CN110596102A - Fluid control device and method for enriching and staining cells - Google Patents
Fluid control device and method for enriching and staining cells Download PDFInfo
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- CN110596102A CN110596102A CN201910846376.7A CN201910846376A CN110596102A CN 110596102 A CN110596102 A CN 110596102A CN 201910846376 A CN201910846376 A CN 201910846376A CN 110596102 A CN110596102 A CN 110596102A
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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
- 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
- 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
- G01N1/34—Purifying; Cleaning
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/645—Specially adapted constructive features of fluorimeters
- G01N21/6456—Spatial resolved fluorescence measurements; Imaging
- G01N21/6458—Fluorescence microscopy
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/84—Systems specially adapted for particular applications
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- Molecular Biology (AREA)
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- Investigating Or Analysing Biological Materials (AREA)
- Sampling And Sample Adjustment (AREA)
Abstract
The invention discloses a fluid control device and a method for enriching and coloring cells. The method of the invention generates pressure difference by pushing and pulling the piston, rotates the filtering cavity to form a channel to flow the fluid into the filtering cavity and carry out filtering, and the filtered cells are enriched on the microporous filter membrane and are colored. The invention adds liquid quantitatively in the coloring process, reduces randomness, improves repeatability, and can avoid the risk caused by the repeated contact of operators with samples. The invention relates to the technical field of medical sample processing.
Description
Technical Field
The invention relates to the technical field of medical sample processing, in particular to a fluid control device and a fluid control method for enriching and coloring cells.
Background
The slide examination and microscopic observation of prokaryotic or eukaryotic cells in a body fluid sample are common means for disease diagnosis in medical examination and important means in biological research, and in order to improve diagnosis sensitivity and facilitate observation, cells in the sample are often subjected to enrichment, staining and other treatments before slide examination. The traditional methods for enriching cells include a centrifugal enrichment method, a biochemical capture method, a membrane filtration enrichment method and the like, wherein the centrifugal method can play a role in enriching cells, but the volume, the form and the weight of the cells are different, so that part of the cells are discarded; the biochemical capture method generally uses biomolecules or chemical molecules capable of binding cell surface molecules, such as antibodies, ligands, polar or non-polar chemical molecules, to be linked to a solid phase, and then captures cells through affinity to achieve enrichment, but capture efficiency is affected by intermolecular affinity, molecular dosage, repeated washing, non-specific adsorption, and the like, so that loss occurs; the membrane filtration enrichment method is an ideal method for carrying out filtration through a filter membrane with the pore diameter smaller than that of cells to be captured, and the cells are completely retained on the surface of the membrane by the filter membrane, but because the filtration pressure is difficult to control, part of the cells are tightly adhered to the membrane and cannot be eluted, and part of the cells are lost.
Staining cells enables medical staff and researchers to obtain the inspection result of the needed cells from microscopic examination, and common staining methods for the cells in a body fluid sample comprise bacterial dye staining, fluorescent antibody combined cell staining and the like, but the common staining methods are greatly influenced by human factors; the staining process needs to repeatedly wash the glass slide, and some cells which are not firmly fixed can be washed away; the coloring liquid is dripped, the elution time and the like depend on the experience of an operator, the randomness is high, the quantification cannot be realized, and the repeatability is difficult; the open mode of operation exposes the operator to danger.
Disclosure of Invention
The present invention is directed to overcoming the drawbacks and disadvantages of the prior art and providing a fluid control device for enriching cells in a sample and quantifying the staining of the cells.
Another object of the present invention is to provide a cell staining method capable of standardizing staining procedures, staining solution amounts, and staining times.
The purpose of the invention can be realized by the following technical scheme: enrichment cell and to colored fluid control device of cell, including filter chamber, carousel, sample room, pre-installed liquid chamber and waste liquid chamber, filter chamber demountable installation is on the carousel, and sample room and a plurality of pre-installed liquid chamber evenly install in filter chamber's circumferencial direction, and waste liquid chamber installs in the bottom of carousel. The filter cavity is of a hollow cylindrical structure, a filter cavity rotating handle is arranged at the top of the filter cavity, two symmetrical convex positions are arranged on the outer side wall surface of the filter cavity, a penetrating filter cavity hole is formed in the wall surface of the filter cavity, a microporous filter membrane is fixed at the bottom of the filter cavity, and the filter cavity is provided with a piston push-pull rod which is matched with the filter cavity in size; the turntable is of a hollow cylindrical structure, and two symmetrical concave positions which are matched with the convex positions are arranged on the inner side wall of the turntable; the inner wall surface of the rotary disc is provided with rotary disc pore channels corresponding to the filter cavity holes, and the rotary disc pore channels penetrate through the top surface and the inner side surface of the rotary disc; holes are formed in the bottoms of the sample chamber and the liquid pre-loading chamber, and the positions of the holes correspond to the holes of the rotary disc pore passage on the top surface of the rotary disc; and the top of the waste liquid chamber is provided with a net structure.
