실험에는 Bacillus subtilis BGA, Bacillus cereus var. mycoides ATCC 11778, Micrococcus luteus ATCC 9341을 상법에 따라 제작하여 muller hinton agar(Difco)에 각각 접종한 뒤, 3가지 조건의 pH가 조정된 검사용 평판배지(plate)인 B. subtilis(pH 6.0), B. cereus(pH 6.0), B. subtilis(pH7.2), B. subtilis(pH 8.0), M. luteus(pH 8.0)의 EEC 4-plate변법을 이용하였다. 넙치에 옥시테트라싸이클린(OTC)을 농도 100 mg/kg/day으로 단 1회 강제 경구 투여한 후 1일, 3일, 5일, 10일, 15일, 20일, 25일 30일마다 3마리씩 채취하여 시료의 처리를 각각 달리한 근육직접법, 추출디스크법, 직접디스크법의 3종류 간이스크리닝법으로써 OTC의 잔존유무를 분석하였다 B. subtilis(pH 6.0)배지는 투여 후 1일째 근육직접법에서만 양성반응으로 의심되는 의양성 반응(${pm}$)을 나타내었을 뿐 30일까지 간이스크리닝법 3종류가 모두 음성 반응을 나타내었다. B. subtilis(7.2), B. subtilis(8.0), M. luteus(8.0)배지는 투여 후 1일째부터 30일까지 간이스크리닝법 3종류가 모두 음성반응을 나타내었다. 그러나 B. cereus(pH 6.0)배지는 1일째부터 15일까지 간이스크리닝법 3종류가 모두 명확한 양성반응, 투여 후 20일째 근육직접법과 직접디스크법은 의양성반응(${pm}$)을 보였으나 추출디스크법은 분명한 음성반응, 그리고 투여 후 25일째 및 30일째는 시험조작법 모두가 음성반응을 나타내었다. 따라서 근육직접법, 추출디스크법, 직접디스크법의 간이스크리닝법 3종류는 OTC의 양성반응 판정에 커다란 차이가 없었으며, B. cereus는 OTC의 모니터링에 유효한 균주라는 사실을 확인하였다.
By standardized method, Bacillus subtilis BGA, Bacillus cereus var. mycoides ATCC 11778, and Micrococcus luteus ATCC 9341 were seeded on the muller hinton agar (Difco) plate, and pH was adjusted to 6.0, 7.2, and 8.0. Five agar plates, B. subtilis (pH 6.0), B. cereus (pH 6.0), B. subtilis (pH 7.2), B. subtilis (pH 8.0), and M. luteus (pH 8.0), were employed as test plates of modified EEC 4-plate method. Oxytetracycline (OTC) with a diet was orally administered to flounder, Paralichthys olivaceus, at 100 mg/kg once a day. After oral administration, modified EEC 4-plate method by the three screening test using muscle-direct, extraction-disk and direct-disk methods was conducted for 3 fish at 1, 3, 5, 10, 15, 20, 25 and 30 days. Muscle-direct treatment of B. subtilis (pH 6.0) was found to be dubious positive (${pm}$) at the 1st day after the administration; thereafter, it was found to be negative to the last day of the experiment. Extraction-disk and direct-disk treatment of B. subtilis (pH 6.0) were found to be negative from the 1st day to the last day after the administration. B. subtilis (pH 7.2), B. subtilis (pH 8.0), and M. luteus (pH 8.0) by the three screening tests, were found to be negative all the way after the administration. On the other hand, B. cereus (pH 6.0) by the three screening tests was clearly found to be positive for the first 15 days after the administration, and then muscle-direct and direct-disk treatment of B. cereus (pH 6.0) were found to be dubious positive at 20th days after the administration. However extraction-disk treatment of B. cereus (pH 6.0) was clearly found to be negative at the same stage; thereafter, the three screening tests of B. cereus (pH 6.0) were found negative to the last of the experiment. These findings showed that to have equal sensitivity to those determination for the residual detection of OTC, and also confirmed that B. cereus was effective test organism for the monitoring of OTC.
