Antibiotic Susceptibility Testing. Part I

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1 CE Update Microbiology I Antibiotic Susceptibility Testing. Part I Patrick R. Murray, PhD W ith the introduction of antimicrobial c h e m o t h e r a p y in t h e 1940s, the hope of eliminating infectious diseases was entertained. However, it was quickly recognized that microorganisms could develop resistance to each newly introduced antimicrobial agent. Today, the pharmaceutical industry has given us an abundance of drugs and nature has responded with an increasingly broader spectrum of resistant pathogens. This, in turn, has presented a challenge to the microbiology laboratory. Antimicrobial susceptibility testing is one of the most important procedures performed in the microbiology laboratory. If accurate results are generated rapidly, specific antimicrobial therapy can be initiated for the appropriate management of serious infections. However, the number of drugs that can or should be tested increase each year, as do the possible test methods. The individual antimicrobics t h a t will be tested in a laboratory should be determined jointly by the infectious disease specialist and the microbiology director. Because this decision is based on the clinical needs of the individual hospital and the technical capabilities of the laboratory, this will not be discussed here. Instead, the various susceptibility testing methods that are currently From the Division of Laboratory Medicine. Washington Univ School of Medicine, Box 8118, 660 S Euclid Ave, St Louis, MO available will be reviewed and the adv a n t a g e s and l i m i t a t i o n s of each method will be discussed. Disk Diffusion Tests Bauer and co-workers 1 published in 1966 a s t a n d a r d i z e d s i n g l e - d i s k method for antibiotic susceptibility tests. This procedure, commonly referred to as the Kirby-Bauer disk diffusion method, forms the foundation of t h e m e t h o d c u r r e n t l y r e c o m mended by the Federal Drug Admini s t r a t i o n (FDA) a n d N a t i o n a l Committee on Clinical Laboratory Standards (NCCLS). 2 The only other diffusion method that gives results comparable to the Kirby-Bauer procedure is the agar overlay method of Barry et al. 3 However, this method is technically more difficult and can only be used for testing Staphylococcus aureus, Enterobacteriaceae, and Pseudomonas aeruginosa. Organisms such as streptococci and Hemophilus cannot be tested. Principle The Kirby-Bauer disk diffusion test is performed by inoculating a standardized suspension of organisms onto specialized susceptibility testing agar (Mueller-Hinton agar). Paper disks impregnated with a specific amount of antibiotic are placed on the agar and the plates are incubated in precisely controlled conditions. After overnight incubation, the zones of in- hibited growth around the disks are measured and correlated with established interpretive standards for organisms susceptible, intermediate, or resistant to the specific antibiotics. The principle of the disk diffusion test lies in the inverse linear relationship between the diameter of the zone of inhibited growth around an antibiotic-saturated disk and the logarithm of the minimum inhibitory concentration (MIC) of the antibiotic. In other words, the size of the zone of inhibition is proportional to the susceptibility of the organism to the tested antibiotic the larger the zone, the more susceptible the organism is to the particular antibiotic (Fig A). Based on this relationship between zone size and MIC, organisms can be classified as resistant or susceptible to a particular antibiotic, or found to be in an indeterminant zone (Fig B). It should be appreciated that the interpretive classification v a r i e s for each a n t i biotic and group of organisms. Thus, an antibiotic with a large zone of inhibited growth may be less active than an antibiotic with a smaller zone. The size of the zone of inhibition is determined by the diffusion coefficient of the antibiotic, and the rate of replication and antibiotic susceptibility of the test organism. T h e diffusion coefficient is an expression of the rate of diffusion of an antibiotic through a specific medium under defined test conditions. LABORATORY MEDICINE VOL. 14, NO. 6, JUNE

