CN103555641A - Mycoplasma hyopneumoniae culture medium and preparation method thereof - Google Patents
Mycoplasma hyopneumoniae culture medium and preparation method thereof Download PDFInfo
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Abstract
The invention discloses a mycoplasma hyopneumoniae culture medium and a preparation method thereof, which belong to the technical field of veterinary biological products. Each 1000 ml of phosphate buffer contains 20-50 g of PPLO (pleuropneumonia-like organism), 2-10 g of yeast powder, 3-15 g of glucose, 0.5-4 ml of 1% phenol red, 2-10 g of amino acid composition, 15-30 g of compound traditional Chinese medicine polysaccharide and 50-100 ml of pig serum. When culturing the mycoplasma hyopneumoniae, the mycoplasma hyopneumoniae culture medium disclosed by the invention can improve the tolerance of the mycoplasma hyopneumoniae and reduce the apoptosis rate of the mycoplasma hyopneumoniae; the mycoplasma hyopneumoniae culture medium disclosed by the invention can increase the amount of culture bacteria of the mycoplasma hyopneumoniae, the concentration of the culture bacteria liquid reaches 10<10-11>/ml, which is increased by more than 5-10 times compared with that of a common culture medium; the mycoplasma hyopneumoniae culture medium disclosed by the invention can inhibit the growth of other bacteria when culturing the mycoplasma hyopneumoniae so as to reduce the culture pollution risk of culturing the mycoplasma hyopneumoniae.
Description
Technical field
The invention belongs to veterinary biologics technical field, be specifically related to a kind of mycoplasma hyopneumoniae culture medium and preparation method thereof.
Background technology
Porcine mycoplasmal pneumonia is a kind of very common respiratory tract disease in pig farm, popular in worldwide, be commonly called as swine enzootic pneumonia, by mycoplasma hyopneumoniae (Mhp), caused, this cause of disease mainly resides in tracheae in pig body, on segmental bronchus and bronchiolar cilium, physics defense mechanism-defence mucous membrane of havoc porcine respiratory, increased the susceptibility of swinery to other respiratory pathogenic agent, as swine influenza virus, pig breeding and respiratory syndrome virus, II type pig circular ring virus, segmental bronchus sepsis bordetella bacilli, actinobacillus pleuropneumoniae and haemophilus parasuis etc., so usually cause the multiplicity of infection of multiple cause of disease, cause porcine respiratory syndrome (PRDC) or other diseases, mycoplasma pneumoniae can reduce pig growth rate 12.7% and reduce the price of deed 13.8%, the development of serious harm pig industry.
Mycoplasma hyopneumoniae is facultative anaerobe, and growth demand is very harsh, separated difficult, and it is higher than general mycoplasma to nutritional requirement.Although mycoplasma hyopneumoniae can utilize the synthetic required material of self growth and breeding of the nutrition in substratum, but due to its genomic molecular weight, the quantity of information of carrying is few, biosynthesis ability is limited, therefore need to be from materials such as external picked-up cholesterol, amino acid, lipid acid, Nucleic acid precurser and VITAMIN, this just impels the medium component more complicated of mycoplasma, must add some special nutrition materials.
Because mycoplasma hyopneumoniae is higher than general mycoplasma to nutritional requirement, medium component more complicated, incubation time is long, and culture concentration is lower, has affected widely people it is studied further, has also hindered i (mycoplasma hyopneumoniae) vaccine exploitation simultaneously and has produced.At present many scholars have designed multiple substratum and have supplied its growth, although they differ from one another, generally speaking all do not solve the difficult problem of cultivating.So also will make further research in the cultivation of mycoplasma hyopneumoniae.
Polysaccharide is one of active ingredient of Chinese herbs, and quantity research shows greatly, and polysaccharide and saccharide complex are not only in vivo as energy resource and constituent material, the more important thing is that it is present in all membrane structures, participates in the comings and goings of cell in biological phenomena.Polysaccharose substance is the important component part of all Living organisms, has the ability of removing free radical, improving activities of antioxidant enzymes and inhibition lipid peroxidation.In addition, herbal polysaccharide has special provide protection to unicellular lower eukaryote.Herbal polysaccharide is rich in hydroxyl, can with the combination of low isoreactivity living organisms, build multidimensional network space hydrogen bond structure, on active bio live body surface, form protective membrane, realize the provide protection to active bio.
