JPH08163997A - Quick testing method of animal tissue number and testing kit therefor - Google Patents

Abstract

PURPOSE: To quickly and simply test animal tissue numbers in a specimen from colored degree of the animal by dying an animal tissue in a specimen with a dilute dying solution and gathering colored animal tissues into a hydrophobic filter with an aspirating filtration or a press filtration. CONSTITUTION: In a testing method of animal tissue numbers in body tissues, etc., of a dairy cattle in a specimen of cow's milk, etc., square polyethylene films 6 respectively having a round hole 8 of a diameter of 3mm are expanded at top and bottom of a plastic filtering tool 1 and a hydrophobic filter 7 made of polypropylene having a diameter of 13mm is inserted between the two films so as to close the holes 8, hooked with a double clip, a filtering vehicle A is fitted on a fitting part 2 of an upper side filtering tool 1A. A specimen and a dilute dying solution (e.g. methyl green) is poured between the films and animal tissues are dyed, the dyed animal tissues are gathered in the hydrophobic filter 7 by an aspirating filtration or a press filtration, then animal tissue numbers in the specimen for diagnosis of mastitis of a dairy cattle are quickly and simply tested from colored degree of animal tissues.

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to a method for rapidly and simply measuring the number of animal cells in a sample by a one-step filtration operation without the need for advanced technology or specialized knowledge. Regarding measurement kit. INDUSTRIAL APPLICABILITY The present invention can be effectively used in a wide range of fields including the field of animal husbandry, the field of urinalysis (diagnosis), the field of hybridoma culture (field of monoclonal antibody production).

[0002]

2. Description of the Related Art Conventionally, a kit (PL tester) for measuring animal cells in a sample has been commercially available. This is a method for determining pH and animal cells from aggregation, but has the drawback of poor accuracy. In addition, although a device (fluorescence optical method) for measuring animal cells in a sample is commercially available, there is a problem that the device is expensive and cannot be used on site.

The applicant of the present invention has previously solved such drawbacks of the prior art, does not require special equipment or specialized knowledge, and is quick (within 30 seconds) and simple in a sample. A method for measuring the number of animal cells and a measuring kit therefor are provided (Japanese Patent Laid-Open No. 6-253893). This method, when measuring the number of animal cells in the sample, the animal cells are collected by suction filtration on a hydrophobic filter and stained, or the animal cells are stained and then collected by suction filtration on a hydrophobic filter, Then, excess dye is removed by washing, and the number of animal cells in the sample is measured from the degree of coloring of animal cells.

According to this method, it becomes possible to measure the number of animal cells in a sample quickly and simply, without requiring high-level technology or specialized knowledge. However, in the above method, since a dyeing solution having a normal concentration is used, the excess dye must be removed by washing after being collected on the hydrophobic filter, which requires a two-step operation. When actually used, there was a problem that the operation was complicated.

The present inventors have conducted extensive studies in order to further simplify the method for measuring the number of animal cells in the above-mentioned sample.
It has been found that the use of a diluted staining solution for staining animal cells enables sufficient staining while omitting the washing step after filtration, and based on this finding, the present invention has been completed. .

[0006]

[Means for Solving the Problems] That is, the present invention provides a method for measuring the number of animal cells in a sample, in which the animal cells are stained with a dilute stain solution and then stained with a hydrophobic filter by suction filtration or pressure filtration. The present invention provides a rapid method for measuring the number of animal cells, which comprises collecting the animal cells and measuring the number of animal cells in the sample from the degree of coloring of the animal cells.

Such a method of the present invention can be easily carried out by using a kit for measuring the number of animal cells, which comprises a hydrophobic filter, a filtering tool to which the hydrophobic filter can be attached, a dilute staining solution and a color control table. can do.

The sample to which the present invention can be applied is not particularly limited as long as it contains animal cells. Specific examples include animal body fluids (blood, lymph, etc.), excrements (urine, etc.), raw milk, and the like. The animal cells targeted by the present invention are not particularly limited. For example, white blood cells in urine or white blood cells,
Targets are red blood cells. Furthermore, hybridomas and the like in the culture medium formed by cell fusion can also be the subject of the present invention. For example, when raw milk of dairy cow is used as a sample, somatic cells of dairy cow present in the raw milk can be mentioned as animal cells. According to the method of the present invention, for example, the somatic cell count of dairy cows in raw milk of such dairy cows can be measured quickly and simply, and can be effectively used for diagnosing mastitis in dairy cows. In addition, since the pore size of the hydrophobic filter may be selected according to the size of the target animal cell, the size of these animal cells is not particularly limited, but the present invention particularly has a diameter of about 5 to 15 μm. Can be effectively used for the determination of animal cells.

