CN116625908A - Method for determining number of particles in unit volume of liquid and application - Google Patents
Method for determining number of particles in unit volume of liquid and application Download PDFInfo
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- CN116625908A CN116625908A CN202310423896.3A CN202310423896A CN116625908A CN 116625908 A CN116625908 A CN 116625908A CN 202310423896 A CN202310423896 A CN 202310423896A CN 116625908 A CN116625908 A CN 116625908A
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- GLNADSQYFUSGOU-GPTZEZBUSA-J Trypan blue Chemical compound [Na+].[Na+].[Na+].[Na+].C1=C(S([O-])(=O)=O)C=C2C=C(S([O-])(=O)=O)C(/N=N/C3=CC=C(C=C3C)C=3C=C(C(=CC=3)\N=N\C=3C(=CC4=CC(=CC(N)=C4C=3O)S([O-])(=O)=O)S([O-])(=O)=O)C)=C(O)C2=C1N GLNADSQYFUSGOU-GPTZEZBUSA-J 0.000 claims description 7
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- ZXJXZNDDNMQXFV-UHFFFAOYSA-M crystal violet Chemical compound [Cl-].C1=CC(N(C)C)=CC=C1[C+](C=1C=CC(=CC=1)N(C)C)C1=CC=C(N(C)C)C=C1 ZXJXZNDDNMQXFV-UHFFFAOYSA-M 0.000 claims description 2
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Classifications
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
- G01N15/10—Investigating individual particles
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/30—Staining; Impregnating ; Fixation; Dehydration; Multistep processes for preparing samples of tissue, cell or nucleic acid material and the like for analysis
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
- G01N15/10—Investigating individual particles
- G01N2015/1006—Investigating individual particles for cytology
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
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- Analytical Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
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Abstract
The invention belongs to the technical field of cell culture, and particularly relates to a method for measuring the number of particles in a unit volume of liquid and application thereof. The method comprises the following steps: (1) The method comprises the steps of dyeing liquid to be detected by using a dyeing agent, sequentially dripping the dyed liquid to be detected and chromatographic separation liquid at one end of filter paper, dispersing particles in the liquid to be detected on the filter paper under the action of capillary chromatography, and counting the number of the particles in the liquid to be detected by naked eyes; (2) And calculating the number of particles in the unit volume of liquid according to the volume of the liquid to be measured. The measuring method of the invention does not need to purchase expensive instruments, does not need to photograph and use image analysis software to carry out auxiliary counting, breaks through the limitation that the blood cell counter cannot measure particles with the particle size larger than 100 mu m, can finish the measurement of the particle number through naked eye observation, and is simple, efficient and low in cost.
Description
Technical Field
The invention belongs to the technical field of cell culture, and particularly relates to a method for measuring the number of particles in a unit volume of liquid and application thereof.
Background
In the field of cell biology, it is common to refer to the study of cell culture and cell counting. The current method is a microscope direct counting method, which is a method of adding a small amount of a cell or microorganism suspension to be measured to a blood cell counting plate having a certain area and volume, directly counting under a microscope, and then estimating the number of cells or microorganisms in the culture solution, which is suitable for cells having a size of 3 to 100 μm, but is not suitable for cells having a size of more than 100 μm. Since the counting chamber size of the blood cell counting plate is only 1x 0.1mm, the height of the counting chamber is fixed, i.e. 0.1mm, limiting the maximum size of particles entering the counting chamber to not exceed 100 μm. Thus, when measuring the number of particles having a diameter of more than 100 μm, the blood cell counting plate will no longer be suitable.
At present, the biology field often needs to count particles with a diameter greater than 100 μm, such as microcarriers used in a stem cell large-scale culture process, the size of each microcarrier is generally 100-300 μm, the microcarriers are used as an auxiliary material necessary for an adherent cell suspension culture technology, and during the culture process, the number of microcarriers per unit volume needs to be known so as to better analyze the relationship between cell growth and the number of microcarriers in real time, separate out single microcarriers and record and analyze the growth state of cells in real time. In the existing method, microscopic photographing and image analysis software is generally used for estimating the number of particles with the size larger than 100 mu m in liquid, so that the method is time-consuming and low in efficiency, and the workload of scientific researchers is greatly increased.
Accordingly, there is a need to provide a method for determining the number of particles in a volume of liquid which allows counting of particles having a particle size of 100-500 μm, which is simple, efficient and low cost.
