JP5083807B2 - Anaerobic bacterial culture kit container, anaerobic bacterial culture kit, anaerobic bacterial culture method, and anaerobic bacterial growth discrimination method - Google Patents

Description

  The present invention relates to an anaerobic bacterial culture kit container, an anaerobic bacterial culture kit, an anaerobic bacterial culture method, and an anaerobic bacterial growth discrimination method.

  In the dental treatment, in the treatment of an infected tooth, so-called carious teeth, a treatment for mechanically cutting and removing the infection source is performed. When infection reaches the pulp, root canal treatment is performed by cutting and expanding the infection source and root canal dentin in the root canal, followed by root canal disinfection and root canal filling. The root canal in need of treatment is filled with tissue pieces, necrotic pieces, bacteria and their produced toxins, spoilage products, and these sources of infection need to be eliminated. One of the purposes of this root canal treatment is sterilization of the root canal, and whether the root canal after sterilization is sufficiently sterilized has a great influence on the subsequent treatment results. Therefore, it is desirable to be able to confirm whether the root canal is sufficiently sterilized.

  It has been reported that 70-80% of the bacterial species detected from the infected root canal are anaerobic bacteria. However, large-scale equipment such as an anaerobic glove box used in a laboratory is required to culture these anaerobic bacteria, particularly obligate anaerobic bacteria that require a high anaerobic degree. This anaerobic glove box has the following disadvantages: (1) equipment is too complex to use on the chair side, (2) cumbersome operation, (3) expensive, etc. Is not realistic.

  Therefore, at present, it is very difficult to objectively indicate the presence or absence of anaerobic bacteria in the root canal at the clinical chair side at the time of root canal filling in infected root canal treatment. It is left to the judgment of the person. In addition, there are many obligately anaerobic bacteria that are strongly suggested to have a poor prognosis in root canal therapy and to be associated with intractable apical periodontitis.

JP 2005-40029 (Patent Document 1) discloses a method, a device and a kit for collecting anaerobic bacteria, and a method and a kit for collecting and culturing anaerobic bacteria.
Japanese Patent Laying-Open No. 2005-40029

  However, the method described in Patent Document 1 is mainly a method of collecting anaerobic bacteria, and the bacteria collected by this method are transferred to a test tube having a rubber stopper and cultured. Although it is described that the inside of the test tube is replaced with anaerobic gas, there is no description of a specific method, and it takes a considerable time to completely replace the inside of the test tube with anaerobic gas. Opening and closing is also necessary, and there is a drawback that it is difficult to maintain anaerobic conditions at the same time.

  For the reasons described above, the present inventors considered that it is important to determine whether or not obligate anaerobic bacteria with high pathogenicity exist in the root canal when performing root canal filling. However, a simple anaerobic culture kit that can easily culture obligate anaerobes on the chair side has not been known so far.

  Therefore, an object of the present invention is to provide a means that allows a culture test for confirmation of sterilization to be performed easily and inexpensively even on the chair side of a general dental clinic.

  A further object of the present invention is to provide a method for culturing anaerobic bacteria and a method for distinguishing growth of anaerobic bacteria using the above means.

  Therefore, the present inventors have studied to achieve the object of the invention and completed the invention.

