This standard specifies the method for the determination of coliform bacteria in food. This standard is applicable to the determination of coliform bacteria in various types of food. GB/T 4789.3-2003 Food hygiene microbiological examination Coliform bacteria determination GB/T4789.3-2003 Standard download decompression password: www.bzxz.net

ICS07.100.30
National Standard of the People's Republic of ChinawwW.bzxz.Net
GB/T4789.3—2003
Replaces GB/T4789.3—1994
Microbiological examination of food hygiene—Detection of Coliform bacteria
Promulgated 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/T4789.3—2003
This standard revise GB/T4789.3-1994 "Microbiological examination of food hygiene—Determination of coliform bacteria". Compared with GB/T4789.3-1994, this standard has the following major revisions: - The standard text format and text are revised in accordance with GB/T1.1--2000. - The "equipment and materials" in the original standard are revised and standardized. From the date of implementation of this standard, GB/T4789.3-1994 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 China Center for Disease Control and Prevention. The main drafters of this standard: Liu Hongdao, Ran Lu, Fu Ping, Yang Baolan, Yao Jinghui. This standard was first issued in 1984, revised for the first time in 1994, and this is the second revision. 14
1 Scope
Food Hygiene Microbiological Examination
Determination of Coliform Group
This standard specifies the method for the determination of coliform group in food. This standard is applicable to the determination of coliform group in various types of food. 2 Normative references
GB/T4789.3—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 reaching 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 examination staining methods, culture media and reagents 3 Terms and definitions
The following terms and definitions apply to this standard. 3.1
Coliform bacteria
A group of Gram-negative, non-spore-forming bacilli that can ferment lactose, produce acid and gas, and are aerobic and facultatively anaerobic. This bacterium mainly comes from human and animal feces, so it is used as a fecal contamination indicator to evaluate the sanitary quality of food and infer whether there is a possibility of intestinal pathogens in the food. The number of coliform bacteria in food is expressed as the most probable number (MPN) of coliform bacteria in 100mL (g) of sample. 4 Equipment and materials
Refrigerator: 0℃～~4℃.
Constant temperature incubator: 36℃±1℃.
Constant temperature water bath: 46℃±1℃.
Microscope: 10×~100×.
Homogenizer or sterile mortar.
Stand-plate pharmaceutical balance: 0g~500g, accurate to 0.5g. Sterile pipette 1mL (with 0.01mL scale), 10mL (with 0.1mL scale). 4.7
Sterile conical flask: 500mL.
Sterile glass beads: diameter of about 5mm.
Sterile culture blood: diameter of 90mm.
Sterile test tube: 16mm×160mm.
Sterile knife, scissors, tweezers, etc.
5 Culture medium and reagents
5.1 Lactose bile salt fermentation tube: in accordance with 4.9 of GB/T4789.28-2003. 5.2 Eosin-methylene blue agar plate: in accordance with 4.25 of GB/T4789.28-2003. 5.3 Lactose fermentation tube: in accordance with 4.10 of GB/T4789.28--2003. 15
GB/T4789.3-2003
5.4 EC broth: in accordance with 4.11 of GB/T4789.28-2003. 5.5 Phosphate buffer: in accordance with 3.22 of GB/T4789.28-2003. 5.6 0.85% sterile saline.
5.7 Gram staining solution: in accordance with 2.2 of GB/T4789.28-2003. 6 Test procedure
See Figure 1 for the coliform group test procedure.
