Kekkaku(Tuberculosis)
Online ISSN : 1884-2410
Print ISSN : 0022-9776
ISSN-L : 0022-9776
Volume 40, Issue 6
Displaying 1-5 of 5 articles from this issue
  • Michio TSUKAMURA, Sumio TSUKAMURA
    1965 Volume 40 Issue 6 Pages 203-205
    Published: June 15, 1965
    Released on J-STAGE: May 24, 2011
    JOURNAL FREE ACCESS
    M. bovis Ravenel and BCG showed only one phenotype of PAS resistance and its pattern resistance development belonged to the “Single-step pattern”.
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  • Ikan YO, Yo TAJIMA, Masaaki IIO, Akio SUGANUMA, Katsuhiko TANIZAKI, To ...
    1965 Volume 40 Issue 6 Pages 207-212
    Published: June 15, 1965
    Released on J-STAGE: May 24, 2011
    JOURNAL FREE ACCESS
    Vertical diffusion method is now generally used for the biological measurement of the drug concentration or the drug resistance. But there still remain some questions for further study, such as, kind of the test tube to be used, amount of inoculum, kind and amount of the culture medium, and the interval period between the inoculation of bacilli and the instillation of the drug. In this paper, results regarding such problems will be presented.
    1) Immediately after instilling 5ml of INH solution into 1020 ordinary test tubes and 448 crook-neckes tubes, both of which had been inoculated with BCG beforehand and kept in an incubator at 37°C in horizontal state for 48 hours, the fluid level was measured from the bcttom of the test tube.The fluctuation of the fluid level was from 1.0cm to 2.1cm in the case of ordinary slant, and from 0.7cm to 1.2cm in the case of crook-neckes tube (Table 1). And even though the constant height and declination of the medium was kept, the fluid level changed considerably (Table 2, 3, 4).
    2) The fluid level after keeping the media in an incubator at 37°C in vertical state for 3 weeks, decreased more than 0.2cm in 19.2% and increased more than 0.2cm in 18% as compared with the state of the medium directly after prepared, in the case of the ordinary slant. Simi larly, in the case of crook-neckes tube, the decrease was found in 7% and the increase in 17.8%. Thus no definite tendency was found in the change of fluid level by the incubation (Table 5).
    3) One microgram of INH was solved in 5 different quantities of water and each was poured carefully into the base of 3% Ogawa medium just inoculated with 0.05mg of BCG. The inhibition zone of each after 3 weeks from the date of the inoculation was almost the same when measured from the bottom of the tube (Table 6A). When the drug weight was 0.2 micro gram, the same result was obtained (Table 6B).
    This experiment demonstrated clearly that the inhibition zone was directly connected with the weight of the drug but not with the solution quantity.
    4) From the above experiments, we can conclude that the length of the inhibition zone can be more accurately indicated when measured from the bottom of the test tube than from the fluid level which is not necessarily constant.
    5) 3% Ogawa media were inoculated with 0.05mg BCG, some of them immediately after and the others after preserving them for 2, 4, 6, 8, 10 days in an incubator at 37, and 0.5ml of one microgram INH solution was poured into the base of the culture medium and the incu bation was continued again. The length of the inhibition zone was each 5.9, 5.4, 4.7, 4.2, 3.7, 2.8cm after 3 weeks of incubation by counting from the date of inoculation and it was almost the same by counting from the date of drug instillation (Table 7).
    This means that the length of inhibition zone becomes shorter accompanying with the elon gation of the period from the inoculation to the instillation of the drug.
    The length of the inhibition zone became also shorter with the elongation of the incubation period. It was 5.9cm after 3 weeks' incubation, and 5.7cm after about 4 weeks in the group. which received the drug solution immediately after inoculation (Table 7).
    Accordingly the period from the inoculation to instillation, and the period of incubation should be always constant. It will be better to instil the drug immediately after inoculation because of the simplicity and the longer inhibition zone.
    6) The inhibition zone of 86 test tubes (4.5%) was un even asshown in Fig. 1 among 1900 tubes, in the case of INH measurement employing the slant inoculated with BCG.
    In these cases, the distance between the highest and the lowest points was from 0.2cm to 2.5cm, but most of them were within the limits of 0.5cm.
    The measurement error between 2 persons was over 0.2cm because of the unevenness in 2 tubes among 60 tubes (3.3%) for the measurement of ethambutol using M. phlei.
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  • Masatoshi TAMURA, Satoshi TAKANO
    1965 Volume 40 Issue 6 Pages 213-218
    Published: June 15, 1965
    Released on J-STAGE: May 24, 2011
    JOURNAL FREE ACCESS
    It is said that Cycloserine (CS) is stable chemically in weak alkaline reaction, while Myc. tuberculosis grows well in acid media. These two matters seem to involve opposite factors in the resistance test of Myc. tuberculosis to CS. Accordingly, studies were made t investigate the influence of pH of media upon the minimal inhibitory concentration of CS to germ.
    Media used were 1% Ogawa's egg slant and Kirchner's semi-liquid agar media. The basal media were adjusted to the pH level of 1.8, 3.2, 5.6, 6.6 and 7.0 with either HCl or NaOH, and the egg were added to them so that the pH level of condense water may become 5.6, 6.2, 6.8, 7.4 and 8.0. They incorporated with CS in the concentrations 0, 10, 20, 30, 50 and 100mcg/ml, respectively. The media were dispensed into test tubes 5ml each. Prior to use, Kirchner's semi-liquid agar media were kept in an incubator at 37°C for 24 hours to confirm sterility.
