National Food Safety Standard Microbiological Examination of Food Hygiene - Examination of Shigella

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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 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 examination: General guidelines examination: 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 examination: Enumeration of coliforms examination: 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 examination: 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 examination: 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 examination: 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 examination: 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 examination: 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 examination: 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 tests (cultivate at 36ºC for 24h~48h). See Table 3 for the biochemical characteristics difference between Shigella and the inactive Escherichia coli, and A-D bacteria. Table 3 Biochemical Characteristics Difference between Shigella, Inactive Escherichia Coli and A-D Bacteria Biochemical reaction Group A: Shigella dysenteriae Group B: Shigella flexneri Group C: Shigella boydii Group D: Shigella sonnei Escherichia coli A-D bacteria Ammonium dextrose Simon citrate d d Mucus acid salt d + d Note 1: + represents positive; - represents negative; d represents different biochemical types. Note 2: in the reactions of ammonium dextrose, Simon citrate and mucus acid salt tests, the Shigella is generally negative, and at least one item of inactive Escherichia coli and A-D (alkali-irregular) bacteria is positive If biochemical identification reagent kit or full automatic microorganism biochemical identification system is selected, it may based on the preliminary judgment result of 5.3.2, use the lawn which is growing on the nutrient agar slant and is cultivated in 5.3.1, and use the biochemical identification reagent kit or full automatic microorganism biochemical identification system to perform identification. 5.5 Serological identification Preparation of antigen Shigella has no power, therefore it has no flagellar antigen. The main antigen in the Shigella is thallus (O) antigen. Thallus (O) antigen may be subdivided to type and group specific antigen. Generally, it shall use 1.2%~1.5% agar culture as the antigen for slide agglutination test. Note 1: If some Shigellas do not have agglutination reaction due to the existence of K antigen, it may pick up lawn into 1mL of normal saline to make concentrated bacteria solution, boil it at 100ºC for 15min~60min to remove the K antigen for re-examination. Note 2: Group D Shigella may be smooth strain or rough strain, which does not have cross reaction with other Shigella group antigen. Different from enterobacteriaceae, the rough strain of Shigella sonnei may not necessarily have autoagglutination. Shigella sonnei does not have K antigen Agglutination reaction Draw out 2 areas of 1cm 2cm on the slide, pick up a loop of bacteria to be tested, place 1/2 loop on the upper part of each area respectively; add a drop of antiserum on the lower part of one area, add a drop of normal saline on the lower part of the other area for comparison. Grind the colonies in the two areas to emulsion respectively with a sterile inoculating loop or needle. Tilt the slide and shake for 1min, observe it against a black background; if particles which condense to blocks occur in the antiserum, and there is no autoagglutination occurs in the normal saline, the agglutination is positive. If agglutinate occurs in the normal Page 12 of 23

10 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