1 Scope
This standard specifies the method for enumeration of coliforms in foods.
In this standard, Method I is applicable to the enumeration of coliforms in foods containing fewer coliforms and Method II to the enumeration of coliforms in foods containing more coliforms.
2 Terms and Definitions
2.1
Coliforms
Aerobic and facultative anaerobic Gram-negative non-spore bacillus that can ferment lactose and produce gas and acid under certain culture conditions.
2.2
Most probable number; MPN
An indirect enumeration method based on Poisson distribution.
3 Examination Principle
3.1 MPN method
MPN method is a quantitative detection method combining statistics with microbiology. After serial dilutions and cultivation, the MPN of coliforms in the to-be-tested sample shall be estimated using statistics and probability theory based on the non-growing minimum degree of dilution and growing maximum degree of dilution.
3.2 Plate counting method
The coliforms ferment lactose and produce acid in solid medium and form red or purple colonies with or without precipitation ring that are countable under the action of indicator.
4 Apparatus and Materials
In addition to the apparatus for conventional sterilization and cultivation in microbiological laboratory, other apparatus and materials are as follows:
4.1 Constant temperature incubator: 36℃±1℃.
4.2 Refrigerator: 2℃~5℃.
4.3 Thermostatic water bath: 46℃±1℃.
4.4 Balance: with sensibility of 0.1g.
4.5 Homogenizer.
4.6 Oscillator.
4.7 Aseptic pipette: 1mL (with scale division of 0.01mL), 10mL (with scale division of 0.1mL) or micropipettor and pipette tips.
4.8 Aseptic conical flask: 500mL.
4.9 Aseptic culture dish: 90mm in diameter.
4.10 pH meter or pH colorimetric tube or precise pH paper.
4.11 Colony counter.
5 Culture Media and Reagents
5.1 Lauryl sulfate tryptose (LST) broth: see A.1.
5.2 Brilliant green lactose bile (BGLB) broth: see A.2.
5.3 Violet red bile agar (VRBA): see A.3.
5.4 Aseptic phosphate buffer solution: see A.4.
5.5 Aseptic normal saline: see A.5.
5.6 1mol/L NaOH solution: see A.6.
5.7 1mol/L HCl solution: see A.7.
Method I MPN Enumeration of Coliforms
6 Examination Procedures
See Figure 1 for the examination procedures of MPN enumeration of coliforms.
Figure 1 Examination Procedures for MPN Enumeration of Coliforms
7 Operation Steps
7.1 Dilution of sample
7.1.1 Solid and semi-solid sample: weight 25g of sample, put in an aseptic homogenizing cup containing 225mL of phosphate buffer solution or normal saline and homogenize at 8 000r/min~10 000r/min for 1min~2min, or put in an aseptic homogenizing bag containing 225mL of phosphate buffer solution or normal saline and flap with a slap type homogenizer for 1min~2min, to prepare the 1:10 homogeneous sample solution.
7.1.2 Liquid sample: use aseptic pipette to pipet 25mL of sample into an aseptic conical flask containing 225mL of phosphate buffer solution or normal saline (proper amount of aseptic glass beads may be put into the flask in advance) or other aseptic container and shake well, or into the mechanical oscillator, shake and mix well, to prepare the 1:10 homogeneous sample solution.
7.1.3 The pH value of homogeneous sample solution shall be 6.5~7.5 and adjusted respectively with 1mol/L NaOH or 1mol/L HCl when necessary.
7.1.4 Use 1mL aseptic pipette or micropipettor to pipet 1mL of 1:10 homogeneous sample solution, slowly inject it into an aseptic test tube containing 9mL of phosphate buffer solution or normal saline along the tube wall (the pipette or pipette tip shall not touch the diluent), shake the test tube or use a new 1mL aseptic pipette and repeatedly insufflate and flap to mix well, to prepare the 1:100 homogeneous sample solution.
7.1.5 Based on the evaluation of sample pollution conditions, prepare the decimal serial dilutions of homogeneous sample solution successively according to the above-mentioned operations. Change a new 1mL aseptic pipette or pipette tip for each decimal serial dilution. The whole process from preparing homogeneous sample solution to completing sample inoculation shall not exceed 15 min.
7.2 Primary fermentation test
For each sample, select the homogeneous sample solution with three appropriate continuous degrees of dilution (the stock solution may be adopted as liquid sample), inoculate three tubes of LST broth at each degree of dilution, 1mL per tube (in case that the inoculum volume exceeds 1mL, double LST broth shall be used), cultivate at 36℃±1℃ for 24h±2h and observe whether there is bubble in the inverted tube; conduct secondary fermentation test (verification test) for those producing gas after 24h±2h and cultivate those free from producing gas until 48h±2h, after that, conduct secondary fermentation test for those producing gas and those free from producing gas are negative.
7.3 Secondary fermentation test (verification test)
Respectively take one loop of the cultures from the LST broth tubes producing gas with inoculating loop, transfer into the BGLB tubes, cultivate at 36℃±1℃ for 48h±2h and observe the gas production condition, those producing gas are positive.
7.4 Report for MPN of coliforms
According to the number of positive BGLB tubes for coliforms as verified in 7.3, retrieve the MPN table (see Appendix B) and report the MPN of coliforms in sample per g(mL).
Contents
Foreword i
1 Scope
2 Terms and Definitions
3 Examination Principle
4 Apparatus and Materials
5 Culture Media and Reagents
6 Examination Procedures
7 Operation Steps
8 Examination Procedures
9 Operation Steps
Appendix A Culture Media and Reagents
Appendix B MPN Retrieval Table of Coliforms
1 Scope
This standard specifies the method for enumeration of coliforms in foods.
