Mycoplasma synoviae culture medium and preparation method thereof

Abstract

The invention belongs to the field of microbial culture media. The invention provides a mycoplasma synoviae culture medium and a preparation method thereof, the culture medium comprises oat hydrolysate, brain heart extract powder, yeast extract powder, beef extract powder and specific nutritional factors, and the specific nutritional factors comprise horse serum, vitamins, arginine and glucose. The invention also provides a preparation method of the culture medium. The culture medium contains basic nutrient substances and specific nutritional factors required by growth of the mycoplasma synoviae, under the synergistic effect of the components, the viable bacteria titer is remarkably improved, high titer is achieved, the serum dosage is remarkably small, the influence of exogenous factors in serum on mycoplasma products is reduced, and the culture medium is more suitable for production.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of microbial culture medium, and particularly relates to a Mycoplasma synoviae culture medium and a preparation method thereof. BACKGROUND

[0002] Mycoplasma synoviae is one of the important poultry pathogens, which can cause poultry upper respiratory tract disease, infectious synovitis and bursitis, reduce egg production, egg quality and hatching rate, cause growth retardation and feed conversion rate reduction, increase carcass waste rate, and bring serious economic losses to the global poultry industry.

[0003] The most effective way at present is Mycoplasma synoviae vaccination prevention, but it is difficult to culture high-titer Mycoplasma synoviae by in vitro culture technology at present.

[0004] Therefore, how to make the titer of Mycoplasma synoviae culture higher and the purity better is particularly important, and it is of great significance to research and develop a good Mycoplasma synoviae culture medium for the research and disease prevention of Mycoplasma synoviae. SUMMARY

[0005] In view of the above problems, the present application provides a Mycoplasma synoviae culture medium and a preparation method thereof. Compared with the existing Mycoplasma culture medium, the titer of the Mycoplasma synoviae culture medium of the present application is significantly improved, and in addition, the serum consumption of the culture medium of the present application is less.

[0006] The object of the present application is to provide a Mycoplasma synoviae culture medium, which comprises oat hydrolysate, brain heart infusion powder, yeast infusion powder, beef infusion powder and specific nutrient factor components, wherein the specific nutrient factor comprises horse serum, vitamins, arginine and glucose.

[0007] The culture medium of the present application not only contains basic nutrients required for the growth of Mycoplasma synoviae, but also contains specific nutrient factors required for the growth of Mycoplasma synoviae. Under the synergistic action of the above components, the culture medium of the present application is beneficial to the growth of Mycoplasma synoviae.

[0008] Further, the culture medium of the present application comprises the following components with the following mass concentration or volume concentration, oat hydrolysate 1-10 g / L; brain heart infusion powder 1-10 g / L; yeast infusion powder 1-10 g / L; beef infusion powder 1-10 g / L; The specific nutrient factor in the culture medium has the following mass concentration or volume concentration of components, horse serum 5-100 ml / L; vitamins 0.1-1 g / L; arginine 0.1-3 g / L; Glucose 1-5 g / L.

[0009] The mycoplasma quantity of the culture medium of the application can reach 10 10 CCU / ml or above, compared with the culture of the existing culture medium, the application significantly improves the viable bacterial titer, and realizes high titer.

[0010] Preferably, the culture medium comprises the following components in mass concentration or volume concentration, Oat hydrolysate 5 g / L; Brain heart infusion powder 5 g / L; Yeast extract powder 5 g / L; Beef infusion powder 5 g / L; Horse serum 50-100 ml / L; Vitamin 0.5 g / L; Arginine 1 g / L; Glucose 2.5 g / L.

