1. Introduction of coliform bacteria
Coliform bacteria is not a nomenclature for bacteriological classification, but a term in the field of hygienic bacteria. It does not represent a certain or a genus of bacteria, but refers to a group of bacteria with certain characteristics related to fecal contamination. These bacteria are biochemical It is not completely consistent with serology. It is defined as: aerobic and facultative anaerobic, Gram-negative non-bacillus bacilli that can decompose lactose to produce acid and gas at 37 ℃. It is generally believed that the bacteria of this flora may include Escherichia coli, Citrobacter, Citrobacter aerogenes, Enterobacter cloacae and the like.
Coliform bacteria are widely distributed in warm-blooded animal feces and in nature. Investigations and studies have shown that coliform bacteria are mostly found in warm-blooded animal feces, places where humans frequently move, and places where fecal pollution occurs. The pollution of human and animal feces to the external environment is the main reason for the existence of coliform bacteria in nature. The feces are mostly dominated by typical E. coli, while other types of coliform bacteria are more in the outside environment.
Coliform bacteria is proposed as an indicator of fecal contamination, mainly based on the detection of the flora to indicate whether there is fecal contamination in food. The number of coliform bacteria indicates the degree of fecal contamination and also reflects the size of the hazard to human health. Feces are feces of human intestines, including feces of healthy people, feces of intestinal patients or carriers, so in addition to normal bacteria, there are also some intestinal pathogens (such as Salmonella, Shiga) Bacteria, etc.), so there is fecal contamination in the food, you can speculate that there is the possibility of intestinal pathogenic bacteria contamination in the food, latent food poisoning and epidemic threats, must be considered as a potential danger to human health .
Coliform bacteria is one of the important indicators to evaluate the quality of food hygiene, and it has been widely used in food hygiene at home and abroad.
2. Inspection method:
Coliform bacteria refers to a group of bacteria with certain characteristics related to fecal pollution, namely: aerobic and facultative anaerobic, Gram-negative non-bacillus bacilli that can decompose lactose to produce acid and gas at 37 ℃. Therefore, the detection of coliform bacteria is generally carried out according to its definition.
At present, the domestic and foreign food coliform detection methods used in China are mainly national standards and industry standards formulated by the former State Administration for Commodity Inspection. The two standard methods differ slightly in the detection procedure.
(1) National standard: The national standard adopts a three-step method, namely: lactose fermentation test, separation culture and confirmation test.
Lactose fermentation test: After the sample is diluted, three dilutions are selected, and each dilution is inoculated with three tubes of lactose bile salt fermentation tubes. Incubate at 36 ± 1 ℃ for 48 ± 2h, and observe whether gas is generated.
Separate culture: Transplant the gas-producing fermentation tube culture on eosin blue agar plate, incubate at 36 ± 1 ℃ for 18-24h, and observe the colony morphology.
Confirmation test: pick up suspicious colonies on the plate and observe by Gram staining. Simultaneously inoculate lactose fermentation tube at 36 ± 1 ℃ for 24 ± 2h to observe gas production.
Report: According to the number of tubes confirmed to be positive for E. coli, check the MPN table and report the MPN value per 100 ml (g) of coliform bacteria.
For specific operations, please refer to GB4789.3-94 "National People's Republic of China National Standards for Food Hygiene Microbiological Examination of Coliforms"
(2) The industry standards formulated by the former State Administration of Commodity Inspection are equivalent to the standard methods of the US FDA for the detection of E. coli in exported food. This method uses a two-step method:
Speculation test: After the sample is diluted, three dilutions are selected, and each dilution is inoculated with three tubes of LST broth. Incubate at 36 ± 1 ℃ for 48 ± 2h, and observe whether gas is generated.
Confirmation test: Inoculate tracheal cultures into BGLB broth tubes, incubate at 36 ± 1 ℃ for 48 ± 2h, and observe whether gas is produced. Positive to BGLB gas production. Check the MPN table and report the MPN value of the coliform bacteria per ml (g) sample.
