CN101591694A - A kind of fluorescent identification method of living cells of Alexandrium tamarense - Google Patents
A kind of fluorescent identification method of living cells of Alexandrium tamarense Download PDFInfo
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- CN101591694A CN101591694A CNA2009101120974A CN200910112097A CN101591694A CN 101591694 A CN101591694 A CN 101591694A CN A2009101120974 A CNA2009101120974 A CN A2009101120974A CN 200910112097 A CN200910112097 A CN 200910112097A CN 101591694 A CN101591694 A CN 101591694A
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Abstract
A kind of fluorescent identification method of living cells of Alexandrium tamarense relates to a kind of fluorescent identification method, especially relates to a kind of fluorescence dye fluorescein diacetate that utilizes poisonous red tide algae Alexandrium tamarense cell is dyeed to differentiate active somatic cell.Provide a kind of fluorescent identification method of living cells of Alexandrium tamarense and active somatic cell examination and have the application of inhibition Alexandrium tamarense cell physiological active screening bioactive compounds in Alexandrium tamarense is cultivated.The fluorescence dye fluorescein diacetate is dissolved in acetone, is mixed with fluorescence dye fluorescein diacetate mother liquor; Get Alexandrium tamarense enchylema in 1.5mL centrifuge tube or 96 orifice plates, add fluorescence dye fluorescein diacetate mother liquor; After room temperature leaves standstill dyeing, get cell suspension, put into the ice chest lucifuge and treat microscopy; The cell suspension of getting after the dyeing is counted frame in frustule, observes under fluorescent microscope blue excitation light and visible light respectively.
Description
Technical field
The present invention relates to a kind of fluorescent identification method, especially relate to and a kind ofly utilize the fluorescence dye fluorescein diacetate (Fluorescein diacetate FDA) dyes to differentiate active somatic cell to poisonous red tide algae Alexandrium tamarense (Alexandrium tamarense) cell.
Background technology
In the red-tide control research field, what of algae bio amount can react by OD value or the frustule number of chlorophyll a content, chlorophyll fluorescence value, specific unicellular algae.At present, the technological method that is used for reacting the algae bio amount mainly contains opticmicroscope direct-counting method, visible spectrophotometry, spectrophotofluorimetry ([1] Hu Xianwen, Deng, visible spectrophotometry is measured the wawter bloom anabena. Hua Zhong Agriculture University's journal, 2002.21 (3): 295-297), chlorophyll a assay method, flow cytometry method, Ku Erte counting process, living body fluorescent detection technique etc.Wherein microscope direct-counting method and chlorophyll a content determination are the basic skills of algae bio flow measurement, be to determine that other measuring method validity is according to ([2] Hou Jianjun, Huang Bangqin, Dai Xianghui, red tide frustule method of counting comparative studies Chinese public health, 2004.20 (8): 907-908).
Because its cytopigment of different algal species are formed and content difference, cellularstructure is not of uniform size, makes to react the index of algae bio amount by detecting chlorophyll a content or frustule OD value, is confined to specific algae and can not get widespread use.Simultaneously, microscope direct-counting method and chlorophyll a content determination can accurately not react the algae cell activity state.
The frustule counting is only applicable to the detection of unicellular algae biomass.Wherein, the counting of live body frustule being had great importance aspect red-tide control method and the laboratory screening algicdal activity material estimating, is the most basic technological method in this field.Upgrowth situation and algae cell density to frustule are accurately monitored and are measured, and need set up a kind of authentication method of the frustule of live body accurately and reliably.
