CN115369040B - 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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CN115369040B
CN115369040B CN202211182578.4A CN202211182578A CN115369040B CN 115369040 B CN115369040 B CN 115369040B CN 202211182578 A CN202211182578 A CN 202211182578A CN 115369040 B CN115369040 B CN 115369040B
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haemophilus parasuis
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strains
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CN115369040A (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 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
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Abstract

The invention discloses a preservation reagent, a kit and a preservation method of haemophilus parasuis strains, which relate to the field of microorganisms, wherein the preservation reagent 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 haemophilus parasuis under the low-20 ℃ environment is 93%, the strains can be preserved for a long time, the biological characteristics and toxicity of the haemophilus parasuis strains are not influenced after the long-term preservation, the problems that other preservation methods are easy to mutate 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, in particular to a preservation reagent, a kit and a preservation method for haemophilus parasuis strains.
Background
Strain preservation is a technology for maintaining biological characteristics, activity and genetic characteristics of microbial strains, and the basic principle is to minimize energy requirements of life activities of the strains and limit metabolism of the microorganisms to a minimum range. The strain preservation method is various, and in the application process, proper methods are selected according to different purposes of laboratory strain preservation.
Haemophilus parasuis genus haemophilus parasuis family, genus haemophilus of the family pasteurellaceae, has gram staining negative, has extramembranous proteins, pili and capsules. The culture condition and the preservation of haemophilus parasuis are very high, meanwhile, the resistance to the external environment is poor, the low-temperature environment can cause the loss of activity, the haemophilus parasuis is not easy to separate clinically, the separation rate is low, the haemophilus parasuis is not easy to preserve, and the inconvenience is brought to the subsequent further identification and research. At present, the preservation of haemophilus parasuis mainly adopts a glycerol normal saline freezing preservation method, a vacuum freeze drying preservation method, a common inclined plane low-temperature preservation method and a liquid paraffin low-temperature preservation method; the glycerol normal saline freezing preservation and the common inclined plane low-temperature preservation are unstable in physical and chemical characteristics of the culture medium, frequent substitution is needed, so that the metabolism of microorganisms is changed, the preserved strains are easy to produce variation, the probability of being polluted by other miscellaneous bacteria is increased, haemophilus parasuis cannot be preserved for a long time, and the survival rate of the recovered strains is low; when the liquid paraffin is used for low-temperature preservation and strain recovery, a culture is taken from the lower side of the liquid paraffin, and when an inoculating loop is burned on flame, the culture is easy to splash together with the residual liquid paraffin to form aerosol, so that a certain difficulty is brought to biological safety protection; the vacuum freeze drying preservation needs special equipment, has great technical difficulty and is expensive, and the vacuum freeze drying preservation is difficult to realize by common units.
Therefore, the preservation method for searching the haemophilus parasuis strain, which is simple and convenient to operate, is not easy to pollute, has high 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.
In view of this, the present invention has been made.
Disclosure of Invention
The invention aims to provide a preservation reagent, a kit and a preservation method for haemophilus parasuis strains.
The invention is realized in the following way:
in a first aspect, embodiments of the present invention provide a preservation reagent for haemophilus parasuis strains, comprising 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, an embodiment of the present invention provides a preservation kit for haemophilus parasuis strains, comprising a preservation reagent for haemophilus parasuis strains as described in the previous embodiment.
In a third aspect, embodiments of the present invention provide a method of preparing a preservation reagent for haemophilus parasuis strains, comprising mixing the components as described in the previous embodiments.
The invention has the following beneficial effects:
(1) The frozen strain adopting the specific components and the specific proportions of the preservation reagent can be repeatedly frozen and thawed for use, so that the change of biological characteristics of the strain and the possibility of contamination of mixed bacteria caused by the passage times of the strain are reduced;
(2) The formula configuration operation is simple, and the recovery survival rate is high;
(3) The method can be used for preserving haemophilus parasuis strains for a long time, and provides a guarantee for subsequent researches on vaccines and strains;
(4) The biological characteristics and toxicity of the haemophilus parasuis are not affected after long-term storage, and the method is a reliable method for storing haemophilus parasuis strains.
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present invention more clear, the technical solutions of the embodiments of the present invention will be clearly and completely described below. The specific conditions are not noted in the examples and are carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.
The embodiment of the invention provides a preservation reagent for haemophilus parasuis strains, which comprises the following components in percentage by weight: 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.
