Transition of Bacterial Length, RNA, DNA and Inorganic Phosphate of Murine Leprosy Bacilli in the Hart-Valentine Medium

Abstract

The in vitro cultivation of the murine leprosy bacillus is being investigated from the aspect of the energy production mechanism and it has been shown that electron transmission system stops at the stage of reduced flavin due to the absence of the cytochrome system. Under such a condition, it would be impossible for the murine leprosy bacillus to grow in vitro. Since the organisms have an enzyme, which promotes production of ATP by accepting a high energy phosphate from polyphosphate, the elongation of the murine leprosy bacillus without increase in number in Hart-Valentine medium, may be explained by a using up of the polyphosphate in the cell. Since the Douglas strain of murine leprosy bacillus was not used, this cannot accurately be considered a reinvestigation but an elongation equal to that reported by Hart & Valentine was not found using the Hawaiian strain. The marked elongation may be due to a difference in method of collection and the condition of the murine leproma. This, however, should not be considered a constant phenomenon and it was impossible to investigate factors essential for energy production or growth by using this phenomenon as an index. Table 1 and Fig. 1 show the average length, RNA, DNA, hydrolyzed inorganic phosphate, free inorganic phosphate and optical density at 260 mμ from 0 to 8 weeks. A slight elongation of the organism is apparent but since the same organism was not followed, it is not clear whether an actual elongation had taken place or short cells had disappeared by autolysis and only one remained. The RNA level showed no change up to 2 weeks but a rapid decrease took place thereafter and a course toward destruction was taken. DNA is relatively stable but there is a gradual decrease. Up to 5 weeks, there is a difference between the hydrolyzed inorganic phosphate and the free inorganic phosphate but after 7 weeks, this difference is no longer apparent. This finding suggests that high energy phosphate is present in the cell up to 5 weeks but is depleted after 7 weeks and a poor condition for survival occurs. Sucrose is one of the constituents in the Hart-Valentine medium and if this aids in the elongation of the murine leprosy bacillus, it may have to be restudied. The cell lysis of the protoplast or spheroplast of other bacteria is prevented by the addition of 20% sucrose to the medium. The cellular components of the murine leprosy bacillus may leak out if biosynthesis in the cell wall dose not parallel the in vitro elongation and fissures occur in the cell wall in medium lacking sucrose. This is an intersting problem in the light of Johne bacillus which is difficult to cultivate in artificial synthetic media. Difco (Bacto) casamino acids and asparagine are used as the nitrogen source in the Hart-Valentine medium but if this medium is utilized for supplying amino acids, casamino acids do not contain tryptophan and tryptophan must be added separately. If it is the aim to supply the materials for biosynthesis of the cell wall, DNA and RNA, bouillion which contains polysaccharides, lipids, purine and pyrimidine derivatives would be much more advantageous.