JP2000262271A - Culture unit - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a culture unit capable of conducting the classified collection of target microorganisms and their culture simultaneously, leading to dispensing with multistep culture operations for testing microorganisms and thereby cutting the test time. SOLUTION: This culture unit is such one as to be characterized by carrying a culture medium containing active ingredients for growing microorganisms on a porous form 2 with an antibody capable of specifically binding microorganisms as object thereto bound to the surface and culturing them after capturing and dispersing them in the interior of the resulting antibody-modified porous form.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

[0001] The present invention relates to the culture of microorganisms.

[0002]

2. Description of the Related Art Generally, a liquid submerged culture method is used for culturing microorganisms. Liquid submerged culture is a culture method in which nutrients and dissolved oxygen are efficiently supplied by shaking a culture flask. In clinical tests, an agar culture method is often used. Culture in microbial testing requires several stages of culture. That is, pre-enrichment cultivation, enrichment cultivation and separation cultivation, and at least three stages of cultivation are required to test one bacterium. Some bacteria also require several stages of culture.

[0003]

In a microorganism test, a target microorganism must be selectively cultured, so that several stages of culture are required as described above. In the culture at each stage, since a medium containing a component that suppresses the growth of microorganisms other than the target is used, the target microorganism can be selectively cultured. However, the inspection requires a considerable number of days. For example, Salmonella requires 2-3 days for culture. Furthermore, since the liquid submerged culture method performed at each stage requires means such as shaking and stirring, an apparatus using this culture method becomes large and complicated. Furthermore, since a liquid medium is handled, caution is required in the operation of injecting and extracting the bacterial solution sample. In addition, when a liquid containing microorganisms is transferred, there is a risk that the handler and the environment will be contaminated by microorganisms. On the other hand, even in the agar culture method used in the stage of separation culture in the microbial test, the contact between the medium and the microorganism is flat, so that the growth of the microorganism is suitable,
When adding microorganisms, it is necessary to perform an operation of spreading and applying thinly. Skilled techniques are required for both liquid submerged culture and agar culture.

[0004]

Means for Solving the Problems To solve the above problems, the present invention provides a porous body having an antibody that specifically binds to a target microorganism on a surface thereof, and a component effective for the growth of the microorganism. The present invention provides a novel culture apparatus for supporting a medium contained therein, dispersing microorganisms inside the antibody-modified porous body, and culturing the same. With this device, the target microorganisms specifically bind to the antibody on the surface of the porous body at the sample sampling stage, so that the microorganisms can be separately collected in the porous body. The antibody-modified porous body is more desirably composed of an elastic material. Thus, the exchange between the sample medium and the liquid medium can be performed by one apparatus. here,
If the medium added after the initial sample volume is reduced to a small amount, the microorganisms are concentrated during this operation, and the culture time can be shortened by increasing the initial concentration.

The present invention comprises an antibody-modified porous body and a case for holding the porous body, and a part of the case has an openable / closable opening so that a sample or a medium can be taken in and out of the opening. Provided is a configured culture device. Thereby, the danger in handling the bacterial solution sample can be reduced. In addition, by providing a region for identifying the culture state of the microorganism in the case, the degree of proliferation can be assayed without extracting a bacterial solution.

[0006] It is preferable that the case has means for pushing out the contents contained in the porous body or the porous body itself to the outside of the case, or sucking the contents from outside the case. This makes it possible to easily and safely obtain the bacterial solution after culturing, and to provide it for inspection work. It is preferable that an agent having a bactericidal action is held in the case, and the agent is brought into contact with the porous body to kill microorganisms after culturing. This makes the operation more secure,
In addition, concerns about environmental pollution after use can be prevented.

[0007]

BEST MODE FOR CARRYING OUT THE INVENTION The porous material used in the present invention is made of a material whose kind, material, or pore size satisfies the necessary and sufficient conditions for culturing by dispersing microorganisms. The types of materials constituting the porous body vary widely. Assuming that the definition of the porous body has pores, a foam having three-dimensionally connected pores, such as zeolite or sponge, a woven or nonwoven fabric of fibers, having a capillary effect, such as It is like paper, cotton or cloth. As the material of the porous body, a material having high hydrophilicity is effective because the culture solution of the microorganism is an aqueous solution, and examples thereof include cellulose, chitin, chitosan, glass, polyvinyl alcohol, and derivatives thereof. Also,
Even in the case of non-hydrophilic plastics and metals, it is effective to use a material that has been made hydrophilic by applying a chemical or plasma treatment to the surface. The pore diameter of the pores of the porous body is
It satisfies at least the necessary and sufficient conditions for dispersing microorganisms. Specifically, when the target is Escherichia coli, a structure having pores having a pore diameter in the range of 10 μm to several mm is desirable. The above purpose is realized by the following configuration.