The present invention is as follows.
[1] A sealed container having an openable / closable external port and a gas injection port sealed with a resilient material,
An intermediate chamber extending from the openable / closable external port into the sealed container, and for transferring the collected sample to the sealed container while suppressing contamination of oxygen in the air ;
The intermediate chamber has an openable / closable internal port leading to the inside of the sealed container,
Inside the sealed container, there is a space for holding the culture medium,
An anaerobic bacterial culture kit container.
[2] The anaerobic bacterial culture kit container according to [1], wherein the sealed container is made of a flexible transparent material.
[3] The anaerobic bacterial culture kit container according to [1] or [2], wherein the gas inlet has a sealing cap.
[4] The anaerobic bacterial culture kit container according to any one of [1] to [3], wherein the openable / closable external port and the openable / closable internal port are both openable and closable with a fastener.
[5] An anaerobic bacterial culture kit container according to any one of [1] to [4],
An anaerobic bacterial culture kit comprising a medium housed in a space for holding the medium in the container and an oxygen scavenger housed in the intermediate chamber.
[6] The anaerobic bacterial culture kit according to [5], wherein the medium is a liquid medium.
[7] The anaerobic bacterium culture kit according to [5] or [6], wherein the medium contains an indicator whose color tone is changed by a substance produced by anaerobic bacteria.
[8] The openable / closable external port and the openable / closable internal port are both openable and closable with fasteners, and the intermediate chamber further contains a lump for assisting the opening of the fasteners. ]-Anaerobic bacteria culture kit in any one of [7].
[9] The anaerobic bacterial culture kit according to [8], wherein the lump is a spherical body.
[10] The anaerobic bacterial culture kit according to any one of [5] to [9], wherein the intermediate chamber further contains an oxygen detection agent.
[ 11 ] A member carrying anaerobic bacteria is housed in the intermediate chamber of the anaerobic bacteria culture kit according to any one of [5] to [ 10 ] in a state in which the inner opening is sealed,
Sealing the outer opening of the intermediate chamber,
Removing oxygen in the intermediate chamber with an oxygen scavenger;
Injecting anaerobic gas into the sealed container through the gas inlet,
Open the internal port and supply the medium carrying anaerobic bacteria to the medium,
An anaerobic bacterial culture method comprising culturing for a predetermined time.
[ 12 ] A member carrying an object to be inspected is housed in the intermediate chamber of the anaerobic bacterial culture kit according to any one of [5] to [ 10 ] in a state in which the inner opening is sealed,
Sealing the outer opening of the intermediate chamber,
Removing oxygen in the intermediate chamber with an oxygen scavenger;
Injecting anaerobic gas into the sealed container through the gas inlet,
Open the internal port and supply the medium carrying anaerobic bacteria to the medium,
Incubate for a predetermined time,
Determining whether or not the test object contains anaerobic bacteria by a change in the color of the medium,
Anaerobic bacteria growth discrimination method.

  ADVANTAGE OF THE INVENTION According to this invention, the kit which can perform the culture | cultivation test of sterilization confirmation simply and cheaply by the chair side of a general dental clinic is provided. As a result of confirming that the root canal has been sterilized by the method for discriminating anaerobic bacteria using the kit, it is possible to proceed to the next treatment and improve the results of root canal treatment. In addition, by using the kit, anaerobic bacteria other than confirmation of sterilization in the root canal can be easily and inexpensively performed.

[Anaerobic Bacteria Culture Kit Container]
The container for an anaerobic bacterial culture kit of the present invention is:
An airtight container having an openable / closable external port and a gas injection port sealed with a resilient material,
Having an intermediate chamber extending from the openable / closable external port into the sealed container;
The intermediate chamber has an openable / closable internal port leading to the inside of the sealed container,
Inside the sealed container, there is a space for holding the culture medium,
It is characterized by that.

The container of the present invention will be described with reference to FIG.
The left figure of FIG. 1 is a front view of the container of the present invention, and the right figure is a side view of the container of the present invention.
The main body of the container of the present invention is a sealed container 10 having an openable / closable external port 20 and a gas injection port 30 sealed with a resilient material.

  The sealed container 10 can be made of a flexible transparent material, and specifically, can be formed from a sheet of a polymer material such as polyethylene or polypropylene. The sealed container 10 is made of a flexible material so that it can be handled and folded and stored, and it is made of a transparent material so that it can be operated while checking the internal state and the culture state can be easily checked visually. There is an advantage that you can.