2 MIC UG/ML ZONE DIAMETER (MM) Figure. Correlation between MICs and known diameters of inhibition. A Distribution of individual test organisms. B Regression analysis plot with theoretical interpretation classification. This rate of diffusion is influenced by the composition of the medium and the physicochemical properties of the antibiotic. If the composition of the m e d i u m is a l t e r e d, e i t h e r d u r i n g preparation or storage, then the rate of antibiotic diffusion will also be affected. However, if the composition of the medium is carefully controlled, the diffusion coefficient can be precisely defined for each antimicrobic. In addition to the diffusion coefficient of the antibiotic, the size of the zone of inhibition is determined by the replication rate of the test organism. If two organisms have the same susceptibility to a particular antibiotic, the organism t h a t grows the fastest will have the smaller zone of inhibition. The two phases of the organism's growth curve t h a t affect the size of the zone of inhibition are the lag phase (the time an organism requires for adjusting to growth on an artificial medium) and the phase of exponential growth. Since these phases are determined by the medium composition, t h e t e s t should only be performed w i t h t h e r e c o m m e n d e d susceptibility test medium (ie, Mueller-Hinton Agar). Finally, the size of the zone of inhibition is determined by the susceptibility of an organism to the critical inhibitory concentration of the antibiotic. This is the minimum concentration that inhibits the growth of a specified number of bacteria. If all testing variables are eliminated or closely controlled, the susceptibility of an organism to a particular antibiotic can be predicted by the size of the zone of inhibition. Factors affecting diffusion test results Numerous variables affect the results of disk diffusion susceptibility tests, including the preparation of the inoculum, the antibiotic content in the disk, the testing medium, incubation conditions, and the characteristics of the zone of inhibition (Table I). The test inoculum is prepared from a suspension of actively growing bacteria t h a t is diluted to a specific concentration (about 10 s bacteria per milliliter). The most common method used for preparing the inoculum is to adjust the inoculum turbidity to be equivalent to a McFarland 0.5 barium sulfate standard. Although this method appears simplistic, it is rapid, accurate, and quite reproducible for aerobic bacteria. It is important that the inoculum be carefully prepared. If the inoculum is too light, the inhibitory concentration of the antibiotic can diffuse further before the zone of inhibition is formed. If the inoculum is too heavy, then the critical number of bacteria is reached before the antibiotic can diffuse and small zones of inhibition are formed. The inoculum density is particularly important with the p-lactam antibiotics (penicillins and cephalosporins). Many itive and gram-negative organisms will appear resistant to these antibiotics at high concentrations due to the production of inducible or constitutive P-lactamases. Table 1: Variables Affecting Susceptibility Test Results Test Variable Inoculum too dense improperly standardized; prolonged delay between standardization of inoculum and inoculation of plate too light improperly standardized Antimicrobics disks not stored at 4 C or with desiccant disks not positioned firmly on agar plate Medium Mueller-Hinton agar (MHA) not used MHA not prepared and/or stored properly Depth of agar less than 4 mm Depth of agar greater than 6 mm Incubation conditions Temperature too high Temperature too low C0 2 or anaerobic atmosphere Measurement of zones Plates not uniformly inoculated Placement of disks on plate results in antagonistic or synergistic interaction between adjacent antimicrobics LABORATORY MEDICINE VOL. 14, NO. 6, JUNE 1983 Downloaded from Effect on Test Results Small zones of inhibition (false resistance) Large zones of inhibition (false susceptibility) Antimicrobic less active small zones Slow, irregular diffusion of antimicrobic small zones Variable growth of test organisms unpredictable zones Unpredictable zones Faster diffusion of antimicrobics large zones Slow diffusion of antimicrobics small zones Methicillin-resistant staphylococci will not be detected Slow growth of test organisms large zones ph changes in media variable effect on zones Irregular zones Unreliable zones

3 The antibiotic content in the test disks is obviously important. The disk content is regulated by the FDA and is required to be within 67% and 150% of the stated content. Although this range of acceptable variation appears to be excessive, it should be noted that two thirds of the disks failed to meet these standards in 1958, compared with 2% in 1970." Furthermore, a twofold variation in disk content will generally cause only small changes in the diameter of inhibited growth and for most antibiotic-organism combinations the interpretation of the test result will not be significantly affected. Bacterial susceptibility is also influenced by the testing medium. 