Evaluation of the Prompt Inoculation System for Preparation

Transcription

1 JOURNAL OF CLNCAL MCROBOLOGY, July 1983, p Vol. 18, No /83/ $02.00/0 Copyright C 1983, American Society for Microbiology Evaluation of the Prompt noculation System for Preparation of Standardized Bacterial nocula MARLYS E. LUND'* AND RONALD W. HAWKNSON2 Medical Products Division1 and Riker Laboratories, nc.,2 3M, St. Paul, Minnesota Received 7 January 1983/Accepted 4 April 1983 Standardized inocula prepared with the 3M Prompt inoculation system were evaluated by (i) performing colony counts on the inocula and (ii) employing these inocula in disk diffusion or broth microdilution susceptibility tests. The inoculation wand delivered 1.85 X 108 ± 0.6 log10 CFU to the various diluents tested. nocula prepared in 1 ml of saline and used to inoculate disk diffusion tests resulted in 96.5% agreement between Prompt and standard tests when Prompt inocula were used within 15 min of preparation and 95.6% agreement between paired tests when Prompt inocula were allowed to sit at room temperature for 2 h before use. With 15-min Prompt inocula, only one (0.04%) major discrepancy between paired tests was observed. The 2-h Prompt inocula gave three (0.13%) major or very major discrepancies. The 15-min Prompt inocula prepared in 30 ml of diluent resulted in 98.2% of Prompt minimal inhibitory concentrations within +1 log2 dilution step of the standard test result, whereas the 2-h inocula resulted in 97.4% agreement between paired minimal inhibitory concentrations at this level. Thus, the Prompt inocula were found to give results equivalent to those obtained from inocula prepared by conventional procedures. Recently D'Amato and Hochstein (3) and Baker and Thornsberry (1) have reported the successful application of a direct suspension method for the preparation of standardized inocula for antimicrobic susceptibility tests (ASTs). Direct suspension of bacteria from an agar plate in a suitable diluent until the turbidity matches that of a 0.5 McFarland standard adds considerable flexibility to the setup procedure for ASTs by eliminating the conventionally required 2- to 8-h incubation period for preparation of a log- or stationary-phase broth culture. The 3M Prompt inoculation system (herein shortened to Prompt) offers this flexibility and in addition elimninates the time-consuming and tedious process of turbidity adjustment. The system includes a special inoculation wand (10) and a selection of prepackaged diluents. The wand picks up atnd delivers a constant number of bacteria. The diluents are packaged to provide (i) an inoculum equivalent to the 0.5 McFarland turbidity standard or (ii) an inoculum suitable for broth microdilution tests that involve a 1:20 dilution of the inoculum in the final inoculation step (e.g., 5,ul of inoculum into 0.1 ml of antimicrobic solution). Furthermore, the diluents have been selected so that the inocula are stable when held at room temperature, obviating the need to use the standardized inocula within 15 min of preparation. This study was undertaken to determine the reproducibility of the inoculation wand's pickup and delivery and to evaluate the performance of inocula prepared with Prompt in antimicrobic susceptibility tests set up (i) within 15 min of inoculum preparation and (ii) 2 h after inoculum preparation. MATERALS AND METHODS Microorganisms. The 100 clinical isolates tested in this study were kindly supplied by C. Thomsberry and C. Baker, Centers for Disease Control, Atlanta, Ga. The organisms were selected to represent a wide variety of susceptibility profiles. Only amikacin and vancomycin were not tested over the entire range of possible response because resistant strains of organisms were not available. Table 1 gives the distribution of the species and also the number of strains subjected to replicate testing. Organisms were maintained at -20 C, frozen in sterile, defibrinated sheep's blood. Before testing, a small portion of a frozen, bacterial suspension was thawed and plated to a sheep blood agar plate. This initial subculture was incubated overnight at 35 ± 2 C. Study subculture plates (SSPs) were prepared by suspending one colony from the initial subculture plate in 5 ml of sterile 0.85% saline; this saline suspension served as the source of inoculafor the requisite number of either blood agar plates for gram-positive bacteria or MacConkey agar plates for gram-negative bacteria. These SSPs were incubated for 18 to 22 h at C. AST. Standard ASTs were performed by the meth- 84

