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Korean Journal of Epidemiology 1986;8(2): 323-329.
접종원, 배지 그리고 배양 후 보관온도에 따른 렙토스피라균의 배양 및 동정 능률에 관한 연구
김정순, 이상신, 허용, 정해원
A study on efficiency of culture by type of medium and inoculum, and of identification by culture temperature in Leptospires isolation for epidemiological mass survey
J.S Kim, S.S Lee, Y Heo, H.W Chung
Abstract
This study was carried out on 37 febrile patients occurred in the endemic area of Korean hemorrhagic fever during the September 1986. The objective of the study was to explore the most efficient method of leptospiral culture and identification by comparing different type of culture medium and different kind of inoculum as well as different culture temperature for epidemiological survey. Fletcher’s medium containing 8% rabbit serum was compared with the newly developed medium by Lee, YUMC-5050A. This is made of MEM tissue culture medium(GIBCO) with 0.45gm Hepes buffer and 1ml 1.2% pyruvate / 50ml of the medium by adding ultra filtered(0.2μm pore size)urine of healthy person in 1:1 ratio. All media prepared were incubated in 30C incubator for 1-2 weeks to be sure that there was no contamination in the process of preparation. Four different kind of inoculum, whold blood, serum separated solid part of blood(blood cells) and hemolyzed precipitate of blood(5000G centrifugation) were all inoculated to rabbit serum containing Fletcher’s and YUMC-5050A medium in duplicate for comparison. Nine strains of the leptospires isolated were inoculated to the semisolid Fletcher’s medium in quadriplicate, cultured in 30°C incubator until the full grown colonies became recognizable, then the half of the culture were kept in 4°C refrigerator and the other half were kept in 30°C continuously(control) and checked morphological changes of the leptospires every six hours. The results obtained are summarized as followings: 1) Among 37 febrill patients 32 revealed leptospires in blood culture, which suggested that most febrile patients were from the leptospiral infection. 2) The YUMC-5050A medium was more efficient for massive leptospiral culture in terms of its cost and leptospiral growth rate than the Fletcher's medium; the leptospiral culture positive rate regardless of inoculum was significantly higher in the YUMC-5050A than Fletcher, and that the YUMC-5050A was less expensive because the rabbit serum is not only expensive but also sometimes difficult to buy immediately. 3) Leptospiral culture positive rate was the highest(86.5%) when the hemolysed precipitates were inoculated among other inocula; whole blood(21.6%), serum(21.6%), blood cells(18.9%). 4) When the fully grown leptospiral culture were kept in 4°C refrigerator for 36 hours, over 95% of the bacteria formed typical spiral forms whereas the control culture kept in 30°C incubator for the same period of time showed spiral form bacteria in only about 10%.
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