  The gas inlet 30 can be a hole filled with an elastic material communicating between the inside of the sealed container 10 and the outside of the container. The elastic material can be, for example, a silicone material (e.g., silicone rubber, silicone resin), and a gas injection needle is inserted into the elastic material, and gas is mixed into the sealed container 10 with air. Can be injected while preventing. Further, the gas inlet 30 may have a mouth sealing cap 31 for the purpose of protecting and sterilizing the gas inlet.

  The sealed container 10 has an intermediate chamber 40 extending from the external port 20 into the sealed container, and the intermediate chamber 40 further has an openable / closable internal port 50 that communicates with the inside of the sealed container 10. The external port 20 and the internal port 50 are not particularly limited as long as they can be opened and closed. However, in consideration of ease of operation, simplicity of structure, ease of creation, etc., both can be opened and closed with fasteners. preferable. In particular, for the purpose of enhancing the hermetic seal with the outside, it is preferable that the external port 20 is provided with double fasteners. Furthermore, since the fastener is a resin rail fastener, it can be opened and closed, can be created in a tightly sealed state, and can be easily installed in a sealed container 10 made of a sheet of polymer material. ,preferable.

  The intermediate chamber 40 is provided for the purpose of transferring the collected sample, for example, a paper point from which the root canal has been wiped, into an airtight container while minimizing the mixing of oxygen in the air. Therefore, it is appropriate that the shape and dimensions of the intermediate chamber 40 be such that the collected sample can be transferred.

  Inside the sealed container 10, there is a space 11 for holding a medium suitable for culturing obligate anaerobic bacteria that may be contained in the collected sample. The amount of the medium may be, for example, about 1 to 50 ml, and the volume of the space 11 can be appropriately determined so that the medium can be accommodated. However, the capacity and shape of the sealed container 10 and the space 11 can be appropriately determined in consideration of convenience of container use and the like. In addition, it is convenient for the inside of the sealed container 10 to be in a reduced pressure or vacuum state until use, including the inside of the intermediate chamber 40, in order to sterilize the inside of the container (excessive in the container). The presence of various bacteria can be reduced).

  The container for an anaerobic bacterial culture kit of the present invention can be supplied in a state of being accommodated in a package, and the packaging is in a state where the container for the anaerobic bacterial culture kit of the present invention is still accommodated in this package. It is preferable that the material has a structure or material that can be sterilized in an autoclave or sterilized with ethylene oxide (EO gas). Such structures and materials can be appropriately selected from known ones.

  As described above, the gas inlet 30 can be provided with a cap 31, but by sterilizing the entire gas inlet 30 with the cap 31 attached, the outer surface of the gas inlet 30 can be aseptically removed. Can keep. Thereby, it is possible to prevent germs from entering the main body when the nozzle is inserted into the gas inlet 30 during gas injection.

  Further, the container for anaerobic bacterial culture kit of the present invention housed in the above-mentioned package includes accessories (for example, a straw for introducing a sterilized liquid medium, and a lump for opening the fastener 51 (for example, Spheroids), oxygen scavengers, oxygen detectors (preferably oxygen detector integrated oxygen scavengers, etc.) are stored together, and can be sterilized together with anaerobic bacterial culture kit containers. it can. It is also possible to sterilize a lump (for example, a spherical body) for opening the fastener 51 separately from the main body and put it in the intermediate chamber 40 together with a paper point with a sample.

[Anaerobic bacteria culture kit]
The present invention also relates to an anaerobic bacterial culture kit.
The culture kit of the present invention is a container for the anaerobic bacterial culture kit of the present invention,
A medium contained in a space for holding the medium in the container, and an oxygen scavenger contained in the intermediate chamber.

