IMPROVEMENTS OF THE TUBE DIFFUSION METHOD FOR DETECTION OF ANTIBIOTICS AND SULFONAMIDES IN RAW-MILK

Recently improvements have been made in the tube diffusion method used in the Netherlands for detecting antibiotics and sulfonamides in raw milk. Samples shown to inhibit growth of Bacillus stearothermophilus v ar. calidolactis in the screening method are now heated for 10 min at 80-degrees-C to exclude the inhibitory effect of any lysozyme and lact oferrin complex. Inhibition of growth in the heated milks, which can b e overcome by adding para-amino benzoic acid (PABA), shows the presenc e of sulfonamides. If no such loss of inhibition occurs, a quantitativ e disc-plate method, including incubation with different concentration s of penase (penicillinase, a beta-lactamase) (EC 3.5.2.6) and differe nt ways of incubation is used to determine concentration of inhibitory substances and the presence of penase-labile and penase-stable beta-l actam antibiotics. This procedure will be modified again in 1993 by th e introduction of a screening test in which the initial pH of the test medium is adjusted to 8.0 rather than 7.0. Streptomycin, neomycin, sp iramycin, erythromycin, dapsone and sulfamethazine are detected to a l ower limit at the higher pH. Benzylpenicillin and ampicillin have some what higher, though acceptable, detection limits. If inhibition remain s in the presence of PABA, the samples will be further tested with add ed penase for the presence of beta-lactam antibiotics with disc-plates at pH 7.0, and with the Charm II test for other (groups of) antibioti cs. In this modified procedure, the concentration of inhibitory substa nces is no longer determined and different groups of beta-lactam antib iotics are no longer distinguished. Possibly some variants of the tube diffusion method will be applied in future as a screening test in ord er to bring the detection limits closer to or beyond the toxicological ly safe levels.