GB 4789.13-2012 PDF EnglishUS$210.00 · In stock · Download in 9 seconds
GB 4789.13-2012: National food safety standard - Microbiological examination of food - Clostridium perfringens inspection Delivery: 9 seconds. True-PDF full-copy in English & invoice will be downloaded + auto-delivered via email. See step-by-step procedure Status: Valid GB 4789.13: Historical versions
Similar standardsGB 4789.13-2012: National food safety standard - Microbiological examination of food - Clostridium perfringens inspection---This is an excerpt. Full copy of true-PDF in English version (including equations, symbols, images, flow-chart, tables, and figures etc.), auto-downloaded/delivered in 9 seconds, can be purchased online: https://www.ChineseStandard.net/PDF.aspx/GB4789.13-2012 NATIONAL STANDARD OF THE PEOPLE’S REPUBLIC OF CHINA National food safety standard - Food microbiological examination -Examination of clostridium perfringens Issued on. MAY 17, 2012 Implemented on. JULY 17, 2012 Issued by. Ministry of Health of PRC Table of ContentsForeword... 3 1 Scope... 4 2 Equipment and materials... 4 3 Media and reagents... 4 4 Inspection procedures... 5 5 Operation steps... 6 6 Results and reports... 7 Appendix A Medium and reagents... 9ForewordThis standard replaces GB/T 4789.13-2003 "Microbiological examination of food hygiene - Examination of Clostridium perfringens". Compared with GB/T 4789.13-2003, the main changes of this standard are as follows. - MODIFY the Chinese name of standard; - MODIFY the sample preparation process; - MODIFY the culture medium and reagents; - MODIFY the operation steps; - MODIFY the calculation of bacterial count; - ADD the Appendix A. National food safety standard - Food microbiological examination -Examination of clostridium perfringens1 ScopeThis standard specifies the inspection methods for Clostridium perfringens in food. This standard applies to the inspection of Clostridium perfringens in food.2 Equipment and materialsIn addition to the routine sterilization and culture equipment in the microbiology laboratory, other equipment and materials are as follows. a) Constant temperature incubator. 36 °C ± 1 °C; b) Refrigerator. 2 °C ~ 5 °C; c) Constant temperature water bath. 50 °C ± 1 °C, 46 °C ± 0.5 °C; d) Balance. The sensitivity is 0.1 g; e) Homogenizer; f) Microscope. 10X ~ 100X;3 Media and reagents3.1 Tryptone-sulfite-cycloserine (TSC) agar. See A.1 in Appendix A. 3.2 Liquid thioglycolate medium (FTG). See A.2 in Appendix A. 3.3 Buffer kinetic-nitrate medium. See A.3 in Appendix A. 3.4 Lactose-gelatin medium. See A.4 in Appendix A. 3.7 Gram staining solution. See A.7 in Appendix A. 3.8 Nitrate reducing reagent. See A.8 in Appendix A. 3.9 Buffered glycerol-sodium chloride solution. See A.9 in Appendix A.4 Inspection proceduresThe test procedure for Clostridium perfringens is as shown in Figure 1.5 Operation steps5.1 Preparation of sample 5.1.1 The samples shall be tested as soon as possible, after collection. If they cannot be tested in time, they can be stored at 2 °C ~ 5 °C. If the test cannot be performed within 8 hours, weigh aseptically 25 g (mL) of sample. Add it into an equal amount of buffered glycerol-sodium chloride solution (add double volume for liquid sample). Store it as soon as possible, in a -60 °C low temperature refrigerator or with dry ice. 5.2 Cultivation 5.2.1 Pipette 1 mL of each dilution into a sterile petri dish. Make two parallels for each dilution. Pour 15 mL of TSC agar, which is cooled to 50 °C (can be kept such temperature, in a constant temperature water bath at 50 °C ± 1 °C) to each plate. Slowly rotate the plate, to mix the dilution and agar well. 5.2.2 After the above agar plates are solidified, add 10 mL of TSC agar, which is cooled to 50 °C (which can be kept in a constant temperature water bath at 50 °C ± 1 °C), to cover the surface of the plate evenly. 5.3 Confirmatory test 5.3.1 Randomly select 5 (if less than 5, select 5) black colonies from a single plate. Inoculate them into FTG medium, respectively, to incubate them at 36 °C ± 1 °C for 18 h ~ 24 h. 5.3.2 Smear with the above-mentioned culture medium. Observe the purity by Gram staining. Clostridium perfringens is a gram-positive stubby bacillus, which has sometimes visible spores. If the culture medium is not pure, it shall be streaked and inoculated on TSC agar plates for purification. 