2 VOL. 18, 1983 TABLE 1. Organisms tested Organism Total no. No. of strains of subjected to strains replicate testing Escherichia coli 20 5 Klebsiella pneumoniae 10 5 Providencia rettgeri 3 2 Morganella morganii 4 2 Proteus vulgaris 3 2 Proteus mirabilis 12 5 Providencia stuartii 5 2 Serratia marcescens 5 2 Enterobacter cloacae 3 2 Enterobacter aerogenes 3 2 Enterobacter agglomerans 2 1 Pseudomonas aeruginosa 5 5 Staphylococcus aureus 12 4 Streptococcus faecalis 4 3 Streptococcus faecium 2 2 Streptococcus faecium var. 2 2 durans Streptococcus bovis 3 3 Staphylococcus epidermidis 2 1 ods recommended by the National Committee for Clinical Laboratory Standards (NCCLS) for disk diffusion (6) or broth microdilution (7) ASTs with the exception that the final diluents for the microdilution tests were those recommended by Micro-Media Systems, nc.: 0.02% polysorbate 80 (P80) and 0.02% polysorbate 80 supplemented with the chloride salts of calcium and magnesium (CSP80). The Prompt tests differed from standard tests only in the preparation and handling of the inocula. noculum preparation and handling. A single SSP served as the source of inocula for all parallel tests. Bacteria taken from three to five colonies were inoculated into 5 ml of soybean-casein digest broth for the standard tests. The broth was incubated for 2 to 4 h at 35 ± 2 C and then was diluted in sterile saline to match the recommended barium sulfate standard. The standardized bacterial suspension was used to inoculate a disk diffusion AST; 0.5-ml portions of the same suspension were diluted 1:30 in P80 and in CSP80. These dilutions were used to inoculate the standard microdilution tests. All standard ASTs were set up within 15 min of inoculum preparation. Prompt inocula were prepared using the Prompt inoculation wand and one of four prepackaged suspending solutions or diluents. The diluent for the disk diffusion ASTs was 1 ml of sterile 0.85% saline. Three diluents-p80, CSP80, and 0.85% saline-in 30-ml portions were tested in broth microdilution ASTs. The wand was used to pick five colonies >1 mm in diameter or 10 colonies 0.5 to 1 mm in diameter from an SSP. The 2-mm tip of the wand served as a guide to colony size. The wand, filled with bacteria, was placed into a diluent vial, and the bacteria were released from the wand by vortexing (disk diffusion test inocula) or shaking vigorously (microdilution test inocula). Each Prompt inoculum was used within 15 min of preparation to set up the time zero (To) test. The wand-stopper was used to recap the vial with its remaining inoculum, and the vial was held at room temperature for 2 h (T2). PROMPT NOCULATON SYSTEM 85 The aged bacterial suspension was then mixed again and used to inoculate the T2 tests. Colony counts. To determine the number of bacteria delivered by the wand to the diluent, Prompt To inocula were diluted, and the dilutions were used to prepare soybean-casein digest pour plates. The 1-ml saline suspensions were diluted 10-'; triplicate 0.1-ml portions of the final dilution were used to inoculate each of three pour plates. The 30-ml suspensions were diluted lo-4 and plated similarly. After overnight incubation of the pour plates, colonies were counted with a Quebec or 3M colony counter. To assess the stability of the bacterial suspensions, the count procedure was repeated on the aged Prompt inocula (T2 counts). T2 