CN112280713A - Special culture medium for clostridium emphysema, preparation method of special culture medium and preparation method of veterinary clostridium emphysema inactivated vaccine - Google Patents

Special culture medium for clostridium emphysema, preparation method of special culture medium and preparation method of veterinary clostridium emphysema inactivated vaccine Download PDF

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CN112280713A
CN112280713A CN202011207013.8A CN202011207013A CN112280713A CN 112280713 A CN112280713 A CN 112280713A CN 202011207013 A CN202011207013 A CN 202011207013A CN 112280713 A CN112280713 A CN 112280713A
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clostridium
emphysema
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罗宏亮
赵丽霞
韩四娥
史文瑞
舒秋婷
李超
姜娟
张一丹
刘国庆
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Inner Mongolia Bo Ao Biotechnology Co ltd
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    • A61K2039/552—Veterinary vaccine

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Abstract

The invention provides a special culture medium for clostridium emphysema and preparation method thereof, and a preparation method of a veterinary clostridium emphysema and gangrene inactivated vaccine, belonging to the technical field of veterinary biological products. The special culture medium for clostridium emphysema includes the following components by mass: 10-15 parts of peptone, 3-5 parts of tryptone, 3-5 parts of casein peptone, 1-3 parts of yeast extract powder, 1-3 parts of NaCl and KH2PO41-3 parts of Na2HPO4·12H210-20 parts of O and 5-10 parts of anhydrous glucose. The special culture medium for clostridium emphysema not only has convenient material acquisition, high quality and low price, but also has stable performance, and overcomes the defects of the traditional culture mediumHas a plurality of disadvantages and is suitable for the large-scale production of the emphysema gangrene inactivated vaccine for animals.

Description

Special culture medium for clostridium emphysema, preparation method of special culture medium and preparation method of veterinary clostridium emphysema inactivated vaccine
Technical Field
The invention belongs to the technical field of biological products for livestock, and particularly relates to a special culture medium for clostridium emphysema and preparation method thereof, and a preparation method of inactivated vaccine for emphysema and gangrene for livestock.
Background
Emphysema and cellulitis commonly called as carbuncle, also called black leg disease, is an acute septicemic zoonotic bacterial infectious disease caused by Clostridium pneumonectum (Clostridium chauvoei). The clostridium emphysema and the spores thereof enter a body through damaged skin or invade into injured mucous membranes of digestive tracts through gastrointestinal tracts, and generate hyaluronidase and toxin in the body in a mass mode, so that the injured tissues have the symptoms of high congestion, bleeding and plasma exudation, and the toxin generated in serious cases can block the operation of central nervous functions, thereby causing fatal toxemia or bacteremia. The disease is mostly caused by ruminants such as cattle, sheep and the like, and brings huge economic loss to the livestock breeding industry in China.
At present, the vaccine for preventing the epidemic disease in China is emphysema gangrene inactivated vaccine (formaldehyde vaccine and alum vaccine). In the preparation of emphysema gangrene inactivated vaccine in the industry, animal raw materials such as beef, beef liver and the like are used for preparing anaerobic pork liver soup (alum vaccine) and peptone pork liver soup (formaldehyde vaccine) culture media, and about 10 percent of liver blocks are usually added in large-scale production. The culture medium prepared by the method has the advantages that because the quality of raw materials is uneven and the difference between batches is large, the residual liver tissues are not easy to remove, the phenomena of unstable toxicity and poor protection of finished seedlings often occur, especially in recent years, due to the development of the domestic dairy industry, the feeding amount of dairy cows is rapidly increased, and accordingly, the quality of the raw materials for preparing the vaccine culture medium is reduced due to the fact that a large amount of eliminated milk beef is filled in the market; meanwhile, the residual drugs in the beef and the beef liver can also inhibit the growth of bacteria, and the quality of the vaccine is seriously influenced by the conditions; in addition, the traditional culture medium is complex in preparation process, long in time consumption, high in price, low in recovery rate, and not suitable for preparation in clean production places, so that huge waste and high cost are brought to vaccine production, and great trouble is caused to vaccine production enterprises. Therefore, there is an urgent need for a culture medium for fusobacterium emphysema, which is easy to obtain, excellent in quality, low in cost and stable in performance, and a culture method suitable for the culture medium.
Disclosure of Invention
In order to solve the problems, the invention provides a special culture medium for clostridium emphysema and a preparation method thereof, and a preparation method of a veterinary clostridium emphysema inactivated vaccine. The special culture medium for clostridium emphysema not only has convenient material acquisition, high quality and low price, but also has stable performance, overcomes a plurality of defects of the traditional culture medium, and is suitable for the large-scale production of the veterinary clostridium emphysema gangrene inactivated vaccine.
In order to achieve the above purpose, the invention provides the following technical scheme:
the invention provides a special culture medium for clostridium emphysema, which comprises the following components in parts by mass: 10-15 parts of peptone, 3-5 parts of tryptone, 3-5 parts of casein peptone, 1-3 parts of yeast extract powder, 1-3 parts of NaCl and KH2PO41-3 parts of Na2HPO4·12H210-20 parts of O and 5-10 parts of anhydrous glucose.
Preferably, the special culture medium for clostridium gangreniformis further comprises water.
Preferably, when the culture medium comprises water, the culture medium comprises the following components by mass per liter: 10-15 g of peptone, 3-5 g of tryptone, 3-5 g of casein peptone, 1-3 g of yeast extract powder, 1-3 g of NaCl and KH2PO4 1～3g、Na2HPO4·12H210-20 g of O and 5-10 g of anhydrous glucose.
Preferably, the pH value of the special culture medium for clostridium emphysema is 7.6-7.8.
The invention provides a preparation method of the special culture medium for clostridium emphysema, which comprises the following steps:
sterilizing a glucose aqueous solution prepared from anhydrous glucose to obtain a culture medium A solution special for clostridium emphysema;
dissolving the rest components with water, adjusting pH, and sterilizing to obtain culture medium B solution special for Clostridium pneumonectasis;
the special culture medium A solution for the clostridium emphysema is mixed with the special culture medium B solution for the clostridium emphysema to obtain the special culture medium for the clostridium emphysema.
The invention provides a culture method of clostridium emphysema, which comprises the following steps: inoculating the clostridium emphysema gangrensis seed liquid into the special culture medium for clostridium emphysema gangrensis in the technical scheme for culture to obtain the clostridium emphysema gangrensis liquid.
Preferably, the viable count of the clostridium emphysema gangrensis seed liquid is 1.0 x 106～2.5*106CFU/ml, the inoculation amount of the clostridium aerocoelions seed liquid is 3-5%.
