CN113866075A - Method for rapidly determining volume of microcystis pseudo-vacuoles by using flow cytometer - Google Patents
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- 241000192701 Microcystis Species 0.000 title claims abstract description 73
- 238000000034 method Methods 0.000 title claims abstract description 44
- 241000195493 Cryptophyta Species 0.000 claims abstract description 63
- 239000003094 microcapsule Substances 0.000 claims description 10
- WSFSSNUMVMOOMR-UHFFFAOYSA-N Formaldehyde Chemical compound O=C WSFSSNUMVMOOMR-UHFFFAOYSA-N 0.000 claims description 9
- 239000008098 formaldehyde solution Substances 0.000 claims description 9
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 8
- 239000008367 deionised water Substances 0.000 claims description 5
- 229910021641 deionized water Inorganic materials 0.000 claims description 5
- 238000012937 correction Methods 0.000 claims description 4
- 238000012417 linear regression Methods 0.000 claims description 2
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 10
- 241000107845 Microcystis aeruginosa PCC 7806 Species 0.000 description 10
- 238000005259 measurement Methods 0.000 description 6
- 229910052757 nitrogen Inorganic materials 0.000 description 5
- 238000012544 monitoring process Methods 0.000 description 4
- 239000011800 void material Substances 0.000 description 4
- 241000192710 Microcystis aeruginosa Species 0.000 description 3
- 238000004458 analytical method Methods 0.000 description 3
- 238000005286 illumination Methods 0.000 description 3
- 238000004364 calculation method Methods 0.000 description 2
- 230000008569 process Effects 0.000 description 2
- 238000000611 regression analysis Methods 0.000 description 2
- 230000005526 G1 to G0 transition Effects 0.000 description 1
- 206010056658 Pseudocyst Diseases 0.000 description 1
- 238000009825 accumulation Methods 0.000 description 1
- 230000004075 alteration Effects 0.000 description 1
- 238000011088 calibration curve Methods 0.000 description 1
- 230000001413 cellular effect Effects 0.000 description 1
- 238000012512 characterization method Methods 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 238000012258 culturing Methods 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 230000007812 deficiency Effects 0.000 description 1
- 230000002950 deficient Effects 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 239000006185 dispersion Substances 0.000 description 1
- 230000002063 effect on algae Effects 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 230000004907 flux Effects 0.000 description 1
- 238000011065 in-situ storage Methods 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 238000004445 quantitative analysis Methods 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 239000002344 surface layer Substances 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
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Abstract
The invention discloses a method for rapidly measuring the volume of a pseudo-vacant cell of microcystis by using a flow cytometer, which belongs to the technical field of quantitative representation of floating capacity of the microcystis and comprises the steps of collecting an algae sample, dispersing a microcystis group into single cells, dividing the microcystis dispersed into the single cells into two parts, completely breaking the pseudo-vacant cell by high pressure for one part, mixing the broken pseudo-vacant cell with another part of algae cell which is not processed by high pressure in different proportions to obtain a mixed algae sample, measuring the volume of the pseudo-vacant cell of the mixed algae sample by using a Walsby capillary pressure tube method, correcting, measuring the side scattering light intensity (called scattering angle for short) of the algae cell in the mixed algae sample by using the flow cytometer, establishing the quantitative relation between the volume of the corrected pseudo-vacant cell and the scattering angle of the cell to obtain a standard curve, and rapidly measuring the volume of the pseudo-vacant cell of the microcystis realized by using the standard curve Accuracy, wide application range and simple operation.
Description
Technical Field
The invention belongs to the technical field of quantitative characterization of buoyancy of microcystis, and relates to a method for rapidly determining the volume of pseudo-vacant cells of microcystis by using a flow cytometer.
Background
The floating accumulation of the microcystis colony is a key stage for forming microcystis bloom and is key information required to be mastered in water bloom monitoring and early warning. The microcystis colony can float to the surface layer of water body because the microcystis cell synthesizes a pseudo-vacuole capable of providing floating capacity, and the pseudo-vacuole can regulate the floating and sinking of the microcystis colony. Therefore, the efficient and accurate quantitative determination of the volume of the microcystis pseudo-vacuole is a key problem to be solved for monitoring and early warning of microcystis bloom.
