JPS59106289A - Multi-layered tool and device for inspecting culture of microorganism - Google Patents

Abstract

PURPOSE:To provide the titled inspection tool capable of separating and culturing bacteria, etc. easily from a specimen containing antibiotic substance, by laminating a water-absorbing member obtained by impregnation with a medium followed by drying, a filter member, and an agent for inactivating the antibiotic substance. CONSTITUTION:A medium for the microorganisms to be inspected is impregnated into a water-absorbing member 1 (e.g. cellulosic filter paper, nonwoven cloth, etc.), and dried. A filter member 3 having a pore diameter of about 0.22-0.75mu and made of a material inert to the specimen (e.g. polycarbonate, a cellulose ester, etc.) is placed on the water-absorbing member 1, and a coating layer 4 consisting of a substance capable of inactivating said antibiotic substance is attached to the filter member 3 to obtain the objective multilayered tool for the inspection of the culture of microoganisms. A substance to inactivate the antibiotic substance is selected from an adsorbent for the antibiotic substance (e.g. ion exchange resin), a chemically bonding agent (e.g. sodium amylosulfate) and a decomposition agent for the antibiotic substance (e.g. enzymes such as penicillinase) according to the kind of the specimen.

Description

Detailed Description of the Invention 10. Background of the Invention (Technical Field) The present invention is a method for separating and culturing microorganisms such as bacteria and fungi from a specimen, which simplifies the culture test operation even when the specimen contains antibiotics. It concerns instruments.

(Prior art and its problems) Conventionally, the culture procedure differs depending on the test material.1 For example, when testing blood bacteria, first, liquid enrichment medium and blood are mixed and cultured, and then blood agar is mixed and cultured. Separation culture is performed using separation media such as flat plates and chocolate agar plates. Thus, it is impossible to directly isolate microorganisms from specimens.

Since it requires an additional operation of enrichment culture, it is quite complicated and time-consuming. Furthermore, if the number of microorganisms in the sample was small, it was sometimes impossible to detect them.

Furthermore, in blood tests for patients suffering from sepsis, the antibiotics administered to the patient are included in the sample. Such antibiotics significantly inhibit the growth of microorganisms that should be detected during culture tests, adversely affecting test results. However, even if the device disclosed in U.S. Pat. Therefore, it has been desired to develop an inspection device that can perform such inspections.

10. Purpose of the Invention Therefore, 1. The purpose of the present invention is to provide a multilayer microbial culture in which microorganisms in a specimen can be collected directly by one mouth passage without requiring additional operations such as enrichment culture of microorganisms in the specimen, and can be isolated and cultured as they are. The aim is to provide testing tools and testing equipment equipped with the same.

Another object of the present invention is to provide a microbial culture test device configured so that an accurate test can be performed without being affected by antibiotics even when the sample contains antibiotics. We are trying to provide testing equipment.

L Specific structure of the invention The multilayer microorganism culture test device according to the present invention includes a water-absorbing member in which a medium suitable for culturing a predetermined microorganism is crushed and dried, and a water-absorbing member that is fixed on the water-absorbing member to collect microorganisms. A filter member having pores large enough to prevent the passage of water is laminated in a sterile state, and the upper surface of the filter member and/or the interface between the filter member and the water-absorbing member contains water that may be present in the sample. A substance that inactivates antibiotics is provided by a method such as passing down a foot. The water-absorbing capacity of the water-absorbing member is approximately equal to the amount of sample to be treated, and the amount of culture medium impregnated and dried in the water-absorbing member is such that when it is dissolved in the sample that has passed through the filter member during filtration, its concentration is substantially It is preferable to adjust the concentration to the concentration normally used for liquid media. The pore size of the filter member is preferably 0.22 to 0.75 microns, allowing liquid to pass through but microorganisms not passing through. The water-absorbing member and the filter member are laminated and fixed, but this fixation is achieved by using polymer fibers such as nylon treated to melt at relatively low temperatures, or water-absorbing polymer adhesives such as starch, cellulose derivatives, etc. Adhesion using a hydrous carbon adhesive, polyvinylpyrrolidone, or the like is preferable because it does not inhibit the flow of liquid between the filter member and the water-absorbing member.