In the present invention, first, animal cells in a sample are stained with a dilute stain solution, and then suction filtration or pressure filtration is performed to
Collect the stained animal cells on a hydrophobic filter. Specifically, the diluted staining solution and the sample are placed in a filter container equipped with a filter equipped with a hydrophobic filter (animal cells in the sample are stained at the same time when they are in contact with each other), and then suction filtration or addition is performed. The animal cells stained with the hydrophobic filter are collected by pressure filtration. As the staining method, there are a method of putting the sample in the diluted staining solution and a method of putting the diluted staining solution in the sample, and either method can be used, but in the embodiment of the present invention, the sample is placed in the diluted staining solution. Was added.

As the diluted staining solution, a staining solution containing a dye that can be used for staining animal cells is used. Examples of the dye include methylene blue, methyl green, and fuchsin, and methylene blue and methyl green are particularly preferable.

In the present invention, the above dye is used as a dye solution (staining solution) using water or various buffer solutions. The usable pH of the buffer solution is 2 to 7, preferably pH 3 to 5. If necessary, a preservative such as ethanol or a surfactant may be added. When ethanol is added to the above dye solution as a preservative, 1
˜20% (v / v) is added, and when a surfactant is added, the amount of the surfactant added is 0.0001 to 1.0% (w
/ V).

Here, the concentration of the dyeing solution is lower than the concentration generally used for dyeing, and usually 0.00001 to 0.02.
%, Preferably 0.0001 to 0.01%, more preferably 0.
0002 to 0.002%. Here, the concentration of the staining solution is 0.0002
If it is less than 0.1%, coloring will be insufficient, while if the concentration of the dyeing solution exceeds 0.02%, excess dye must be washed, which is not preferable. In the case of methyl green staining, the concentration of the staining solution was 0.001%.

The ratio of the dye liquid amount to the sample liquid amount is 1 or more, preferably 5 or more. If the ratio of the dye liquid amount to the sample liquid amount is less than 1, coloring is insufficient, which is not preferable. In the case of methyl green staining, the dye liquid amount ratio per sample liquid amount was set to 10.

After the animal cells in the sample are stained with the diluted stain solution as described above, the hydrophobically-filtered animal cells are collected by suction filtration or pressure filtration. Specifically, a diluting dyeing solution and a sample are put into a filter container equipped with a filter equipped with a hydrophobic filter to stain animal cells in the sample, and then the hydrophobic filter is obtained by suction filtration or pressure filtration. The animal cells stained with are collected. In the present invention, a hydrophobic filter is used as a filter for collecting animal cells. Examples of the hydrophobic filter include hydrophobic filters such as nylon filters, fluorine filters such as polytetrafluoroethylene (tetrafluoroethylene resin), and polyolefin filters such as polypropylene. Among these filters, a hydrophobic filter made of polytetrafluoroethylene or polypropylene is particularly preferable because it allows bacteria and proteins in the sample to pass through. Hydrophilic filters are not preferred because they adsorb bacteria and proteins. However, if a hydrophilic material such as a nitrocellulose filter is made hydrophobic by surface treatment, it can be used as a hydrophobic filter. Here, the surface treatment is coating or the like, and as the material used for coating, the above-mentioned nylon-based, fluorine-based, or polyolefin-based materials can be used.

The filtration pore size of this hydrophobic filter may be appropriately selected according to the type of the target animal cell. For example, somatic cells (about 5 to 20 μm) in raw milk of dairy cows are collected by the hydrophobic material. In that case, it is preferable to use a filter having a pore size of 3 to 10 μm. Moreover, the size of the suction or pressure filtration area (area of the colored region) of the hydrophobic filter is not particularly limited as long as it is easy to see. The shape of the portion to be suctioned or filtered under pressure is not particularly limited, but a circular shape is preferable from the viewpoint of visibility, and a circular shape having a diameter (diameter) of about 2 to 7 mm is preferable. The size of the entire hydrophobic filter is not particularly limited. In addition, the shape of the entire hydrophobic filter is preferably circular in terms of visibility, and its diameter (diameter)
Can be arbitrarily selected, but one having a thickness of about 13 to 25 mm is particularly preferable.

