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Mycoplasma synoviae culture method

A technology of mycoplasma synovialum and culture methods, applied in the direction of microorganism-based methods, biochemical equipment and methods, microorganisms, etc., can solve the problems of long time consumption and low content of viable bacteria, reduce the possibility of pollution and shorten the cultivation time Time, the effect of reducing the error

Inactive Publication Date: 2016-08-10
YEBIO BIOENG OF QINGDAO
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  • Description
  • Claims
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Problems solved by technology

At present, there is neither live vaccine nor inactivated vaccine for preventing Mycoplasma gallinarum on the market in China. Due to the difficulty in cultivating Mycoplasma gallinarum, the traditional static culture process takes a long time and the content of live bacteria is not high, so it cannot Used for large-scale production of Mycoplasma bursa vaccine

Method used

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  • Mycoplasma synoviae culture method
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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0017] Embodiment 1 prepares culture medium of the present invention according to different formulations

[0018] 1 Culture medium preparation

[0019] 1.1 Preparation of base liquid Take 21.0g of PPLO broth, 5.0g of glucose, add to 1000ml of deionized water, 1.0ml of 1% phenol red solution, mix and dissolve the above ingredients, autoclave at 116°C for 30 minutes, cool and set aside for 2-8 Store at ℃.

[0020] 1.2 Culture medium preparation After cooling the base solution, aseptically add different amounts of inactivated horse serum, 25% yeast extract, 1% coenzyme I solution, 1% L-cysteine ​​solution, and 800,000 units / ml penicillin solution in 1.2 ml, adjust the pH value to 7.6 with 1mol / L sodium hydroxide solution, and prepare 6 kinds of liquid culture medium. The specific formula is shown in Table 1.

[0021] Table 1: Liquid medium preparation method

[0022]

Embodiment 2

[0023] The screening of embodiment 2 different formula liquid culture medium:

[0024] Inoculate the above-mentioned 6 kinds of liquid culture medium with 10% mycoplasma synovialum bacteria liquid respectively, place at 37°C, shake culture at 150r / min, culture time is 36 hours, and take samples at different culture time to count the viable bacteria. The screening test results of liquid culture media with different formulas show that the liquid culture media prepared according to formula 2, formula 3, formula 5 and formula 6 have better culture effects, and the number of viable bacteria in 24 to 30 hours is not less than 10 9 CCU / ml, formula 3 and formula 6, due to the high amount of supplementary liquid components such as serum in the medium, the medium is too rich in nutrients, resulting in the YBF-MS1 strain growing too fast and aging quickly; and the supplementary liquid such as serum The addition of liquid ingredients is high, which also increases the production cost, and ...

Embodiment 3

[0028] Embodiment 3: Culture medium comparison of the present invention and improved Frey's liquid medium

[0029] Mycoplasma gallinarum YBF-MS1 strain (YBF-MS1 strain has been preserved in the Chinese Type Culture Collection Center located in Wuhan, Wuhan University, the preservation date is March 3, 2016, and the preservation number is: CCTCC M 2016080.) After the freeze-dried seeds are revived, inoculate the seed solution with 10% of the improved Frey's liquid medium and the liquid medium of the formula five of the present invention respectively, place them at 37°C, shake and cultivate at 150r / min for 36 hours, and take samples at different times of cultivation Perform a viable count. The test results show that the amount of viable bacteria at different culture times is higher than that of the improved Frey's liquid medium with different culture times when the culture medium of the present invention is used. The results are shown in Table 3.

[0030] Table 3: Results of t...

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Abstract

The invention provides a mycoplasma synoviae culture method including three steps of first-level seed breeding, seed solution preparation and bacterial liquid preparation. A used liquid culture medium comprises the components: PPLO broth with the concentration of 21.0 g / L, glucose with the concentration of 5.0 g / L, inactivated horse serum with the concentration of 200 ml / L, a 25% yeast extract liquid with the concentration of 200 ml / L, a 1% phenol red solution with the concentration of 1.0 ml / L, a 1% coenzyme I solution with the concentration of 30 ml / L, and a 1% L-cysteine solution with the concentration of 30 ml / L, wherein the pH value is adjusted to 7.6-7.8. The process method not only shortens the culture time of mycoplasma synoviae, but also reduces the procedures of multiple sampling and detection during culture, reduces the possibility of pollution in the culture process, reduces errors caused by manual operation, and has advancement compared with the prior art.

Description

technical field [0001] The invention belongs to the technical field of pathogenic bacteria cultivation, and in particular relates to a method for cultivating Mycoplasma gallinarum. Background technique [0002] Mycoplasma synoviae (Mycoplasma synoviae, MS), also known as chicken infectious synovitis, is an acute or chronic infectious disease caused by Mycoplasma synoviae in chickens and turkeys. Chicken infectious synovitis was first reported by Olson et al. (1964). The disease mainly affects joint synovial fluid and tendon sheath, which is characterized by swelling of joints, tendon sheaths and soles. Chicken flocks infected with the disease can cause obvious lameness, growth retardation and carcass degradation, etc. The disease can be transmitted vertically, causing the disease in chicks hatched from eggs produced during the disease period of the breeder chickens. Mycoplasma synovialis infection has spread all over the world and is distributed worldwide. It mainly affect...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/20C12R1/35
CPCC12N1/20
Inventor 马爽于国营胡潇郭莉莉邹桂荣陶晓珊宋新宇宫晓王丽萍
Owner YEBIO BIOENG OF QINGDAO
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