Preferably, the temperature of the culture is 36-37 ℃; the culture time is 24-48 h.
The invention provides a preparation method of a clostridium emphysema gangrensis inactivated vaccine, which comprises the following steps: inactivating the clostridium emphysema bacterium liquid obtained by the culture method in the technical scheme to obtain an inactivated bacterium liquid; and preparing the inactivated bacterial liquid into an inactivated vaccine.
Preferably, the preparation method of the inactivated vaccine comprises directly preparing the inactivated bacterial liquid or mixing the inactivated bacterial liquid with an adjuvant.
Has the advantages that:
the invention provides a special culture medium for clostridium emphysema, which comprises the following components in parts by mass: 10-15 parts of peptone, 3-5 parts of tryptone, 3-5 parts of casein peptone, 1-3 parts of yeast extract powder, 1-3 parts of NaCl and KH2PO41-3 parts of Na2HPO4·12H210-20 parts of O and 5-10 parts of anhydrous glucose. The special culture medium for clostridium emphysema does not need to be limited to beef, beef liver and other raw materials which are difficult to store, high in cost and inconvenient to obtain and are used in the traditional anaerobic pork liver soup culture medium, can greatly reduce the production cost and simplify the operation steps; at the same timeThe special culture medium for clostridium emphysema does not contain the medicine and animal material residues carried by the traditional culture medium raw materials, has no inhibiting effect on the cultured bacteria, is convenient for purifying and concentrating thalli and toxoid, and provides possibility and convenience for preparing toxoid with low immune dose, toxoid plus thalli inactivated vaccine; in addition, the toxicity of the clostridium emphysema grade bacterial liquid obtained by the special culture medium for clostridium emphysema is more stable than that of the bacterial liquid prepared by the traditional culture medium, the batch difference is small, the cultured clostridium emphysema grade has stronger toxicity production capacity no matter the culture is carried out in a 500ml bottle or a fermentation tank, the toxicity production capacity is stable, the quality is controllable, and the guarantee is provided for the subsequent vaccine preparation. The results of the embodiment show that the clostridium pneumonectum cultured by the special culture medium for clostridium pneumonectum provided by the invention has stable virulence, and the virulence of three batches of continuous fermentation all meets the virulence standard of 'veterinary biological product regulation'.
Furthermore, the invention provides a preparation method of the clostridium emphysema gangrene inactivated vaccine, and the preparation method provided by the invention overcomes the problems of unstable quality and poor protective performance of finished seedlings caused by uneven quality of culture medium raw materials, large batch-to-batch difference, difficult removal of residual animal raw materials and the like. The results of the embodiment show that the safety of the clostridium emphysema inactivated vaccine provided by the invention is good, and the injection parts of the prepared 4 inactivated vaccines are not necrotized after subcutaneous injection of guinea pigs; meanwhile, the protective efficacy of 4 inactivated vaccines is evaluated on guinea pigs, and the inactivated vaccines have better protective power and reach and exceed the standard of Chinese veterinary biological product regulation.
Detailed Description
The invention provides a special culture medium for clostridium emphysema, which comprises the following components in parts by mass: 10-15 parts of peptone, 3-5 parts of tryptone, 3-5 parts of casein peptone, 1-3 parts of yeast extract powder, 1-3 parts of NaCl and KH2PO41-3 parts of Na2HPO4·12H210-20 parts of O and 5-10 parts of anhydrous glucose.
The special culture medium for clostridium emphysema includes, by mass, 10-15 parts of peptone, more preferably 10-12 parts, and still more preferably 10 parts. The mass concentration of the peptone is preferably 10-15 g/L, more preferably 10-12 g/L, and even more preferably 10 g/L. The source of the peptone has no special requirement, and the peptone can be obtained by adopting common commercial peptone, such as Boaoxing biotechnology, Limited liability company. The peptone is a water-soluble mixture of peptone, mainly peptone, polypeptide, dipeptide and amino acid, obtained by hydrolyzing ox bone, bone fascia and residual beef through enzyme, acid and alkali, and can provide nitrogen source, partial trace elements and growth factors for growth of clostridium tetani.
The special culture medium for clostridium emphysema includes, by mass, 3-5 parts of tryptone, preferably 4-5 parts, and more preferably 5 parts. The mass concentration of the tryptone is preferably 3-5 g/L, more preferably 4-5 g/L, and even more preferably 5 g/L. The invention has no special requirement on the source of the tryptone, and can be realized by adopting the common commercially available tryptone, such as Boaoxing Biotechnology, Inc. The tryptone in the special culture medium for clostridium emphysema is rich in trace elements and growth factors required by clostridium emphysema and promotes the growth of clostridium emphysema.
The special culture medium for clostridium emphysema includes, by mass, 3-5 parts of casein peptone, preferably 4-5 parts, and more preferably 5 parts. The mass concentration of the casein peptone is preferably 3-5 g/L, more preferably 4-5 g/L, and even more preferably 5 g/L. The invention has no special requirement on the source of the casein peptone, and the casein peptone can be obtained from common commercial casein peptone, such as Boaoxing Biotechnology, Inc. The casein peptone in the special culture medium for clostridium emphysema contains rich amino acids, particularly some restrictive amino acids, and provides necessary nutrient elements for the growth of clostridium emphysema and clostridium gangreniformis.
The special culture medium for clostridium emphysema includes, by mass, 3-5 parts of yeast extract powder, more preferably 3-4 parts of yeast extract powder, and even more preferably 3 parts of yeast extract powder. The mass concentration of the yeast extract powder is preferably 3-5 g/L, more preferably 3-4 g/L, and even more preferably 3 g/L. The invention has no special requirement on the source of the yeast extract powder, and can be obtained by adopting common commercially available yeast extract powder, such as OXOID company. The yeast extract powder in the special culture medium for clostridium emphysema can provide nutrients such as vitamins and the like required by growth for the clostridium emphysema.
The special culture medium for clostridium emphysema includes, by mass, 1-3 parts of NaCl, preferably 1-2.5 parts, and more preferably 2.5 parts. The mass concentration of NaCl in the invention is preferably 1-3 g/L, more preferably 1-2.5 g/L, and even more preferably 2.5 g/L. The invention has no special requirement on the source of NaCl, and can be obtained by adopting common NaCl sold in the market, such as Nippon Fine chemical development Co., Ltd. The NaCl in the special culture medium for clostridium tetani regulates the osmotic pressure of the clostridium tetani so that the clostridium tetani keeps good activity.
