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A method for isolation and identification of sea cucumber pathogenic bacteria

An identification method, a technology of pathogenic bacteria, applied in the field of microorganisms, can solve the problems that restrict the sustainable and stable development of the industry, economic losses, etc., and achieve the effects of rapid separation and identification of bacteria, early diagnosis, and simple process

Inactive Publication Date: 2011-12-28
DALIAN UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

It has caused heavy economic losses to the vast sea cucumber farmers, and has seriously restricted the sustainable and stable development of the industry.

Method used

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  • A method for isolation and identification of sea cucumber pathogenic bacteria
  • A method for isolation and identification of sea cucumber pathogenic bacteria
  • A method for isolation and identification of sea cucumber pathogenic bacteria

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0017] Embodiment 1, separation and purification of dominant bacteria in the lesion

[0018] Take samples of sea cucumbers suffering from rotting skin syndrome (see figure 1 ) in the ultra-clean workbench, cut out 1cm × 1cm × 1cm tissue pieces, put them in a 5mL sterile centrifuge tube, and use sterilized ophthalmic scissors to homogenize to make a bacterial suspension. Streak on solid agar medium, culture in 24-28°C incubator for 48-72 hours, separate the colonies grown on the petri dish again by specific TCBS plate streak separation method, and culture in 24-28°C incubator Observe after 48-72, select a single colony according to the colony shape, color and bacterial culture characteristics, and then store the pure culture of the bacteria.

[0019] Results: The colony was in the shape of fried egg, flat, about 3-5mm in diameter, with raised surface, corn yellow, opaque, odorless, not easy to stir up the bacteria, and the colony was smooth and sticky. (See figure 1 )

Embodiment 2

[0020] Embodiment 2, strain preservation

[0021] Inoculate the single colony of the pure pathogenic bacteria prepared by the above method on the plate directly on the 2216E solid slope, place it in an incubator at 28°C for 24 hours, and then store it in a refrigerator at 4°C;

[0022] Long-term storage: culture the bacteria in 2216E liquid medium for about 5 to 6 hours, and the concentration of bacteria reaches the logarithmic growth phase OD 600 When = 0.2, add glycerin, adjust the volume concentration of glycerin to about 30-50%, and store in a -80°C refrigerator.

Embodiment 3

[0023] Embodiment 3, artificial inoculation infection experiment of pathogenic bacteria

[0024] (1) Preparation before the experiment

[0025] The plastic basin with a volume of about 8000mL was cleaned with detergent and then wiped and disinfected with 75% alcohol. After the alcohol evaporated, it was rinsed three times with filter-sterilized PBS. Inject 5000mL of seawater into each basin, and ventilate with the inflation valve. The temperature of the seawater is maintained at 18-25°C.

[0026] (2) Pretreatment of sea cucumber

[0027] The sea cucumbers retrieved from the farm were domesticated for 7 days in a laboratory environment with filter-sterilized seawater, and healthy individuals of similar size were selected, washed twice with sterilized PBS, and 10 were placed in each pot.

[0028] (3) Preparation of bacterial suspension

[0029] The isolated dominant strains were plated, cultured at 28°C for 48 hours, and the colonies were washed with filter-sterilized PBS to ...

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Abstract

The invention discloses a method for separating and identifying pathogenic bacteria of sea cucumber, which is characterized in that the tissue of sea cucumber containing target pathogenic bacteria is sterilized with 75% alcohol for 60-90 seconds, and then washed with sterile sea water. Homogenize the lesion tissue, add sterile PBS buffer solution dropwise, use an inoculation loop to dip the bacteria-containing solution and streak it on the agar medium suitable for the growth of the pathogenic bacteria of Apostichopus rot skin syndrome, cultivate it at 24-28°C, and then pass it through a specific The corresponding target bacteria can be obtained by purifying and screening a single colony in TCBS culture medium. The pathogenicity of the isolated bacteria is verified through the animal experiment of A. japonicus inoculation and the morphological structure of the bacteria is observed by scanning electron microscope. The 16srDNA PCR technology is further used for identification. Isolated pathogenic bacteria. The invention has the advantages of simple operation and easy method, can quickly and effectively screen out the pathogenic bacteria of sea cucumber rotting skin syndrome, and simultaneously provides ideas for rapid screening of pathogenic bacteria for other aquatic animal diseases.

Description

technical field [0001] The invention relates to a method for isolating and identifying sea cucumber pathogenic bacteria, which belongs to the technical field of microorganisms. Background technique [0002] Due to the rapid development and non-standard operation of sea cucumber cultivation, the problem of diseases has become increasingly prominent, and various obvious diseases and large-scale deaths have appeared. The most serious disease in sea cucumber farming. It has caused heavy economic losses to the vast number of japonicus cultivators, and has seriously restricted the sustainable and stable development of this industry. In order to ensure the healthy development of the sea cucumber aquaculture industry, active research on the epidemiology and etiology of "rotting skin syndrome" is an essential basic work for the comprehensive prevention and treatment of the disease. [0003] "Rotten skin syndrome" is the most common and most serious disease in sea cucumber cultivati...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/20C12Q1/68C12Q1/04C12R1/01
Inventor 金礼吉谭德猛徐永平李晓宇
Owner DALIAN UNIV OF TECH
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