JP4570032B2 - Anaerobic culture test apparatus using flexible culture bag - Google Patents

Description

The present invention is flexible culture bags and, anaerobic culture test device the liquid while maintaining the anaerobic conditions by anoxic gas supply combined injectable and the dispensing device in a flexible culture bag to conduct culture assays anaerobic microorganisms It is about. This device is useful for indoor experiments to evaluate the cleaning effect of the bar Iore Medellin Activation especially.

  In recent years, bioremediation promoting technology has been widely used as a technology for purifying soil and groundwater contamination by organic chlorine compounds (for example, trichlorethylene, tetrachloroethylene, or various organic solvents) and restoring the environment. This is by injecting nutrient sources (such as electron donors) of underground microorganisms into the ground and activating the natural purification function by local anaerobic microorganisms (microorganisms that propagate in anaerobic conditions) and purifying pollution in situ. It is a technology to plan. In such bioremediation promotion technology, in order to enhance the purification effect, it is important to select an appropriate type of nutrient source according to the contamination status and inject an appropriate amount into the ground. In other words, it is necessary to quantitatively evaluate what kind of microorganisms should be activated and what kind of nutrient source should be injected at what rate (rate and concentration). . This is because, depending on the nutrient source to be input and the amount thereof, microorganisms other than the target may propagate and the target microorganism may be inhibited from growing.

  In order to achieve quantitative evaluation, it is desirable to bring the environment where local anaerobic microorganisms live into the room so that the culture test can be easily performed. However, when testing with anaerobic microorganisms that inhabit soil or water, special anaerobic culture techniques must be used because oxygen inhibits growth. Conventionally, techniques for culturing and testing anaerobic microorganisms include a roll tube method and a glove box method (see, for example, Non-Patent Document 1).

  The roll tube method uses a test tube with a butyl rubber stopper, injects an oxygen-free mixed gas (hydrogen: nitrogen: carbon dioxide, etc.), dispenses the test tube while blocking oxygen, and inspects it. is there. This method is not suitable for an inspection in which a volatile substance is contained in a sample in order to spray an oxygen-free mixed gas and the concentration change thereof is examined. In addition, the sample for confirming the propagation status of microorganisms can be taken out only once because it is a sealed container (the inside of the sealed container is depressurized and the culture environment is changed by taking out the sample). Therefore, when used for such purposes, it is necessary to prepare a large number of test tubes in advance, which requires time, labor and skill.

  The glove box method is a method of dispensing and culturing a sample using rubber gloves attached to a box wall in an airtight box filled with oxygen-free gas. In order to make the entire box oxygen-free, the vacuum suction by the vacuum pump and the injection of the oxygen-free mixed gas are repeated several times, and finally the trace amount of remaining oxygen is hydrogenated by the deoxygenation catalyst (palladium). To maintain an anaerobic state. In anaerobic culture and inspection, it is important to operate anaerobically and how to maintain the anaerobic state. Therefore, time, labor, skill, and large-scale equipment are required.

  Recently, an incubator that does not need to be dispensed into a culture vessel has been developed for anaerobic microorganism culture tests. For example, there is an aseptic medium packaging body having a structure in which a bottle is filled with a medium and tightly fitted with a cap that can be aseptically inserted into an injection needle (see Patent Document 1). This is because it is not necessary to open a sterile medium package in use, take out the medium in a sterile atmosphere, and dispense it into another culture container, so that complicated and troublesome dispensing work can be eliminated. Has been.

  However, in the case of such a sterile medium package, it cannot be applied to a method of use in which groundwater containing anaerobic microorganisms is injected into the bottle. This is because the anaerobic microorganisms to be cultured are also killed by the heat treatment. In this case, a complicated and troublesome aseptic dispensing operation as described in the prior art is required.

