Feasibility Study of Disk Diffusion Susceptibility Tests with Mueller- Hinton Broth Solidified with Gelrite, an Agar Substitute

Transcription

1 JOURNAL OF CLINICAL MICROBIOLOGY, Mar. 1985, p /85/ $02.00/0 Copyright (O 1985, American Society for Microbiology Vol. 21, No. 3 Feasibility Study of Disk Diffusion Susceptibility Tests with Mueller- Hinton Broth Solidified with Gelrite, an Agar Substitute A. L. BARRY,'* L. CREITZ,2 AND R. R. PACKER1 The Clinical Microbiology Institute' and Prepared Media Laboratory,2 Tualatin, Oregon Received 13 August 1984/Accepted 3 December 1984 Feasibility studies were done to determine whether a new agar substitute, Gelrite gellan gum, could be used to prepare a solid Mueller-Hinton medium for disk diffusion susceptibility tests. Mueller-Hinton broth was combined with 0.43% of the gellan gum and 0.75% KCI. The resulting medium had performance characteristics similar to those of Mueller-Hinton agars; however, zones on the gellan gum media tended to be a little larger. Significant differences among Mueller-Hinton broths and among Mueller-Hinton agars from different manufacturers were documented: zones on different lots of the gellan gum were more consistent. The Mueller-Hinton broth-gellan gum medium appears to represent a satisfactory alternative to agar media. However, because somewhat larger zones were seen on the gellan gum plates, further study will be needed to develop quality control limits and interpretive zone size standards for tests on this new medium. In the process of standardizing methods for susceptibility testing, batch-to-batch and lot-to-lot variability in test reagents must be kept to a minimum. Mueller-Hinton agar is the standard medium for disk diffusion susceptibility tests (1, 14, 15), but Mueller-Hinton agars from different manufacturers have been shown to produce significantly different results (2, 13, 18, 20) as have different lots from the same manufacturer (13, 17, 18). The differences among media are more pronounced with some antimicrobial agents than with others and probably relate to specific drug-medium or microorganism-medium interactions. For example, the concentration of free, unbound metallic cations can influence the activity of the aminoglycosides, tetracyclines, and polymyxins against Pseudomonas aeruginosa (1, 5, 6, 10, 17, 18, 20, 21). In addition to directly influencing the activity of the antimicrobial agent, the gel strength of the agar medium can influence the diffusion rate of the agent, and the nutritive capacity of the medium influences the growth rate of the test strain. These factors all influence the size of a zone of inhibition in a standardized disk diffusion test (1). Agar is a natural product which may influence the activity and diffusability of antimicrobial agents other than the aminoglycosides (4, 6, 8, 11, 13, 16). Because agar can vary from season to season and from lot to lot, alternative solidifying agents deserve serious consideration. Two such products that have been evaluated previously are Separan NP10 (12) and Neutra-gel (3) but neither has been made available commercially. Gelrite is the microbiological grade of a gellan gum (formerly known as PS-60 or S-60) which is being developed as an agar substitute by Kelco, Div. of Merck and Co. (San Diego, Calif.). It is a naturally derived fermentation product of a Pseudomonas species and is highly purified and chemically defined (9). It is a heterosaccharide which, in the presence of cations, has the ability to form a gel with an agar-like rigidity. The gel is thermostable, optically clear, and compatible with a variety of nutrient additives (19). The present report describes our efforts to evaluate the feasibility of using Mueller-Hinton broth solidified with gellan gum to perform a standardized disk diffusion susceptibility test. * Corresponding author. MATERIALS AND METHODS Susceptibility test. The Kirby-Bauer disk diffusion test was used