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Culture medium for separation and purification of mycoplasma bovirhinis, preparation method and application of culture medium

A technology of separation and purification and liquid culture medium, which is applied in the field of culture medium for separation and purification of Mycoplasma bovis and its preparation, which can solve the problems of poor reproductive ability, easy misdiagnosis, and low titer of viable bacteria, and achieve rapid growth and high titer , the effect of high success rate

Active Publication Date: 2017-05-10
LANZHOU INST OF VETERINARY SCI CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The identification of Mycoplasma bovis mainly relies on the identification of physiological and biochemical characteristics, polymerase chain reaction (Kobayashi H et al, 1998; Miles K et al. 2004) and immune tests including growth inhibition test, metabolic inhibition test, immunofluorescence test, However, polymerase chain reaction and immunoassays all involve the isolation and cultivation of the pure culture of Mycoplasma bovis; while the current isolation of Mycoplasma bovis is mainly GS medium and SP-4 medium, and there are still low titers of viable bacteria, reproduction Poor ability and other problems are easy to cause missed diagnosis; therefore, there is an urgent need in this field for efficient and rapid isolation and diagnosis of Mycoplasma bovis and the method for separating and purifying Mycoplasma bovis, and then carry out pathogenic characteristics, culture characteristics, pathogenic mechanism, vaccine preparation, and so on of Mycoplasma bovis. Research on diagnosis and prevention

Method used

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  • Culture medium for separation and purification of mycoplasma bovirhinis, preparation method and application of culture medium
  • Culture medium for separation and purification of mycoplasma bovirhinis, preparation method and application of culture medium
  • Culture medium for separation and purification of mycoplasma bovirhinis, preparation method and application of culture medium

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] The preparation of substratum of the present invention:

[0042] 1, Mycoplasma bovis substratum of the present invention is made up of liquid medium and solid medium:

[0043] The formula of 1000ml liquid medium is: PPLO broth 10.5 g / L, brain heart extract 8.0 g / L, sodium chloride 4.0 g / L, magnesium sulfate 0.05 g / L, potassium chloride 0.2 g / L, calcium chloride 0.09 g / L, disodium hydrogen phosphate 0.024 g / L, potassium dihydrogen phosphate 0.03 g / L, glucose 5.0 g / L, hydrolyzed milk protein 5.0 g / L, 25% fresh yeast extract 60 ml / L, 1% phenol red 2.5ml / L, sterile pig serum 100 ml / L, sterile horse serum 100 ml / L, ampicillin 100 mg / L, 10% thallium acetate 1 ml / L, and deionized water as the balance.

[0044] The method for preparing 1000 ml of the liquid culture medium of the present invention is as follows: take by weighing 8.0 g of brain heart extract, 4.0 g of sodium chloride (NaCl), magnesium sulfate (MgSO 4 .7H 2 O) 0.05 g, potassium chloride (KCl) 0.2 g, calcium chl...

Embodiment 2

[0049] The comparison test of culture medium of the present invention and several culture mediums of prior art:

[0050] 1, culture medium of the present invention and prior art several culture medium viable bacteria titer comparative tests:

[0051] 1.1 The type strain of Mycoplasma bovis PG43 (purchased from American Culture Collection Center ATCC, Wuhan University, Bayi Road, Hongshan District, Wuhan City, Hubei Province, Wuhan University, China Type Culture Collection Center, Zip Code 430072, number ATCC 27748) and Mycoplasma bovis self-isolated strains GS01 strain (CCTCC, China Center for Typical Culture Collection, No. M2016393, date of preservation: July 14, 2016) was inoculated with the Mycoplasma bovis culture medium of the present invention and the two culture mediums of the prior art. After the seeds were subcultured and rejuvenated, 10% Inoculate the corresponding culture medium at the ratio of (V / V) and incubate at a constant temperature of 37°C. When the color of...

Embodiment 4

[0068] Application example:

[0069] The method for separating and purifying Mycoplasma bovis from the prepared medium is as follows:

[0070] 1. Take the diseased lung tissue under aseptic conditions, cut it into pieces to the size of rice grains, take 0.5 g of the chopped diseased lung tissue and inoculate it into 4.5 ml of liquid medium, and make at least three ten-fold dilutions with the liquid medium, and dilute to 10 -3 , put the gradient dilution samples in a constant temperature incubator at 37°C and cultivate them, and observe the color change of the liquid medium and the presence or absence of flocs every day. After 5 days, no matter whether the color of the liquid culture changes or not, carry out blind passage, and inoculate the liquid culture into the solid medium for cultivation; if turbidity occurs after 24 hours, the culture can be filtered through a 0.45 μm filter membrane and then transplanted. Transfer to a new liquid medium at a ratio of 1:10 or use an in...

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Abstract

The invention discloses a culture medium for separation and purification of mycoplasma bovirhinis. Every 1000ml of culture medium comprises, by weight 10.5g of PPLO (pleuropneumonia-like organism) broth, 8,0g of cerebrocardiac leach liquor, 4.0g of sodium chloride, 0.05g of magnesium sulfate, 0.2g of potassium chloride, 0,09g of calcium chloride, 0.024g of disodium hydrogen phosphate, 0,03g of monopotassium phosphate, 5.0g of glucose, 5.0g of lactoalbumin hydrolysate, 60ml of 25% of fresh yeast leach liquor, 100ml of aseptic swine serum, 100ml of aseptic horse serum, 100mg of ampicillin and 1ml of 10% of thallium acetate. The invention further provides a preparation method and application of the culture medium. Compared with the prior art, the culture medium for separation and purification of the mycoplasma bovirhinis, the preparation method and the application of the culture medium have the advantages of high growing speed and titer and high mycoplasma bovirhinis separation success rate.

Description

technical field [0001] The invention relates to the field of veterinary biotechnology, in particular to a culture medium for separating and purifying Mycoplasma bovis, a preparation method and application thereof. Background technique [0002] Mycoplasma bovis ( Mycoplasma bovirhinis ), can cause bovine pneumonia, mastitis, conjunctivitis and inflammation of the reproductive tract. Mycoplasma bovis is an important but often overlooked pathogen. As early as 1947, Edward reported that a strain of Mycoplasma was isolated from the reproductive tract of aborted dairy cows (Edward, D. G et al. 1947), which was later identified and named Mycoplasma bovis (Edward and Freundt, 1956). Mycoplasma bovis has a worldwide distribution. my country's Chen Jiadi et al. reported in 1985 that the strain was isolated from a dairy farm in Shanghai, and its physiological and biochemical characteristics were basically consistent with those of the established strain (Chen Jiadi et al., 1985). In...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/20C12N1/02C12R1/35
CPCC12N1/02C12N1/20
Inventor 郝华芳陈胜利储岳峰赵萍刘永生
Owner LANZHOU INST OF VETERINARY SCI CHINESE ACAD OF AGRI SCI
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