The integrity of cytolemma is basic sign ([3] Gonzalez of viable cell, J.E.and R.Y.Tsien, Improvedindicators of cell membrane potential that use fluorescence resonance energy transfer.Chemistry﹠amp; Biology, 1997.4 (4): 269-277).For example traditional vital stain such as trypan blue, integrity based on cytolemma is judged cell activity, trypan blue can not be by the cytolemma of viable cell by the cytolemma of dead cell, thereby dead cell microscopically show blue viable cell do not develop the color ([4] Zhang Xianjie etc. trypan blue dyeing and the two dyeing of FDA/PI are to detecting the evaluation of liver cell motility rate. Capital University of Medical Sciences's journal, 2000,21 (3)).Yet this method can not illustrate the cell interior physiological situation, can not the accurate response cell viability.Along with the widespread use of Flow Cytometry, select for use according to the difference of fluorescence dye, 3 classes occurred in context of detection and detected index: 1. cell leakage viable cell; 2. intracellular enzyme activity; 3. microbial film current potential.FDA dyestuff among the present invention is based on the glimmering material of a kind of live body that active two indexs of cell membrane integrity and intracellular enzyme detect algae cell activity.Its principle is that the easy permeate through cell membranes of FDA enters in the cell, under the hydrolytic action of born of the same parents' lactonase, discharge fluorescein, and being difficult for permeate through cell membranes, fluorescein accumulates in born of the same parents, (send bright green (515～525nm) fluorescence under 470～495nm) the exciting light effects blue, under fluorescent microscope Olympus BX41, observe, this type of cell is accredited as viable cell ([5] Garveyl, M., B.M.andU.P., Applicability of the FDA assay to determine the viability of marine phytoplankton underfifferent environmental conditions.MARINE ECOLOGY PROGRES S SERIES Mar Ecol Prog Ser, 2007.352:17-26).
Summary of the invention
The fluorescent identification method that the purpose of this invention is to provide a kind of living cells of Alexandrium tamarense.
Another object of the present invention is the examination that the fluorescent identification method of described living cells of Alexandrium tamarense is applied to active somatic cell in the Alexandrium tamarense cultivation.
Another object of the present invention is that the fluorescent identification method of described living cells of Alexandrium tamarense is applied to have the screening that suppresses Alexandrium tamarense cell physiological active active substance.
The present invention includes following steps:
1) the fluorescence dye fluorescein diacetate is dissolved in acetone, is mixed with fluorescence dye fluorescein diacetate mother liquor;
2) get Alexandrium tamarense enchylema in 1.5mL centrifuge tube or 96 orifice plates, add fluorescence dye fluorescein diacetate mother liquor;
3) after room temperature leaves standstill dyeing, get cell suspension, put into the ice chest lucifuge and treat microscopy;
4) cell suspension of getting after the dyeing is counted frame in frustule, observes under fluorescent microscope blue excitation light and visible light respectively, and the blue excitation light wavelength is 470～495nm.
The concentration of described mother liquor is preferably 1mg/mL, and mother liquor preferably places 4 ℃ of refrigerators to keep in Dark Place.
It is 50～500 μ g/mL that described adding fluorescence dye fluorescein diacetate mother liquor preferably makes its final concentration.
The fluorescent identification method of described living cells of Alexandrium tamarense can be used for the examination of active somatic cell in the Alexandrium tamarense cultivation.
The fluorescent identification method of described living cells of Alexandrium tamarense can be used for having the screening that suppresses Alexandrium tamarense cell physiological active active substance.
The invention provides a kind ofly, and live body Alexandrium tamarense cell carried out accurate counting, thereby be applied to the Related Research Domain research of this algae poisonous red tide algae Alexandrium tamarense live body frustule authentication method accurately and effectively.
The present invention relates to fluorescence dye FDA, when it dyes to the Alexandrium tamarense cell, this dyestuff descends in live body frustule born of the same parents lactonase hydrolytic action and explains the discharging fluorescence element, fluorescein can not permeate through cell membranes and is accumulated in the born of the same parents, through the blue excitation optical excitation, the live body frustule sends bright green fluorescence.Under fluorescent microscope, observe, can utilize frustule counting frame to count, thereby realize purpose of the present invention.
The poisonous tower agate of the dyeing target algae Alexandria red tide algae that the present invention relates to, be that (cell length is at 20～55 μ m for the relatively big unicellular algae of a kind of cell, width is at 17～45 μ m), under forfeiture illumination cultivation condition sedimentation taking place assembles, can not form homogeneous frustule suspension, and frustule concentration can only reach 10 under laboratory condition
4～10
5Cells/mL can not satisfy the detection requirement of flow cytometer fast high-flux, makes Flow Cytometry be not suitable for the detection counting of this algae, therefore, need utilize the artificial microscopy counting of fluorescent microscope.
Description of drawings
Fig. 1 is in the observations of light field work/dead cell mixing after FDA dyeing.
Fig. 2 is in the observations of fluorescence work after the match/dead cell mixing after FDA dyeing.
Fig. 3 is that final concentration is the Color of the FDA of 2.5 μ g/mL to frustule.
Fig. 4 is that final concentration is the Color of the FDA of 25 μ g/mL to frustule.
