GB 4789.38-2012 (GB4789.38-2012)

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GB 4789.38-2012: PDF in English

NATIONAL STANDARD
OF THE PEOPLE’S REPUBLIC OF CHINA
National Food Safety Standard
Microbiological Examination of Food Hygiene -
Enumeration of Escherichia Coli
ISSUED ON. MAY 17, 2012
IMPLEMENTED ON. JULY 17, 2012
Issued by the Ministry of Health of the People's Republic of China
GB
Foreword
This standard replaces "Microbiological Examination of Food Hygiene - Enumeration of
Escherichia Coli" (GB/T 4789.38-2008).
The main changes in this standard over GB/T 4789.38-2008 are as follows.
- Chinese name of this standard was modified;
- Culture mediums and reagents were modified;
- "Method II Plate count method for Escherichia coli" was modified as "plate count
method for Escherichia coli (Method II)";
- "Method III PetrifilmTM Escherichia coli count plate method" was deleted;
- Appendix A was modified.
Contents
1 Scope ... 1
2 Terms and Definitions ... 1
3 Equipment and Materials ... 1
4 Culture Medium and Reagent ... 2
5 MPN for Counting Escherichia Coli (Method I) ... 2
6 Plate Count Method for Escherichia Coli (Method II) ... 5
Appendix A Culture Medium and Reagent... 7
Appendix B Most Probable Number (MPN) Search List of Escherichia Coli ... 13
1National Food Safety Standard
Microbiological Examination of Food Hygiene -
Enumeration of Escherichia Coli
1 Scope
The standard specifies the counting method of Escherichia coli in food.
This standard is applicable to the counting of Escherichia coli in food. Among them,
plate count method for Escherichia coli (Method II) is not applicable to shellfish products.
2 Terms and Definitions
2.1 Escherichia coli
Escherichia coli
It extensively exists in the intestinal tract of humans and warm-blooded animals and is
able to ferment lactose and generate acid and gas at 44.5 . That it is presented as ℃ + + - or - +
- Gram negative bacilli in IMViC biochemical test (indole, methyl red, VP test and citrate) is
taken as fecal pollution index to assess the hygienic condition of food and deduce the
possibility of enteropathogenic bacteria pollution in food.
2.2 Most probable number
It is an indirect counting method based on poisson distribution and referred to as MPN.
3 Equipment and Materials
In addition to the conventional sterilization and cultivation equipment in microbiological
laboratory, other equipment and materials needed are as follows.
a) Thermostatic incubator. 36 ±1 ;℃ ℃
b) Refrigerator. 2 ~5 ;℃ ℃
c) Thermostatic water bath. 44.5 ±0.2 ;℃ ℃
d) Balance. sensibility 0.1g;
c) Homogenizer;
d) Oscillator;
e) Aseptic pipette. 1mL (with 0.01mL scale), 10mL (with 0.1mL scale) or
micropipettor and sucker head;
2f) Aseptic conical flask. capacity, 500 mL;
g) Sterile Petri dish. 90 mm in diameter;
h) PH meter or pH colorimetric tube or precise pH paper;
i) Colony counter;
j) Ultraviolet lamp. wavelength 360nm ~ 366nm; power ≤6W.
4 Culture Medium and Reagent
4.1 Lauryl sulfate tryptone (LST) broth. see A.1 in Appendix A.
4.2 EC broth (E.coli broth). see A.2 of Appendix A.
4.3 Peptone water. see A.3 of Appendix A.
4.4 Buffer glucose peptone water [used in methyl red (MR) and V-P tests]. see A.4 of
Appendix A.
4.5 Simmons citrate culture medium. see A.5 of Appendix A.
4.6 Phosphate buffer. see A.6 of Appendix A.
4.7 Eosin methylene blue (EMB) agar. see A.7 of Appendix A.
4.8 Nutrient agar slant. see A.8 of Appendix A.
4.9 Violet red bile agar (VRBA). see A.9 of Appendix A.
4.10 Violet red bile agar-4-methyl umbellulone-β-D glucoside (VRBA-MUG). see A.10 of
Appendix A.
4.11 Gram staining solution. see A.11 of Appendix A.
4.12 Kovacs indole reagent. see A.12 of Appendix A.
4.13 Aseptic 1mol/L NaOH. see A.13 of Appendix A.
4.14 Aseptic 1mol/L HCl. see A.14 of Appendix A.
5 MPN for Counting Escherichia Coli (Method I)
5.1 Test procedures
Test procedures of MPN for counting Escherichia coli are illustrated in Figure 1.
