CN105713838A - High-vitality cell preservation method for thalassiosira pseudonana - Google Patents
High-vitality cell preservation method for thalassiosira pseudonana Download PDFInfo
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- CN105713838A CN105713838A CN201610294363.XA CN201610294363A CN105713838A CN 105713838 A CN105713838 A CN 105713838A CN 201610294363 A CN201610294363 A CN 201610294363A CN 105713838 A CN105713838 A CN 105713838A
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- cell preservation
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- 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
- C12N1/12—Unicellular algae; Culture media therefor
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- 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
- C12N1/04—Preserving or maintaining viable microorganisms
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Abstract
The invention discloses a high-vitality cell preservation method for thalassiosira pseudonana. The high-vitality cell preservation method comprises the following steps: 1) inoculating microalgae in a culture medium so as to proliferate microalga cells to 10<6>-10<7> pieces/ml; 2) performing gradient cooling treatment on the cultured alga solution; 3) reducing the lighting intensity of lighting while cooling, wherein the reduction amplitude is 1000-2000lx/day; and 4) preserving the alga solution under a dark condition of 4-5 DEG C. The invention discloses the high-vitality cell preservation method for high-quality bait thalassiosira pseudonana, gradient cooling, light shielding and suspension alga solution high-vitality cell preservation are adopted, and the high-vitality cell preservation method is high in microalga survival rate, good in rejuvenation property, unlikely to cause pollution and simple, convenient and feasible to operate.
Description
Technical field
The invention belongs to microalgae method for preserving, be specifically related to a kind of bait micro-algae Thalassiosira pseudonana high density high
Vigor method for preserving.
Background technology
Bait micro-algae comprehensive nutrition, optimum is as the fresh food of aquatic animal；Aquatic animal can be improved
Survival rate, reduces seedling cost, meets nutrition and the growth demand of nursery particular stage；And water can be purified
Matter, it is achieved the sustainable development of aquaculture, therefore becomes irreplaceable role in culture fishery.Water
Produce in cultivated animals nursery, generally require the supply ensureing a large amount of high-quality bait micro-algaes.But artificial culture's microalgae
Time, often can face growth and breeding slow, and easily be surmounted its proliferative advantage, as by miscellaneous by competitive plankton
Algae pollution or prey, cause and breed unsuccessfully.One of them important reason is to breed initial period bait algae
Cell number few, lack competitive advantage.The advantage propagation how realizing high-quality bait is that a letter is to be solved
Problem.Being affected by conditions such as season, weather, aquaculture organism growth differences, offer in good time, enough can
The high density height vigor of rejuvenation energy fast breeding initiates algae source rapidly is to cultivate successful important process.Wherein
One of be the high vigor high density method for preserving of bait micro-algae.
Thalassiosira pseudonana is first eucaryon planktonic diatom being used for genome sequencing, makees afterwards
Numerous necks such as metabolism, heredity, toxicity, ecology and energy microalgae screening it are widely used in for model organism
The research in territory；This algae is nutritious, and can breed at rainy weather, is the one in aquaculture nursery
Important high-quality bait micro-algae.
Summary of the invention
It is an object of the invention to provide the high vigor high density method for preserving of a kind of Thalassiosira pseudonana, use by
Level cooling, fall light, carry out high vigor preservation under optimum density.The microalgae survival rate of the inventive method is high,
Rejuvenation ability is strong, is difficult to pollute, and easy to operation.
The method of the present invention, comprises the following steps that
1) microalgae is inoculated in culture medium, makes microalgae cell number propagation reach 106-107Individual/ml,
2) algae solution after cultivating carries out gradient cooling process；
Described gradient cooling processes, and is by cultivation temperature from culture environment temperature, reduces after every 24 hours
5℃；Until being reduced to 4-5 DEG C；
3) intensity of illumination when cooling reduces illumination simultaneously, reduction amplitude is 1000-2000lx/ days.
4) algae solution is placed in 4-5 DEG C of dark condition preserve.
