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National food safety standard - Food microbiological examination - Enumeration of coliforms
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National food safety standard - Food microbiological examination - Enumeration of coliforms
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National food safety standard -- Food microbiological examination: Enumeration of coliforms
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| GB/T 4789.3-2008 | English | 439 |
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Microbiological examination of food hygiene -- Enumeration of coliforms
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Microbiological examination of food hygiene -- Detection of coliform bacteria
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| GB 4789.3-1994 | English | RFQ |
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Microbiological examination of food hygiene. Detection of Coliform bacteria
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Microbiological examination of food hygiene--Detection of coliform bacteria
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GB 4789.3-2025: PDF in English GB 4789.3-2025
GB
NATIONAL STANDARD OF THE
PEOPLE’S REPUBLIC OF CHINA
National Food Safety Standard – Food Microbiological
Examination – Enumeration of Conliforms
ISSUED ON. MARCH 16, 2025
IMPLEMENTED ON. SEPTEMBER 16, 2025
Issued by. National Health Commission of the People’s Republic of China;
State Administration for Market Regulation.
Table of Contents
Foreword... 3
1 Scope... 4
2 Terms and Definitions... 4
3 Equipment and Materials... 4
4 Culture Medium and Reagents... 5
5 Inspection Procedures... 5
6 Operation Steps... 6
7 Results and Report... 8
8 Inspection Procedures... 8
9 Operation Steps... 8
10 Results and Report... 9
Appendix A Culture Medium and Reagents... 11
Appendix B Retrieval Table for Most Probable Number (MPN) of Coliforms.. 14
Appendix C Method for Determining the Most Appropriate 3 Continuous
Dilutions... 15
Appendix D Results Processing and Examples of Coliform Plate Count... 16
National Food Safety Standard – Food Microbiological
Examination – Enumeration of Conliforms
1 Scope
This Standard specifies the method for enumeration of coliforms in food.
The first method of this Standard is applicable to the enumeration of coliforms in foods with
low coliform content; and the second method is applicable to the enumeration of coliforms in
foods with high coliform content.
2 Terms and Definitions
2.1 Coliforms
An aerobic and facultative anaerobic Gram-negative spore-less bacterium that can ferment
lactose, produce acid and gas under certain culture conditions.
2.2 Most probable number (MPN) technique
A quantitative test method that combines statistics and microbiology. After the sample to be
tested is serially diluted and cultured, the most likely number of coliforms in the sample is
calculated using statistical probability theory based on the lowest dilution at which no growth
occurs and the highest dilution at which growth occurs.
3 Equipment and Materials
In addition to routine sterilization and culture equipment for microbiological laboratories, other
equipment and materials are as follows.
3.1 Constant temperature incubator. 36℃ ± 1℃, 30℃ ± 1℃.
3.2 Refrigerator. 2℃ ~ 8℃.
3.3 Constant temperature device. 48℃ ± 2℃.
3.4 Balance. With sensitivity of 0.1 g.
3.5 Homogenizer and sterile homogenizing bag, homogenizing cup, oscillator.
3.6 Test tube. 15 mm×150 mm, 18 mm×180 mm or other suitable specifications; as well as
small inverted tube (Durham’s tube) or other suitable gas collection device.
3.7 Sterile pipette. 1 mL (with 0.01 mL scale), 2 mL (with 0.02 mL scale), 5 mL (with 0.05 mL
scale), 10 mL (with 0.1 mL scale) or micropipette and sterile sucker.
3.8 Sterile conical flask. With capacity of 500 mL.
3.9 Sterile culture dish. With diameter of 90 mm.
3.10 pH meter or precision pH test paper.
3.11 Sterile inoculation loop. 10 μL (about 3 mm in diameter).
3.12 Magnifying glass or colony counter.
4 Culture Medium and Reagents
4.1 Phosphate buffer. See A.1 in Appendix A.
4.2 Physiological saline. See A.2.
4.3 Lauryl sulfate tryptone (LST) broth. See A.3.
4.4 Brilliant green lactose bile (BGLB) broth. See A.4.
4.5 Crystal red bile agar (VRBA). See A.5.
4.6 1 mol/L NaOH. See A.6.
4.7 1 mol/L HCl. See A.7.
4.8 Coliform count test piece (the positive result of the production of acid and gas by
fermentation of lactose shall be considered as the judgment basis; and the performance shall
meet the quality requirements of relevant culture medium in GB 4789.28).
