(1) test method
1. Lactose fermentation test. Take samples by aseptic operation, and determine the dosage and dilution multiple according to the requirements of national or local health standards and the pollution of samples. Inoculate the sample to be tested into a lactose bile salt fermentation tube. If the inoculation amount is greater than l m J, use a double lactose bile salt fermentation tube, and if l m is less than 1 and 1mI, use a single lactose bile salt fermentation tube. Inoculate 3 tubes at each dilution, put them in an incubator at 36+1℃, and cultivate for 24+/-2 hours. If all lactose bile salt fermentation tubes do not produce gas, they can be reported as negative for coliform bacteria. If gas is generated, follow the following procedure.
2. Isolation and culture. Transfer the gas-producing fermentation tube to eosin methylene blue agar plate, put it in an incubator at 36 L℃ for 65438 08 ~ 24 hours, then take it out, observe the colony morphology, and carry out Gram staining and confirmation test.
3. Confirm the test. On the above-mentioned plate, L ~ 2 suspected coliforms were selected for Gram staining, and at the same time, lactose fermentation tubes were inoculated, and cultured in a constant temperature box at 36 65438 0℃ for 24±2h hours, and the gas production was observed. Lactose tube produces gas, leather
Gram-negative Bacillus-free bacteria can be reported as positive for Escherichia coli.
4. Report. According to the number of coliform positive test tubes, check MPN key table and report the most possible coliform count per 100 ml (g).
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(2) culture medium
① Lactose bile salt fermentation medium
Ingredients: peptone: 20g
Pig bile salt (or cattle and sheep bile salt): 5g.
Lactose: 10g
0.04% bromocresol violet aqueous solution: 25m 1
Distilled water: 1 000m 1
Methods: Dissolve peptone, bile salt and lactose in water, adjust the pH value, add indicator, pack them in each tube 10 ml, put them in an inverted small test tube, and sterilize them at high pressure 1 15℃ 15 minutes.
Note: Except distilled water, the composition of the fermentation medium of bile salt with double lactose is doubled.
Usage: Used in lactose fermentation test.
Principle: Bacteria break down sugars through the action of various enzymes produced by bacterial cells. Different kinds of bacteria produce different enzymes, which can be used to identify bacteria.
Lactose is disaccharide, and the enzymes that bacteria decompose disaccharide are mostly extracellular enzymes. Lactose is hydrolyzed by lactase into glucose and galactose. Glucose can be directly used by bacteria, while galactose needs to be converted into glucose in cells to be used.
Sugar fermentation test is mainly to determine the acid produced by bacteria after decomposing sugar, and the decrease of pH value of culture medium (indicator discoloration) is taken as the index of observation results.
In acidic environment, bacteria such as Escherichia coli can decompose formic acid into CO2 and H2 due to the action of formate dehydrogenase, and generate a large amount of gas in the culture medium, which enters the inverted tube for observation.
Note: Various sugars, alcohols and glycosides commonly used in fermentation experiments (see Table 3- 1).
② eosin methylene blue agar medium
Ingredients: peptone10g
Lactose 10g
Dipotassium hydrogen phosphate 2g
Agar 17g
20 ml of 2% Eosin Y solution
0.65% methylene blue solution 10ml
Distilled water 1000 ml
pH7. 1
Methods: L-peptone, phosphate and agar were dissolved in distilled water, and the pH was adjusted. They were packaged in a flask and autoclaved 12 1℃ 15 minutes for later use. When in use, lactose is added, agar is heated to dissolve, cooled to 50-55 DEG C, eosin and methylene blue solution are added, shaken evenly, and poured into a plate.
Principle: Escherichia coli bacteria ferment lactose to produce acid, which makes eosin and methylene blue combine to form black compounds, so the colonies are dark purple and sometimes produce metallic luster. The degree of blackness and luster is related to the contrast between eosin and methylene blue. Bacteria that do not ferment lactose (such as Salmonella and Shigella) are colorless colonies.
③ Lactose fermentation medium
Ingredients: peptone 20g.
Lactose 10g
25ml of 0.04%% bromocresol violet aqueous solution.
Distilled water 1000 ml
pH7.4
Methods: Dissolve peptone and lactose in water, adjust the pH, add indicator, subpackage 30ml, 10ml or 3ml according to the inspection requirements, put them in a small inverted tube, and autoclave 1 15℃ 15 minutes.
Note:'
(1) The dual-material lactose fermentation tube has two components besides distilled water.
(2)30ml and 10ml lactose fermentation tubes are specially used for soy sauce and sauce inspection. 3 ml lactose fermentation tube was used for coliform bacteria.
Used for verification testing.
4. Peptone water
Ingredients: peptone (or pancreatic peptone) 20g.
Sodium chloride 5g
Distilled water 1 000 ml pH 7.4