Disclosure of Invention
The present invention is directed to solving one or more of the problems of the prior art and providing at least one of a beneficial choice or creation of conditions. The invention provides a method for measuring the number of particles in a unit volume of liquid, which can count particles with the particle size of 100-500 mu m, and is simpler, more efficient and lower in cost.
The invention is characterized in that: the method comprises the steps of dyeing liquid to be detected by using a dyeing agent, dropwise adding the liquid to be detected and chromatographic separation liquid at one end of filter paper, dispersing the particles on the filter paper through chromatographic separation, comparing the dark particles with white filter paper, and counting the particles by naked eyes on the filter paper, wherein the number of the particles in the liquid to be detected in unit volume is calculated according to the volume of the liquid to be detected, so that the limit that the particle size of the particles which cannot be detected by a blood cell counter is larger than 100 mu m is broken through, and counting the particles with the particle size of 100-500 mu m can be realized.
Accordingly, in a first aspect the invention provides a method of determining the number of particles in a unit volume of liquid.
Specifically, the method comprises the following steps:
(1) The method comprises the steps of dyeing liquid to be detected by using a dyeing agent, sequentially dripping the dyed liquid to be detected and chromatographic separation liquid at one end of filter paper, dispersing particles in the liquid to be detected on the filter paper under the action of capillary chromatography, and counting the number of the particles in the liquid to be detected by naked eyes;
(2) And calculating the number of particles in the unit volume of liquid according to the volume of the liquid to be measured.
The liquid to be tested in the step (1) is to take a proper amount of liquid to be tested from the total volume of the liquid to be tested, and the total particle number in the whole liquid to be tested can be further calculated according to the particle number in the liquid per unit volume and the total volume of the liquid to be tested.
Preferably, in the step (1), if the particle concentration in the liquid to be measured is greater than or equal to 50 particles/10 mu L, diluting the liquid to be measured before dyeing; sequentially dripping diluted and dyed diluent and chromatographic separation liquid at one end of the filter paper, and dispersing particles in the diluent on the filter paper to obtain the number of particles in the diluent; and calculating the number of particles in the unit volume of the liquid to be measured according to the multiple of dilution, the volume of the diluent and the number of particles in the diluent.
Preferably, the coloring agent is any coloring agent capable of coloring particles; the coloring agent is at least one selected from trypan blue, crystal violet, hematoxylin, eosin, gentian violet, methyl green, congo red, methyl blue, sudan III, safranine, fast green, basic fuchsin, neutral red and magenta.
Specifically, the coloring agent dyes particles in the liquid to be measured into dark colors to obtain dark-colored particles, so that the number of the particles can be directly counted by naked eyes.
Preferably, in step (1), the concentration of the coloring agent is 0.4-4%; the dyeing time is 30-60s.
Specifically, in the actual measurement process, the concentration of the dye and the dyeing time can be adjusted correspondingly according to specific conditions, so long as the particles can be easily distinguished after dyeing.
Preferably, the particles are dispersed with a chromatographic separation liquid selected from at least one of water and an inorganic salt buffer.
Further preferably, the inorganic salt buffer is at least one selected from the group consisting of phosphate buffer, tris-HCl buffer, and citrate buffer.
Preferably, in step (1), the filter paper is filter paper.
Specifically, after the liquid to be detected and the chromatographic separation liquid are dripped at one end of the filter paper, particles in the liquid can be gradually dispersed through capillary chromatographic separation, and because the dark-colored particles and the white filter paper have clear contrast in color, macroscopic single particles can be obtained on the filter paper, so that the particles can be counted more simply and rapidly without a microscope or a counter.
Preferably, in the step (1), the particles are selected from any one of microcarriers, hydrogel microspheres, drug slow release particles and hemostatic powder particles.
The method of the present invention is not limited to determining the number of particles in a unit volume of liquid, but may provide an effective counting method for determining the number of particles in a unit mass of particles or powder, as long as the unit mass of powder is dispersed in a solvent to form a liquid in which the particles are uniformly dispersed.
Preferably, in step (1), the particles have a particle size of 100 to 500. Mu.m.
Specifically, the particles are particles that are freely and uniformly dispersible in a solvent.
Preferably, the liquid to be detected and the chromatographic separation liquid obtained in the step (1) are respectively and sequentially sucked by a pipette, and then are sequentially dripped at one end of the filter paper.
Specifically, the pipettor is a common instrument for molecular cell biology experiments, and has higher accuracy.
Preferably, in the step (1), if the concentration of particles in the liquid to be measured is greater than 50 particles/10 μl, the liquid to be measured is diluted with a solvent.
