JPH0147994B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a microorganism culture device filled with a medium, and particularly to a microorganism culture device filled with a medium capable of culturing both aerobic bacteria and anaerobic bacteria. Conventionally, when attempting to culture aerobic bacteria and anaerobic bacteria using a culture device, it was necessary to remove the seal from the culture device and inject a medium suitable for culturing each type of bacteria. However, in this case, bacteria from the outside air could be mixed in, and in some cases it was difficult to distinguish them from the bacteria being cultured. Therefore, it has been considered to enclose a culture medium in advance in a culture device. but,
It is necessary to use a culture device filled with a medium suitable for each of aerobic bacteria and anaerobic bacteria, making the culture method extremely complicated and not a rational and reliable culture method. Therefore, there has been a demand for a microorganism culture device filled with a medium capable of cultivating both anaerobic bacteria and aerobic bacteria. The present invention was made in view of the above circumstances, and its purpose is to be able to completely seal a microorganism culture device before use, while having a simple structure, and to allow culture using the same stopper and culture medium. Cultivate aerobic bacteria and anaerobic bacteria rationally and reliably using equipment,
The purpose of the present invention is to provide microorganism culture equipment that can be used and handled. In order to achieve the above object, there is provided a microbial culture device comprising a container filled with a culture medium and a stopper that fits into the opening of the container to seal the opening. A microbial culture device comprising yeast extract, liver hydrolyzate, glucose, potassium hydrogen phosphate, L-cysteine hydrochloride, P-aminobenzoic acid, sodium polyanethole sulfonate, hemin, agar, and gelatin. It provides: Furthermore, the stopper is made of an elastic body that can be penetrated by a sample injection needle, and has a body that fits into and seals the opening of the container, and further has an outer diameter larger than an inner diameter of the opening. The present invention provides a microorganism culturing device according to claim 1, wherein the stopper has a head having a shape, and the internal atmosphere surrounded by the container and the stopper is in a reduced pressure state. 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 onto the stopper 4 attached to the mouth portion 3 of the culture device 1.
The 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. 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. 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 culture device with only a comparatively long mouth portion, or a bolt-shaped culture device. It's okay. Next, the culture medium will be explained. The same medium can be used for both anaerobic and aerobic culture, and the following media are 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, measure gelatin and dissolve it completely in 500 ml of distilled water while heating. After mixing both well, cool to room temperature, adjust the pH to 7.3±0.1 with an alkaline solution, and prefilter using a degreased wire. 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.
Obtain a plug. Next, if necessary, a needle having a predetermined shape and size is heated, and a hole is made in the stopper body using the needle, or a hole is made 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 dispense a specified amount of culture medium (18 ml for a 20 ml tube, 45 ml for a flask or bottle) into a 20 ml tube (15.5 mm × 165 mm) or a suitable flask-like or bottle-like container. After depressurizing and degassing the inside of the device, a mixed gas (nitrogen:carbon dioxide = 9:1) is gently fed to near normal pressure. Then,
Reduce the pressure to the specified amount of water absorption, and then press the stopper under reduced pressure to seal it. Then, sterilize the culture equipment in a high-pressure steam sterilizer 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 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 outside the mouth of the culture device, and then culture is performed. Experimental 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 in the culture equipment was examined, as shown in Table 2 below.

【table】

[Table] Experimental 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, it is possible to completely seal a microorganism culture device before use, and it is possible to rationally and reliably control aerobic and anaerobic bacteria with a culture device using the same stopper and medium. Sexual bacteria can be successfully cultured. Furthermore, if the internal atmosphere surrounding the stopper is kept in a reduced pressure state, a sample can be automatically collected by piercing the stopper with a sample injection needle.

[Brief explanation of drawings]

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 part of culture device, 4... Stopper, 5... Stopper head, 6... Stopper body, 7
... recessed part of the trunk, 10... hole, 11... annular groove.

Claims (1)

[Scope of Claims] 1. A microorganism culture device comprising a container filled with a medium and a stopper that fits into an opening of the container to seal the opening, wherein the medium contains tryptone, soybean peptone, meat extract, A microbial culture device comprising yeast extract, liver hydrolyzate, glucose, potassium hydrogen phosphate, L-cysteine hydrochloride, P-aminobenzoic acid, sodium polyanethole sulfonate, hemin, agar, and gelatin. . 2. The stopper is made of an elastic body that can be penetrated by a sample injection needle, has a body that fits into and seals the opening of the container, and further has an outer diameter larger than the inner diameter of the opening. 2. The microorganism culturing device according to claim 1, wherein the stopper has a head having a shape, and an internal atmosphere surrounded by the container and the stopper is in a reduced pressure state.