Milk bile salt fermentation tube
36℃±1c,24h±2h
No gas production
Coliform group negative
Gram staining
Strawberry positive
Coliform group negative
7 Operating steps
7.1 Sample dilution
Eosin-methylene blue agar plate
36℃±1,24 h±2 h
Gram-negative, no bacillus
Escherichia coli negative
Dairy fermentation tube
36c±1c,24h±2h
No gas
Large bacteria negative
7.1.1Put 25g (mL) of the sample in a sterile glass bottle (with appropriate amount of glass beads in the bottle) or sterile mortar containing 225mL of sterile physiological saline or other diluent by aseptic operation, and shake or grind it thoroughly to make a 1:10 homogenous dilution. It is best to use a homogenizer at a speed of 8000r/min~10000r/min for 1min to make a 1:10 homogenous dilution. 16
GB/T4789.3—2003
7.1.2 Use a 1mL sterile pipette to draw 1mL of the 1:10 dilution solution and inject it into a test tube containing 9mL of sterile saline or other diluent. Shake the test tube to mix and make a 1:100 dilution solution. 7.1.3 Take another 1mL sterile pipette and make 10-fold incremental dilutions in sequence according to the above operation. After each incremental dilution, use a 1mL sterile pipette.
7.1.4 According to the requirements of food hygiene standards or the estimation of the contamination of the test sample, select three dilutions and inoculate three tubes for each dilution. 7.2 Lactose fermentation test
Inoculate the sample to be tested into a lactose bile salt fermentation tube. If the inoculation volume is more than 1mL, use a double-layer lactose bile salt fermentation tube. If the inoculation volume is 1mL or less, use a single-layer lactose bile salt fermentation tube. Inoculate three tubes for each dilution, place in a 36℃±1℃ incubator, and culture for 24h±2h. If all lactose bile salt fermentation tubes do not produce gas, they can be reported as negative for coliform bacteria. If there are gas-producing ones, follow the following procedures. 7.3 Separation and Culture
Transfer the gas-producing fermentation tubes to eosin-methylene blue agar plates, place in a 36℃±1℃ incubator, culture for 18h~24h, then take them out, observe the colony morphology, and perform Gram staining and confirmation tests. 7.4 Confirmation Test
Pick 1~2 suspected coliform bacteria colonies on the above plates for Gram staining, and inoculate the lactose fermentation tubes at the same time, place in a 36℃±1℃ incubator for 24h±2h, and observe the gas production. Any lactose tube that produces gas and is Gram-negative without spores can be reported as positive for coliform bacteria.
7.5 Report
According to the number of tubes confirmed as positive for coliform bacteria, check the MPN retrieval table and report the MPN value for each 100mL (g) of coliform bacteria. 8 Faecal coliform bacteria (Faecal coliform bacteria) 8.1 Use an inoculation loop to transfer all gas-producing lactose bile salt fermentation tube cultures (see 7.2) into EC broth tubes, place in a 44.5℃±0.2℃ water bath (the water level in the water bath should be higher than the EC broth liquid level), and culture for 24h±2h. After culture, if all EC broth tubes do not produce gas, they can be reported as negative; if there are gas-producing ones, transfer all gas-producing EC broth tubes to eosin-methylene blue agar plates respectively, and culture for 18h~24h. Those with typical colonies on the plates are confirmed to be positive for fecal coliform bacteria. 8.2 Result report
According to the number of positive tubes confirmed as fecal coliforms, check the MPN retrieval table and report the MPN value of fecal coliforms per 100mL (g) (see Table 1).
Table 1 Most Probable Number (MPN) Retrieval Table for Coliforms Number of Positive Tubes
1 mL(g)×3
0.1mL(g)×3
0.01mL(g)×3
100mL(g)
95% Confidence Limit
GB/T4789.32003
Table 1 (Continued)
95% Confidence Limit
Number of Positive Tubes
100mL(g)
0.01 mL(g)×3
0.1 mL(g)X3
1 mL(g)×3
≥24000
Note 1: This table uses three dilutions (1mL(g), 0.1mL(g) and 0.01mL(g)), with three tubes for each dilution. Note 2: If the sample plates listed in the table are changed to 10mL(g), 1mL(g) and 0.1mL(g), the numbers in the table should be reduced by 10 times accordingly. If they are changed to 0.1mL(g), 0.01mL(g) and 0.001mL(g), the numbers in the table should be increased by 10 times accordingly. The rest can be deduced by analogy. 18
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