Fig. 5 is that final concentration is the Color of the FDA of 600 μ g/mL to frustule.
Embodiment
Following examples are to further specify of the present invention, but the invention is not restricted to following embodiment.
Embodiment 1: the FDA dyeing concentration of Alexandrium tamarense cell is determined
1) FDA is dissolved in the acetone, is made into the staining fluid that concentration is 25mg/mL, place 4 ℃ of refrigerators to keep in Dark Place.
2) the FDA stock solution of dilution 25mg/mL is to 10mg/mL, 5mg/mL, 2.5mg/mL, 1mg/mL, 0.1mg/mL, 0.01mg/mL.
3) get 200 μ L exponential phases even frustule nutrient solutions in the 1.5mL centrifuge tube.
4) add 25mg/mL stock solution and step 2 respectively) in each FDA diluent 5 μ L, FDA dye liquor final concentration is respectively: 625 μ g/mL, 250 μ g/mL, 125 μ g/mL, 62.5 μ g/mL, 25 μ g/mL, 2.5 μ g/mL, 0.25 μ g/mL, each concentration gradient establish 3 parallel.
5) room temperature leaves standstill dyeing 2min, puts into the ice chest lucifuge and treats microscopy.
6) behind the stained specimens mixing, the cell suspension of getting after the 10 μ L dyeing is counted frame in frustule, (observes under 470～495nm), determines that frustule is sent and stablize the FDA dyeing concentration scope of bright green fluorescence in the fluorescent microscope blue excitation light.
7) FDA dyeing final concentration scope: 2.5～600 μ g/mL.Color is seen Fig. 3～5.
Embodiment 2:FDA is to the Color of work/dead cell mixing suspension
1) gets 10mL exponential phase frustule liquid in the 50mL triangular flask, add fixedly 10min of 1mL Shandong Ge Shi solution, the deactivation frustule.
2) the centrifugal 10min of 1000 * g, colourless with the algae culture base f/2 repetitive scrubbing of sterilization to supernatant.
3) 10mLf/2 algae culture medium re-suspended cell is made the dead cell suspension of same concentrations.
4) get 10mL exponential phase frustule liquid as the viable cell suspension.
5) cell mixes in varing proportions anyway, makes the viable cell ratio be respectively 0,20%, 50%, 80%, 100%.
6) get 200 μ L exponential phases even frustule nutrient solutions in the 1.5mL centrifuge tube, it is described to press embodiment 1, and the FDA stock solution 5 μ L that add 1mg/mL make its final concentration reach best FDA dyeing concentration 25 μ g/mL.
7) room temperature leaves standstill dyeing 2min, puts into the ice chest lucifuge and treats microscopy.
8) behind the stained specimens mixing, the cell suspension of getting after 10 μ L dye is counted frame in frustule, observes counting respectively under fluorescent microscope blue excitation light and visible light.
9) calculate FDA dyeing efficient according to following formula:
Dyeing efficient (%)=(N
1/ N
2) * 100
N in the formula
1Be fluorescence cell count after the match, N
2Be cell count under the light field.
10) 3 parallel laboratory test groups are set, carry out dyeing counting.Experimental result sees Table 1.
The FDA dyeing efficient of the work of table 1 different ratios/dead cell mixing suspension
Embodiment 3:FDA is to the Color of different concns frustule
1) gets exponential phase Alexandrium tamarense liquid 10ml, frustule number in the microscopically counting 20ul algae liquid.
2) by centrifugal, the f/2 algae culture medium is resuspended and the method for gradient dilution, adjusts frustule number to 1 * 10
3Cells/mL, 2 * 10
3Cells/mL, 1 * 10
4Cells/mL, 2 * 10
4Cells/mL.
3) it is described to press embodiment 1, to the frustule suspension dyeing of different concns.
4) 3 parallel laboratory test groups are set, count.Experimental result sees Table 2.
The FDA dyeing efficient of table 2 different concns frustule
Embodiment 4: illumination and temperature are to the influence of Color
1) it is described to press embodiment 1, and the cell suspension after the FDA dyeing is divided into two parts, and portion places on ice and keeps in Dark Place, and another part is not made any lucifuge and handled room temperature preservation.
2) respectively at dyeing back 30min, 60min, 90min, 120min, 150min, 180min and 210min sampling are counted.
3) 3 experimental group are set.Experimental result sees Table 3.
