NATIONAL STANDARD OF THE PEOPLE S REPUBLIC OF CHINA. National Food Safety Standard

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1 GB NATIONAL STANDARD OF THE PEOPLE S REPUBLIC OF CHINA GB National Food Safety Standard Microbiological Examination of Food Hygiene - Enumeration of Escherichia Coli 食品安全国家标准食品微生物学检验大肠埃希氏菌计数 GB How to BUY & immediately GET a full-copy of this standard? Search --> Add to Cart --> Checkout (3-steps); 3. No action is required - Full-copy of this standard will be automatically & immediately delivered to your address in 0~60 minutes. 4. Support: Wayne, Sales manager Issued on: May 17, 2012 Implemented on: July 17, 2012 Issued by the Ministry of Health of the People's Republic of China

2 Contents 1 Scope Terms and Definitions Equipment and Materials Culture Medium and Reagent MPN for Counting Escherichia Coli (Method I) Plate Count Method for Escherichia Coli (Method II)... 5 Appendix A Culture Medium and Reagent... 7 Appendix B Most Probable Number (MPN) Search List of Escherichia Coli... 13

3 National Food Safety Standard Microbiological Examination of Food Hygiene - Enumeration of Escherichia Coli 1 Scope The standard specifies the counting method of Escherichia coli in food. This standard is applicable to the counting of Escherichia coli in food. Among them, plate count method for Escherichia coli (Method II) is not applicable to shellfish products. 2 Terms and Definitions 2.1 Escherichia coli Escherichia coli It extensively exists in the intestinal tract of humans and warm-blooded animals and is able to ferment lactose and generate acid and gas at That it is presented as or Gram negative bacilli in IMViC biochemical test (indole, methyl red, VP test and citrate) is taken as fecal pollution index to assess the hygienic condition of food and deduce the possibility of enteropathogenic bacteria pollution in food. 2.2 Most probable number It is an indirect counting method based on poisson distribution and referred to as MPN. 3 Equipment and Materials In addition to the conventional sterilization and cultivation equipment in microbiological laboratory, other equipment and materials needed are as follows: a) Thermostatic incubator: 36 ±1 ; b) Refrigerator: 2 ~5 ; c) Thermostatic water bath: 44.5 ±0.2 ; d) Balance: sensibility 0.1g; c) Homogenizer; d) Oscillator; e) Aseptic pipette: 1mL (with 0.01mL scale), 10mL (with 0.1mL scale) or micropipettor and sucker head; 1

4 f) Aseptic conical flask: capacity, 500 ml; g) Sterile Petri dish: 90 mm in diameter; h) PH meter or ph colorimetric tube or precise ph paper; i) Colony counter; j) Ultraviolet lamp: wavelength 360nm ~ 366nm; power 6W. 4 Culture Medium and Reagent 4.1 Lauryl sulfate tryptone (LST) broth: see A.1 in Appendix A. 4.2 EC broth (E.coli broth): see A.2 of Appendix A. 4.3 Peptone water: see A.3 of Appendix A. 4.4 Buffer glucose peptone water [used in methyl red (MR) and V-P tests]: see A.4 of Appendix A. 4.5 Simmons citrate culture medium: see A.5 of Appendix A. 4.6 Phosphate buffer: see A.6 of Appendix A. 4.7 Eosin methylene blue (EMB) agar: see A.7 of Appendix A. 4.8 Nutrient agar slant: see A.8 of Appendix A. 4.9 Violet red bile agar (VRBA): see A.9 of Appendix A Violet red bile agar-4-methyl umbellulone-β-d glucoside (VRBA-MUG): see A.10 of Appendix A Gram staining solution: see A.11 of Appendix A Kovacs indole reagent: see A.12 of Appendix A Aseptic 1mol/L NaOH: see A.13 of Appendix A Aseptic 1mol/L HCl: see A.14 of Appendix A. 5 MPN for Counting Escherichia Coli (Method I) 5.1 Test procedures Test procedures of MPN for counting Escherichia coli are illustrated in Figure 1. 2

