Marine-derived vibrio bacteriophage, microecological preparation and preparation method and application of marine-derived vibrio bacteriophage
A technology of micro-ecological preparations and phages, applied in the direction of microorganism-based methods, biochemical equipment and methods, phages, etc., to achieve the effects of preventing abuse, easy acquisition, saving economic costs and time costs
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Embodiment 1
[0025] Example 1 Cracking Marine Vibrio Phage Isolation and Purification
[0026] 1. Recovery culture of Vibrio cyanophage E-110:
[0027] Vibrio cyanophagee strain E-110 was isolated from Litopenaeus vannamei; Vibrio E-110 was streaked on a TCBS (Thiosulfate Citrate Bile Salts Sucrose, BD) plate and cultured at 30°C for 12 hours; a single clone was picked and placed in 1 mL LB ( Luria Bertani) + 2% NaCl liquid medium, culture overnight at 30°C with shaking (200rpm) for 12h; transfer the overnight culture solution to 100mL fresh liquid medium at a ratio of 1:100, and shake until the OD600 is about 0.4.
[0028] 2. Preliminary isolation of marine phage:
[0029] 500mL seawater samples were subjected to high-speed centrifugation (10000rpm) at 4°C for 10min, and the supernatant was passed through a 0.45μm filter membrane; the filtrate was centrifuged at high speed (30000rpm) for 3h; 300μL sterile seawater was resuspended. Add 100 μL of the resuspension to 1% diluted Vibrio E-11...
Embodiment 2
[0032] Example 2 Cracking Marine Vibrio Phage Amplification and Electron Microscope Detection Observation
[0033] 1. Amplification of a single phage virus strain HH-109:
[0034] A single phage virus strain derived from phage plaques was inoculated in the culture medium of Vibrio alginolyticus E-110 in the logarithmic growth phase at a ratio of 1:10 (V / V), cultured with shaking at 30°C, centrifuged at 10,000rpm for 10min to collect the lysate, and passed 0.45 μm filter membrane to obtain a single phage HH-109 strain stock solution.
[0035] 2. Detection of bacteriophage HH-109 by transmission electron microscopy:
[0036]200mL single phage virus strain HH-109 stock solution was subjected to high-speed centrifugation (10000rpm) at 4°C for 10min, and the supernatant was passed through a 0.22μm filter membrane; the filtrate was centrifuged at high-speed (30000rpm) for 2.5h; resuspended concentrated phage. Add 10 μL of concentrated phage dropwise to a 200-mesh copper mesh, abso...
Embodiment 3
[0037] Example 3 Effect evaluation of bacteriophage HH-109 cracking Vibrio alginolyticus
[0038] The culture solution of Vibrio alginolyticus E-110 in the logarithmic growth phase was inoculated into the well plate (150 μL / well); different volumes of phage were added in different proportions (phage HH-109 volume: bacterial solution volume), as shown in the table Shown in 1; Vibrio growth positive control without phage HH-109; add LB+2% to each well so that the total volume of each well is 300μL; the negative control is 300μL LB+2%NaCl liquid medium; for detailed experimental group information, see Table 1.
[0039] Table 1 The design of each experimental group for the detection of the effect of phage lysing Vibrio
[0040] Total volume 300μL 1:1 1:2 1:5 1:10 1:20 1:50 1:100 Negative Positive E-110 bacteria solution (μL) 150 150 150 150 150 150 150 0 150 HH-109 (μL) 150 75 30 15 7.5 3 1.5 0 0 LB+2%NaCl (μL) 0 75 120 13...
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