CN113046272B - Low-temperature preservation method of pathogenic strains - Google Patents
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Abstract
The invention relates to a low-temperature preservation method of pathogenic strains, belonging to the technical field of pathogenic strain preservation. The invention provides a bacterium for low-temperature preservation of yersinia, which comprises the following steps: mixing a low-temperature protective agent with the bacterial suspension, uniformly shaking, and preserving at-70 ℃ to-90 ℃, wherein the low-temperature protective agent comprises: glycerol, sucrose, L-carnitine and PBS buffer solution, wherein the molar ratio of the glycerol to the sucrose to the L-carnitine is (2-9): (0.2-0.9): (0.5-2). In the low-temperature preservation method of pathogenic bacteria, the single protective agent glycerol, sucrose and L-carnitine are mixed to serve as the composite protective agent, and the preservation effect is obviously better than that of the single protective agent, so that the low-temperature protective agent provided by the invention has higher survival rate on low-temperature preserved plague bacilli, and meanwhile, the low-temperature preservation method of pathogenic bacteria provided by the invention not only can reduce the toxicity of a system, but also has better preservation effect, and thus, the plague bacilli can be effectively preserved.
Description
Technical Field
The invention belongs to the technical field of pathogenic strain preservation, and particularly relates to a low-temperature preservation method of a pathogenic strain.
Background
The preservation method of microbial (toxin) species can be divided into four types on the whole: subculture preservation method, drying preservation method, freeze drying method and low temperature preservation method. The cryopreservation method is a reliable method for long-term preservation of most microorganisms, morphological and physiological characteristics are more stable after cryopreservation, and no infrastructure (except a refrigerator at-80 ℃ or liquid nitrogen (196 ℃)) is required, so the cryopreservation method is most commonly used in laboratory research, and has the advantages of long preservation time, wide application range and low biological information variation rate.
Yersinia pestis (Yersiniapestis, abbreviated as plague) is the causative agent of human and animal plagues, and its main host is rodent mammal, and the vector is blood sucking insect. Plague bacilli can exist in different forms in soil for a long time, so that researches on the preservation of plague bacilli in soil under natural and laboratory conditions are more. At present, most of laboratory preserved strains are preserved in a semi-solid state, the method has short preservation time, needs periodic subculture, is easy to mutate, and has less related researches on low-temperature preservation of plague bacillus in a laboratory.
Disclosure of Invention
The present invention has been made to solve the above problems, and an object of the present invention is to provide a method for cryopreservation of pathogenic bacterial species.
The invention provides a method for the cryopreservation of pathogenic bacterial species, for the cryopreservation of yersinia bacteria, characterized in that it comprises the following steps: mixing a low-temperature protective agent with the bacterial suspension, uniformly shaking, and preserving at-70 ℃ to-90 ℃, wherein the low-temperature protective agent comprises: glycerol, sucrose, L-carnitine and PBS buffer solution, wherein the molar ratio of the glycerol to the sucrose to the L-carnitine is (2-9): (0.2-0.9): (0.5-2).
The method for low-temperature preservation of pathogenic bacteria provided by the invention also has the following characteristics: wherein the volume ratio of the cryoprotectant to the bacterial suspension is 1: (0.5-2), preferably 1:1.
the method for low-temperature preservation of pathogenic bacteria provided by the invention is also characterized in that a 2mL freezing tube is taken, 0.8mL of each of the cryoprotectant and the bacterial suspension with certain concentration is taken for 1:1 equal volume, shaking, and storing in a refrigerator at-80 deg.C or liquid nitrogen.
The method for low-temperature preservation of pathogenic bacteria provided by the invention also has the following characteristics: wherein the concentration of the bacterial suspension is 10 7 cfu/mL-10 9 cfu/mL, preferably 10 8 cfu/mL。
The method for low-temperature preservation of pathogenic bacteria provided by the invention also has the following characteristics: wherein the mol ratio of the glycerol to the sucrose to the L-carnitine is (8-9): (0.2-0.4): (0.5-0.7).
The method for preserving pathogenic bacteria at low temperature provided by the invention also has the following characteristics: wherein the molar volume ratio of the glycerol to the sucrose to the L-carnitine to the PBS buffer solution is (2-9) mol: (0.2-0.9) mol: (0.5-2) mol:1L of the compound.
The method for low-temperature preservation of pathogenic bacteria provided by the invention also has the following characteristics: wherein the Yersinia bacterium is Yersinia pestis.
