr (1/24) P. mirabilis 99.8 P. vulgaris 99.1 carb (1/9) M. morganii 97.9 cefo (2/12) P. rettgeri 98.2 cefo (1/9) ampi (1/9) P. stuartii 93.3 tobr (1/15) cefam (2/15), chlo (2/15), gent (2/15), tobr (4/15) S. marcescens 97.2 cefo (1/15) P. aeruginosa 97.8 amik (3/15) All gram-positive 96.7 O.% 3.3% S. aureus 99.3 kana (3/12) S. epidermidis 1. S. faecalis 84.1 ceph (7/12), eryt (6/12), kana (4/12), peni (3/12) S. faecium 97. ceph (1/6), kana (1/6) S. durans 97. peni (2/6) S. bovis 99. gent (1/9), st (1/9) All "fastidious" 97.8.%7 2.2% H. influenzae 98.7 N. gonorrhoeae 1. N. meningitidisb 76.2 sulf (5/21) S. pneumoniae 98.3 a Discrepancies: very major = resistant by standard method and susceptible by test method; Major = susceptible by standard method and resistant by test method; Minor = one method yielded an intermediate result. Numbers in parentheses indicate number of discrepancies/total number tested. Minor discrepancies listed occurred 1%o of the time with these antibiotics: amikacin (amik), ampicillin (ampi), carbenicillin (carb), cefamandole (cefam), cefoitin (cefo), cephalothin (ceph), chloramphenicol (chlo), erythromycin (eryt), gentamicin (gent), kanamycin (kana), nitrofurantoin (nitr), penicillin (peni), tetracycline (tetr), tobramycin (tobr), sulfadiazine (sulf), and sulfamethoazole-trimethoprim (st). b Disk diffusion test not recommended, ecept for sulfadiazine. mm. The number of minor discrepancies with enterococci is so high that the agreement is only 84.1%, but this same problem has been demonstrated in other studies (11, 14). The only other species with overall agreement below 9% was N. meningitidis with 76.2%. In this case, the disk diffusion test is not a recommended procedure ecept for sulfadiazine (4). Since we routinely perform MIC tests on these organisms, we have little eperience from which to speculate on the cause for this discrepancy. However, the zone sizes obtained are very large and may be more difficult to reproduce. We did not categorize the MIC susceptibilities, but the correlation would have had to be impressive because there was such a high percentage of agreement at the +1 log2 dilution level. Our results are in agreement with those of D'Amato and Hockstein, who prepared their inoculum by removing some growth from an agar plate, suspending it in diluent, and adjusting the turbidity to be equivalent to a.5 McFarland standard (7). In the standard methods, the desired inoculum number is obtained by adjusting the turbidity of broth cultures to that of a.5 McFarland (BaSO4) standard. The actual number of organisms is supposed to be approimately 18 CFU/ml. In most of the inocula prepared by either the standard method or by the RISS, the number of organisms was within ±.5 log1o of 18 CFU/ml. The eceptions (Fig. 1) were with some staphylococci (inocula prepared by the standard method) and pneumococci (inocula prepared by both methods). The pneumococci problem may have been due to lysis of the bacteria or difficulty in suspending the organism. The control S. aureus ATCC 25923 was also consistently low (always <5 17 CFU/ml) when the inoculum was prepared by the standard method, but it was almost 1 logl higher and much closer to 18 CFU/ml when prepared by the RISS. However, the difference in zone

456 BAKER, THORNSBERRY, AND HAWKINSON TABLE 5. Comparison of microdilution tests: RISS versus Std % Agreement at Organism ±1 ±2 Drug with > ±2 dilutiona dilution dilution All gram-negative 97.3 99.2 E. coli 96.7 99.9 tetr (1/6) K. pneumoniae 98.9 99.7 ampi (1/3) Enterobacter sp. 99. 1. P. mirabilis 96. 99.5 amik (1/36), gent (1/36) P. vulgaris 97.2 1. M. morganii 95.8 99.3 cefam (1/12) P. rettgeri 96.3 98.1 ampi (1/9), tobr (1/9) P. stuartii 96.7 98.3 ceph (2/15), tobr (1/15) S. marcescens 97.8 1. P. aeruginosa 99.4 1. All gram-positive 97.7 99.2 S. aureus 96.8 98.8 ampi (1/36), peni (3/36), kana (1/36) S. epidermidis 1. S. faecalis 99.3 1. S. faecium 98.6 1. S. durans 97.2 98.6 st (1/6) S. bovis 97.2 99.1 st (1/9) All "fastidious" 96.2 99.8 H. influenzae 91.9 99.5 cefam (1/3) N. gonorrhoeaeb 1. N. meningitidis 1. S. pneumoniae 1. a Numbers in parentheses indicate the number of organisms with > ±2 dilutions/total number tested. Abbreviations are defined in footnote a to Table 4. b MIC test was performed by agar dilution. sizes was no more than 1 to 2 mm and had no bearing on the category of susceptibility (Table 1). The average difference in zone diameters for all staphylococci was.6 mm. Since inocula