JP2003225083A - Disc-shaped culturing medium - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a culturing technology capable of widely simplifying a culturing work of germs, etc. <P>SOLUTION: This disk-shaped culturing medium 1 is equipped with at least a layered structure consisting of a disk-shaped substrate 2 formed of a synthetic resin, etc., a culturing medium layer 3 applied on the substrate 2 and solidified, and a thin thickness cover sheet 4 covering the culturing medium 3. <P>COPYRIGHT: (C)2003,JPO

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a technique for culturing organisms such as bacteria. More specifically, the present invention relates to a disc-shaped culture medium having a layered structure in which a thin layer medium having a uniform thickness is provided in an inner layer portion, a culture method using the culture medium, and a method for producing the disc-shaped culture medium.

[0002]

2. Description of the Related Art Conventionally, it has been common practice to culture cells, tissues, organs, organisms such as microorganisms and fungi using a culture instrument such as a petri dish or a flask containing a predetermined medium. There is.

FIG. 9 attached herewith outlines a general procedure of a bacterial culture technique using a solid medium. First, the petri dish 100 is sterilized in a sterilization chamber under predetermined temperature and time conditions, and agar serving as a medium is separately placed in a container 101 such as Kolben to dissolve it, and sterilized by heating under pressure in an autoclave. Next, the sterilized petri dish 10
The liquid medium 102a, which is liquid with respect to 0, is carefully poured and solidified so as to have a uniform thickness and not to mix bubbles. Subsequently, the platinum loop 103 is sterilized, and the bacteria to be cultured are scooped with the tip of the platinum loop 103 to solidify the medium 10.
It is applied on 2b and cultured at a predetermined temperature and time.
If bacteria are growing in the medium 102b, colonies are formed. When observing this with the optical microscope 104, the culture medium 102b is cut into a predetermined size to prepare an observation sample on the slide glass 105, and the cover glass 107 is placed on the observation sample to perform observation.

[0004]

However, the above-mentioned conventional culture technique has the following technical problems.

(1) It was necessary to sterilize the petri dish used each time. (2) It was difficult to prepare a medium having a uniform thickness on a petri dish. (3) It took skill to be able to apply the bacteria uniformly on the medium using the platinum loop.
(4) When preparing a plurality of culture systems, it was necessary to repeat the culture procedure individually using separate Petri dishes, which was troublesome. (5) The observation work of the cultured bacteria is
It was troublesome because the work of cutting out the medium and the predetermined preparation work using the slide and the cover glass were required.

Therefore, a main object of the present invention is to provide a culture technique capable of dramatically simplifying the work of culturing organisms such as bacteria.

[0007]

In order to solve the above technical problems, first of all, the present invention comprises a substrate, a medium layer provided on the substrate, and a cover sheet covering the medium layer. Provided is a disc-shaped culture medium having at least a layered structure.

That is, the disc-shaped culture medium is a medium layer for culturing organisms such as cells, tissues, organs, microorganisms and fungi, and a synthetic resin having a function of directly or indirectly holding the medium layer. A disc-shaped culture medium having an outer appearance, comprising at least a layered structure portion comprising a substrate formed in (1) and a cover sheet for covering the medium layer. Since this culture medium is obtained in a sterilized state during the manufacturing process, it has the advantage that it can be used as it is without sterilization.

The medium layer interposed in the middle comprises a solid medium which can be applied in a solution state on the upper surface of the substrate and can be solidified after application. In this medium layer,
Regardless of the specific form, devise so that the culture solution can be fed from the outside. The thickness of the medium layer is appropriately selected according to the conditions such as the organism to be cultured and the required amount of medium. Further, it is desirable to use a polycarbonate sheet having a thin thickness of about 100 μm as the cover sheet, but the thickness and material of the cover sheet can be appropriately selected according to the purpose.

In this disc-shaped culture medium, since the medium layer is preliminarily set to have a predetermined thickness between the substrate and the cover sheet, the culture medium is laborious as in the conventional case in a series of culture operations. It has a great advantage that the manufacturing work becomes unnecessary.

Here, in the disc-shaped culture medium according to the present invention, a structure in which the substrate and the cover sheet are bonded together via the solidified medium layer can be adopted. That is, the cover sheet can be directly adhered and fixed to the surface of the medium layer by utilizing the solidifying action of the liquid medium layer applied on the surface of the substrate. By this means, the disc-shaped culture medium according to the present invention can have a simple three-layer structure composed of a substrate, a medium layer and a cover sheet.

