CN109294955A - A kind of mycoplasma hyopneumoniae Pseudonocardia and preparation method thereof - Google Patents
A kind of mycoplasma hyopneumoniae Pseudonocardia and preparation method thereof Download PDFInfo
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- CN109294955A CN109294955A CN201811280614.4A CN201811280614A CN109294955A CN 109294955 A CN109294955 A CN 109294955A CN 201811280614 A CN201811280614 A CN 201811280614A CN 109294955 A CN109294955 A CN 109294955A
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- mycoplasma hyopneumoniae
- pseudonocardia
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/02—Separating microorganisms from their culture media
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
- C12Q1/04—Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor
- C12Q1/045—Culture media therefor
Abstract
The invention discloses a kind of mycoplasma hyopneumoniae Pseudonocardias and preparation method thereof, belong to veterinary biologics field.Mycoplasma hyopneumoniae Pseudonocardia of the invention, including following components: brain heart oxoid, PPLO meat soup, 10 × Hank ' s liquid, yeast leachate, lactoalbumin hydrolysate, amino acid composition, glucose, penicillin, Nysfungin, 0.1% phenol red, inactivation Swine serum, specific inhibit serum, deionized water；Mycoplasma hyopneumoniae Pseudonocardia disengaging time of the invention is short, and Success rate of virus isolation is high, and viable bacteria titre and isolated sensibility are much higher than the culture medium of the prior art, is suitble to mycoplasma hyopneumoniae fast-growth.
Description
Technical field
The present invention relates to a kind of veterinary biologics field more particularly to a kind of mycoplasma hyopneumoniae selective separation cultures
Base and preparation method thereof.
Background technique
Porcine mycoplasmal pneumonia is a kind of extremely common respiratory disease in pig farm, popular in worldwide, is commonly called as pig
Asthma, by mycoplasma hyopneumoniae (Mhp) caused by, which mainly resides in trachea-bronchial epithelial cell and ramuscule gas in pig body
On the cilium of pipe, the serious physics defense mechanism-defence mucous membrane for destroying porcine respiratory increases swinery to other respiratory cause of diseases
The neurological susceptibility of body causes porcine respiratory syndrome (PRDC) or other diseases so often resulting in the compound infection of a variety of cause of diseases
Disease seriously endangers the development of pig breeding industry.
Mycoplasma hyopneumoniae is facultative anaerobic bacteria, and growth demand is extremely harsh, and separation is extremely difficult, it is to nutritional requirement ratio
General mycoplasma is high.Although mycoplasma hyopneumoniae can breed required object using the nutrition synthesis own growth in culture medium
Matter, but since the molecular weight of its genome is smaller, the information content of carrying is few, and biosynthesis ability is limited, it is therefore desirable to from body
The substances such as outer intake cholesterol, amino acid, fatty acid, Nucleic acid precurser and vitamin, this just promotes the medium component of mycoplasma
It is more complicated, it is necessary to add some special dietary substances.The speed of growth of mycoplasma hyopneumoniae in the medium is slower, living simultaneously
Bacterium titre is 107-108CCU/ml or so, viable bacteria titre is low, not ideal enough.
Mycoplasma is a kind of small prokaryotes, and cell-free wall can be grown on artificial medium, forms petite, but
Condition of culture requirement is very harsh, and the speed of growth is slow, and mycoplasma hyorhinis and mycoplasma hyopneumoniae often exist simultaneously the body in pig
It is interior, and its speed of growth is above mycoplasma hyopneumoniae, pollutes often to carry out mycoplasma hyopneumoniae separation identification, and
Lead to mycoplasma hyopneumoniae separation failure, difficulty is caused to the research of prevention and treatment swine enzootic pneumonia in this way.
Summary of the invention
It is an object of the invention in view of the above shortcomings of the prior art, provide the pig that a kind of growth is fast, viable bacteria titre is high
Mycoplasma pneumoniae Pseudonocardia.
To solve the above problems, the technical solution used in the present invention is:
A kind of mycoplasma hyopneumoniae Pseudonocardia, including following components: brain heart oxoid 5.0-10.0g, PPLO meat
Soup 5.0-10.0g, 10 × Hank ' s liquid 2.0-5.0ml, yeast leachate 3.0-5.0g, lactoalbumin hydrolysate 2.0-4.0g, amino
Acid composition 3.0-5.0g, glucose 10.0-20.0g, penicillin 200-400U/ml, Nysfungin 0.001-0.005g, 0.1%
Phenol red 1-20ml, the Swine serum 150-200ml of inactivation, specific inhibit serum 15-20ml, deionized water 600-800ml.