Substances that inactivate antibiotics and do not inhibit the growth of microorganisms in the sample include substances that physically adsorb antibiotics, substances that chemically bind to antibiotics, and substances that degrade antibiotics. Can list substances.

The multilayer microbial culture test device of the present invention includes the multilayer microbial test tool described above, a container for supporting the test tool, and a support container for supporting the test tool at a predetermined height position from the bottom thereof. The support container includes an inspection container having an upper lid that can be attached to the support container, and the support container is composed of upper and lower halves separated at a predetermined height position, and a test tool is sandwiched between the upper and lower halves and can be mounted. It is better to configure. The test device and the support container can be attached to each other by fusing the constituent materials of the support container using ultrasonic sealing or heat sealing, or using a solvent or This can also be carried out by fusing the constituent materials of the support container with an adhesive obtained by dissolving the constituent materials of the support container in a solvent. and.

Preferably, the test container is transparent or translucent.

Next, a multilayer microorganism culture test device according to the present invention and a test device equipped with the same will be described in detail with reference to preferred embodiments shown in the accompanying drawings, taking blood culture as a representative example.

The multilayer microbial culture test device according to the present invention has a water-absorbing member 1 impregnated with and dried a medium suitable for culturing predetermined bacteria.
As described in the above-mentioned U.S. patent, filter members 3 having a large number of pores 2 large enough to prevent microorganisms from passing through are laminated and fixed together and sterilized by an appropriate method, which is known in the art (No.
(see figure), for specimens that may contain antibiotics, such as blood cultures from patients with sepsis.

As shown in FIG.

Alternatively, as shown in FIG. 3, it is preferable to form a coating 4 of a substance that inactivates antibiotics on the interface between the filter member 3 and the water-absorbing member 1. In addition, in FIG. 1, the coating 4 is simply shown.

The above-mentioned water-absorbing member may be made of any material as long as it can absorb an aqueous solution, but cellulose-based paper, nonwoven fabric, glass fiber, etc., which have rapid absorption ability, are preferable.

The filter member may be made of any material as long as it is inert to the sample, but typical examples include polycarbonate,
Examples include polyamide, cellulose ester, etc. Commercially available products include millibore membrane filters.
(Product name, manufactured by Millibore Corporation), Met 11 Cell (Product name, manufactured by Kerman Instrument Company)
, Sartorius membrane filter (trade name, manufactured by Carl Zeiss Corporation), TOYO membrane filter (trade name, Toyo Roshi ■), etc.

The pore size of this filter member is optimally 0.22 microns, which is able to filter even the smallest microorganism, Bacillus cerevisiae, if the function of treating all microorganisms is important, but the pore size of the filter member is Even if the microorganisms are larger than the target microorganisms, not all the microorganisms will pass through, so the microorganisms can be substantially captured, and the specimen can pass through the water-absorbing member quickly.
The thickness should preferably be 75 microns or less, preferably 0.45 microns or less.

In addition, the upper surface of the filter member, that is, the surface opposite to the water-absorbing material, may be coated with a hemolytic agent such as saponin in an amount of 3 to 5 tq per 1 of blood as a specimen, or heparin or An anticoagulant such as sodium citrate may be coated in an amount of 0.1 to 1 q per ml of blood as a specimen.

In this case, it is possible to omit the blood processing operation before starting the culture, and it is also possible to prevent clogging of the filter member.

Blood samples from patients suffering from septicemia usually contain antibiotics. When performing culture tests on such specimens,
If it inactivates the function of antibiotics.

