CN103421693A - Bradyrhizobium japonicum vitrified cryopreservation liquid and method for vitrified cryopreservation of bradyrhizobium japonicum - Google Patents
Bradyrhizobium japonicum vitrified cryopreservation liquid and method for vitrified cryopreservation of bradyrhizobium japonicum Download PDFInfo
- Publication number
- CN103421693A CN103421693A CN2013104003184A CN201310400318A CN103421693A CN 103421693 A CN103421693 A CN 103421693A CN 2013104003184 A CN2013104003184 A CN 2013104003184A CN 201310400318 A CN201310400318 A CN 201310400318A CN 103421693 A CN103421693 A CN 103421693A
- Authority
- CN
- China
- Prior art keywords
- rihizobium japonicum
- stock solution
- japonicum
- vitrification
- frozen stock
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Landscapes
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Agricultural Chemicals And Associated Chemicals (AREA)
Abstract
The invention discloses bradyrhizobium japonicum vitrified cryopreservation liquid and a method for vitrified cryopreservation of bradyrhizobium japonicum and relates to microorganism vitrified cryopreservation liquid and a method for vitrified cryopreservation of microorganisms. The bradyrhizobium japonicum vitrified cryopreservation liquid contains ethanediol and is simple in compound composition. The bradyrhizobium japonicum vitrified cryopreservation liquid is composed of glycerinum, ethanediol, DMSO and distilled water. According to the method for vitrified cryopreservation of the bradyrhizobium japonicum, bradyrhizobium japonicum liquid is added into the bradyrhizobium japonicum vitrified cryopreservation liquid, and then the bradyrhizobium japonicum vitrified cryopreservation liquid is put into liquid nitrogen for fast vitrified cryopreservation. The bradyrhizobium japonicum vitrified cryopreservation liquid and the method for vitrified cryopreservation of the bradyrhizobium japonicum are used in the field of microorganism preservation.
Description
Technical field
The present invention relates to a kind of microorganism vitrification frozen stock solution and glass frozen preservation method of microorganism.
Background technology
Root nodule bacterium are that a class is lived in the Gram-negative rod-shaped bacterium in soil, and under suitable condition, root nodule bacterium can be infected leguminous plants and be carried out with it symbiotic nodulation and nitrogen fixation.According to estimates, the fixing nitrogen of root nodule bacterium accounts for 65% of biological nitrogen fixation total amount, in agriculture production, plays an important role.Microorganism resource is the important component part of the natural resources treasure-house of China's abundant, development along with biology techniques, the utilization of germ plasm resource plays a part more and more important to the research of life science, cell preservation technique has also been proposed to more and more higher requirement.The excellent species for aspects such as scientific research and industrial production obtained for the wild-type bacterial classification that nature separated obtain and artificially breeding plays a role as far as possible for a long time, need to adopt various suitable methods properly to preserve to guarantee that bacterial classification survives stable with inherited character, makes it not occur or the least possible morphing.Therefore, culture presevation is an important element task.
Cryogenic freezing is preserved as brand-new store method, day by day is subject to the mankind's attention, and wherein the glass freezing Techniques of preserving is a simple and direct and very potential method.The glass frozen preservation technology refers to that the cryoprotectant that utilizes multiple high density combines the glass freezing protection liquid protection suspension cell of formation; direct plunge into Liquid Nitrogen carries out frozen method; the cell suspension freezed with this kind of method does not have the formation of ice crystal, and directly liquid rotating becomes amorphous solidification process.Utilize method for vitrification generally to succeed in the animal and plant cell is preserved, but extremely rare in microorganism preservation field; And in existing vitrification frozen stock solution, ingredient is various, except water, majority is comprised of 5～6 kinds of compounds.Because ethylene glycol is toxic to animal, so basically all using polyoxyethylene glycol etc. as the vitrification frozen stock solution moiety.
Summary of the invention
The invention provides a kind of method that ethylene glycol and compound form simple rihizobium japonicum vitrification frozen stock solution and glass frozen preservation rihizobium japonicum that contains.
The rihizobium japonicum vitrification frozen stock solution is comprised of 28%～32% glycerine, 12%～18% ethylene glycol, 12%～18% DMSO and the distilled water of surplus by mass percentage.