Further proportion optimizing is as follows: brain heart oxoid 7.0g, PPLO meat soup 8.0g, 10 × Hank ' s liquid 3.5ml, ferment
Female leachate 4.0g, lactoalbumin hydrolysate 3.0g, amino acid composition 4.0g, glucose 15.0g, penicillin 300U/ml, Nysfungin
0.003g, 0.1% phenol red 10ml, the Swine serum 175ml of inactivation, specific inhibit serum 17ml, deionized water 700ml.
Further, the amino acid composition contains L-arginine, glycine, tyrosine, L-cysteine and silk ammonia
Acid.
A kind of preparation method of mycoplasma hyopneumoniae Pseudonocardia as described above, comprising the following steps:
A, brain heart oxoid and PPLO meat soup are measured according to the ratio, are dissolved in deionized water, are mixed evenly, are allowed to be completely dissolved,
115 DEG C of sterilizing 20min, it is spare after cooling；
B, 10 × Hank ' s liquid, yeast leachate, lactoalbumin hydrolysate, amino acid composition, glucose, mould are weighed according to the ratio
Element, Nysfungin and 0.1% are phenol red, are dissolved in deionized water, are mixed evenly, be allowed to be completely dissolved, save backup at 4 DEG C；
C, after mixing, the Swine serum and specific for adding inactivation inhibit blood to the solution for obtaining step a and step b
Clearly, pH value is adjusted to 7.4-7.8, with 0.22 μm of membrane filtration degerming to get mycoplasma hyopneumoniae choosing with 1mol/L NaOH solution
Selecting property isolation medium.
The beneficial effects of adopting the technical scheme are that
The sensibility that mycoplasma hyopneumoniae Pseudonocardia of the invention separates mycoplasma hyopneumoniae is strong, can be used in
The quick separating of mycoplasma hyopneumoniae identifies that disengaging time is short, and Success rate of virus isolation is high；Mycoplasma hyopneumoniae selectivity of the invention
Isolation medium is the culture medium for being suitble to mycoplasma hyopneumoniae fast-growth, which can significantly improve mycoplasma hyopneumoniae
Viable bacteria titre (CCU), viable bacteria titre and isolated sensibility are significantly larger than the culture medium of the prior art.
Specific embodiment
Embodiments of the present invention are described in further detail below with reference to embodiment.Following embodiment is for illustrating this
Invention, but cannot be used to limit the scope of the invention.
Embodiment 1
A kind of mycoplasma hyopneumoniae Pseudonocardia, including following components: brain heart oxoid 6.0g, PPLO meat soup
6.0g, 10 × Hank ' s liquid 2.0ml, yeast leachate 3.0g, lactoalbumin hydrolysate 2.0g, amino acid composition 3.0g, glucose
10.0g, penicillin 250U/ml, Nysfungin 0.002g, 0.1% phenol red 5ml, the Swine serum 150ml of inactivation, specific suppression
Serum 15ml processed, deionized water 600ml.
Further, the amino acid composition contains L-arginine, glycine, tyrosine, L-cysteine and silk ammonia
Acid, L-arginine, glycine, tyrosine, L-cysteine and serine mass ratio be 2:1:1:1:1.
Further, specific of the present invention inhibits serum to refer to the specific suppression for inhibiting mycoplasma hyorhinis
Serum processed.
Preparation method, comprising the following steps:
A, brain heart oxoid 6.0g and PPLO meat soup 6.0g are measured according to the ratio, are dissolved in 300ml deionized water, are mixed evenly,
It is allowed to be completely dissolved, 115 DEG C of sterilizing 20min are spare after cooling；
B, 10 × Hank ' s liquid 2.0ml, yeast leachate 3.0g, lactoalbumin hydrolysate 2.0g, amino acid composition are weighed according to the ratio
3.0g, glucose 10.0g, penicillin 250U/ml, the phenol red 5ml of Nysfungin 0.002g and 0.1%, are dissolved in 300ml deionized water, mix
Conjunction stirs evenly, and is allowed to be completely dissolved, and saves backup at 4 DEG C；
C, the solution for obtaining step a and step b after mixing, adds Swine serum 150ml and the specific suppression of inactivation
Serum 15ml processed adjusts pH value to 7.4, with 0.22 μm of membrane filtration degerming to get pig pneumonia branch original with 1mol/L NaOH solution
Body Pseudonocardia.
