CN115369040A - Preservation reagent, kit and preservation method for haemophilus parasuis strain - Google Patents

Preservation reagent, kit and preservation method for haemophilus parasuis strain Download PDF

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CN115369040A
CN115369040A CN202211182578.4A CN202211182578A CN115369040A CN 115369040 A CN115369040 A CN 115369040A CN 202211182578 A CN202211182578 A CN 202211182578A CN 115369040 A CN115369040 A CN 115369040A
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haemophilus parasuis
preservation
reagent
preserving
parasuis strain
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CN115369040B (en
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邓红玉
张蓉
沈俊杰
凌勇
丁能水
邓政
龙毅
张国辉
吴有林
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Fujian Aoxin Biotechnology Group Co ltd
Fujian Aoxin Seed Industry Technology Group Co ltd
Guangxi Kexin Yuanyuan Pig Co ltd
Ji'an Aobao Biotechnology Co ltd
Taihe Aomu Breeding Co ltd
Fujian Aonong Biological Technology Group Co Ltd
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Fujian Aoxin Seed Industry Technology Group Co ltd
Guangxi Kexin Yuanyuan Pig Co ltd
Ji'an Aobao Biotechnology Co ltd
Taihe Aomu Breeding Co ltd
Fujian Aonong Biological Technology Group Co Ltd
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Abstract

The invention discloses a preservation reagent, a kit and a preservation method for haemophilus parasuis strains, and relates to the field of microorganisms, the preservation reagent provided by the invention comprises 50-150 g/L of skimmed milk powder, 1-10 g/L of sucrose, 1-10 g/L of glucose, 1-50 g/L of sodium chloride, 20-80 ml/L of serum and 1-10 g/L of L-arginine hydrochloride, the components of the reagent are simple to prepare, the preserved strains have high recovery survival rate, the 3-year survival rate of the haemophilus parasuis is 93% when the haemophilus parasuis is preserved in a low-temperature environment of-20 ℃, the strains can be preserved for a long time, the biological characteristics and the toxicity of the haemophilus parasuis strains are not influenced after the long-term preservation, infectious microbes are not easily polluted, the problems that other preservation methods are easy to change and the recovery survival rate is low are solved, and a way is provided for the long-term preservation of the haemophilus parasuis strains.

Description

Preservation reagent, kit and preservation method for haemophilus parasuis strain
Technical Field
The invention relates to the field of microorganisms, and in particular relates to a preservation reagent, a kit and a preservation method for a haemophilus parasuis strain.
Background
The preservation of strains is a technology for maintaining the biological characteristics, activity and genetic characteristics of microbial strains, and the basic principle is to minimize the energy requirement of the life activities of the strains and limit the metabolism of the microorganisms to a minimum range. The strain preservation method is various, and in the application process, a proper method is selected according to the preservation purpose of different strains in a laboratory.
The haemophilus parasuis, haemophilus suis of the pasteurellaceae family, is gram negative, with extra-membrane proteins, pili and a capsule. The Haemophilus parasuis has high requirements on culture conditions and storage, has poor resistance to the external environment, can lose activity in a low-temperature environment, is not easy to separate clinically, has low separation rate, is not easy to store, and brings inconvenience to subsequent further identification and research. At present, the haemophilus parasuis is mainly preserved by adopting a glycerin physiological saline freezing preservation method, a vacuum freezing drying preservation method, a common inclined plane low-temperature preservation method and a liquid paraffin low-temperature preservation method; the glycerol physiological saline is frozen and preserved, and the common inclined plane is preserved at low temperature, because the physical and chemical characteristics of the culture medium are unstable, frequent generation change is needed, so that the microorganism metabolism is changed, the preserved strains are easy to generate variation, the chance of being polluted by other mixed bacteria is increased, haemophilus parasuis can not be preserved for a long time, and the survival rate of the recovered strains is not high; when the liquid paraffin is used for low-temperature preservation of the strain for recovery, a culture is taken from the lower side of the liquid paraffin, and when the inoculating loop is burnt on flame, the culture is easy to splash together with residual liquid paraffin to form aerosol, so that certain difficulty is brought to biosafety protection; the vacuum freeze drying preservation requires special equipment, has large technical difficulty and is expensive, and is difficult to realize by a general unit.