As a preferable technical scheme, the filtering cavity hole is arranged on one convex position, and the rotary disc pore passage is arranged on one concave position corresponding to the convex position. The filtering cavity is arranged on the convex position, so that the use amount of the silica gel layer can be reduced, and the cost is reduced.
As a preferred technical scheme, silica gel layers are arranged at the joints of the filtering cavity and the rotary disc, and the joints of the rotary disc, the sample chamber and the prefilled chamber. The silica gel layer can guarantee the inside leakproofness of device.
As a preferred technical scheme, a sand chip is arranged on the net-shaped structure. When the waste liquid chamber is connected with the rotary disc, the sand core sheet props against the microporous filter membrane, so that the microporous filter membrane can be better supported.
As the preferred technical scheme, the microporous filter membrane is fixed between the silica gel sealing ring and the membrane installing ring, and the silica gel sealing ring is connected with the bottom of the filtering cavity. The microporous filter membrane is a track-etched polycarbonate membrane (PCTE membrane) or a track-etched polyethylene terephthalate membrane (PETTE membrane). The microporous filter membrane is characterized in that heavy ions are punched in an accelerated manner, micropores vertically penetrate through the membrane, and the aperture of each pore is fixed, so that cells larger than the aperture can be completely intercepted.
As a preferable technical scheme, the top of the sample chamber is provided with a sample adding port, and the top of the liquid pre-loading chamber is provided with a liquid pre-loading port. And sealing films are arranged on the sample adding port and the liquid pre-assembling ports, position marks are arranged on the sealing films, and blank position marks are arranged between the sample adding port and the liquid pre-assembling ports and between the two liquid pre-assembling ports.
The other purpose of the invention is realized by the following technical scheme: a method of enriching and staining cells comprising the steps of: aligning the hole channel of the rotary disc with the hole of the sample chamber, opening a sealing film of the sample adding port, adding a sample into the sample chamber, pulling the piston push-pull rod upwards, enabling the sample to flow into the filtering cavity, rotating the filtering cavity to a blank position, enabling the hole channel of the rotary disc to be staggered with the hole of the sample chamber, pressing the piston push-pull rod downwards, and filtering the sample; rotating the filter cavity to make the rotary disc pore passage align with the first prefill liquid chamber hole, pulling the piston push-pull rod upwards, making the first prefill liquid flow into the filter cavity, rotating the filter cavity to a blank position to make the rotary disc pore passage staggered with the bottom hole of the first prefill liquid chamber, pressing the piston push-pull rod downwards, and filtering the first prefill liquid; and (4) repeatedly filtering other pre-filled liquid, and finishing the coloring process after all the pre-filled liquid is filtered.
As the preferred technical scheme, after coloring, the filtering cavity is taken out, and the microporous filtering membrane can be microscopically inspected after being taken out. The samples were treated blood, sputum, spinal fluid.
The working principle of the fluid control device is as follows: collecting a body fluid sample, adding corresponding sample treatment fluid to decompose impurities or non-target cells in the sample, and easily filtering; positive pressure is generated through the piston effect to filter a sample, and all target cells in the sample are trapped above the filter membrane; adding a quantitative coloring liquid corresponding to the target cells to perform steps of coloring, decoloring, cleaning and the like on the cells on the filter membrane, wherein after each step is completed, positive pressure is generated by the action of a piston to discharge the liquid and the like, and the target cells on the membrane are all colored; the filter membrane is taken out for direct microscopic observation and counting.
Compared with the prior art, the invention has the following advantages and beneficial effects:
1. the fluid control device of the invention takes the motion of the piston as the driving force, and the push-and-pull piston can control the inflow and filtration of the fluid; the microporous filter membrane is used for enriching cells in a sample and is colored in situ, the sample amount, the treatment liquid, the usage amount of each component of the coloring liquid and the like are standardized in the coloring process, influence factors on results are reduced to the minimum, and the microporous filter membrane is controlled in a totally-enclosed manner and has the advantages of safety, automation, simplicity and convenience in operation and high detectable rate.
2. The method for enriching and coloring the cells can enrich the target cells on one filter membrane, and the cells are retained by the filter membrane in the coloring process and cannot be washed away and lost, so that the enrichment degree and the diagnosis sensitivity of the cells can be effectively improved.
Drawings
FIG. 1 is a front cross-sectional view of a fluid control device in an embodiment of the present invention;
FIG. 2 is a cross-sectional view A-A of FIG. 1;
FIG. 3 is a cross-sectional view B-B of FIG. 1;
FIG. 4 is a top view of a fluid control device in an embodiment of the present invention.
