GB 4789.9: Evolution and historical versions
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National food safety standard - Food microbiological examination - Examination of Campylobacter jejuni and Campylobacter coli
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| GB 4789.9-2014 | English | 130 |
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Microbiological examination of food hygiene - Examination of Campylobacter jejuni
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GB 4789.9-2014
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| GB/T 4789.9-2008 | English | 599 |
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Microbiological examination of food hygiene -- Examination of campylobacter jejuni
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GB/T 4789.9-2008
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| GB/T 4789.9-2003 | English | 239 |
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Microbiological examination of food hygiene -- Examination of Campylobacter jejuni
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| GB 4789.9-1994 | English | RFQ |
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Microbiological examination of food hygiene. Examination of Campylobacter jejuni
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| GB 4789.9-1984 | English | RFQ |
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Microbiological examination of food hygiene--Examination of campylobacter jejuni
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GB 4789.9-1984
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Basic data | Standard ID | GB 4789.9-2025 (GB4789.9-2025) | | Description (Translated English) | National food safety standard - Food microbiological examination - Examination of Campylobacter jejuni and Campylobacter coli | | Sector / Industry | National Standard | | Classification of Chinese Standard | C53 | | Word Count Estimation | 16,171 | | Date of Issue | 2025-09-02 | | Issuing agency(ies) | National Health Commission; State Administration for Market Regulation | GB 4789.9-2014: Microbiological examination of food hygiene - Examination of Campylobacter jejuni
---This is a DRAFT version for illustration, not a final translation. Full copy of true-PDF in English version (including equations, symbols, images, flow-chart, tables, and figures etc.) will be manually/carefully translated upon your order.
Microbiological examination of food hygiene - Examination of Campylobacter jejuni
National Standards of People's Republic of China
National Food Safety Standard
Food Microbiology testing Campylobacter
Issued on. 2014-12-01
2015-05-01 implementation
People's Republic of China
National Health and Family Planning Commission released
Foreword
This standard replaces GB/T 4789.9-2008 "Microbiological examination of food hygiene inspection Campylobacter jejuni."
This standard compared with GB/T 4789.9-2008, the main changes are as follows.
--- Modify the standard Chinese name;
--- Modify the scope;
--- Modify the equipment and materials;
--- Modify the media and reagents;
--- Modify the sample;
--- Remove the drug susceptibility testing;
--- Remove the second law of automatic immunoassay analyzer ELFA screening.
National Food Safety Standard
Food Microbiology testing Campylobacter
1 Scope
This standard specifies the foods Campylobacter jejuni (Campylobacterjejuni) test method.
This standard applies to food inspection in C. jejuni.
2 Equipment and Materials
In addition to the microbiological laboratory conventional sterilization and cultivation equipment, other equipment and materials as follows.
a) incubator. 25 ℃ ± 1 ℃, 36 ℃ ± 1 ℃, 42 ℃ ± 1 ℃;
b) Refrigerator. 2 ℃ ~ 5 ℃;
c) Shaking Incubator. 36 ℃ ± 1 ℃, 42 ℃ ± 1 ℃;
d) balance. a sense of the amount of 0.1g;
e) homogenizer and supporting homogeneous bags;
f) oscillator;
g) sterile pipette. 1mL (with 0.01mL scale), 10mL (with 0.1mL scale) or micro pipettes and tips;
h) sterile conical flask. capacity 100mL, 200mL, 2000mL;
i) sterile Petri dish. diameter 90mm;
j) pH meter or pH colorimetric tubes or precision pH test paper;
k) water bath. 36 ℃ ± 1 ℃, 100 ℃;
l) micro-aerobic cultivation means. providing microaerobic conditions (5% oxygen, 10% carbon dioxide and 85% nitrogen);
m) filtration apparatus and membrane (0.22μm, 0.45μm);
n) Microscope. 10 to 100 times, there is a difference function;
o) centrifuges. centrifugal speed ≥20000g;
p) turbidity meter;
q) biochemical microbial identification system.
3 media and reagents
3.1 Bolton broth (Boltonbroth). A A.1 in the appendix.
3.2 Improved CCD agar (modifiedCharcoalCefoperazoneDeoxycholateAgar, mCCDA). See Appendix A A.2.
3.3 Colombia blood agar (Columbiabloodagar). See Appendix A in A.3.
3.4 Brucella Broth (Brucelabroth). See Appendix A A.4.
3.5 oxidase reagent. A A.5 in the appendix.
