FUNDAMENTAL STUDY ON THE VERTICAL DIFFUSION METHOD (I-Report)

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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.

FUNDAMENTAL STUDY ON THE VERTICAL DIFFUSION METHOD (I-Report)

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