In this standard, Method I is applicable to the enumeration of coliforms in foods containing fewer coliforms and Method II to the enumeration of coliforms in foods containing more coliforms.
2 Terms and Definitions
2.1
Coliforms
Aerobic and facultative anaerobic Gram-negative non-spore bacillus that can ferment lactose and produce gas and acid under certain culture conditions.
2.2
Most probable number; MPN
An indirect enumeration method based on Poisson distribution.
3 Examination Principle
3.1 MPN method
MPN method is a quantitative detection method combining statistics with microbiology. After serial dilutions and cultivation, the MPN of coliforms in the to-be-tested sample shall be estimated using statistics and probability theory based on the non-growing minimum degree of dilution and growing maximum degree of dilution.
3.2 Plate counting method
The coliforms ferment lactose and produce acid in solid medium and form red or purple colonies with or without precipitation ring that are countable under the action of indicator.
4 Apparatus and Materials
In addition to the apparatus for conventional sterilization and cultivation in microbiological laboratory, other apparatus and materials are as follows:
4.1 Constant temperature incubator: 36℃±1℃.
4.2 Refrigerator: 2℃~5℃.
4.3 Thermostatic water bath: 46℃±1℃.
4.4 Balance: with sensibility of 0.1g.
4.5 Homogenizer.
4.6 Oscillator.
4.7 Aseptic pipette: 1mL (with scale division of 0.01mL), 10mL (with scale division of 0.1mL) or micropipettor and pipette tips.
4.8 Aseptic conical flask: 500mL.
4.9 Aseptic culture dish: 90mm in diameter.
4.10 pH meter or pH colorimetric tube or precise pH paper.
4.11 Colony counter.
5 Culture Media and Reagents
5.1 Lauryl sulfate tryptose (LST) broth: see A.1.
5.2 Brilliant green lactose bile (BGLB) broth: see A.2.
5.3 Violet red bile agar (VRBA): see A.3.
5.4 Aseptic phosphate buffer solution: see A.4.
5.5 Aseptic normal saline: see A.5.
5.6 1mol/L NaOH solution: see A.6.
5.7 1mol/L HCl solution: see A.7.
Method I MPN Enumeration of Coliforms
6 Examination Procedures
See Figure 1 for the examination procedures of MPN enumeration of coliforms.
Figure 1 Examination Procedures for MPN Enumeration of Coliforms
7 Operation Steps
7.1 Dilution of sample
7.1.1 Solid and semi-solid sample: weight 25g of sample, put in an aseptic homogenizing cup containing 225mL of phosphate buffer solution or normal saline and homogenize at 8 000r/min~10 000r/min for 1min~2min, or put in an aseptic homogenizing bag containing 225mL of phosphate buffer solution or normal saline and flap with a slap type homogenizer for 1min~2min, to prepare the 1:10 homogeneous sample solution.
7.1.2 Liquid sample: use aseptic pipette to pipet 25mL of sample into an aseptic conical flask containing 225mL of phosphate buffer solution or normal saline (proper amount of aseptic glass beads may be put into the flask in advance) or other aseptic container and shake well, or into the mechanical oscillator, shake and mix well, to prepare the 1:10 homogeneous sample solution.
7.1.3 The pH value of homogeneous sample solution shall be 6.5~7.5 and adjusted respectively with 1mol/L NaOH or 1mol/L HCl when necessary.
7.1.4 Use 1mL aseptic pipette or micropipettor to pipet 1mL of 1:10 homogeneous sample solution, slowly inject it into an aseptic test tube containing 9mL of phosphate buffer solution or normal saline along the tube wall (the pipette or pipette tip shall not touch the diluent), shake the test tube or use a new 1mL aseptic pipette and repeatedly insufflate and flap to mix well, to prepare the 1:100 homogeneous sample solution.
7.1.5 Based on the evaluation of sample pollution conditions, prepare the decimal serial dilutions of homogeneous sample solution successively according to the above-mentioned operations. Change a new 1mL aseptic pipette or pipette tip for each decimal serial dilution. The whole process from preparing homogeneous sample solution to completing sample inoculation shall not exceed 15 min.
7.2 Primary fermentation test
For each sample, select the homogeneous sample solution with three appropriate continuous degrees of dilution (the stock solution may be adopted as liquid sample), inoculate three tubes of LST broth at each degree of dilution, 1mL per tube (in case that the inoculum volume exceeds 1mL, double LST broth shall be used), cultivate at 36℃±1℃ for 24h±2h and observe whether there is bubble in the inverted tube; conduct secondary fermentation test (verification test) for those producing gas after 24h±2h and cultivate those free from producing gas until 48h±2h, after that, conduct secondary fermentation test for those producing gas and those free from producing gas are negative.
7.3 Secondary fermentation test (verification test)
Respectively take one loop of the cultures from the LST broth tubes producing gas with inoculating loop, transfer into the BGLB tubes, cultivate at 36℃±1℃ for 48h±2h and observe the gas production condition, those producing gas are positive.
7.4 Report for MPN of coliforms
According to the number of positive BGLB tubes for coliforms as verified in 7.3, retrieve the MPN table (see Appendix B) and report the MPN of coliforms in sample per g(mL).
Contents of GB 4789.3-2016
Contents
Foreword i
1 Scope
2 Terms and Definitions
3 Examination Principle
4 Apparatus and Materials
5 Culture Media and Reagents
6 Examination Procedures
7 Operation Steps
8 Examination Procedures
9 Operation Steps
Appendix A Culture Media and Reagents
Appendix B MPN Retrieval Table of Coliforms
