CN104017856A - Mycoplasma hyopneumoniae selective separation medium and preparation method thereof - Google Patents
Mycoplasma hyopneumoniae selective separation medium and preparation method thereof Download PDFInfo
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- CN104017856A CN104017856A CN201410233471.7A CN201410233471A CN104017856A CN 104017856 A CN104017856 A CN 104017856A CN 201410233471 A CN201410233471 A CN 201410233471A CN 104017856 A CN104017856 A CN 104017856A
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Abstract
The invention belongs to the technical field of veterinary biology, and relates to a mycoplasma hyopneumoniae selective separation medium and a preparation method thereof. The mycoplasma hyopneumoniae selective separation medium comprises the main components of lactoalbumin hydrolysate, yeast extract powder, MEM (minimum essential medium), Hank 's solution, 0.1% phenol red solution, healthy horse serum, penicillin, deionized water, and specific growth inhibition serum. The mycoplasma hyopneumoniae selective separation medium can be used for the rapid separation and identification of mycoplasma hyopneumoniae, the preparation method is simple and feasible, experimental results are reliable, the separation time is shortened to 103h-122h, the time is shortened to 17%-23%, the separation success rate is as high as 75% above, and is more than 10 times of the separation success rate of mediums in the prior art.
Description
Technical field
The invention belongs to animal doctor's biology techniques field, relate to a kind of mycoplasma hyopneumoniae Pseudonocardia and preparation method thereof.
Background technology
Mycoplasma hyopneumoniae is one of the important pathogen that causes the contact chronic respiratory tract disease of pig, in the whole world, extensively exist, serious harm pig industry develops in a healthy way, its main harm is that the growth retardation, the feed conversion rate that cause pig decline to a great extent, and secondary causes the generation of other diseases and increases the weight of disease symptom.Porcine mycoplasmal pneumonia inactivated vaccine can reduce the generation of the disease of breathing, but the prerequisite of high-quality vaccine need to have a kind of good vaccine strain and regional mycoplasma hyopneumoniae prevalence situation to support.Substratum is that the various nutritive substances of synthetic are for the matrix of microorganism growth.Mycoplasma is a kind of small prokaryotic organism, acellular wall, can on artificial medium, grow, form small colonies, but it is very harsh that culture condition requires, and the speed of growth is slow, mycoplasma hyorhinis, mycoplasma hyosynoviae, mycoplasma flocculare and mycoplasma hyopneumoniae are often present in the body of pig simultaneously, and its speed of growth is all higher than mycoplasma hyopneumoniae, thereby carrying out mycoplasma hyopneumoniae isolation identification pollutes often, and cause the separated failure of mycoplasma hyopneumoniae, like this research of control swine enzootic pneumonia is caused to difficulty.In actual production process, mycoplasma hyopneumoniae classical symptom piglet, the separation rate of the mycoplasma hyopneumoniae of pathological material of disease is extremely low, and even the pollution separation rate due to pathogenic micro-organisms such as mycoplasma hyosynoviaes is low to moderate 1～5%, and urgently a kind of new experimental program solves the separation problem of mycoplasma hyopneumoniae.
Summary of the invention
The object of the present invention is to provide a kind of applicable mycoplasma hyopneumoniae growth, and growth is fast, viable bacteria titre is relatively high and can suppress the mycoplasma hyopneumoniae Pseudonocardia of mycoplasma hyorhinis, mycoplasma hyosynoviae, mycoplasma flocculare growth, use this Pseudonocardia for clinical separation and Culture mycoplasma hyopneumoniae, improve the mycoplasma speed of growth in sepn process, improve the separation rate of mycoplasma hyopneumoniae, specificity and susceptibility significantly improve, for the research of the aspects such as mycoplasma hyopneumoniae separation and Culture, biochemical character, heredity, immunity.
A kind of mycoplasma hyopneumoniae Pseudonocardia provided by the invention, its main component is lactoalbumin hydrolysate, yeast soaks powder, MEM substratum, Hank ' s liquid, 0.1% phenol red solution, healthy horse serum, penicillin and deionized water, specific suppresses serum (mycoplasma hyorhinis, mycoplasma hyosynoviae, mycoplasma flocculare).
