Vacuum freeze-drying protective agent for Edwardsiella tarda and freeze-drying method thereof

A freeze-vacuum drying and protective agent technology, applied in microorganism-based methods, biochemical equipment and methods, preservation of microorganisms, etc., can solve problems such as difficulty in learning from preservation methods, and achieve good preservation effect, long preservation period, and direct isolation. contact effect

Active Publication Date: 2013-12-25
YELLOW SEA FISHERIES RES INST CHINESE ACAD OF FISHERIES SCI
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  • Abstract
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Problems solved by technology

However, due to the specificity of Edwardsiella tarda's own biological characteristics, habitat conditions, nutritional requirements, and artificial cultivation methods, it is difficult to l

Method used

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  • Vacuum freeze-drying protective agent for Edwardsiella tarda and freeze-drying method thereof
  • Vacuum freeze-drying protective agent for Edwardsiella tarda and freeze-drying method thereof

Examples

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Embodiment 1

[0019] Several turbot fish with "spleen and kidney nodule" were collected from a turbot breeding plant in Weihai City, Shandong Province, and they were packed and oxygenated and brought back to the laboratory of the Yellow Sea Fisheries Research Institute of the Chinese Academy of Fishery Sciences for pathogen analysis. separation. A few spleen and kidney tissues of the diseased fish were cut aseptically, put into a sterile petri dish and washed several times with sterilized 1.5% NaCl solution, and then the tissues were cut into pieces. A small amount of chopped tissue was picked with an inoculating loop and streaked on tryptone soy broth agar to isolate bacteria. The streaked medium was placed in a constant temperature incubator at 28°C for 48h, and a single colony was selected from the dominant colonies with the same morphology, and then transferred to a new tryptone soybean broth agar medium for streak culture. Repeat the above steps 2 to 3 times until pure culture colonie...

Embodiment 2

[0028] In 2012, the Yellow Sea Fisheries Research Institute of the Chinese Academy of Fishery Sciences was exchanged and presented a strain of Edwardsiella blunted by Ocean University of China, which was used in the Luria-Bertani agar medium (Luria-Bertani agar medium ( LB medium) isolated by streak culture method can cause "ascites disease" of flounder, resulting in serious death.

[0029]In the laboratory, this strain was transferred with tryptone soy broth agar medium, and the pure culture of this strain of Edwardsiella tarda was obtained by streak culture. Pick a single colony of pure culture and inoculate it into tryptone soybean broth liquid medium, place it in a biochemical incubator at 28°C and 150rpm for 18h, then centrifuge at 4000r / min for 8min, discard the supernatant, and collect the bacterial cells Precipitate, add a sterile NaCl solution with a concentration of 1.5% that is about 15 times the volume of the cell body, and fully shake it evenly, centrifuge again a...

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Abstract

The invention provides a vacuum freeze-drying protective agent for Edwardsiella tarda and a freeze-drying method thereof, and belongs to the technical field of vacuum freeze-drying preservation of marine bacteria. The vacuum freeze-drying protective agent comprises the following components: dried skim milk, lactose, mycose, vitamin C, NaCl and sterile water. The viable bacterium suspension of the Edwardsiella tarda is sufficiently and evenly mixed with the freeze-drying protective agent in the volume ratio of 1: 4 to 1: 1 under a sterile condition; the mixed solution is subpackaged, and quickly frozen for 5-6 hours under the condition of -80 DEG C and then freeze-dried for 18 hours under the conditions of the vacuum degree of 0.28 mbar and the freeze-drying chamber temperature of -54 DEG C; next, the degree of vacuum is reduced to 0.034 mbar, and the subpackaged solutions are continuously freeze-dried for 2 hours at the temperature of -54 DEG C. After the completion of freeze-drying, 99.99% dry carbon dioxide gas is introduced into the freeze-drying chamber to achieve the standard atmospheric pressure and then the freeze-drying chamber is sealed with a gland. According to the invention, a technical method for long-term vacuum freeze-drying preservation of the Edwardsiella tarda is established, and technical support is provided for carrying out the related research of the Edwardsiella tarda in a deep-going way.

Description

technical field [0001] The invention belongs to the technical field of freeze-drying preservation of marine bacteria, and in particular relates to a vacuum freeze-drying protective agent suitable for Edwardsiella indolus and a freeze-drying method thereof. Background technique [0002] Edwardsiella tarda, also known as Edwardsiella tarda, is widely distributed in marine and freshwater environments worldwide. It is an important pathogen of aquatic organisms and can cause fish, amphibians and other aquatic economic animals. The disease poses a serious threat to aquaculture production (Zheng Dahai et al. 2004). Therefore, the research on Edwardsiella tarda has gradually become a hot spot in the field of aquaculture etiology. Scholars at home and abroad have conducted in-depth research on the classification, pathogenic mechanism, antigenic characteristics and vaccines of Edwardsiella tarda ( Matt J. Griffin, et al. 2013, Jong Earn Yu, et al. 2013, Meng Zhang, et al. 2012, Seung...

Claims

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Application Information

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IPC IPC(8): C12N1/04C12R1/01
Inventor 张正王印庚于永翔廖梅杰荣小军史秀清李彬王岚
Owner YELLOW SEA FISHERIES RES INST CHINESE ACAD OF FISHERIES SCI
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