GB/T 4789.12-2003 Microbiological examination of food hygiene - Examination of Clostridium botulinum and botulinum toxin
Some standard content:
ICS07.100.30
National Standard of the People's Republic of China
GB/T4789.12—2003
Replaces GB/T4789.12—1994
Microbiological examination of food hygiene—Examination of Clostridium botulinum and botulinus toxin
Microbiological examination of food hygiene—Examination of Clostridium botulinum and botulinus toxinPromulgated on August 11, 2003
Ministry of Health of the People's Republic of China
National Standardization Administration of China
Implementation on January 1, 2004
GB/T4789.12—2003
This standard revise GB/T4789.12—1994 "Microbiological examination of food hygiene—Examination of Clostridium botulinum and botulinus toxin". Compared with GB/T4789.12-1994, this standard has the following major revisions: The standard text format and text are revised in accordance with GB/T1.1-2000. --- Modify and standardize the "equipment and materials" in the original standard. From the date of implementation of this standard, GB/T4789.12-1994 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 and Yao Jinghui. This standard was first issued in 1984, revised for the first time in 1994, and this is the second revision. 84
1 Scope
Food Hygiene Microbiological Examination
Clostridium botulinum and botulinum toxin examination
This standard specifies the test methods for Clostridium botulinum and botulinum toxin. This standard is applicable to the test of Clostridium botulinum and botulinum toxin in various types of food and food poisoning samples. 2 Normative references
GB/T 4789.12-2003
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 an agreement 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 microbiology inspection staining method, culture medium and reagents 3 Equipment and materials
3.1 Refrigerator: 0℃~4℃.
3.2 Constant temperature incubator: 30℃±1℃, 35℃±1℃36℃±1℃. 3.3 Centrifuge: 3000r/min.
3.4 Microscope: 10×~100×.
3.5 Phase contrast microscope.
3.6 Homogenizer or sterile mortar.
3.7 Scale plate pharmaceutical balance: 0g~500g, accurate to 0.5g. 3.8 Anaerobic culture device: room temperature catalytic deoxygenation type or alkaline pyro-mercapto acid deoxygenation type. 3.9 Sterile pipette: 1mL (with 0.01mL scale), 10mL (with 0.1mL scale). 3.10 Sterile plate: 90mm in diameter.
Sterile conical flask: 500mL.
Sterile syringe: 1mL.
3.13 White mouse: 12g~15g.
4. Culture medium and reagentsbzxz.net
4.1 Meat culture medium: 4.67 in GB/T4789.28-2003. 4.2 Egg yolk agar culture medium: 4.68 in GB/T4789.28-2003. 4.3 Gelatin phosphate buffer: 3.23 in GB/T4789.28-2003. 4.4 Botulinum typing antitoxin diagnostic serum.
4.5 Pancreatic enzyme: activity 1:250.
4.6 Gram staining solution: 2.25 Inspection procedure in GB/T4789.28-2003
See Figure 1 for the inspection procedure for Clostridium botulinum and botulinum toxin. 85
GB/T4789.12-2003
Toxin detection
Toxin sac detection
(Food)
Toxin detection
Cultivation for bacterial growth and toxin production
30℃, 5d
Culture inspection
Note 1: Report (I): The sample contains a certain type of botulinum toxin. Note 2: Report (II): The sample contains a certain type of Clostridium botulinum. Note 3: Report (III): The strain isolated from the sample is a certain type of Clostridium botulinum. Bacteria enrichment and toxin production culture
30℃, 5d
Observe the growth
characteristics, staining microscopy
Separation culture
Observe the characteristics of the colony,
staining microscopy
As shown above, the sample is homogenized and inoculated and cultured in time for bacteria enrichment and toxin production, and toxin detection test is performed at the same time. The results of the toxin detection test can prove whether there is botulinum toxin in the sample and what type of botulinum toxin exists. For the bacteria enrichment and toxin production culture, on the one hand, general growth characteristics are observed, and the production of botulinum toxin is detected at the same time. The results obtained can prove whether there is Clostridium botulinum in the sample and what type of Clostridium botulinum exists. For other special purposes, if you want to obtain pure strains, you can use the bacteria enrichment and toxin production culture for separation and culture, observe the morphology, culture characteristics, etc. of the obtained pure strains, and perform toxin detection. The results can prove what type of Clostridium botulinum the obtained pure bacteria is. Operation steps
6.1 Botulinum toxin test
Liquid samples can be directly centrifuged. Solid or semi-fluid samples must be added with an appropriate amount (e.g., equal, double, 5 times, 10 times) of gelatin phosphate buffer, soaked, crushed, and then centrifuged. Take the supernatant for testing. Take another part of the supernatant, adjust the pH to 6.2, add 1 part of 10% pancreatic enzyme (activity 1:250) aqueous solution to every 9 parts, mix well, stir gently, and act at 37℃ for 60 minutes before testing.