  Any medium can be used as long as it can cultivate anaerobic bacteria. For example, BHI (Brain Heart Infusion) is preferable because many kinds of bacteria in the oral cavity are most likely to grow. Moreover, Tryptic soy broth to which Yeast extract or menadione is added can also be used. The medium is suitably a liquid medium when the presence or absence of bacterial growth is observed by a color change caused by a pH indicator. If necessary, an oxygen absorbing material can coexist in the culture medium, and the kit of the present invention can further contain an oxygen absorbing material. By coexisting the oxygen absorbing material, it becomes easy to maintain the anaerobic degree of the culture medium (lower the redox potential). Examples of the oxygen absorbing material include moisture-dependent ageless (label type and card type) (both manufactured by Mitsubishi Gas Chemical Co., Ltd.). The anaerobic degree can also be increased by using a sheet bag-shaped Ageless Omak (Mitsubishi Gas Chemical Co., Ltd.) having a layer structure of an iron-based oxygen absorbent as the container material of this kit.

  However, when the anaerobic bacterial culture kit container of the present invention is simply used for culturing anaerobic bacteria, a solid medium can be used. For example, a solid medium containing petri dish agar having a diameter of about 3 cm can be prepared, and a clinical sample attached to a paper point can be rubbed on the surface, and then the individual medium can be placed in this kit and cultured. In that case, the external port 20 of the container 10 is adjusted to a size that allows the solid medium to be easily carried into the container. Alternatively, a solution as a raw material for a solid medium containing agar can be introduced into the space 11 in the container 10 and solidified there to obtain a solid medium. In that case, the outer port 20 of the container 10 may be of a size that can introduce a solution that is a raw material of the solid medium, and may have the shape shown in FIG.

  It is preferable to add glucose to the medium. Addition of glucose makes it easier for bacteria to produce acids (lactic acid, acetic acid, etc.), and BCG in the medium changes from green to yellow due to the change in pH caused by the acid produced, and the presence of anaerobic bacteria can be detected .

  The medium preferably contains an indicator whose color changes depending on the substance produced by the anaerobic bacteria in order to determine on the spot whether or not the anaerobic bacteria have propagated. As the medium, for example, based on the BHI medium that can cultivate the largest number of oral bacteria, with the addition of hemin / vitamin K, glucose to promote bacterial metabolism, and BCG, a pH indicator can do. In this medium, BCG reacts to the decrease in pH due to organic acids produced by bacteria, and the color of the medium changes from green to yellow. This color change makes it possible to confirm the presence or absence of anaerobic bacteria. Examples of indicators other than BCG include BTB (bromtimole blue), MR (methyl red), and BPB (bromphenol blue), but depending on the indicator, the change in color tone due to pH is similar to the color tone of the medium itself. Some are doing it. In that case, it is difficult to clearly determine a change in color tone. Therefore, the pH indicator added to the medium of this kit is appropriately considered depending on the medium used.

  When both the outer opening 20 and the inner opening 50 are openable and closable by the fasteners 21 and 51, the intermediate chamber 40 further contains a lump 60 for assisting the opening of the fastener. Is preferred. This lump can be, for example, a spherical body. By pressing the lump with the finger against the fastener, the fastener can be easily opened. The spherical body can be about 1 cm in diameter, for example.

  An oxygen scavenger 41 is accommodated in the intermediate chamber 40. Examples of the oxygen scavenger include “oxygen detector-integrated AGELESS: product code SAPE-A” (manufactured by Mitsubishi Gas Chemical Company, Inc.). This oxygen detector-integrated oxygen scavenger is sold as a vacuum pack in a sterilized state and can be used as it is.

  By storing the oxygen scavenger 41 in the intermediate chamber 40, the sample collected in the intermediate chamber 40 is temporarily stored in the intermediate chamber 40 and then deoxygenated in the intermediate chamber 40 after a predetermined time. Thus, by transferring the sample collected from the intermediate chamber 40 to the medium in the sealed container 10, the anaerobic environment in the sealed container 10 can be maintained.

  In addition to the oxygen scavenger 41, the oxygen detector 42 is preferably accommodated in the intermediate chamber 40. By storing the oxygen detection agent 42 in the intermediate chamber 40, after the sample collected in the intermediate chamber 40 is temporarily stored, the state of deoxygenation in the intermediate chamber 40 can be detected. After the inside has surely become an anaerobic environment, the sample collected in the medium in the sealed container 10 can be transferred from the intermediate chamber 40. As an example of the oxygen detector, an oxygen detector-integrated oxygen scavenger can be used. As an example, the aforementioned oxygen detector-integrated AGELESS can be used.

