GB 4789.41-2016 PDF English
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National food safety standard - Microbiological Examination of Food Hygiene - Examination of Enterobacteriaceae
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GB 4789.41-2016: National food safety standard - Microbiological Examination of Food Hygiene - Examination of Enterobacteriaceae
---This is an excerpt. Full copy of true-PDF in English version (including equations, symbols, images, flow-chart, tables, and figures etc.), auto-downloaded/delivered in 9 seconds, can be purchased online: https://www.ChineseStandard.net/PDF.aspx/GB4789.41-2016
GB
NATIONAL STANDARD OF THE
PEOPLE’S REPUBLIC OF CHINA
National Food Safety Standard -
Microbiological Examination of Food Hygiene -
Examination of Enterobacteriaceae
Issued on. AUGUST 31, 2016
Implemented on. MARCH 1, 2017
Issued by. National Health and Family Planning Commission of the
People’s Republic of China
Table of Contents
1 Application Scope... 3
2 Terms and Definitions... 3
3 Apparatus and Materials... 3
4 Culture Media and Reagents... 4
5 Test Procedures... 5
6 Operating Procedures... 5
7 Report... 9
8 Test Procedures... 9
9 Operating Procedures... 11
10 Report... 11
Annex A Examples for Calculation of Results... 12
Annex B Culture Media and Reagents... 14
Annex C Retrieval Table of Enterobacteriaceae Most Probable Numbers
(MPNs)... 18
National Food Safety Standard -
Microbiological Examination of Food Hygiene -
Examination of Enterobacteriaceae
1 Application Scope
This Standard specifies the method for the test of Enterobacteriaceae in foods.
The first method of this Standard applies to the bacterial counts of Enterobacteriaceae
in the foods which has a high content of Enterobacteriaceae; and the second method
applies to the bacterial counts of Enterobacteriaceae in the foods which has a low
content of Enterobacteriaceae.
2 Terms and Definitions
2.1 Enterobacteriaceae
The oxidase-negative aerobic or facultative anaerobic Gram-negative non-spore-
bearing bacillus, which ferments glucose producing acid under given conditions.
2.3 most probable number; MPN
An indirect enumeration method based on Poisson's distribution.
3 Apparatus and Materials
In addition to the conventional sterilization and culture apparatus in the microbiological
laboratories, the other apparatus and materials are as follows.
3.1 Thermostatic incubator. 36°C ± 1°C.
3.2 Refrigerator. 2°C ~ 5°C.
3.3 Water bath. 46°C ± 1°C.
3.6 Homogenizer.
3.7 Shaker.
3.8 Sterile pipettes. 1 ml (having graduates of 0.01 ml), 10 ml (having 0.1 ml) or
micropipettors and tips.
3.9 Sterile conical flasks or equivalent vessels. capacities 150 ml and 500 ml.
3.12 pH meters or pH colorimetric tubes or precise pH test paper.
4 Culture Media and Reagents
4.1 Buffered peptone water (BPW). see B.1.
4.2 Buffered glucose brilliant green bile broth (EE broth). see B.2.
4.5 Glucose agar. see B.5.
4.6 Gram stain solution. see B.6.
4.7 Oxidase reagent. see B.7.
4.8 Sterile 1 mol/l NaOH. see B.8.
5 Test Procedures
See Figure 1 for the test procedures for the Enterobacteriaceae plate count method.
6 Operating Procedures
6.1 Specimen dilution
Test specimen
6.1.1 Solid and semi-solid specimens. take 25 g of specimen to place into a sterile
homogeneous cup containing 225 ml of BPW, homogenize for 1 min ~ 2 min at 8 000
r/min ~ 10 000 r/min, or place into a homogeneous bag containing 225 ml of BPW,
6.1.2 Liquid specimens. use a sterile pipette to absorb 25 ml of specimen to pour into
a sterile conical flask (in which an appropriate quantity of sterile glass beads are
provided in the flask in advance) containing 225 ml of BPW, mix up, and make
homogeneous specimen solutions of 1.10.
6.2 Pout plate and culture
6.2.1 In accordance with the estimation of the contamination of specimens and
relevant limit requirements, select 2 ~ 3 homogeneous specimen solutions (liquid
specimens may be raw solutions) of appropriate continuous dilution and inoculate the
solutions of each dilution degree to 2 sterile plates. And Meanwhile, absorb 1 ml of
BPW each to add to two sterile plates as blank control.
