A variety of different media for the cultivation of mycobacteria have been described but a few of them are in use today. Those currently used can be characterized by three basic types. The first is egg-based media represented by Ogawa and LOwenstein-Jensen. The second type is agar-based media the most common one are Middlebrook 7H10 and 7H11. The third type is liquid media such as Middlebrook 7H9.
Several weeks of incubation may be required for the isolation of M. tuberculosis on solid media. Substantial improvement in the time to detection and the recovery rate was realized by using broth-based culture system such as the BACTEC 460TB, Septi-Chek AFB, MGIT and BACTEC 9000.
In the BACTEC 460TB system, the mycobacteria is detected radiometrically. The processed specimen is added to a modified 7H9 medium (BACTEC 12B) containing ¹⁴ C-labeled palmitic acid and an antibiotic complex, PANTA. Mycobacterial growth can be ascertained by the liberation of ¹⁴ CO₂ and detected by BACTEC 460TB instrument.
The Septi-Chek AFB is a biphasic medium which combines broth and solid media. The liquidmedium is a modified Middlebrook 7H9 in a carbon-dioxide-enriched culture bottle. After inoculation of the sample, the bottle is capped with a slide consisting of three solid media a non-selective Middlebrook 7H11 agar, an egg-based medium, and chocolate agar.
A novel system is the MGIT, which is a nonradiometric broth method for the detection of mycobacteria from clinical specimens. The MGIT consists of a modified Middlebrook 7H9 broth and a sensor embedded in silicone on the bottom of a tube. The appearance of orange-colored fluorescence in the sensor when excited indicates the growth of mycobacteria.
MB Redox is a modified, serum-supplemented Kirchner medium containing p-indonitrotetrazolium violet (INT) as an indicator of microbial growth. The INT is reduced by the redox system of the mycobacteria to deep violet-colored formazan. This substance is water insoluble and is reduced to the cell surface, by which bacterial clamps can be easily detected by their violet color.
At present, the egg-based media are the first choice for the culture of clinical samples. However, there are advantages to each type of medium and not all strains of mycobacteria can be recovered on a single medium. Therefore, it is recommended that one representative of each type of medium be used for primary isolation ; one example in Japan may be Ogawa egg medium in combination with Middlebrook 7H11 and MGIT.

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An intratracheal infection method of experimental murine mycobacteriosis was developed for an in vivo study of antimycobacterial agents.
Two models of intratracheal (IT) and intravenous (IV) routes of infection with mycobacteria of the same inoculum dose were compared in terms of the mean survival days of mice or bacterial loads in organs during the course of infection. IT model with either of M. bovis Ravenel, M. tuberculosis Kurono, M. tuberculosis H37Rv or M. intracellulare N-256 exhibited a much more distinct lung-specific infection than IV model with the same dose of respective mycobacterial strains.
The intratracheal infection method presented in this report dose not require any special equipment and is a much safer method for the researcher than airborne infection. In this model, following slight anesthetizing of mice, bacillary suspension was injected quantitatively into lungs through the mouth and trachea by using a specially modified needle set with a short fine polyethylene tube.
This IT model may be useful not only for the in vivo assessment of anti-mycobacterial agents but also for the comparison of virulence among various mycobacterial strains.

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This study was carried out to clarify the diagnostic usefulness of the skin tests of purified protein derivatives from M.intracellulare (PPD-B) and M. tuberculosis (PPDs). Study subjects consisted of 41 patients with primary infection type of M. avium complex (MAC) disease, 36 patients with pulmonary tuberculosis (TB) and 29 patients with other bacterial respiratory infections including COPD (OB). The patients were sorted out to middle (50-69 y.o.) and old (70-89 y.o.) age groups of each disease. The size of skin redness elicited 48 hours after the PPD-B and PPDs intradermal injections were compared among them.
The results were as follows.
1) Both PPD-B and PPDs skin reactions were larger in the middle age group than in the old one for each disease.
2) In PPDs skin tests, the reaction of TB group was the largest among the three diseases. in PPD-B skin tests, that of MAC was the largest.
3) In TB group, PPDs skin reaction was significantly larger than that of PPD-B, while in MAC and OB groups there was no significant difference between the skin reactions of PPDs and PPD-B.
4) Defining significant positive reaction to PPD-B as PPD-B skin reaction exceeding 10mm and larger than that of PPDs skin reaction, the rate of significant positive reaction to PPD-B was significantly higher in MAC than TB in both age groups.
These results showed that the simultaneous skin tests of PPD-B and PPDs were a usefulaid in the diagnosis of MAC infection disease when mycobacterial infection diseases were clinically suspected by bacteriological or chest readiographic examinations.

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In the past decade, the non-tuberculous mycobacterial (NTM) infection was a rare disease but now is the common and increasing pulmonary infection in Japan. It is suspected that about 16% of patients with positive AFB in their sputa are the NTM patients. It is well known that disseminated M. avium complex infection is an important complication of HIV disease. It will be worth to summarize some basic and clinical topics of this infectious disease in this symposium.
The profiles of the antimicrobial action of microbicidal effector molecules against Mycobacterium avium complex (MAC) were presented by Dr. Tomioka, who reported the collaborating effect of reactive nitrogen intermediates with free fatty acids is crucial for macrophage-mediated intracellular killing of AFB. In addition, the auther discussed some important roles of type II alveolar epithelial cells as a target cell for primary invasion of AFB in the host lungs.
Dr. Koga reviewed the clinical usefullness of Amplicor, one of commercially available PCR detection kits for MAC. The MAC was detected in clinical samples such as sputum, bronchial washing fluids and gastric juice with 5 to 10% in frequency. The 60% to 80% of MAC positive cases were finally diagnosed as MAC disease and treated. He recommended that the positive result of genetic diagnostic method such as Amplicor should be carefully considered for clinical use.
Dr. Kurashima summarized the short term effects of chemotherapy for pulmonary MAC infection. The auther analysed the 170 MAC cases treated with multi-drug chemotherapies which had not changed for 6 months or more. At 2 months after starting chemotherapy, 57.1% of patients obtained negative conversion but at 6 months only 28.9% remained bacilli negative. He recommended the regimen with combination of more than 3 drugs including aminoglycosid and clarithromycin.
Two authers supplemented additional comments. Dr. Shigeto reported the long-term prognosis of MAC disease, indicating 64.7% obtained negative conversion and 35.5% remained bacteriologically negative in 71 patients who treated with at least 3 drugs and for more than 12 months. In addition, in the 12 patients who underwent surgical therapy, only one patient deteriorated.
Dr. Kawahara precisely reported the basic preclinical evaluation of promising new drugs including new quinolones and new macrolides and Rifamycins. He also reviewed the clinical impact of these drugs.
The present status of HIV infection and NTM in Japan was presented by Dr. Mizutani. All of 32 reported cases are male, in which 23 were with MAC and 6 were with M. kansasii disease. The 3 cases in 4 without bacteremia were patients with M. kansasii. The Mean count of CD 4+ lymphocytes in the peripheral bloods of these 32 patients was only 11/mm³.
As additional comments, the geographical distribution in environment and serovars of MAC were reported by Dr. Saito. It was not found that the difference in the geographic distribution between soil MAC and disease-associated MAC. The 55% of soil MAC reacted with the AccuProbe MAC but not with the AccuProbe M. avium and M. intracellulare. The auther also demonstrated that 38 MAC strains isolated from 38 Japanese AIDS patients were all M. avium and serovars of Japanese MAC from AIDS patients are similar to those of the American MAC strains from such patients.

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