CN104642135A - Large-scale manufacturing method for high-precision BL culture medium - Google Patents
Large-scale manufacturing method for high-precision BL culture medium Download PDFInfo
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- CN104642135A CN104642135A CN201510082272.5A CN201510082272A CN104642135A CN 104642135 A CN104642135 A CN 104642135A CN 201510082272 A CN201510082272 A CN 201510082272A CN 104642135 A CN104642135 A CN 104642135A
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- 238000004519 manufacturing process Methods 0.000 title abstract 5
- 239000001963 growth medium Substances 0.000 title abstract 3
- 238000002360 preparation method Methods 0.000 claims abstract description 15
- 238000005303 weighing Methods 0.000 claims abstract description 12
- 239000012153 distilled water Substances 0.000 claims abstract description 7
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 7
- 238000001556 precipitation Methods 0.000 claims abstract description 4
- 239000011159 matrix material Substances 0.000 claims description 26
- 239000012452 mother liquor Substances 0.000 claims description 21
- NLKNQRATVPKPDG-UHFFFAOYSA-M potassium iodide Chemical compound [K+].[I-] NLKNQRATVPKPDG-UHFFFAOYSA-M 0.000 claims description 18
- XEEYBQQBJWHFJM-UHFFFAOYSA-N Iron Chemical compound [Fe] XEEYBQQBJWHFJM-UHFFFAOYSA-N 0.000 claims description 12
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 claims description 12
- PVNIIMVLHYAWGP-UHFFFAOYSA-N Niacin Chemical compound OC(=O)C1=CC=CN=C1 PVNIIMVLHYAWGP-UHFFFAOYSA-N 0.000 claims description 12
- ARUVKPQLZAKDPS-UHFFFAOYSA-L copper(II) sulfate Chemical compound [Cu+2].[O-][S+2]([O-])([O-])[O-] ARUVKPQLZAKDPS-UHFFFAOYSA-L 0.000 claims description 12
- 229910000366 copper(II) sulfate Inorganic materials 0.000 claims description 12
- FGIUAXJPYTZDNR-UHFFFAOYSA-N potassium nitrate Chemical compound [K+].[O-][N+]([O-])=O FGIUAXJPYTZDNR-UHFFFAOYSA-N 0.000 claims description 12
- 239000003153 chemical reaction reagent Substances 0.000 claims description 11
- 239000007787 solid Substances 0.000 claims description 11
- 238000000034 method Methods 0.000 claims description 10
- 239000000203 mixture Substances 0.000 claims description 7
- PAWQVTBBRAZDMG-UHFFFAOYSA-N 2-(3-bromo-2-fluorophenyl)acetic acid Chemical compound OC(=O)CC1=CC=CC(Br)=C1F PAWQVTBBRAZDMG-UHFFFAOYSA-N 0.000 claims description 6
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 claims description 6
- SQUHHTBVTRBESD-UHFFFAOYSA-N Hexa-Ac-myo-Inositol Natural products CC(=O)OC1C(OC(C)=O)C(OC(C)=O)C(OC(C)=O)C(OC(C)=O)C1OC(C)=O SQUHHTBVTRBESD-UHFFFAOYSA-N 0.000 claims description 6
- 125000000254 aspartoyl group Chemical group 0.000 claims description 6
- KGBXLFKZBHKPEV-UHFFFAOYSA-N boric acid Chemical compound OB(O)O KGBXLFKZBHKPEV-UHFFFAOYSA-N 0.000 claims description 6
- 239000004327 boric acid Substances 0.000 claims description 6
- LLSDKQJKOVVTOJ-UHFFFAOYSA-L calcium chloride dihydrate Chemical compound O.O.[Cl-].[Cl-].[Ca+2] LLSDKQJKOVVTOJ-UHFFFAOYSA-L 0.000 claims description 6
- 229940052299 calcium chloride dihydrate Drugs 0.000 claims description 6
- 229940009662 edetate Drugs 0.000 claims description 6
- CDAISMWEOUEBRE-GPIVLXJGSA-N inositol Chemical compound O[C@H]1[C@H](O)[C@@H](O)[C@H](O)[C@H](O)[C@@H]1O CDAISMWEOUEBRE-GPIVLXJGSA-N 0.000 claims description 6
- 229960000367 inositol Drugs 0.000 claims description 6
- 229910052742 iron Inorganic materials 0.000 claims description 6
- 239000007788 liquid Substances 0.000 claims description 6
- 229910052943 magnesium sulfate Inorganic materials 0.000 claims description 6
- 235000019341 magnesium sulphate Nutrition 0.000 claims description 6
- 235000007079 manganese sulphate Nutrition 0.000 claims description 6
- BZDIAFGKSAYYFC-UHFFFAOYSA-N manganese;hydrate Chemical compound O.[Mn] BZDIAFGKSAYYFC-UHFFFAOYSA-N 0.000 claims description 6
- 229910000402 monopotassium phosphate Inorganic materials 0.000 claims description 6