3.6 Sodium hippurate hydrolysis reagents. See Appendix A A.6.
3.7 Skirrow blood agar (Skirrowbloodagar). See Appendix A A.7.
3.8 indole acetate paper. See Appendix A A.8.
3.9 0.1% peptone water. See Appendix A, A.9.
3.10 1mol/L sodium thiosulfate (Na2S2O3) solution. See Appendix A A.10.
3.11 3% hydrogen peroxide (H2O2) solution. A in Appendix A.11.
3.12 Campylobacter jejuni chromogenic medium.
3.13 biochemical identification kit or biochemical identification card.
4 inspection procedures
Campylobacter jejuni test program shown in Figure 1.
Figure 1 Campylobacter jejuni inspection procedures
5 steps
5.1 Sample Preparation
5.1.1 General Sample
Take 25g (mL) sample (fruit, vegetables, aquatic products 50g) was added broth containing 225mLBolton have homogeneous filter bags
In (if it is not homogeneous filter bags can be filtered using sterile gauze), with slap-type homogenizer homogeneous 1min ~ 2min, through strainer or sterile gauze
Filtered and the filtrate was cultured.
5.1.2 samples such as whole birds
With 200mL0.1% peptone water to fully rinse inside and outside of the sample and shaken 2min ~ 3min, sterile gauze to filter
250mL centrifuge tube after centrifugation 15min 16000g supernatant was discarded and precipitated with 10mL0.1% peptone water suspension, draw 3mL
100mLBolton cultured in broth.
5.1.3 Shellfish
Sample taken at least 12 shell, the shell will remove all the contents of the bag into the homogeneous, with slap-type homogenizer homogeneous 1min ~
2min, take 25g sample to 225mLBolton broth (1.10 dilution), and then transfer the full shock 25mL to 225mL
Bolton broth (100 dilution), and the 1.10 100 dilution of Bolton broth culture at the same time.
5.1.4 yolk egg liquid or slurry
Take 25g (mL) sample was 125mLBolton broth and mix (1.6 dilution), and then transferred to 25mL 100mLBolton
Broth and mix (1.30 dilution), while 1.6 and diluted 1.30 in Bolton broth culture.
5.1.5 milk, ice cream, cheese, etc.
If liquid dairy products take 50g; if taken as solid dairy products containing added 50g 50mL0.1% peptone water with a strainer homogeneous Bag
In with slap-type homogenizer homogeneous 15s ~ 30s, retain the filtrate. If necessary, adjust the pH to 7.5 ± 0.2, or filtered liquid dairy products
After the liquid was centrifuged 30min 20000g supernatant was discarded, the precipitate was suspended with 10mLBolton broth (avoid into the reservoir), and then transferred
To 90mLBolton cultured broth.
5.1.6 required coated swab samples tested
Sterile swab test sample surface (an area of at least 100cm2 or more), the swab head scissors fell 100mLBolton broth
Cultured.
5.1.7 water samples
The 4L water (chlorine for water treatment, prior to filtration of water per liter of sodium thiosulfate solution was added 5mL1mol L /) through 0.45μm
Filter membrane, the membrane is immersed in 100mLBolton cultured broth.
5.2 pre-enrichment and enrichment
Under microaerophilic conditions, 36 ℃ ± 1 ℃ cultured 4h, such as the conditions allow with 100r/min speed oscillation. Determination of necessary
Enrichment broth and adjust the pH to 7.4 ± 0.2,42 ℃ ± 1 ℃ cultured 24h ~ 48h.
5.3 Separation
The 1.50 dilution enrichment broth 24h, 48h and corresponding enrichment broth were streaked on blood agar and mCCDA Skirrow
On agar plates, micro-aerobic conditions under 42 ℃ ± 1 ℃ cultured 24h ~ 48h. Also choose to use color flatbed Campylobacter jejuni as
supplement.
Culture and colony morphology was observed 24h 48h cultured agar plates, suspected colonies on the agar plate mCCDA usually pale
Gray, with metallic luster, damp, flat, showing a tendency diffusion growth. The first type of suspicious colonies on blood agar plates Skirrow gray,
Flat, moist and shiny, showing a tendency outward diffusion line along the inoculation; the second type of suspicious colonies dispersed single colony often has a raised edge and tidy,
Shiny. Campylobacter jejuni chromogenic media on suspected colonies determined in accordance with the instructions.
