GB/T 4789.14-2003 Microbiological examination of food hygiene - Examination of Bacillus cereus

This standard specifies the test method for Bacillus cereus. This standard is applicable to the test of Bacillus cereus in various types of food and food poisoning samples. GB/T 4789.14-2003 Food Hygiene Microbiological Test Bacillus cereus Test GB/T4789.14-2003 Standard Download Decompression Password: www.bzxz.net

ICS 07.100.30
National Standard of the People's Republic of China
GB/T4789.14--2003
Replaces GB/T4789.14—1994
Microbiological examination of food hygiene
Bacillus cereus examination
Microbiological examination of food Hygiene-Examination of Bacillus cereus
2003-08-11 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/T4789.14—2003
This standard amends GB/T4789.14—1994 "Microbiological Examination of Food Hygiene". The format and text of the standard text are amended in accordance with GB/T1.1-2000. The "equipment and materials" in the original standard are amended and standardized. From the date of implementation of this standard, GB/T4789.14--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. The drafting unit of this standard: Nutrition and Food Safety Institute of Chinese Center for Disease Control and Prevention. The main drafters of this standard are Bai Jingyu, Fu Ping, Yang Baolan and Yao Jinghui. This standard was first issued in 1984, revised for the first time in 1994, and this is the second revision. 96
1 Scope
Microbiological examination of food hygiene
Bacillus cereus examination
This standard specifies the examination method of Bacillus cereus. This standard is applicable to the examination of Bacillus cereus in various types of food and food poisoning samples. 2 Normative references
GB/T4789.14—2003wwW.bzxz.Net
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, the latest version shall apply to this standard. GB/T4789.2 Food hygiene microbiological examination Determination of total colony count GB/T4789.28—2003 Food hygiene microbiological examination Staining method, culture medium and reagents 3 Equipment and materials
3.1 Refrigerator: 0℃~4℃.
3.2 Constant temperature incubator: 36℃±1℃.
3.3 Constant temperature water bath: 46℃±1℃.
3.4 ​​Microscope: 10X100×.
3.5 Homogenizer or sterile mortar.
3.6 Rack-plate drug balance: 0g~500g, accurate to 0.5g. 3.7 Sterile conical flask: 500mL.
3.8 Sterile test tube: 16mm×160mm.
Sterile pipette: 1mL (with 0.01mL scale), 10mL (with 0.1mL scale). 3.93
Sterile flat culture blood: diameter 90mm.
Sterile knife, scissors, tweezers, etc.
4 Culture medium and reagents
4.1 Meat extract broth culture medium: 4.1 in GB/T4789.28-2003. 4.2 Casein agar culture medium: 4.65 in GB/T4789.28-2003. 4.3 Power-nitrate culture medium: 4.72 in GB/T4789.28--2003. 4.4 Buffered glucose protein water: 3.4 in GB/T4789.28-2003. 4.5 Blood agar culture medium: 4.6 in GB/T4789.28-2003. 4.63% hydrogen peroxide solution.
4.7 Naphthylamine-acetic acid solution: 3.17 in GB/T4789.28--2003. 4.8 p-Aminobenzenesulfonic acid-acetic acid solution: 3.17 in GB/T4789.28-2003. 4.9 Gram staining solution: 2.2 in GB/T4789.28-2003. 4.10 Mannitol yolk polymyxin (MYP) agar medium: GB/T4789 .28—2003, 4.64.4.110.5% alkaline fuchsin staining solution: 2.8.97 in GB/T4789.28—2003
GB/T4789.14—2003
4.12 Xylose-gelatin culture medium: 4.66 in GB/T4789.28—2003.5 Inspection procedure
The inspection procedure for Bacillus cereus is shown in Figure 1.