Preferably, the solvent is at least one selected from water and inorganic salt buffer.
Preferably, the inorganic salt buffer is at least one selected from phosphate buffer, tris-hydrochloric acid buffer and citrate buffer.
Specifically, the dilution factor can be determined according to the concentration of the liquid to be measured, and in general, when the concentration of the liquid to be measured is greater than 50/10 μl, the liquid to be measured needs to be diluted, because when the number of particles is large, counting by naked eyes is easy to make mistakes, and the time taken is long. In addition, for judging whether the liquid to be measured needs to be diluted or not and the multiple of the dilution, the liquid to be measured can be directly dyed and counted without being diluted, and if the number is found to be too large (more than 50/10 mu L), the particle number can be measured after the liquid to be measured is diluted by the corresponding multiple according to the estimated number.
Preferably, the addition amount of the coloring agent is 1-10% of the volume of the diluent.
Preferably, the concentration of the diluent is less than 50/10. Mu.L.
Specifically, the concentration of the diluted diluent is less than 50/10 mu L, so that the error in counting caused by excessive particle number can be avoided, and the accuracy of the visual measurement timing of the particle number can be ensured.
In a second aspect the invention provides the use of a method for determining the number of particles per volume of liquid in the field of cell biology.
Compared with the prior art, the technical scheme provided by the invention has the following beneficial effects:
(1) According to the method, the particle size of the counting object particles is larger than 100 mu m, the liquid to be measured is dyed by using the coloring agent, the particles in the liquid to be measured are dyed into dark color, then the liquid to be measured and the chromatographic separation liquid are dripped at one end of the filter paper, the particles are dispersed through the chromatographic separation effect, the dyed dark color particles are compared with the white filter paper, and the macroscopic single particles can be obtained on the filter paper, so that the particles can be simply and rapidly counted without a microscope and a counter. The measuring method of the invention does not need to purchase expensive instruments, does not need to photograph and use image analysis software to carry out auxiliary counting, breaks through the limitation that the blood cell counter cannot measure particles with the particle size larger than 100 mu m, can finish the measurement of the particle number through naked eye observation, and is simple, efficient and low in cost.
(2) The method of the invention not only can be used for measuring the number of particles in unit volume of liquid, but also can be used for providing an effective counting method for measuring the number of particles in unit mass of particles or powder, and has wider application range.
Drawings
FIG. 1 is a schematic diagram of the present invention;
FIG. 2 is a diagram showing the dilution and staining process of the liquid to be measured in example 1 of the present invention;
FIG. 3 is a distribution diagram of hydrogel microspheres before and after chromatographic separation of the diluent in example 1 of the present invention;
FIG. 4 is a chart showing microcarrier profiles of 6 random wells in a 96-well plate according to example 2 of the present invention;
FIG. 5 is a state diagram of a circular hydrophobic region on a comparative example 1 slide of the present invention;
FIG. 6 is a view showing a state after a dilution liquid is dropped into the water repellent region of comparative example 1 of the present invention;
FIG. 7 is a view showing the state of the cover slip of comparative example 1 after the diluted liquid is squeezed out of the water repellent region;
FIG. 8 is a state diagram showing that hydrogel microsphere particles of comparative example 1 of the present invention remain in the hydrophobic region;
FIG. 9 is a graph showing the microscopic distribution of hydrogel microspheres in the hydrophobic region of comparative example 1 of the present invention.
Detailed Description
In order to make the technical solutions of the present invention more apparent to those skilled in the art, the following examples will be presented. It should be noted that the following examples do not limit the scope of the invention.
The starting materials, reagents or apparatus used in the following examples are all available from conventional commercial sources or may be obtained by methods known in the art unless otherwise specified.
The principle of the invention is as follows: the dyed particles are dispersed on the filter paper by using a chromatographic separation method, the dyed dark particles and the white filter paper form clear contrast, and macroscopic single particles can be obtained on the filter paper, so that the particles can be simply and rapidly counted, and the schematic diagram of the invention is shown in figure 1.
