GB 4789.36-2016National food safety standard - Microbiological Examination of Food Hygiene Examination of Escherichia Coli O157:H7/NM (English PDF)

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.

2.9 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.

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.

3.8 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.

5.1 Enrichment

Take 25 g (or 25 ml) of test specimen by sterile operation to add to a homogeneous

bag containing 225 ml of mEC+n broth and homogenize continuously for 1 min ~ 2 min

on a vibrating homogenizer;

5.2 Isolation

Take the mEC+n broth enriched, conduct streak inoculation on the CT-SMAC plate and

Escherichia coli O157 chromogenic agar plate, culture for 18 h ~ 24 h at 36°C ± 1°C

and observe the colonial morphologies. On the CT-SMAC plate, the typical colonies

Test specimen 25 g (or 25 ml) + 225 ml mEC+n broth, homogenized

5.3 Preliminary biochemical test

Take 5 ~ 10 suspicious colonies respectively from the CT-SMAC and Escherichia coli

O157 chromogenic agar plates, inoculate TSI agar respectively, and meanwhile,

inoculate MUG-LST broth, use Escherichia coli strain (ATCC25922 or equivalent

standard strains) as positive control and Escherichia coli O157.H7 (NCTC12900 or

equivalent standard strains) as negative control, and culture for 18 h ~ 24 h at 36°C ±

1°C. Conduct oxidase test and Gram staining if necessary. I

5.4 Identification

5.4.1 Serological test

Pick analytically pure colonies on the nutrient agar plate and use O157 and H7

diagnostic serum or O157 latex agglutination reagent for the slide agglutination test.

For the non-agglutination serum because of H7 factor, puncture the inoculation semi-

solid agar, check the dynamics, and determined it as a nondynamic strain when all

dynamic tests turn out to be negative after continuous 3 passages. If diagnostic serums

or latex agglutination reagents produced by different companies are used, the product

manuals shall be followed.

5.4.2 Biochemical test

5.4.2.2 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;

8.1 Enrichment

As in 5.1.

8.2 Immunomagnetic beads capture and separation

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.

8.2.5 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.

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.

8.3 Colony recognition

The characteristics of the colonies on the Escherichia coli O157.H7/NM plate and

Escherichia coli O157 chromogenic agar plate are as in 5.2.

8.4 Preliminary biochemical test

As in 5.3.

8.5 Identification

As in 5.4.