The content of deoxyribunucleic acids (DNA) was compared in various mycobacteria by Tsukamura, but, since the data were presented inadequately, it was desired again to estimat e the DNA content, applying a more appropriate method, in various mycobacteria and nocardias.
Twelve strains of 7 species of Mycobacterium and 12 strains of 5 species of Nocardia were used (Table 1 and 2). The strains had been maintained on Ogawa egg medium at -20°C. After subcultureing test strains on Ogawa egg medium at 37°C (Mycobacterium)or at 28°C(Nocardia), growing organisms were harvested, washed three times in distilled water, and then used for estimation of DNA, ribonucleic acid (RNA) and protein contents. Rapid-growing mycobacteria (Table 3) and nocardias were harvested after incubation for 7 days, slow-growing mycobacteria (Table 3) after incubation for 14 days, and very slowly growing mycobacteria (Table 3) after incubation for 21 days. The organisms were fractionated according to the procedures of Schneider or of Ogur-Rosens. The DNA content was measured by the diphenylamine reaction, the RNA content by the oricinol reactive, and the protein content by the Folin phenol reagent. The estimation was made on three different samples and the results were expressed as μg DNA per mg protein.
Influ e nce of the Fractionation Method on DNA Content Estimation
The DNA content varied depending on the method of fr a ctionation. Some species showed similar values in both methods, while some showed markedly different ones (Tables 1 and 2). It was noticed that the influence of the fractionation method be consistent in species groups (Table 3). It was suggested that the effect of extraction agents on the DNA extraction be correlated with some taxonomic group (Table 3). The difference of the DNA content between the two fractionation methods would have been produced by either the use of hot trichloroacetic acid (Schneider method) or the use of hot perchloric acid (Ogur-Rosen method) for extraction of the DNA. The effect may be essentially related to the cell wall or cell membrane structure of organisms.
RNA content appeared relatively low in the Ogur-Rosen method (Tables 1 and 2).
DNA Content in Various Taxonomic Groups
1. When observed in the values obta i ned by the Schneider method, the DNA content of mycobacteria were considerably uniform, whereas that of nocardias differed from strain to strain (Tables 1 and 2). Especially the content in N. polychromogenes was significantly lower than the others (Table 2).
2. When observed i n the values obtained by the Ogur-Rosen method, the DNA content in slow-growing mycobacteria was significantly higher than that in rapid-growing mycbbacteria. The content in M. tuberculosis and M. bovis was low. The nocardias were grouped into two subgroups; one showing low values (M, polychromogenes, N. asteroides and N. farcinica) and another showing high values of the DNA content(N. brasiliensis and N. caviae)(Tab.1 and 2).
In view of the results obtained, the DNA content estimated using the Ogur-Rosen meth o d seemed to be more related to taxonomic grouping, and it was believed that the comparison of the DNA content between two fractionation methods, Schneider and Ogur-Rosen methods, contributes to the taxonomy ofkn-abstract=