[0008] In a preferred embodiment of the present invention, the antibody bound to the porous body is made to stay on the porous body surface against the flow of water when a sample is taken in and out. Most conveniently, the antibody may be physically adsorbed on the porous body. Further, preferably, an amino group or a hydroxyl group,
Alternatively, the antibody is chemically bonded to the porous body using a chemically active functional group such as a carboxyl group. In this case, the porous body can be used repeatedly.

In another embodiment, the antibody-modified porous body is made of a resilient material. Thus, for example, the porous body is placed in a syringe-like container, and an aqueous sample is first sucked by the operation of the piston, and the microorganisms are adsorbed on the surface of the porous body via the antibody. The microorganisms can then be extracted and captured from the sample by compressing the piston. After this, by the operation of the piston,
By inhaling a medium optimized for the growth of microorganisms,
The exchange between the sample medium and the liquid medium is made possible with one device. For this purpose, it is more preferable that the inside of the porous body has such a property that moisture moves at a speed higher than that of microorganisms. Here, if the medium added after the initial sample volume is reduced to a small amount, the microorganisms are concentrated during this operation, and the culturing time can be shortened by increasing the initial concentration.

In still another embodiment, microorganisms to be cultured, for example, bacteria such as salmonella, vibrio parahaemolyticus, staphylococcus, pathogenic Escherichia coli, botulinum, cereus, or Welsh, which are food poisoning bacteria, or viruses, An antibody of a microorganism that specifically binds to Rickettsia or the like is bound to the surface of the porous body. In another embodiment, by sampling a sample on the antibody-modified porous body, only microorganisms that specifically bind to the antibody are captured in the porous body, and impregnated with a medium,
Only the target microorganism can be cultured.

[0011] In a preferred embodiment of the present invention, the type, material, or pore size of the porous body modified with the antibody satisfies the necessary and sufficient conditions for dispersing and culturing microorganisms. It is made of elastic material. Assuming that the definition of porous body has pores, a foam having three-dimensionally connected pores,
A transition woven or nonwoven fabric with a capillary effect. As the elastic material, since the culture solution of the microorganism is an aqueous solution, a material having high hydrophilicity is effective, for example, cellulose or a derivative thereof,
Even for non-hydrophilic materials, those whose surfaces have been rendered hydrophilic by chemical treatment or plasma treatment are effective. The pore diameter of the pores of the porous body satisfies at least a necessary and sufficient condition for dispersing microorganisms. Specifically, if the subject is E. coli,
A structure having pores with pore diameters ranging from micron to several mm is desirable. By using the antibody-modified porous body having elasticity, it is possible to extract only the medium after sampling the sample and then to inhale the liquid medium. By using the antibody-modified porous body having elasticity as described above, only the liquid can be exchanged.

In another embodiment, the antibody-modified porous body contains a medium that provides an environment suitable for the growth of microorganisms or a medium that promotes or suppresses the growth of microorganisms.
In still another embodiment, after capturing a target microorganism using any of the antibody-modified porous bodies, the microorganism is impregnated and cultured in a container such as a test tube or a petri dish.

[0013] In another embodiment, the porous body is sealed in a case made of, for example, plastics, and has a structure in which there is no risk of infection to a user in a culture process and microorganisms do not go outside. In addition, the structure does not allow foreign substances to enter. In another embodiment, the case is structured to facilitate sample sampling. That is, the case has an openable / closable opening so that the sample can be impregnated from the opening into the antibody-modified porous body in the case. Alternatively, the antibody-modified porous body is exposed at the opening, and the exposed part is immersed in the sample, or by wiping the sample attached to the holder,
Sampling can be performed.

In still another embodiment, a porous body impregnated with a medium and an antibody-modified porous body for culturing are placed in a case, and a sample or a medium is circulated between the porous bodies. Then, a filter is provided in a part of the circulation path, and by circulating the sample or the medium, it is possible to remove large contaminants higher than microorganisms in the sample.

In another embodiment, the case is provided with a transparent region so that the porous body can be observed from the outside. When identifying the growth or detection of microorganisms, the bacterial solution after cultivation or the antibody-modified porous body containing this bacterial solution,
It can be identified in a transparent area of the case without taking it out of the case. Alternatively, an openable door is provided in the case, and when the door is opened, the antibody-modified porous body is visible. Further, in order to facilitate identification of the growth or detection of the microorganism, a reagent capable of easily determining the growth of the microorganism, for example, an antibody-modified porous body containing a luminescent reagent or a staining reagent is present separately. Alternatively, the color of the antibody-modified porous body is a color that facilitates identification of the growth or detection of microorganisms. In other embodiments, not only identify the inside of the case, but directly from it,
The identification area is openable and closable so that the bacterial solution after culturing or the antibody-modified porous body containing the bacterial solution can be taken out.

