CN106399207B - A kind of Mycoplasma bovis culture medium - Google Patents
A kind of Mycoplasma bovis culture medium Download PDFInfo
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- CN106399207B CN106399207B CN201611072162.1A CN201611072162A CN106399207B CN 106399207 B CN106399207 B CN 106399207B CN 201611072162 A CN201611072162 A CN 201611072162A CN 106399207 B CN106399207 B CN 106399207B
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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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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/02—Bacterial antigens
- A61K39/0241—Mollicutes, e.g. Mycoplasma, Erysipelothrix
Abstract
The invention discloses a kind of Mycoplasma bovis culture mediums and preparation method thereof, belong to technical field of veterinary biology.Mycoplasma bovis culture medium of the invention is made of basal medium and auxiliary culture medium mixing, wherein the composition of culture medium are as follows: PPLO meat soup, Sodium Pyruvate, glucose, fresh yeast leachate, L-Glutamine, L-cysteine, lactoalbumin hydrolysate, horse serum, penicillin, phenol red, deionized water.The present invention both can be reduced amount of serum, also significantly improve the viable bacteria titre of Mycoplasma bovis, lay a good foundation for the development of the high-quality vaccine of Mycoplasma bovis.
Description
Technical field
The present invention relates to a kind of Mycoplasma bovis culture medium and preparation method thereof, being exactly in its component of one kind includes:
PPLO meat soup, Sodium Pyruvate, glucose, fresh yeast leachate, horse serum, penicillin, phenol red, deionized water Mycoplasma bovis
Culture medium and preparation method thereof.
Background technique
Mycoplasma bovis (Mycoplasma bovis,M.bovis) ox pneumonia, arthritis, mammitis, cornea knot can be caused
Film inflammation, otitis, genital inflammation, miscarriage and a variety of diseases such as infertile.Disease caused by the cause of disease worldwide prevalence simultaneously
Cause serious financial consequences.Mycoplasma bovis is the Etiological of ox respiratory disorder syndrome.China is from 2008 for the first time from Hubei
It has saved since being separated to Mycoplasma bovis with outburst necrotizing pneumonia beef cattle, then constantly has Niu Zhiyuan in the multiple province ,city and areas in the whole nation
The report of body case, Mycoplasma bovis has become universal Infection Status in China now, and Mycoplasma bovis, which also has become, threatens China to support
One of important pathogen of Niu Ye.
The prevention and control of Mycoplasma bovis disease need comprehensive measures for the prevention and control, and vaccine immunity is prevention and control Mycoplasma bovis pathogenetic one
A important means, and high-quality and efficient vaccine needs high-quality culture medium as support.The culture medium of Mycoplasma bovis mainly has ox at present
Heart soup culture medium, improvement KM2 culture medium, improvement Thiaucourt ' s culture medium etc..Prior art culture medium culture Mycoplasma bovis
There are incubation times it is longer, viable bacteria titre is lower, fertility is poor the problems such as.In addition, serum contains in prior art culture medium
Amount generally between 10%~20%, not only increases seedling cost, and the vaccine of excessive serum preparation undoubtedly increases alloplasm
To the allergy stress reaction of ox body, the final immune effect for influencing vaccine.It is simple to reduce serum content in prior art culture medium
It will lead to low 10~100 times of Mycoplasma bovis antigen titre or so of culture, immune required antigen dose be not only not achieved, but also need to improve dense
Demagnification number, actually increases production cost.
Chinese invention patent 2011100012310 discloses a kind of mycoplasma hyopneumoniae culture medium and preparation method thereof, this is specially
Benefit basal medium constituent are as follows: 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, be added 140 before using~
The healthy horse serum of 200ml, and need to add agar.According to disclosure of which, with the pig of the culture medium culture of the 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
Fastly, the characteristics of sensibility separated is strong, preparation method simple process, strong operability, is suitble to industrialized production.
A kind of mycoplasma capri goat pneumonia subspecies in vitro culture disclosed in Chinese invention patent application 2015100244778
Base by PPLO meat soup 21g/L, glucose 2g/L, Sodium Pyruvate 2g/L, the yeast extract 100ml/L of mass volume ratio 25%,
0.4% phenol red 0.18ml/L, the penicillin 200IU/ml for inactivating horse serum 100mL/L and water dissolution are constituted, pH value 7.4
~7.6.Using the culture medium culture mycoplasma capri goat pneumonia subspecies, growth titre can reach 4 × 10 within 56 hours9Ccu,
While reaching original culture basal growth titre, incubation time shortens 10 hours, while can reduce the cost of culture medium.