Table 3 illumination and temperature are to the influence of Color
Alexandrium tamarense cell dyeing result of the present invention is referring to Fig. 1 and Fig. 2.The work that Fig. 1 observes down for light field/dead cell mixing, the cellular form basically identical can't be distinguished work/dead cell.The work that Fig. 2 observes after the match for fluorescence/dead cell mixing, viable cell (arrow A indication) is dyed bright green, dead cell (arrow B indication) is then because the stack of the green of self spontaneous red fluorescence and background becomes yellow, therefore can be well with live/dead cell distinguishes.
The working concentration scope of fluorescence dye fluorescein diacetate FDA is 2.5～600 μ g/mL.Fig. 3～5 provide the Color of different final concentration FDA.Fig. 3 is the Color of the FDA of final concentration 2.5 μ g/mL to frustule, and the very weak explanation of the green of viable cell FDA concentration is not high enough; Fig. 4 is the Color of the FDA of 25 μ g/mL to frustule, and viable cell bright green explanation FDA concentration is enough; Fig. 5 is the Color of the FDA of 600 μ g/mL to frustule, and at this moment, FDA crystallization explanation FDA excessive concentration occurring can not dissolve fully, but still can make normal cell send bright green fluorescence this moment.
The difference that different algal species autofluorescence intensity and intracellular enzyme are formed, the method that makes fluorescence vital staining of the present invention detect algae cell activity are not had a universality.Therefore, need be applied to the feasibility that living cells of Alexandrium tamarense detects to this method verifies.The present invention after inquiring into the feasibility that this dyeing process is used to characterize living cells of Alexandrium tamarense: A. dyeing aspect following three, dead, the viable cell of differentiation that must can be simple and effective; B. coloration result should be accurately, can repeat and false positive can not occur; C. after the dyeing, fluorescence can be stablized the not cancellation of maintenance time enough, counts for microscopy.At above three aspects, contrived experiment is verified respectively: 1. a series of concentration known ratios of preparation is dead/the frustule mixing suspension of living, utilize this fluorescent staining method dyeing back microscopy counting.The microscopy result shows that this fluorescent staining method can make frustule alive send bright green fluorescence under blue-light excited, and dead cell is green-emitting fluorescence not.2. the frustule suspension alive of preparation different concns utilizes this fluorescent staining method dyeing back microscopy counting.The microscopy result shows, this fluorescent staining method frustule alive that can more accurately dye.3. the frustule suspension after the dyeing places respectively on ice and keeps in Dark Place and lucifuge room temperature preservation not.On ice under the preservation condition, with the growth of shelf time, observed bright green cell number is kept balance substantially under the fluorescent microscope in lucifuge, and the background green fluorescence intensity remains unchanged substantially, can guarantee the observation and the counting needs of testing like this.And handle and preservation cell suspension at ambient temperature without lucifuge, growth with the shelf time, observed bright green cell number reduces gradually under the fluorescent microscope, and the brightness of background green fluorescence strengthens gradually, illustrates that the fluorescein in the frustule film leaks or fluorescent quenching.
Experimental result shows: utilize the fluorescent staining method of fluorescein diacetate FDA dyeing Alexandrium tamarense cell, can identify live body Alexandrium tamarense cell.
The integrity of cytolemma is the basic sign of viable cell.Only judge the dyeing process of cytoactive, can not the characterize cells internal physiology change and the accurate response cell viability based on the integrity of cytolemma.Fluorescence vital staining method based on cell membrane integrity and active two indexs of intracellular enzyme can characterize the intracellular enzyme activity, thus the accurate response cell viability.Its principle is that the release fluorescein is separated in the effect decline of non-fluorogenic substrate relevant enzyme in born of the same parents, and fluorescein sends fluorescence at intracellular accumulation under the exciting light effect.
Vital dye fluorescein diacetate FDA (Fluorescein diacetate) is the epipolic apolar substance of tool not, and itself does not fluoresce, and can pass freely come in and go out cell and can not accumulating in cell of cytolemma.In viable cell, FDA is decomposed into fluorescein through esterase, and the latter is the polar material with fluorescence, the cytolemma of can not freely coming in and going out, thus in cell, accumulate, make cell under blue excitation light, send green fluorescence.