In addition, after filling the medium with the culture medium, it is necessary to perform autoclaving and sterilization, and therefore, a vial having pressure resistance and heat resistance must be used as the bottle. Therefore, if a large number of bottles are prepared for the test, they are bulky, heavy and difficult to handle. Also, during the test, storage facilities such as a thermostatic chamber will be a major issue. Furthermore, when a liquid sample is injected or taken out with a syringe or the like, the amount of liquid changes accordingly, so the internal pressure of the bottle increases or decreases, and the culture conditions (culture environment) also change. . In order to avoid this, it is necessary to perform a cumbersome operation such as inserting a needle into the cap and penetrating it, and pushing the cylinder of the syringe to inject the sample into the bottle while raising and lowering the cylinder and degassing the bottle. It becomes.
JP 2001-114339 A New edition "Soil Microbial Experiment Method" Soil Microbial Society published by Yokendo Co., Ltd. 1992

  The problem to be solved by the present invention is that in the prior art, when a sample is injected or taken out with a syringe, the internal pressure of the bottle increases or decreases accordingly, and the culture conditions (culture environment) change. When preparing a bottle, it is bulky and difficult to handle, and with conventional technology, dispensing work in an absolute anaerobic state cannot be easily performed, and skill is required, so it takes a lot of labor and time for a culture test, etc. is there.

In the present invention , a bag-like body made of a flexible material and containing a liquid therein, an infusion sleeve with a sealable cock airtightly attached to the bag-like body, and a needle for injecting and sucking liquid Jo tube that a not include a hermetically stab narrowing-extractable packing with sealing plug Ru using the full Rekishiburu culture bag.

  Here, the bag-like body is preferably made of a multilayer material in which a resin film is laminated on the front and back so that the aluminum foil is positioned in the middle, for example. Furthermore, it is also effective to use a multilayer material in which a cloth-like sheet made of a fibrous material is attached as the outer layer and the bending strength is reinforced.

The present invention comprises a combination of a flexible culture bag for culturing anaerobic microorganisms in groundwater having the above structure and a dispensing device for injecting a liquid while maintaining an anaerobic state in the flexible culture bag. The injection device includes a rigid container having a rigid structure for containing a liquid therein, a connecting portion having a shuttable valve provided in the hermetic container, and a transfer pipe continuously reaching the lower part of the sealed container from the connecting portion. And an oxygen-free gas supply system to the hermetic container, which is airtight by opening and closing the cock and the valve between the infusion sleeve with the cock of the flexible culture bag and the connecting portion having the valve of the dispensing device. and freely connected and separated to the water sampling groundwater in a sealed container, while enabling dispensing the flexible culture bag while maintaining the anaerobic conditions depending on the pressurization by free oxygen gas, A flexible culture bags sewage has been dispensed, is anaerobic culture test device being characterized in that the separable from the dispensing device while maintaining the anaerobic conditions.

  Here, the sealed container has a decompression port and an oxygen-free gas supply port at the top, a vacuum suction pump is connected to the decompression port, and an oxygen-free gas supply system is connected to the oxygen-free gas supply port, It is preferable to enable an anaerobic atmosphere in the sealed container by alternately performing vacuum suction and oxygen-free gas supply. Moreover, it is preferable to equip the bottom part of an airtight container with the magnetic stirrer for stirring.

The flexible culture bag used in the present invention can be freely deformed because the bag-like body is made of a flexible material, and the internal pressure always automatically matches the atmospheric pressure even if the amount of liquid inside changes. Adjusted. Therefore, the pressure condition (pressure environment) does not change at all even if liquids such as solutions are injected prior to or during the culture, or the liquid is periodically withdrawn as a sample during the culture. The culture can be continued under the same conditions. For this reason, even when it is desired to investigate changes in the culture state over time, it is economical because it is only necessary to periodically take out a liquid sample sequentially from one flexible culture bag, and the thermostat used can be downsized. Furthermore, since it can be stored in a crushed state before use, it can be stored compactly and is easy to handle lightly.

  The anaerobic culture test apparatus according to the present invention is composed of a combination of a flexible culture bag and a dispensing apparatus, but the dispensing apparatus can easily create an absolute anaerobic state without taking time and effort. Since the bag can be connected and separated in an airtight manner with respect to the dispensing device, the solution in the sealed container can be easily dispensed (sorted) into a number of flexible culture bags while maintaining an anaerobic state. .