But since above-mentioned patent is for cultivating mycoplasma hyopneumoniae and mycoplasma capri goat pneumonia subspecies, cultivate Niu Zhiyuan
Body titre is not higher than 109 CCU/ml；In addition, horse serum amount required by above-mentioned patent is larger, horse serum content 10% and
More than；Therefore, it develops the low blood serum medium of efficient Mycoplasma bovis and has become in mycoplasma bovis vaccine research and production and be badly in need of solution
Major issue certainly.
Summary of the invention
The present invention provides a kind of low serum efficient culture medium of Mycoplasma bovis that can be overcome the shortage of prior art, and the present invention is another
Purpose is to provide the preparation method of the culture medium.
This Mycoplasma bovis culture medium of the invention is made of basal medium and auxiliary culture medium mixing, wherein every liter
The composition of basal medium in culture medium are as follows:
(1) 22.0~24.0 g of PPLO meat soup
(2) 5.0~6.0 g of Sodium Pyruvate
(3) 6.0~8.0 g of glucose
(4) phenol red 2.0~2.5 ml that mass concentration is 1%
(5) 750 ml of deionized water；
Assist the composition of culture medium are as follows:
(6) 110~120 ml of fresh yeast leachate that mass concentration is 25%
(7) 16~17 ml of L-Glutamine that mass concentration is 3%
(8) 4.0~6.0 ml of L-cysteine that mass concentration is 10%
(9) 32.0~35.0 ml of lactoalbumin hydrolysate that mass concentration is 15%
(10) penicillin (200,000 IU/ml) 0.25 ml
(11) 50 ml of sterile horse blood serum
The pH value of culture medium is 7.2~7.4.
Mycoplasma bovis culture medium of the invention the preparation method comprises the following steps: by the component of basal medium dissolve in one by one 750ml go from
In sub- water, dry in the air after 115 DEG C of high pressure sterilization 20min spare to room temperature；The each component for assisting culture medium is filtered through 0.22 micron respectively
It is sufficiently mixed after film filtration sterilization；Under aseptic condition, basal medium and auxiliary culture medium are mixed, with aqua sterilisa polishing constant volume
To 1000ml, 100 ml deionized waters are dissolved in the 1M NaOH(4 g of sterilizing) adjust pH value to 7.2-7.4, it sufficiently shakes up, dispenses,
4 DEG C are set to save backup.
The low serum efficient culture medium culture Mycoplasma bovis of Mycoplasma bovis of the invention can be prepared in mycoplasma bovis vaccine antigen
In application.
The invention has the benefit that
The low serum efficient culture medium of Mycoplasma bovis of the invention is according to the growth characteristics of Mycoplasma bovis, by different carbon
The combination of many nutrition compositions such as source, nitrogen source, protein, inorganic salts, cholesterol is screened, at the same to the pH value of culture medium,
Osmotic pressure, ionic strength etc. compare analysis, have investigated the low serum efficient culture medium for being suitble to culture Mycoplasma bovis.It should
PPLO meat soup is mycoplasma growth basal liquid in culture medium；Sodium Pyruvate can be used as the substitution carbon source in Culture Mycoplasma；Culture
Glucose in base provides energy for Mycoplasma bovis growth；By adding fresh yeast leachate, provided for Mycoplasma bovis growth
The nutritional ingredients such as required nitrogen source, electrolytes and minerals；Addition L-Glutamine can promote microbial metabolism, promote albumen
Matter synthesis；Addition L-cysteine and lactoalbumin hydrolysate grow for Mycoplasma bovis provides suitable amino acid；It is a small amount of by adding
Horse serum, cholesterol and required saturation or unsaturated fatty acid needed for providing growth to Mycoplasma bovis；By adding mould
Element can inhibit varied bacteria growing, to mycoplasma unrestraint effect, can avoid culture medium pollution and extends the culture medium holding time；Addition
It is phenol red to be used as pH indicator, it can determine whether the upgrowth situation of mycoplasma.In the formula of the culture medium in addition to fundamental component, cultivating
It also added fresh yeast leachate, L-Glutamine, L-cysteine and lactoalbumin hydrolysate in base, the addition of mentioned component was both
It can be reduced amount of serum, also significantly improve the viable bacteria titre of Mycoplasma bovis；Through repeatedly it is experimentally confirmed that viable bacteria is dripped before being not added with
Degree is 108-109CCU/ml, viable bacteria titre is up to 10 after addition10CCU/ml.And the present invention is another advantage is that culture
Low serum content in base, it is the 1/2~1/4 of prior art culture medium serum content that amount of serum, which is only 5%, in culture medium；With this
The Mycoplasma bovis semi-finished product bacterium solution titre of inventive method preparation is up to 1010CCU/ml, viable bacteria titre are apparently higher than improvement KM2 culture
Base (107 CCU/ml), cattle heart soup culture medium (108 CCU/ml) and improvement Thiaucourt ' s culture medium (108~109CCU/ml).
