National Food Safety Standard Microbiological Examination of Food Hygiene - Examination of Shigella 食品安全国家标准食品微生物学检验志贺氏菌检验

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1 Translated English of Chinese Standard: GB Translated by: Wayne Zheng et al. NATIONAL STANDARD GB OF THE PEOPLE S REPUBLIC OF CHINA GB National Food Safety Standard Microbiological Examination of Food Hygiene - Examination of Shigella 食品安全国家标准食品微生物学检验志贺氏菌检验 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: Ministry of Health of the People s Republic of China Page 1 of 23

2 Tips - GB 4789 Series (Not part of this Standard) Standard ID GB GB Standard Name General guidelines Aerobic plate count Issued Date Enforced Date New Version (Click to check)? GB GB GB GB/T GB/T GB/T GB/T GB GB/T GB/T Enumeration of coliforms Salmonella National Food Safety Standard Microbiological Examination of Food Hygiene - Examination of Shigella diarrheogenic Escherichia coli Microbiological examination of food hygiene. Examination of Vibrio parahaemolyticus Microbiological examination of food hygiene.examination of Yersinia enterocolitica Microbiological examination of food hygiene.examination of Campylobacter jejuni Staphylococcus aureus streptococcus hemolyticus Clostridium botulinum and botulinus toxin Page 2 of 23

3 GB GB/T GB National Food Safety Standard Microbiological Examination of Food Hygiene - Examination of Clostridium Perfringens Bacillus cereus Enumeration of moulds and yeasts GB/T GB/T GB GB/T GB/T GB/T GB/T GB/T GB/T GB/T Microbiological examination of food hygiene Identification of common mycotoxin producing fungi meat and meat products Milk and milk products egg and egg products aquatic product foods frozen drinks and cold drinks flavourings cold dish and bean products candy, cake and preserved fruits wines Page 3 of 23

4 GB/T commercial sterilization of canned food GB/T Microbiological examination of food hygiene.examination of residue of antibiotics in fresh milk GB/T Microbiological examination of food hygiene Staining methods, culture mediums and reagents GB/T pseudomonas cocovenenans subsp. farinofermentans GB Listeria monocytogenes GB/T GB/T GB/T GB salmonellae, shigellae, and diarrhoea causative Escherichia coli by means of the diagnostic typing phage set for enterobacteriaceae Microbiological examination for food hygiene rapid detection of coliform bacteria cereal, fruit and vegetable National Food Safety Standard Microbiological Examination of Food Hygiene - Examination of Bifidobacterium GB Lactic acid bacteria GB/T Microbiological examination of food hygiene.examination of Escherichia coli O157: H7/NM Page 4 of 23

5 GB/T Microbiological examination of food hygiene.examination of Staphylococcus aureus GB GB/T GB National Food Safety Standard Microbiological Examination of Food Hygiene - Enumeration of Escherichia Coli Microbiological examination of food hygiene. Enumeration of faecal coliforms Enterobacter sakazakii Page 5 of 23

6 Table of Contents Foreword Scope Devices and Materials Culture Medium and Reagents Examination Procedures Operational Procedures Appendix A Page 6 of 23

7 Foreword This Standard replaces GB/T "Microbiological Examination of Food Hygiene - Examination of Shigella". Compared with GB/T , main changes in this Standard are as follows: - The standard name was modified; - Culture mediums and reagents were modified; - Enrichment part, biochemical test and additional biochemical test part in the operational procedures were modified; - Table 2 was modified; - Table 4 was modified. Page 7 of 23

