CN218026026U - Single bacterium separation trapping apparatus - Google Patents

Abstract

The utility model relates to the technical field of single bacterium separation and capture, and discloses a single bacterium separation and capture device. The single bacterium separation and capture device comprises a microscope (1), a stage (2), a widening platform (3), a micromanipulator (4) and a single bacterium picking structure (5), wherein the stage ( 2) a coated glass slide (21) is arranged on the covered glass slide (21), and an agar microarray chip (22) is placed on the covered glass slide (21), and the microscope (1) includes a microarray chip (22) located on the agar microarray chip (22). ), the widened platform (3) is arranged side by side with the stage (2), the micromanipulator (4) is installed on the widened platform (3), and the single bacterial challenge The structure (5) is installed on the micromanipulator (4). The device for separating and capturing single bacteria described in the utility model can realize precise picking of single cells of bacteria.

Description

A device for separating and capturing single bacteria

technical field

The utility model relates to the technical field of single bacterium separation and capture, in particular to a single bacterium separation and capture device.

Background technique

The classic bacterial population culture method cannot satisfy the study of bacterial heterogeneity. Single-cell biology is a very popular research direction at present. It is of great significance to reveal heterogeneity at the single-cell level, but isolating a single bacterium is still very technically challenging. At present, there are several methods for isolation and capture of single bacteria, including limiting dilution method, micromanipulation method, fluorescence flow sorting method, microfluidic technology, laser microdissection technology, etc.

Among them, the micromanipulation method is to place the bacterial suspension under a microscope to select a single bacterium, generally using a capillary. The method is intuitive. However, the bacterial suspension has strong bacterial mobility under the microscope, which requires very high technical requirements for operators.

Utility model content

The purpose of the utility model is to provide a device for separating and capturing single bacteria in order to overcome the problem in the prior art that micromanipulation is used to select single bacteria and the requirements for operators are relatively high.

In order to achieve the above object, the utility model provides a single bacteria separation and capture device, including a microscope, a stage, a widening platform, a micromanipulator and a single bacteria picking structure, wherein the stage is equipped with a cover A film slide, an agar microarray chip is placed on the film cover slide, the microscope includes an eyepiece located above the agar microarray chip, the widening platform is arranged side by side with the stage, and the micro A manipulator is mounted on the widened platform, and the single bacteria picking structure is mounted on the micromanipulator.

Preferably, an aseptic collection device is also arranged on the stage.

Preferably, the single-bacteria picking structure includes a syringe and a needle provided on the syringe, or the single-bacteria picking structure is a glass capillary.

Preferably, the microscope is an inverted fluorescence microscope.

Preferably, the single bacteria picking structure is mounted on the micromanipulator via an adapter clamp.

Preferably, the agar microarray chip has 500-5000 round holes, the diameter of the round holes is 10-50 microns, the distance between adjacent round holes is 5-15 microns, and the depth of the round holes is 8-20 microns .

Preferably, the size of the agar microarray chip is 15mm×15mm.

Preferably, the coated glass slide is a laser microdissected coated glass slide covered with a transparent film.

Preferably, the overall size of the coated glass slide is 75×24 mm, wherein the size of the coated transparent film is 45×16 mm.

Preferably, the micromanipulator is electric or manual.

According to the single bacterium separation and capture device described in the utility model, when the microscope is observed, the bacteria are coated on the agar microarray chip, and the coated glass slide is used to utilize the microscope, the widening platform next to the stage and the micromanipulator , which can accurately pick out single cells of bacteria. The device described in the utility model has the following advantages: (1) bacteria are coated in the agar microarray chip for screening and cultivation, which is convenient for microscope observation; Configure different medium components for bacterial screening; (3) the agar microarray chip is placed on the coated glass slide, and the coated glass slide has an ultra-thin film with strong light transmission, which is conducive to microscopic observation. When the bacterial picking structure (such as a syringe needle or glass capillary) picks up bacteria, the small hole covered with the film will not damage the syringe needle or glass capillary, and will not affect the picking operation; (4) Electric and manual micromanipulators are more efficient than manual picking. Bacteria are more precise.