Embodiment 2
A kind of mycoplasma hyopneumoniae Pseudonocardia, including following components: brain heart oxoid 7.0g, PPLO meat soup
8.0g, 10 × Hank ' s liquid 3.5ml, yeast leachate 4.0g, lactoalbumin hydrolysate 3.0g, amino acid composition 4.0g, glucose
15.0g, penicillin 300U/ml, Nysfungin 0.003g, 0.1% phenol red 10ml, the Swine serum 175ml of inactivation, specific suppression
Serum 17ml processed, deionized water 700ml.
Further, the amino acid composition contains L-arginine, glycine, tyrosine, L-cysteine and silk ammonia
Acid, L-arginine, glycine, tyrosine, L-cysteine and serine mass ratio be 1:1:1:2:1.
Further, specific of the present invention inhibits serum to refer to the specific suppression for inhibiting mycoplasma hyorhinis
Serum processed.
Preparation method, comprising the following steps:
A, brain heart oxoid 8.0g and PPLO meat soup 8.0g are measured according to the ratio, are dissolved in 350ml deionized water, are mixed evenly,
It is allowed to be completely dissolved, 115 DEG C of sterilizing 20min are spare after cooling；
B, 10 × Hank ' s liquid 3.0ml, yeast leachate 4.0g, lactoalbumin hydrolysate 3.0g, amino acid composition are weighed according to the ratio
4.0g, glucose 15.0g, penicillin 300U/ml, Nysfungin 0.003g, lemon acid diamine 0.003g and 0.1% phenol red 10ml, it is molten
It in 350ml deionized water, is mixed evenly, is allowed to be completely dissolved, saved backup at 4 DEG C；
C, the solution for obtaining step a and step b after mixing, adds Swine serum 175ml and the specific suppression of inactivation
Serum 17ml processed adjusts pH value to 7.6, with 0.22 μm of membrane filtration degerming to get pig pneumonia branch original with 1mol/L NaOH solution
Body Pseudonocardia.
Embodiment 3
A kind of mycoplasma hyopneumoniae Pseudonocardia, including following components: brain heart oxoid 10.0g, PPLO meat soup
10.0g, 10 × Hank ' s liquid 5.0ml, yeast leachate 5.0g, lactoalbumin hydrolysate 4.0g, amino acid composition 5.0g, glucose
20.0g, penicillin 400U/ml, Nysfungin 0.005g, 0.1% phenol red 18ml, the Swine serum 200ml of inactivation, specific suppression
Serum 20ml processed, deionized water 800ml.
Further, the amino acid composition contains L-arginine, glycine, tyrosine, L-cysteine and silk ammonia
Acid, L-arginine, glycine, tyrosine, L-cysteine and serine mass ratio be 1:1:1:1:1.
Further, specific of the present invention inhibits serum to refer to the specific suppression for inhibiting mycoplasma hyorhinis
Serum processed.
Preparation method, comprising the following steps:
A, brain heart oxoid 10.0g and PPLO meat soup 10.0g are measured according to the ratio, is dissolved in 400ml deionized water, are mixed equal
It is even, it is allowed to be completely dissolved, 115 DEG C of sterilizing 20min are spare after cooling；
B, 10 × Hank ' s liquid 5.0ml, yeast leachate 5.0g, lactoalbumin hydrolysate 4.0g, amino acid composition are weighed according to the ratio
5.0g, glucose 20.0g, penicillin 400U/ml, Nysfungin 0.005g, lemon acid diamine 0.003g and 0.1% phenol red 18ml, it is molten
It in 400ml deionized water, is mixed evenly, is allowed to be completely dissolved, saved backup at 4 DEG C；
C, the solution for obtaining step a and step b after mixing, adds Swine serum and the suppression of 200ml specific of inactivation
Serum 20ml processed adjusts pH value to 7.8, with 0.22 μm of membrane filtration degerming to get pig pneumonia branch original with 1mol/L NaOH solution
Body Pseudonocardia.