As the color of the hydrophobic filter, a color that can be easily determined may be set in consideration of the dye used. A white hydrophobic filter is preferable for easily determining the degree of coloring. A transparent or semi-transparent hydrophobic filter can also be used, and the determination becomes easy when the hydrophobic filter is placed on a white sheet for determination.

Usually, a diluting dyeing solution and a sample are put in a filtration container equipped with a filtering tool having the above-described hydrophobic filter to stain animal cells in the sample. The filtering tool is not particularly limited, but, for example, one having a shape as shown in FIG. 1 is used. FIG. 1 is a front view showing one embodiment of a filtering tool used in the method of the present invention, and FIG. 2 is a plan view thereof.

In the figure, reference numeral 1 is a filtering tool, reference numeral 1A is the upper surface side of the filtering tool, and reference numeral 1B is the lower surface side of the filtering tool. Reference numeral 2 is a mounting portion provided on the upper surface side 1A of the filtering tool, and a filtering container (for example,
The tip of a syringe capable of pressure filtration or a suction filtration device capable of suction filtration is attached. A state in which the filtration container (a syringe in the figure) A is thus mounted is shown in FIG.
The filtering tool 1 is usually made of plastic or the like, and the shape thereof is not particularly limited, and various shapes such as a quadrangular shape (shape when viewed from a plane) as shown in the drawing and a circular shape can be used. Any thing can be used. The upper surface side 1A and the lower surface side 1B of the filter usually have substantially the same shape as illustrated. A through hole 3 is provided through the mounting portion 2 on the upper surface side 1A of the filtering tool. Also, reference numeral 4
Is a discharge or suction unit provided on the lower surface side 1B of the filter, and is a discharge unit in the case of pressure filtration and a suction unit in the case of suction filtration. Also on the lower side 1B of the filtering tool,
Through holes 5 are provided through the discharge or suction unit 4.
Further, reference numeral 6 is a polyethylene sheet, and reference numeral 7 is a hydrophobic filter, which is inserted between the upper surface side 1A and the lower surface side 1B of the filtering tool so as to close the hole 8 formed in the polyethylene sheet 6. It is used by fastening with a double clip. As a result, a passage for the dyeing solution or the sample is formed from the through hole 3 in the mounting portion 2 on the upper surface side 1A of the filtering tool to the through hole 5 in the discharging or suction portion 4 on the lower surface side 1B of the filtering tool. .

As shown in FIG. 3, a dilute staining solution and a sample are put into a filtration container A, for example, a syringe, in which the filtration tool 1 having the hydrophobic filter 7 as described above is mounted.
After staining the animal cells in the sample, suction filtration or pressure filtration is performed without performing a washing operation. As a result, the stained animal cells are collected by the hydrophobic filter 7.

In the method of the present invention, since the dilute staining solution is used as described above, the dye does not become excessive, and the animal cells in the sample can be effectively stained without the usual washing operation. However, it is possible to measure the number of animal cells in the sample. The suction filtration or the pressure filtration is preferably performed after stirring the mixed solution of the diluted staining solution and the animal cell sample. The filtration method may be pressure filtration or suction filtration. With the syringe, either pressure filtration or suction filtration can be performed.

In the case of suction filtration, usually, a hydrophobic filter for collecting animal cells is attached to a predetermined suction filter, and the sample and the diluted staining solution are put into this suction filter, and then suction filtration is performed. To collect the stained animal cells on the hydrophobic filter. Here, the suction filter is usually composed of a filter equipped with a hydrophobic filter for collecting animal cells and mainly responsible for filtration, and a suction tool mainly responsible for suction. It is possible to use a combination of a suction pump and a funnel type filter which can be fitted with a collecting hydrophobic filter. Also,
A vacuum line may be used instead of the suction pump.

The number of animal cells in the sample is determined from the degree of coloring of the animal cells in the sample collected and stained by the hydrophobic filter in this manner. For the measurement of the number of animal cells, it is most convenient to visually compare (1) a color control table, but (2) by a colorimetric quantification method by optical density (OD) measurement. You can also do it. In the case of visual judgment, not only the front surface of the hydrophobic filter,
It can also be measured from the degree of coloring on the back surface. In these cases, a color control table for each and a calibration curve for the absorbance and the number of animal cells may be prepared.