As described above, more accurate inspection can be performed. Representative substances that inactivate the functions of antibiotics include the following:

(1) Antibiotic adsorbent (IL) Cation exchange resin polystyrene sulfonic acid type, phenol methylene sulfonic acid type, etc. These adsorb cationic antibiotics and inhibit their function.

(b) Anion exchange resins such as polystyrene amine type and phenol formaldehyde polyamine type, which adsorb anionic antibiotics and inhibit their function.

(2) Chemical binder (a) Sodium am
ylo-sulfate (SAS) Chemically binds to many antibiotics such as streptomycin, kanamycin, polymyxin B, and other aminoglucosides to inactivate their effects.

(b) Sodium polyanethole sulfate (3odium sulfate)
olyanethol 5ulfonate: SP
S) Chemically binds to and inactivates the action of antibiotics, similar to the case of SAS. However, in the case of sps, since the growth of some bacterial species is suppressed, gelatin is added to neutralize this.

(3) Degrading agent (a) Penicillinase Penicillinase, an antibiotic-degrading enzyme that specifically degrades penicillin, eliminates its action.

Cephalosporinase Cephalosporinase is a special enzyme that breaks down cephalosporinase antibiotics.

The medium impregnated with the water-absorbing material may be any medium suitable for the specimen, but preferably a medium in which anaerobic bacteria and aerobic bacteria grow through anaerobic culture and aerobic culture, respectively. In the case of III liquid culture, a typical composition example of a medium suitable for anaerobic and aerobic culture is shown in Table 1 below. In addition, add 60 to 700 gelatin to the medium as necessary.
It doesn't matter if 119 is added. Two multi-layered microbial culture test tools impregnated with such a medium can be prepared at the time of testing, and the sample can be poured into each and filtered, and one can be used for anaerobic culture and the other for aerobic culture, making it convenient. be.

Table 1 Example of composition of thin liquid culture medium (when filtering the sample in 5-) It is best to adhere the water-absorbing member impregnated with the culture medium and dried to the filter member by adhesion.
Polymer fibers made of nylon or the like that have been melted and vine-treated at a relatively low temperature, hydrophilic polymer adhesives such as starch, hydrated carbon adhesives such as cellulose derivatives, and polyvinylpyrrolidone (PVP') are suitable.

The shape of the multilayer microorganism culture test device of the present invention configured as described above is not particularly limited.

Most commonly used application devices such as petri dishes are circular, so it is most desirable to use a circular shape.

In addition, the size is not particularly limited, but for example, when dropping along the surface as a sample, the diameter should be 50 to 601111, and the thickness of the medium-immersed part should be 2 to 3. ■It is better to do so.

Further, the above-mentioned test tool is preferably mounted in a container having an appropriate shape and structure for convenient testing, and a representative example thereof is shown in FIGS. 5 and 6. As shown in the sectional view of FIG. 5, the test container to which the test tool is attached includes a support container 6 and an upper lid 8. A gap 9 for ventilation must be formed between the support container 6 and the upper lid 8 for culture purposes. The test tool is firmly attached to a recess 7 formed at a predetermined height from the bottom of the support container 6. The mounting method may be to fit the test tool into the recess 7, specifically, the support container 6.

It is composed of an upper half part 10 and a lower half part 11 with a recess as a boundary, and an inspection tool is held in the recess 7 between the upper half part 10 and the lower half part 11, and the upper and lower halves are fixed.

There are various methods of fixing the upper and lower halves 10 and 11 of the support container 6 while holding the test tool. For example, by fusing the rear half of the test tool held by the test tool using ultrasonic sealing or heat sealing, the sample can be fixed. It can adhere to microorganisms without any adverse effects.

A solvent that can dissolve the constituent material of the support container, or an adhesive in which a part of the constituent material of the support container is dissolved in this solvent, is applied between the upper and lower halves 10.11 to partially dissolve the constituent material of the support container. Can be melted and bonded. Of course, the above solvent must not inhibit the growth of microorganisms.
Methylene chloride, xylene and the like are particularly suitable.