counts were not determined for the 30-ml saline diluent. Reproducibility study. One-half of the bacterial isolates-35 gram-negative strains and 15 gram-positive strains-were subjected to replicate testing (Table 1). Both ASTs and counts were replicated. For any given organism replicate tests were set up on the same day on the same lots of materials. Triplicate tests were performed for the 1-ml saline and 30-ml P80 and CSP80 diluents, and duplicate tests were performed for the 30-ml saline diluent. Parallel standard tests were also replicated. RESULTS The overall mean, based upon 742 counts, for the wand's pickup and delivery was 1.85 x 108 CFU. The 95% confidence interval for an individual pick was 4.66 x 107 to 7.36 x 108 CFU, a range of 1.2 log1o. Data from five counts derived from colonies of either Staphylococcus epidermidis or Klebsiella pneumoniae which had not reached the requisite minimum size (0.5 mm) at the time of inoculum preparation have been excluded. Counts for the excluded data ranged from 1.0 x 106 to 1.4 x 107 CFU. Figure 1 shows the mean wand delivery and 95% confidence interval for the various genera and related bacterial groups tested. The mean To and T2 inoculum densities for the 1-ml saline and 30-ml P80 and CSP80 diluents appear in Fig. 2. n the saline diluent, counts remained essentially stable. n P80, there was a significant tendency (paired Student's t test, P < 0.001) for decreased counts at T2 with gramnegative organisms. The most extreme decrease in counts was seen with strains of Pseudomonas aeruginosa. T2 counts for these organisms dropped, on the average, to 47.3% of the starting value. The Proteus and Enterobacter-Serratia group counts dropped to approximately 65% of the starting value, and those for the Morganella- Providencia group dropped to approximately 73%. Escherichia coli, Klebsiella pneumoniae, and the gram-positive organisms remained relatively unaffected by the P80 diluent. n the cation-supplemented system, the opposite effect was noted. That is, counts increased significantly (P < 0.001). All organism groups except P.

3 86 LUND AND HAWKNSON 1O10- U. E0' C) E ( =No. ofolony countspsdomsd OVrsS K. psumonds orqsnsl P. sruglnoss Str u (742) 4J( owh nd 0 (52) (114) ELoN Ent_ Po-S tspotus Stsphyloccw (115) (101 (107) (9) FG. 1. Mean number and 95% confidence interval for viable microorganisms picked up by the Prompt inoculation wand. aeruginosa showed a significant increase in the T2 counts. The magnitude of the increase was, however, much smaller than that of the decrease seen in the unsupplemented system. The average increase in counts was only 12.7%. J. CLN. MCROBOL. Disk diffusion susceptibility results were categorized as susceptible, intermediate, or resistant according to the published zone diameter break points (8). Disagreements between paired standard and Prompt results were scored in the traditional manner into three categories of discrepancy: (i) minor, intermediate in one system and either susceptible or resistant in the other; (ii) major, resistant by the Prompt system and susceptible by the standard method; and (iii) very major, susceptible by the Prompt system and resistant by the standard method. The data for the To comparison are summarized in Table 2, and those for the T2 comparison are summarized in Table 3. The results were in total accord for 96.54% of To paired tests and 95.57% of T2 paired tests. At To only one major and no very major errors were observed in 2,396 paired tests (agreement plus minor discrepancies or essential accord, 99.96%). At T2 there were one very major and two major errors in 2,395 paired tests (essential accord, 99.87%). Only the combination of penicillin and streptococci at To and penicillin and streptococci and kanamycin and streptococci at T2 showed significantly less than 90% agreement. For those antimicrobic agentorganism combinations essential accord equalled 100%; i.e., all discrepancies between Prompt and