The special culture medium for clostridium gangreniformis comprises, by mass, 1-3 parts of KH2PO4More preferably 1 to 2 parts, and still more preferably 1 part. KH is described in the invention2PO4The mass concentration of (A) is preferably 1 to 3g/L, more preferably 1 to 2g/L, and still more preferably 1 g/L. The invention is to KH2PO4The source of the KH-containing natural product is not particularly required, and common commercial KH-containing natural product is adopted2PO4Namely, such as Tianjin chemical industry trade company, Ltd.
The special culture medium for clostridium emphysema contains 10-20 parts by mass of Na2HPO4·12H2O is more preferably 15 to 20 parts, and still more preferably 20 parts. Na as described in the invention2HPO4·12H2The mass concentration of O is preferably 10 to 20g/L, more preferably 15 to 20g/L, and still more preferably 20 g/L. The invention is to Na2HPO4·12H2The source of O has no special requirement, and common commercial Na is adopted2HPO4·12H2And O, such as Tianjin chemical industry trade company Limited.
In the present invention, the KH is2PO4And Na2HPO4·12H2O constitutes a buffer system of the culture medium and mainly plays a role in stabilizing pH.
The special culture medium for clostridium gangreniformis comprises, by mass, 5-10 parts of anhydrous glucose, preferably 5-8 parts of anhydrous glucose, and more preferably 5 parts of anhydrous glucose. The mass concentration of the anhydrous glucose is preferably 5-10 g/L, more preferably 5-8 g/L, and even more preferably 5 g/L. The invention has no special requirement on the source of the anhydrous glucose, and can be obtained by adopting the anhydrous glucose sold in the market, such as Tianjin scientific union chemical industry trade company Limited. The glucose provides a main carbon source for clostridium emphysema and provides guarantee for the growth of the clostridium emphysema.
The special culture medium for clostridium aerocoelions preferably also comprises water. The water of the present invention is preferably water for injection. The invention has no special requirement on the source of the water for injection, and can be prepared by common commercial or conventional preparation methods. The water is mainly used as a solvent to dissolve all components in the culture medium.
When the medium comprises water, the medium preferably comprises the following components by mass per liter: 10-15 g of peptone, 3-5 g of tryptone, 3-5 g of casein peptone, 3g of yeast extract powder, 2.5g of NaCl and KH2PO4 1g、Na2HPO4·12H2O20 g and anhydrous glucose 5 g; more preferably 10g of peptone, 5g of tryptone, 5g of casein peptone, 1-3 g of yeast extract, 1-3 g of NaCl, and KH2PO4 1～3g、Na2HPO4·12H210-20 g of O and 5-10 g of anhydrous glucose.
In the invention, the pH value of the special culture medium for clostridium emphysema is preferably 7.6-7.8. The pH value of the invention is very suitable for the growth of clostridium tetani, and provides a good environment for the growth of clostridium tetani.
The special culture medium for clostridium emphysema replaces the raw materials of beef, beef liver and the like with the raw materials of which the original quality is uncontrollable by using finished products of commercial peptone, yeast extract powder and the like as the raw materials, and ensures that the toxin production capacity of the culture medium and the protection rate of finished seedlings reach or even exceed the original regulation standard by screening the formula of the culture medium and the optimal content of each component and optimizing the culture conditions, almost has no batch difference, and is very suitable for preparing the clostridium emphysema liquid for livestock; in addition, the special culture medium for clostridium emphysema has strong toxin production capacity no matter the culture medium is cultured by using a 500ml bottle or a fermentation tank, and the toxin production capacity is stable and the quality is controllable; the special culture medium used in the invention has the advantages of convenient material acquisition and simple preparation, and omits the processes of cold soaking, boiling, filtering and the like of the anaerobic pork liver soup, thereby greatly shortening the preparation time and reducing the production cost; meanwhile, the special culture medium for clostridium emphysema does not contain animal raw material residues and drug residues, so that the pressure of developing and producing low-immune-dose inactivated vaccines and purifying and concentrating the inactivated vaccines is reduced. The special culture medium for clostridium emphysema has wide prospect for replacing the existing culture medium for anaerobic pork liver soup and peptone pork liver soup to produce the inactivated vaccine for emphysema and gangrene.
The invention also provides a preparation method of the special culture medium for clostridium emphysema, which comprises the following steps:
sterilizing a glucose aqueous solution prepared from anhydrous glucose to obtain a culture medium A solution special for clostridium emphysema;
dissolving the rest components with water, adjusting pH, and sterilizing to obtain culture medium B solution special for Clostridium pneumonectasis;
the special culture medium A solution for the clostridium emphysema is mixed with the special culture medium B solution for the clostridium emphysema to obtain the special culture medium for the clostridium emphysema.
The invention prepares the glucose aqueous solution by the anhydrous glucose for sterilization to obtain the culture medium A solution special for the clostridium gangreniformis. The invention preferably prepares the anhydrous glucose into a glucose solution with the mass volume percentage of 25.0-50.0%, and further preferably 50.0%, and then sterilizes to obtain the special culture medium A solution for the clostridium gangreniformis. The solvent of the anhydrous glucose solution of the present invention is preferably water for injection. The sterilization method is preferably high-pressure steam sterilization, and the high-pressure steam sterilization is preferably carried out under the conditions of 108-110 ℃ and 30.0-40.0 min; further preferably, the sterilization is carried out by autoclaving at 110 ℃ for 30 min. The glucose solution is sterilized at 110 ℃ for 30min, so that the glucose is prevented from being carbonized and damaged at high temperature to the maximum extent. And (4) after sterilization, obtaining the special culture medium A solution for clostridium emphysema.
The invention uses water to dissolve the other components, adjusts the pH value, and sterilizes to obtain the special culture medium B solution of clostridium gangreniformis. The invention uses water for injection to dissolve other raw materials except glucose, wherein the other raw materials are peptone, tryptone, casein peptone, yeast extract powder, NaCl and KH2PO4、Na2HPO4·12H2And O. The amount of the water for injection for dissolution of the present invention is preferably 80.0% to 90.0%, and more preferably 80.0% of the total volume of the water for injection. After dissolution, the pH value is preferably adjusted to 7.6-7.8 by using NaOH solution, and the pH value is further preferably 7.7; the concentration of the NaOH solution is preferably 2-10 mol/L, and more preferably 2 mol/L. After the pH value is adjusted, the invention performs constant volume and sterilization on the solution after the pH value is adjusted. The invention has no special requirements on the volume fixing method, and the conventional volume fixing method is adopted. The sterilization method is preferably high-pressure steam sterilization, and the conditions of the high-pressure steam sterilization are preferably 116.0-121.0 ℃ and 30.0-40.0 min; further preferably, the sterilization is carried out by autoclaving at 116 ℃ for 30 min. And (4) after sterilization, obtaining the special culture medium B solution for clostridium emphysema.
