Comparison of media for agar dilution susceptibility testing ofBordetella pertussis andBordetella parapertussis

Antimicrobial susceptibility testing of the fastidious speciesBordetella pertussis andBordetella parapertussis is not standardized. In an attempt to find the optimal medium for agar dilution testing, the activity of erythromycin againstBordetella pertussis andBordetella parapertussis (34 isolates each) was assessed using homologous broth/agar combinations of Bordet-Gengou, charcoal, Iso-Sensitest (Oxoid) and Mueller-Hinton media. Each medium was supplemented with 5 % and 20 % whole defibrinated horse blood. Mueller-Hinton medium supplemented with 5 % horse blood performed best overall.     

Comparison of three media for agar dilution susceptibility testing ofBordetella pertussis using six antibiotics

Since antimicrobial susceptibility testing of the fastidious speciesBordetella pertussis is not standardized, the most suitable medium for agar dilution testing of this species has not yet been determined. In the present study, Mueller-Hinton, Bordet-Gengou, and Oxoid charcoal agars (each supplemented with 5% horse blood) were evaluated for agar dilution susceptibility testing ofBordetella pertussis against ampicillin, chloramphenicol, ciprofloxacin, doxycycline, erythromycin, and trimethoprim-sulfamethoxazole. Mueller-Hinton agar was the most suitable medium.     

Interpretive criteria for susceptibilities of Haemophilus influenzae to ampicillin, amoxicillin, and amoxicillin-clavulanic acid.

One hundred fifty isolates of Haemophilus influenzae (including 30 beta-lactamase-positive strains and 23 beta-lactamase-negative, ampicillin-resistant strains) were tested for susceptibilities to ampicillin, amoxicillin, and amoxicillin-clavulanic acid (A/C) by the broth microdilution method in Haemophilus test medium (HTM) and in Mueller-Hinton medium with lysed horse blood and by the disk diffusion method on HTM agar. Our results support the use of HTM for susceptibility testing of H. influenzae but raise a number of questions regarding the interpretive criteria currently in use, particularly with respect to the fourfold difference in MIC susceptibility breakpoints for ampicillin and A/C and a resulting high proportion of A/C-susceptible beta-lactamase-negative, ampicillin-resistant strains.     

Quantitative antimicrobial susceptibility testing of Haemophilus influenzae and Streptococcus pneumoniae by using the E-test.

The E-test (PDM Epsilometer; AB Biodisk, Solna, Sweden) is an antimicrobial agent gradient-coated plastic test strip which allows MIC determinations on agar media. The test is performed in a manner similar to the agar disk diffusion procedure. A collection of Haemophilus influenzae and Streptococcus pneumoniae strains possessing various resistance mechanisms was used to evaluate the E-test method. H. influenzae strains were tested with both Haemophilus test medium (HTM) and PDM ASM II chocolate agar, while the S. pneumoniae strains were tested on Mueller-Hinton sheep blood agar. E-test MICs for a total of 10 antimicrobial agents were compared with broth microdilution MICs determined according to National Committee for Clinical Laboratory Standards methods. In general, E-test MICs for both species were quickly and easily interpreted and agreed within one log2 MIC increment in 89.8% of tests with H. influenzae and in 80.4% of pneumococcal tests. The majority of disagreements between the E-test and conventional MICs occurred with trimethoprim-sulfamethoxazole because of trailing and diffuse E-test MIC endpoints with both species. Ampicillin MICs for beta-lactamase-producing H. influenzae determined by the E-test differed at times from those determined by conventional testing because of the vagaries of interpreting colonies growing within the E-test inhibition ellipses. E-test penicillin MICs for pneumococci tended to be 1 to 2 log2 dilutions lower than those determined by using Mueller-Hinton broth supplemented with lysed horse blood. Nevertheless, strains of both species with documented resistance to the study drugs were detected by E-tests, i.e., 0.7% of the tests had very major errors with H. influenzae and 0.8% had very major errors with S. pneumoniae. Thus, the E-test represents a potential alternative method for antimicrobial susceptibility testing of these two fastidious bacterial species.     

Detection of penicillin and extended-spectrum cephalosporin resistance among Streptococcus pneumoniae clinical isolates by use of the E test.