5.3.4 Use an inoculating loop (needle), to take the FTG culture medium, for puncture and inoculate the buffered kinetic-nitrate medium. Incubate it at 36 °C ± 1 °C, for 24 h. The growth of bacteria along the puncture line is examined under transmitted light, to determine the presence or absence of motility. 5.3.5 Use an inoculating loop (needle), to puncture the FTG culture medium and inoculate the lactose-gelatin medium. Incubate it at 36 °C ± 1 °C, for 24 h. Observe the results. If gas production is found and the medium turns from red to yellow, it indicates that lactose is fermented and acid is produced. The test tube is placed at about 5 °C for 1 h, to check the liquefaction of gelatin. If the medium is solid, incubate it for another 24 h at 36 °C ± 1 °C. Repeat the check for gelatin liquefaction. Clostridium perfringens can ferment lactose and liquefy gelatin.6 Results and reports6.1 Typical colony count Select a plate, which has a typical number of colonies, between 20 CFU and 200 CFU, to count the number of typical colonies. if. 6.2 Result calculation 6.1 The counting result is calculated according to formula (1). 6.3 Report According to the typical colony count of Clostridium perfringens on the TSC agar plate, make calculation according to the formula in 6.2;Appendix AMedium and reagents A.1 Tryptone-sulfite-cycloserine (TSC) agar A.1.2 D-cycloserine solution Dissolve 1 g of D-cycloserine, in 200 mL of distilled water. Sterilize it by membrane filtration. Store it at 4 °C for later use. A.1.3 Preparation method Heat and boil the basic ingredients, until they are completely dissolved. Adjust the pH. Divide it into 500 mL flasks, 250 mL per flask. Sterilize it by autoclaving, at 121 °C for 15 min. Keep it at 50 °C ± 1 °C, for later use. Before use, add 20 mL of D-cycloserine solution to every 250 mL of base solution. Mix well. Pour it on a plate. A.2 Liquid thioglycolate medium (FTG) A.2.2 Preparation method Heat and boil the above ingredients, until they are completely dissolved. Adjust the pH after cooling. Divide it into test tubes, 10 mL per tube. Sterilize it, by autoclaving at 121 °C for 15 min. Before use, boil or heat with flowing steam for 15 min. Quickly cool it to the inoculation temperature. A.3 Buffer kinetic-nitrate medium A.3.1 Composition Peptone. 5.0 g Beef powder. 3.0 g Potassium nitrate. 5.0 g Disodium hydrogen phosphate. 2.5 g Galactose. 5.0 g Glycerin. 5.0 mL Agar. 3.0 g Distilled water. 1000.0 mL pH 7.3 ± 0.2 A.3.2 Preparation method Heat and boil the above ingredients, until they are completely dissolved. Adjust the pH. Divide it into test tubes, 10 mL per tube. Sterilize it, by autoclaving at 121 °C for 15 min. If not in use on the same day, refrigerate it at about 4 °C. Before use, boil or heat with flowing steam for 15 min. Quickly cool to the inoculation temperature. A.4 Lactose-gelatin medium A.4.1 Composition Peptone. 15.0 g Yeast powder. 10.0 g Lactose. 10.0 g Phenol red. 0.05 g Gelatin. 120.0 g Distilled water. 1000.0 mL pH. 7.5 ± 0.2 A.4.2 Preparation method Heat to dissolve peptone, yeast powder, gelatin in 1000 mL of distilled water. Adjust pH. Add lactose and phenol red. Divide it into test tubes, 10 mL per tube. Sterilize it by autoclaving at 121 °C for 10 min. If not in use on the same day, refrigerate it at about 4 °C. Before use, boil or heat with flowing steam for 15 min. Quickly cool to the inoculation temperature. A.5 Iron-containing milk medium A.8 Nitrate reducing reagent A.8.1 Solution A (p-aminobenzenesulfonic acid solution) Dissolve 8 g of p-aminobenzenesulfonic acid, in 1000 mL of 5 mol/L acetic acid. A.8.2 Solution B (α-naphthol acetic acid solution) Dissolve 5 g of α-naphthol, in 1000 mL of 5 mol/L acetic acid. A.9 Buffered glycerol-sodium chloride solution A.9.1 Composition Glycerin. 100.0 mL Sodium chloride. 4.2 g Dipotassium hydrogen phosphate (anhydrous). 12.4 g Potassium dihydrogen phosphate (anhydrous). 4.0 g Distilled water. 900.0 mL pH.7.2 ± 0.1 A.9.2 Preparation method Heat the above components, to complete dissolution. Adjust pH. Autoclave it at 121 °C for 15 min. When preparing a double-buffered glycerol solution, use 200 mL of glycerol and 800 mL of distilled water. ......Source: Above contents are excerpted from the full-copy PDF -- translated/reviewed by: www.ChineseStandard.net / Wayne Zheng et al. 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