GB/T 4789.9-2003 Food hygiene microbiology test Campylobacter jejuni test

This standard specifies the test method for Campylobacter jejuni. This standard is applicable to the test for Campylobacter jejuni in overstocked food and food poisoning samples. GB/T 4789.9-2003 Food Hygiene Microbiological Test Campylobacter jejuni Test GB/T4789.9-2003 Standard Download Decompression Password: www.bzxz.net

ICS07.100.30
National Standard of the People's Republic of China
GB/T4789.9—2003
Replaces GB/T4789.9—1994
Microbiological examination of food hygieneExamination of Campylobacter jejuniPromulgated on August 11, 2003
Ministry of Health of the People's Republic of China
Administrative Committee of Standardization of the People's Republic of China
Implementation on January 1, 2004
GB/T4789.9-—2003
This standard revise GB/T4789.9—1994 "Microbiological examination of food hygieneExamination of Campylobacter jejuni". Compared with GB/T4789.9-1994, this standard has the following major revisions: - The format and text of the standard text are revised according to GB/T1.1-2000. - The "equipment and materials" in the original standard are revised and standardized. GB/T4789.9-1994 will be abolished as of the date of implementation of this standard. This standard is proposed and managed by the Ministry of Health of the People's Republic of China. The drafting units of this standard are: China Import and Export Commodity Inspection Technology Research Institute, Nutrition and Food Safety Institute of China Center for Disease Control and Prevention. The main drafters of this standard are: Meng Hongde, Ran Lu, Fu Ping, Yao Jinghui. This standard was first issued in 1984, revised for the first time in 1994, and this is the second revision. 60
1 Scope
Food Hygiene Microbiological Examination
Campylobacter jejuni Examination
This standard specifies the test method for Campylobacter jejuni. This standard is applicable to the test of Campylobacter jejuni in various types of food and food poisoning samples. 2 Normative references
GB/T4789.9—2003
The clauses in the following documents become the clauses of this standard through reference in this standard. For all dated references, all subsequent amendments (excluding errata) or revisions are not applicable to this standard. However, parties to an agreement based on this standard are encouraged to study whether the latest versions of these documents can be used. For all undated references, the latest versions are applicable to this standard. GB/T4789.28—2003 Food hygiene microbiology inspection staining method, culture medium and reagents 3 Equipment and materials
3.1 Refrigerator: 0℃~4℃.
Constant temperature incubator: 42℃±1℃, 36℃±1℃, 25℃±1℃. 3.2
3.3 Constant temperature water bath: 37℃±1℃.
3.4 ​​Microscope: 10×~100×.
3.5 Phase contrast microscope
3.6 Centrifuge: 3000r/min.
Homogenizer or sterile mortar.
Anaerobic tank: with a two-phase pressure gauge.
3.9 Liquid nitrogen tank.
3.10 Air bag.
Sterile thick-walled capillary.
Sterile culture III: 90mm in diameter.
Sterile conical flask: 500mL.
Sterile knife, scissors, tweezers, etc.
4 Culture medium and reagents
Protein stale water: in accordance with 3.13 of GB/T4789.28-2003. 4.1
4.2Cary-Blair's transport medium: in accordance with 4.52 of GB/T4789.28-2003. 4.3 Modified Camp-BAP medium: in accordance with 4.53 of GB/T4789.28-2003. 4.4 Skirrow's medium: in accordance with 4.54 of GB/T4789.28-2003. 4.5 Oxidase reagent: in accordance with 3.18 of GB/T4789.28-2003. 4.63% hydrogen peroxide solution: in accordance with 3.20 of GB/T4789.28-2003. 4.71% glycine medium: in accordance with 4.56 of GB/T4789.28-2003. 4.830μg nalidixic acid circular filter paper.
4.9 Trisaccharide iron medium: in accordance with 4.26 of GB/T4789.28-2003. 4.10 Gram staining solution: in accordance with 2.2 of GB/T4789.28-2003. 61
GB/T4789.9-—2003
TTC agar: in accordance with 4.55 of GB/T4789.28-2003. Sodium hippurate culture medium: in accordance with 3.9 of GB/T4789.28-2003. 4.12
Ferric trifluoride: in accordance with 3.9 of GB/T4789.28-2003. Rapid hydrogen sulfide (HzS) test agar: in accordance with 4.50 of GB/T4789.28-2003. 4.14
5DNAse methyl green agar: in accordance with 4.51 of GB/T4789.28-2003. 4.15
5 Test procedureWww.bzxZ.net
The test procedure for Campylobacter jejuni is shown in Figure 1.