  The medium is supplied to the space 11 for holding the medium inside the sealed container 10 via the intermediate chamber 40. When supplying the medium, the intermediate chamber 40 may contain the oxygen scavenger 41 and the oxygen detector 42. However, the medium should be supplied before the oxygen absorber 41 and the oxygen detector 42 are stored. Is preferred. It is preferable that the supply of the medium is completed, the internal port 50 is closed, and then the external port 20 is closed after the oxygen scavenger 41 and the oxygen detector 42 are accommodated in the intermediate chamber 40. The oxygen scavenger 41 and the oxygen detector 42 may be collected in the intermediate chamber 40 after the collected sample is stored in the intermediate chamber 40, or in the intermediate chamber 40 in which the oxygen absorber 41 and the oxygen detector 42 are stored. The collected sample may be accommodated.

  The container for anaerobic bacteria culture kit and the anaerobic bacteria culture kit of the present invention can be used for culturing anaerobic bacteria, and further, can be used for testing specimens that may contain anaerobic bacteria. it can.

[Anaerobic bacteria culture method]
The anaerobic bacterial culture method of the present invention comprises:
A member carrying anaerobic bacteria is housed in the intermediate chamber of the anaerobic bacteria culture kit of the present invention in a state where the inner opening is sealed,
Sealing the outer opening of the intermediate chamber,
Removing oxygen in the intermediate chamber with an oxygen scavenger;
Injecting anaerobic gas into the sealed container through the gas inlet,
Open the internal port and supply the medium carrying anaerobic bacteria to the medium,
Incubating for a predetermined time.

  The anaerobic bacterium that can be cultured by the anaerobic bacterium culture method of the present invention is not particularly limited, and may be an anaerobic bacterium other than the obligate anaerobic bacterium present in the human oral cavity. The member for supporting anaerobic bacteria is not particularly limited as long as it is a paper point or any other material suitable for supporting anaerobic bacteria.

  In the anaerobic bacterium culture method of the present invention, a member carrying anaerobic bacteria is accommodated in the intermediate chamber of the anaerobic bacterium culture kit of the present invention with the internal port sealed and the external port opened. Next, the external port of the intermediate chamber is sealed, and oxygen in the intermediate chamber is removed with an oxygen scavenger. Completion of removal of oxygen in the intermediate chamber can be determined from the color of the oxygen detector when the oxygen detector is accommodated in the intermediate chamber. When the oxygen detection agent is not stored in the intermediate chamber, for example, the time during which oxygen can be removed is measured in advance using the oxygen detection agent, and a predetermined time is set in advance based on the measured time. I can keep it.

  Next, an anaerobic gas is injected into the inside of the sealed container through a gas injection port. The anaerobic gas can be injected without waiting for completion of oxygen removal in the intermediate chamber. Preferably, the anaerobic gas is injected while oxygen is being removed from the intermediate chamber. There are no particular restrictions on the anaerobic gas. For example, three types of mixed gas (80% nitrogen, 10% hydrogen, 10% carbon dioxide) used in many laboratories for the culture of obligate anaerobic bacteria are common. is there. However, depending on the type of bacteria to be cultured, two types of mixed gas of nitrogen and hydrogen, or hydrogen, nitrogen or carbon dioxide alone can be used.

  Although the culture medium is previously stored in the sealed container before the anaerobic gas is injected, the space in the sealed container other than the culture medium can be filled with the anaerobic gas in advance. In addition, an oxygen absorbing material can be added to the medium in advance. By adding an oxygen absorbing material to the medium in advance, anaerobic bacteria can be easily cultured in a medium having a high anaerobic degree.

