Purpose: Mannitol-yolk-polymyxin B agar (MYPA) was modified by supplementation with trimethoprim. The ability of the supplemented medium to select for and recover Bacillus cereus from pure cultures and food samples with high background microflora was compared to MYPA.
Methods: For modified MYPA (mMYPA), trimethoprim dissolved in sterilized distilled water with 5 mM HCL was added to cooled MYPA to achieve 32 μg/ml final concentrations. B. cereus was experimentally spiked into red pepper powder, soybean paste, vegetable salad, and radish sprout, and then recovered on MYPA and mMYPA for comparison. Twenty-five grams of foods were spiked with 3-5 log CFU/g of B. cereusATCC 14579. Each spiked food sample was suspended in 225 ml of Butterfield’s phosphate-buffered water and then homogenized by a stomacher. After stomaching, 0.1 ml of homogenate was serially diluted in 0.85% saline, and then 0.1 ml of each dilution was inoculated onto the two plating media in duplicate. Plates were incubated at 30°C for 24 h and suspected colonies were enumerated. Plates that contained estimated 15-150 suspicious colonies were selected and 5 typical colonies on each plate were sub-cultured. Suspected colonies were finally confirmed with colony PCR. All experiments were repeated three times. Colony counts in food samples were converted to log CFU/g, and analyzed by using ANOVA (Duncan multiple range test).
Results: In all food samples, there was no difference in recoverability (P > 0.05) between MYPA (soybean paste, 3.33 ± 0.43; red pepper powder, 3.18 ± 0.20; radish sprouts, 3.33 ± 0.31; vegetable salad, 3.36 ± 0.19) and mMYPA (soybean paste, 3.52 ± 0.47; red pepper powder, 3.34 ± 0.24; radish sprouts, 3.32 ± 0.40; vegetable salad, 3.51 ± 0.23). However, the mMYPA exhibited better selectivity than MYPA, because differentiation and picking of suspected colonies for confirmation of B. cereuson MYPA was significantly hindered by dense competing microflora occupying all parts of the plates.
Significance: Addition of trimethoprim to conventional media could be a useful option to improve selectivity in foods with high background microflora.