Therefore, the search for a preservation method which is simple and convenient to operate, not easy to pollute and high in survival rate and can preserve the haemophilus parasuis strain for a long time is very necessary for the subsequent pathological research, vaccine development and the like of the separated haemophilus parasuis disease.
In view of the above, the present invention is particularly proposed.
Disclosure of Invention
The invention aims to provide a preservation reagent, a kit and a preservation method for a haemophilus parasuis strain.
The invention is realized in the following way:
in a first aspect, the embodiment of the present invention provides a preservation reagent for haemophilus parasuis strains, which comprises the following components and final concentrations of the components: 50-150 g/L of skimmed milk powder, 1-10 g/L of sucrose, 1-10 g/L of glucose, 1-50 g/L of sodium chloride, 20-80 ml/L of serum and 1-10 g/L of L arginine hydrochloride.
In a second aspect, the embodiments of the present invention provide a kit for preserving a haemophilus parasuis strain, which comprises a preserving agent for a haemophilus parasuis strain as described in the previous embodiments.
In a third aspect, the embodiments of the present invention provide a method for preparing a preservation reagent for haemophilus parasuis strains, which comprises mixing the components as described in the previous embodiments.
The invention has the following beneficial effects:
(1) The strain frozen by the specific components and the specific proportion of the preservation reagent can be repeatedly frozen and thawed for use, so that the change of the biological characteristics of the strain and the chance of contaminating mixed bacteria caused by the passage times of the strain are reduced;
(2) The formula configuration is simple to operate, and the resuscitation survival rate is high;
(3) The method can be used for storing the haemophilus parasuis strain for a long time, and provides guarantee for the subsequent research of vaccines and strains;
(4) The biological characteristics and the toxicity of the haemophilus parasuis are not influenced after long-term storage, and the method is a reliable method for storing the haemophilus parasuis strain.
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.
The embodiment of the invention provides a preservation reagent for haemophilus parasuis strains, which comprises the following components in final concentration: 50-150 g/L of skimmed milk powder, 1-10 g/L of sucrose, 1-10 g/L of glucose, 1-50 g/L of sodium chloride, 20-80 ml/L of serum and 1-10 g/L of L arginine hydrochloride.
And (3) skim milk powder: providing growth factors, carbon sources, nitrogen sources, energy sources, inorganic salts and the like for the preserved haemophilus parasuis; the main components of the skimmed milk powder are protein and saccharide, wherein the diameter of the protein is between 1nm and 2nm, the aqueous solution of the skimmed milk powder is a colloidal solution, and the protein molecules are much smaller than the bacteria, so that the bacterial particles are surrounded by the protein molecules, not only can prevent the bacterial polymerization, but also are beneficial to absorption and transformation, maintain the bacterial activity, stabilize the cell membrane in a low-temperature environment, provide a protective coat for the cells, and avoid the damage to the preserved haemophilus parasuis in the low-temperature environment.
In some embodiments, the final concentration of the skim milk powder may be specifically any one or a range between any two of 50g/L, 60g/L, 70g/L, 80g/L, 90g/L, 100g/L, 110g/L, 120g/L, 130g/L, 140g/L, and 150 g/L.
Sucrose: the small molecular membrane external protective agent can inhibit the phase change of cell membranes and keep the liquid crystal state.
In some embodiments, the final concentration of sucrose may be any one or a range between any two of 1g/L, 2g/L, 3g/L, 4g/L, 5g/L, 6g/L, 7g/L, 8g/L, 9g/L, and 10g/L.
Glucose: can provide energy for cell metabolism, and can also reduce the temperature of membrane phase transition and inhibit the fusion of biological membranes in the freeze-drying process.
In some embodiments, the final concentration of glucose may specifically be any one or a range between any two of 1g/L, 2g/L, 3g/L, 4g/L, 5g/L, 6g/L, 7g/L, 8g/L, 9g/L, and 10g/L.
L arginine hydrochloride: the molecular structure and charge distribution of arginine can form multiple hydrogen bonds, so that arginine can be combined with molecules with negative charges to maintain the stability of protein.
In some embodiments, the final concentration of L arginine hydrochloride can be specifically any one or a range between any two of 1g/L, 2g/L, 3g/L, 4g/L, 5g/L, 6g/L, 7g/L, 8g/L, 9g/L, and 10g/L.