  After injecting the anaerobic gas, the internal port is opened and the member carrying the anaerobic bacteria is supplied to the medium, and then cultured for a predetermined time. The culture conditions (temperature and time) can be appropriately determined according to the type of the desired anaerobic bacteria. For example, it can be placed in a heat retaining device at 37 ° C. and cultured for 3-5 days. The cultured anaerobic bacteria can be used for, for example, the next test.

  In the anaerobic bacteria culturing method of the present invention, the liquid medium for culturing anaerobic bacteria can be adjusted to an anaerobic degree at which the anaerobic bacteria can suitably grow. When anaerobic bacteria are supplied to the liquid medium using the above procedure, the anaerobic degree of the liquid medium can be displayed as an oxidation-reduction potential by using a triple gas mixture (80% nitrogen, 10% hydrogen, 10% carbon dioxide). It can be adjusted to about -100 to -150mV. In particular, the anaerobic degree commonly used for anaerobic bacterial culture of about −110 to −130 mV can be easily achieved. In addition, when hydrogen, nitrogen or carbon dioxide is used alone instead of the three kinds of mixed gas, the anaerobic degree of the liquid medium can be displayed as a redox potential and can be adjusted to, for example, about -30 to -100 mV. . In anaerobic bacterial culture using a normal anaerobic glove box, a catalyst that promotes the reaction between oxygen and hydrogen (for example, platinum alumina pellet) is used to remove the remaining oxygen by reacting it with water to form water. To do. However, in the culture method using the kit of the present invention, anaerobic degree in the above range can be realized without using the catalyst.

  In addition, by using an oxygen absorbent such as the moisture-dependent ageless (label type and card type) (both manufactured by Mitsubishi Gas Chemical Co., Ltd.), the oxidation-reduction potential can be further reduced by about 50 to 80 mV. . Similarly, the oxidation-reduction potential can also be lowered by using a sheet bag-shaped Ageless Omak (manufactured by Mitsubishi Gas Chemical Co., Ltd.) having a layer structure of an iron-based oxygen absorbent as the container material of this kit.

  However, when anaerobic bacteria that require a higher anaerobic degree are cultured, it is necessary to increase the anaerobic degree of the liquid medium. In such a case, a catalyst (for example, platinum alumina pellets) that promotes the reaction between oxygen and hydrogen is accommodated in the intermediate chamber, and a higher anaerobic degree is obtained by using a gas containing hydrogen as an anaerobic gas. It can also be realized. Therefore, the kit of the present invention can also contain a catalyst that promotes the reaction between oxygen and hydrogen, if necessary.

[Analysis method of anaerobic bacteria]
The method for determining the growth of anaerobic bacteria according to the present invention includes housing a member carrying an object to be tested in the intermediate chamber of the anaerobic bacteria culture kit according to the present invention in a state where the internal opening is sealed,
Sealing the outer opening of the intermediate chamber,
Removing oxygen in the intermediate chamber with an oxygen scavenger;
Injecting anaerobic gas into the sealed container through the gas inlet,
Open the internal port and supply the medium carrying anaerobic bacteria to the medium,
Incubate for a predetermined time,
This includes determining whether or not the object to be inspected contains anaerobic bacteria based on a change in the color tone of the medium.

  The growth determination method for anaerobic bacteria of the present invention can be carried out in the same manner as the anaerobic bacteria culture method of the present invention by using a member carrying an object to be tested instead of a member carrying an anaerobic bacterium. Specifically, the member carrying the object to be inspected can be a paper point that wipes off a predetermined site in the human oral cavity, for example, the root canal.

  In the method for discriminating anaerobic bacteria according to the present invention, a medium containing an indicator whose color tone is changed by a substance produced by anaerobic bacteria is used as the medium. Whether or not the object to be inspected contains anaerobic bacteria is determined based on the degree of change in color tone. When the medium contains BCG, a pH indicator, if anaerobic bacteria are present, the anaerobic bacteria produce acid (lactic acid, acetic acid, etc.), and the BCG in the medium changes from green to yellow due to the change in pH caused by the generated acid. Change.

