WO2022199145A1

WO2022199145A1 - Anaerobic microbial culture and real-time observation device

Info

Publication number: WO2022199145A1
Application number: PCT/CN2021/137926
Authority: WO
Inventors: 黄术强; 于跃; 马智鑫; 邓宇芳; 温慧; 李思宏
Original assignee: 中国科学院深圳先进技术研究院
Priority date: 2021-03-24
Filing date: 2021-12-14
Publication date: 2022-09-29
Also published as: CN112899124A

Abstract

The present invention belongs to the technical field of microbial culture, and provides an anaerobic microbial culture and real-time observation device, and a culture method for observing anaerobic microorganisms in real time by using the device. The device comprises a base, an upper lid, a micro-fluidic chip and a culture solution storage chamber, wherein the base and the upper lid are snap-fitted with each other in a sealed manner; a first slot is used for accommodating the culture solution storage chamber; a second slot is used for providing a culture space for anaerobic microorganisms; a third slot is used for accommodating the micro-fluidic chip, and a horizontal slot face of the third slot is provided with a first window; a fourth slot is disposed opposite the second slot, and a fifth slot is disposed opposite the first slot; a horizontal slot face of the fourth slot is provided with a second window; the first window and the second window are arranged opposite each other; and the culture solution storage chamber is provided with a liquid output hole, and a side wall of the upper lid is provided with a gas inlet, a gas outlet and a liquid outlet. The device has excellent gas tightness, and can provide a stable anaerobic gas atmosphere for long-term stable liquid culture of anaerobic microorganisms, and the gas atmosphere is controllable; and experimental requirements such as high throughput, large-range multi-site sampling and high-magnification photography are met.

Description

An anaerobic microorganism culture and real-time observation device

technical field

The invention belongs to the technical field of microorganism culture, and in particular relates to an anaerobic microorganism culture and real-time observation device.

Background technique

The long-term follow-up observation of a large number of single bacterial cells has become an important means of microbial physiology research. Because microfluidic technology has the advantages of flexible design, convenient and controllable, automatic integration, real-time analysis, single-cell manipulation, saving samples and reagents, etc., it has been applied to a variety of microbial physiology research. , Design related microfluidic chips. No matter how the chip is designed, its micro-scale characteristics enable researchers to use a special fixing device (CN112113901A) to fix it and combine it with a time-lapse microscopic imaging system to realize long-term autofocus shooting and data. collection.

Regarding time-lapse imaging technology, in addition to designing and building an auto-focusing optical imaging system, some high-end scientific research-grade commercial microscopes have integrated time-lapse photography systems and high-precision motorized displacement while providing high-power oil lenses. Platforms, autofocus systems, etc. (such as the Nikon ECLIPSE Ti2 series). In addition, some commercially available temperature control devices can be integrated into specific models of microscopes to control the temperature around the cell sample to a set value.

The microorganisms cultivated by the current application of the original technology basically have no special requirements for the gas atmosphere. However, in nature, there are a variety of microorganisms that have strict requirements on the culture gas atmosphere, especially many anaerobic microorganisms that are closely related to human health, but there is a lack of related microfluidic culture and time-lapse imaging systems for them. . At present, there are no related reports that simultaneously satisfy the combination of culture gas atmosphere control, single-cell microfluidic technology, and time-lapse microscopic imaging technology.

The microfluidic chip is mainly made of polydimethylsiloxane (PDMS), which is integrally sealed on a glass slide. The chip structure is mainly composed of a main channel that communicates with many sub-channels that communicate with it vertically. The culture medium flows from the main channel. The inlet flows in and the outlet flows out, providing nutrients to the microbial cells growing in the sub-channels through diffusion. The chip itself made of polydimethylsiloxane has good air permeability, which can ensure the oxygen required for the growth of general microorganisms. This microfluidic chip is directly exposed to the air atmosphere for time-lapse microscopic imaging, and the strict gas atmosphere cannot be controlled. This method is widely used in general microorganism culture, but cannot meet the microorganisms with special requirements for gas atmosphere, especially It is a long-term culture requirement for strict anaerobic microorganisms.

technical problem

Finevest, A. et al. adopted the solid culture method of agarose plate for anaerobic microbial culture, specifically: the microbial cells were placed between the glass slide and the plate formed by the solidification of agarose, and the top transparent lid was sealed by a gasket, The device also includes an inlet and an outlet for the anaerobic mixture. Since this method uses a solid medium, it can only ensure the short-term growth of anaerobic microorganisms, and the physiological state of the microbial cells is not stable, so it cannot be cultured for a long time like the continuous perfusion liquid culture, and the microbial cells are in a stable growth state. However, liquid culture is an important prerequisite for the study of microbial physiology. Although the Desulfovibrio successfully cultivated in this institute is an anaerobic bacteria, in fact, many anaerobic bacteria can still survive under a certain oxygen concentration (2%~8%), but experiments show that it is difficult to be cultivated for a long time. To ensure the steady-state growth (logarithmic growth) of anaerobic bacteria, the mortality rate will be greatly increased; for facultative anaerobic bacteria, under low oxygen, they are in different oxygen concentrations (such as <0.1%, 0.1%~15%, 2-10%), the physiological state is very different; the limitations of its device design cannot achieve a strict anaerobic degree, and can not achieve further liquid culture.

technical solutions

Based on the problems existing in the prior art, the purpose of the present invention is to provide an anaerobic microorganism cultivation and real-time observation device; the present invention also aims to provide a cultivation and real-time observation of anaerobic microorganisms using the anaerobic microorganism cultivation and real-time observation device method.

