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GB 4789.9-2014 English PDF (GB4789.9-2014)

GB 4789.9-2014 English PDF (GB4789.9-2014)

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GB 4789.9-2014: Microbiological examination of food hygiene -- Examination of Campylobacter jejuni

This Standard specifies the examination method of Campylobacter jejuni in food. This Standard is applicable to the examination of Campylobacter jejuni in food.
GB 4789.9-2014
GB
NATIONAL FOOD SAFETY STANDARD OF
THE PEOPLE REPUBLIC OF CHINA
GB/T 4789.9-2014
Microbiological examination of food hygiene -
Examination of Campylobacter jejuni
ISSUED ON. DECEMBER 1, 2014
IMPLEMENTED ON. MAY 1, 2015
Issued by. National Health and Family Planning Commission of the
People 's Republic of China
Table of Contents
Foreword ... 3
1 Scope ... 4
2 Equipment and materials ... 4
3 Media and reagents ... 5
4 Examination procedures ... 5
5 Operation steps ... 6
Annex A Media and reagents ... 11
Foreword
This Standard replaces GB/T 4789.9-2008 Microbiological examination of food hygiene - Examination of Campylobacter jejuni.
Compared with GB/T 4789.9-2008, the main changes of this Standard are as follows.
- modified the Chinese name of the standard;
- modified the scope;
- modified the equipment and material;
- modified the media and reagents;
- modified sample treatment;
- deleted the drug sensitivity test;
- deleted Method 2 - Automatic enzyme - linked immunosorbent assay.
Microbiological examination of food hygiene -
Examination of Campylobacter jejuni
1 Scope
This Standard specifies the examination method of Campylobacter jejuni in food.
This Standard is applicable to the examination of Campylobacter jejuni in food. 2 Equipment and materials
In addition to microbiology laboratory routine sterilization and culture equipment, other equipment and materials are as follows.
a) constant temperature incubator. 25??C ?? 1??C, 36??C ?? 1??C, 42??C ?? 1??C;
b) refrigerator. 2??C ~ 5??C;
c) constant temperature oscillation incubator. 36??C ?? 1??C, 42??C ?? 1??C;
d) balance. a sense of 0.1g;
e) homogenizer and matching homogeneous bag;
f) oscillator;
g) sterile pipette. 1 mL (with 0.01 mL scale), 10 mL (with 0.1 mL scale) or micropipette and tip;
h) sterile Erlenmeyer flask. capacity of 100 mL, 200 mL, 2000 mL;
i) sterile petri dish. 90 mm in diameter;
j) pH meter or pH colorimetric tube or precision pH test paper;
k) water bath device. 36??C ?? 1??C, 100??C;
l) micro-aerobic incubator. providing micro-aerobic conditions (5% oxygen, 10% carbon dioxide and 85% nitrogen);
m) filter device and filter membrane (0.22 ??m, 0.45 ??m);
5.1.1 General sample
Take 25 g (mL) of sample (50 g for fruits, vegetables, aquatic products) into the homogeneous bag with screen filled with 225 mL of Bolton broth (it may use sterile gauze for filtration if there is no homogeneous bag with screen). Use rapping homogenizer to homogenize for 1 min ~ 2 min. Filter it through strainer or sterile gauze. Cultivate the filtrate.
5.1.2 Whole poultry and other samples
Use 200 mL of 0.1% peptone water to rinse the inside and outside of the sample sufficiently. Oscillate it for 2 min ~ 3 min. Filter it through sterile gauze into a 250 mL centrifuge tube. After 16000 g is centrifuged for 15 min, discard the supernatant liquid. Use 10 mL of 0.1% peptone water to conduct suspension precipitation. Pipet 3 mL into 100 mL of Bolton broth to cultivate.
5.1.3 Shellfish
Table at least 12 samples with shell. After removal of the housing, all contents are put into a homogeneous bag. Use rapping homogenizer to homogenize for 1 min ~ 2 min. Take 25 g of sample into 225 mL of Bolton broth (1.10 dilution). After sufficient shaking, transfer 25 mL to 225 mL of Bolton broth (1.100 dilution). Cultivate the Bolton broth diluted in 1.10 and the Bolton broth diluted in 1.100 simultaneously.
5.1.4 Egg yolk liquid or egg slurry
Take 25 g (mL) of sample into 125 mL of Bolton broth and well mix it (1.6 dilution). Transfer 25 mL into 100 mL of Bolton broth and well mix it (1.30 dilution). Cultivate the Bolton broth diluted in 1.6 and the Bolton broth diluted in 1.30 simultaneously.
5.1.5 Fresh milk, ice cream, cheese, etc.
If it is liquid dairy product, take 50 g. If it is solid dairy product, take 50 g into the homogeneous bag with screen filled with 50 mL of 0.1% peptone water. Use rapping homogenizer to homogenize for 15 s ~ 30 s. Retain the filtrate. When necessary, adjust pH to 7.5 ?? 0.2. After 20000 g of liquid diary product or filtrate is centrifuged for 30 min, discard the supernatant liquid. Use 10 mL of Bolton broth to conduct suspension precipitation (try to avoid into the reservoir). Transfer to 90 mL of Bolton broth to cultivate.
5.1.6 Samples to be smeared on the surface
