Culture medium applicable to mycoplasma bovis culture as well as preparation method and application of culture medium

Abstract

The invention relates to the technical field of veterinary microbiology, in particular to a culture medium suitable for mycoplasma bovis culture and a preparation method of the culture medium. The culture medium is composed of a basic part and an auxiliary part, the basic part is prepared from 15-16 g / L of PPLO powder, 2-3 g / L of yeast extract, 2-3 g / L of sodium pyruvate, 1-1.5 g / L of glucose and 20-60 ml / L of phosphate buffer solution and ultrapure water (0.06-0.12 g / L of agar is added to a solid culture medium), and the auxiliary part is prepared from a proper amount of sodium chloride and a proper amount of sodium chloride. The auxiliary part comprises 1 to 1.5 ml / L of 10 * MEM, 8 to 12 ml / L of cholesterol with a mass concentration of 0.5%, 8 to 12 ml / L of glutamine with a mass concentration of 1%, 8 to 12 ml / L of crotonic acid with a mass concentration of 4%, 8 to 12 ml / L of malic acid with a mass concentration of 0.5%, 2 to 6 ml / L of a deoxynucleotide sodium salt solution with a molar concentration of 5 mmol / L, 40 to 60 ml / L of bovine serum and ultrapure water; the invention also provides a preparation method and application of the culture medium, which are simple and convenient, small in batch difference, simplified in production process and improved in product stability. According to the culture medium, stable mycoplasma bovis with high viable count can be obtained in a short time, and meanwhile, the problems of long detection period and the like of an existing quantitative bacterium concentration method can be effectively solved.

Description

Technical Field

[0001] The present invention relates to the technical field of veterinary microbiology, and in particular to a culture medium suitable for culturing Mycoplasma bovis, and a preparation method and application thereof. Background Art

[0002] Mycoplasma bovis, also known as bovine mycoplasma, is one of the most important pathogens currently causing infections in cattle. Currently, M. bovis has been shown to cause a variety of diseases, including pneumonia, mastitis, and arthritis, as well as keratoconjunctivitis, otitis, genital inflammation, miscarriage, and infertility.

[0003] Because the culture conditions of mycoplasmas are much more stringent than those of bacteria, their own biosynthetic ability is weak, the bacteria are small and the yield is low, and most species find it difficult to form typical colonies on solid culture media. Therefore, the existing quantitative bacterial concentration is mostly based on the CCU method. However, although the CCU method can roughly assess the viable bacterial titer in the culture, it is not accurate enough. The difference between two adjacent titers is 10 times, which makes it difficult to meet the needs of some experiments with more stringent requirements on the number of viable bacteria. This method also has disadvantages such as long detection cycle, time and labor consuming, easy to cause identification and counting errors, easy contamination during the experimental process and volatile results.

[0004] Prior art culture of M. bovis using M. bovis culture media presents challenges such as complex composition, high serum content, long culture cycles, and the predominantly CCU method for detection, which prevents accurate viable counts. Chinese invention patent CN103667154A discloses a M. bovis culture medium containing pig lung digestion broth. Chinese invention patent CN103937728A prepares a culture medium using healthy, fat-free, tendon-free bovine heart and lung tissue. Chinese invention patent CN106635891A discloses a M. bovis culture medium containing ox heart broth and bovine lung digestion fluid. These culture media have complex components and preparation methods, require long preparation times, and are prone to large batch-to-batch variability, leading to unstable culture results.

[0005] Although the bovine Mycoplasma culture media of Chinese invention patents CN106350472A, CN106399207A, and CN106399206A avoid problems such as complex culture medium components, large batch differences, and unstable culture effects, their culture cycles are relatively long, and the CCU method detection cycle is also relatively long, which cannot meet the time requirements of relevant scientific research.

[0006] Therefore, at this stage, there is an urgent need for a bovine Mycoplasma culture medium with a simple, efficient, stable preparation process, simple components, low serum addition, and the ability to form single colonies, which can be used in the production, scientific research, and detection of bovine Mycoplasma-related vaccine antigens. Summary of the Invention

[0007] The technical problem to be solved in the first aspect of the present invention is to provide a liquid culture medium suitable for the cultivation of Mycoplasma bovis, in view of the shortcomings of the existing technology of Mycoplasma bovis culture medium, such as complex components, large batch-to-batch variability, unstable culture effect, long culture cycle, high serum dosage, low viable bacterial titer, and complex preparation method.

[0008] The technical problem to be solved in the second aspect of the present invention is to provide a solid culture medium suitable for culturing Mycoplasma bovis.

[0009] The technical problem to be solved in the third aspect of the present invention is to provide a method for preparing a liquid culture medium suitable for culturing Mycoplasma bovis.

