Translated English of Chinese Standard: GB4789.3-2010 Translated by: www.chinesestandard.net Wayne Zheng et al. Email: Sales@ChineseStandard.net NATIONAL STANDARD GB OF THE PEOPLE S REPUBLIC OF CHINA GB 4789.3-2010 National Food Safety Standard Food Microbiological Examination: Enumeration of Coliforms Issued on: March 26, 2010 Implemented on: June, 1, 2010 Issued by: Ministry of Health of the People s Republic of China www.chinesestandard.net Page 1 of 17
Tips - GB 4789 Series (Not part of this Standard) Standard ID GB 4789.1-2010 GB 4789.2-2010 Standard Name examination: General guidelines examination: Aerobic plate count Issued Date Enforced Date New Version (Click to check)? 2010-03-26 2010-06-01 http://www.chinesestandard.net/default.aspx?stdid=gb%204789.1 2010-03-26 2010-06-01 http://www.chinesestandard.net/default.aspx?stdid=gb%204789.2 GB 4789.3-2010 GB 4789.4-2010 GB 4789.5-2012 GB/T 4789.6- GB/T 4789.7- GB/T 4789.8- GB/T 4789.9- GB 4789.10-2010 GB/T 4789.11- GB/T 4789.12- examination: Enumeration of coliforms examination: Salmonella National Food Safety Standard Microbiological Examination of Food Hygiene - Examination of Shigella diarrheogenic Escherichia coli Vibrio parahaemolyticus Yersinia enterocolitica Campylobacter jejuni examination: Staphylococcus aureus streptococcus hemolyticus Clostridium botulinum and botulinus toxin 2010-03-26 2010-06-01 http://www.chinesestandard.net/default.aspx?stdid=gb%204789.3 2010-03-26 2010-06-01 http://www.chinesestandard.net/default.aspx?stdid=gb%204789.4 2012-05-17 2012-07-17 http://www.chinesestandard.net/default.aspx?stdid=gb%204789.5-08-11 2004-01-01 http://www.chinesestandard.net/default.aspx?stdid=gb/t%204789.6-05-16-11-01 http://www.chinesestandard.net/default.aspx?stdid=gb/t%204789.7-11-21 2009-03-01 http://www.chinesestandard.net/default.aspx?stdid=gb/t%204789.8-11-21 2009-03-01 http://www.chinesestandard.net/default.aspx?stdid=gb/t%204789.9-11-21 2009-03-01 http://www.chinesestandard.net/default.aspx?stdid=gb%204789.10-08-11 2004-01-01 http://www.chinesestandard.net/default.aspx?stdid=gb/t%204789.11-08-11 2004-01-01 http://www.chinesestandard.net/default.aspx?stdid=gb/t%204789.12 www.chinesestandard.net Page 2 of 17
GB 4789.13-2012 GB/T 4789.14- GB 4789.15-2010 National Food Safety Standard Microbiological Examination of Food Hygiene - Examination of Clostridium Perfringens Bacillus cereus examination: Enumeration of moulds and yeasts 2012-05-17 2012-07-17 http://www.chinesestandard.net/default.aspx?stdid=gb%204789.13-08-11 2004-01-01 http://www.chinesestandard.net/default.aspx?stdid=gb/t%204789.14 2010-03-26 2010-06-01 http://www.chinesestandard.net/default.aspx?stdid=gb%204789.15 GB/T 4789.16- GB/T 4789.17- GB 4789.18-2010 GB/T 4789.19- GB/T 4789.20- GB/T 4789.21- GB/T 4789.22- GB/T 4789.23- GB/T 4789.24- GB/T 4789.25- Microbiological examination of food hygiene Identification of common mycotoxin producing fungi meat and meat products examination: Milk and milk products egg and egg products aquatic product foods frozen drinks and cold drinks flavourings cold dish and bean products candy, cake and preserved fruits wines -08-11 2004-01-01 http://www.chinesestandard.net/default.aspx?stdid=gb/t%204789.16-08-11 2004-01-01 http://www.chinesestandard.net/default.aspx?stdid=gb/t%204789.17 2010-03-26 2010-06-01 http://www.chinesestandard.net/default.aspx?stdid=gb%204789.18-08-11 2004-01-01 http://www.chinesestandard.net/default.aspx?stdid=gb/t%204789.19-08-11 2004-01-01 http://www.chinesestandard.net/default.aspx?stdid=gb/t%204789.20-08-11 2004-01-01 http://www.chinesestandard.net/default.aspx?stdid=gb/t%204789.21-08-11 2004-01-01 http://www.chinesestandard.net/default.aspx?stdid=gb/t%204789.22-08-11 2004-01-01 http://www.chinesestandard.net/default.aspx?stdid=gb/t%204789.23-08-11 2004-01-01 http://www.chinesestandard.net/default.aspx?stdid=gb/t%204789.24-08-11 2004-01-01 http://www.chinesestandard.net/default.aspx?stdid=gb/t%204789.25 www.chinesestandard.net Page 3 of 17
