JPS6243664B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention allows aerobic bacteria and anaerobic bacteria culture equipment to be completely sealed before use, and also allows gas generated within the culture equipment to be released while maintaining the culture environment during use. This invention relates to a microorganism culture device fitted with a stopper. Conventionally, in culturing anaerobic bacteria, an elastic stopper such as a rubber stopper, such as a screw cap or a seamer, has been used to completely seal the culture apparatus. However, in culturing this anaerobic bacteria, when transplanting after culturing, when the sampling needle is inserted into the stopper, the medium may blow out due to the internal pressure of the generated gas, or when the screw cap or seamer is removed, etc. Caps and stoppers often fly off, putting users at risk. Moreover, as the opening of the culture device that was opened at that time came into contact with the outside air, bacteria from the outside air entered the culture device and contaminated the culture medium. In addition, if a stopper that uses a simple tightening member is used, the gas produced by anaerobic bacteria will increase the internal pressure of the culture device, causing the stopper to fly off during culture, making it impossible to maintain anaerobic conditions, and furthermore, the culture medium may be contaminated by bacteria in the outside air. ,
Furthermore, due to the anaerobic bacteria that flew away with the stopper,
There was a problem that the surrounding environment was contaminated. On the other hand, for culturing aerobic bacteria, a permeable stopper such as a Morton stopper, cotton stopper, aluminum cap, or paper stopper is used for the culture apparatus. However, these stoppers do not allow storage of the culture medium due to their air permeability, and opening the stopper when collecting samples can easily contaminate the culture medium with bacteria in the outside air.
The culture medium leaks during transportation and cannot be easily transported.
Moreover, it has the disadvantage that the user is exposed to danger. Furthermore, when collecting a sample, it was necessary to remove the stopper, and at that time, outside air entered and bacteria in the outside air were also cultured, and it was sometimes difficult to distinguish them from bacteria that had already been cultured. Furthermore, when trying to cultivate aerobic bacteria and anaerobic bacteria using the same culture device, aerobic bacteria are difficult to grow in the culture device with the above-mentioned airtight stopper, while culture devices with an air-permeable stopper do not. There was a problem that it was difficult for anaerobic bacteria to grow. For this reason, it is necessary to use two culture devices for each culture of aerobic bacteria and anaerobic bacteria, making the culture method extremely complicated. The law was impossible. Therefore, a culture device filled with a culture medium capable of cultivating anaerobic bacteria and aerobic bacteria is used to form holes for gas venting to enable anaerobic culture, and holes to enable aerobic culture. Therefore, a microorganism culturing device is required that has a closed stopper formed with a part. The present invention was made in order to improve the drawbacks related to the above-mentioned circumstances, and its purpose is to be able to completely seal microbial culture equipment before use, while having a simple structure, and to During culture, gas generated in the culture equipment is removed without using special equipment or accessories, and aerobic bacteria and anaerobic bacteria can be grown rationally, safely, and reliably using culture equipment that uses the same stopper and culture medium. It is an object of the present invention to provide a microorganism culturing device capable of cultivating and handling bacteria. The object of the present invention is to have a container filled with a culture medium, a body that fits into an opening of the container to seal the opening, and further has an outer diameter larger than an inner diameter of the opening of the container. In the microorganism culture device, the atmosphere inside the container and the stopper is in a reduced pressure state;
The stopper is made of an elastic body that can be penetrated by a sample injection needle, and has a body that is pushed up by an increase in the internal pressure of the atmosphere due to gas generated by culture after sample collection, and the body is A microorganism culturing device, characterized in that it has a hole that is opened by the container and that communicates the inside of the container with the outside by pushing up the container to release the gas inside the container to the outside.
Our goal is to provide the following. Still another object of the present invention is to provide the microorganism culture device described above, in which the stopper has a recess on the outer head end of the container and an annular groove on the side end of the body on the side of the container. Still another object of the present invention is to provide the above microorganism culture device in which the stopper is made of a rubber elastic material. Still another object of the present invention is to provide the microorganism culturing device, wherein the medium is an anaerobic bacterial culture medium. Still another object of the present invention is to provide the microorganism culture device, wherein the medium is an aerobic bacteria culture medium. Still another object of the present invention is to provide the microorganism culturing device, wherein the medium is an anaerobic or aerobic bacterial culture medium. Still another object of the present invention is that the medium contains tryptone, soybean peptone, meat extract, yeast extract,