[0010] The technical problem to be solved in the fourth aspect of the present invention is to provide a method for preparing a solid culture medium suitable for culturing Mycoplasma bovis.

[0011] The technical problem to be solved in the fifth aspect of the present invention is to provide a use of the liquid culture medium or the solid culture medium in culturing bovine Mycoplasma.

[0012] The technical problem to be solved in the sixth aspect of the present invention is to provide a use of the liquid culture medium or the solid culture medium in the preparation of a bovine Mycoplasma vaccine.

[0013] In order to solve the above technical problems, the technical solutions adopted by the present invention are as follows:

[0014] In a first aspect, the present invention provides a liquid culture medium suitable for culturing Mycoplasma bovis, wherein the liquid culture medium comprises a basic part and an auxiliary part.

[0015] The components of the basic part are as follows: 15-16 g / L PPLO powder, 2-3 g / L yeast extract, 2-3 g / L sodium pyruvate, 1-1.5 g / L glucose and 20-60 ml / L phosphate buffer, and ultrapure water is added to 800 ml / L.

[0016] Preferably, the base part comprises the following components: 15.5 g / L PPLO powder, 2.5 g / L yeast extract, 2.5 g / L sodium pyruvate, 1.25 g / L glucose and 40 ml / L phosphate buffer, and ultrapure water is added to 800 ml / L.

[0017] The auxiliary part comprises the following components: 1-1.5 ml / L 10×MEM, 8-12 ml / L 0.5% cholesterol, 8-12 ml / L 1% glutamine, 8-12 ml / L 4% crotonic acid, 8-12 ml / L 0.5% malic acid, 2-6 ml / L 5 mmol / L sodium deoxynucleotide solution, 40-60 ml / L bovine serum, and ultrapure water to 200 ml / L.

[0018] Preferably, the components of the auxiliary part are as follows: 1.25 ml / L 10×MEM, 10 ml / L cholesterol with a mass concentration of 0.5%, 10 ml / L glutamine with a mass concentration of 1%, 10 ml / L crotonic acid with a mass concentration of 4%, 10 ml / L malic acid with a mass concentration of 0.5%, 4 ml / L deoxynucleotide sodium salt solution with a molar concentration of 5 mmol / L, 50 ml / L bovine serum, and ultrapure water is added to 200 ml / L.

[0019] Furthermore, in some embodiments, the auxiliary part may also be added with a pH indicator and / or an antibiotic; preferably, the pH indicator is phenol red, which is used to determine whether Mycoplasma bovis is growing and the growth status; and the antibiotic is sodium penicillin, which is used to inhibit bacterial growth.

[0020] In a second aspect, the present invention provides a solid culture medium suitable for culturing Mycoplasma bovis, wherein the solid culture medium comprises a basic part and an auxiliary part.

[0021] The components of the base part are as follows: 15-16 g / L PPLO powder, 2-3 g / L yeast extract, 2-3 g / L sodium pyruvate, 1-1.5 g / L glucose, 20-60 ml / L phosphate buffer and 0.06-0.12 g / L agar, and ultrapure water is added to 800 ml / L.

[0022] Preferably, the base part comprises the following components: 15.5 g / L PPLO powder, 2.5 g / L yeast extract, 2.5 g / L sodium pyruvate, 1.25 g / L glucose, 40 ml / L phosphate buffer and 0.08 g / L agar, and the mixture is filled to 800 ml / L with ultrapure water.

[0023] The auxiliary part comprises the following components: 1-1.5 ml / L 10×MEM, 8-12 ml / L 0.5% cholesterol, 8-12 ml / L 1% glutamine, 8-12 ml / L 4% crotonic acid, 8-12 ml / L 0.5% malic acid, 2-6 ml / L 5 mmol / L sodium deoxynucleotide solution, 40-60 ml / L bovine serum, and ultrapure water to 200 ml / L.

[0024] Preferably, the components of the auxiliary part are as follows: 1.25 ml / L 10×MEM, 10 ml / L cholesterol with a mass concentration of 0.5%, 10 ml / L glutamine with a mass concentration of 1%, 10 ml / L crotonic acid with a mass concentration of 4%, 10 ml / L malic acid with a mass concentration of 0.5%, 4 ml / L deoxynucleotide sodium salt solution with a molar concentration of 5 mmol / L, 50 ml / L bovine serum, and ultrapure water is added to 200 ml / L.

[0025] Furthermore, in some embodiments, the auxiliary part may also be added with a pH indicator and / or an antibiotic; preferably, the pH indicator is phenol red, and the antibiotic is sodium penicillin.

