GB 4789.9-2025National food safety standard - Food microbiological examination - Examination of Campylobacter jejuni and Campylobacter coli (English PDF)

2.5 Electronic balance. Sensitivity

0.1 g, sensitivity

0.01 g, sensitivity

0.001 g.

2.6 Constant temperature water bath or metal bath. 36 °C ~ 100 °C.

2.8 Sterile petri dishes. 60 mm and 90 mm in diameter.

2.9 Sterile hydrophilic filter membrane. 47 mm in diameter, 0.45 µm pore size.

2.10 Microaerobic culture apparatus. Provides microaerophilic conditions (5 % oxygen, 10 % carbon dioxide, and 85 % nitrogen).

2.11 Microscope. 10× ~ 1000×.

2.12 Low-temperature high-speed centrifuge. 8000 g ~ 20000 g, 4 °C.

2.13 pH meter or pH colorimetric tube or precision pH test paper.

2.14 Real-time fluorescence PCR instrument.

2.15 Real-time fluorescence PCR reaction tube.

2.16 Micropipettes and tips. 0.1 µL ~ 2.5 µL, 0.5 µL ~ 10 µL, 2 µL ~ 20 µL, 20 µL ~ 200 µL, 100 µL ~ 1000 µL.

2.17 Sterile centrifuge tubes. 50 mL, 100 mL, or larger capacity.

2.20 Sterile pipettes. 1 mL (with

0.01 mL graduations), 10 mL (with

0.1 mL graduations).

2.21 Microbial biochemical identification system.

2.22 Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS).

5.1 Sample preparation

5.1.1 General samples Take 25 g (mL) of sample [50 g for fruits, vegetables, and aquatic products (excluding shellfish)] and add it to a homogenizing bag with filter containing 100 mL of sterile 0.1 % peptone water, homogenize using a flapping homogenizer for 1 min ~ 2 min (or thoroughly knead and oscillate for 5 min ~ 10 min), filter the homogenate into a centrifuge tube, centrifuge the filtrate at 12000 g at 4 °C for 15 min and discard the supernatant (multiple centrifugations can be performed for enrichment), resuspend the precipitate with 1 mL of sterile physiological saline, take 1 mL of the suspension and add it to 9 mL of Campylobacter enrichment broth for enrichment culture.

5.1.2 Samples of whole poultry, etc. Thoroughly rub the inside and outside of the sample with 200 mL ~ 400 mL of sterile 0.1 % peptone water, oscillate for 5 min ~ 10 min, then filter through sterile gauze or a homogenizing bag with filter into a centrifuge tube, centrifuge the filtrate at 12000 g 4 °C for 15 min and discard the supernatant (multiple centrifugations can be performed for enrichment), resuspend the precipitate with 1 mL of sterile physiological saline, take 1 mL of the suspension and add it to 9 mL of Campylobacter enrichment broth for enrichment culture.

5.1.3 Shellfish (shelled) Take 100 g ~ 200 g of sample and place it into a sterile homogenizing bag, homogenize using a flapping homogenizer for 1 min ~ 2 min, take 25 g of the contents and add it to a homogenizing bag with filter containing 100 mL of sterile 0.1 % peptone water, homogenize using a flapping homogenizer for 1 min ~ 2 min (or oscillate thoroughly for 5 min ~ 10 min) and filter through a filter into a centrifuge tube, centrifuge the filtrate at 12000 g at 4 °C for 15 min and discard the supernatant (multiple centrifugations can be performed for enrichment), resuspend the precipitate with 1 mL of sterile physiological saline, take 1 mL of the suspension and add it to 9 mL of Campylobacter enrichment broth for enrichment culture.

5.1.4 Liquid egg white or whole egg mixture Take 25 g (mL) of sample and add it to 75 mL of sterile 0.1 % peptone water, mix the sample thoroughly, transfer it to a centrifuge tube, centrifuge at 20000 g at 4 °C for 15 min and then discard the supernatant (multiple centrifugations can be performed for enrichment), resuspend the precipitate with 1 mL of sterile physiological saline, take 1 mL of the suspension and add it to 9 mL of Campylobacter enrichment broth for enrichment culture.

5.1.5 Fresh milk and other dairy products For liquid dairy products, take 50 g directly; for solid dairy products, chop them and take 50 g, add them to a homogenizing bag with filter containing 100 mL of sterile 0.1 % peptone water, homogenize using a flapping homogenizer for 15 s ~ 30 s and then filter through a filter into a centrifuge tube. Centrifuge the liquid dairy product or filtrate at 20000 g at 4 °C for 30 min and then discard the supernatant (multiple centrifugations can be performed for enrichment), resuspend the precipitate with 1 mL of sterile physiological saline (avoid introducing the lipid layer), take 1 mL of the suspension and add it to 9 mL of Campylobacter enrichment broth for enrichment culture.

5.1.6 Samples requiring surface swab testing Wipe the surface of the test sample (at least 100 cm^2) with a sterile swab moistened with sterile physiological saline, cut off the swab tip and place it into 9 mL of Campylobacter enrichment broth for enrichment culture.

5.1.7 Water sample Take 4 L of water (for chlorinated water, add 5 mL of 1 mol/L sodium thiosulfate solution per liter of water before centrifugation), centrifuge at 12000 g at 4 °C for 30 min, discard the supernatant (multiple centrifugations can be performed for enrichment), resuspend the precipitate with 1 mL of sterile physiological saline, and take 1 mL of the suspension and add it to 9 mL of Campylobacter enrichment broth for enrichment culture.

5.2 Enrichment Loosen the cap of the screw-cap tube containing the enrichment broth, under microaerophilic conditions, incubate at 42 °C ± 1 °C for 24 h ± 2 h.

5.3 Separation

5.3.1 Membrane application After Columbia blood agar plates and Carmalai blood agar plates have equilibrated to room temperature, use sterile forceps to pick up a 0.45 µm sterile filter membrane along the edge, and apply it to the surface of both types of plates (in the center of the plate), ensuring full adhesion between the filter membrane and the culture medium surface.

5.3.2 Sample addition Take 300 µL of enrichment broth, drop to the filter membrane surface (droplets shall be within the filter membrane edge) in 4 times ~ 6 times (4 drops ~ 6 drops each time, avoiding droplet merging), let stand to allow the droplets to fully permeate the filter membrane (let stand for 45 min ~ 60 min, under microaerophilic or normal gas conditions), perform aseptic operation, and remove the filter membrane. Invert the plate, under microaerophilic conditions, incubate at 42 °C ± 1 °C for 24 h ~ 48 h.