standard tests were minor ones. Figures 3 and 4 show, for the combined diluent systems, the percentages of paired minimum Downloaded from TABLE 2. nterpretive agreement between standard method and Prompt To disk diffusion susceptibility tests Gram-negative species Staphylococcus spp. Streptococcus spp. Antimicrobial % % Majorb % % Major % % Major agent Agree- Minor or very nd Agree- Minor or very n Agree- Minor or very n ment majorc ment major ment major Amikacin Ampicillin Carbenicillin Cefamandole Cefoxitin Cephalothin Chloramphenicol Clindamycin Erythromycin Gentamicin Kanamycin Nitrofurantoin Oxacillin Penicillin e Tetracycline TMSf Tobramycin Vancomycin a A minor error is classified as intermediate by one system and either susceptible or resistant by the other. b A major error is classified as resistant by Prompt and susceptible by the standard method. c A very major error is classified as susceptible by Prompt and resistant by the standard method. d n, Number of paired tests. ' Significantly less than 90%o agreement. f TMS, Trimethoprim-sulfamethoxazole. on July 22, 2018 by guest

4 VOL. 18, 1983 TABLE 3. PROMPT NOCULATON SYSTEM 87 nterpretive agreement between the standard method and Prompt T2 disk diffusion susceptibility testsa Gram-negative species Staphylococcus spp. Streptococcus spp. Antimicrobial % % Major % % Major % % Major agent Agree- Minor or very n Agree- Mm or very n Agree- M or very n ment major ment or major ment Minor major Amikacin Ampicillin Carbenicillin % Cefamandole Cefoxitin Cephalothin Chloramphenicol Clindamycin Erythromycin Gentamicin Kanamycin b Nitrofurantoin Oxacillin Penicillin b Tetracycline TMSC Tobramycin Vancomycin a See footnotes a through d of Table 2 for an explanation of the headings. b Significantly less than 90%o agreement. c TMS, Trimethoprim-sulfamethoxazole. inhibitory concentrations (MCs) which agreed was 98.2% at the one-dilution step level and within ±1 and ±2102 dilution steps. The overall 99.8% at the two-step level. The respective agreement between 7,074 pairs of MCs at To values for the agreement between the 7,046 pairs a 1XU A= To * =T, SeNs aev z 4 S~~~~4 E Morgene. 5x' 0 K.P.eu U. ELoN l(- 2 x 10" f bh 30 ml Poorbute 80 Prot f im P. ootee eruakweae8rpt" ou seh.h ce." _ A A * A *A A. A E - A 2 x101 0 o Ousral K. Kpn.umtonime Morg6n.b- A'een;kare A A at A A 2x10- FG. 2. Geometric mean density of freshly prepared (T0) and aged (T2) inocula prepared with the Prompt inoculation system.

5 88 LUND AND HAWKNSON J. CLN. MCROBOL. Percent Agreement oi111co1 t oiid i Amikacln ON lz.- P;v Ampicillin Carbenicillin Cefamandole Cefotaxime Cefoxitin Cephalothin Chloramphenicol Colistin Gentamicin Kanamycin Nitrofurantoin Tetracycline Tobramycin Trimethoprimn Sulfamethoxazole FG. 3. NE amememen m MMx mm"mmmm ap"m,"q."." MEMEN \ln "m 'm 1; RE &'10 "WNNUMMMMMMMM 'mil tlil.' 1; le ; Jui &mmm -4-1 r'. _ 5 a :J 1"Wa a g\\\\\\\\\\\\\ 1 t- Percent agreement between standard and Prompt MCs for gram-negative organisms. le c -4 at T2 were 97.4 and 99.6%. These data include agreement between systems for off-scale as well as on-scale endpoints. At To there were 2,194 MC pairs with on-scale endpoints. Of these pairs, 97.0% agreed within one dilution step and 99.5% agreed within two dilution steps. At T2 there were 2,126 on-scale paired MCs with 94.4% (not significantly <95%) agreement at one dilution step and 99.2% agreement at two dilution steps. MC pairs showing significant disagreementi.e., paired results greater than 2 log2 dilution steps apart-were tested for significant skewness. Because so few tests (<1%) showed significant disagreement, the data were pooled over time (i) by Gram stain reaction of the organisms within each Prompt diluent system and (ii) by each of the organism groups over all diluent systems. For the diluent systems, the T2 Prompt results for the P80 system showed significant bias toward low MCs with gram-negative organisms. On the other hand, the staphylococci