Embodiment 4
A kind of mycoplasma hyopneumoniae Pseudonocardia, including following components: brain heart oxoid 7.0g, PPLO meat soup
8.0g, 10 × Hank ' s liquid 3.5ml, yeast leachate 4.0g, lactoalbumin hydrolysate 3.0g, amino acid composition 4.0g, glucose
15.0g, penicillin 300U/ml, Nysfungin 0.003g, 0.1% phenol red 10ml, the Swine serum 175ml of inactivation, specific suppression
Serum 17ml processed, deionized water 700ml.
Further, mycoplasma hyopneumoniae Pseudonocardia of the invention further includes lemon acid diamine 0.003g.
Further, the amino acid composition contains L-arginine, glycine, tyrosine, L-cysteine and silk ammonia
Acid, L-arginine, glycine, tyrosine, L-cysteine and serine mass ratio be 1:1:1:2:1.
Further, specific of the present invention inhibits serum to refer to the specific suppression for inhibiting mycoplasma hyorhinis
Serum processed.
Preparation method, comprising the following steps:
A, brain heart oxoid 8.0g and PPLO meat soup 8.0g are measured according to the ratio, are dissolved in 350ml deionized water, are mixed evenly,
It is allowed to be completely dissolved, 115 DEG C of sterilizing 20min are spare after cooling；
B, 10 × Hank ' s liquid 3.0ml, yeast leachate 4.0g, lactoalbumin hydrolysate 3.0g, amino acid composition are weighed according to the ratio
4.0g, glucose 15.0g, penicillin 300U/ml, Nysfungin 0.003g, lemon acid diamine 0.003g and 0.1% phenol red 10ml, it is molten
It in 350ml deionized water, is mixed evenly, is allowed to be completely dissolved, saved backup at 4 DEG C；
C, the solution for obtaining step a and step b after mixing, adds Swine serum 175ml and the specific suppression of inactivation
Serum 17ml processed adjusts pH value to 7.6, with 0.22 μm of membrane filtration degerming to get pig pneumonia branch original with 1mol/L NaOH solution
Body Pseudonocardia.
1. the culture medium for using the embodiment of the present invention 1, embodiment 2, embodiment 3 and embodiment 4 to select as the present invention, makes
It uses KM2 culture medium as control medium, 50 parts of pig lungs pathological material of diseases of the mycoplasma hyopneumoniae infection positive is confirmed using PCR method
Mycoplasma hyopneumoniae separation identification is carried out respectively, as a result such as table 1.
Table 1
It can be seen that mycoplasma hyopneumoniae Pseudonocardia can be used in the quick separating mirror of mycoplasma hyopneumoniae from table 1
Fixed, disengaging time is short, and Success rate of virus isolation is high.
2. culture medium of the present invention and two kinds of common culture medium viable bacteria titre comparative tests in the prior art
2.1 according to animal doctor institute of academy of agricultural sciences of Jiangsu Province 168 plants of cultural methods of mycoplasma hyopneumoniae, by mycoplasma hyopneumoniae (purchased from expensive
State Foster Biotechnology Co., Ltd) it is inoculated with mycoplasma hyopneumoniae Pseudonocardia embodiment 4 of the invention respectively
It with 2 kinds of common culture mediums of the prior art, after seed subculture rejuvenation, is inoculated with respectively in the ratio of 10% (V/V), 37 DEG C of cultures,
When the colour changed into yellow of culture medium, pH are down to 6.5~6.9 by 7.6, sterile taking-up culture.
2.2 viable bacteria titres (CCU) measure each bacterial strain and take 12 sterile test tubes, and every pipe dress 4.5ml contains mycoplasma hyopneumoniae
The well-grown culture of 0.5ml is added in the 1st pipe in Pseudonocardia, after mixing, draws 0.5ml and is added the
2 root canals so carry out 10 times and are serially diluted to the pipe of most end 1, while setting the mycoplasma hyopneumoniae Selective Separation training for not adding bacterium solution
Base is supported as negative control.Developmental tube sets three repetitions.Later, test tube is placed into static gas wave refrigerator in 37 DEG C of insulating boxs, it is daily to see
It examines 1 time, the color change and turbidity of main detection culture medium, it is 14 that the time, which is observed continuously, and color change finally occurs
Tubule dilution is the CCU titre of the culture.This test has carried out 3 times altogether.168 plants of mycoplasma hyopneumoniae inoculations, 3 kinds of trainings
It supports 3 secondary growth of base test CCU (color changing units) measurement result and is shown in Table 2.