Method I Most Probable Number (MPN) Technique
of Coliforms
5 Inspection Procedures
The inspection procedures for most probable number (MPN) technique of coliforms can refer
to Figure 1.
number of sterile glass beads can be pre-placed in the flask) and mix thoroughly; or place it in
a sterile homogenizing bag containing 225 mL of phosphate buffer or physiological saline and
beat it with a slapping homogenizer for 1min ~ 2min to make a 1.10 sample homogenate. When
the sample is not suitable for volume sampling, follow 6.1.1.
6.1.3 If necessary, use 1 mol/L NaOH or 1 mol/L HCl to adjust the pH of the sample
homogenate or liquid sample stock solution to 6.5 ~ 7.5.
6.1.4 Use a sterile pipette or micro-pipettor to draw 1 mL of 1.10 sample homogenate; and
slowly inject it into a sterile test tube containing 9 mL of phosphate buffer or physiological
saline along the tube wall (be careful not to let the pipette or sucker touch the surface of the
diluent); and oscillate the test tube on an oscillator to mix well and make a 1.100 sample
homogenate.
6.1.5 Based on the estimation of the sample contamination status, refer to the operation of 6.1.4
and make a 10-fold incremental serial dilution of the sample homogenate. After each
incremental dilution, replace a sterile pipette or sucker.
6.2 Primary fermentation test
For each sample, select 3 appropriate sample homogenates with continuous dilutions (the stock
solution can be selected for liquid samples); inoculate 3 tubes of LST broth for each dilution;
and inoculate 1 mL of sample homogenate into each tube of LST broth (if the inoculation
volume exceeds 1 mL, add it to an equal volume of double-material LST broth). The whole
process from the preparation of sample homogenate to the completion of inoculation into LST
broth shall not exceed 15 min. Place the LST broth tube with the inoculated sample at 36℃ ±
1℃ to culture for 24 h±2 h; and check the gas production. If bubbles are generated in the small
inverted tube or the gas collection device, or if fine bubbles are continuously rising in the test
tube when the LST broth tube is gently shaken, it is judged to be gas production; and the gas
production is subjected to a re-fermentation test (confirmation test). If no gas is produced,
continue culturing for 48 h±2 h and check the gas production again. If gas is produced, conduct
a re-fermentation test; if no gas is produced after culturing for 48 h±2 h, it is judged as negative
coliforms. If no gas is produced in all LST broth tubes after culturing for 48 h±2 h, report the
MPN value of coliforms per gram (ml) of sample according to the MPN retrieval table in
Appendix B, expressed as MPN/g(mL).
6.3 Re-fermentation test (confirmation test)
Gently shake each gas-producing LST broth tube; take 1 loop of culture with an inoculation
loop, respectively; transfer it to the BGLB broth tube; and culture it at 36℃±1℃ for 24h±2h.
Check the gas production. If it produces gas, it is judged as positive coliforms; if it does not
produce gas, continue to culture it for 48h±2h and check the gas production again. If it produces
gas, it is judged as positive coliforms; and if it still does not produce gas, it is judged as negative
coliforms.
9.2.1 Based on the estimation of the sample contamination status, select 2~3 appropriate sample
homogenates with continuous dilutions (the stock solution can be selected for liquid samples);
and inoculate 2 sterile culture dishes for each dilution; 1 mL in each dish. At the same time,
take phosphate buffer or physiological saline and add it to 2 sterile culture dishes as blank
controls; 1 mL in each dish.
9.2.2 Pour VRBA cooled to 48℃ ± 2℃ into the culture dishes as soon as possible; 15 mL~20
mL in each dish. Carefully rotate the culture dishes to mix the culture medium and the
inoculated sample homogenate thoroughly; and let it stand horizontally until it solidifies. The
whole process from preparing the sample homogenate to pouring VRBA shall not exceed 15
min. After the agar solidifies, evenly cover the entire surface of the plate with 3 mL ~ 4 mL
VRBA. After the agar layer solidifies, turn the plate over and culture it at 36℃ ± 1℃ for 18 h
~ 24 h. For milk and dairy products, they shall be cultured at 30℃ ± 1℃ for 18 h~24 h.