The present method for determining the pseudo-vacuole volume of microcystis is the capillary pressure tube method invented by british scientist a.e. walsby, 1982. Although the method can accurately measure the volume of the microcystis pseudohollow cells, the method is difficult to realize high-efficiency and rapid analysis of the microcystis pseudohollow cells due to the complex operation, the need of keeping constant temperature in the measurement process and the like. In addition, the Walsby capillary pressure tube method needs more algae suspension samples for each measurement when the pseudo-empty cells of the microcysts are measured, and the volume of the measured pseudo-empty cells needs to be converted into the volume of the pseudo-empty cells of single algae cells according to the concentration of the algae cells, so that the Walsby capillary pressure tube method cannot directly analyze the volume of the pseudo-empty cells of the microcysts at a cellular level.
Based on the analysis, the method for rapidly and accurately measuring the volume of the pseudo-vacant cells of the microcystis directly from the cell level has important practical significance for improving the monitoring and early warning precision of the microcystis bloom.
Disclosure of Invention
The technical problem to be solved by the invention is to provide a method for rapidly determining the volume of microcystis pseudo-empty cells by using a flow cytometer, aiming at the defects of the prior art, and the following technical scheme is provided: a method for rapidly measuring the volume of microcystis pseudocyst by using a flow cytometer comprises the following steps,
collecting microcystis, dispersing microcystis groups into single-cell microcystis samples, and fixing the microcystis samples by using formaldehyde solution;
step two: dividing the microcystis unicellular sample into two parts on average, wherein one part is subjected to high pressure to completely break pseudo-vacant cells, and the other part is reserved for the pseudo-vacant cells;
step three: mixing the two algae samples according to different proportions to prepare mixed algae samples with different proportions;
step four: measuring the pseudo-vacuole volume of the mixed algae sample by adopting a Walsby capillary pressure tube method, and correcting; measuring the side angle scattering light intensity of the mixed algae sample by using a flow cytometer;
step five: after the mixed algae sample is measured, establishing a standard curve of the pseudo-vacuole volume and the cell side angle scattering light intensity;
step six: and (4) measuring the side angle scattering light intensity of the microcystis to be measured by a flow cytometer, and calculating the pseudo-empty cell volume of the microcystis to be measured according to the standard curve obtained in the step five.
Preferably, the first step is collecting the microcystis in the lake water by using a plankton enrichment net.
Preferably, the first step is to put the microcapsule algae population into deionized water, place for 1-360min, shake and disperse into single-cell microcapsule algae-like.
Preferably, the first step is to put the microcapsule algae population into deionized water, place for 10-180min, shake and disperse into single-cell microcapsule algae-like.
Preferably, the final concentration of the formaldehyde solution in the first step is 0.1-5%, and the fixing time is 10-60 min.
Preferably, the pressure intensity of the high-pressure rupture pseudo-empty cells in the step two is 0.5-2.5 MPa. .
Preferably, the number of the mixed algae in the third step is 6, wherein the concentration of the single-cell microcystis-like subjected to high-pressure rupture of pseudo-vacant cells is 100%, 80%, 60%, 40%, 20% and 0%.
Preferably, the pseudo-vacuole volume correction in the fourth step is to subtract the background value of the pseudo-vacuole volume of the mixed algae sample measured by the Walsby capillary pressure tube method to obtain the corrected pseudo-vacuole volume; the background value is the pseudo-vacuole volume value of the high pressure complete rupture pseudo-vacuole algae-like measured by the Walsby capillary pressure tube method.
Preferably, the linear regression equation of the standard curve in the step five is as follows: Y-0.0423X-2.0274.
Has the advantages that: the method is simple, easy to operate and very wide in applicability; the microcystis pseudo-vacant cells are quantitatively analyzed in an in-situ damaged manner by adopting a flow cytometer, so that real-time online analysis can be realized, and accurate quantitative analysis is directly carried out at a high flux from the cell level; provides a method for monitoring and early warning the microcystis bloom and provides a necessary means for researching the generation mechanism of the microcystis bloom.
Drawings
FIG. 1 is a diagram of the gradual dispersion of microcystis population into single cells;
FIG. 2 is a standard graph of scattering angle of algal cells versus uncorrected pseudo-void cell volume;
FIG. 3 is a standard graph of scattering angle of algal cells versus corrected pseudo-void cell volume.