The method for low-temperature preservation of pathogenic bacteria provided by the invention also has the following characteristics: the preparation method of the low-temperature protective agent comprises the following steps: dissolving glycerol in PBS buffer solution, and sterilizing to obtain solution A; dissolving sucrose in PBS buffer solution, and sterilizing to obtain solution B; dissolving L-carnitine in PBS buffer solution, and sterilizing to obtain solution C; and uniformly mixing the solution A, the solution B and the solution C to obtain the cryoprotectant.
The method for low-temperature preservation of pathogenic bacteria provided by the invention also has the following characteristics: the specific preparation method of the low-temperature protective agent comprises the following steps: 8M to 9M of glycerol is sterilized by high temperature and high pressure for 15min at 121 ℃,0.2M to 0.4M of sucrose is sterilized by high temperature and high pressure for 15min at 115 ℃, and 0.5M to 0.7M of L-carnitine is filtered and sterilized by a filter membrane with the aperture of 0.22 mu M; respectively preparing raw materials, and performing 1:1:1 equal volume is mixed evenly to obtain the low temperature protective agent.
The method for low-temperature preservation of pathogenic bacteria provided by the invention also has the following characteristics: wherein the volume ratio of the solution A to the solution B to the solution C is 1: (0.5-2): (0.5-2).
The method for low-temperature preservation of pathogenic bacteria provided by the invention also has the following characteristics: wherein the volume ratio of the solution A to the solution B to the solution C is 1: (0.8-1.2): (0.8-1.2).
The method for low-temperature preservation of pathogenic bacteria provided by the invention also has the following characteristics: the cryoprotectants included 8.46M glycerol, 0.584M sucrose, 0.62M l-carnitine, and Phosphate Buffered Saline (PBS).
The method for preserving pathogenic bacteria at low temperature provided by the invention also has the characteristics that: the enrichment of the plague bacillus thallus comprises the following specific steps: inoculating plague bacillus into a BHI liquid culture medium prepared in advance, loosening a circle of a centrifugal tube bottle cap after inoculation, placing the centrifugal tube bottle cap in an air shaking table, placing the plague bacillus in the air shaking table at 28 ℃ for overnight culture until the growth logarithmic phase, centrifuging by a low-temperature centrifuge 5411Xg for 10min, removing supernatant, and reserving thalli; adding 20ml PBS, blowing, shaking, resuspending, centrifuging to remove supernatant, retaining thallus, blending bacterial suspension with PBS to make the bacterial liquid concentration reach preservation concentration of 10 8 cfu/mL。
The method for the low-temperature preservation of pathogenic bacteria provided by the invention is also characterized by comprising the following steps: and (3) recovering the pestilence bacillus after low-temperature preservation, wherein when the pestilence bacillus is recovered, the frozen tube is taken out and is rewarmed for 3-5min by adopting a water bath at 35-40 ℃ until the solution system is completely melted, and then the strain is subcultured in a super clean workbench, wherein the water bath recovery temperature of the strain is preferably 37 ℃.
Action and Effect of the invention
The invention relates to a method for the cryopreservation of pathogenic bacterial species, which is used for the cryopreservation of yersinia bacteria, and comprises the following steps: mixing a low-temperature protective agent with the bacterial suspension, uniformly shaking, and preserving at-70 ℃ to-90 ℃, wherein the low-temperature protective agent comprises: glycerol, sucrose, L-carnitine and PBS buffer solution, wherein the molar ratio of the glycerol to the sucrose to the L-carnitine is (2-9): (0.2-0.9): (0.5-2). In the low-temperature preservation method of the pathogenic bacteria, the single protective agent glycerol, the sucrose and the L-carnitine are mixed to be used as the composite protective agent, and the preservation effect is obviously better than that of the single protective agent, so that the low-temperature protective agent provided by the invention has higher survival rate for low-temperature preservation of plague bacillus, and meanwhile, the low-temperature preservation method of the pathogenic bacteria not only can reduce the toxicity of a system, but also has better preservation effect, and therefore, the plague bacillus can be effectively preserved.
Furthermore, glycerol can be combined with hydrogen bonds of water molecules, so that the concentration of the solution is increased, the free diffusion movement of the water molecules in the solution is slowed down, the formation of ice crystals is inhibited, a cluster structure formed by hydrophobic interaction among hydroxyl groups is favorable for decomposing clusters among the water molecules, and the capacity of the water molecules for forming the ice crystals is weakened. Sucrose is a hydrophilic substance that can combine with free water to increase the concentration of the solution and replace the hydrated layer that is lost during freezing. The L-carnitine can form zwitterion molecules, and the positive and negative electric groups of the L-carnitine and water molecules can form ion solvation stronger than the acting force of hydrogen bonds.