9.2.3 If the coliforms count test piece is used, operate according to its instructions.
9.3 Selection of plate colony count
9.3.1 Observe and count the colonies, using a magnifying glass or colony counter when
necessary. Select all plates with colony counts between 15 CFU ~ 150 CFU; and count the
typical and suspicious coliforms colonies on the plates. Typical colonies are red to purple-red;
with a red precipitation ring around the colonies; and the colony diameter is generally greater
than 0.5 mm. Suspicious colonies are red to purple-red, and the colony diameter is generally
less than 0.5 mm.
9.3.2 If there are 2 dilutions with plate colony counts between 15 CFU ~ 150 CFU and other
situations, the selection of colony counts shall be carried out in accordance with the provisions
of Appendix D.
9.4 Confirmation test
Respectively pick 5 typical and suspicious colonies from the VRBA plate of the same dilution.
If there are less than 5 typical or suspicious colonies, pick all the colonies. Inoculate each colony
into 1 BGLB broth tube and culture at 36℃ ± 1℃ for 24 h ± 2 h. Check the gas production. If
it produces gas, it is positive coliforms. If it does not produce gas, continue to culture for 48 h
± 2 h and observe again. If it produces gas, then it is positive coliforms; if it still does not
produce gas, then it is negative coliforms.
10 Results and Report
10.1 The coliform colony count is the average sum of the product of the typical colony count/the
suspicious colony count of the selected dilution and the positive rate of coliforms, multiplied
by the dilution factor. When the coliform colony count is less than 100 CFU, it shall be rounded
off according to the principle of "rounding off" and reported as an integer. When the coliform
colony count is greater than or equal to 100 CFU, the third digit is rounded off according to the
Appendix A
Culture Medium and Reagents
A.1 Phosphate buffer
A.1.1 Compositions
Potassium dihydrogen phosphate. 34.0 g
Distilled water. 500 mL
A.1.2 Preparation method
Stock solution. Weigh 34.0 g of potassium dihydrogen phosphate and dissolve it in 500 mL of
distilled water (or other experimental water that meets the requirements, the same below);
adjust the pH to 7.2±0.2 with about 175 mL of 1 mol/L sodium hydroxide solution; dilute to
1,000 mL with distilled water; and sealed it and store in a refrigerator. Working solution. Take
1.25 mL of stock solution; dilute to 1,000 mL with distilled water; divide into appropriate
containers; autoclave at 121℃ for 15 min; and use for sample dilution.
A.2 Physiological saline
A.2.1 Compositions
Sodium chloride. 8.5 g
Distilled water. 1,000 mL
A.2.2 Preparation method
Dissolve sodium chloride in distilled water and autoclave at 121 ℃ for 15 min.
A.3 Lauryl sulfate tryptone (LST) broth
A.3.1 Compositions
Tryptone or casein tryptone. 20.0 g
Sodium chloride. 5.0 g
Lactose. 5.0 g
Dipotassium hydrogen phosphate. 2.75 g
Potassium dihydrogen phosphate. 2.75 g
Sodium lauryl sulfate. 0.1 g
Distilled water. 1,000 mL
A.3.2 Preparation method
Add all compositions (except distilled water for double-composition LST broth, double the
amounts of other compositions) to distilled water and heat to dissolve. Adjust pH if necessary.
Dispense into test tubes with small inverted tubes; 10 mL per tube. After autoclaving at 121℃
for 15 min; the pH of the culture medium at 25 ℃ is 6.8±0.2.
A.4 Brilliant green lactose bile (BGLB) broth
A.4.1 Compositions
Peptone. 10.0 g
Lactose. 10.0 g
Ox bile powder. 20.0 g
Brilliant Green. 0.0133 g
Distilled water. 1,000 mL
A.4.2 Preparation method
Dissolve each component in distilled water by heating; and adjust pH if necessary. Dispense
into test tubes with small inverted tubes; 10 mL per tube. Autoclave at 121 ℃ for 15 min. The
pH of the sterilized culture medium at 25 ℃ is 7.2±0.2.
A.5 Violet red bile agar (VRBA)
A.5.1 Compositions
Peptone. 7.0 g
Yeast extract powder. 3.0 g
Lactose. 10.0 g
Sodium chloride. 5.0 g
Bile salt or No. 3 bile salt. 1.5 g
Neutral red. 0.03 g
Crystal violet. 0.002 g
......
Source: Above contents are excerpted from the PDF -- translated/reviewed by: www.chinesestandard.net / Wayne Zheng et al.
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