噬菌蛭弧菌的分离及初步鉴定

采用双层平板法,以滤膜过滤的方法来收集噬菌蛭弧菌,以嗜水气单胞菌(Aeromonas hudrophila)、荧光假单胞菌(Pseudomonas fluorescent)和绿脓杆菌(Pseudomonas aeruginosa)为宿主菌,进行噬菌蛭弧菌的分离研究;并在此基础上,通过接触酶检测和寄生性确认对噬菌蛭弧菌(Bdellovibrio bacteriovorus)进行了初步的鉴定.结果表明未使用滤膜过滤,采用自来水琼脂双层平板法分离噬菌蛭弧菌的效果较好;并经过接触酶和寄生性检测初步鉴定此BD-SPOI菌株为噬菌蛭弧菌.

The isolation and the elementary identification of Bdellovibrio bacteriovorus

Bdellovibrio bacteriovorus is a small gram-negative bacterium, and distributes over soil, freshwater, seawater and sewage. With regard to its great ability to lyse other gram-negative bacteria, the application of this microecological preparation to aquaculture is well expected. No identification, however, it has been given by other scholars previously. In the present study, we isolated the B. bacteriovorus and tried to confirm its identity.Double layer plant was used to isolate B. bacteriovorus from sewage water. The sewage water was collected from six different pools of the sewage disposal plant of Wuhan, which were intake sump, anaerobic tank, anoxic pond, aeration tank, outlet sump and primary settling tank orderly. We did the identification of the bacterium for H_2O_2 activity and parasitical activity, and took the photo of the bacterium of SEM and the stain of gram after the isolation of the B. bacteriovorus.In the isolation experiment, Aeromonas hudrophila, Pseudomonas fluorescent and Vibrio anguillarum were used as the host bacteria, and the six kinds of sewage water were filtered through 3.0 μm, 1.2 μm, 0.8 μm pore-size filters to collect the B. bacteriovorus individually. Then 100 μL host bacteria were added to 200 μL filtered water which mixed with 4mL molten top-layer culture medium, and were plated on bottom layer and incubated at 301. After the filtration through 0.8 μm filter, some portion of the left water passed through 0.22 μm filter. The filter membrane was mixed with 100μL host bacteria and the molten top layer culture medium plated on bottom layer and incubated at 301. The Bdellovobrio appeared as plaques, and then chosed one transparent and round plaque to purify for obtaining the pure Bdellovobrio after 2-4 days. We could obtain the pure B. bacteriovorus after 4 times purification. We isolated one strain of B. bacteriovorus from aeration tank with Pseudomonas fluorescent as host bacterium using the tap water plant of two layers. And the sewage water was not filtrated with any filter membrane.Then we did the elementary identification of H_2O_2 activity and parasitical activity. Three percents H_2O_2 solution was dropped on the microscope slide and one plaque was cut out from top layer and immerged in the solution with the invertion side. If bladder appeared, the bacterium should be masculine of H_2O_2; if not, the bacterium should be feminine of H_2O_2 On the other hand, the isolated bacterium with no host bacteria was incubated at 301 to test its parasitical characteristic. If there was no bacterium grown on the plant, the isolated bacterium was obligate parasitical bacterium. If not, the isolated bacterium was not the obligate parasitical bacterium. The two identification results showed that this bacterium was masculine of H_2O_2 activity and parasitical activity.Some plaques were cut out from the one plant to a 1.5mL EP cannulation and dipped in 1mL water for 30min to dissociate the B. bacteriovorus from the plaque. After that, one blob of the dissociated bacterium was dropped on the microscope slide and air-dried the blob, then fixed. The bacterium on the microscope slide was stained as the following step: firstly, stained the bacterium with crystal purple for 1 min and wash; secondly, stained with iodine solution for 1 min and wash; thirdly, decolored with 95% ethanol for 30s; fourthly, stained with safranine solution for 2-3min, washed and air-dried. Finally, it was observed by oil lens. The result of this identification proved the isolated bacterium was negative of Gram.Besides, the SEM photo of this B. bacteriovorus had been taken. The photo showed that the bacterium was rod shaped and had one flagellum on one side. The size of the bacterium was 2.0 um.It was substantiated that the isolated bacterium was Bdellovobrio bacteriovorus.