violet-colored formazan. This formazan is water-insoluble and secreted at the cell surface in a granular form. As M. tuberculosis and some other mycobacteria usually grow in a serum-supplemented medium in the form of macroscopically visible cell particles, these may be easily detected by the naked eye according to the basis of their formazan color. At high cell concentrations, formazan-stained, as well as unstained white particles are visible. The staining of the particles is stable for 2 weeks, after which a decolorization may occur.

　

1-6.　Evaluation of the MGIT system

Two systems, the newly developed MGIT and biphasic Septi-Chek AFB based on liquid media, proved to be significantly better than the egg-based solid media for the isolation of mycobacteria from clinical specimens³⁾.　The difference in the rates of isolation of bacteria between the two groups of media was more remarkable with smear-negative specimens (Table 1).　The isolation of the M. tuberculosis complex by MGIT occurred 8 days previous to the isolation by the conventional Ogawa method.　The mean time for detecting M. tuberculosis complex by Septi-Chek AFB was similar to that of the Ogawa method.　A greater difference in isolation time was observed for mycobacteria other than M. tuberculosis (MOTT) isolates (Table 2).　These results indicate that the MGIT and Septi-Chek AFB systems based on liquid media are efficient for the recovery of mycobacteria.

Table 1.　Recovery of Mycobacteria from 305 Smear-positive and Smear-negative specimens with Different Media

Table 2.　Mean Time of Detection for Patients Who Were Positive for All Methods

2.　Simple differentiation between TB and MOTT bacilli

　

2-1.　Niacin test

Niacin (nicotinic acid) is produced by mycobacteria and used in the synthesis of NAD and NADP.　Most mycobacteria possess an enzyme that converts free niacin to nicotinic acid mononucleotides.　Organisms that lack the enzyme accumulate niacin in the medium.　M. tuberculosis, and some of strains of M. simiae, M. chelonae, and M. marinum can not convert niacin and therefore accumulate it.　The niacin is excreted into the egg or agar media, from which it can be extracted and detected. Only＞3-week-old cultures grown on egg-based or Lowenstein-Jensen agar slants and showing heavy growth should be used for the test.

　

2-2.　BACTEC NAP test⁵⁾

NAP (p-nitro-α-acetylamino-β-hydroxypropiophenone) is an intermediate in the synthesis of chloramphenicol.　Mycobacteria belonging to the M. tuberculosis complex are inhibited by the presence of NAP, while MOTT are inhibited by the slightly or not at all.　Growth in the radiometric BACTEC 12B medium is detected by monitoring ¹⁴CO₂ production, which is measured in terms of growth index units.　If NAP (5μg/ml) is present, growth of mycobacteria belonging to the M. tuberculosis complex is inhibited, resulting in a reduction of ¹⁴CO₂ production, which can be detected by the daily growth index readings.　If there is no inhibition, as in the case of other mycobacteria, the ¹⁴CO₂ output during growth is uninhibited.　Using this system, the M. tuberculosis complex and MOTT can be differentiated within 5 days of isolation.

　

2-3.　DNA probe test^{6, 7)}

A commercially manufactured system, Accuprobe Culture Confirmation Tests (Gene-Probe, Inc., San Diego, Calif.), is available for identification of isolates of several species of mycobacteria.　This nucleic acid hybridization test is based on the ability of complementary nucleic acid strands to specifically align and associate to form stable double-stranded complexes.　The Accuprobe system uses a chemiluminescence (acridinium ester) labeled single stranded DNA probe, that is complementary to

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