CN110951639A

CN110951639A - In-vitro culture medium for mycoplasma ovipneumoniae and preparation method thereof

Info

Publication number: CN110951639A
Application number: CN201911237916.8A
Authority: CN
Inventors: 武军元; 杜奕州; 郗宇
Original assignee: Tarim University
Current assignee: Tarim University
Priority date: 2019-11-30
Filing date: 2019-11-30
Publication date: 2020-04-03

Abstract

The invention discloses a mycoplasma in vitro culture medium and a preparation method thereof. The preparation method of the mycoplasma in vitro culture medium comprises the following steps: PPLO bath, glucose, sodium pyruvate, 25% yeast extract and phenol red are mixed according to a certain proportion and then are sterilized under high pressure, after the temperature is stabilized below 37 ℃, inactivated healthy horse serum and 50mg/mL ampicillin dissolved by autoclaved water are added, and the pH value is adjusted to 7.6-7.8 by 1mol/L NaOH to obtain the culture medium. The invention can reduce the time required by culture, obviously improve the live bacteria titer of the mycoplasma ovipneumoniae and lay the foundation for the development of high-quality vaccines of the mycoplasma ovipneumoniae.

Description

In-vitro culture medium for mycoplasma ovipneumoniae and preparation method thereof

Technical Field

The invention relates to a bacterial culture medium and a preparation method thereof, in particular to an in vitro culture medium for mycoplasma ovipneumoniae and a preparation method of the culture medium.

Background

Mycoplasma ovipneumoniae (Mycoplasma ovinepneumoniae. mo) was originally isolated by Mackay et al (1963) from sheep lungs with lung adenocarcinoma to obtain a cell-wall-free prokaryotic pathogenic microorganism between bacteria and viruses, which is a pathogen causing contagious pleuropneumonia of sheep, and after the sheep are infected by respiratory tract, the sheep can show fever, cough, abdominal respiration, progressive emaciation, hyperplastic and purulent pneumonia, the morbidity is 20-30%, the fatality rate is generally 30-50%, and some are as high as 80%, which causes massive death of sheep, especially lambs, and causes serious harm to sheep industry. In China, mycoplasma ovipneumoniae is separated from diseased sheep by Hujing Shao and the like (1982) at first, and then Xinjiang, Sichuan, Yunnan, Liaoning, Jiangsu, Gansu and the like have cases of separating Mo from pneumonia-infected sheep lungs, so that the mycoplasma ovipneumoniae is a high-incidence high-harmful infectious disease of domestic large-scale house-feeding sheep at present. Therefore, the research on the mycoplasmosis of sheep is increasingly paid attention by people.

In recent years, with the rapid development of sheep raising industry in China, especially in the production of mutton sheep mainly in a large-scale whole-barn feeding breeding mode, the contagious pleuropneumonia of sheep caused by mycoplasma ovipneumoniae is increasingly harmful and more serious in regional distribution and prevalence intensity. The sheep farming industry is an important component in livestock farming in China, and is greatly helpful for pulling the overall economic development of the livestock farming in China. However, some problems still occur in the actual sheep breeding process, the infection of epidemic diseases can bring great harm to the body health of sheep, so that great economic loss is caused to sheep farmers, mycoplasma pneumonia of sheep is one of key diseases, vaccination is the most effective way for preventing and treating the disease, and the bottleneck restricting the development of the vaccine industry is the problem that large amount of Mo is difficult to culture in vitro, so that how to improve the growth titer of Mo is an urgent problem to be solved.

Disclosure of Invention

The invention provides a rapid and efficient in-vitro culture medium for culturing mycoplasma ovipneumoniae and a preparation method thereof. Solves the problems of low bacteria culture quantity and poor efficiency of the existing culture medium. Also provides a preparation method of the culture medium.

The preparation method comprises the following steps:

21-25g/L of PPLO Both, 10-12g/L of glucose, 10-12g/L of sodium pyruvate, 80-100mL/L of 25% yeast extract and 2-3mL/L of 1% phenol red, adjusting the pH value to 7.6-7.8 by using 1M NaOH, uniformly mixing and then carrying out autoclaving. After the temperature of the mixed solution is stabilized at 37 ℃, 200 mL/L of inactivated healthy horse serum and 220mL/L of ampicillin dissolved in autoclaved water and 1mL of ampicillin are added to obtain the culture medium.

Repeated tests show that the growth titer and the growth speed of the culture medium are better than those of a KM2 culture medium, and compared with the prior art, the culture medium has the technical innovation points that:

1. compared with the prior culture medium KM2, the culture quantity of the mycoplasma ovipneumoniae is 10⁷CCU/ml. The maximum mycoplasma ovipneumoniae cultured by the culture medium can reach 10¹⁰CCU/ml；

2. Compared with the prior culture medium KM2, the preparation method of the culture medium is simpler and more convenient, and MEM is not required to be prepared;

3. the mycoplasma ovipneumoniae cultured by the culture medium can grow only in 12 hours, and the peak value is reached in 72 hours.