Another object of the present invention is to provide the preparation method for this mycoplasma hyopneumoniae culture medium.
The object of the invention is to be achieved through the following technical solutions:
A kind of mycoplasma hyopneumoniae Pseudonocardia, it is characterized in that, according to following proportioning, make: lactoalbumin hydrolysate 40～50g, yeast soaks powder 40～50g, MEM substratum 40～50g, 10 * Hank ' s liquid, 20～60mL, 0.1% phenol red solution 10～20mL, penicillin 500～800Wan unit, healthy horse serum 1000～2000mL, specific suppresses serum 25～45mL, and deionized water is adjusted to 10000mL.
The proportioning of further optimizing is as follows: lactoalbumin hydrolysate 45g, and yeast soaks powder 45g, MEM substratum 49g, 10 * Hank ' s liquid 50mL, 0.1% phenol red solution 10mL, penicillin 600Wan unit, healthy horse serum 1500mL, specific suppresses serum 30mL, and deionized water is adjusted to 10000mL.
The present invention for the preparation method of mycoplasma hyopneumoniae Pseudonocardia, is characterized in that, according to the following step, prepares:
(1) by proportioning, take that lactoalbumin hydrolysate 40～50g, yeast soak powder 40～50g, MEM substratum 49g is dissolved in deionized water 5000mL, stir, make it to dissolve completely, add 10 * Hank ' s liquid, 20～60mL, 0.1% phenol red solution 10～20mL, penicillin 500～800Wan unit, with 0.45 μ m membrane filtration degerming, 4 ℃ save backup.
(2) before use, add healthy horse serum 1000～2000mL, specific to suppress serum 25～45mL, with 1mol/L sodium hydroxide solution, adjust pH to 7.6～7.8, use deionized water to be adjusted in right amount cumulative volume 10000mL, obtain mycoplasma hyopneumoniae selective separation liquid nutrient medium.
Specific of the present invention suppresses serum and refers to that the specific that simultaneously suppresses mycoplasma hyorhinis, mycoplasma hyosynoviae, mycoplasma flocculare suppresses serum.
Main points of the present invention are to select suitable component to prepare conventional mycoplasma hyopneumoniae special culture media, and on this basis, further add the three species specificity growth-inhibiting serum such as mycoplasma hyorhinis, mycoplasma hyosynoviae, mycoplasma flocculare, be prepared into mycoplasma hyopneumoniae Pseudonocardia, for the separation and Culture of mycoplasma hyopneumoniae.On this substratum, after mycoplasma hyopneumoniae inoculation, well-grown.The growth-inhibiting serum that adds preparation before use, can suppress the growth of mycoplasma hyorhinis, mycoplasma hyosynoviae, mycoplasma flocculare, optionally separation and Culture mycoplasma hyopneumoniae.
The present invention has the following advantages: mycoplasma hyopneumoniae selective medium can be used in the sharp separation of mycoplasma hyopneumoniae and identifies, compound method simple possible, test-results is reliable, disengaging time shortens to 103h～122h, time shorten 17%～23%, Success rate of virus isolation, up to more than 75%, is the more than 10 times of existing substratum Success rate of virus isolation.
Embodiment
Below in conjunction with example, the present invention is described in further detail.
According to the listed proportioning of following formula and according to the described preparation method of invention, make formula one, formula two, formula three, specific as follows:
Formula one: lactoalbumin hydrolysate 40g, yeast soaks powder 40g, MEM substratum 40g, 10 * Hank ' s liquid 20mL, 0.1% phenol red solution 10mL, penicillin 500Wan unit, healthy horse serum 1000mL, specific suppresses serum 25mL, and deionized water is adjusted to 10000mL.