The invention mixes the culture medium A solution special for clostridium emphysema and the culture medium B solution special for clostridium emphysema to obtain the culture medium special for clostridium emphysema. In the invention, the special culture medium A solution for clostridium emphysema is preferably added into the special culture medium B solution for clostridium emphysema and mixed to ensure that the concentration of anhydrous glucose is 5-10 g/L, and more preferably 5g/L, so as to obtain the special culture medium for clostridium emphysema.
The solution A and the solution B of the special culture medium for clostridium emphysema are prepared respectively and are mixed when in use, so that the nutrition of the culture medium can be fully ensured, the operation steps are simplified, and the labor cost for preparing the culture medium is reduced.
The invention provides a culture method of clostridium emphysema, which comprises the following steps: inoculating the seed liquid of clostridium gangreniformis to the gas in the technical schemeCulturing in special culture medium for clostridium gangreniformis to obtain clostridium gangreniformis bacterial liquid. In the invention, the clostridium emphysema gangreniformis strain C54-1 has the strain number CVCC60001 (purchased from China veterinary microbial strain preservation management center). The viable count of the clostridium emphysema seed liquid in the invention is preferably 1.0 x 106～2.5*106CFU/ml, more preferably 2.0 x 106CFU/ml. In the present invention, the inoculation amount of the clostridium pneumonectum seed liquid is preferably 3% to 5%, and more preferably 4%. In the invention, the temperature of the culture is preferably 35-37 ℃, and more preferably 37.0 ℃; the culture time is preferably 24-48 h, and more preferably 36 h. The invention further improves the toxin producing capability of the clostridium emphysema and gangrene and improves the immune effect by optimizing the culture condition of the clostridium emphysema and gangrene. In the invention, the clostridium emphysema gangrene seed liquid is preferably obtained by culturing anaerobic pork liver soup, and the anaerobic pork liver soup is used for culturing the seed liquid, so that the thallus activity is better, the toxicity is ensured, and the subsequent further expansion fermentation is smoothly carried out.
The invention provides a preparation method of a clostridium emphysema gangrensis inactivated vaccine, which comprises the following steps: inactivating the clostridium emphysema bacterium liquid obtained by the culture method in the technical scheme to obtain an inactivated bacterium liquid; and preparing the inactivated bacterial liquid into an inactivated vaccine.
The invention inactivates the clostridium emphysema bacterium liquid obtained by the culture method in the technical scheme to obtain the inactivated bacterium liquid. In the invention, the toxicity of the clostridium emphysema bacterium liquid is preferably less than or equal to 0.3ml of 1 MLD. The method of inactivation according to the invention preferably comprises formaldehyde inactivation.
After the inactivated bacterial liquid is obtained, the inactivated bacterial liquid is prepared into an inactivated vaccine. In the present invention, the preparation method of the inactivated vaccine preferably includes directly preparing the inactivated bacterial liquid or mixing the inactivated bacterial liquid with an adjuvant. In the present invention, the adjuvant is preferably aluminum potassium sulfate and aluminum hydroxide gel. The invention preferably prepares the formaldehyde vaccine directly from the inactivated bacteria liquid, or mixes the inactivated bacteria liquid with the aluminum potassium sulfate solution to prepare the alum vaccine, or mixes the inactivated bacteria liquid with the aluminum hydroxide glue to prepare the aluminum glue vaccine, or mixes the inactivated bacteria liquid after purification and concentration with the sodium hydroxide aluminum glue to prepare the concentrated vaccine. When formaldehyde seedlings are prepared, the pH of the inactivated bacterial liquid is preferably adjusted to 6.8-7.2 by using NaOH solution to obtain the formaldehyde seedlings; when preparing the alum vaccine, the inactivated vaccine is mixed with the aluminum potassium sulfate solution to obtain the alum vaccine; in the invention, the concentration of the aluminum potassium sulfate solution is preferably 10.0-12.0%, and more preferably 10.0%; the volume ratio of the inactivated bacterial liquid to the aluminum potassium sulfate solution is preferably 8: 1-12: 1; more preferably 9: 1. when preparing the aluminum glue vaccine, the inactivated vaccine is preferably mixed with the aluminum hydroxide glue solution to obtain the aluminum glue vaccine; in the invention, the volume ratio of the inactivated bacterial liquid to the aluminum hydroxide gel solution is preferably 4-5: 1, more preferably 5: 1. when concentrated seedlings are prepared, the method preferably adopts a centrifugation and ultrafiltration concentration method to concentrate 6-8 times of bacterial liquid; then, the clostridium emphysema concentrated bacterial liquid and the sterile aluminum hydroxide glue are mixed to obtain concentrated seedlings, the invention has no special requirement on the aluminum hydroxide glue, and the invention meets the good quality version P431 of the veterinary biological product regulation of the people's republic of China; in the invention, the volume ratio of the clostridium pneumonectum concentrated bacterial liquid to the aluminum hydroxide gel solution is preferably 4-5: 1, more preferably 5: 1. the clostridium emphysema inactivated vaccine provided by the invention has good safety, strong protection capability, stable quality and small batch difference, and can reach and exceed the standard of Chinese veterinary biological product code.
In order to further illustrate the present invention, the following examples are provided to describe the culture medium specifically for clostridium emphysema, the preparation method thereof, and the preparation method of the inactivated vaccine for emphysema for livestock, which should not be construed as limiting the scope of the present invention.
Example 1
A special culture medium for clostridium emphysema is composed of the following components in mass concentration: peptone 15g/L, tryptone 5g/L, casein peptone 5g/L, yeast extract 2.5g/L, NaCl 2.5.5 g/L, KH g/L2PO4 3g/L、Na2HPO4·12H2O10 g/L and anhydrous glucose 5 g/L. Wherein, peptone and tryptoneThe casein peptone and casein peptone are from Boaoxing Biotechnology, Inc., the yeast extract is from OXOID, glucose, KH2PO4And Na2HPO4·12H2O is purchased from Tianjin scientific union chemical industry trade Co., Ltd, and NaCl is purchased from Beilian Fine chemical development Co., Ltd, Tianjin City.
The preparation method of the special culture medium for clostridium gangreniformis comprises the following steps:
preparing anhydrous glucose into a glucose solution with the mass volume percentage of 50%, sterilizing with high-pressure steam at 110 ℃ for 30min to obtain a special culture medium A solution for clostridium emphysema and clostridium gangreniformis, and storing at 37 ℃ for later use;
mixing 15g peptone, 5g tryptone, 5g casein peptone, 2.5g yeast extract powder, 2.5g NaCl, 3g KH2PO4And 10g of Na2HPO4·12H2Dissolving O in 800ml of water for injection, adjusting pH to 7.6 with 2mol/L NaOH, diluting to 1000ml with water for injection, sterilizing with high pressure steam at 116 deg.C for 30min to obtain culture medium B solution special for Clostridium aeronevum, and storing at 37 deg.C;
before use, the special culture medium A solution for the clostridium tetani is added into the special culture medium B solution for the clostridium tetani according to the volume percentage of 1 percent to obtain the special culture medium for the clostridium tetani.