Serum: providing nutritional factors required for survival for preserved haemophilus parasuis: such as plasma proteins, fats, carbohydrates, hormones, polypeptides, etc., which are indispensable for the growth of Haemophilus parasuis.
In some embodiments, the serum comprises calf serum.
In some embodiments, the final concentration of the serum may be any one or a range between any two of 20ml/L, 25ml/L, 30ml/L, 35ml/L, 40ml/L, 45ml/L, 50ml/L, 55ml/L, 60ml/L, 65ml/L, 70ml/L, 75ml/L, and 80 ml/L.
Sodium chloride: maintaining osmotic pressure balance inside and outside the cell.
In some embodiments, the final concentration of sodium chloride can be any one or a range between any two of 1g/L, 5g/L, 10g/L, 15g/L, 20g/L, 25g/L, 30g/L, 35g/L, 40g/L, 45g/L, and 50 g/L.
In some embodiments, the preservation reagent further comprises a solvent for dissolving the component.
In some embodiments, the solvent comprises ddH 2 O。
The components of the preservation reagent provided by the invention are simple to prepare, and under the preparation of the specific components and the specific proportion thereof, the defatted milk powder, the glucose and the sucrose provide required nutritional factors for the growth and survival of the haemophilus parasuis, and simultaneously, the shape of a cell membrane is maintained, so that the cell membrane is prevented from finding phase change under a low-temperature environment; the hydrogen ions in the L arginine hydrochloride can maintain the stability of the skimmed milk powder protein, can be repeatedly frozen and thawed for use, reduces the change of the biological characteristics of the strain and the chance of contaminating other bacteria caused by the passage times of the strain, has high recovery survival rate of the stored strain, does not influence the biological characteristics and the toxicity of the haemophilus parasuis strain after long-term storage, and provides a way for the long-term storage of the haemophilus parasuis strain.
In some preferred embodiments, the preservation reagent comprises the following components and final concentrations of the components: 80-120 g/L of skimmed milk powder, 1-5 g/L of sucrose, 2-8 g/L of glucose, 20-40 g/L of sodium chloride, 40-60 ml/L of serum and 1-5 g/L of L-arginine hydrochloride.
In some preferred embodiments, the components and final concentrations of the components are included as follows: 90-110 g/L of skimmed milk powder, 1-4 g/L of sucrose, 2-6 g/L of glucose, 25-35 g/L of sodium chloride, 45-55 ml/L of serum and 1-5 g/L of L-arginine hydrochloride.
In some embodiments, the preservation reagent further comprises a pH adjuster.
In some embodiments, the pH adjusting agent is added in an amount to adjust the pH of the preservation agent to 6.5 to 7.5. Alternatively, the pH adjuster may specifically adjust the pH of the preservative agent to any one of pH 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4 and 7.5 or a range between any two of them.
In some preferred embodiments, the pH adjusting agent is added in an amount to adjust the pH of the preservation agent to 6.8 to 7.2.
In some embodiments, the pH adjusting agent comprises K 2 HPO 4 . K2HPO4 can act as a buffer and adjust pH.
The embodiment of the invention also provides a kit for preserving the haemophilus parasuis strain, which comprises a preserving reagent for the haemophilus parasuis strain as described in any of the preceding embodiments.
In some embodiments, the preservation kit further comprises reagents and/or equipment for culturing, inoculating, or both culturing and inoculating a haemophilus parasuis strain.
The embodiment of the invention also provides a preparation method of the preservation reagent for the haemophilus parasuis strain, which comprises mixing the components as described in any embodiment.
In some embodiments, when a pH adjusting agent is present, the step of mixing comprises: according to the final concentration, firstly, the skimmed milk powder, the cane sugar, the glucose, the sodium chloride and the L-arginine hydrochloride are mixed, then the pH value of the mixed solution is adjusted to 6.5-7.5 by adopting a pH adjusting reagent, and then the serum is added for mixing. The adjustment of the pH can be carried out in particular as described with reference to any of the preceding examples.
In some embodiments, the method further comprises sterilizing the pH-adjusted mixed solution prior to adding the serum.
In some embodiments, the sterilization conditions are 110 to 130 ℃ for 5 to 20min. Specifically, the temperature for sterilization may be any one or a range between any two of 110 ℃, 115 ℃, 120 ℃, 125 ℃ and 130 ℃; the time for sterilization may be any one or a range between any two of 5min, 10min, 15min, and 20min.
