JPH0146109B2 - - Google Patents

Description

[Detailed description of the invention]

TECHNICAL FIELD The present invention relates to a microorganism culture device. 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 collection 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. Caps and stoppers often fly off, putting users at risk. Moreover, when 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, with a stopper that uses a simple tightening member, the gas produced by anaerobic bacteria increases the internal pressure of the culture device, causing the stopper to fly off during culture, contaminating the culture medium with bacteria in the outside air, and even flying off with the stopper. There was a problem that the surrounding environment was contaminated by anaerobic bacteria. 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 breathability, 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. In addition, when collecting samples, it was necessary to remove the stopper, and at that time, outside air entered and bacteria in the outside air were also cultured, making it 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, which makes the culture method extremely complicated. A proper culture method was not possible. Therefore, a culture device filled with a culture medium capable of cultivating anaerobic bacteria and aerobic bacteria is used, a gas vent hole is formed to enable anaerobic culture, and a culture medium is used to allow aerobic bacteria to grow. A microorganism culture device is required that has a closed stopper with a hole formed therein. The present invention was made to improve the drawbacks related to the above-mentioned circumstances, and its purpose is to be able to culture both anaerobic bacteria and aerobic bacteria, and to be able to culture more types of bacteria. Another object of the present invention is to provide a microorganism culturing device capable of culturing bacteria in a shorter time. Another object of the present invention is to be able to completely seal a microbial culture device before use, and to prevent the generation of microorganisms in the culture device during anaerobic culture without using any special equipment or accessories. Remove the gas,
Reasonable with culture equipment using the same stopper and medium.
Another object of the present invention is to provide a microorganism culture device that can safely and reliably culture and handle aerobic bacteria and anaerobic bacteria. In a microbial culture device comprising a container filled with a medium and a stopper that fits into the opening of the container, the medium contains tryptone, soybean peptone, meat extract, yeast extract, liver hydrolyzate, glucose, potassium hydrogen phosphate. , L-cysteine hydrochloride, p-aminobenzoic acid, sodium polyanetholesulfonate, hemin, gelatin, and sodium bicarbonate. Another object of the present invention is to provide the above-mentioned microorganism culture device, in which the body of the stopper is provided with a hole that communicates with the atmosphere inside the container. Another object of the present invention is to provide the above-mentioned microorganism culture device, in which the stopper has a recessed portion on the end face of the inner body of the container, and the atmosphere inside the container is in a reduced pressure state. 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. Next, an embodiment of the present invention is shown in FIG. 1 for explaining its specific configuration with drawings, in which an assembly of a microorganism culture device 1 in which a stopper 4 made of rubber such as butyl rubber or styrene-butadiene rubber is fitted is shown. Indicates the condition. The inside of the glass culture device 1 is filled with a medium 2 for culturing aerobic bacteria and anaerobic bacteria. A stopper 4 is attached to the opening 3 of the culture device 1, so that the culture device 1 is sealed tightly. The stopper 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 6 inside the container. In addition, a plug 4 attached to the mouth part 3 of the culture device 1
A cover 8 is fitted over. This cover 8
The cap may be made of plastic, a Morton cap, an aluminum cap, or an aluminum foil, and is fitted from the lower tip 9 of the head 5 of the cap 4 with a slight gap. FIG. 2 shows an embodiment of the structure of the stopper, in which a hole 10 communicating with a recess 7 is formed in the body 6 of the stopper 4. As shown in FIG. Further, an annular groove 11 is formed at the side end of the body 6 on the container side. When the internal pressure of the culture device 1 reaches a certain level, the annular groove 11 pushes up the stopper 4 and the stopper 4 rises, allowing gas to be released. When the mm-wide groove 11 reaches the tip of the mouth 3 of the culture device 1, its rubber elasticity 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, the stopper 4 is pushed up and the hole 10 comes to the tip of the mouth 3 of the culture device 1. In addition to degassing this gas, it also serves as a ventilation hole in aerobic culture. The shape of the hole 10 is 0.3 mm to 3 mm in diameter.
It has a circular shape of about mm or an oval shape with a width of 0.3 mm to 2 mm and a length of 2 mm to 3 mm. Plug body 4
The holes 10 are provided in the body part 6, but it is best if two circular holes with a diameter of 0.7 mm to 1.5 mm are placed symmetrically at a distance of 3 mm to 5 mm from the upper end of the body part 6. A recess 12 is formed in the outer end surface of the container of the head 5 of the stopper 4 to facilitate penetration 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, samples can be collected while maintaining airtight conditions even in anaerobic culture and aerobic culture. However, in anaerobic culture, gas is generated and the culture equipment 1
When the internal pressure exceeds a certain level, the plug body 4 is pushed up,
As a result, the hole 10 reaches the tip of the opening 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 not touch the tip of the opening 3 of the culture device 1. in contact with the stopper 4 due to rubber elasticity.
It is designed to stop. After degassing, the inside of the culture device 1 can be kept airtight again by pushing down by hand so that the hole 10 comes into contact with the inner wall of the mouth 3 of the culture device 1. 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, the plug body 4 is connected to the mouth part 3 of the culture device 1.
Ventilation can be performed even when the device is installed in the air, 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. Conventional culture media are shown in Table 1 below for comparison.
Table 2 shows the culture medium used in the present invention.