GB/T 4789.26- commercial sterilization of canned food -08-11 2004-01-01 http://www.chinesestandard.net/default.aspx?stdid=gb/t%204789.26 GB/T 4789.27- residue of antibiotics in fresh milk -11-21 2009-03-01 http://www.chinesestandard.net/default.aspx?stdid=gb/t%204789.27 GB/T 4789.28- Microbiological examination of food hygiene Staining methods, culture mediums and reagents -08-11 2004-01-01 http://www.chinesestandard.net/default.aspx?stdid=gb/t%204789.28 GB/T 4789.29- pseudomonas cocovenenans subsp. farinofermentans -08-11 2004-01-01 http://www.chinesestandard.net/default.aspx?stdid=gb/t%204789.29 GB 4789.30-2010 examination: Listeria monocytogenes 2010-03-26 2010-06-01 http://www.chinesestandard.net/default.aspx?stdid=gb%204789.30 GB/T 4789.31- GB/T 4789.32-2002 GB/T 4789.33- GB 4789.34-2012 salmonellae, shigellae, and diarrhoea causative Escherichia coli by means of the diagnostic typing phage set for enterobacteriaceae Microbiological examination for food hygiene rapid detection of coliform bacteria cereal, fruit and vegetable National Food Safety Standard Microbiological Examination of Food Hygiene - Examination of Bifidobacterium -08-11 2004-01-01 http://www.chinesestandard.net/default.aspx?stdid=gb/t%204789.31 2002-06-13 2002-10-01 http://www.chinesestandard.net/default.aspx?stdid=gb/t%204789.32-08-11 2004-01-01 http://www.chinesestandard.net/default.aspx?stdid=gb/t%204789.33 2012-05-17 2012-07-17 http://www.chinesestandard.net/default.aspx?stdid=gb%204789.34 GB 4789.35-2010 examination: Lactic acid bacteria 2010-03-26 2010-06-01 http://www.chinesestandard.net/default.aspx?stdid=gb%204789.35 GB/T 4789.36- Escherichia coli O157: H7/NM -05-16 -11-01 http://www.chinesestandard.net/default.aspx?stdid=gb/t%204789.36 www.chinesestandard.net Page 4 of 17
GB/T 4789.37- Staphylococcus aureus -11-21 2009-03-01 http://www.chinesestandard.net/default.aspx?stdid=gb/t%204789.37 GB 4789.38-2012 GB/T 4789.39- GB 4789.40-2010 National Food Safety Standard Microbiological Examination of Food Hygiene - Enumeration of Escherichia Coli Microbiological examination of food hygiene. Enumeration of faecal coliforms examination: Enterobacter sakazakii 2012-05-17 2012-07-17 http://www.chinesestandard.net/default.aspx?stdid=gb%204789.38-11-21 2009-03-01 http://www.chinesestandard.net/default.aspx?stdid=gb/t%204789.39 2010-03-26 2010-06-01 http://www.chinesestandard.net/default.aspx?stdid=gb%204789.40 www.chinesestandard.net Page 5 of 17
Table of Contents Foreword... 7 1 Scope... 8 2 Terms and Definitions... 8 3 Devices and Materials... 8 4 Culture Medium and Reagents... 9 5 Examination Procedure... 9 6 Operational Procedure... 10 7 Examination Procedure... 12 8 Operational Procedure... 12 Annex A... 14 Annex B... 17 www.chinesestandard.net Page 6 of 17
Foreword This Standard replaces GB/T 4789.3- Microbiological Examination of Food Hygiene Enumeration of Coliforms. The main changes in this Standard over GB/T 4789.3- are as follows: - Modify standard name of both English and Chinese; - Modify the selection range of plate colony count of Method 2: Coliform Plate Count Method into 15 CFU ~ 150 CFU ; - Delete Method 3: Coliforms Pertifilm TM Test Strip Method. Annex A and Annex B of this Standard are normative. The historical editions replaced by this Standard are as follows: - GB 4789.3-1984, GB 4789.3-1994, GB 4789.3-, and GB 4789.3-. www.chinesestandard.net Page 7 of 17
examination: Enumeration of Coliforms 1 Scope This Standard specifies the methods of enumeration of coliforms in food. This Standard is applicable to the enumeration of coliforms in food. 2 Terms and Definitions 2.1 Coliforms Aerobic and facultative anaerobic gram-negative asporous bacilli that can ferment lactose and are acid-producing and aerogenic, under certain culture conditions. 2.2 Most probable number (MPN) An indirect enumeration method based on Poisson distribution. 3 Devices and Materials In addition to conventional sterilization and culture devices in the microbiology laboratory, other devices and materials are as follows: 3.1 Thermostat incubator: 36 ºC ± 1 ºC. 3.2 Refrigerator: 2 ºC ~ 5 ºC. 3.3 Thermostat water bath: 46 ºC ± 1 ºC. 3.4 Balance: sensibility 0.1 g. 3.5 Homogenizer. 3.6 Oscillator. 3.7 Sterile sucking tubes: 1 ml (accurate to 0.01 ml), 10 ml (accurate to 0.1 ml) or micropipette with a sucker. 3.8 Sterile conical flask: 500 ml. 3.9 Sterile petri dish: diameter 90 mm. 3.10 ph meter or ph cuvette tube or precision ph test paper. 3.11 Colony counter. www.chinesestandard.net Page 8 of 17
