ISO 17995:2019 was used as a reference method for the isolation of campylobacters from wastewater and surface water samples [27 ]. Briefly, water samples (500 mL) were prefiltered (1.4 μm glass filter; Duren, Macherey Nagel, Germany) for quick removal of mechanical impurities and filtered (0.22 μm, mixed cellulose ester filter; Merk, Darmstadt, Germany); the filters were then transferred into 2 campylobacter selective broths (Preston and Bolton broth) for enrichment and incubated at 42 °C in an anaerostat (AnaeroJar, Oxoid, Basingstoke, UK) under a microaerobic atmosphere (CampyGen 3.5 L, Oxoid, Basingstoke, UK). After 44 ± 4 h of incubation, the inoculum was cultivated on Campylobacter blood-free selective agar (modified charcoal–cefoperazone–deoxycholate agar (mCCDA), Oxoid, Basingstoke, UK) and incubated for another 44 ± 4 h. After incubation under a microaerobic atmosphere at 42 °C, isolation of presumptive colonies on nonselective agar (blood agar) and mCCDA was performed under the same conditions. Finally, Campylobacter spp. were identified (see below).
Mccda
Manufactured by Thermo Fisher Scientific
Sourced in United Kingdom
MCCDA is a culture medium used for the selective isolation and enumeration of Campylobacter species from clinical and food samples. It supports the growth of Campylobacter while inhibiting the growth of other bacteria.
Automatically generated - may contain errors
Lab products found in correlation
Bolton broth, by Thermo Fisher Scientific (4 mentions)
Sr0155e, by Thermo Fisher Scientific (2 mentions)
Campygen, by Thermo Fisher Scientific (2 mentions)
Charcoal broth, by Merck Group (1 mentions)
Campygen sachet, by Thermo Fisher Scientific (1 mentions)
Amphotericin, by Thermo Fisher Scientific (1 mentions)
Cefoperazone, by Thermo Fisher Scientific (1 mentions)
Potassium clavulanate, by Merck Group (1 mentions)
Campygen cn25, by Thermo Fisher Scientific (1 mentions)
Microaerophilic workstation, by Don Whitley Scientific (1 mentions)
Mueller hinton broth mh, by Thermo Fisher Scientific (1 mentions)
Lysed horse blood, by Thermo Fisher Scientific (1 mentions)
Sr0155, by Thermo Fisher Scientific (1 mentions)
Brucella broth, by Thermo Fisher Scientific (1 mentions)
Anaerojar, by Thermo Fisher Scientific (1 mentions)
Campygen 3.5 l, by Thermo Fisher Scientific (1 mentions)
Sheep s blood, by BD (1 mentions)
15 protocols using mccda
1
Isolation of Campylobacter from Water Samples
2
Cultivation of Pathogenic Isolates
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Purified isolates (Table 1 ) that were previously confirmed by PCR [5 (link)] were recovered from defibrinated horse blood at −80 °C and cultivated in modified charcoal cefoperazone agar (mCCDA, Oxoid, Basingstoke, UK), supplemented with mCCDA selective supplement (SR0155E, Oxoid, Basingstoke, UK) at 37 °C for 48 h microaerobically. They were subsequently sub-cultured onto the chosen inoculation medium for each assay and incubated for 48 h under the same conditions.
3
Isolation and Identification of Campylobacter
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Following incubation, the faecal samples in charcoal broth (Sigma-Aldrich, St. Louis, MO) were filtered through a 0.65 µm cellulose nitrate filter (Sartorius Stedim Biotech, Germany) onto mCCDA (Blood-Free Agar) (Oxoid). Approximately 500 µL of the incubated charcoal broth was evenly distributed over the filter aseptically, once the liquid had been filtered through, forceps were used to aseptically remove the filter. The culture plates were then set in an inverted position in an anaerobic jar containing an atmosphere generation system (CampyGen sachet, Oxoid) and then incubated at 37 °C for 48 h. Subsequent to incubation, DNA was isolated for the purposes of species identification using PCR targeting the hipO gene specific for C. jejuni (Marinou et al. 2012 (link)) and the asp gene specific for C. coli (Table 1 ) (Al Amri et al. 2013 ).
4
Antimicrobial-Supplemented Culture Media for Campylobacter
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Bolton broth was prepared following the manufacturer’s instruction. It contained 20 mg/L cefoperazone, 20 mg/L vancomycin, 20 mg/L trimethoprim lactate, 10 mg/L amphotericin B, and 5% lysed defibrinated horse blood. C-Bolton broth was prepared using Bolton broth supplemented with potassium clavulanate (Sigma-Aldrich, St. Louis, MO, USA) to a final concentration of 2 mg/L [5 (link)]. Modified charcoal cefoperazone deoxycholate agar (mCCDA, Oxoid) was prepared per the manufacturer’s recommendations using mCCDA antibiotic supplement (32 mg/L cefoperazone and 10 mg/L amphotericin; Oxoid). C-mCCDA was generated by adding 0.5 mg of potassium clavulanate to 1 L of cooled mCCDA [15 (link)].
5
Enumeration of Campylobacter in Fecal/Caecal Samples
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Enumeration was carried out in accordance with ISO 10272-2:2016. To 25g of each pooled faecal or caecal sample, 225ml of Bolton Broth (Oxoid, Basingstoke, United Kingdom) was added. Samples were stomached for 60s and serially diluted 10-fold in maximum recovery diluent (MRD, Oxoid, Basingstoke, United Kingdom). Each dilution was spread on modified charcoal cefoperazone deoxycholate agar (mCCDA, Oxoid, Basingstoke, United Kingdom) and incubated at 42°C microaerobically (10% CO2, 5% O2 and balancing N2) in a controlled atmosphere incubator (MACS VA-500, Don Whitley) for 48h.