8 National Food Safety Standard Microbiological Examination of Food Hygiene - Examination of Shigella 1 Scope This Standard specifies the methods to examine the Shigella in foods. This Standard is applicable to the examination of Shigella in foods. 2 Devices and Materials In addition to the conventional sterilization and cultivation devices in the microbiological laboratory, other devices and materials needed for the examination are as follows: a) Thermostatic incubator: 36ºC±1ºC; b) Refrigerator: 2ºC~5ºC; c) Membrane filter system; d) Anaerobic cultivation device: 41.5ºC±1ºC; e) Electronic balance: sensibility of 0.1g; f) Microscope: 10x~100x; g) Homogenizer; h) Oscillator; i) Sterile pipette: 1mL (with 0.01mL scale), 10mL (with 0.1mL scale) or micro-pipettor and sucker head; j) Aseptic homogenizing cup or aseptic homogenizing bag: capacity of 500mL; k) Sterile culture dish: diameter of 90 mm; l) PH meter or ph colorimetric tube or precise ph test paper; m) Full automatic microorganism biochemical identification system. 3 Culture Medium and Reagents 3.1 Enriched Shigella broth - novobiocin: see A.1 in Appendix A. 3.2 MAC agar: see A.2 in Appendix A. 3.3 XLD agar: see A.3 in Appendix A. 3.4 Shigella chromogenic medium. 3.5 TSI agar: see A.4 in Appendix A. 3.6 Nutrient agar slant: see A.5 in Appendix A. 3.7 Semi-solid agar: see A.6 in Appendix A. 3.8 Ammonium dextrose medium: see A.7 in Appendix A. 3.9 Urea agar: see A.8 in Appendix A. Page 8 of 23

9 Result of Shigella grouping and typing Figure 1 Shigella examination procedures 5 Operational Procedures 5.1 Enrichment Take 25g (ml) of examined sample by sterile operation, add it into a homogenizing cup containing 225mL of sterile enriched Shigella broth, homogenize it with a spinning-blade homogenizer at 8000r/min~10000r/min; or add it into a homogenizing bag containing 225mL of enriched Shigella broth, continuously homogenize it with a slapping homogenizer for 1min~2min, liquid sample shall be mixed and oscillated uniformly. Cultivate the examined sample in an anaerobic condition at 41.5ºC±1ºC for 16h~20h. 5.2 Separation Take the enriched Shigella enrichment liquid, and inoculate it on XLD agar plate and MAC agar plate or Shigella chromogenic medium plate respectively. Cultivate it at 36ºC±1ºC for 20h~24h, observe the morphology of colonies growing on each plate. The diameter of single Shigella sonnei colony shall be larger than other Shigella's. If the appeared colonies are not typical or the colonies are too small to observe easily, then continue to cultivate for 48h before observation. See Table 1 for the colony characteristics of Shigella on different selective agar plates. Table 1 Colony Characteristics of Shigella on Different Selective Agar Plates Selective agar plate MAC agar XLD agar Shigella chromogenic medium Colony characteristics of Shigella Colorless to light-pink, semi-transparent, smooth, wet, round, regular or irregular edges Pink to colorless, semi-transparent, smooth, wet, round, regular or irregular edges Judgment is carried out according to the description of chromogenic medium 5.3 Preliminary biochemical test Pick up more than 2 typical or questionable bacterial colonies from the selective agar plate, inoculate them on a tube of TSI, semi-solid and nutrient agar slant respectively, cultivate them at 36ºC±1ºC for 20h~24h, observe the results respectively. Page 10 of 23

10 3b III (3,4),6 (+) + - 4a Ⅳ 3, b Ⅳ c Ⅳ 7, a V (3,4) (+) - - 5b V 7, VI X - 7, Y - 3, Note: + represents agglutination; - represents no agglutination; () represents either one. 5.6 Result report Summarize above biochemical test and serological identification results, report if Shigella is detected in the 25g (ml) of samples. Page 14 of 23

11 Appendix A Culture Medium and Reagents A.1 Enriched Shigella broth - novobiocin (Shigella broth) A.1.1 Enriched Shigella broth A Ingredients Tryptone 20.0g Glucose 1.0g Dipotassium hydrogen phosphate 2.0g Potassium dihydrogen phosphate 2.0g Sodium chloride 5.0g Tween mL Distilled water mL A Preparation Mix above ingredients and dissolve them by heating; after the mixture is cooled to around 25ºC, calibrate the ph to 7.0±0.2, dispense them into appropriate containers, sterilize the mixture at 121ºC for 15min. Take them out and cool them to 50ºC~55ºC, add sterilized and filtered novobiocin solution (0.5μg/mL), and dispense them into 225mL for use. Note: If the broth is not used immediately, it may be stored at 2ºC~8ºC for a month. A.1.2 Novobiocin solution A Ingredients Novobiocin 25.0mg Distilled water mL A Preparation Dissolve the novobiocin into the distilled water, remove the bacteria with 0.22μm membrane filter; if the solution is not used immediately, it may be stored at 2ºC~8ºC for a month. A.1.3 5mL of novobiocin solution (A.1.2) is added in per 225mL of enriched Shigella broth (A.1.1) and mixed uniformly before use. A.2 MAC agar A.2.1 Ingredients Peptone 20.0g Lactose 10.0g No.3 bile salt 1.5g Sodium chloride 5.0g Neutral red 0.03g Page 15 of 23