6.2.4 Turn over the plate after the upper-layer agar solidifies on the VRBGA plate and
culture for 18 h ~ 24 h at 36°C ± 1°C.
6.3 Enumeration and confirmation of typical colonies
6.3.1 The typical colonies of Enterobacteriaceae are pink to red or violet colonies
with or without precipitation rings. Select the plates with typical colony count between
15 CFU ~ 150 CFU and without spreading growth of colonies and only enumerate the
typical colonies. The colony counting is expressed by the colony-forming unit, CFU.
6.3.5 Confirmation of typical colonies.
6.4 Calculation of results
6.4.1 General principles
If the plate counts of typical colonies of two continuous dilution degrees are within the
appropriate counting range, calculate in accordance with Formula (1). See A.1, A.2,
A.3 and A.4 for the examples.
6.4.2 Small colony counts
If all the typical colony counts of the plate of minimum dilution degree (including the
raw solution of the liquid specimen) are less than 15 CFU and there are confirmed
Enterobacteriaceae colonies, then multiply the confirmed colony count by the minimum
dilution factor.
6.4.3 Special circumstances
If all the typical colony counts on the plate of the first dilution degree are greater than
150 CFU and there are confirmed Enterobacteriaceae colonies; and if there is no
confirmed Enterobacteriaceae colony on the plate of the second dilution degree or the
typical colony counts are not between 15 CFU ~ 150 CFU, then multiply the confirmed
colony count by the first dilution factor. See A.5 for the example.
7 Report
7.1 When the colony count is less than 100, it shall be reported with integers.
7.2 When the colony count is greater or equal to 100, take the first two digits after
rounding off the third digit, and use 0 to replace the places after it;
7.3 The numbers shall be rounded off to the nearest whole numbers.
7.4 If it is impossible to count because of spreading colonies on all the plates, then
report the colony spreading.
8 Test Procedures
See Figure 2 for the test procedures for the Enterobacteriaceae most probable number
method.
9 Operating Procedures
9.1 Specimen dilution
As in 6.1.
9.2 Inoculation and culture
9.2.1 Nonselective pre-enrichment
In accordance with the estimation of the contamination of specimens and relevant limit
requirements, select 3 homogeneous specimen solution of appropriate continuous
dilution degrees, 3 test tubes for each dilution degree and altogether 9 test tubes of
BPW, and culture for 18 h ± 2 h at 36°C ± 1°C.
9.3 Confirmation of typical colonies
See 6.3 for the typical colonies.
10 Report
Enterobacteriaceae is Gram-negative non-spore-bearing bacillus, which ferments
glucose producing acid and is oxidase negative. Once one colony is confirmed
Enterobacteriaceae, the EE tube represented by it is Enterobacteriaceae positive.
Annex A
Examples for Calculation of Results
A.1 Both of two continuous dilution degrees include appropriate typical colony
counting plates and contain confirmed Enterobacteriaceae colonies. See Table A.1 for
the data.
A.2 If both of two continuous dilution degrees include appropriate typical colony
counting plates, including one plate colony count of some dilution degree not within
the appropriate range or one plate colony count on each of two dilution degrees not
within the appropriate range, then the corresponding plates shall not be used as
counting plates. See Table A.2 for the data.
A.3 If both of two continuous dilution degrees include appropriate colony counting
plates, including one plate on which there is not confirmed Enterobacteriaceae colony,
the corresponding plates are not used as counting plates. See Table A.3 for the data.
A.5 If the colony counts on the plate of the first dilution degree are greater than 150
CFU and there are confirmed Enterobacteriaceae colonies; and if there is no confirmed
Enterobacteriaceae colony on the plate of the second dilution degree or the typical
colony counts are not between 15 CFU ~ 150 CFU, then multiply the confirmed colony
count by the first dilution factor. See A.5 for the example.
Annex B
Culture Media and Reagents
B.1 Buffered peptone water (BPW)
B.2 Buffered glucose brilliant green bile broth (EE broth)
B.3 Crystal violet neutral red bile salt glucose agar (VRBGA)
B.4 Nutrient agar (NA)
B.5 Glucose agar
B.6 Gram stain solutions
B.6.1 Crystal violet stain solution
Annex C
Retrieval Table of Enterobacteriaceae Most Probable
Numbers (MPNs)
......
Source: Above contents are excerpted from the full-copy PDF -- translated/reviewed by: www.ChineseStandard.net / Wayne Zheng et al.
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