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

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

Active Publication Date: 2015-03-25
YELLOW SEA FISHERIES RES INST CHINESE ACAD OF FISHERIES SCI
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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 learn from the preservation methods of other microbial strains. The method is a technical problem that must be solved to achieve freeze-drying and long-term preservation of Edwardsiella tarda

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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Effect test

Embodiment 1

[0019] Several diseased turbot fish suffering from "spleen-kidney sarcoidosis" were collected from a turbot breeding factory in Weihai City, Shandong Province. 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 pathogenic testing. separate. Aseptically cut the spleen and kidney tissues of a little diseased fish, put them into a sterile petri dish, wash them gently with sterilized 1.5% NaCl solution several times, and then cut the tissues into pieces. Use an inoculation loop to pick a small amount of chopped tissue, and streak the bacteria on the tryptone soybean broth agar medium. The streaked culture medium was placed in a constant temperature incubator at 28°C for 48 hours, and a single colony was selected from the dominant colonies with consistent morphology, and then transferred to a new tryptone soybean broth agar medium for streak culture. Repeat the above steps...

Embodiment 2

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

[0029]In the laboratory, the strain was cultured with tryptone soybean broth agar medium, and the pure culture of this strain of Edwardsiella tarda was obtained by streak culture. Pick a purely cultured single colony and inoculate it in tryptone soybean broth liquid medium, place it in a biochemical incubator and culture it at 28°C and 150rpm for 18h, centrifuge at 4000r / min for 8min, discard the supernatant, and collect the bacteria For precipitation, add a sterile NaCl solution with a concentration of 1.5% that is about 15 times the volume of the bacteria, shake it well, and then centrifuge at 4000r / min for 8 minutes, discard the supernatant, collect the sedim...

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Abstract

The invention discloses a freeze-vacuum drying protection agent for Edwardsiella tarda and a freeze-drying method thereof, belonging to the technical field of freeze-vacuum drying preservation of marine bacteria. The ingredients are: skimmed milk powder, lactose, trehalose, vitamin C, NaCl and sterile water. Under aseptic conditions, mix the live bacterial suspension of Edwardsiella tarda and the above-mentioned freeze-drying protectant solution in a volume ratio of 1:4 to 1:1, divide them into separate packages, and quickly freeze them at -80°C After 5 to 6 hours, freeze-dry for 18 hours at a vacuum degree of 0.28mbar and a freeze-drying chamber temperature of -54°C for 18 hours, then reduce the vacuum degree to 0.034mbar and continue to freeze-dry for 2 hours at a temperature of -54°C. After the freeze-drying is completed, fill the freeze-drying cabin with 99.99% dry carbon dioxide gas to standard atmospheric pressure and seal the cover. The invention establishes a technical method for long-term preservation of Edwardsiella tarda by freeze-drying and vacuum drying, and provides technical support for in-depth related research on Edwardsiella tarda.

Description

technical field [0001] The invention belongs to the technical field of marine bacteria freeze-drying and vacuum-drying preservation, and in particular relates to a vacuum freeze-drying protective agent suitable for Edwardsiella tarda and a freeze-drying method thereof. Background technique [0002] Edwardsiella tarda (Edwardsiella tarda), also known as Edwardsiella tarda, is widely distributed in sea and freshwater environments worldwide. The disease poses a serious threat to the breeding 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 Hyuk Choi, et al. 2011). [0003] In the past ten years, with the rapid developmen...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12R1/01C12N1/04
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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