The 1% egg yolk mediums¹⁾ is prepared as follows: The salt solution consists of KH₂PO₄, 1.0g; sodium glutamate, 1.0g; distilled water, 100ml. Added 6 ml glycerol, 6ml of a 2 per cent malachite green solution and 200ml egg yolk to the salt solution, mixed well, and distributed in sterile test tubes (170mm long, 18mm in diameter) in 5ml amounts. The medium is then coagulated in a slanting position at 90°C for 60 minutes. Reaction of the medium is about 6.1.
Despite the fact that only the egg yolk medium supports gross visible growth of the acid-fast organisms supposedly murine leprosy bacilli, the ability of the medium proved not so satisfactory. As previously described²⁾, all of the ingredients in the medium appeared to be necessary for growth of the organism. This paper aimed for the improvement of the medium.
The following modified egg yolk media were prepared by varying the concentration of the salts and the glycerol or by varying the pH of the medium. The test media in Experiments I and II had the same composition as the original (standard) one except that the amount of monopotassium phosphate in the salt solution was varied to give concentrations ranging from 0.3 to 3 per cent. As a result, these slants possessed different pH values from that of the standard (Table 1). In Experiment III, similarly, the test media had the same formula as the standard one except the concentration of sodium glutamate in the salt solution was altered from 1 to 0.5 or 2 per cent (Table 2). The glycerol was decreased or increased to give final concentrations ranging from 0.5 to 16 per cent in the case of the test media in Experiments IV and V (Table 3).
In Experiments VI-IX, the pH of the medium was adjusted to 6.4 to 7.0 either by adding dibasic sodium phosphate to the salt solution (Table 4) or by adding an aqueous sodium hydroxide solution to the medium before inspissation (Table 5).
A total of 169 subcultures of the supposed Hawaiian strain was used for these growth experiments. Small portions of the growth were transferred by a loop lightly on the middle of the surface of media and then the tubes were incubated at 37°C for 3 months. Observations were repeated weekly or biweekly to compare the size of the gross visible growth on the standard and modified egg yolk media with that of the negative control growth on Ogawa's egg medium inoculated in parallel.
Evaluation of the modified media was made according to whether the frequency of the positive growth on them is higher or not than that of the positive growth on the standard egg yolk medium. The results showed, however, that none of the modified egg yolk media tested were supperior to the original one.

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According to almost the same method¹⁾ as that employed for the primary isolation of the acid-fast organisms, supposedly murine leprosy bacillus, from mice which has been infected with the Hawaiian strain, a similar attempt was made to isolate mycobacteria from tissue specimens of the mice with the Keishicho strain.
Male mice, ddN strain, were inoculated intravenously or subcutaneously with 0.1ml each of a bacterial suspension prepared from a subcutaneous leproma (Keishicho strain). At various intervals, 3 to 11 months after inoculation, they were sacrificed to obtain various infected specimens including lungs, liver, spleen, kidneys, lymph nodes and local lesion.
Each of the materials was removed aseptically, ground in a mortar, treated with an equal or a half amount of one per cent sodium hydroxide solution, and then inoculated by loop onto both of Ogawa's 1% egg yolk and 1% egg media. The tubes were incubated at 37°C for over 3 months.
Table 2 presents the results of the experiment in a summary. Of a total of 63 specimens inoculated, 25 (40 per cent) gave positive cultures of slow growing acid-fast bacilli. All culture-positive specimens were derived from the mice sacrificed at 6 months or later, at which time gross lesions could be distinguished easily. The positive cultures were obtained more frequently in the mice intravenously inoculated than in those subcutaneously inoculated. Table 3 shows that the specimens with great number of stainable acid-fast bacilli tend to give more positive results in primary cultures. In view of the fact that these results are quite similar to those of the previous report dealing with the Hawaiian strain, the method employed seemed to have a high degree of reproducibility. The colony of the organisms grown on the 1% egg yolk medium is rough, slightly moist, and buff in color. The cells are acid-fast bacilli. Growth occurs only on egg yolk media such as the 1% egg yolk medium, with no growth on Ogawa's 1% egg medium. The growth on subcultivation, however, could be recognized only when the organisms were inoculated by loop as a small but visible colonial fragment, and not when inoculated in the form of a bacterial suspension. Niacin formation is negative and catalase activity is weakly positive. Thus, the characteristics of the organisms appears to be the same as those of the acid-fast organisms isolated from the mice with the Hawaiian strain.
At present, the eighth generations are growing and a reproduction test of the disease in mice are now in progress.