throughout this study, as outlined by the National Committee for Clinical Laboratory Standards (NCCLS) (14, 15). Commercially prepared (BBL Microbiology Systems, Cockeysville, Md.) antimicrobial disks were used throughout the study. Each lot of disks contained 100 to 130% of labeled potency by independent assay. Mueller-Hinton agars were those collected for a separate study (H. M. Pollock, A. L. Barry, T. L. Gavan, P. C. Fuchs, S. Hanson, C. Thornsberry, and H. Frankel, manuscript in preparation). One lot of Mueller-Hinton agar was obtained from each of the seven media manufacturers that sell Mueller-Hinton agar intheunitedstates. Acumedia(Baltimore,Md.),BBLMicrobiology Systems, Difco Laboratories (Detroit, Mich.), GIBCO Laboratories (Madison, Wis.), Inolex Spectrum Diagnostics (Glenwood, Ill.), Oxoid Ltd. (Columbia, Md.) and Scott Laboratories (Fiskeville, R.I.) all contributed the agar media. Plates containing each of the seven media were prepared at the same time, and control strains were tested on all seven media at the same time to permit comparison of zones on different media. Each strain was tested with five separate inocula and the mean of 5 zones on each medium was compared to those on other media tested at the same time. Gellan gum media. A series of preliminary trials suggested that 21 g of Mueller-Hinton broth, 7.5 g of KCI and 4.3 g of the gellan gum per liter would produce a solid medium with performance characteristics similar to that of Mueller-Hinton agar. The gel strength and performance can be modified by adjusting the concentration of the gellan gum and the concentration of KCI. The gellan gum media were sterilized at 121 C for 15 min. Thermocouples recorded the temperature of each medium over the entire sterilization and pouring process. The media were exposed to temperatures over 100 C for ca. 35 min. A pipetting machine was used to distribute 60 ml of medium into each 150-mm petri plate. Because the medium tends to solidify at temperatures just below 60 C, the pipetting process was initiated when the temperature of the medium was 90 C; the temperature dropped to 71 to 80 C before the pipetting was complete. Once solidified, the plated media were sealed in plastic bags and refrigerated until needed (less than 7 days). 409

2 410 BARRY, CREITZ, AND PACKER For the current study, gellan gum media were prepared with 11 different Mueller-Hinton broths from Acumedia (lot no and ), BBL Microbiology Systems (lot no. H5B MQV and D60 NHG), GIBCO Laboratories (lot no ), Scott Laboratories (lot no ) and Difco Laboratories (lot no , , , , and ). All 11 broths were solidified by adding 0.75% KCI and 0.43% gellan gum (lot no A). In addition, one Mueller-Hinton broth (Difco lot no ) was solidified with five different gellan gums (lot no A, as above, and 67001A, 76004A, 78007A, and 79008A). This provided 15 different gellan gum-solidified media, which were all tested in parallel. The ph of each solidified medium varied from ph 7.1 to 7.5. Five media had ph values just outside of the accepted range of ph 7.2 to 7.4 that is often cited for Mueller-Hinton agar (14). Statistical analysis. For each microorganism-drug combination, the mean and range of zone diameters were evaluated. The mean of five zone diameters was calculated for each medium and compared to those on other media and to the quality control limits that are recommended by the NCCLS for tests with agar media (15). Mean zone diameters and ranges of zone diameters on gellan gum media were compared with those calculated from tests on the seven Mueller-Hinton agars. Differences between zone diameters on different types of media were tested by applying a oneway analysis of variance. Mean squares between media and within tests (test error) were compared, and an F ratio was calculated. Differences between media being compared were considered significant if the F ratio was greater than the value which is significant at the 1% level