This standard specifies the basic requirements, operating procedures and result judgment of the inspection of Pseudomonas cocovenenans subspecies. This standard is applicable to the etiological diagnosis of food poisoning caused by fermented rice flour, spoiled fresh Tremella and other starchy fermented foods, as well as the routine inspection and strain identification of Pseudomonas cocovenenans subspecies. GB/T 4789.29-2003 Food Hygiene Microbiological Inspection Inspection of Pseudomonas cocovenenans subspecies GB/T4789.29-2003 Standard Download Decompression Password: www.bzxz.net

ICS07.100.30
National Standard of the People's Republic of China
GB/T4789.292003
Replaces GB/T4789.29-1994
Microbiological examination of food hygiene
Examination of Pseudomonas cocovenenans subsp. farinofermentans
Microbiological examination of food hygieneExamination of Pseudomonas cocovenenans subsp. farinofermentans2003-08-11Promulgated
Implementation on 2004-01-01
Ministry of Health of the People's Republic of China
Standardization Administration of the People's Republic of China
GB/T4789.29—2003
This standard revise GB/T4789.29—1994 "Microbiological examination of food hygiene".
Compared with GB/T4789.29-1994, the main modifications of this standard are as follows: "Test for Pseudomonas aeruginosa subspecies fermented rice flour" is modified in accordance with GB/T1.1-2000. "Equipment and materials" in the original standard are standardized. GB/T4789.29-1994 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 unit of this standard: Institute of Nutrition and Food Safety, Chinese Center for Disease Control and Prevention. The main drafters of this standard are Liu Xiumei, Bai Jingyu, Fu Ping, and Yang Baolan. This standard was first issued in 1994, and this is the first revision. 234
1 Scope
Microbiological examination of food hygiene
Examination of Pseudomonas cocovenenans subspecies fermented rice flour
GB/T4789.29—2003
This standard specifies the basic requirements, operating procedures and result determination of the examination of Pseudomonas cocovenenans subspecies fermented rice flour. This standard applies to the etiological diagnosis of food poisoning caused by fermented rice flour, spoiled fresh Tremella and other starchy fermented foods, and the routine examination and identification of Pseudomonas cocovenenans subspecies fermented rice flour. 2
Normative references
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 Food hygiene microbiology test staining method, culture medium and reagents GB/T11675 Tremella fuciformis hygiene standard
3 Equipment and instruments
3.1 Refrigerator: 4℃~-20℃.
3.2 Constant temperature incubator: 26℃±1℃, 36℃±1℃. 3.3 Constant temperature water bath: 46℃±1℃.
3.4 ​​Microscope: 10×~100×.
3.5 Centrifuge: 4000r/min.
3.6 Rack-plate drug balance: 0g~500g, accuracy 0.5g. 3.7 Conical flask: 100mL, 500mL.
3.8 Sterile pipette: 1mL (with 0.01mL scale), 10mL (with 0.1mL scale). 3.9 Sterilized flat III: diameter 90mm, 120mm. 3.10
Sterilized test tube: 16mm×160mm.
Centrifuge tube: 30mmX100mm.
Small test tube: 10mm×100mm.
Sterilized L-shaped glass rod.
Sterilized transparent glass paper, filter paper.
Gastric gavage: 1mL.
3.16 White mice: 18g~20g.
4 Culture medium and reagents
4.1 Gram staining solution: prepared according to GB/T4789.28. 4.2 Oxidase test: carried out according to GB/T4789.28. 4.3 Potato dextrose agar (PDA): prepared according to GB/T4789.28. 4.4 Potato dextrose semisolid agar: According to the PDA formula in 4.3, the amount of agar is reduced to 0.5g/100mL. 4.5 Potato dextrose water (PD water): According to the PDA formula in 4.3, without adding agar. 235
GB/T 4789.29--2003
4.6 GVC enrichment solution (for enrichment of Pseudomonas cocovenenans subspecies): In the autoclaved PD water (4.5), add gentian violet aqueous solution (final concentration of 1/100,000) and chloranthone aqueous solution (final concentration of 20μg/mL) in a sterile manner, mix well, dispense and place at 4℃ for later use. 4.7 Egg yolk agar: Prepared according to GB/T4789.28. 4.8 SS agar: Prepared according to GB/T4789.28. 4.9 Hugh-Leifson medium (for O/F test): prepared according to GB/T4789.28. 4.10
Strong white aged water (for indigo matrix test): prepared according to GB/T4789.28. Buffered glucose protein aged water (for MR and VP test): prepared according to GB/T4789.28. Simon's citrate medium: prepared according to GB/T4789.28. Phenylalanine medium: prepared according to GB/T4789.28. Sugar fermentation tube: prepared according to GB/T4789.28. Semisolid agar: prepared according to GB/T4789.28. Anti-O multivalent serum, type-specific factor serum (O-Ⅲ, OV, OV, O-И type, O-colonial type). Test procedure
The test procedure for Pseudomonas cerebrovenans subsp. cerebrovenans is shown in Figure 1. Test sample
Starchy food 25g+225mLGVC enrichment solutionFresh Tremella fuciformis 1g+20mLGVC enrichment solution
36℃±148h
PDA plate
36℃±
Apple Blue stain
Serotyping
Oxidized alcohol test
24h48h
Egg yolk agar
36℃±i
PDA slope
36℃±1℃| |tt||Biochemical test
24h48h
SS agar plate
Toxicity testbZxz.net
Myric acid determination
6 Operation steps
GB/T4789.29--2003
Take 25g of sample with aseptic operation, add it to a conical flask containing 225mL of enrichment solution (take 1g of fresh Tremella sample, cut it into pieces with scissors, add it to a conical flask containing 20mL of enrichment solution) and culture it at 36℃±1℃ for 48h. 6.2 Isolation and purification culture
Take an inoculation loop of enrichment solution, streak and inoculate it on PDA plate, and culture it at 36℃±1℃ for 24h~48h. Observe the morphology of the growing colonies on the plate, pick out suspicious single colonies, and perform Gram staining and oxidase test. Gram staining negative and oxidase test negative colonies were then spotted on egg yolk agar plates and SS agar plates and cultured at 36℃±1℃ for 48h and 24h respectively. The colony characteristics of Pseudomonas cocovenenans subspecies fermented rice flour on different separation plates are shown in Table 1.
