Simplified classification method for residual antibacterial agents in meat by microbiological assay

A simplified classification method was developed for the detection of residual antibacterial agents in meat by microbiological assay. Bioassay was carried out by a pulp disc method using Bacillus subtilis ATCC 6633, Micrococcus luteus ATCC 9341 and Bacillus cereus var. mycoides ATCC 11778. Antibacterial agents were grouped into type I including penicillins (PCs) and macrolide antibiotics (MLs), type II including aminoglycoside antibiotics (AGs), type III including tetracyclines (TCs), and type IV including sulfa drugs (SAs), according to the patterns of growth inhibition of these three strains. Animal tissues were homogenized in 0.01M di-Na EDTA Mcllvaine buffer (pH 4.0) and centrifuged. Hexane was added to the supernatant and the mixture was centrifuged The aqueous layer 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 followed by a BAKER 10 SPE COOH column. The Sep-Pak C18 cartridge column was washed with water and adsorbed antibacterial agents were eluted with methanol. The eluate was evaporated to dryness. The residue was dissolved in phosphate buffer (pH 4.5) to prepare fraction B. The antibacterial agents on the BAKER 10 SPE COOH column were eluted with 5ml of Mcllvaine buffer (pH 3.0). The eluate was adjusted to pH 8.0 with 1N NaOH to prepare fraction C. Fractions A, B and C contained MLs and SAs, TCs and PCs, and AGs, respectively. Antibacterial agents in each fraction were confirmed by the different patterns if growth inhibition of the above three strains.