A typical example of the flexible culture bag used in the present invention is shown in FIG. As shown to A, the flexible culture bag 10 consists of a flexible material, and the bag-like body 12 of the airtight structure which can accommodate the liquid (liquid culture medium) containing a nutrient source inside is attached to this bag-like body 12 airtightly And an injection sleeve 14 with a cock that can be sealed, and a sealing plug 16 with packing capable of hermetically inserting and extracting a needle tube (injection needle or the like) for liquid injection and suction.

  Here, the bag-like body 12 has a structure in which two rectangular sheets 18 are welded on four sides. For example, as shown in B, it is made of a multilayer material in which a resin film 22 is laminated on the front and back so that the aluminum foil 20 is located in the middle. A sheet 18 having a higher bending strength is applied. When the aluminum foil 20 is sandwiched in this way, the permeation of an organic chlorine compound or the like can be blocked with high efficiency, and is particularly suitable for use in a bioremediation promoting technique. Further, since the light shielding property is obtained, there is an effect that the internal alteration can be prevented. In the present invention, since the liquid is contained in the bag-like body 12, the aluminum foil may be bent and damaged at the same place due to its weight, but the cloth-like sheet 24 is stuck as a reinforcing material as described above. Thus, such inconvenience can be solved.

  The constituent material of the bag-like body is not limited to the above-described configuration, and can be made of a single layer or multiple layers of transparent or opaque resin sheets made of various materials. When the transparent resin sheet is used, the cultured microorganism colonies can be counted from the outside.

  An injection sleeve 14 with a cock and a sealing plug 16 with packing are provided in an airtight manner at two arbitrary locations on the bag-like body 12. The cocked sleeve 14 has a structure in which the flow path can be closed and opened by manually rotating the cock 26.

  The sealing plug 16 with packing has a structure in which a cylindrical packing 28 is sandwiched between a hollow joint 30 and a cylindrical packing presser 32 and tightened by a screw method, as shown in C, for example. Here, the packing 28 is made of a material capable of airtightly inserting and removing a needle-like tube (injection needle or the like). For example, butyl rubber or the like is preferable. When the needle tube is inserted and then extracted, the puncture site is naturally reduced and blocked due to the flexibility and elasticity of the packing. Further, when the liquid is injected or taken out, the bag 12 is deformed and the internal pressure always matches the atmospheric pressure. By these, the airtightness of the flexible culture bag is maintained.

  FIG. 2 is a schematic view showing an example of the anaerobic culture test apparatus according to the present invention. This anaerobic culture test apparatus comprises a combination of the flexible culture bag 10 and the dispensing apparatus 40 as described above. The dispensing apparatus 40 includes a sealed container 42 having a rigid structure for containing a liquid therein, a connection portion 46 provided with a sealable valve 44 provided in the sealed container 42, and a sealed container continuously from the connection portion 46. A transfer pipe 48 reaching the lower part of the inside and an oxygen-free gas supply system 50 to the sealed container 42 are provided. And between the infusion sleeve 14 with a cock of the flexible culture bag 12, and the connection part 46 of the dispensing apparatus 40 are airtightly connectable / separable. “Rigidity” is not flexible and means that it does not deform at atmospheric pressure even if the internal pressure changes, and is made of, for example, glass or stainless steel.

  The inside of the sealed container 42 is anaerobic. By supplying oxygen-free gas from the oxygen-free gas supply system 50 and pressurizing the liquid surface, the liquid in the sealed container 42 can be injected into the flexible culture bag 10 while maintaining an anaerobic state. Whether or not the state is anaerobic may be determined by adding a redox indicator (for example, resazurin) to the liquid and observing the color change. By replacing the flexible culture bags one after another, the liquid in the sealed container can be easily dispensed into a large number of flexible culture bags. If a flow meter is provided at the connection portion, a predetermined amount can be easily injected.

  An embodiment of the anaerobic culture test apparatus according to the present invention is shown in FIG. The anaerobic culture test apparatus mainly comprises a combination of the flexible culture bag 10 and the dispensing device 40. Both can be connected and disconnected in an airtight manner.