Use sterile cotton swab to wipe the surface of the test sample (the area is at least greater than 100 cm2). Cut the cotton swab head into 100 mL of Bolton broth to cultivate.
5.4.2.2 Sodium hippurate hydrolysis test
Pick colonies. Add into a test tube filled with 0.4 mL of 1% sodium hippurate to make bacterial suspension. After mixing evenly, incubate at 36??C ?? 1??C water bath for 2 h or 36??C ?? 1??C incubator for 4 h. Along with the tube wall, slowly add into 0.2 mL of ninhydrin solution. Do not oscillate. Incubate at 36??C ?? 1??C water bath or incubator for another 10 min. Then read the results. If it appears purple, it shall be positive. If it appears light purple or there is no color change, it shall be negative.
5.4.2.3 Indole acetate hydrolysis test
Pick colonies to indole acetate tablet. Add 1 drop of sterile water. If indole acetate hydrolyzes, it shall appear dark blue within 5 min ~ 10 min. If there is no color change, there shall be no hydrolysis. The identification of
Campylobacter jejuni is shown in Table 2.
Table 2 Identification of Campylobacter jejuni
Characteristics C. jejuni C. coli C. lari C. upsaliensis
Catalase test + + + - or weak
Sodium hippurate hydrolysis test + - - -
Indole acetate hydrolysis test + + - +
NOTE + indicates positive; - indicates negative.
5.4.2.4 Alternative test
For colonies identified as Campylobacter spp, it may use biochemical
identification kit or biochemical identification card to replace the identification in 5.4.2.1 ~ 5.4.2.3.
5.5 Results report
Combining the aforementioned rest results, it shall report that Campylobacter jejuni is detected or not in the sample.
A.1.4.1 Composition
Basal medium 1000.0 mL
Sterile lysis defibrillation sheep or horse blood 50.0 mL
Antibiotic solution 5.0 mL
A.1.4.2 Preparation method
When the basal medium temperature is around 45??C, sterilely add sheep or horse blood and antibiotic solution. Well mix. Calibrate pH to 7.4 ?? 0.2 (25??C). Placement at room temperature shall not exceed 4 h. Or storage in darkness at 4??C must not exceed 7 d.
A.2 modified Charcoal Cefoperazone Deoxycholate Agar, mCCDA
A.2.1 Basal medium
A.2.1.1 Composition
Meat infusion 10.0 g
Animal tissue hydrolyzate 10.0 g
Sodium chloride 5.0 g
Charcoal 4.0 g
Casein hydrolyzate 3.0 g
Sodium deoxycholate 1.0 g
Ferrous sulfate 0.25 g
Sodium pyruvate 0.25 g
Agar 8.0 g ~ 18.0 g
Distilled water 1000.0 mL
A.2.1.2 Preparation method
Dissolve the compositions in A.2.1.1 into distilled water. Sterilize at 121??C for 15 min, ready for use.
A.2.2 Antibiotic solution
A.2.2.1 Composition
cefoperazone 0.032 g
amphotericin B 0.01 g
rifampicin 0.01 g
ethanol / sterile water (50 / 50, volume fraction) 5.0 mL
A.2.2.2 Preparation method
Dissolve the compositions in A.2.2.1 into ethanol / sterile water mixed solution. medium into sterile plate. Place still till medium is solidified. When the plate for preparation is not dry, placement at room temperature shall not exceed 4 h, or refrigeration at around 4??C must not exceed 7 d.
A.4 Brucella broth
A.4.1 Composition
Casein hydrolyzate 10.0 g
Animal tissue hydrolyzate 10.0 g
Glucose 1.0 g
Yeast extract 2.0 g
Sodium chloride 5.0 g
Sodium bisulfite 0.1 g
Distilled water 1000.0 mL
A.4.2 Preparation method
Dissolve the compositions in A.4.1 into distilled water. Calibrate pH to 7.3 ?? 0.2 (25??C). Sterilize at 121??C for 15 min, ready for use.
A.5 Oxidase reagent
A.5.1 Composition
Tetramethyl-p-phenylenediamine hydrochloride 1.0 g
Distilled water 100.0 mL
A.5.2 Preparation method
Before use, dissolve the compositions in A.5.1 into distilled water.
A.6 Sodium hydrogen succinate hydrolysis reagent
A.6.1 Hippuric acid sodium solution
A.6.1.1 Composition
Sodium hippurate 10.0 g
Phosphate buffer (PBS) components.
Sodium chloride 8.5 g
Disodium hydrogen phosphate 8.98 g
Sodium dihydrogen phosphate 2.71 g
Distilled water 1000.0 mL
A.6.1.2 Preparation method
Dissolve sodium hippurate into phosphate buffer solution. Filter and sterilize. A.7.3.1 Composition
cefoperazone 0.032 g
amphotericin B 0.01 g
rifampicin 0.01 g
ethanol / sterile water (50 / 50, volume fraction) 5.0 mL
A.7.3.2 Preparation method
Dissolve the compositions in A.7.3.1 into ethanol / sterile water mixture. A.7.4 Sterile defibrinated sheep blood
Under aseptic operating conditions, pour sheep's blood into the container containing sterilized glass beads. Shake it for about 10 min. After still placement, remove the glass beads with blood fiber.
A.7.5 Complete medium
A.7.5.1 Composition
Basal medium 1000.0 mL
FBP solution 5.0 mL
Antibiotic solution 5.0 mL
Sterile defibrinated sheep blood 50.0 mL
A.7.5.2 Preparation method
When the temperature of basal medium is around 45??C, add FBP solutio...

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