[0026] Furthermore, the phosphate buffer comprises 10 g of sodium chloride, 5 g of potassium chloride, 1.25 g of magnesium sulfate heptahydrate, 1.25 g of disodium hydrogen phosphate hexahydrate, 1.75 g of anhydrous calcium chloride, 0.75 g of potassium dihydrogen phosphate dihydrate, 0.75 g of disodium hydrogen phosphate hexahydrate and 100 ml of ultrapure water.

[0027] Furthermore, the 5 mmol / L deoxynucleotide sodium salt solution includes 0.0982 g dAMP, 0.0964 g dGMP, 0.0694 g dCMP, 0.0614 g dTMP and 40 ml ultrapure water.

[0028] Furthermore, the bovine serum is fetal bovine serum or newborn bovine serum inactivated at 56°C.

[0029] In a third aspect, the present invention provides a method for preparing the liquid culture medium suitable for culturing Mycoplasma bovis, comprising the following steps:

[0030] (a) Base: Dissolve the base components one by one in 500–600 ml of ultrapure water, stir well, adjust the pH to 7.6–8.0, and make up the remaining volume to 800 ml with ultrapure water. Autoclave and allow to cool to room temperature before use.

[0031] (b) Mix the components of the auxiliary part and stir evenly, adjust the pH to 7.6-8.0, and make up the remaining volume to 200 ml with ultrapure water. After filtration and sterilization, thoroughly mix the base part and the auxiliary part to prepare a liquid medium suitable for culturing Mycoplasma bovis, and store at 4°C until used.

[0032] or,

[0033] The components of the basic part and the auxiliary part were directly mixed thoroughly, and the remaining volume was made up to 1000 ml with ultrapure water. The liquid culture medium suitable for the culture of Mycoplasma bovis was prepared by filtration and sterilization, and stored at 4°C for future use.

[0034] In a fourth aspect, the present invention provides a method for preparing the solid culture medium suitable for culturing Mycoplasma bovis, comprising the following steps:

[0035] (A) Base: Dissolve the base components one by one in 500–600 ml of ultrapure water, stir well, adjust the pH to 7.6–8.0, add agar, and mix thoroughly. Make up the remaining volume to 800 ml with ultrapure water. Autoclave and place in a 50–55°C water bath until ready for use.

[0036] (B) The components of the auxiliary part are mixed and stirred uniformly, and the pH is adjusted to 7.6-8.0. The remaining volume is made up to 200 ml with ultrapure water. After filtration and sterilization, the base part and the auxiliary part are thoroughly mixed in a water bath at 50-55°C for 20-40 minutes. After cooling, a solid medium suitable for the cultivation of M. bovis is prepared and stored at 4°C until use.

[0037] or,

[0038] Under sterile conditions, directly add the components of the auxiliary part to the basic part, and make up the remaining volume to 1000 ml with ultrapure water. Mix thoroughly in a water bath at 50-55°C for 20-40 minutes. After cooling, a solid culture medium suitable for the culture of Mycoplasma bovis is prepared and stored at 4°C for later use.

[0039] In a fifth aspect, the present invention provides use of the liquid culture medium suitable for culturing Mycoplasma bovis or the solid culture medium suitable for culturing Mycoplasma bovis in culturing Mycoplasma bovis.

[0040] In a sixth aspect, the present invention provides use of the liquid culture medium suitable for culturing Mycoplasma bovis or the solid culture medium suitable for culturing Mycoplasma bovis in preparing a Mycoplasma bovis vaccine.

[0041] Beneficial effects:

[0042] (1) The present invention provides a liquid / solid culture medium suitable for culturing Mycoplasma bovis. Using this culture medium, Mycoplasma bovis with a high and stable viable count can be obtained in a short period of time. After about 16 hours of culture, the viable count of Mycoplasma bovis can reach 1.0×10 10 The results of CFU / ml, live bacteria and separation sensitivity are much higher than those of various culture media in the existing technology, and are more suitable for the isolation, identification and vaccine production of bovine Mycoplasma.

[0043] (2) The solid culture medium suitable for culturing Mycoplasma bovis prepared by the present invention can form single colonies of Mycoplasma bovis visible to the naked eye in about 3 days. It has high sensitivity, a short detection cycle, and accurate quantification of live bacteria. It can effectively solve the problems of the existing quantitative bacterial concentration method (CCU method) for Mycoplasma bovis, such as long detection cycle, time and labor consumption, identification and counting errors, and contamination during the experimental process. It is of great significance for the rapid quantitative detection of Mycoplasma bovis culture and vaccine semi-finished products.

[0044] (3) The culture medium of the present invention has a high viability of bovine Mycoplasma and a relatively clear degree. The growth of bovine Mycoplasma can be determined without the aid of any indicator, which significantly improves the production efficiency of bovine Mycoplasma.

