Half fraser broth

L. monocytogenes was isolated and identified, according to ISO 11290-1 [18 ]. Briefly, the samples were inoculated into a selective primary enrichment medium (half Fraser broth, Oxoid) and incubated at 30°C for 24 h, followed by inoculation of 0.1 μL of the incubated broth into a full-strength secondary liquid enrichment medium (full Fraser broth, Oxoid). After incubation at 37°C for 24 h, the broth was streaked on Agar Listeria according to Ottaviani and Agosti (ALOA) medium and Oxford agar (LabM and Oxoid) and incubated at 37°C for 24-48 h. The typical colonies were blue-green surrounded with or without an opaque halo on ALOA agar and olive green with a black halo on Oxford agar. Then, the Listeria strains were identified using API strips (BioMérieux, France).

Listeria spp. were isolated and detected using ISO11290-1 method [22 ]. Briefly, samples were pre-enriched by half Fraser broth (Oxoid, Basingstoke, UK) and enriched by Fraser broth (Oxoid, Basingstoke, UK) for 48 and 24 h at 37 °C, respectively. Finally, the enriched Fraser broth-culture was streaked onto Palcam agar (Oxoid, Basingstoke, UK) and Oxford agar (Oxoid, Basingstoke, UK) followed by 24 to 48 h incubation at 37 °C. The presumed colonies were verified by biochemical tests and API Listeria (bioMérieux, Marcy l’Étoile, France). The isolates of Listeria spp. were then further confirmed by PCR [23 (link)].

Isolation was performed on the basis of the ISO 11290–1:2017 standard. Each 25 g food sample was inoculated in 225 mL half Fraser broth (Oxoid, Basingstoke, United Kingdom) as primary selective enrichment and homogenized in a stomacher (Seward 400, Radnor, PA, United States). Incubation was performed at 30 ± 1°C for 25 ± 1 h; the second enrichment consisted of 0.1 mL of the broth culture inoculated in 10 mL of full-strength Fraser broth, which was cultured at 37°C for 24 ± 2 h. A loopful of each of the half- and full-strength Fraser broths were plated on the Listeria chromogenic agar base according to Ottaviani and Agosti (ALOA) (Merck, Darmstadt, Germany). These plates were incubated at 37°C for 24–48 h. Five typical colonies from each ALOA agar plate were restreaked on tryptic soy agar supplemented with 0.6% yeast extract (TSA-YE) (Sigma, Darmstadt, Germany) as a non-selective medium, and these were incubated at 37°C for 24–48 h. Colonies from the TSA-YE were verified by Gram staining, catalase reactions, oxidase tests, carbohydrate utilization test, CAMP tests, and motility at 20–25°C. These colonies were stored for further study.

Isolation was performed according to the ISO 11290–1:2017 method with two-stage enrichment. First, 25 g of each food sample was inoculated in 225 mL half Fraser broth (Basingstoke, Oxoid, UK) for initial selective enrichment and homogenized in a stomacher (Seward 400, Radnor, PA, USA). Incubation followed at 30 ± 1 °C for 25 ± 1 h and 0.1 mL of the broth culture was inoculated in 10 mL of full-strength Fraser broth for the second enrichment, which was cultured at 37 °C for 24 ± 2 h. A loopful (1 mL) of each of the half- and full-strength Fraser broths were plated on the chromogenic Listeria agar Ottaviani and Agosti (ALOA agar) (Merck, Darmstadt, Germany). The plates were incubated at 37 °C for 24 to 48 h. Five typical colonies from each ALOA agar were restreaked on tryptic soy agar supplemented with 0.6% yeast extract (TSA-YE) (Sigma, Darmstadt, Germany) as a nonselective medium and incubated at 37 °C for 24 to 48 h. The colonies from TSA-YE were verified by Gram staining, catalase reactions, oxidase tests, carbohydrate utilization, CAMP tests, and motility at 20 to 25 °C. Listeria monocytogenes was enumerated in the samples according to the methodology described in the ISO 11,290–2:2017 Microbiology of the food chain standard—Horizontal method for the detection and enumeration of L. monocytogenes and Listeria spp.—Part 2: Enumeration method [17 ,18 ].

Detection of L. monocytogenes was performed as described by ISO 11290-1 for the isolation of such bacteria from food [20 (link)]. First for the enrichment of L. monocytogenes, 25 g of each poultry sample was diluted in 225 ml of Half Fraser broth (Oxoid, UK) and homogenized in a blender for 2 minutes. The hand swab samples were transferred to 10 ml of Half Fraser broth. Homogenates of poultry samples and swabs were incubated at 30 °C for 24 h. After that, 0.1 ml of pre-enriched culture was added to 10 ml of Fraser broth and incubated at 30 °C for 24 h. Each Fraser broth culture was streaked onto Palcam agar (Oxoid) and incubated at 37 °C for 48 h. Approximately five colonies of the growing Listeria specieswere purified and underwent further biochemical identification using catalase test, oxidase test, sugar fermentation test, and evaluation of hemolysis type [21 (link)]. The biochemically confirmed strains of L. monocytogenes in the present study were further verified using the API Listeria test (BioMerieux).