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 var. calidolactis in the screening method are now heated for 10 min at 80-degrees-C to exclude the inhibitory effect of any lysozyme and lactoferrin complex. Inhibition of growth in the heated milks, which can be overcome by adding para-amino benzoic acid (PABA), shows the presence of sulfonamides. If no such loss of inhibition occurs, a quantitative disc-plate method, including incubation with different concentrations of penase (penicillinase, a beta-lactamase) (EC 3.5.2.6) and different ways of incubation is used to determine concentration of inhibitory substances and the presence of penase-labile and penase-stable beta-lactam antibiotics. This procedure will be modified again in 1993 by the 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, spiramycin, erythromycin, dapsone and sulfamethazine are detected to a lower limit at the higher pH. Benzylpenicillin and ampicillin have somewhat higher, though acceptable, detection limits. If inhibition remains in the presence of PABA, the samples will be further tested with added 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) antibiotics. In this modified procedure, the concentration of inhibitory substances is no longer determined and different groups of beta-lactam antibiotics are no longer distinguished. Possibly some variants of the tube diffusion method will be applied in future as a screening test in order to bring the detection limits closer to or beyond the toxicologically safe levels.
