GB/T 4789.6-2003 Microbiological examination of food hygiene - Examination of diarrhea-causing Escherichia coli
Some standard content:
ICS07.100.30
National Standard of the People's Republic of China
GB/T4789.6—2003
Replaces GB/T4789.6—1994
Microbiological examination of food hygiene-Examination of diarrheogenic Escherichia coli2003-08-11 Issued
Ministry of Health of the People's Republic of China
Standardization Administration of the People's Republic of China
2004-01-01 Implementation
GB/T4789.6—2003
This standard amends GB/T4789.6--1994 "Test for Diarrhea Escherichia coli". The format and text of the standard text are modified in accordance with GB/T1.1-2000. - Modify and standardize "Equipment and Materials" in the original standard. From the date of implementation of this standard, GB/T4789.6-1994 will be abolished at the same time. This standard is proposed and managed by the Ministry of Health of the People's Republic of China. Drafting units of this standard: Jiangxi Provincial Health and Epidemic Prevention Station, Nutrition and Food Safety Institute of Chinese Center for Disease Control and Prevention. The main drafters of this standard are He Xiaoqing, Ran Lu, Fu Zang, and Yao Jinghui. This standard was first issued in 1984, revised for the first time in 1994, and this is the second revision. 40
1 Scope
Microbiological examination of food hygiene
Testing for diarrhea-causing Escherichia coli
This standard specifies the test method for diarrhea-causing Escherichia coli in food. This standard is applicable to the test of diarrhea-causing Escherichia coli in food and food poisoning samples. 2 Normative references
GB/T4789.6—2003
The clauses in the following documents become the clauses of this standard through reference in this standard. For any dated referenced document, all subsequent amendments (excluding errata) or revisions are not applicable to this standard. However, the parties to an agreement based on this standard are encouraged to study whether the latest versions of these documents can be used. For any undated referenced document, the latest version shall apply to this standard. GB/T4789.28—2003 Food hygiene microbiology inspection staining method, culture medium and reagents 3 Equipment and materials
Refrigerator: 0℃~4℃.
3.2 Constant temperature incubator: 36℃±1℃, 42℃. 3.3 Constant temperature water bath: 100℃, 65℃～68℃, 50℃. 3.4 Microscope: 10X~100×.
Centrifuge: 3000r/min.
3.6 Microplate reader.
Homogenizer or sterilized mortar.
Stand-plate drug balance: 0g～500g, accurate to 0.5g. 3.94Www.bzxZ.net
Bacteria concentration turbidimetric tube: MacFarland No. 3. 3.10 Sterilized wide-mouth bottle: 500mL.
3.11 Sterile conical flask: 500mL, 250mL. 3.12 Sterile pipette: 1mL (with 0.01mL scale), 5mL (with 0.1mL scale). 3.13 Sterile culture III: 90mm in diameter.
3.14 Sterile test tube: 10mm×75mm.16mm×160mm. 3.15 Syringe: 0.25mL, connected to a small plastic tube with an inner diameter of 1mm. 3.16 Sterile knives, scissors, tweezers, etc. 3.17 White mice: 1 to 4 days old.
