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 p1ant 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 H2O2 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 30℃.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 30℃.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 H2O2 activity and parasitical activity.Three percents H2O2 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 H2O2;if not,the bacterium should be feminine of H2O2.On the other hand,the isolated bacterium with no host bacteria was incubated at 30℃ 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 H2O2 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 1min and wash;secondly,stained with iodine solution for 1min 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 μm.It was substantiated that the isolated bacterium was Bdellovobrio bacteriovorus.