JPS621717B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION BACKGROUND OF THE INVENTION 1. Field of the Invention This invention relates to a microbial grafting device particularly suitable for anaerobic bacteria.

Prior art and problems In general, in order to identify microorganisms such as pathogenic bacteria,
BACKGROUND ART A liquid culture solution containing cultured microorganisms, such as a blood culture solution, is transferred from a culture tube to a plate medium and cultured to form a colony.
Conventionally, this transfer is done by opening the lid of the culture tube, inserting a platinum loop into it to attach the culture medium, transferring it to a plate medium, and streaking, or by inserting a transfer needle into the lid of the culture tube. This was done by dropping one or two drops of the culture solution onto the plate medium through the needle and spreading it with a platinum loop or the like. In these conventional methods, the transplanting operation is complicated, and since the transplanting is done in an open system, anaerobic culture cannot be carried out as it is, and there is a high risk of contamination with bacteria from the atmosphere.

For this reason, the following two devices have been proposed for subplanting and culturing in a somewhat closed system. One of them is a culture bottle in which the inside of the culture bottle is partitioned with a flat agar medium to form two chambers that communicate with each other at the top.The culture solution is poured into the first chamber, and after culturing, the bottle is turned over to drain the culture solution. Transfer to the second chamber and contact with the surface of the plate agar medium. again,
The culture solution is returned to the first chamber and culture is performed to form colonies.

The second device consists of a flat plate agar medium formed on one side of the culture bottle, and after adding the culture solution and culturing, turn the bottle on its side so that the plate medium is on the bottom side, and remove the entire medium. Contact with culture solution. Thereafter, the bottle is rotated so that the medium is placed on top, and a culture for colony formation is placed.

With these devices, it is necessary to carry out culture for colony formation while a large amount of the culture solution once cultured remains present. In addition, when separating formed colonies, the operation is not only troublesome because a platinum loop is placed in the bottle, but also because there is a large amount of culture solution in the bottle, the culture medium is always in a moist state, and colonies are separated. are easy to move and difficult to extract independent colonies.

A transplanting device has also been proposed that has a flat culture medium suspended within the body from one end of the cylindrical body. The other end of this cylindrical body has a threaded groove formed therein. After culturing in a culture bottle with a thread groove corresponding to this thread groove, the above-mentioned transplanting device is screwed onto the culture bottle, and the culture solution is poured from the bottle into the transplanting device by overturning them. Transfer and contact with plate medium. After inverting the bottle again, return the culture solution to the bottle, and then culture for colony formation either as is or after removing the transplanting device and sealing the bottle.

Since this device is temporarily opened, it is not possible to perform anaerobic culture using only this device, and there is a risk of contamination with bacteria.

OBJECTS OF THE INVENTION Accordingly, an object of the present invention is to provide a microbial subculture device that can perform subplanting and culturing with simple operations and in a sealed state, and also allows easy anaerobic culture.

According to this invention, in order to maintain the internal atmosphere at a predetermined degree of reduced pressure, the opening is closed by an airtight pierceable closing member having a concave portion extending from the bottom surface to the top surface. a substrate supported in the container by the closure member, and a microorganism culture medium formed in a flat plate shape on at least one surface of the substrate, the substrate being configured to allow gas to pass through but not to allow liquid to pass therethrough. There is provided a microorganism subculturing device characterized in that a membrane filter is installed to define the recess of the closing member and the hollow chamber, and an oxygen scavenger is housed in the hollow chamber.

In the above-described subculture culture device of the present invention, the closing member is usually made of rubber. In addition, the culture medium is tyokolate agar medium, pine conkey agar medium,
Any medium can be used, such as malt agar medium, deoxycholate agar medium, Drigaski agar medium, or a medium obtained by replacing the agar of these mediums with a hydrophilic synthetic polymer.

DETAILED DESCRIPTION OF THE INVENTION The present invention will be described below with reference to the accompanying drawings.

