Tryptone salt broth

Enumeration of EHEC O157:H7 was performed in a liquid medium by using a most probable number method. One gram of feces, stored at 4°C, was 10-fold serial diluted in tryptone salt broth (Oxoid, Dardilly, France). One milliliter of each dilution was added, in duplicate, to 9 ml of modified tryptone soya broth supplemented with novobiocin at 16 mg L⁻¹. Broths were incubated overnight at 37°C. In order to isolate EHEC O157:H7 from positive (turbid) broths, IMS-based isolations were performed. Twenty-five microliters of immunoconcentrated bacteria were plated onto CT-SMAC agar and ChromoID O157:H7 agar. For each positive broth, five plates of each agar were plated, and all media were incubated overnight at 37°C. Suspect colonies were tested by PCR for the presence of stx, eae-γ1, and rfbE_O157 genes. Enumeration was finally calculated from the number of EHEC O157:H7-positive duplicates or each dilution according to McCrady's tables, and expressed as the most probable number per gram of feces (MPN/g) (17 ). In order to perform generic E. coli counts, 10 g of feces was homogenized in 90 ml of saline solution, and 10-fold serial dilutions were prepared. Decimal dilutions were plated onto Petrifilm^TM Select E. coli (Grosseron, Saint Herblain, France). Enumerations were performed after incubation at 42°C during 24 h.

Feces (10 g) were mixed with 90 mL of a tryptone salt broth (Oxoid). Suspensions were transferred to sterile stomacher bags and homogenized for 2 min in a stomacher blender. Serial dilutions were made in tryptone salt broth. Dilutions were inoculated in Slanetz-Bartley Agar (Oxoid) (37 °C, 24–48 h). Up to four red-colored colonies were selected to collect a variety of enterococcus strains. For further identification, catalase, growth in Bile Esculin Agar (Oxoid), and trypticase soy broth + 6.5% NaCl tests were done. If there was any doubt about the identification, the API STREP (BioMérieux, France) was used.

The analysis was carried out using 25 g of homogenized food in 225 mL of pre-enrichment diluent tryptone-salt broth (Oxoid, Dardilly, France) using a Stomacher-type homogenizer. Further decimal dilutions were carried up to 10⁻⁵. Thereafter, the corresponding dilutions were plate-counted in accordance with the standard reference culture method recommended by the International Organization for Standardization [91 ] for the enumeration of coagulase-positive staphylococci, using Baird Parker with egg yolk emulsion (BPEY) incubated at 37 °C for 24–48 h. From each food sample processed, one presumptive Staphylococcus aureus colony was subcultured on BPEY (Oxoid, Dardilly, France) and purified by repeated streaking. The pure cultures were stored at −80 °C in brain heart infusion broth (BHI; Conda Pronadisa, Madrid, Spain), amended with 0.6% yeast extract (Biolife Italiana, Milano, Italy) added with 20% glycerol.