8.3 Selection of plate colony count Select the plates with colony counts of 15 CFU 150 CFU and enumerate typical and suspicious coliform colonies on the plates respectively. Typical colonies are claret, around which is zone of precipitated red bile salt. The colonies have a diameter of 0.5 mm or above. 8.4 Confirmation test Pick up 10 typical and suspicious colonies of different types from the plates, then inoculate into BGLB broth tubes respectively, culture at 36 ºC ± 1 ºC for 24 h 48 h and observe the aerogenesis. The BGLB broth tubes with aerogenesis can be reported as coliform positive. 8.5 Reporting of plate enumeration of coliforms The number of coliform groups in each gram (ml) sample is obtained by multiplying the ratio of test tubes which are confirmed to be coliform positive BY the plate colony count in 8.3, and further multiplied by the dilution multiple. For example, 1 ml 10-4 sample diluent has 100 typical and suspicious colonies on the VRBA plate, ten of them are selected to inoculate the BGLB broth tube, six tubes are confirmed to be positive, then the coliform group count of the sample is 100 6/10 10 4 /g(ml) = 6.0 10 5 CFU/g (ml). www.chinesestandard.net Page 13 of 17
Annex A (Normative) Culture Medium and Reagents A.1 Lauryl sulfate tryptose (LST) broth A.1.1 Ingredients Tryptone or tryticase Sodium chloride Lactose Pdipotassium hydrogen phosphate (K 2 HPO 4 ) Potassium dihydrogen phosphate (KH 2 PO 4 ) Sodium lauryl sulfate Distilled water 20.0 g 5.0 g 5.0 g 2.75 g 2.75 g 0.1 g 1 000 ml ph 6.8 ± 0.2 A.1.2 Method of preparation Dissolve the ingredients into the distilled water, adjust ph value, pour the solution into test tubes with an inverted vial, 10 ml for each tube, and perform autoclaving at 121 ºC for 15 min. A.2 Brilliant green lactose bile (GGLB) broth A.2.1 Ingredients Peptone Lactose Oxgall or oxbile powder solution 10.0 g 10.0 g 200 ml 0.1% Brilliant green water solution 13.3 ml Distilled water 800 ml ph 7.2 ± 0.1 A.2.2 Method of preparation www.chinesestandard.net Page 14 of 17
Annex B (Normative) Search-table to Most Probable Numbers (MPN) of Coliforms B.1 Search-table to Most Probable Numbers (MPN) of coliforms See Table B.1 for most probable numbers (MPN) of coliforms in 1 g (ml) of sample. Table B.1 Search-table to Most Probable Numbers (MPN) of Coliforms Number of positive tubes 0.10 0.01 0.001 MPN Lower limit 95% confidence level Upper limit Number of positive tubes 0.10 0.01 0.001 MPN Lower limit 95% confidence level 0 0 0 < 3.0-9.5 2 2 0 21 4.5 42 0 0 1 3.0 0.15 9.6 2 2 1 28 8.7 94 0 1 0 3.0 0.15 11 2 2 2 35 8.7 94 0 1 1 6.1 1.2 18 2 3 0 29 8.7 94 0 2 0 6.2 1.2 18 2 3 1 36 8.7 94 0 3 0 9.4 3.6 38 3 0 0 23 4.6 94 Upper limit 1 0 0 3.6 0.17 18 3 0 1 38 8.7 110 1 0 1 7.2 1.3 18 3 0 2 64 17 180 1 0 2 11 3.6 38 3 1 0 43 9 180 1 1 0 7.4 1.3 20 3 1 1 75 17 200 1 1 1 11 3.6 38 3 1 2 120 37 420 1 2 0 11 3.6 42 3 1 3 160 40 420 1 2 1 15 4.5 42 3 2 0 93 18 420 1 3 0 16 4.5 42 3 2 1 150 37 420 2 0 0 9.2 1.4 38 3 2 2 210 40 430 2 0 1 14 3.6 42 3 2 3 290 90 1 000 2 0 2 20 4.5 42 3 3 0 240 42 1 000 2 1 0 15 3.7 42 3 3 1 460 90 2 000 2 1 1 20 4.5 42 3 3 2 1 100 180 4 100 2 1 2 27 8.7 94 3 3 3 > 1 100 420 - Note 1: There are three dilution rates in this table [0.1 g(ml), 0.01 g(ml) and 0.001 g(ml)] and each dilution rate is inoculated with three tubes. Note 2: If the amounts of samples in the table are changed into 1 g(ml), 0.1 g(ml) and 0.01 g(ml), figures in the table shall be decreased by 10 times accordingly; if the amounts are changed into 0.01 g(ml), 0.001 g(ml) and 0.0001 g(ml), figures in the table shall be increased by 10 times accordingly, the rest uses similar method. www.chinesestandard.net Page 17 of 17