5.4 Identification
5.4.1 Identification of Campylobacter
5.4.1.1 Overview
Picked five (if less than five are all picked) or more suspicious colonies were inoculated onto Columbia blood agar plates, micro-aerobic conditions
At 42 ℃ ± 1 ℃ cultured 24h ~ 48h, in accordance with 5.4.1.1 ~ 5.4.1.5 were identified, in line with the results in Table 1 of suspicious colonies determined to be bent
Spp.
Table 1 Identification of Campylobacter
Campylobacter project characteristics
Morphology
Gram-negative bacteria such as small comma-shaped bend, the end of the two-phase cells when the S-shaped spiral
Or like a gull-shaped wings
Power observe a spiral motion b
Oxidase test positive
Microaerophilic conditions of 25 ℃ ± 1 ℃ growth test does not grow
Under aerobic conditions 42 ℃ ± 1 ℃ growth test does not grow
Some form of a strain of atypical.
Some sports b strains not obvious.
5.4.1.2 morphology
Suspected colonies were picked Gram staining, microscopy.
5.4.1.3 Dynamic Observation
Suspected colonies were picked with 1mL Brandt broth suspension with a phase contrast microscope to observe the state of motion.
5.4.1.4 oxidase test
Platinum/iridium ring vaccination or glass rod suspected colonies were picked to the oxidase reagent wet filter paper, if there is in the 10s mauve, purple
Roland or dark blue as positive.
5.4.1.5 Under microaerophilic conditions 25 ℃ ± 1 ℃ growth test
Suspected colonies were picked, inoculated on Columbia blood agar under microaerophilic conditions 25 ℃ ± 1 ℃ culture 44h ± 4h, observe fine
Bacteria growth.
5.4.1.6 under aerobic conditions 42 ℃ ± 1 ℃ growth test
Suspected colonies were picked, inoculated on Columbia blood agar plates under aerobic conditions 42 ℃ ± 1 ℃ culture 44h ± 4h, observe bacteria
growing situation.
5.4.2 Identification of Campylobacter jejuni
5.4.2.1 catalase test
Colonies were picked, is applied to a clean glass slides 3% hydrogen peroxide solution, if it is determined that the bubbles in the 30s the result is positive.
5.4.2.2 Sodium hippurate hydrolysis test
Colonies were picked and added containing 0.4mL1% sodium hippurate tubes made of bacterial suspension. After mixing in 36 ℃ ± 1 ℃ water bath
Incubated for 2h or 36 ℃ ± 1 ℃ incubator incubated for 4h. 0.2mL was added slowly along tube wall ninhydrin solution, do not oscillate, in
Water bath or incubator 36 ℃ ± 1 ℃ incubated for 10min in the interpretation of the results after. If there is a dark purple was positive; if there is lavender or
No color change was negative.
5.4.2.3 indole acetic ester hydrolysis test
Colonies were picked to indole on acetate sheets, then add 1 drop of sterile water. If the indole acetic acid ester hydrolysis, within 5min ~ 10min
Appear dark blue; if there is no color change indicates hydrolysis. Campylobacter jejuni identification results are shown in Table 2.
Table 2 Identification of Campylobacter jejuni
feature
Campylobacter jejuni
(C.jejuni)
Campylobacter
(C.coli)
Seagull Campylobacter
(C.lari)
Uppsala Campylobacter
(C.upsaliensis)
Catalase test - or weak
Hippurate hydrolysis test - - -
Indole acetic acid lipid hydrolysis test -
Note. Indicates positive; - indicates negative.
5.4.2.4 Alternative test
For identified as Campylobacter colonies, use biochemical identification kit or biochemical identification card instead of 5.4.2.1 ~ 5.4.2.3 be
Identification.
5.5 Results Report
Based on the above test results, the test sample reporting units detected or not detected Campylobacter jejuni.
Appendix A
Media and reagents
A.1 Bolton broth (Boltonbroth)
A.1.1 basal medium
A.1.1.1 ingredient
Animal tissue hydrolysates 10.0g
Lactalbumin hydrolyzate 5.0g
Yeast extract 5.0g
Sodium chloride 5.0g
0.5g sodium pyruvate
Sodium bisulfite 0.5g
Sodium carbonate 0.6g
α- ketoglutarate 1.0g
Distilled water 1000.0mL
A.1.1.2 Method
A.1.1.1 The respective components dissolved in distilled water, 121 ℃ sterilization 15min, standby.
A.1.2 split release fibers Sterile sheep or horse blood
Sterile defibrinated sheep or horse blood lysed by repeated freeze-thaw or saponin lysed.