5 An ideal susceptibility testing medium should h a v e a chemically defined composition, support the growth of most important organisms, and be buffered to minimize ph changes during growth. The medium should not antagonize antibiotic activity, should be isotonic, and should have acceptable batch-to-batch reproducibility. Mueller-Hinton agar, which is the only medium approved for disk diffusion tests in this country, satisfies most of these requirements. This medium, originally developed for the isolation of Neisseria gonorrhoeae, has a simple formulation: beef infusions (the leastdefined ingredient) are a source of some peptones, nucleic acids, and vitamins; acid hydrolysate of casein is added as a peptone source; corn starch is used to neutralize toxic fatty acids; and agar is used to solidify the medium. Most organisms grow well on this medium, although blood is frequently added for the more fastidious bacteria. Even with the addition of blood, however, some bacteria, particularly streptococci, do not grow adequately and cannot be tested by this method. The buffering capacity of Mueller-Hinton agar is sufficient to prevent ph shifts during bacterial growth. This is important because some antibiotics, such as the aminoglycosides and erythromycin, are more active at alkaline phs and antibiotics such as tetracycline and methicillin are more active in acidic media. Antibiotic activity is antagonized in some media. For example, if thymidine is present in the medium, the sulfonamides which interfere with folic acid synthesis are ineffective. Mueller- Hinton agar does not antagonize antibiotic activity. In addition, it is isotonic, which allows the medium to be enriched with blood to support the growth of fastidious organisms. Finally, reproducibility of tests performed with Mueller-Hinton agar is reasonably good, although some problems have been encountered with lotto-lot variations in the concentration of free cations. 6 This variation can significantly affect test results for aminoglycosides with Pseudomonas and for tetracycline. 7 9 Pseudomonas takes up aminoglycosides by oxygendependent transport through the cell wall. The integrity of the cell wall is maintained by divalent cations, such as calcium, m a g n e s i u m, and zinc. Therefore, as the cation concentration is increased, the Pseudomonas cell wall becomes more stable, and the antibiotic artificially appears less active. Tetracycline is also affected by the cation concentration, but by a different mechanism. Tetracycline is inact i v a t e d by chelation with cations; therefore, as the cation concentration increases, tetracycline appears less active. The concentration of divalent cations differs from lot-to-lot of MuellerH i n t o n broth and Mueller-Hinton agar. The concentration in agar is between that found in broth medium and in h u m a n serum. It had been proposed that agar media should be supplemented with cations, but that is a problem because insoluble complexes of phosphates and cations are formed during autoclaving. The best solution to this problem is to run performance tests before the media are used. Test a number of batches with control organisms of known susceptibility patterns, select the medium with the appropriate test results, and then use only t h a t m e d i u m. My laboratory r o u t i n e l y tests five to t e n lots of Mueller-Hinton agar at the beginning of each year and then selects one lot from which our commercial sup- plier will prepare all testing media for t h a t year. In our experience the majority of lots fail these quality control t e s t s. Because my laboratory performs a large number of tests, we are able to perform these quality control tests and insist that we receive media from a single lot. However, most smaller laboratories do not have the resources to perform these quality control tests and must use the media supplied to them by their commercial vendor. I believe this is one of the major causes of poor test reproducibility. The incubation atmosphere must also be properly regulated for reliable test results. If the temperature exceeds 35 C, m e t h i c i l l i n - r e s i s t a n t staphylococci will not be detected. If the temperature is too low, bacteria will grow slowly and false susceptibility may result. Finally, tests performed in a n a n a e r o b i c or C 0 2 e n r i c h e d a t m o s p h e r e can significantly affect antimicrobial activity. For example, aminoglycosides are inactive when tested in an anaerobic atmosphere. A final variable which can influence the results of disk diffusion tests is the measurement of the zone of inhibition. 10 When the zone of inhibition is formed, a gradient is created from complete inhibition of growth to no inhibition (or even stimulation) of growth. Thus, the zone of inhibition must be carefully measured for the test results to be reproducible. In addition, if the plates are incubated for a prolonged period, the areas of partial inhibition and delayed growth will eventually grow when the concentration of antibiotic is reduced either by continued diffusion or deterioration of the antibiotic. This is particularly common with bacteriostatic antibiotics such as tetracycline. Because the disk diffusion test was developed originally to be used for specific antimicrobial susceptibility tests, the limitations of the test must be considered (Table II). Only those Table II: Limitations of Disk Diffusion Tests Only bacteria that grow rapidly on Mueller-Hinton agar can be tested. Capnophilic or anaerobic bacteria cannot be tested. Only a pure growth of bacteria should be tested. Only antibiotics for which interpretive standards have been established can be tested. LABORATORY MEDICINE VOL. 14, NO. 6, JUNE 1983