For specific operations, please refer to SN0169-92 "Inspection Methods for Coliforms, Fecal Coliforms and Escherichia coli in Exported Foods of the People's Republic of China Import and Export Commodity Inspection Industry Standard"
3. Description:
1. MPN search form:
MPN is the abbreviation of Most Probable Number. In this method, the samples are serially diluted, and culture medium is added for cultivation. From the occurrence rate of the number of positive tubes with a predetermined reaction, the probability theory is used to calculate the most approximate number of bacteria in the sample.
The MPN search table only gives three dilutions. If you use a different dilution, the number in the table should be reduced or increased by a factor of 10 accordingly. Note that the dilutions used in the MPN table attached to the national standard and the industry standard are different, and the result reporting unit is also different.
2. Initial fermentation and confirmation test:
Both the national standard three-step method and the industry standard two-step method used lactose fermentation tubes for two fermentation tests. The preparation of the medium is slightly different, but it is to confirm whether the culture meets the coliform flora Definition, that is, "decompose lactose at 37 ° C to produce acid and gas".
The initial fermentation positive tube is not surely the coliform bacteria. After the confirmation test, it may sometimes become negative. There are data showing that the conformity rate of the inspection procedure of coliform bacteria in food, the initial fermentation is quite different from the confirmation test. Therefore, in the actual inspection work, confirmation test is necessary.
3. Gas production and down tube:
In the experiment of lactose fermentation, very few bubbles (sometimes smaller than the grains of rice) can often be seen in the fermentation tube. Sometimes, small bubbles that produce acid during the initial fermentation or slowly rise along the tube wall can be encountered. Experiments have shown that the gas production of coliform bacteria can fill all the fermentation tubes with gas, and the smaller ones can produce bubbles smaller than millet grains. If there is any doubt about the fermentation of lactose that produces acid but not gas, the test tube can be gently moved by hand. If there are bubbles floating along the wall of the tube, it should be considered that there may be gas generation, and further tests should be conducted.
4. Pick the colony:
In the national standard, it is necessary to inoculate the eosin blue plate to isolate the positive fermentation positive culture, and observe and verify the typical and suspicious colonies. Coliform bacteria is a general term for a group of bacteria. Larger coliform bacteria are more complex and diverse in terms of color and morphology of flat coliform colonies, and are closely related to the detection rate of coliform bacteria. The national standard method stipulates that the Eosin Methylene blue plate is the separation medium. On this plate, the detection rate of the coliform colonies is black-purple shiny or dull; the detection rate of red and pink colonies is low.
In addition, the number of colonies picked is closely related to the detection rate of coliform bacteria. Only one colony is picked. Due to the probability problem, especially when the colonies are not typical, it is difficult to avoid the occurrence of false negatives. So pick colonies must pick typical colonies, if there are no typical colonies, you should pick a few more to avoid false negatives.
5. Bacteriostatic agent:
Bacteriostatic agents commonly used in the inspection of coliform bacteria include bile salts, sodium lauryl sulfate, washing powder, brilliant green, gentian violet, and malachite green. The main role of bacteriostatic agents is to inhibit the growth of other miscellaneous bacteria, especially Gram-positive bacteria.
The national standard lactose bile salt fermentation tube uses bile salt as a bacteriostatic agent, the industry standard LST broth uses sodium lauryl sulfate as a bacteriostatic agent, and the BGLB broth uses brilliant green and bile salt as a bacteriostatic agent.
Although bacteriostatic agents can inhibit some of the bacteria in the sample, which is beneficial to the growth and selection of coliform bacteria, it also sometimes produces some inhibitory effects on certain strains of coliform bacteria. Some bacteriostats are used in very small amounts, and a slight error in weighing can have an effect on the bacteriostasis. Therefore, the addition of bacteriostat should be carried out in strict accordance with standard methods.
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