Sterile buffered peptone water

All formulations’ organoleptic characteristics, such as appearance, color, odor, and pH, were evaluated. The pH values were determined at room temperature using a pH-meter (Mettler Toledo, Columbus, OH, USA) in the production day (T₀) and 6 days after production (T₆).
To evaluate the microbiological contamination of all formulations, microbiological control tests were carried out using a spread-plating method, always in sterile conditions. In this assay, Trypticase Soy Agar (TSA) (BIOKAR Diagnostics) and Sabouraud Dextrose Agar (SDA) (BIOKAR Diagnostics) were used as media. After autoclaving, the media were distributed by Petri plates with 90 cm in diameter (VWR). The formulations were diluted 1:10 and 1:1000 in sterile Buffered Peptone Water (BIOKAR Diagnostics) pH 7.0. The TSA and SDA media plates were inoculated with 100 μL of each sample dilution (n = 1) and were incubated at 30 °C for the SDA medium and at 37 °C for the TSA media, for 3–5 days. The number of colony-forming units (CFU) that developed during the incubation period (3–5 days) was evaluated and recorded. The count of total microorganisms corresponds to the average of CFU present in the TSA and SDA plates multiplied by the dilution factor.

One gram of homogenised faecal sample was added to 10 mL of sterile buffered peptone water (Biokar diagnostics) and plated onto SuperPolymyxin medium [19 (link)], an eosin methylene blue agar (Biokar diagnostics) supplemented with 3.5 μg/ml colistin (Sigma-Aldrich), 10 μg/ml daptomycin (Glentham Life Sciences), and 5 μg/ml amphotericin B (Glentham Life Sciences), and incubated for 24 h at 37°C. For each faecal sample, up to five colonies with a E. coli phenotype were isolated (i.e. metallic green sheen in SuperPolymyxin medium).

A total of 156 samples of sausages distributed as follow: 60 of turkey sausages, 60 of beef sausages, and 36 of “Merguez” sausages were randomly collected from various local supermarkets, weekly market, butcheries, and street vendors. The collection was carried out during 1 year from March 2014 to February 2015. The samples were aseptically collected, and each sample was placed in a separate, sterile plastic bag. The samples were brought under refrigeration to the laboratory and analyzed within the following 2 h. The samples (25 g) were weighed into sterile stomacher bags diluted with 225 mL sterile buffered peptone water (Biokar) and homogenized in a stomacher for about 1 min; 0.1 mL was streaked on Baird-Parker (BP) agar (Biokar) supplemented with egg yolk tellurite emulsion and incubated at 37°C for 24-48 h. Strains cultured on BP agar medium were identified as S. aureus if growth was observed and the colonies showed the typical morphologic characteristics (black colonies with an opaque precipitation halo). The tube coagulate test was determined and evaluated for coagulation after 3 and 24 h of incubation.