Farm milk samples, which were positive in the routine assay for inhibitory substances detected by the Arla microtest but negative in the Delvotest SP, were used in the study. All samples analyzed gave positive results in the determination of tetracyclines and macrolides by the Charm II microbial receptor tests. To confirm the presence of tetracyclines, liquid chromatography and the Charm II immuno-receptor test were used. All samples, except one, showed negative results in the analyses by both these methods. This suggested that some other factor(s) was causing the observed positive results for tetracyclines and macrolides. Circumstantial evidence indicated that free fatty acids (FFA) may be a causative factor. Consequently, the influence of FFA on the Arla microtest, Valio T101, Delvotest SP, and the tetracycline and macrolide determinations by the Charm II test was investigated. Lipolysis of milk fat was induced by the addition of human blood serum, followed by storage for 24 and 48 h at 4°C. Samples were found to be inhibitory in the Arla microtest and the Valio T101 test at FFA levels of 4–5 mM, whereas the results of the Delvotest SP remained negative. The Charm II counts for tetracyclines and macrolides as determined by the microbial receptor tests were also highly correlated with FFA. It was concluded that lipolysis of milk fat may cause positive results in the microbial nonagar assays and interfere in the confirmation by the Charm II test.

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