A kind of vibrio phage and its application
A phage and Vibrio technology, applied in the field of microorganisms, can solve the problems of narrow host spectrum and inability to effectively control multiple bacterial diseases, and achieve the effect of wide host spectrum, high safety, and small toxic and side effects
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Embodiment 1
[0032] The isolation and purification of embodiment 1 phage
[0033] Sewage samples collected from the sewage outlet of Guangzhou Aquatic Products Trading Market were preliminarily filtered with multi-layer gauze, and then the filtrate was centrifuged at 10,000g at 4°C for 15min. Take 20 mL of the supernatant and add it to a pre-sterilized Erlenmeyer flask, add 20 mL of double-concentrated 2216E liquid medium and 400 μL of Vibrio alginolyticus H29 bacterial solution in the logarithmic phase, and culture overnight at 37 °C. The next day, the culture solution was centrifuged at 5000r / min for 10min, and the supernatant was filtered with a disposable filter with a pore size of 0.22μm, and the filtrate was placed at 4°C for later use to obtain the phage stock solution 0, which was collected in a sterile centrifuge tube, Label it well and store it at 4°C for later use.
[0034] Take 200 μL of phage 0 stock solution, add 20 mL of double-concentrated 2216E liquid medium and 400 μL of...
Embodiment 2
[0039] The amplification of embodiment 2 phage
[0040] The phage purified in Example 1 was inoculated into 1 mL of sterile SM buffer, and kept overnight at 4°C. Take 500 μL of phage-containing SM buffer and 500 μL of H29 bacteria in logarithmic phase growth in 5 mL of 2216E liquid medium, and shake at 37 ° C for 6 h. The culture solution was collected and centrifuged at 4°C at 10,000×g for 15 minutes, and the supernatant was filtered through a disposable filter with a pore size of 0.22 μm. The filtrate was the phage proliferation solution and stored at 4°C for use.
Embodiment 3
[0041] Example 3 Phage Titer Determination
[0042]Take 600 μL of the bacteria H29 cultured to the logarithmic phase and 60 μL of the phage prepared in Example 2 and add them to the 2216E broth, and amplify for about 6 hours. Take 1 mL of the phage proliferation solution and filter it with a 0.22 μm filter to obtain the phage stock solution. Use SM buffer to serially dilute the phage proliferation solution by 10 times, take the appropriate dilution of the phage dilution solution and mix it with the bacterial solution, let it stand for 3-5 minutes to lay double-layer plates, and culture it in a 37°C incubator for 8 hours before performing phage plaques. Counting results showed that the phage titer reached 10 11 More than pfu / mL.
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