1 Scope
This standard specifies the examination method of Escherichia coli O157:H7/NM in foods.
This standard applies to the examination of Escherichia coli O157:H7/NM in foods.
2 Apparatus and Materials
In addition to the apparatus for conventional sterilization and cultivation in microbiological laboratory, other apparatus and materials are as follows:
2.1 Constant temperature incubator: 36℃±1℃.
2.2 Refrigerator: 2℃~5℃.
2.3 Thermostatic water bath: 46℃±1℃.
2.4 Balance: with sensibility of 0.1g, 0.01g.
2.5 Homogenizer.
2.6 Microscope: 10x~100x.
2.7 Aseptic pipette: 1mL (with scale of 0.01mL), 10mL (with scale of 0.1mL) or pipettor and pipette tip.
2.8 Aseptic homogenizing cup or aseptic homogenizing bag: with volume of 500mL.
2.9 Aseptic culture dish: with diameter of 90mm.
2.10 pH meter or precision pH paper.
2.11 Ultraviolet lamp with long wave: 365nm, power ≤ 6W.
2.12 Microcentrifuge tube: 1.5mL or 2.0mL.
2.13 Magnetic sheet, magnetic sheet frame, sample mixer.
2.14 Microbial identification system.
3 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 Triple sugar iron agar (TSI): see A.3.
3.4 Nutrient agar: see A.4.
3.5 Semi-solid agar: see A.5.
3.6 Lauryl sulfate tryptone 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 washing liquid: see A.9.
3.10 Potassium tellurite(Grade AR).
3.11 Cefixime.
3.12 Chromogenic medium of Escherichia coli O157.
3.13 Diagnostic serum of Escherichia coli O157 and H7 or latex agglutination reagent of O157.
3.14 Identification reagent kit.
3.15 Anti-E.coli O157 immunomagnetic beads.
Method I Routine Culture Method
4 Examination Procedures
See Figure 1 for the examination procedures of Escherichia coli O157:H7/NM with routine culture method.
Figure 1 Examination Procedures for Escherichia Coli O157:H7/NM by Routine Culture Method
5 Operation Steps
5.1 Enrichment
Take 25g (or 25mL) of examined sample by aseptic technique, add it to the homogenizing bag containing 225mL of mEC+n broth, continuously homogenize them on the slap type homogenizer for 1min~2 min; or put the examined sample into the homogenizing cup containing 225mL of mEC+n broth to homogenize at 8000r/min~10000r/min for 1min~2 min. Culture it at 36℃±1℃ for 18h~24h.
5.2 Isolation
Take enrichened mEC+n broth to undergo streak inoculation on the CT-SMAC plate and Escherichia coli O157 chromogenic agar plate, culture it at 36℃±1℃ for 18h~24h, and observe the colony shape. Typical colony on the CT-SMAC plate is round, smooth, small and colorless, with darker taupe in the center; for the colony on Escherichia coli O157 chromogenic agar plate, its characteristics are judged according to product instructions.
5.3 Preliminary biochemical test
Respectively pick 5~10 suspicious colonies from CT-SMAC plate and Escherichia coli O157 chromogenic agar plate to inoculate TSI agar and MUG-LST broth simultaneously, and adopt Escherichia coli strain (ATCC 25922 or equivalent standard strain) to carry out positive control and adopt Escherichia coli O157:H7 (NCTC 12900 or equivalent standard strain) to carry out negative control, and then culture them at 36℃±1℃ for 18h~24h. If necessary, conduct oxidase test and gram stain. In TSI agar, typical strain presents yellow inclined plane and bottom layer, aerogenesis or an-aerogenesis, no hydrogen sulfide (H2S) generated. Observe the MUG-LST broth tube under long-wave ultraviolet light, the MUG-positive Escherichia coli strain - shall generate fluorescence, while MUG-negative strain shall be free from fluorescence, and the Escherichia coli O157:H7/NM is MUG-negative test without fluorescence. Pick suspected colony to clone on nutrient agar plate, culture it at 36℃±1℃ for 18h~24h, and carry out the following identifications.
5.4 Identification
5.4.1 Serological test
Pick cloned colony from nutrient agar plate to carry out slide agglutination test with O157 and H7 diagnostic serums or O157 latex agglutination reagent. For H7 factor with inagglutinable serum, it shall be stabbed to inoculate semi-solid agar, inspect power, and carry out serial passage for three times, if all the power tests are negative, it is determined as non-powered strain. If diagnostic serums or latex agglutination reagents from different companies are adopted, the test shall be conducted according to the product instructions.