Next, the method for discriminating anaerobic bacteria of the present invention will be described more specifically.
A polyethylene bag was processed to produce an anaerobic culture kit of the present invention of about 15 × 10 cm as shown in FIG. This kit consists of a polyethylene bag 10 as the main body, a sample intake (external port) 20 at the top of the main body, and three mixed gases for culturing obligate anaerobic bacteria (80% nitrogen, 10% hydrogen, 10% carbon dioxide) %) A structure in which an inlet 30 is provided. Furthermore, the prepared liquid culture medium 70 is contained inside the main body. The sample inlet 20 is made of polyethylene and has a bag-like structure with two fasteners 21 at the top and bottom. And inside it is an oxygen scavenger 40 (integrated with an oxygen detector that changes its color in response to oxygen) and a resin-made ball 1 cm in diameter. It is possible to open the lower fastener even from the top.

  The three-type mixed gas inlet 30 is provided with a resin cap 31, and when the cap is opened, the inside is filled with silicone (not shown). With this structure, when filling three kinds of mixed gas into the main body of the container 10 from the handy spray can 90, the mixed gas does not leak and oxygen does not enter the inside of the container 10. Gas can be easily filled into the container 10 from the nozzle. Further, the inside of the container 10 before use is sterilized and in a reduced pressure state. Further, the polyethylene container 10 is processed into a shape such that the bottom can be cultured in a standing state in an incubator while being a bag. The medium was autoclaved and then dispensed into EOG sterilized container 10.

FIG. 2 shows a culture method of obligate anaerobic bacteria using the anaerobic culture kit of the present invention.
(Step 1) Collect bacteria using sterile paper point 80.
(Step 2) The collected paper point 80 is put into the intermediate chamber 40 containing the oxygen absorber 41, and the fastener 21 is closed to make the intermediate chamber anaerobic.
(Step 3) The three types of mixed gas filled in the handy spray can 90 from the gas inlet 30 are filled into the kit container 10.
(Step 4) Open the fastener 51 and drop the paper point into the medium.
(Step 5) The whole kit is placed in an incubator and cultured.
(Step 6) After 72 hours, the presence or absence of anaerobic bacteria is confirmed by changing the color of the medium.
When the presence of obligate anaerobic bacteria is actually confirmed at the clinical chair side, a sample is collected by inserting a sterilized paper point into the root canal.

Experiments using the anaerobic culture kit of the present invention First, five bacterial species including obligate anaerobes that are frequently isolated from infected root canals and are relatively highly pathogenic to living organisms (S. Mutans ( S. mutans ), E. limosum ( E. limosum ), E. faecalis ( P. gingivalis ), F. nucleatum ( F. nucleatum )) were selected. Then, the same five bacterial species were cultured at 37 ° C. for 72 hours using the simplified anaerobic culture kit of the present invention. After the culture, changes in the pH of the medium and changes in the color of the indicator were measured, and the presence or absence of growth of each bacterium was examined. The redox potential of the culture medium after the culture was also measured. Furthermore, using a large anaerobic glove box at the laboratory level that can cultivate obligate anaerobic bacteria in a strict anaerobic environment, the same 5 types of bacteria are cultured in the same medium (including indicator), and simplified anaerobic The results were compared with the results from the culture kit. In addition, clinical samples were collected, and anaerobic bacteria were cultured using the anaerobic culture kit of the present invention.