Increasing penicillin resistance and the initial recognition of resistance to extended-spectrum cephalosporins among Streptococcus pneumoniae isolates have placed greater emphasis on accurate methods for susceptibility testing of clinical isolates. This study has evaluated the use of the E test (AB Biodisk NA, Piscataway, N.J.) for the detection of penicillin and cefotaxime resistance among 147 pneumococcal clinical isolates in three geographically separate laboratories. These included 42 penicillin-resistant (MIC, > or = 2 micrograms/ml) and 14 cefotaxime-resistant (defined here as an MIC of > or = 2 micrograms/ml) isolates. E test strips were applied to the surface of Mueller-Hinton sheep blood agar plates and incubated at 35 degrees C in 5% CO2 for 20 to 24 h. E test MICs were compared with MICs determined with lysed horse blood-supplemented Mueller-Hinton broth in a microdilution format as recommended by the National Committee for Clinical Laboratory Standards. Penicillin MICs agreed within one log2 dilution for 136 of 147 (92.5%) isolates, and cefotaxime MICs agreed within one log2 dilution for 142 of 147 (96.6%) isolates. No very major or major interpretive errors occurred with either penicillin or cefotaxime E test MIC results. There were 9.5 and 5.4% minor interpretive category errors with penicillin and cefotaxime E test MICs, respectively. These data indicate that the E test represents a convenient and reliable method for the detection of penicillin or cephalosporin resistance in pneumococci.     

Two percent sodium chloride is required for susceptibility testing of staphylococci with oxacillin when using agar-based dilution methods.

The need to add NaCl to agar media to ensure accuracy of results when testing staphylococci with oxacillin was investigated. The results of four antimicrobial susceptibility testing methods (agar and broth dilution, E test, and disk diffusion) in which the growth medium contained 0, 2, 4, or 5% NaCl were compared with the results of a hybridization assay using a mec gene probe. We tested 223 strains of staphylococci, 128 of which were mec gene positive. A total of 7 of the 128 positive strains were coagulase-negative staphylococci with 24-h oxacillin MICs of < or = 2 micrograms/ml. Ninety-five isolates were mec gene negative, including seven strains of Staphylococcus aureus with oxacillin MICs of > or = 4 micrograms/ml. The oxacillin MICs for mec gene-positive, oxacillin-resistant strains of staphylococci increased two- to fourfold with the addition of NaCl to the test medium, while the MICs for mec gene-negative strains did not change in the presence of added salt. Very major error rates for the agar dilution and E test methods in the absence of salt ranged from 18.2 to 20.2%. Major error rates for mec gene-negative S. aureus isolates were > 17% for all test methods when 4 or 5% NaCl was added to the test medium. The addition of 2% NaCl to Mueller-Hinton agar for testing of oxacillin resulted in very major error rates of < 1% for the agar dilution and E test methods although the major error rates for the two methods with added NaCl were 8.5 and 6.9%, respectively. The disk diffusion test did not perform well in this study, showing essential error rates of > or = 18.3%. We recommended the addition of 2% NaC1 to Mueller-Hinton agar when testing staphylococci with oxacillin by either the agar dilution or E test method. NaC1 should not be added for the disk diffusion test.     

Variability of clarithromycin and erythromycin susceptibility tests with Haemophilus influenzae in four different broth media and correlation with the standard disk diffusion test.

Four separate laboratories performed antimicrobial susceptibility tests with 40 Haemophilus influenzae isolates, each tested in triplicate. Erythromycin and a new macrolide, clarithromycin (A-56268; TE-031), were tested by the disk diffusion method, by the agar dilution procedure in two different media, and by broth microdilution tests in four different media. Erythromycin MICs for 90% of the strains were 16 micrograms/ml in Mueller-Hinton broth with 3% lysed horse blood and NAD, 4.0 micrograms/ml in hemophilus test medium, and 2.0 micrograms/ml in supplemented Schaedler broth or in the fastidious broth medium from Beckman Instruments, Inc. Clarithromycin MICs were generally 1 doubling dilution greater than erythromycin MICs in each of the media. Erythromycin disk tests corresponded best with MICs determined in the fastidious broth medium. In that same medium, clarithromycin MICs were about 1 doubling dilution greater than what would be expected from the results of disk tests. Because there were fewer growth failures, hemophilus test medium is recommended for microdilution tests with H. influenzae. Incubation of all tests for a full 24 h without an increased CO2 atmosphere was needed to achieve maximal precision of the tests. Interlaboratory and intralaboratory reproducibility of all tests was satisfactory.     

