Mycoplasma hyopneumoniae culture medium and preparation method thereof

Abstract

The invention provides a mycoplasma hyopneumoniae culture medium and a preparation method thereof, belonging to the technical field of veterinary biology. The mycoplasma hyopneumoniae liquid culture medium comprises the components as follows: brain heart infusion, lactalbumin hydrolysate, PPLO (pleuropneumonia-like organism) broth, yeast extract powder, proteose peptone, sodium thiosulfate, Hank's liquid, sodium pyruvate, 0.1% phenol red solution, penicillin and deionized water. The preparation method comprises the following steps of: adding health horse serum before using, and adding agar into the liquid culture medium to obtain a solid culture medium of mycoplasma hyopneumoniae. The viable bacteria titer of the mycoplasma hyopneumoniae culture medium can reach 1*109CCU / ml-1*1010CCU / ml; the viable bacteria titer and the separation sensibility are far higher than those of the existing culture medium, and the mycoplasma hyopneumoniae is fast in growth speed and high in the separation sensibility; and the preparation method of the culture medium is simple in technology, strong in operability, and suitable for industrial large-scale production.

Description

technical field

[0001] The invention belongs to the technical field of veterinary biology, and relates to a culture medium for mycoplasma hyopneumoniae and a preparation method thereof. Background technique

[0002] Mycoplasma hyopneumoniae is one of the important pathogens that cause contact chronic respiratory diseases in pigs, and it is one of the main swine disease pathogens that are widespread in the world and seriously endanger the healthy development of the pig industry. The incidence rate dropped sharply, secondary to the occurrence of other diseases and aggravated symptoms. Inactivated vaccine against Mycoplasma swine pneumonia can reduce the occurrence of panting disease, but the premise of high-quality vaccine needs to be supported by a good culture medium.

[0003] The culture medium is a matrix in which various nutrients are artificially synthesized for the growth of microorganisms. Mycoplasma is a tiny prokaryotic organism without a cell wall. It can grow on ...

Examples

Embodiment 1

[0018] The preparation of embodiment 1 culture medium of the present invention

[0019] Liquid medium:

[0020] Liquid A: 2.0g brain heart extract, 5.0g PPLO broth, 300ml deionized water

[0021] Mix and stir the above ingredients to dissolve them completely, autoclave at 116°C for 20 minutes, and cool for later use;

[0022] Solution B: 5.0ml of 10× Hank's solution, 1.0g of whey protein hydrolyzate, 5.0g of yeast extract, 0.8g of sodium pyruvate, 3.0g of peptone, 0.1g of sodium thiosulfate, 10ml of 0.1% phenol red, 400U of penicillin / ml, deionized water 545ml

[0023] Mix and stir the above ingredients to dissolve them completely, filter and sterilize with a 0.22 μm filter membrane, and store at 4°C for later use;

[0024] Solution C: healthy horse serum 140ml

[0025] Thoroughly mix liquid A, liquid B and liquid C evenly, and adjust the pH to 7.6 with 1mol / L sodium hydroxide solution to prepare a liquid culture medium for Mycoplasma hyopneumoniae.

[0026] Solid medium...

Embodiment 2

[0028] The preparation of embodiment 2 medium of the present invention

[0029] Liquid medium:

[0030] Liquid A: 4.5g brain heart extract, 4.0g PPLO broth, 260ml deionized water

[0031] Mix and stir the above ingredients to dissolve them completely, autoclave at 116°C for 20 minutes, and cool for later use;

[0032] Solution B: 3.0ml of 10× Hank's solution, 1.25g of whey protein hydrolyzate, 4.5g of yeast extract, 1.0g of sodium pyruvate, 1.5g of peptone, 0.135g of sodium thiosulfate, 17ml of 0.1% phenol red, 200U of penicillin / ml, deionized water 560ml

[0033] Mix and stir the above ingredients to dissolve them completely, filter and sterilize with a 0.22 μm filter, and store at 4°C for later use;

[0034] Liquid C: healthy horse serum 160ml

[0035] Thoroughly mix liquid A, liquid B and liquid C uniformly, and adjust the pH to 7.8 with 1 mol / L sodium hydroxide to prepare a liquid culture medium for Mycoplasma hyopneumoniae.

[0036] Solid medium:

[0037] On the ba...

Embodiment 3

[0038] The preparation of embodiment 3 medium of the present invention

[0039] Liquid medium:

[0040] Liquid A: 5.0g brain heart extract, 1.5g PPLO broth, 200ml deionized water

[0041] Mix and stir the above ingredients to dissolve them completely, autoclave at 116°C for 20 minutes, cool and store at 4°C for later use;

[0042] Solution B: 1.0ml of 10× Hank's solution, 3.0g of whey protein hydrolyzate, 3.0g of yeast extract, 2.5g of sodium pyruvate, 1.0g of peptone, 0.5g of sodium thiosulfate, 20ml of 0.1% phenol red, 200U of penicillin / ml, deionized water 579ml

[0043] Mix and stir the above ingredients to dissolve them completely, filter and sterilize with a 0.22 μm filter membrane, and store at 4°C for later use;

[0044] Solution C: healthy horse serum 200ml

[0045] Fully mix liquid A, liquid B and liquid C evenly, and adjust the pH to 7.7 with 1mol / L sodium hydroxide solution to prepare a liquid culture medium for Mycoplasma hyopneumoniae.