(result)
As a result of culturing five bacterial species including obligate anaerobic bacteria using the anaerobic culture kit of the present invention, the pH of the medium decreased and the color of the medium changed from green to yellow. Moreover, this result became the same as the result of culturing in a large anaerobic glove box at the laboratory level. Also, the relatively high anaerobic degree requirement E. Rimosumu (E. limosum), F. Nukureitamu (F. nucleatum) was also possible cultured in anaerobic culture kit of the present invention. From these results, it was found that the presence or absence of obligate anaerobic bacteria in the root canal can be easily confirmed using the anaerobic bacteria culture kit of the present invention. The redox potential of the medium measured after culturing using the anaerobic culture kit of the present invention was in the range of −110 to −130 mV. Furthermore, the oxidation-reduction potential when the oxygen absorbent Ageless Omak (Mitsubishi Gas Chemical Co., Ltd.) was used was in the range of -170 to -180 mV. Further, in a culture experiment using a clinical sample presumed to contain many bacterial species other than the five bacterial species that were purely cultured, a change in the color of the medium was also observed. From the above, the usefulness of the anaerobic culture kit (anaerobic bacterial culture kit) of the present invention was confirmed.

  By using the container and kit of the present invention and the method, a culture test for confirming sterilization can be performed easily and inexpensively even on the chair side of a general dental clinic. This increases the accuracy of treatment and leads to prevention of recurrence.

Explanatory drawing of the culture kit of this invention. Explanatory drawing of the culture | cultivation method of obligate anaerobic bacteria using the anaerobic culture kit of this invention.

Claims (12)

An airtight container having an openable / closable external port and a gas injection port sealed with a resilient material,
An intermediate chamber extending from the openable / closable external port into the sealed container, and for transferring the collected sample to the sealed container while suppressing contamination of oxygen in the air ;
The intermediate chamber has an openable / closable internal port leading to the inside of the sealed container,
Inside the sealed container, there is a space for holding the culture medium,
An anaerobic bacterial culture kit container. 2. The container for an anaerobic bacteria culture kit according to claim 1, wherein the sealed container is made of a flexible transparent material. 3. The anaerobic bacterial culture kit container according to claim 1 or 2, wherein the gas inlet has a sealing cap. The anaerobic bacterial culture kit container according to any one of claims 1 to 3, wherein both the openable / closable external opening and the openable / closable internal opening are openable and closable with a fastener. An anaerobic bacterial culture kit container according to any one of claims 1 to 4,
An anaerobic bacterial culture kit comprising a medium housed in a space for holding the medium in the container and an oxygen scavenger housed in the intermediate chamber. 6. The anaerobic bacteria culture kit according to claim 5, wherein the medium is a liquid medium. The anaerobic bacteria culture kit according to claim 5 or 6, wherein the medium contains an indicator whose color tone changes depending on a substance produced by anaerobic bacteria. 8. The openable / closable external port and the openable / closable internal port are both openable and closable by fasteners, and the intermediate chamber further contains a lump for assisting the opening of the fasteners. The anaerobic bacterial culture kit according to any one of the above. 9. The anaerobic bacterial culture kit according to claim 8, wherein the mass is a spherical body. The anaerobic bacteria culture kit according to any one of claims 5 to 9, wherein an oxygen detection agent is further accommodated in the intermediate chamber. The member carrying the anaerobic bacteria is accommodated in the intermediate chamber of the anaerobic bacteria culture kit according to any one of claims 5 to 10 in a state where the inner opening is sealed,
Sealing the outer opening of the intermediate chamber,
Removing oxygen in the intermediate chamber with an oxygen scavenger;
Injecting anaerobic gas into the sealed container through the gas inlet,
Open the internal port and supply the medium carrying anaerobic bacteria to the medium,
An anaerobic bacterial culture method comprising culturing for a predetermined time. A member carrying an object to be inspected is accommodated in the intermediate chamber of the anaerobic bacteria culture kit according to any one of claims 5 to 10 in a state in which an inner opening is sealed,
Sealing the outer opening of the intermediate chamber,
Removing oxygen in the intermediate chamber with an oxygen scavenger;
Injecting anaerobic gas into the sealed container through the gas inlet,
Open the internal port and supply the medium carrying anaerobic bacteria to the medium,
Incubate for a predetermined time,
Determining whether or not the test object contains anaerobic bacteria by a change in the color of the medium,
Anaerobic bacteria growth discrimination method.