Modified Camp-BAP
Bruceella culture medium
42℃, 48h, microaerobic
6 Operation steps
Serotyping
6.1 Collection and processing of samples
Blood agar plate
42℃, 48h, microaerobic
Biological typing
Food samples should be tested as soon as possible after collection. If samples must be transported and stored, Cary-Blair semi-solid medium should be used. The survival time of Campylobacter depends on temperature. At 25℃, its survival time is less than 24h or even shorter, so samples should be kept in a refrigerator or cold place for 62
before initial isolation.
6.2 Preparation of microaerobic conditions
The best microaerobic conditions are 5% oxygen, 10% carbon dioxide, and 85% nitrogen. GB/T4789.9--2003
6.2.1 Anaerobic tank with a two-phase pressure gauge (which can indicate positive and negative pressure): After installing III seal, first evacuate the air in the tank to a negative pressure of 7.3×10\Pa (550mmHg), and input the above three mixed gases to restore the pressure in the tank to zero; then evacuate the gas in the tank to a negative pressure of 7.3×10*Pa (550mmHg) and input the above gases to zero, and then place it in an incubator for cultivation. 6.2.2 Air bag method: Only use a gas generator without a catalyst during operation. 6.2.3 Double plate method: Take two plates, one plate is inoculated with known facultative anaerobic bacteria (such as Proteus), and the other plate is inoculated with samples. Remove the lid of the dish, put the two plates containing agar together and seal them with tape, and place them in a sealed airtight jar (or bag) for cultivation. Due to the metabolic effect of the growth of facultative anaerobic bacteria, the oxygen concentration in the microenvironment can be reduced and the carbon dioxide concentration can be increased. 6.2.4 Candle jar method: Place the plate inoculated with bacteria in a 2000mL ground-mouth specimen jar or desiccator. Apply vaseline to the jar cover and the mouth of the jar, put a small piece of lit candle in the jar (do not get close to the jar wall to avoid heating the jar wall and bursting), and cover the jar cover tightly. The candle is burned in the jar for about 0.5min~1min, and it will extinguish itself due to the reduction of oxygen. At this time, the container contains about 5%~10% carbon dioxide. Finally, place it together with the container in a 42℃±1℃ incubator for cultivation.
6.3 Isolation and culture
6.3.1 Swabs or liquids can be streaked directly on selective culture media such as modified Camp-BAP Brucella culture media or Skirrow's culture media plates. After culturing at 42℃ for 48 hours, observe the suspected colonies. 6.3.2 Type I colonies are non-hemolytic, gray, flat, moist, shiny, and look like water droplets, with a tendency to spread outward from the inoculation line. Type II colonies are also non-hemolytic, often appearing as scattered, raised single colonies (1mm2mm in diameter), with complete, shiny edges. 6.4 Preliminary identification
Submit the suspected colonies for oxidase and catalase tests. If positive, smear the smear for Gram staining. This bacterium is a Gram-negative bacterium with a size of (0.3μm~0.4μm)×(1.5μm3μm), and is S-shaped, spiral or spindle-shaped. If cultured on solid culture media for too long or under inappropriate conditions, it often appears as spherical bacteria. Observe under dark field or phase contrast microscope, the motility is obvious. According to the colony growth characteristics, bacterial staining, motility, oxidase and catalase test, those who are positive can be preliminarily determined to be Campylobacter. 6.5 Definitive identification
After preliminary identification, the following tests are still required (the following tests must be cultured in a microaerobic environment with 5% oxygen). 6.5.1 Glycine tolerance test: Inoculate in glycine culture medium and culture for 48 hours. If it is positive and there is cloudy growth on the surface of the culture medium, it is Campylobacter jejuni subspecies.