Skimmed 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 skim milk powder are protein and sugar, wherein the diameter of the protein is between 1nm and 2nm, the aqueous solution of the skim milk powder is colloid solution, and protein molecules are much smaller than bacteria, so that bacterial particles are in the surrounding of the protein molecules, thereby not only preventing the bacterial cells from polymerizing, but also facilitating the absorption and transformation, keeping the bacterial activity, simultaneously stabilizing the cell membrane in a low-temperature environment, providing a protective coating for the cells, and avoiding the damage to the stored haemophilus parasuis in the low-temperature environment.
In some embodiments, the final concentration of the skim milk powder may specifically be in a range between any one or 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 cell membrane from undergoing phase change so as to maintain the liquid crystal state.
In some embodiments, the final concentration of sucrose may be in a range between any one or 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, 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 in a range between any one or 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 may specifically be in a range between any one or 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 the preserved haemophilus parasuis with the nutritional factors required for survival: such as plasma proteins, fats, carbohydrates, hormones, polypeptides, etc., which are essential for the growth of haemophilus parasuis.
In some embodiments, the serum comprises calf serum.
In some embodiments, the final concentration of serum may be in a range between any one or 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 the osmotic pressure balance inside and outside the cell.
In some embodiments, the final concentration of sodium chloride may be in a range between any one or 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 components.
In some embodiments, the solvent comprises ddH 2 O。
The preservation reagent provided by the invention has simple component preparation, and under the preparation of the specific component and the specific proportion, the skim milk powder, glucose and sucrose together provide necessary nutrition factors for the growth and survival of haemophilus parasuis, and simultaneously maintain the shape of cell membranes, so as to prevent the cell membranes 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 caused by the passage times of the strain and the possibility of contaminating other bacteria, has high recovery survival rate of the preserved strain, does not influence the biological characteristics and toxicity of the haemophilus parasuis strain after long-term preservation, and provides a way for long-term preservation 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 included are 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 adjuster is added in an amount to adjust the pH of the preservation reagent to 6.5 to 7.5. Alternatively, the pH adjuster may specifically adjust the pH of the preservation reagent to any one or a range between any two of pH's of 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, and 7.5.
In some preferred embodiments, the pH adjustor is added in an amount to adjust the pH of the preservation reagent to between 6.8 and 7.2.
In some embodiments, the pH adjuster comprises K 2 HPO 4 . K2HPO4 can act as a buffer and adjust pH.
The embodiment of the invention also provides a preservation kit of the haemophilus parasuis strain, which comprises the preservation reagent of the haemophilus parasuis strain in any embodiment.
In some embodiments, the preservation kit further comprises reagents and/or equipment for culturing, inoculating a haemophilus parasuis strain.
The embodiment of the invention also provides a preparation method of the preservation reagent of the haemophilus parasuis strain, which comprises the steps of mixing the components in any embodiment.
In some embodiments, when a pH adjuster is included, the step of mixing includes: according to the final concentration, firstly mixing skimmed milk powder, sucrose, glucose, sodium chloride and L-arginine hydrochloride, then adopting a pH adjusting reagent to adjust the pH of the mixed solution to 6.5-7.5, and then adding serum for mixing. The pH adjustment is specifically as described in any of the previous examples.
In some embodiments, the method of preparing further comprises sterilizing the pH adjusted mixed solution prior to adding serum.
In some embodiments, the sterilization conditions are 110 to 130 ℃,5 to 20 minutes. Specifically, the sterilization temperature may be in a range between any one or any two of 110 ℃, 115 ℃, 120 ℃, 125 ℃, 130 ℃; the sterilization time may be in a range between any one or 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 adopting the preserving reagent of the haemophilus parasuis strain in any embodiment or the preserving kit of the haemophilus parasuis strain in any embodiment.
In some embodiments, the preservation method comprises: mixing haemophilus parasuis strain with a preservation reagent of the 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 specifically any one or any range between two of-40 ℃, -35 ℃, -30 ℃, -25 ℃, -20 ℃, -15 ℃, -10 ℃, -5 ℃ and ℃ inclusive.
Preferably, the preservation temperature is-25 ℃ to-15 ℃.
The features and capabilities of the present invention are described in further detail below in connection with the examples.
Experimental materials:
the standard strain Nagasaki of Bacillus parasuis used in the examples and test examples below was stored in the laboratory.