  The flexible culture bag 10 has a bag-like body 12 made of a flexible sheet, an infusion sleeve 14 with a cock that is hermetically attached to the bag-like body 12, and a needle-like tube for injecting and aspirating liquid. It is the structure provided with the sealing stopper 16 with packing which can be stabbed and pulled out. This is the same as that shown in FIG.

  The dispensing device 40 includes a stainless steel sealed container 42 that contains a liquid therein. Here, the sealed container 42 includes a container main body 42a and a lid 42b, which are configured to abut each other via a seal member 52 at a flange portion 42c, and the inside can be sealed by tightening a fastener 54. It has a simple structure. In the sealed container 42, a pressure reducing port 60 and an oxygen-free gas supply port 62 are provided in the lid 42b. A valve 61 is provided at the decompression port 60, a vacuum suction pump (not shown) is connected, and a valve 63 is also provided at the oxygen-free gas supply port 62, and an oxygen-free gas supply system is connected (not shown). . As the oxygen-free gas, for example, any single gas such as hydrogen, nitrogen, carbon dioxide, or a mixed gas of two or more thereof is used. Further, a connecting portion 46 having a valve 44 that can be closed, and a transfer pipe 48 that continuously reaches the lower portion of the sealed container 42 from the connecting portion 46 are provided. The transfer pipe 48 is provided so as to penetrate the lid 42b in an airtight manner. A magnetic stirrer 68 is provided for stirring and mixing the liquid 66 in the sealed container. The magnetic stirring bar 68a is put in the sealed container 42, and the sealed container 42 is placed on the magnet stirrer main body 68b. In addition, although the pressure gauge 70 is provided in the decompression port 60 and illustration is abbreviate | omitted in the connection part 46, it is desirable to incorporate the flowmeter.

  Since the cocked sleeve 14 of the flexible culture bag 10 is airtightly connectable and detachable to and from the connecting portion 46 of the dispensing device 40, the anaerobic state is maintained by closing and opening the valve 44. However, it can be sequentially replaced with another flexible culture bag, and the liquid 66 in the sealed container 42 can be dispensed (sorted). Moreover, it can seal by turning the cock 26 of the sleeve 14 with a cock, and can isolate | separate from the dispensing apparatus 40 as it is. After that, by inserting a needle-like tube (such as an injection needle) into the sealing plug 16 with packing, while keeping the anaerobic state, chemicals, nutrients, microorganisms, etc. are injected from the outside in a solution state with a syringe. It is possible to take out a part of the solution inside.

This anaerobic culture test apparatus is particularly effective for, for example, a culture test for examining a concentration change by a bioremediation promotion technique (microorganism purification of soil or groundwater by organic chlorine compounds). An example of the operation method in that case is shown below.
(1) Collect groundwater in anaerobic condition. Therefore, anaerobic microorganisms are present in the collected water.
(2) The inside of the sealed container 42 is made anaerobic by repeated decompression and gas injection. That is, vacuum suction is performed from the decompression port 60 to decompress the inside of the sealed container 42, and then oxygen-free gas is injected from the oxygen-free gas supply port 62. By repeating these operations, the inside of the sealed container 42 is in an oxygen-free state (anaerobic state).
(3) Place the groundwater sample and various nutrients in a sealed container. For this purpose, the connecting portion 46 and the transfer pipe 48 may be used. Here, if necessary, the magnet stirrer 68 is driven to stir and mix the liquid in the sealed container. Whether or not an anaerobic state is present can be determined by adding a redox indicator. For example, when resazurin is used, it is red if oxygen is present, but is colorless if oxygen is not present. Therefore, it can be determined that the anaerobic state is obtained by visually confirming the discolored state.
(4) The flexible culture bag 10 and the dispensing device 40 are connected using the sleeve 14 with a cock and the connecting portion 46. The inside of the sealed container is pressurized to about 2 atm.
(5) Open the valve 46 connected to the flexible culture bag 10. Then, since the inside of the sealed container is pressurized, the liquid 66 in an anaerobic state in the sealed container is injected into the flexible culture bag 10 through the transfer tube 48 and the sleeve 14 with the cock. If a flow meter is installed, the injection volume can be measured.
(6) The valve 44 is closed and the cock 26 is also closed to separate the cocked sleeve 14 from the connecting portion 46. Thereby, the flexible culture bag 10 can be removed from the dispensing apparatus 40 in an anaerobic state after solution injection. Thus, by repeating the operations (5) to (6), the liquid in the sealed container can be dispensed (sorted) into a plurality of flexible culture bags.
(7) Since the flexible culture bag 10 is provided with a sealing plug 16 with packing, a solution (nutrient source, organic chlorine compound, etc.) can be obtained using a needle tube (such as an injection needle) pierced through the packing. A predetermined amount can be additionally injected.
(8) Many types of flexible culture bags having different contents such as ground water and organochlorine compounds to be tested are placed in a thermostatic chamber at 20 ° C., for example, and microorganisms are cultured. In addition, when it is necessary to stir the contents, it can be easily performed by bending or flexing the flexible culture bag.
(9) In the case of the bioremediation promotion test, a part of the contents is taken out from the flexible culture bag as a sample periodically (for example, once a day for several weeks), and the organochlorine compound in the solution is removed. Measure and record the mass balance (degraded material status). The flexible culture bag from which the sample has been extracted is returned again to a constant temperature bath at 20 ° C. and left to stand until the next sample is extracted. Thereby, the purification effect can be quantitatively determined.