Example 2
A special culture medium for clostridium emphysema is composed of the following components in mass concentration: peptone 15g/L, tryptone 5g/L, casein peptone 5g/L, yeast extract 2.5g/L, NaCl 2.5.5 g/L, KH g/L2PO4 3g/L、Na2HPO4·12H2O10 g/L and anhydrous glucose 10 g/L. The raw materials are from the same sources as in example 1.
The preparation method of the special culture medium for clostridium emphysema is the same as that in example 1, except that the special culture medium A solution for clostridium emphysema is added into the special culture medium B solution for clostridium emphysema in an amount of 2% by volume.
Example 3
A special culture medium for clostridium emphysema is composed of the following components in mass concentration: peptone 10g/L, tryptone 3g/L3g/L of casein peptone and 1g/L, NaCl 1g/L, KH g of yeast extract powder2PO4 3g/L、Na2HPO4·12H2O15 g/L and anhydrous glucose 10 g/L. The raw materials are from the same sources as in example 1.
The preparation method of the special culture medium for clostridium emphysema is the same as that in the example 1, except that the dosage of each component is adjusted in the special culture medium B solution for clostridium emphysema; the special culture medium A solution for the clostridium tetani is added into the special culture medium B solution for the clostridium tetani according to the volume percentage of 2 percent.
Example 4
A special culture medium for clostridium emphysema is composed of the following components in mass concentration: peptone 15g/L, tryptone 3g/L, casein peptone 5g/L, yeast extract 3g/L, NaCl 2.5.5 g/L, KH2PO4 3g/L、Na2HPO4·12H2O10 g/L and anhydrous glucose 10 g/L. The raw materials are from the same sources as in example 1.
The preparation method of the special culture medium for clostridium emphysema is the same as that in the example 1, except that the dosage of each component is adjusted in the special culture medium B solution for clostridium emphysema; the special culture medium A solution for the clostridium tetani is added into the special culture medium B solution for the clostridium tetani according to the volume percentage of 2 percent.
Example 5
A special culture medium for clostridium emphysema is composed of the following components in mass concentration: 10g/L of peptone, 5g/L of tryptone, 5g/L of casein peptone and 3g/L, NaCl 3g/L, KH of yeast extract powder2PO4 1g/L、Na2HPO4·12H2O20 g/L and anhydrous glucose 10 g/L. The raw materials are from the same sources as in example 1.
The preparation method of the special culture medium for clostridium emphysema is the same as that in the example 1, except that the dosage of each component is adjusted in the special culture medium B solution for clostridium emphysema; the special culture medium A solution for the clostridium tetani is added into the special culture medium B solution for the clostridium tetani according to the volume percentage of 2 percent.
Comparative example 1
The anaerobic pork liver soup is prepared from 1L of culture medium, wherein the dosage of each component is as follows: 250g of beef, 250g of beef liver, 10g of peptone, 5g of NaCl, 2g of glucose and 1000ml of distilled water. The beef and beef liver are purchased in the market, the peptone is purchased from Boaoxing biotechnology, Inc., the NaCl is purchased from Beilian fine chemical development, Inc. in Tianjin, and the glucose is purchased from Tianjin element chemical industry and trade, Inc.
The anaerobic pork liver soup is prepared according to the composition and the preparation method of Chinese veterinary biological product code.
The preparation method of the anaerobic pork liver soup comprises the following steps:
1. removing fat and fascia from beef, mincing with meat mincer, mixing with cut liver pieces of about 100g, adding distilled water, stirring, and cold soaking for 22 hr.
2. Boiling for 30min, supplementing water, filtering with white cloth, removing meat residue, and taking out liver pieces.
3. Adding peptone and NaCl into the filtrate, heating to melt, adjusting pH to 8.0 with NaOH solution, and boiling for 20 min.
4. Filtering with filter paper or flannel, adding glucose, and stirring to melt.
5. Cleaning the filtered liver blocks, cutting into small blocks, washing with distilled water, and packaging into test tubes or neutral glass bottles in an amount of 1/10.
6. Packaging the filtrate into neutral container (such as test tube, and appropriate amount of liquid paraffin), and sterilizing with high pressure steam at 116 deg.C for 30-40 min.
Comparative example 2
A special culture medium for clostridium emphysema is composed of the following components in mass concentration: peptone 15g/L, tryptone 5g/L, casein peptone 5g/L, yeast extract 2.5g/L, NaCl 2.5.5 g/L, KH g/L2PO4 3g/L、Na2HPO4·12H2O10 g/L and anhydrous glucose 2 g/L. The raw materials are from the same sources as in example 1.
The preparation method of the special culture medium for clostridium emphysema is the same as that in example 1, except that the special culture medium A solution for clostridium emphysema is added into the special culture medium B solution for clostridium emphysema in an amount of 0.4% by volume.
Example 7
The culture method of clostridium emphysema includes the following steps:
the ampoule containing the freeze-dried clostridium aerocoes strain with good vacuum degree is opened by a method of aseptic burst. The clostridium emphysema grade producing strain adopted by the invention is clostridium emphysema grade C54-1 strain, the strain number is CVCC60001, and the strain is purchased from China veterinary microorganism strain preservation management center.
Dissolving appropriate amount of sterile nutrient broth, inoculating into 6 min anaerobic pork liver soup test tube, standing at 37 deg.C for 22h, and simultaneously detecting and detecting toxicity, wherein the pure and qualified product is Clostridium pneumoneyi seed liquid.
Inoculating the clostridium gangreniformis seed liquid into a special culture medium for clostridium gangreniformis in a 500ml bottle according to the inoculation amount of 4% (v/v), standing and culturing at 37 ℃ for 48h, sampling the obtained culture, and measuring the toxicity to obtain qualified clostridium gangreniformis liquid.
In the present invention, the methods and standards for pure detection and virus detection refer to the "veterinary biological product code of the people's republic of China" 2000 edition and the "pharmacopoeia of the people's republic of China" 2015 edition.