Example 1
A preservation reagent for a haemophilus parasuis strain, comprising: skimmed milk powder 50g/L, sucrose 3g/L, glucose 4g/L, sodium chloride 30g/L, calf serum 50ml/L, L arginine hydrochloride 3g/L and K 2 HPO 4 (reagent pH was adjusted to 7.0).
It will be appreciated that the concentrations of the above components are the final concentrations of the components in the preserving agent.
Example 2
A preservation reagent for a haemophilus parasuis strain, comprising: 100g/L of skimmed milk powder, 3g/L of sucrose, 4g/L of glucose, 30g/L of sodium chloride and 5g of calf serum0ml/L, L arginine hydrochloride 3g/L and K 2 HPO 4 (reagent pH was adjusted to 7.0).
Example 3
A preservation reagent for a haemophilus parasuis strain, comprising: skimmed milk powder 150g/L, sucrose 3g/L, glucose 4g/L, sodium chloride 30g/L, calf serum 50ml/L, L arginine hydrochloride 3g/L and K 2 HPO 4 (reagent pH was adjusted to 7.0).
Test example 1
3 test groups were set up, each prepared according to the above components of examples 1 to 3, in a ratio of ddH per 1L 2 Adding the above 3 groups of skimmed milk powder, 0.3g sucrose, 0.4g glucose, 3g sodium chloride, 0.3g L arginine hydrochloride into O, and then using K 2 HPO 4 Adjusting the pH of the reagent to 7.0; sterilizing at 121deg.C for 15min, cooling, and aseptically adding calf serum 50mL; respectively taking 3 groups of 100mL of prepared preservation reagent of the invention, placing the 100mL of prepared preservation reagent in 2mL sterile freezing pipes, 1.5mL of prepared preservation reagent is taken out from each pipe, 60 pipes are taken out altogether, then picking up fresh colony 2 of haemophilus parasuis, placing the fresh colony 2 in the freezing pipes in a ring, and preserving the colony at-20 ℃; the haemophilus parasuis preserved by the 3 group method is prepared by randomly selecting 15 tubes for resuscitating treatment after 6 months, 12 months, 18 months, 24 months, 30 months and 36 months after freezing, wherein the resuscitating method comprises the steps of rapidly thawing the frozen tubes in a 37 ℃ water bath, placing the thawed tubes in serum broth for culturing for 24 hours, then picking up resuscitated bacterial liquid by an inoculating loop, uniformly coating the resuscitated bacterial liquid on an improved TSA culture medium, culturing for 24 hours at 37 ℃, observing whether bacterial colony is generated, and calculating the survival rate of the bacterial liquid. The calculation formula of the survival rate is as follows: survival = number of surviving tubes/number of tubes selected, test results are shown in table 1.
TABLE 1 recovery survival rates (%)
Group of 6 months of For 12 months 18 months of 24 months of For 30 months For 36 months
50g/L skimmed milk powder group (example 1) 100％ 100％ 90％ 80％ 70％ 40％
100g/L skimmed milk powder group (example 2) 100％ 100％ 100％ 100％ 93％ 93％
150g/L skimmed milk powder group (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, compared with the 100g/L skimmed milk powder group, the preservation method is more beneficial to long-term preservation of Hps strains.
Test example 2
The experiments are divided into 3 groups, namely an experiment group, a glycerol low-temperature preservation group and a liquid paraffin low-temperature preservation group; and (5) performing preservation test comparison of haemophilus parasuis strains.
(1) Experimental group: taking 30mL of prepared preservation reagent of the embodiment 1 of the invention, respectively placing the 30mL of prepared preservation reagent into 2mL sterile freezing pipes, 1.5mL of prepared preservation reagent is added into 91 pipes, then picking fresh colony 2 of haemophilus parasuis, placing the fresh colony 2 in the freezing pipes in a ring, and preserving the colony at-20 ℃;
(2) Glycerol cryopreservation group: preparing a preservation solution by glycerol and normal saline according to the proportion of 8:2, and sterilizing for later use. After the Hps strain is liquid-cultured to an excellent state, under the aseptic condition, mixing a culture medium and a preservation solution according to the proportion of 5:2, subpackaging into 10mL centrifuge tubes, adding 91 tubes, and preserving in a refrigerator at the temperature of minus 20 ℃;
(3) Liquid paraffin low-temperature preservation group: after autoclaving liquid paraffin, inoculating the separated and purified haemophilus parasuis colony onto slant culture medium with an inoculating loop, culturing at 37deg.C for 24 hr, adding sterile liquid paraffin, sealing 1cm above culture medium, and preserving at-20deg.C.
