Welch D F, Guruswamy A P, Sides S J, Shaw C H, Gilchrist M J
Clinical Microbiology Laboratories, University of Oklahoma Health Science Center, Oklahoma City 73126.
J Clin Microbiol. 1993 Aug;31(8):2178-84. doi: 10.1128/jcm.31.8.2178-2184.1993.
For the isolation of mycobacteria from clinical specimens, we evaluated a method that used a thinly poured Middlebrook 7H11 agar plate (10 by 90 mm) that was examined microscopically. Inoculated plates were sealed, incubated, and examined at regular intervals for the appearance of microcolonies. Plates were examined microscopically, while still sealed, by focusing on the agar surface through the bottom of the plate and 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 method that used macroscopic examination of standard mycobacterial media. By using all specimens submitted for mycobacterial culture over the duration of the study, the method was evaluated until 270 isolates of mycobacteria (Mycobacterium tuberculosis, n = 103; M. avium-M. intracellulare, n = 115; miscellaneous, n = 52) were detected. While the conventional method required an average of 23 days to the time of first detection of mycobacteria, the experimental method required an average of only 11 days. When limited to acid-fast stain-positive specimens that were culture positive for M. tuberculosis, the average interval to positivity was 7 days for the microcolony method compared with 17 days for the conventional method. With the experimental method, the microscopic colonial morphology allowed for the presumptive identification of M. tuberculosis colonies, which were distinguished by cording, and M. avium-M. intracellulare colonies, which were smooth and entire. Presumptive identification was complete for 83.5% of the M. tuberculosis isolates within 10 days and for 85% of the M. avium-M. intracellulare isolates within 11 days after inoculation. If the microcolony method was combined with a conventional tube medium, the composite would optimize for speed of recovery while providing the full sensitivity of the conventional method. In addition to reducing the interval to positivity, the microcolony method allows for the easy detection of mixed mycobacterial infections and yields a presumptive identification that facilitates the selection of a confirmatory gene probe test.
为了从临床标本中分离分枝杆菌，我们评估了一种方法，该方法使用薄倾注的Middlebrook 7H11琼脂平板（10×90毫米），并进行显微镜检查。接种后的平板密封、培养，并定期检查微菌落的出现情况。平板仍密封时，通过平板底部和琼脂聚焦于琼脂表面进行显微镜检查。平板先用低倍镜（总放大倍数为40倍）扫描，然后用中倍镜（放大倍数为100至180倍）确认菌落形态。将该方法与使用标准分枝杆菌培养基进行宏观检查的传统方法进行比较。在研究期间，通过使用所有提交进行分枝杆菌培养的标本，对该方法进行评估，直至检测到270株分枝杆菌（结核分枝杆菌，n = 103；鸟分枝杆菌-胞内分枝杆菌，n = 115；其他，n = 52）。传统方法首次检测到分枝杆菌平均需要23天，而实验方法平均仅需11天。当仅限于结核分枝杆菌培养阳性的抗酸染色阳性标本时，微菌落法阳性的平均间隔时间为7天，而传统方法为17天。采用实验方法，显微镜下的菌落形态有助于对结核分枝杆菌菌落进行初步鉴定，其特征为索状，以及鸟分枝杆菌-胞内分枝杆菌菌落，其菌落光滑完整。接种后10天内，83.5%的结核分枝杆菌分离株和11天内85%的鸟分枝杆菌-胞内分枝杆菌分离株完成了初步鉴定。如果将微菌落法与传统的试管培养基相结合，这种组合将在提高恢复速度的同时，提供传统方法的全部灵敏度。除了缩短阳性间隔时间外，微菌落法还便于检测混合分枝杆菌感染，并产生初步鉴定结果，有助于选择确证性基因探针检测。