Further, in the disc-shaped culture medium according to the present invention,
The cover sheet can be formed of a synthetic resin having a light transmitting property at least in the visible light region. By this means, the culture state of the medium layer can be directly observed through a cover sheet or visually observed through a microscope. In particular, in microscopic observation, the disc-shaped culture medium according to the present invention itself can be directly installed on a preparation stand of a microscope for observation, and thus the conventional preparation work of an observation sample using a preparation is unnecessary.

Here, in the disc-shaped culture medium according to the present invention, a liquid reservoir portion which is recessed in the central portion in the diameter direction of the culture medium and stores the culture fluid, and which is opened in the liquid reservoir portion, It is devised so as to provide a groove of an internal cavity extending to the medium layer, and further, the groove is arranged radially when viewed from above. The volume and size of the liquid reservoir are appropriately selected mainly depending on the required amount of the culture solution for the medium layer. Further, the number of the grooves is also a density number such that colonies spreading around each groove do not come into contact with each other, and may be appropriately selected as necessary. In the specification of the present application, the "groove" means a groove extending in the streak regardless of its width or depth, and the "culture liquid" contains an organism such as a bacterium to be cultured. Means a solution prepared as above.

In the structure having the above-mentioned liquid reservoir and the groove,
It is possible to inject a predetermined volume of the culture solution into the liquid reservoir and to deliver a predetermined amount of the culture solution to each groove formed in the liquid reservoir by utilizing the capillary phenomenon. As a result, in the case of bacterial culture,
A colony is formed so as to extend substantially along each groove, and a substantially radial colony can be formed as a whole. By dividing the liquid reservoir into a plurality of chambers and injecting different kinds of culture liquids into the respective chambers, a plurality of culture systems can be formed in one disc-shaped culture medium, and therefore, depending on the culture purpose. It's very convenient.

By forming a medium layer in the groove, the culture in the groove can be surely performed. This makes it easier to obtain more linear colonies along the groove.

Further, the liquid reservoir is devised so that a sealing valve for preventing the backflow of the culture solution can be attached, so that the culture solution is sent to each groove utilizing the capillary phenomenon. Work can be performed more smoothly.

That is, according to the disk-shaped culture medium having such a liquid reservoir and the groove, the culture liquid injection procedure for injecting the culture liquid into the liquid reservoir having the above-mentioned structure and the groove having the above-mentioned structure It is possible to provide a simple culture medium introduction method including a liquid feeding procedure for feeding the culture medium by the capillary phenomenon. Then, by performing the liquid feeding procedure while rotating the disc-shaped culture medium, it becomes possible to reliably feed the culture liquid to the outer peripheral side end portion of the groove by the centrifugal force accompanying the rotation. Further, in the culture using the above disc-shaped culture medium, a culture state confirmation method characterized by directly observing the disc-shaped culture medium can be provided.

The disc-shaped culture medium according to the present invention described above can be manufactured by the following steps (a) to (d). That is, (a) a step of injection-molding a synthetic resin to form a disk-shaped substrate having grooves arranged radially from above, (b) a step of applying a medium to the substrate, (c) an application The disc-shaped culture medium according to the present invention can be produced by the step of placing a disc-shaped cover sheet on the medium, and (d) the step of solidifying the medium and bonding the substrate and the cover sheet together. . More preferably, in the step (b), the medium is applied to the substrate using a spin coater.

The "spin coater" means means for continuously discharging the coating material (liquid medium in the present invention) in an equal amount, and a disc-shaped object to be coated (substrate in the present invention) rotating at a predetermined speed. And a means for causing the coating means to be provided at least, and is generally used in the step of attaching recording media such as optical disks.

The manufacturing process will be described more specifically. First, in the step (a), a synthetic resin is injection-molded under high temperature conditions to manufacture a substrate, so that there is an advantage that a sterilized substrate can be obtained from the manufacturing process. (B)
In the step, a liquid culture medium containing a predetermined nutrient source or the like is discharged (or dropped) onto the upper surface of the sterilized substrate obtained in the step (a), and applied so as to have a uniform thickness.
Preferably, the spin coater is used to thinly apply a medium having a uniform thickness to the upper surface of the substrate while surely preventing the inclusion of air bubbles. In this step (b), it is particularly important that air bubbles are not mixed in the applied medium and that the medium has a uniform thickness. In the step (c), a thin cover sheet is placed on the upper surface of the culture medium applied on the substrate. On this occasion,
A mounting means that does not leave air on the upper surface of the medium is suitable. In the step (d), the cover medium and the substrate are attached to each other via the medium layer by converting the liquid medium into a solid medium.