The low serum efficient culture medium of Mycoplasma bovis significantly reduces alloplasm serum in vaccine and improves to the allergy stress reaction of ox body
Bio-safety also improves viable bacteria bacterium solution titre, reduces production cost, has established base for the development of the high-quality vaccine of Mycoplasma bovis
Plinth.
Specific embodiment
Embodiment 1:
1, the preparation of culture medium of the present invention:
Basal medium:
(1) 22.0 g of PPLO meat soup
(2) 5.0 g of Sodium Pyruvate
(3) 6.0 g of glucose
(4) phenol red 2.0 ml that mass concentration is 1%
(5) deionized water 750ml.
115 DEG C of sterilizing 20min are aseptically added following auxiliary medium component, the present invention are made after cooling
Culture medium.
Assist culture medium:
(6) 110 ml of fresh yeast leachate that mass concentration is 25%
(7) 17.0 ml of L-Glutamine that mass concentration is 3%
(8) 5.0 ml of L-cysteine that mass concentration is 10%
(9) 33.0 ml of lactoalbumin hydrolysate that mass concentration is 15%
(10) penicillin (200,000 IU/ml) 0.25ml
(11) 50 ml of sterile horse blood serum
(1) in basal medium~(4) ingredient is dissolved in one by one in 750ml deionized water (5), 115 DEG C of high pressure sterilizations
It dries in the air after 20min spare to room temperature.It will assist (6)~(11) ingredient of culture medium respectively after 0.22 micron membrane filter filtration sterilization
It is sufficiently mixed；Under aseptic condition, basal medium and auxiliary culture medium are mixed, are settled to 1000ml with aqua sterilisa polishing, is used
The 1M NaOH(4 g of sterilizing is dissolved in 100 ml deionized waters) adjust pH value to 7.2-7.4, it sufficiently shakes up, dispenses, it is standby to set 4 DEG C of preservations
With.
2, mass concentration be 25% fresh yeast leachate the preparation method comprises the following steps:
500 g of yeast cake is taken, is added in 2000 ml of deionized water, stirring and dissolving, with concentrated hydrochloric acid: deionized water is with=1:1
(volume ratio) adjusts pH value to 4.5-5.0,80 DEG C water-bath (temperature in bottle) 30 minutes, 3000 revs/min are centrifuged 20 minutes, take supernatant
Liquid.Supernatant is dissolved in 100 ml deionized waters with 1 M NaOH(4 g) adjust pH value to 7.8-8.0, it boils, sets after room temperature cools
It is filtered with double-layer filter paper, then mends deionized water to 2000 ml, -20 DEG C save backup.
3, the preparation for the phenol red solution that mass concentration is 1%:
Phenol red 1.0 g is weighed, is set in glass mortar, 0.1M NaOH(0.4 g is added dropwise and is dissolved in 10 ml deionized waters),
It is ground to and is completely dissolved.In the 100 ml measuring bottle of phenol red sucking of dissolution, will carefully be washed in lower mortar with deionized water remain it is phenol red
Liquid finally adds deionized water to 100 ml into measuring bottle.
Embodiment 2:
The preparation of culture medium of the present invention:
Basal medium:
(1) 22.0 g of PPLO meat soup
(2) 5.0 g of Sodium Pyruvate
(3) 8.0 g of glucose
(4) 1% phenol red 2.0 ml
(5) deionized water 750ml.
115 DEG C of sterilizing 20min are aseptically added following auxiliary medium component, the present invention are made after cooling
Culture medium.
Assist culture medium:
120 ml of (6) 25% fresh yeast leachate
(7) 17.0 ml of L-Glutamine that mass concentration is 3%
(8) 5.0 ml of L-cysteine that mass concentration is 10%
(9) mass concentration is 15% lactoalbumin hydrolysate, 33.0 ml
(10) penicillin (200,000 IU/ml) 0.25ml
(11) 50 ml of sterile horse blood serum
(1) in basal medium~(4) ingredient is dissolved in one by one in 750ml deionized water (5), 115 DEG C of high pressure sterilizations
It dries in the air after 20min spare to room temperature.It will assist (6)~(11) ingredient of culture medium respectively after 0.22 micron membrane filter filtration sterilization
It is sufficiently mixed；Under aseptic condition, basal medium and auxiliary culture medium are mixed, are settled to 1000ml with aqua sterilisa polishing, is used
The 1M NaOH(4 g of sterilizing is dissolved in 100 ml deionized waters) adjust pH value to 7.2-7.4, it sufficiently shakes up, dispenses, it is standby to set 4 DEG C of preservations
With.