3.18 Nitrocellulose filter membrane 150mm×50mm, o.45μm. Cut into two pieces before use, each 75mm×50mm, mark with a pencil, each grid 6mm×6mm, 10 grids per row, divided into 6 rows. Sterilize and set aside. 41
GB/T4789.6—2003
4 Culture medium and reagents
Lactose bile salt fermentation tube: in accordance with 4.9 of GB/T4789.28—2003. 4.1
4.2 Nutrient broth: in accordance with 4.8 of GB/T4789.28—2003. 4.3 Enterobacteria enrichment broth: in accordance with 4.18 of GB/T4789.28--2003. MacConkey agar: in accordance with 4.24 of GB/T4789.28—2003. 4.4
Eosin methylene blue agar (EMB): in accordance with 4.25 of GB/T4789.28—2003. 4.5
4.6 Trisaccharide iron agar (TSI): in accordance with 4.26 and 4.27 of GB/T4789.28--2003. 4.7 Klein double sugar iron agar (KI): in accordance with 4.28 and 4.29 of GB/T4789.28--2003. Sugar fermentation tube (lactose, rhamnose, xylose and mannitol): in accordance with 3.2 of GB/T4789.28-2003. 4.8
Lysine decarboxylase test medium: in accordance with 3.12 of GB/T4789.28--2003. 4.9
4.10 Urea agar (pH7.2): in accordance with 3.15 of GB/T4789.28--2003. 4.11 Potassium cyanide (KCN) culture medium: in accordance with 3.16 of GB/T4789.28-2003. Protein water, indigo matrix reagent: in accordance with 3.13 of GB/T4789.28-2003. 4.12
Semisolid agar: in accordance with 4.30 of GB/T4789.28-2003. 4.13
4.14 Honda's toxigenic broth: in accordance with 4.34 of GB/T4789.28-2003. 4.15 Elek's culture medium: in accordance with 4.35 of GB/T4789.28-2003. 4.16 Oxidase reagent: in accordance with 3.18 of GB/T4789.28-2003. 4.17 Gram staining solution: in accordance with 2.2 of GB/T4789.28-2003. 3 Pathogenic Escherichia coli diagnostic serum, invasive Escherichia coli diagnostic serum, enterotoxigenic Escherichia coli diagnostic serum, hemorrhagic Escherichia coli diagnostic serum. 4.18
4.19 Enterotoxigenic Escherichia coli LT and ST enzyme-labeled diagnostic kit. 4.20 Standard strains of enterotoxigenic LT and ST Escherichia coli. 4.21 Anti-LT antitoxin.
Polymyxin B paper strip: 300U, 16mm.
4.23 0.1% thimerosal solution.
4.24 2% Evans blue solution.
5 Test procedure
The test procedure for diarrheagenic Escherichia coli is shown in Figure 1. 42
Xie E. coli MPV
Milk essence fermentation positive
Fermentation tube
Serious contamination of sample liquid
Ling Kangkai
18h~24h
36±1r
GB/T4789.6—2003
25g+nutrient broth 225mL
Enterobacterial enrichment broth
3~5 lactose fermenting or non-fermenting colonies, oxidized alcohol, Gram-negative bacteria
TSI, indigo matrix, pH7.2 urea,
KCN, knockout amino acid, power
TSI bottom layer+, HS-
KCN-, urea-
Serological test
Enterotoxin+
Enterotoxigenic
Enterotoxin-producing
Escherichia coli
EPEC+ or
EHEC+(O157)
Lysine-, indole-
Motility-(0124 motility+/-)
Enteropathogenic Escherichia coli
Enterohemorrhagic Escherichia coli
Enteroinvasive
Escherichia coli
Not as described on the left
Various nests
Non-diarrhea
Escherichia coli
Not as described on the left
Various reaction structures
Non-coli
Escherichia coli
GB/T4789.6-—2003
6 Operation steps
6.1 Enrichment
The sample should be tested as soon as possible after collection. Except for perishable foods that are pre-refrigerated before testing, it is generally not refrigerated. Take 25g (mL) of the sample with aseptic operation, add it to 225mL nutrient broth, and break it with a homogenizer for 1min or grind it with sterilized sand in a mortar. Take out an appropriate amount and inoculate it with lactose bile salt medium to determine the MPN of coliform bacteria. The rest is transferred to a 500mL wide-mouth bottle and cultured at 36℃±1℃ for 6h. Pick 1 loop and inoculate it into 1 tube of 30mL enteric bacteria enrichment broth, and culture it at 42℃ for 18h. 6.2 Separation
The lactose bile salt fermentation tube and enrichment solution that are positive for lactose fermentation are streaked and inoculated on MacConkey or eosin-methylene blue agar plates respectively: For severely contaminated samples, the sample liquid can be directly streaked and inoculated on MacConkey or eosin-methylene blue plates, and cultured at 36℃±1℃ for 18h~24h to observe the colonies. Not only should we pay attention to the lactose fermenting colonies, but also the lactose non-fermenting and slow fermenting colonies. 6.3 Biochemical test
6.3.1 Pick several colonies directly from the identification plate and inoculate them with trisaccharide iron agar (TSI) or Klebsiella bisugar iron agar (KI). At the same time, inoculate these tower cultures with peptone vine water, semi-solid, pH7.2 urea agar, KCN broth and lysine decarboxylase test medium. All the above cultures are cultured at 36℃ overnight.
