GB Translated English of Chinese Standard: GB NATIONAL STANDARD OF THE

Transcription

1 Translated English of Chinese Standard: GB GB NATIONAL STANDARD OF THE PEOPLE S REPUBLIC OF CHINA GB National food safety standard Food microbiological examination Enumeration of coliforms Issued on: December 23, 2016 Implemented on: June 23, 2017 Issued by: National Health and Family Planning Commission of the People s Republic of China; China Food and Drug Administration. Page 1 of 15

2 Table of Contents Foreword Scope Terms and definitions Test principle Apparatus and materials Culture media and reagents... 5 The first method The MPN count method for coliforms Test procedure Operating procedure... 7 The second method The plate count method for coliforms Test procedure Operating procedure... 9 Annex A Culture media and reagents Annex B Coliform most probable number (MPN) key Page 2 of 15

3 Foreword This Standard replaces GB , National food safety standard Food microbiological examination Enumeration of coliforms, GB/T , Microbiological examination for food hygiene Rapid detection of coliform bacteria, and SN/T , Determination of coliform, fecal coliform and escherichia coli in food for import and export. Compared with GB , the main changes of this Standard are as follows: -- it adds the test principle; -- it modifies the application scope; -- it modifies the description of morphology of typical colony; -- it modifies the selection of the second method for plate colony count; -- it modifies the second method for confirmatory test; and -- it modifies the report for the second method for plate count. Page 3 of 15

4 National food safety standard Food microbiological examination Enumeration of coliforms 1 Scope This Standard specifies the method for the enumeration of coliforms in food. The first method of this Standard applies to the enumeration of coliforms in food having a low content of coliforms; the second method applies to the enumeration of coliforms in food having a high content of coliforms. 2 Terms and definitions 2.1 coliforms aerobic and facultative gram-negative budless bacillus which could ferment lactose and produce acids and gas. 2.2 most probable number; MPN an indirect count method based on Poisson distribution 3 Test principle 3.1 MPN method The MPN method is a quantitative test method combining statistics and microbiology. 3.2 Plate count method Coliforms ferment lactose and produce acids in a solid medium, and form under the action of indicator countable red or purple bacterial colonies, with or without precipitation ring. Page 4 of 15

5 4 Apparatus and materials In addition to conventional sterilization and culture equipment in a microbiological laboratory, the other equipment and materials are as follows: 4.1 Constant temperature incubator: 36 C ± 1 C. 4.2 Refrigerator: 2 C to 5 C. 4.3 Constant-temperature water bath: 46 C ± 1 C. 4.4 Balance: sensitivity 0.1 g. 4.5 Homogenizer. 4.6 Oscillator. 4.7 Sterile pipette: 1 ml (having 0..1 ml scale), 10 ml (having 0.1 ml scale) or micropipette and tip. 4.8 Sterile conical flask: volume 500 ml. 4.9 Sterile culture dish: diameter 90 mm PH meter Bacterial colony counter. 5 Culture media and reagents 5.1 Lauryl sulfate tryptose (LST) broth: see A Brilliant green lactose bile (BGLB) broth: see A Violet red bile agar (VRBA): see A Sterile phosphate buffer: see A Sterile normal saline: see A mol/l NaOH solution: see A mol/l HCl solution: see A.7. Page 5 of 15

6 7.2 Primary fermentation test For each sample, select 3 continuous diluent sample homogenates (select original liquid for liquid samples), inoculate for each dilution degree 3 pipettes of lauryl sulfate tryptose (LST) broth, with each pipette inoculating 1 ml (if the incubation amount exceeds 1 ml, then use double LST broth), incubate at 36 C ± 1 C for 24 h ± 2 h, observe whether there is bubble produced in the inverted tube, and carry out secondary fermentation test (confirmatory test) for that producing gas for 24 h ± 2 h; or if no gas is produced, continue to incubate until 48 h ± 2 h, and carry out secondary fermentation test for that producing gas. One producing no gas is coliform negative. 7.3 Secondary fermentation test (confirmatory test) Use an incubating loop to take a loop of culture from the LST broth producing gas; transfer to a test tube of brilliant green lactose bile (BGLB) broth; incubate at 36 C ± 1 C for 48 h ± 2 h; and observe the gas production. One producing gas is an escherichia coli positive tube. 7.4 Report of most probable number (MPN) of coliforms In accordance with the number of the coliform BGLB positive tubes confirmed in 7.3, look up in the MPN table (see Annex B) and report the MPN value of coliforms in each g (ml) of sample. The second method The plate count method for coliforms 8 Test procedure See Figure 2 for the test procedure for the plate count method for coliforms. Page 8 of 15

7 Lactose Sodium chloride Bile or bile salt No.3 Nuetral red Crystal violet Agar Distilled water 10.0 g 5.0 g 1.5 g 0.03 g g 15 g to 18 g ml A.3.2 Preparation Dissolve the constituents above into distilled water; allow to stand for several minutes; mix up; and adjust the ph value to 7.4 ± 0.1. Boil the solution for 2 min; melt culture medium and maintain to constant temperature 45 C to 50 C before pouring onto the plates. Prepare before use; do not exceed 3 h. A.4 Phosphate buffer A.4.1 Constituents Monopotassium phosphate Distilled water 34.0 g 500 ml A.4.2 Preparation Stock solution: weigh 34.0 g of monopotassium phosphate to dissolve in 50 ml of distilled water; use approximately 175 ml of 1 mol/l sodium hydroxide solution to adjust the ph value to 7.2 ± 0.2; and use distilled water to dilute to ml before stocking in a refrigerator. Diluent: take 1.25 ml of stock solution; use distilled water to dilute to ml; transfer to proper containers separately; and carry out autoclaved sterilization at 121 C for 15 min. A.5 Sterile normal saline A.5.1 Constituents Sodium chloride Distilled water 8.5 g ml A.5.2 Preparation Weigh 8.5 g of sodium chloride to dissolve in ml of distilled water; carry out autoclaved sterilization at 121 C for 15 min. A.6 1 mol/l NaOH solution A.6.1 Constituents NaOH Distilled water 40.0 g ml A.6.2 Preparation Page 12 of 15

8 F Upper limit G Lower limit H Upper limit I NOTE 1 This table employs 3 dilution degrees [0.1 g (ml), g (ml) and g (ml)], each dilution degree inoculating 3 tubes. NOTE 2 When the sample amounts listed are changed into 1 g (ml), 0.1 g (ml) and 0.01 g (ml), the figures in the table shall be reduced by 10 times accordingly; and when changed into 0.01 g (ml), g (ml) and g (ml), the figures in the table shall be increased by 10 times accordingly; and so on. END Page 15 of 15