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Occasionally one may encounter primary culture in which both tubercle bacilli and other acid-fast organisms are growing simultaneously. Such a mixed culture, however, is particularly difficult to recognize provided that the two types of acid-fast orgnaisms, especially of slow-growers, make entirely confluent colonies each other. If there is any sign of being mixed, the cultures should be subjected to certain procedures for separation. The purpose of this report is to introduce a method for separating mycobacterial mixed culture into its components of pure cultures.
1) Suspectable features of a mixed culture: In the experiment shown in Table 1, artificially mixed cultures of tubercle bacilli strain H₃₇Rv and nonphotochromogenic strain Mori were prepared by inoculating the mixture of the two bacterial suspensions in various proportions. The table shows that among their cultural characteristics colonial appearance or niacin test may serve as an aid for suspecting a culture of mixed one. However, it seems likely that most of the cases reveal no sign of being a mixed population.
2) Method for separating tubercle bac i lli and atypical mycobacteria from their mixed culture (Table 2 and 3): A 0.1 mg/ml aqueous suspension was made from each of the cultures of the tubercle bacilli and the atypical mycobacteria and, as shown in Table 2 and Table 3, they were mixed each other in varying proportions. Serial 10-fold dilutions were made up from these mixtures using distilled water, 4% NaOH solution and 4% H₂SO₄ solution, respectively. These dilutions were inoculated in an amount of 0.1 ml each into the following media: the aqueous dilution was inoculated into four 1% (KH2PO4) Ogawa egg slants two of which were incubated at 37°C and the other two at 22°C; the alkaline and acidic dilutions were inoculated into 3%(KH₂PO₄) Ogawa egg slants and 3% Na₂HPO₄ egg slants, respectively. These were incubat ed 37°C. Such an alkaline or acidic treatment aimed at recovering tubercle bacilli from the mixed culture where atypical mycobacteria were considered predominant.
Well-isolated colonies were obtained from each of the su ff iciently diluted suspensions and distinguished by virtue of colonial appearance and growth temperature: rough colony grown at 37°C is evidence of the tubercle bacilli and smooth form of colony at 22°C or 37°C indicates the atypical mycobacteria.
3) Confirmation (Table 4): To make certain of the above differentiation, two types of separate colonies as differentiated above were removed and streaked on Ogawa egg slants. The data in Table 4 clearly showed that the colonies suspected of the tubercle bacilli gave no growth at 22°C and good growth at 37°C which proved niacin-positive and that the colonies suspected of atypical mycobacteria yielded niacin-negative cultures at both 22°C and 37°C. These results suggest that the above procedure including confirmation test may be useful for the aforesaid purpose in routine laboratories.

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A certain: Minowa, a 30-year-old company employee, was admitted to our hospital with diagnosis of the relapse of pulmonary tuberculosis in October 1965. Acid-fast organisms w ere isolated twice from his sputum; one rough colony was obtained in the above month w hich proved niacin-positive and smooth orange-colored fast-growing mycobacteria, 33 colonie s per tube, were isolated in March of the next year.
Drug sensitivity test shown in the Table 1 revealed that some culture of the latter strain 'contained a few rough colonies among smooth ones. All of the six cultures, therefore, were submitted to examination for purity. On preliminary subcultivation (Table 2) it was found that out of six cultures at 37°C two (# 5 and # 6) contained rough colonies and four (# 1, 4, 5 and #6) were niacin-positive. Further, the method described in the preceding paper wa#s applied to these cultures: a 1m g/ml aqueous suspension was prepared from each of the grow th 'of cultures. From these suspensions serial 10-fold dilutions were made up using distilled water, 4% NaOH solution, and 4% H₂SO₄ solution, respectively. Each aqueous dilution was inoculated into four 1% Ogawa egg slants two of which were incubated at 37°C and the other two at: 22°C. The alkaline and acidic dilutions were inoculated into 3% Ogawa egg slants and 3% Na₂HPO₄ egg slants, respectively. They were incubated at 37°C. After one-month incubation the resulting growth was read on the assumptions that rough colony was tubercle b acilli and smooth orange-colored one as atypical mycobacteria. The number of colonies per tube of the two types organisms was recorded in Table 3. It will be seen that all substrains except 5# produced a few rough colonies in slants inoculated with the aqueous or alkaline dilution. On the other hand, smooth pigmented colonies were obtained on most of the slants at either temperature.
For identification colonial subcultures on Ogawa egg medium were made of these rough and smooth colonies differentiated as above. The growth characteristics and niacin test shown in Table 4 indicate that all subcultures of the rough colony except one are the pure culture of the tubercle bacilli and all of the smooth colony are the same atypical mycobacteria as read before. One subculture which was derived from the aqueous suspension of the rough colonies of strain Minowa # 6 was found to be a mixed population of tubercle bacilli and atypical mycobacteria.
Drug sensitivity test was carried out on these purified cultures. Table 5 shows the result of the test being compared with that of the test on the tubercle bacilli which were isolated from the lung lesion of the patient. It will be concluded that the method applied above is useful for such cases.