Wherein: 1: a filtering cavity, 2: piston, 3: push-pull rod, 4: filter chamber rotating handle, 5: filter cavity holes, 6: convex position, 7: silica gel sealing ring, 8: film-mounting ring, 9: microfiltration membrane, 10: sand chip, 11: sample addition port, 12: sample chamber, 13: sample cell aperture, 14: pre-filled liquid port, 15: prefilled liquid chamber, 15-1: prefill 1 chamber, 15-2: prefill 2 chamber, 15-3: prefill 3 chamber, 15-4: liquid pre-filling 4 chamber, 15-5: prefill 5 chamber, 15-6: prefill 6 chamber, 15-7: prefill 7 chamber, 15-8: prefill 8 chamber, 15-9: prefill 9 chamber, 16: prefill chamber hole, 17: turntable, 18: carousel aperture, 19: waste liquid chamber, 20: sealing film, 21: silica gel layer, 22: and (5) marking the position.
Detailed Description
The present invention will be described in further detail with reference to examples and drawings, but the present invention is not limited thereto.
The fluid control device for enriching and coloring cells comprises a filtering cavity, a rotating disc, a sample chamber, a prefilled chamber and a waste liquid chamber.
Filter cavity demountable installation on the carousel, filter the cavity and be hollow cylinder structure, the top is equipped with filters the rotatory handle of cavity, is equipped with two symmetrical strip protruding positions on the outside wall, and strip protruding position is pasted on the outside wall. One convex position is provided with a through filtering cavity hole. The bottom of the filtering cavity is fixed with a microporous filter membrane which is fixed between a silica gel sealing ring and a membrane installing ring, and the silica gel sealing ring is connected with the bottom of the filtering cavity. The microporous filter membrane is in a disc shape with the diameter of 13mm, and the aperture is 0.2-8 μm. The microporous filter membrane can be selected from a track-etched polycarbonate membrane (PCTE membrane) or a track-etched polyethylene terephthalate membrane (PETTE membrane). The microporous filter membrane is characterized in that heavy ions are punched in an accelerated manner, micropores vertically penetrate through the membrane, and the aperture of each pore is fixed, so that cells larger than the aperture can be completely intercepted. The filtering cavity is provided with a piston and a push-pull rod, the piston and the push-pull rod are matched with the cavity in size, the piston is fixed at the bottom of the push-pull rod, and the push-pull rod can drive the piston to move up and down.
The turntable is of a hollow cylindrical structure, and two symmetrical concave positions which are matched with the convex positions are arranged on the inner side wall; and a rotary disc pore passage corresponding to the filter cavity hole is arranged on one of the concave positions, the rotary disc pore passage is L-shaped, and the pore passage penetrates through the top surface and the inner side surface of the rotary disc.
The fluid control device is provided with 1 sample chamber and 9 pre-filled liquid chambers, and the sample chamber and the pre-filled liquid chambers are uniformly arranged in the circumferential direction of the filtering cavity. The bottom of the sample chamber and the bottom of the liquid prefilling chamber are both provided with holes, and the positions of the holes correspond to the holes of the rotary disc pore passage on the top surface of the rotary disc. The top of the sample chamber is provided with a sample adding port, and the top of the liquid pre-loading chamber is provided with a liquid pre-loading port. Sealing films cover the sample adding port and the liquid pre-loading port. In order to accurately control the addition of the pre-filled liquid, a position mark is arranged at the sealing film, the mark 0 indicates that the hole of the rotary disc is aligned with the hole of the sample chamber, the sample can flow into the filtering cavity at the moment, the mark □ indicates a vacant position, and the hole of the rotary disc is staggered with the hole of the sample chamber at the moment. Reference numeral 1 indicates alignment of the disk channel with the pre-loaded chamber 1 chamber bottom hole and the adjacent □ indicates a void where the disk channel is offset from the pre-loaded chamber hole. The meaning of other labels can be analogized.
The waste liquid chamber is arranged at the bottom of the rotary disc, the top of the waste liquid chamber is provided with a net structure, and filtrate can flow into the waste liquid chamber from the net structure. In order to better support the microporous filter membrane, a sand core sheet is also supported on the net structure. When the waste liquid chamber is connected with the rotary disc, the sand core sheet props against the bottom of the microporous filter membrane to support the microporous filter membrane.
In order to ensure the sealing performance of the whole fluid control device, silica gel layers are arranged at the joint of the filtering cavity and the rotary disc, the joint of the rotary disc and the sample chamber and the joint of the rotary disc and the pre-filled liquid chamber.
The method for enriching and staining cells by using the fluid control device comprises the following steps:
(1) the initial position of the device is that the opening of the pore passage of the rotary disc is aligned with the hole of the sample chamber, namely the rotary handle of the filtering cavity rotates to 0 position.
(2) And opening the sealing film of the sample adding port of the device, and adding 1-2mL of sample into the sample chamber.