Use of Haemophilus test medium for broth microdilution antimicrobial susceptibility testing of Streptococcus pneumoniae.

A recently described medium (Haemophilus test medium [HTM]) for antimicrobial susceptibility testing of Haemophilus influenzae was evaluated in this study for broth microdilution testing of Streptococcus pneumoniae. A total of 137 clinical isolates was tested against 11 antimicrobial agents, using Mueller-Hinton broth supplemented with 3% lysed horse blood in parallel with HTM. Inocula of 5 X 10(5) CFU/ml and incubation for 20 to 24 h were used with both media. All isolates of S. pneumoniae produced acceptable growth in both media, and MICs determined in HTM agreed closely with those determined in lysed horse blood. Drugs which provided a MIC within 1 log2 concentration difference in both media included penicillin (100%), ampicillin (98.0%), amoxicillin-clavulanate (100%), ampicillin-sulbactam (100%), cephalexin (98.9%), cefaclor (96.8%), cefuroxime (99.0%), chloramphenicol (96.2%), tetracycline (96.2%), and erythromycin (100%). HTM MICs with trimethoprim-sulfamethoxazole were 1 to 2 log2 concentration increments higher in 92.0% of isolates than MICs determined in lysed horse blood. Based on the results of this study, HTM appears to represent a promising alternative medium for broth microdilution susceptibility testing of S. pneumoniae.     

Effects of various test media on the activities of 21 antimicrobial agents against Haemophilus influenzae

As considerable variation in the antimicrobial susceptibility of Haemophilus influenzae has been reported, the effects of various test media on the susceptibility of H. influenzae were studied. MICs were determined by three laboratories for 21 antimicrobial agents against a panel of 100 selected isolates. Testing was performed using a reference NCCLS frozen broth microdilution method with Haemophilus test medium (HTM) broth and dried commercial MIC trays rehydrated with the following media: in-house and commercially prepared HTM broth, Mueller-Hinton broth with 2% lysed horse blood and NAD, IsoSensitest broth with 2% lysed horse blood and NAD, and IsoSensitest broth-based HTM. Overall, all results were very reproducible, with the MIC at which 50% of the isolates tested are inhibited (MIC(50)), MIC(90), and geometric mean MIC being within one doubling dilution by all six methods and at all three testing centers for 15 of the 21 agents tested. Interlaboratory differences were more marked than intralaboratory differences or differences among media. Cefprozil, cefaclor, and trimethoprim-sulfamethoxazole results differed the most, while results for ampicillin, amoxicillin-clavulanic acid, cefdinir, cefixime, ceftriaxone, and clarithromycin were the most reproducible. However, these variations in results caused considerable differences in susceptibility rates for agents for which NCCLS susceptible breakpoints were close to the geometric mean MIC, particularly for cefaclor and cefprozil. This was much less of a problem when pharmacokinetic-pharmacodynamic breakpoints were used. Reproducible susceptibility results were obtained for a wide range of agents against H. influenzae in three laboratories using a variety of media that support the growth of this fastidious species.     

Comparative evaluation of the E test, agar dilution, and broth microdilution for testing susceptibilities of Helicobacter pylori strains to 20 antimicrobial agents.

The Epsilometer test (E test; AB Biodisk, Solna, Sweden), a new quantitative technique for the determination of antimicrobial susceptibility, was compared to reference methods (agar dilution and broth microdilution) for the antimicrobial susceptibility testing of Helicobacter pylori. Seventy-one H. pylori strains isolated from patients with duodenal ulcers were tested against 20 antimicrobial agents. The E test and the agar dilution method were carried out on Mueller-Hinton agar; the broth microdilution method was performed with Mueller-Hinton broth. The E-test results showed excellent correlation with the agar dilution results, with 91.3 and 98.8% agreement within 1 and 2 log2 dilution steps, respectively, in a total of 1,350 tests. The correlation between the E-test results and the broth microdilution results was slightly higher, with 91.6 and 99.1% agreement within 1 and 2 log2 dilution steps, respectively, in a total of 1,317 tests. There were six major errors and two very major errors by the metronidazole E test compared to the results obtained by reference methods. Excellent agreement between E-test, agar dilution, and broth microdilution results was found for resistance to erythromycin (8%), clarithromycin (6%), and tetracycline (6%). Our results confirm that the E test is comparable to standardized methods for susceptibility testing. Therefore, the E test is a reliable and alternative method for testing H. pylori susceptibility to a wide range of antimicrobial agents in clinical practice.     

