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GB 4789.36-2016 (GB4789.36-2016)

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GB 4789.36-2016: PDF in English

GB 4789.36-2016
National Food Safety Standard -
Microbiological Examination of Food Hygiene
Examination of Escherichia Coli O157.H7/NM
Issued by. National Health and Family Planning Commission of the
People’s Republic of China;
China Food and Drug Administration.
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Table of Contents
Foreword ... 3 
1 Application Scope ... 4 
2 Apparatus and Materials ... 4 
3 Culture Media and Reagents ... 5 
4 Test Procedures ... 5 
5 Operating Procedures ... 6 
6 Result Reporting ... 8 
7 Test Procedures ... 8 
8 Operating Procedures ... 9 
9 Result Reporting ... 11 
Annex A Culture Media and Reagents ... 12 
This Standard replaces GB/T 4789.36-2008, Microbiological Examination of Food
Hygiene – Examination of Escherichia Coli O157.H7/NM.
Compared with GB/T 4789.36-2008, the major changes of this Standard are as follows.
-- the standard name is modified into “National Food Safety Standard –
Microbiological Examination of Food Hygiene – Examination of Escherichia Coli
-- the application scope of the standard is modified;
-- the apparatus and materials are modified;
-- the text descriptions of culture media and biochemical reactions are modified;
-- “Method II The Principle of the Immunomagnetic Beads Capture Method” is
-- “Method III Full-automatic Enzyme-linked Fluorescence Immunoassay
Instrument Screening Method” is deleted;
-- “Method IV Full-automatic Pathogenic Bacteria Detecting System Screening
Method” is deleted.
National Food Safety Standard -
Microbiological Examination of Food Hygiene
Examination of Escherichia Coli O157.H7/NM
1 Application Scope
This Standard specifies the test method of Escherichia coli o157.h7/nm in foods.
This Standard applies to the test of Escherichia coli o157.h7/nm in foods.
2 Apparatus and Materials
In addition to the conventional sterilization and culture equipment in the microbiological
laboratory, other apparatus and materials are as follows.
2.1 Thermostatic incubator. 36°C ± 1°C.
2.2 Refrigerator. 2°C ~ 5°C.
2.3 Thermostatic water bath. 46°C ± 1°C.
2.4 Balance. sensitivities 0.1 g and 0.01 g.
2.5 Homogenizer.
2.6 Microscope. 10X ~ 100X.
2.7 Sterile pipettes. 1 ml (having graduates of 0.01 ml), 10 ml (having graduates of
0.1 ml) or pipettes and tips.
2.8 Sterile homogeneous cups or sterile homogeneous bags. capacity 500 ml.
2.9 Sterile culture dishes. diameter 90 mm.
2.10 pH meters or precise pH test paper.
2.11 Long-wave UV lamps. 365 nm, power ≤ 6 W.
2.12 Microcentrifuge tubes. 1.5 ml or 2.0 ml.
2.13 Magnetic plate, magnetic plate frame, specimen mixer.
2.14 Microbiological identification system.
3 Culture Media and Reagents
3.1 Modified EC broth (mEC + n). see A.1.
3.2 Modified Sorbitol-MacConkey agar (CT-SMAC). see A.2.
3.3 Trisaccharide iron agar (TSI). see A.3.
3.4 Nutrient agar. see A.4.
3.5 Semi-solid agar. see A.5.
3.6 Lauryl sulphate peptone broth – MUG (MUG-LST). see A.6.
3.7 Oxidase reagent. see A.7.
3.8 Gram stain solution. see A.8.
3.9 PBS-Tween 20 lotion. see A.9.
3.10 Potassium tellurite (analytical reagent).
3.11 Cefixime.
3.12 Chromogenic medium for Escherichia coli O157.
3.13 Diagnostic serum for Escherichia coli O157 and H7 or latex agglutination
reagent for O157.
3.14 Identification kit.
3.15 Anti-E. coli O157 immunomagnetic beads.
Method I The Routine Culture Method
4 Test Procedures
See Figure 1 for the test procedures of the routine culture method of Escherichia coli
O157. H7/NM.
Simmons citrate Negative
Lysine decarboxylase Positive (purple)
Ornithine decarboxylase Positive (purple)
Cellobiose fermentation Negative
Raffinose fermentation Positive
MUG test Negative (no fluorescence)
Dynamic test Dynamic or non-dynamic If the biochemical identification kit or microbiological identification system is
selected, pick colonies from the nutrient agar plate, use the diluent to make the
bacterial suspension of appropriate turbidity and use the biochemical identification kit
or microbiological identification system for identification.
