GB/T 4789.10-2003 Microbiological examination of food hygiene - Examination of Staphylococcus aureus
Some standard content:
ICS 07.100.30
National Standard of the People's Republic of China
GB/T4789.10—2003
Replaces GB/T4789.10-—1994
Microbiological examination of food hygiene—Examination of Staphylococcus aureus
Microbiological examination of food hygiene-Examination of Staphylococcus aureusPromulgated on August 11, 2003
Ministry of Health of the People's Republic of China
Standardization Administration of the People's Republic of China
Implementation on January 1, 2004
GB/T4789.10—2003
This standard revise GB/T4789.10—1994 "Microbiological examination of food hygiene—Examination of Staphylococcus aureus". Compared with GB/T4789.10-1994, the main changes of this standard are as follows: The format and text of the standard text are modified in accordance with GB/T1.1-2000. The "equipment and materials" in the original standard are modified and standardized. The concentration of reagent A.2.15 in Appendix A is changed from 0.008mol/L to 0.2mol/L. From the date of implementation of this standard, GB/T4789.10-1994 will be abolished at the same time. Appendix A of this standard is a normative appendix.
This standard is proposed and managed by the Ministry of Health of the People's Republic of China. The drafting units of this standard: Beijing Municipal Health and Anti-epidemic Station, Institute of Nutrition and Food Safety, Chinese Center for Disease Control and Prevention. The main drafters of this standard: Liu Yi, 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. 66
1 Scope
Microbiological examination of food hygiene
Testing of Staphylococcus aureus
This standard specifies the test method for Staphylococcus aureus in food. This standard is applicable to the test of Staphylococcus aureus in various types of food and food poisoning samples. Normative references
GB/T4789.10—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 the agreement based on this standard are encouraged to study whether the latest versions of these documents can be used. For any undated referenced document, its latest version is applicable to this standard. GB/T4789.28—2003 Microbiological examination of food hygiene Staining methods, culture media and reagents 3 Equipment and materials
3.1 Refrigerator: 0℃~4℃.
3.2 Constant temperature incubator: 36℃±1℃.
3.3 Microscope: 10×100×.
3.4 Homogenizer or sterile mortar.
3.5 Drug volume on tray: 0g~500g, accurate to 0.5g. 3.6 Sterile test tube: 10mm×100mm, 16mm×160mm. 3.7 Sterile pipette: 1mL (with 0.01mL scale), 10mL (with 0.1mL scale). 3.8 Sterile conical flask: 500mL, 100mL. 3.9 Sterile culture III: diameter 90mm.
3.10 Syringe: 0.5mL.
Sterile L-shaped coating rod.
Sterile knife, scissors, tweezers, etc.
Culture medium and reagents
Tryptic soy broth: in accordance with 4.59 of GB/T4789.28-2003. 4.27.5% sodium chloride broth: in accordance with 4.61 of GB/T4789.28-2003. 4.3 Blood agar plate: in accordance with 4.6 of GB/T4789.28-2003. 4.4 Baird-Parker agar plate: in accordance with 4.60 of GB/T4789.28-2003. 4.5 Meat extract broth: in accordance with 4.1 of GB/T4789.28-2003. 4.60.85% sterile saline.
4.7 Rabbit plasma: in accordance with 4.63 of GB/T4789.28-2003. Test procedure
The test procedure for Staphylococcus aureus is shown in Figure 1.67
GB/T4789.10—2003
Direct counting method
Baird-Parker plate
0.3mL, 0.3mL, 0.4mL
36℃±1℃
Blood plate
Removal and staining
6 Operation steps
6.1 Enrichment culture method
25g(mL)+225mL Sterilization Physiological saline
Observe bleeding
Bacterial enrichment culture method
7.5% sodium chloride broth or
tryptic soy broth
36c+1c24h
Blood agar plate, Baird-Parker agar plate
36±1℃
Plasma coagulase test
6.1.1 Sample handling: Take 25g (mL) of sample according to aseptic operation, add 225mL sterile physiological saline, grind the solid sample or put it in a homogenizer to make a suspension.