By these steps (a) to (d), it is possible to manufacture a disk-shaped culture medium having a layered structure in which a cover sheet, a medium layer and a substrate are laminated in this order from the upper layer.

As described above, the present invention provides a new-type culture tool having a layered structure in which a thin solid medium layer is interposed, and a suitable method for producing and using this culture tool (method for introducing a culture solution, It has the technical significance of providing a culture state confirmation method) to related industries.

[0023]

Preferred embodiments of the present invention will be described below with reference to the accompanying drawings.

FIG. 1 is an external perspective view of a disc-shaped culture medium according to the present invention, FIGS. 2A and 2B are longitudinal sectional views of a portion indicated by reference numeral X in FIG. 1, and FIG. FIG. 3 is a plan view of a substrate constituting the same disk-shaped culture medium as viewed from above.

The disc-shaped culture medium indicated by reference numeral 1 in FIG. 1 and the like (hereinafter, simply referred to as “culture medium”) has a disc-shaped (disk-shaped) form with a thickness of about several millimeters. A substrate 2 is provided in the lowermost layer thereof, and a plurality of fine grooves 21 are formed on the upper surface of the substrate 2, and a circular liquid reservoir portion 22 is formed in a concave shape at a central portion in the diametrical direction. (Mainly refer to FIG. 3).

On the upper surface including the groove 21 of the substrate 2, a solid medium layer 3a is provided with a uniform thickness, and the medium layer 3 is fixed from the light-transmitting cover sheet 4 fixedly arranged on the uppermost layer.
The structure a is transparent. Note that in FIG. 1 and the like, the size of the groove 21 is exaggerated for convenience of description.

Further, the structure of the culture medium 1 will be described in detail with reference to FIG.
A substrate 2 formed of a synthetic resin such as an acrylic resin, a solid-state medium layer 3a laminated on the substrate 2, and a light-transmitting cover sheet 4 laminated on the medium layer 3a; At least a layered structure composed of

It is essential that the layer structure includes at least the substrate 2, the medium layer 3a, and the cover sheet 4, and the other intermediate layers (for example, the substrate 2).
It is not intended to exclude the presence of a coating layer that may be formed on the surface).

The substrate 2 has a diameter of 8 in this embodiment.
cm, thickness 1.2 mm, obtained by injection molding synthetic resin. Needless to say, the size of the substrate 2 is not limited to the above size and can be appropriately selected according to the application.

On the upper surface 2a of the substrate 2, there are linear grooves 21 extending in a streak pattern from the liquid reservoir 22 at the center of the substrate 2 toward the outer peripheral side and arranged radially at an equal interval in the circumferential direction. However, a total of eight are provided (see FIGS. 1 and 3). The number of the groove 21 can be appropriately selected according to the application and is not limited to eight.

The structure of the groove 21 will be described in more detail with reference to FIG. 3. Each groove 21 has an opening formed in the inner peripheral wall surface 22a of the liquid reservoir 22 formed to have a diameter of 10 mm and a depth of 1 mm. 21a and is formed at a position close to the outer peripheral wall 23 of the substrate 2 or at the end. The groove 21 has a capillary function capable of delivering the liquid medium to be injected into the liquid reservoir 22 in the outer peripheral direction by utilizing the capillary phenomenon, and is capable of culturing a desired organism. It can be formed into a size. For example, the width is 1 μm or more and the depth is 1
It is formed to a thickness of μm or more.

The groove 21 is not limited to the embodiment shown in FIG.
It is also possible to employ a modified form in which the cover sheet 4 is recessed as shown in (B). Even when the groove 21 is formed on the cover sheet 4 side, the medium layer 3a along the inner wall surface of the groove 21 may be formed by an appropriate method.

FIG. 4 is a plan view of the cover sheet constituting the culture medium 1 according to the present invention, as viewed from above.

The cover sheet 4 is a light-transmissive thin sheet having a substantially donut shape or a compact disc (CD) shape when viewed from above. The cover sheet 4 is formed of a synthetic resin having a light transmitting property at least in the visible light region, and its preferable thickness is about 100 μm.