of confidence, for the appropriate degrees of freedom. RESULTS The overall mean zone diameters and ranges of zone sizes observed on different types of gellan gum media and on the seven Mueller-Hinton agars are described in Table 1. On the gellan gum media, mean zone sizes were equal to or greater than those on the agar media. On the three different types of gellan gum media, zone diameters were not remarkably different; these differences were later tested for statistical significance, but most of the differences had little practical importance (generally <1.0-mm differences between means). To compare different types of media, the mean of five replicate tests was used to express the performance of each medium with each drug-microorganism combination. These means were compared rather than the individual zone measurements. Table 2 lists the ranges of the mean values for the 15 different gellan gum media and for the seven different agar media. These mean values are compared to established NCCLS control limits for means of five tests on Mueller- Hinton agar. In general, zone diameters on gellan gum media tended to be slightly larger than those on agar media but most gellan gum media performed within NCCLS control limits; the agar media did not perform as well. One or more of the seven agar media produced mean zones outside the NCCLS control limits for 13 of the 27 drug-microorganism combinations that could be evaluated. On gellan gum media, mean zone diameters that were outside of these control limits were observed with 22 of the 28 microorganism-drug combinations that could be evaluated. When was tested against polymyxin B on gellan gum media, zones were more than 6 mm larger than those that would be expected on agar media. Polymyxin B disks were not tested on the agar media. J. CLIN. MICROBIOL. With, only polymyxin B produced exceptionally large zones on gellan gum media. However, the Escherichia coli control strain produced unusually large zones with tetracycline, chloramphenicol, cephalothin, cefotaxime, and sulfisoxazole. The Staphylococcus aureus strain provided zones more than 1 mm larger than the upper control limit with all nine drugs that were tested. The differences between overall mean zones on gellan gum media and those on the agar media (Table 2) are exaggerated because the agar media tended to provide zones in the lower end of the NCCLS control limits. The midpoint between the upper and lower control limits listed in the NCCLS document (15) may be considered the theoretical ideal zone-size value for each drug-microorganism combination. Overall mean zones on gellan gum media were closer to the theoretical target value than were the overall mean zones on the agar media. Deviation from the target value ranged from -0.5 to +5.4 mm for tests on gellan gum media and from -4.0 to +1.2 mm for tests on agar media. A one-way analysis of variance was used to evaluate the significance of differences between mean zone diameters on similar types of media. Table 3 presents the results of such analyses. Eleven different lots of Mueller-Hinton broth (five manufacturers) were all solidified with the same lot of gellan gum. The broths provided significantly different results with 21 of the 29 microorganism-drug combinations. Of the 19 antimicrobial agents, 16 demonstrated significant differences with one or more test strains., cefoperazone, and cefoxitin were the only drugs that demonstrated no significant differences in zone sizes when tested on all 11 broths. On one lot of Mueller-Hinton broth, the strain produced an unacceptably heavy haze of growth within the zones of inhibition around sulfisoxazole and trimethoprimsulfamethoxazole disks; the outer zones were measured and used for this anlaysis. Included in the 11 lots of broth described above were 5 different lots of Mueller-Hinton broth from the same manufacturer (Difco Laboratories). Only 5 of the 29 microorganism-drug combinations