Determination of Trimethoprim-Sulfamethoxazole Resistance in Streptococcus pneumoniae by Using the E Test with Mueller-Hinton Agar Supplemented with Sheep or Horse Blood May Be Unreliable

An international, multicenter study compared trimethoprim-sulfamethoxazole MICs for 743 Streptococcus pneumoniae isolates (107 to 244 isolates per country) by E test, using Mueller-Hinton agar supplemented with 5% defibrinated horse blood or 5% defibrinated sheep blood, with MICs determined by the National Committee for Clinical Laboratory Standards broth microdilution reference method. Agreement within 1 log₂ dilution and minor error rates were 69.3 and 15.5%, respectively, on sheep blood-supplemented agar and 76.9 and 13.6%, respectively, with horse blood as the supplement. Significant interlaboratory variability was observed. E test may not be a reliable method for determining the resistance of pneumococci to trimethoprim-sulfamethoxazole.     

Haemophilus test medium versus Mueller-Hinton broth with lysed horse blood for antimicrobial susceptibility testing of four bacterial species

Studies were undertaken to determine whether broth microdilution susceptibility tests could be standardized by using a single medium for testing fastidious respiratory pathogens. Mueller-Hinton broth with lysed horse blood and the broth version of Haemophilus Test Medium (HTM) were directly compared. Ten orally administered agents were found to give essentially identical results in both media but minor differences were noted. Because the tests are easier to read when HTM broth is used, that medium is to be preferred for routine testing ofHaemophilus influenzae, Streptococcus pneumoniae, Streptococcus pyogenes andMoraxella catarrhalis isolates by the microdilution procedure.     

Quantitative antimicrobial susceptibility test for Streptococcus pneumoniae using inoculum supplemented with whole defibrinated sheep blood.

The National Committee for Clinical Laboratory Standards recommends the use of lysed horse blood-supplemented Mueller-Hinton broth for determining the quantitative antimicrobial susceptibility of Streptococcus pneumoniae. This procedure may be difficult for laboratories using previously prepared or commercial MIC systems. Therefore, a study was undertaken to determine whether previously prepared microdilution trays containing Mueller-Hinton broth without blood could be used for determining the antimicrobial susceptibility of S. pneumoniae by adding whole defibrinated sheep blood to the bacterial suspension used to inoculate the trays. The presence of alpha-hemolysis was used as an indicator of bacterial growth. One hundred isolates of S. pneumoniae selected to represent a distribution of susceptibility patterns were tested by the National Committee for Clinical Laboratory Standards method and the sheep blood-supplemented-inoculum method. Greater than 94% agreement between the two methods was achieved. The sheep-blood-supplemented-inoculum procedure was highly reproducible and easy to perform and provides an acceptable alternative for determining the MICs for S. pneumoniae for laboratories using previously prepared or commercial microdilution systems.     

Standardization of Broth Microdilution and Disk Diffusion Susceptibility Tests for Actinobacillus pleuropneumoniae and Haemophilus somnus: Quality Control Standards for Ceftiofur, Enrofloxacin, Florfenicol, Gentamicin, Penicillin, Tetracycline, Tilmicosin, and Trimethoprim-Sulfamethoxazole

Quality control (QC) standards for the in vitro antimicrobial susceptibility testing of two fastidious veterinary pathogens, Actinobacillus pleuropneumoniae and Haemophilus somnus, were developed in a multilaboratory study according to procedures established by the National Committee for Clinical Laboratory Standards for broth microdilution and disk diffusion testing. The medium recommended for the broth microdilution testing is cation-adjusted Mueller-Hinton broth supplemented with 2% lysed horse blood, 2% yeast extract, and 2% supplement C. This medium has been designated veterinary fastidious medium. The medium recommended for the disk diffusion testing is chocolate Mueller-Hinton agar. The recommended QC organisms are A. pleuropneumoniae ATCC 27090 and H. somnus ATCC 700025. The QC MICs of ceftiofur, enrofloxacin, florfenicol, gentamicin, penicillin, tetracycline, tilmicosin, and trimethoprim-sulfamethoxazole were determined for each isolate, as were the zone size ranges. Of the results from the participating laboratories, 94.0% of the zone diameter results and 97.0% of the MIC results fell within the suggested QC ranges for all compounds. These QC guidelines should allow greater accuracy in interpreting results when testing these antimicrobial agents against fastidious pathogens.     