6 VOL. 18, 1983 ApcillinE Cephalothin PROMPT NOCULATON SYSTEM 89 Percent Agreement t1 co co taon -A SD 1i _ M11 C C." 0 Saa X 5co c s 8 a_ knvnn ll PS" `, N Rl"ll 1.M t1 - Chioraheicol j1 Clindamycin Erythromycin Gentamicin Methicillin or Nalcillin Nitrofurantoin Penicillin Tetracycilne Trimethoprim/ Sulfamethoxazole Vancomycin FG. 4. E~~~~~~~~~ E---E-E ~~~~~ME EE EEE\\\\\ 4- _~~~~~~~~~-----,\\\\\\\\ \\\\L MiME;E 1:" "N0N, 11"." "''M 1,.,11,,.sl" '. S" S' 'S ",.".,'.,.,"S'"lls "S "n M lgm llsll, Ss Percent agreement between standard and Prompt MCs for gram-positive organisms. showed a significant tendency for MCs higher than the standard test in both Prompt To and T2 tests. The data from the intrasystem reproducibility studies appear in Tables 4 and 5. For the disk diffusion susceptibility test (Table 4), each system showed, overall, >97% of replicate tests in total accord and >99.7% in essential accord. No significant difference in reproducibility was detected between systems. Similarly, no difference in reproducibility was detected between systems in the broth microdilution test (Table 5) at the 1 and 2 log2 dilution step levels. The Prompt To test, however, gave the same MC significantly more times than did the standard test (P < 0.05). Nevertheless, all test systems showed excellent intrasystem reproducibility, with >98% of replicate tests falling within one dilution step of each other. "MMON, NNNN" EMMEN -1:.4 c c az 5 fcfix+:.-wz -4-4 V&-'4 _ 3 =3 C a- 3 t DSCUSSON As Barry points out (2), the density of the inoculum in any AST is "extremely critical" and "must be controlled in order to obtain reliable results." The importance of the organism's phase of growth has been less certain. The standard test procedures of the NCCLS give as the growth phase of choice the log phase. Stationary-phase cultures of rapidly growing bacteria and direct suspension offastidious organisms taken from an overnight agar culture are recognized as acceptable alternative sources for standardized inocula in the agar disk diffusion test (6). Recently, D'Amato and Hochstein (3) reported the equivalence of the direct suspension method to the traditional preparation of a standardized suspension of organisms in the log phase of growth in disk diffusion ASTs. The work of Baker and Thornsberry (1) confirms this ; -4

7 90 LUND AND HAWKNSON TABLE 4. ntrasystem reproducibility for disk diffusion susceptibility testsa Total No. (%) Organism Test system no. of No. (%) T major or group compari- minor very sons majorb Gram NCCLS 1, (2.07) 1 (0.08) negative Prompt ToC 1, (2.38) 0 (0) Prompt T2d 1, (1.91) 2 (0.16) Gram NCCLS (3.35) 0 (0) positive Prompt To (2.22) 0 (0) Prompt T (4.09) 2 (0.37)e Totals NCCLS 1, (2.45) 1 (0.06) Prompt To 1, (2.33) 0 (0) Prompt T2 1, (2.56) 4 (0.22) a See footnotes a through c of Table 2 for an explanation of the headings. b The usual distinction between major and very major errors cannot be made in an intrasystem comparison. c Prompt tests set up within 15 min of inoculum preparation. d Prompt tests set up with an inoculum aged for 2 h at room temperature. e Errors are traceable to the appearance of resistant variants in one in a set of triplicate tests for S. aureus and clindamycin. observation and extends the successful application of direct suspension to the microdilution procedure. The current study reconfirms the efficacy of direct suspension of organisms from an agar plate culture as a method of preparing standardized inocula for either disk diffusion or microdilution susceptibility tests. Furthermore, the T2 data indicate that if such a suspension is prepared in a suitable nonnutritive diluent, one is no J. CLN. MCROBOL. longer constrained to use the standardized inoculum within 15 min of preparation. That is not to say that the bacteria may be allowed to remain in the starvation environment indefinitely. How long the inoculum will remain suitable for susceptibility testing is both