The embodiment of the invention also provides a method for preserving the haemophilus parasuis strain, which comprises the step of preserving the haemophilus parasuis strain by using the preservation reagent for the haemophilus parasuis strain as described in any embodiment or the preservation kit for the haemophilus parasuis strain as described in any embodiment.
In some embodiments, the saving method comprises: mixing a haemophilus parasuis strain with a preservation reagent for said haemophilus parasuis strain.
In some embodiments, the preservation method further comprises preserving the mixed product at a preservation temperature of-40 ℃ to 0 ℃. The preservation temperature can be any one or any two of-40 deg.C, -35 deg.C, -30 deg.C, -25 deg.C, -20 deg.C, -15 deg.C, -10 deg.C, -5 deg.C and deg.C.
Preferably, the preservation temperature is-25 ℃ to-15 ℃.
The features and properties of the present invention are described in further detail below with reference to examples.
Experimental materials:
the standard strain Nagasaki of Haemophilus parasuis used in the following examples and test examples was stored in the laboratory.
Example 1
A preservation reagent for haemophilus parasuis strains, comprising: 50g/L skimmed milk powder, 3g/L sucrose, 4g/L glucose, 30g/L sodium chloride, 50ml/L calf serum, 3g/L arginine hydrochloride and K 2 HPO 4 (adjust reagent pH to 7.0).
It will be understood that the concentrations of the components described above are the final concentrations of the components in the preservative.
Example 2
A preservation reagent for a haemophilus parasuis strain comprising: 100g/L skimmed milk powder, 3g/L sucrose, 4g/L glucose, 30g/L sodium chloride, 50ml/L calf serum, 3g/L arginine hydrochloride and K 2 HPO 4 (reagent pH was adjusted to 7.0).
Example 3
A preservation reagent for haemophilus parasuis strains, comprising: 150g/L skimmed milk powder, 3g/L sucrose, 4g/L glucose, and chlorineSodium chloride 30g/L, calf serum 50ml/L, L arginine hydrochloride 3g/L and K 2 HPO 4 (adjust reagent pH to 7.0).
Test example 1
3 test groups were set, each prepared according to the above-described group allocation of examples 1 to 3, at a ratio of ddH per 1L 2 Adding skimmed milk powder of the above group 3, sucrose 0.3g, glucose 0.4g, sodium chloride 3g, arginine hydrochloride 0.3g into the mixture, and adding K 2 HPO 4 Adjusting the pH value of the reagent to 7.0; sterilizing at 121 ℃ for 15min under high pressure, cooling, and aseptically adding 50mL of calf serum; respectively taking 3 groups of prepared 100mL of preservation reagent of the invention, placing the 3 groups of the preservation reagent in 2mL of sterile cryopreservation tubes, wherein each tube is 1.5mL, and the total number of the tubes is 60, and then picking 2 rings of fresh colonies of haemophilus parasuis, placing the 2 rings in the cryopreservation tubes, and preserving the colonies at the temperature of minus 20 ℃; the haemophilus parasuis preserved by the 3 groups of methods are randomly selected from 15 tubes for resuscitation respectively at 6 months, 12 months, 18 months, 24 months, 30 months and 36 months after cryopreservation, the resuscitation method comprises the steps of unfreezing the cryopreserved tubes by 37 ℃ water bath, then culturing in serum broth for 24 hours, then selecting the resuscitative bacterial liquid by using an inoculating loop, uniformly coating the resuscitative bacterial liquid on an improved TSA culture medium, culturing for 24 hours at 37 ℃, observing whether bacterial colonies are generated, and calculating the survival rate of the haemophilus parasuis. The survival rate is calculated as follows: survival = number of surviving tubes/number of selected tubes, and the results are shown in table 1.
TABLE 1 resuscitative survival rate (%) of Hps strain after preservation of different skim milk powder contents
Group of 6 months old 12 months old 18 months old 24 months 30 months old 36 months old
50g/L skimmed milk powder set (example 1) 100％ 100％ 90％ 80％ 70％ 40％
100g/L skimmed milk powder set (example 2) 100％ 100％ 100％ 100％ 93％ 93％
150g/L skimmed milk powder set (example 3) 100％ 100％ 80％ 50％ 0％ 0％
The survival rate of the three groups is 100% when the three groups are stored for 12 months, but the survival rate of the 150g/L skimmed milk powder group and the 50g/L skimmed milk powder group is lower than that of the 100g/L skimmed milk powder group after 18 months, which shows that the storage method of the 100g/L skimmed milk powder group is more favorable for long-term storage of the Hps strain.