Comparative test
Using culture medium of the present invention, improvement Thiaucourt ' s culture medium, cattle heart soup culture medium and improvement KM2 culture medium pair
PG45 plants of Mycoplasma bovis compare test, and test and result are as follows.
One, prepared by culture medium
1, the preparation of culture medium of the present invention
Basal medium:
(1) 22.0 g of PPLO meat soup
(2) 5.0 g of Sodium Pyruvate
(3) 6.0 g of glucose
(4) phenol red 2.5 ml that mass concentration is 1%
(5) deionized water 750ml.
115 DEG C of sterilizing 20min are aseptically added following auxiliary medium component, the present invention are made after cooling
Culture medium.
Assist culture medium:
(6) 110 ml of fresh yeast leachate that mass concentration is 25%
(7) 16.7 ml of L-Glutamine that mass concentration is 3%
(8) the L-cysteine 5.0ml that mass concentration is 10%
(9) 33.0 ml of lactoalbumin hydrolysate that mass concentration is 15%
(10) penicillin (200,000 IU/ml) 0.25ml
(11) 50 ml of sterile horse blood serum
(1) in basal medium~(4) ingredient is dissolved in one by one in 750ml deionized water (5), 115 DEG C of high pressure sterilizations
It dries in the air after 20min spare to room temperature.It will assist (6)~(11) ingredient of culture medium respectively after 0.22 micron membrane filter filtration sterilization
It is sufficiently mixed；Under aseptic condition, basal medium and auxiliary culture medium are mixed, are settled to 1000ml with aqua sterilisa polishing, is used
The 1M NaOH(4 g of sterilizing is dissolved in 100 ml deionized waters) adjust pH value to 7.2-7.4, it sufficiently shakes up, dispenses, it is standby to set 4 DEG C of preservations
With.
2, the preparation of Thiaucourt ' s culture medium is improved
Basal liquid:
21.0 g of PPLO meat soup
2.0 g of Sodium Pyruvate
1.0 g of glucose
0.4% phenol red 4.5 ml
Deionized water 700ml.
115 DEG C of high pressure sterilization 20min.
Culture medium:
Basal liquid 700ml
25% fresh yeast leachate, 100 ml
Penicillin (200,000 IU/ml) 1ml
10% thaliium acetate, 1 ml
200 ml of sterile horse blood serum
It is dispensed after 0.22 micron membrane filter filtration sterilization spare with the 1M NaOH tune pH value of sterilizing to 7.4 after mixing.
3, the preparation of cattle heart soup culture medium
1% lactoalbumin hydrolysate Hank ' s liquid, 562.5 ml
337.5 ml of cattle heart soup
25% fresh yeast leachate, 20 ml
Penicillin (200,000 IU/ml) 1ml
1% phenol red 2.5 ml
10% thaliium acetate, 1 ml
100 ml of sterile horse blood serum
It is dispensed after 0.22 micron membrane filter filtration sterilization spare with the 1M NaOH tune pH value of sterilizing to 7.4 after mixing.
4, the preparation of KM2 culture medium is improved
MEM 5 g
0.4 g of glucose
0.2 g of Sodium Pyruvate
5.1 g of lactoalbumin hydrolysate
1% phenol red 2.5 ml
25% fresh yeast leachate, 20 ml
Penicillin (200,000 IU/ml) 1ml
10% thaliium acetate, 1 ml
200 ml of sterile horse blood serum
Mixing, is settled to 1000ml with deionized water, with the 1M NaOH tune pH value of sterilizing to 7.4, filters through 0.22 micron
It is dispensed after film filtration sterilization spare.
Two, the culture of Mycoplasma bovis
It is inoculated with this hair respectively by PG45 plants of Mycoplasma bovis (being purchased from American Type Culture Collecti ATCC, number ATCC 25523)
Bright culture medium (serum content 5%), cattle heart soup culture medium (serum content 10%), improvement Thiaucourt ' s culture medium (blood
Clear content is 20%) and to improve KM2 culture medium (serum content 20%), after seed subculture rejuvenation, respectively by 10% (V/V's)
Ratio, which is inoculated with, corresponds to culture medium, 37 DEG C of constant temperature incubations, when the colour changed into yellow of culture medium, pH value are down to 6.8~6.9 by 7.4,
Sterile taking-up culture.
