Phage isolation steps

Phage isolation steps

The steps of the phage isolation process corresponding to some strains:

1. Phage isolation
(1) Add 4g of fresh pigeon dung to 100ml of ordinary liquid medium, and put it into a 37 ℃ incubator for 24h.
(2) The next day, take out 10ml of it and heat it at 70°C for 30min, centrifuge at 2000r/10min, filter the supernatant with a filter and store it in a 4°C refrigerator for later use.
(3) Mark No. 1, No. 2, No. 3 on the bottom of the ordinary agar plate.
(4) Plate No. 1 was inoculated with the pre-cultured 6-hour juvenile Bacillus paratyphoid with an inoculation loop; plate No. 2 was inoculated with the inoculation loop of the juvenile Shigella flexneri; plate No. 3 was inoculated with the juvenile bacterium Escherichia coli, the bacteria of each plate were densely spread on the surface of the agar plate.
(5) Then use a sterile inoculating loop to take the pigeon feces culture filtrate, add dropwise to the center of the three inoculated bacteria plates, and incubate them in a 37 ℃ incubator for 24 hours.

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2. Phage isolation and identification
A total of 158 types of seafood were collected from farmers’ markets, and 15 strains of Pseudomonas were used as indicator bacteria. A total of 27 strains of bacteriophage were isolated, with an isolation rate of 1711%. The detected bacteriophages were identified by repeated proliferation and lysis experiments in the laboratory, and 9 strains of phages with strong lysis ability and typical biological characteristics were selected from them, and were purified and cultured by single-spot purification by double-layer agar method.
The plaques of these 9 phage strains were round and transparent, with some neat and some slightly irregular edges, and the diameters were all 110-210 mm; the morphology under the electron microscope showed that there were heads and tails, all of which were tadpole-shaped; The titer can reach 10829pfuˆml.

3. Phage isolation
On the double-layer agar plate with indicator bacteria, add the standby phage solution to be tested one by one, and carry out the cross-lysis test, that is, each strain of bacteria is added dropwise to all the phage solutions to be tested one by one.
After the dishes were dry, they were incubated at 37 °C overnight. If there are corresponding phages, lysis plaques (that is, positive) can appear on the plate, and those without lysis are negative. Each isolated phage was isolated and purified by single plaque for more than 3 times, and stored in a refrigerator at 4 ℃ after proliferation.