Example 1
A method of determining the number of particles in a unit volume of liquid comprising the steps of:
(1) Preparing 3mL of a to-be-detected liquid containing hydrogel microspheres, a plurality of filter papers and a trypan blue coloring agent with the concentration of 4% (w/v), wherein the preparation method of the trypan blue coloring agent with the concentration of 4% (w/v) comprises the steps of adding 1mL of 1xPBS solution (phosphate buffer solution with 1-time concentration) into 0.04g of trypan blue solid, oscillating and mixing to obtain the preparation method;
(2) Adding 10 mu L of liquid to be measured into a 1.5mL centrifuge tube, then adding 80 mu L of water and 10 mu L of trypan blue dye solution with the concentration of 4% (w/v) obtained in the step (1) into the centrifuge tube to obtain a 10-fold diluted solution, wherein the specific dilution and the dyeing process are shown in figure 2;
(3) Cutting filter paper into a strip shape, dripping 10 mu L of the diluted solution obtained in the step (2) to one end of the filter paper, dripping 200 mu L of 1xPBS solution on the diluted solution, gradually dispersing the black-dyed hydrogel microspheres dyed by trypan blue coloring agent on the filter paper by utilizing the chromatographic separation effect, and displaying the distribution state of the hydrogel microspheres on the filter paper before and after chromatographic separation as shown in figure 3, wherein figure 3 (a) is a distribution state diagram of the hydrogel microspheres on the filter paper before chromatographic separation, and figure 3 (b) is a distribution state diagram of the hydrogel microspheres on the filter paper after chromatographic separation; after the particles are dispersed, the hydrogel microspheres dyed into black on the filter paper can be observed and counted by naked eyes;
(4) And (3) counting the diluted solution obtained in the step (2) for 5 times (unit/unit), taking an average value, wherein the number of hydrogel microspheres in the diluted solution is respectively 11, 17, 10, 19 and 14, and the average value is 14.2, namely the number of hydrogel microspheres in 10 mu L of diluted solution, and then calculating to obtain the number of hydrogel microspheres in the unit volume of liquid and the total number of hydrogel microspheres in the liquid to be tested according to the dilution multiple and the total volume of the liquid to be tested, wherein the calculation results of the number of the specific hydrogel microspheres are shown in the table 1.
TABLE 1
Example 2
In cell microcarrier suspension culture experiments, it is often necessary to isolate individual microcarriers according to the experimental requirements in order to observe the growth and migration of cells on the microcarrier surface. In this case, the method of the present invention may be used to obtain the number of microcarriers per unit volume of the culture medium, then the culture medium may be subjected to a precise dilution operation according to the number of microcarriers per unit volume of the culture medium, and then the diluted culture medium may be added to a multi-well plate. Due to the relative accuracy of dilution, each well in the well plate can have an average of one microcarrier, which is beneficial for subsequent investigation of cells on a single microcarrier.
Accordingly, there is provided the use of a method for determining the number of particles in a volume of liquid in cell culture, comprising the steps of:
(1) The number of particles in the liquid to be measured per unit volume was measured in the same manner as in example 1, specifically, 10-fold dilution was performed on the culture solution to obtain a diluted solution, 10. Mu.L of the diluted solution was respectively taken and then 5 times of counting (units/unit) were performed, an average was taken, the number of particles of the microcarriers was 41, 37, 53, 38, 40, respectively, the average was 41.8, i.e., the number of microcarriers in 10. Mu.L of the diluted solution was 41.8, and then the number of microcarriers in the culture solution per unit volume and the number of microcarriers in 1mL of the culture solution to be measured were calculated according to the dilution factor, and the calculation results of the number of particular microcarriers were shown in Table 2;
(2) mu.L of microcarriers was added to a volume of 5mL of culture medium such that there were only 1 microcarrier per 100. Mu.L of culture medium on average, and 100. Mu.L was the volume of culture medium required to be added to one well of a 96-well plate. Thus, the experimental results were obtained with an average of only one microcarrier per well by dividing 5mL of the dilution into a number of 100. Mu.L of separate wells, and the distribution of microcarriers in 6 random wells in a 96-well plate, as shown in FIG. 4.