As shown in FIG. 1, the microorganism subculturing device of the present invention is equipped with an open-mouthed container, such as a bottomed tube 11. The container may be made of any material such as glass or plastic as long as it can withstand reduced pressure and maintenance of reduced pressure. In particular, it is preferably made of a transparent material. The internal atmosphere of this container 11 is maintained at a predetermined degree of reduced pressure.

The mouth portion 11a of the container 11 is closed by a closing member 12 to maintain the internal atmosphere of the container in the reduced pressure state. The material of the closing member is not limited as long as it is airtight and can be pierced, but it is preferably made of rubber such as styrene-butadiene rubber or butyl rubber. This closure member 12 is formed with a recess 12a extending from its bottom surface to its top surface in order to facilitate piercing.

A substrate 13 is supported within the container 11 by a closing member 12 so as not to contact the bottom of the container 11 . A protrusion 13a is formed at one end of this substrate 13, and this protrusion 13a is connected to the closing member 12.
A substrate 13 is supported by being inserted therein, preferably at the end of the closure member.
The substrate 13 may be made of any material such as plastic or glass, but plastic is suitable.

On the surface of the substrate 13, a thiokolate agar medium,
A medium 14 suitable for the culture conditions, such as a pine conkey agar medium, a malt agar medium, a deoxycholate agar medium, a Dorigalsky agar medium, or a medium obtained by replacing the agar of these mediums with a hydrophilic synthetic polymer, is formed in a flat plate shape. There is. This medium 14 may be formed on both sides of the substrate.

A membrane filter 15, such as an acetyl cellulose filter or a nitrocellulose filter, which allows gas to pass through but not liquid, is installed to define the recess 12a of the closing member 12 and the hollow chamber 16, and an oxygen scavenger 16a is installed in the hollow chamber 16. For example, iron () compounds are contained. Due to the presence of the oxygen scavenger 16a, even if the atmosphere inside the container is initially composed of an oxygen-containing gas such as air, oxygen is eliminated, so that anaerobic culture can be performed and the operation becomes easy.

In the microorganism subculturing device having the above configuration, the inside of the container 11 is maintained at a predetermined degree of vacuum as described above, that is, a degree of vacuum sufficient to aspirate a predetermined amount (for example, 1 to 3 ml) of the culture solution in the operations described below. However, since the subculture culture device of the present invention contains an oxygen scavenger, there is no particular need to exclude oxygen from the internal atmosphere from the beginning. Good too.
Of course, the internal atmosphere may be composed of a normal anaerobic atmosphere (for example, a mixed gas of carbon dioxide and nitrogen).

Note that the structure of the substrate is not limited to a flat plate as shown in FIG. 1, but as shown in FIG.
In this case, different media can be formed in a flat plate shape on each surface of the substrate, and desired culture suitable for each can be performed.

Specific Effects of the Invention In order to carry out subculturing of microorganisms using the microorganism subculturing device of the present invention having the above configuration, as shown in FIG. Needle 17 on stopper 20 of bottle 18
Prick one end of the needle, and keep the needle tip inside the stopper 17 without penetrating into the bottle. Next, the other end of the needle 17 is inserted into the container 1 into the closing member 12 of the microorganism subculturing device.
Pierce until it reaches the inside of 1. Thereafter, the culture bottle 18 is turned upside down as shown in FIG. 2 so that the culture medium 14 is at the bottom, and the needle 17 is inserted into the bottle 18.
Then, the culture solution 19 is easily transferred from the bottle 18 in an amount corresponding to the degree of vacuum in the container 11 (for example, 1 to 3 ml).
sucked inside. After aspirating the culture solution 19,
Remove the needle 17 from the closure member 12 (Fig. 3),
By shaking the subculture device, the stored culture solution 19' is brought into contact with the medium 14, and the culture solution 19' is placed in the culture device as it is to perform culture for forming colonies 20. After culturing, the closing member 12 is placed in the container 1.
1, it is convenient because the culture medium in which colonies 20 have been formed can also be removed together.