Drawings
FIG. 1 is a graph showing the results of a cooling rate test of a plague bacillus vaccine strain in test example 3 of the present invention; and
FIG. 2 is a graph showing the results of the repeated freeze-thaw test of the vaccine strain of plague bacillus in test example 4 of the present invention.
Detailed Description
In order to make the technical means, creation features, achievement objects and effects of the present invention easy to understand, the method for low-temperature preservation of a pathogenic bacterial species of the present invention is specifically described below with reference to the following embodiments and accompanying drawings.
The following examples adopt the plague bacillus vaccine strains (gram negative bacteria) provided by the Chinese disease prevention and control center, and further test the influence of the cryoprotectant and the cryopreservation method of pathogenic bacteria provided by the following examples and test examples on the survival rate of the plague bacillus vaccine strains after preservation.
< example >
Low-temperature preservation method of pathogenic strains
The method specifically comprises the following steps:
step 1, preparing a cryoprotectant, comprising the following substeps:
preparing a glycerol solution: taking 10mL, 20mL and 30mL of glycerol by using a pipette, respectively placing the glycerol into a 100mL conical flask, adding 30mL of PBS solution, stirring to dissolve, adding the PBS solution to a constant volume in a 50mL volumetric flask, and sterilizing at 121 ℃ under high temperature and high pressure for 15min to obtain 2.82M, 5.64M and 8.46M glycerol.
Preparing a sucrose solution: weighing 5g, 10g and 15g of sucrose by an electronic balance, respectively placing the sucrose in a 100mL conical flask, adding 30mL of PBS solution, stirring to dissolve, adding the PBS solution to fix the volume in a 50mL volumetric flask, and sterilizing at 115 ℃ for 15min at high temperature and high pressure to obtain 0.292M, 0.584M and 0.876M sucrose.
Preparing an L-carnitine solution: 5g, 10g and 15g of L-carnitine are weighed by an electronic balance and respectively placed in a 100mL conical flask, and filtration sterilization is carried out by adopting a filter membrane with the aperture of 0.22 mu M to obtain 0.62M, 1.24M and 1.86M L-carnitine.
Preparing a composite protective agent: using minitab design orthogonal experiments, the separately formulated cryoprotectants were subjected to 1:1:1, uniformly mixing to obtain the mixed composite protective agent.
a small amount of the strain was picked up by an inoculating loop and applied to a 1% hemolytic Hach plate by three-region streaking. Another 1% hemolysis Henschel plate is coated with the whole plate by adopting the dense scribing method, a drop of phage is dropped on the upper side of the plate by using a 10mL pipette, and the plate is erected to leave a trace on the plate by the phage drop. Place the plate in CO 2 Culturing in an incubator at 28 deg.C for 48 hr to obtain opaque colony. A single colony of the plague bacillus vaccine strain is taken from a 1% hemolytic hertzian medium plate and inoculated in a BHI liquid medium, and is cultured for 24 hours overnight under the conditions that the rotating speed is 200r/min and the temperature is 28 ℃. According to the following steps of 1: transferring 100 vol% to BHI liquid culture medium, culturing to logarithmic growth phase, centrifuging for 10min with 5411Xg high speed centrifuge, removing supernatant,PBS was washed 3 times to leave the cells. Blending bacterial suspension with PBS to make bacterial liquid concentration reach 10 8 cfu/mL。
taking 2mL of a freezing storage tube, mixing the low-temperature protective agent with the concentration of 10 8 0.8mL of each cfu/mL of bacterial suspension was subjected to 1:1 equal volume, shaking, and storing in a refrigerator at-80 deg.C or liquid nitrogen.
And 4, recovering the pestis bacillus subjected to low-temperature preservation, which comprises the following specific steps:
when the recovery is used, taking out 2mL of the freezing tube, rewarming for 3min-5min by adopting a water bath at 37 ℃ until the solution system is completely melted, and then carrying out subculture on the strain.
< test example 1>
Survival rate test of single protective agent on plague bacillus vaccine strain
The test method comprises the following steps: the cryoprotectant in step 3 of the example was replaced with a single cryoprotectant of different concentrations as in table 1, and the survival rate effect test was performed after cryopreservation of plague bacillus according to steps 3 and 4.
The test results are shown in table 1.