[0045] (4) All components in the culture medium of the present invention are commercial reagents with no special requirements. The preparation method of the culture medium is simple and convenient, with minimal batch-to-batch variability, which greatly simplifies the production process and improves product stability. Furthermore, the use of components such as serum is reduced, which reduces the stress response of cattle to foreign matter and reduces the production costs of the enterprise. BRIEF DESCRIPTION OF THE DRAWINGS

[0046] In order to more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the specific embodiments or the description of the prior art. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0047] Figure 1 It is a single colony formed by the growth of Mycoplasma bovis on the Mycoplasma bovis solid culture medium 4 of the present invention. DETAILED DESCRIPTION

[0048] The present invention will be further described below in conjunction with specific embodiments, and the above and / or other advantages of the present invention will become more apparent.

[0049] The experimental methods described in the following examples are conventional methods unless otherwise specified; the reagents and materials are commercially available unless otherwise specified.

[0050] Example 1: Preparation of Mycoplasma bovis culture medium

[0051] 1. Preparation of Mycoplasma bovis liquid culture medium

[0052] Table 1 Mycoplasma bovis culture medium (1L)

[0053]

[0054]

[0055] Eight groups of Mycoplasma bovis liquid culture media were prepared according to the composition in Table 1.

[0056] (a) Base: Dissolve the components of the base one by one in 500-600 ml of ultrapure water, stir evenly, adjust the pH to 7.6-8.0, and make up the remaining volume to 800 ml with ultrapure water. Sterilize by autoclaving at 115°C for 20 min and allow to cool to room temperature before use.

[0057] (b) Mix the components of the auxiliary part and stir evenly, adjust the pH to 7.6-8.0, make up the remaining volume to 200 ml with ultrapure water, and sterilize by filtration through 0.22 μm to prepare the auxiliary part. Thoroughly mix the base part and the auxiliary part to prepare the Mycoplasma bovis liquid culture medium, which is stored at 4°C until used.

[0058] Alternatively, the components of the base part and the auxiliary part are directly mixed thoroughly, the remaining volume is made up to 1000 ml with ultrapure water, and then filtered and sterilized to prepare the bovine Mycoplasma liquid culture medium, which is stored at 4°C for future use.

[0059] 2. Preparation of solid culture medium for Mycoplasma bovis

[0060] (A) Basic part: Dissolve the components of the basic part one by one in 500-600 ml of ultrapure water, stir evenly, adjust the pH to 7.6-8.0, add 0.06-0.12 g / L agar and mix well. Make up the remaining volume to 800 ml with ultrapure water. Sterilize at 115°C for 20 min and transfer to a 50-55°C water bath for use.

[0061] (B) The components of the auxiliary part were mixed and stirred uniformly. The pH was adjusted to 7.6-8.0. The remaining volume was made up to 200 ml with ultrapure water. After sterilization through a 0.22 μm filter, the base part and the auxiliary part were thoroughly mixed in a water bath at 50-55°C for approximately 30 minutes. After cooling, the M. bovis solid culture medium was prepared and stored at 4°C until use.

[0062] Under sterile conditions, directly add the components of the auxiliary part to the basic part, and make up the remaining volume to 1000 ml with ultrapure water. Mix thoroughly in a water bath at 50-55°C for 30 minutes. After cooling, a solid culture medium suitable for the culture of Mycoplasma bovis is prepared and stored at 4°C for later use.

[0063] Example 2: Detection of viable Mycoplasma bovis count using different Mycoplasma bovis culture media

[0064] 1. Culture of Mycoplasma bovis

[0065] The Mycoplasma bovis HM strain stored in the laboratory was taken out of a refrigerator below -70°C and dissolved. The inoculum was then inoculated into the Mycoplasma bovis liquid culture media 1 to 8 described in Example 1 at an inoculum size of 2-5% v / v. The culture was shaken and cultured at 37°C and 50-100 r / min for 24 hours. The culture fluid was then inoculated into the corresponding Mycoplasma bovis liquid culture media 1 to 8 at an inoculum size of 2-5% v / v. The culture fluid was shaken and cultured at 37°C and 50-100 r / min for 24 hours. The Mycoplasma bovis bacterial liquid was harvested. During this period, samples were taken at 12 hours, 16 hours, 20 hours, and 24 hours for viable cell count detection.