TABLE 2
Comparative example 1
The liquids to be measured in comparative example 1 and example 1 were taken from the same sample and the volumes of the liquids to be measured were 3mL, and the number of particles in the liquid per unit volume was directly measured in comparative example 1 using a microscope, and the measurement method thereof comprises the steps of:
(1) Preparing a glass slide, a cover glass and an immunohistochemical pen, and drawing a circular hydrophobic area with the diameter of 1cm at the middle position of the glass slide by using the immunohistochemical pen, as shown in figure 5;
(2) Taking 10 mu L of liquid to be measured, adding 90 mu L of water, and obtaining a diluted solution after 10 times dilution;
(3) Taking 10 mu L of the diluent obtained in the step (2), and dripping the diluent into the middle position of a circular hydrophobic area on a glass slide, wherein a state diagram of the diluent in the hydrophobic area is shown in fig. 6, a cover glass is lightly covered on the glass slide, the diluent is observed to be extruded out of the hydrophobic area, as shown in fig. 7, and hydrogel microsphere particles still remain in the hydrophobic area, as shown in fig. 8;
(4) Placing the slide glass of the step (3) under a common microscope, and observing the distribution state of hydrogel microsphere particles in a hydrophobic area, as shown in fig. 9; and the hydrogel microsphere particles are respectively counted for 5 times (unit/unit), the average value is taken, the particle numbers are respectively 15, 16, 15, 17 and 14, the average value is 15.4, the number of the hydrogel microsphere particles in 10 mu L of diluent is 15.4, then the number of the hydrogel microsphere particles in the unit volume of liquid and the total number of the hydrogel microsphere particles in the liquid to be tested are calculated according to the dilution multiple and the total volume of the liquid to be tested, and the calculation results of the specific particle numbers are shown in table 3.
TABLE 3 Table 3
As can be seen from tables 1 and 3, when the number of hydrogel microspheres in the same sample is counted by the method of the invention and the method of directly counting by the microscope of comparative example 1, the numbers of hydrogel microsphere particles in each 10 mu L of diluent are 14.2 and 15.4 respectively, and the difference is not large, which indicates that the determination method of the invention can directly count the number of particles in the unit volume of liquid instead of the microscope and has the advantages of rapidness, simple operation and no need of instrument. In addition, when the microscope is adopted for direct counting, the volume of liquid sucked is 10 mu L, when the volume of liquid sucked is larger than the upper limit, hydrogel microsphere particles are easy to overflow the hydrophobic region, so that the number of the obtained particles is not the actual number of the particles, and the accuracy of final counting is affected.
The above embodiments are only for illustrating the technical solution of the present invention and not for limiting the scope of the present invention, and although the present invention has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that the technical solution of the present invention may be modified or substituted without departing from the spirit and scope of the technical solution of the present invention.
Claims (10)
1. A method for determining the number of particles in a volume of liquid comprising the steps of:
(1) The method comprises the steps of dyeing liquid to be detected by using a dyeing agent, sequentially dripping the dyed liquid to be detected and chromatographic separation liquid at one end of filter paper, dispersing particles in the liquid to be detected on the filter paper under the action of capillary chromatography, and counting the number of the particles in the liquid to be detected by naked eyes;
(2) And calculating the number of particles in the unit volume of liquid according to the volume of the liquid to be measured.
2. The method according to claim 1, wherein in step (1), if the particle concentration in the liquid to be measured is >50 particles/10 μl, the liquid to be measured is diluted before dyeing; sequentially dripping diluted and dyed diluent and chromatographic separation liquid at one end of the filter paper, and dispersing particles in the diluent on the filter paper to obtain the number of particles in the diluent; and calculating the number of particles in the unit volume of the liquid to be measured according to the multiple of dilution, the volume of the diluent and the number of particles in the diluent.
3. The method of claim 1, wherein in step (1), the stain is any stain capable of staining the particle, the stain being selected from at least one of trypan blue, crystal violet, hematoxylin, eosin, gentian violet, methyl green, congo red, methyl blue, sudan III, safranin, fast green, basic fuchsin, neutral red, and magenta.
4. The method of claim 1, wherein in step (1), the concentration of the dye is 0.4-4%; the dyeing time is 30-60s.
5. The method of claim 1, wherein in step (1), the particles are selected from any one of microcarriers, hydrogel microspheres, drug-eluting particles, and hemostatic powder particles.
6. The method of claim 5, wherein the particles have a particle size of 100-500 μm.
7. The method according to claim 1 or 2, wherein the particles are dispersed with a chromatographic separation liquid selected from at least one of water, inorganic salt buffer; the inorganic salt buffer solution is at least one selected from phosphate buffer solution, tris-hydrochloric acid buffer solution and citrate buffer solution.
8. The method according to claim 2, wherein the liquid to be tested is diluted with a solvent selected from at least one of water, an inorganic salt buffer; the inorganic salt buffer solution is at least one selected from phosphate buffer solution, tris-hydrochloric acid buffer solution and citrate buffer solution.
9. The method according to claim 2, wherein the amount of the dye added is 1-10% of the volume of the diluent.
10. Use of the method according to any one of claims 1-9 in the fields of metering tests, cell biology.
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