The cover sheet 4 serves to cover and protect the medium layer 3. In addition, this cover sheet 4
By controlling the gas permeability of
Since any anaerobic culture condition can be freely created, the culture medium 1 has versatility as a culture tool.

Since the cover sheet 4 is light-transmissive, organisms and colonies grown on the medium layer 3 can be observed from the outside. Reference numeral 41 shown in FIG. 4 is a circular hole formed in the central portion of the cover sheet 4, and the cover sheet 4 is positioned so that the circular hole 41 exactly overlaps the upper part of the liquid reservoir 22 of the substrate 2. Then, they are stacked on the medium layer 3 (see FIG. 1).

Next, FIG. 5 is a diagram showing manufacturing steps of the method for manufacturing the culture medium 1. The method for producing the culture medium 1 according to the present invention is characterized by including at least the steps shown by P _{1 to} P ₄ shown in FIG. Hereinafter, each step P ₁
~ P ₄ will be described in order.

<Regarding Step P ₁ > In the step P ₁ , a synthetic resin is injection-molded, and the grooves 2 are arranged radially in a top view.
It is a step of integrally molding the disk-shaped substrate 2 provided with 1.
Although the specific method of the injection molding is not particularly limited, the injection molding is performed at a resin temperature of about 300 ° C. and a mold temperature of about 120 ° C.
The substrate 2 to be molded is obtained in a sterilized state since the above process is performed. As a result, the substrate 2 and the culture medium 1 for culturing organisms are suitable members.

<Regarding Process P ₂ > The process P ₂ is a process of applying the liquid medium 3a to the substrate 2 obtained from the process P ₁ . Specifically, first, the substrate 2
Is held by the rotating body 5a, and while the substrate 2 is rotated by the driving of the rotating body 5a (see arrow Y), the liquid medium 3b is discharged from the nozzle 5b arranged at a predetermined position above the substrate 2.
Is discharged.

That is, using a so-called spin coater device having at least the rotating body 5a and the nozzle 5b, the culture medium 3a is provided on the upper surface of the substrate 2 with a uniform thickness (for example, 1).
00 μm). The composition of the medium 3a to be applied can be appropriately selected according to the purpose of culturing and may be at least a solid medium (including a semi-solid medium), and further suitable for application by a spin coater. If

<Regarding Step P ₃ > In the step P ₃ , the disc-shaped cover sheet 4 is formed on the upper surface of the applied culture medium 3 a.
Is the step of placing. Specifically, after holding the cover sheet 4 with an air suction pad or the like and transferring it to a position above the substrate 2, the holding state is released, and the cover sheet 4 is placed on the upper surface of the medium 3a applied to the substrate 2. Place it. At this time, in order to prevent air bubbles from remaining or mixing between the cover sheet 4 and the culture medium 3a, the cover sheet 4 is held so as to be warped upward and air is utilized by utilizing the returning action of the warp. You may make it mount, pushing out to the outer peripheral side.

Here, when the cover sheet 4 is placed on the liquid medium 3a applied to the upper surface of the substrate 2, the medium 3a formed along the wall surface of the groove 21 by the surface tension.
(See FIG. 2) may be pulled upward, and the cavity S (see FIG. 2) of the groove 21 may be closed. When the cavity S is closed, there arises a problem that a culture solution described later cannot be sent to the groove 21.

As a method for preventing the occurrence of this problem, a method of appropriately selecting the shape of the groove 21 can be considered. That is, by forming the groove 21 deep,
The medium 3a can be retained in the groove 21. As an example, by forming the groove 21 so that the aspect ratio (ratio of the width W of the groove to the height H: H / W, see FIG. 2) is 1.0 or more, the occurrence of the above problem is effective. Can be prevented. An appropriate aspect ratio may be selected according to the type of medium 3a.

<Regarding Process P ₄ > The process P ₄ is a process of bonding the substrate 2 and the cover sheet 4 by solidifying the liquid medium 3 a applied to the substrate 2. Specifically, by heating under pressure in an autoclave (not shown) and then leaving it at room temperature until the medium 3a is solidified, a solid medium layer 3a is formed between the substrate 2 and the cover sheet 4. The cover sheet 4 is adhered to the medium layer 3. That is, through the medium layer 3, the substrate 2
And the cover sheet 4 are pasted together. In this way, the culture medium 1 according to the present invention can be reliably manufactured.