Botulinum toxin detection is based on the intraperitoneal injection method in mice as the standard method, GB/T4789.12—2003
6.1.1 Detection test: Take the above centrifuged supernatant and its pancreatic enzyme activation treatment solution and inject them into three mice, 0.5mL each, and observe for 4 days. If botulinum toxin is present in the injection, mice generally become ill or die within 24 hours after injection. The main symptoms are erect hair, paralysis of the limbs, difficulty breathing, bellows-like breathing, sunken waist, like a wasp waist, and eventually death from respiratory paralysis. In case of sudden death of mice and no obvious symptoms, the injection solution can be appropriately diluted and the test can be repeated. 6.1.2 Confirmatory test: Regardless of the supernatant or its pancreatic enzyme activation treatment solution, if it can cause illness and death in mice, take samples and divide them into three parts for testing. One part is added with an equal amount of multi-type mixed botulinum anti-toxin diagnostic serum, mixed, and exposed at 37℃ for 30 minutes; one part is added with an equal amount of gelatin phosphate buffer, mixed, and boiled for 10 minutes; one part is added with an equal amount of gelatin phosphate buffer, mixed, and no other treatment is done. The three mixed solutions are injected into two mice, each with 0.5mL, and observed for 4 days. If the mice injected with the diagnostic serum and the two mixed solutions heated by boiling are protected and survive, and only the mice injected with the mixed solution without other treatment die with specific symptoms, it can be determined that botulinum toxin exists in the sample, and toxicity determination and typing test should be carried out if necessary.
6.1.3 Toxicity determination: Take the supernatant of the centrifuged sample that has been determined to contain botulinum toxin, and use gelatin phosphate buffer to make 50-fold, 500-fold and 5000-fold dilutions, and inject them into two mice, 0.5 mL each, and observe for 4 days. According to the death of the animals, calculate the general toxicity of the botulinum toxin contained in the sample (MLD/mL or MLD/g). For example, if all animals died after 5-fold, 50-fold and 500-fold dilutions, but all animals survived after being injected with 5000-fold dilutions, it can be roughly determined that the toxicity of the toxin contained in the supernatant of the sample is 1000MLD/mL to 10000MLD/mL.
6.1.4 Typing test: According to the toxicity test results, the supernatant of the sample is diluted with gelatin phosphate buffer to a toxicity of the toxin contained in the range of 10MLD/mL to 1000MLD/mL, and then mixed with equal amounts of each monotype botulinum anti-diagnostic serum, and incubated at 37℃ for 30 minutes. Two mice are injected with each 0.5mL, observe for 4 days. At the same time, replace the diagnostic serum with gelatin phosphate buffer and mix it with the diluted toxin solution in equal amounts as a control. The diagnostic serum type that can protect animals from disease and death is the type of botulinum toxin contained in the test sample. Note 1: If the toxin detection test or confirmation test of the test sample that has not been activated by pancreatic enzymes is positive, the toxicity determination and typing test of the pancreatic enzyme activation solution can be omitted.
Note 2: In order to gain time and get results as soon as possible, the customer tests of toxin detection can also be carried out at the same time. Note 3: According to specific conditions and possibilities, types C, D, F and G can be omitted in the typing test as appropriate. Note 4: When conducting neutralization tests such as confirmation and typing, the dilution of the test sample should refer to the titer of the botulinum diagnostic serum used. Note 5: The observation of the test animal can be terminated at any time according to the appearance of positive results to shorten the observation time; only when a negative result appears, sufficient observation time should be reserved.
6.2 Detection of Clostridium botulinum (toxin production culture test) Take three tubes of meat culture medium, boil for 10min~15min, and do the following: a) First tube: Rapidly cool, inoculate 1mL~2mL of the test sample homogenate; b) Second tube: Cool to 60℃, inoculate the test sample, continue to keep warm at 60℃ for 10min, and rapidly cool; c) Third tube: inoculate the test sample, continue to boil and heat for 10min, and rapidly cool. The above inoculum is cultured at 30℃ for 5d. If there is no growth, it can be cultured for another 10d. At the end of the culture period, if there is growth, take the culture medium and centrifuge it, and use its supernatant for toxin detection test, the method is the same as 6.1, and a positive result proves the presence of Clostridium botulinum in the test sample. 6.3 Isolation and culture
Select the aforementioned enriched and toxin-producing culture (if necessary, repeat the appropriate heating treatment) that has been confirmed to contain Clostridium botulinum through the toxin detection test and inoculate it on the egg yolk agar plate, and culture it anaerobically at 35℃ for 48h. When Clostridium botulinum grows on the egg yolk agar plate, the colony and the surrounding culture medium surface are covered with a unique iridescent (or pearl-like) thin layer, but G-type bacteria do not have this phenomenon. Select suspicious colonies based on the colony morphology and bacterial morphology, inoculate them with blister culture medium, culture them at 30℃ for 5d, and conduct toxin detection and culture characteristic inspection confirmation tests.
6.3.1 Toxin detection: The test method is the same as 6.1.87
GB/T4789.12—2003
6.3.2 Culture characteristic inspection: Inoculate the egg yolk agar plate, divide it into two parts, and culture them under aerobic and anaerobic conditions at 35℃ for 48h, respectively, and observe the growth and colony morphology. Clostridium botulinum can only grow on egg yolk agar plates under anaerobic conditions and form colonies with the above characteristics, but it will not grow under aerobic conditions. Note: To detect Clostridium botulinum in honey, the honey sample needs to be preheated to 37℃ (fluid honey) or 52℃~53℃ (crystalline honey). After fully stirring, 20g is weighed immediately and dissolved in 100mL sterile distilled water (37℃ or 52℃~53℃), stirred and diluted, centrifuged at 8000r/min~10000r/min for 30min (20℃), precipitated, added 1mL of sterile distilled water, shaken thoroughly, divided equally, inoculated into each half of the meat culture medium (8mL~10mL), and cultured anaerobically at 30℃ and 37℃ for 7d, respectively, and tested for botulinum toxin according to 6.2.
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