Liver hydrolyzate, glucose, potassium hydrogen phosphate, L
- cysteine hydrochloride, P-aminobenzoic acid, sodium polyanethole sulfonate, hemin,
An object of the present invention is to provide the microorganism culture device containing agar and gelatin. Still another object of the present invention is to provide the microorganism culturing device, wherein the body of the stopper is coated with silicone oil. Next, a specific configuration of an embodiment of the present invention will be explained with reference to the drawings. FIG. 1 shows the assembled state of the microorganism culture device 1 with a rubber stopper 4 fitted therein. The inside of the glass culture device 1 is filled with a medium 2 for culturing aerobic bacteria and anaerobic bacteria. Culture equipment 1
A stopper 4 is attached to the opening 3 of the culture device 1 to seal the culture device 1. The plug body 4 consists of an integrally formed head 5 and a body 6 having a slightly smaller diameter than the head 5, and has a recess 7 on the end surface of the body inside the container.
have. Further, a cover 8 is fitted over the stopper 4 attached to the mouth portion 3 of the culture device 1.
This cover 8 may be made of plastic, a Morton stopper, an aluminum cap, or an aluminum foil, and is fitted over the lower tip 9 of the head 5 of the stopper 4 with a slight gap. FIG. 2 shows an example of the structure of the plug, in which a hole 10 communicating with a recess 7 is formed in the body 6 of the plug 4. As shown in FIG. Further, an annular groove 11 is formed at the side end of the body 6 on the container side. This annular groove 11 is formed when the internal pressure of the culture device 1 reaches a certain level or more.
Pushing up the stopper 4 causes the stopper 4 to rise, allowing gas to be released. However, if the stopper 4 were pushed up further and the groove 11 came to the tip of the opening 3 of the culture device 1,
The elasticity of the rubber serves to prevent the stopper 4 from coming off the mouth 3. On the other hand, when the internal pressure rises above a certain level due to the gas generated by anaerobic culture and the stopper 4 is pushed up, and the hole 10 comes to the tip of the mouth 3 of the culture device 1, the hole 10 Along with degassing the gas,
It also plays a role as a ventilation hole in aerobic culture. The shape of the hole 10 is circular with a diameter of about 0.3 mm to 3 mm, or with a width of 0.3 mm to 2 mm and a length of 2 mm.
It has an oval shape of mm to 3 mm. The plug body 4 is provided with a hole 10, which has a diameter of 0.7 mm to 1.5 mm.
The best results are obtained when two circular holes of mm diameter are placed symmetrically at positions of 3 mm to 5 mm from the upper end of the body 6. A recess 12 is formed in the outer end surface of the container of the head 5 of the stopper 4 to facilitate insertion of a sample injection needle and a bacteria collection needle during bacterial transplantation. On the other hand, when collecting a sample, if the inside of the culture device 1 is kept in a reduced pressure state, injection can be performed automatically by piercing the sample injection needle. In this way, both anaerobic and aerobic cultures
Samples can be collected while maintaining airtight conditions. However, in anaerobic culture, gas is generated and the culture equipment 1
When the internal pressure reaches a certain level, the plug body 4 is pushed up,
As a result, the hole 10 reaches the tip of the mouth 3 of the culture device 1 or above it, and the stopper 4 stops. At this time, the generated gas flows out through the hole 10, passes between the opening 3 of the culture device 1 and the cover 8, and is degassed to the outside of the cover 8. Note that even if the stopper 4 is pushed up further by the generated gas, the annular groove 11 provided at the side end of the body 6 on the container side will keep the culture device 1
The stopper 4 is made to come into contact with the tip of the opening 3 of the stopper 4 and stop the stopper 4 due to its rubber elasticity. And after degassing,
By manually pushing down the hole 10 so that it comes into contact with the inner wall of the opening 3 of the culture device 1, the inside of the culture device 1 can be maintained in an airtight state again. Therefore, gas generated inside the culture device 1 can be vented while maintaining an airtight state inside the culture device 1, and there is no possibility that the stopper 4 will fly off due to internal pressure or the culture solution will overflow during or after culture. There is nothing to do and it is extremely safe. On the other hand, in the case of aerobic culture, after sample collection, the plug 4
After lifting the culture device 1 so that the hole 10 is above the tip of the mouth 3 of the culture device 1, the cover 8 is attached and culture is performed. In other words, ventilation can be carried out with the stopper 4 attached to the opening 3 of the culture device 1, and contamination by bacteria in the outside air does not occur. Although a test tube-shaped culture device was used in the above example, the present invention is not limited to this, and may be a flask-shaped or bottle-shaped culture device with only a relatively long mouth portion. It's okay. Next, the culture medium to be used will be explained. Similar to the plug according to the present invention, the same medium can be used in both anaerobic and aerobic culture, and the following medium is suitable.