FIG. 6 shows a liquid reservoir 22 of the culture medium 1 according to the present invention formed by the above-mentioned manufacturing process P (P _{1 to} P ₄ ).
FIG. 7 is a partially enlarged view of a partial cross section for explaining the configuration of the sealing valve 5 attached to the liquid storage device, FIG. 7 is a view showing a state of injecting the culture solution 7 into the liquid storage part 22, and FIG. It is a figure which shows the mode that the culture solution 7 inject | poured into 22 progressed in the groove 21 in the outer peripheral direction.

A method for introducing the culture solution into the culture medium 1 will be described with reference to FIGS. 6 to 8. First, the substrate 2
The liquid reservoir 22 in the central portion of (1) functions as a place where the culture liquid 7 containing the bacteria for culturing is injected (see FIG. 7) and the culture liquid 7 is temporarily stored.

Reference numeral 25 shown in FIG. 6 is a recess formed in the center of the lower surface of the substrate 2. The recess 25 is formed on the rotating body 5a constituting the spin coater and the culture medium 1 is formed.
It is a part used when holding.

Here, the external small disc-shaped sealing valve 5 shown in FIG. 6 is constructed so as to be fitted inside the circular hole 41 formed in the central portion of the cover sheet 4, and the liquid reservoir portion. It serves to seal 22. The method of fixing the sealing valve 5 can be appropriately selected.

<Regarding the procedure for injecting the culture solution> With respect to the sealed liquid reservoir 22, the predetermined culture solution 7 is supplied from the needle stick portion 51 formed in the center of the sealing valve 5 by the injector 6 as shown in FIG. Is injected in the required amount. The needle stick part 51
Is formed of, for example, a flexible rubber material,
The distal end of the injector 6 is introduced into the liquid reservoir 22 by the needle puncturing operation by the injector 6, and when the injector 6 is pulled out, the flexibility of the material seals the puncture needle to prevent the liquid reservoir 22 from leaking. The airtightness is configured to be kept constant.

<Regarding liquid feeding procedure> The culture liquid 7 is the liquid reservoir 2
When the medium 7 is filled and filled, the culture solution 7 flows into each groove 21 from the opening 21a of the liquid reservoir 22 by the capillary phenomenon and proceeds toward the outer peripheral side of the substrate (see arrow Z in FIG. 8). ). At this time, since the liquid reservoir 22 is sealed by the sealing valve 5, the culture liquid 7 does not flow backward and each groove 21
The solution is smoothly sent to.

Further, this liquid feeding procedure is described by arrows Y, Y in FIG.
As shown in FIG. 4, when the culture medium 1 is horizontally rotated, a centrifugal force is added to the culture solution 7 in addition to the capillary phenomenon, so that the culture solution 7 can be more smoothly transferred from the liquid reservoir 22. It is preferable because the liquid is fed to the groove 21.

When a desired organism is cultured using the culture medium 1 described above, the organism grows along the groove 21, and in the case of bacterial culture, colonies are formed. After the culturing work is completed, the culturing state of the culturing medium 1 is visually checked or using a microscope, a CCD camera, or the like.
It can be confirmed by directly observing.

[0053]

According to the disc-shaped culture medium of the present invention,
Since sterilization work of the equipment used for culture and culture medium formation work are unnecessary, the culture work of various organisms can be dramatically simplified. Further, the disc-shaped culture medium according to the present invention,
Since it is formed of a synthetic resin and a medium, the material cost and the manufacturing cost are low, and thus a low-cost disposable type culture medium can be provided.

Since the medium layer interposed in the disc-shaped culture medium has no air bubbles and has a uniform thickness, a uniform and stable culture system can be provided. Further, since the thickness of the medium layer can be freely controlled or adjusted in the manufacturing process by changing the amount of the medium applied to the substrate, it is possible to provide a culture medium having a medium layer suitable for various culture systems. It is convenient for

The medium layer of the disc-shaped culture medium according to the present invention is provided with an internal hollow groove, and the culture solution is sent to the groove by utilizing the capillary phenomenon (and centrifugal force). Therefore, the work of introducing the organism into the medium layer is facilitated, and even a beginner can easily perform the work. Further, in the disc-shaped culture medium according to the present invention, the organisms grow along the grooves of the medium layer, and in the case of bacterial culture, colonies are formed, so that the growth area of the organisms is patterned and cultivated. The state can be easily observed.

By carrying out the culturing work using the disc-shaped culturing medium according to the present invention, the work of cutting off the medium and the work of preparing the sample for observation on the preparation are completely unnecessary, and the culturing medium can be used with a microscope or a CCD camera. It is very convenient because it can be observed directly, and bacteria tests can be performed quickly.

