The concentration, microculture, and sensitivity testing of M. tuberculosis.

The Tuberculosis Chemotherapy Trials Committee, in the summary of their second report to the Medical Research Council (1953), state that "there is a great need for simple, reliable, and speedy techniques for the culture of tubercle bacilli and for the determination of their sensitivity to chemotherapeutic agents." Since Pryce (1941) introduced the method of culturing M. tuberculosis in smears of sputum on glass slides, in a liquid medium, many workers have reported their success with a variety of technical modifications. Rubbo and Morris (1951) applied the microculture technique to streptomycin sensitivity tests. The great advantage of microculture is the speed with which the growth of the organisms can be detected. Particularly in general hospital practice, the possibility of a diagnostic or sensitivity report in two to five days instead of in two to five weeks is a great advance. We set out to investigate the reliability of the method. The need is for a technique which is simple, reliable, and relatively inexpensive, equally good for concentrates of urine, pus, etc., as for direct smears of sputum. Most published reports on microculture are restricted to work on direct smears of sputum, which are notoriously easy to handle and which give a high proportion of successes. After trying the trisodium phosphate, sulphuric acid, and Jungmann's methods of concentration we had, by 1951, gone back to Petroff's technique, which in our hands gave the best results (cf. Medical Research Council, 1952). But, especially in the early stages, we found difficulty with the Petroff concentrates in the micromethod, as the smears tend to wash off the cover-glasses on which, for economy and convenience, we make our preparations. One of us had, some years ago, experimented with enzymes, and had found pancreatin to be a very useful concentrating agent, superior to pepsin, trypsin, and papain. A series of cultures using pancreatin and Petroff concentrates in parallel was therefore made. This has yielded markedly better results with pancreatin. Growth on LoewensteinJensen medium has been both more rapid and more profuse-presumably largely because more viable organisms are inoculated (Tab'es I and II). Pancreatin has also been found to give excellent results in the microculture method. Smears of concentrates from sputum, pus, urine, etc., readily adhere to the cover-slips. Our practice has been to prepare smears for microculture and to make Loewenstein-Jensen cultures from the same concentrate. In this way we have been able to compare the efficiency of microand macro-culture.