CN1318570C - Medium formula and preparation method suitable for large scale production of mycobacterium - Google Patents
Medium formula and preparation method suitable for large scale production of mycobacterium Download PDFInfo
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- CN1318570C CN1318570C CNB2004100581472A CN200410058147A CN1318570C CN 1318570 C CN1318570 C CN 1318570C CN B2004100581472 A CNB2004100581472 A CN B2004100581472A CN 200410058147 A CN200410058147 A CN 200410058147A CN 1318570 C CN1318570 C CN 1318570C
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- potato
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- mycobacterium
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Abstract
The present invention provides a formula for preparing mycobacterium culture medium and a preparation method. The formula of the culture medium comprises the following components of 9.0 to 11 g of sodium glutamate (or asparagine), 3.0 to 3.1 g of K2HPO4, 0.9 to 1.0 g of KH2PO4, 0.002 to 0.001 g of ZnSO4, 0.002 to 0.001 g of CuSO4, 0.5 to 0.6 g of MgSO4, 1.7 to 1.8 g of Na3C6H5O7.2H2O, 0.02 to 0.06 g of Fe(NH4)3(C6H5O7)2, 80 to 90.0 ml of glycerol, 250 to 270g of potato (or potato powder), 6.0 to 4.0g of agar powder and 1000.0 ml of distilled water. The preparation method comprises the steps: the components of the sodium glutamate, the K2HPO4, the KH2PO4, the glycerol, the potatoes (or potato powder) and the distilled water are uniformly mixed to be heated and boiled, then other components of the formula are added to be fully and uniformly mixed with a stirrer, then the culture medium is quantitatively filled into a culture tube to be sterilized under high pressure after a plug is added, and the culture medium is cooled to be used for culturing mycobacteria. Compared with the prior art, the formula has the advantages of easy preparation method, low production cost, stable pH, favourable culture effect and high production capacity of mycobacteria, the filling with mechanization can be realized, and the formula is suitable for mass production of all mycobacteria (except for leprae mycobacteria).
Description
Technical field: the culture medium prescription and the compound method that the present invention relates to a kind of vaccine.Say that definitely this case relates to culture medium prescription and the compound method (not comprising Mycobacterium leprae) of a kind of mass production mycobacterium.
Background technology: mycobacterium is the actinomycetes Mycobacterium, mainly contains mycobacterium tuberculosis complex, non-tuberculous mycobacteria and Mycobacterium leprae.With the human health relation the closest to belong to mycobacterium tuberculosis and Mycobacterium bovis.20 beginning of the century French scientist Calmette and Guerin go down to posterity from the isolated Mycobacterium bovis in cow's milk room a strain at glycerin-potato medium, strong malicious Mycobacterium bovis is become avirulent strains through 13 years 230 generations.Most widely used in the world now prevention bacille Calmette-Guerin vaccine lungy just.
Bacille Calmette-Guerin vaccine prevents the tuberculosis except that being used to, and is also extensive at home and abroad from the sixties in 20th century Be applied to other treatment of diseases.Use the shallow bladder cancer of bacille Calmette-Guerin vaccine bladder instillation to treat table as Moralcs first report in 1976, bacille Calmette-Guerin vaccine has been obtained good result aspect the tumor of bladder preventing and treating as a kind of effective immunological adjuvant.Domestic bacille Calmette-Guerin vaccine prevent and treat upper respiratory tract infection, asthma, allergic rhinitis, ephritis, etc. also obtained obvious curative effects in the disease.Along with in recent years to mycobacterium (mainly being bacill calmette-guerin and cow tubercule bacillus) disease preventing and treating mechanism progress of research, confirmed that bacill calmette-guerin and cow tubercule bacillus be not only a kind of good immunostimulant, and be a kind of good immunomodulator, up-to-date achievement in research shows that bacill calmette-guerin and composition extract thereof have boundless application prospect in the control of anaphylactic disease.