Table 2
From mycoplasma hyopneumoniae using culture medium prepared by the present invention compared with the viable bacteria titre of 2 kinds of common culture mediums in technology
(CCU) from the point of view of measurement result, under same experimental condition, mycoplasma hyopneumoniae Pseudonocardia bacterial strain of the invention
3 test CCU measurement results are 109More than；And 2 kinds of common culture mediums of the prior art, 3 test CCU measurement results are
107~108.Illustrate that mycoplasma hyopneumoniae Pseudonocardia of the invention has growth rapidly, the high spy of viable bacteria titre
Point.
Finally, it should be noted that the above embodiments are merely illustrative of the technical solutions of the present invention, rather than its limitations；Although
Present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that: it still may be used
To modify to technical solution documented by previous embodiment or equivalent replacement of some of the technical features；And
These are modified or replaceed, the spirit and model of technical solution of the embodiment of the present invention that it does not separate the essence of the corresponding technical solution
It encloses.
Claims (4)
1. a kind of mycoplasma hyopneumoniae Pseudonocardia, which is characterized in that including following components: brain heart oxoid 5.0-
10.0g, PPLO meat soup 5.0-10.0g, 10 × Hank ' s liquid 2.0-5.0ml, yeast leachate 3.0-5.0g, lactoalbumin hydrolysate
2.0-4.0g, amino acid composition 3.0-5.0g, glucose 10.0-20.0g, penicillin 200-400U/ml, Nysfungin
0.001-0.005g, 0.1% phenol red 1-20ml, the Swine serum 150-200ml of inactivation, specific inhibit serum 15-20ml,
Deionized water 600-800ml.
2. a kind of mycoplasma hyopneumoniae Pseudonocardia according to claim 1, which is characterized in that including following
Component: brain heart oxoid 7.0g, PPLO meat soup 8.0g, 10 × Hank ' s liquid 3.5ml, yeast leachate 4.0g, lactoalbumin hydrolysate
3.0g, it amino acid composition 4.0g, glucose 15.0g, penicillin 300U/ml, Nysfungin 0.003g, 0.1% phenol red 10ml, goes out
Swine serum 175ml living, specific inhibit serum 17ml, deionized water 700ml.
3. a kind of mycoplasma hyopneumoniae Pseudonocardia according to claim 1, which is characterized in that the amino
Acid composition contains L-arginine, glycine, tyrosine, L-cysteine and serine.
4. a kind of preparation method of mycoplasma hyopneumoniae Pseudonocardia as described in claim 1, which is characterized in that
The following steps are included:
A, brain heart oxoid and PPLO meat soup are measured according to the ratio, are dissolved in deionized water, are mixed evenly, are allowed to be completely dissolved,
115 DEG C of sterilizing 20min, it is spare after cooling；
B, 10 × Hank ' s liquid, yeast leachate, lactoalbumin hydrolysate, amino acid composition, glucose, mould are weighed according to the ratio
Element, Nysfungin and 0.1% are phenol red, are dissolved in deionized water, are mixed evenly, be allowed to be completely dissolved, save backup at 4 DEG C；
C, after mixing, the Swine serum and specific for adding inactivation inhibit blood to the solution for obtaining step a and step b
Clearly, pH value is adjusted to 7.4-7.8, with 0.22 μm of membrane filtration degerming to get mycoplasma hyopneumoniae choosing with 1mol/L NaOH solution
Selecting property isolation medium.
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| CN110938560A (en) * | 2019-11-19 | 2020-03-31 | 成都天邦生物制品有限公司 | Rapid and efficient mycoplasma hyopneumoniae culture medium 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 |
| CN111635876A (en) * | 2020-06-16 | 2020-09-08 | 武汉科前生物股份有限公司 | Mycoplasma hyopneumoniae culture medium and preparation method and application thereof |
| CN114836341A (en) * | 2022-04-15 | 2022-08-02 | 辽宁益康生物股份有限公司 | Mycoplasma hyopneumoniae culture medium and preparation method thereof |
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Application publication date: 20190201 |