Repeated freeze thawing survival conditions: the haemophilus parasuis preserved by the 3 group method is randomly selected from 1 tube to carry out repeated freeze thawing test, wherein the test method is that the preserved haemophilus parasuis is firstly placed at the temperature of minus 20 ℃ for 48 hours and then placed in a water bath at the temperature of 37 ℃ for quick thawing; after complete thawing, placing at-20 ℃ for 48 hours, and then rapidly thawing in a water bath at 37 ℃; the procedure was repeated for 2, 4 and 6 times, and the resuscitated bacterial liquid was picked up with an inoculating loop, spread evenly on the modified TSA medium, cultured at 37℃for 24 hours, and observed for colony formation.
The results show that haemophilus parasuis stored in the experimental group still generates bacterial colonies after the experimental group is repeatedly frozen and thawed for 6 times, and the glycerol low-temperature preservation group and the liquid paraffin low-temperature preservation group do not generate bacterial colonies after the experimental group is repeatedly frozen and thawed for 2 times, so that the experimental group has the advantages of being capable of being used in anti-thawing and reducing strain passage compared with the other two groups.
Survival rate detection: the haemophilus parasuis preserved by the 3 group method is prepared by randomly selecting 15 tubes for resuscitating treatment after 6 months, 12 months, 18 months, 24 months, 30 months and 36 months after freezing, wherein the resuscitating method comprises the steps of rapidly thawing the frozen tubes in a 37 ℃ water bath, placing the thawed tubes in serum broth for culturing for 24 hours, then picking up resuscitated bacterial liquid by an inoculating loop, uniformly coating the resuscitated bacterial liquid on an improved TSA culture medium, culturing for 24 hours at 37 ℃, observing whether bacterial colony is generated, and calculating the survival rate of the bacterial liquid. The calculation formula of the survival rate is as follows: survival = number of surviving tubes/number of tubes selected, test results are shown in table 2.
TABLE 2 recovery survival rate of Hps strain after preservation (%)
Group of 6 months of For 12 months 18 months of 24 months of For 30 months For 36 months
Experimental group 100％ 100％ 100％ 100％ 93％ 93％
Glycerol low-temperature preservation group 100％ 80％ 33％ 13％ 0％ 0％
Liquid paraffin low-temperature preservation group 100％ 86％ 40％ 13％ 0％ 0％
The recovery survival rate of Hps strains in the glycerol low-temperature storage group and the liquid paraffin low-temperature storage group which are stored for 12 months, 18 months and 24 months is reduced, and the recovery survival rate of Hps strains in 30 months and 36 months is 0, but the survival rate of the strains in the experimental group is 93% in 36 months, which is compared with the method for storing the experimental group, which is more beneficial to long-term storage of Hps strains.
Detection of biological properties of Hps strains: before the strains are subjected to the preservation test and after the strains are preserved for the longest time by using each preservation method, the strains are respectively taken for biological property detection, including hemolysis, NAD-dependence, gram staining, urease, indole, hydrogen sulfide, nitrate reduction test and contact enzyme test, and the difference of the biological properties of Hps strains under each method is compared, and is shown in Table 3.
TABLE 3 results of detection of biological Properties of Hps Strain before and after preservation
Project Before preservation Experimental group Glycerol low-temperature preservation group Liquid paraffin low-temperature preservation group
Hemolysis treatment + + + +
NAD dependence + + + +
Gram staining Positive and negative Positive and negative Positive and negative Positive and negative
Urease enzyme - - - -
Indole compounds - - - -
Hydrogen sulfide - - - -
Nitrate reduction - - - -
Contact enzyme + + + +
The biological properties of the Hps strains in the 3 groups are almost consistent with those of the Hps strains before preservation, which shows that the formula components in the experimental groups have no influence on the biological properties of the Hps strains, and the Hps strains can be preserved at low temperature.
Stored strain virulence detection: before the strain is subjected to a preservation test and after the strain is preserved for the longest time by using each preservation method, the strain is respectively inoculated on a modified TSB culture medium after being activated, the strain is cultured for 17 hours at 37 ℃, the concentration is adjusted to be 1.2 multiplied by 10CFU/mL, 10 healthy Balb/c mice (weight 20 g) are respectively subjected to intraperitoneal injection, each 0.5mL, and the toxicity change condition of the preserved strain is judged according to the death rate of the mice (repeated 3 times, and an average value is obtained). The test results are shown in Table 4.