The present invention is based on above-mentioned technical background, propose a kind of mycoplasma culture medium, specifically a kind of substratum that is specially adapted to mycoplasma hyopneumoniae, has improved mycoplasma hyopneumoniae bacterial concentration.
Summary of the invention
The problem existing for prior art, the object of the invention is to design provides the technical scheme of a kind of mycoplasma hyopneumoniae culture medium and preparation method thereof, by this culture medium culturing mycoplasma hyopneumoniae, can improve mycoplasma hyopneumoniae bacterial concentration, improve mycoplasma hyopneumoniae Yield of Antigen.
Described a kind of mycoplasma hyopneumoniae culture medium, is characterized in that the component that contains following weight proportion in every 1000ml phosphoric acid buffer:
PPLO 20～50g
Yeast powder 2～10g
Glucose 3～15g
1% phenol red 0.5～4ml
Amino acid combination 2～10g
Herbal mixture polysaccharide 15～30g
Porcine blood serum 50～100ml;
In described amino acid combination, contain L-arginine, glycine, tyrosine, Cys and Serine;
Described herbal mixture polysaccharide extracts and obtains from following Chinese medicine composition: the coptis, Ginkgo Leaf, Radix Achyranthis Bidentatae, Radix Codonopsis, Poria cocos, matrimony vine, Radix Glycyrrhizae and the red sage root.
Described a kind of mycoplasma hyopneumoniae culture medium, is characterized in that the component that contains following weight proportion in every 1000ml phosphoric acid buffer:
PPLO 30～40g
Yeast powder 4～8g
Glucose 5～10g
1% phenol red 1～3ml
Amino acid combination 4～8g
Herbal mixture polysaccharide 20～25g
Porcine blood serum 60～80ml.
Described a kind of mycoplasma hyopneumoniae culture medium, is characterized in that described amino acid combination contains L-arginine 10～30%, glycine 5～15%, tyrosine 10～30%, Cys 20～40% and Serine 10～30%.
Described a kind of mycoplasma hyopneumoniae culture medium, is characterized in that described herbal mixture polysaccharide extracts and obtains from following Chinese medicine composition: 5～15 parts of 5～15 parts of the coptiss, 10～30 parts of Ginkgo Leaves, 10～20 parts of Radix Achyranthis Bidentataes, 10～30 parts of Radix Codonopsis, 5～50 parts, Poria cocos, 10～30 parts of matrimony vines, 10～20 parts, Radix Glycyrrhizae and the reds sage root.
Described a kind of mycoplasma hyopneumoniae culture medium, is characterized in that described herbal mixture polysaccharide makes by following steps:
A, take each Chinese medicine material, chopping, by cold water soak, spend the night after cleaning, then add the purified water of 15 times of raw material weights, water-bath to 90 ℃, and remain on 90 ℃, and boil 2 hours, boil in process every 10 minutes and stir once;
B, discard a Chinese medicine slag, collect liquid, room temperature is cooling discards precipitation by centrifugal 10 minutes of 10000rpm, collects supernatant;
C, supernatant in b is carried out to alcohol precipitation, precipitation is rough herbal polysaccharide;
D, with rough herbal polysaccharide in sterilizing purified water washing c, after washing, with 42 ℃ of sterilizing purified water of 20 times of amounts, dissolves, after dissolving, in 0.5% ratio, add 4 ℃ of absorption of gac to spend the night, absorption, by centrifugal 10 minutes of 10000rpm, discards precipitation, collection supernatant;
E, by supernatant in d after the degerming of 0.22um membrane filtration, carry out 10 times concentrated, make herbal polysaccharide concentrated solution;
F, herbal polysaccharide concentrated solution in e is carried out to vacuum lyophilization obtain herbal mixture polysaccharide.
The preparation method of described a kind of mycoplasma hyopneumoniae culture medium, is characterized in that comprising following processing step:
1) preparation phosphate buffer solution;
2) from 5～15 parts of 5～15 parts of the coptiss, 10～30 parts of Ginkgo Leaves, 10～20 parts of Radix Achyranthis Bidentataes, 10～30 parts of Radix Codonopsis, 5～50 parts, Poria cocos, 10～30 parts of matrimony vines, 10～20 parts, Radix Glycyrrhizae and the reds sage root, extract and obtain herbal mixture polysaccharide;
3) take PPLO 20～50g, yeast powder 2～10g, glucose 3～15g, amino acid combination 2～10g, herbal mixture polysaccharide 15～30g, be dissolved in the 1000mL phosphate buffer solution that step 1) makes, add 1% phenol red 0.5～4ml fully to mix, with acid or alkali, regulate between pH to 7.4～7.8, under aseptic condition, pass through 0.22 μ m filter membrane, rearmounted 4 ℃ of filtration sterilization saves backup, and adds health pig serum 50～100ml before using, and obtains mycoplasma hyopneumoniae culture medium.