    The following 5 strains were used for the studies: H37Rv, Aoyama-B, a strain isolated from sputum of a pulmonary tuberculosis patient never treated with CS previously and 2 strains from those treated with CS. From the 3 weeks old cultures of the primary isolation on 1% Ogawa's egg slant, emulsions were made in a concentration of approximately 1mg/ml employing electric photometer and were diluted 10 times, respectively. Then 0.1ml each of the emulsions was inoculated into media. Incubation period was 6 weeks at 37°C.
    Results revealed that the most suitable pH level for the resistance test to CS was 6.8 in both kinds of media. The bacteriostatic effect of CS against Myc. tuberculosis seemed to be stronger in acid media than in alkaline ones. In both media, the minimal inhibitory concen tration of CS to the susceptible strain of Myc. tuberculosis was shown at the concentration of 20 to 30mcg/ml at a pH of 6.8.
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  • Sumio TSUKAMURA
    1965 Volume 40 Issue 6 Pages 219-222
    Published: June 15, 1965
    Released on J-STAGE: May 24, 2011
    JOURNAL FREE ACCESS
    It is known that most bacteria maintain their viability at freezing state if they are suspended in 5 to 20% glycerol (1.2). Postgate and Hunter (2) reported that storage of Aerobacter aerogenes at -20°C depended on the protective agent used; only glycerol permitted extended storage. They observed that these organisms maintained their viability at 85% after 40 days of freezing at -20°C. Since similar studies on mycobacteria have not been made, the present study has concerned with this, and the storage of mycobacterial strains was extended to 6 months of freezing. The purpose of the study is to find a conventional method for maintainance of stock cultures of mycobacteria.
    M. smegmatis Jucho, M. tuberculosis H37Rv, M. bovis BCG, M. kansasii Forbes 84, nonpho tochromogen N 100616, and scotochromogen P-5 were used. E. coli K-12 also was used.
    M. smegmatis Jucho was suspended in various solutions at concentration of 5. 5 mg (wet weight) /ml and stored in a refrigerator at -15°C. At different intervals, one tube of each group was taken and diluted with saline to 1: 1 to 1: 1, 000, 000 dilutions. Aliquots (0.02mlsamples) of these dilutions were inoculated to Löwenstein-Jensen medium and the number of colonies was counted after five days of incubation at 37°C. The results are shown in Table 1. When suspended in saline, the organisms decreased their viable numbers, but when suspended in Sauton medium, 3% glycerol broth, and 10% glycerol, all of which contained glycerol, they maintained their viability even after 42 days of storage. Similar experiments were conducted using E. coli. Determination of viable numbers was made similarly but in use of nutrient broth agar medium for counting. The results are shown in Table 2. The resutls showed that stor age in 10% glycerol was most satisfactory to maintain the viability. Suspension of nutrient broth without glycerol decreased the viable number markedly.
    In view of the above results, storage of five strains at -20°C was examined in 10% glycerol (aquaeous solution). The observation was made until the end of the sixth month. The results are shown in Table 3. This method of storage maintained the viability of the test strains almost completely until the end of the third month. After six months of storage also, the viability was maintained fairly well in M. tuberculosis and M. bovis and almost satis factorily in other three strains. In view of the results obtained, storage of mycobacterial strains in 10% glycerol at -20°C was considered to be useful to maintain the stock cultures in laboratories.
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  • Masako UESUGI
    1965 Volume 40 Issue 6 Pages 223-230
    Published: June 15, 1965
    Released on J-STAGE: May 24, 2011
    JOURNAL FREE ACCESS
    Eighty nine hundred fifty-six pupils of 17 primary schools in Kawasaki city, who entered school during the years 1955-1958, were followed up till their graduation. A total of 81 tuber culosis morbid cases were found in their school life. The relationship between the incidence of tuberculosis morbid case and the maximal intensity of tuberculin reaction before detection of their diseases was as follows: 5.2% in marked positive group (redness over 10mm in di ameter with induration and duplicate redness against 1: 2000 OT), 1.0% in moderate positive group (redness over 10mm with induration), and 0.1% in feeble positive group (redness over 10mm only). A total of 147 cases with calcified primary pulmonary and hilar nodes foci were found during the period of observation. The incidence of the cases with calcified foci in relation to the maximal intensity of tuberculin reaction before detection of their diseases indicated similar result: 7.3% in marked, 1.7% in moderate, and 0.2% in feeble positive group respectively. Those who showed feeble positive reaction alone before detection of thei r diseases were only 7 cases in number, 6 of these showed moderate or marked positive reaction afterdetection of their diseases. Another case showed feeble positive reaction through his school life, however he showed redness over 30mm 1 year before the detection of disease. Of the cases with calcified foci, 3 cases showed feeble positive reaction through their school life. One out of these 3 also showed redness over 30mm. After entrance in junior high school, both of these 2 cases showed induration by the unused site examination.
    As to the relationship between the size of redness and the appearance of induration, 403 cases with redness over 10mm were examined. Those with redness less than 14mm showed no induration. On the contrary, of those with redness over 30mm, 91% showed induration. Four hundred thirty-nine cases, who showed induration at least once or more times through their primary school life, were tested by the unused site injection in the 1st grade of the junior high school. Of them 65% showed induration. Of 593 cases who showed redness only through their primary school life, 21% showed induration by the unused site injection in the 1st grade of the junior high school. The correlation of the size of redness in the used site and the appearance of induration in the unused site, was not present in the former. However, the correlation was present, to some degree, in the latter.
    Of 81 tuberculosis morbid cases of the primary schools, 43.2% had the history of previous BCG inoculation. Thirty morbid cases and 15 morbid cases, detected in 1963 respectively from all the junior high schools and 4 high schools in Kawasaki city, had been inoculated with BCG in 26.3% and 33.3% respectively. Of the morbid cases of the junior high schools, 57% had calcified foci, and none had the history of BCG inoculation.
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