[0046] Solid medium...

technical field

[0001] The invention belongs to the technical field of veterinary biology, and relates to a culture medium for mycoplasma hyopneumoniae and a preparation method thereof. Background technique

[0002] Mycoplasma hyopneumoniae is one of the important pathogens that cause contact chronic respiratory diseases in pigs, and it is one of the main swine disease pathogens that are widespread in the world and seriously endanger the healthy development of the pig industry. The incidence rate dropped sharply, secondary to the occurrence of other diseases and aggravated symptoms. Inactivated vaccine against Mycoplasma swine pneumonia can reduce the occurrence of panting disease, but the premise of high-quality vaccine needs to be supported by a good culture medium.

[0003] The culture medium is a matrix in which various nutrients are artificially synthesized for the growth of microorganisms. Mycoplasma is a tiny prokaryotic organism without a cell wall. It can grow on ...

Examples

Embodiment 1

[0018] The preparation of embodiment 1 culture medium of the present invention

[0019] Liquid medium:

[0020] Liquid A: 2.0g brain heart extract, 5.0g PPLO broth, 300ml deionized water

[0021] Mix and stir the above ingredients to dissolve them completely, autoclave at 116°C for 20 minutes, and cool for later use;

[0022] Solution B: 5.0ml of 10× Hank's solution, 1.0g of whey protein hydrolyzate, 5.0g of yeast extract, 0.8g of sodium pyruvate, 3.0g of peptone, 0.1g of sodium thiosulfate, 10ml of 0.1% phenol red, 400U of penicillin / ml, deionized water 545ml

[0023] Mix and stir the above ingredients to dissolve them completely, filter and sterilize with a 0.22 μm filter membrane, and store at 4°C for later use;

[0024] Solution C: healthy horse serum 140ml

[0025] Thoroughly mix liquid A, liquid B and liquid C evenly, and adjust the pH to 7.6 with 1mol / L sodium hydroxide solution to prepare a liquid culture medium for Mycoplasma hyopneumoniae.

[0026] Solid medium...

Embodiment 2

[0028] The preparation of embodiment 2 medium of the present invention

[0029] Liquid medium:

[0030] Liquid A: 4.5g brain heart extract, 4.0g PPLO broth, 260ml deionized water

[0031] Mix and stir the above ingredients to dissolve them completely, autoclave at 116°C for 20 minutes, and cool for later use;

[0032] Solution B: 3.0ml of 10× Hank's solution, 1.25g of whey protein hydrolyzate, 4.5g of yeast extract, 1.0g of sodium pyruvate, 1.5g of peptone, 0.135g of sodium thiosulfate, 17ml of 0.1% phenol red, 200U of penicillin / ml, deionized water 560ml

[0033] Mix and stir the above ingredients to dissolve them completely, filter and sterilize with a 0.22 μm filter, and store at 4°C for later use;

[0034] Liquid C: healthy horse serum 160ml

[0035] Thoroughly mix liquid A, liquid B and liquid C uniformly, and adjust the pH to 7.8 with 1 mol / L sodium hydroxide to prepare a liquid culture medium for Mycoplasma hyopneumoniae.

[0036] Solid medium:

[0037] On the ba...

Embodiment 3

[0038] The preparation of embodiment 3 medium of the present invention

[0039] Liquid medium:

[0040] Liquid A: 5.0g brain heart extract, 1.5g PPLO broth, 200ml deionized water

[0041] Mix and stir the above ingredients to dissolve them completely, autoclave at 116°C for 20 minutes, cool and store at 4°C for later use;

[0042] Solution B: 1.0ml of 10× Hank's solution, 3.0g of whey protein hydrolyzate, 3.0g of yeast extract, 2.5g of sodium pyruvate, 1.0g of peptone, 0.5g of sodium thiosulfate, 20ml of 0.1% phenol red, 200U of penicillin / ml, deionized water 579ml

[0043] Mix and stir the above ingredients to dissolve them completely, filter and sterilize with a 0.22 μm filter membrane, and store at 4°C for later use;

[0044] Solution C: healthy horse serum 200ml

[0045] Fully mix liquid A, liquid B and liquid C evenly, and adjust the pH to 7.7 with 1mol / L sodium hydroxide solution to prepare a liquid culture medium for Mycoplasma hyopneumoniae.

[0046] Solid medium...

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