Detailed Description
The present invention is described in further detail below with reference to the attached drawings and the specific preferred embodiments, but those skilled in the art will understand that the following described embodiments are a part of the embodiments of the present invention, rather than the whole embodiments, and are only used for illustrating the present invention and should not be construed as limiting the scope of the present invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.
Example 1:
the invention provides a technical scheme, in particular to a method for rapidly measuring the volume of microcystis pseudo-vacant cells by using a flow cytometer, which comprises the following steps,
collecting microcystis, adopting a plankton enrichment net with the aperture of 63 mu m to enrich microcystis groups in Wuxi Taihu Meilianwan to obtain enriched microcystis samples, storing the enriched microcystis samples at the low temperature of 4 ℃, and carrying the enriched microcystis samples back to a laboratory for separation operation; dispersing the microcystis colony into a single-cell microcystis sample, and fixing the microcystis sample by using a formaldehyde solution; taking a proper amount of the algae sample, placing in deionized water for 180min to gradually disperse the microcystis colony into single cells as shown in figure 1, and then adding formaldehyde solution for fixing for 30min to obtain final concentration of 1.6%.
Step two: dividing the microcystis unicellular sample into two parts, wherein one part is used for completely breaking pseudo-vacant cells under the high pressure of 1.5MPa, and the other part is not treated and remains the pseudo-vacant cells;
step three: mixing two algae samples according to different proportions to prepare mixed algae samples with 6 proportions: respectively 100%, 80%, 60%, 40%, 20% and 0%, wherein the content of the proportion is as follows: the proportion of the single-cell microcystis-like sample with 1.5MPa high pressure for completely breaking the pseudo-vacuoles in the total concentration of the mixed algae-like sample;
step four: determining the pseudo-air cell volumes of the 6 mixed algae samples by adopting a Walsby capillary pressure tube method to obtain the directly measured pseudo-air cell volumes; subtracting the background value of the pseudo-vacuole volume measured by the Walsby capillary pressure tube method, wherein the background value is the value of the algae sample with completely broken pseudo-vacuoles measured by the Wals by capillary pressure tube method, and obtaining the corrected measured pseudo-vacuole volume; the results are shown in Table 1; measuring the side angle scattering light intensity of 6 mixed algae samples by using a flow cytometer, which is called scattering angle for short and is called scattering angle for short in the drawing; the mixed algae sample with the same proportion is measured for three times, and the measurement result is shown in table 1;
step five: establishing a quantitative relationship between the directly measured pseudo-vacuole volume and the scattering angle of the algae cells by regression analysis to obtain an uncorrected standard curve equation as follows: Y0.0423X-1.519, R2 0.99, X scattering angle of algal cells, Y uncorrected pseudo-vacuole volume of algal cells, see figure 2,
establishing a quantitative relation between the corrected actually-measured pseudo-empty cell volume and the scattering angle of the algae cells through regression analysis to obtain a corrected standard curve equation as follows: y is 0.0423X-2.0274, R2 is 0.99, X is the scattering angle of the algal cells, and Y is the corrected pseudo-vacuole volume of the algal cells, as shown in fig. 3.
TABLE 1 Mixed algal-like pseudo-vacuole volume and side scatter light intensity measurements
Step six: measuring the side angle scattering light intensity of the microcystis to be measured by a flow cytometer, and calculating the pseudo-empty cell volume of the microcystis to be measured according to the standard curve obtained in the step five:
(1) the microcystis to be detected is microcystis aeruginosa (the serial number of the strain is PCC 7806):
culturing Microcystis aeruginosa (strain number: PCC7806) with BG-11 culture solution in conical flask at 25 deg.C under 45 μmol m illumination intensity-2s-1The light-dark ratio is 12h:12 h. The microcystis aeruginosa PCC7806 is a single-cell type microcystis with pseudo-vacuoles and can be suspended in a culture solution; fixing the obtained algae sample by using a formaldehyde solution, wherein the final concentration is 1.6%, preparing a mixed algae sample of microcystis aeruginosa PCC7806 according to the second step and the third step, measuring the scattering angle of the mixed algae sample by using a flow cytometer, respectively calculating the pseudo-air cell volume of the algae sample according to the uncorrected standard curve and the corrected standard curve in the fifth step, and obtaining the pseudo-air cell volume calculated by the uncorrected standard curve and the pseudo-air cell volume calculated by the corrected standard curve, which are shown in a table 2;
pseudo-vacuole volume of the mixed algae sample was measured by the Walsby capillary pressure tube method and corrected according to the method described above, and the results are shown in Table 3;
by comparing the calculated pseudo-vacant cell volume with the actually measured pseudo-vacant cell volume, the corrected standard curve can be effectively applied to the microcystis aeruginosa PCC7806, and the corrected standard curve can better calculate the pseudo-vacant cell volume of the algae cells; the calculated dummy cell volume obtained from the uncorrected calibration curve is generally higher than the actual dummy cell volume.
