Evaluation of a single plate microbiological growth inhibition assay as a screening test for the presence of antimicrobial agents in compound animal feedingstuffs at therapeutic and contaminating concentrations

The Inhibitory Substance Test (IST), a microbiological growth inhibition te st, is used for screening animal feedingstuffs for the presence of (contami nating) antimicrobial compounds. The effectiveness of the IST was establish ed for 33 compounds that may be incorporated ill feeding stuffs. Minimum de tectable concentrations (MDCs) for standard solutions were established and compared with those obtained following solvent extraction of an antimicrobi al-free compound feedingstuff spiked with each compound at 0-20 mg/kg. Of t he 33 standard solutions examined the test organism was not sensitive to II and the MDC for one was greater than its maximum inclusion rate in complet e feedingstuffs. Following routine extraction (25%) acetone-phosphate buffe r of feedingstuffs spiked with each of the 22 compounds to which the organi sm was sensitive, 10 were not detected, 15 were detectable at both minimum and maximum feed-inclusion rates and four were only detectable at their max imum feed-inclusion rates. Extraction with methanol (25%) had a deleterious effect with 12 compounds not detected, nine detectable at both minimum and maximum feed-inclusion rates and five detectable at their maximum feed-inc lusion rates. Increasing acetone and methanol concentrations to 40 and 55% respectively resulted in larger inhibitory zones for antibiotic-free feedin gstuff (25.3 + 2.43 mm vs 21.1 + 1.02 mm) compared with both 25% acetone (1 1.3 + 0.22 mm) and 25% methanol (11.2 + 0.22 mm), requiring the establishme nt of greater threshold zone diameters and negating any advantage in increa sing the solvent concentration under these test conditions. If is concluded that the IST may be particularly useful for detection of a number of the z ootechnical feed-additives recently, banned in the EU, which, if used illeg ally, may be present at sufficiently high inclusion rates to facilitate det ection. Further alteration of extraction conditions may improve the scape o f the assay.