JPH01312991A - Inspection apparatus for microorganism in liquid - Google Patents

Abstract

PURPOSE:To provide an apparatus for easy and sure inspection of microorganisms by providing a 1st member holding a filter for capturing microorganisms, a 2nd member for forming an inspection liquid receiving chamber, an air-introduction port for the introduction of outer air and attached with an air-filter, a covering member, etc. CONSTITUTION:A covering member 36 of an inspection apparatus for microorganisms is removed from a 2nd member 34, the 2nd member 34 is turned upside down in a state bonded to a 1st member 32 and a bonding wall 72 is bonded in the inverted state to the circumference of the mouth of a beer bottle 74 containing beer which is the liquid to be inspected. The above apparatus is turned upside down together with the bottle 74 to normal state and the lower end of the bottom ring 42 is supported via an O ring 87 of a supporting table 86. The specimen liquid in the bottle 74 is discharged through an inspection liquid receiving chamber 70, etc., outer air is introduced through the air filter 78 into the bottle 74 and the inspection liquid in the bottle 74 is completely discharged through a membrane filter 48. The 1st member 32 is removed from the bottle 74, etc., and a medium is poured on the member 32, covered with the covering member 36 and cultured at a prescribed temperature for a prescribed period. The inspection of microorganism is carried out by counting the number of colonies formed on the filter 48.

Description

DETAILED DESCRIPTION OF THE INVENTION [Field of Industrial Application] The present invention relates to a microbial testing device for confirming and quantifying microorganisms in liquids such as drinking water and alcoholic beverages.

[Background technology]

In recent years, the membrane tearing method has been widely used in the food, pharmaceutical, semiconductor, and other fields as a means of confirming and quantifying microorganisms such as bacteria, mold, and yeast contained in liquids such as beverages, injections, and water. .

Various types of devices and filtration forms have been proposed for use in this membrane filtration method, but basically they are as shown in FIG. That is, the suction flask 16 in which the filter 12 is set is connected to a vacuum filter, and the test liquid 18 is poured into the funnel 14 from a test tube 19 or the like to perform suction filtration. If necessary, wash and filter using sterile water, etc., then remove filter 1.
2 is taken out with tweezers 24 and placed on an absorbent pad 28 in a Petri dish soaked with a medium in advance, and then the colonies grown on the filter are confirmed and counted.

Screening for microorganisms by such a membrane filtration method is particularly frequently used to confirm and quantify microorganisms in a test solution with a dilute bacterial concentration, as the microorganisms may be concentrated and complemented by the membrane. The vacuum filtration method, which is often used to measure microorganisms, has simple equipment and operation, but it is difficult to perform the operation in a completely closed system, so there is a possibility of cross-contamination of microorganisms. always includes. For this reason, it is necessary to perform the filtration operation in a clean room or clean bench.

Regardless of the type of filtration, when testing is performed multiple times, the primary system, including the membrane, must be sterilized each time. In the case of the typical vacuum filtration system mentioned above, a funnel (liquid reservoir) 14 of about 300 m1 is used to hold the test liquid.
is placed on the primary side of the filter, and the test liquid is passed through the funnel 14.
After pouring into water, filter under reduced pressure. If the test is to be carried out many times, sterilized funnels 14 must be prepared for the number of tests.

As for cross-contamination, it is always necessary to pour the test liquid into the funnel once, or several times if the test liquid exceeds the capacity of the funnel, which increases the possibility of contamination of the test liquid.

In addition, after filtration, it is essential to remove the filter from the filter and place it on the culture medium in order to multiply the complemented microorganisms on the filter and perform counting measurements. Failure to do so may cause cross-contamination. When placing the filter on the medium, care must be taken to avoid air bubbles between the filter and the medium.

This is because the air bubbles prevent the diffusion of medium components from the medium to the filter surface, thereby preventing the normal growth of microorganisms on the filter surface.

In order to improve these drawbacks, a disposable sterilized filter unit is known in which a filter and a medium absorption band are set in a plastic container 29 having an entrance and exit, as shown in FIG.

With this product, after filtration, the culture medium can be poured into the absorption pad and cultured as is, so there is no need to remove the filter from the device, and since it is disposable and sterilized,
It is a simple device and is widely used for screening microorganisms.

However, as shown in Figure 8, the downstream side is an open system, and the structure is such that the test liquid is guided to the filter through a tube 30 that connects the filtration stock solution to the device, so there is no possibility of cross-contamination. It still exists. In addition, since the liquid culture medium is injected into the absorption pad from the outlet on the secondary side, it takes time to inject the culture medium into the absorption pad that is already moistened with the filtrate of the test liquid. Diluted with filtrate. Forcibly injecting with a syringe or the like can cause air bubbles to form between the pad and filter or damage the filter, so the structure is such that the injection is done slowly over time.

[Purpose of the invention]

The present invention has been made in consideration of the above-mentioned facts, and an object of the present invention is to obtain a microbial testing device that is free from cross-contamination and can perform testing reliably and easily.

