Palcam agar

Microbial analysis was performed at intervals of 5 days until 60 days of ripening and at intervals of 10 days from 70 to 160 days of ripening. The 25 g of cheese was cut aseptically from the cheese block and placed into a filter bag (3M^™, St. Paul, MN, USA). Fifty milliliters of 0.1% buffered peptone water (BPW; Becton, Dickinson and Company) was added to the filter bag, and the bag's contents were homogenized for 60 s using a pummeller (BagMixer^® 400W, Interscience, St. Nom, France). Homogenates were serially diluted with 0.1% BPW, and the 0.1 ml dilutions were plated on PALCAM agar (Oxoid Ltd., Basingstoke, Hampshire, UK) with 0.4% of PALCAM supplement. The plates were incubated at 30°C for 48 hr for L. monocytogenes enumeration. To enumerate Lactobacillus spp., the dilutions were also plated on Lactobacilli MRS agar (Becton, Dickinson and Company) and incubated at 37°C for 24 hr.

Salad samples (25 g) were added to 225 mL of Fraser Broth (Oxoid) supplemented with Fraser Selective Supplement SRO156E (Oxoid) and homogenized in a stomacher (BagFilter, Interscience, FR) for 2 min. Triplicate series of tubes containing decimal serial dilution from 10 to 10⁻⁵ grams of homogenate were incubated for 48 hours at 37°C in the same media. After incubation, aliquots of enrichment broth were taken from dark tubes (containing presumptive Listeria spp.) and streaked onto Oxford agar plates (Oxoid) and PALCAM agar (Oxoid). Plates were incubated at 37°C for 48 h and five typical colonies were picked for purification on TSA + 0.6% yeast extract plates (Oxoid). Then, plates were incubated for 24 h at 37°C and Gram-positive, catalase positive colonies were streaked on blood agar (37°C, 24 h). Hemolytic colonies were identified as L. monocytogenes using API Listeria strips (Biomerieux, Marcy l'Etoile, FR). The MPN value and 95% confidence intervals were determined by the number of positive tubes obtained in serial dilutions as reported by the USDA guidelines [31 ].

To assess the practical feasibility of the novel MCDA method, we employed the L. monocytogenes-MCDA assay to the routine detection for 55 pork samples, and compared the detection results with the conventional PCR and ISO 11290-1 standard methods. A PCR assay, which was developed by Wang Y et al., was carried out to verify the presence of L. monocytogenes strains in food samples13 (link). The culture-biotechnical detection of L. monocytogenes was performed according to the manufacturer’s instructions of ISO 11290-1 method. In brief, 25 g of each pork sample was added into 225 ml Listeria enrichment broth (Half Fraser’s broth, Oxoid, Hampshire, UK), homogenized and incubated at 30 °C for 24 h. Then, 0.1 ml of each sample was placed into 10 ml of Fraser’s broth (FB) in a culture tube and incubated at 37 °C with shaking (250 rpm) for 48 h. Aliquots (0.05 ml) of positive FB cultures were plated on PALCAM agar (Oxoid), followed by incubating at 37 °C for 24 or 48 h. The identification of L. monocytogenes strains was confirmed by subsequent morphological, biotechnical and serological tests. Aliquots (1 ml) of the enriched cultures were subjected to extracted DNA, which was used as the template in the PCR and MCDA assays.

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)].

This investigation used three L. monocytogenes (ATCC 15313, ATCC 19115, and ATCC 1917) strains. In a 50 mL conical tube (SPL Life Sciences Co., Ltd., Gyeonggi-do, Republic of Korea), 100 µL of aliquot cultures (10⁸ to 10⁹ CFU/mL) was inoculated into 10 mL of tryptic soy broth (TSB; BD Difco, Sparks, NV, USA) at 30 °C for 24 h at 220 rpm in a shaking incubator (VS-8480, Vision Scientific, Gyeonggi-do, Republic of Korea). A portion of the cultured culture (100 µL) was placed in 10 mL TSB for another 24 h. After centrifuging at 10,000× g for 10 min at 4 °C, the samples were washed with Dulbecco’s phosphate-buffered saline (DPBS; Sigma-Aldrich, St Louis, MO, USA), and then diluted in peptone water (PW; BD Diagnostics, Franklin Lakes, NJ, USA). The mixed culture was made by mixing equal quantities of each strain. The mixed culture was used to make a suspension of 10⁵ CFU/mL and cultured on PALCAM agar (Oxoid, Hampshire, UK) medium at 30 °C for 24 h.

The following microorganisms were tested: Staphylococcus aureus ATCC 6538P, Escherichia coli ATCC 25922, Salmonella enteritidis ATCC 13076 and Listeria monocytogenes ATCC 19114. Each strain was grown in a test tube containing 10 mL sterile nutrient broth (Oxoid Ltd., Basingstoke, Hampshire, UK) at 37 °C for 24 h. The purity of the inoculum was confirmed by microscopic examination of the Gram-stained smear. A loopful of inoculum was transferred to selective medium: Baird-Parker agar base supplemented with egg yolk tellurite emulsion for S. aureus, TBX agar for E. coli, XLD agar for Salmonella enteritidis (Oxoid Ltd., Basingstoke, Hampshire, UK) and Palcam agar (Oxoid Ltd., Basingstoke, Hampshire, UK) for Listeria monocytogenes. Plates were incubated for 24 h at 37 °C. Bacterial morphology was confirmed by optical microscopy. Several colonies were transferred to sterile saline solution (8.5 g/L), and adjusted to match the turbidity of McFarland 0.5 standard (1.5 × 10⁸ CFU/mL) [36 (link),37 (link)].