1971 Volume 40 Issue 3 Pages 149-155
We have been, for the purpose of isolation culture of murine leprosy bacilli obtained from mice infected by Mycobacterium lepraemurium, trying to make the isolation culture more firmer one by conductig various preliminary treatment, as stated in Report No. 1, and further after that by trying various kinds of preliminary treatments. We compared the contamination rate and positive rate of the supposed Hawaiian strain and observed the effect of preliminary treatments of only those cases on which two different kinds of preliminary treatments were conducted at the same time on the same material.
As the materials for inoculation, using organs, superficial lymph nodes and subcutaneous leprous nodules obtained aseptically from the mice in dd-N strain which were infected by the supposed Hawaiian strain we isolated, subcutaneously or intravenously and autopsied in various stages, (1) Nontreated: materials were ground in a mortar and were inoculated by smearing, (2) Treatment with 1% NaOH solution: small portion of 1% NaOH solution was added to the material prepared by (1) and was homogenized and was inoculated by smearing, (3) Treatment with 1% H2SO4 solution was used. Other process followed (2). (4) Treatment with 1% NaOH solution and 10 times diluted: materials were weighed beforehand and were ground and 10 times diluted 1% NaOH solution was added and homogenized and 0.1ml each of the homogenate was inoculated. (5) Neutralizing treatment: after neutralizing the homogenate prepared in (4) by addition of 2% HCl solution, 0.1ml each of them was inoculated.
Two kinds of methods of the preliminary treatments above mentioned and inoculation were conducted for each same material at the same time. The materials were inoculated onto 1% egg yolk media on which the supposed Hawaiian strain grows, and onto 1% Ogawa's whole egg media and on which the bacilli does not grow, then were incubated for more than 3 months at 37 C. By this method, contamination rate of saprophyte and positive rate of the supposed Hawaiian strain was compared. The result was as follows.
1) Contamination rate: (Table 1 and Table 2).
In 1% egg yolk media, by (1), it was about a half or more than a half of the number of media used, and by (2), it decreased to about one-sixth of that of the (1) There was no big difference between (2) and (3), and in (4) and (5), invasion of no saprophyte was found. Also in 1% Ogawa's whole egg media, comparing with 1% egg yolk media, is generally the contamination was smaller.
2) Positive rate of the supposed Hawaiian strain: (Table 3 and Table 4).
Excluding the facts that it was assumed that some times, due to the invasion of small amount of saprophyte, the supposed Hawaiian strain did not grow, and that the positive rate of (3) was lower than that of (2), no big difference was found in the positive rate of those preliminary treatment. Also, when observed the process of growth of colonies, it was assumed that these solutions used for preliminary treatments had slightly disturbing effect for growth.
