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We demonstrated previously that susceptibility testing of Mycobacterium tuberculosis could be accomplished within 24 h after the organisms were incubated with antituberculosis agents by using fluorescein diacetate (FDA) staining and flow cytometry. Continued studies have now shown that assay suspensions containing M. avium, M. fortuitum, M. gordonae, or M. marinum incubated with various concentrations of ciprofloxacin, clarithromycin, erythromycin, kanamycin, rifampin, tobramycin hydrolyzed less FDA than drug-free controls. Suspensions of treated and nontreated mycobacteria could be easily differentiated at 6 and 24 h after the initiation of the susceptibility assays by using FDA staining and flow cytometry. In addition, multiplication of the mycobacteria was not required to discern differences between drug-free suspensions of mycobacteria and those treated with antimycobacterial agents. The flow cytometric assay is simple, reproducible, and rapid.

The resurgence of tuberculosis has caused considerable effort to be focused on the development of rapid methods for determining the susceptibility of Mycobacterium tuberculosis to antimycobacterial agents. We demonstrated that susceptibility testing of M. tuberculosis can be accomplished rapidly by using flow cytometry. Results of tests were available within 24 h after M. tuberculosis organisms were incubated with ethambutol, isoniazid, rifampin, or streptomycin. The method was based on the ability of viable M. tuberculosis organisms to hydrolyze fluorescein diacetate (FDA) and the detection of fluorescent mycobacteria by flow cytometric analysis. The assay system also did not require multiplication of the mycobacteria. In contrast, M. tuberculosis organisms exposed to antimycobacterial agents hydrolyzed significantly less FDA. The use of flow cytometry and FDA staining shows considerable promise as a rapid method for obtaining susceptibility test results.

The reliability of the 10-micrograms clindamycin disk was evaluated for susceptibility testing of anaerobes by the aerobic thioglycolate broth disk method. A good correlation between the aerobic thioglycolate broth disk method and the reference agar dilution procedure of the National Committee for Clinical Laboratory Standards was obtained by using a 4-microgram/ml breakpoint. Improved correlation was obtained when the medium of the National Committee for Clinical Laboratory Standards was buffered.

We evaluated the aerobic thioglycolate broth disk and the vaspar overlay broth disk methods for antibiotic susceptibility testing of 144 strains of anaerobes. For penicillin, carbenicillin, chloramphenicol, and metrionidazale, both broth disk methods yielded at least 95% agreement with results obtained by the National Committee for Clinical Laboratory Standards reference agar dilution procedure. For cefoxitin and clindamycin, the agreement was ca. 90%. Overall, the aerobic thioglycolate broth disk and vaspar overlay broth disk methods yielded agreements of 93.3 and 93%, respectively, with the National Committee for Clinical Laboratory Standards method.

The primary plate bacitracin differentiation disk susceptibility test identified 85% of group A streptococci from throat cultures on SXT-BA(CO2) plates within 24 h, as compared to only 26% on a conventional sheep blood agar plate.