The preparation method of described a kind of mycoplasma hyopneumoniae culture medium, is characterized in that described step 2) in herbal mixture polysaccharide by following steps, make:
A, take each Chinese medicine material, chopping, by cold water soak, spend the night after cleaning, then add the purified water of 15 times of raw material weights, water-bath to 90 ℃, and remain on 90 ℃, and boil 2 hours, boil in process every 10 minutes and stir once;
B, discard a Chinese medicine slag, collect liquid, room temperature is cooling discards precipitation by centrifugal 10 minutes of 10000rpm, collects supernatant;
C, supernatant in b is carried out to alcohol precipitation, precipitation is rough herbal polysaccharide;
D, with rough herbal polysaccharide in sterilizing purified water washing c, after washing, with 42 ℃ of sterilizing purified water of 20 times of amounts, dissolves, after dissolving, in 0.5% ratio, add 4 ℃ of absorption of gac to spend the night, absorption, by centrifugal 10 minutes of 10000rpm, discards precipitation, collection supernatant;
E, by supernatant in d after the degerming of 0.22um membrane filtration, carry out 10 times concentrated, make herbal polysaccharide concentrated solution;
F, herbal polysaccharide concentrated solution in e is carried out to vacuum lyophilization obtain herbal mixture polysaccharide.
The preparation method of described a kind of mycoplasma hyopneumoniae culture medium, is characterized in that the amino acid combination described in described step 3) contains L-arginine 10～30%, glycine 5～15%, tyrosine 10～30%, Cys 20～40% and Serine 10～30%.
The preparation method of described a kind of mycoplasma hyopneumoniae culture medium, in step 3) described in it is characterized in that, take PPLO 30～40g, yeast powder 7～8g, glucose 5～10g, amino acid combination 4～8g, herbal mixture polysaccharide 20～25g, be dissolved in the 1000mL phosphate buffer solution that step 1) makes, add 1% phenol red 1～3ml fully to mix, with acid or alkali, regulate between pH to 7.4～7.8, under aseptic condition, pass through 0.22 μ m filter membrane, rearmounted 4 ℃ of filtration sterilization saves backup, and adds health pig serum 60～80ml before using.
In the present invention, PPLO is that PPLO substratum is buied by Sen Beijia (Nanjing) bio tech ltd.
Compared with prior art, the present invention has following beneficial effect:
(1) the prepared mycoplasma hyopneumoniae culture medium of the present invention can improve mycoplasma hyopneumoniae tolerance when cultivating mycoplasma hyopneumoniae, reduces mycoplasma hyopneumoniae apoptosis rate.
(2) the prepared mycoplasma hyopneumoniae culture medium of the present invention, can improve mycoplasma hyopneumoniae breeding bacteria, cultivates bacterial concentration and reaches 10
10-11/ ml(ccu counting process), than ordinary culture medium, improve more than 5-10 times.
(3) the prepared mycoplasma hyopneumoniae culture medium of the present invention can suppress other bacterial growths when cultivating mycoplasma hyopneumoniae simultaneously, reduces and cultivates the Pollution risk that mycoplasma hyopneumoniae is cultivated.
Embodiment
In order to make the present invention easier to understand, below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment are only not used in and limit the scope of the invention for the present invention is described.
Embodiment 1
, the component that contains following weight proportion in the every 1000ml phosphoric acid buffer of this substratum:
PPLO 20g
Yeast powder 6g
Glucose 8g
1% phenol red 4ml
Amino acid combination 4g
Herbal mixture polysaccharide 15g
Porcine blood serum 50ml
A preparation method for mycoplasma hyopneumoniae culture medium, the steps include:
(1) preparation of phosphate buffer solution: accurately take sodium-chlor 8g, Repone K 0.2g, Sodium phosphate dibasic 1.15g, potassium primary phosphate 0.2g, calcium chloride 0.1g, the magnesium chloride 0.1g that contains 6 crystal water is dissolved in 1000ml distilled water, under aseptic condition, by 0.22 μ m filter membrane, filtration sterilization is standby.