6.1.2 Enrichment and isolation culture: Take 5mL of the above suspension and inoculate it into 50mL of 7.5% sodium chloride broth or fern casein soy broth, culture it at 36℃±1℃ for 24h, transfer it to blood plate and Baird-Parker plate, culture it at 36℃±1℃ for 24h, pick the golden yellow (sometimes white) colonies on the blood plate for Gram staining and plasma coagulase test. 6.1.3 Morphology: This bacterium is a Gram-positive coccus, arranged in a staphylococcal shape, without spores and capsules. The pathogenic staphylococci are small, with a diameter of about 0.5μm to 1μm.
6.1.4 It grows turbidly in broth, sometimes the liquid is clear in pancreatic casein soy broth, and it grows turbidly when the amount of bacteria is large. The colonies on the blood plate are golden yellow, sometimes white, large and protruding, round, opaque, smooth on the surface, and surrounded by a hemolytic zone. On the Baird-Parker plate, it is round, smooth, convex, moist, 2mm to 3mm in diameter, gray to black in color, with a light edge, surrounded by a turbid band, and a transparent ring on the outer layer. When the colony is touched with an inoculation needle, it feels like the hardness of butter gum, and occasionally you may encounter similar colonies that are not fat-soluble; but there is no turbid band or transparent pattern. The colonies isolated from frozen or dried foods that have been stored for a long time are lighter in black than those produced by typical colonies, and may appear rough and dry.
6.1.5 Plasma coagulase test: Take 0.5mL of 1:4 fresh immune plasma and put it into a small test tube, then add 0.5mL of Staphylococcus aureus meat extract broth culture that has been cultured for 24 hours, shake well, place in a 36℃±1℃ incubator or water bath, observe every half an hour, and observe for 6 hours. If it is coagulated, that is, when the test tube is tilted or inverted, a clot is formed, which is considered a positive result. At the same time, known positive and negative Staphylococcus aureus strains and broth are used as controls.
6.2 Direct counting method
6.2.1 Take the above 1:10 suspension and dilute it 10 times. According to the contamination of the sample, select different concentrations of dilution 68
GB/T4789.10-2003
1mL, add it to three Baird-Parker plates, and the inoculation volume of each plate is 0.3mL, 0.3mL and 0.4mL respectively, and then use a sterilized L stick to coat the entire plate. If it is difficult to absorb too much water, the plate can be placed at 36℃±1℃ for 1h, and after the water evaporates, it can be inverted and placed at 36℃1℃ for incubation.
6.2.2 Count the black colonies with turbid bands around them on the three plates, and select five colonies from them, inoculate them on blood plates respectively, and incubate them at 36℃±1℃ for 24h before staining and microscopic examination and plasma coagulase test. The steps are the same as the enrichment culture method. 6.2.3 Colony count: Add the number of suspected black colonies of Staphylococcus aureus in the three plates, multiply by the number of plasma coagulase-positive colonies, divide by 5, and then multiply by the dilution factor to calculate the number of Staphylococcus aureus per gram of sample. 69
GB/T4789.10—2003
A.1 Equipment and materials
A.1.1 Refrigerator: 0℃~4℃.
A, 1.2 Constant temperature incubator: 36℃±1℃. Appendix A
(Normative Appendix)
Staphylococcal enterotoxin test
A.1.3 Oscillating incubator or ordinary incubator: 36℃±1℃. A.1.4 Microplate reader.
A.1.5 Centrifuge: 8000r/min.
A.1.6Homogenizer or sterile mortar.
Chromatography column: 40mm× (20mm~25mm). A.1.7
Micropipette: 200μL, 50μL.
A,1t.9Thin dropper.
Separating funnel.
Dialysis bag.
2Washing bottle.
Sterile pipette: 1mL (with 0.01mL scale), 10mL (with 0.1mL scale). A.1.13
Sterile culture III: 150mm in diameter (or sugar porcelain plate with lid). Sterile conical flask: 250mL
Organic glass template.