【table】

[Table] Prepare the culture medium using the following method. Weigh out the medium components shown in Table 1, excluding gelatin, and dissolve them in 500 ml of distilled water. However, L-cysteine hydrochloride and hemin are dissolved in advance and added. Separately, weigh the gelatin and dissolve it completely in 500 ml of distilled water while heating. After mixing the two well, cool to room temperature, adjust the pH to 7.3±0.1 with an alkaline solution, and prefilter using absorbent cotton. The liquid obtained from this was further filtered under reduced pressure through a glass filter,
The obtained liquid is a medium. The plug is manufactured by the following method. A rubber-like material that has been heated and melted is poured into a mold and solidified and molded.
A plug without a hole is obtained, and a needle having a predetermined shape and size is heated, and a hole is made in the plug with this, or a hole is punched with something such as a punch. After washing, the culture device stopper obtained above is dried naturally or by heating. This is coated with silicone oil. Next, combine the culture device and the stopper. The method is to use a 20ml tube (15.5mmφ
x 165 mm) or a specified amount of culture medium in a suitable flask-like or bottle-like container (20 ml tube is
18ml, 45 in a flask or bottle-like container
ml). After depressurizing and degassing the inside of the device, a mixed gas (nitrogen:carbon dioxide = 9:1) is gently fed to near normal pressure. Next, the pressure is reduced to a specified amount of water absorption, and the stopper is pushed in under reduced pressure to seal the stopper. Then, sterilize it in a high-pressure steam sterilizer.
Sterilize the culture equipment at 115°C for 15 minutes. Next, the effect of the present invention will be explained in the case where a vacuum blood collection tube holder (manufactured by Terumo Corporation) is used. After disinfecting the stopper part of the culture device with alcohol, iodine, etc., insert the culture device into a holder equipped with a sample injection needle. Next, after puncturing the patient's vein with the sample injection needle, the culture device is inserted sufficiently deeply into the holder, and since the inside of the culture device is under reduced pressure, a predetermined amount of sample is injected into the culture device. In addition, the present invention can also be practiced using a syringe. After collecting the sample with a syringe, insert the syringe needle into the center of the stopper of the culture device whose stopper part has been sterilized with alcohol, iodine tincture, etc. At this time, since the inside of the culture device is under reduced pressure, a predetermined amount of sample is automatically aspirated from the syringe and injected. After this, culturing is carried out as follows. In the case of anaerobic culture, a predetermined amount of sample is aspirated using the sample collection method and then cultured at 27° to 37°C for 1 to 14 days. Cultivation can be continued further if necessary. Incidentally, if the stopper is rotated once just before the start of culture, degassing will be performed well. In the case of aerobic culture, samples are collected in the same manner as in anaerobic culture, and after mixing, the stopper is lifted so that part or all of the hole is exposed to the outside of the mouth of the culture device, and then culture is performed. Example 1 First, various gas-producing bacteria were suspended in an appropriate amount of sterile distilled water in an anaerobic culture, adjusted to a concentration of 10 ¹ to 10 ² bacteria/ml, collected, and then cultured at 37°C. . This was observed every day and the growth status of the bacteria inside the culture device and the movement of the stopper due to gas pressure were investigated, as shown in Table 2 below. The movement of the stopper is expressed as the number of days required for part or all of the hole in the stopper to move to the tip of the mouth of the culture device after the start of culture. Even if the culture was continued from the day on which the plug body moved to this point, no further movement of the plug body was observed.