4 bacteria that grow rapidly on Mueller-Hinton agar can be tested. Slowgrowing organisms or organisms with unpredictable growth characteristics must be tested by another method, such as broth dilution tests. Likewise, if the test organism requires a specialized atmosphere, such as an anaerobic or capnophilic environment, an alternative method must be found." A pure growth of the test organism must be used. Thus, direct testing of clinical specimens is generally unreliable. E l l n e r and Johnson 1 2 h a v e demonstrated this with wound cultures and Hollick and Washington 13 with urine cultures. The only specimens for which direct susceptibility tests have proven to be reliable are positive blood cultures. 11 This is because large numbers of bacteria are generally present when the positive culture is detected and the cultures are usually monomicrobic. Finally, diffusion tests can only be performed with antibiotics for which interpretive standards have been established. This presents a particular problem for some of the newer antim i c r o b i c s. However, t h e FDA r e quires t h a t preliminary standards must be established at the time new drugs are approved for clinical use. Reproducibility of disk diffusion tests Although numerous technical variations can influence the KirbyBauer test, the test results are rem a r k a b l y reproducible if test performance is carefully monitored. In two large multilaboratory studies, the standard deviations of intra- and interlaboratory diffusion tests were between 1 and 2 mm In addition, o t h e r i n v e s t i g a t o r s h a v e demonstrated that the reproducibility of the interpretive categories (susceptible, intermediate, resistant) is greater than 95%, w i t h significant changes in interpretation (ie, between susceptible and resistant) occurring in only 1% of the t e s t s. " This relative lack of significant test-to-test variation is a major virtue of the disk diffusion test. It should be obvious that the reproducibility of the disk diffusion test is influenced by the distribution of the organism-antibiotic combination in r e l a t i o n to the i n t e r p r e t i v e breakpoint. Thus, combinations which are clustered near the breakpoints (eg, Pseudomonas aeruginosa and genta- micin) are strongly influenced by minor t e c h n i c a l c h a n g e s, w h e r e a s combinations distributed away from the breakpoints (eg, Staphylococcus aureus and tetracycline) are not significantly affected.17 Quantitative Dilution Tests Quantitative dilution tests are used to determine the minimum concent r a t i o n of a n t i m i c r o b i a l a g e n t required to inhibit or to kill a particular organism. The test is performed by diluting antibiotics in medium and then inoculating the medium with a standardized suspension of the patient's o r g a n i s m. After o v e r n i g h t incubation, the lowest concentration of antimicrobic that inhibits the growth of the organism is called the MIC. If the tests are performed in broth medium, the MBC (minimum bactericidal concentration) can be determined by subc u l t u r i n g all t h e b r o t h s w i t h no macroscopic evidence of growth onto antibiotic-free medium. After incubation for one to two days, the lowest concentration of antimicrobic t h a t killed the initial inoculum is called the MBC. Quantitative dilution tests are performed in either agar or broth. Agar dilution tests are relatively inexpensive when large numbers of tests are performed but are impractical for testing a few organisms. In addition, agar dilution plates must be made weekly and the method cannot be used for determining MBCs. Broth dilution tests are performed either in tubes or in microdilution trays. The macrotube dilution method is practical for laboratories that test a few isolates or infrequently used antibiotics, but the method is too cumbersome and expensive for testing many organisms. The microdilution method is well-suited for large numbers of tests. Many antibiotic trays can be prepared at one time, or purchased from commercial vendors, and then stored for many weeks. The major limitations with the microdilution method are that only predetermined panels of antibiotics can be tested and the measurement of MBCs is more difficult in comparison with the macrotube dilution method. Quantitative susceptibility tests have a number of important advantages in comparison with the qualitative disk diffusion tests. Virtually LABORATORY MEDICINE VOL. 14, NO. 6, JUNE 1983 Downloaded from any antibiotic and organism can be t e s t e d by t h e q u a n t i t a t i v e t e s t. Whereas the disk test cannot be used for antimicrobial agents that diffuse slowly in agar or for slow-growing, capnophilic, or anaerobic organisms, the quantitative test is not affected by these limitations. Furthermore, the quantitative dilution