In another embodiment, after the sample is sampled, the powdered medium is dispersed in the antibody-modified porous body so that the inside of the antibody-modified porous body inside the case can be efficiently placed in an environment suitable for the growth of microorganisms. Have been. Alternatively, the liquid medium is in a state of permeating the inside of the antibody-modified porous body. Alternatively, a capsule packed with a powdered medium is inside the antibody-modified porous body. The capsule referred to here is, for example, a capsule medicine used for pharmaceuticals and the like, and the material is desirably water-soluble.
The size or number of the capsules is such that they can fit inside the porous body to be cultured, and when water is contained in the porous body, the capsules dissolve and are necessary and sufficient to impregnate the porous body with the medium. Size or quantity that satisfies various conditions.

[0017] In still another embodiment, the case has a structure capable of extracting a bacterial solution after culturing in the antibody-modified porous body inside the case, such as a syringe-like structure. In another embodiment, the structure is such that the microorganism is killed by the drug as the treatment after the culture. For example, a structure in which a surfactant impregnated with a surfactant is incorporated therein and brought into contact with the antibody-modified porous body after culturing, so that the surfactant can be sent to the antibody-modified porous body. Alternatively, there is a surfactant in a glass sealed tube, and the structure is such that the antibody-modified porous body after culturing can be immersed in the surfactant by breaking the glass sealed tube. In another embodiment, a function for controlling the temperature of the culture is attached, for example, a Peltier device is contained in a case.

Next, some embodiments will be described with reference to the drawings. The culturing apparatus shown in FIG. 1 comprises a plastic syringe type case 1 and a cellulose porous body 2 placed under the case. An antibody to which a target microorganism specifically binds is bound to the porous body 2 and is impregnated with a medium. In the case 1, a piston 3 with a rubber plate 6 is inserted above the antibody-modified porous body 2. A sample injection port 4 is provided at the center of the piston 3, and the injection port is sealed with a cap 5 except when a sample is injected. The lower part of the case 1 has a sample extraction port 7 which is always closed with a cap 8. In order to culture microorganisms using this apparatus, first, the cap 5 is opened, the microorganisms are introduced from the injection port 4, and the microorganisms are captured by the antibody-modified porous body 2. Next, the culture medium is inhaled and the culture is impregnated into the antibody-modified porous body. Then, the culture is carried out under culture conditions, for example, at a predetermined temperature. After the cultivation, the porous body 2 is compressed by the piston 3 to extract the microorganism after the growth from the extraction port 7.

The culturing apparatus shown in FIG. 2 has a case 11 having an opening at the lower end and an antibody-modified porous body 12 with its tip protruding outside through the opening. Sampling can be performed by immersing the tip of the porous body in the sample or wiping the sample attached to the holder. After sampling the sample in this way, a cap 13 having a size to cover the protruding portion of the porous body is fitted to the tip of the case 11.
The sample is cultured in this state.

The culture apparatus shown in FIG. 3 is a combination of a case 21 containing an antibody-modified porous body 25 and a case 29 containing a porous body 28 carrying a medium or a bactericide. The case 21 is provided with a piston 22 having a sample injection port 31, a rubber plate 24 attached to the lower end thereof, and an extraction port 26 for extracting a sample after culturing. On the other hand, a piston 23 having a rubber plate 30 is fitted into the case 29, and a nozzle 27 is provided at the tip. This nozzle 23
Can be inserted into the opening 32 provided in the case 21 and combined as shown in the figure, and the medium or the germicide can be injected from the case 29 into the case 21. That is, the medium or the germicide impregnated in the elastic porous body 28 is added to the antibody-modified porous body 25 in the case 21 from the nozzle 27 by compressing the porous body 28 with the piston 23.

The culture apparatus shown in FIG.
, A partition wall 4 having fine pores
This is an example in which a case 42 accommodating a porous body 45 is combined with a case 6. A rubber plate 44 and a piston 41 are inserted into the case 42, and a culture medium or a bactericide impregnated in the porous body 45 is appropriately passed through the pores of the partition wall 46 to the antibody-modified porous body 47 by operating the piston 41. Can be added. Reference numeral 48 denotes an extraction port, which is always sealed with a cap.