12 Ammonium dihydrogen phosphate 1.0g Dipotassium hydrogen phosphate 1.0g Sodium citrate 5.0g Agar 20g 0.2% bromothymol blue solution 40.0mL Distilled water mL A.12.2 Preparation Firstly dissolve the salts in the water, calibrate ph to 6.8±0.2, add agar and heat them to dissolve. And then add indicator, mix them to uniform and dispense into test tubes, sterilize them at 121ºC for 15min. Make the solution to be slant for use. A.12.3 Test methods Pick up a small amount of agar culture to inoculate, cultivate the culture at 36ºC±1ºC for 4d, observe the result everyday. For positive ones, there are colonies growing on the slant, the medium turns from green to blue. A.13 Mucus acid salt medium A.13.1 Test broth A Ingredients Casein peptone 10.0g Bromothymol blue solution 0.024g Distilled water mL Mucic acid 10.0g A Preparation Gradually add 5mol/L sodium hydroxide to dissolve the mucic acid, mix them to uniform. Heat the rest ingredients to dissolve, add above-mentioned mucic acid, cool them to around 25ºC, calibrate ph to 7.4±0.2, dispense the solution into test tubes, 5mL for each tube, sterilize them under high-pressure at 121ºC for 10min. A.13.2 Quality control broth A Ingredients Casein peptone 10.0g Bromothymol blue solution 0.024g Distilled water mL A Preparation Heat all ingredients to dissolve, cool them to around 25ºC, calibrate ph to 7.4±0.2, dispense the solution into test tubes, 5mL for each tube, sterilize them under high-pressure at 121ºC for 10min. Page 22 of 23

13 A.13.3 Test methods Inoculate the fresh culture to be tested into the test broth (A.13.1) and quality control broth (A.13.2) respectively, cultivate it them at 36ºC±1ºC for 48h, observe the result, if the blue color of broth does not change, the result is negative; if the broth turns to yellow or strawyellow, the result is positive. A.14 Peptone water and indole reagent A.14.1 Ingredients Peptone (or tryptone) 20.0g Sodium chloride 5.0g Distilled water mL ph7.4 A.14.2 Preparation Prepare with above-mentioned ingredients, dispense them in small test tubes, sterilize under high-pressure at 121ºC for 15min. Note: This reagent may be stored at 2ºC~8ºC for one month. A.14.3 Indole reagent A Kovacs reagent: dissolve 5g of paradime thylaminobenzaldehyde into 75mL of pentyl alcohol. Then slowly add 25mL of concentrated hydrochloric acid. A Ehrlich's reagent: dissolve 1g of paradime thylaminobenzaldehyde into 95mL of 95% ethanol. Then slowly add 20mL of concentrated hydrochloric acid. A.14.4 Test methods Pick up small amount of culture to inoculate, cultivate at 36ºC±1ºC for 1d~2d, cultivate for 4d~5d if necessary. Add about 0.5mL of Kovacs reagent, shake the test tube gently, for positive ones, the reagent layer will show carmine. Or add about 0.5mL of Ehrlich s reagent, flow it down along the tube wall, cover the culture solution s surface, for positive ones, the contacting area with the solution s surface will show rose-red. Note: Peptone contains abundant tryptophan. After the peptone is purchased, each batch of peptone shall be identified by known strain before use. This reagent may be stored at 2ºC~8ºC for one month. Page 23 of 23

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