The object of the present invention is achieved through the following technical solutions:

On the one hand, an anaerobic microorganism cultivation and real-time observation device of the present invention comprises:

Base, upper cover, microfluidic chip and culture medium storage chamber;

The base and the upper cover are sealed and buckled with each other, and the base is provided with a first groove and a second groove; the first groove is used for accommodating the culture liquid storage chamber, and the second groove The groove is used to provide a culture space for anaerobic microorganisms; a third groove is opened on the horizontal groove surface of the second groove, and the third groove is used for accommodating the microfluidic chip, and the A first window for image acquisition is provided on the horizontal groove surface of the third groove;

The upper cover is provided with a fourth groove and a fifth groove; the fourth groove is arranged relative to the second groove to provide a culture space for anaerobic microorganisms; the fifth groove is opposite to the first groove A second viewing window is provided on the horizontal groove surface of the fourth groove for the incident light source; the first viewing window and the second viewing window are arranged opposite to each other ;

The culture liquid storage chamber is provided with a liquid outlet hole, which is used to communicate with the inlet end of the microfluidic chip through a pipeline; the side wall of the upper cover is provided with a gas inlet, a gas outlet and a liquid outlet , the liquid outlet is used to communicate with the outlet end of the microfluidic chip through a pipeline.

The anaerobic microorganism culture and real-time observation device of the present invention has excellent air tightness, wherein the culture liquid storage chamber and the microfluidic chip are in a strictly sealed environment, and the continuous flow of gas in the device can be realized through the gas inlet and the gas outlet. Specific gas atmosphere flow (such as nitrogen, carbon dioxide and other gases) to achieve a strict anaerobic environment in the device to ensure that the liquid medium in the microfluidic chip and the culture liquid storage chamber is in a stable anaerobic gas atmosphere; in addition The device is also provided with a window for light source incidence and acquisition and imaging. It integrates microfluidic chips and time-lapse microphotography technology. With the better anaerobic gas atmosphere of the present invention, it can realize long-term (more than 200h) liquid culture, and maintain a steady state of cell growth. The gas inlet of the anaerobic microorganism culture and real-time observation device of the present invention can be designed with at least one gas inlet, for example, two gas inlets are designed to prepare for the requirement of positive pressure feeding into the culture medium; in general use, only one gas inlet is required Gas inlet, alternate gas inlet can be sealed by sealing cock or photosensitive glue.

The microfluidic chip in the anaerobic microorganism cultivation and real-time observation device of the present invention is a conventional microfluidic chip in the field, which is composed of a glass slide and a chip structure sealed on the glass slide, and the chip structure is The main channel communicates with many sub-channels perpendicular to it, and the inlet end and the outlet end communicate with the main channel. Before the microfluidic chip is assembled in the device of the present invention, the seed liquid of anaerobic microorganisms has been built in. The microfluidic chip can be accommodated in the third groove, and the size of the glass slide matches the size of the horizontal groove surface of the third groove.

In the above-mentioned anaerobic microorganism cultivation and real-time observation device, preferably, the device further comprises a chip fixing plate;

The chip fixing plate is used for fixing the microfluidic chip in the third groove, and a hollow window is opened on the chip fixing plate; The second window is set relatively.

In the above-mentioned apparatus for culturing and real-time observation of anaerobic microorganisms, preferably, a plurality of first screw holes are formed on the chip fixing plate around the hollow window, and the lower ends of the first screw holes are provided with first elastic holes. One end of the first elastic member is in contact with the chip fixing plate on the edge of the first screw hole, and is fixed by adhesive; the other end is connected with the microfluidic control plate in the third groove The chips are in contact with each other; the periphery of the chip fixing plate is fixed with the horizontal groove surface of the second groove.

In the present invention, the lower glass slide of the microfluidic chip is pressed against the first window at the bottom of the third groove through the chip fixing plate and the first elastic member, so as to realize the stable assembly between the chip fixing plate and the microfluidic chip. .

In the above-mentioned anaerobic microorganism cultivation and real-time observation device, preferably, the number of the first screw holes is at least 4, which are evenly arranged on the chip fixing plate around the hollow window; The number of elastic members is at least four.

