Fluid culture medium and method for isolated-culturing mycoplasma gallisepticum using same

A liquid culture medium, the technology of Mycoplasma gallisepticum, which is applied in the field of veterinary biology, can solve the problems of inability to guarantee the integrity of animals, consumption of large culture medium, low separation rate, etc., to promote rapid growth and metabolism, facilitate rapid growth, improve The effect of separation rate

Active Publication Date: 2019-06-21
HUAZHONG AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Clinical isolation of strains is a method of obtaining purified single colonies of target strains from miscellaneous flora through professional separation and culture techniques. The traditional method uses dissected lungs and other tissues to isolate Mycoplasma gallisepticum, which cannot guarantee the integrity of animals. And the medium used to cultivate Mycoplasma gallisepticum needs 3-7 days for each generation of culture. Since the isolated strains need multiple generations of continuous screening, a large amount of medium is consumed, which also causes problems such as long time-consuming and low isolation rate.

Method used

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  • Fluid culture medium and method for isolated-culturing mycoplasma gallisepticum using same
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preparation example Construction

[0041] The invention provides a method for preparing a liquid culture medium. After dissolving and mixing various components of the basic culture medium according to the proportion, adjust the pH to 7.8-8.2 with 1mol / L NaOH, seal it, and steam under high pressure at 105°C Sterilize for 20 minutes and store at 0-4°C;

[0042] The phenol red, arginine, cysteine ​​and coenzyme I in the auxiliary medium were dissolved into solutions with a mass concentration of 1%, 2%, 1% and 1% respectively, and penicillin was dissolved into 10 6 IU / mL, and then each component in the auxiliary medium is sterile filtered and sealed for storage, and mixed with the basic medium according to the addition ratio of the auxiliary medium when used.

[0043] In the above scheme, the liquid culture medium prepared by the above method is wine red. When using this culture medium to separate and screen Mycoplasma gallisepticum, the wine red color becomes orange-yellow, that is, it is judged that the discolora...

Embodiment 1

[0062] Utilize the method for separating and cultivating Mycoplasma gallisepticum in liquid medium, comprising the following steps:

[0063] Step 1: Collect throat swabs of live chickens suspected of respiratory disease with disposable sterile nasal flocking swabs, and immediately transfer the mucus on the throat swabs into liquid culture medium (not exceeding 20s), and statically at 0°C. Leave it overnight to obtain a culture solution;

[0064] Step 2: The culture solution is filtered with a 0.22 μm sterile filter membrane, the filtrate is collected, and the filtrate is inoculated into the liquid medium at a volume ratio of 10%, at 37° C., 5% CO 2 Carry out liquid culture under certain conditions until the color of the medium changes, and obtain the first generation of color-changing culture;

[0065] Step 3: Inoculate the first-generation color-changing culture into the liquid medium at 10% by volume, at 37°C, 5% CO 2 Passage and enrich the bacteria under certain condition...

Embodiment 2

[0071] Utilize the method for separating and cultivating Mycoplasma gallisepticum in liquid medium, comprising the following steps:

[0072] Step 1: Disposable sterile nasal cavity flocking swabs were collected from throat swabs of live chickens suspected of respiratory diseases, and the mucus on the throat swabs was immediately transferred to liquid medium (not exceeding 20s), and statically placed at 4°C. Leave it overnight to obtain a culture solution;

[0073] Step 2: The culture solution is filtered with a 0.22 μm sterile filter membrane, the filtrate is collected, and the filtrate is inoculated into the liquid medium at a volume ratio of 12%, at 37.5°C, 5% CO 2 Carry out liquid culture under certain conditions until the color of the medium changes, and obtain the first generation of color-changing culture;

[0074] Step 3: Inoculate the first-generation color-changing culture in the liquid medium at a volume ratio of 12%, at 37.5°C and 6% CO 2 Subculture and enrichment...

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Abstract

The invention discloses a fluid culture medium. The fluid culture medium is prepared by mixing a basic culture medium and an auxiliary culture medium. The basic culture medium contains the following components by mass concentration (g/L): 3-7 g/L of sodium chloride, 0.3-0.6 g/L of potassium chloride, 1.5-3.5 g/L of sodium phosphate dibasic dodecahydrate, 0.1-0.4 g/L of magnesium sulfate heptahydrate, 0.1-0.2 g/L of potassium dihydrogen phosphate, 10-15 g/L of glucose, 6-8 g/L of lactoalbumin hydrolysate and 6-7 g/L of yeast extract powder; and the auxiliary culture medium contains the following components by volume concentration (mL/L): 40-80 mL/L of fetal calf serum, 1-2 mL/L of penicillin, 2-3 mL/L of phenol red, 40-80 mL/L of a DMEM high glucose culture medium, 8-10 mL/L of arginine, 8-10 mL/L of cysteine and 8-10 mL/L of coenzyme I. The invention further discloses a preparation method for the culture medium and a method for isolated-culturing mycoplasma gallisepticum using the culture medium. The fluid culture medium is capable of providing the more excellent growing conditions, accelerating metabolism, advantageously shortening isolation time of the mycoplasma gallisepticum and improving an isolating rate.

Description

technical field [0001] The invention relates to the technical field of veterinary biology, in particular to a liquid culture medium and a method for isolating and culturing Mycoplasma gallisepticum using it. Background technique [0002] At present, the diseases with latent infection in the breeding industry are mainly mycoplasma gallisepticum, which is a kind of Mycoplasma gallisepticum. The common pathogenic mycoplasma in poultry can be classified into three categories: Mycoplasma gallisepticum, Mycoplasma synovialis and Mycoplasma turkey, among which Mycoplasma gallisepticum is the most harmful to poultry farming. Mycoplasma gallisepticum infection of chickens can cause outbreaks of chronic respiratory diseases, mainly sinusitis, bronchitis and air sacculitis, accompanied by secondary symptoms. The lethality rate of Mycoplasma gallisepticum is low, which usually leads to slow growth of sick chickens, lower feed conversion rate, lower carcass quality of broiler chickens, ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/20C12N1/02C12R1/35
Inventor 袁宗辉郝海红王淑歌毕丁仁胡思顺王玉莲黄玲利王旭彭大鹏陈冬梅陶燕飞潘源虎谢书宇程古月瞿玮谷宇锋
Owner HUAZHONG AGRI UNIV
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