6.3.2 Cultures that produce acid or no acid on the TSI slant, produce acid on the bottom layer, are H, S negative, KCN negative and urea negative are Escherichia coli. Cultures that do not produce acid on the TSI bottom layer, or are positive for any one of H2S, KCN and urea are not Escherichia coli. Perform oxidase test and Gram staining when necessary.
6.4 Serological test
6.4.1 Presumptive test: Select agar culture confirmed to be Escherichia coli by biochemical test, and use polyvalent 0 serum of pathogenic Escherichia coli, invasive Escherichia coli and enterotoxigenic Escherichia coli and O157 serum of hemorrhagic Escherichia coli for slide agglutination test. When agglutination with a certain polyvalent ○ serum is observed, the test is then conducted with the monovalent O serum contained in the polyvalent serum. The O antigen group included in diarrheagenic Escherichia coli is shown in Table 1. If a strong agglutination reaction is observed with a certain monovalent ○ serum, the presumptive test is positive. Table 1 0 antigen groups included in diarrhea-causing Escherichia coli Types of Escherichia coli
0 antigen groups included
026055086011lab011401190125ac01270128ab01420158
028ac029
0112ac0115012401350136 014301440167
06011015
06307808501140115
0128ac014801490159
0166Q167
6.4.2 Confirmation test: Prepare 0 antigen suspension and dilute it to a concentration equivalent to that of MacFarland No. 3 turbidity tube. The original titer of 0 serum is (1:160)~1:320), and it is diluted to 1:40 with 0.5% saline. The diluted serum and antigen suspension are mixed in equal amounts in a 10mm×75mm test tube, and a single tube agglutination test is performed. After mixing, place it in a 50℃ water bath and observe the results after 16 hours. If agglutination occurs, it can be confirmed to be the ○ antigen.
6.5 Enterotoxin test
6.5.1 Enzyme-linked immunosorbent assay to detect LT and ST6.5.1.1 Toxigenic culture: Inoculate the test strain and the positive and negative control strains into 0.6 mL CAYE medium, shake and culture at 37°C overnight. Add 0.05 mL of 20,000 IU/mL polymyxin B, incubate at 37°C for 1 hour, centrifuge at 4,000 r/min for 15 minutes, separate the supernatant, add 0.05 mL of 0.1% thimerosal, and store at 4°C for use. 6.5.1.2 LT detection method (double antibody sandwich method): Coating: first take out the LT antibody tube for coating from the enterotoxigenic Escherichia coli LT and ST enzyme-labeled diagnostic kit, add 0.5mL of a)
GB/T4789.6-2003
coating solution, mix it and aspirate it all into 3.6mL of coating solution, mix it evenly, add 100μL per well to a 40-well polystyrene hard reaction plate, leave the first well blank as a control, and place it in a 4℃ refrigerator wet box overnight. Wash the plate: shake off the solution in the plate, wash it three times with washing solution I, shake off all the liquid, turn the reaction plate over, tap it on absorbent paper, and remove all the b)
residual liquid in the well.
Blocking: add 100μL of blocking solution to each well and place it in a 37℃ water bath for 1h. c)
Wash the plate: wash it three times with washing solution IⅡ, and operate as above. d)
e) Add sample: add 100μL of toxin-producing culture fluid of various test strains to each well, and place in a 37℃ water bath for 1h. Wash plate: wash three times with washing solution Ⅱ, and operate as above. f)
Add enzyme-labeled antibody: first add 0.5mL of diluent to the enzyme-labeled LT antibody tube, mix and aspirate all into 3.6mL of diluent g)
Mix, add 100μL to each well, and place in a 37℃ water bath for 1h. Wash plate: wash three times with washing solution Ⅱ, and operate as above. h)
Enzyme substrate reaction: add 100μL of matrix solution to each well (including the first well), incubate in the dark for 5min10min at room temperature, and add 50L of stop solution i)
.
