GB 4789.41-2016 PDF in English
GB 4789.41-2016 (GB4789.41-2016) PDF English
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National Food Safety Standard -- Food Microbiological Examination -- Enterobacteriaceae
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Standards related to: GB 4789.41-2016
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GB 4789.41-2016: PDF in English GB 4789.41-2016
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
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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.
2.2
Enumeration of Enterobacteriaceae
The enumeration of Enterobacteriaceae per gram or per millimeter in accordance with
the method specified in this Standard.
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.
3.4 Balance. sensitivity 0.1 g.
3.5 Microscope. 10X ~ 100X.
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.
3.10 Sterile culture dishes. diameter 90 mm.
3.11 Sterile test tubes. 18 mm × 180 mm and 15 mm × 150 mm.
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.
4.3 Crystal violet neutral red bile salt glucose agar (VRBGA). see B.3.
4.4 Nutrient agar (NA). see B.4.
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.
4.9 Sterile 1 mol/l HCl. see B.9.
Method I The Enterobacteriaceae Plate Count
Method
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, use
a vibrating homogenizer to vibrate for 1 min ~ 2 min, and make homogeneous
specimen solutions of 1.10.
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.
6.1.3 Use a 1-ml sterile pipette or micropipette to absorb 1 ml of specimen
homogeneous solution of 1.10, pour slowly along the wall of the sterile test tube
containing 9 ml of BPW (attention is drawn to that the pipette or the tip shall not contact
with the diluent surface), vibrate the test tube or replace one 1-ml sterile pipette to blow
and beat repeatedly to mix up, and make homogeneous specimen solutions of 1.100.
6.1.4 In accordance with the operating procedures of 6.1.3, make tenfold increasing
serial diluted specimen homogeneous solutions. For each time of increasing dilution,
replace one 1-ml sterile pipette or tip. The whole process from the preparation of the
specimen homogeneous solutions to the completion of inoculation of specimens shall
not exceed 15 min.
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.
6.2.2 Pour 10 ml ~ 15 ml of VRBGA (which may be placed in a thermostatic water
bath for heat preservation at 46°C ± 1°C) cooled to 46°C to pour on each plate. Rotate
the plates carefully to mix up the specimen homogeneous solutions and culture media.
6.2.3 After the agar solidifies, pour a thin layer of the same culture medium to cover
the surface of the plates. Prevent the spreading growth and make the colony
characteristics more obvious.
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.
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.
9.2.2 Selective enrichment
Transfer 1 ml of culture from each culture tube of BPW, inoculate in 10 ml of EE broth,
and culture for 24 ± 2 h at 36°C ± 1°C.
9.2.3 Separation
Use an inoculating loop to take one loop of culture from all EE broths, conduct streak
inoculation on the BRBGA plate, culture at 24 ± 2 h at 36°C ± 1°C and observe whether
there is any typical colony on the plate.
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.
Look up in the MPN table (see Annex C) in accordance with the EE positive tube counts
and report the MPN values per gram (millimeter) of specimen. Report the sampling by
weight in MPN/g and the sampling by volume in MPN/ml.
Annex B
Culture Media and Reagents
B.1 Buffered peptone water (BPW)
B.1.1 Ingredients
Peptone 10.0 g
Sodium chloride 5.0 g
Disodium phosphate 3.5 g
Potassium dihydrogen phosphate 1.5 g
Distilled water 1 000.0 ml
B.1.2 Preparation
Heat the ingredients in B.1.1 to dissolve, adjust the pH to 7.2 ± 0.2 at 25°C, load
separately into 500-ml wide-mouthed bottles with 225 ml each bottle, and conduct
autoclaved sterilization for 15 min at 121°C.
B.2 Buffered glucose brilliant green bile broth (EE broth)
B.2.1 Ingredients
Peptone 10.0 g
Glucose 5.0 g
Disodium phosphate (anhydrous) 6.45 g
Potassium dihydrogen phosphate 2.0 g
Bile salt 20.0 g
Brilliant green 0.013 5 g
Distilled water 1 000.0 ml
B.2.2 Preparation
Dissolve the ingredients in B.2.1 in water, heat to boiling for full dissolution (heating is
preferably less than 30 min), cool the culture medium rapidly, adjust the pH to 7.2 ±
0.2 at 25°C, and load separately in sterile test tubes of 18 mm × 180 mm with 10 ml
each tube. They require no autoclaved sterilization and may be stored for one month
at 5°C ± 3°C.
B.3 Crystal violet neutral red bile salt glucose agar (VRBGA)
B.3.1 Ingredients
Peptone 7.0 g
Yeast extract 3.0 g
Bile salt No. 3 1.5 g
Glucose 10.0 g
B.6 Gram stain solutions
B.6.1 Crystal violet stain so...
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Source: Above contents are excerpted from the PDF -- translated/reviewed by: www.chinesestandard.net / Wayne Zheng et al.
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