1. Enrichment
The samples should be inspected as soon as possible after collection. Except that perishable foods should be pre-refrigerated before inspection, they are generally not refrigerated.
Weigh 25g sample in aseptic procedure, add it to 225ml nutrient broth, break it with homogenizer for 1 minute or grind it with mortar and sterilized sand.
Take out an appropriate amount and inoculate lactose bile salt medium (culture medium for measuring coliform bacteria) to determine the coliform bacteria MPN, and transfer the rest into a 500ml wide-mouth bottle and incubate at 36 ℃ ± 1 ℃ for 6 hours. Pick 1 ring, inoculate 1 tube of 30ml intestinal bacteria enrichment broth, and incubate at 42 ℃ for 18 hours. 2. Separation
Streak the lactose bile salt fermentation tube and the enrichment solution that are positive for lactose fermentation and inoculate MacConkey or Eosin Blue agar plates; for samples with serious contamination, you can inoculate MacConkey directly with the homogenate of the test sample
Or eosin blue plate, cultivate at 36 ℃ ± 1 ℃ for 18-24 hours, and observe the colony. Pay attention not only to the colonies of lactose fermentation, but also to the colonies of lactose non-fermentation and slow fermentation.
3. Biochemical test
1. Directly pick several colonies from the identification plate and inoculate trisaccharide iron agar (TSI) or Kjeldahl iron agar (KI).
At the same time, these cultures were inoculated with peptone water, semi-solid, pH 7.2 urea agar, KCN broth and lysine decarboxylase test medium. The above cultures were incubated overnight at 36 ° C. 2. The culture of TSI (triose iron agar) slant producing acid or no acid, bottom layer producing acid, H2S negative, KCN negative and urea negative is Escherichia coli.
TSI (triose iron agar) bottom layer does not produce acid, or H2S, KCN, urea have any one of the positive cultures, are not Escherichia coli. If necessary, do oxidase test and Gram stain.
4. Serological test
1. Assuming that the test picks agar culture confirmed by biochemical tests as Escherichia coli,
Use â—‡
1 Pathogenic Escherichia coli, â—‡ 2 invasive E. coli and â—‡ 3 enterotoxigenic Escherichia coli multivalent O serum and â—‡ 4 hemorrhagic Escherichia coli O157 serum for slide agglutination test. When agglutinating with a certain kind of multivalent O serum, the monovalent O serum contained in the multivalent serum is tested. If there is a strong agglutination reaction with a certain monovalent O serum, it is assumed to be positive. 2. Confirm that the test prepared an O antigen suspension and dilute it to a concentration equivalent to that of Mac Farland No. 3 Turbidity Tube.
The original serum with a titer of 1: 160 to 1: 320 was diluted with 0.5% saline to 1:40. Dilute serum and antigen suspension were mixed in equal amount in 10mm × 75mm test tube for single tube agglutination test. After mixing, place in a 50 ° C water bath and observe the results after 16 hours. If agglutination occurs, it can be confirmed as the O antigen.
Puyang Optimus Household Products Co., Ltd , https://www.magicspongecn.com