Utilize the method for above-mentioned rihizobium japonicum vitrification frozen stock solution glass frozen preservation rihizobium japonicum to carry out according to the following steps:
Rihizobium japonicum bacterium liquid is added in the rihizobium japonicum vitrification frozen stock solution, and the volume ratio of rihizobium japonicum bacterium liquid and rihizobium japonicum vitrification frozen stock solution is 2:3; Then rapid glass in rihizobium japonicum vitrification frozen stock solution input liquid nitrogen is thawed and deposits;
The rihizobium japonicum vitrification frozen stock solution is comprised of 28%～32% glycerine, 12%～18% ethylene glycol, 12%～18% DMSO and the distilled water of surplus by mass percentage;
The glass frozen preservation cooling rate is greater than 2000 ℃/min.
Rihizobium japonicum also is different from other microorganisms, and the outer mucus of born of the same parents that it can produce a great deal of, therefore hindered and had at present the frozen effect of vitrification frozen stock solution, causes a large amount of inactivations of rihizobium japonicum, and survival rate is low; The at present existing frozen rihizobium japonicum of vitrification frozen stock solution, it preserves the survival rate less than 70% after 1 year.The present invention adopts glycerine, ethylene glycol and DMSO(dimethyl sulfoxide (DMSO)) as vitrification frozen stock solution, can effectively penetrate the outer mucus of born of the same parents of rihizobium japonicum, thereby rihizobium japonicum is carried out to glass frozen preservation.
Through the soybean of glass frozen preservation of the present invention living root nodule bacterium and the knurl bacteria strain of taking root slowly soon, preservation after 12 months its bacterial strain survival rate all can reach more than 85%, and nitrogenase activity does not obviously descend.Vitrification frozen stock solution of the present invention and method for glass frozen preservation have excellent preservation effect to rihizobium japonicum.
Experiment does not find have any unusual phenomenon to occur through the rihizobium japonicum of glass frozen preservation of the present invention, illustrates that the ethylene glycol in vitrification frozen stock solution has no adverse effects to rihizobium japonicum.
Embodiment
Technical solution of the present invention is not limited to following cited embodiment, also comprises the arbitrary combination between each embodiment.
Embodiment one: present embodiment rihizobium japonicum vitrification frozen stock solution is comprised of 28%～32% glycerine, 12%～18% ethylene glycol, 12%～18% DMSO and the distilled water of surplus by mass percentage.
Embodiment two: present embodiment rihizobium japonicum vitrification frozen stock solution is comprised of 30% glycerine, 15% ethylene glycol, 15% DMSO and 40% distilled water by mass percentage.
Embodiment three: present embodiment rihizobium japonicum vitrification frozen stock solution is comprised of 29% glycerine, 14% ethylene glycol, 14% DMSO and 43% distilled water by mass percentage.
Embodiment four: present embodiment rihizobium japonicum vitrification frozen stock solution is comprised of 31% glycerine, 14% ethylene glycol, 16% DMSO and 39% distilled water by mass percentage.
Embodiment five: the method for present embodiment glass frozen preservation rihizobium japonicum is carried out according to the following steps:
Rihizobium japonicum bacterium liquid is added in the rihizobium japonicum vitrification frozen stock solution, and the volume ratio of rihizobium japonicum bacterium liquid and rihizobium japonicum vitrification frozen stock solution is 2:3; Then rapid glass in rihizobium japonicum vitrification frozen stock solution input liquid nitrogen is thawed and deposits;
The rihizobium japonicum vitrification frozen stock solution is comprised of 28%～32% glycerine, 12%～18% ethylene glycol, 12%～18% DMSO and the distilled water of surplus by mass percentage;
The glass frozen preservation cooling rate is greater than 2000 ℃/min.
Embodiment six: the difference of present embodiment and embodiment five is: rihizobium japonicum is cultured to the logarithmic growth later stage, then adds in proportion rihizobium japonicum bacterium liquid vitrification frozen stock solution.Other step and parameter are identical with embodiment five.
Embodiment seven: the method for present embodiment glass frozen preservation rihizobium japonicum is carried out according to the following steps:
Rihizobium japonicum bacterium liquid is added in the rihizobium japonicum vitrification frozen stock solution, and the volume ratio of rihizobium japonicum bacterium liquid and rihizobium japonicum vitrification frozen stock solution is 2:3; Then rapid glass in rihizobium japonicum vitrification frozen stock solution input liquid nitrogen is thawed and deposits;
The rihizobium japonicum vitrification frozen stock solution is comprised of 30% glycerine, 15% ethylene glycol, 15% DMSO and the distilled water of surplus by mass percentage;
The glass frozen preservation cooling rate is greater than 2000 ℃/min.
