Abstract: Here, we adopted microbiological cylinder plate method to detectβ-lactamase in milk. Since penicillin inhibits the growth of Micrococcus luteus, producing a zone of inhibition, the difference between the diameters of inhibition zone produced by penicillin alone and together with sulbactam, which specifically inhibits the activity ofβ-lactamase, will indirectly suggest whether or not a sample contains the enzyme. The optimum working concentration of penicillin G determined in this study was 0.5μg/mL. The influence ofβ-lactamase products from different manufacturers with different given units on the antimicrobial activity of penicillin G in liquid skim milk was evaluated, and the results showed that the limits of detection of the developed method for them were different. Addition of sulbactam at 50μg/mL provided optimum detection ofβ-lactamase. Based on the above investigations, the optimum protocol for detectingβ-lactamase by cylinder plate method in milk was that the presence or not ofβ-lactamase was identified based on differences in the diameter of inhibition zone among additions of penicillin G alone or in combination with sulbactam or/andβ-lactamase to one sample together with the results from constructed positive and negative controls.

Key words: β-lactamaseccy, linder plate method, penicillin G, sulbactam, milk