5.4.3 Virulence genes test (optional item)
When Escherichia coli O157. H7 or O157. NM is detected in the specimen, it may be
tested by inoculating Vero cells or Hela cells and observing the cytopathic effects if the
existence of Vero cytotoxin requires further tests; it may also be tested by using the
gene probe tests or polymerase chain reaction (PCR) method for Shiga toxin genes
(stx1 and stx2), eae, hly and so on. If kits are used for the test of the above-mentioned
genes, the product manuals shall be followed.
6 Result Reporting
Summarize the biochemical and serological test results and report whether
Escherichia coli O157.H7 or Escherichia coli O157.NM is detected or not detected in
the 25 g (or 25 ml) of specimen.
Method II The Immunomagnetic Beads Capture
7 Test Procedures
See Figure 2 for the test procedures of the immunomagnetic beads capture method of
Escherichia coli O157.H7/NM.
8.2.2 Number the microcentrifuge tubes in accordance with the specimens and
quality-control strains, use one microcentrifuge tube for each specimen, and then
insert into the magnetic plate frame. Vibrate the Escherichia coli O157
immunomagnetic beads suspension gently on the vortex mixer, use an opener to open
the cap of each microcentrifuge tube, and add 20 μL of Escherichia coli O157
immunomagnetic beads suspension in each tube.
8.2.3 Take 1 ml of mEC+n broth enrichment medium, add to the microcentrifuge tube,
put on the cap, and then vibrate gently for 10 s. Replace one specimen adding tip for
each specimen, and the quality-control strains shall be separated from the specimen
to prevent cross-contamination.
8.2.4 Binding. at 18°C ~ 30°C, rotate the above-mentioned microcentrifuge tube
together with the magnetic plate frame on the specimen mixer or use hands to rotate
gently for 10 min to make Escherichia coli O157 fully contact with the immunomagnetic
8.2.5 Capture. insert the magnetic plate into the concentrated magnetic beads in the
magnetic plate frame. Tilt the magnetic plate frame within 3 min to ensure the
immunomagnetic beads in the suspension and on the cap are fully collected. Then,
rounded or oval brown accumulation becomes visible in the middle of the wall of the
microcentrifuge tubes.
8.2.6 Absorption of supernatant. take one sterile elongated pipette and absorb gently
the supernatant by inserting deep into the liquid from the immunomagnetic beads
accumulation opposite to the liquid level. When the liquid is absorbed until the liquid
level passes through the immunomagnetic accumulation, slow down to ensure no
immunomagnetic beads are absorbed away. If the supernatant absorbed contains
magnetic beads, place them back in the microcentrifuge tube, and repeat the
procedures in 8.2.5. Replace one sterile elongated tube for each specimen.
Fall of immunomagnetic beads. the magnetic beads accumulation of some specimens,
especially those specimens containing much fat is liable to fall to the bottom. When
absorbing the supernatant, it is hard to prevent the loss of magnetic beads; and under
such circumstances, 50 μL ~ 100 μL of supernatant may be reserved in the
microcentrifuge tube. If it is operated like this in the later washing process, the effects
of fat will become smaller and the purpose of full capture may also be achieved.
8.2.7 Washing. move away the magnetic plate from the frame, add 1 ml of PBS-
Tween 20 lotion in each microcentrifuge tube, and rotate on a specimen mixer or use
hand to rotate gently for 3 min to wash the immunomagnetic beads mixture. Repeat
the above-mentioned procedures 8.2.5 ~ 8.2.7.
8.2.8 Repeat the above-mentioned procedures 8.2.5 ~ 8.2.6.
8.2.9 Immunomagnetic beads suspension. move away the magnetic plate and make
the immunomagnetic beads suspend in 100 μL of PBS-Tween 20 lotion once again.
Annex A
Culture Media and Reagents
A.1 Modified EC broth (mEC+n)
A.1.1 Ingredients
Tryptone 20.0 g
Bile salt No.3 1.12 g
Lactose 5.0 g
K2HPO4·7H2O 4.0 g
KH2PO4 1.5 g
NaCl 5.0 g
Novobiocin sodium salt solution (20 mg/ml) 1.0 ml
Distilled water 1 000 ml
A.1.2 Preparation
Except novobiocin, dissolve all ingredients in the water, heat to boiling, calibrate the
pH to 6.9 ± 0.1 at 20°C ~ 25°C, and load separately. Conduct autoclaved sterilization
for 15 min at 121°C as standby. Prepare novobiocin stock solution of concentration 20
mg/ml and conduct disinfection by filtering. Wait until the temperature ...
Source: Above contents are excerpted from the PDF -- translated/reviewed by: www.chinesestandard.net / Wayne Zheng et al.