Preparation method: (1) takes lactoalbumin hydrolysate 40g, yeast soaks powder 40g, MEM substratum 40g is dissolved in deionized water 5000mL, stir, make it to dissolve completely, add 10 * Hank ' s liquid 20mL, 0.1% phenol red solution 10mL, penicillin 500Wan unit, with 0.45 μ m membrane filtration degerming, 4 ℃ save backup; (2) add healthy horse serum 1000mL, specific to suppress serum 25mL, with 1mol/L sodium hydroxide solution, adjust pH to 7.6, use deionized water to be adjusted to cumulative volume 10000mL, obtain mycoplasma hyopneumoniae selective separation liquid nutrient medium one.
Formula two: lactoalbumin hydrolysate 50g, yeast soaks powder 50g, MEM substratum 50g, 10 * Hank ' s liquid 60mL, 0.1% phenol red solution 20mL, penicillin 800Wan unit, healthy horse serum 2000mL, specific suppresses serum 45mL, and deionized water is adjusted to 10000mL.
Preparation method: (1) takes lactoalbumin hydrolysate 50g, yeast soaks powder 50g, MEM substratum 50g is dissolved in deionized water 5000mL, stir, make it to dissolve completely, add 10 * Hank ' s liquid 60mL, 0.1% phenol red solution 20mL, penicillin 800Wan unit, with 0.45 μ m membrane filtration degerming, 4 ℃ save backup.(2) add healthy horse serum 2000mL, specific to suppress serum 45mL, with 1mol/L sodium hydroxide solution, adjust pH to 7.8, use deionized water to be adjusted to cumulative volume 10000mL, obtain mycoplasma hyopneumoniae selective separation liquid nutrient medium two.
Formula three: lactoalbumin hydrolysate 45g, yeast soak that powder 45g, MEM substratum 49g, 10 * Hank ' s liquid 50mL, 0.1% phenol red solution 10mL, penicillin 600Wan unit, healthy horse serum 1500mL, specific suppress serum 30mL, deionized water is adjusted to 10000mL.
Preparation Method: (1) takes lactoalbumin hydrolysate 45g, yeast soaks powder 45g, MEM substratum 49g is dissolved in deionized water 5000mL, stir, make it to dissolve completely, add 10 * Hank ' s liquid 50mL, 0.1% phenol red solution 100mL, penicillin 600Wan unit, with 0.45 μ m membrane filtration degerming, 4 ℃ save backup.(2) add healthy horse serum 1500mL, specific to suppress serum 30mL, with 1mol/L sodium hydroxide solution, adjust pH to 7.6, use deionized water to be adjusted to cumulative volume 10000mL, obtain mycoplasma hyopneumoniae selective separation liquid nutrient medium three.
The substratum that uses substratum one of the present invention, substratum two, substratum three to select as the present invention, use KM2, two kinds of substratum of A26 substratum in contrast, carry out clinical symptom typical case and use PCR method to confirm that 71 parts of pathological material of diseases of the mycoplasma hyopneumoniae infection positive have carried out respectively mycoplasma hyopneumoniae pathogen separation and identified, result is as shown in control experiment data sheet (table 1).
Claims (4)
1. a mycoplasma hyopneumoniae Pseudonocardia, it is characterized in that, by following component, according to proportioning, made: lactoalbumin hydrolysate 40～50g, yeast soaks powder 40～50g, MEM substratum 40～50g, 10 * Hank ' s liquid, 20～60mL, 0.1% phenol red solution 10～20mL, penicillin 500～800Wan unit, healthy horse serum 1000～2000mL, specific suppresses serum 25～45mL, and deionized water is adjusted to 10000mL.
2. a kind of mycoplasma hyopneumoniae Pseudonocardia according to claim 1, it is characterized in that, the proportioning of described component is lactoalbumin hydrolysate 45g, and yeast soaks powder 45g, and MEM substratum is 49g, 10 * Hank ' s liquid 50mL, 0.1% phenol red solution 10mL, penicillin 600Wan unit, healthy horse serum 1500mL, specific suppresses serum 30mL, and deionized water is adjusted to 10000mL.