Test example 2
The test is divided into 3 groups, namely an experimental group, a glycerin low-temperature preservation group and a liquid paraffin low-temperature preservation group; and (5) carrying out a haemophilus parasuis strain preservation test comparison.
(1) Experimental groups: taking 30mL of prepared preservation reagent of the invention in the embodiment 1, respectively placing the preservation reagent in 2mL of sterile cryopreservation tubes, wherein each tube is 1.5mL, and the number of the tubes is 91, then picking up a fresh colony 2 ring of haemophilus parasuis, placing the colony in the cryopreservation tubes, and preserving at-20 ℃;
(2) Glycerol cryopreservation group: and (3) preparing a preservation solution from glycerol and normal saline according to the proportion of 8. After the Hps strain liquid is cultured to be in an excellent state, under the aseptic condition, taking a culture medium and a preservation solution to mix according to the proportion of 5;
(3) Liquid paraffin low-temperature storage group: after the liquid paraffin is sterilized by high pressure, the separated and purified haemophilus parasuis colony is inoculated on a slant culture medium by an inoculating loop, 91 parts in total are cultured for 24 hours at 37 ℃, then aseptic liquid paraffin is added, and the mixture is sealed by 1cm higher than the culture medium and is preserved at the temperature of minus 20 ℃.
Repeated freeze-thaw survival status: randomly selecting 1 tube of Haemophilus parasuis stored by the 3 groups of methods for repeated freeze thawing test, wherein the test method comprises the steps of placing the preserved Haemophilus parasuis at-20 ℃ for 48h, and then placing the Haemophilus parasuis in a water bath at 37 ℃ for rapid thawing; completely unfreezing, placing at-20 ℃ for 48h, and then quickly unfreezing in 37 ℃ water bath; the freeze thawing is repeated for 2 times, 4 times and 6 times, then the recovered bacteria liquid is picked by an inoculating loop and evenly coated on an improved TSA culture medium, the bacteria liquid is cultured for 24 hours at 37 ℃, and whether the bacteria liquid is generated or not is observed.
The results show that the haemophilus parasuis stored in the experimental group still generates colonies after the experimental group is repeatedly frozen and thawed for 6 times, and neither the glycerol low-temperature storage group nor the liquid paraffin low-temperature storage group generates colonies after being repeatedly frozen and thawed for 2 times.
And (3) survival rate detection: and randomly selecting 15 tubes of haemophilus parasuis stored by the 3 groups of methods for resuscitation respectively at 6 months, 12 months, 18 months, 24 months, 30 months and 36 months after cryopreservation, and performing resuscitation treatment, wherein the resuscitation method comprises the steps of quickly thawing the cryopreserved tubes by using a water bath at 37 ℃, then placing the cryopreserved tubes into serum broth for culturing for 24 hours, then selecting the resuscitation bacteria liquid by using an inoculating loop, uniformly coating the resuscitation bacteria liquid on an improved TSA culture medium, culturing for 24 hours at 37 ℃, observing whether colonies are generated or not, and calculating the survival rate of the haemophilus parasuis. The survival rate is calculated as follows: survival = number of surviving tubes/number of selected tubes, and the results are shown in table 2.
TABLE 2 Resuscitation survival (%)
Group of 6 months old 12 months old 18 months old 24 months 30 months old 36 months old
Experimental group 100％ 100％ 100％ 100％ 93％ 93％
Glycerol cryopreservation group 100％ 80％ 33％ 13％ 0％ 0％
Liquid paraffin low-temperature storage group 100％ 86％ 40％ 13％ 0％ 0％
The recovery survival rates of the Hps strains of the glycerol cryopreservation group and the liquid paraffin cryopreservation group are reduced at 12 months, 18 months and 24 months, the recovery survival rates of the Hps strains at 30 months and 36 months are 0, but the survival rate of the strains of the experimental group is 93% at 36 months, compared with the fact that the preservation method of the experimental group is more beneficial to long-term preservation of the Hps strains.