5 in aseptic homogenizing bag filled with 225mL phosphate buffer and pat for 1min ~ 2min with patting-type homogenizer to prepare 1:10 sample homogeneous solution Liquid sample: pipet 25mL sample by aseptic pipette; inject it in aseptic conical flask (proper quantity of aseptic glass bead is preset within) filled with 225mL phosphate buffer and mix uniformly to prepare 1:10 sample homogeneous solution The ph value of the sample homogeneous solution shall be between 6.5 and 7.5; if necessary, the sample homogeneous solution shall be adjusted by NaOH (1 mol/l) or HCl (1 mol/l) respectively Pipette 1mL of 1:10 sample homogeneous solution with the 1mL aseptic pipette or the micropipettor and inject it slowly along the tube wall into the aseptic test tube containing 9 ml phosphate buffer (the sucker or the sucker head tip shall not touch the diluent), mix the solution by shaking the test tube or beating upon the tube repeatedly with another 1 ml aseptic pipette or sucker head, and finally prepare the 1:100 sample homogeneous solution According to the estimation for the sample contamination condition, prepare the sample homogeneous solution with dilution increasing successively until 10 times basing on the above-described operations. Change one 1 ml aseptic pipette or sucker head each time the solution is diluted. The entire process shall not exceed 15 min from the preparation of the sample homogeneous solution to the finishing of sample inoculation Primary fermentation test Choose 3 kinds of sample homogeneous solution with appropriate continuous dilutability (the original solution may be chosen for the liquid sample) for each sample. Inoculate three tubes of LST brouth for each dilutability, with 1 ml inoculation in each tube (e.g. if the inoculation exceeds 1 ml, then the double-material LST broth shall be applied); cultivate the LST broth for 24h±2h at 36 ± 1 and observe the small inverted tube; if bubbles are generated in 24h±2h, fermentation test shall be performed again; if not, then the LST broth shall be cultivated for another 48 h±2 h. If bubbles are generated, then fermentation test shall be carried out again. If no bubble is observed in all LST broth tubes, report the MPN result of Escherichia coli Secondary fermentation test Take one loop of culture from each aerogenic LST broth tube with inoculating loop to transfer in EC broth tube pre-heated to 45 ; place in the lid-provided water bath with a temperature of 44.5 ±0.2. The water surface of the bath shall be higher than the solution level of the broth culture medium; cultivate for 24h±2h and observe whether bubbles are generated in the small inverted tube; if not continue to cultivate to 48±2h. Record the number of EC broth tubes which generate bubbles in 24h and 48h. If none of the EC broth tube generate bubble, MPN result of Escherichia coli may be reported; any tube generates bubbles shall be conducted with EMB plate isolated culture Eosin methylene blue plate isolated culture Gently shake the aerogenic tube; take the culture with inoculating loop to steak-inoculate it in EMB plate respectively and cultivate for 18h~24h at 36 ±1. Observe whether lustrous or luster-lacking typical colony with black center exists in the plate Nutrient agar slant or plate culture Pick 5 typical colonies from each plate or pick questionable one in case of no typical colony existing. Touch the center of colony by inoculating needle and subculture on nutrient 4

6 Polypeptone 7.0g Glucose 5.0g Dipotassium hydrogen phosphate (K 2 HPO 4 ) 5.0g mL ph7.0 A.4.2 Preparation Dissolve the above-mentioned compositions in distilled water and adjust ph. Sub-inject 1mL in each test tube; autoclave for 15min at 121 for use. A.4.3 Methyl red (MR) test A Methyl red reagent A Composition Methyl red 10mg 95% ethanol 30.0mL 20.0mL A Preparation Dissolve 10mg of methyl red in 30mL of 95% ethanol and then add 20mL of distilled water. A Test method Inoculate proper quantity of agar culture in buffer glucose peptone water and cultivate for 2d ~ 5d at 36 ±1. Add a drop of methyl red reagent, and immediately observe the result. Cardinal red represents positive and yellow represents negative. A.4.4 V-P test A % α-naphthol-ethanol solution Composition and preparation: take 6.0g of α-naphthol; add absolute ethyl alcohol to dissolve and scale the volume to 100mL. A % potassium hydroxide solution Composition and preparation: take 40g of potassium hydroxide; add distilled water to dissolve and scale the volume to 100mL. A Test method Inoculate proper quantity of agar culture in buffer glucose peptone water and cultivate for 2d ~ 4d at 36 ±1. Add 0.5mL of 6% α-naphthol-ethanol solution and 0.2mL of 40% potassium hydroxide solution, sufficiently shake test tube, and observe the result. For positive reaction, red will appear immediately or within a few minutes; if it is negative, cultivate for another 4h at 36 ±1, and then observe. A.5 Simmons citrate culture medium A.5.1 Composition Sodium citrate 2.0g Sodium chloride 5.0g Dipotassium hydrogen phosphate 1.0g Ammonium dihydrogen phosphate 1.0g Magnesium sulfate 0.2g Bromine thymol blue 0.08g Agar 8.0g~18.0g mL 8

7 A Composition Iodine 1.0g Potassium iodide 2.0g 300.0mL A Preparation Firstly mix the iodine with potassium iodide, add a small amount of distilled water into the mixture and shake sufficiently, wait till the mixture dissolve completely, and add distilled water till 300mL. A.11.3 Safranin counterstain A Composition Safranin 0.25g 95% ethanol 10.0mL 90.0mL A Preparation Dissolve safranin in ethanol and dilute with distilled water. A.11.4 Stainning method A Fix the smear over the flame, add crystal violet staining reagent by drops, stain for 1min and wash the smear with water. A Drop gram iodine solution to react for 1min then wash with water. A Add 95% ethanol by drops to fade for about 15s~30s till the staining solution is washed off; don't fade excessively and finally wash with water. A Drop counterstain to redye for 1min; wash, dry and conduct microscopic examination. A.12 Kovacs indole reagent A.12.1 Composition Paradime thylaminobenzaldehyde 5.0g Pentanol 75.0mL Hydrochloric acid (concentrated) 25.0mL A.12.2 Preparation Dissolve paradime thylaminobenzaldehyde in pentanol and then slowly add concentrated hydrochloric acid. A.12.3 Test method Inoculate culture in peptone water and cultivate for 24h± 2h at 36 ±1 ; add 0.2mL ~ 0.3mL of Kovacs indole reagent. That red is observed on the upper layer is a representation of indole positive reaction. A.13 Aseptic 1mol/L NaOH: A.13.1 Composition NaOH 40.0g mL A.13.2 Preparation: Dissolve 40g of sodium hydroxide in 1000mL of distilled water and autoclave for 15min at 121. A.14 Aseptic 1mol/L HCl: A.14.1 Composition 11

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