demonstrated significant differences in the performance of these broths. Amikacin, gentamicin, tetracycline, and oxacillin were the only drugs that performed differently on plates prepared with broth from the same manufacturer. Variability in broth from different manufacturers appears to be more significant than lot-to-lot variation in media from one of the manufacturers. However, the magnitude of the differences between the mean zone sizes observed on different gellan gum media was generally of little practical consequence (Table 1) although they were statistically significant. Mueller-Hinton agars from seven different manufacturers demonstrated significantly different results with 18 of the 28 microorganism-drug combinations. Chloramphenicol, piperacillin, penicillin, vancomycin, and cefoperazone were the only drugs that failed to demonstrate significant differences in the performance of the agar media. Because the gellan gums are currently being produced by a single manufacturer and are made under strictly controlled conditions, lot-to-lot variability should be minimal. One lot of Mueller-Hinton broth was solidified with five different lots of gellan gum, and the data obtained with those five media were analyzed. Only 7 of the 29 microorganism-drug combinations provided significant differences among media. The greatest differences were seen when amikacin and gentamicin were tested against or. The differences were not due to a single atypical lot of gellan gum. Tobramycin was also tested against and

3 VOL. 21, 1985 GELRITE FOR SUSCEPTIBILITY TESTS 411 TABLE 1. Ranges of zone diameters on Mueller-Hinton broth solidified with a gellan gum and on Mueller-Hinton agars Mean (range) zone diameters (mm) on each medium type Antimicrobial agent Test strain' 11 MHBs Difco 5 gellan gellan gumb MHBs + 1 gums s' gellan gum' MHB" Amikacin 21.6 (19-24) 22.0 (20-23) 22.4 (20-25) 21.3 (19-24) 21.8 (19-25) 23.6 (22-25) 23.4 (23-24) 19.9 (18-22) Chloramphenicol Piperacillin Penicillin G Oxacillin Vancomycin Cephalothin Cefoxitin Cefoperazone Polymyxin Sulfisoxazole Trimethoprimsulfamethoxazole! Erythromycin Clindamycin 17.6 (16-19) 21.9 (18-25) 22.8 (20-28) 13.2 (11-15) 26.6 (25-28) 31.0 (27-35) 27.6 (27-29) 24.8 (22-28) 21.8 (21-23) 28.4 (27-30) 30.9 (29-33) 36.2 (33-39) 25.1 (23-28) 19.8 (19-23) 22.6 (20-25) 37.6 (34-41) 27.7 (27-29) 20.9 (18-22) 35.1 (33-37) (21-24) 34.4 (33-36) 27.9 (26-30) 19.9 (18-21) 23.7 (20-27) 29.5 (28-31) 28.7 (27-32) 28.0 (25-32) 18.2 (17-19) 23.6 (22-25) (21-24) 13.2 (11-14) 26.3 (25-28) 29.8 (27-32) 27.5 (27-28) 24.1 (22-26) 21.8 (21-23) 28.2 (27-29) 31.2 (30-33) 35.0 (33-37) 24.1 (23-25) 19.7 (19-21) 22.7 (20-25) 36.5 (34-38) 27.8 (27-29) 20.7 (18-22) 34.8 (33-36) 22.7 (22-24) 34.4 (33-36) 28.0 (27-30) 20.2 (20-21) 24.0 (23-25) 30.0 (29-31) 28.2 (27-29) 27.6 (26-29) 18.7 (17-21) 23.5 (23-24) 22.2 (19-23) 14.1 (13-16) 27.6 (26-29) 31.2 (30-34) 28.0 (27-29) 24.6 (23-26) 21.8 (21-23) 28.6 (28-30) 31.3 (30-33) 35.9 (34-38) 24.8 (23-26) 20.2 (20-21) 22.7 (21-25) 37.3 (35-39) 28.1 (27-29) 21.3 (18-23) 35.2 (34-36) 23.2 (22-24) 34.7 (34-36) 27.9 (27-29) 20.6 (20-22) 24.9 (24-27) 30.7 (30-32) 28.4 (27-29) 27.2 (26-29) 18.5 (17-21) 20.3 (19-22) (20-26) 11.1 (8-15) 20.5 (18-23) 24.0 (21-26) (21-24) 21.9 (20-24) 20.3 (18-23) 22.9 (20-25) 27.8 (25-31) 30.1 (28-32) 20.5 (18-22) 17.3 (16-18) 20.2 (18-23) 29.8 (27-32) 22.2 (20-25) 20.0 (19-22) 28.7 (26-30) 20.7 (19-22) 28.7 (26-30) 24.6 (21-27) Not tested 19.9 (17-23) 26.2 (24-28) 24.2 (22-26) 24.0 (22-26) " (ATCC 27853), (ATCC 25922). or (ATCC 25923). Five separate tests were performed with each strain with each medium. " Eleven lots of Mueller-Hinton broth (MHB) from five different manufacturers, each solidified with the same lot of gellan gum. ' Five different lots of Mueller-Hinton broth from Difco Laboratories, each solidified with the same lot of gellan gum (data included in the above group). " One lot of Mueller-Hinton broth (Difco