TIMELY CULTURE FOR MYCOBACTERIA WHICH UTILIZES A MICROCOLONY METHOD

For the isolation of mycobacteria from clinical specimens, we evaluate d a method that used a thinly poured Middlebrook 7H11 agar plate (10 b y 90 mm) that was examined microscopically. Inoculated plates were sea led, incubated, and examined at regular intervals for the appearance o f microcolonies. Plates were examined microscopically, while still sea led, by focusing on the agar surface through the bottom of the plate a nd the agar. Plates were scanned at low power (x40 total magnification ), and colony morphology was confirmed at intermediate power (x100 to x180 magnification). This method was compared with a traditional metho d that used macroscopic examination of standard mycobacterial media. B y using all specimens submitted for mycobacterial culture over the dur ation of the study, the method was evaluated until 270 isolates of myc obacteria (Mycobacterium tuberculosis, n = 103; M. avium-M. intracellu lare, n = 115; miscellaneous, n = 52) were detected. While the convent ional method required an average of 23 days to the time of first detec tion of mycobacteria, the experimental method required an average of o nly 11 days. When limited to acid-fast stain-positive specimens that w ere culture positive for M. tuberculosis, the average interval to posi tivity was 7 days for the microcolony method compared with 17 days for the conventional method. With the experimental method, the microscopi c colonial morphology allowed for the presumptive identification of M. tuberculosis colonies, which were distinguished by cording, and M. av ium-M. intracellulare colonies, which were smooth and entire. Presumpt ive identification was complete for 83.5% of the M. tuberculosis isola tes within 10 days and for 85% of the M. avium-M. intracellulare isola tes within 11 days after inoculation. If the microcolony method was co mbined with a conventional tube medium, the composite would optimize f or speed of recovery while providing the full sensitivity of the conve ntional method. In addition to reducing the interval to positivity, th e microcolony method allows for the easy detection of mixed mycobacter ial infections and yields a presumptive identification that facilitate s the selection of a confirmatory gene probe test.