The FDA dyestuff is to the judgement of the optimum concn of Alexandrium tamarense, is according under fluorescent microscope, and viable cell sends that the intensity of green fluorescence determines, the dead cell of spontaneous red fluorescence is as the criterion can obviously distinguish on every side.The difference that different algae kind autofluorescence intensity and intracellular enzyme are formed makes that using method that the fluorescence vital staining detects algae cell activity does not have a universality.Therefore, need be applied to the feasibility that living cells of Alexandrium tamarense detects to this method verifies.
This dyeing process whether can be applied to Alexandrium tamarense belong in other algae kinds, based on 2 prerequisites: 1, the green fluorescence that sends of frustule can not covered by background fluorescence.This requires the frustule can not be too little, otherwise is difficult for differentiating counting at microscopically, can use flow cytometer and count, and this is not the problem that the present invention discussed.2, the green fluorescence that sends of frustule can not covered by spontaneous red fluorescence.This requirement excite excitation wavelength that pigment in the frustule sends intense red fluorescence with excite the FDA degraded product to send the excitation wavelength of green fluorescence can not be in same scope, otherwise green fluorescence can be covered.
Claims (6)
1. the fluorescent identification method of a living cells of Alexandrium tamarense is characterized in that may further comprise the steps:
1) (Fluorescein diacetate FDA) is dissolved in acetone, is mixed with fluorescence dye fluorescein diacetate mother liquor with the fluorescence dye fluorescein diacetate;
2) get Alexandrium tamarense enchylema in 1.5mL centrifuge tube or 96 orifice plates, add fluorescence dye fluorescein diacetate mother liquor;
3) after room temperature leaves standstill dyeing, get cell suspension, put into the ice chest lucifuge and treat microscopy;
4) cell suspension of getting after the dyeing is counted frame in frustule, observes under fluorescent microscope blue excitation light and visible light respectively, and the blue excitation light wavelength is 470～495nm.
2. the fluorescent identification method of a kind of living cells of Alexandrium tamarense as claimed in claim 1, the concentration that it is characterized in that described mother liquor is 1mg/mL.
3. the fluorescent identification method of a kind of living cells of Alexandrium tamarense as claimed in claim 1 or 2 is characterized in that mother liquor places 4 ℃ of refrigerators to keep in Dark Place.
4. the fluorescent identification method of a kind of living cells of Alexandrium tamarense as claimed in claim 1 is characterized in that it is 50～500 μ g/mL that described adding fluorescence dye fluorescein diacetate mother liquor makes its final concentration.
5. the fluorescent identification method of a kind of living cells of Alexandrium tamarense as claimed in claim 1 is used for the examination that Alexandrium tamarense is cultivated active somatic cell.
6. the fluorescent identification method of a kind of living cells of Alexandrium tamarense as claimed in claim 1 is used to have the screening that suppresses Alexandrium tamarense cell physiological active active substance.
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| CN102426126A (en) * | 2011-08-29 | 2012-04-25 | 西北农林科技大学 | Differential staining method for inner cell mass cells and trophoblastic cells of cattle blastulae |
| CN102426126B (en) * | 2011-08-29 | 2013-04-24 | 西北农林科技大学 | Differential staining method for inner cell mass cells and trophoblastic cells of cattle blastulae |
| WO2018041215A1 (en) * | 2016-08-31 | 2018-03-08 | 威海中远造船科技有限公司 | Fast detection method and device for organisms of 10-50 μm in ship ballast water |
| CN106244669A (en) * | 2016-10-18 | 2016-12-21 | 上海海洋大学 | A kind of method of 10 50 microns of living body biologicals in quick discriminating water |
| CN112782137A (en) * | 2020-12-24 | 2021-05-11 | 自然资源部第一海洋研究所 | Detection method of living plankton living cell flow cytometer with 10-50 mu m |
| CN113295665A (en) * | 2021-05-26 | 2021-08-24 | 苏州科技大学 | Pressurizing device, blue algae liquid pretreatment method based on pressurizing device and blue algae living cell rapid fluorescent staining counting method |
| CN114292753A (en) * | 2021-12-23 | 2022-04-08 | 南京大学 | Method for separating cells and discharging nanoparticles from cells |
| CN114292753B (en) * | 2021-12-23 | 2023-06-16 | 南京大学 | Method for separating cells and discharging nanoparticles from cells |
| CN117589729A (en) * | 2023-03-13 | 2024-02-23 | 大理大学 | Method for detecting active oxygen scavenging effect of flavonoid compounds in living cells |
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