A.1.3 antibiotic solution
A.1.3.1 ingredient
Cefoperazone (cefoperazone) 0.02g
Vancomycin (vancomycin) 0.02g
Three Trimethoprim lactate (trimethoprimlactate) 0.02g
Amphotericin B (amphotercinB) 0.01g
Polymyxin B (polymyxinB) 0.01g
Ethanol/sterile water (50/50, volume fraction) 5.0mL
A.1.3.2 Method
The A.1.3.1 each ingredient is dissolved in ethanol/sterile water mixed solution.
A.1.4 complete medium
A.1.4.1 ingredient
Basal Medium 1000.0mL
Split release sterile fiber sheep or horse blood 50.0mL
Antibiotic solution 5.0mL
A.1.4.2 Method
When the temperature of the basal medium of about about 45 ℃, added sterile sheep or horse blood and antibiotic solution, mix, correcting pH to 7.4
± 0.2 (25 ℃), placed at room temperature should not exceed 4h, or stored in the dark at about 4 ℃ shall not exceed 7d.
A.2 improved CCD agar (modifiedCharcoalCefoperazoneDeoxycholateAgar, mCCDA)
A.2.1 basal medium
A.2.1.1 ingredient
Meat infusion 10.0g
Animal tissue hydrolysates 10.0g
Sodium chloride 5.0g
Charcoal 4.0g
Casein hydrolyzate 3.0g
1.0g sodium deoxycholate
Ferrous sulfate 0.25g
0.25g sodium pyruvate
Agar 8.0g ~ 18.0g
Distilled water 1000.0mL
A.2.1.2 Method
A.2.1.1 The respective components dissolved in distilled water, 121 ℃ sterilization 15min, standby.
A.2.2 antibiotic solution
A.2.2.1 ingredient
Cefoperazone (cefoperazone) 0.032g
Amphotericin B (amphotericinB) 0.01g
Rifampin (rifampicin) 0.01g
Ethanol/sterile water (50/50, volume fraction) 5.0mL
A.2.2.2 Method
The A.2.2.1 each ingredient is dissolved in ethanol/sterile water mixed solution.
A.2.3 complete medium
A.2.3.1 ingredient
Basal Medium 1000.0mL
Antibiotic solution 5.0mL
A.2.3.2 Method
When the temperature of the basal medium is about 45 ℃, added antibiotic solution and mix. Correcting pH to 7.4 ± 0.2 (25 ℃). pour
Note 15mL sterile petri dish and allowed to stand until the medium solidified. Pre-dried before use tablet. Flat when not prepared to put dried at room temperature
Set shall not exceed 4h, or refrigerated at 4 ℃ shall not exceed 7d.
A.3 Columbia blood agar (Columbiabloodagar)
A.3.1 basal medium
A.3.1.1 ingredient
Animal tissue hydrolysates 23.0g
Starch 1.0g
Sodium chloride 5.0g
Agar 8.0g ~ 18.0g
Distilled water 1000.0mL
A.3.1.2 Method
The A.3.1.1 ingredients dissolved in distilled water, 121 ℃ sterilization 15min, standby.
A.3.2 sterile defibrinated sheep blood
Under aseptic conditions, the sheep blood poured into a sterilized glass beads vessel and shaken for about 10min, after the standing was removed with fibrin
Glass beads can be.
A.3.3 complete medium
A.3.3.1 ingredient
Basal Medium 1000.0mL
Sterile defibrinated sheep blood 50.0mL
A.3.3.2 Method
When the temperature of the basal medium is about 45 ℃, the sterile sheep blood was added, and mix. Correcting pH to 7.3 ± 0.2 (25 ℃). pour
15mL culture based entirely sterile petri dish and allowed to stand until the medium solidified. Prepared flat when not dried left at room temperature should not exceed 4h,
Or refrigerated at 4 ℃ shall not exceed 7d.
A.4 Brucella Broth (Brucelabroth)
A.4.1 ingredients
Casein hydrolyzate 10.0g
Animal tissue hydrolysates 10.0g
Glucose 1.0g
Yeast extract 2.0g
Sodium chloride 5.0g
Sodium bisulfite 0.1g
Distilled water 1000.0mL
A.4.2 Method
The A.4.1 of each component is dissolved in distilled water, pH correction to 7.0 ± 0.2 (25 ℃), 121 ℃ sterilization 15min, standby.
A.5 oxidase reagent
A.5.1 ingredients
Tetramethyl-p-phenylenediamine hydrochloride 1.0g
Distilled water 100.0mL
A.5.2 Method
A.5.1 before use each component is dissolved in distilled water.