Detection of biological characteristics of Hps strains: before the strains are subjected to preservation tests and after the strains are preserved for the longest time by using the preservation methods, the strains are respectively taken to be subjected to biological characteristic detection, including hemolysis, NAD-dependency, gram stain, urease, indole, hydrogen sulfide, nitrate reduction tests and catalase tests, and the differences of the biological characteristics of the Hps strains under the methods are compared, and the differences are shown in Table 3.
TABLE 3 examination results of biological characteristics of Hps strains before and after preservation
Item Before preservation Experimental group Glycerol cryopreservation group Liquid paraffin low-temperature storage group
Hemolytic property + + + +
NAD dependence + + + +
Blue dyeing Positive for Positive for Positive for Positive for
Urease - - - -
Indoles - - - -
Hydrogen sulfide - - - -
Nitrate reduction - - - -
Contact enzyme + + + +
It can be seen from table 3 that the biological properties of the Hps strains of the 3 groups are almost identical to those of the strains before preservation, indicating that the formulation components of the experimental group have no influence on the biological properties of the Hps strains and can be used for low-temperature preservation of the Hps strains.
And (3) detecting the toxicity of the preserved strain: before the strains are subjected to preservation test and after the strains are preserved for the longest time by using each preservation method, respectively taking the activated strains, inoculating the activated strains on an improved TSB culture medium, culturing for 17 hours at 37 ℃, adjusting the concentration to be 1.2 multiplied by 10CFU/mL, respectively injecting the activated strains into the abdominal cavity of 10 healthy Balb/c mice (with the weight of 20 g) with each mouse being 0.5mL, and judging the toxicity change condition of the preserved strains according to the death rate of the mice (repeating for 3 times and taking an average value). The test results are shown in Table 4.
TABLE 4 detection results of Hps strains virulence
Figure BDA0003865858230000101
The average lethality of the haemophilus parasuis stored in the experimental group to the mice after the preservation reaches 90%, the average lethality of the glycerol low-temperature storage group and the liquid paraffin low-temperature storage group to the mice is respectively 50% and 30%, and the results are obviously lower than that of the experimental group, which indicates that the virulence of the haemophilus parasuis stored in the experimental group is basically not changed obviously, and the average lethality of the haemophilus parasuis stored in the experimental group for 3 years is still 90%.
Example 4
A preservation reagent for a haemophilus parasuis strain comprising: 100g/L skimmed milk powder, 3g/L sucrose, 4g/L glucose, 30g/L sodium chloride, 50ml/L calf serum, 1g/L arginine hydrochloride and K 2 HPO 4 (adjust reagent pH to 7.0).
It will be understood that the concentrations of the components described above are the final concentrations of the components in the preservative.
Example 5
A preservation reagent for haemophilus parasuis strains, comprising: 100g/L skimmed milk powder, 3g/L sucrose, 4g/L glucose, 30g/L sodium chloride, 50ml/L calf serum, 3g/L arginine hydrochloride and K 2 HPO 4 (adjust reagent pH to 7.0).
Example 6
A preservation reagent for haemophilus parasuis strains, comprising: 100g/L skimmed milk powder, 3g/L sucrose, 4g/L glucose, 30g/L sodium chloride, 50ml/L calf serum, 5g/L arginine hydrochloride and K 2 HPO 4 (adjust reagent pH to 7.0).
Test example 3
Setting 3 test groups, preparing according to the components of the above examples 1-3, respectively, adding 100g/L skimmed milk powder, 0.3g of sucrose, 0.4g of glucose, 3g of sodium chloride and arginine hydrochloride of the above 3 groups into 1L ddH2O, and then adjusting the pH of the reagent to 7.0 by using K2HPO 4; sterilizing at 121 ℃ under high pressure for 15min, cooling, and aseptically adding 5mL of calf serum; respectively taking 3 groups of prepared 100mL of preservation reagent of the invention, placing the 3 groups of the preservation reagent in 2mL of sterile cryopreservation tubes, wherein each tube is 1.5mL, and the total number of the tubes is 60, and then picking 2 rings of fresh colonies of haemophilus parasuis, placing the 2 rings in the cryopreservation tubes, and preserving the colonies at the temperature of minus 20 ℃; and randomly selecting 15 tubes of haemophilus parasuis stored by the 3 groups of methods for resuscitation respectively at 6 months, 12 months, 18 months, 24 months, 30 months and 36 months after cryopreservation, and performing resuscitation treatment, wherein the resuscitation method comprises the steps of quickly thawing the cryopreserved tubes by using a water bath at 37 ℃, then placing the cryopreserved tubes into serum broth for culturing for 24 hours, then selecting the resuscitation bacteria liquid by using an inoculating loop, uniformly coating the resuscitation bacteria liquid on an improved TSA culture medium, culturing for 24 hours at 37 ℃, observing whether colonies are generated or not, and calculating the survival rate of the haemophilus parasuis. The survival rate is calculated as follows: survival = number of surviving tubes/number of selected tubes, and the results are shown in table 5.
