CN106350472A - Liquid culture medium for culturing mycoplasma bovis and preparation method of liquid culture medium - Google Patents
Liquid culture medium for culturing mycoplasma bovis and preparation method of liquid culture medium Download PDFInfo
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Abstract
The invention discloses a liquid culture medium for mycoplasma bovis and a preparation method of the liquid culture medium, and relates to the field of veterinary biology. The liquid culture medium for the mycoplasma bovis, disclosed by the invention, is formed through mixing a basal culture medium and an auxiliary culture medium, and is prepared from the following ingredients: peptone, glucose, sodium pyruvate, fresh yeast lixivium, L-glutamine, L-cysteine, lactoalbumin hydrolysate, transferrin, insulin, a small volume of horse serum, penicillin, phenol red and deionized water. The culture medium for the mycoplasma bovis, disclosed by the invention, is beneficial to the growth of the mycoplasma bovis, and the titer of viable bacteria reaches 10<10>CCU/ml, and is apparently higher than that of culture media in the prior art. In addition, the content of serum in the culture medium disclosed by the invention is only 5%, and is 1/4 to 1/2 that of the serum content of the culture media in the prior art. Compared with the prior art, the culture medium disclosed by the invention has the characteristics that the serum content is low, the growth speed is high, and the titer of viable bacteria is high.
Description
Technical field
The present invention relates to a kind of Mycoplasma bovis fluid medium and preparation method thereof, it is exactly that its component a kind of includes
Have: peptone, glucose, Sodium Pyruvate, fresh yeast leachate, l- L-Glutamine, a small amount of horse serum, penicillin, phenol red, go
Ionized water is used for cultivating Mycoplasma bovis fluid medium and preparation method thereof.
Background technology
Mycoplasma bovis (mycoplasma bovis,m.bovis) belong to Mollicutes, Mycoplasmas microorganism, Neng Gouyin
Play multiple diseases such as pulmonis Bovis seu Bubali inflammation, arthritis, mastitiss.This cause of disease is the main pathogen of cattle respiratory disorder syndrome, is to threaten to educate
Cattle and a kind of important pathogen of milk cow health.This disease is popular in worldwide at present, and the serious health threatening cattle-raising is sent out
Open up and cause serious financial consequences.
Vaccine immunity is prevention and controls the pathogenetic important means of Mycoplasma bovis.Mycoplasma bovis vaccine produces and grinds
One important foundation of system is the preparation of high-quality culture medium.Fluid medium currently used for culture Mycoplasma bovis mainly has Cor Bovis seu Bubali
Soup culture medium, improvement km2 culture medium and improvement thiaucourt ' s culture medium etc..Prior art culture medium culturing Mycoplasma bovis are deposited
Incubation time longer, viable bacteria titre is relatively low, fertility is poor the problems such as.Additionally, serum content in prior art culture medium
Higher, typically between 10%～20%, on the one hand increased seedling cost, on the other hand, the vaccine of excessive serum preparation increases
Having added allogeneic serum affects the immune effect of vaccine to the allergy stress of cattle body.The serum content reducing in culture medium is got over
More paid close attention to by manufacturing enterprise and researcher.In simple reduction prior art culture medium, serum content can lead to culture cattle to prop up
Low 10～100 times about of mycoplasma antigen titre, had not both reached antigen dose needed for immunity, need to improve cycles of concentration, actually again
Increased production cost.
Chinese invention patent 2011100012310 discloses a kind of mycoplasma hyopneumoniae culture medium and preparation method thereof, and this is special
Profit basal medium constituent be: brain heart infusion, lactalbumin hydrolysate, pplo meat soup, yeast extract, show peptone,
Sodium thiosulfate, hank ' s liquid, Sodium Pyruvate, 0.1% phenol red solution, penicillin and deionized water, with using front add 140～
The healthy horse serum of 200ml is and it needs to add agar.According to disclosure of which, with the pig of the culture medium culturing of this patent
Mycoplasma pneumoniae culture medium viable bacteria titre is up to 1 × 109Ccu/ml～1 × 1010Ccu/ml, and there is the mycoplasma speed of growth
Hurry up, detached sensitivity strong, preparation method process is simple, workable, the feature of suitable industrialized great production.
