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食肉中に残留する抗菌性物質を微生物学的方法により簡易, 迅速に系統別に推定する検査法の開発をした. すなわち, 厚生省法の分別同定法を改良した簡易な方法で抽出. 分画し, その分画液を試験液とし, これに対するB. subtilis, M. luteus及びB. cereusの3試験菌の感受性パターンから抗菌性物質を系統別に推定する方法, すなわち, 簡易系統別検査法を考案した. 本法によれば, MLsとSAsはクロロホルム層に抽出される分画液A, TCsとPCsはC18カラムで抽出される分画液B, AGsは COOH型カラムで抽出される分画液Cにそれぞれ分画された.分画液Aに対してM. luteus>B. subtilisの順に感受性を示す場合はMLs, B. subtilis (SAs用平板) のみが感受性を示す場合はSAsであった. 分画液Bに対してB. cereus>B. subtilisの順に感受性を示す場合はTCs, M. luteus>B. subtilisの順に感受性を示す場合はPCsであった. 分画液Cに対してB. subtilis>B. cereusの順に感受性を示す場合はAGsであった.本法の検出感度は, MLs, TCs及びAGsは分別同定法と同等であり, PCs及びSAsは残留抗菌性物質検査としては十分満足できる値であった.本法を屠畜場で注射痕を認めたウシ及びブタの筋肉及び腎臓に応用した結果, PCs, TCs及びSAsがそれぞれ単剤で, SAs及びTCsあるいはPCs及びAGsが複合でそれぞれ検出され, 分別同定法及び簡易検査法より高い確率で検出できた.

A simplified method was developed for the detection of residual tetracyclines and sulfa drugs in honey by microbiological assay. A honey sample was homogenized in 0.01M di-Na EDTA McIlvaine buffer (pH 4.0) and centrifuged. The supernatant was mixed well with chloroform and centrifuged. The chloroform layer was evaporated to dryness and the residue was dissolved in phosphate buffer (pH 8.0) to prepare fraction A. The aqueous layer was passed through a Sep-pak C18 cartridge column. The Sep-pak C18 cartridge column was washed with water and absorbed antibacterial agents were eluted with methanol. The eluate was evaporated to dryness, and the residue was dissolved in phosphate buffer (pH 4.5) to prepare fraction B.Fractions A and B contained sulfa drugs and tetracyclines, respectively. The pulp disk method with Bacillus subtilis ATCC 6633 and Bacillus cereus var. mycoides ATCC 11778 as test organisms was employed for the assay of sulfa drugs and tetracyclines. As little as 0.05μg/g (oxytetracycline), 0.01μg/g (chlortetracycline) or 0.1μg/g (sulfa drugs) was detectable.Thin layer chromatographic identification was performed for positive test solutions. The test solution A was spotted on an aluminum oxide plate (Merck 5550) and a silica gel plate (Merck 5553), then developed in chloroform-methanol (7:3) and ethyl ether-chloroform-n-butanol (5:1:1), respectively. The test solution B was spotted on a cellulose plate (Merck 5552), which was developed in n-butanol saturated with water. Rf values were determined by bioautography with B. subtilis and B. cereus as the test organisms. Microbioautography was suitable for separation and identification of sulfa drugs and tetracyclines in honey. We used this method to identify sulfa drugs and tetracyclines detected in 22 samples out of 297 commercial honeys. It was found that 8 specimens contained oxytetracycline, 12 specimens chlortetracycline, 1 specimen sulfadimethoxine, and 1 specimen sulfisomidine.