organism and diluent dependent. For example, dilute, aqueous P80 is obviously a bad environment for bacteria. Although the T2 Prompt microdilution tests employing this diluent still give satisfactory results, the inimical effect of the diluent is apparent in the trends toward decreased counts and significantly lower MCs for the gram-negative organisms. The effect of this nonionic surfactant, particularly upon gram-negative organisms, is not surprising; one would expect the hydrophobic portion of the polysorbate molecule to interact with and disrupt the lipopolysaccharides and lipoproteins of the gram-negative cell wall. t would appear that 2 h represents the outer limit of exposure to the dilute P80 environment for susceptibility test inocula, and shorter exposure times are undoubtedly preferable. For susceptibility test systems requiring the absence of cations in the diluent, saline proved to be superior to P80, especially over prolonged periods of time. No trend in either change of inoculum density or skewness in MC results developed for inocula held for 2 h in saline. Thus, the outer limit of exposure time to the saline diluent has not been defined, nor has this limit been defined for the cation-supplemented P80 system. The trend toward increased counts over time in the CSP80 illustrates the sparing effect of divalent cations, especially Mg2+, on bacteria in a starvation environment (4). How long the bacteria can maintain the vigor exhibited in the supplemented diluent is uncertain. The trend for significantly higher MCs with the Prompt and staphylococci is attributable to the inoculum effect rather than to the phase of growth or the diluent. The significant disagree- TABLE 5. ntrasystem reproducibility for broth microdilution susceptibility tests Cumulative % agreement between tests at the Organism group Test comparisons following log2 dilution steps: >2 Gram negative Standard 2, Prompt To 2, Prompt T2 2, Gram positive Standard Prompt To Prompt T Overall totals Standard 3, Prompt To 3, Prompt T2 3, a See footnotes c and d of Table 4.

8 VOL. 18, 1983 ments between standard and Prompt tests occurred with penicillin or ampicillin and betalactamase-producing strains of S. aureus. Tilton et al. (9) note that these particular antimicrobic agent-organism combinations are especially subject to inoculum effect, but that reasonable reproducibility of MCs may be expected if the inoculum density is maintained between 105 to 106 CFU/ml in the susceptibility test itself. When the contents of the growth control well in the microdilution tray were quantitatively cultured, the inoculum density in the standard tests was found to range from 2.4 x 104 to 6.7 x 104 CFU/ml for the staphylococci. Corresponding densities for the Prompt tests were 2.8 x 105 to 6.1 x 105 CFU/ml. The tendency for conventionally standardized suspensions of staphylococci to err on the low side of the ideal density may be noted in other studies (3, 5). The excellent agreement (>95%) between Prompt and standard tests reflects the performance of the inoculation wand. The pickup and delivery of the wand were quite consistent. One often hears that a bacterial suspension matching a 0.5 McFarland turbidity standard will contain between 5 x 107 to S x 108 (1.6 x log10) CFU/ml. Although this may represent the ideal situation, little if any data have been published which support this claim. f, for example, one determines the 95% confidence interval for the 24 counts reported by Matsen et al. (5), one finds the span of the interval to be 1.4 log10 when a variety of organisms are tested. n this light, the 1.2 log10 span for the wand's delivery with a large variety of organisms seems very respectable. To have the wand perform properly, it is important to follow the simple strictures in the directions for use. Data in this study, the excluded counts, indicate that when colonies are less than 0.5 mm in diameter, the process of touching 10 colonies will not fill