This standard specifies the test method for Clostridium perfringens. This standard is applicable to the test for Clostridium perfringens in various types of food and food poisoning samples. GB/T 4789.13-2003 Food Hygiene Microbiology Test Clostridium perfringens Test GB/T4789.13-2003 Standard download decompression password: www.bzxz.net

ICS07.100.30
National Standard of the People's Republic of China
GB/T4789.13—2003
Replaces GB/T4789.13-1994
Microbiological examination of food hygiene—Examination of Clostridium perfringensbzxZ.net
Microbiological examination of food hygiene—Examination of Clostridium perfringensPromulgated on August 11, 2003
Ministry of Health of the People's Republic of China
Standardization Administration of the People's Republic of China
Implementation on January 1, 2004
GB/T4789.13—2003
This standard revise GB/T4789.13--1994 "Microbiological examination of food hygiene—Examination of Clostridium perfringens". Compared with GB/T4789.13--1994, this standard has the following major revisions: - The format and text of the standard text are revised in accordance with GB/T1.1-2000. - The "equipment and materials" in the original standard are revised and standardized. From the date of implementation of this standard, GB/T4789.13-1994 will be abolished at the same time. This standard is proposed and managed by the Ministry of Health of the People's Republic of China. The drafting units of this standard are: Lanzhou Institute of Biological Products of the Ministry of Health, Institute of Nutrition and Food Safety of Chinese Center for Disease Control and Prevention. The main drafters of this standard are: Wang Chenghuai, Ran Lu, Fu Ping, Yao Jinghui. This standard was first issued in 1984, revised for the first time in 1994, and this is the second revision. 90
1 Scope
Food Hygiene Microbiological Examination
Test for Clostridium perfringens
This standard specifies the test method for Clostridium perfringens. This standard is applicable to the test for Clostridium perfringens in various types of food and food poisoning samples. 2 Normative references
GB/T4789.13-2003
The clauses in the following documents become the clauses of this standard through reference in this standard. For all dated references, all subsequent amendments (excluding errata) or revisions are not applicable to this standard. However, parties to agreements based on this standard are encouraged to study whether the latest versions of these documents can be used. For all undated references, the latest versions are applicable to this standard. GB/T4789.28-2003 Food hygiene microbiological examination staining methods, culture media and reagents 3 Equipment and materials
3.1 Refrigerator: 0℃~4℃.
3.2 Constant temperature incubator: 36℃±1℃.
3.3 Constant temperature water bath: 46℃±1℃.
3.4 ​​Microscope: 10×～100×.
3.5 Homogenizer or sterile mortar.
3.6 Anaerobic culture device: room temperature catalytic deoxygenation type or alkaline pyro-petroleum acid deoxygenation type. 3.7 Sterile pipette: 1mL (with 0.01mL scale), 10mL (with 0.1mL scale). 3.8 Sterile test tube: 16mm×160mm.
3.9 Sterile blood: diameter 90mm.
3.10 Sterile conical flask: 500mL.
4 Culture media and reagents
4.1 Blister culture medium: 4.67 in GB/T4789.28-2003. 4.2 Sulfite-polymyxin-sulfadiazine agar (SPS): 4.69 in GB/T4789.28-2003. 4.3 Liquid thioglycollate culture medium (FT): 4.70 in GB/T4789.28-2003. 4.4 Power-nitrate culture medium: 4.72 in GB/T4789.28--2003. 4.5 Iron-containing milk culture medium Culture medium: 4.71 in GB/T4789.28-2003. 4.6 Egg yolk agar medium: 4.68 in GB/T4789.28--2003. 4.7 0.1% protein aged water: 4.85 in GB/T4789.28--2003. 4.8 Nitrate reducing agent: 3.17 in GB/T4789.28--2003. 4.9 Gram staining solution: 2.25 in GB/T4789.28-2003. Test procedure
The test procedure for Clostridium perfringens is shown in Figure 1.
GB/T4789.13—2003
Microscopic morphology
6 Operating steps
6.1 Live bacteria count culture
Dilution
Agar mixture
Injection
25g (mL) test sample + 225mL 0.1% protein aging agent Homogenize, centrifuge, dilute
Dilution
Agar mixture
Pour
Dilution
Agar mixture
Injection
Dilution
Agar mixture
Pour
Anaerobic culture, 36℃±1℃, 24h, black colony count Pick 10 black colonies at random and inoculate FT medium respectively Confirmation test
Nitrate reduction
Counting of bacterial count
Dilution
Agar mixture
Pour
Milk fermentation
Lecithin decomposition
6.1.1 According to aseptic operation, weigh 25g (mL) of food test sample and put it into homogenizer, add 225mL of 0.1% protein water, stir at low speed for 1min~2min to homogenize it, and make it as 1:10 dilution. 6.1.2 Add 9mL of 0.1% protein water to 1mL of the above 1:10 diluted homogenate to make a series of dilutions of 10-2~10-. 6.1.3 Take 1ml of each dilution and put it into two sterile plates respectively. Pour 15mL~20mL of SPS agar at about 50℃ on each plate, and carefully turn the plate to mix the diluent and agar thoroughly. 6.1.4 After the above agar plate solidifies, place it upside down in an anaerobic culture device and culture it at 36℃±1℃ for 24h. 6.1.5 Select a plate with 30~300 black colonies and count the number of black colonies. 92
6.2 Confirmation test
GB/T4789.13—2003
6.2.1 Take 10 black colonies from the plate at random, and culture them at 36℃±1℃ for 18h~24h according to the FT culture medium. 6.2.2 Use the above culture solution to smear and examine under Gram staining microscope. Clostridium perfringens is a Gram-positive large rod. Its heat-resistant strain may form oval spores, which are located in the center or proximal end of the bacteria and are generally not wider than the bacteria. 6.2.3 Use an inoculation loop (needle) to puncture the motility-nitrate culture medium, and culture it at 36℃±1℃ for 24h. Observe the growth of the inoculation line to determine whether it has motility. Then, add 0.5mL of naphthylamine solution and p-aminobenzenesulfonic acid solution respectively to observe whether nitrate is reduced. Clostridium perfringens has no motility and can reduce nitrate to nitrite. 6.2.4 Take 1mL of the vigorously growing FT culture medium and inoculate it into the iron-containing milk culture medium. After culturing it in a 46℃ water bath for 2h, observe whether there is "violent fermentation". Those that do not ferment within 5h are negative. Clostridium perfringens ferments lactose, coagulates casein and produces a lot of gas, showing "violent fermentation" phenomenon, but the culture medium does not turn black.
6.2.5 Use an inoculation loop to take the FT culture medium and inoculate it on the egg yolk agar plate (at least 10 points can be inoculated on each plate), and incubate it anaerobically at 36℃±1℃ for 24h, and observe the changes in the inoculation points. Clostridium perfringens produces lecithinase, which decomposes the lecithin in the egg yolk, forming a milky white turbid band at the bottom and around the inoculation point.
6.3 Calculation of bacterial count
Calculate the number of bacteria per gram (ml) of food sample based on the count of black colonies (6.1.5) and the results of the confirmation test (6.2). For example, if there are 100 black colonies on the plate of the 10 dilution solution, and 7 of the 10 colonies used for the confirmation test are confirmed to be Clostridium perfringens, then the number of Clostridium perfringens per gram (ml) of food sample is: 100×0.7×104=7×10
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