Example 8
Evaluation of virulence effect of clostridium pneumonectum bacterial liquid cultured by special culture medium for clostridium pneumonectum
The special culture medium for clostridium emphysema and gangrene in examples 1-6 and the anaerobic pork liver soup in comparative example 1 are used for culturing clostridium emphysema and gangrene, the clostridium emphysema and gangrene seed liquid is the clostridium emphysema and gangrene seed liquid obtained in example 7, the clostridium emphysema and gangrene seed liquid is inoculated in each culture medium in a 350ml/500ml bottle according to the inoculation amount of 4% (v/v), the culture medium is placed at 37 ℃ for standing culture for 48h, the obtained culture is sampled and tested for toxicity, each experimental condition is repeated for three times, the toxicity of clostridium emphysema and gangrene bacterial liquid obtained by different culture media is inspected, and the detection result is shown in table 1.
TABLE 1 comparison of virulence (guinea pig MLD) effects of Clostridium pneumonectasis bacteria liquid cultured by the culture medium special for Clostridium pneumonectasis of examples 1-6 and the anaerobic pork liver soup of comparative example 1
Figure BDA0002757182840000121
Figure BDA0002757182840000131
As can be seen from the data in table 1, the toxicity of 3 batches of clostridium emphysema bacterium liquid cultured in the clostridium emphysema-suffering special culture medium of examples 1 to 5 is qualified, and reaches the clostridium emphysema-suffering toxicity standard in "clostridium emphysema inactivated vaccine" specified in the "clostridium emphysema-suffering biological product code" (hereinafter referred to as code) 2000 edition: 1MLD: 0.1-0.3 ml, wherein the virulence effect of the embodiment 1 is best, the clostridium emphysema is well developed, the fermentation virulence is most stable, and the highest virulence can reach 1MLD: 0.15ml, 2 times higher toxicity than qualified standard; comparative example 1 (anaerobic liver broth group) culture of clostridium pneumonectum has unstable virulence effect, and only 1 batch of 3 batches has virulence capable of reaching 1MLD:0.3ml, the traditional anaerobic pork liver soup culture medium has poor and unstable toxicity effect; comparative example 2 cultured Clostridium pneumonectasis has low toxicity, and after inoculation, Clostridium pneumonectasis develops very weakly and does not meet the standard. The special culture medium for clostridium emphysema does not need to be limited to beef, beef liver and other raw materials which are difficult to store, high in cost and inconvenient to obtain and are used in the traditional anaerobic pork liver soup culture medium, the preparation process is simple and convenient, the time for preparing the culture medium is greatly saved, and the cost is reduced to 1/3-1/2 of the traditional anaerobic pork liver soup, which is shown in table 2.
TABLE 2 comparison of the parameters relating to the process of the invention with those of the conventional process (10000ml scale)
Figure BDA0002757182840000132
Figure BDA0002757182840000141
Example 9
Preparation of clostridium emphysema bacterium liquid and evaluation of virulence effect
The invention adopts the special culture medium for clostridium tetani to ferment and culture the clostridium tetani by 3 times of repeated expansion culture. The method comprises the following specific steps:
1 first amplification of fermentation culture
The clostridium emphysema gangrensis seed liquid obtained in the example 7 is used as a primary seed liquid and is inoculated into a 350ml/500ml bottle of anaerobic pork liver soup according to the inoculation amount of 4% (v/v), the anaerobic pork liver soup is placed at 37 ℃ for static culture for 22h, and the pure and qualified virulence is used as a secondary seed liquid after pure inspection and toxicity measurement.
Inoculating qualified secondary seed liquid into 12000ml of a toxin-producing culture medium of clostridium pneumonectum according to the inoculation amount of 3-5% (v/v), and standing and culturing for 24-48 h at 37 ℃.
Sampling is carried out after 24h, 36h and 48h of culture respectively, the sample bacterial liquid is injected into 2 guinea pigs of 350-450 g through muscle on the inner sides of the thighs according to 0.2ml and 0.3ml respectively, the toxicity of the bacterial liquid to the guinea pigs in different periods of culture is measured, and the test results are shown in table 3 to determine the optimal toxicity production time.
TABLE 3 first virulence determination table of Clostridium verrucosum bacterial liquid (12000ml scale)
Figure BDA0002757182840000142
From table 3 above, it can be seen that: culturing for 24h, sampling and determining virulence 1MLD to be less than or equal to 0.2 ml; culturing for 36h, sampling and determining virulence 1MLD to be less than or equal to 0.3 ml; culturing for 48h, sampling and determining virulence 1MLD:0.3 ml.
2 second time of enlarged fermentation culture
The Clostridium pneumophilum seed liquid obtained in example 7 was used as a primary seed liquid, inoculated into 350ml/500ml bottles of anaerobic meat-liver soup at an inoculation amount of 4% (v/v), left to stand at 37 ℃ for 22 hours, and subjected to pure inspection and toxicity measurement, and the pure and toxicity-qualified product was used as a secondary seed liquid.
Inoculating the second-level seed liquid into 7000mL/10000mL anaerobic pork liver soup according to the inoculation amount of 4%, standing at 37 deg.C for 22h, and performing pure inspection and toxicity measurement to obtain pure and qualified product as the third-level seed liquid.
Inoculating qualified third-stage seed liquid into 100000ml of clostridium pneumonectum toxin-producing culture medium according to the inoculation amount of 4%, and standing and culturing at 37 ℃ for 24-48 h.
Sampling is carried out after 24h, 36h and 48h of culture respectively, the sample bacterial liquid is injected into 2 heads of guinea pigs with the weight of 350-450 g through muscle injection on the inner sides of the thighs according to the volume of 0.2ml and the volume of 0.3ml respectively, the toxicity of the bacterial liquid to the guinea pigs during different periods of culture is measured, and the test results are shown in table 4, so as to determine the optimal toxicity production time.
TABLE 4 Clostridium tetani bacterial liquid second virulence determination table (100000ml scale)
Figure BDA0002757182840000151
As can be seen from the above Table 4, the virulence 1MLD is determined to be less than or equal to 0.2ml by sampling after 24h of culture; culturing for 36h, sampling and determining virulence 1MLD to be less than or equal to 0.2 ml; culturing for 48h, sampling and determining virulence 1MLD:0.3 ml.
3 third time of enlarged fermentation culture
The Clostridium pneumophilum seed liquid obtained in example 7 was used as a primary seed liquid, inoculated into 350ml/500ml bottles of anaerobic meat-liver soup at an inoculation amount of 4% (v/v), left to stand at 37 ℃ for 22 hours, and subjected to pure inspection and toxicity measurement, and the pure and toxicity-qualified product was used as a secondary seed liquid.
Inoculating the second-level seed liquid into 7000mL/10000mL anaerobic pork liver soup according to the inoculation amount of 4%, standing at 37 deg.C for 22h, and performing pure inspection and toxicity measurement to obtain pure and qualified product as the third-level seed liquid.
Inoculating qualified third-stage seed liquid into 400000ml of clostridium pneumonectum toxin-producing culture medium according to the inoculation amount of 4%, and standing and culturing at 37 ℃ for 24-48 h.