3Figure 1 Test Procedures of MPN for Counting Escherichia Coli
5.2 Operation procedures
5.2.1 Dilution of sample
5.2.1.1 Solid sample and semi-solid sample. weigh 25 g sample, inject it into the aseptic
homogeneity cup filled with 225mL phosphate buffer and homogenize for 1min ~ 2min with
the rate of 8000r/min ~ 10000r/min to prepare 1.10 sample homogeneous solution, or inject it
Tested sample
25 g(mL) sample + 225 ml diluent, homogenize
10-time series dilution
Adopt 3 suitable sample homogeneous solution of continuous dilutability and
inoculate in LST broth tube
36 ±1℃ ℃ 48h±2h
Bubbles are
observed
No bubbles are
observed
EC broth
48h±2h44.5℃±0.2℃
Bubbles are
observed
No bubbles are
observed
EMB plate
streaking
36℃±1℃ 48h±2h
Subcultivate questionable colonies
in nutrient agar slant or plant
36℃±1℃ 48h±2h
IMViC biochemical test, gram staining
Negative tube Positive tube
Check MPN table
Report
4in aseptic homogenizing bag filled with 225mL phosphate buffer and pat for 1min ~ 2min
with patting-type homogenizer to prepare 1.10 sample homogeneous solution.
5.2.1.2 Liquid sample. pipet 25mL sample by aseptic pipette; inject it in aseptic conical
flask (proper quantity of aseptic glass bead is preset within) filled with 225mL phosphate
buffer and mix uniformly to prepare 1.10 sample homogeneous solution.
5.2.1.3 The pH value of the sample homogeneous solution shall be between 6.5 and 7.5; if
necessary, the sample homogeneous solution shall be adjusted by NaOH (1 mol/L) or HCl (1
mol/L) respectively.
5.2.1.4 Pipette 1mL of 1.10 sample homogeneous solution with the 1mL aseptic pipette or
the micropipettor and inject it slowly along the tube wall into the aseptic test tube containing
9 mL phosphate buffer (the sucker or the sucker head tip shall not touch the diluent), mix the
solution by shaking the test tube or beating upon the tube repeatedly with another 1 mL
aseptic pipette or sucker head, and finally prepare the 1.100 sample homogeneous solution.
5.2.1.5 According to the estimation for the sample contamination condition, prepare the
sample homogeneous solution with dilution increasing successively until 10 times basing on
the above-described operations. Change one 1 mL aseptic pipette or sucker head each time the
solution is diluted. The entire process shall not exceed 15 min from the preparation of the
sample homogeneous solution to the finishing of sample inoculation.
5.2.2 Primary fermentation test
Choose 3 kinds of sample homogeneous solution with appropriate continuous dilutability
(the original solution may be chosen for the liquid sample) for each sample. Inoculate three
tubes of LST brouth for each dilutability, with 1 mL inoculation in each tube (e.g. if the
inoculation exceeds 1 mL, then the double-material LST broth shall be applied); cultivate the
LST broth for 24h±2h at 36 ±℃ 1 and observe the small inverted tube; if bubbles are ℃
generated in 24h±2h, fermentation test shall be performed again; if not, then the LST broth
shall be cultivated for another 48 h±2 h. If bubbles are generated, then fermentation test shall
be carried out again. If no bubble is observed in all LST broth tubes, report the MPN result of
Escherichia coli.
5.2.3 Secondary fermentation test
Take one loop of culture from each aerogenic LST broth tube with inoculating loop to
transfer in EC broth tube pre-heated to 45 ; place in the lid℃ -provided water bath with a
temperature of 44.5 ±0.2 . The water surface of the bath shall be higher than the solution ℃ ℃
level of the broth culture medium; cultivate for 24h±2h and observe whether bubbles are
generated in the small inverted tube; if not continue to cultivate to 48±2h. Record the number
of EC broth tubes which generate bubbles in 24h and 48h. If none of the EC broth tube
generate bubble, MPN result of Escherichia coli may be reported; any tube generates bubbles
shall be conducted with EMB plate isolated culture.
5.2.4 Eosin methylene blue plate isolated culture
Gently shake the aerogenic tube; take the culture with inoculating loop to steak-inoculate
it in EMB plate respectively and cultivate for 18h~24h at 36 ±1 . Observe whether ℃ ℃
lustrous or luster-lacking typical colony with black center exists in the plate.
5.2.5 Nutrient agar slant or plate culture
Pick 5 typical colonies from each plate or pick questionable one in case of no typical
colony existing. Touch the center of colony by inoculating needle and subculture on nutrient
5agar slant or plate and cultivate for 18h~24h at 36 ±1 . Take culture to conduct gram ℃ ℃
staining and biochemical tests.
5.2.6 Assessment
Take culture to conduct indole test, MR-VP test and citrate-utilization test. Biochemical
assessment of Escherichia coli and other bacteria are detailed in Table 1.
Table 1 Biochemical Assessment of Escherichia Coli and Other Bacteria
Indole (I) Methyl red (MR) VP test (VP) Citrate (C) Assessment (type)
Typical Escherichia coli
Atypical Escherichia coli
Typical central type
Atypical central type
Typical enteroaerogen
Atypical enteroaerogen
Note 1. the emergence of biochemical reaction types not included in Table 1 indicates that culture may not be pure. Thus re-steak
separation shall be conducted and rete...
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(Above excerpt was released on 2017-12-02, modified on 2021-06-07, translated/reviewed by: Wayne Zheng et al.)
Source: https://www.chinesestandard.net/PDF.aspx/GB4789.38-2012