[0022]

EXAMPLE An example of culture using an antibody-modified porous body will be described. A sponge made of cellulose was used for the porous body. The cellulose porous body (51) was reacted with 3-aminopropyltriethoxysilanate (52) to introduce an amino group. Furthermore, carboxyl diimidazole (5
In the presence of 3), the mixture was reacted with an anti-Escherichia coli antibody (54) to obtain a porous body (55) made of an anti-Escherichia coli antibody-modified cellulose (FIG. 5). This was placed in a syringe type case having the structure shown in FIG. 1, and Escherichia coli was actually cultured. First, the cap 5 was opened, Escherichia coli solution was injected from the inlet, and Escherichia coli was captured by the anti-Escherichia coli antibody-modified porous body 2. Subsequently, the medium was inhaled, and the porous body was impregnated with the medium. In this state, the cells were cultured at 37 ° C. for 8 hours. As a result, it was confirmed that E. coli was grown.

[0023]

According to the antibody-modified porous body of the present invention, it is possible to simultaneously carry out the separate collection and cultivation of the target microorganism, eliminating the necessity of performing several stages of cultivation in the microbial test. It contributes to shortening. In addition, by providing additional functions such as a combination of the antibody-modified porous body and the case and a piston, a series of operations in culturing, such as sampling, sorting, culturing, and extraction, can be performed by one apparatus. There is no danger such as contamination due to the use of the device, and it becomes a culture device for general use, and greatly contributes to the field of microbial research or microbial testing. Further, the present invention can be applied to a home inspection system.

[Brief description of the drawings]

FIG. 1 is a longitudinal sectional view of a syringe-type culture apparatus according to one embodiment of the present invention.

FIG. 2 is a longitudinal sectional view of a culture apparatus according to another embodiment of the present invention.

FIG. 3 is a longitudinal sectional view of a culture apparatus according to still another embodiment of the present invention.

FIG. 4 is a longitudinal sectional view of a culture apparatus according to still another embodiment of the present invention.

FIG. 5 is a diagram showing a reaction process for producing a porous body modified with an anti-Escherichia coli antibody.

[Explanation of symbols]

 DESCRIPTION OF SYMBOLS 1 Case 2 Porous body 3 Piston 4 Injection port 5 Cap 6 Rubber plate 7 Extraction port 8 Cap

──────────────────────────────────────────────────の Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) // (C12N 1/20 C12R 1: 185) (72) Inventor Hitoshi Miyazaki Kazuma 1006 Kadoma, Kadoma City, Osaka Address Matsushita Electric Industrial Co., Ltd. F term (reference) 4B029 AA07 BB02 CC04 CC10 FA01 4B033 NA02 NA12 NA43 NB45 NB62 NB68 NC03 NC04 NC12 ND06 ND12 NE02 NF03 NF06 4B065 AA26X BC01 BC42 BD13 CA46

Claims (11)

[Claims] 1. A medium containing a component effective for growth of a microorganism is carried on a porous body having an antibody to which a target microorganism specifically binds bound on the surface thereof. A culture device for capturing and dispersing and culturing the same. 2. A porous body having elasticity, an antibody bound to the porous body and specifically binding a microorganism of interest, and a component carried inside the porous body and effective for growth of the microorganism. A medium, and a case for holding the porous body therein; a part of the case having an openable / closable opening; and a sample or a medium subjected to a test for the presence or absence of microorganisms is taken in and out of the opening. Culture device configured as described above. 3. The culture apparatus according to claim 2, wherein the interior of the antibody-modified porous body having elasticity has such a property that water moves at a higher speed than the microorganism. 4. A method for capturing only microorganisms contained in an aqueous sample by expanding and contracting an elastic antibody-modified porous body,
The culture device according to claim 2, wherein the environment in which the microorganisms grow can be replaced by separately sucking a medium effective for growing the microorganisms. 5. The medium according to claim 1, wherein the medium contains a component that promotes or suppresses the growth of a specific microorganism.
The culture apparatus according to claim 1. 6. The culture apparatus according to claim 2, wherein an antibody-modified porous body is arranged facing the opening of the case, and a sample and a medium are taken in and out through the antibody-modified porous body. 7. The culture apparatus according to claim 2, wherein the case includes an area having means for identifying a culture state of the microorganism. 8. The culture apparatus according to claim 1, wherein the antibody-modified porous body or a medium carried on the antibody-modified porous body is impregnated with a reagent for identifying the degree of growth of microorganisms. 9. The culture apparatus according to claim 1, further comprising at least two antibody-modified porous bodies, and means for circulating at least one of a medium and a sample between the two antibody-modified porous bodies. 10. The culture apparatus according to claim 2, wherein the case has a means for pushing out the contents contained in the antibody-modified porous body or the porous body itself out of the case, or sucking the contents from outside the case. 11. The culture apparatus according to claim 2, wherein a drug having a bactericidal action is held in the case, and a means for killing microorganisms after culturing by bringing the drug into contact with the porous body is provided. .