4. The culture medium does not need to be added with thallium acetate, is more environment-friendly and harmless to human bodies.

Detailed Description

Example 1:

1. preparation of the culture medium of the invention:

the chemical reagents used in the following examples are conventional and are commercially available.

Preparing 1000mL of mycoplasma ovipneumoniae culture medium:

(1)PPLO Both 21g

(2) glucose 10g

(3) Sodium pyruvate 10g

(4) 100mL of 25% yeast extract

(5) 3mL of 1% phenol Red

(6) 200mL of sterile horse serum

(7)50mg/mL ampicillin 1mL

The reagents (1) - (5) are respectively and fully mixed in 700mL of single distilled water, the pH value is adjusted to 7.8, the autoclave sterilization is carried out for 20 minutes at the temperature of 115 ℃, and the mixture is taken out and cooled to the temperature below 37 ℃. Adding (6) and (7).

2. Preparing a phenol red solution with the mass concentration of 1%:

phenol red 1.0g was weighed into a mortar, 6mL (1 mL each) of 1M NaOH (4.0g in 100mL of single distilled water) was added in 6 portions and ground to dissolve completely. The dissolved phenol red was taken in a 100mL volumetric flask, and the phenol red liquid remaining in the mortar was washed with single distilled water and then the volume of 100mL was made up with single distilled water.

3. Preparing a yeast leaching solution with the mass concentration of 25%:

500g of fresh yeast is stirred and dissolved in 2000mL of double distilled water, and the mixture is subjected to water bath at 80 ℃ for 30 minutes. After cooling to room temperature, the supernatant was collected by centrifugation at 3000 rpm for 20 minutes. The supernatant was adjusted to pH 7.8-8.0 with 2M NaOH and boiled. Standing at room temperature, cooling, filtering with double-layer filter paper, and storing at-20 deg.C.

Example 2:

1. comparative test

Preparation of KM2 culture medium:

MEM 5g, glucose 0.4g, sodium pyruvate 0.2g, hydrolyzed milk protein 5.1g, 1% phenol red 2.5mL, 25% fresh yeast extract 20mL, penicillin (20 ten thousand IU/mL)1mL, 10% thallium acetate 1mL, sterile horse serum 200mL, mixing. Adding deionized water to 1000ml, adjusting pH to 7.4 with sterilized 1M NaOH, filtering with 0.22 μ M filter membrane, and sterilizing.

2. Detection of

Viable bacterial titer (CCU) assay. The method comprises the following steps: taking 12 test tubes, adding 4.5mL of corresponding culture medium into each test tube, adding 0.5mL of well-grown mycoplasma ovipneumoniae culture into the 1 st test tube, fully and uniformly mixing the mixture by using an oscillator, replacing a new pipette, sucking 0.5mL from the 1 st test tube, adding the 0.5mL into the 2 nd test tube, sequentially diluting the mixture by 10 times to the 11 th test tube, and discarding 0.5mL of culture solution in the 11 th test tube; the obtained culture solution was diluted to 10 degrees^-1-10^-11And tube 12 is the corresponding media control. The test tubes were replicated in 3 replicates. And (3) placing the test tube in a constant-temperature incubator at 37 ℃ for static culture, observing the color change every day for 10 days continuously, wherein the highest dilution of the color change is the viable bacteria titer of the culture and is expressed by a Color Change Unit (CCU). The test was repeated 3 times

3. Results

The results of three tests show that the results of 3 times of CCU (common cycle analysis) tests on the mycoplasma ovipneumoniae cultured by the KM2 culture medium under the same conditions are all 10⁷CCU/ml, the mycoplasma ovipneumoniae is cultured by using the culture medium of the invention, and the results of 3 times of CCU measurement are 10¹⁰CCU/ml, which is much higher than the viable bacteria titer of the KM2 culture medium for culturing the mycoplasma ovipneumoniae.

Claims

1. A preparation method of a mycoplasma ovipneumoniae in-vitro culture medium is characterized by comprising the following steps: PPLO bath 21-25g/L, glucose 10-12g/L, sodium pyruvate 10-12g/L, 25% yeast extract 80-100mL/L, 1% phenol red 2-3mL/L, adjusting pH to 7.6-7.8 with 1M NaOH, mixing well, and autoclaving. After the temperature of the mixed solution is stabilized at 37 ℃, 200 mL/L of inactivated healthy horse serum and 220mL/L of ampicillin dissolved in autoclaved water and 1mL of ampicillin are added to obtain the culture medium.

2. The method for preparing the mycoplasma ovipneumoniae in vitro culture medium according to claim 1. The application in the preparation of mycoplasma ovipneumoniae vaccine antigens.