[Summary and operation of the invention]

The present invention includes a first member that holds a microorganism-trapping filter, and a second member that is separably coupled to the first member and forms a test liquid inflow chamber together with the first member and the filter. , a test liquid container connecting portion provided on the second member and communicating with the test liquid inflow chamber, and a test liquid container connecting portion provided on the second member and communicating with the test liquid inflow chamber;
A test liquid inflow chamber/an air inlet with an air filter for guiding outside air, which is provided in the member, and a test liquid inflow chamber which can be connected to the second member and the first member and when the second member is removed. It is characterized by having a lid member for closing the lid.

The air inlet may be on the test liquid inflow side.

In the present invention, the lid member is removed from the second member, and the test liquid container connecting portion of the second member is connected to the test liquid container such as a beer bottle. In this case, the test liquid container is placed in an upright position with its spout facing upward, and its stopper has been removed in advance to open the spout.

When the test liquid container connecting portion is connected to the spout of a beer bottle or the like, the test device is turned upside down together with the test liquid container such as a beer bottle in this state. Then, the sample liquid such as a beer bottle enters the sample liquid inflow chamber due to its own weight and is discharged through the filter. However, due to atmospheric pressure, the liquid inside the beer bottle or the like may not be completely drained and the liquid may stop midway, or even if it is drained, it may take a long time, so the secondary side is reduced in pressure. As a result, the atmosphere enters the inside of the beer bottle or the like from the air inlet on the next side through the test liquid inflow chamber, so that back pressure acts on the test liquid and all of it is discharged through the filter.

Here, since living microorganisms are attached to the filter after being filtered, the test container is removed from the beer bottle, etc., and after separating the second member from the first member, a liquid medium is injected onto the filter. After the culture medium is uniformly distributed over the filter surface, a lid member is connected to this first member. Here, this device is incubated at a predetermined temperature for a predetermined period of time, and the amount of microorganisms is measured.

Preferably, the first member is provided with a sealing member that makes the joint part liquid-tight when the first member is joined to the second member and the lid member. As a result, when the lid member is fitted after injecting the liquid medium into the filter, the pressure inside the test liquid inflow chamber increases due to the piston effect, ensuring that the test liquid passes through the filter and the medium reaches the entire area of the absorption pad. I will cross.

In addition, by forming a ring groove with a narrow width toward the bottom of the groove on the outer periphery between the first member and the second member, by inserting an opening arm or the like into this ring groove, the first
and the second member are subjected to forces that separate them, which facilitates the application of equipment for automatically carrying out this microbiological test.

[Embodiments of the invention]

As shown in FIGS. 1 and 4, the testing device of this embodiment includes a first member 32 as a filtration container, a second member 34 as a sample liquid introduction part, and a lid member 36. These are cylindrical and coaxially arranged with each other.

The first member 32 has a bottom ring 42 and an intermediate ring 44, and a retaining plate 46 is integrally formed on the inner periphery of the axially intermediate portion of the bottom ring 42.

On this holding plate 46, a membrane filter 48 and an absorbent pad 50 are placed one on top of the other.

The filter 48 is of a type that is generally widely used for analyzing microorganisms, and the pore size is selected depending on the representative organism. The material is usually cellulose nitrate, with a pore size of 0.45 μm for bacteria, and a 0.8 μm pore size for molds and yeast. Colonies grown on filters produce various pigments depending on the type of microorganism, so colored filters are often used to make their identification easier.

The absorbent pad 50 is made of 100% cellulose pure filter paper because it is required to absorb the liquid medium well and be free of eluates that inhibit the growth of microorganisms. The above device is assembled as shown in FIG. 1 and is provided sterilized.

On the side where the absorbent pad 50 is mounted, a plurality of grooves 52 are formed radially and concentrically on the surface of the holding plate 46 to guide the sample liquid. Furthermore, a through hole 54 is formed in the axial center of the holding plate 46 for discharging the sample liquid. A sleeve 56 protrudes from the holding plate 46 coaxially with the through hole 54 on the opposite side of the absorbent pad 50, and a check valve 58 is attached to the sleeve 56. This check valve 58 is formed into a cap shape from a flexible synthetic resin material, and has a notch 58A formed in the center. For this reason, the liquid passes from the top to the bottom in Figure 1, but when the liquid tries to move from the bottom to the top, the pressure of this liquid causes the cut 5.
8A is brought into close contact to perform a check function.

This check valve 58 may be a simple cap without the notch 58A, but in this case it will be removed when filtering the test liquid.

A ring-shaped protrusion 4 is provided on the outer circumference of the bottom ring 42 at an axially intermediate portion.
A ring-shaped projection 43A is also formed at one end in the axial direction, which is the upper end in FIG.

This ring-shaped protrusion 43A has a tapered shape whose width becomes narrower toward the tip.

The intermediate ring 44 is press-fitted into the inner periphery of the upper end of the bottom ring 42, and the lower end thereof holds a filter 48 between it and a retaining plate 46 to hold the filter 48 and the absorbent pad 50 in the first
It is fixed to the member 32 of.

Directly attach the bottom ring 4 around the filter 48 by ultrasonic welding etc.
2, the middle ring 44 is fixed to the bottom ring 42.
The structure does not have to be inserted deeply into the body.