A simplified method for testing Bordetella pertussis for resistance to erythromycin and other antimicrobial agents

Present methods of antimicrobial susceptibility testing of Bordetella pertussis are time consuming and require specialized media that are not commercially available. We tested 52 isolates of B. pertussis for resistance to erythromycin, trimethoprim-sulfamethoxazole, chloramphenicol, and rifampin by agar dilution with Bordet-Gengou agar (BGA) containing 20% horse blood (reference method), Etest using BGA and Regan-Lowe agar without cephalexin (RL-C), and disk diffusion using BGA and RL-C. The organisms tested included four erythromycin-resistant isolates of B. pertussis from a single patient, a second erythromycin-resistant strain of B. pertussis from an unrelated patient in another state, and 47 nasopharyngeal surveillance isolates of B. pertussis from children in the western United States. The results of agar dilution testing using direct inoculation of the organisms suspended in Mueller-Hinton broth were within +/-1 dilution of those obtained after overnight passage of the inoculum in Stainer-Scholte medium, which is the traditional method of testing B. pertussis. The Etest method produced MICs similar to those of the agar dilution reference method for three of the four antimicrobial agents tested; the trimethoprim-sulfamethoxazole results were lower with Etest, particularly when the direct suspension method was used. Most of the Etest MICs, except for that of erythromycin, were on scale. Disk diffusion testing using RL-C medium was helpful in identifying the erythromycin-resistant strains, which produced no zone of inhibition around the disk; susceptible isolates produced zones of at least 42 mm. Thus, the antimicrobial susceptibility testing of B. pertussis can be simplified by using the Etest or disk diffusion on RL-C to screen for erythromycin-resistant isolates of B. pertussis.     

Susceptibility of Neisseria meningitidis to 16 antimicrobial agents and characterization of resistance mechanisms affecting some agents

Neisseria meningitidis represents a pathogen of great public health importance in both developed and developing countries. Resistance to some antimicrobial agents used either for therapy of invasive infections or for prophylaxis of case contacts has long been recognized, although specific guidelines for susceptibility testing have not been fully developed. We have examined the susceptibilities of a collection of 442 meningococcal clinical isolates from 15 countries to 16 antimicrobial agents. These included isolates recovered between 1917 and 2004, with representatives of all major serogroups. All isolates were tested by the Clinical and Laboratory Standards Institute (formerly NCCLS) broth microdilution method using Mueller-Hinton lysed horse blood broth, while a subset of 102 isolates was tested by agar dilution using Mueller-Hinton sheep blood agar. Most isolates provided adequate growth for MIC determinations by both broth and agar methods. Growth in broth was enhanced by CO(2) incubation and was required for two strains (1.7%). MICs of the study drugs compared favorably between the broth and agar methods (79 to 100% essential agreement), and MICs also generally agreed closely (92 to 100% essential agreement, excluding azithromycin) between broth tests incubated in the two different atmospheres. Elevated penicillin and ampicillin MICs (> or =0.12 microg/ml and > or =0.25 microg/ml, respectively) occurred in 14.3% and 8.6% of strains and were associated with polymorphisms of the penA gene encoding a modified penicillin-binding protein 2. None of the 442 isolates produced beta-lactamase. Elevated tetracycline and doxycycline (but not minocycline) MICs were associated with efflux-mediated resistance encoded by tet(B) in 13 strains. Resistance to sulfisoxazole in 21.7% of strains and to trimethoprim-sulfamethoxazole in 21.0% resulted from polymorphisms of folP encoding a modified dihydropteroate synthetase. Seven strains were resistant to rifampin due to mutations in the rpoB gene, and two strains were resistant to chloramphenicol due to production of chloramphenicol acetyltransferase mediated by catP. Two strains had reduced quinolone susceptibility due to mutations of gyrA. The determination of the susceptibilities of a large group of meningococcal strains (including strains with characterized resistance mechanisms) to 16 antimicrobial agents has served as the essential first step in defining susceptibility testing breakpoints specific for this organism.     