Laboratories) solidified with each of five different lots of gellan gums. ' Seven different Mueller-Hinton agars (s) from seven different maunfacturers. ' Trimethoprim/sulfamethoxazole ratio, 1:19. neomycin was tested against (data not shown). No significant differences were observed among lots of gellan gum with the latter drugs. DISCUSSION To improve the precision of the disk diffusion susceptibility test, the performance of agar media must be standardized. Others have previously noted significant differences among Mueller-Hinton agars obtained from different manufacturers (2, 13, 17, 18, 20) and that observation is confirmed by the findings included in the present study. Efforts are currently underway to develop a physical standard to be used by manufacturers of dehydrated agar media to help them provide a more uniform product (Pollock et al., in preparation). The agar portion of Mueller-Hinton agar is an ill-defined component which can lead to lot-to-lot variability. The current study was undertaken to determine whether satisfactory results could be obtained with an alternative solidifying agent which is manufactured under well-controlled

4 412 BARRY, CREITZ, AND PACKER TABLE 2. Zone diameters on Mueller-Hinton broths solidified with gellan gum and on Mueller-Hinton agars Zone size Test strain Type of agent medium Range of Limitsd imt Overall means' mean Amikacin Piperacillin Cefoperazone Polymyxin B Amikacin Cephalothin Cefoxitin Sulfisoxazole conditions and, therefore, should be more uniform. When appropriately adjusted, the gellan gum performed quite well, providing a solid medium which was more uniform than Mueller-Hinton agar. With further experience, it should be possible to eliminate almost all lot-to-lot variability in the gellan gum component. Differences in Mueller-Hinton broths from different manufacturers are similar to differences in Mueller-Hinton agars. It might be possible to develop a physical standard for guiding the manufacture of Mueller-Hinton broth in a manner similar to that which is'currently being developed for Mueller-Hinton agar. Lot-to-lot reproducibility of results on solidified broth from a single manufacturer was much more encouraging. In practice, the manufacturer-to-manufacturer variable can be eliminated by simply using a single source of broth. In that case, results of diffusion tests with a Mueller- Hinton broth-gellan gum medium should be quite reproducible. The experimental design of this study does not permit definition of control limits, but we can conclude that all Mueller-Hinton agars do not perform within the established limits. Furthermore, quality control limits for tests on agar media will need to be modified when gellan gum media are used. Tentative control limits for tests on gellan gum media can be calculated from data included on Table 2. Our overall Chloramphenicol Trimethoprim-sulfamethoxazole NTf Midpoint' J. CLIN. MICROBIOL. TABLE 2-Continued Zone size Test strain Type of agent medium Range of Limitsd Overall means' mean Midpoint' Cephalothin Chloram phenicol Erythromycin Clindamycin Vancomycin Penicillin Oxacillin (ATCC 27853), (ATCC 25922), or (ATCC 25923). Five separate tests were performed with each strain with each medium. b Fifteen different Mueller-Hinton broth-plus-gellan gum combinations () and seven different Mueller-Hinton agars from seven manufacturers (). C Minimum and maximum mean zone diameters (millimeters) for five separate tests on each medium. d NCCLS-recommended control limits for the means of five tests (from reference 15). Control limits for polymyxin B are control limits for a single test (from reference 14). No control limits are available for tetracycline and P. aeruginosa. e Midpoint between the upper and lower control limits: this is the theoretical target value that should be produced when tests are performed on agar media and should be reasonably close to the overall mean values. f NT, Not tested.