Three, it detects
Viable bacteria titre (CCU) measurement.Method is as follows: taking 12 test tubes, every pipe adds corresponding 4.5 ml of culture medium, in the 1st pipe
The middle addition well-grown M. bovis culture of 0.5 ml, is mixed well, the pipette renewed with oscillator, is inhaled from the 1st pipe
It takes 0.5 ml to be added in the 2nd pipe, successively carries out 10 times and be diluted to the 11st pipe, discard 0.5 ml culture solution in the 11st pipe；It obtains
The dilution of culture solution is respectively 10-1-10-11, the 12nd pipe is corresponding culture medium control.Developmental tube sets 3 repetitions.Test tube is set
Stationary culture in 37 DEG C of constant incubators, observes color change daily, is observed continuously 10 days, and the highest dilution of color change occurs
Degree is the viable bacteria titre of the culture, is indicated with color changing units (CCU).Test is repeated 3 times.
Four, test result:
Mycoplasma bovis is inoculated with 4 kinds of 3 secondary growth of culture medium test CCU measurement results and is shown in Table 1.
It is shown through 3 test results, under similarity condition, uses the growth time of culture medium culture Mycoplasma bovis of the present invention
It is 1 day, the growth time of improvement Thiaucourt ' s culture medium culture Mycoplasma bovis is 1 day, cattle heart soup culture medium and improvement KM2
The growth time of culture medium culture Mycoplasma bovis is 2 days；Improveing 3 CCU measurement results of Thiaucourt ' s culture medium is 108~
109CCU/ml, 3 CCU measurement results of cattle heart soup culture medium are 108 CCU/ml improves 3 CCU measurement results of KM2 culture medium
It is 107 CCU/ml.Using culture medium culture Mycoplasma bovis of the present invention, 3 times CCU measurement result is 1010CCU/ml(table
1), hence it is evident that higher than the viable bacteria titre of prior art culture medium culture Mycoplasma bovis.As a result illustrate, Mycoplasma bovis culture of the invention
Base has the characteristics that serum content is low, growth is rapid and viable bacteria titre is high.
Claims (5)
- It include: that PPLO meat soup, Sodium Pyruvate, glucose, fresh yeast leach in composition 1. a kind of Mycoplasma bovis culture medium Liquid, horse serum, penicillin, phenol red, deionized water, it is characterised in that the culture medium is mixed by basal medium and auxiliary culture medium It constitutes, wherein the composition of the basal medium in every liter of culture medium are as follows:(1) 22.0~24.0 g of PPLO meat soup(2) 5.0~6.0 g of Sodium Pyruvate(3) 6.0~8.0 g of glucose(4) phenol red 2.0~2.5 ml that mass concentration is 1%(5) 750 ml of deionized water；Assist the composition of culture medium are as follows:(6) 110~120 ml of fresh yeast leachate that mass concentration is 25%(7) 16~17 ml of L-Glutamine that mass concentration is 3%(8) 4.0~6.0 ml of L-cysteine that mass concentration is 10%(9) 32.0~35.0 ml of lactoalbumin hydrolysate that mass concentration is 15%0.25 ml of penicillin of (10) 20 ten thousand IU/ml(11) 50 ml of sterile horse blood serumThe pH value of culture medium is 7.2~7.4.
- 2. a kind of preparation method of Mycoplasma bovis culture medium described in claim 1, it is characterised in that by the group of basal medium Divide and dissolved in 750ml deionized water one by one, is dried in the air after 115 DEG C of high pressure sterilization 20min spare to room temperature；It will assist each group of culture medium Divide and is sufficiently mixed after 0.22 micron membrane filter filtration sterilization respectively；Under aseptic condition, basal medium and auxiliary culture medium are mixed It closes, is settled to 1000ml with aqua sterilisa polishing, with the 1M NaOH tune pH value of sterilizing to 7.2-7.4, sufficiently shakes up, dispense, set 4 It DEG C saves backup.
- 3. application of a kind of Mycoplasma bovis culture medium in culture Mycoplasma bovis as described in claim 1.
- 4. a kind of Mycoplasma bovis culture medium as described in claim 1 is preparing the application in mycoplasma bovis vaccine.
- 5. a kind of Mycoplasma bovis culture medium as described in claim 1 is preparing the application in Mycoplasma bovis antigen.
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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 |
| CN103074246A (en) * | 2012-08-31 | 2013-05-01 | 南京天邦生物科技有限公司 | Low serum efficiency mycoplasma gallisepticam attenuated strain 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 |
| CN103074246A (en) * | 2012-08-31 | 2013-05-01 | 南京天邦生物科技有限公司 | Low serum efficiency mycoplasma gallisepticam attenuated strain culture medium and preparation method thereof |
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