Standardized disk-diffusion susceptibility test for Haemophilus influenzae.

The emergence of ampicillin-resistant strains of Haemophilus influenzae has emphasized the need for an improved practical method for routine susceptibility testing of clinical isolates. We have previously described a simplified medium for quantitative dilution susceptibility testing that is composed of Mueller-Hinton medium plus Supplement C (Difco). In the present study, paired broth-dilution and disk-diffusion susceptibility tests with ampicillin and chloramphenicol were performed on 100 strains of Haemophilus (95 H. influenzae and five H. parainfluenzae), including 30 strains with previously documented ampicillin resistance. Disk-diffusion tests were performed in exactly the same manner as the standardized Kirby-Bauer procedure used for less fastidious organisms, except that supplemented Mueller-Hinton agar plates were incubated in an increased-CO2 atmosphere. Using this method, ampicillin-susceptible strains of Haemophilus produced zone diameters of 22 mm or more, while ampicillin-resistant strains produced zones of 18 mm or less. All strains were chloramphenicol-susceptible and produced zone diameters of 30 mm or more. This method would allow routine disk-diffusion testing of isolates of H. influenzae by hospital diagnostic laboratories, using a clear medium that closely resembles unsupplemented Mueller-Hinton agar.     

Aerococcus urinae and trimethoprim-sulfamethoxazole

Aerococcus urinae has been described as resistant to trimethoprim-sulfamethoxazole (SXT), but the test medium may affect this observation. Twenty-seven clinical isolates of A. urinae tested susceptible to SXT in cation-adjusted Mueller-Hinton broth (CAMHB) plus lysed horse blood and resistant in CAMHB plus lysed sheep blood.     

Development of interpretive criteria and quality control limits for broth microdilution and disk diffusion antimicrobial susceptibility testing of Streptococcus pneumoniae.

A five-center collaborative study was undertaken to develop quality control and specific interpretive criteria for susceptibility testing of Streptococcus pneumoniae against 12 antimicrobial agents. MICs were determined for 248 pneumococcal clinical isolates (with an emphasis on resistant strains) by use of the National Committee for Clinical Laboratory Standards (NCCLS)-recommended broth microdilution procedure incorporating lysed horse blood-supplemented Mueller-Hinton broth. NCCLS disk diffusion testing was also performed for each isolate by using Mueller-Hinton sheep blood agar incubated in 5% CO2. Repetitive testing of S. pneumoniae ATCC 49619 with different sources and lots of media and disks allowed development of quality control ranges which encompassed approximately 95% of MIC and zone size values observed in the study. Good intra- and interlaboratory reproducibilities were seen with these testing methods and all of the drugs examined. On the basis of the results of this study, MIC interpretive criteria are proposed for 11 agents. Comparisons of MICs and disk diffusion zone sizes allowed disk diffusion zone size interpretive criteria to be proposed for five drugs and confirmed the use of the oxacillin disk test for prediction of penicillin susceptibility among pneumococci. Excessive numbers of minor-category interpretive errors precludes recommendation at this time of the disk diffusion method for testing of pneumococci against five of the drugs. Use of these proposed quality control and interpretive criteria should provide for reproducible test results and allow recognition of recently emerging resistance among pneumococcal clinical isolates.     

Evaluation of a proprietary broth medium for microdilution susceptibility testing of nutritionally fastidious bacteria.

For microdilution susceptibility tests with nutritionally fastidious microorganisms, a new clear broth medium developed at Micro-Media Systems, Inc., Potomac, Md., was evaluated in a three-laboratory collaborative study. Replicate tests were performed with 80 isolates (51 Streptococcus spp., 27 Haemophilus influenzae isolates, and 2 Neisseria meningitidis isolates) against 15 antimicrobial agents. In standard 100-microliters volumes, results of tests in the new broth medium were comparable to those in the reference medium (Mueller-Hinton broth with 2 to 3% lysed horse blood), but MICs were somewhat easier to read in the new broth medium. Results of similar tests in smaller panels, containing 40 microliters in each well, were less satisfactory; i.e., growth failures and poorly defined endpoints were more commonly encountered. With drugs other than erythromycin or clindamycin, the 40-microliters panels provided MICs which compared favorably with those obtained by standard reference methods.