TABLE 4 Hps Strain virulence detection results
The average mortality rate of the haemophilus parasuis preserved in the experimental group after preservation to mice reaches 90%, the average mortality rates of the glycerol low-temperature preservation group and the liquid paraffin low-temperature preservation group to the mice are respectively 50% and 30%, which are obviously lower than those of the experimental group, and the average mortality rate of the haemophilus parasuis preserved in the experimental group after preservation for 3 years still reaches 90%.
Example 4
A preservation reagent for a haemophilus parasuis strain, comprising: skimmed milk powder 100g/L, sucrose 3g/L, glucose 4g/L, sodium chloride 30g/L, calf serum 50ml/L, L arginine hydrochloride 1g/L and K 2 HPO 4 (reagent pH was adjusted to 7.0).
It will be appreciated that the concentrations of the above components are the final concentrations of the components in the preserving agent.
Example 5
A preservation reagent for a haemophilus parasuis strain, comprising: skimmed milk powder 100g/L, sucrose 3g/L, glucose 4g/L, sodium chloride 30g/L, calf serum 50ml/L, L arginine hydrochloride 3g/L and K 2 HPO 4 (reagent pH was adjusted to 7.0).
Example 6
A preservation reagent for a haemophilus parasuis strain, comprising: skimmed milk powder 100g/L, sucrose 3g/L, glucose 4g/L, sodium chloride 30g/L, calf serum 50ml/L, L arginine hydrochloride 5g/L and K 2 HPO 4 (reagent pH was adjusted to 7.0).
Test example 3
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 arginine hydrochloride of the above 3 groups into ddH2O of 1L, and then regulating the pH of the reagent to 7.0 by using K2HPO 4; sterilizing at 121deg.C for 15min, cooling, and aseptically adding calf serum 5mL; respectively taking 3 groups of 100mL of prepared preservation reagent of the invention, placing the 100mL of prepared preservation reagent in 2mL sterile freezing pipes, 1.5mL of prepared preservation reagent is taken out from each pipe, 60 pipes are taken out altogether, then picking up fresh colony 2 of haemophilus parasuis, placing the fresh colony 2 in the freezing pipes in a ring, and preserving the colony at-20 ℃; the haemophilus parasuis preserved by the 3 group method is prepared by randomly selecting 15 tubes for resuscitating treatment after 6 months, 12 months, 18 months, 24 months, 30 months and 36 months after freezing, wherein the resuscitating method comprises the steps of rapidly thawing the frozen tubes in a 37 ℃ water bath, placing the thawed tubes in serum broth for culturing for 24 hours, then picking up resuscitated bacterial liquid by an inoculating loop, uniformly coating the resuscitated bacterial liquid on an improved TSA culture medium, culturing for 24 hours at 37 ℃, observing whether bacterial colony is generated, and calculating the survival rate of the bacterial liquid. The calculation formula of the survival rate is as follows: survival = number of surviving tubes/number of tubes selected, test results are shown in table 5.
TABLE 5 recovery survival rates (%)
Group of 6 months of For 12 months 18 months of 24 months of For 30 months For 36 months
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 storage method of the L-arginine hydrochloride of 3g/L is more beneficial to long-term storage of Hps strains compared with the method.
Example 7
A preservation reagent for a haemophilus parasuis strain, comprising: skimmed milk powder 100g/L, sucrose 3g/L, glucose 4g/L, sodium chloride 30g/L, calf serum 20ml/L, L arginine hydrochloride 3g/L and K 2 HPO 4 (reagent pH was adjusted to 7.0).
It will be appreciated that the concentrations of the above components are the final concentrations of the components in the preserving agent.
Example 8
A preservation reagent for a haemophilus parasuis strain, comprising: skimmed milk powder 100g/L, sucrose 3g/L, glucose 4g/L, sodium chloride 30g/L, calf serum 50ml/L, L arginine hydrochloride 3g/L and K 2 HPO 4 (reagent pH was adjusted to 7.0).