[0011] The application also aims to provide a preparation method of the synovial mycoplasma culture medium, comprising the following steps, (1) Preparation of the basic culture medium: oat hydrolysate, yeast extract powder, beef infusion powder, and glucose are weighed according to the above proportions and added into deionized water, and 1% phenol red is dissolved in the deionized water, and after high-pressure sterilization at 100-150°C for 10-45 min, the mixture is cooled to room temperature for standby; (2) Preparation of the auxiliary culture medium: brain heart infusion powder, vitamin, arginine, horse serum, and penicillin are weighed according to the above proportions, filtered with a 0.1-0.5 micron filter to remove bacteria, and mixed thoroughly to obtain the auxiliary culture medium; (3) Preparation of the culture medium: under sterile conditions, the above basic culture medium and auxiliary culture medium are mixed, sterilized water is added to make up the volume, a sterilized NaOH solution is used to adjust the pH to 7.4-7.6, and the mixture is shaken thoroughly to obtain the synovial mycoplasma culture medium.

[0012] The above synovial mycoplasma culture medium or the synovial mycoplasma culture medium prepared by the above preparation method is applied to the preparation of a mycoplasma vaccine.

[0013] Compared with the prior art, the application has the following beneficial effects: (1) The culture medium of the application contains basic nutrients and specific nutrients required for the growth of synovial mycoplasma, and under the synergistic effect of the above components, the culture medium of the application is beneficial to the growth of synovial mycoplasma, and the mycoplasma quantity can reach 10 10 CCU / ml or above, the application significantly improves the viable bacterial titer, improves the low-titer status of the existing synovial mycoplasma culture medium, realizes high titer, and is more suitable for production. (2) The serum dosage is significantly less, and the influence of exogenous factors in the serum on mycoplasma products is reduced; (3) The harm caused by the use of thallium acetate is avoided, and the unsafe adverse factors of the traditional culture medium containing thallium acetate to experimenters and animals are reduced; (4) The fresh yeast extract is replaced by yeast extract powder, time and labor are saved, and the labor cost and the cost of machine equipment are greatly reduced. DETAILED DESCRIPTION

[0014] The technical solutions of the present application will be clearly and completely described below in combination with the embodiments of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor fall within the scope of protection of the present application.

[0015] Example 1 Preparation of avian synovial capsule mycoplasma culture medium 1. Preparation of the basic culture medium: 5.0 g of oat hydrolysate, 5.0 g of yeast extract powder, 5.0 g of beef extract powder and 2.5 g of glucose were weighed, mixed with 850 ml of deionized water, 1.0 ml of 1% phenol red was added, the pH was adjusted to 7.4-7.6 with 1M NaOH, and then sterilized at 115°C and 70kPa for 30 min, and cooled to room temperature for standby; 1. Preparation of 1% phenol red solution: 1.0 g of phenol red was placed in a glass mortar, 0.1M NaOH was added dropwise, and grinding was performed until complete dissolution. The dissolved phenol red was sucked into a 100 ml volumetric flask, and the residual phenol red in the mortar was carefully washed with deionized water into the volumetric flask. Finally, deionized water was added to 100 ml.

[0016] 2. Preparation of the auxiliary culture medium: 50 ml of 0.1g / ml brain heart infusion powder, 10 ml of 0.05g / ml vitamin, 10 ml of 0.1g / ml arginine, 50 ml of horse serum and 1 ml of penicillin (100,000 IU) filtered and sterilized by 0.22 micron filter were mixed to obtain the auxiliary culture medium.

[0017] 3. Under sterile conditions, the basic culture medium obtained in step 1 and the auxiliary culture medium obtained in step 2 were mixed, and the volume was adjusted to 1000 ml with sterile water. The pH value was adjusted to 7.4-7.6 with sterilized 1M NaOH (4 g dissolved in 100 ml deionized water), and then shaken well and stored at 4°C for standby.

[0018] Example 2 Preparation of avian synovial capsule mycoplasma culture medium 1. Preparation of the base medium: Take oat hydrolysate 5.0 g, yeast extract 5.0 g, beef extract 5.0 g, glucose 2.5 g, and mix with 800 ml of deionized water. Add 1% phenol red (prepared in the same way as in Example 1) 1.0 ml into the above 800 ml of deionized water, adjust the pH to 7.4-7.6 with 1 mol NaOH, and sterilize at 115°C for 30 min, then cool to room temperature for standby use. 2. Preparation of the auxiliary medium: Take 10% brain heart infusion powder 50 ml, 0.05 g / ml vitamin 10 ml, 0.1 g / ml arginine 10 ml, horse serum 100 ml, and penicillin (100,000 IU) 1 ml, which have been sterilized by 0.22 micron filter membrane, and mix thoroughly to obtain the auxiliary medium.