In the method for producing a disc-shaped culture medium according to the present invention, a substrate, a medium layer having a uniform thickness, a cover glass,
It is possible to reliably mass-produce a disc-shaped culture medium including at least a layered structure including.

According to the present invention, it is possible to provide a culture instrument having a layered structure, a novel culture method and a culture state confirmation method, which are completely different in concept from conventional culture instruments such as petri dishes and flasks.

[Brief description of drawings]

FIG. 1 is an external perspective view of a disc-shaped culture medium (1) according to the present invention.

FIG. 2 (A) is a vertical cross-sectional view of a portion indicated by reference numeral X in FIG. (B) Vertical cross-sectional view of a modified form of the X portion

FIG. 3: Substrate (2) constituting the same disk-shaped culture medium (1)
Top view of the

FIG. 4 is a plan view of a cover sheet (4) constituting the same disk-shaped culture medium (1) viewed from above.

FIG. 5 is a view showing manufacturing steps of the method for manufacturing the same culture medium (1).

FIG. 6 is a partially enlarged view of a partial cross section for explaining the configuration of the sealing valve (5) mounted on the liquid reservoir (22) of the culture medium (1).

FIG. 7 is a view showing a state in which the culture solution (7) is injected into the liquid reservoir (22) of the substrate (2).

FIG. 8 is a view showing a state in which the culture solution (7) injected into the liquid reservoir (22) proceeds in the groove (21) in the outer peripheral direction.

FIG. 9 is a procedure flow chart showing a conventional general culture process.

   ─────────────────────────────────────────────────── ─── Continued front page    F term (reference) 4B029 AA03 AA08 BB01 CC01 CC02                       CC07 GA08 GB01 GB02 GB05                       GB06 GB07 GB09 GB10                 4B065 AA01X AA57X AA87X BC46                       BC50 CA46

Claims (12)

[Claims] 1. A disc-shaped culture medium comprising at least a layer structure comprising a substrate, a medium layer provided on the substrate, and a cover sheet covering the medium layer. 2. The disc-shaped culture medium according to claim 1, wherein the substrate and the cover sheet have a configuration in which they are bonded together via the solidified medium layer. 3. The disc-shaped culture medium according to claim 1, wherein the cover sheet is made of a synthetic resin having a light transmissive property in at least a visible light region. 4. A liquid reservoir portion that is recessed in the central portion in the diametrical direction and stores a culture fluid, and an internal cavity that is opened in the liquid reservoir portion and is formed in either the substrate or the cover sheet. 2. The disc-shaped culture medium according to claim 1, further comprising: 5. The disc-shaped culture medium according to claim 4, wherein the liquid reservoir is equipped with a sealing valve for preventing backflow of the culture liquid. 6. The disc-shaped culture medium according to claim 4, wherein the groove is formed radially when viewed from above. 7. The disc-shaped culture medium according to claim 4, wherein the medium layer is formed in the groove. 8. A liquid reservoir formed at the center in the diametrical direction of a disc-shaped culture medium, which comprises at least a layered structure comprising a substrate, a medium layer formed on the substrate, and a cover sheet covering the medium layer. Culture solution injection procedure for injecting the culture solution into the part, and a solution delivery procedure for sending the culture solution by capillary action to the groove of the internal cavity that opens in the solution reservoir and extends to the medium layer Liquid introduction method. 9. The culture medium introduction method according to claim 8, wherein the liquid feeding procedure is performed while rotating the disc-shaped culture medium. 10. A substrate, a medium layer formed on the substrate, and a cover sheet made of a light-transmissive resin covering the medium layer,
A method for confirming a culture state, which comprises culturing an organism in a disc-shaped culture medium having at least a layered structure comprising, and then directly observing the disc-shaped culture medium. 11. A method for producing a disc-shaped culture medium, which comprises at least the following steps (a) to (d). (A) A step of forming a disc-shaped substrate made of a synthetic resin, which is provided with stripe grooves radially arranged when viewed from above. (B) A step of applying a culture medium to the substrate. (C) A step of placing a disk-shaped cover sheet on the applied culture medium. (D) A step of solidifying the medium and bonding the substrate and the cover sheet together. 12. The method for producing a disk-shaped culture medium according to claim 11, wherein the step (b) is performed using a spin coater.