In addition, the constituent materials of the support container 6 and the top lid 8 are transparent so that they can be observed from the outside of the container, have a certain degree of hardness, have good moldability, and are inexpensive. polystyrene, polyethylene, hard vinyl chloride,
Polycarbonate, methacrylic resin.

Examples include glass. Upper and lower halves of the container 10
When bonding and 11 by ultrasonic sealing method,
As shown in FIG. 8, the upper and lower halves 10 and 11 are provided with aligned downward circumferential flanges 13 and 14, respectively.

In addition, a vent hole 1 is provided in the lower half of the container 11 to allow air pushed out from the water-absorbing member when the sample liquid is poured into the container to escape to the outside.
It is preferable to form at least one number 2 (see FIGS. 5 and 7). In addition, as shown in FIG.
By providing a vent hole 12, the air can flow smoothly.
can lead to.

(6) Specific Functions of the Invention The method of using the multilayer microorganism culture test device shown in FIGS. 5 to 7 of the present invention will be explained by taking a liquid culture as an example. The specimen (thin liquid) is anticoagulated with an appropriate anticoagulant, and a solubility inhibitor such as saponin is added aseptically to cause total hemolysis of the red blood cells, which is then placed on the multilayer microbial culture test device of the present invention. dripping into. Microorganisms in the blood are separated by the filter member 3.

Other substances pass through the pores 2 of the filter member 3 and are adsorbed by the water-absorbing member 1, dissolving the culture medium impregnated and dried in the water-absorbing member. As mentioned above, the water-absorbing member 1 is configured to have an appropriate water-absorbing capacity and the culture medium is configured to have a concentration that is normally used by administering a sample of a predetermined species. The microorganisms separated onto the filter member are cultured on a medium under appropriate conditions.

This is cultured under appropriate culture conditions. This culture.

As mentioned above, prepare two specimens and two test devices.

The microorganisms cultured in this way, which are preferably carried out aerobically on one side and anaerobically on the other, form colonies 5 on the filter member 3 as shown in FIG. Furthermore, if a test tool such as that shown in FIG. 2 or 3 is used, even if antibiotics are contained in the blood, they will be adsorbed and removed, so that they will not adversely affect the culture of microorganisms.

(8) Specific Effects of the Invention As is clear from the above explanation, the multilayer microbial culture test device according to the present invention has many advantages as described below.

(1) There is no need for enrichment culture as in conventional culture tests, and direct isolation and culture is possible, and at the same time, the culture work is extremely simplified.

(2) Since the culture medium is immersed in a water-absorbing material and dried,
The stability of the medium is high, and the dissolution from a dry state to a normal state is always constant because the water absorption capacity of the water absorbing member and the amount of medium contained can be adjusted appropriately. and do not allow variations in culture conditions.

(3) Even if the concentration of microorganisms is small, detection is possible with the product of the present invention. (4) No special handling is required even if the sample contains antibiotics.

[Brief explanation of the drawing]

FIG. 1 is an exaggerated diagrammatic cross-sectional view of a known multilayer microbial culture test device, FIGS. 2 and 3 are diagrammatic cross-sectional views of a configuration example of the multilayer microbial culture test device of the present invention, and FIG. Diagrammatic sectional view showing the culture state of the multilayer microorganism culture test device of the present invention, No. 5
The figure is a diagrammatic cross-sectional view of the upper and lower halves of a container fused together by the multiple ultrasonic sealing method of the present invention. Explanation of symbols 1...Water-absorbing member% 2...Hole, 3...Filter member. 4...Antibiotic inactivating agent coating, 5...Microorganism (7) co=+, 6-+・Support container% 7...
Recess, 8-4 lid, 9... Gap % 10... Upper half % 11-- Lower half. 12...Vent, 13.14...Flange, 15-
...Radial groove patent applicant Terumo Corporation