Present embodiment is to fast-growing Rhizobium---Sinorhizobium fredii (Sinorhizobium fredii) HN01 and give birth to slowly type rihizobium japonicum (Bradyrhizobium japonicum) USDA110 and carry out the glass frozen preservation test.
By rihizobium japonicum, to cultivate in 5% inoculum size switching YM liquid nutrient medium, (the YM liquid nutrient medium is by 0.5gK
2HPO
4, 10.0g N.F,USP MANNITOL, 0.2g MgSO
47H
2O, 10g NaCl, 1.0g yeast extract and 1000ml distilled water form, and adjust pH to 7.0.), 28 ℃ of cultivations are standby.
Adopt the serial dilution method to measure rihizobium japonicum bacterium number: take 10ml soybean nodulation bacteria culture fluid and add in the sterilized water of 90mL, standing 20min, the 200r/min 30min that fully vibrates on rotary shaking table, the mother liquor bacteria suspension.Draw respectively 5.0mL mother liquor bacteria suspension and add in the 45mL sterilized water, by 1:10, carry out serial dilution, obtain respectively 1:1 * 10
1, 1:1 * 10
2, 1:1 * 10
3, 1:1 * 10
4The bacteria suspension of dilution.Get 3 serial dilution degree, draw respectively bacteria suspension 0.1mL, be applied to that on previously prepared good YMA solid medium flat board, (the YMA solid medium is by 0.5g K
2HPO
4, 10.0g N.F,USP MANNITOL, 0.2gMgSO
47H
2O, 10g NaCl, 1.0g yeast extract, 20g agar and 1000ml distilled water form, and adjust pH to 7.0.), under 28 ℃ of conditions, cultivate.The dilution flat board that 20～300 colony numbers occur of take is the counting standard, and living bacteria count is by calculating:
Nv-volume living bacteria count, hundred million/mL;
-bacterium colony mean number, individual; K-extension rate; v
1-mother liquor bacteria suspension volume, mL; v
2-bacteria suspension add-on, mL; v
0-sample size, mL.
Bacterial strain survival rate through present embodiment glass frozen preservation fast-growing Rhizobium (Sinorhizobium fredii (Sinorhizobium fredii) HN01) is as shown in table 1, and test minute is carried out for 3 times.
Table 1
The bacterial strain survival rate of giving birth to slowly type rihizobium japonicum (Bradyrhizobium japonicum) USDA110 through the present embodiment glass frozen preservation is as shown in table 2, and test minute is carried out for 3 times.
Table 2
Can find out by above bacterial strain survival rate testing data, preserve after glass frozen preservation that within 1 year, no matter the Fast-growing Soybean rhizobia bacterium still gives birth to the type rihizobium japonicum slowly all can keep for a long time higher cell survival rate under present embodiment glass frozen preservation condition.Fast-growing Soybean rhizobia bacterium particularly, cell survival rate reaches more than 94%.
After the present embodiment glass frozen preservation, adopt the Acetylene Reduction method to measure nitrogenase activity the bacterial strain after frozen activation.
Nitrogenase activity after the bacterial strain glass frozen preservation activation of fast-growing Rhizobium (Sinorhizobium fredii (Sinorhizobium fredii) HN01) is as shown in table 3, and test minute is carried out for 3 times.
Table 3 nitrogenase activity (nmol/mgh)
Slowly the nitrogenase activity after the activation of the bacterial strain glass frozen preservation of living type rihizobium japonicum (Bradyrhizobium japonicum) USDA110 is as shown in table 4, and test minute is carried out for 3 times.
Table 4 nitrogenase activity (nmol/mgh)
Experimental data shows that the nitrogenase activity of rihizobium japonicum after the present embodiment glass frozen preservation does not obviously descend, almost maintain its initial nitrogenase activity level, and the difference between two strain bacterial strain nitrogenase activities may be because give birth to slowly type rihizobium japonicum poor growth, cause its in same time nitrogenase activity lower than the Fast-growing Soybean rhizobia bacterium.