6.5.2 Hydrogen sulfide growth test: Inoculate on the TSI slant and hang a lead acetate filter paper strip at the mouth of the tube. After 48h72h of culture, Campylobacter jejuni generally grows a small amount on the slant, and its reaction is alkaline/alkaline (K/K). The filter paper strip turns black, but the bottom of the culture medium does not turn black due to hydrogen sulfide.
6.5.3 Tolerance test for 3.5% sodium chloride: Inoculate on 3.5% salt broth medium, after 48h of culture, Campylobacter jejuni does not grow.
6.5.4 42℃ and 25℃ growth test: Inoculate the culture into two tubes of Brucella broth, one tube is placed at 42℃, and the other tube is placed at 25℃ for 48h. If it grows at 42℃ but not at 25℃, it is Campylobacter jejuni. 6.5.5 Sodium hippurate hydrolysis test: Inoculate the culture into sodium hippurate medium, culture at 42℃ for 48h, centrifuge the sediment and take out part of the supernatant. Add ferric nitride reagent. If permanent precipitate appears, it is positive. Campylobacter jejuni shows positive reaction. 6.5.6 Nalidixic acid test: Spread suspected Campylobacter jejuni on the blood plate, and then stick filter paper containing 30μg nalidixic acid on the plate. Campylobacter jejuni is sensitive but Campylobacter intestinalis is not sensitive. 6.5.7 TTC (2,3,5-chloro-blue benzotetrazolyl) test: Campylobacter jejuni is positive on TTC culture medium, while Campylobacter fetus is negative. The positive one is purple and shiny. 6.5.8 Nitrate reduction test: Take the concentrated bacterial suspension of the culture, place it at 37℃ for 2h, add the reagent, observe the color reaction, and Campylobacter jejuni shows a red positive reaction.
GB/T4789.9—-2003
The biochemical identification of Campylobacter is shown in Table 1.
Table 1 Biochemical properties of various species of Campylobacter
Calatase
Oxidase
1% glycine test
Hydrogen sulfide test (paper strip method)
3.5% sodium chloride tolerance test
25℃ growth test
42℃ growth test
Nitrate reduction test
Nalidixic acid test||tt| |TTC test
Sodium hippurate hydrolysis
Fetus subspecies
Campylobacter fetus
Colic subspecies
Jejunal subspecies
Campylobacter sputum
Sputum subspecies
Bovine subspecies
Note: +90% and above 90% positive; dSome negative, some positive; (i) Generally no growth; -90% and above 90% negative;? Unknown. 6.6 Biotyping
Campylobacter faecalis
6.6.1 Rapid sodium hippurate hydrolysis test: Inoculate the bacteria in 0.4mL of 1% sodium hippurate, bathe in 37℃ water for 2h, then add 0.2mL of 3.5% benzyltrione in acetone and butanone solution, add slowly to avoid mixing, and observe the results after 10min. Dark purple is positive, light purple or colorless is negative.
6.6.2 Rapid hydrogen sulfide (HS) test: Take a loop of bacteria and suspend it in one-third of the rapid hydrogen sulfide semisolid agar, and place it in a water bath at 37℃ for 2h. Observe the results. If the area around the bacterial mass turns black, it is positive. 6.6.3 DNA enzyme hydrolysis test: The bacteria are spotted on a DNA enzyme methyl green agar plate and cultured in a microaerobic environment for 3d to 5d. If a colorless transparent ring appears around the bacterial lawn, it is positive. The results of biological typing are shown in Table 2. Table 2 Biological typing of Campylobacter jejuni, etc.
Rapid sodium hippurate test
Rapid sulfide oxygen test
DNA enzyme hydrolysis
6.7 Serological typing
Campylobacter jejuni
Aspergillus li
Campylobacter jejuni O factor typing should use the indirect hemagglutination method, and H or K factor typing can use the slide agglutination method. 6.8 Preservation of strains
Thermophilic Campylobacter
Campylobacter jejuni should be inoculated on the modified Camp-BAP slant medium (the cotton plug should not be too tight), cultured at 42℃～43℃ microaerobic for 48h, and then stored in a 4℃ refrigerator in microaerobic for 10d~14d. It can be transferred once if necessary, and freeze-dried for long-term preservation. 6.9 Precautions
When testing Campylobacter jejuni, aseptic operation should be strictly implemented to prevent self-infection.
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