5 VOL. 21, 1985 GELRITE FOR SUSCEPTIBILITY TESTS 413 TABLE 3. One-way analysis of variance comparing zone diameters on Mueller-Hinton broths solidified with gellan gum and on Mueller- Hinton agars F ratio comparing variances among media and within tests' Antimicrobial agent Test strain" 11 MHBs plus 5 Difco MHBs plus 5 gellan gums 7 s one gellan gum' one gellan gumd plus one MHB' Amikacin Chloramphenicol Piperacillin Penicillin G Oxacillin Vancomycin Cephalothin Cefoxitin Cefoperazone Polymyxin B Sulfisoxazole Trimethoprim-sulfamethoxazoleh Erythromycin Clindamycin " (ATCC 27853), (ATCC 25922), or (ATCC 25923). Five separate tests were performed with each strain with each medium. "For the applicable degrees offreedom, F0.99 values are as follows: 11 MHBs plus one gellan gum, >2.80; five Difco MHBs plus one gellan gum, >4.43; five gellan gums plus one MHB, >4.43; seven s, >3.53. F ratios that exceed the designated F(, 99 values are significant at the 1% level of confidence (between-media variances exceed within-media variances). ' Eleven lots of Mueller-Hinton broth (MHB) from five different manufacturers, each solidified with the same lot of gellan gum. d Five different lots of Mueller-Hinton broth from Difco Laboratories, each solidified with the same lot of gellan gum (data included in the above group). eone lot of Mueller-Hinton broth (Difco Laboratories) solidified with each offive different lots of gellan gums. f Seven different Mueller-Hinton agars from seven different manufacturers. NT, Not tested. 1Trimethoprim/sulfamethoxazole ratio, 1: mean zone diameters on all gellan gum media are the best available estimates of central tendency (midpoint value), and the midpoint between the upper and lower NCCLS control limits (15) represents an idealized target value for tests on agar media. The range of acceptable variability on both media should be similar since the ranges of zone sizes on the two types of media were not subtantially different (Tables 1 and 2). Thus, the control limits may be adjusted by adding the differences between estimated midpoint' values for the two types of media. Only three tests on gellan gum media gave mean zone diameters outside of such adjusted control limits. Supportive information from a multilaboratory study will be needed to test the applicabiity of the adjusted control limits. The type of protocol defined by Gavan et al. (7) could

6 414 BARRY, CREITZ, AND PACKER be modified to evalute quality control parameters for this new product. Although gellan gum media tended to give zones slightly larger than those seen on different agar media, the overall mean zone sizes for tests on gellan gum media were closer to the theoretical target value that was determined from the current NCCLS control limits (15). Some of the agar media tended to give zones that were too small. On gellan gum media, mean zone sizes of -2.0 mm above the theoretical target value were observed with piperacillin, polymyxin B, tetracycline, chloramphenicol, carbenicillin, cephalothin, cefotaxime, moxalactam, sulfisoxazole, erythromycin, vancomycin, penicillin, and oxacillin. With those drugs, appropriate studies should be performed to determine whether the interpretive zone-size breakpoints need to be adjusted for tests on gellan gum media. The aminoglycosides appear to perform well on gellan gum media, but that needs to be confirmed by additional supportive data. In summary, it is possible to solidify Mueller-Hinton broth with 0.43% gellan gum and 0.75% KCI to develop a solid medium with disk diffusion susceptibility testing performance characteristics similar to those of most Mueller-Hinton agars. LITERATURE CITED 1. Barry, A. L Procedure for testing antibiotics in agar media: theoretical considerations, p In V. Lorian (ed.), Antibiotics in laboratory medicine. The Williams & Wilkins Co., Baltimore. 