tests can precisely define the relative susceptibility of a n o r g a n i s m to a panel of antimicrobics. Yet, the quantitative tests are not without problems. Although an international collaborative group 1 8 in 1971 and t h e N a t i o n a l Committee for Clinical Laboratory Standards 1 9 in 1980 proposed standard agar and broth test methods, major methodologic variations continue to persist The preparation of the antimicrobial solutions, standardization and concentration of the test inoculum, test medium and incubation conditions, and determinations of MIC and MBC endpoints are major variables that continue to plague the tests. All of these variables can influence the test results. For example, most clinical laboratories use Mueller-Hinton medium for MIC tests but many do not perform adequate quality control tests to evaluate the med i u m ' s performance. Performance testing of the medium is critical for accurate test results with both qualitative and quantitative susceptibility tests. Another example of the lack of standardized testing methods for quantitative susceptibility tests is seen with the determination of MBC endpoints. The MBC endpoint is traditionally defined as t h e m i n i m u m concentration of antibiotic required to kill at least 99.9% of the original bacterial population. A survey of 41 major laboratories in the United States revealed that all of these laboratories accepted this as the appropriate test endpoint. However, 68% of the laboratories that performed MBC tests selected a volume of the original broth that prevented an accurate determination of 99.9% cell death. 21 One effort to standardize the results of quantitative susceptibility tests has been the commercial development of both quantitative microdilution trays and the instruments to inoculate the trays and interpret the results (Table III). Microdilution trays are filled with small volumes (eg, 50 to 100 u.l) of

5 Table III: Microdilution Susceptibility Test Systems Results System (manufacturer) Microdilution trays Available trays Tray inoculation Incubation time (hours) Interpretation Reporting Micro Media (Micro Media Systems) 96 wells, frozen Micro-Coder I Micro Scan (American Scientific) 96 wells, frozen gram-neg* anaerobes gram-neg* / Auto-Scan-3 / Sensititre Computer Reader/Recorder gram-neg urine Sceptor 84 wells, dried (BBL) gram-neg urine gram-negative trays include antimicrobics for ui inary tract pathogens Sensititre (Gibco) 96 wells, dried antimicrobics diluted in the susceptibility testing medium. Twofold dilutions of 8 to 12 antibiotics are generally tested in each microdilution tray. Because of the convenience and economy of performing tests by microdilution, commercial production of these trays has developed rapidly. Initially the trays were filled with antimicrobic dilutions, frozen, and then shipped to the laboratory. Now many companies prepare trays with dried stabilized antimicrobic dilutions which are rehydrated in the laboratory with a suspension of the test organism. This procedure extends the stability and shelf life of the trays. Instruments have also been developed to inoculate the trays, which is particularly important with the dried trays, and to assist in the interpretation of the MIC endpoints. Although the instruments designed to inoculate the trays have been shown to be accurate, the accuracy of interpretation of MIC endpoints remains to be completely evaluated.22 The accuracy and reproducibility of tests performed with commercially prepared microdilution trays are equivalent to tests with trays prepared in the laboratory. Limitations of quantitative tests Results of quantitative susceptibility tests are traditionally compared with the concentration of drug expected at the focus of infection. This comparison is an oversimplification. The quantitative tests are usually performed by evaluating the organism's susceptibility to twofold dilutions of the antibiotic. For example, the organism may be tested against 1, 2, 4, 8, 16, and 32 ixg/ml of Drug A. If the organism grows in the presence of 1, 2, 4 and 8 u,g/ml and is inhibited by 16 and 32 u-g/ml, then the MIC of Drug A (the lowest concentration that inhibits the organism) is defined as 16 u.g/ml. However, because the dilution scheme does not discriminate between an MIC of 9 u.g/ ml and 16 u-g/ml, the reported result may be misleading. Furthermore, the susceptibility test methods can influence the numerical value. Microdilution test results are generally one to three twofold dilutions lower than macrodilution results.2324 Therefore, microdilution tests underestimate resistance. The testing medium and other variables can also significantly affect the test results, as described above. Finally, the tests are intended to be performed under well-defined conditions. Unfortunately, when the tests are performed in the presence of serum (as would be more analogous to the clinical situation), the results are significantly different.25 With this multitude of variables