TABLE 1 survival rate of single protectant against a vaccine strain of pestivirus after cryopreservation
From table 1, it can be seen that the survival rate of the plague bacillus vaccine strain can be significantly improved by using any single protectant, the survival rate of the plague bacillus vaccine strain is only 5.66% in the PBS control group without using the low-temperature protectant, and the low-temperature preservation effect is similar in the experimental group with the added glycerol, sucrose and l-carnitine as the protectant, and the survival rate is over 50%, which is 5.66% higher than that of the PBS control group.
< test example 2>
Survival rate test of composite protective agent on plague bacillus vaccine strain
The test method comprises the following steps: the cryoprotectant in step 3 of the example was replaced with the compound protectant with different formulations as in table 2, and the orthogonal experiment and survival rate impact test were performed as in table 2 after the plague bacillus was cryopreserved according to step 3 and step 4.
The test results are shown in table 2.
TABLE 2 survival rate of the compound protectant against the low-temperature preservation of plague bacillus vaccine strains
Wherein a, b, c, d mark significant differences.
As can be seen from table 2, using formulation 8: the survival rate of the plague bacillus vaccine strain can reach 90.73 percent at most when the glycerol is 8.46M, the sucrose is 0.584M and the L-carnitine is 0.62M.
The results of the orthogonal experiments were statistically analyzed according to table 2, and the results are shown in table 3.
TABLE 3 statistical analysis of plague bacilli
As can be seen from Table 3, the preservation effect of the mixed protectant is better than that of the single protectant, the primary and secondary sequence influencing the cryopreservation survival rate of the plague bacillus vaccine strain is L-carnitine > glycerol > sucrose, and the proportion of the optimal compound protectant system for low-temperature preservation of the plague bacillus vaccine strain is L-carnitine 0.62M, glycerol 8.46M and sucrose 0.292M.
Formulation verification of optimal composite protectant
An optimal compound protective agent system of the plague bacillus vaccine strain is as follows: l-carnitine of 0.62M, glycerol of 8.46M, and sucrose of 0.292M was tested according to the test method in test example 2, and the survival rate of 88.26% was obtained. The validation result is close to the highest survival rate of 90.73% obtained in the orthogonal experiment, and the survival rates are all higher than that of a single protective agent.
< test example 3>
Cooling rate test of plague bacillus vaccine strains
The test method comprises the following steps: selecting single protective agent glycerol with the best preservation effect as a protective agent, adopting a temperature acquisition instrument and a thermocouple building test bed to measure the cooling rate, setting the temperature acquisition instrument to record temperature change once every 15 seconds, obtaining a cooling curve, and obtaining the cooling rate of-1.635 ℃/min, -2.775 ℃/min, -3.677 ℃/min and-110 ℃/min through first-order derivation.
The test results are shown in fig. 1.
FIG. 1 is a graph showing the results of the cooling rate test of the vaccine strains of plague bacillus in this test example 3.
As can be seen from FIG. 1, the temperature with the best effect of low-temperature preservation of the plague bacillus vaccine strain is-110 ℃/min, and is-3.677 ℃/min, under the four cooling rates, the survival rate of the strains can reach more than 70% under the two cooling rates.
< test example 4>
Repeated freeze-thaw test of plague bacillus vaccine strains
The test method comprises the following steps: in a repeated freeze-thaw experiment, the temperature reduction rate of the bacterial suspension added with the protective agent is reduced to-80 ℃ by adopting-3.677 ℃/min, then the bacterial suspension is placed in a refrigerator at-80 ℃ for freezing storage, taken out every 48 hours and placed in a water bath kettle at 37 ℃, and the bacterial suspension is completely recovered and counted by adopting the rewarming rate of 25 ℃/min, so that the descending trend of live bacteria of the plague bacillus vaccine strain in the repeated freeze-thaw process is obtained. 2.82M glycerol, 0.292M sucrose, 0.31M L-carnitine and PBS buffer were subjected to 4 repeated freeze-thaw tests on the plague bacillus vaccine strain, and the test results are shown in FIG. 2.
FIG. 2 is a graph showing the results of the repeated freeze-thaw test of the vaccine strain of plague bacillus in this test example 4.
As can be seen from FIG. 2, the survival rates of the plague bacillus vaccine strains are all reduced along with the increase of the number of repeated freeze thawing times, the survival rate of the PBS control group after 4 repeated freeze thawing is almost 0,2.82M glycerol, the reduction range is the largest, and the preservation effect of 0.292M sucrose is the best. This may be related to the molecular structure of sucrose itself and the nutrition provided, and in most microorganisms during preservation, the small temperature change easily causes repeated freezing and thawing of the solution and damages cells, thereby affecting the survival rate of the bacterial strains.