Bacill calmette-guerin and cow tubercule bacillus substratum and cultural method also adopt the logical liquid nutrient medium of traditional Soviet Union to cultivate mostly at present, output is subjected to certain restriction, be difficult to satisfy on the medical market the demand of bacill calmette-guerin and extract and cow tubercule bacillus, domestic have only a spot of bacill calmette-guerin extract of seldom several manufacturer production and cow tubercule bacillus goods supply the market.
The logical liquid nutrient medium of Soviet Union
Composition:
Potassium primary phosphate 0.5g
Sal epsom 0.5g
Citric acid 2.0g
Ferric ammonium citrate 0.05g
Sodium Glutamate 4.0g
Glycerine 30.0ml
Distilled water 970.0ml
The agar,bile salt substratum
Composition:
Cholate 1.5g
Peptone 20.0g
Sodium Fluoride 5.0g
Lactose 10.0g
Distilled water 1000.0ml
Agar 20.0g
Viola crystallina 0.001g
Toluylene red 0.03g
The logical liquid nutrient medium of improvement Soviet Union
Composition:
L-asparagine 2.0g
Potassiumphosphate 1.6g
Sal epsom (7H
2O) 0.25g
Glycerine 20.0ml
Trisodium Citrate (2H
2O) 1.25g
Distilled water adds to 950.0ml
Ferric ammonium citrate 0.05g
Calf serum 50.0ml
Sodium phosphate dibasic 3.4g
The logical potato culture of Soviet Union
Composition: with the logical liquid nutrient medium of improvement Soviet Union, wherein the L-asparagine is used monosodium glutamate instead and is replaced, in every 1000ml liquid nutrient medium with monosodium glutamate 16.0g.Be sub-packed in after the heating for dissolving in the special-purpose culture tube of bacill calmette-guerin, every pipe 30.0ml, and then add one of treated potato plug, 121 ℃ of autoclavings 30 minutes take out that to put cold back standby.
The potato plug preparation process is loaded down with trivial details also to make the production in enormous quantities of bacill calmette-guerin substratum be restricted, in this process, stainless steel tube with a 2.6cm diameter makes it into cylindrical in the potato punching of cleaning peeling, the diagonal angle is cut into two inclined-planes then, putting into 1% sodium carbonate solution soaked 2～3 hours, with tap water flushing 2～3 hours, take out potato plug and put into the distillation bubbly water 30 minutes.
Though liquid nutrient medium can adopt the automatization can to produce in enormous quantities, but it is big to take the incubator area during with flask culture, and bacill calmette-guerin must be inoculated in fluid surface, can not growth and breeding in case the bacill calmette-guerin of inoculation sinks in the nutrient solution, so the inoculation operation easier is big, and must put into incubator gently.
The logical potato culture of Soviet Union is with the inoculation shovel bacill calmette-guerin evenly to be smeared to be inoculated on the potato plug, tiltedly is placed on the shelf.Making wherein the logical liquid nutrient medium of Soviet Union be immersed in potato plug around and can not soak the bacill calmette-guerin of inoculation.Put into incubator gently.Complex operation, the technical requirements height.
People expect to have a kind of making in enormous quantities that is easy to always for a long time, and compound method is easy, all mycobacteriums (except the Mycobacterium leprae) can both culture medium prescription and compound method well-grown, that also facilitate for subsequent operationss such as inoculation, results.
Mycobacterium culture medium prescription provided by the invention and compound method can be used for the production in enormous quantities (except the Mycobacterium leprae) of all mycobacteriums.