TABLE 2 measurement of scattering angle of mixed algae sample of Microcystis aeruginosa PCC7806 and calculation of pseudo-vacant cell
TABLE 3 determination of the pseudo-void cell volume of Microcystis aeruginosa PCC7806
(2) The microcapsule algae to be detected is microcapsule algae (the serial number of the algae strain is HUB 524):
microcystis (strain number: HUB524) is cultured in conical flask with BG-11 culture solution at 25 deg.C and illumination intensity of 45 μmol m to logarithmic phase-2s-1The light-dark ratio is 12h:12 h. The strain is in a single cell form, but pseudo-vacuoles are degenerated, and the algal cells are always deposited at the bottom of the conical flask in the culture process; fixing the algae sample obtained after the culture by using a formaldehyde solution to obtain a final concentration of 1.6%, and preparing a mixed algae sample of the microcystis HUB524 according to the second step and the third step; measuring the scattering angle of the mixed algae sample by using a flow cytometer, respectively calculating the pseudo-air cell volume of the algae sample according to the uncorrected standard curve and the corrected standard curve in the step five, and obtaining the pseudo-air cell volume calculated by the uncorrected standard curve and the pseudo-air cell volume calculated by the corrected standard curve, wherein the result is shown in a table 4;
pseudo-vacuole volume of the mixed algae sample was measured by the Walsby capillary pressure tube method and corrected according to the method described above, the results are shown in Table 5;
by comparing the calculated pseudo-vacuole volume with the actually measured pseudo-vacuole volume, the pseudo-vacuole volumes calculated by the two standard curves are both negative values, and the actual pseudo-vacuole volume is about equal to 0, which shows that the two standard curves have underestimation effect on algae samples without pseudo-vacuoles. This underestimated effect is also present in microcystis aeruginosa PCC7806 algal-like with completely ruptured pseudovacuoles. Therefore, when the calculated dummy cell is negative, the negative should be zero, which can better reflect the actual situation.
TABLE 4 measurement of the scattering angle of the mixed algal-like samples of the microcystis HUB524 and calculation of the pseudoeukarya
TABLE 5 determination of the pseudo-vacuole volume of the microcystis HUB524
Example 2:
checking whether the corrected standard curve is suitable for microcystis cultured under different conditions, and carrying out the following experiment:
algae-like culture and preparation: taihu lake algae strain (colony type microcystis separated and purified in the first step of example 1) and microcystis aeruginosa PCC7806 are selected to be cultured normally and in nitrogen deficiency respectively.
During normal culture, the Taihu lake algae strain and the microcystis aeruginosa PCC7806 are respectively cultured in a conical flask by BG-11 culture solution to logarithmic phase, the culture temperature is 25 ℃, and the illumination intensity is 45 mu mol m-2s-1The light-dark ratio is 12h:12 h.
During nitrogen-deficient culture, in order to ensure that the algae cells are in a nitrogen limitation state, the nitrogen concentration in BG-11 culture solution is reduced to 5mg/L, and the Taihu lake algae strains and the microcystis aeruginosa PCC7806 are respectively cultured in conical flasks to the initial stage of the stationary phase (namely, when the algae cells just stop growing, the growth shows that the algae cells are limited by nitrogen), and other culture conditions are the same as normal culture conditions.
Preparing an alga sample to be detected from the cultured Taihu lake alga sample by adopting the method in the first step of the embodiment 1; fixing the microcystis aeruginosa PCC7806 obtained after the culture by adopting a formaldehyde solution with the final concentration of 1.6 percent to obtain the algae sample to be detected.
And (3) measuring the scattering angle of the algae cells in the algae sample to be measured by adopting a flow cytometer, and obtaining the pseudo-void cell volume calculated by the standard curve after correction through the standard curve equation after correction in the fifth step of the embodiment 1.