【table】

【table】

[Table] The culture medium is prepared according to the composition shown in Table 1 or 2 by the following method. Weigh out the medium components shown in Tables 1 and 2, excluding gelatin, and dissolve them in 500 ml of distilled water. However, L-cysteine hydrochloride and hemin are added after being dissolved in advance. Separately, weigh gelatin and dissolve it completely while heating it in 500 ml of distilled water. After mixing both well, cool to room temperature,
Even with an alkaline solution, the pH is 7.3± with the composition shown in Table 1.
0.1, adjust the composition to 7.3±0.2 according to Table 2, and pre-filter using absorbent cotton. The liquid obtained from this is further filtered under reduced pressure through a glass filter, and the obtained liquid is a culture medium. The plug is manufactured by the following method. A rubber-like material that has been heated and melted is forced into a mold, solidified and molded to obtain a plug with no holes, and a needle with a predetermined shape and size is heated to make a hole in the plug, or Or make a hole with something like a punch. After washing the culture device stopper obtained above, it is dried naturally or by heating. Silicone oil is added to this for coating. Next, combine the culture device and the stopper. The method is to use a 20ml tube (15.5mmφ x
165 mm) or a suitable flask-like or bottle-like container, etc., with the specified amount of culture medium (18 mm for a 20 ml tube).
ml, 45ml in flask or bottle-like containers)
Dispense. 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 the culture equipment in a high-temperature autoclave 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 tincture, 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 deep enough into the holder, and since the pressure inside the culture device is reduced, 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. Test 1 First, in an anaerobic culture, various gas-producing bacteria were suspended in an appropriate amount of sterile distilled water, and the bacteria concentration was adjusted to 10 ¹ to ^{10 2} cells/ml, collected, and then cultured at 37°C. When this was observed every day and the growth status of the bacteria within the culture device and the movement of the stopper due to gas pressure were examined, the results are shown in Tables 3 and 4 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 when the plug body moved to this point, no further movement of the plug body was observed.

【table】

【table】

[Table] Test 2 On the other hand, in aerobic culture, various bacterial strains are suspended in an appropriate amount of sterile distilled water, and approximately 1 × 10 ¹ to 5 × 10 ¹ cell/
ml, then collect the bacteria. Lift the stopper so that the hole in the stopper is above the mouth of the culture device, then start culturing at 37℃, and after 2 days culture. When the number of viable bacteria in the liquid was measured according to the usual method, it was 10 ⁷ ~ as shown in Tables 5 and 6 below.
They were proliferating well at ¹⁰⁹ cells/ml. In addition, a control culture device in which no bacteria were collected was placed in the same aerated state.
Even when cultured at ℃ for 21 days, no contamination of bacteria from the outside air was observed with or without a cover.

【table】

【table】

[Table] * Bacterial liquid was prepared using human blood instead of sterile distilled water.
Test 3 Various bacterial strains were suspended in an appropriate amount of sterile distilled water, and the bacterial concentration was adjusted to approximately 1×10 ¹ to 5×10 ² cells/ml before use in the experiment. In the case of aerobic culture, lift the stopper so that the hole in the stopper is above the mouth of the culture device, and in the case of anaerobic culture, start culturing at 37℃ and collect live bacteria every day. The number of bacteria was measured according to a conventional method, and the number of days required for the number of viable bacteria to reach approximately 5 x ¹⁰⁷ cells/ml or more was investigated.
As shown in the table (in the case of aerobic culture), superior results were obtained for some bacterial strains with the medium composition shown in Table 2.

【table】

【table】

[Table] Prepare a bacterial solution.
*2) Observation for 7 days In addition, instead of using the tube-shaped culture device used above, culture can be performed using a flask-shaped container with a relatively long cylindrical mouth or a bottle-shaped container. However, the results were the same as those obtained above. As described above, according to the present invention, both anaerobic bacteria and aerobic bacteria can be cultured, and more types of bacteria can be cultured than when conventional culture media are used. Furthermore, bacteria can be cultured in a shorter time and microorganisms can be cultured efficiently. Also,
By providing a hole in the body of the stopper that communicates with the atmosphere of the container, gas generated by anaerobic culture can be easily removed from the hole in the stopper.
Furthermore, aerobic culture can be performed by lifting the stopper so that the hole in the stopper is above the mouth of the culture device. 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 drawings]

FIG. 1 is a partially cutaway sectional view showing the microorganism culture device of the present invention in a state before use, and FIG. 2 is a partially sectional view of the stopper. 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 microorganism culture device comprising a container filled with a culture medium and a stopper that fits into an opening of the container,
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, gelatin, and sodium bicarbonate. A microorganism culture device comprising: 2. The microorganism culture device according to claim 1, wherein the stopper has a hole in its body that communicates with the atmosphere inside the container. 3. The stopper has a recess on the end face of the inner body of the container, and the atmosphere inside the container is in a reduced pressure state.
Microorganism culture equipment as described in section. 4. The microorganism culture device according to claim 2, 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. 5. The microorganism culture device according to claim 1 or 4, wherein the stopper is made of a rubber elastic material. 6. The microorganism culture device according to claim 1, wherein the medium is an anaerobic or aerobic bacterial culture medium.