3. for the preparation method of a kind of mycoplasma hyopneumoniae Pseudonocardia of claim 1, it is characterized in that, according to the following step, prepare:
(1) by proportioning, take that lactoalbumin hydrolysate 40～50g, yeast soak powder 40～50g, MEM substratum 49g is dissolved in deionized water 5000mL, stir, make it to dissolve completely, add 10 * Hank ' s liquid, 20～60mL, 0.1% phenol red solution 10～20mL, penicillin 500～800Wan unit, with 0.45 μ m membrane filtration degerming, 4 ℃ save backup;
(2) before use, add healthy horse serum 1000～2000mL, specific to suppress serum 25～45mL, with 1mol/L sodium hydroxide solution, adjust pH to 7.6～7.8, use deionized water to be adjusted in right amount cumulative volume 10000mL, obtain mycoplasma hyopneumoniae selective separation liquid nutrient medium.
4. for the preparation method of a kind of mycoplasma hyopneumoniae Pseudonocardia of claim 2, it is characterized in that, according to the following step, prepare:
(1) take lactoalbumin hydrolysate 45g, yeast soaks powder 45g, and MEM substratum 49g is dissolved in deionized water 5000mL, stirs, make it to dissolve completely, add 10 * Hank ' s liquid 50mL, 0.1% phenol red solution 100mL, penicillin 600Wan unit, with 0.45 μ m membrane filtration degerming, 4 ℃ save backup;
(2) add healthy horse serum 1500mL, specific to suppress serum 30mL, with 1mol/L sodium hydroxide solution, adjust pH to 7.6, use deionized water to be adjusted to cumulative volume 10000mL, obtain mycoplasma hyopneumoniae selective separation liquid nutrient medium.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106399203A (en) * | 2016-11-17 | 2017-02-15 | 山东省滨州畜牧兽医研究院 | Selective separation medium for caprine mycoplasmal pneumonia subspecies and preparation method of selective separation medium |
| CN109294955A (en) * | 2018-10-30 | 2019-02-01 | 南京大爻网络科技有限公司 | A kind of mycoplasma hyopneumoniae Pseudonocardia and preparation method thereof |
| CN111154696A (en) * | 2020-02-12 | 2020-05-15 | 石河子大学 | Mycoplasma hyopneumoniae culture medium and preparation method thereof |
| CN113444666A (en) * | 2021-07-14 | 2021-09-28 | 武汉轻工大学 | Method for efficiently separating mycoplasma hyopneumoniae |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102154167A (en) * | 2011-01-05 | 2011-08-17 | 北京大北农科技集团股份有限公司 | Mycoplasma hyopneumoniae culture medium and preparation method thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102154167A (en) * | 2011-01-05 | 2011-08-17 | 北京大北农科技集团股份有限公司 | Mycoplasma hyopneumoniae culture medium and preparation method thereof |
Non-Patent Citations (2)
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| 郭福全等: "支原体培养基的改良及其效果评价", 《微生物学免疫学进展》 * |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106399203A (en) * | 2016-11-17 | 2017-02-15 | 山东省滨州畜牧兽医研究院 | Selective separation medium for caprine mycoplasmal pneumonia subspecies and preparation method of selective separation medium |
| CN109294955A (en) * | 2018-10-30 | 2019-02-01 | 南京大爻网络科技有限公司 | A kind of mycoplasma hyopneumoniae Pseudonocardia and preparation method thereof |
| CN111154696A (en) * | 2020-02-12 | 2020-05-15 | 石河子大学 | Mycoplasma hyopneumoniae culture medium and preparation method thereof |
| CN113444666A (en) * | 2021-07-14 | 2021-09-28 | 武汉轻工大学 | Method for efficiently separating mycoplasma hyopneumoniae |
| CN113444666B (en) * | 2021-07-14 | 2023-05-30 | 武汉轻工大学 | Method for efficiently separating mycoplasma hyopneumoniae |
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Effective date of registration: 20170317 Address after: 256600 the Yellow River Road, Shandong, No. two, No. 169, Binzhou Patentee after: Shandong Lvdu Bio Sicience & Technology Co., Ltd. Address before: 256600 the Yellow River Road, Shandong, No. two, No. 169, Binzhou Patentee before: Binzhou, Shandong Province animal and veterinary research institute |