5.4.2 Biochemical test
5.4.2.1 Pick colony from nutrient agar plate to carry out biochemical test. See Table 1 for the characteristics of biochemical reaction of Escherichia coli O157:H7/NM.
Table 1 Characteristics of Biochemical Reaction of Escherichia Coli O157:H7/NM
Biochemical test Characteristic reaction
Triple sugar iron agar Yellow bottom layer and inclined plane, negative H2S
Sorbitol Negative or late fermentation
Indole Positive
Methyl red-voges proskauer test (MR- VP) Positive MR, negative VP
Oxidase Negative
Simmons Citrate Negative
Lysine decarboxylase Positive (purple)
Ornithine decarboxylase Positive (purple)
Cellobiose fermentation Negative
Raffinose fermentation Positive
MUG test Negative (without fluorescence)
Power test With power or without power
5.4.2.2 If biochemical identification kit or microbial identification system is adopted, the colony shall be picked from nutrient agar plate to prepare with diluent to bacterial suspension of appropriate turbidity, and then identify by biochemical identification kit or microbial identification system.
5.4.3 Virulence gene determination (optional)
Where the Escherichia coli O157:H7 or O157:NM is detected in sample, if further examination is required to examine Vero cell toxin gene, Vero cell or HeLa cell may be inoculated to judge via observing cytopathic effect; besides, gene probe detecting or polymerase chain reaction (PCR) may also be adopted to detect such genes as Shiga toxin gene (stx1, stx2), eae and hly. If the above genes are detected by reagent kit, the product instructions shall apply.
6 Result Report
According to the results of biochemical and serological test, report whether Escherichia coli O157:H7 or Escherichia coli O157:NM is detected or not in 25g (or 25mL) of sample.
Foreword I
1 Scope
2 Apparatus and Materials
3 Media and Reagents
4 Examination Procedures
5 Operation Steps
6 Result Report
7 Examination Procedures
8 Operation Steps
9 Result Report
Appendix A Media and Reagents
1 Scope
This standard specifies the examination method of Escherichia coli O157:H7/NM in foods.
This standard applies to the examination of Escherichia coli O157:H7/NM in foods.
2 Apparatus and Materials
In addition to the apparatus for conventional sterilization and cultivation in microbiological laboratory, other apparatus and materials are as follows:
2.1 Constant temperature incubator: 36℃±1℃.
2.2 Refrigerator: 2℃~5℃.
2.3 Thermostatic water bath: 46℃±1℃.
2.4 Balance: with sensibility of 0.1g, 0.01g.
2.5 Homogenizer.
2.6 Microscope: 10x~100x.
2.7 Aseptic pipette: 1mL (with scale of 0.01mL), 10mL (with scale of 0.1mL) or pipettor and pipette tip.
2.8 Aseptic homogenizing cup or aseptic homogenizing bag: with volume of 500mL.
2.9 Aseptic culture dish: with diameter of 90mm.
2.10 pH meter or precision pH paper.
2.11 Ultraviolet lamp with long wave: 365nm, power ≤ 6W.
2.12 Microcentrifuge tube: 1.5mL or 2.0mL.
2.13 Magnetic sheet, magnetic sheet frame, sample mixer.
2.14 Microbial identification system.
3 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 Triple sugar iron agar (TSI): see A.3.
3.4 Nutrient agar: see A.4.
3.5 Semi-solid agar: see A.5.
3.6 Lauryl sulfate tryptone 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 washing liquid: see A.9.
3.10 Potassium tellurite(Grade AR).
3.11 Cefixime.
3.12 Chromogenic medium of Escherichia coli O157.
3.13 Diagnostic serum of Escherichia coli O157 and H7 or latex agglutination reagent of O157.
3.14 Identification reagent kit.
3.15 Anti-E.coli O157 immunomagnetic beads.
Method I Routine Culture Method
4 Examination Procedures
See Figure 1 for the examination procedures of Escherichia coli O157:H7/NM with routine culture method.