【table】

[Table] Example 2 On the other hand, in aerobic culture, various bacterial strains are suspended in an appropriate amount of sterile distilled water, the bacterial concentration is adjusted to approximately 1 × 10 ¹ cells/ml, and then collected. After lifting the stopper so that the hole in the body was above the mouth of the culture device, culture was started at 37°C, and two days later, the number of viable bacteria in the culture solution was measured according to the standard method. , 10 ⁷ to 10 ⁹ cells/ml, showing good proliferation. Also,
Even when culturing was carried out at 37°C for 21 days in the same aerated state using a control culture device in which bacteria were not collected, no bacteria from the outside air was observed either with or without a cover. .

【table】

[Table] Note that even if you do not use the tube-shaped culture device used above and instead use a flask-shaped or bottle-shaped container with only a relatively long opening, the above results will not occur. The results were the same as those obtained. As described above, according to the present invention, the gas generated during anaerobic culture can be easily removed from the hole in the stopper, and the hole in the stopper is positioned above the mouth of the culture device. In addition, aerobic culture is also possible if the plug is lifted. In other words, even anaerobic and aerobic bacteria can be cultured using a culture device with the same stopper and medium, and the stopper and medium do not scatter during or after cultivation, making it extremely safe. Moreover, culture can be performed reliably. Further, since no other accessories are required for degassing or ventilation, it is easy to manufacture and has various effects such as low cost.

[Brief explanation of the drawing]

FIG. 1 is a partially cutaway sectional view of the assembly of the plug and the culture device before use, and FIG. 2 is a partially sectional view of the plug. DESCRIPTION OF SYMBOLS 1... Culture device, 2... Culture medium, 3... Mouth of culture device, 4... Plug, 5... Plug head, 6... Plug body, 7... Recess in body. 10...hole, 11
...A circular groove.

Claims (1)

[Scope of Claims] 1. A container filled with a culture medium, a body that fits into an opening of the container to seal the opening, and further has an outer diameter larger than an inner diameter of the opening of the container. In a microbial culture device comprising a stopper having a head, the atmosphere inside the container and the stopper is under reduced pressure, and the stopper is made of an elastic material that can be penetrated by a sample injection needle. , has a body that is pushed up by an increase in the internal pressure of the atmosphere due to gas generated by culturing after sample collection, and the body is closed by the container before culturing and is pushed up by the container. A microorganism culturing device characterized by having a hole that communicates the inside of the container with the outside and releases gas inside the container to the outside. 2. The microorganism culture device according to claim 1, wherein the stopper has a recess on the outer head end surface of the container and an annular groove on the side end of the body on the side of the container. 3. The microorganism culture device according to claim 1 or 2, wherein the stopper is made of a rubber elastic material. 4. The microorganism culture device according to claim 1, wherein the medium is an anaerobic bacteria culture medium. 5. The microorganism culture device according to claim 1, wherein the medium is an aerobic bacteria culture medium. 6. The microorganism culture device according to claim 1, wherein the medium is an anaerobic or aerobic bacterial culture medium. 7 The medium contains tryptone, soybean peptone, meat extract, yeast extract, liver hydrolyzate, glucose, potassium hydrogen phosphate, L-cysteine hydrochloride, P-
The microorganism culture device according to claim 1, which contains aminobenzoic acid, sodium polyanethole sulfonate, hemin, agar, and gelatin. 8. The microorganism culturing device according to any one of claims 1 to 7, wherein the body of the stopper is coated with silicone oil.