Low serum high-efficient culture mycoplasma hyopneumoniae culture medium and its system disclosed in Chinese invention patent 201210318816x
Preparation Method, this patent includes (1) basal medium (2) auxiliary culture medium, wherein mainly has mem, yeast extract powder, Trypsin
Peptone, glucose, inorganic salt etc. forms, although the porcine blood serum that the culture medium of this patent uses is less, remains a need for 170ml and arrives
180ml.
But because above-mentioned patent is used for cultivating mycoplasma hyopneumoniae, during culture Mycoplasma bovis, viable bacteria titre is all not higher than
109Ccu/ml, additionally, serum amount needed for above-mentioned patent (horse serum or porcine blood serum) is larger, serum content is all more than 10%；Cause
This, study a kind of energy high-efficient culture Mycoplasma bovis at this stage and can use the culture medium of less serum, it has also become Mycoplasma bovis
It is badly in need of the major issue solving in vaccine research and production.
Content of the invention
The present invention provides one kind can overcome deficiency of the prior art, the not only efficient but also Mycoplasma bovis liquid of low serum content
Culture medium, and the method preparing this culture medium.
The present invention is made up of basal medium and auxiliary culture medium mixing for cultivating Mycoplasma bovis fluid medium, its
In the consisting of of basal medium in every liter of culture medium:
(1) peptone 28.0～33.0 g/l,
(2) glucose 6.0～8.0 g/l,
(3) Sodium Pyruvate 5.0～6.0 g/l,
(4) mass concentration is 25% fresh yeast leachate 110～130 ml/l,
(5) mass concentration is 1% phenol red 2.0～2.5 ml/l,
(6) 750ml deionized water；
The consisting of of auxiliary culture medium in every liter of culture medium:
(7) mass concentration is 3% l- L-Glutamine 16～17 ml/l,
(8) mass concentration is 10% l- cysteine 4.0～6.0 ml/l,
(9) mass concentration is 15% lactoalbumin hydrolysate 32.0-35.0 ml/l,
Transferrinss 0.8～1.5 ml/l of (10) 10 mg/ml,
(11) 10 mg/ml insulin 0.8～1.5 ml/l,
(12) horse serum 50 ml/l,
Penicillin 0.25 ml/l of (13) 20 ten thousand iu/ml,
The ph value of culture medium is adjusted to 7.2～7.4.
The culture medium preparation method of the present invention is:
A. its preparation is cultivated on basis
Weigh and measure peptone, glucose, Sodium Pyruvate, fresh yeast leachate in proportion, and the phenol red 750ml that dissolves in is gone
In ionized water, it is cooled to room temperature after 115 DEG C of autoclaving 20min standby；
B. assist the preparation of culture medium
Weigh in proportion and measure l- L-Glutamine, l- cysteine, lactoalbumin hydrolysate, transferrinss, insulin, horse serum,
Penicillin, with 0.22 micron membrane filter Entkeimung, is sufficiently mixed, obtains final product auxiliary culture medium,
C. the preparation of culture medium
Under aseptic condition, basic liquid-based and auxiliary culture medium mixing are settled to 1000 ml with aquesterilisa polishing, with sterilizing
1m naoh adjusts ph value to 7.2-7.4, fully shakes up, puts 4 DEG C and save backup.
Application in culture Mycoplasma bovis for the fluid medium of the present invention.
A kind of fluid medium for cultivating Mycoplasma bovis of the present invention passes through each needed for supplementary Mycoplasma bovis growth
Plant nutritional labeling, by the ph value in sodium hydroxide effective control culture medium, be conducive to the growth of Mycoplasma bovis, viable bacteria titre reaches
1010Ccu/ml is hence it is evident that (improvement km2 culture medium culturing M. bovis viable titre is 10 higher than prior art culture medium7ccu/
Ml, Cor Bovis seu Bubali soup culture medium is 108Ccu/ml, improves thiaucourt ' s culture medium 108～109Ccu/ml).Additionally, the present invention
In culture medium, serum content is only 5%, is the 1/4～1/2 of prior art culture medium serum content, low blood serum medium can mitigate
The allergy stress to cattle body for the allogeneic serum, improves bio-safety, reduces production cost simultaneously.
Specific embodiment
Embodiment 1:
1st, the preparation of culture medium of the present invention:
(1) preparation of basal medium
Take peptone 31.0 g, glucose 6.5 g, Sodium Pyruvate 5.0 g, 25% fresh yeast leachate 120 ml, 1% phenol red
2.5 ml dissolve in 750ml deionized water, are cooled to room temperature standby after 115 DEG C of autoclaving 20min.