Hereinafter, an example will be described in which microbial subculturing was performed using the microorganism subculturing device of the present invention.

Clostridium Sporogenes IAM19235 was cultured in blood culture tubes. After culturing, the bacterial concentration in the culture solution was approximately 1×10 ⁴ /ml.

As a subculture culture device, one having a configuration similar to that shown in FIG. 1 was used. The filter was made of acetate and had a pore size of 45 μm (manufactured by Fuji Film Photo Industries, Ltd.), and the oxygen scavenger was Ageless (manufactured by Mitsubishi Gas Chemical Co., Ltd.). The inside of the container consisted of a 1:9 mixture of carbon dioxide and nitrogen, and the pressure was reduced to such an extent that 1 ml of liquid could be aspirated. A 1×8 cm, 2 mm thick Chiyocoat agar medium was used as a medium. After aspirating 1 ml of the culture solution from the culture tube into the subculture device under the conditions shown in FIG. 2, the culture tube was removed and cultured at 37° C. for 1 day. As a control, a similar experiment was conducted using a subculture device that did not use Ageless. For those using oxygen absorbers,
Approximately 140 colonies were formed on the medium. In addition, about 100 colonies were obtained in the control without oxygen scavenger. Colonies could be easily isolated using a platinum loop after removing the plate medium together with the closure member.

Specific Effects of the Invention As described above, in the microorganism subculturing device according to the present invention, the inside of the container is maintained at a predetermined reduced pressure state with a penetrable closing member, so the needle can be inserted from the culture bottle. The operation is extremely simple, as the culture medium can be stored simply by passing the medium through the medium, and the microorganisms can be inoculated into the medium simply by shaking. In addition, since the transplanting can be done in a sealed state, there is no chance of contamination with bacteria from the outside air. Furthermore, since an oxygen scavenger is present, even if the inside of the container is initially configured with an atmosphere in which oxygen is present, it immediately becomes an anaerobic atmosphere, and the desired culture can be performed as it is, which is convenient.

[Brief explanation of the drawing]

Fig. 1 is a partial cross-sectional view of a microorganism subculturing device according to an embodiment of the present invention, Fig. 2 is a diagram showing a state in which a culture solution is subcultured to the device shown in Fig. FIG. 4 is a perspective view showing a modified example of the substrate of the microorganism transplantation and culture device of the present invention. 11... Container, 12... Closing member, 11a...
Mouth part, 12a... recessed part, 13... substrate, 14...
Medium, 13a... Protrusion, 13'... Flat plate, 1
3″...Flat plate, 15...Filter, 16...Hollow chamber, 16a...Oxygen absorber, 17...Piercing needle, 1
8...Culture bottle, 19...Culture solution, 19'...Culture solution, 20...Colony.

Claims (1)

[Scope of Claims] 1. The opening is an airtight pierceable closing member having a concave portion extending from the bottom to the top so that the internal atmosphere is maintained at a predetermined degree of reduced pressure. It comprises a closed container, a substrate supported in the container by the closing member, and a microorganism culture medium formed in a flat plate shape on at least one surface of the substrate, which allows gas to pass through but not liquid. 1. A microorganism transplantation and culturing device characterized in that a membrane filter that does not allow the passage of microorganisms is installed so as to define the recess of the closing member and the hollow chamber, and an oxygen scavenger is housed in the hollow chamber. 2. The microorganism subculturing device according to claim 1, wherein the closing member is made of rubber. 3. Claim 1, wherein the medium is a thiocholate agar medium, a pine conkey agar medium, a malt agar medium, a deoxycholate agar medium, a Drigalusky agar medium, or a medium obtained by replacing the agar of these mediums with a hydrophilic synthetic polymer. Or the microbial subculture device described in item 2.