6
Campylobacter Detection in Food Samples
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After transferring carcasses into a rinse solution, massaging, and shaking, as previously described, 10 ml was added into 10 ml of Bolton broth (Oxoid) supplemented with a selective supplement (SR0155 E; Oxoid). Samples were incubated at 42°C for 48 hr under microaerophilic conditions generated by a gas‐generating pack (Campygen CN25; Oxoid). Following incubation, a loopful of Bolton's broth was streaked onto modified cefoperazone charcoal deoxycholate agar plate (mCCDA; Oxoid) supplemented with a selective supplement (SR0155 E; Oxoid) and incubated at 42°C for 48 hr under microaerophilic conditions generated by a gas‐generating pack (Campygen CN25; Oxoid).
7
Enumeration of P. aeruginosa using mCCDA
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After incubation each biostud was removed and rinsed with phosphate buffer saline (PBS; 1st BASE, Singapore) before being placed in a 15 ml centrifuge tube containing 1 ml of PBS. Each tube was then sonicated at room temperature at a frequency of 35 kHz for 10 min using a water bath soniator (LC-130H; ELMA, Germany). The tube was then vortexed for 1 min before serial diluted in PBS and plated on modified cefoperazone charcoal deoxylate agar (mCCDA; Oxoid, UK). The plates were incubated at 42°C for 48 h before enumeration. Growth of P. aeruginosa is inhibited on mCCDA.
8
Cultivation of E. coli and Campylobacter
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Escherichia coli XL1 (Stratagene, USA) was used for maintenance of plasmid constructs and E. coli Rosetta BL21 pLysE (Merck Millipore, UK) was used for protein expression. E. coli strains were grown in Luria Bertani (LB) broth or agar at 37 °C, unless otherwise indicated, with shaking at 200 rpm for liquid cultures. C. jejuni M1 was used as a source of DNA for gene cloning and as the challenge strain in vaccination experiments as described [12] (link). C. jejuni 11168H was used to assess the cross-reactivity and subcellular localisation of SodB. C. jejuni strains were grown on modified charcoal-cephoperazone-deoxycholate agar (mCCDA) (Oxoid, UK) or in Mueller-Hinton Broth (MH; Oxoid), at 37 °C in a microaerophilic workstation (Don Whitley Scientific, UK) in a low oxygen atmosphere (5% O2, 5% CO2 and 90% N2). Liquid cultures of Campylobacter were grown with shaking at 400 rpm using a table top shaker (IKA, Germany) under low oxygen conditions as above. Antibiotics were used at the final concentrations of 100 μg/ml ampicillin and 34 μg/ml chloramphenicol where appropriate.
9
Campylobacter Isolation from Greek Pigs
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A total of 200 Campylobacter isolates, originating from 16 commercial pig farms from six different Greek regions were examined. The median size of the farms was 550 breeding sows (230–1600). Among these isolates, 100 have been previously identified as C. jejuni and 100 as C. coli. Speciation of Campylobacter isolates has been made according to a multiplex PCR protocol [20 ]. All samples were cryopreserved at −70 °C, in Brucella broth (OXOID, UK) supplemented with 5% lysed horse blood (OXOID, UK) and 15% glycerol.
The cryopreserved Campylobacter samples were defrosted, revived, and inoculated on to modified charcoal cefoperazone deoxycholate agar (mCCDA-OXOID, UK) with the selective supplement SR0155 (OXOID, UK). The inoculated plates were incubated under microaerobic conditions (85% N2, 10% CO2, and 5% O2) at 41.5 °C for 48 h.
The cryopreserved Campylobacter samples were defrosted, revived, and inoculated on to modified charcoal cefoperazone deoxycholate agar (mCCDA-OXOID, UK) with the selective supplement SR0155 (OXOID, UK). The inoculated plates were incubated under microaerobic conditions (85% N2, 10% CO2, and 5% O2) at 41.5 °C for 48 h.
10
Campylobacter Isolation from Livestock
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Livestock isolates of C. jejuni and C. coli from faecal samples and carcasses were collected at farms and slaughterhouses by Wageningen Bioveterinary Research (WBVR) and Wageningen Food Safety Research (WFSR), in collaboration with the RIVM and the Netherlands Food and Consumer Product Safety Authority (NVWA). This was done within the framework of established and nationally representative surveillance programs for zoonotic agents ( Opsteegh et al., 2018 ) and antimicrobial resistance ( de Greeff et al., 2019 ) (link) in food-producing animals, as well as routine inspection and testing activities by veterinary services, in the Netherlands during 2014-2019. Additional isolates from small ruminants (sheep and goats) were obtained through a small-scale internal project including ad-hoc sampling events at farms in the Netherlands conducted by engaging field veterinarians collaborating with the Veterinary Microbiological Diagnostic Centre (VMDC) of Utrecht University.
Isolates were obtained from faecal samples analysed without enrichment by direct streaking onto mCCDA (Oxoid) plates following the ISO 10272-1:2017 procedure, whereas carcass samples were analysed with an enrichment step in accordance with the same procedure. Species identification was performed using MALDI-TOF MS.
Isolates were obtained from faecal samples analysed without enrichment by direct streaking onto mCCDA (Oxoid) plates following the ISO 10272-1:2017 procedure, whereas carcass samples were analysed with an enrichment step in accordance with the same procedure. Species identification was performed using MALDI-TOF MS.
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