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A middle lobe syndrome having a traction oesophageal diverticulum in a leprosy patient was presented. By the way, the author described its pathogenesis, clinical symptoms, diagnosis and treatment in details.

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In 1969, Ogawa and Motomura¹⁾ isolated slow growing acid-fast bacilli from mice previously inoculated with the Hawaiian strain of Mycobacterium lepraemurium. The 4th subcultures of the organisms, designated here as strain HI of Ogawa's bacillus, were given through the courtesy of Dr. Ogawa and submitted to the present experiment in which comparative studies with other known strains of slow growing mycobacteria were carried out.
Growth characteristics in vitro: When heavily transferred by loop (Table 1), the organisms grew very slowly on Ogawa's 1% egg yolk medium¹⁾ but not on the 1% egg medium⁴⁾, the egg yolk medium deprived of glycerol, or the Morrison⁶⁾ and Smith⁷⁾ agar media containing mycobactin. The addition of heat-killed M. phlei cells to the egg yolk medium did not enhance the growth of the organisms. Small inocula from the filtered bacterial suspension, 0.01mg to 0.3mg (moist weight), yielded isolated colonies on the egg yolk medium; growth became barely visible to the naked eye after approximately 40 weeks' incubation at 37° (Table 2).
Animal test: Male mice of dd-Y strain, weighing 20 to 22g, were inoculated intravenously with an amount of 0.2 or 0.02mg of the 4th subculture and utopsied 5 to 10 months later. As shown in Table 3, the organisms gave rise to a progressive murine leprosy-like disease with severe lesions involving liver, spleen, and skin; the lungs were less severely affected. Most of the smears from these tissues revealed vast number of acid-fast bacilli. For isolation weighed tissue was ground in a mortar and treated with 5 to 10 volumes of 1 per cent sodium hydroxide; 0.1-ml amounts (20 to 10mg of tissue) of the resulting suspension were pipetted into Ogawa's egg yolk and egg slants. Of a total of 59 specimens inoculated, 35 gave positive cultures on the egg yolk medium, 19 negative and 5 contaminated. No positive cultures were obtained with the egg medium. All of the cultures recovered were identified with the organisms employed for injection.
It seems reasonable to conclude that strain HI is very characteristic in vitro and in experimental animals, and definitely distinguishable from other known culturable mycobacteria. Some similarities between Ogawa's bacillus and murine leprosy bacillus has briefly discussed.

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Recently, Ogawa and Hiraki²⁾ reported that according to their method they had isolated slow growing acid-fast bacilli also from mice previously infected with the Keishicho (the Metropolitan Police Department in English) strain of Mycobacterium lepraemurium. The 2nd subcultures of the organisms were given by Dr. Ogawa, which were designated in our laboratory as strain MPD of Ogawa's bacillus. Thus, the present paper was concerned with drug susceptibility and biochemical reaction of the two strains of HI¹⁾ and MPD. In an effort at differentiation, comparative studies with other known strains were carried out.
Drug susceptibilities: Despite the fact that some difficulties were encountered in performing drug susceptibility test of Ogawa's bacilli, the following feature was revealed (Table 1). Both strains were highly susceptible to INH, ETA, RFP, and DDS, moderately susceptible to CS, but resistant to PAS, EB, and TbI. On the other hand, there was some difference in susceptibilities to the remaining drugs between the two strains; strain MPD was moderately or slightly susceptible to all of the five drugs, while strain HI was slightly susceptible only to DHSM, KM, and PZ but resistant to VM and CPM. Among the most distinctive characters of Ogawa's bacilli was high susceptibility to both INH and DDS (Tables 1 and 2).
It seems likely that the in vitro growth inhibitory effects of these drugs on strain HI are comparable to the suppressive effects of the drugs on the murine leprosy due to the Hawaiian strain (Table 4).
Qualitatiue differential test: Ogawa's bacilli gave a positive catalase test at room temperature, but negative tests for heat-stable catalase, niacin, nitrate reductase, arylsulfatase, and Tween 80 hydrolysis (Table 3).
The morphologic and cultural characteristics of strain MPD were also quite similar to those of strain HI. The final conclusion can be expressed as follows: It is very possible that Ogawa's bacillus is Mycobacterium lepraemurium.

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