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Induction rate of resistance to kanamycin (KM), viomycin (VM) and capreomycin (CPM)and cross-resistance among these drugs were studied in tubercle bacilli.
A streptomycin (SM)-sensitive strain of H₃₇Rv and the SM-resistant strain isolated as one step mutant of the former were employed in the present study.
Increasing degrees of resistance to KM, VM and CPM we r e induced by succesive culture of above strains in the 1% Ogawa's egg media containing an increasing amount of each drug.
1) Induction rate of resistance to KM, VM and CPM was much higher in the SM-resis tant strain than in the SM-sensitive strain.
2) In both SM-sensitive strain and SM-resistant strain, there were complete cross-resistance between VM and CPM, and partial cross-resistance between KM and VM, as well as between KM and CPM.
3) I n both SM-sensitive strain and SM-resistant strain, the increase of resistance to KM, VM and CPM was always associated with the increasing resistance to SM.

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A study was made to intensify the clinical effects of initial chemotherapy for pulmonary tuberculosis. Since side effects of PAS such as gastrointestinal disturbance and allergic reaction are frequently seen, and its anti-tuberculous effect in vivo is less than that in vitro, a trial was made to substitute PAS by EB as one of the primary drugs in the initial combined treatment with SM and INH.
One hundre d and thirty patients with newly found active pulmonary tuberculosis were devided at random into two groups; 66 cases treated with SM, INH and EB (Group I) and 64 cases treated with SM, INH and PAS (Group II). The dose of the drugs was as follows: 1 gm SM per day twice a week, 400 mg INH divided in two doses daily, 10 gm PAS divided in four doses daily and 1 gm EB daily.
No significant difference wa s found in the background factors of patients before starting chemotherapy between both groups, and the details are presented in Fig.1. The sputum positivity was 48.4% in group I and 26.5% in group II by smear, and about 70% in both groups by culture.
Roentgenographic examinations of the chest by routine PA films and tomograms were made in each case before and every month after starting treatment. Bacteriologic examinations were done every month for three consecutive days with sputum or specimens collected by gastric lavage if sputum was not available. Clinical signs and symptoms were checked monthly in respect of body weight, temperature, erythrocyte sedimentation rate, cough, sputum and appetite.
During the treatment, all the cases were subjected to laboratory examinations once or twice a month to detect any toxic side effect of drugs on liver, kidney, eye and other organs by means of hematologic examinations, liver function test by protein fractionation, routine test of urine and faeces. In addition to these tests, ophthalmologic examinations including determination of visual acuity, visual field, color vision and fundscopy were conducted for patients in group I

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The changes of tuberculosis mortality in Japan were observed on 23 successive cohorts born from 1915 to 1959, analyzing the age specific cohort mortality rates and their accumulations calculated by life-table procedure.
The following results were obtained from these observations.
1. The age distribution of the mortality from t u berculosis showed the common pattern among the generations born before 1930, rising up from the age of puberty followed by the peak at early adult age and finally falling down in old ages. Such pattern, however, changed in younger generations born after 1930, particularly in those born after 1936, which were at the highest rates of the mortality from tuberculosis at the age under 5 years. One of the main factors influencing upon the change of age pattern was reasonably thought to be the development of chemotherapeutics.
2. The accumulated mortality rates from tuberculosis increased rapidly in younger ages almost identically among each generation born before 1928, but more and more gradually in generations born from 1929 to 1959. Such factors as BCG vaccination, improvement of living standard and chemotherapy may have caused these changes.
3. Sex difference of the accumulated mortality rate from tuberculosis was observed. The rates in male which were lower than those of female in young ages exceeded female at age 25or later in the generations born before 1924. For the generations born between 1927 and 1939, the rates in male did not exceed those in female, and no significant difference were found between both sex in the generations born after 1940. Such fact suggested that male was benefited by the chemotherapeutics much more than the female.
4. The accumulated mortality rates from tuberculosis at 15 years of age were found to be in decrease even in the generations before the popularization of BCG vaccination.
5. A proportion of the death due to tuberculosis under 20 years o f age among those under 40 years was constantly higher in female than male in all generations. This may be partly related to the difference of physiological resistance between both sex.

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