GB 4789.3-2025 National food safety standard - Food microbiological examination -Enumeration of coliforms
1 Scope
This standard specifies the method for the enumeration of coliforms in food.
Method I in this standard is applicable to the enumeration of coliforms in foods with low coliform content, while Method II is applicable to the enumeration of coliforms in foods with high coliform content.
2 Terms and definitions
2.1
coliforms
gram-negative, non-spore-forming bacilli that are aerobic or facultatively anaerobic and capable of fermenting lactose with the production of acid and gas under specified culture conditions
2.2
most probable number (MPN) technique
a quantitative detection method combining statistics and microbiology, that is, after the sample to be tested is serially diluted and incubated, the most probable number of coliforms in the sample is estimated using statistical probability theory, based on the lowest dilution showing no growth and the highest dilution showing growth
3 Equipment and materials
In addition to conventional sterilization and incubation equipment used in microbiological laboratories, other equipment and materials required are as follows:
3.1 Constant temperature incubators: 36℃±1℃, and 30℃±1℃.
3.2 Refrigerator: 2℃~8℃.
3.3 Thermostat: 48℃±2℃.
3.4 Balance: With a sensitivity of 0.1g.
3.5 Homogenizer, aseptic homogenizing bag, homogenizing cup, and oscillator.
3.6 Test tubes: With sizes of 15mm×150mm, 18mm×180mm, or other appropriate sizes; and inverted tubes (Durham tubes) or other suitable gas collection devices.
3.7 Aseptic pipettes: 1mL (graduated to 0.01mL), 2mL (graduated to 0.02mL), 5mL (graduated to 0.05mL), 10mL (graduated to 0.1mL), or micropipettors with aseptic tips.
3.8 Aseptic conical flask: With a capacity of 500mL.
3.9 Aseptic culture dish: With a diameter of 90mm.
3.10 pH meter or precision pH test paper.
3.11 Aseptic inoculation loop: 10μL (with a diameter of about 3mm).
3.12 Magnifying glass or colony counter.
4 Culture media and reagents
4.1 Phosphate buffer solution: See Annex A, A.1.
4.2 Normal saline: See A.2.
4.3 Lauryl sulfate tryptose (LST) broth: See A.3.
4.4 Brilliant green lactose bile (BGLB) broth: See A.4.
4.5 Violet red bile agar (VRBA): See A.5.
4.6 1mol/L NaOH: See A.6.
4.7 1mol/L HCl: See A.7.
4.8 Coliform count test film (A result shall be considered positive if lactose is fermented with the production of acid and gas, and its performance shall meet the quality requirements for relevant culture media specified in GB 4789.28).
Method I Enumeration of coliforms by MPN technique
5 Examination procedures
The examination procedures for the enumeration of coliforms by the MPN technique is shown in Figure 1.
Foreword I
1 Scope
2 Terms and definitions
3 Equipment and materials
4 Culture media and reagents
5 Examination procedures
6 Operation steps
7 Results and report
8 Examination procedures
9 Operation steps
10 Results and report
Annex A Culture media and reagents
Annex B Retrieval table for the most probable number (MPN) of coliforms
Annex C Method for determining 3 most appropriate consecutive dilutions
Annex D Processing and examples of coliform plate count results