TABLE 5 Resuscitation survival (%) -of Hps strains after preservation of different L-arginine hydrochloride content
Group of 6 months old 12 months old 18 months old 24 months 30 months old 36 months old
L arginine hydrochloride 1g/L (example 4) 100％ 98％ 86％ 70％ 0％ 0％
L arginine hydrochloride 3g/L (example 5) 100％ 100％ 100％ 100％ 93％ 93％
L arginine hydrochloride 5g/L (example 6) 100％ 97％ 80％ 60％ 0％ 0％
The survival rate of the three groups is 100% when the three groups are stored for 6 months, but the survival rate of the L arginine hydrochloride of 1g/L and 3g/L is obviously reduced after 12 months, and the survival rate is reduced to 0% after 30 months, so that the method for storing the L arginine hydrochloride of 3g/L is more favorable for the long-term storage of the Hps strain.
Example 7
A preservation reagent for haemophilus parasuis strains, comprising: 100g/L skimmed milk powder, 3g/L sucrose, 4g/L glucose, 30g/L sodium chloride, 20ml/L calf serum, 3g/L arginine hydrochloride and K 2 HPO 4 (adjust reagent pH to 7.0).
It will be understood that the concentrations of the individual components described above are the final concentrations of the individual components in the preservative.
Example 8
A preservation reagent for a haemophilus parasuis strain comprising: 100g/L skimmed milk powder, 3g/L sucrose, 4g/L glucose, 30g/L sodium chloride, 50ml/L calf serum, 3g/L arginine hydrochloride and K 2 HPO 4 (adjust reagent pH to 7.0).
Example 9
A preservation reagent for a haemophilus parasuis strain comprising: 100g/L skimmed milk powder, 3g/L sucrose, 4g/L glucose, 30g/L sodium chloride, 80ml/L calf serum, 3g/L arginine hydrochloride and K 2 HPO 4 (adjust reagent pH to 7.0).
Test example 4
Setting 3 test groups, respectively preparing according to the components of the above examples 1-3, adding 100g/L skimmed milk powder, 0.3g sucrose, 0.4g glucose, 3g sodium chloride and 3g/L arginine hydrochloride of the above 3 groups into 1L ddH2O, and then adjusting the pH of the reagent to 7.0 by using K2HPO 4; sterilizing at 121 deg.C under high pressure for 15min, cooling, and adding calf serum according to the above addition; respectively taking 3 groups of prepared 100mL of preservation reagent of the invention, placing the 3 groups of the preservation reagent in 2mL of sterile cryopreservation tubes, wherein each tube is 1.5mL, and the total number of the tubes is 60, and then picking 2 rings of fresh colonies of haemophilus parasuis, placing the 2 rings in the cryopreservation tubes, and preserving the colonies at the temperature of minus 20 ℃; and randomly selecting 15 tubes of haemophilus parasuis stored by the 3 groups of methods for resuscitation respectively at 6 months, 12 months, 18 months, 24 months, 30 months and 36 months after cryopreservation, and performing resuscitation treatment, wherein the resuscitation method comprises the steps of quickly thawing the cryopreserved tubes by using a water bath at 37 ℃, then placing the cryopreserved tubes into serum broth for culturing for 24 hours, then selecting the resuscitation bacteria liquid by using an inoculating loop, uniformly coating the resuscitation bacteria liquid on an improved TSA culture medium, culturing for 24 hours at 37 ℃, observing whether colonies are generated or not, and calculating the survival rate of the haemophilus parasuis. The survival rate is calculated as follows: survival = number of surviving tubes/number of selected tubes, and the results are shown in table 5.