Claims (1)

[Scope of Claims] (1) A water absorbent member impregnated with a medium suitable for culturing a predetermined microorganism and dried, and a water absorbent member coated on the water absorbent member,
A filter member having pores large enough to prevent microorganisms from passing through, and an antibiotic inactivator provided on the upper surface of the filter member and/or at the interface between the filter member and the water-absorbing member are sterilized. A multilayer microbial culture test device characterized by: (2) The multilayer microorganism culture test device according to claim 1, wherein the water absorbing capacity of the water absorbing member is approximately equal to the amount of the sample to be administered. (3) The amount of the medium to be impregnated and dried in the water-absorbing member is set so that when it is dissolved in the sample that has passed through the filter member during administration, its concentration is substantially the same as that normally used as a liquid medium. A multilayer microbial culture test device according to claim 2. (4) The pore diameter of the filter member is 0.22 to 0.75.
The multilayer microbial culture test device according to any one of claims 1 to 3, which is micron. (Group) The antibiotic inactivating agent is an antibiotic adsorbent. The multilayer microbial culture test device according to any one of claims 1 to 4, which is a chemical binder with antibiotics or an antibiotic decomposing agent. (6) The multilayer microbial culture test device according to claim 5, wherein the antibiotic adsorbent is a cation exchange resin or an anion exchange resin. (η the chemical binder is aminosulfate) 11 um or sodium polyanethole sulfate. (8) The multilayer microbial culture test device according to claim 5, wherein the decomposing agent is an antibiotic-degrading enzyme. (9) a water absorbent member impregnated with a medium suitable for culturing a predetermined microorganism and dried;
A filter member having pores having a size that does not allow microorganisms to pass therethrough; and an antibiotic inactivating agent layer provided on the upper surface of the filter member and/or at the interface between the filter member and the water absorbing member. a container for supporting the test tool, the test container having a support container that supports the test tool at a predetermined height position from the bottom of the support container, and a top lid that can be attached to the support container; A multilayer microbial culture test device characterized by: αO) The support container is configured to have upper and lower halves separated at the predetermined height position, and the test tool is sandwiched between the upper and lower halves so as to be mounted thereon.
The multilayer microbial culture test device described in section. 0υ The multilayer microorganism culture test device according to claim 10, wherein the test tool and the support container are attached by fusing the constituent materials of the support container by ultrasonic sealing or heat sealing. Q21 The test tool and the support container are attached by dissolving the constituent material of the support container and using a solvent that has little effect on microorganisms or an adhesive in which the constituent material of the support container is dissolved in the solvent. The multilayer microorganism culture test device according to claim 10, which is performed by fusing materials. α3) The multilayer microorganism culture test device according to any one of claims 9 to 12, wherein the test container is transparent or translucent. 04) The multilayer microbial culture test device according to claim 9, wherein the water absorbing capacity of the water absorbing member of the testing tool is approximately equal to the amount of the sample to be administered, (1) the water absorbing member of the testing tool Claim 9: The amount of the medium to be impregnated and dried is such that when it is dissolved in the sample that has passed through the filter member during administration, its concentration is substantially the same as that normally used as a liquid medium. The multilayer microbial culture test device described in section. α6) The pore diameter of the filter member is 0.22 to 0.75
The multilayer microbial culture test device according to claim 9, which is micron. α force The multilayer microbial culture test device according to claim 9, wherein the antibiotic inactivating agent is an antibiotic adsorbent, a chemical binder with antibiotics, or an antibiotic decomposing agent. The multilayer microbial culture test device according to claim 17, wherein the antibiotic adsorbent is a cation exchange resin or an anion exchange resin. 09) The chemical binder is aminosulfate, sodium chloride or sodium polyanethole sulfate. A multilayer microbial culture test device according to claim 17. G! Φ The multilayer microbial culture test device according to claim 17, wherein the decomposing agent is an antibiotic degrading enzyme.