[0066] 2. Determination of live bacteria of Mycoplasma bovis using CFU method

[0067] 0.1 ml of M. bovis bacterial suspension harvested at different times was added to the first 2 ml EP tube containing 0.9 ml of M. bovis liquid culture medium 1 and vortexed to mix thoroughly. The pipette tip was replaced, and 0.1 ml of the mixture from the first tube was transferred to the second 2 ml EP tube containing 0.9 ml of M. bovis liquid culture medium 1. This step was repeated until the dilution reached the seventh tube. The mixture was vortexed to mix thoroughly. 0.1 ml of the mixture from the seventh tube was transferred and evenly spread onto each of the seven tubes containing M. bovis solid culture medium 1. Repeat this process for three tubes containing M. bovis solid culture medium 1. The tubes were placed in a 37°C, 5% CO2 incubator and incubated statically. Colonies were counted for approximately three days.

[0068] The process of detecting the viable count of Mycoplasma bovis HM strain using Mycoplasma bovis liquid culture medium 2-8 and Mycoplasma bovis solid culture medium 2-8 is the same as above. Figure 1 It is a single colony formed by the growth of Mycoplasma bovis on the Mycoplasma bovis solid culture medium 4 of the present invention.

[0069] Under the same conditions, the viable bacterial counts (CFU method) of Mycoplasma bovis at different times were determined using different Mycoplasma bovis culture media as shown in Table 2. As can be seen from Table 2, the viable bacterial counts of Mycoplasma bovis in liquid and solid culture media 1-2 took slightly longer to reach 6.0×10 at 24 hours. 9CFU / ml or above; the effect of culturing Mycoplasma bovis using liquid and solid culture medium for 3-5 days is better, and the number of viable bacteria in 16-24 hours is not less than 9.5×10 9 CFU / ml, among which the liquid and solid medium 4 for Mycoplasma bovis had the best effect in culturing Mycoplasma bovis, reaching 1.03×10 in 16 hours. 10 CFU / ml; However, because the liquid and solid culture media of bovine Mycoplasma 6-8 contain a high content of auxiliary components, the culture media are too nutritious, which leads to the rapid growth of bovine Mycoplasma and its aging is also fast (reaching 9.5×10 in 16 hours). 9 After that, the CFU / ml dropped rapidly to 5.0~6.0×10 9 CFU / ml). After comprehensive consideration, the bovine M. bovis liquid and solid culture medium 4 were finally selected to verify the culture effect of bovine M. bovis in the later stage.

[0070] Table 2 The number of viable bacteria of Mycoplasma bovis cultured in different culture media of the present invention at different times

[0071]

[0072]

[0073] Example 3: Comparison of the culture medium of the present invention and the commonly used culture medium for culturing Mycoplasma bovis

[0074] 1. Cultivation of Mycoplasma bovis strains

[0075] The laboratory-stored Mycoplasma bovis HM strain was taken out of a refrigerator below -70°C and dissolved. The culture was then inoculated at an inoculum size of 2-5% v / v into Mycoplasma bovis liquid culture medium 4 (serum content: 5% v / v), modified Thiaucourt's medium (serum content: 20% v / v), modified KM2 medium (serum content: 20% v / v), and beef heart broth medium (serum content: 10% v / v), respectively. The culture was shaken and cultured at 37°C and 50-100 r / min for 24 hours. The culture was then inoculated at an inoculum size of 2-5% v / v into the corresponding Mycoplasma bovis liquid culture medium 4, modified Thiaucourt's medium, modified KM2 medium, and beef heart broth medium, respectively. The culture was shaken and cultured at 37°C and 50-100 r / min for 24 hours. The Mycoplasma bovis bacterial liquid was harvested. During this period, samples were taken at 12 hours, 16 hours, 20 hours, and 24 hours for detection of viable cell count and protein content.

[0076] 2. Determination of live bacteria of Mycoplasma bovis using CFU method

[0077] 0.1 ml of M. bovis bacterial suspension harvested at different times was added to a first 2 ml EP tube containing 0.9 ml of M. bovis liquid culture medium 4 and vortexed to mix thoroughly. A pipette tip was replaced, and 0.1 ml of the mixture from the first tube was pipetted into a second 2 ml EP tube containing 0.9 ml of M. bovis liquid culture medium 4. This step was repeated until the appropriate dilution was achieved. The mixture was vortexed to mix thoroughly, and 0.1 ml of the diluted mixture was pipetted and evenly spread onto each of the M. bovis solid culture medium 4. This process was repeated three times for M. bovis solid culture medium 4. The tubes were placed in a 37°C, 5% CO2 incubator and incubated for approximately 3 days. Colonies were counted.

[0078] The process of detecting the viable count of Mycoplasma bovis HM strain using modified Thiaucourt's medium, modified KM2 medium and ox heart soup medium is the same as above.

