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The rate of recovery and time to the detection of mycobacteria from clinical specimens were measured for biphasic (MB-Check; Nippon Roche Co., Ltd., Tokyo, Japan) and radiometric (BACTEC; Nippon Becton Dickinson Co., Ltd., Tokyo, Japan) liquid-based culture systems and egg-based media (3% Ogawa and Ogawa K). From the 245 sputum specimens processed, a total of 86 (35.1%) mycobacterial isolates were detected. Of these, 81 (94.2%) and 80 (93.01%) isolates were detected with the MB-Check and BACTEC systems, respectively, and 65 (75.6%) isolates were detected with the 3% Ogawa egg method. The difference in the percentages of positive cultures between the two systems based on liquid media and the 3% Ogawa egg method was significant (P < 0.01). This difference was even greater among smear-negative specimens. The detection time was shorter with the liquid-based systems. The mean times to the detection of the Mycobacterium tuberculosis complex were 19.1 days with the MB-Check system, 13.4 days with the BACTEC system, and 21.7 days with the 3% Ogawa egg method. These results indicate that both the MB-Check and the BACTEC systems, based on liquid media, are efficient for the recovery of mycobacteria.

Restriction fragment length polymorphism (RFLP) analysis of a large number of Japanese isolates of Mycobacterium tuberculosis, containing isolates from small outbreaks of M. tuberculosis infection, and clinical isolates of M. bovis BCG, was carried out using a DNA probe derived from the insertion sequence IS986. Clinical isolates of M. tuberculosis had a high degree of RFLP. The occurrences of the IS element varied from 1 to 19, the majority of isolates having 8 to 15 copies. Very similar fingerprints, however, were seen among strains isolated in the Kanto district. In particular, 3 strains were of the same pattern with or without an additional band. Similarity of the banding patterns of strains islated in the same district was observed in other areas. Six groups of strains, each group arising from a suspected common source of infection, were analyzed. Of these, 5 showed identical fingerprints within each group, but one showed different fingerprints. RFLP patterns of three strains isolated from individuals with lymphadenitis developed about two months after BCG vaccination, and one strain isolated from a bladder cancer patient with BCG instillation therapy were identical to those of BCG-Tokyo which had been used for the vaccination and therapy. These results confirm that RFLP analysis using IS986 is a suitable tool for epidemiology of tuberculosis.

Peritoneal exudate cells of mice were stimulated with a streptomycin-dependent Mycobacterium tuberculosis strain, 18b. Gamma interferon production by natural killer cells depending on interleukin-12 and interleukin-18 was induced only in the presence of a high dose of streptomycin. This study suggested the requirement of active bacterial metabolism for this host response.Mycobacterium tuberculosis is a causative agent of tuberculosis in human being. One-third of the world's population is currently infected with tubercle bacilli, and the global incidence of active tuberculosis per year is around 8 million cases (2). It is of great importance to understand the basic mechanism for the induction of the immune response of the host against M. tuberculosis. The generation of protective immunity against tuberculosis is dependent on the induction of CD4 ϩ Th1 cells capable of producing gamma interferon (IFN-␥) upon stimulation with specific antigen. IFN-␥ contributes to the development of acquired resistance via the activation of macrophages (3). The importance of IFN-␥ in the resistance of mice to M. tuberculosis has been confirmed by utilizing IFN-␥ knockout mice and IFN-␥ receptor knockout mice (1, 4, 11). IFN-␥ is crucial also for the development of protective T cells. In our previous study, the treatment of mice with anti-IFN-␥ antibody during primary immunization with viable cells of M. bovis bacillus Calmette-Guérin reduced the number of antigen-specific IFN-␥-producing cells and abolished the generation of protective immunity (26). Thus, IFN-␥ is indispensable for both induction and expression of protective immunity against tuberculosis.It has been shown that CD4 ϩ protective T cells are generated after infection with a sublethal dose of M. tuberculosis or M. bovis bacillus Calmette-Guérin, whereas such effector T cells are hardly induced by immunization with killed bacteria (14). We have found that the failure of killed bacteria to induce effective protective immunity in mice is due to the absence of an IFN-␥-inducing ability that is observed exclusively in viable bacilli (25,26). Killed M. tuberculosis has been prepared generally by treatment with heating, germicides, or irradiation (7,16,20,24), but such treatment may affect several bacterial components physically or chemically. In order to address whether the significant difference in the IFN-␥-inducing abilities of viable and killed M. tuberculosis is due to some undesirable changes introduced during the killing process or actually due to the viability itself, we have employed a streptomycin (SM)-dependent M. tuberculosis strain, 18b, in this study.This particular strain, originally isolated in 1955 (6), has been maintained as a stock for a long time at the National Institute of Infectious Diseases in Japan, so we first confirmed whether this strain maintained the original SM dependency. On a Middlebrook 7H10 agar plate, strain 18b never grew during 5 weeks of observation in the absence of SM (Fig. 1). However, the addition of SM at concentrations of 50 g/ml...