Summary of the invention:
Culture medium prescription provided by the invention and compound method are applicable to and produce mycobacterium in enormous quantities that wherein term " potato " means potato and cleans the chopping of peeling back.Compare with prior art, the culture medium prescription and the compound method that provide are provided, cost is low, and PH is stable, prepare simple and easy to doly, can realize the mechanize can, and culture effect is good, is applicable to the production in enormous quantities mycobacterium.The substratum most preferably every pipe of scheme can produce bacill calmette-guerin 2.5～3.5 grams, and the comparatively preferred every pipe of scheme can produce bacill calmette-guerin 2.0～2.5 grams; The every pipe of preferred version can produce 1.5 grams.Liquid nutrient medium is led in the Soviet Union that adopts at most at present, and every bottle can be produced bacill calmette-guerin 4.0 grams, but culture technique is loaded down with trivial details, culturing room's area is big, and the manpower and materials input is many, and cost is higher than the several times of the method for the invention provides.
Embodiment: culture medium prescription of the present invention is as follows:
Composition:
(1) sodium glutamate (or asparagine) 10～15g
(2)K
2HPO
4 2.8～3.5g
(3)KH
2PO
4 0.8～1.2g
(4)ZnSO
4 0.004～0.001g
(5)CuSO
4 0.004～0.001g
(6)MgSO
4 0.3～0.9g
(7)Na
3C
6H
5O
7.2H
2O 1.4～2.0g
(8) Fe (NH
4)
3(C
6H
5O
7)
20.02-0.10 gram
(9) glycerol 70～100.0ml
(10) potato (or potato powder) 200～300g
(11) agar powder 9.0～3.0g
(12) distilled water 1000.0ml
A kind of substratum screening formulation of mass production mycobacterium is as follows:
Composition:
(1) sodium glutamate (or asparagine) 12～14g
(2)K
2HPO
4 2.9～3.3g
(3)KH
2PO
4 0.9～1.1g
(4)ZnSO
4 0.003～0.002g
(5)CuSO
4 0.003～0.002g
(6)MgSO
4 0.4～0.7g
(7)Na
3C
6H
5O
7.2H
2O 1.6～1.9g
(8) Fe (NH
4)
3(C
6H
5O
7)
20.04-0.08 gram
(9) glycerol 80～90.0ml
(10) potato (or potato powder) 240～280g
(11) agar powder 8.0～4.0g
(12) distilled water 1000.0ml
The most preferably prescription of a kind of substratum of mass production mycobacterium is as follows:
Composition:
(1) sodium glutamate (or asparagine) 9.0～11g
(2)K
2HPO
4 3.0～3.1g
(3)KH
2PO
4 0.9～1.0g
(4)ZnSO
4 0.002～0.001g
(5)CuSO
4 0.002～0.001g
(6)MgSO
4 0.5～0.6g
(7)Na
3C
6H
5O
7.2H
2O 1.7～1.8g
(8) Fe (NH
4)
3(C
6H
5O
7)
20.02-0.06 gram
(9) glycerol 80～90.0ml
(10) potato (or potato powder) 250～270g
(11) agar powder 6.0～4.0g
(12) distilled water 1000.0ml
Mass production mycobacterium substratum compound method provided by the invention is, after mentioned component (1) (2) (3) (9) (10) (12) mixing heated and boiled, add again in the prescription other compositions with the abundant mixing of stirrer after quantitative filling in culture tube, 15 pounds of sterilizations in 30 minutes of high pressure promptly can be used for mycobacterium and cultivate usefulness after jumping a queue after the cooling.
Mass production mycobacterium culture medium prescription provided by the invention and compound method not only make the making of substratum reduce cost, and are that the mass-produced subsequent operations of mycobacterium (as inoculation operation, results operation) provides great convenience.Over half than the employed manpower minimizing of traditional method, but also can make the output of mycobacterium improve 5 times.The present invention plays an important role to the imbalance between supply and demand of alleviating medical market mycobacterium and extract formulation thereof.
After the scientific and technical personnel of this area watch this description, can carry out various modifications and changes, but any modification or change are all within the protection domain of claims that application is awaited the reply to culture medium prescription.
