Bdellovibrio preparation for preventing frog wryneck diseases and application thereof
A technology for phagocytosis of Bdellovibrio and Bdellovibrio, which is applied in the field of freshwater aquaculture, can solve the problems of introducing pathogenic bacteria, lack of function of Bdellovibrio, and potential safety hazards, and achieve safe and efficient use, conducive to growth and reproduction, and low cost of use Effect
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Embodiment 1
[0038] Embodiment 1 effective phage Bdellovibrio isolation test
[0039] This example is used to illustrate the source of Bdellovibrio and its acquisition method of the present invention.
[0040] Sewage samples were obtained from the drainage ditch of a bullfrog farm, centrifuged at 3000r / min for 5min, the supernatant was taken, and 100mL was divided into 250mL triangular flasks, and 4 small bottles were added with a bacterial concentration of 10 8 CFU / mL Flavobacterium meningitidis, Elisabeth mil, Pseudomonas aeruginosa, Aeromonas hydrophila enriched culture, 30 ℃, 100r / min aerobic culture for 48h. After the enrichment culture, the tap water agar double-layer plate was used for separation and detection, and Flavobacterium meningitidis, Elisabeth mil, Pseudomonas aeruginosa, and Aeromonas hydrophila were used as host bacteria for testing, and the plates were cultured at 30°C for 2 days , multiple transparent plaques appeared. Pick multiple upper layer of phage plaque cultur...
Embodiment 2
[0041] Embodiment 2 preparation of phage Bdellovibrio bacterial liquid
[0042] This example is used to illustrate the bacteriophage Bdellovibrio liquid provided by the present invention and its preparation method.
[0043] The Bdellovibrio phage described in this example is provided by Example 1.
[0044] 1. Preparation of inactivated host bacteria concentrate: Inoculate Flavobacterium meningitidis, Elizabeth mil, Pseudomonas aeruginosa, Aeromonas hydrophila respectively in 10g / L peptone, 3g / L beef extract, 5g / L L sodium chloride, pH7.0-7.5 culture medium, after aerobic culture at 30°C for 24 hours, collect the sludge by centrifugation, mix and place in a water bath at 60°C for 30 minutes, and cool for later use.
[0045] 2. Preparation of bacteriophage Bdellovibrio bacteria liquid: Take the uninactivated mixed host bacteria in the above step 1 and dilute them with sterilized physiological saline to make the bacterial concentration at 10 9 CFU / mL was inoculated with Bdellov...
Embodiment 3
[0046] Preparation of Example 3 Bdellovibrio Phage Preparation 1
[0047] This example is used to illustrate a phage Bdellovibrio preparation and its preparation method for preventing bullfrog tilted head disease provided by the present invention.
[0048] The phage Bdellovibrio bacterial liquid obtained in Example 2 was inserted into the inactivated host bacterium, and the bacterial concentration was about 10 6 CFU / mL, after aerobic culture for 2 hours, add 2% (V / V) glycerol, stir evenly, and then mix with freeze-dried carrier (sucrose: skimmed milk powder = 6%: 94%) at a liquid-solid mass ratio of 1:0.5 Proportionally mixed to obtain a mixture to be freeze-dried.
[0049] The above-mentioned mixed solution to be freeze-dried was divided into stainless steel shallow pans, placed in a vacuum freeze dryer at -40°C, pre-frozen for 3 hours, and then started to vacuumize. First raise the temperature to 0°C within 24 hours, then gradually raise the temperature to 10°C in the next...
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