- 235000019796 monopotassium phosphate Nutrition 0.000 claims description 6
- 239000011664 nicotinic acid Substances 0.000 claims description 6
- 229960003512 nicotinic acid Drugs 0.000 claims description 6
- 235000001968 nicotinic acid Nutrition 0.000 claims description 6
- PJNZPQUBCPKICU-UHFFFAOYSA-N phosphoric acid;potassium Chemical compound [K].OP(O)(O)=O PJNZPQUBCPKICU-UHFFFAOYSA-N 0.000 claims description 6
- 235000007715 potassium iodide Nutrition 0.000 claims description 6
- 239000004323 potassium nitrate Substances 0.000 claims description 6
- 235000010333 potassium nitrate Nutrition 0.000 claims description 6
- CDAISMWEOUEBRE-UHFFFAOYSA-N scyllo-inosotol Natural products OC1C(O)C(O)C(O)C(O)C1O CDAISMWEOUEBRE-UHFFFAOYSA-N 0.000 claims description 6
- RWVGQQGBQSJDQV-UHFFFAOYSA-M sodium;3-[[4-[(e)-[4-(4-ethoxyanilino)phenyl]-[4-[ethyl-[(3-sulfonatophenyl)methyl]azaniumylidene]-2-methylcyclohexa-2,5-dien-1-ylidene]methyl]-n-ethyl-3-methylanilino]methyl]benzenesulfonate Chemical compound [Na+].C1=CC(OCC)=CC=C1NC1=CC=C(C(=C2C(=CC(C=C2)=[N+](CC)CC=2C=C(C=CC=2)S([O-])(=O)=O)C)C=2C(=CC(=CC=2)N(CC)CC=2C=C(C=CC=2)S([O-])(=O)=O)C)C=C1 RWVGQQGBQSJDQV-UHFFFAOYSA-M 0.000 claims description 6
- NWONKYPBYAMBJT-UHFFFAOYSA-L zinc sulfate Chemical compound [Zn+2].[O-]S([O-])(=O)=O NWONKYPBYAMBJT-UHFFFAOYSA-L 0.000 claims description 6
- 229910000368 zinc sulfate Inorganic materials 0.000 claims description 6
- 239000011686 zinc sulphate Substances 0.000 claims description 6
- 235000009529 zinc sulphate Nutrition 0.000 claims description 6
- 235000010338 boric acid Nutrition 0.000 claims description 3
- 238000010438 heat treatment Methods 0.000 claims description 3
- 239000011702 manganese sulphate Substances 0.000 claims description 3
- 239000012530 fluid Substances 0.000 claims description 2
- 239000002609 medium Substances 0.000 abstract description 6
- 239000003814 drug Substances 0.000 abstract description 4
- 238000004161 plant tissue culture Methods 0.000 abstract description 2
- 239000000758 substrate Substances 0.000 abstract description 2
- JZCCFEFSEZPSOG-UHFFFAOYSA-L copper(II) sulfate pentahydrate Chemical compound O.O.O.O.O.[Cu+2].[O-]S([O-])(=O)=O JZCCFEFSEZPSOG-UHFFFAOYSA-L 0.000 abstract 2
- 229940079593 drug Drugs 0.000 abstract 1
- 238000009630 liquid culture Methods 0.000 abstract 1
- 238000007789 sealing Methods 0.000 abstract 1
- 241000196324 Embryophyta Species 0.000 description 2
- 239000002689 soil Substances 0.000 description 2
- 239000011573 trace mineral Substances 0.000 description 2
- 235000013619 trace mineral Nutrition 0.000 description 2
- 229920001817 Agar Polymers 0.000 description 1
- 208000035143 Bacterial infection Diseases 0.000 description 1
- 244000228451 Stevia rebaudiana Species 0.000 description 1
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 description 1
- 229930006000 Sucrose Natural products 0.000 description 1
- 239000008272 agar Substances 0.000 description 1
- 208000022362 bacterial infectious disease Diseases 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 238000009395 breeding Methods 0.000 description 1
- 230000001488 breeding effect Effects 0.000 description 1
- 230000019771 cognition Effects 0.000 description 1
- 239000003086 colorant Substances 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 238000005286 illumination Methods 0.000 description 1
- RBTARNINKXHZNM-UHFFFAOYSA-K iron trichloride Chemical compound Cl[Fe](Cl)Cl RBTARNINKXHZNM-UHFFFAOYSA-K 0.000 description 1
- 239000012120 mounting media Substances 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- HELXLJCILKEWJH-NCGAPWICSA-N rebaudioside A Chemical compound O([C@H]1[C@H](O)[C@@H](CO)O[C@H]([C@@H]1O[C@H]1[C@@H]([C@@H](O)[C@H](O)[C@@H](CO)O1)O)O[C@]12C(=C)C[C@@]3(C1)CC[C@@H]1[C@@](C)(CCC[C@]1([C@@H]3CC2)C)C(=O)O[C@H]1[C@@H]([C@@H](O)[C@H](O)[C@@H](CO)O1)O)[C@@H]1O[C@H](CO)[C@@H](O)[C@H](O)[C@H]1O HELXLJCILKEWJH-NCGAPWICSA-N 0.000 description 1
- 230000001954 sterilising effect Effects 0.000 description 1