(3) And pulling the push-pull rod upwards, enabling the sample fluid to enter the filtering cavity, rotating the filtering cavity to a blank position (□ position), staggering the opening of the hole passage of the rotary disc and the hole of the sample chamber, pressing the push-pull rod downwards, and filtering the sample.
(4) After filtration, the rotary handle of the filtration cavity is rotated to align the opening of the rotary disc pore passage with the hole (1 position) of the chamber 1 of the pre-filled liquid chamber, the push-pull rod is pulled upwards, the component fluid pre-filled in the chamber 1 of the pre-filled liquid chamber enters the filtration cavity, the filtration cavity is rotated to a blank position (□ position), at the moment, the opening of the rotary disc pore passage is staggered with the hole of the chamber 1 of the pre-filled liquid chamber, the push-pull rod 3 is pressed downwards, and the fluid pre-filled liquid 1 is.
(5) After the filtration, the filter cavity is rotated to align the opening of the pore passage of the rotary disc with the hole (2 position) of the liquid pre-loading chamber 2, the push-pull rod is pulled upwards, the component fluid pre-loaded in the liquid pre-loading chamber 2 enters the filter cavity, the filter cavity is rotated to a blank position (□ position), the opening of the pore passage of the rotary disc is staggered with the hole of the liquid pre-loading chamber 2, the push-pull rod is pressed downwards, and the liquid pre-loading chamber 2 is filtered.
(6) And (5) repeating the step (5), finishing the coloring process after all the pre-installed components are used, taking out the filtering cavity at the moment, taking down the microporous filtering membrane and placing the microporous filtering membrane on a glass slide for microscopic examination.
The first embodiment is as follows:
this example used the fluid control device of the present invention to stain bacteria using a gram stain method. The microporous filter membrane used in the fluid control device is a track etched polyethylene terephthalate (PETTE) membrane with the aperture of 0.45 mu m, and each component of gram staining solution is pre-filled in a pre-filling solution 1-9 chamber respectively, and the pre-filling solution sequentially comprises ammonium oxalate crystal violet solution, ultrapure water, iodine solution, ultrapure water, 95% alcohol, ultrapure water, lycopene solution and ultrapure water.
Collection and processing of samples
(1) The patient expectorated approximately 5mL of sputum into a sterile 50mL centrifuge tube.
(2) 5mL of 4% NaOH solution was added, shaken for 1 minute, and allowed to stand for 15 minutes.
(3) 0.067mol/L Phosphate Buffered Saline (PBS) was added to 50mL, and centrifuged at 3000Xg for 20 minutes.
(4) The supernatant was decanted and 2mL of resuspended pellet was added.
Secondly, the fluid control device is used for enriching bacteria and gram staining the bacteria
(1) The initial position of the device is that the opening of the turntable pore channel is aligned with the hole of the sample chamber (0 position).
(2) And the sealing film of the sample addition port of the device was opened, and 2mL of the sample was added to the sample chamber.
(3) And pulling the push-pull rod upwards, enabling the sample to enter the filtering cavity, rotating the filtering cavity to a blank position (□ position), pressing the push-pull rod downwards, and filtering the sample.
(4) After the filtration, rotating the rotary handle of the filtration cavity to align the opening of the pore passage of the rotary disc with the hole (1 position) of the liquid pre-loading chamber 1, pulling the push-pull rod upwards, allowing the ammonium oxalate crystal violet solution pre-loaded in the liquid pre-loading chamber 1 to enter the filtration cavity, rotating the filtration cavity to a blank position (□ positions), waiting for 1 minute, pressing the push-pull rod downwards, and filtering the ammonium oxalate crystal violet solution.
(5) After the filtration is finished, the rotary handle of the filtration cavity is rotated to enable the opening of the pore passage of the rotary disc to be aligned with the hole (2 position) of the liquid 2 chamber, the push-pull rod is pulled upwards, the ultrapure water pre-filled in the liquid 2 chamber enters the filtration cavity, the filtration cavity is rotated to a blank position (□ position), the push-pull rod is pressed downwards, and the ultrapure water is filtered.
(6) After the filtration, the rotating handle of the filtering cavity is rotated to align the opening of the pore passage of the turntable with the hole (3 positions) of the 3 chambers of the pre-filled liquid, the push-pull rod is pulled upwards, the pre-filled iodine liquid in the 3 chambers of the pre-filled liquid enters the filtering cavity, the filtering cavity is rotated to a blank position (□ positions), the filtering cavity is rotated to wait for 1 minute, the push-pull rod is pressed downwards, and the iodine liquid is filtered.
(7) After the filtration is finished, the rotating handle of the filtering cavity is rotated to enable the opening of the pore passage of the rotary disc to be aligned with the hole (4 positions) of the liquid pre-loading chamber 4, the push-pull rod is pulled upwards, the ultrapure water pre-loaded in the liquid pre-loading chamber 4 enters the filtering cavity, the filtering cavity is rotated to a blank position (□ positions), the push-pull rod is pressed downwards, and the ultrapure water is filtered.