Puncher: 2.5mm in diameter.
Glass slide, rubber ring.
Sterilized glass paper, triangular rods, forges, etc.
A.2 Culture medium and reagents
A.2.1 Enterotoxin production culture medium: in accordance with 4.86 of GB/T4789.28-2003. A.2.2 Nutrient agar: in accordance with GB/T4789.28--2003. A.2.3 1% agarose (prepared with 0.9% saline) solution. A.2.4 0.2mol/L pH7.5 phosphate buffer. A.2.5 Trifluoromethane.
A.2.6 6mol/L hydrochloric acid solution.
A.2.75mol/L sodium hydroxide.
A.2.8 0.85% saline.
A.2.9 1% acetic acid solution.
00.1% thiazine red R or amido black B.
A.2.11 Silica gel or vaseline.
A.2.12 Staphylococcal enterotoxins and antiserum of type A, B, C, D. A.2.13
Carboxymethylcellulose (CM22 or CM11 Whatman) A.2.14
0.2mol/LpH6.8 phosphate buffer.
Enzyme-labeled enterotoxin antiserum of type A, B, C, D or ELISA kit. 0.1mol/LpH9.5 carbonate buffer.
0.05%0.02mol/LpH7.2 Tween-20 buffer. O-phenylenediamine enzyme substrate.
2 mol/L sulfuric acid.
Test procedure
Detection of enterotoxins from strains
The procedure for detecting enterotoxins from strains is shown in Figure A, 1. Smear
nutrient agar slant at 36±1℃ for 18h24hbZxz.net
wash off the bacterial lawn with salt water and put it into toxin-producing culture medium. Peristaltic culture method: culture at 36℃±1℃ for 48h. Liquid culture method: culture at 36℃±1℃ with shaking for 48h. Centrifuge at 8000r/min for 20min, take the supernatant, heat at 100C for 10min. Centrifuge at 8000/min for 10min, and concentrate. Biphasic agar diffusion: microslide method or slide method for enterotoxin detection. Report
A.3.2 Extraction and detection of enterotoxin from food samples. A.3.2.1 Microslide method for enterotoxin detection (concentration method and chromatography method). See Figure A.2 for the procedure. GB/T4789.10-—2003
GB/T4789.10—2003
Add 100 mL of 0.2 mol/L pH 7.5 phosphate buffer to 100 g of solid sample and soak at 4°C for 18 h to 24 h: Directly absorb 100 mL of solid sample and filter with gauze
Liquid sample: Centrifuge at 8000 t/min for 20 min and take the supernatant; Solid sample: Centrifuge at 8000 r/min for 20 min and take the supernatant and add 10 mL of trichloromethane. 15mL, 10min, let stand, discard diazomethane, add 6mol/L hydrochloric acid solution to adjust pH to 4.5, 8000r/min 20min, take the supernatant, add 5mol/L sodium hydroxide solution, adjust pH to 7.5. 8000r/min, 20min, take the supernatant, put it into a dialysis bag, add polyethylene glycol or blow it with an electric fan, concentrate to 1mL2mL, use the microslide method to detect enterotoxin
Concentration method
Wash the concentrate with distilled water
Put it into a dialysis bag, and use 0. 0.008mol/LpH5.6 phosphate buffer was balanced and passed through a CM chromatography column (flow rate 1mL/min～2mL/min). About 100mL of 0.008mol/LpH5.6 phosphate buffer was used for elution. Enterotoxin was eluted with 0.2mol/LpH6.8 phosphate buffer. Enterotoxin was detected by microslide method
Chromatography
Enzyme-linked immunosorbent assay for enterotoxin detection. The procedure is shown in Figure A, 3. Serum was diluted with 0.1mol/LpH9.5 carbonate grade buffer, 0.2mL of styrene was added to the plate, 36℃±1℃30min, the upper liquid was discarded, and 0.05% 0.02mol/LpH7.2 buffer was washed five times at -20℃. Liquid sample was directly added to the plate well with 0.2mL; solid sample 100g + 100mL0.2mol/LpH7.5 phosphate buffer, homogenized and 0.2mL of filtrate was taken, 36±1℃30min Remove the supernatant, wash five times with 0.05% 0.02mol/L pH7.2 Tween-20 buffer, add 0.2mL enzyme antibody, and place at 36℃±1℃ for 30min. Discard the supernatant, wash five times with 0.05% 0.02mol/L pH7.2 Tween-20 buffer, add 0.2mL o-phenylenediamine alcohol substrate, and place at room temperature for 30min. Add 0.05mL 2mol/L sulfuric acid, and immediately compare the color.