Wherein step 1) condition of culture, temperature 23-25 DEG C, intensity of illumination 4000-5000lx, periodicity of illumination:
12h:12h；
Wherein step 1) culture medium: 20mg/L Na2EDTA,2.5mg/L FeSO4·7H2O,100mg/L KNO3,
10mg/L K2HPO4,0.25mg/L MnSO4,0.5×10-3Mg/L vitamin B12,5×10-3Mg/L vitamin
B1。
Utilize said method, it is possible to achieve Thalassiosira pseudonana keeps high survival rate and quickly 2-6 middle of the month
Rejuvenation multiplication capacity, for providing high vigor highdensity bait algae source in good time in production practices.
Accompanying drawing explanation
Rejuvenation proliferation process figure after Fig. 1: conservation February；
The Photosynthetic capacity variation diagram of the rejuvenation cell after Fig. 2: conservation February.
Detailed description of the invention
The high vigor high density method for preserving of the present invention, cultivates Thalassiosira pseudonana to the platform initial stage, carries out ladder
Suspension algae solution live body preservation is carried out after degree cooling fall light.After certain time, its survival rate is detected, and
Preservation algae solution is reactivated, utilizes blood counting chamber that its cell proliferation density is counted；Utilize water sample
Luminoscope is tracked monitoring to its photosynthetic parameters Fv/Fm.According to propagation speed after algae solution reactivates after preservation
Rate, fluorescence parameter Fv/Fm result, to determine Thalassiosira pseudonana vigor, survival rate and rejuvenation ability.
Term description for relating in description of the invention is as follows:
1, the microalgae platform initial stage: referring to that Thalassiosira pseudonana growth conditions is stable, algae solution orders of density is 106-107
Individual/ml；The breeding of this step and this culture medium and condition of culture can ensure cell tool in preservation term algae solution
There is high vigor.
2, gradient cooling, refers to lower the temperature being in algae solution under suitable cultivation temperature 24 hours in 20 DEG C of environment,
The most gradually carry out ambient temperature cooling process；
3, reactivate, refer to quantitatively take out preservation algae solution and be re-seeded into culture fluid and cultivate；
4, survival rate, refers in algae solution after preservation cell number ratio before intact living cells number and preservation；
5, upgrowth situation and cell viability, refers to reactivate rear algae solution cell proliferation rate and propagation density,
Chlorophyll fluorescence parameters Fv/Fm (the most photosynthetic potential), has reacted cell viability.
Below in conjunction with embodiment, the present invention is described in detail.
Embodiment 1
Preserving process:
1) Thalassiosira pseudonana is inoculated in fills culture fluid (its content is as follows: 20mg/L ethylenediaminetetraacetic acid
Disodium (Na2EDTA), 2.5mg/L ferrous sulfate heptahydrate (FeSO4·7H2O), 100mg/L potassium nitrate (KNO3),
10mg/L dipotassium hydrogen phosphate (K2HPO4), 0.25mg/L manganese sulfate (MnSO4), 0.5 × 10-3Mg/L vitamin
B12, 5 × 10-3Mg/L vitamin B1) conical flask in, be positioned over culturing room (temperature 23 DEG C, intensity of illumination
5000lx, periodicity of illumination 12h:12h) middle cultivation；
2) treat that Thalassiosira pseudonana is bred to 106-107Individual/ml.Two groups respectively: maintain this algae solution density, or
This algae is concentrated into 109Individual/ml order of magnitude.Algae solution is placed in incubator and carries out gradient cooling
(20-15-10-5 DEG C each 24 hours), fall light (reduction amplitude is 1000-2000lx/ days), until lucifuge processes；
3) preserve 4-5 DEG C of lucifuge condition；
Survival rate and rejuvenation propagation detection:
1) respectively the part algae solution of preservation under the conditions of the 4-5 DEG C of lucifuge of the 2-6 month is taken out, utilize I-KI
Reagent dyeing method carries out survival rate detection.(I-KI preparation of reagents method: potassium iodide 3g；Distilled water 100ml；
Iodine 1g, is first dissolved in potassium iodide in distilled water, adds iodine after all dissolving again, and vibration is dissolved.Note: by this
Liquid is saved in Brown Glass Brown glass bottles and jars only)；
2) Tibetan algae solution of going bail for reactivates: be incubated in 250ml triangular pyramidal bottle, constant volume 150ml,
Initial live algae cell density about 104-105Individual/ml, respectively arranges 3 parallel；
3) utilize blood counting chamber that frustule is counted, utilize chlorophyll fluorescence instrument to detect Fv/Fm parameter.