TABLE 6 Resuscitation survival Rate (%)
Group of 6 months old 12 months old 18 months old 24 months 30 months old 36 months old
20mL Calf serogroup (example 7) 100％ 77％ 50％ 0％ 0％ 0％
50mL Calf serogroup (example 8) 100％ 100％ 100％ 100％ 93％ 93％
80mL Calf serogroup (example 9) 100％ 87％ 40％ 0％ 0％ 0％
The survival rate of the three groups is 100% when the three groups are stored for 6 months, but the survival rate of the calf serum groups of 20mL and 80mL after 12 months is obviously reduced, and the survival rate of the calf serum groups of 24 months is reduced to 0%, so that the method for storing the calf serum groups of 50mL is more favorable for the long-term storage of the Hps strain.
The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims (10)

1. A preservation reagent for Haemophilus parasuis strains is characterized by comprising the following components in final concentration: 50-150 g/L of skimmed milk powder, 1-10 g/L of sucrose, 1-10 g/L of glucose, 1-50 g/L of sodium chloride, 20-80 ml/L of serum and 1-10 g/L of L arginine hydrochloride.
2. The agent for preserving a haemophilus parasuis strain according to claim 1, wherein the components and the final concentrations of the components are as follows: 80-120 g/L of skimmed milk powder, 1-5 g/L of sucrose, 2-8 g/L of glucose, 20-40 g/L of sodium chloride, 40-60 ml/L of serum and 1-5 g/L of L arginine hydrochloride;
preferably, the components and the final concentrations of the components are as follows: 90-110 g/L of skimmed milk powder, 1-4 g/L of cane sugar, 2-6 g/L of glucose, 25-35 g/L of sodium chloride, 45-55 ml/L of serum and 1-5 g/L of L arginine hydrochloride.
3. The agent for preserving a haemophilus parasuis strain according to claim 1 or 2, characterized in that the agent for preserving further comprises a pH adjusting agent;
preferably, the addition amount of the pH adjuster is to adjust the pH of the preservative agent to 6.5 to 7.5;
preferably, the addition amount of the pH regulator is to regulate the pH of the preservation reagent to 6.8-7.2;
preferably, the pH adjusting agent comprises K 2 HPO 4 。
4. The agent for preserving a haemophilus parasuis strain according to claim 1 or 2, wherein the serum comprises calf serum;
preferably, the preservation reagent further comprises a solvent for dissolving the component;
preferably, the solvent comprises ddH 2 O。
5. A kit for preserving a Haemophilus parasuis strain, characterized in that it comprises the preserving agent for a Haemophilus parasuis strain according to any one of claims 1 to 4.
6. A preparation method of a preservation reagent for a haemophilus parasuis strain is characterized by comprising the following steps: mixing the components of any one of claims 1 to 4.
7. The method for producing a preservation reagent for a haemophilus parasuis strain according to claim 6, wherein when a pH adjuster is contained, the step of mixing comprises: according to the final concentration, firstly, mixing the skimmed milk powder, the sucrose, the glucose, the sodium chloride and the L-arginine hydrochloride, then adjusting the pH of the mixed solution to 6.5-7.5 by adopting a pH adjusting reagent, and then adding the serum for mixing;
preferably, before the serum is added, the preparation method further comprises sterilizing the mixed solution after the pH adjustment;
preferably, the sterilization condition is 110-130 ℃ for 5-20 min.
8. A method for preserving a Haemophilus parasuis strain, characterized in that it comprises preserving a Haemophilus parasuis strain with the preservation reagent for a Haemophilus parasuis strain according to any one of claims 1 to 4 or the preservation kit for a Haemophilus parasuis strain according to claim 5.
9. The method for preserving a haemophilus parasuis strain according to claim 8, wherein the preservation method comprises: mixing a haemophilus parasuis strain with a preservation reagent for said haemophilus parasuis strain.
10. The method for preserving a haemophilus parasuis strain according to claim 8 or 9, further comprising preserving the mixed product at a preservation temperature of-40 ℃ to 0 ℃;
preferably, the preservation temperature is-30 ℃ to-20 ℃;
preferably, the preservation temperature is-25 ℃ to-15 ℃.
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