Phage typing of M. tuberculosis isolated from the patients was performed to identify the source of tuberculous infection in families.
1) The phages used were derived from the National Institute of Health in Japan. One RTD was tested by spotting 0.01m
l of ten fold dilutions starting from 1×10
8/m
l onto RVA plates overlaid with semisolid RVA containing 0.5% agar and host bacilli. Complete lysis was observed by 100 particles per 0.01m
l or more for all phages.
Thus it was defined that one RTD in our laboratory was 10
4 particles per milliliter for each phages.
In 1954, Dr. S. Froman originally reported the phage D34. However the D34 which was given to me by Dr. Murohashi did not lyse H
37Rv. Consequently this particular phage has been termed D34'.
2) Using one RTD, each phage was spotted on the 160 strains isolated from pulmonary tuberculosis patients. As for susceptibility, the common receptors were observed among DS6A, Y10, C3, B1, GS4E and BK
1 phages each other. D34' phage showed the most specific lysis. Accordingly it was suggested that strains of M. tuberculosis were divided into two lytic patterns. In one pattern, the strains show susceptibility to some of the DS6A, Y10, C3, B1, GS4E and BK
1 phages. In the other pattern, only one strain was susceptible to D34'. Since the phage types, to which the least number of strains show susceptible, seemed very useful for epidemiological studies, the BK1 and D34' were selected for the nexexperiments.
3) For the reason just stated, BK
1 and D34' were selected and applied to the typing of isolated strains, and further phage typing were carried out by the following procedure. About 0.5mg of semi-dried isolated strains in 0.5m
l of saline and 3m
l of RVA were poured over a plate of 20m
l of solidified RVA. After incubation at 37°C for three days, 0.01m
l of phage suspensions containing 1×10
4 and 1×10
3/m
l were spotted. After additional incubation at 37°C for another 11 days, the degree of lysis was recorded.
Final reading were recorded by classifying the strains “highly susceptible”, “intermediately susceptible” and “less susceptible” to each phage. That is, two concentrations of one RTD and 1/10 RTD were spotted on each bacterial lawn of the strains to be tested. When 10 phages (1/10 RTD) spotted produced plaques, such a strain was regarded as “highly susceptible” to the phage; and if 100 phage particles (1 RTD) produced no plaques, such a strain was regarded as less susceptible to the phage; the other strains were regarded as having intermediate susceptibility to the phage.
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