(2) preparation of auxiliary culture medium:
3% l- L-Glutamine, 17.0 ml, 10%l- cysteine 5.0 ml of 0.22 micron membrane filter Entkeimung of learning from else's experience, 15% water
Solution lactoprotein 33.0ml, transferrinss (10 mg/ml) 0.8 ml, insulin (10 mg/ml) 1.5 ml, horse serum 50 ml, green grass or young crops
Mycin (200,000 iu/ml) 0.25 ml, is sufficiently mixed, obtains final product auxiliary culture medium.
(3) preparation of culture medium
Under aseptic condition, basal liquid and auxiliary culture medium mixing are settled to 1000ml with aquesterilisa polishing, with the 1m of sterilizing
Naoh(4 g is dissolved in 100 ml deionized waters) adjust ph value to 7.4, fully shake up, put 4 DEG C and save backup.
, 25% preparation method of fresh yeast leachate be:
Take fresh yeast 500 g, add in deionized water 2000 ml, stirring and dissolving, with concentrated hydrochloric acid: deionized water is with=1:1(volume
Than) adjust ph value to 4.5-5.0,80 DEG C of water-baths (temperature in bottle) 30 minutes, 3000 revs/min are centrifuged 20 minutes, take supernatant.Will
Supernatant is dissolved in 100 ml deionized waters with 1 m naoh(4 g) adjust ph value to 7.8-8.0, boil, put use after room temperature cools double
Layer filter paper filtering, then mend deionized water to 2000 ml, -20 DEG C save backup.
, 1% phenol red solution compound method:
Weigh phenol red 1.0 g, put in glass mortar, be added dropwise over 0.1m naoh(0.4 g and be dissolved in 10 ml deionized waters), grind
To being completely dissolved.By in the phenol red suction 100 ml measuring bottle of dissolving, deionized water is carefully washed in lower mortar and is remained phenol red liquid extremely
In measuring bottle, finally add deionized water to 100 ml.
Embodiment 2:
The preparation of culture medium of the present invention:
(1) preparation of basal medium
Take peptone 30.0 g, glucose 6.5 g, Sodium Pyruvate 5.5 g, 25% fresh yeast leachate 120 ml, 1% phenol red
2.5 ml dissolve in 750 ml deionized waters, are cooled to room temperature standby after 115 DEG C of autoclaving 20 min.
(2) preparation of auxiliary culture medium:
3% l- L-Glutamine, 16.5 ml, 10%l- cysteine 6.0 ml of 0.22 micron membrane filter Entkeimung of learning from else's experience, 15% water
Solution lactoprotein 32.0 ml, transferrinss (10 mg/ml) 1.2 ml, insulin (10 mg/ml) 1.2 ml, horse serum 50 ml,
Penicillin (200,000 iu/ml) 0.25 ml, is sufficiently mixed, obtains final product auxiliary culture medium.
(3) preparation of culture medium
Under aseptic condition, basal liquid and auxiliary culture medium mixing are settled to 1000ml with aquesterilisa polishing, with the 1m of sterilizing
Naoh(4 g is dissolved in 100 ml deionized waters) adjust ph value to 7.4, fully shake up, put 4 DEG C and save backup.
Contrast test
Apply culture medium of the present invention, Cor Bovis seu Bubali soup culture medium, improvement km2 culture medium, improvement thiaucourt ' s culture medium that cattle is propped up
Substance pg45 strain is compared test, and test and result are as follows.
First, culture medium preparation
1st, the preparation of culture medium of the present invention
(1) preparation of basal medium
Take peptone 30.0 g, glucose 6.0 g, Sodium Pyruvate 5.0 g, 25% fresh yeast leachate 110 ml, 1% phenol red
2.5 ml dissolve in 750ml deionized water, are cooled to room temperature standby after 115 DEG C of autoclaving 20min.
(2) preparation of auxiliary culture medium:
3% l- L-Glutamine, 16.5 ml, 10%l- cysteine 6.0 ml of 0.22 micron membrane filter Entkeimung of learning from else's experience, 15% water
Solution lactoprotein 32.0ml, transferrinss (10 mg/ml) 1.0 ml, insulin (10 mg/ml) 1.2 ml, horse serum 50 ml, green grass or young crops
Mycin (200,000 iu/ml) 0.25 ml, is sufficiently mixed, obtains final product auxiliary culture medium.
