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Strain preservation method of anaerobic bacteria

A technology for strain preservation and anaerobic bacteria, which is applied in the field of microorganisms, can solve the problems of short preservation period, high preservation cost, and high risk factor of liquid nitrogen, and achieve the effect of reducing preservation cost

Inactive Publication Date: 2019-07-26
ANHUI RUISIWEIER TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although the method of regular transplantation and preservation is simple to operate, the preservation period is relatively short, usually transferred once every 1 to 3 months, and it is easy to contaminate bacteria or cause death, and the strains are also prone to mutation; although the preservation method of freeze-drying method has a long period of preservation , but the survival rate of strictly anaerobic bacteria is not high due to the easy contact with oxygen during the operation; while the liquid nitrogen cryopreservation method has a longer storage period, because the liquid nitrogen is extremely volatile, the storage cost is higher, and the risk factor of liquid nitrogen Higher; and -80 ℃ cryopreservation is relatively general in the operation technology of anaerobic bacteria preservation

Method used

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  • Strain preservation method of anaerobic bacteria

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Experimental program
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Effect test

Embodiment 1

[0026] Embodiment 1: the preparation of liquid culture medium

[0027] Weigh 0.5g MES, 0.5g KH 2 PO 4 , 0.2g Na 2 SO 4 , 1.0g NaCl, 0.4g MgCl 2 ·6H 2 O, 0.3gNaHCO 3 , 0.15g CaCl 2 2H 2 O, 1.0g yeast extract, 1.5g peptone, 0.5g D-glucose, 1.0g L-cysteine ​​hydrochloride, 6mg resazurin were added to the beaker, and then 5g soluble starch was weighed and added to the small beaker Heat an appropriate amount of pure water until completely dissolved, pour 200mL pit mud extract, 100mL yellow water and completely dissolved starch into a beaker, add water to 1L, mix well and dissolve, then adjust the pH value between 6.5-7 During the period, the medium was divided into 100mL anaerobic bottles with butyl stoppers, and N 2 Remove oxygen until the color of the indicator disappears, and sterilize at 115°C for 20 minutes to obtain a liquid medium.

Embodiment 2

[0029] The present embodiment is the preservation method of single anaerobic strain, has the following steps:

[0030] (1) Inoculate the anaerobic bacteria Oscillibacter valericigenes in the liquid medium prepared in Example 1 and cultivate to the logarithmic phase;

[0031] (2) Use a sterile syringe to draw 1mL of Oscillibacter valericigenes bacterial solution into a sterile deoxygenated 5mL cryopreservation tube, take 1mL of antifreeze and add it to the cryopreservation tube, turn the cryopreservation tube upside down several times to make the antifreeze and bacterial solution fully Mix well, then add 0.5mL liquid paraffin after deoxygenation and sterilization to isolate the contact between the bacteria liquid and oxygen;

[0032] (3) Preserve the strain Oscillibacter valericigenes in 5 tubes, mark the cryopreservation tubes, and store them in a -80°C ultra-low temperature freezer.

[0033] (4) When resuscitating, take out a cryopreservation tube from the refrigerator and s...

Embodiment 3

[0036] Ruminococcaceae bacterium, Clostridium guangxiense, Clostridium saccharobutylicum, Clostridium celerecrescens and other anaerobic bacteria were preserved according to the strain preservation method in Example 2, and the survivability of the preserved strains after recovery in different time periods within two years was detected, and the results are shown in Table 1.

[0037] Table 1 Detection of strain viability in different storage periods

[0038]

[0039] It can be seen from Table 1 that all the strains of anaerobic bacteria preserved by the method of the present invention survived after recovery at different time periods within two years.

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Abstract

The invention discloses a strain preservation method of anaerobic bacteria. The strain preservation method comprises the steps of firstly inoculating a strain to be preserved into an anaerobic liquidculture medium, executing culturing to enable the strain to grow to a logarithmic phase; transferring the log-phase fresh culture bacterial solution into a strain preservation and freezing storage tube in an anaerobic workstation by using a syringe, adding an anti-freezing agent, oscillating and uniformly mixing the bacterial solution; and finally adding a liquid paraffin-oxygen isolating agent and then executing cryopreservation at the temperature of -80 DEG C. The strain preservation method is suitable for long-term preservation of anaerobic bacteria (facultative anaerobic bacteria / absoluteanaerobic bacteria). By adopting the preservation method , the preservation cost of the strain can be greatly reduced, the strain can be effectively preserved for a long time under an anaerobic condition, and the research and utilization of anaerobic microorganisms are facilitated.

Description

technical field [0001] The invention relates to a method for preserving strains of anaerobic bacteria, which belongs to the technical field of microorganisms. Background technique [0002] Microbial strain preservation technology is a very basic and important technology in the field of microbiology. Anaerobic microorganisms are a type of microorganisms that need to grow and reproduce normally under the condition of no molecular oxygen or low redox potential. Due to the physiological toxicity of oxygen to anaerobic microorganisms, the key to its cultivation and preservation technology is to keep such microorganisms in an environment with no oxygen or low redox potential. Therefore, the long-term preservation technology of anaerobic bacteria has the problems of relatively cumbersome operation and relatively low success rate. [0003] Existing strain preservation methods for anaerobic bacteria, such as: regular transplantation preservation method, freeze-drying method, liquid...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/04
CPCC12N1/04
Inventor 孟雅静王艳丽张会敏李静心何宏魁刘国英王录
Owner ANHUI RUISIWEIER TECH
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