Investigations on the reproducibility of microbial inhibitor test results in raw milk after sample storage

Foissy, H.; Lindner, G.; Fugger, F.; Wagner, M.

Wiener Tierärztliche Monatsschrift 92(5): 126-130


ISSN/ISBN: 0043-535X
Accession: 012919399

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Introduction: In Austria the determination of antibiotic residues in bulk raw milk is mainly done on a monthly basis by using the brilliant black reduction test and with Bacillus stearothermophilus var. calidolactis as an indicator organism. The presence of beta -lactam antibiotics and sulfonamides is confirmed in subsequent individual tests. In presumptive positive cases when the milk producers insist on a reevaluation using an additional sample the reproducibility of the earlier test result may be ambiguous due to possible changes occurring in the parallel sample during sampling and storage. Material and methods: In model experiments, 9 conventional antibiotics were used and serially diluted in raw milk up to the extinction limit of the positive signal in the BR-TestReg. AS-Brilliant from DSM (Deft, The Netherlands). The diluted samples were then stored at +6 degrees C and -18 degrees C and tested for the lowest concentration showing a positive result (all samples), as well as for the concentration second to the lowest concentration (6 samples) in the brillant black reduction test. The samples were initially reinvestigated daily for 3 days, when stored at +6 degrees C, or initially daily and then weekly, when stored at -18 degrees C. Results: Already the first day after the beginning of the experiments the results of 11 out of 15 samples were non-reproducible. In the frozen samples, 2 and another 3 samples gave negative results on the first and the second day after the beginning of the experiment, respectively. After a week, the results of 8 samples did not comply with the results found at the starting point of the analyses. Only in 4 samples stored at -18 degrees C, the results remained reproducible over a 4 week period. Conclusion: These data prove that a reexamination of presumptive positive results obtained in the brilliant black reduction test may lead to a bias in data interpretation, especially when low concentrations are present. Only positive results serve as a proof of antibiotic residues in milk, but negative results are not prone to confirm a negative result in the preceding investigation.