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A kind of culture medium for isolating and cultivating mycoplasma heteroplasma and its preparation method

A technology of separation culture and liquid culture medium, which is applied in the field of culture medium and its preparation for the separation and culture of Mycoplasma dissimilarum, which can solve the problems of long culture time, complex configuration, poor reproductive ability, etc.

Active Publication Date: 2019-07-26
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

Growth is demanding on nutrients. In addition to basic nutrition, it is often necessary to add various ingredients such as yeast liquid, in addition to adding 10% to 20% animal serum, and other ingredients, and its configuration is complicated.
At present, the isolation and culture media of Mycoplasma heterogenes are mainly GS medium and modified Friis medium, but there are still problems such as long culture time, low titer of viable bacteria, and poor reproductive ability.

Method used

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  • A kind of culture medium for isolating and cultivating mycoplasma heteroplasma and its preparation method
  • A kind of culture medium for isolating and cultivating mycoplasma heteroplasma and its preparation method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] 1. Test reagent:

[0026]PPLO Broth is a product of BD Company in the United States, and the article number is 2625084.

[0027] Brain-heart extract is a product of BD Company in the United States, and the product number is 237500.

[0028] Phenol red is a product of sigma company, the article number is P3532.

[0029] Ampicillin is a product of Sigma Company, the product number is A0166.

[0030] The horse serum is the product of Hyclone Company, the article number is SH30074.03.

[0031] Pig serum is the product of GIBCO Company, the product number is 2625084.

[0032] Fresh yeast is the product of Angel Company.

[0033] Sodium chloride, magnesium sulfate, potassium chloride, calcium chloride, disodium hydrogen phosphate, potassium dihydrogen phosphate, sodium bicarbonate, glucose and other reagents are domestic analytical reagents.

[0034] , Configure 1000 ml of liquid culture medium of the present invention:

[0035] Weigh 8.5 g of brain heart extract and ad...

Embodiment 2

[0045] The comparison test of culture medium of the present invention and several culture mediums of prior art:

[0046] 1, culture medium of the present invention and prior art several culture medium viable bacteria titer comparative tests:

[0047] 1.1 Mycoplasma heteroplasma GS01 strain (CCTCC, China Type Culture Collection Center, Wuhan University, Bayi Road, Hongshan District, Wuhan City, Hubei Province, China Type Culture Collection Center, Zip Code 430072, No. M 2016394, storage date July 14, 2016 ) to inoculate the Mycoplasma heteroplasma liquid culture medium of the present invention and the two culture mediums of the prior art, after the seeds are subcultured and rejuvenated, inoculate the corresponding culture medium at a ratio of 10% (V / V), and culture at a constant temperature of 37°C. Remove the culture aseptically when the color turns yellow and the pH value drops from 7.2 to 6.1-6.2.

[0048] 1.2 Determination of viable bacterial titer (CCU). The method is as...

Embodiment 3

[0055] Sensitivity test of culture medium of the present invention and several kinds of medium of prior art heteromycoplasma separation:

[0056] Under the same conditions, 15 bovine lung disease materials that were identified as positive for Heteromycoplasma by PCR were separated by using the Heteromycoplasma culture medium provided by the present invention and the two culture media of the prior art. The specific method is as follows: cut the positive disease material of Mycoplasma heterogenes into pieces to the size of rice grains, put into the Mycoplasma heterogenes culture medium provided by the present invention and the culture medium of the prior art (both contain thallium acetate and penicillin which inhibit the growth of miscellaneous bacteria in the culture medium. or ampicillin), 10 small pieces in each tube, make 3 ten-fold dilutions with the corresponding liquid medium, and dilute to 10 -3 , cultured in a constant temperature incubator at 37°C, and observed the col...

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Abstract

The invention discloses a culture medium for isolated culture of mycoplasma dispar. Each 1000 ml of the culture medium comprises: 8.5 g of PPLO broth, 8.5 g of brain heart infusion, 2.0 g of glucose, 500 ml of a Hank's balanced salt solution, 60 ml of a 25% fresh yeast extraction liquid, 100 ml of sterile swine serum, 100 ml of sterile horse serum, 100 mg of ampicillin, and 1 ml of 10% thallium acetate. The invention also provides a preparation method of the culture medium. The culture medium, compared with culture mediums in the prior art, reaches 10<10> CCU / ml in living bacteria titer, while the growth titers of the culture mediums in the prior art are 10<7>-10<9> CCU / ml. In subculture of the mycoplasma dispar, the culture medium requires 2-3 days for one passage, while the culture mediums in the prior art require 3-5 days usually for one passage; the culture medium is fully proved to have quick growth and high culture titer, and is suitable for the isolated culture of the mycoplasma dispar.

Description

technical field [0001] The invention relates to the technical field of veterinary biology, in particular to a culture medium for the isolation and cultivation of Mycoplasma heteroplasma and a preparation method thereof. Background technique [0002] Mycoplasma heterogenes ( Mycoplasma dispar ), formerly known as Mycoplasma heteromycetes. Gourlay RN and Leach RH first isolated the bacterium from the lung of a calf suffering from pneumonia in England in 1969 (Gourlay RN and Leach RH, 1970), and then reported in the United States, Australia, New Zealand, Japan, and Denmark. The bacterium hardly grows on commonly used media and requires special media to grow. It takes 7-14 days to isolate the bacteria in the first generation (the longest period is 4-23 days), and the growth rate of the strain becomes faster after adaptation to the laboratory medium, generally within 2 weeks. The colony morphology of the bacteria was atypical for mycoplasma without a central bulge. Zhang Daoy...

Claims

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Application Information

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