  As described above, the anaerobic culture test apparatus according to the present invention can be used in various ways and forms according to the purpose of the culture test.

Explanatory drawing which shows the typical example of the flexible culture bag which concerns on this invention. Schematic which shows an example of the anaerobic culture test apparatus based on this invention. Explanatory drawing which shows the Example of the anaerobic culture test apparatus which concerns on this invention.

Explanation of symbols

DESCRIPTION OF SYMBOLS 10 Flexible culture bag 12 Bag-like body 14 Sleeve with cock 16 Sealing stopper with packing 18 Multi-layer material 20 Aluminum foil 28 Packing 40 Dispensing device 42 Sealed container 44 Valve 46 Connection part 48 Transfer pipe

Claims (4)

A flexible culture bag for performing a culture test of anaerobic microorganisms in groundwater and a dispensing device for injecting a liquid while maintaining the anaerobic state in the flexible culture bag. A bag-like body having a sealed structure and containing a liquid therein, an infusion sleeve with a sealable cock airtightly attached to the bag-like body, and a needle-like tube for injecting and aspirating liquid The dispensing device is provided with a sealing plug with packing that can be inserted and withdrawn, and the dispensing device includes a sealed container having a rigid structure for containing a liquid therein, and a connection part including a closeable valve provided in the sealed container When, the the transfer tube which continuously reaches the bottom of the sealed container from the connection portion has a free oxygen gas supply system to the closed container, before and cock injection sleeve of the flexible culture bag Between the connecting part with a valve of the dispensing device, the cock is freely air tightly connected and separated by the opening and closing of the valve, the water sampling groundwater in a sealed container, pressurization by anoxic gas thereby enabling dispensing the flexible culture bag while maintaining the anaerobic conditions depending on a feature that the ground water is a flexible culture bags dispensed, and can be separated from the dispensing device while maintaining the anaerobic conditions An anaerobic culture test device. The sealed container has a decompression port and an oxygen-free gas supply port at the top, a vacuum suction pump is connected to the decompression port, and an oxygen-free gas supply system is connected to the oxygen-free gas supply port. together to enable the anaerobic atmosphere of the sealed container by performing the oxygen-free gas supply alternately, anaerobic culture test device according to claim 1, wherein magnetic stirrer for agitation in the bottom portion of the sealed container is equipped. The anaerobic culture test apparatus according to claim 1 or 2 , wherein the flexible culture bag is made of a multilayer material in which a resin film is laminated on both sides so that an aluminum foil is located in the middle. The anaerobic culture test apparatus according to claim 3 , wherein the bag-like body is made of a multilayer material in which a cloth-like sheet made of a fibrous material is attached as an outer layer to reinforce the bending strength.

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