Claims (3)
1. be applicable to a kind of substratum of mass production bacill calmette-guerin, it is characterized in that:
Composition: (1) sodium glutamate or asparagine 10～15g
(2)K
2HPO
4 2.8～3.5g
(3)KH
2PO
4 0.8～1.2g
(4)ZnSO
4 0.004～0.001g
(5)CuSO
4 0.004～0.001g
(6)MgSO
4 0.3～0.9g
(7)Na
3C
6H
5O
7.2H
2O 1.4～2.0g
(8) Fe (NH
4)
3(C
6H
5O
7)
20.02-0.10 gram
(9) glycerol 70～100.0ml
(10) potato or potato powder 200～300g
(11) agar powder 9.0～3.0g
(12) distilled water 1000.0ml
2. a kind of substratum that is applicable to the mass production bacill calmette-guerin according to claim 1 is characterized in that:
Composition: (1) sodium glutamate or asparagine 12～14g
(2)K
2HPO
4 2.9～3.3g
(3)KH
2PO
4 0.9～1.1g
(4)ZnSO
4 0.003～0.002g
(5)CuSO
4 0.003～0.002g
(6)MgSO
4 0.4～0.7g
(7)Na
3C
6H
5O
7.2H
2O 1.6～1.9g
(8) Fe (NH
4)
3(C
6H
5O
7)
20.04-0.08 gram
(9) glycerol 80～90.0ml
(10) potato or potato powder 240～280g
(11) agar powder 8.0～4.0g
(12) distilled water 1000.0ml
3. a kind of substratum that is applicable to the mass production bacill calmette-guerin according to claim 1 is characterized in that:
Composition: (1) sodium glutamate or asparagine 9.0～11g
(2)K
2HPO
4 3.0～3.1g
(3)KH
2PO
4 0.9～1.0g
(4)ZnSO
4 0.002～0.001g
(5)CuSO
4 0.002～0.001g
(6)MgSO
4 0.5～0.6g
(7)Na
3C
6H
5O
7.2H
2O 1.7～1.8g
(8) Fe (NH
4)
3(C
6H
5O
7)
20.02-0.06 gram
(9) glycerol 80～90.0ml
(10) potato or potato powder 250～270g
(11) agar powder 6.0～4.0g
(12) distilled water 1000.0ml
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB2004100581472A CN1318570C (en) | 2004-08-17 | 2004-08-17 | Medium formula and preparation method suitable for large scale production of mycobacterium |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB2004100581472A CN1318570C (en) | 2004-08-17 | 2004-08-17 | Medium formula and preparation method suitable for large scale production of mycobacterium |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN1737115A CN1737115A (en) | 2006-02-22 |
| CN1318570C true CN1318570C (en) | 2007-05-30 |
Family
ID=36080064
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CNB2004100581472A Expired - Fee Related CN1318570C (en) | 2004-08-17 | 2004-08-17 | Medium formula and preparation method suitable for large scale production of mycobacterium |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN1318570C (en) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| FR2939447B1 (en) * | 2008-12-05 | 2011-01-07 | Univ Aix Marseille Ii | MEDIUM AND METHOD FOR CULTIVATING AND IDENTIFYING MYCOBACTERIA INCLUDING MYCOBACTERIA OF THE MYCOBACTERIUM TUBERCULOSIS COMPLEX. |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1506115A (en) * | 2002-12-09 | 2004-06-23 | 北京大经万易生物医药科技发展有限公 | Calmette-Guerin bacillus prepn |
-
2004
- 2004-08-17 CN CNB2004100581472A patent/CN1318570C/en not_active Expired - Fee Related
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1506115A (en) * | 2002-12-09 | 2004-06-23 | 北京大经万易生物医药科技发展有限公 | Calmette-Guerin bacillus prepn |
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| Publication number | Publication date |
|---|---|
| CN1737115A (en) | 2006-02-22 |
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Granted publication date: 20070530 Termination date: 20100817 |