In the above-mentioned apparatus for culturing and real-time observation of anaerobic microorganisms, preferably, the first elastic member includes a syringe and a needle movably arranged in the syringe, a spring is arranged in the syringe, and the needle The spring can move elastically, the needle cylinder is abutted with the chip fixing plate through the first screw hole, and the needle can be elastically abutted on the microfluidic chip.

The elastic member of the present invention is designed as a pogo pin group, which provides a uniform and gentle pressing and fixing method, which can effectively prevent the glass sheet with a thickness of more than 0.1 mm from breaking when it is pressed and fastened. In addition, the fixed method of this design can be combined with the microscope for real-time culture observation and data collection, and the time-lapse microscopic imaging of single-cell anaerobic microorganisms does not lose focus and horizontal position shift exceeding 2 μm within 200 hours.

In the above-mentioned anaerobic microorganism culture and real-time observation device, preferably, a plurality of second screw holes are opened around the chip fixing plate, and the horizontal groove surface of the second groove around the third groove is surrounded. There are a plurality of third screw holes, the second screw holes correspond to the holes of the third screw holes, and the fixed connection between the chip fixing plate and the horizontal groove surface of the second groove is realized by screws. . The third screw hole does not penetrate the bottom plate.

In the above-mentioned anaerobic microorganism culture and real-time observation device, preferably, the number of the second screw holes is at least 4, which are evenly arranged around the chip fixing plate; the number corresponding to the third screw holes is at least 4. 4.

In the above-mentioned anaerobic microorganism cultivation and real-time observation device, preferably, first support beams are arranged on the surrounding edges of the horizontal groove surface of the third groove, and the surrounding edges of the first viewing window are connected with the first support beam through an adhesive. A support beam is sealed and fixedly connected. In order to ensure that the device has excellent air tightness under the premise of excellent light permeability.

In the above-mentioned anaerobic microorganism cultivation and real-time observation device, preferably, the peripheral edge of the horizontal groove surface of the fourth groove is provided with a second support beam, and the peripheral edge of the second viewing window is connected to the first window through an adhesive. The two supporting beams are sealed and fixedly connected. In order to ensure that the device has excellent air tightness under the premise of excellent light permeability.

In the above-mentioned apparatus for culturing and real-time observation of anaerobic microorganisms, preferably, the binder includes a photosensitive adhesive.

In the above-mentioned apparatus for culturing and real-time observation of anaerobic microorganisms, preferably, the first viewing window and the second viewing window comprise glass plates or acrylic plates with good light permeability.

In the above-mentioned anaerobic microorganism culture and real-time observation device, preferably, the shape of the first window and the second window includes a rectangle, a circle or an ellipse; the area of the second window is larger than that of the first window area.

By designing the first viewing window and the second viewing window of transparent material, and the area of the second viewing window is larger than that of the first viewing window, it is convenient for inverted microscope observation, and can realize various imaging such as brightfield/phase contrast epi-light, laser-excited fluorescence, etc. This mode meets the needs of epi-light illumination and the need for sampling and shooting of at least 100 points with an oil immersion objective up to 100x magnification.

In the above-mentioned anaerobic microorganism cultivation and real-time observation device, preferably, the culture liquid storage chamber is composed of a storage chamber body and a storage chamber upper cover, and the liquid outlet is opened on the storage chamber upper cover and communicates with the storage chamber. The pipe at the inlet end of the microfluidic chip goes deep into the bottom of the culture medium storage chamber through the liquid outlet. The culture fluid storage chamber of the present invention can provide sufficient liquid culture medium for the long-term culture of anaerobic microorganisms in the microfluidic chip.

In the above-mentioned anaerobic microorganism culture and real-time observation device, preferably, the storage chamber body and the storage chamber upper cover are respectively provided with a plurality of screw holes that are evenly distributed and corresponding to each other, and the storage chamber body is realized by screws. A fixed connection to the upper cover of the storage compartment.

In the above-mentioned apparatus for culturing and real-time observation of anaerobic microorganisms, preferably, at least one air hole is further opened on the upper cover of the storage chamber.

The arrangement of the air holes in the invention can ensure that the upper air pressure of the liquid medium in the culture medium storage chamber is the same as the air pressure inside the device, so that the negative pressure of the liquid outlet can smoothly draw out the liquid medium in the culture medium storage room, so as to provide a stable flow rate .

In the above-mentioned anaerobic microorganism cultivation and real-time observation device, preferably, a plurality of evenly distributed and corresponding screw holes are respectively opened on the edge of the contact surface where the base and the upper cover are engaged with each other, and the screw holes are realized by screws. The base is fastened with the seal of the upper cover.

In the above-mentioned anaerobic microorganism cultivation and real-time observation device, preferably, an annular groove is provided on the edge of the contact surface of the base, and the annular groove is used for accommodating a rubber ring to realize the connection between the base and the upper part. Further sealing of the lid. Ensure that the device has excellent air tightness.