influencing the final quantitative result, it is not surprising that the numerical laboratory result should not be directly correlated with the antibiotic level achieved in the patient. Rather, the quantitative results, in conjunction with a knowledge of the pharmacokinetic properties of the tested antibiotics, should be used to compare the relative susceptibility of an isolate to a group of antibiotics. References 1. Bauer AW, Kirby WMM, Sherris JC, et al: Antibiotic susceptibility testing by a standardized single disk method. Am J Clin Pathol 1966; 36: National Committee for Clinical Laboratory Standards. Performance standards for antimi- crobial disk susceptibility tests. Chicago, Barry AL, Garcia F, Thrupp LD: Improved single-disk method for testing the antibiotic susceptibility of rapidly growing pathogens. Am J Clin Pathol 1970;53: Wright WW: FDA actions on antibiotic susceptibility disks, in Balows A led): Current Techniques for Antibiotic Susceptibility Testing. Springfield, IL. C C Thomas, 1974, pp Brenner VC, Sherris JC: Influence of different media and bloods on the results of diffusion antibiotic susceptibility tests. Antimicrob Agents Chemother 1972;1: Pollock HM, Minshew BH, Kenny MA, et al: Effect of different lots of Mueller-Hinton agar on interpretation of the gentamicin susceptibility of Pseudomonas aeruginosa. Antimicrob Agents Chemother 1978;14: Reller LB, Schoenknecht FD. Kenny MA, et al: Antibiotic susceptibility testing of Pseudomonas aeruginosa: selection of control strain and criteria for magnesium and calcium content in media. J Infect Dis 1974;130: Kenny MA, Pollock HM, Minshew BH, et al: Cation components of Mueller-Hinton agar affecting testing of Pseudomonas aeruginosa susceptibility to gentamicin. Antimicrob Agents Chemother 1980:17: Washington JA II, Snyder RJ, Kohner PC, et al: Effect of cation content of agar on the activity of gentamicin. tobramycin, and amikacin against Pseudomonas aeruginosa. J Infect Dis 1978; 137: Barry AL, Coyle MB, Thornsberry C, et al: Methods of measuring zones of inhibition with the Bauer-Kirby disk susceptibility test. J Clin Microbiol 1979:10: Verklin RM Jr, Mandell GL: Alteration of effectiveness of antibiotics by anaerobiosis. J Lab Clin Med 1977;89: Ellner PD, Johnson E: Unreliability of direct antibiotic susceptibility testing on wound exudates. Antimicrob Agents Chemother 1976;9: Hollick GE. Washington JA II: Comparison of direct and standardized disk diffusion susceptibility testing of urine cultures. Antimicrob Agents Chemother 1976;9: Mirrett S, Reller LB: Comparison of direct and standard antimicrobial disk susceptibility testing for bacteria isolated from blood. J Clin Microbiol 1979;10: Barry AL, Schoenknecht FD, Norton R, et al: Inter- and intralaboratory variability in antibiotic susceptibility tests with Pseudomonas aeruginosa and Enterobacteriaceae. J Infect Dis 1976;134: Thornsberry C, Gavan TL, Sherris JC, et al: Laboratory evaluation of a rapid, susceptibility study. Antimicrob Agents Chemother 1975;7: Murray PR, Zeitinger JR, Krogtad DJ: Reliability of disk diffusion susceptibility testing. Infect Control 1982;3: LABORATORY MEDICINE VOL. 14, NO. 6, JUNE

6 18. Ericsson HM, Sherris JC: Antibiotic sensitivity testing. Report of an international collaborative study. Acta Pathol Microbiol Scand IB] 1971;217(suppl):l National Committee for Clinical Laboratory Standards. Proposed standard PSM-7: Standard methods for dilution antimicrobial susceptibility tests for bacteria which grow aerobically. Chicago, Branch A, Starkey DH, Power EE: Diversifications in tube dilution tests for antibiotic sensitivity of microorganisms. Appl Microbiol 1965:13: Murray PR, Jorgensen JH: Quantitative susceptibility test methods in major US medical centers. Antimicrob Agents Chemother 1981;20: Genta VM, McCarthy LR, Herrick M, et al: Evaluation of the Titertek Multiskan for determining minimal inhibitory concentration endpoints. Antimicrob Agents Chemother 1982;22: Barry AL, Jones RN, Gavan TL: Evaluation of the Micro-Media system for quantitative antimicrobial drug susceptibility test: a collaborative study. Antimicrob Agents Chemother 1978;13: Peterson LR, Gerling DN, Johnson MM, et al: Evaluation of a commercial microdilution system for quantitative susceptibility testing of aminoglycosides against multidrug-resistant, gram-negative bacilli. Antimicrob Agents Chemother 1980;17: Reimer LG, Stratton CW, Reller LB: Minimum inhibitory and bactericidal concentrations of 44 antimicrobial agents against three standard control strains in broth with and without human serum. Antimicrob Agents Chemother 1981;19: Category A-l continuing education credit is available to anyone who studies a CIE Update series and completes a written exam. Exams will appear with the final article of each series in LABORATORY MEDI CINE. After receipt of a completed answer sheet and exam fee at ASCP, a certificate of credit will be awarded to each participant. 350 LABORATORY MEDICINE VOL. 14, NO. 6, JUNE 1983