(2) preparation of herbal mixture polysaccharide
By 15 parts of 15 parts of the coptiss, 30 parts of Ginkgo Leaves, 10 parts of Radix Achyranthis Bidentataes, 10 parts of Radix Codonopsis, 20 parts, Poria cocos, 10 parts of matrimony vines, 10 parts, Radix Glycyrrhizae and the reds sage root, take each Chinese medicine material, after shredding, cleaning, by cold water soak, spend the night, the purified water that adds again 15 times of raw material weights, water-bath to 90 ℃, and remain on 90 ℃, boil 2 hours, boil in process every 10 minutes and stir once;
B, discard a Chinese medicine slag, collect liquid, room temperature is cooling discards precipitation by centrifugal 10 minutes of 10000rpm, collects supernatant;
C, supernatant in b is carried out to alcohol precipitation, precipitation is rough herbal polysaccharide;
D, with rough herbal polysaccharide in sterilizing purified water washing c, after washing, with 42 ℃ of sterilizing purified water of 20 times of amounts, dissolves, after dissolving, in 0.5% ratio, add 4 ℃ of absorption of gac to spend the night, absorption, by centrifugal 10 minutes of 10000rpm, discards precipitation, collection supernatant;
E, by supernatant in d after the degerming of 0.22um membrane filtration, carry out 10 times concentrated, make herbal polysaccharide concentrated solution;
F, herbal polysaccharide concentrated solution in e is carried out to vacuum lyophilization obtain herbal mixture polysaccharide.
(3) preparation of mycoplasma hyopneumoniae culture medium: take in proportion PPLO 20g, yeast powder 6g, glucose 8g, 1% phenol red 4ml, amino acid combination 4g(L-arginine 0.8g, glycine 0.4g, tyrosine 1.0g, Cys 1.2g and Serine 0.6g), herbal mixture polysaccharide 15g, be dissolved in the phosphate buffer solution that step (1) makes, add 1% phenol red 4mL fully to mix, with acid or alkali, regulate between pH to 7.4～7.8, under aseptic condition, pass through 0.22 μ m filter membrane, rearmounted 4 ℃ of filtration sterilization saves backup, and adds health pig serum 50ml before using.
Embodiment 2
, the component that contains following weight proportion in the every 1000ml phosphoric acid buffer of this substratum:
PPLO 35g
Yeast powder 2g
Glucose 15g
1% phenol red 0.5ml
Amino acid combination 10g
Herbal mixture polysaccharide 30g
Porcine blood serum 80ml.
A preparation method for mycoplasma hyopneumoniae culture medium, its step is with embodiment 1.Wherein herbal mixture polysaccharide extracts and obtains from 5 parts of 10 parts of the coptiss, 20 parts of Ginkgo Leaves, 15 parts of Radix Achyranthis Bidentataes, 20 parts of Radix Codonopsis, 50 parts, Poria cocos, 20 parts of matrimony vines, 15 parts, Radix Glycyrrhizae and the reds sage root.
Embodiment 3
, the component that contains following weight proportion in the every 1000ml phosphoric acid buffer of this substratum:
PPLO 50g
Yeast powder 10g
Glucose 3g
1% phenol red 2.5ml
Amino acid combination 2g
Herbal mixture polysaccharide 25g
Porcine blood serum 100ml.
A preparation method for mycoplasma hyopneumoniae culture medium, its step is with embodiment 1.Wherein herbal mixture polysaccharide extracts and obtains from 10 parts of 5 parts of the coptiss, 10 parts of Ginkgo Leaves, 20 parts of Radix Achyranthis Bidentataes, 30 parts of Radix Codonopsis, 5 parts, Poria cocos, 30 parts of matrimony vines, 20 parts, Radix Glycyrrhizae and the reds sage root.
The substratum that comparative example 1 the present invention is prepared and conventional substratum comparison test
1 Mhp bacterial strain and culture condition
Mycoplasma hyopneumoniae J strain purchased from ATCC is inoculated respectively to the mycoplasma hyopneumoniae culture medium of embodiment 1 and 2 kinds of mycoplasma hyopneumoniae culture mediums of prior art, in 5%(V/V) ratio Pigs Inoculated mycoplasma pneumoniae in each substratum, 37 ℃ of cultivations, when substratum its colour changed into yellow, when pH is down to 6.4-6.8, aseptic taking-up culture.