Present embodiment vitrification frozen stock solution and method for glass frozen preservation do not change gene order and the gene regulating factor of rihizobium japonicum, so the nifHDK in the soybean nodulation bacteria strain can normal expression.
Test does not find that ethylene glycol is on soybean nodulation bacteria strain toxic side effect and impact in the glass frozen preservation process.
Claims (4)
1. rihizobium japonicum vitrification frozen stock solution, is characterized in that the rihizobium japonicum vitrification frozen stock solution is comprised of 28%～32% glycerine, 12%～18% ethylene glycol, 12%～18% DMSO and the distilled water of surplus by mass percentage.
2. rihizobium japonicum vitrification frozen stock solution according to claim 1, is characterized in that the rihizobium japonicum vitrification frozen stock solution is comprised of 30% glycerine, 15% ethylene glycol, 15% DMSO and 40% distilled water by mass percentage.
3. utilize the method for claim 1 rihizobium japonicum vitrification frozen stock solution glass frozen preservation rihizobium japonicum, it is characterized in that glass frozen preservation rihizobium japonicum according to the following steps:
Rihizobium japonicum bacterium liquid is added in the rihizobium japonicum vitrification frozen stock solution, and the volume ratio of rihizobium japonicum bacterium liquid and rihizobium japonicum vitrification frozen stock solution is 2:3; Then rapid glass in rihizobium japonicum vitrification frozen stock solution input liquid nitrogen is thawed and deposits;
The rihizobium japonicum vitrification frozen stock solution is comprised of 28%～32% glycerine, 12%～18% ethylene glycol, 12%～18% DMSO and the distilled water of surplus by mass percentage;
The glass frozen preservation cooling rate is greater than 2000 ℃/min.
4. the method for glass frozen preservation rihizobium japonicum according to claim 3, is characterized in that rihizobium japonicum is cultured to the logarithmic growth later stage, then add in proportion rihizobium japonicum bacterium liquid vitrification frozen stock solution.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310400318.4A CN103421693B (en) | 2013-09-05 | 2013-09-05 | The method of rihizobium japonicum vitrification frozen stock solution and glass frozen preservation rihizobium japonicum |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310400318.4A CN103421693B (en) | 2013-09-05 | 2013-09-05 | The method of rihizobium japonicum vitrification frozen stock solution and glass frozen preservation rihizobium japonicum |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN103421693A true CN103421693A (en) | 2013-12-04 |
| CN103421693B CN103421693B (en) | 2019-10-15 |
Family
ID=49647088
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201310400318.4A Active CN103421693B (en) | 2013-09-05 | 2013-09-05 | The method of rihizobium japonicum vitrification frozen stock solution and glass frozen preservation rihizobium japonicum |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN103421693B (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103718830A (en) * | 2013-12-27 | 2014-04-16 | 湖南农业大学 | Ultra-low-temperature Pleurotus eryngii spawn preservation method for factory production |
| CN105524868A (en) * | 2016-01-18 | 2016-04-27 | 黑龙江省科学院微生物研究所 | Slow-growing soybean rhizobia liquid protective agent |
| CN108024545A (en) * | 2015-09-11 | 2018-05-11 | 诺维信生物农业公司 | Stable inoculation compositions and its production method |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1217873A (en) * | 1997-11-20 | 1999-06-02 | 杭州大学 | Method for glass frozen preservation of plant sample |
| CN101253856A (en) * | 2008-04-11 | 2008-09-03 | 中山大学 | a vitrification liquid |
-
2013
- 2013-09-05 CN CN201310400318.4A patent/CN103421693B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1217873A (en) * | 1997-11-20 | 1999-06-02 | 杭州大学 | Method for glass frozen preservation of plant sample |
| CN101253856A (en) * | 2008-04-11 | 2008-09-03 | 中山大学 | a vitrification liquid |
Non-Patent Citations (1)
| Title |
|---|
| GAYLE M. VOLK ET AL: "Survival of mint shoot tips after exposure to cryoprotectant solution components", 《CRYOBIOLOGY》 * |
Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103718830A (en) * | 2013-12-27 | 2014-04-16 | 湖南农业大学 | Ultra-low-temperature Pleurotus eryngii spawn preservation method for factory production |