(8) After the filtration is finished, the rotating handle of the filtering cavity body is rotated to enable the opening of the pore passage of the rotary disc to be aligned with the hole (5 positions) of the liquid pre-loading 5 chamber, the push-pull rod is pulled upwards, 95% alcohol pre-loaded in the liquid pre-loading 5 chamber enters the filtering cavity body, the filtering cavity body is rotated to a blank position (□ positions), the push-pull rod is pressed downwards after 20 seconds, and the 95% alcohol is filtered.
(9) After the filtration is finished, the rotating handle of the filtering cavity is rotated to enable the opening of the pore passage of the rotary disc to be aligned with the hole (6 position) of the liquid pre-loading 6 chamber, the push-pull rod is pulled upwards, the ultrapure water pre-loaded in the liquid pre-loading 6 chamber enters the filtering cavity, the filtering cavity is rotated to a blank position (□ position), the push-pull rod is pressed downwards, and the ultrapure water is filtered.
(10) After the filtration is finished, the rotating handle of the filtering cavity is rotated to enable the opening of the pore passage of the rotary disc to be aligned with the hole (7 position) of the liquid pre-loading 7 chamber, the push-pull rod is pulled upwards, the ultrapure water pre-loaded in the liquid pre-loading 7 chamber enters the filtering cavity, the filtering cavity is rotated to a blank position (□ position), the push-pull rod is pressed downwards, and the ultrapure water is filtered.
(11) After the filtration is finished, a rotating handle of the filtering cavity body is rotated to enable the opening of a pore passage of the rotary disc to be aligned with a hole (8 positions) of the liquid pre-loading chamber 8, the push-pull rod is pulled upwards, the lycopene solution pre-loaded in the liquid pre-loading chamber 8 enters the filtering cavity body, the filtering cavity body is rotated to a blank position (□ positions), the filtering cavity body is waited for 1 minute, the push-pull rod is pressed downwards, and the lycopene solution is filtered.
(12) After the filtration is finished, the rotating handle of the filtering cavity is rotated to enable the opening of the pore passage of the rotary disc to be aligned with the hole (9 position) of the liquid pre-loading 9 chamber, the push-pull rod is pulled upwards, the ultrapure water pre-loaded in the liquid pre-loading 9 chamber enters the filtering cavity, the filtering cavity is rotated to a blank position (□ position), the push-pull rod is pressed downwards, and the ultrapure water is filtered.
(13) And at this moment, the filtering cavity can be taken out, the microporous filtering membrane is taken down and placed on a glass slide for microscopic examination, and gram-positive bacteria are purple bacteria and gram-negative bacteria are red bacteria.
Example two:
in this example, acid-fast staining was performed by using the fluid control apparatus of the present invention to stain acid-fast bacilli. The filter membrane used in the fluid control device is a track etched polyethylene terephthalate (PETTE) membrane with the aperture of 0.45 mu m, and the pre-filling liquid 1-9 chambers are respectively pre-filled with the components of acid-resistant staining liquid, namely carbolic acid reddish water solution, ultrapure water, acidic alcohol, ultrapure water, methylene blue solution, ultrapure water and ultrapure water in sequence.
Collection and processing of samples
(1) The patient expectorated approximately 5mL of sputum into a sterile 50mL centrifuge tube.
(2) 5mL of 4% NaOH solution was added, shaken for 1 minute, and allowed to stand for 15 minutes.
(3) 0.067mol/L Phosphate Buffered Saline (PBS) was added to 50mL, and centrifuged at 3000Xg for 20 minutes.
(4) The supernatant was decanted and 2mL of resuspended pellet was added.
Secondly, the fluid control device of the invention is used for enriching acid-fast bacilli and coloring the acid-fast bacilli
(1) The initial position of the device is that the opening of the turntable pore channel is aligned with the hole of the sample chamber (0 position).
(2) And the sealing film of the sample addition port of the device was opened, and 2mL of the sample was added to the sample chamber.
(3) And pulling the push-pull rod upwards, enabling the sample to enter the filtering cavity, rotating the filtering cavity to a blank position (□ position), pressing the push-pull rod downwards, and filtering the sample.
(4) After the filtration is finished, the filter cavity is rotated to enable the opening of the pore channel of the rotary disc to be aligned with the hole (1 position) of the liquid pre-loading chamber 1, the push-pull rod is pulled upwards, the carbolic acid reddish solution pre-loaded in the liquid pre-loading chamber 1 enters the filter cavity, the filter cavity is rotated to a blank position (□ positions), the filter cavity is waited for 10 minutes, the push-pull rod is pressed downwards, and the carbolic acid reddish solution is filtered.
