灿烂弧菌鉴定及溶血素检测-预防兽医学专业论文.docxVIP

海洋灿烂弧菌的鉴定及溶血素的检测 海洋灿烂弧菌的鉴定及溶血素的检测 PAGE PAGE 4 agar-solidified medium, added 1% of fresh egg yolk, and made the surface flat. The colonies that exhibited ivory translucent circles around them were considered positive for phospholipase activity. the radius of the translucent cycle was 0.5 mm. Pathogenicity test: For the pathogenicity test, fish were divided into six groups, including five experimental groups and one control group. Each group included 10 healthy guppies, and each group was maintained in different aquariums. The bacterial preparation was diluted to 2 × 104, 2 × 105, 2 × 106, 2 × 107, and 2 × 108 cfu/mL, and the dilutions were injected into the experimental guppies (200 μL for each), whereas the fish in the control group were injected with normal saline solution. When a guppy died, bacteria were isolated from the fish. Of note, the water temperature was kept at 27.4-28°C. We observed and recorded the morbidity and mortality daily, and subsequently calculated the LD50 according to the simplified Korbor’s formula method. Based on statistical analysis of the number of the deaths in the fish, we calculated the LD50 using Korbor’s approach, and determined that the LD50 was 5.62×105 cfu/mL. 3 The V. splendidus strain was inoculated into a seawater medium AGAR plate, and was kept at 28°C for 24 h. We picked a single colony and inoculated it into 5 ml of seawater medium, subsequently optimized the conditions according to a previous study, and kept the preparation at 28°C for 18 h. A 1% inoculate was prepared in 1L seawater medium, and was maintained with 200 r/m shaking for 48 h. Ammonium sulfate precipitation: The culture was centrifuged at 8,000 rpm for 30 min, the supernatant was collected, and solid ammonium sulfate was added into the supernatant at 70% saturation. Furthermore, the precipitate was solubilized at a concentration of 50 mmol/L in Tris-HCL in (pH 7.8) buffer fluid, and was kept in the same buffer for dialysis at 4°C for 48 h. The enrichment o