[0019] 3. Under sterile conditions, mix the base medium obtained in step 1 and the auxiliary medium obtained in step 2, and make up to 1000 ml with sterile water. Adjust the pH to 7.4-7.6 with sterilized 1M NaOH (4 g dissolved in 100 ml of deionized water), shake thoroughly, and store at 4°C for standby use.

[0020] Preparation of modified KM2 medium Dissolve MEM 5 g, glucose 0.4 g, sodium pyruvate 0.2 g, hydrolyzed milk protein 5.1 g, and 1% phenol red solution 2.5 ml in 700 ml of deionized water, add 25% fresh yeast extract 20 ml, penicillin (200,000 IU / ml), 10% thallium acetate 1 ml, and sterile horse serum 200 ml, mix thoroughly, make up to 1000 ml with deionized water, adjust the pH to 7.4 with sterilized 1M NaOH, filter sterilize with a 0.22 micron filter membrane, and store at 4°C for standby use. Preparation of modified Thiaucourt's medium 1. Preparation of the base solution: Take PPLO broth 21.0 g, sodium pyruvate 2.0 g, glucose 1.0 g, and 0.4% phenol red 4.5 ml, and dissolve in 700 ml of deionized water. Sterilize at 115°C for 30 min, then cool to room temperature for standby use.

[0021] 2. Take 700 ml of the base solution prepared in step 1, and mix with 25% fresh yeast extract 100 ml, penicillin (200,000 IU) 1 ml, 10% thallium acetate 1 ml, and sterile horse serum 200 ml. Adjust the pH to 7.4-7.6 with 1M NaOH, filter sterilize with a 0.22 micron filter membrane, and store at 4°C for standby use.

[0022] Note: Preparation of 25% yeast extract in Comparative Example 1 and Comparative Example 2: 500 g of fresh yeast was added to 2000 ml of deionized water, dissolved by stirring, and the pH value was adjusted to 4.5-5.0 with concentrated hydrochloric acid: deionized water at a ratio of 1:1 (by volume), and then 80°C water bath (bottle temperature) for 30 min, and centrifuged at 3000 rpm / min for 20 min. The supernatant was taken, the pH value was adjusted to 7.8-8.0 with 1 M NaOH, boiled, and then cooled at room temperature. The supernatant was filtered with double-layer filter paper, and then deionized water was added to 2000 ml. The solution was stored at 2-8°C for standby use.

[0023] Application Example Culture of Mycoplasma synoviae Mycoplasma synoviae MS-w01 strain was inoculated into the culture medium prepared in Example 1, the modified KM2 culture medium prepared in Comparative Example 1, and the modified Thiaucourt's culture medium prepared in Comparative Example 2, respectively. After seed subculture and rejuvenation, each culture medium was inoculated with 10% (V / V) of the corresponding culture medium, and incubated at 37°C. When the color of the culture medium changed to yellow and the pH value changed from 7.4-7.6 to 6.8-6.9, the culture was taken out aseptically.

[0024] Viable bacterial titer (CCU) test 1. Determination method Twelve sterile test tubes were taken, and 4.5 ml of the corresponding culture medium was added to each tube. In the first tube, 0.5 ml of well-grown Mycoplasma synoviae culture was added, and mixed well with a shaker. A new pipette was used to take 0.5 ml from the first tube and add it to the second tube, and then 10-fold dilution was performed in turn to the eleventh tube. In the eleventh tube, 0.5 ml of the culture solution was discarded. The dilution of the culture solution was 10-1-10-11, and the twelfth tube was the corresponding culture medium control. Three replicates were set for the test tubes. The test tubes were placed in a 37°C constant temperature incubator for static culture. The color change was observed every day, and the observation was continued for 10 days. The highest dilution with color change was the viable bacterial titer of the culture, which was expressed by color change unit (CCU). The test was repeated three times.