(3) preparation of culture medium
Under aseptic condition, basal liquid and auxiliary culture medium mixing are settled to 1000ml with aquesterilisa polishing, with the 1m of sterilizing
Naoh(4 g is dissolved in 100 ml deionized waters) adjust ph value to 7.4, fully shake up, rearmounted 4 DEG C of subpackage saves backup.
2nd, the preparation (1000ml) of Cor Bovis seu Bubali soup culture medium: take 1% lactoalbumin hydrolysate hank ' s liquid 562.5 ml, be mixed into cattle
Heart soup 337.5ml, 25% fresh yeast leachate 20 ml, penicillin (200,000 iu/ml) 1ml, 1% phenol red 2.5 ml, 10% acetic acid
Thallium 1 ml, sterile horse blood serum 100 ml, adjust ph value to 7.4 with the 1m naoh of sterilizing after being sufficiently mixed, through 0.22 micron membrane filter
Subpackage after Entkeimung, puts 4 DEG C and saves backup.
3rd, improve the preparation (1000ml) of km2 culture medium: by mem 5 g, glucose 0.4 g, Sodium Pyruvate 0.2 g, water
Solution lactoprotein 5.1 g, 1% phenol red 2.5 ml dissolve in 700ml deionized water, add 25% fresh yeast leachate 20 ml, penicillium sp
Plain (200,000 iu/ml) 1ml, 10% thaliium acetate 1 ml, sterile horse blood serum 200 ml, are sufficiently mixed, deionized water is settled to
1000ml, adjusts ph value to 7.4 with the 1m naoh of sterilizing, and subpackage after 0.22 micron membrane filter Entkeimung puts 4 DEG C of preservations standby
With.
4th, improve the preparation (1000ml) of thiaucourt ' s culture medium: (1) basal liquid is prepared: takes pplo meat soup 21.0
G, Sodium Pyruvate 2.0 g, glucose 1.0 g, 0.4% phenol red 4.5 ml are dissolved in deionized water 700ml, 115 DEG C of autoclavings
20min, is cooled to room temperature standby.(2) take basal liquid 700ml, with 25% fresh yeast leachate 100 ml, penicillin (200,000
Iu/ml) 1ml, 10% thaliium acetate 1 ml, sterile horse blood serum 200 ml, adjusts ph value to arrive with the 1m naoh of sterilizing after being sufficiently mixed
7.4, subpackage after 0.22 micron membrane filter Entkeimung, put 4 DEG C and save backup.
5th, the culture of Mycoplasma bovis by Mycoplasma bovis pg45 strain (purchased from American Type Culture Collecti atcc, numbering atcc
25523) culture medium of the present invention, Cor Bovis seu Bubali soup culture medium, improvement km2 culture medium and improvement thiaucourt ' s culture are inoculated respectively
Base, after seed subculture rejuvenation, respectively in the corresponding culture medium of ratio inoculation of 10% (v/v), 37 DEG C of constant temperature culture, works as culture medium
Its colour changed into yellow, ph value is when being down to 6.8～6.9 by 7.4, aseptic taking-up culture.
3rd, detect
Viable bacteria titre (ccu) measures.Method is as follows: takes 12 test tubes, often pipe plus corresponding culture medium 4.5 ml, add in the 1st pipe
Enter the well-grown M. bovis culture of 0.5 ml, fully mixed with agitator, the pipet renewing, draw 0.5 from the 1st pipe
Ml is added in the 2nd pipe, carries out 10 times successively and is diluted to the 11st pipe, discards 0.5 ml culture fluid in the 11st pipe；Obtain culture fluid
Dilution factor be respectively 10-1-10-11, the 12nd manages as corresponding culture medium comparison.Developmental tube sets 3 repetitions.Test tube is put 37 DEG C of perseverances
Quiescent culture in warm incubator, observes color change, Continuous Observation 10 days daily, occurs the highest dilution of color change to be
The viable bacteria titre of this culture, is represented with color changing units (ccu).Test is repeated 3 times.
4th, result:
Mycoplasma bovis inoculate culture medium of the present invention, Cor Bovis seu Bubali soup culture medium, improvement km2 culture medium and improvement thiaucourt ' s culture
Base 3 secondary growth tests ccu measurement result, is shown in Table 1.