The above-mentioned (1) visual measurement of the number of animal cells is carried out by specifically measuring the degree of coloring of the animal cells present on the hydrophobic filter, that is, the intensity of the color, using a sample having a known number of animal cells. It may be performed by comparing with a color comparison table prepared in advance. The color control table uses a sample in which the number of animal cells is known, and takes a color photograph of the hydrophobic filter stained by the method of the present invention, or a filter paper or the like is used in the same degree as the stained hydrophobic filter. It can be created by coloring or printing the same color on paper. For example, when measuring animal cells in raw milk, a color control table is prepared as follows. That is, for example, a hybridoma flask culture solution (GI
The number of cells in T medium for 3 days was calculated by the hemocytometer method ("Tissue culture technology", 2nd edition, page 25, Asakura Shoten, 1988), and after centrifugation, 10 ml of milk was added. When the number of cells is 100,000 cells / ml,
Dispense the culture solution into a centrifuge tube at about 300,000 cells / ml and about 500,000 cells / ml, centrifuge at 1500 rpm for 5 minutes, then discard the supernatant and shake the centrifuge tube to loosen the cells. Then add 10 ml of milk to each centrifuge tube
Then, three kinds of standard samples may be prepared.

When using the color comparison table as described above,
The hydrophobic filter described above, a filter that can be fitted with the hydrophobic filter, a diluted staining solution, a color control table, and a sampling pipette, if necessary, are further combined to rapidly and rapidly determine the number of animal cells in the sample. It can be a measurement kit that can be easily measured. The filtering tool is not particularly limited as long as it can be filtered in combination with a hydrophobic filter. Further, as the material, either glass or plastic can be used. Furthermore, the hydrophobic filter and this filter can be integrated.

Further, the optical density (OD) of the above (2).
The colorimetric quantification by measurement is performed by eluting a dye coloring the animal cells present on the hydrophobic filter with an organic solvent, measuring the degree of coloring of the eluate by absorbance, and preparing an optical density (OD) prepared in advance. )) And the number of animal cells to be applied for quantification. The wavelength at the time of measuring the absorbance may be appropriately determined depending on the dye used. As the organic solvent, various alcohols can be used, but ethanol is particularly preferable.

The calibration curve may be created, for example, as follows. That is, when methyl green was used as the dye, the degree of coloring of the sample diluted to various concentrations was measured by using a microplate reader.
The optical density (OD) of 2 nm was used for measurement, while the number of animal cells in the sample solution of the same sample diluted with various concentrations was calculated by the above-mentioned blood cell counter method. A calibration curve of animal cell number and optical density may be created from the results of both.

As mentioned above, since the method of the present invention is simple, it is easy to automate the measurement. Specifically, for example, a cell harvester that collects cells labeled with a radioisotope on a filter paper can be used, and the filter paper is colored in substantially the same manner except that the cells are stained with a dye instead of labeling with the radioisotope. The collected cells can be collected. If a 96-well plate is used, it is possible to process 96 samples at a time.

Automation is also possible when measuring with optical density. That is, if the measurement and recording of the optical density are automated by a microplate reader or the like, the error due to the individual difference is reduced and it is convenient for management. Furthermore, when measuring a large number of samples, it is possible to measure by using a conventional dot blotter and changing the hydrophilic filter to a hydrophobic filter.

[0029]

EXAMPLES Next, the present invention will be described in detail with reference to Examples. Example 1 (1) Preparation of Color Control Table The number of cells in the hybridoma flask culture solution (cultivated in GIT medium for 3 days) was determined by the hemocytometer method (“Tissue culture technology”, 2nd edition, page 25, Asakura Shoten). , 1988), and when 10 ml of milk was added after centrifugation, the number of cells was 100,000 cells / ml or less, 100,000 cells / m 2, respectively.
The culture solution was dispensed into a centrifuge tube at about 1, 300,000 cells / ml, 500,000 cells / ml, 500,000 cells / ml or more, and centrifuged at 1500 rpm for 5 minutes. After centrifugation, discard the supernatant, shake the centrifuge tubes to loosen the cells, and add 10 ml of milk to each centrifuge tube.
A variety of standard samples were prepared.

Hybridomas were stained and collected using the filter 1 and the hydrophobic filter 7 shown in FIGS. That is, the plastic filter 1 has a diameter of 3 m.
As shown in the figure, a square polyethylene membrane (polyethylene sheet 6) having a side of 2 cm with a circular hole 8 of m is placed up and down, and a polypropylene filter having a diameter of 13 mm (filtering pore diameter 7 μm) (hydrophobic) Filter 7)
Was inserted so as to close the hole 8 and fastened with a double clip.

