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).
Campygen 3.5 l
Manufactured by Thermo Fisher Scientific
Sourced in United Kingdom
The CampyGen 3.5 L is a compact anaerobic gas generation system designed for the incubation of microaerophilic organisms such as Campylobacter species. The device generates a controlled atmosphere with a pre-set gas mixture to support the growth of these fastidious bacteria.
Automatically generated - may contain errors
Lab products found in correlation
Anaerojar, by Thermo Fisher Scientific (2 mentions)
Mccda, by Thermo Fisher Scientific (1 mentions)
75 cm2 cell culture flask, by Corning (1 mentions)
50 ml centrifuge tube, by Corning (1 mentions)
Gaspak, by BD (1 mentions)
Campylobacter selective supplement, by Thermo Fisher Scientific (1 mentions)
4 protocols using campygen 3.5 l
1
Isolation of Campylobacter from Water Samples
2
Comparative Growth of C. hepaticus
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Costar® 24-well cell culture plates (CCP24), Corning® 50 mL centrifuge tubes (CT50) with a vented cap (0.2 μm pore size), Corning® 75cm2 cell culture flask (TCF75) with a vented cap (0.2 μm pore size) and Erlenmeyer flasks (250 mL) (EF) were used to compare the growth of C. hepaticus in Brucella broth (BD BBL™). The volume of culture media used in CCP24 was 1 mL, 25 mL (CT50 and TCF) and 40 mL (EF). All vessels were placed in BD GasPak™ EZ container, charged with CampyGen 3.5 L (Oxoid) to produce microaerobic conditions and then incubated at 37 °C for 48 h. The growth rate of C. hepaticus HV10 was determined by the plate count method on HBA plates.
3
Detecting Thermotolerant Campylobacter in Water
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The standard ISO 17995 (Water quality—Detection and enumeration of thermotolerant Campylobacter spp.) (International Organization for Standardisation, 2019 ) was used for the detection of thermotolerant Campylobacter spp. with a slight modification (Strakova et al., 2021 (link)). Briefly, prefilters with a pore size of 1.4 µm (glass filter; Macherey Nagel) were used to remove mechanical particles from water before sample filtration. Thereafter, water samples (500 ml) were filtered (0.22 µm, mixed cellulose ester filter; Millipore Sigma), and the filters were transferred into two selective broths (Preston and Bolton broth) for enrichment and incubated at 42℃ in an anaerostat (AnaeroJar; Oxoid) under a microaerobic atmosphere (CampyGen 3.5 L; Oxoid). After 44 ± 4 h of incubation, inoculi were cultivated on Campylobacter blood‐free selective agar (mCCDA; Modified charcoal‐cefoperazone‐deoxycholate agar) and incubated for another 44 ± 4 h under a microaerobic atmosphere at 42℃, followed by the isolation of presumptive colonies on a nonselective blood agar and species level identification.
4
Isolation of Campylobacter hepaticus from Environmental Samples
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To isolate C. hepaticus, bile samples were directly streaked onto horse blood agar (HBA) plates [Brucella broth (BBL) supplemented with 1.5% agar (BBL) and 5% defibrinated horse blood (Equicell)], as described previously (9 (link)). A combination of filter membrane and Campylobacter selective media approaches (called the motile-filter method in this paper) were used for the isolation of C. hepaticus from fecal, caecal, and soil samples. Fifty milligrams of samples were resuspended in 200 μl sterile Milli-Q water and 50 μl of the mix was spotted onto 0.65 μm cellulose acetate filter membranes (Sartorius Stedim Biotech) and placed on the surface of HBA plates supplemented with Campylobacter selective supplement (Skirrow, Oxoid) (HBAS) and left for 30 min. The filter was then removed and the plate incubated. Motile organisms, including C. hepaticus, can move through the membrane whereas non-motile organisms are trapped on top and removed with the filter. Isolation of C. hepaticus from environmental samples was attempted by suspension of samples in Brucella broth (10 times dilution) and direct plating onto HBAS plates as well as using the motile-filter method. All plates were incubated at 37°C for 3–5 days under microaerobic conditions using CampyGen 3.5L (Oxoid, CN0035A) in an anaerobic jar.
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