(5) After the filtration is finished, the filter cavity is rotated to enable the opening of the pore passage of the rotary disc to be aligned with the hole (2 position) of the liquid 2 pre-loading chamber, the push-pull rod is pulled upwards, the ultrapure water pre-loaded in the liquid 2 pre-loading chamber enters the filter cavity, the filter cavity is rotated to a blank position (□ position), the push-pull rod is pressed downwards, and the ultrapure water is filtered.
(6) After the filtration is finished, the filter cavity is rotated to enable the opening of the pore passage of the rotary disc to be aligned with the hole (3 bit) of the liquid pre-loading chamber 3, the push-pull rod is pulled upwards, the ultrapure water pre-loaded in the liquid pre-loading chamber 3 enters the filter cavity, the filter cavity is rotated to a blank position (□ bit), the push-pull rod is pressed downwards, and the ultrapure water is filtered.
(7) After the filtration, the filter cavity is rotated to enable the opening of the pore passage of the rotary disc to be aligned with the hole (4 positions) of the liquid pre-loading chamber 4, the push-pull rod is pulled upwards, the acidic alcohol pre-loaded in the liquid pre-loading chamber 4 enters the filter cavity, the filter cavity is rotated to a blank position (□ positions), the filter cavity is rotated for 2 minutes, the push-pull rod is pressed downwards, and the acidic alcohol is filtered.
(8) After the filtration is finished, the filter cavity is rotated to enable the opening of the pore passage of the rotary disc to be aligned with the hole (5 bit) of the liquid pre-loading 5 chamber, the push-pull rod is pulled upwards, the ultrapure water pre-loaded in the liquid pre-loading 5 chamber enters the filter cavity, the filter cavity is rotated to a blank position (□ bit), the push-pull rod is pressed downwards, and the ultrapure water is filtered.
(9) After the filtration is finished, the filter cavity is rotated to enable the opening of the pore passage of the rotary disc to be aligned with the hole (6 position) of the liquid pre-loading 6 chamber, the push-pull rod is pulled upwards, the ultrapure water pre-loaded in the liquid pre-loading 6 chamber enters the filter cavity, the filter cavity is rotated to a blank position (□ position), the push-pull rod is pressed downwards, and the ultrapure water is filtered.
(10) After the filtration, the filter cavity is rotated to enable the opening of the pore channel of the rotary disc to be aligned with the hole (7 position) of the liquid pre-loading chamber 7, the push-pull rod is pulled upwards, methylene blue solution pre-loaded in the liquid pre-loading chamber 7 enters the filter cavity, the filter cavity is rotated to a blank position (□ position), the push-pull rod is pressed downwards after 30 seconds, and the methylene blue solution is filtered.
(11) After the filtration is finished, the filter cavity is rotated to enable the opening of the pore passage of the rotary disc to be aligned with the hole (8 bit) of the liquid pre-loading chamber 8, the push-pull rod is pulled upwards, the ultrapure water pre-loaded in the liquid pre-loading chamber 8 enters the filter cavity, the filter cavity is rotated to a blank position (□ bit), the push-pull rod is pressed downwards, and the ultrapure water is filtered.
(12) After the filtration is finished, the filter cavity is rotated to enable the opening of the pore passage of the turntable to be aligned with the hole (9 bit) of the liquid pre-loading 9 chamber, the push-pull rod is pulled upwards, the ultrapure water pre-loaded in the liquid pre-loading 9 chamber enters the filter cavity, the filter cavity is rotated to a blank position (□ bit), the push-pull rod is pressed downwards, and the ultrapure water is filtered.
(13) And at this moment, the filter cavity can be taken out, the microporous filter membrane is taken down and placed on a glass slide for microscopic examination, and the acid-resistant bacillus is red rod-shaped thallus and the mixed bacteria is blue thallus.
Example three:
in this example, acid-fast bacilli were subjected to fluorescence by the gold-amine O fluorescence staining method using the fluid control apparatus of the present invention. The filter membrane used in the fluid control device is a track etched polyethylene terephthalate (PETTE) membrane with the aperture of 0.45 mu m, and the pre-filling liquid 1-9 chambers are respectively pre-filled with the components of an acid-resistant dyeing liquid, namely a auramine O dyeing liquid, ultrapure water, acidic alcohol, ultrapure water, a potassium permanganate solution, ultrapure water and ultrapure water.
Collection and processing of samples
(1) The patient expectorated approximately 5mL of sputum into a sterile 50mL centrifuge tube.
(2) 5mL of 4% NaOH solution was added, shaken for 1 minute, and allowed to stand for 15 minutes.
(3) 0.067mol/L Phosphate Buffered Saline (PBS) was added to 50mL, and centrifuged at 3000Xg for 20 minutes.
(4) The supernatant was decanted and 2mL of resuspended pellet was added.
