Separation of Mycobacterium lepraemurium from the subcutaneous tissues of the rat

In an effort to obtain cell suspensions free from blood cells which interfere in spectrophotometric studies, a method was devised by which relatively large quantities of whole cell suspensions of M. lepraemurium are obtained for experimental purposes. We have routinely employed this procedure which is quite reliable, technically simple, moderate in equipment requirements, and in a matter of five minutes it can be ascertained whether or not the preparations are free from contaminants which render the purified bacillary suspensions unsuitable for studies involving spectrophotometric technics. However, in this procedure, certain points are essential in order to obtain large quantities of purified bacillary suspensions. It is important to homogenize the lepromata for only a few seconds at brief intervals. Excessive homogenization may cause considerable damage to the bacilli and it may be difficult to separate the bacilli from the host tissues. It is also important to use transparent bottles and tubes during centrifugation as the material can be seen and thus each fraction can be separated easily. Very few bacilli are lost in the discarded material and we have repeatedly obtained 2.5 to 3 gm wet weight of M. lepraemurium from one leproma weighing 25 gm to 30 gm.