[0079] Under the same conditions, M. bovis was cultured in M. bovis Medium 4 (5% v / v serum content), modified Thiaucourt's Medium (20% v / v serum content), modified KM2 Medium (20% v / v serum content), and Beef Heart Broth Medium (10% v / v serum content). The results of viable bacterial counts (CFU method) at different times are shown in Table 3. As can be seen from Table 3, the viable bacterial counts of M. bovis ranged from 5.68 to 11.0 × 10 after 12 to 24 hours of culture in M. bovis Medium 4. 9 CFU / ml; cultured in modified Thiaucourt's medium for 16 to 24 hours, the number of viable bacteria was 2.24 to 2.64 × 10 9 CFU / ml (approximately 1 / 5 to 1 / 2 times the result of culture using bovine Mycoplasma medium 4); after culturing in modified KM2 medium for 12 to 24 hours, the number of viable bacteria was 6.71 to 9.24×10 7 CFU / ml (approximately 1 / 164 to 1 / 61 times the result of culture using bovine Mycoplasma culture medium 4); after culturing in ox heart broth medium for 12 to 24 hours, the number of viable bacteria is 1.08 to 8.36 × 10 8 CFU / ml (approximately 1 / 101 to 1 / 7 times the result of culture using bovine Mycoplasma culture medium 4).

[0080] 3. Determination of bovine mycoplasma protein content

[0081] Take 1 ml of the Mycoplasma bovis bacterial liquid harvested at different times from each sample to be tested, centrifuge at 12000 r / min for 30 min, discard the supernatant, and draw an equal volume of PBS buffer (pH 7.2-7.4, 0.01 mol / L) to blow on the precipitate to suspend and mix it. After centrifugation at 12000 r / min for 30 min, discard the supernatant, wash twice in the same way, centrifuge and discard the supernatant, resuspend the precipitate with 1 ml of buffer, and finally determine the total protein amount at the corresponding time point using a BCA protein quantitative analysis kit.

[0082] As shown in Table 3, the protein content of M. bovis cultured in M. bovis Medium 4 reached 0.6076 mg / ml at 12 hours and remained at 0.9805-1.1048 mg / ml from 16 to 24 hours. In contrast, the maximum protein contents of M. bovis cultured in modified Thiaucourt's medium, modified KM2 medium, and Beef Heart Broth medium during the 24-hour period were 0.3235 mg / ml (approximately 1 / 3 the protein content of M. bovis cultured in M. bovis Medium 4), 0.0854 mg / ml (approximately 1 / 12 the protein content of M. bovis cultured in M. bovis Medium 4), and 0.1549 mg / ml (approximately 1 / 7 the protein content of M. bovis cultured in M. bovis Medium 4), respectively.

[0083] Table 3 The number of viable bacteria and protein content of Mycoplasma bovis cultured with the medium of the present invention and the commonly used medium at different times

[0084]

[0085] 4. Cost accounting

[0086] The cost of the bovine Mycoplasma culture medium 4 of the present invention is 130.21 yuan / liter, the modified Thiaucourt's medium is 434.21 yuan / liter (approximately 3.3 times the cost of the culture medium of the present invention), the modified KM2 medium is 366.76 yuan / liter (approximately 2.8 times the cost of the culture medium of the present invention), and the ox heart broth medium is 229.19 yuan / liter (approximately 1.8 times the cost of the culture medium of the present invention).

[0087] In summary, the Mycoplasma bovis culture medium 4 prepared by the present invention is more suitable for the growth of Mycoplasma bovis. According to the inoculation ratio of 2-5% v / v, the number of viable bacteria can reach 10 after about 16 hours of culture. 10 CFU / ml, the protein content reaches 1.0 mg / ml, and the culture medium preparation process is simple and low in cost.

[0088] Example 4: Verification of the effect of fermentation and amplification of Mycoplasma bovis culture medium

[0089] 1. Cultivation of Mycoplasma bovis strains

[0090] The Mycoplasma bovis HM strain stored in the laboratory was taken out of a refrigerator below -70°C and dissolved. The inoculum was then inoculated with Mycoplasma bovis liquid culture medium 3-5 at a volume of 2-5% v / v. The culture was shaken and cultured at 37°C and 50-100 r / min for 24 hours. The culture was then inoculated with Mycoplasma bovis liquid culture medium 4 at a volume of 2-5% v / v. The culture was shaken and cultured at 37°C and 50-100 r / min. After culturing for 16-18 hours, the new culture was inoculated with a volume of 2-5% v / v into a 100 L fermenter containing 50-70 L of Mycoplasma bovis liquid culture medium 3-5. The fermenter was sealed, and sterile air or sterile air + oxygen was introduced to maintain a dissolved oxygen value of 10-30%. The culture was stirred and cultured at 37°C and 50-100 r / min for 24 hours. During this period, samples were taken at 12 hours, 16 hours, 20 hours, and 24 hours for detection of viable bacterial count and protein content. Bovine Mycoplasma culture media 3 to 5 were all amplified and cultured in 4 batches.