j) Result determination: Measure the absorbance OD value at a wavelength of 492nm with an enzyme marker. The OD value of the sample to be tested is more than 3 times that of the negative control, and the color is orange or significantly higher than the negative control. 6.5.1.3ST detection method (antigen competition method): Coating: First add 0.5mL of coating solution to the ST antigen tube for coating, mix well, and then aspirate all of it into 1.6mL of coating solution and mix well. a
Add 50μL per well to a 40-well polystyrene soft reaction plate. After adding the liquid, tap the plate gently to make the liquid cover the bottom of the well. Leave the first well empty as a control and place it in a 4℃ refrigerator wet box overnight. Wash the plate: Wash three times with washing solution I, and operate as above. b)
Blocking: Add 100μL of blocking solution to each well and place in a 37℃ water bath for 1h. e
Wash the plate: Wash three times with washing solution II, and operate as above. d)
Add samples and ST monoclonal antibodies: add 50μL of toxin-producing culture fluid of each test strain to each well, and 50μL of diluted ST monoclonal antibodies (first add 0.5mL of diluent to the ST monoclonal antibody tube, mix well, and then aspirate all into 1.6mL of diluent, mix well for use), and place in a water bath at 37℃ for 1h.
f) Wash the plate: wash three times with washing solution Ⅱ, and operate as above. Add enzyme-labeled rabbit anti-mouse Ig complex: first add 0.5mL of diluent to the enzyme-labeled rabbit anti-mouse Ig complex tube, mix well, and then aspirate all into 3.6mL of diluent, mix well, add 100μL to each well, and place in a water bath at 37℃ for 1h. Wash the plate: wash three times with washing solution ⅡI, and operate as above. h)
Enzyme substrate reaction, add 100μL of matrix solution to each well (including the first well), keep away from light at room temperature for 5min~10min, and then add 50μL of stop solution.
j) Result determination: Use a microplate reader to measure the absorbance (OD) value at a wavelength of 492nm, and calculate as shown in formula (1): Absorbance = OD value of negative control stop sample × 100% OD value of negative control
Absorbance greater than or equal to 50% is positive. Visual inspection is colorless or obviously lighter than the negative control is positive. 6.5.2 Two-way agar diffusion test detection L1
...(1)
Inoculate the test strain on Elek's culture medium in a five-point circle. Perform the same operation in duplicate and culture at 36℃ for 48h. Place a polymyxin B paper sheet on the bacterial lawn of each strain, and incubate at 36°C for 5h~6h to allow the enterotoxin to penetrate into the agar. Dig a 4mm diameter circular hole in the center of each of the five circular bacterial lawns at 5mm, and use a drop of agar as a bottom. Add 30μL of LT antitoxin to the central hole of the plate, use known LT-producing and non-toxin-producing strains as controls, and observe the results at 36°C for 15h~20h. A white precipitation band appears between the bacterial plaque and the antitoxin hole, which is positive, and a white precipitation band is negative. 6.5.3 Test for ST in suckling mice by oral gavage: Inoculate the strain to be tested into Honda's toxin-producing broth, incubate at 36℃ for 24h, centrifuge at 3000r/min for 30min, take the supernatant 45
GB/T4789.6-2003
, filter it through a membrane filter, heat it at 60℃ for 30min, and add 0.02mL of 2% Evans blue solution to every 1mL of filtrate. Inject 0.1mL of this filtrate into the stomach of suckling mice aged 1 to 4 days with a plastic tube, and inoculate 3 to 4 mice at the same time. After fasting for 3h to 4h, anesthetize them with chloroform, remove all the intestines, weigh the weight of the intestines (including the effusion) and the remaining body weight. The ratio of the weight of the intestine to the remaining body weight is greater than 0.09, which is positive, and 0.07~0.09 is suspicious.
6.6 Result report
Make a report based on the above biochemical tests, serological tests, and enterotoxin tests. 46
Tip: This standard content only shows part of the intercepted content of the complete standard. If you need the complete standard, please go to the top to download the complete standard document for free.