Evaluation of a modified EC Four Plate Method to detect antimicrobial drugs

This paper describes a modification of the EC Four Plate Method based on microbial growth inhibition of Bacillus subtilis on agar medium at pH 6.0, 7.2 and 8.0 and Micrococcus luteus at pH 8.0 developed to cope with large numbers of samples. The method's performance was evaluated by determining the Minimum Inhibitory Concentrations of 66 commonly used drugs and determining the between-assay variation of antimicrobial control standards. The modified method proved particularly sensitive for beta-lactams, tetracyclines, quinolones, marcrolides and lincosamides and least sensitive for anticoccidials and nitrofurans. The pH 6.0 and 7.2 plates were more sensitive for 39 of the 66 antimicrobials (59%) whereas the two pH 8.0 plates (B. subtilis, M. luteus) were the most sensitive for 27 (41%). Muscle samples were taken from 1830 routine meat inspection investigations between 1994 and 1996. Of the 38 (2%) positive meat inspection carcasses, the following antimicrobials were confirmed above the MRL: penicillin G (10), oxytetracycline (16), sulphadimidine and sulphadiazine in combination (4) and chlortetracycline (1). The method as described is technically simple, cost effective, robust, suitable for large sample throughput and for frozen, thawed or fresh tissues. When all four plates are used the pattern of inhibition can reduce unnecessary confirmatory assays by indicating the antimicrobial group most likely to be present.