Description of drawings

Fig. 1 is the structural representation of the single bacterium separation and capture device described in the utility model;

Fig. 2 is the physical partial picture of the agar microarray chip described in the utility model;

Fig. 3 is a picture of an agar microarray chip observed under a 40X eyepiece by the device for separating and capturing single bacteria described in the present invention.

Explanation of reference signs

1. Microscope; 2. Stage; 3. Widening platform; 4. Micromanipulator; 5. Single bacteria picking structure; 21. Covered glass slide; 22. Agar microarray chip; 23. Sterile collection device.

detailed description

The specific embodiment of the utility model will be described in detail below in conjunction with the accompanying drawings. It should be understood that the specific embodiments described here are only used to illustrate and explain the utility model, and are not intended to limit the utility model.

In the description of the present application, the term "comprising" and any variations thereof mean non-exclusive inclusion, the possible existence or addition of one or more other features, integers, steps, operations, units, components and/or combinations thereof.

In addition, terms such as "upper", "lower", "left", "right", "inner", "outer" and other indicated orientations or positional relationships are described based on the orientations or relative positional relationships shown in the drawings, The simplified description is only for the convenience of describing the present application, and does not indicate that the device or element referred to must have a specific orientation, be constructed and operate in a specific orientation, and thus should not be construed as limiting the present application.

In addition, unless otherwise clearly specified and limited, the terms "mounted", "connected" and "connected" should be interpreted in a broad sense, for example, it can be a fixed connection, a detachable connection, or an integral connection; it can be a mechanical connection , can also be an electrical connection; it can be a direct connection, an indirect connection through an intermediary, or an internal communication between two components. Those of ordinary skill in the art can understand the specific meanings of the above terms in this application according to specific situations.

As shown in Figure 1, the single bacterium separation and capture device described in the utility model includes a microscope 1, an object stage 2, a widening platform 3, a micromanipulator 4 and a single bacterium picking structure 5, wherein the object stage 2 is equipped with a coated glass slide 21 on which an agar microarray chip 22 is placed, the microscope 1 includes eyepieces positioned above the agar microarray chip 22, and the widened platform 3 Arranged side by side with the stage 2 , the micromanipulator 4 is mounted on the widened platform 3 , and the single bacteria picking structure 5 is mounted on the micromanipulator 4 .

In the device for separating and capturing single bacteria described in the present invention, a sterile collecting device 23 is also arranged on the stage 2 . In a specific embodiment, the aseptic collection device 23 has a built-in sterile nutrient solution or solid medium for placing the picked bacteria.

In the single bacterium separation and capture device of the present invention, the single bacterium picking structure 5 includes a syringe and a needle provided on the syringe, or the single bacterium picking structure 5 is a glass capillary.

In the device for separating and capturing single bacteria described in the present invention, the microscope 1 is used to observe the bacteria on the agar microarray chip 22 so as to pick the bacteria. In a more preferred embodiment, the microscope 1 is an inverted fluorescence microscope.

In the single bacterium separation and capture device described in the present invention, the single bacterium picking structure 5 is installed on the micromanipulator 4 through an adapter fixture.

In the device for separating and capturing single bacteria described in the present invention, the agar microarray chip 22 can be designed as circular holes with different depths and diameters according to experimental requirements. In a more preferred embodiment, the agar microarray chip 22 has 500-5000 round holes, the diameter of the round holes is 10-50 microns, and the distance between adjacent round holes is 5-15 microns, and the diameter of the round holes is 5-15 microns. The depth is 8-20 microns. The size of the agar microarray chip 22 is 15mm×15mm. As an example, Fig. 2 has shown the actual partial picture of the agar microarray chip of above-mentioned preferred embodiment, in this example, the size of the agar microarray chip is the circular hole pattern of 15mm * 15mm, and each circular hole diameter is 20 microns, The distance between the cylinders is 8 microns, and the depth of the cylinders is 10 microns.

In the utility model, agar is a hydrogel material, which is a polysaccharide extracted from seaweed, a kind of seaweed gel, a colorless solid without a fixed shape, soluble in hot water, and used as a bacterial culture medium. The agar microarray chip 22 refers to a bacterial solid culture medium microarray chip containing agar, which is used for bacterial microscopic observation and culture screening.