A.4 Enterotoxin detection
A.4.1 Method for extracting enterotoxin from strains
GB/T4789.10-2003
A.4.1.1 Liquid dialysis culture method: Use a dialysis bag with a width of 2.5 cm and a length of 80 cm to fill 60 mL of toxin-producing culture medium, tie both ends tightly, put the dialysis bag into a 250 mL conical flask, add 15 mL of sterile saline, leave both ends of the dialysis bag at the mouth of the flask, plug it with cotton plugs, sterilize it at 121℃ for 30 minutes, and inoculate the strain to be tested with nutrients. Incubate on agar slant (test tube 18mmX180mm) at 37℃ for 24h, wash the colony with 5mL of physiological saline and pour into the above-mentioned culture bottle, one bottle for each bacterial species, and culture at 37℃ with shaking for 48h at a vibration speed of 100 times/min. Aspirate the bacterial solution and centrifuge at 8000r/min for 30min. Take the supernatant for two-phase agar diffusion. If it is negative, put it into a dialysis bag and blow it with an electric fan, or use polyethylene glycol to concentrate it to 1mL~2mL, and then do agar diffusion. A.4.1.2 Solid dialysis culture method: Pour about 100mL~120mL of sterile toxin-producing culture medium into a sterile flat dish or a covered porcelain dish with a diameter of 150mm. After solidification, spread a sterile glass paper on the surface. Inoculate the strain to be tested on nutrient agar and culture at 37℃ for 24h. Wash the bacterial moss with about 3mL of sterile saline, pour it on the glass paper, and spread the blood with a sterile triangular stick. Culture at 37℃ for 48h. Add 10mL~20mL of sterile saline, scrape the bacterial moss with a sterile triangular stick, absorb the bacterial solution and centrifuge it. The following steps are the same as the liquid dialysis culture method. A.4.2 Method for extracting enterotoxin from food
A.4.2.1 Direct concentration method: Take 100g of food sample, add sterile 0.2mol/LpH7.5 phosphate buffer, homogenize into a homogenate, soak at 4℃ for 18h24h, filter the filtrate with gauze, centrifuge at 8000r/min for 20min, take the supernatant, put it into a separatory funnel, add 73
GB/T4789.10—2003
10mL of chloroform, shake for 10min, let it stand, and discard the chloroform on the bottom layer (if no stratification occurs, centrifuge at 8000r/min for 20min). Add 6mol/L hydrochloric acid solution to adjust pH to 4.5, centrifuge at 8000r/min for 20min, take the supernatant, add 5mol/L sodium hydroxide solution, adjust pH to 7.5, centrifuge to take the supernatant, put it into dialysis bag or cellophane, blow dry with an electric fan, or add polyethylene glycol to concentrate to 1mL~2mL, and do micro slide two-way agar diffusion.