Effects and effects of the embodiments
The method for cryopreservation of a pathogenic bacterial species according to this embodiment for cryopreservation of yersinia bacteria, comprises the steps of: mixing a low-temperature protective agent with the bacterial suspension, uniformly shaking, and preserving at-70 ℃ to-90 ℃, wherein the low-temperature protective agent comprises: glycerol, sucrose, L-carnitine and PBS buffer solution, wherein the molar ratio of the glycerol to the sucrose to the L-carnitine is (2-9): (0.2-0.9): (0.5-2). In the low-temperature preservation method of the pathogenic bacteria, the single protective agent glycerol, the sucrose and the L-carnitine are mixed to be used as the composite protective agent, and the preservation effect is obviously better than that of the single protective agent. Therefore, the cryoprotectant provided by the embodiment has a high survival rate for cryopreserving the plague bacillus, and meanwhile, the cryopreservation method of pathogenic bacteria provided by the embodiment can not only reduce the toxicity of a system, but also has a good preservation effect, so that the plague bacillus can be effectively preserved.
Furthermore, the optimal compound protective agent system of the plague bacillus vaccine strain comprises 0.62M L-carnitine, 8.46M glycerol and 0.292M sucrose.
Furthermore, the low-temperature preservation method of pathogenic bacteria provided by the embodiment can be used for future research and application of plague bacillus, thereby exerting the research value thereof to the maximum extent and having important significance on the work of preserving the natural biological resources with high quality.
The above embodiments are preferred examples of the present invention, and are not intended to limit the scope of the present invention.
Claims (4)
1. A method for cryopreservation of pathogenic bacterial species for the cryopreservation of yersinia spp, comprising the steps of:
mixing the cryoprotectant with the bacterial suspension, shaking uniformly, preserving at-70 to-90 ℃,
the cryoprotectant is composed of glycerol, sucrose, L-carnitine and PBS buffer solution, and is prepared by a cryoprotectant preparation method, wherein the cryoprotectant preparation method comprises the following steps:
dissolving the glycerol in the PBS buffer solution, and sterilizing to obtain solution A, wherein the concentration of the solution A is 2-9 mol/L;
dissolving the sucrose in the PBS buffer solution, and sterilizing to obtain a solution B, wherein the concentration of the solution B is 0.2-0.9 mol/L;
dissolving the L-carnitine in the PBS buffer solution, and sterilizing to obtain a solution C, wherein the concentration of the solution C is 0.5-2 mol/L;
and uniformly mixing the solution A, the solution B and the solution C in equal volume to obtain the cryoprotectant.
2. The method for the cryopreservation of pathogenic bacterial species according to claim 1, characterized in that:
wherein the volume ratio of the cryoprotectant to the bacterial suspension is 1: (0.5-2).
3. The method for the cryopreservation of pathogenic bacterial species according to claim 1, characterized in that:
wherein the concentration of the bacterial suspension is 10 7 cfu/mL-10 9 cfu/mL。
4. The method for the cryopreservation of pathogenic bacterial species as claimed in claim 1, characterized in that:
wherein the bacterium of the genus Yersinia is a Yersinia pestis.
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| DE102005060741A1 (en) * | 2005-12-16 | 2007-06-21 | Erbacher Gmbh & Co. Betriebs Kg | Instant dry sauce base and instant sauce base preparation process |
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| US20060257842A1 (en) * | 2003-05-29 | 2006-11-16 | Pettegrew Jay W | Cryopreservation media and molecules |
| CN105685016B (en) * | 2016-04-08 | 2018-06-08 | 天津大学 | Cell cryopreservation protection composition, the composition purposes and cell cryopreservation method |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| DE102005060741A1 (en) * | 2005-12-16 | 2007-06-21 | Erbacher Gmbh & Co. Betriebs Kg | Instant dry sauce base and instant sauce base preparation process |
| CN103301450A (en) * | 2013-06-04 | 2013-09-18 | 中国食品药品检定研究院 | Plague live vaccine freezing-drying protecting agent for percutaneous scarification |
| CN110476957A (en) * | 2019-09-12 | 2019-11-22 | 徐宁 | A kind of Cortex Eucommiae suspension cell cryopreservation method |
| CN111346222A (en) * | 2020-03-30 | 2020-06-30 | 中国人民解放军军事科学院军事医学研究院 | Preparation method of plague attenuated live vaccine dry powder |
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