TABLE 6 calculated pseudo-vacuoles under different culture conditions
Meanwhile, the Walsby capillary pressure tube method is adopted to measure the pseudo-empty cell volume of the algae sample, the pseudo-empty cell volume is corrected by the method of the step four in the embodiment 1, and the corrected measured pseudo-empty cell volume is obtained.
While the present invention has been described in detail with reference to the preferred embodiments, it should be understood that the above description should not be taken as limiting the invention. Various modifications and alterations to this invention will become apparent to those skilled in the art upon reading the foregoing description. Within the technical idea of the invention, various equivalent changes can be made to the technical scheme of the invention, and the equivalent changes all belong to the protection scope of the invention.
Claims (9)
1. A method for rapidly measuring the volume of microcystis pseudo-vacuoles by using a flow cytometer is characterized by comprising the following steps: comprises the following steps of (a) carrying out,
collecting microcystis, dispersing microcystis groups into single-cell microcystis samples, and fixing the single-cell microcystis samples by using a formaldehyde solution;
step two: dividing the microcystis unicellular sample into two parts on average, wherein one part is subjected to high pressure to completely break pseudo-vacant cells, and the other part is reserved for the pseudo-vacant cells;
step three: mixing the two algae samples according to different proportions to prepare mixed algae samples with different proportions;
step four: measuring the pseudo-vacuole volume of the mixed algae sample by adopting a Walsby capillary pressure tube method, and correcting; measuring the side angle scattering light intensity of the mixed algae sample by using a flow cytometer;
step five: after the mixed algae sample is measured, establishing a standard curve of the pseudo-vacuole volume and the cell side angle scattering light intensity;
step six: and (4) measuring the side angle scattering light intensity of the microcystis to be measured by a flow cytometer, and calculating the pseudo-empty cell volume of the microcystis to be measured according to the standard curve obtained in the step five.
2. The method for rapidly determining the volume of the pseudo-empty cells in the microcystis by using a flow cytometer as claimed in claim 1, wherein: and step one, collecting the microcystis in the lake water by adopting a plankton enrichment net.
3. The method for rapidly determining the volume of the pseudo-empty cells in the microcystis by using a flow cytometer as claimed in claim 1, wherein: the first step is to put the microcapsule algae group in deionized water, place for 1-360min, shake and disperse into single cell microcapsule algae sample.
4. The method for rapidly determining the volume of the pseudo-empty cells in the microcystis by using the flow cytometer as claimed in claim 3, wherein: the first step is to put the microcapsule algae group into deionized water, place for 10-180min, shake and disperse into single cell microcapsule algae sample.
5. The method for rapidly determining the volume of the pseudo-empty cells in the microcystis by using a flow cytometer as claimed in claim 1, wherein: in the first step, the final concentration of the formaldehyde solution is 0.1-5%, and the fixing time is 10-60 min.
6. The method for rapidly determining the volume of the pseudo-empty cells in the microcystis by using a flow cytometer as claimed in claim 1, wherein: and in the second step, the pressure strength of the high-pressure rupture pseudo-empty cells is 0.5-2.5 MPa.
7. The method for rapidly determining the volume of the pseudo-empty cells in the microcystis by using a flow cytometer as claimed in claim 1, wherein: the number of the mixed algae in the third step is 6, wherein the concentration of the single-cell microcystis-like subjected to high-pressure rupture of pseudo-vacuoles accounts for 100%, 80%, 60%, 40%, 20% and 0%.
8. The method for rapidly determining the volume of the pseudo-empty cells in the microcystis by using a flow cytometer as claimed in claim 1, wherein: the pseudo-vacuole volume correction in the fourth step is to subtract the background value of the pseudo-vacuole volume of the mixed algae sample measured by the Walsby capillary pressure tube method to obtain the corrected pseudo-vacuole volume; the background value is the pseudo-vacuole volume value of the high pressure complete rupture pseudo-vacuole algae-like measured by the Walsby capillary pressure tube method.
9. The method for rapidly determining the volume of the pseudo-empty cells in the microcystis by using a flow cytometer as claimed in claim 1, wherein: the linear regression equation of the standard curve in the step five is as follows: Y-0.0423X-2.0274.
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