In the above-mentioned anaerobic microorganism cultivation and real-time observation device, preferably, a plurality of fourth screw holes are opened on the side edge of the second groove, and a plurality of corresponding threaded holes are arranged on the outer edge of the second groove. The second elastic member of the fourth screw hole; the fixing of the second elastic member and the fourth screw hole is realized by the adhesive; the second elastic member is used to fit and fix the microscope standard stage . The second elastic member of the present invention can also be selected from the same elastic needle assembly as the first elastic member.

In addition, by changing the outer contour size and shape of the upper cover and base, it can be matched with different types and models of motorized stage adapters, and also with different models and brands of microscopes.

In the above-mentioned anaerobic microorganism cultivation and real-time observation device, preferably, the material of the base includes aluminum alloy; the material of the upper cover includes resin; and the material of the culture liquid storage chamber includes polyether ether ketone.

In the above-mentioned apparatus for culturing and real-time observation of anaerobic microorganisms, preferably, the material of the chip fixing plate includes aluminum alloy.

In the above-mentioned anaerobic microorganism cultivation and real-time observation device, preferably, both the gas inlet and the gas outlet are connected to an external gas pipeline (to prevent gas leakage) through a screw plug and a joint edge ring, and the gas pipeline is arranged on the There is a stop valve. Setting a stop valve can prevent the device from leaking when no gas is being introduced.

In the above-mentioned apparatus for culturing and real-time observation of anaerobic microorganisms, preferably, a membrane pump is connected to the liquid outlet, and the membrane pump is used to provide negative pressure. By setting the membrane pump, the liquid medium in the culture medium storage chamber can smoothly flow through the microfluidic chip and then flow out under the action of the pressure difference between the air pressure in the device and the negative pressure of the liquid outlet.

On the other hand, the present invention also provides a cultivation method for real-time observation of anaerobic microorganisms, which adopts the above-mentioned anaerobic microorganism cultivation and real-time observation device to operate, comprising the following steps:

Prepare a microfluidic chip with built-in anaerobic microbial seed solution. The microfluidic chip is composed of a glass slide and a chip structure sealed on the glass slide. channel, the inlet end and the outlet end are connected with the main channel; the micro-control flow chip is placed in the third groove, the inlet end is connected with the liquid outlet hole of the culture medium storage chamber through the pipeline; the outlet end is connected with the side wall of the upper cover The liquid outlet on the top is connected; after the pipeline is connected, the top cover and the bottom plate are buckled and sealed to complete the assembly of the device;

The device is set up on the microscope and fixed with the microscope stage adapter; the specific gas atmosphere flow of anaerobic microorganisms is introduced through the gas inlet on the side wall of the upper cover, and the continuous cultivation of anaerobic microorganisms in an anaerobic environment is carried out. The microscope light source and objective lens position are used for real-time culture imaging observation.

beneficial effect

The anaerobic microorganism culture and real-time observation device of the invention has excellent air tightness, can provide a stable anaerobic gas atmosphere for long-term stable liquid culture of anaerobic microorganisms, the gas atmosphere is controllable, and the anaerobic microorganism can last for more than 200 hours. In addition, with the chip fixing plate of the present invention and the designed window structure, it can ensure that the long-time time-lapse shooting will not lose focus and the horizontal position shift of more than 2 μm, and at the same time meet the requirements of A series of experimental requirements, such as high-throughput multi-site sampling, large-scale multi-site sampling, and high-magnification shooting, improve the experimental throughput while ensuring data quality.

Description of drawings

The drawings described herein are for explanatory purposes only and are not intended to limit the scope of the present disclosure in any way. In addition, the shapes and proportions of the components in the figures are only schematic and are used to help the understanding of the present invention, and do not specifically limit the shapes and proportions of the components of the present invention. Under the teachings of the present invention, those skilled in the art can select various possible shapes and proportions according to specific conditions to implement the present invention.

Fig. 1 is a schematic diagram of the partially disassembled structure of the anaerobic microorganism culture and real-time observation device in Example 1 of the present invention (without including the microfluidic chip, connecting pipeline, fixing screw, second elastic member, rubber ring and other components);

2 is a schematic diagram of the base structure in the anaerobic microorganism cultivation and real-time observation device in Example 1 of the present invention;

3 is a schematic diagram of the structure of the upper cover in the anaerobic microorganism cultivation and real-time observation device in Example 1 of the present invention;

4 is a schematic structural diagram of a chip fixing plate in the anaerobic microorganism culture and real-time observation device in Example 1 of the present invention;

5 is a schematic diagram of the assembly structure of the first elastic member in the anaerobic microorganism culture and real-time observation device in Example 1 of the present invention;

6 is a schematic diagram of the structure of the storage chamber body of the culture liquid storage chamber in the anaerobic microorganism cultivation and real-time observation device in Example 1 of the present invention;

7 is a schematic diagram of the structure of the upper cover of the storage chamber of the culture liquid storage chamber in the anaerobic microorganism cultivation and real-time observation device in Example 1 of the present invention;

8 is a schematic structural diagram of a microfluidic chip in the anaerobic microorganism culture and real-time observation device in Example 1 of the present invention;

Fig. 9 is a bright field phase contrast image (A), aerobic culture fluorescence field image (B) and anaerobic culture fluorescence field image (C) actually photographed by microscopy during the culture process in Example 2 of the present invention;

FIG. 10 is a graph showing the results of oxygen indicator detection in the tightness detection experiment in Example 3 of the present invention.

Description of the symbols in the drawings:

1, base; 11, first groove; 12, second groove; 13, third groove; 14, first window; 15, annular groove; 16, fourth screw hole; 17, screw hole; 18 , the third screw hole; 19, the first support beam; 2, the upper cover; 21, the fifth groove; 22, the fourth groove; 23, the second window; 24, the gas inlet; 25, the second support beam; 26, screw hole; 27, gas outlet; 28, liquid outlet; 3, chip fixing plate; 31, hollow window; 32, second screw hole; 33, first screw hole; 34, first elastic part; 341, Needle; 342, syringe; 4, storage chamber body; 41, screw hole; 5, storage chamber upper cover; 51, screw hole; 52, air hole; 53, liquid outlet; 61, slide glass; 62, main channel ; 63, sub-channel; 64, inlet end; 65, outlet end.

Embodiments of the present invention

In order to have a clearer understanding of the technical features, purposes and beneficial effects of the present invention, the technical solutions of the present invention are now described in detail below, but should not be construed as limiting the scope of implementation of the present invention. Under the teaching of the present invention, the skilled person can conceive any possible modifications based on the present invention, and these should be regarded as belonging to the scope of the present invention. It should be noted that when an element is referred to as being "disposed on" another element, it can be directly on the other element or intervening elements may also be present. When an element is referred to as being "connected" to another element, it can be directly connected to the other element or intervening elements may also be present. The terms "installed", "connected" and "connected" should be understood in a broad sense, for example, it can be a mechanical connection or an electrical connection, or it can be the internal communication between two components, it can be directly connected, or it can be indirectly connected through an intermediate medium, For those of ordinary skill in the art, the specific meanings of the above terms can be understood according to specific situations. The terms "vertical", "horizontal", "upper", "lower", "left", "right" and similar expressions used herein are for the purpose of illustration only and do not represent the only embodiment.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terms used herein in the description of the present invention are for the purpose of describing specific embodiments only, and are not intended to limit the present invention. As used herein, the term "and/or" includes any and all combinations of one or more of the associated listed items.

Example 1:

This embodiment provides an anaerobic microorganism cultivation and real-time observation device, as shown in FIG. 1 , which includes:

Base 1 (as shown in Figure 2), upper cover 2 (as shown in Figure 3), microfluidic chip, chip fixing plate 3 (as shown in Figure 4), and culture fluid storage chamber (storage chamber body 4 and storage chamber The upper cover 5 is formed, as shown in Figures 6 and 7).

The edges of the contact surfaces of the base 1 and the upper cover 2 are respectively provided with 8 evenly distributed and corresponding screw holes 17 and 26, and the base 1 and the upper cover 2 are sealed and buckled by screws; the contact of the base 1 The edge of the surface is provided with an annular groove 15, which is used for accommodating a rubber ring to further seal the base 1 and the upper cover 2 and ensure the airtightness of the device.

The base 1 is provided with a first groove 11 and a second groove 12; the first groove 11 is used for accommodating a culture liquid storage chamber, and the second groove 12 is used to provide a culture space for anaerobic microorganisms; the second groove 12 is provided with a third groove 13 on the horizontal groove surface, the third groove 13 is used for accommodating the microfluidic chip, and the horizontal groove surface of the third groove 13 is provided with a first window 14 for capturing and imaging . The material of the base 1 is aluminum alloy, which is machined by a CNC machine tool.

The upper cover 2 is provided with a fourth groove 22 and a fifth groove 21; the fourth groove 22 is arranged relative to the second groove 12 to provide a culture space for anaerobic microorganisms; the fifth groove 21 is opposite to the first groove 11 is provided for clamping and accommodating the culture medium storage chamber; the horizontal groove surface of the fourth groove 22 is provided with a second window 23 for the light source to enter; the first window 14 and the second window 23 are arranged opposite. The material of the upper cover 2 is resin, which is obtained by 3D resin printing.

The microfluidic chip is a conventional microfluidic chip in the field. As shown in FIG. 8 , it is a chip structure composed of a glass slide 61 and sealed on the glass slide 61 , and the chip structure is communicated by the main channel 62 Numerous sub-channels 63 perpendicular to it, the inlet end 64 and the outlet end 65 communicate with its main channel 62; the seed liquid of anaerobic microorganisms has been built into it before being assembled in the device of the present invention.

The chip fixing plate 3 is used for fixing the microfluidic chip in the third groove 13 , and the chip fixing plate 3 is provided with a hollow window 31 ; The material of the chip fixing plate 3 is aluminum alloy, which is machined by a numerical control machine tool.

The chip fixing plate 3 surrounding the hollow window 31 is provided with 20 first screw holes 33 evenly distributed, and the lower ends of the first screw holes 33 are provided with 20 components of the first elastic members 34 (as shown in FIG. 5 ). The first elastic member 34 includes a needle cylinder 342 and a needle head 341 movably arranged in the needle cylinder 342. A spring is provided in the needle cylinder 342, the needle head 341 can be elastically moved by the spring, and the needle cylinder 342 is connected to the needle cylinder 342 through the first screw hole 33. The chip fixing plate 3 is in contact with each other, and is fixed by photosensitive adhesive, and the needle 341 can elastically abut on the slide glass 61 of the microfluidic chip.

Four second screw holes 32 are evenly distributed around the chip fixing plate 3, and the horizontal groove surface of the second groove 12 around the third groove 13 is provided with a one-to-one correspondence with the center of the second screw holes 32. The four third screw holes 18 are used to realize the fixed connection between the chip fixing plate 3 and the horizontal groove surface of the second groove 12 through screws.

A first support beam 19 is disposed on the peripheral edge of the horizontal groove surface of the third groove 13 , and the peripheral edge of the first viewing window 14 is sealed and fixedly connected to the first support beam 19 through photosensitive adhesive. A second support beam 25 is arranged on the peripheral edge of the horizontal groove surface of the fourth groove 22 , and the peripheral edge of the second viewing window 23 is sealed and fixedly connected to the second support beam 25 through photosensitive adhesive. The material of the first window 14 and the second window 23 is a glass plate (or acrylic plate) with good light permeability, the shape of the first window 14 and the second window 23 is a rectangle, and the area of the second window 23 is larger than that of the first window 23. The area of a window.

The culture liquid storage chamber is composed of the storage chamber body 4 and the storage chamber upper cover 5, the liquid outlet 53 is opened on the storage chamber upper cover 5, and the pipeline connected to the inlet end 64 of the microfluidic chip goes deep into the culture through the liquid outlet 53. bottom of the liquid storage chamber. The storage chamber body 4 and the storage chamber upper cover 5 are respectively provided with four evenly distributed screw holes 41 and 51 corresponding to each other, and the storage chamber body 4 and the storage chamber upper cover 5 are fixedly connected by screws. The upper cover 5 of the storage chamber is also provided with four evenly distributed air holes 52 .

The arrangement of the air holes in the invention can ensure that the upper air pressure of the liquid medium in the culture medium storage chamber is the same as the air pressure inside the device, so that the negative pressure of the liquid outlet can smoothly draw out the liquid medium in the culture medium storage room, so as to provide a stable flow rate . The material of the culture medium storage chamber is polyetheretherketone (PEEK), which is prepared by 3D printer printing and can provide enough liquid medium for the long-term culture of anaerobic microorganisms in the microfluidic chip.

The side wall of the upper cover 2 is provided with 2 gas inlets 24 (one of the gas inlets is a spare gas inlet, which is sealed by photosensitive adhesive when not in use), a gas outlet 27 and a liquid outlet 28, and the liquid outlet 28 It is used to communicate with the outlet end 65 of the microfluidic chip through a pipeline. Both the gas inlet 24 and the gas outlet 27 are connected to the external thin gas pipeline through a screw plug and a joint edge ring, and a flow stop valve is arranged on the thin gas pipeline. Setting a stop valve can prevent the device from leaking when no gas is being introduced. The liquid outlet 28 is connected to a membrane pump. The membrane pump is used to provide negative pressure, so that the liquid medium in the culture medium storage chamber can smoothly flow through the microfluidic control under the action of the pressure difference between the air pressure in the device and the negative pressure of the liquid outlet. The chip is then flowed out.

Sixteen fourth screw holes 16 are formed on the side edge of the second groove 12 , and sixteen elastic members (same as the first elastic member) corresponding to the fourth screw holes 16 are arranged on the outer edge of the second groove 12 . ; The fixing of the elastic member and the fourth screw hole 16 is realized by photosensitive adhesive. An elastic piece is arranged on the side edge of the second groove, which can be adapted to and fixed with a standard microscope stage, so as to facilitate multi-point time-lapse shooting.

Example 2:

The present embodiment provides a real-time observation and cultivation method for anaerobic microorganisms, which adopts the anaerobic microorganism cultivation and real-time observation device of Embodiment 1 for operation, and includes the following steps:

Prepare a microfluidic chip with built-in anaerobic microorganism (facultative anaerobic bacteria Escherichia coli) seed solution, the microfluidic chip is composed of a glass slide and a chip structure sealed on the glass slide , the chip structure is composed of a main channel that communicates with many sub-channels perpendicular to it, and the inlet end and the outlet end are connected to the main channel; the micro-control flow chip is placed in the third groove, and the inlet end is connected to the culture medium storage chamber through the pipeline. The outlet end of the device is connected with the liquid outlet on the side wall of the upper cover; after the pipeline is connected, the upper cover and the bottom plate are buckled and sealed and fixed to complete the assembly of the device; among them, the micro-control flow chip The fixing is achieved by the chip fixing plate and its first elastic member.

Set up the device on the microscope and fix it with the microscope stage adapter; pass the gas atmosphere flow (95%N ₂ and 5% CO ₂ ) specific to anaerobic microorganisms through the gas inlet on the side wall of the upper cover to conduct anaerobic Continuous cultivation of anaerobic microorganisms in the environment, adjust the position of the microscope light source and objective lens, and conduct real-time imaging observation. The experimental results are shown in Figure 9.

It can be seen from Figure 9 that since the fluorescent protein can be excited to produce fluorescence under the oxygen concentration of 0.00005%, the fluorescent protein expressed by the microorganisms cultivated in this device is excited and has no obvious fluorescence (see C in Figure 9). ), indicating that the oxygen concentration around the microbial cells is extremely low, reaching a strictly anaerobic level.

Example 3:

In this implementation, the air-tightness verification test of the anaerobic microorganism culture and real-time observation device of Example 1 was carried out. When gas was continuously supplied, the entire device was placed in water without visible gas leakage, indicating that its air-tightness was excellent.

For the verification of anaerobic atmosphere, Mitsubishi MGC oxygen indicator is used, and the experimental results are shown in Figure 10. It can be seen intuitively from Figure 10 that the oxygen concentration in the cultured gas environment is less than 0.1%, which is lower than the minimum detection limit.

Claims

An anaerobic microorganism cultivation and real-time observation device is characterized in that, comprising:

Base, upper cover, microfluidic chip and culture medium storage chamber;

The base and the upper cover are sealed and buckled with each other, and the base is provided with a first groove and a second groove; the first groove is used for accommodating the culture liquid storage chamber, and the second groove The groove is used to provide a culture space for anaerobic microorganisms; a third groove is opened on the horizontal groove surface of the second groove, and the third groove is used for accommodating the microfluidic chip, and the A first window for image acquisition is provided on the horizontal groove surface of the third groove;

The upper cover is provided with a fourth groove and a fifth groove; the fourth groove is arranged relative to the second groove to provide a culture space for anaerobic microorganisms; the fifth groove is opposite to the first groove A second viewing window is provided on the horizontal groove surface of the fourth groove for the incident light source; the first viewing window and the second viewing window are arranged opposite to each other ;

The culture liquid storage chamber is provided with a liquid outlet hole, which is used to communicate with the inlet end of the microfluidic chip through a pipeline; the side wall of the upper cover is provided with a gas inlet, a gas outlet and a liquid outlet , the liquid outlet is used to communicate with the outlet end of the microfluidic chip through a pipeline.
The anaerobic microorganism culture and real-time observation device according to claim 1, characterized in that: the device further comprises a chip fixing plate;

The chip fixing plate is used for fixing the microfluidic chip in the third groove, and a hollow window is opened on the chip fixing plate; The second window is set relatively.
The apparatus for culturing and real-time observation of anaerobic microorganisms according to claim 2, wherein a plurality of first screw holes are formed on the chip fixing plate around the hollow window, and the lower ends of the first screw holes are provided with a plurality of first screw holes. A first elastic member is provided, one end of the first elastic member is in contact with the chip fixing plate on the edge of the first screw hole, and is fixed by adhesive; the other end is connected with the chip fixing plate in the third groove. The microfluidic chips are in contact with each other; the periphery of the chip fixing plate is fixed with the horizontal groove surface of the second groove.
The apparatus for culturing and real-time observation of anaerobic microorganisms according to claim 3, wherein the number of the first screw holes is at least 4, which are evenly arranged on the chip fixing plate around the hollow window; The number corresponding to the first elastic members is at least four.
The apparatus for culturing and real-time observation of anaerobic microorganisms according to claim 3 or 4, wherein the first elastic member comprises a syringe and a needle movably arranged in the syringe, and the syringe A spring is provided, the needle can elastically move through the spring, the needle cylinder is in contact with the chip fixing plate through the first screw hole, and the needle can elastically abut on the microfluidic on the chip.
The apparatus for culturing and real-time observation of anaerobic microorganisms according to claim 4, wherein a plurality of second screw holes are formed around the chip fixing plate, and the second concave holes around the third groove are formed. The horizontal groove surface of the groove is provided with a plurality of third screw holes, the second screw holes correspond to the holes of the third screw holes, and the level of the chip fixing plate and the second groove is realized by screws Fixed connection of groove face.
The anaerobic microorganism culture and real-time observation device according to claim 6, wherein the number of the second screw holes is at least 4, which are evenly arranged around the chip fixing plate; corresponding to the third screw holes The number of holes is at least four.
The apparatus for culturing and real-time observation of anaerobic microorganisms according to claim 1, characterized in that: the peripheral edge of the horizontal groove surface of the third groove is provided with a first support beam, and the peripheral edge of the first viewing window is bonded by bonding The agent is sealed and fixedly connected to the first support beam.
The device for culturing and real-time observation of anaerobic microorganisms according to claim 1, characterized in that: the peripheral edge of the horizontal groove surface of the fourth groove is provided with a second support beam, and the peripheral edge of the second viewing window is bonded by bonding The agent is sealed and fixedly connected with the second support beam.
The device for culturing and real-time observation of anaerobic microorganisms according to claim 3, 8 or 9, characterized in that: the binder comprises a photosensitive adhesive.
The anaerobic microorganism cultivation and real-time observation device according to claim 1, wherein the first viewing window and the second viewing window comprise glass plates or acrylic plates with good light permeability.
The device for culturing and real-time observation of anaerobic microorganisms according to claim 1 or 11, wherein the shape of the first window and the second window comprises a rectangle, a circle or an ellipse; The area is larger than the area of the first viewing window.
The apparatus for culturing and real-time observation of anaerobic microorganisms according to claim 1, wherein the culture liquid storage chamber is composed of a storage chamber body and a storage chamber upper cover, and the liquid outlet is opened on the storage chamber upper cover On the upper part, the pipeline connected to the inlet end of the microfluidic chip goes deep into the bottom of the culture medium storage chamber through the liquid outlet.
The anaerobic microorganism culture and real-time observation device according to claim 13, wherein the storage chamber body and the storage chamber upper cover are respectively provided with a plurality of screw holes that are evenly distributed and corresponding to each other, and are realized by screws. The storage chamber body is fixedly connected with the upper cover of the storage chamber.
The apparatus for culturing and real-time observation of anaerobic microorganisms according to claim 13, wherein at least one air hole is further opened on the upper cover of the storage chamber.
The anaerobic microorganism cultivation and real-time observation device according to claim 1, wherein a plurality of screw holes evenly distributed and corresponding to each other are respectively opened on the edge of the contact surface of the base and the upper cover that are engaged with each other, and The base is sealed with the upper cover through screws.
The apparatus for culturing and real-time observation of anaerobic microorganisms according to claim 16, characterized in that: an annular groove is formed on the edge of the contact surface of the base, and the annular groove is used for accommodating a rubber ring to realize the The base is further sealed with the upper cover.
The apparatus for culturing and real-time observation of anaerobic microorganisms according to claim 5, wherein a plurality of fourth screw holes are opened on the side edge of the second groove, and are arranged on the outer edge of the second groove There are a plurality of second elastic members corresponding to the fourth screw holes; the second elastic member and the fourth screw holes are fixed by the adhesive; the second elastic member is used for the microscope standard load The table is adapted and fixed.
The anaerobic microorganism cultivation and real-time observation device according to claim 1, wherein: the material of the base comprises aluminum alloy; the material of the upper cover comprises resin; the material of the culture solution storage chamber comprises polyether ether ketone.
The anaerobic microorganism culture and real-time observation device according to claim 2, wherein the material of the chip fixing plate comprises aluminum alloy.
The anaerobic microorganism cultivation and real-time observation device according to claim 1, wherein the gas inlet and the gas outlet are connected to an external gas pipeline through a screw plug and a joint edge ring, and the gas pipeline is arranged on the There is a stop valve.
The apparatus for culturing and real-time observation of anaerobic microorganisms according to claim 1, wherein a membrane pump is connected to the liquid outlet, and the membrane pump is used to provide negative pressure.
A cultivation method for real-time observation of anaerobic microorganisms, which adopts the anaerobic microorganism cultivation and real-time observation device according to any one of claims 1 to 22 to operate, comprising the following steps:

Prepare a microfluidic chip with built-in anaerobic microbial seed solution. The microfluidic chip is composed of a glass slide and a chip structure sealed on the glass slide. channel, the inlet end and the outlet end are connected with the main channel; the micro-control flow chip is placed in the third groove, the inlet end is connected with the liquid outlet hole of the culture medium storage chamber through the pipeline; the outlet end is connected with the side wall of the upper cover The liquid outlet on the top is connected; after the pipeline is connected, the top cover and the bottom plate are buckled and sealed to complete the assembly of the device;

The device is set up on the microscope and fixed with the microscope stage adapter; the specific gas atmosphere flow of anaerobic microorganisms is introduced through the gas inlet on the side wall of the upper cover, and the continuous cultivation of anaerobic microorganisms in an anaerobic environment is carried out. The microscope light source and objective lens position are used for real-time culture imaging observation.