A.4.2.2 Chromatography: If you need to extract purer enterotoxin, you can wash the above concentrate with distilled water, put it into a dialysis bag, balance it with 0.008mol/LpH5.6 phosphate buffer, add it to the CM chromatography column, flow rate 1mL/min~2mL/min, elute with 0.008mol/LpH5.6 phosphate buffer, and then elute the enterotoxin with 0.2mol/LpH6.8 phosphate buffer. The eluate is placed in a dialysis bag and concentrated to 1 mL using an electric fan or polyethylene glycol. The microslide bidirectional agar diffusion test is performed to detect enterotoxins. A.4.3 Bidirectional agar diffusion test for enterotoxins
A.4.3.1 Microslide method: Wipe the slide soaked in 95% ethanol with clean gauze, absorb the dissolved 0.2% agarose (prepared with distilled water) is dripped on the glass slide, and the remaining agarose is allowed to flow down. It is placed in a dust-free environment to dry. First, a thin plastic plate is placed on the glass slide, and then a thin layer of silica gel or vaseline is applied to the edge of the organic glass template with holes, and it is placed on the plastic plate, and the two sides are fastened with rubber bands to fix it. 1% agarose is absorbed and immediately added from the middle hole of the template between the glass slide and the template until it is full of agarose. After solidification, the agarose in the hole is picked out with a syringe needle, and antiserum is dripped into the middle hole, and the toxin-producing liquid of the strain or food extract is dripped around it. It is placed in a container with wet cotton balls, and the result is observed at 25℃~30℃ for 18h~24h. It can be observed under light and against a dark background. An obvious precipitation line is shown between the antiserum and the extract. If the precipitation line is only faintly visible, it can be stained. For details, see Figure A, 4. ?
Control enterotoxin
Tested sample
Anti-blood-disinfection
Tested sample
A——The tested sample has no enterotoxin, which is negative
Tested sample
Tested samples 3 and 5 contain enterotoxin, which is positive: 4 has no enterotoxin, which is negative. B
Control enterotoxin
Test sample
Antiserum
Test sample
Intercepted sample
A.4.3.2 Slide method: Take 2.5mL of dissolved 1% agarose and spread it on a clean slide. After solidification, use a metal puncher with a diameter of 2.5mm to punch it into a radial shape with a hole spacing of 2.5mm. Add human enterotoxin antiserum to the center hole and human strain or food enterotoxin extract to the six surrounding holes. Put it in a container with a wet cotton ball of 2/10000 sodium trinitride to maintain the mixing degree. Place it at 25℃～30℃ for 18h～20h and observe the results. If there is a clear precipitation line between the extracts of the antiserum, it is positive. A.4.3.3 Staining method: Use the microslide method to remove the tape and organic glass template. The slide method can be directly placed in distilled water and soaked for 4h~8h, changing the water 2 times or 3 times in the middle, and soaked in the following liquids for 10min: 1% thiazine red R (or amino black) and 1% acetic acid in 10% acetic acid; 1% acetic acid containing 1% glycerol. If the decolorization is not complete, continue to soak. After taking it out, cover it with a filter paper, absorb the excess liquid, and dry it at room temperature or 35℃. The precipitate of positive is stained and can be stored for a long time. A, 4.4 Enzyme-linked immunosorbent assay for enterotoxin (double antibody method) A, 4.4.1 Coating antibody: Dilute the enterotoxin antiserum with 0.1mol/LpH9.5 carbonate buffer to 5μg/mL, add it to the cleaned styrene concave plate, 0.2mL per well, place it at 36℃±1℃ for 30min, and discard the upper liquid. A.4.4.2 Washing: Wash five times with 0.05% 0.02mol/L pH7.2 Tween-20 buffer. A.4.4.3 Add test sample: If it is a liquid, add 0.2mL directly. Take 100g of solid sample, add 100mL of 0.2mol/L pH7.5 phosphate buffer, homogenize and take 0.2mL of filtrate. A.4.4.4 Washing: Same as A.4.4.2.
GB/T4789.10—2003
Add 0.2mL of enzyme antibody to each well, place at 36℃±1℃ for 30min, and do positive and negative controls at the same time. Discard the upper liquid. A.4.4.5
A.4.4.6 Washing: Same as A.4.4.2.
A.4.4.7 Add 0.2mL of o-phenylenediamine enzyme substrate solution to each well, place at room temperature for 30min. Add 0.05 mL of 2 mol/L sulfuric acid to each well and immediately place it on a microplate reader for colorimetry. A,4.4.8
Result determination: The OD value of the sample is compared with the negative control. If the ratio is greater than 2, it is positive, and less than 2, it is negative. A.4.4.9
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.