for disk diffusion and microdilution tests can be prepared by these methods, we presume that inocula for other antimicrobial susceptibility tests can also be prepared in this manner. Our conclusion is that inocula for antimicrobial susceptibility tests can be prepared from bacterial colonies taken from overnight agar cultures and can be epected to yield susceptibility results comparable to those with the standard method. We also conclude that the 3M RISS is a reliable and efficacious method for adjusting inocula to approimately 18 CFU/ml, the desired number of organisms. We recommend that the use of overnight cultures be adopted as an optional method for preparing inocula in the standard methods for disk diffusion and dilution tests. The NCCLS subcommittees on disk diffusion and dilution standards have tentatively endorsed this proposal for inclusion in the appropriate standards when they are revised. ACKNOWLEDGMENT We thank Rosemary Nave and Joan Garrison for their administrative assistance. J. CLIN. MICROBIOL. ADDENDUM Since this study was finished, the 3M system we called the Rapid Inoculum Standardization System (RISS) has been given the trade name PROMPT. LITERATURE CITED 1. Barry, A. L., D. Amsterdam, M. B. Coyle, E. H. Gerlach, C. Thornsberry, and R. W. Hawkinson. 1979. Simple inoculum standardizing system for antimicrobial disk susceptibility tests. J. Clin. Microbiol. 1:91-918. 2. Barry, A. L., F. Garcia, and L. D. Thrupp. 197. An improved single disk method for testing the antibiotic susceptibility of rapidly-growing pathogens. Am. J. Clin. Pathol. 53:149-159. 3. Bauer, A. W., W. M. M. Kirby, J. C. Sherris, and M. Turck. 1966. Antibiotic susceptibility testing by a standardized single disk method. Am. J. Clin. Pathol. 45:493-496. 4. Bennett, J. V., H. M. Camp, and T. C. Eickhoff. 1968. Rapid sulfonamide disc sensitivity test for meningococci. Appl. Microbiol. 16:156-16. 5. Biddle, J. W., J. M. Swenson, and C. Thornsberry. 1978. Disc agar diffusion antimicrobial susceptibility tests with,-lactamase producing Neisseria gonorrhoeae. J. Antibiot. 31:352-358. 6. Cooksey, R. C., R. R. Facklam, and C. Thornsberry. 1978. Antimicrobial susceptibility patterns of Streptococcus pneumoniae. Antimicrob. Agents Chemother. 13:645-648. 7. D'Amato, R. F., and L. Hochstein. 1982. Evaluation of a rapid inoculum preparation method for agar disk diffusion susceptibility testing. J. Clin. Microbiol. 15:282-285. 8. Kirven, L. A., and C. Thornsberry. 1974. In vitro susceptibility of Haemophilus influenzae to trimethoprim-sulfa-

VOL. 17, 1983 methoazole. Antimicrob. Agents Chemother. 6:869-87. 9. National Committee for Clinical Laboratory Standards. 1979. Performance standards for antimicrobic disc susceptibility tests. National Committee for Clinical Laboratory Standards, Villanova, Pa. 1. National Committee for Clinical Laboratory Standards. 198. NCCLS proposed standard: PSM-7. Standard methods for dilution antimicrobial susceptibility tests for bacteria which grow aerobically. National Committee for Clinical Laboratory Standards, Villanova, Pa. 11. Thornsberry, C., J. P. Anhalt, J. A. Washington II, L. R. McCarthy, F. D. Schoenknecht, J. C. Sherris, and H. J. Spencer. 198. Clinical laboratory evaluation of the Abbott MS-2 automated antimicrobial susceptibility testing system: report of a collaborative study. J. Clin. Microbiol. 12:375-39. 12. Thornsberry, C., C. N. Baker, and L. A. Kirven. 1978. In vitro activity of antimicrobial agents on Legionnaires INOCULUM STANDARDIZATION 457 disease bacterium. Antimicrob. Agents Chemother. 13:78-8. 13. Thornsberry, C., T. L. Gavan, and E. H. Gerlach. 1977. Cumitech 6, New developments in antimicrobial agent susceptibility testing. Coordinating ed., J. C. Sherris. American Society for Microbiology, Washington, D.C. 14. Thornsberry, C., T. L. Gavan, J. C. Sherris, A. Balows, J. M. Matsen, L. D. Sabath, F. Schoenknecht, L. D. Thrupp, and J. A. Washington II. 1975. Laboratory evaluation of a rapid, automated susceptibility testing system: report of a collaborative study. Antimicrob. Agents Chemother. 7:466-48. 15. Thornsberry, C., and J. M. Swenson. 1978. Antimicrobial susceptibility testing of anaerobes. Lab. Med. 9:43-48. 16. Wright, W. W. 1974. FDA actions on antibiotic susceptibility tests, p. 26-46. In A. Balows (ed.), Current techniques for antibiotic susceptibility testing. Charles C. Thomas, Publisher, Springfield, Ill. Downloaded from http://jcm.asm.org/ on April 8, 218 by guest