- 239000005720 sucrose Substances 0.000 description 1
- 239000002699 waste material Substances 0.000 description 1
Classifications
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- Y02P60/216—
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- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention discloses a large-scale manufacturing method for a high-precision BL culture medium, and belongs to the field of plant biotechnology. The large-scale manufacturing method comprises the following steps: firstly, calculating required amount, which is increased by 1000 times, of 16 elements contained in the medium as well as the required amount, which is increased by 10000 times, of CuSO4.5H2O and CoCl2.6H2O which have low requirements; accurately weighing the drugs; mixing the 16 elements according to the required amount, and sealing; dissolving CuSO4.5H2O and CoCl2.6H2O with 1L of distilled water for later use; respectively opening according to the needs for use. The substrate increased by 1000 times prepared by the large-scale manufacturing method disclosed by the invention has the characteristics of being high in precision, convenient and quick to operate, simple to apply, and small in errors of different batches and within the batches, phenomena of large errors, precipitation, mildewing and the like caused by manual operation and external factor interferences during the preparation of high-precision BL liquid culture medium are avoided. The large-scale manufacturing method can be applied to the biotechnical purposes of plant tissue culture and the like.
Description
Technical field
The invention discloses a kind of scale preparation method of high accuracy BL culture matrix, belong to plant biotechnology field.Be more particularly one include accurate calculating, accurate weighing, first few after how mix, seal, mother liquor preparation separately, six steps such as use of breaking seal, to acquisition different batches and batch between the minimum culture matrix of error, minimizing experimental error method.
Background technology
Plant tissue culture technique or cultivation technique without soil need preparation medium mother liquor usually, and namely preparation expands certain multiple, liquid containing all kinds reagent.But, because mother liquor contains dissimilar reagent, be usually divided into the large class of macroelement, trace element, molysite and organic element four, and the multiple that four large classes expand is different, causes medicine service property (quality) inconsistent, easily occurs the error of calculation first; Second the producer of medicine, quality are inconsistent, even if to calculate, weigh consistent, also can affect result of the test; Three due to mother liquor be liquid condition, at the end of spring and the beginning of summer season, because the temperature rises and humidity increases, extremely easily cause mother liquor bacterial infection, mould, cause the phenomenon presenting different colours in mother liquor or occur precipitation, waste raw material; Comparatively big error can be there is to the difference of the cognition of container scale (especially trace element), instrument precision in four fundamental rules because of weighing person.
In view of the above-mentioned shortcoming of mother liquor; with the mother liquor medium of routine; usually there will be the failed phenomenon that maybe cannot repeat of the result of the test caused because of factors such as weighing person, medicine, seasons; have impact on the process using Plant Tissue Breeding or cultivation technique without soil to carry out test greatly, hit the confidence of experimenter.
Summary of the invention
A scale preparation method for high accuracy BL culture matrix, is characterized in that, include in described method accurate calculating, accurate weighing, first few after how mix, seal, prepared by independent mother liquor, six steps such as use of breaking seal.