the wand. Baker and Thornsberry (1) found it necessary to touch up to 30 colonies to fill the wand with some of the fastidious organisms they tested. The report of Wicks et al. (10) indicates that touching five colonies is sufficient to fill the wand when colonies are larger than 1 mm in diameter. t is also important that organisms be taken from fresh (<24-h) plate cultures to assure that viable organisms are selected and to prevent the occurrence of an unduly long lag phase in the susceptibility test. The wand may be depressed to the surface of the agar, but it must not penetrate the surface. The mixing procedure must be vigorous because gentle inversion of the vial will not release the organisms from the wand's tip. The foaming that occurs upon shaking the surfactant-containing diluents will not interfere with susceptibility tests because the bubbles formed tend to be PROMPT NOCULATON SYSTEM 91 trapped in the bottle when the inoculum is dispensed. f one follows the directions outlined above, the wand will pick up and deliver a consistent number of bacteria, 1.85 X loglo CFU. The desired density of the inoculum to be prepared is controlled by the amount of diluent into which the wand is placed. Within practical limits, it is possible to package diluents to provide standardized suspensions of bacteria over a range of concentrations while saving time and eliminating the tedium of manual turbidity adjustment. The nature of the diluent may be dictated by the test procedure in which the inoculum is to be used. The 1-ml system described in this study provides inocula functionally equivalent to those matching a 0.5 McFarland turbidity standard and gives reliable results (>99%o essential accord with standard tests) in agar disk diffusion susceptibility tests. The 30-ml systems described here, with >95% paired MCs within ±1 log2 dilution of the standard test, provide inocula appropriate for those broth microdilution systems with inoculator pegs delivering approximately 5,ul per well. ACKNOWLEDGMENTS We are especially grateful to J. M. Kelly and J. H. Wicks for their technical assistance. LTERATURE CTED 1. Baker, C. N., and C. Thornsberry noculum standardization in antimicrobial susceptibility testing: evaluation of overnight agar cultures and the rapid inoculum standardization system. J. Clin. Microbiol. 17: Barry, A. L The antimicrobic susceptibility test: principles and practice. Lea & Febiger, Philadelphia, Pa. 3. D'Amato, R. F., and L. HochsteLn Evaluation of a rapid inoculum preparation method for agar disk diffusion susceptibility testing. J. Clin. Microbiol. 15: Dawes, E. A Endogenous metabolism and survival, p n T. R. G. Gray and J. R. Postgate (ed.), The survival of vegetative microbes. Cambridge University Press, New York. 5. Matsen, J. M., M. E. Lund, and D. C. Brooker Comparison and evaluation of carbenicillin disks in diffusion susceptibility testing. Antimicrob. Agents Chemother. 5: National Committee for Clinical Laboratory Standards Performance standards for antimicrobic disc susceptibility tests. Approved standard: ASM-2, 2nd ed. National Committee for Clinical Laboratory Standards, Villanova, Pa. 7. National Committee for Clinical Laboratory Standards Standard methods for dilution antimicrobial susceptibility tests for bacteria which grow aerobically. Proposed standard: PSM-7. National Committee for Clinical Laboratory Standards, Villanova, Pa. 8. National Committee for Clnia; Laboratory Standards Performance standards. for antimicrobic disc susceptibility tests, supplement 1, p National Committee for Clinical Laboratory Standards, Villanova, Pa. 9. Tilton, R. C., L. Lieberman, and E. H. Gerlach Microdilution antibiotic susceptibility test: examination of certain variables. Appl. Microbiol. 26: Wicks, J. H., R. L. Nelson, and G. E. Krejcarek A rapid inoculum standardization system: a novel device for standardization of inocula in antimicrobial susceptibility testing. J. Clin. Microbiol. 17: