Microorganism observation device and microorganism observation method

Abstract

Provided is a technology relating to a microfluidic device capable of detecting or quantifying a substance produced by microorganisms without affecting the growth of the microorganisms. This device for observing microorganisms includes a laminate of a first substrate and a second substrate. A recessed portion, a first groove, and a second groove are provided in the surface of the second substrate facing the first substrate. An inflow hole, an outflow hole, and a reagent introduction hole are provided in the first substrate or the second substrate. The recessed portion is connected to the first groove and constitutes a culture portion for culturing microorganisms. One end of the first groove is connected to the inflow hole, and the other end of the first groove is connected to the outflow hole. One end of the second groove is connected to the reagent introduction hole, and the other end of the second groove is connected to the first groove. The first substrate and / or the second substrate is transparent in an observation area corresponding to a portion of the first groove.

Description

[Technical Field]

[0001] The present invention relates to a device for observing microorganisms and a method for observing microorganisms. [Background technology]

[0002] Microfluidic devices for culturing cells such as microorganisms are known. The microfluidic devices are, for example, as large as a glass slide and include channels with widths of several tens of micrometers and cell culture spaces therein, and have inlet and outlet holes on the surface that form the ends of the channels.

[0003] When microorganisms are cultured in a microfluidic device, the activity of the microorganisms can be observed by measuring the amount of the products produced by the microorganisms (hereinafter referred to as "microorganism-produced substances"). In this case, it is necessary to add a reagent to the microbial culture space to measure the amount of the microbial-produced substances, but there is a concern that the reagent may affect the growth of the microorganisms. [Prior art documents] [Non-patent literature]

[0004] [Non-Patent Document 1] Tarryn E. Miller et al. 2020. “Light-Powered CO2 Fixation in a Chloroplast Mimic with Natural and Synthetic Parts.” Science 368 (6491): 649-654. Summary of the Invention [Problem to be solved by the invention]

[0005] An object of the present invention is to provide a technique relating to a microparticle device that can detect or quantify substances produced by microorganisms without affecting the growth of the microorganisms. [Means for solving the problem]

[0006] According to a first aspect of the present invention, a laminate of a first substrate and a second substrate, a recess, a first groove, and a second groove are provided on a surface of the second substrate facing the first substrate; an inlet, an outlet, and a reagent introduction hole are provided in the first substrate or the second substrate; the recess is connected to the first groove and constitutes a culture section for culturing microorganisms; The first groove has one end connected to the inlet and the other end connected to the outlet, one end of the second groove is connected to the reagent introduction hole and the other end is connected to the first groove; There is provided a microorganism observation device, wherein at least one of the first substrate and the second substrate is optically transparent in an observation region corresponding to a part of the first groove.

[0007] According to a second aspect of the present invention, A method for observing microorganisms using a microorganism observation device according to the first aspect, comprising: supplying a culture medium from the inlet to the first groove; Culturing the microorganism in the presence of the culture solution in the culture section; allowing a product produced by the microorganism to flow out from the culture section into the first groove; supplying a reagent from the reagent introduction hole to the second groove; observing the culture solution containing the product and the reagent at the position of the observation area; An observation method is provided, which includes: [Effects of the Invention]

[0008] According to the present invention, a technique is provided for a microparticle device that can detect or quantify a substance produced by a microorganism without affecting the growth of the microorganism. [Brief explanation of the drawings]

[0009] [Figure 1]FIG. 1 is a top view of a microorganism observation device according to an embodiment. [Figure 2] FIG. 2 is a cross-sectional view taken along line II-II of the device for observing microorganisms shown in FIG. DETAILED DESCRIPTION OF THE INVENTION

[0010] The following describes embodiments of the present invention. The embodiments described below are more specific embodiments of any of the above aspects. The following features can be incorporated into each of the above aspects, either singly or in combination.

[0011] 1.Device for observing microorganisms A microorganism observation device according to an embodiment will be described with reference to Fig. 1 and Fig. 2. Fig. 1 is a top view of the microorganism observation device according to an embodiment. Fig. 2 is a cross-sectional view of the microorganism observation device shown in Fig. 1 taken along line II-II.

[0012] As shown in FIGS. 1 and 2, the microorganism observation device 1 includes a laminate of a first substrate 1A and a second substrate 1B. Each of the first substrate 1A and the second substrate 1B is optically transparent in an observation region 100 corresponding to the observation portion of the third flow path 10C. It is not necessary for both the first substrate 1A and the second substrate 1B to be optically transparent in the observation region 100 corresponding to the observation portion of the third flow path 10C; it is sufficient for either one of them to be optically transparent in the observation region 100 corresponding to the observation portion of the third flow path 10C. Furthermore, at least one of the first substrate 1A and the second substrate 1B may be optically transparent throughout. Here, "optical transparency" refers to a property that allows observation of a culture solution containing a microbial product and a reagent. "Optical transparency" refers to, for example, visible light transparency.

[0013] The first substrate 1A and the second substrate 1B can be made of polydimethylsiloxane (PDMS), glass, etc. The microorganism observation device 1 has, for example, the same overall size as a glass slide.

[0014] The first substrate 1A is provided with an inlet 11, an outlet 12, and a reagent introduction hole 14. The inlet 11, the outlet 12, and the reagent introduction hole 14 may be provided on the second substrate 1B. The inlet 11 is a hole for introducing a liquid such as a culture medium from outside the microorganism observation device 1 into a main channel 10, which will be described later. The outlet 12 is a hole for discharging a liquid such as a culture medium from the main channel 10 to outside the microorganism observation device 1. The reagent introduction hole 14 is a hole for introducing a reagent from outside the microorganism observation device 1 into a first sub-channel 10E, which will be described later.

[0015] A recess, a first groove, a second groove, and a third groove are provided on the surface of second substrate 1B facing first substrate 1A. The recess constitutes culture section 13 for culturing microorganisms 20. The first groove constitutes main channel 10. The second groove constitutes first sub-channel 10E. The third groove constitutes second sub-channel 10D.

[0016] The culture unit 13 is a chamber for culturing the microorganisms 20. The culture unit 13 contains a microbial suspension containing the microorganisms 20 and a culture solution 30. The microorganisms 20 are, for example, soil microorganisms. The culture solution 30 is a liquid containing nutrients for the microorganisms 20. The culture unit 13 is connected to the main channel 10 via a second sub-channel 10D. The second sub-channel 10D functions to allow a portion of the culture solution 30 supplied from the inlet 11 to the main channel 10 to flow into the culture unit 13, and to allow the culture solution containing products produced by the microorganisms 20 to flow out from the culture unit 13 to the main channel 10. The second sub-channel 10D can also be omitted, and the culture unit 13 can be directly connected to the main channel 10.

[0017] When viewed from a direction perpendicular to the main surface of first substrate 1A, culture section 13 has a length of, for example, 10 μm to 1 mm in the width direction of second sub-channel 10D and a length of, for example, 10 μm to 1 mm in the direction perpendicular to the width direction of second sub-channel 10D. Culture section 13 has a depth of, for example, 1 μm to 1 mm.

[0018] The width W2 of the second sub-channel 10D is smaller than the dimension W1 of the culture section 13 in the width direction of the second sub-channel 10D. By reducing the width W2 of the second sub-channel 10D in this manner, it is possible to make it more difficult for the microorganisms 20 to flow out from the culture section 13 to the main channel 10. The width W2 of the second sub-channel 10D is, for example, 5 μm to 0.5 mm, and the ratio W2 / W1 of the width W2 to the width W1 is, for example, 0.2 to 0.5.

[0019] The length of second sub-channel 10D is, for example, 2 μm to 0.5 mm. By shortening the length of second sub-channel 10D in this manner, it becomes easier for the culture solution containing the product produced by microorganisms 20 to flow from culture section 13 to main channel 10.

[0020] One end of the main flow path 10 is connected to an inlet 11, and the other end is connected to an outlet 12. The main flow path 10 is composed of a first flow path 10A, a second flow path 10B, and a third flow path 10C. The first flow path 10A, the second flow path 10B, and the third flow path 10C are arranged in this order from the inlet 11 toward the outlet 12.

[0021] The first flow path 10A is connected to the culture section 13 via the second sub-flow path 10D. The first flow path 10A is also connected to the reagent introduction hole 14 via the first sub-flow path 10E. This allows a culture solution containing a product produced by the microorganism 20 to flow out from the culture section 13 to the first flow path 10A, and also allows a reagent to be supplied to the first flow path 10A from the reagent introduction hole 14. The reagent is, for example, a substrate that reacts with the product produced by the microorganism 20 to produce a color or emit light. The first flow path 10A has a width of, for example, 10 to 800 μm, a depth of, for example, 10 to 1000 μm, and a length of, for example, 1 to 50 mm.

[0022] The second flow path 10B is located downstream of the first flow path 10A and forms a meandering section. The second flow path 10B may not be meandering, but may be straight. In the second flow path 10B, the culture solution containing the microbial production substance and the reagent are mixed. The second flow path 10B has a width of, for example, 10 to 800 μm, a depth of, for example, 10 to 1000 μm, and a length of, for example, 1 to 50 mm.

[0023] The third flow path 10C is located downstream of the second flow path 10B and includes an observation section. As shown in Figure 2, in this device, the area where the color or light produced by the reagent reacting with the microbial product is observed is called the observation section 100. The part of the third flow path 10C corresponding to the observation section 100 is called the observation section.

[0024] 2, the depth D2 of the observation portion is greater than the depth D1 of the third flow path 10C other than the observation portion. That is, the depth D2 of the main flow path 10 (specifically, the third flow path 10C) at the position corresponding to the observation region 100 is greater than the depth D1 of the main flow path 10 (specifically, the third flow path 10C) at other positions. Increasing the depth of the observation portion in this way makes it easier to observe the turbidity of the liquid present in the observation portion and the diffusion of light.

[0025] The depth D2 of the observation portion is, for example, 10 to 3000 μm. The depth D1 of the third flow path 10C other than the observation portion is, for example, 10 to 1000 μm. The ratio D2 / D1 of the depth D2 to the depth D1 is, for example, 2 to 5. Note that while FIG. 2 shows a case where the depth D2 of the observation portion is greater than the depth D1 of the third flow path 10C other than the observation portion, the depth D2 may be the same as the depth D1. The third flow path 10C including the observation portion has a width of, for example, 10 to 800 μm and a length of, for example, 1 to 50 mm.

[0026] As shown in Fig. 2, inclined portions are provided at both ends of the observation section. That is, the depth of the main channel 10 (specifically, the third channel 10C) gradually increases from a position other than the observation region 100 toward a position corresponding to the observation region 100. Providing inclined portions in this manner prevents liquid from stagnating in the observation section. The inclination angle θ of the inclined portions is, for example, 45 to 60°. Note that in Fig. 2, the inclined portions (i.e., the surfaces connecting the bottom surfaces of the main channel 10 and the observation section) are flat, but they may also be curved.

[0027] As described above, in the microorganism observation device 1 shown in Figure 1, the portion to which the culture section 13 (specifically, the second sub-channel 10D) is connected, the portion to which the first sub-channel 10E is connected, the serpentine portion, and the observation portion are arranged in this order from the inlet 11 toward the outlet 12.

[0028] (effect) As described above, according to the microorganism observation device 1, a portion of the culture solution 30 contained in the culture section 13 is discharged into the main flow path 10, and the culture solution 30 that has discharged into the main flow path 10 is mixed with a reagent introduced from the reagent introduction hole 14 in the serpentine section, and then the microbially produced substances contained in the culture solution 30 can be detected or quantified in the observation section.

[0029] In conventional microfluidic devices, it was necessary to add a reagent to the microbial culture space (culture section) to detect or quantify microbially produced substances. In contrast, in the microorganism observation device 1, a portion of the culture solution 30 contained in the culture section 13 is discharged into the main channel 10, and the discharged culture solution 30 is mixed with a reagent to detect or quantify the microbially produced substances contained in the culture solution 30, making it possible to observe the status of the microorganisms without affecting their growth.

[0030] Furthermore, when a substrate that becomes fluorescent upon reacting with a microbial product is used as a reagent, conventional microfluidic devices add the reagent to the microbial culture space (culture section), which causes the fluorescence to fade in the culture section over time, making long-term observation impossible.In contrast, with the microorganism observation device 1, a portion of the culture solution 30 contained in the culture section 13 is discharged into the main channel 10 and used as a sample, so new samples can be sent to the observation section even as time passes, making long-term observation possible without causing fluorescence fading.

[0031] 2. How to observe microorganisms According to another aspect, there is provided a method for observing microorganisms using the above-mentioned microorganism observation device. According to one embodiment, the method for observing microorganisms using the above-mentioned microorganism observation device includes: Supplying the culture solution 30 from the inlet 11 to the main channel 10; Cultivating microorganisms 20 in the presence of a culture solution 30 in a culture unit 13; Allowing the product produced by the microorganisms 20 to flow out from the culture section 13 to the main channel 10; supplying a reagent from the reagent introduction hole 14 to the first sub-channel 10E; The culture solution 30 containing the microbial product and the reagent is observed at the position of the observation area 100. Includes:

[0032] This method can be carried out, for example, as follows: First, a microbial suspension containing microorganisms 20 and a culture solution 30 is added to a recess in the second substrate 1B for forming the culture section 13. Then, the first substrate 1A is attached onto the second substrate 1B to form the microorganism observation device 1.

[0033] Thereafter, the culture solution 30 is supplied from the inlet 11 to the main flow channel 10. A portion of the culture solution 30 supplied to the main flow channel 10 flows into the culture section 13, and a portion of the culture solution 30 contained in the culture section 13 flows out into the main flow channel 10. As a result, the product produced by the microorganisms 20 flows out from the culture section 13 to the main flow channel 10. Here, it is possible that only the culture solution 30 flows out from the culture section 13 to the main flow channel 10, and that the microorganisms 20 do not flow out, or that the culture solution 30 flows out from the culture section 13 to the main flow channel 10, and some of the microorganisms 20 also flow out.

[0034] Meanwhile, a reagent is supplied from the reagent introduction hole 14 to the first sub-channel 10E. The reagent is generally supplied in the form of a reagent-containing liquid. As described above, the reagent can be a substrate that reacts with a microbial product to develop a color or emit light that can be observed under visible light or fluorescent observation. The reagent can be a substrate that develops a color by reacting with a product produced by the microorganism, a substrate that becomes chemiluminescent by reacting with a product produced by the microorganism, or a substrate that becomes fluorescent by reacting with a product produced by the microorganism. For example, Salzmann's reagent can be used as a reagent that develops a color by reacting with nitrite.

[0035] The culture solution 30 that has flowed into the main channel 10 is mixed with a reagent in the second channel (serpentine section) 10B and thoroughly stirred. The stirring can be performed by alternately switching the flow of the liquid in the main channel 10 between the forward direction and the reverse direction to stir the liquid contained in the serpentine section.

[0036] After stirring, the mixed sample present in the serpentine section (i.e., the culture medium containing the reaction product of the microbially produced substance and the reagent) is sent to the observation section of the third flow path 10C. The observation section can be observed under a microscope, and the microbially produced substance can be detected based on the presence or absence of color or fluorescence. The amount of color or fluorescence can also be measured in the observation section, and the amount of the microbially produced substance can be determined based on the measured value. Once the observation and / or measurement is complete, the mixed sample in the observation section is discharged through the outlet 12.

[0037] (effect) In conventional microorganism observation methods using microfluidic devices, it was necessary to add a reagent to the microbial culture space (culture section) to detect or quantify substances produced by the microorganisms. In contrast, in the microorganism observation method using the microorganism observation device 1, a portion of the culture solution 30 contained in the culture section 13 is discharged into the main channel 10, the discharged culture solution 30 is mixed with a reagent, and the substances produced by the microorganisms contained in the culture solution 30 are detected or quantified, making it possible to observe the status of the microorganisms without affecting their growth.

[0038] Furthermore, when a substrate that becomes fluorescent upon reacting with a microbial product is used as a reagent, conventional microorganism observation methods using microfluidic devices add the reagent to the microbial culture space (culture section), which causes the fluorescence to fade in the culture section over time, making long-term observation impossible. In contrast, in a microorganism observation method using the microorganism observation device 1, a portion of the culture solution 30 contained in the culture section 13 is flowed into the main channel 10 and used as a sample, so new samples can be sent to the observation section even as time passes, making long-term observation possible without causing the fluorescence to fade. [Explanation of symbols]

[0039] 1. Microorganism observation device 1A...First board 1B…Second board 10...Main flow path 10A...First flow path 10B...Second flow path 10C...Third flow path 10D: Second sub-channel 10E...First sub-channel 100...Observation area 11...Inflow hole 12...Outflow hole 13…Cultivation Department 14...Reagent introduction hole 20…Microorganisms 30…Culture solution

Claims

1. a laminate of a first substrate and a second substrate, a recess, a first groove, and a second groove are provided on a surface of the second substrate facing the first substrate; an inlet, an outlet, and a reagent introduction hole are provided in the first substrate or the second substrate; the recess is connected to the first groove and constitutes a culture section for culturing microorganisms; The culture section contains microorganisms and a culture solution, The first groove has one end connected to the inlet hole and the other end connected to the outlet hole, one end of the second groove is connected to the reagent introduction hole and the other end is connected to the first groove; At least one of the first substrate and the second substrate is optically transparent in an observation region corresponding to a part of the first groove.

2. The microorganism observation device according to claim 1 , wherein the first groove includes a meandering portion.

3. A microorganism observation device as described in claim 2, wherein the portion of the first groove to which the recess is connected, the portion of the first groove to which the other end of the second groove is connected, the serpentine portion, and the portion of the first groove corresponding to the observation area are arranged in this order between the inlet and the outlet.

4. 4. The microorganism observation device according to claim 1, wherein the depth of the first groove at the position corresponding to the observation region is greater than the depth of the first groove at other positions.

5. 5. A microorganism observation device according to claim 4, wherein the depth of the first groove gradually increases from the other position toward the position corresponding to the observation region.

6. A microorganism observation device described in any one of claims 1 to 5, wherein the recess and the first groove are connected via a third groove provided on the surface of the second substrate, and the width of the third groove is smaller than the dimension of the recess in the width direction of the third groove.

7. A method for observing microorganisms using the microorganism observation device according to any one of claims 1 to 6, comprising: supplying a culture medium from the inlet to the first groove; Culturing the microorganism in the presence of the culture solution in the culture section; allowing a product produced by the microorganism to flow out from the culture section into the first groove; supplying a reagent from the reagent introduction hole to the second groove; observing the culture solution containing the product and the reagent at the position of the observation area; An observation method that includes:

8. The observation method described in claim 7, wherein the reagent is a substrate that reacts with the product to produce a color, chemiluminescence, or fluorescence, and the observation of the culture solution containing the product and the reagent includes measuring light intensity.

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