A.6 Sodium hippurate hydrolysis reagent
A.6.1 sodium hippurate
A.6.1.1 ingredient
10.0g of sodium hippurate
Phosphate buffered saline (PBS) Component.
Sodium chloride 8.5g
Disodium hydrogen phosphate 8.98g
2.71g sodium dihydrogen phosphate
Distilled water 1000.0mL
A.6.1.2 Method
Sodium hippurate is dissolved in the phosphate buffer, filtered sterilization. Aseptic packaging, each tube 0.4mL, stored at -20 ℃.
A.6.2 3.5% (hydrated) ninhydrin solution (mass/volume)
A.6.2.1 ingredient
(Hydrated) ninhydrin (ninhydrin) 1.75g
Acetone 25.0mL
Butanol 25.0mL
A.6.2.2 Preparation
The (hydrated) ninhydrin dissolved in acetone/butanol mixture. The solution was frozen in the dark when no more than 7d.
A.7 Skirrow blood agar (Skirrowbloodagar)
A.7.1 basal medium
A.7.1.1 ingredient
Peptone 15.0g
Tryptone 2.5g
Yeast extract 5.0g
Sodium chloride 5.0g
Agar 15.0g
Distilled water 1000.0mL
A.7.1.2 Method
A.7.1.1 The respective components dissolved in distilled water, 121 ℃ sterilization 15min, standby.
A.7.2 FBP solution
A.7.2.1 ingredient
0.25g sodium pyruvate
0.25g sodium metabisulfite
Ferrous sulfate 0.25g
Distilled water 100.0mL
A.7.2.2 Method
The A.7.2.1 of each component is dissolved in distilled water, filter-sterilized through 0.22μm membrane. FBP according to requirements using now, in
-70 ℃ storage no more than three months or stored at -20 ℃ not more than one month.
A.7.3 antibiotic solution
A.7.3.1 ingredient
Cefoperazone (cefoperazone) 0.032g
Amphotericin B (amphotericinB) 0.01g
Rifampin (rifampicin) 0.01g
Ethanol/sterile water (50/50, volume fraction) 5.0mL
A.7.3.2 Method
The A.7.3.1 each ingredient is dissolved in ethanol/sterile water mixed solution.
A.7.4 sterile defibrinated sheep blood
Under aseptic conditions, the sheep blood poured into a sterilized glass beads vessel and shaken for about 10min, after the standing was removed with fibrin
Glass beads can be.
A.7.5 complete medium
A.7.5.1 ingredient
Basal Medium 1000.0mL
FBP solution 5.0mL
Antibiotic solution 5.0mL
Sterile defibrinated sheep blood 50.0mL
A.7.5.2 Method
When the temperature of the basal medium is about 45 ℃, added FBP solution, antibiotic solution and freeze-thaw sterile defibrinated sheep blood,
Mix well. Correcting pH to 7.4 ± 0.2 (25 ℃). Pour 15mL sterile petri dish and allowed to stand until the medium solidified. Plates previously prepared
When unseasoned place shall not exceed 4h at room temperature or refrigerated at 4 ℃ shall not exceed 7d.
A.8 paper indole acetate
A.8.1 ingredients
Indole acetic acid lipid 0.1g
Acetone 1.0mL
A.8.2 Method
Indole acetic acid resin dissolved in acetone draw 25μL ~ 50μL solution on a blank piece of paper (diameter 0.6cm ~ 1.2cm). Room temperature
Dried, brown tube with a silica gel plug/bottle at 4 ℃.
A.9 0.1% peptone water
A.9.1 ingredients
Peptone 1.0g
Distilled water 1000.0mL
A.9.2 Method
The peptone was dissolved in distilled water, correcting the pH to 7.0 ± 0.2 (25 ℃), 121 ℃ autoclave 15min.
A.10 1mol/L sodium thiosulfate (Na2S203) solution of
A.10.1 ingredient
Sodium thiosulfate (anhydrous) 160.0 g of
Sodium carbonate (anhydrous) 2.0 g of
Distilled water 1000.0mL
A.10.2 Method
Weigh 160g of anhydrous sodium thiosulfate, was added 2g of anhydrous sodium carbonate was dissolved in 1000mL of water, slowly boiled 10min, cooled.
A.11 3% hydrogen peroxide (H2O2) solution of
A.11.1 ingredient
30% hydrogen peroxide (H2O2) solution 100.0mL
Distilled water 900.0mL
A.11.2 Method
Draw 100mL30% hydrogen peroxide (H2O2) solution, dissolved in 900mL distilled water, mix, sub-equipment use.
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