Example 9
A preservation reagent for a haemophilus parasuis strain, comprising: skimmed milk powder 100g/L, sucrose 3g/L, glucose 4g/L, sodium chloride 30g/L, calf serum 80ml/L, L arginine hydrochloride 3g/L and K 2 HPO 4 (reagent pH was adjusted 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 ddH2O of 1L, and then regulating the pH of the reagent to 7.0 by using K2HPO 4; sterilizing at 121deg.C for 15min, cooling, and adding calf serum aseptically according to the above steps; respectively taking 3 groups of 100mL of prepared preservation reagent of the invention, placing the 100mL of prepared preservation reagent in 2mL sterile freezing pipes, 1.5mL of prepared preservation reagent is taken out from each pipe, 60 pipes are taken out altogether, then picking up fresh colony 2 of haemophilus parasuis, placing the fresh colony 2 in the freezing pipes in a ring, and preserving the colony at-20 ℃; the haemophilus parasuis preserved by the 3 group method is prepared by randomly selecting 15 tubes for resuscitating treatment after 6 months, 12 months, 18 months, 24 months, 30 months and 36 months after freezing, wherein the resuscitating method comprises the steps of rapidly thawing the frozen tubes in a 37 ℃ water bath, placing the thawed tubes in serum broth for culturing for 24 hours, then picking up resuscitated bacterial liquid by an inoculating loop, uniformly coating the resuscitated bacterial liquid on an improved TSA culture medium, culturing for 24 hours at 37 ℃, observing whether bacterial colony is generated, and calculating the survival rate of the bacterial liquid. The calculation formula of the survival rate is as follows: survival = number of surviving tubes/number of tubes selected, test results are shown in table 5.
TABLE 6 recovery survival rates (%)
Group of 6 months of For 12 months 18 months of 24 months of For 30 months For 36 months
20mL calf serum group (example 7) 100％ 77％ 50％ 0％ 0％ 0％
50mL calf serum group (example 8) 100％ 100％ 100％ 100％ 93％ 93％
80mL calf serum group (example 9) 100％ 87％ 40％ 0％ 0％ 0％
The survival rate of the three groups is 100% when the groups are stored for 6 months, but the survival rate of the calf serogroup with 20mL and 80mL is obviously reduced after 12 months and after 24 months, and the survival rate is reduced to 0%, so that the preservation method of the calf serogroup with 50mL is more beneficial to long-term preservation of Hps strains compared with the preservation method of the calf serogroup with 50 mL.
The above description is only of the preferred embodiments of the present invention and is not intended to limit the present invention, but various modifications and variations can be made to the present invention by those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims (8)

1. A preservation reagent for haemophilus parasuis strains, which is characterized in that the components and the final concentration of the components are as follows: pH regulator, skimmed milk powder 90-110 g/L, sucrose 1-4 g/L, glucose 2-6 g/L, sodium chloride 25-35 g/L, serum 45-55 ml/L, L arginine hydrochloride 2-4 g/L and ddH 2 O; the serum is calf serum, and the pH regulator is K 2 HPO 4 The pH regulator is added in an amount to regulate the pH of the preservation reagent to 6.8-7.2.
2. A preservation kit for a haemophilus parasuis strain, comprising the preservation reagent for a haemophilus parasuis strain according to claim 1.
3. The method for preparing a preservation reagent for haemophilus parasuis strain according to claim 1, comprising: all the components described in claim 1 are mixed.
4. The method for preparing a preservation reagent for haemophilus parasuis strain according to claim 3, wherein the step of mixing comprises: according to the final concentration, firstly mixing skimmed milk powder, sucrose, glucose, sodium chloride and L-arginine hydrochloride, then adopting a pH adjusting reagent to adjust the pH of the mixed solution to 6.8-7.2, and then adding serum for mixing.
5. The method for preparing a preservation reagent for a haemophilus parasuis strain according to claim 4, wherein the method further comprises sterilizing the mixed solution after the pH adjustment before adding the serum.
6. The method for preparing a preservation reagent for haemophilus parasuis strain according to claim 5, wherein the sterilization condition is 110-130 ℃ for 5-20 min.
7. A method for preserving a haemophilus parasuis strain, comprising preserving the haemophilus parasuis strain with a preserving reagent of the haemophilus parasuis strain according to claim 1 or a preserving kit of the haemophilus parasuis strain according to claim 2, wherein the preserving temperature is-25 ℃ to-15 ℃.
8. The preservation method of haemophilus parasuis strain according to claim 7, characterized in that the preservation method comprises: mixing the haemophilus parasuis strain with the preservation reagent of the haemophilus parasuis strain of claim 1.
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