After the syringe (filtration container A) was attached to the mounting portion 2 of the filtration tool 1A on the upper surface side, 2 ml of methyl green dyeing solution having a dye concentration of 0.001% and 200 μm of the standard sample were placed in the syringe (filtration container A). After putting in and stirring lightly, the syringe of the syringe was pushed (pressurized), the aqueous solution was pushed out from the sample discharge part (discharge or suction part 5), and the stained hybridoma was collected on the hydrophobic filter 7. The hydrophobic filter 7 was taken out from the filter tool 1 and the degree of coloring was visually observed, and the results are shown in Table 1 below. In addition, 2 ml of methyl green with a dye concentration of 0.001% and 200 μl of a standard sample were put in a test tube in advance, and after agitating, aspirating from a sample aspirating part (discharging or aspirating part 5) with a syringe (filter container A) ( The results were exactly the same, even if suction filtration was used instead of pressure filtration.

[0032]

[Table 1]

These filters were color photographed as standard samples and used as a color control table.

(2) Determination of the number of animal cells in raw milk For 5 samples of raw milk, the same operation was carried out using the pressurizing method with a syringe as in the case of preparing the color control table of (1) above, and the hydrophobicity was measured. The degree of coloration of the animal cells present on the sex filter 7 was visually observed and compared with the color control table prepared in (1) above to make a visual determination. The results are shown in Table 2 together with the number of animal cells measured by the hemocytometer method. The calculation of the animal cells in the raw milk by the blood cell counter method was performed by centrifuging the raw milk (1500 rpm, 10 minutes), collecting the cells as a precipitate, and washing the cells twice with physiological saline.

[0035]

[Table 2]

[0036]

EFFECTS OF THE INVENTION According to the method of the present invention, animal cells can be stained and collected by one-step filtration operation, and the number of animal cells in a sample can be measured quickly and simply. . According to the method of the present invention, the determination can be usually made within 30 seconds. Moreover, according to the method of the present invention, it is possible to measure the number of animal cells in a sample without requiring specialized technology or knowledge. Further, the method of the present invention can be applied to all animal cells by selecting the filtration pore size of the hydrophobic filter, so that the application range is extremely wide. Furthermore, since the method of the present invention does not require special equipment,
It can be measured by small hospitals, clinics, and even ordinary people. According to the method of the present invention, for example, animal cells present in raw milk can be measured rapidly and easily, and thus can be effectively used for diagnosing mastitis in dairy cows and is useful in the field of animal husbandry. . Further, the measurement kit of the present invention is extremely simple and inexpensive, and does not require any special equipment, so that it has an advantage that it can be used in any field. Therefore, the present invention includes the fields of livestock and urinalysis (diagnosis),
It can be widely used for determining the culture state of hybridomas that are effective for producing monoclonal antibodies used for various treatments.

[Brief description of drawings]

FIG. 1 is a front view showing an aspect of a filtering tool.

FIG. 2 is a plan view showing an aspect of a filtering tool.

FIG. 3 is an explanatory view showing one embodiment of a filtration container equipped with a filtration tool having a hydrophobic filter.

[Explanation of symbols]

 1 Filtering Tool 1A Upper Side of Filtering Tool 1B Lower Side of Filtering Tool 2 Mounting Part 3 Through Hole 4 Discharging or Suction Section 5 Through Hole 6 Polyethylene Sheet 7 Hydrophobic Filter 8 Hole Opened in Polyethylene Sheet A Filter Container

Claims (7)

[Claims] 1. A method for measuring the number of animal cells in a sample, which comprises staining the animal cells with a dilute stain and collecting the stained animal cells on a hydrophobic filter by suction filtration or pressure filtration, A rapid method for measuring the number of animal cells, which comprises measuring the number of animal cells in a sample from the degree of coloring of the animal cells. 2. The method according to claim 1, wherein the dye colored in the animal cells is eluted with an organic solvent and the degree of coloring of the eluate is colorimetrically determined by optical density measurement. 3. The method according to claim 1, wherein the sample is raw milk and the animal cells are somatic cells of dairy cows. 4. The concentration of the diluted dyeing solution is 0.00001 to
The method according to claim 1, which is 0.02%. 5. The concentration of the diluted dyeing solution is 0.0001-0.
The method according to claim 1, which is 01%. 6. A diluted dyeing solution having a concentration of 0.0002 to 0.
The method according to claim 1, which is 002%. 7. A kit for measuring the number of animal cells, which comprises a hydrophobic filter, a filter which can be fitted with the hydrophobic filter, a dilute staining solution and a color control table.