Sampling is carried out after 24h, 36h and 48h of culture respectively, the sample bacterial liquid is injected into 2 heads of guinea pigs with the weight of 350-450 g through muscle injection on the inner sides of the thighs according to the volume of 0.2ml and the volume of 0.3ml respectively, the toxicity of the bacterial liquid to the guinea pigs during different periods of culture is measured, and the test results are shown in table 5, so as to determine the optimal toxicity production time.
TABLE 5 Clostridium tetani bacterial liquid third virulence determination table (400000ml scale)
Figure BDA0002757182840000161
As can be seen from the above Table 5, the virulence 1MLD is determined to be less than or equal to 0.2ml by sampling after 24h of culture; culturing for 36h, sampling and determining virulence 1MLD to be less than or equal to 0.3 ml; culturing for 48h, sampling and determining virulence 1MLD:0.3 ml.
The results shown in the tables 3 to 5 show that the clostridium emphysema and gangrene toxin producing culture medium is adopted to carry out fermentation culture on clostridium emphysema and gangrene toxin producing culture for 3 times respectively through repeated amplification culture, when the fermentation culture time is 24 hours, 36 hours and 36 hours, the toxicity of clostridium emphysema and gangrene bacteria liquid obtained through fermentation culture is all 1MLD which is less than or equal to 0.3ml, and the toxicity reaches the qualified standard of clostridium emphysema and gangrene toxicity in the 'clostridium emphysema inactivated vaccine' specified in the 'biological product code for Chinese veterinary use' in the version of 2000: 1MLD: 0.1 to 0.3 ml. When the fermentation culture time is 24-36h, the results of 1MLD (maximum residual stress) of less than or equal to 0.2ml appear in all three times of amplification culture tests. Is the lower limit of toxicity standard of the clostridium emphysema gangrene in the ' emphysema gangrene inactivated vaccine ' specified in the code ' 2000 edition: 1MLD: more than 1.5 times of 0.3 ml. Therefore, the preferable fermentation culture time is 24-36h when the clostridium emphysema is subjected to fermentation culture, so that the toxicity standard specified in the regulation can be met or exceeded, and the toxicity production effect of the clostridium emphysema-gangreniformis toxicity production culture medium is stable, and the obtained clostridium emphysema-gangreniformis bacterial liquid has stable and stronger toxicity, and can be effectively used for preparing the clostridium emphysema-gangreniformis inactivated vaccine.
Example 10
Preparation and efficacy evaluation of emphysema gangrene inactivated vaccine
1, preparing the emphysema gangrene inactivated vaccine:
adding 0.5% formaldehyde solution (40% concentration) into the cultured clostridium emphysema and gangrene culture according to volume, and inactivating and detoxifying at 37 ℃ for 72h (stirring for 4 times per day at the stirring speed of 100-150 rpm for 20min) to obtain an inactivated bacterial liquid. Inactivation test: a, inoculating the inactivated bacterial liquid into an anaerobic pork liver soup large tube, a TSB small tube, a tyrose agar inclined plane small tube and a blood inclined plane small tube, culturing for 72h at 37 ℃, and observing aseptic growth within 3 days; b, transplanting TG. small tubes, TSB small tubes, tyrose agar slant small tubes and blood slant small tubes into the anaerobic pork liver soup large tube cultured for 72h, culturing for 72h at 37 ℃, observing aseptic growth within 3 days, and indicating complete inactivation.
And (4) taking the completely inactivated bacterial liquid, placing the bacterial liquid in a proper sterilization container, and storing the bacterial liquid at the temperature of 2-8 ℃ for later use.
Taking a proper amount of aluminum hydroxide gel (purchased from inner Mongolia Synoli biological Co., Ltd.) and placing in a glass bottle, adding a proper amount of water for injection (sterilization evaporation amount), and sterilizing for later use.
Taking a proper amount of aluminum potassium sulfate (purchased from Daoyou chemical reagent factory in Tianjin) to prepare a 10% solution, placing the solution in a glass bottle, and sterilizing for later use.
Taking a proper amount of anhydrous sodium carbonate to prepare a 25% solution, placing the solution in a glass bottle, and sterilizing for later use.
Preparation of potassium alum vaccine: taking a proper amount of clostridium pneumonectum bacterial liquid which is completely inactivated and stored at the temperature of 2-8 ℃, putting the clostridium pneumonectum bacterial liquid into a sterilized glass bottle, and adjusting the pH value to 7.9-8.1 by using a sterile 25% anhydrous sodium carbonate solution; mixing the clostridium emphysema bacterium liquid and 10% sterile aluminum potassium sulfate solution according to the volume ratio of 9:1, shaking and mixing uniformly to obtain potassium alum vaccine.
Preparing formaldehyde seedlings: and (3) taking a proper amount of clostridium pneumonectum bacterial liquid which is completely inactivated and stored at the temperature of 2-8 ℃, putting the clostridium pneumonectum bacterial liquid into a sterilized glass bottle, adjusting the pH to 6.8-7.2 by using a sterile 2mol/L NaOH solution, shaking and uniformly mixing to obtain the formaldehyde seedling.
Preparing the aluminum glue seedling: taking a proper amount of clostridium pneumonectum bacterial liquid which is completely inactivated and stored at the temperature of 2-8 ℃, putting the clostridium pneumonectum bacterial liquid into a sterilized glass bottle, and adjusting the pH to 6.8-7.2 by using a sterile 2mol/L NaOH solution; mixing the clostridium emphysema bacterium liquid and the sterile aluminum hydroxide gel according to the volume ratio of 5:1, shaking and uniformly mixing to obtain the aluminum gel seedlings.
Preparing the alumina gel concentrated seedling: taking a proper amount of clostridium pneumonectum bacterial liquid which is completely inactivated and stored at the temperature of 2-8 ℃, putting the clostridium pneumonectum bacterial liquid into a sterilized glass bottle, and adjusting the pH to 6.8-7.2 by using a sterile 2mol/L NaOH solution; concentrating the bacterial liquid by 6-8 times by a centrifugal and ultrafiltration concentration method; mixing the clostridium emphysema concentrated bacterial liquid and sterile aluminum hydroxide gel according to the volume ratio of 5:1, shaking and uniformly mixing to obtain the aluminum gel concentrated seedling.
2 evaluation of safety and efficacy of inactivated vaccine for emphysema:
50 guinea pigs of 350 to 450g were prepared, 8 of which were used for safety evaluation and 42 of which were used for efficacy evaluation.