In the present utility model, the agar microarray chip 22 can be prepared according to the following process:

(1) Prepare a polydimethylsiloxane (PDMS) chip: the chip size is 1.5cm×1.5cm, the diameter of each protruding cylinder is 20 μm, the distance between the cylinders is 8 μm, and the depth of the cylinder is 10 μm, that is, the PDMS chip is about It consists of 2000 cylindrical protrusions, each with a height of 10 μm. The production process of PDMS chips includes: mask design, silicon wafer cleaning, silicon wafer drying, coating uniformity, pre-baking, exposure, post-baking, developing, and film hardening.

(2) Preparation of agar fixed medium: prepare different bacterial solid medium according to experimental needs, for example, the composition of LB solid medium (1L volume) is: 10g tryptone, 5g yeast extract, 10g sodium chloride, 15g agar.

(3) Preparation of agar microarray chips: the basic principle is that 2000 cylindrical PDMS chips of 1.5cm×1.5cm are "stamps". array chip. The specific process is to use the gel plate and gel cover for protein electrophoresis to complete the preparation of the agar microarray chip. The gel thickness of 1 mm or 1.5 mm can be routinely prepared between the glass plate and glass cover for protein electrophoresis. The method is as follows: wash the glass plate and glass cover, wipe with alcohol and dry them. Place the PDMS chip on the glass plate with the cylindrical pattern facing up. After heating the LB solid medium into a liquid in a microwave oven, pour it over the PDMS chip on the glass plate, and then cover it with a glass cover. After waiting for a few minutes, the medium is completely condensed, remove the glass cover, and place a 1.5cm×1.5 The cm agar microarray chip was cut out with a small scalpel blade, moved to a clean and sterile glass, and the bacteria were spread on the agar microarray chip.

In the device for separating and capturing single bacteria described in the present utility model, the coated glass slide 21 is a coated glass slide of laser microdissection, and the coated glass slide of laser microdissection is covered with a transparent film. . When in use, the film-coated slide 21 is placed on the stage 2, and one side of the agar microarray chip 21 containing bacteria is attached to the transparent film of the film-covered slide 21, because the light transmission of the transparent film is strong , ultra-thin needle piercing holes, convenient for picking single bacteria.

In a preferred embodiment, the overall size of the coated glass slide 21 is 75×24 mm, and the size of the coated transparent film is 45×16 mm.

In the device for separating and capturing single bacteria described in the present invention, the micromanipulator 4 can be electric or manual.

According to a specific embodiment of the present invention, as shown in Figure 1, the single bacteria separation and capture device includes a microscope 1, a stage 2, a widening platform 3, a micromanipulator 4 and a single bacteria picking structure 5, Wherein, the described stage 2 is equipped with a coated glass slide 21 and a sterile collection device 23, an agar microarray chip 22 is placed on the covered coated glass slide 21, and the microscope 1 includes a chip located on the agar microarray. The eyepiece above the array chip 22, the widened platform 3 is arranged side by side with the object stage 2, the micromanipulator 4 is installed on the widened platform 3, and the single bacteria picking structure 5 passes through the adapter clamp Installed on the micromanipulator 4, the micromanipulator 4 is electric or manual, wherein the single bacterium picking structure 5 includes a syringe and a needle arranged on the syringe, or the single bacterium picking Structure 5 is a glass capillary, the microscope 1 is an inverted fluorescence microscope, and the coated glass slide 21 is a laser microdissected coated glass slide covered with a transparent membrane.

In the single bacterium separation and capture device of the above-mentioned specific embodiment, bacteria are coated on the agar microarray chip 22, and the agar microarray chip 22 coated with bacteria is placed on the film-coated slide 21, and the film-coated slide 21 is placed On the stage 2, the bacteria on the agar microarray chip 22 are observed through a microscope; on the stage 2, a widening platform 3 is installed next to it, and an electric or manual micromanipulator 4 is installed on the widening platform 3 , an adapter fixture is installed on the micromanipulator 4, and the syringe and needle or glass capillary are clamped by the adapter fixture; after finding the target bacteria in the agar microarray chip 22 under the microscope, the micromanipulator 4 is controlled electrically or manually, and the The needle or glass capillary accurately picks out single cells of bacteria or target flora, and places the picked single cells of bacteria or target flora in the aseptic collection device 23 for subsequent culture analysis and the like. Figure 3 shows a picture of the agar microarray chip observed by the single bacterium isolation and capture device under a 40X eyepiece.