25g(mL) add 225mL physiological saline
micro dilution of 101~10-5
37℃,18h~24h
selective culture medium
(bacterial count determination)
12h~20h
bacterial count calculation
growth and colony observation
6 operation steps
staining microscopy
select Selective culture medium
(isolation culture)
36℃±1
12h~20h
Nutrient agar medium
Biochemical test
Strain preservation
6.1 Determination of bacterial count
Use sterile operation to make 10-1～10- dilutions with sterile saline or phosphate buffer for 25g (mL) of the test sample and determine according to GB/T4789.2. Take 0.1mL of each dilution and inoculate it on two selective culture media - mannitol egg yolk polymyxin (MYP) agar medium, spread it on the entire surface with an L-shaped stick, and incubate it at 36℃±1℃ for 12h~20h. Select a plate with an appropriate number of colonies for counting. The colonies of Bacillus cereus on this culture medium are pink (indicating that mannitol is not fermented) with a pink halo around them (indicating the production of lecithinase). After counting, pick five such colonies for confirmation test. Calculate the number of colonies on the plate based on the confirmed number of Bacillus cereus colonies, and then multiply it by the dilution factor to get the number of Bacillus cereus contained in each gram (ml) of sample. For example: 0.1mL 10-4 sample dilution is spread on the MYP plate, and there are 25 suspected colonies. Five are taken for identification, and four colonies are confirmed to be Bacillus cereus. Then the number of Bacillus cereus contained in 1g (mL) of the test sample is: 25×4/5×10°×10=2×106
6.2 Isolation and culture
Take the test sample or dilution and streak it on the selective culture medium (MYP), culture it at 37℃ for 12h~20h, pick the suspected Bacillus cereus colonies (see 6.1) and inoculate them into broth and nutrient agar to make pure culture, and then do the confirmation test. 6.3 Confirmation test
6.3:1 Morphological observation
This bacterium is a large Gram-positive bacillus with a width of 1μm or more than 1um. The spores are oval and do not protrude from the body. They are mostly located in the center or slightly to the end of the body.
6.3.2 Culture characteristics
This bacterium grows turbid in broth, often with a slight bacterial film or wall ring, and is easily emulsified by shaking; on ordinary agar plates, its colonies are opaque, with a rough surface, like frosted glass or molten wax, and irregular edges. 6.3.3 Biochemical properties and biochemical typing
6.3.3.1 Biochemical properties
This bacterium is motile: it can produce lecithinase and casein; it is positive in catalase test; it is hemolytic; it does not ferment mannitol and xylose, but can often liquefy gelatin and reduce nitrate; it can ferment glucose under anaerobic conditions. 6.3.3.2 Biochemical typing
According to the tests of citrate utilization, nitrate reduction, starch hydrolysis, VP reaction, and gelatin liquefaction of Bacillus cereus, it is divided into different types, see Table 1.
Table 1 Biochemical typing of Bacillus cereus
Gelatin liquefaction
VP reaction
Starch hydrolysis
Nitrate reduction
Citrate utilization
Identification from similar bacteria
The identification of this bacterium from other similar bacteria is shown in Table 2. Table 2 Identification of Bacillus cereus from other similar bacteria
Bacillus anthracis
Bacillus thuringiensis
Bacillus cereus
Bacillus megaterium
Calatase
Nitrate reduction
Casein decomposition
Yolk reaction
GB/T4789.14—2003
Glucose utilization (anaerobic)
Mannitol
Known pathogenic bacteria characteristics
Bacillus megaterium
Table 2 (continued)
Bacillus cereus
Enterotoxin production
Bacillus thuringiensis
Pathogenic to insects
Endotoxin crystals
Bacillus phleiformis
Rhizoid growth
Bacillus anthracis
Pathogenic to animals and humans
Note: +90% to 100% of strains are positive; -90% to 100% of strains are negative! ±Most strains are positive; most strains are negative. This bacterium is very similar to Bacillus thuringiensis in biochemical characteristics, but the latter can be distinguished by the production of protein toxin crystals in the cell. The inspection method is as follows: take a small amount of pure culture on nutrient agar, add a small amount of distilled water and apply it on a glass slide. After natural drying, fix it with a weak flame, add methanol to the glass slide, pour off the methanol after half a minute, dry it on the flame, then add 0.5% alkaline fuchsin solution, and heat it with an alcohol lamp until the steam is slightly visible and maintain it for 1.5 minutes. Remove the alcohol lamp, leave the glass slide for half a minute, pour off the dye solution, rinse it with clean tap water, dry it, and examine it under a microscope. Check for free spores and dark-stained diamond-shaped red crystalline bodies under an oil immersion microscope (if free spores are not formed, the culture should be kept at room temperature for another 1d to 2d before inspection). If there are, it is Bacillus thuringiensis, and the inspection of Bacillus cereus is negative. 100
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