[0025] 2. Test results Mycoplasma synoviae was inoculated into the culture medium prepared in Example 1, the modified KM2 culture medium prepared in Comparative Example 1, and the modified Thiaucourt's culture medium prepared in Comparative Example 2, respectively. Each group was subjected to three growth tests for CCU determination, and the determination results are shown in the following table.

[0026]

[0027] As can be seen from the data in the table, under the same conditions, the culture medium prepared in Example 1 of the present application was used to culture Mycoplasma synoviae for three tests, and the CCU determination results were all 10 10CCU / ml. The Mycoplasma synoviae was cultured in the modified KM2 medium for three times, and the CCU determination results were all 10 8 CCU / ml. The Mycoplasma synoviae was cultured in the modified KM2 medium for three times, and the CCU determination results were all 10 9 CCU / ml.

[0028] Therefore, the titer of the medium of the present application is obviously higher than that of the modified KM2 medium and the modified Thiaucourt's medium. In addition, the serum content of the medium of the present application is 5%, which is 1 / 4 of the modified KM2 medium and the modified Thiaucourt's medium, and the yeast extract powder is used to replace 25% of the fresh yeast extract, which is more convenient.

[0029] From the above results, it can be seen that the medium of the present application has the characteristics of high titer of live bacteria, low serum content and convenience for the growth and culture of the Mycoplasma synoviae, and is suitable for the growth and culture of the Mycoplasma synoviae.

[0030] The above examples are only used to illustrate the present application, but not to limit the present application. Those skilled in the art can make various changes or modifications without departing from the spirit and scope of the present application. Therefore, all equivalent technical solutions should belong to the scope of the present application, which is defined by the claims.

Claims

1. A culture medium for Mycoplasma synoviae, characterized in that, It includes oat hydrolysate, brain and heart extract powder, yeast extract powder, beef extract powder, and specific nutrient components, wherein the specific nutrient components include horse serum, vitamins, arginine, and glucose.

2. The Mycoplasma synoviae culture medium according to claim 1, characterized in that, Including the following components with mass or volume concentrations, Oat hydrolysate 1-10 g / L; Brain and heart extract powder 1-10g / L; Yeast extract 1-10g / L; Beef extract powder 1-10g / L; The specific nutrient factor, in the culture medium, has a component mass concentration or volume concentration of [missing information]. Horse serum 5-100 ml / L; Vitamins 0.1-1 g / L; Arginine 0.1-3 g / L; Glucose 1-5 g / L.

3. The Mycoplasma synoviae culture medium according to claim 2, characterized in that, Including the following components with mass or volume concentrations, Oat hydrolysate 5g / L; Brain and heart extract powder 5g / L; Yeast extract 5g / L; Beef extract powder 5g / L; Horse serum 50-100 ml / L; Vitamin 0.5g / L; Arginine 1g / L; Glucose 2.5g / L.

4. A method for preparing the Mycoplasma synoviae culture medium as described in claim 1, characterized in that, Includes the following steps, (1) Preparation of basic culture medium: Weigh oat hydrolysate, yeast extract, beef extract and glucose according to the proportion and add them to deionized water. Dissolve 1% phenol red in the above deionized water. Autoclave at 100-150℃ for 10-45 min and cool to room temperature for later use. (2) Preparation of auxiliary culture medium: Weigh out brain heart extract powder, vitamins, arginine, horse serum and penicillin according to the proportion, filter them with a 0.1-0.5 micrometer filter membrane to remove bacteria, mix them thoroughly and the auxiliary culture medium is obtained. (3) Preparation of culture medium: Under aseptic conditions, the basic culture medium obtained in step (1) and the auxiliary culture medium obtained in step (2) are mixed, the volume is made up with sterile water, the pH is adjusted to 7.4~7.6 with sterile NaOH solution, and the mixture is shaken thoroughly to obtain Mycoplasma synoviae culture medium.

5. The use of the Mycoplasma synovitis culture medium as described in claim 1 in the preparation of mycoplasma vaccines.

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