Secondly, the fluid control device of the invention is used for enriching acid-fast bacilli and coloring the acid-fast bacilli
(1) The initial position of the device is that the opening of the turntable pore channel is aligned with the hole of the sample chamber (0 position).
(2) And the sealing film of the sample addition port of the device was opened, and 2mL of the sample was added to the sample chamber.
(3) And pulling the push-pull rod upwards, enabling the sample to enter the filtering cavity, rotating the filtering cavity to a blank position (□ position), pressing the push-pull rod downwards, and filtering the sample.
(4) And after filtering, rotating the filtering cavity to enable the opening of the pore passage of the rotary disc to align with the hole (1 position) of the liquid pre-loading chamber 1, pulling the push-pull rod upwards, enabling the auramine O dye liquid pre-loaded in the liquid pre-loading chamber 1 to enter the filtering cavity, rotating the filtering cavity to a blank position (□ position), waiting for 10 minutes, pressing the push-pull rod downwards, and filtering the auramine O dye liquid.
(5) After the filtration is finished, the filter cavity is rotated to enable the opening of the pore passage of the rotary disc to be aligned with the hole (2 position) of the liquid 2 pre-loading chamber, the push-pull rod is pulled upwards, the ultrapure water pre-loaded in the liquid 2 pre-loading chamber enters the filter cavity, the filter cavity is rotated to a blank position (□ position), the push-pull rod is pressed downwards, and the ultrapure water is filtered.
(6) After the filtration is finished, the filter cavity is rotated to enable the opening of the pore passage of the rotary disc to be aligned with the hole (3 bit) of the liquid pre-loading chamber 3, the push-pull rod is pulled upwards, the ultrapure water pre-loaded in the liquid pre-loading chamber 3 enters the filter cavity, the filter cavity is rotated to a blank position (□ bit), the push-pull rod is pressed downwards, and the ultrapure water is filtered.
(7) After the filtration, the filter cavity is rotated to enable the opening of the pore passage of the rotary disc to align with a hole (4 positions) of a liquid pre-loading chamber 4, the push-pull rod is pulled upwards, the acidic alcohol pre-loaded in the liquid pre-loading chamber 4 enters the filter cavity, the filter cavity is rotated to a blank position (□ positions), the filter cavity is rotated for 3 minutes, the push-pull rod is pressed downwards, and the acidic alcohol is filtered.
(8) After the filtration is finished, the filter cavity is rotated to enable the opening of the pore passage of the rotary disc to be aligned with the hole (5 bit) of the liquid pre-loading 5 chamber, the push-pull rod is pulled upwards, the ultrapure water pre-loaded in the liquid pre-loading 5 chamber enters the filter cavity, the filter cavity is rotated to a blank position (□ bit), the push-pull rod is pressed downwards, and the ultrapure water is filtered.
(9) After the filtration is finished, the filter cavity is rotated to enable the opening of the pore passage of the rotary disc to be aligned with the hole (6 position) of the liquid pre-loading 6 chamber, the push-pull rod is pulled upwards, the ultrapure water pre-loaded in the liquid pre-loading 6 chamber enters the filter cavity, the filter cavity is rotated to a blank position (□ position), the push-pull rod is pressed downwards, and the ultrapure water is filtered.
(10) After the filtration, the filter cavity is rotated to enable the opening of the pore passage of the rotary disc to be aligned with the hole (7 bit) of the liquid pre-loading chamber 7, the push-pull rod is pulled upwards, the potassium permanganate solution pre-loaded in the liquid pre-loading chamber 7 enters the filter cavity, the filter cavity is rotated to a blank position (□ bit), the filter cavity is rotated to wait for 2 minutes, the push-pull rod is pressed downwards, and the potassium permanganate solution is filtered.
(11) After the filtration is finished, the filter cavity is rotated to enable the opening of the pore passage of the rotary disc to be aligned with the hole (8 bit) of the liquid pre-loading chamber 8, the push-pull rod is pulled upwards, the ultrapure water pre-loaded in the liquid pre-loading chamber 8 enters the filter cavity, the filter cavity is rotated to a blank position (□ bit), the push-pull rod is pressed downwards, and the ultrapure water is filtered.
(12) After the filtration is finished, the filter cavity is rotated to enable the opening of the pore passage of the turntable to be aligned with the hole (9 bit) of the liquid pre-loading 9 chamber, the push-pull rod is pulled upwards, the ultrapure water pre-loaded in the liquid pre-loading 9 chamber enters the filter cavity, the filter cavity is rotated to a blank position (□ bit), the push-pull rod is pressed downwards, and the ultrapure water is filtered.
(13) And at this moment, the filter cavity can be taken out, the microporous filter membrane is taken down and placed on a glass slide for fluorescence microscopy, and the acid-resistant bacillus is bright yellow or yellow green rod-shaped thallus under the excitation of ultraviolet light.
The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the present invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.