[0091] 2. Determination of live bacteria of Mycoplasma bovis by CFU method in amplified culture

[0092] 0.1 ml of M. bovis bacterial suspension harvested at different times was added to the first 2 ml EP tube containing 0.9 ml of M. bovis liquid medium 4 and vortexed to mix thoroughly. The pipette tip was replaced, and 0.1 ml of the mixture from the first tube was transferred to the second 2 ml EP tube containing 0.9 ml of M. bovis liquid medium 4. This step was repeated until the dilution reached the seventh tube. The mixture was vortexed to mix thoroughly. 0.1 ml of the mixture from the seventh tube was transferred and evenly spread onto each of the seven tubes of M. bovis solid medium 4. Repeat this process for three tubes of M. bovis solid medium 4. The tubes were then placed in a 37°C, 5% CO2 incubator for static culture and colonies were counted for approximately three days.

[0093] The process of detecting the viable count of Mycoplasma bovis HM strain using Mycoplasma bovis liquid culture medium 3 and 5 and Mycoplasma bovis solid culture medium 3 and 5 is the same as above.

[0094] 3. Determination of protein content of Mycoplasma bovis in amplified culture

[0095] Take 1 ml of the Mycoplasma bovis bacterial liquid harvested at different times from each sample to be tested, centrifuge at 12000 r / min for 30 min, discard the supernatant, and draw an equal volume of PBS buffer (pH 7.2-7.4, 0.01 mol / L) to blow on the precipitate to suspend and mix it. After centrifugation at 12000 r / min for 30 min, discard the supernatant, wash twice in the same way, centrifuge and discard the supernatant, resuspend the precipitate with 1 ml of buffer, and finally determine the total protein amount at the corresponding time point using a BCA protein quantitative analysis kit.

[0096] 4. Results of fermentation and amplification of Mycoplasma bovis culture medium

[0097] The results are shown in Tables 4 to 6. It can be seen from the table that the present invention used 3 to 5 culture mediums of Mycoplasma bovis to ferment and amplify 4 batches of Mycoplasma bovis. In 16 to 22 hours, the number of viable bacteria in each batch reached 10 10 CFU / ml or more, and the protein content reaches 1.0 mg / ml or more. This shows that using the bovine Mycoplasma culture medium 3-5 of the present invention can reproduce large-volume culture, and has good reproducibility and stability between batches.

[0098] Table 4 Live cell count and protein content of Mycoplasma bovis at different times of fermentation and amplification culture of Mycoplasma bovis culture medium 3 of the present invention

[0099]

[0100] Table 5 Live cell counts and protein contents of Mycoplasma bovis at different times of fermentation and amplification culture of Mycoplasma bovis culture medium 4 of the present invention

[0101]

[0102] Table 6 The number of viable cells and protein content of Mycoplasma bovis at different times during the fermentation and amplification culture of Mycoplasma bovis culture medium 5 of the present invention

[0103]

[0104] The present invention provides a culture medium suitable for culturing Mycoplasma bovis, as well as a method for its preparation and application. There are numerous methods and approaches for implementing this technical solution. The above description is merely a preferred embodiment of the present invention and represents only a portion of the embodiments of the present invention. It should be noted that those skilled in the art may make various improvements and modifications without departing from the principles of the present invention, and such improvements and modifications are also within the scope of protection of the present invention. Any components not specified in this embodiment may be implemented using existing technologies.

Claims

1. A liquid culture medium suitable for culturing Mycoplasma bovis, characterized in that: The liquid culture medium consists of a basic part and an auxiliary part; The components of the base part are as follows: 15-16 g / L PPLO powder, 2-3 g / L yeast extract, 2-3 g / L sodium pyruvate, 1-1.5 g / L glucose and 20-60 ml / L phosphate buffer, and ultrapure water is added to 800 ml / L; The auxiliary part comprises the following components: 1-1.5 ml / L 10×MEM, 8-12 ml / L 0.5% cholesterol, 8-12 ml / L 1% glutamine, 8-12 ml / L 4% crotonic acid, 8-12 ml / L 0.5% malic acid, 2-6 ml / L 5 mmol / L deoxynucleotide sodium salt solution, 40-60 ml / L bovine serum, and ultrapure water to 200 ml / L.