Survival rate and rejuvenation propagation testing result:
Algae density is 106-107The preservation algae solution of the individual/ml survival rate through February is 100%, through survival rate in June
It is 60%.Cell viability is high, and rejuvenation propagation rapidly, can quickly enter cell proliferation；Algae density is 109Individual/ml
The preservation algae solution of the order of magnitude is through the survival rate in February less than 1%, and the survival rate through June is 0.01%.Cell is lived
Power is low, and slowly, the maximum cell number that can reach is low for rejuvenation propagation.
Fig. 1 shows the rejuvenation proliferation process after conservation February.Fig. 2 show conservation February after answer
The Photosynthetic capacity change of strong cell.From result, the cell density order of magnitude is 106-107The preservation of individual/ml
Group, survival rate is high, cell proliferation is rapid, cell viability is high, for suitable high vigor preservation density.
Claims (4)
1. the high vigor cell method for preserving of a Thalassiosira pseudonana, it is characterised in that described method includes
The steps:
1) microalgae is inoculated in culture medium, makes microalgae cell number propagation reach 106-107Individual/ml,
2) algae solution after cultivating carries out gradient cooling process；
3) intensity of illumination when cooling reduces illumination simultaneously, reduction amplitude is 1000-2000lx/ days；
4) algae solution is placed in 4-5 DEG C of dark condition preserve.
2. the method for claim 1, it is characterised in that described step 1) condition of culture such as
Under: temperature 23-25 DEG C, intensity of illumination 4000-5000lx, periodicity of illumination: 12h:12h.
3. the method for claim 1, it is characterised in that described step 1) in culture medium composition
As follows: 20mg/L Na2EDTA,2.5mg/L FeSO4·7H2O,100mg/L KNO3,10mg/L K2HPO4,
0.25mg/L MnSO4,0.5×10-3Mg/L vitamin B12,5×10-3Mg/L vitamin B1。
4. the method for claim 1, it is characterised in that described step 2) gradient cooling at
Reason, is by cultivation temperature from culture environment temperature, within every 24 hours, reduces by 5 DEG C；Until being reduced to 4-5 DEG C.
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| CN201610294363.XA CN105713838B (en) | 2016-05-05 | 2016-05-05 | A kind of high vigor cell method for preserving of Thalassiosira pseudonana |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106244512A (en) * | 2016-08-24 | 2016-12-21 | 湖南文理学院 | A kind of preparation method of high activity Microcystis aeruginosa hypopus algae mud |
| CN106867953A (en) * | 2017-03-15 | 2017-06-20 | 哈尔滨工业大学 | A kind of method that microalgae processes molasses containing waste water synchronization production capacity under cryogenic |
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| CN102311919A (en) * | 2010-07-07 | 2012-01-11 | 中国石油化工股份有限公司 | Microalgae species preserving method |
| CN104651235A (en) * | 2015-03-02 | 2015-05-27 | 宁波大学 | Thalassiosira pseudonana and application of thalassiosira pseudonana as mercenaria mercenaria larva breeding bait |
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2016
- 2016-05-05 CN CN201610294363.XA patent/CN105713838B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102311919A (en) * | 2010-07-07 | 2012-01-11 | 中国石油化工股份有限公司 | Microalgae species preserving method |
| CN104651235A (en) * | 2015-03-02 | 2015-05-27 | 宁波大学 | Thalassiosira pseudonana and application of thalassiosira pseudonana as mercenaria mercenaria larva breeding bait |
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| 俞建中等: "两种微藻浓缩液保藏效果的比较", 《现代食品科技》 * |
| 刘平怀: "《生物工艺实验》", 31 March 2012, 南京大学出版社 * |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106244512A (en) * | 2016-08-24 | 2016-12-21 | 湖南文理学院 | A kind of preparation method of high activity Microcystis aeruginosa hypopus algae mud |
| CN106244512B (en) * | 2016-08-24 | 2019-10-01 | 湖南文理学院 | A kind of preparation method of high activity Microcystis aeruginosa hypopus algal gel |
| CN106867953A (en) * | 2017-03-15 | 2017-06-20 | 哈尔滨工业大学 | A kind of method that microalgae processes molasses containing waste water synchronization production capacity under cryogenic |
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