Modified charcoal cefoperazone deoxycholate agar plates

Thermophilic Campylobacter was quantitatively detected from poultry neck
skin samples essentially according to ISO 10272-2:2017^{8 )}. Briefly, after homogenization with 225 mL of buffered
peptone water (Oxoid, Hampshire, UK), a total of 1 mL of the homogenates and serial
dilutions were spread on modified charcoal-cefoperazone-deoxycholate agar plates (Oxoid),
followed by microaerophilic incubation at 41.5 °C for 48 h using an AnaeroPack-Microaero
(Mitsubishi Gas Chemicals, Tokyo, Japan). Per sample, five suspected colonies grown on the
plates were subjected to PCR-based identification using C. jejuni- and
C. coli-specific primers, as previously described^{19 (link))}.

Cecal samples were homogenized with Campylobacter enrichment medium (25 ) and incubated at 37°C for 48 h in a microaerobic environment for enrichment. A loopful of each homogenate was streaked on modified charcoal cefoperazone deoxycholate agar plates (Oxoid, Basingstoke, UK). After an incubation at 37°C for 48 h in a microaerobic environment, white-colored colonies assumed to be Campylobacter isolates were identified at the species level using multiplex PCR (53 (link)).

For Campylobacter spp. isolation, the obtained specimen in Bolton broth was incubated at 42 °C/24–48 h in darkness in a microaerophilic atmosphere (85% N₂, 10% CO₂, and 5% O₂) utilizing an anaerobic jar (Sigma-Aldrich, St. Louis, MO, USA) and CampyGen sachets (Oxoid, Cambridge, UK). Subsequently, 0.1 mL of the inoculated Bolton broth was inoculated onto the surface of supplemented modified charcoal cefoperazone deoxycholate agar (mCCDA) plates (Oxoid, Cambridge, UK), then the inoculated plates were incubated at 42 °C/48 h in a microaerophilic atmosphere. For further purification, the suspected campylobacter colonies were cultivated on 5% sheep blood agar plates (Oxoid, Cambridge, UK), then incubated at 42 °C/48 h in a microaerophilic atmosphere. Next, the suspected colonies were presumably identified through their culture characters on mCCDA and blood agar, Gram’s staining, motility test, susceptibility to nalidixic acid and cephalothin, and biochemical identification procedures, including oxidase, catalase, rapid sodium hippurate, and indoxyl acetate hydrolysis [23 (link)].