Murine and human leprosy bacilli are obligate parasites, usually occurring intracellularly, which have not been cultivated in vitro. Conventional methods of determining nutritional requirements by growth studies, therefore, are not applicable. In the experimental work here reported, an approach has been made to the cultivation problem by the only available rational method, i.e., by a study of the metabolism of Mycobacterium lepraemurium separated from infected tissue. METHODS The source of

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... li was the infected rat testicle which Hanks and Backerman (1950) have shown to be the favored site for growth of these bacilli. Bacilli of comparable activity can be obtained from subcutaneous lepromas but in lower yields and with greater difficulty. Rats were inoculated four to six months before use with the Hawaiian strain of M. lepraemurium. Infected testicles usually increase several fold in size and average 2 X 1011 bacilli per gram of tissue. Bacilli were freed from tissue components and tissue oxidative enzymes aseptically by centrifuging homogenates of infected tissue through dense layers of hypertonic sucrose and of KCI solutions. This procedure incorporates principles used in the Hogeboom, Schneider, and Palade (1948) method for isolating mitochondria and other cellular particulates and in the albumin method developed by Ferrebee and Geiman (1946) for the separation of blood cells, and is described elsewhere (Hanks, 1951a) . The bacilli were suspended in potassium phosphate buffer M/60 at pH 7.5 and standardized turbidimetrically so that a 1:100 dilution permitted 80 per cent light transmission at 420 my. and corresponded to nephelometer 80 on the Mac-Farland scale. A 0.6 ml aliquot of suspension which contained, on an average, 0.36 mg of nitrogen was added to each Warburg flask. Small flasks (5 ml) and manometers (3 mm bore) were used to provide a system which was two to three times as sensitive as standard flasks. Respiration was measured by the "direct method" (Umbreit et al., 1949 ) at 37 C. EXPERIMENTAL RESULTS Biochemical purity of bacilli. Early in these studies and before the purification procedure reached its present state of development, the author occasionally obtained succinoxidase activity in bacillary suspensions. This was not surprising since the most stable oxidative system in preparations from tissue is the succinoxi-305 on May 9, 2020 by guest