PM Indicator Agar

Principle And Interpretation

PM Indicator Agar is used for rapid detection of trace amounts of penicillin in milk where AOAC has recommended Bacillus stearothermophilus qualitative discs (1). PM Indicator Agar is designed according to the formula published by the 18th Annual Meeting of the National Mastitis Council (2). This method is a modification of the method approved by the International Dairy Federation for the qualitative detection of penicillin in milk (3). Originally a medium called Reductase Medium was suggested by Reid and Brewer for detecting penicillin in milk by using Bacillus subtilis (4). The present medium is better, faster and more reliable than that of Reid and Brewer (This medium is designed to support and demonstrate the growth and acid formation by B. stearothermophilus, which is sensitive to penicillin and b-lactam residues). To demonstrate the presence of traces of penicillin in milk; the qualitative disc method is found to be more suitable which is also recommended by AOAC.

Inoculate the medium with 1 ml of uniformly dispersed B. stearothermophilus suspension prepared as per AOAC (1). Prepare dilutions of standard penicillin G (positive control) to give concentrations in the range of 0.005 to 0.1 units/ml. Use a suspension of inhibitor free non-fat dry milk for negative control.

For Screening Assay: Place blank control disc, negative control disc, positive control disc on agar surface. Invert the plate and incubate till 17-20 mm zones of inhibition are obtained around positive control disc.

Interpretation of Screening Assay

Test Sample Positive Negative Interpretation Disc Control Disc Control Disc No zone Clear zone No zone Negative test indicating 17 - 20 mm that significant amounts of inhibiting substances are not present.

Zone Clear zone No zone Negative test indicating < 14mm 17-20 mm that significant amounts of inhibitory substances are not present.

Clear zone Clear zone No zone Positive test indicating > 14 mm 17-20 mm that an inhibitor is present in test sample. Perform CONFIRMING ASSAY to determine if inhibitor is a beta-lactam residue.

Any reaction Any reaction Any zone Result indicates error in the test system. Any reaction No zone Any reaction Determine source of error.

For Confirming Assay: Inactivate milk sample by heating at 82°C for 2 minutes. Cool promptly to room temperature. Invert the plate and incubate as in screening assay.

Interpretation of Confirming Assay

Penicillinase/ Interpretation Inactivated Positive Inactivated Test Control Disc Milk Sample Disc Sample DiscClear zone Clear zone No zone Positive test > 14 mm 17-20 mm indicating the presence of beta- lactam residues.

Clear zone Clear zone Clear zone, Positive test > 14 mm 17-20 mm same size as indicating the test sample presence of inhibitor(s) other than beta-lactam residues.

Clear zone Clear zone Clear zone Positive test > 14 mm 17-20 mm substan- indicating presence tially smaller of beta-lactam than 14 mm residues as well as other inhibitors.