2. Barry, A. L., and L. J. Effinger Performance of Mueller- Hinton agars prepared by three manufacturers. Am. J. Clin. Pathol. 62: Barry, A. L., and L. J. Effinger Antimicrobic diffusion susceptibility tests on a defined medium solidified with a synthetic polymer (Neutra-Gel). Curr. Microbiol. 1: Brenner, V. C., and J. C. Sherris Influence of different media and bloods on the results of diffusion antibiotic susceptibility tests. Antimicrob. Agents Chemother. 1: D'Amato, R. F., and C. Thornsberry Calcium and magnesium in Mueller-Hinton agar and their influence on disc diffusion susceptibility test results. Curr. Microbiol. 2: D'Amato, R. F., C. Thornsberry, C. N. Baker, and L. A. Kirven Effect of calcium and magnesium ions on the susceptibility of Pseudomonas species to tetracycline, gentamicin, polymyxin B, and carbenicillin. Antimicrob. Agents Chemother. 7: Gavan, T. L., R. N. Jones, A. L. Barry, P. C. Fuchs, E. H. Gerlach, J. M. Matsen, L. B. Relier, C. Thornsberry, and L. D. Thrupp Quality control limits for ampicillin, carbenicillin, mezlocillin, and piperacillin disk diffusion susceptibility tests: a J. CLIN. MICROBIOL. collaborative study. J. Clin. Microbiol. 14: Hanus, F. J., J. G. Sands, and E. O. Bennett Antibiotic activity in the presence of agar. Appl. Microbiol. 15: Kang, K. S., G. T. Veeder, P. J. Mirrasoul, T. Kaneko, and I. W. Cottreli Agar-like polysaccharide produced by a Pseudomonas species: production and basic properties. Appl. Environ. Microbiol. 43: Kenney, M. A., H. M. Pollock, B. H. Minshew, E. Casillas, and F. D. Schoenknecht Cation components of Mueller-Hinton gar affecting testing of Pseudomonas aeruginosa susceptibility to gentamicin. Antimicrob. Agents Chemother. 17: Kunin, C. M., and W. P. Edmondson Inhibitor of antibiotics in bacteriologic agar. Proc. Soc. Exp. Biol. Med. 129: Lorian, V., and N. Gray Separan NP10, a viscosity-producing substance in culture media. Appl. Microbiol. 14: Murray, P. R., and J. R. Zeitinger Evaluation of Mueller-Hinton agar for disk diffusion susceptibility tests. J. Clin. Microbiol. 18: National Committee for Clinical Laboratory Standards Performance standards for antimicrobic disk susceptibility tests. Approved Standard, ASM-2 (2nd edition). National Committee for Clinical Laboratory Standards, Villanova, Pa. 15. National Committee for Clinical Laboratory Standards Second informational supplement, performance standards for antimicrobic disk susceptibility tests. Supplement to approved standard M2-A2S. National Committee for Clinical Laboratory Standards. Villonova, Pa. 16. Newton, B. A Reversal of antibacterial activity of polymyxin by divalent cations. Nature (London) 172: Pollock, H. M., B. H. Minshew, M. A. Kenney, and F. D. Schoenknecht Effect of different lots of Mueller-Hinton agar on the interpretation of the gentamicin susceptibility of Pseudomonas aeruginosa. Antimicrob. Agents Chemother. 14: Relier, L. B., F. D. Schoenknecht, M. A. Kenney, and J. C. Sherris Antibiotic susceptibility testing of Pseudomonas aeruginosa: selection of a control strain and criteria for magnesium and calcium content in media. J. Infect. Dis. 130: Shungu, D., M. Valiant, V. Tutlane, E. Weinberg, B. Weissberger, L. Koupal, H. Gadebusch, and E. Stapley GEL- RITE as an agar substitute in bacteriological media. Appl. Environ. Microbiol. 46: Washington, J. A., R. J. Snyder, P. C. Kohner, G. G. Wiltse, D. M. Ilstrup, and J. T. McCall Effect of cation content on the activity of gentamicin, tobramycin and amikacin against Pseudomonas aeruginosa. J. Infect. Dis. 137: Zimelis, V. M., and E. G. Jackson Activity of aminoglycoside antibiotics against Pseudomonas aeruginosa: specificity and site of calcium and magnesium antagonism. J. Infect. Dis. 127: