Polymyxin b supplement

All Staphylococcus strains were grown in tryptic soy broth (TSB; Difco, Detroit, MI, USA) at 37 °C. All Bacillus, Listeria, Streptococcus strains were grown in brain heart infusion (BHI; Difco) broth at 37 °C. Gram-negative bacteria were grown in LB broth at 37 °C. All tested bacteria were grown under aerobic conditions. Escherichia coli DH5α and BL21 (DE3) star strains were used in the cloning and expression of proteins, respectively. Baird-Parker agar plates with egg yolk tellurite (BPA; Difco) were used for selective enumeration of S. aureus and a Bacillus cereus selective agar plate with egg yolk emulsion and polymyxin B supplement (Oxoid) was used for selective enumeration of B. cereus.

All chemicals used were of analytical grade. Potassium chromate, 0.1 N Sodium hydroxide, silver nitrate, methyl alcohol, ethyl alcohol, and sodium chloride were obtained from Duksan Pure Chemicals (Ansan, Gyeonggi-do, Korea). Sodium hydroxide, sodium hydrogen carbonate, and ammonium hydroxide were purchased from Junsei Chemicals (Tokyo, Japan). Formalin solution, standard methanol, ethanol, pentanol, propanol, and butanol were purchased from Sigma-Aldrich (St. Louis, MO, USA). Plate count agar, nutrient agar, potato dextrose agar (PDA), and potato dextrose broth were purchased from Difco (Becton, Dickinson and Company, Sparks, MD, USA). Mannitol egg yolk polymyxin agar (MYP), egg yolk emulsion, and polymyxin B supplement were purchased from Oxoid LTD (Basingstoke, Hampshire, UK). 3M Yeast and Mold Petrifilm was purchased from 3M Health Care (St. Paul, MN, USA). API 50CHB and API 20E were obtained from bioMerieux (Marcy I’Etoile, France).

Detection of presumptive B. cereus was performed as previously described [47 (link)]. In brief, 25 g of selected food product was placed in a sterile filter stomacher and enrichment broth composed of Tryptone Soy Broth (TSB) (Oxoid, UK) and 50,000 IU of polymyxin B supplement (Oxoid, Hampshire, UK) was added to the total of 250 g. Enrichment of homogenized samples was performed at 30 °C for 24 h, after which 0.1 mL was inoculated onto a mannitol-egg yolk-phenol-red polymyxin-agar medium (Oxoid, Hampshire, UK), supplemented with polymyxin B according to manufacturer instructions and incubated at 30 °C for 48 h.