Method according to claim 1, is characterized in that, six described step concrete operations are as follows:
1. accurately calculate: 16 kinds of elements contained by BL minimal medium are expanded 1000 times, i.e. Sodium Molybdate Dihydrate 0.25g, nicotinic acid 0.5g, potassium iodide 0.83g, boric acid 6.2g, white vitriol 10.6g, four water manganese sulphate 22.3g, iron edetate 36.5g, inositol 100g, aspartoyl 100g, potassium dihydrogen phosphate 170g, epsom salt 370g, calcium chloride dihydrate 440g, ammonium nitrate 1650g, potassium nitrate 1900g; 2 kinds of element CoCL2 6H2Os and cupric sulfate pentahydrate expand 10000 times, i.e. each 0.25g;
2. accurate weighing: according to the result precise of 1. middle calculating, especially with CoCL2 6H2O and the cupric sulfate pentahydrate of each 0.25g of ten thousand/balance precise;
3. how to mix after first few: mix according to few priority of carrying out of amount more, namely according to Sodium Molybdate Dihydrate, nicotinic acid, potassium iodide, boric acid, white vitriol, four water manganese sulphates, iron edetate, inositol, aspartoyl, potassium dihydrogen phosphate, epsom salt, calcium chloride dihydrate, ammonium nitrate, the order of potassium nitrate mixes, and amounts to 4707.73g;
4. seal: the requirement of mixed solid matrix according to 250g every bottle is sub-packed in plastic containers, and seals;
5. mother liquor preparation separately: CoCL2 6H2O and cupric sulfate pentahydrate respectively weigh 0.25g, are dissolved in 1L distilled water, are mixed with 10000 times of mother liquors;
6. to break seal use: as required, add the above-mentioned solid matrix 4.7g of precise in every 1L distilled water, heating adds the independent mother liquor of 100 μ L CoCL2 6H2Os and cupric sulfate pentahydrate after it dissolves completely, and then adds reagent needed for other.
Beneficial effect of the present invention: the scale preparation method of this high precision culture matrix has following characteristics, i.e. without the error of calculation between the matrix of different batches; Because 16 kinds of reagent all expand 1000 times, 2 kinds of reagent expand 10000 times, and the weighing error between the matrix of different batches is minimum, and precision is high; In theory, due to the usage amount (calculating with each 1L) that solid matrix is 1000 times at every turn, namely the above-mentioned formula of 1000L can be prepared, liquid mother liquor only needs 100 μ L at every turn, and batch inside there will not be the human error caused because using the reagent of different manufacturers, different times; Solid matrix after expansion is that every 250g is sealed in plastic containers, can avoid because the impact of the extraneous factor such as weather, temperature, moisture causes occurring the common precipitation of liquid mother liquor, the phenomenon such as mouldy, and fluid matrix is only the mixing of two kinds of elements.After expanding 10000 times, precision is high; Above-mentioned plurality of advantages is all the follow-up test carried out for matrix with this medium, obtains result of the test accurately and lays the foundation.。
Embodiment:
Below in conjunction with specific embodiment, the present invention is conducted further description.
The scale preparation method of a kind of high accuracy BL (carbonaceous sources and mounting medium) solid culture matrix in the present embodiment, include accurate calculating, accurate weighing, first few after how mix, seal, prepared by independent mother liquor, six steps such as use of breaking seal.
Above-mentioned six step concrete operations are as follows:
1. accurately calculate: 16 kinds of elements contained by BL minimal medium are expanded 1000 times, i.e. Sodium Molybdate Dihydrate 0.25g, nicotinic acid 0.5g, potassium iodide 0.83g, boric acid 6.2g, white vitriol 10.6g, four water manganese sulphate 22.3g, iron edetate 36.5g, inositol 100g, aspartoyl 100g, potassium dihydrogen phosphate 170g, epsom salt 370g, calcium chloride dihydrate 440g, ammonium nitrate 1650g, potassium nitrate 1900g; 2 kinds of element CoCL2 6H2Os and cupric sulfate pentahydrate expand 10000 times, i.e. each 0.25g;
2. accurate weighing: according to the result precise of 1. middle calculating, especially with CoCL2 6H2O and the cupric sulfate pentahydrate of each 0.25g of ten thousand/balance precise;
3. how to mix after first few: mix according to few priority of carrying out of amount more, namely according to Sodium Molybdate Dihydrate, nicotinic acid, potassium iodide, boric acid, white vitriol, four water manganese sulphates, iron edetate, inositol, aspartoyl, potassium dihydrogen phosphate, epsom salt, calcium chloride dihydrate, ammonium nitrate, the order of potassium nitrate mixes, and amounts to 4707.73g;
4. seal: the requirement of mixed solid matrix according to 250g every bottle is sub-packed in plastic containers, and seals;
5. mother liquor preparation separately: CoCL2 6H2O and cupric sulfate pentahydrate respectively weigh 0.25g, are dissolved in 1L distilled water, are mixed with 10000 times of mother liquors;
6. to break seal use: as required, add the above-mentioned solid matrix 4.7g of precise in every 1L distilled water, heating adds the independent mother liquor of 100 μ L CoCL2 6H2Os and cupric sulfate pentahydrate after it dissolves completely, and then adds reagent needed for other.