Claims (11)
1. The fluid control device for enriching and coloring cells is characterized by comprising a filtering cavity, a rotary disc, a sample chamber, a pre-loaded liquid chamber and a waste liquid chamber, wherein the filtering cavity is detachably arranged on the rotary disc, the sample chamber and the pre-loaded liquid chambers are uniformly arranged in the circumferential direction of the filtering cavity, and the waste liquid chamber is arranged at the bottom of the rotary disc;
the filter cavity is of a hollow cylindrical structure, a filter cavity rotating handle is arranged at the top of the filter cavity, two symmetrical convex positions are arranged on the outer side wall surface of the filter cavity, a penetrating filter cavity hole is formed in the wall surface of the filter cavity, a microporous filter membrane is fixed at the bottom of the filter cavity, and the filter cavity is provided with a piston push-pull rod which is matched with the filter cavity in size;
the turntable is of a hollow cylindrical structure, and two symmetrical concave positions which are matched with the convex positions are arranged on the inner side wall of the turntable; the inner wall surface of the rotary disc is provided with rotary disc pore channels corresponding to the filter cavity holes, and the rotary disc pore channels penetrate through the top surface and the inner side surface of the rotary disc;
holes are formed in the bottoms of the sample chamber and the liquid pre-loading chamber, and the positions of the holes correspond to the holes of the rotary disc pore passage on the top surface of the rotary disc;
and the top of the waste liquid chamber is provided with a net structure.
2. The fluid control device according to claim 1, wherein the filter cavity aperture is disposed in one of the raised locations and the disk aperture is disposed in a recessed location corresponding to the raised location.
3. The fluid control device according to claim 1, wherein the joint between the filtration chamber and the rotary disk, and the joint between the rotary disk and the sample chamber and the pre-liquid chamber are provided with silica gel layers.
4. The fluid control device according to claim 1, wherein the mesh structure is provided with sand chips.
5. The fluid control device according to claim 1, wherein the microporous filter membrane is fixed between a silica gel sealing ring and a membrane mounting ring, and the silica gel sealing ring is connected with the bottom of the filter cavity.
6. The fluid control device according to claim 1 or 5, wherein the microfiltration membrane is a track etched polycarbonate membrane or a track etched polyethylene terephthalate membrane.
7. The fluid control device of claim 1, wherein the sample chamber has a sample loading port at a top thereof, and the pre-loaded chamber has a pre-loading port at a top thereof.
8. The fluid control device according to claim 7, wherein the sample port and the pre-loading port are provided with sealing films, the sealing films are provided with position marks, and blank position marks are provided between the sample port and the pre-loading port and between the two pre-loading ports.
9. A method of enriching and staining cells using the fluid control device of any of claims 1-8, comprising the steps of:
aligning the hole channel of the rotary disc with the hole of the sample chamber, opening a sealing film of the sample adding port, adding a sample into the sample chamber, pulling the piston push-pull rod upwards, enabling the sample to flow into the filtering cavity, rotating the filtering cavity to a blank position, enabling the hole channel of the rotary disc to be staggered with the hole of the sample chamber, pressing the piston push-pull rod downwards, and filtering the sample;
rotating the filter cavity to make the rotary disc pore passage align with the first prefill liquid chamber hole, pulling the piston push-pull rod upwards, making the first prefill liquid flow into the filter cavity, rotating the filter cavity to a blank position to make the rotary disc pore passage staggered with the bottom hole of the first prefill liquid chamber, pressing the piston push-pull rod downwards, and filtering the first prefill liquid;
and repeatedly filtering other coloring liquid, and finishing the coloring process after all the coloring liquid is filtered.
10. The method of claim 9, wherein the filter chamber is removed after staining is complete, and the microporous filter membrane is removed for microscopic examination.
11. The method of enriching and staining cells of claim 9, wherein the sample is processed blood, sputum, spinal fluid.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN111643948A (en) * | 2020-06-09 | 2020-09-11 | 长江水利委员会长江科学院 | Device and method suitable for simultaneously filtering leaching liquor of multiple groups of soil or sediments |
| CN113832028A (en) * | 2020-06-24 | 2021-12-24 | 上海医药集团生物治疗技术有限公司 | Full-automatic cell culture system and culture method |
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| CN210923495U (en) * | 2019-09-09 | 2020-07-03 | 暨南大学 | Fluid control device for enriching and staining cells |
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| JPH0792160A (en) * | 1991-08-15 | 1995-04-07 | La Mina Ltd | Sampling device, testing sampling device, testing method and measuring method |
| CN1646222A (en) * | 2002-02-25 | 2005-07-27 | 西菲伊德公司 | Fluid processing and control |
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| CN111643948A (en) * | 2020-06-09 | 2020-09-11 | 长江水利委员会长江科学院 | Device and method suitable for simultaneously filtering leaching liquor of multiple groups of soil or sediments |
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