The characteristics of the single bacteria separation and capture device described in the utility model are as follows:

(1) The bacterial liquid is coated on the agar microarray chip. Agar is commonly used as one of the solid medium components for cultivating bacteria in the laboratory. Different solid medium microarray chips can be prepared according to the experimental requirements; the bacteria are distributed in the agar microwells It is possible to pick single bacteria, observe growth and division, or screen out the desired bacteria by bacterial phenotype.

(2) Ordinary micromanipulation uses traditional slide glass and cover glass. If you use traditional hard slide glass, the agar microarray chip has appropriate hardness, and the needle or glass capillary is also hard. Inadvertent removal may easily lead to bending of the needle or breaking of the glass capillary or failure of picking bacteria. And what adopted in the utility model is coated slide glass, is special-purpose on the laser micro-cutting instrument, and the overall size of size is 75 * 24mm, and wherein the size of the transparent film that covers is 45 * 16mm; Because coating is transparent , strong light transmission, even if a small hole is punctured when picking bacteria, it can still support the agar microarray chip stably; in addition, the coating is ultra-thin, does not damage the needle or glass capillary, and does not affect the picking of single bacteria.

(3) In the utility model, an inverted fluorescence microscope, a widening platform, and an electric or manual micromanipulator are used. An electric or manual micromanipulator is installed on the widened platform. After the adapter fixture is installed on the micromanipulator, syringes of different volumes can be clamped. The syringe can be equipped with a fine needle or a micro glass capillary. The purpose is to realize rapid capture of the target bacteria by using an electric or manual micromanipulator after the target bacteria are observed under the microscope. In addition, liquid medium or other liquid components can be added to the syringe according to the needs of the experiment. After the needle captures the target bacteria, manipulate the micromanipulator to move the needle into the sterile collection device, push the liquid in the syringe to the sterile collection hole, and flush the bacteria into the sterile vial.

The preferred embodiments of the present invention have been described in detail above, but the present invention is not limited thereto. Within the scope of the technical concept of the utility model, various simple modifications can be made to the technical solution of the utility model, including the combination of various technical features in any other suitable manner, and these simple variations and combinations should also be regarded as disclosed by the utility model. All the contents belong to the protection scope of the present utility model.

Claims (10)

1. The single-bacterium separation and capture device is characterized by comprising a microscope (1), an object stage (2), a widened platform (3), a micromanipulator (4) and a single-bacterium picking structure (5), wherein the object stage (2) is provided with a film-coated glass slide (21), an agar microarray chip (22) is placed on the film-coated glass slide (21), the microscope (1) comprises an ocular lens positioned above the agar microarray chip (22), the widened platform (3) and the object stage (2) are arranged in parallel, the micromanipulator (4) is installed on the widened platform (3), and the single-bacterium picking structure (5) is installed on the micromanipulator (4).

2. The single bacteria separation and capture device of claim 1, wherein the stage (2) is further provided with a sterile collection device (23).

3. The single bacteria separating and capturing device according to claim 1, wherein the single bacteria picking structure (5) comprises a syringe and a needle provided on the syringe, or the single bacteria picking structure (5) is a glass capillary.

4. The single bacteria separation and capture device of claim 1, wherein the microscope (1) is an inverted fluorescence microscope.

5. The single bacteria separation trap of claim 1, wherein the single bacteria picking structure (5) is mounted on the micromanipulator (4) by means of an adapter fixture.

6. The single bacteria separating and capturing device of claim 1, wherein the agar microarray chip (22) has 500-5000 circular holes, the diameter of the circular holes is 10-50 microns, the distance between adjacent circular holes is 5-15 microns, and the depth of the circular holes is 8-20 microns.

7. The single bacteria separating and capturing device of claim 6, wherein the agar microarray chip (22) has a size of 15mm x 15mm.

8. The single bacteria separation capture device of claim 1, wherein the coated slide (21) is a laser microdissected coated slide coated with a transparent film.

9. The single bacteria separation and capture device of claim 8, wherein the coated slide (21) has an overall dimension of 75 x 24mm, and wherein the coated transparent membrane has a dimension of 45 x 16mm.

10. The single bacteria separation and capture device of claim 1, wherein the micromanipulator (4) is electrically or manually operated.

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