| Abstract |
Viable potential of Mycobacterium smegmatis, a slow grower in vitro cultivation and of M. leprae, an obligate intracellular parasitic bacterium, which can not be cultured yet in vitro was assessed by fluorospectrophotometry. Bacterial cells in different numbers and under various physiological status were incubater with fluorescein diacetate(FDA). After an incubation of the bacterial preparations with FDA at specified conditions, amount of fluorescein inside bacteria was measured by a fluorospectrophotometer at 470nm and 510nm of excitation and emission wavelengths, respectively. Fluorounit given by such bacteria showed a correlation with assessment of viability of the same preparations made by other methods, such as optical density and colony forming units of M. smegmatis and intracellular ATP content of M. leprae. The possible use of fluorospectrophotometry in assessing viability or physiological potential of bacteria, particularly intracellular parasites and fastidious organisms to culture in vitro is discussed in relation to other methods. In order to develope interspecific fusion of the genus Cellulomonas capable of assimilation cellulose, the optimun conditions for the protoplast formation was investigated to examine the susceptibility of cell wall, between different species of the same genus using scanning electron microscope. The variation in the susceptibilities of Cellulomonas sp. CS 1-1 and C. flavigena to lysozyme treatment were considerably remarkable, although they belong to the same genus. The rate of protoplast formation of CS1-1 was 99.9% being treated with lysozyme (100㎍/㎖) for 30 minute and that of C. flavigena was about 80% being treated at the concentration of 600㎍/㎖ of lysozyme for 6 hours. The susceptibility of cell wall to the lysozyme treatment on protoplast formation of the strain, CS1-1 seems not to be depend on the cultural periods of cells. On the contrary, that of C. flavigena was considerably depend on the periods. Cells of C. flavigena at mid exponential phase could be more efficiently converted to protoplast cells than those at late exponential phase be done. The rate of the protoplast formation was 95%, when cells of C. flavigena at mid exponential phase were treated with lysozyme 600㎍/㎖ for 6 hours and observed by SEM. In the evalution of protoplast formation of the CS1-1 results of counting method in plate after osmotic shock treatment were similar to the results of the direct observation method by means of SEM. But in the case of C. flavigena the latter method was much more reliable than the former, because the differences between the number of spheroplasts and protoplasts were not able to figure out on counting the number of protoplast after osmotic shock tretment. |