2 live bacterial counts (CCU method)
Each strain culture is got 14 sterile test tube, every pipe dress 4.5ml is containing mycoplasma hyopneumoniae culture medium, at the 1st pipe, add the well-grown culture of 0.5ml, after mixing, draw 0.5ml and add the 2nd by all means, so carry out 10 times of serial dilutions and manage to last, establish simultaneously do not add bacterium liquid mycoplasma hyopneumoniae culture medium as negative control.Developmental tube is done three repetitions.After serial dilution, test tube is placed to static cultivation in 37 ℃ of constant incubators, observe once every day, main colour-change and opacity of observing substratum, the Continuous Observation time is 14, the tubule extent of dilution that colour-change finally occurs is the CCU titre of this culture.
3 results
3 kinds of substratum of 3.1 mycoplasma hyopneumoniae Z strain inoculations, 3 secondary growth experiment CCU measurement results are in Table 1.
3 kinds of substratum of table 1 mycoplasma hyopneumoniae Z strain inoculation, 3 secondary growth experiment CCU measurement results
The mycoplasma hyopneumoniae culture medium that the present invention is prepared, can improve mycoplasma hyopneumoniae breeding bacteria, cultivates bacterial concentration and reaches 10
10-11/ ml(ccu counting process), than ordinary culture medium, improve more than 5-10 times.
Mycoplasma hyopneumoniae culture medium in embodiment 2 and 3 carries out the application identical with comparative example 1, and it finally also can reach the technique effect identical with comparative example 1.
Claims (9)
1. a mycoplasma hyopneumoniae culture medium, is characterized in that the component that contains following weight proportion in every 1000ml phosphoric acid buffer:
PPLO 20～50g
Yeast powder 2～10g
Glucose 3～15g
1% phenol red 0.5～4ml
Amino acid combination 2～10g
Herbal mixture polysaccharide 15～30g
Porcine blood serum 50～100ml;
In described amino acid combination, contain L-arginine, glycine, tyrosine, Cys and Serine;
Described herbal mixture polysaccharide extracts and obtains from following Chinese medicine composition: the coptis, Ginkgo Leaf, Radix Achyranthis Bidentatae, Radix Codonopsis, Poria cocos, matrimony vine, Radix Glycyrrhizae and the red sage root.
2. a kind of mycoplasma hyopneumoniae culture medium as claimed in claim 1, is characterized in that the component that contains following weight proportion in every 1000ml phosphoric acid buffer:
PPLO 30～40g
Yeast powder 4～8g
Glucose 5～10g
1% phenol red 1～3ml
Amino acid combination 4～8g
Herbal mixture polysaccharide 20～25g
Porcine blood serum 60～80ml.
3. a kind of mycoplasma hyopneumoniae culture medium as claimed in claim 1 or 2, is characterized in that described amino acid combination contains L-arginine 10～30%, glycine 5～15%, tyrosine 10～30%, Cys 20～40% and Serine 10～30%.
4. a kind of mycoplasma hyopneumoniae culture medium as claimed in claim 1 or 2, is characterized in that described herbal mixture polysaccharide extracts and obtains from following Chinese medicine composition: 5～15 parts of 5～15 parts of the coptiss, 10～30 parts of Ginkgo Leaves, 10～20 parts of Radix Achyranthis Bidentataes, 10～30 parts of Radix Codonopsis, 5～50 parts, Poria cocos, 10～30 parts of matrimony vines, 10～20 parts, Radix Glycyrrhizae and the reds sage root.
5. a kind of mycoplasma hyopneumoniae culture medium as claimed in claim 1, is characterized in that described herbal mixture polysaccharide makes by following steps:
A, take each Chinese medicine material, chopping, by cold water soak, spend the night after cleaning, then add the purified water of 15 times of raw material weights, water-bath to 90 ℃, and remain on 90 ℃, and boil 2 hours, boil in process every 10 minutes and stir once;
B, discard a Chinese medicine slag, collect liquid, room temperature is cooling discards precipitation by centrifugal 10 minutes of 10000rpm, collects supernatant;
C, supernatant in b is carried out to alcohol precipitation, precipitation is rough herbal polysaccharide;
D, with rough herbal polysaccharide in sterilizing purified water washing c, after washing, with 42 ℃ of sterilizing purified water of 20 times of amounts, dissolves, after dissolving, in 0.5% ratio, add 4 ℃ of absorption of gac to spend the night, absorption, by centrifugal 10 minutes of 10000rpm, discards precipitation, collection supernatant;
E, by supernatant in d after the degerming of 0.22um membrane filtration, carry out 10 times concentrated, make herbal polysaccharide concentrated solution;
F, herbal polysaccharide concentrated solution in e is carried out to vacuum lyophilization obtain herbal mixture polysaccharide.
