Toyoho Murohashi

Cultivation of Mycobacterium leprae in cell-free, semi-synthetic soft agar media. 1. First attempt to cultivate the organism from leprous nodules.

One-tenth ml each of bacterial suspensions derived from nodules of 4 different L-type patients was inoculated into 0.1% semiliquid agar media of various semi-synthetic base compositions and incubated at 37°C. The results revealed that rather large colony-like masses of bacteria had appeared, in most cases, after about 20 weeks and persisted even after about one year of incubation at 37°C. These masses varied in size and shape. Generally, they were composed of smaller, round bacterial masses of 10 to 20μ in diameter, confluent with one another. Each mass seemed to have boundary around it. It consisted of strongly acid-fast rods, stainable with malachite green. It was suggested that the bacterial masses might be colonial growths.Bacterial suspensions prepared from nearly one-year-old cultures by centrifugation induced essentially the same intradermal reaction in leprous patients as Dharmendras antigen. Therefore, it was revealed clearly that the bacterial masses appearing in culture tubes were composed of M. leprae.

Attempts at cultivation of Mycobacterium lepraemurium in cell-free, semi-synthetic soft agar medium.

A biological significance was suggested to be shown by an acid-fastness map devised from investigation of the effect of ultraviolet irradiation on the acid-fastness of mycobacteria. Then, attempts were made to cultivate Mycobacterium lepraemurium Hawaii from nodules of infected mice in a cell-free, semi-synthetic soft agar medium. The basic composition of this medium was modified to some extent from that used for the successul cultivatioh of M. paratuberculosis Johnei.Experiments were carried out repeatedly five times. Those organisms which had multiplied even to a limited extent were observed to form microcolony-like masses, which were composed of elongated, strongly acid-fast rods. It was interesting to note that when a medium contained both glycerol and glucose as carbon sources in addition to ferricion, it seemed to support the growth of M. lepraemurium to some extent.

Epidemiological Meaning of Reaction due to Dharmendra Antigen on Survey of Leprosy

It was reported previously that guinea pigs sensitized with M. leprae were converted to positive reaction with the use of not only Dharmendra antigen but also tuberculin and guinea pigs sensitized with human tubercle bacilli or BCG too were converted to positive reaction due to tuberculin and Dharmendra antigen. On the other hand, it was found that the positive conversion of reaction due to Dharmendra antigen were primarily effected with BCG vaccination and secondarly with the natural infection of tuberculosis, investigating on the results of reaction due to Dharmendra antigen among general population. Then, for the investigating on the problem whether the infection of leprosy could be detected with the use of Dharmendra antigen, the results of reaction due to Dharmendra antigen among people of leprosy-household were compared with those in the othe populations. The results obtained were as follows; 1) The ratio of Dharmendra positive rate to tuberculin positive rate and the

[RELATION BETWEEN THE LOSS OF ACID-FASTNESS DUE TO THE UV IRRADIATION AND THE VIRULENCE OF TUBERCLE BACILLI].

As reported in the previous paper tubercle bacilli are deprived of their acid-fast stainability completely by the UV irradiation for a certain time period. Obviously longer time period of UV irradiation is required for the complete loss of acid-fastness in the virulent type strains than in the a- or low-virulent ones. This fact seems to suggesst the difference in the cell wall structure between these two types of tubercle bacilli and is likely to enable us to attribute the virulence of tubercle bacilli primarily to the proper cell wall structure. Based on the results obtasned a very simple method to distinguish, even roughly, these two types of strain by the staining of smears was devised. Further, the relation between the virulence and the cell wall structure of tubercle bacilli was discussed.

Studies on the adsorption of mycobacteriophage

Effect of the abortive infection of mycobacteriophage was studied on the series of adsorption tubes set up at varying multiplicities of infection in the system of D 29 and Myc. phlei-Yoken.After 60 minutes of adsorption at 37°C, assay was made on the total infectious centers, the number of unadsorbed free phage particles and the surviving bacteria. It was observed that nearly 90% of the phage particles was adsorbed and the number of unadsorbed phage particles was almost equal to that of the total infectious centers regardless of the phage imput ratio. This fact indicates that all of the adsorbed phage particles were inactivated, even though nothing was known on what step of infection the inactivation took place.On the other hand, the number of survival bacteria decreased obviously in accordance with the increasing of the multiplicity of infection. The expected number of surviving bacteria calculated by means of binominal distribution in assumption of that the all adsorbed phage particles resulted in killing of the bacteria was not equal to the actual one. It seems likely that this result is due to the unavoidable clumping growth of Myc. phlei in liquid medium. From these results it was revealed that the abortive infection of phage D 29 to Myc. phlei-Yoken resulted in not only inactivation of the phage particles but also the death of the host cells.The relation between the host killing effect of phage and the spotting method of phage typing of Inycobacteria was discussed.