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Since the success of the multiplication of Mycobacterium lepraemurium in cell-free liquid medium by the author in 1972, various factors affecting its growth have been reported. In particular, it was emphasized that the key essential factor for the growth was alpha-ketoglutaric acid(alpha-kg). However, recent data now indicate that the critical factor for the growth of M. lepraemurium is not alpha-kg, but the optimal pH of the liquid culture medium, quite similar to the condition of egg-yolk solid medium. The recent experimental results clearly indicated that an abundant multiplication of M. lepraemurium took place in an acid liquid medium containing egg-yolk extract, without alpha-kg. This finding means that the cells of M. lepraemurium can multiply in a liquid medium, if the pH of the medium is adjusted to 6.0-6.2. The reason why the cells of M. lepraemurium multiplied in NC medium, and NC-5 medium, containing alpha-kg was that the pH of neutral liquid base medium of the NC or NC-5 medium, was adjusted to 6.0-6.2, optimal for the growth of M. lepraemurium by addition of the 10% alpha-kg (where 10% alpha-kg is strongly acidic; pH:1.22). Other supplements, such as cytochrome c, l-cysteine, and hemin, which are routinely added to the base medium of NC and NC-5 medium, have shown stimulatory effects on multiplication of M. lepraemurium, rather than a key essential effect. In addition, recent experimental data have suggested a second key essential component for growth present in egg-yolk extract.

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For the Growth of Mycobacterium lepraemurium in Cell-Free Liquid Medium, the Key Essential Factor may be the pH(Optimal 6.0-6.2) of the Culture Medium, rather than the Presence of .ALPHA.-Ketoglutaric Acid.