6. a preparation method for mycoplasma hyopneumoniae culture medium, is characterized in that comprising following processing step:
1) preparation phosphate buffer solution;
2) from 5～15 parts of 5～15 parts of the coptiss, 10～30 parts of Ginkgo Leaves, 10～20 parts of Radix Achyranthis Bidentataes, 10～30 parts of Radix Codonopsis, 5～50 parts, Poria cocos, 10～30 parts of matrimony vines, 10～20 parts, Radix Glycyrrhizae and the reds sage root, extract and obtain herbal mixture polysaccharide;
3) take PPLO 20～50g, yeast powder 2～10g, glucose 3～15g, amino acid combination 2～10g, herbal mixture polysaccharide 15～30g, be dissolved in the 1000mL phosphate buffer solution that step 1) makes, add 1% phenol red 0.5～4ml fully to mix, with acid or alkali, regulate between pH to 7.4～7.8, under aseptic condition, pass through 0.22 μ m filter membrane, rearmounted 4 ℃ of filtration sterilization saves backup, and adds health pig serum 50～100ml before using, and obtains mycoplasma hyopneumoniae culture medium.
7. the preparation method of a kind of mycoplasma hyopneumoniae culture medium as claimed in claim 6, is characterized in that described step 2) in herbal mixture polysaccharide by following steps, make:
A, take each Chinese medicine material, chopping, by cold water soak, spend the night after cleaning, then add the purified water of 15 times of raw material weights, water-bath to 90 ℃, and remain on 90 ℃, and boil 2 hours, boil in process every 10 minutes and stir once;
B, discard a Chinese medicine slag, collect liquid, room temperature is cooling discards precipitation by centrifugal 10 minutes of 10000rpm, collects supernatant;
C, supernatant in b is carried out to alcohol precipitation, precipitation is rough herbal polysaccharide;
D, with rough herbal polysaccharide in sterilizing purified water washing c, after washing, with 42 ℃ of sterilizing purified water of 20 times of amounts, dissolves, after dissolving, in 0.5% ratio, add 4 ℃ of absorption of gac to spend the night, absorption, by centrifugal 10 minutes of 10000rpm, discards precipitation, collection supernatant;
E, by supernatant in d after the degerming of 0.22um membrane filtration, carry out 10 times concentrated, make herbal polysaccharide concentrated solution;
F, herbal polysaccharide concentrated solution in e is carried out to vacuum lyophilization obtain herbal mixture polysaccharide.
8. the preparation method of a kind of mycoplasma hyopneumoniae culture medium as claimed in claim 6, is characterized in that the amino acid combination described in described step 3) contains L-arginine 10～30%, glycine 5～15%, tyrosine 10～30%, Cys 20～40% and Serine 10～30%.
9. the preparation method of a kind of mycoplasma hyopneumoniae culture medium as claimed in claim 6, in step 3) described in it is characterized in that, take PPLO 30～40g, yeast powder 7～8g, glucose 5～10g, amino acid combination 4～8g, herbal mixture polysaccharide 20～25g, be dissolved in the 1000mL phosphate buffer solution that step 1) makes, add 1% phenol red 1～3ml fully to mix, with acid or alkali, regulate between pH to 7.4～7.8, under aseptic condition, pass through 0.22 μ m filter membrane, rearmounted 4 ℃ of filtration sterilization saves backup, and adds health pig serum 60～80ml before using.
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| CN104560835A (en) * | 2015-01-27 | 2015-04-29 | 新疆天康畜牧生物技术股份有限公司 | Culture medium for culturing mycoplasma hyopneumoniae and preparation method thereof |
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| CN110607260A (en) * | 2019-09-23 | 2019-12-24 | 山东甲骨文生物科技有限公司 | Culture medium and method for low-serum culture of mycoplasma hyopneumoniae |
| CN110804563A (en) * | 2019-11-13 | 2020-02-18 | 山东滨州沃华生物工程有限公司 | Culture medium for low-serum culture of mycoplasma hyopneumoniae |
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