2. The liquid culture medium according to claim 1, characterized in that The liquid culture medium consists of a basic part and an auxiliary part; The base part consists of the following components: 15.5 g / L PPLO powder, 2.5 g / L yeast extract, 2.5 g / L sodium pyruvate, 1.25 g / L glucose, and 40 ml / L phosphate buffer, and ultrapure water is added to 800 ml / L. The auxiliary part comprises the following components: 1.25 ml / L 10×MEM, 10 ml / L 0.5% cholesterol, 10 ml / L 1% glutamine, 10 ml / L 4% crotonic acid, 10 ml / L 0.5% malic acid, 4 ml / L 5 mmol / L sodium deoxynucleotide solution, 50 ml / L bovine serum, and ultrapure water to 200 ml / L.

3. A solid culture medium suitable for culturing Mycoplasma bovis, characterized in that: The solid culture medium consists of a basic part and an auxiliary part; The components of the base part are as follows: 15-16 g / L PPLO powder, 2-3 g / L yeast extract, 2-3 g / L sodium pyruvate, 1-1.5 g / L glucose, 20-60 ml / L phosphate buffer and 0.06-0.12 g / L agar, and ultrapure water is added to 800 ml / L; The auxiliary part comprises the following components: 1-1.5 ml / L 10×MEM, 8-12 ml / L 0.5% cholesterol, 8-12 ml / L 1% glutamine, 8-12 ml / L 4% crotonic acid, 8-12 ml / L 0.5% malic acid, 2-6 ml / L 5 mmol / L deoxynucleotide sodium salt solution, 40-60 ml / L bovine serum, and ultrapure water to 200 ml / L.

4. The solid culture medium according to claim 3, characterized in that The solid culture medium consists of a basic part and an auxiliary part; The base part consists of the following components: 15.5 g / L PPLO powder, 2.5 g / L yeast extract, 2.5 g / L sodium pyruvate, 1.25 g / L glucose, 40 ml / L phosphate buffer, and 0.08 g / L agar, and the mixture is made up to 800 ml / L with ultrapure water. The auxiliary part comprises the following components: 1.25 ml / L 10×MEM, 10 ml / L 0.5% cholesterol, 10 ml / L 1% glutamine, 10 ml / L 4% crotonic acid, 10 ml / L 0.5% malic acid, 4 ml / L 5 mmol / L sodium deoxynucleotide solution, 50 ml / L bovine serum, and ultrapure water to 200 ml / L.

5. The method for preparing the liquid culture medium according to any one of claims 1 to 2, characterized in that: The steps include: (a) Base: Dissolve the base components one by one in 500–600 ml of ultrapure water, stir well, adjust the pH to 7.6–8.0, and make up the remaining volume to 800 ml with ultrapure water. Autoclave and allow to cool to room temperature before use. (b) Mix the components of the auxiliary part and stir evenly, adjust the pH to 7.6-8.0, and make up the remaining volume to 200 ml with ultrapure water. After filtration and sterilization, thoroughly mix the base part and the auxiliary part to prepare a liquid medium suitable for culturing Mycoplasma bovis, and store at 4°C until used. or, The components of the basic part and the auxiliary part were directly mixed thoroughly, and the remaining volume was made up to 1000 ml with ultrapure water. The liquid culture medium suitable for the culture of Mycoplasma bovis was prepared by filtration and sterilization, and stored at 4°C for future use.

6. The method for preparing the solid culture medium according to any one of claims 3 to 4, characterized in that: The steps include: (A) Base: Dissolve the base components one by one in 500–600 ml of ultrapure water, stir well, adjust the pH to 7.6–8.0, add agar, and mix thoroughly. Make up the remaining volume to 800 ml with ultrapure water. Autoclave and place in a 50–55°C water bath until ready for use. (B) The components of the auxiliary part are mixed and stirred uniformly, and the pH is adjusted to 7.6-8.

0. The remaining volume is made up to 200 ml with ultrapure water. After filtration and sterilization, the base part and the auxiliary part are thoroughly mixed in a water bath at 50-55°C for 20-40 minutes. After cooling, a solid medium suitable for the cultivation of M. bovis is prepared and stored at 4°C until use. or, Under sterile conditions, directly add the components of the auxiliary part to the basic part, and make up the remaining volume to 1000 ml with ultrapure water. Mix thoroughly in a water bath at 50-55°C for 20-40 minutes. After cooling, a solid culture medium suitable for the culture of Mycoplasma bovis is prepared and stored at 4°C for later use.

7. Use of the liquid culture medium according to any one of claims 1 to 2 in culturing Mycoplasma bovis.

8. Use of the solid culture medium according to any one of claims 3 to 4 in culturing Mycoplasma bovis.

9. Use of the liquid culture medium according to any one of claims 1 to 2 in the preparation of a Mycoplasma bovis vaccine.

10. Use of the liquid culture medium according to any one of claims 3 to 4 in the preparation of a Mycoplasma bovis vaccine.

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