2.1, safety test in guinea pigs:
and 4 prepared emphysema gangrene inactivated vaccines are taken, 2 guinea pigs of 350-450 g are injected subcutaneously at the back of each neck, 2 mL/vaccine of formaldehyde vaccine, potassium alum vaccine and aluminum gel vaccine are injected subcutaneously, and 0.4 mL/vaccine of concentrated aluminum gel vaccine is injected subcutaneously. The observation period was 10 days, and the results are shown in Table 6 below.
TABLE 6 safety evaluation results of inactivated vaccine for emphysema and cellulitis
Figure BDA0002757182840000181
As can be seen from table 6 above, 8 guinea pigs injected were healthy and alive for 10 days, and no necrotic reaction occurred in the injection site, and were judged to be: the safety inspection of the prepared 'emphysema gangrene inactivated vaccine' meets the regulation.
2.2, evaluation of efficacy of the emphysema gangrene inactivated vaccine:
and injecting 350-450 g of guinea pigs into the inner left thigh muscle of the prepared emphysema gangrene inactivated vaccine, wherein each guinea pig is 8, each formaldehyde vaccine, each potassium alum vaccine and each aluminum glue vaccine is 1mL, and each concentrated aluminum glue vaccine is 0.2 mL. After 21 days of immunization, 0.3ml of the virulent strain solution for emphysema and gangrene in 22h was attacked in control guinea pigs under the same conditions for 22h, and the results of observation for 10 days are shown in Table 7.
TABLE 7 evaluation of efficacy of inactivated vaccine for emphysema in Guinea pig
Figure BDA0002757182840000182
According to the regulation of Chinese veterinary biological product code in 2000 edition: 4 guinea pigs with the weight of 350-450 g are inoculated with the emphysema inactivated vaccine, each guinea pig is injected with 1mL intramuscular, 21 days later, the control guinea pigs with the same conditions are injected with at least 0.2mL of emphysema and carbuncle virulent strain liquid for 24h for each intramuscular, 10 days of observation are carried out, the control guinea pigs die in 72h, and the immunized guinea pigs are protected by at least 3 guinea pigs, namely, the condition is judged to be qualified. According to the data in the above table 7, it can be seen that the control guinea pig was 2/2 dead within 72h, and 4/4 healthy guinea pigs were observed 10 days after the challenge of each immunized group of the killed control guinea pigs, in the inactivated vaccine for emphysema and gangrene prepared by using the inactivated bacterial liquid of clostridium emphysema obtained by fermentation culture of the toxigenic medium in this example. The formaldehyde vaccine attacks 0.5mL of emphysema gangrene virulent bacterial liquid, and 4/4 healthy bacteria are observed for 10 days, which shows that the emphysema gangrene inactivated vaccine prepared by using the special culture medium for clostridium gangrene has better protective power, reaches and exceeds the protection regulation of 3/4 edition in 2000 of Chinese veterinary biological product code, and the culture medium has low cost and stable toxicity production performance, and reduces or avoids the generation of unqualified bacterial liquid in large-scale production.
The results of the above embodiments show that the special culture medium for clostridium emphysema does not need to be limited to beef, beef liver and other raw materials which are difficult to store, high in cost and inconvenient to obtain and are used in the traditional anaerobic pork liver soup culture medium, so that the production cost is greatly reduced, and the operation steps are simplified; meanwhile, the special culture medium for clostridium emphysema is stable in performance and is slightly influenced by the quality of raw materials, so that the influence of residual drugs in animal raw materials on clostridium emphysema is eliminated, the toxicity stability of clostridium emphysema is improved, and a guarantee is provided for subsequent vaccine preparation; the inactivated vaccine prepared by the special culture medium for clostridium emphysema has good safety and strong protection capability, and avoids the generation of unqualified bacterial liquid in large-scale production.
Although the present invention has been described in detail with reference to the above embodiments, it is only a part of the embodiments of the present invention, not all of the embodiments, and other embodiments can be obtained without inventive step according to the embodiments, and the embodiments are within the scope of the present invention.

Claims (10)

1. The special culture medium for clostridium emphysema is characterized by comprising the following components in parts by mass: 10-15 parts of peptone, 3-5 parts of tryptone, 3-5 parts of casein peptone, 1-3 parts of yeast extract powder, 1-3 parts of NaCl and KH2PO41-3 parts of Na2HPO4·12H210-20 parts of O and 5-10 parts of anhydrous glucose.
2. The special culture medium for clostridium emphysema according to claim 1, wherein the special culture medium for clostridium emphysema further comprises water.
3. According toThe special culture medium for clostridium emphysema of claim 2, wherein when the culture medium comprises water, the culture medium comprises the following components by mass per liter: 10-15 g of peptone, 3-5 g of tryptone, 3-5 g of casein peptone, 1-3 g of yeast extract powder, 1-3 g of NaCl and KH2PO4 1～3g、Na2HPO4·12H210-20 g of O and 5-10 g of anhydrous glucose.
4. The special culture medium for clostridium emphysema according to claim 1, wherein the pH value of the special culture medium for clostridium emphysema is 7.6-7.8.
5. The method for preparing the culture medium special for clostridium emphysema of any one of claims 1 to 4, comprising the following steps:
sterilizing a glucose aqueous solution prepared from anhydrous glucose to obtain a culture medium A solution special for clostridium emphysema;
dissolving the rest components with water, adjusting pH, and sterilizing to obtain culture medium B solution special for Clostridium pneumonectasis;
the special culture medium A solution for the clostridium emphysema is mixed with the special culture medium B solution for the clostridium emphysema to obtain the special culture medium for the clostridium emphysema.
6. A method for culturing clostridium emphysema is characterized by comprising the following steps: inoculating the clostridium emphysema gangrenia seed solution into the special culture medium for clostridium emphysema gangrenia of claim 1 or 2 for culture to obtain clostridium emphysema gangrenia bacterial solution.
7. The culture method according to claim 6, wherein the viable count of the Clostridium pneumonectans seed fluid is 1.0 x 106～2.5*106CFU/ml, the inoculation amount of the clostridium aerocoelions seed liquid is 3-5%.
8. The culture method according to claim 6, wherein the temperature of the culture is 36 to 37 ℃; the culture time is 24-48 h.
9. A preparation method of a clostridium emphysema inactivated vaccine comprises the following steps: inactivating the clostridium pneumonectum bacterial liquid obtained by the culture method according to any one of claims 6 to 8 to obtain an inactivated bacterial liquid; and preparing the inactivated bacterial liquid into an inactivated vaccine.
10. The method according to claim 9, wherein the inactivated vaccine is prepared by directly preparing the inactivated bacterial liquid or by mixing the inactivated bacterial liquid with an adjuvant.
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杨本升: "《动物微生物学》", 31 August 1995 *

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