3 times result of the test shows, under similarity condition, culture medium of the present invention and improvement thiaucourt ' s culture medium culturing
The growth time of Mycoplasma bovis is 1 day, and the growth time of Cor Bovis seu Bubali soup culture medium and improvement km2 culture medium culturing Mycoplasma bovis is
2 days；Cor Bovis seu Bubali 3 ccu measurement results of soup culture medium culturing Mycoplasma bovis are 108Ccu/ml, km2 culture medium culturing cattle props up for improvement
3 ccu measurement results of substance are 107Ccu/ml, improvement 3 ccu of thiaucourt ' s culture medium culturing Mycoplasma bovis measure
Result is 108～109ccu/ml.Culture medium culturing Mycoplasma bovis of the present invention, 3 times ccu measurement result is 1010Ccu/ml, training
Foster M. bovis viable titre is apparently higher than Cor Bovis seu Bubali soup culture medium, improvement km2 culture medium and improvement thiaucourt ' s culture medium.
Additionally, culture medium serum content of the present invention is 5%, it is the 1/2 of Cor Bovis seu Bubali soup culture medium (serum content is 10%) serum content, for changing
Blood in good km2 culture medium (serum content is 20%) and improvement thiaucourt ' s culture medium culturing base (serum content is 20%)
The 1/4 of clear content.As can be seen from the above results, culture medium culturing Mycoplasma bovis of the present invention have that growth is rapid, culture viable bacteria
The feature that titre is high, serum content is low, is suitable for the grown cultures of Mycoplasma bovis.
Claims (3)
1. it is used for cultivating the fluid medium of Mycoplasma bovis, its composition includes: peptone, glucose, Sodium Pyruvate, fresh
Yeast leachate, l- L-Glutamine, a small amount of horse serum, penicillin, phenol red, deionized water are it is characterised in that described culture medium
It is made up of basal medium and auxiliary culture medium mixing, the consisting of of the basal medium in wherein every liter of culture medium:
(1) peptone 28.0～33.0 g/l,
(2) glucose 6.0～8.0 g/l,
(3) Sodium Pyruvate 5.0～6.0 g/l,
(4) mass concentration is 25% fresh yeast leachate 110～130 ml/l,
(5) mass concentration is 1% phenol red 2.0～2.5 ml/l,
(6) 750ml deionized water；
The consisting of of auxiliary culture medium in every liter of culture medium:
(7) mass concentration is 3% l- L-Glutamine 16～17 ml/l,
(8) mass concentration is 10% l- cysteine 4.0～6.0 ml/l,
(9) mass concentration is 15% lactoalbumin hydrolysate 32.0-35.0 ml/l,
Transferrinss 0.8～1.5 ml/l of (10) 10 mg/ml,
(11) 10 mg/ml insulin 0.8～1.5 ml/l,
(12) horse serum 50 ml/l,
Penicillin 0.25 ml/l of (13) 20 ten thousand iu/ml,
The ph value of culture medium is adjusted to 7.2～7.4.
2. the culture medium preparation method described in claim 1 it is characterised in that:
A. its preparation is cultivated on basis
Weigh and measure peptone, glucose, Sodium Pyruvate, fresh yeast leachate in proportion, and the phenol red 750ml that dissolves in is gone
In ionized water, it is cooled to room temperature after 115 DEG C of autoclaving 20min standby；
B. assist the preparation of culture medium
Weigh in proportion and measure l- L-Glutamine, l- cysteine, lactoalbumin hydrolysate, transferrinss, insulin, horse serum,
Penicillin, with 0.22 micron membrane filter Entkeimung, is sufficiently mixed, obtains final product auxiliary culture medium,
C. the preparation of culture medium
Under aseptic condition, basic liquid-based and auxiliary culture medium mixing are settled to 1000 ml with aquesterilisa polishing, with sterilizing
1m naoh adjusts ph value to 7.2-7.4, fully shakes up, puts 4 DEG C and save backup.
3. application in culture Mycoplasma bovis for the Mycoplasma bovis fluid medium described in claim 1.
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| CN112410248A (en) * | 2020-11-09 | 2021-02-26 | 山东滨州沃华生物工程有限公司 | Mycoplasma synoviae culture medium and preparation method thereof |
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| CN102344892A (en) * | 2010-06-11 | 2012-02-08 | 中国农业科学院哈尔滨兽医研究所 | Chinese isolate of Leachiii mycoplasma, isolation medium and purpose thereof |
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