1L solution after above-mentioned substrate preparation is added sucrose and the 5g agar powder of 30g, and then add the KOH of 1N of 1ml, dissolve completely and be sub-packed in the vial of 30 200ml, after 121 DEG C of sterilizing 20min.On superclean bench, inoculate stevia stem section, after cultivating 28d under 25 DEG C of illumination 12h conditions, occur axillalry bud and take root good.
Claims (3)
1. a scale preparation method for high accuracy BL culture matrix, is characterized in that, include in described method accurate calculating, accurate weighing, first few after how mix, seal, prepared by independent mother liquor, six steps such as use of breaking seal.
2. method according to claim 1, is characterized in that, six described step concrete operations are as follows:
1. accurately calculate: 16 kinds of elements contained by BL minimal medium are expanded 1000 times, i.e. Sodium Molybdate Dihydrate 0.25g, nicotinic acid 0.5g, potassium iodide 0.83g, boric acid 6.2g, white vitriol 10.6g, four water manganese sulphate 22.3g, iron edetate 36.5g, inositol 100g, aspartoyl 100g, potassium dihydrogen phosphate 170g, epsom salt 370g, calcium chloride dihydrate 440g, ammonium nitrate 1650g, potassium nitrate 1900g; 2 kinds of element CoCL2 6H2Os and cupric sulfate pentahydrate expand 10000 times, i.e. each 0.25g;
2. accurate weighing: according to the result precise of 1. middle calculating, especially with CoCL2 6H2O and the cupric sulfate pentahydrate of each 0.25g of ten thousand/balance precise;
3. how to mix after first few: mix according to few priority of carrying out of amount more, namely according to Sodium Molybdate Dihydrate, nicotinic acid, potassium iodide, boric acid, white vitriol, four water manganese sulphates, iron edetate, inositol, aspartoyl, potassium dihydrogen phosphate, epsom salt, calcium chloride dihydrate, ammonium nitrate, the order of potassium nitrate mixes, and amounts to 4707.73g;
4. seal: the requirement of mixed solid matrix according to 250g every bottle is sub-packed in plastic containers, and seals;
5. mother liquor preparation separately: CoCL2 6H2O and cupric sulfate pentahydrate respectively weigh 0.25g, are dissolved in 1L distilled water, are mixed with 10000 times of mother liquors;
6. to break seal use: as required, add the above-mentioned solid matrix 4.7g of precise in every 1L distilled water, heating adds the independent mother liquor of 100 μ L CoCL2 6H2Os and cupric sulfate pentahydrate after it dissolves completely, and then adds reagent needed for other.
3. method according to claim 1, is characterized in that, the scale preparation method of this high precision culture matrix has following characteristics, i.e. without the error of calculation between the matrix of different batches; Because 16 kinds of reagent all expand 1000 times, 2 kinds of reagent expand 10000 times, and the weighing error between the matrix of different batches is minimum, and precision is high; In theory, due to the usage amount (calculating with each 1L) that solid matrix is 1000 times at every turn, namely the above-mentioned formula of 1000L can be prepared, liquid mother liquor only needs 100 μ L at every turn, and batch inside there will not be the human error caused because using the reagent of different manufacturers, different times; Solid matrix after expansion is that every 250g is sealed in plastic containers, can avoid because the impact of the extraneous factor such as weather, temperature, moisture causes occurring the common precipitation of liquid mother liquor, the phenomenon such as mouldy, and fluid matrix is only the mixing of two kinds of elements; After expanding 10000 times, precision is high; Above-mentioned plurality of advantages is all the follow-up test carried out for matrix with this medium, obtains result of the test accurately and lays the foundation.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2006254832A (en) * | 2005-03-18 | 2006-09-28 | Sanei Gen Ffi Inc | Solid medium |
| CN101070531A (en) * | 2006-05-09 | 2007-11-14 | 陈曦 | Dry-powder-type culturing substrate and preparing method |
-
2015
- 2015-02-16 CN CN201510082272.5A patent/CN104642135A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2006254832A (en) * | 2005-03-18 | 2006-09-28 | Sanei Gen Ffi Inc | Solid medium |
| CN101070531A (en) * | 2006-05-09 | 2007-11-14 | 陈曦 | Dry-powder-type culturing substrate and preparing method |
Non-Patent Citations (1)
| Title |
|---|
| 仪宏等: "脱水植物组织培养基的制备", 《河北轻化工学院学报》, vol. 19, no. 2, 30 June 1998 (1998-06-30), pages 80 - 82 * |
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Application publication date: 20150527 |