Table 1 Colony characteristics of Pseudomonas cocovenenans subspecies fermented rice flour on different separation plates Culture medium
PDA plate
Egg yolk agar plate
SS agar plate
Colony characteristics
Colony 1mm~2mm, gray or milky white, smooth, moist, with neat edges. After 48 hours of cultivation, there is a bulge in the center, which is in the shape of a straw hat. The inherent yellow-green pigment around the colony diffuses into the matrix, and the surface of the colony is smooth and moist. After 48 hours, a milky white turbid ring is formed around the colony. Under oblique sunlight, the surface of the ring is iris-colored.
No growth
Pick a single colony with positive lecithinase and iris ring from the egg yolk agar plate, inoculate the PDA slope, and culture it at 36℃±1℃ for 24 hours for the next test.
6.3 Biochemical test
Pick a small amount of bacterial lawn from the pure culture PDA slope, inoculate Hugh-Leifson medium, protein water, buffered protein water, Simon's citrate medium, sugar fermentation tube and phenylalanine medium, culture at 36℃±1℃, and observe them according to the requirements of the single test. The biochemical characteristics of Pseudomonas cerebrolyticus are shown in Table 2. Table 2 Biochemical characteristics of Pseudomonas cocovenenans subspecies fermented rice flour positive
O/F test (O type)
Glucose
Galactine
Arabinose
Mannitol
Dysostigmine
Nitrate reduction
6.4 Serological typing assessment
6.4.10 Preparation of antigens
Gelatin liquefaction
Lecithinase
Adonitol
Citrate utilization
Arginine
Litmus milk
37 growth
Oxidase
Indigo matrix
H,S production
No growth at 5℃
No growth at 41℃
Wash the potato dextrose agar (PDA) slant at 36℃±1℃, 24h culture with sterile saline, boil for 2h, centrifuge and discard the supernatant, then dilute with sterile saline to 500 million/mL~1 billion/mL bacterial suspension as antigen for agglutination test. 6.4.20 Identification of antigens
Use polyvalent serum for slide agglutination test, and use saline as control. For those that agglutinate with polyvalent serum, use OⅢ, O-IV, 0237
GB/T4789.29--2003
V, OM, OI type, O-type factor serum in turn for test tube agglutination test. According to the test results, determine the O antigen type. Those that self-agglutinate in physiological saline cannot be typed. Those whose biochemical properties meet the requirements but cannot agglutinate with the above sera need to be retained for further identification. 6.5 Toxicity test
6.5.1 Toxicity culture
Take the strain identified as Pseudomonas cocovenenans subspecies fermented rice flour and inoculate it on the PDA slant, culture it at 36℃±1℃ for 24h, add 3mL of sterile physiological saline to make a bacterial suspension of about 10 billion/mL, take 0.5mL with a sterile pipette, drop it on a semi-solid PDA plate covered with sterile glass paper, spread it evenly with a sterile L-shaped glass rod, and culture it at 26℃±1℃ for 5d. Remove the bacterial cellophane and place the semi-solid plate in a sterilizer, sterilize with flowing steam at 100℃ for 30min. After cooling to room temperature, place in a 10℃～20℃ refrigerator overnight. Thaw the frozen semi-solid plate at room temperature, take out the thawed liquid with a sterilized pipette, filter it through filter paper and place it in a sterilized test tube or conical bottle, and store it at 4℃ away from light. This is the crude toxin.
6.5.2 Toxicity determination
Take 0.5mL of the crude toxin or the 5-10 times concentrated solution evaporated by water bath, gavage three mice weighing 18g～20g, and observe for 7 days. If the strain produces fumonisin, the mice will become ill and die within 20min～24h after gavage. The main symptoms are erect hair, atrophy, followed by restlessness, strabismus, limb paralysis, limpness, twitching, opisthotonos, rapid breathing, and death. 6.5.3 Determination of fumonisin
shall be carried out in accordance with GB/T11675.
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