| CN103718830B (en) * | 2013-12-27 | 2015-08-19 | 湖南农业大学 | A kind of factorial praluction pleurotus eryngii quel strains cryopreservation method |
| CN108024545A (en) * | 2015-09-11 | 2018-05-11 | 诺维信生物农业公司 | Stable inoculation compositions and its production method |
| US11472981B2 (en) | 2015-09-11 | 2022-10-18 | Novozymes Bioag A/S | Stable inoculant compositions and methods for producing same |
| US12116488B2 (en) | 2015-09-11 | 2024-10-15 | Novonesis Plant Biosolutions A/S | Stable inoculant compositions and methods for producing same |
| US12173167B2 (en) | 2015-09-11 | 2024-12-24 | Novozymes Bioag A/S | Stable inoculant compositions and methods for producing same |
| CN105524868A (en) * | 2016-01-18 | 2016-04-27 | 黑龙江省科学院微生物研究所 | Slow-growing soybean rhizobia liquid protective agent |
| CN105524868B (en) * | 2016-01-18 | 2019-02-26 | 黑龙江省科学院微生物研究所 | A kind of Slow-growing Soybean rhizobia liquid protectant |
Also Published As
| Publication number | Publication date |
|---|---|
| CN103421693B (en) | 2019-10-15 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN104082277B (en) | A kind of freezing protective agent of human peripheral blood single nucleus cell and store method | |
| CN113373096B (en) | Bacillus cereus and application thereof in relieving plant salt stress | |
| CN113943660B (en) | Talaromyces fungus NJAU-L8 for preventing and controlling continuous cropping soil-borne blight and application thereof | |
| Makowski et al. | Integration of tissue culture and cryopreservation methods for propagation and conservation of the fern Osmunda regalis L. | |
| CN103070163B (en) | The cryopreservation method and activity identification method of protoplasts of Dalilia yunnanensis | |
| Fenwick et al. | Cryopreservation of Tetraselmis suecica cultured under different nutrients regimes | |
| CN108102984A (en) | Raw Bacillus megatherium BM18-2 and its application in one plant of somatotrophic hybrid Chinese pennisetum of cadmium | |
| CN103421693A (en) | Bradyrhizobium japonicum vitrified cryopreservation liquid and method for vitrified cryopreservation of bradyrhizobium japonicum | |
| CN104450550A (en) | Screening method and application of Raoultella planticola strain SRPG-4 producing ACC (1-aminocyclopropane-1-carboxylic acid) deaminase activity | |
| Ma et al. | Enhancing the cryopreservation system of larch embryogenic culture by optimizing pre-culture, osmoprotectants, and rapid thawing | |
| CN109430055B (en) | Low-temperature preservation method for gene resources of potato virus-free test-tube plantlet | |
| Sato et al. | A modified perlite protocol with a mixed dimethyl sulfoxide and trehalose cryoprotectant improves the viability of frozen cultures of ectomycorrhizal basidiomycetes | |
| CN103650977B (en) | Method for improving survival rate of afforestation of non-woven fabric container seedlings in arid mountains | |
| CN106222121A (en) | A kind of bacillus megaterium bacterial strain, biocontrol agent and preparation method and application | |
| Satchuthananthavale et al. | Carbohydrate nutrition of some nematode-trapping fungi | |
| CN106244480B (en) | A strain of Pseudogrignon pallidum and its application in the control of plant parasitic nematodes | |
| Kaur et al. | Storage and preservation of temperate mushroom cultures, Agaricus bisporus and Pleurotus florida | |
| CN114214201B (en) | Method for long-term stable preservation of marine nannochloropsis | |
| CN116987632A (en) | Soil microorganism probiotic combination for increasing resistance of banana rhizosphere pathogenic bacteria and application thereof | |
| Tu et al. | Effects of arbuscular mycorrhizal inoculation on osmoregulation and antioxidant responses of blueberry plants | |
| Sopher et al. | Quantitative relationships of Pseudomonas chlororaphis 63-28 to Pythium root rot and growth in hydroponic peppers | |
| CN108117993B (en) | Preservation method of denitrifying bacteria | |
| CN105585135A (en) | Method for controlling cyanobacteria in aquaculture water | |
| CN107881114A (en) | A kind of method of fast-propagation paramecium | |
| Hurst et al. | The effect of temperature on the growth of Candida sake isolated from the leaves of a subantarctic grass |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |