A method for determining the number of particles in a unit volume of liquid and applications thereof

Abstract

The application belongs to the technical field of cell culture, and particularly relates to a method for determining the number of particles in a unit volume of liquid and application. The method comprises the following steps: (1) using a dye to dye the liquid to be measured, and sequentially dropping the dyed liquid to be measured and a chromatographic separation liquid at one end of filter paper, the particles in the liquid to be measured are dispersed on the filter paper under the action of capillary chromatography, and the number of particles in the liquid to be measured is obtained by naked-eye counting; (2) according to the volume of the liquid to be measured, the number of particles in a unit volume of liquid is calculated. The determination method of the application does not need to purchase expensive instruments, does not need to take photos and use image analysis software for auxiliary counting, breaks through the limitation that a blood cell counter cannot determine particles with a particle size greater than 100 mu m, and can complete the determination of the number of particles by naked-eye observation, which is simple, efficient and low in cost.

Description

Technical Field

[0001] This invention belongs to the field of cell culture technology, and specifically relates to a method and application for determining the number of particles in a unit volume of liquid. Background Technology

[0002] In the field of cell biology, research typically involves cell culture and cell counting. Currently, the commonly used method is direct microscopic counting, which involves adding a small amount of the cells or microbial suspension to a hemocytometer with a defined area and volume, counting the cells directly under a microscope, and then estimating the number of cells or microorganisms in the culture medium. This method is suitable for cells with a size of 3–100 μm, but not for cells larger than 100 μm. This is because the counting chamber of the hemocytometer is only 1*1*0.1 mm in size, and the height of the counting chamber is fixed at 0.1 mm, limiting the maximum size of particles entering the counting chamber to no more than 100 μm. Therefore, the hemocytometer is no longer suitable for determining the number of particles with a diameter greater than 100 μm.

[0003] Currently, the biological field frequently requires counting particles larger than 100 μm in diameter. For example, microcarriers used in large-scale stem cell culture processes typically range in size from 100 to 300 μm. As an essential auxiliary material for adherent cell suspension culture, it's crucial to know the number of microcarriers per unit volume during the culture process to better analyze the relationship between cell growth and microcarrier quantity in real time, and to isolate individual microcarriers for real-time recording and analysis of cell growth status. Existing methods usually involve using microscopy and image analysis software to estimate the number of particles larger than 100 μm in liquids. These methods are not only time-consuming and inefficient, but also significantly increase the workload for researchers.

[0004] Therefore, there is an urgent need to provide a method for determining the number of particles in a unit volume of liquid. This method can count particles with a diameter of 100-500 μm and is simple, efficient, and low in cost. Summary of the Invention

[0005] This invention aims to solve one or more technical problems existing in the prior art, and at least provide a beneficial alternative or create conditions. This invention provides a method for determining the number of particles in a unit volume of liquid, which can count particles with a diameter of 100-500 μm, and is simpler, more efficient, and lower in cost.

[0006] The inventive concept of this invention is as follows: A dye is used to stain the test liquid, turning the particles in the liquid a dark color. Then, the test liquid and the chromatographic separation liquid are dropped onto one end of filter paper. Through chromatographic separation, the particles disperse on the filter paper. The dark-colored particles contrast with the white filter paper, resulting in visible individual particles on the filter paper, allowing for particle counting. Finally, based on the volume of the test liquid, the number of particles per unit volume of the test liquid is calculated. This invention overcomes the limitation of hematology counters, which cannot measure particles larger than 100 μm. It can count particles with a diameter of 100-500 μm, and is simpler, more efficient, and lower in cost.

[0007] Therefore, a first aspect of the present invention provides a method for determining the number of particles in a unit volume of liquid.

[0008] Specifically, the method includes the following steps:

[0009] (1) The test liquid is stained with a dye, and the stained test liquid and the chromatographically separated liquid are dropped onto one end of the filter paper in sequence. The particles in the test liquid are dispersed on the filter paper under the action of capillary chromatography, and the number of particles in the test liquid is obtained by visual counting.

[0010] (2) Calculate the number of particles per unit volume of liquid based on the volume of the liquid to be tested.

[0011] In step (1), the test liquid is an appropriate amount of the test liquid taken from the total volume of the test liquid. Based on the number of particles in the unit volume of liquid and the total volume of the test liquid, the total number of particles in the entire test liquid can be further calculated.

[0012] Preferably, in step (1), if the particle concentration in the test liquid is ≥50 particles / 10μL, the test liquid is further diluted before staining; the diluted and stained diluent and the chromatographic separation liquid are sequentially dropped onto one end of the filter paper, and the particles in the diluent are dispersed on the filter paper to obtain the number of particles in the diluent; the number of particles in a unit volume of the test liquid is calculated based on the dilution factor, the volume of the diluent and the number of particles in the diluent.

[0013] Preferably, the dye is any dye capable of coloring particles; the dye is selected from at least one of trypan blue, crystal violet, hematoxylin, eosin, gentian violet, methyl green, Congo red, methylene blue, Sudan III, safranin, fast green, basic fuchsin, neutral red, and magenta.

[0014] Specifically, the dye stains the particles in the test liquid into a dark color, making it easier to count the number of particles directly with the naked eye.

[0015] Preferably, in step (1), the concentration of the dye is 0.4-4% and the dyeing time is 30-60s.

[0016] Specifically, in the actual measurement process, the concentration of the dye and the dyeing time can be adjusted according to the specific situation, as long as the particles can be easily distinguished after dyeing.

[0017] Preferably, the particles are dispersed using a chromatography separation liquid, wherein the chromatography separation liquid is selected from at least one of water and inorganic salt buffer solution.

[0018] More preferably, the inorganic salt buffer is selected from at least one of phosphate buffer, Tris-hydrochloric acid buffer, and citrate buffer.

[0019] Preferably, in step (1), the filter paper is a filter paper for filtration.

[0020] Specifically, when the test liquid and the chromatographic separation liquid are dropped onto one end of the filter paper, the particles in the liquid will gradually disperse through capillary chromatographic separation. Because the dark-colored particles and the white filter paper have a sharp color contrast, individual particles that are visible to the naked eye can be obtained on the filter paper, making it easier and faster to count the particles without the need for a microscope or counter.

[0021] Preferably, in step (1), the particles are selected from any one of microcarriers, hydrogel microspheres, drug sustained-release particles, and hemostatic powder particles.

[0022] The method of the present invention is not limited to determining the number of particles in a unit volume of liquid, but can also provide an effective counting method for determining the number of particles in a unit mass of particles or powder, simply by dispersing a unit mass of powder in a solvent to form a liquid in which the particles are uniformly dispersed.

[0023] Preferably, in step (1), the particle size is 100-500 μm.

[0024] Specifically, the particles are particles that can be freely and uniformly dispersed in a solvent.

[0025] Preferably, the test liquid and the chromatography separation liquid obtained in step (1) are sequentially aspirated using a pipette and then sequentially added to one end of the filter paper.

[0026] Specifically, pipettes are commonly used instruments in molecular and cell biology experiments and have high accuracy.

[0027] Preferably, in step (1), if the particle concentration in the liquid to be tested is greater than 50 particles / 10μL, the liquid to be tested is diluted with a solvent.

[0028] Preferably, the solvent is selected from at least one of water and inorganic salt buffer solution.

[0029] Preferably, the inorganic salt buffer is selected from at least one of phosphate buffer, Tris-hydrochloric acid buffer, and citrate buffer.

[0030] Specifically, the dilution factor can be determined based on the concentration of the test liquid. Generally, when the concentration of the test liquid is greater than 50 particles / 10μL, the test liquid needs to be diluted because counting particles visually when the number of particles is large is prone to errors and is time-consuming. Alternatively, to determine whether the test liquid needs dilution and the dilution factor, the test liquid can be stained and counted directly without dilution. If the number is found to be excessive (exceeding 50 particles / 10μL), the test liquid can be diluted again by the appropriate factor based on the estimated number before measuring the particle count.

[0031] Preferably, the amount of the dye added accounts for 1-10% of the volume of the diluent.

[0032] Preferably, the concentration of the diluent is less than 50 cells / 10 μL.

[0033] Specifically, the concentration of the diluted solution is less than 50 particles / 10μL, which can avoid errors in counting due to an excessive number of particles and ensure the accuracy of particle count determination.

[0034] A second aspect of the present invention provides an application of a method for determining the number of particles in a unit volume of liquid in the field of cell biology.

[0035] Compared with the prior art, the beneficial effects of the technical solution provided by the present invention are as follows:

[0036] (1) The method of this invention is for counting particles with a diameter greater than 100 μm. A staining agent is used to stain the test liquid, turning the particles in the liquid a dark color. Then, the test liquid and the chromatographic separation liquid are dropped onto one end of filter paper. Through chromatographic separation, the particles are dispersed. The dark-stained particles contrast with the white filter paper, allowing for the observation of individual particles visible to the naked eye. This enables simple and rapid particle counting without the need for a microscope or counter. This method eliminates the need for expensive instruments, photography, and image analysis software for auxiliary counting, overcoming the limitation of hematology counters in detecting particles larger than 100 μm. The particle count can be determined by visual observation, making it simple, efficient, and low-cost.

[0037] (2) The method of the present invention can not only determine the number of particles in a unit volume of liquid, but also provide an effective counting method for determining the number of particles in a unit mass of particles or powder, and its application scope is relatively wide. Attached Figure Description

[0038] Figure 1 This is a schematic diagram of the invention;

[0039] Figure 2 This is a process diagram of the dilution and staining of the test liquid in Example 1 of the present invention;

[0040] Figure 3 This is a diagram showing the distribution of hydrogel microspheres before and after chromatographic separation using the dilution solution in Example 1 of the present invention.

[0041] Figure 4 This is a microcarrier distribution diagram of 6 random wells in a 96-well plate in Embodiment 2 of the present invention;

[0042] Figure 5 This is a diagram showing the state of the circular hydrophobic region on the glass slide of Comparative Example 1 of the present invention;

[0043] Figure 6 This is a diagram showing the state of the hydrophobic region after adding diluent to Comparative Example 1 of the present invention;

[0044] Figure 7 This is a diagram showing the state of the diluent after it was squeezed out of the hydrophobic area after the coverslip was placed on Comparative Example 1 of the present invention;

[0045] Figure 8 This is a diagram showing the state of the hydrogel microspheres retained in the hydrophobic region in Comparative Example 1 of the present invention;

[0046] Figure 9 This is a microscopic distribution diagram of the hydrogel microspheres in the hydrophobic region of Comparative Example 1 of the present invention. Detailed Implementation

[0047] To enable those skilled in the art to more clearly understand the technical solutions described in this invention, the following embodiments are provided for illustration. It should be noted that the following embodiments do not constitute a limitation on the scope of protection claimed by this invention.

[0048] Unless otherwise specified, the raw materials, reagents or devices used in the following examples are available from conventional commercial sources or can be obtained by existing known methods.

[0049] The principle of this invention is as follows: Chromatographic separation is used to disperse the dyed particles on filter paper. The dark-colored particles and the white filter paper create a sharp contrast, resulting in individual particles visible to the naked eye on the filter paper. This allows for simple and rapid particle counting. A schematic diagram of this invention is shown below. Figure 1 As shown.

[0050] Example 1

[0051] A method for determining the number of particles in a unit volume of liquid includes the following steps:

[0052] (1) Prepare 3 mL of test liquid containing hydrogel microspheres, several filter papers, and 4% (w / v) trypan blue staining agent. The 4% (w / v) trypan blue staining agent is prepared by adding 1 mL of 1xPBS solution (1-fold diluted phosphate buffer) to 0.04 g trypan blue solid, shaking and mixing.

[0053] (2) Add 10 μL of the test liquid to a 1.5 mL centrifuge tube, then add 80 μL of water and 10 μL of trypan blue solution with a concentration of 4% (w / v) obtained in step (1) to the centrifuge tube to obtain a 10-fold diluted solution. The specific dilution and staining process is as follows: Figure 2 As shown;

[0054] (3) Cut the filter paper into strips. Add 10 μL of the diluent obtained in step (2) to one end of the filter paper. Then add 200 μL of 1xPBS solution to the diluent. Using chromatographic separation, the hydrogel microspheres stained black by trypan blue gradually disperse on the filter paper. The distribution of the hydrogel microspheres on the filter paper before and after chromatographic separation is as follows: Figure 3 As shown, where, Figure 3 (a) is a diagram showing the distribution of hydrogel microspheres on filter paper before chromatographic separation. Figure 3 (b) is a diagram showing the distribution of hydrogel microspheres on filter paper after chromatographic separation; after the particles are dispersed, the hydrogel microspheres that are stained black on the filter paper can be observed and counted with the naked eye;

[0055] (4) The diluent obtained in step (2) was counted 5 times (unit / piece), and the average value was taken. The number of hydrogel microspheres in the diluent was 11, 17, 10, 19 and 14, respectively, with an average value of 14.2, which is the number of hydrogel microspheres in 10 μL of diluent. Then, based on the dilution factor and the total volume of the liquid to be tested, the number of hydrogel microspheres in a unit volume of liquid and the total number of hydrogel microspheres in the liquid to be tested were calculated. The specific calculation results of the number of hydrogel microspheres are shown in Table 1.

[0056] Table 1

[0057]

[0058] Example 2

[0059] In cell microcarrier suspension culture experiments, it is usually necessary to isolate individual microcarriers according to experimental requirements to observe cell growth and migration on the microcarrier surface. In this case, the method of this invention can be used to first determine the number of microcarriers per unit volume of culture medium. Then, the culture medium is precisely diluted according to the number of microcarriers per unit volume of culture medium before being added to a multi-well plate. Due to the relative accuracy of the dilution, each well in the plate can contain an average of one microcarrier, which is beneficial for subsequent studies of cells on individual microcarriers.

[0060] Therefore, a method for determining the number of particles per unit volume of liquid is provided for application in cell culture, comprising the following steps:

[0061] (1) The number of particles in a unit volume of the test liquid was determined using the same method as in Example 1. Specifically, the culture medium was diluted 10 times to obtain a diluted solution. 10 μL of the diluted solution was taken and counted 5 times (unit / particle). The average value was taken. The number of microcarrier particles were 41, 37, 53, 38 and 40, with an average value of 41.8. That is, the number of microcarriers in 10 μL of diluted solution was 41.8. Then, based on the dilution factor, the number of microcarriers in a unit volume of culture medium and the number of microcarriers in 1 mL of the test culture medium were calculated. The specific calculation results of the number of microcarriers are shown in Table 2.

[0062] (2) Take 12 μL of microcarrier and add it to 5 mL of culture medium, so that there is only one microcarrier per 100 μL of culture medium on average. 100 μL is the volume of culture medium required to add to one well of a 96-well plate. Therefore, by dividing the 5 mL dilution into many 100 μL portions and adding them to different wells, the experimental result of having only one microcarrier per well on average can be obtained. The distribution of microcarriers in the 6 random wells of the 96-well plate is as follows: Figure 4 As shown.

[0063] Table 2

[0064]

[0065] Comparative Example 1

[0066] In Comparative Example 1 and Example 1, the test liquids were taken from the same sample, and the volume of the test liquid was 3 mL in both cases. In Comparative Example 1, the number of particles per unit volume of liquid was directly determined using a microscope. The determination method included the following steps:

[0067] (1) Prepare a glass slide, a coverslip, and an immunohistochemistry pen. Use the immunohistochemistry pen to draw a circular hydrophobic area with a diameter of 1 cm in the center of the glass slide, as shown below. Figure 5 As shown;

[0068] (2) Take 10 μL of the liquid to be tested and add 90 μL of water to obtain a 10-fold diluted solution;

[0069] (3) Take 10 μL of the diluent obtained in step (2) and drop it onto the center of the circular hydrophobic region on the glass slide. The state of the hydrophobic region after adding the diluent is shown in the figure below. Figure 6 As shown, by gently covering the slide with a coverslip, it can be observed that the diluent is squeezed out of the hydrophobic area, as... Figure 7 As shown, the hydrogel microspheres remain within the hydrophobic region, as... Figure 8 As shown;

[0070] (4) By placing the slide from step (3) under a regular microscope, the distribution of hydrogel microspheres in the hydrophobic region can be observed, such as... Figure 9 As shown in the figure, the hydrogel microspheres were counted 5 times (unit / particle), and the average number of particles was taken. The particle counts were 15, 16, 15, 17, and 14, with an average of 15.4 particles. The number of hydrogel microspheres in 10 μL of diluted solution was 15.4. Then, based on the dilution factor and the total volume of the liquid to be tested, the number of hydrogel microspheres per unit volume of liquid and the total number of hydrogel microspheres in the liquid to be tested were calculated. The specific particle count calculation results are shown in Table 3.

[0071] Table 3

[0072]

[0073] As shown in Tables 1 and 3, when counting the number of hydrogel microspheres in the same sample using the method of the present invention and the direct microscopic counting method of Comparative Example 1, the number of hydrogel microsphere particles per 10 μL of dilution was 14.2 and 15.4, respectively, which is not significantly different. This indicates that the determination method of the present invention can replace the microscope to directly calculate the number of particles in a unit volume of liquid, and has the advantages of being fast, simple to operate, and requiring no instruments. In addition, when using the microscope for direct counting, there is an upper limit of 10 μL for the volume of liquid absorbed. When the volume of liquid absorbed is greater than the upper limit, it is easy for hydrogel microsphere particles to overflow the hydrophobic region, so that the number of particles obtained is not the actual number of particles, affecting the accuracy of the final count. However, the determination method of the present invention does not have an upper limit on the liquid volume, and it is more conducive to improving the accuracy of counting when absorbing a larger volume for measurement. The determination method of the present invention has a wider range of applications than the microscope counting method.

[0074] The above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the essence and scope of the technical solutions of the present invention.

Claims

1. A method for determining the number of particles in a unit volume of liquid, characterized in that, Includes the following steps: (1) The test liquid is stained with a dye, and the stained test liquid and the chromatographic separation liquid are dropped onto one end of the filter paper in sequence. The particles in the test liquid are dispersed on the filter paper under the action of capillary chromatography, and the number of particles in the test liquid is obtained by visual counting; wherein, the chromatographic separation liquid is used to disperse the particles. (2) Calculate the number of particles per unit volume of liquid based on the volume of the liquid to be tested.

2. The method according to claim 1, characterized in that, In step (1), if the particle concentration in the test liquid is >50 particles / 10μL, the test liquid is further diluted before staining; the diluted and stained diluent and the chromatographic separation liquid are sequentially dropped onto one end of the filter paper, and the particles in the diluent are dispersed on the filter paper to obtain the number of particles in the diluent; the number of particles in a unit volume of the test liquid is calculated based on the dilution factor, the volume of the diluent and the number of particles in the diluent.

3. The method according to claim 1, characterized in that, In step (1), the dye is any dye that can color the particles, and the dye is selected from one of trypan blue, crystal violet, hematoxylin, eosin, gentian violet, methyl green, Congo red, methylene blue, Sudan III, safranin, fast green, basic fuchsin, neutral red and magenta.

4. The method according to claim 1, characterized in that, In step (1), the concentration of the staining agent is 0.4-4%; the staining time is 30-60s.

5. The method according to claim 1, characterized in that, The particle size is 100-500 μm.

6. The method according to claim 1 or 2, characterized in that, The chromatography separation liquid is selected from water and inorganic salt buffer; the inorganic salt buffer is selected from phosphate buffer, Tris-hydrochloric acid buffer, and citrate buffer.

7. The method according to claim 2, characterized in that, The test liquid is diluted with a solvent selected from water and inorganic salt buffer; the inorganic salt buffer is selected from phosphate buffer, Tris-hydrochloric acid buffer, and citrate buffer.

8. The method according to claim 2, characterized in that, The amount of the dye added is 1-10% of the volume of the diluent.

9. The application of the method according to any one of claims 1-8 in the fields of metrology and cell biology.

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