Figure 1 Examination Procedures for Escherichia Coli O157:H7/NM by Routine Culture Method
5 Operation Steps
5.1 Enrichment
Take 25g (or 25mL) of examined sample by aseptic technique, add it to the homogenizing bag containing 225mL of mEC+n broth, continuously homogenize them on the slap type homogenizer for 1min~2 min; or put the examined sample into the homogenizing cup containing 225mL of mEC+n broth to homogenize at 8000r/min~10000r/min for 1min~2 min. Culture it at 36℃±1℃ for 18h~24h.
5.2 Isolation
Take enrichened mEC+n broth to undergo streak inoculation on the CT-SMAC plate and Escherichia coli O157 chromogenic agar plate, culture it at 36℃±1℃ for 18h~24h, and observe the colony shape. Typical colony on the CT-SMAC plate is round, smooth, small and colorless, with darker taupe in the center; for the colony on Escherichia coli O157 chromogenic agar plate, its characteristics are judged according to product instructions.
5.3 Preliminary biochemical test
Respectively pick 5~10 suspicious colonies from CT-SMAC plate and Escherichia coli O157 chromogenic agar plate to inoculate TSI agar and MUG-LST broth simultaneously, and adopt Escherichia coli strain (ATCC 25922 or equivalent standard strain) to carry out positive control and adopt Escherichia coli O157:H7 (NCTC 12900 or equivalent standard strain) to carry out negative control, and then culture them at 36℃±1℃ for 18h~24h. If necessary, conduct oxidase test and gram stain. In TSI agar, typical strain presents yellow inclined plane and bottom layer, aerogenesis or an-aerogenesis, no hydrogen sulfide (H2S) generated. Observe the MUG-LST broth tube under long-wave ultraviolet light, the MUG-positive Escherichia coli strain - shall generate fluorescence, while MUG-negative strain shall be free from fluorescence, and the Escherichia coli O157:H7/NM is MUG-negative test without fluorescence. Pick suspected colony to clone on nutrient agar plate, culture it at 36℃±1℃ for 18h~24h, and carry out the following identifications.
5.4 Identification
5.4.1 Serological test
Pick cloned colony from nutrient agar plate to carry out slide agglutination test with O157 and H7 diagnostic serums or O157 latex agglutination reagent. For H7 factor with inagglutinable serum, it shall be stabbed to inoculate semi-solid agar, inspect power, and carry out serial passage for three times, if all the power tests are negative, it is determined as non-powered strain. If diagnostic serums or latex agglutination reagents from different companies are adopted, the test shall be conducted according to the product instructions.
5.4.2 Biochemical test
5.4.2.1 Pick colony from nutrient agar plate to carry out biochemical test. See Table 1 for the characteristics of biochemical reaction of Escherichia coli O157:H7/NM.
Table 1 Characteristics of Biochemical Reaction of Escherichia Coli O157:H7/NM
Biochemical test Characteristic reaction
Triple sugar iron agar Yellow bottom layer and inclined plane, negative H2S
Sorbitol Negative or late fermentation
Indole Positive
Methyl red-voges proskauer test (MR- VP) Positive MR, negative VP
Oxidase Negative
Simmons Citrate Negative
Lysine decarboxylase Positive (purple)
Ornithine decarboxylase Positive (purple)
Cellobiose fermentation Negative
Raffinose fermentation Positive
MUG test Negative (without fluorescence)
Power test With power or without power
5.4.2.2 If biochemical identification kit or microbial identification system is adopted, the colony shall be picked from nutrient agar plate to prepare with diluent to bacterial suspension of appropriate turbidity, and then identify by biochemical identification kit or microbial identification system.
5.4.3 Virulence gene determination (optional)
Where the Escherichia coli O157:H7 or O157:NM is detected in sample, if further examination is required to examine Vero cell toxin gene, Vero cell or HeLa cell may be inoculated to judge via observing cytopathic effect; besides, gene probe detecting or polymerase chain reaction (PCR) may also be adopted to detect such genes as Shiga toxin gene (stx1, stx2), eae and hly. If the above genes are detected by reagent kit, the product instructions shall apply.
6 Result Report
According to the results of biochemical and serological test, report whether Escherichia coli O157:H7 or Escherichia coli O157:NM is detected or not in 25g (or 25mL) of sample.
Contents of GB 4789.36-2016
Foreword I
1 Scope
2 Apparatus and Materials
3 Media and Reagents
4 Examination Procedures
5 Operation Steps
6 Result Report
7 Examination Procedures
8 Operation Steps
9 Result Report
Appendix A Media and Reagents