A ring-shaped protrusion 44A is formed on the outer periphery of the intermediate ring 44 at an axially intermediate portion. This ring-shaped protrusion 44Δ
Similarly to the ring-shaped protrusion 43A, the protrusion has a tapered shape in which the width gradually decreases toward the tip. In addition, a rectangular groove is formed on the outer periphery of the lower end of the intermediate ring 44.
A ring 62 is retained.

The second member 34 is provided with an enlarged diameter portion 34A at its lower end, and is dimensioned such that the upper end of the intermediate ring 44 fits into this enlarged diameter portion 34A. Therefore, the inner periphery of the enlarged diameter portion 34A and the outer periphery of the intermediate ring 44 are in a liquid-tight state. A ring-shaped protrusion 34B is also formed on the outer periphery of the lower end of this enlarged diameter portion 34A, and the width dimension gradually decreases toward the tip. Therefore, between the ring-shaped projection 43A and the ring-shaped projection 44A and between the ring-shaped projection 44A and the ring-shaped projection 34B are formed as ring grooves 64, 66, and an opening arm is inserted between these from the outside. Then, a force is generated that separates the bottom ring 42 and the intermediate ring 44 and the intermediate ring 44 and the second member 34 from each other in the axial direction, so that they can be easily disconnected and separated.

A retaining plate 68 is integrally formed on the inner periphery of the axially intermediate portion of the second member 34, and a through hole 68A is formed in the axial center portion. This holding plate 68 defines a test liquid inflow chamber 70 together with the second member 34, the intermediate ring 44, and the holding plate 46. The test liquid is inflowed through the through hole 68A, passes through the filter 48, and the check valve 58 It is designed to be discharged from

A coupling wall 72 having a larger diameter than the through hole 68A protrudes from the holding plate 68 in a cylindrical shape toward the opposite side of the test liquid inflow chamber 70. Is this joint wall 72 a protrusion on the inner periphery of the axially intermediate portion? 2A is formed so that an external pouring spout of a beer bottle 74 (see FIG. 5), which is a test liquid container, can be connected. For this reason, materials such as PE and PP that can be appropriately expanded and contracted are used. If the external spout of the test liquid container has a male thread, a female thread may be formed on the inner periphery of the connecting wall 72 to be screwed therewith.

Also, an O-ring 76 is attached to the inside of the connecting wall 72 on the retaining plate 68.
are arranged so that a liquid-tight state can be maintained between the spout of the combined beer bottle 74 and the holding plate 68.

An air filter unit 78 having an air inlet on the outer periphery is coupled to the second member 34 . That is, this air filter unit 78 has an air filter (PTF) inside.
A membrane filter made of an aqueous material with a pore diameter of 0.5 μm such as E or polypropylene is preferably arranged, and the sleeve 78A is inserted into the introduction hole 82 in the thick wall portion 68B formed in the radial direction of the second member 34. has been done.

The leading end of the introduction hole 82 opens near the through hole 68Δ, and has the role of introducing outside air into the beer bottle 74. If this air filter is hydrophilic, when the filter becomes wet with the test liquid, gas cannot pass through unless the differential pressure increases above the bubble point pressure of the filter.

Normally, the bubble point pressure of a filter with a pore size of 0.5 μm used for this purpose is around 2 kg/cffI, so that gas such as atmospheric air cannot be introduced in normal vacuum filtration.

In addition, if this water-repellent material air filter has a pore diameter of 0.5 μm, if it has an effective filtration area of about 0.5 cut,
Sufficient air introduction allows rapid filtration.

Further, the second member 34 has a ring-shaped protrusion 34C protruding from the outer periphery of the axially intermediate portion, and corresponds to the ring-shaped protrusion 36A formed at the lower end of the lid member 36. That is, these ring-shaped protrusions 34C136A have opposing surfaces that are sloped to form a ring groove 84, and the ring groove 84, like the ring grooves 64 and 66, has a groove width that gradually becomes narrower toward the groove bottom. There is. Therefore, when an opening arm (not shown) is inserted into the ring groove 84, the lid member 36 is easily separated from the second member 34.

The inner diameter of the lid member 36 is the enlarged diameter portion 34A of the second member 34.
It is assumed that the inner diameter is the same as that of the Therefore, as shown in FIG. 3, after the second member 34 is removed from the first member 32, the lid member 36 can be fitted into the upper end of the intermediate ring 44. In this case as well, the lid member 36
An O-ring 62 maintains a liquid-tight state between the inner periphery and the intermediate ring 44 .

A lid plate portion 36B is formed at the upper end of the lid member 36, and as shown in FIG.
6 is in a connected state, the inside of the second member 34 is closed. Moreover, this cover plate part 36B is made of transparent material (PS, AS, P
It is preferable that the third material is made of
In the state in which it is connected to the intermediate ring 44 shown in the figure, microorganisms on the filter 48 can be observed through the cover plate portion 36B.

Furthermore, a recess 36C is formed at the upper end of the lid plate part 36B, into which the lower end of the bottom ring 42 disposed above enters, as shown by the imaginary line when a plurality of inspection devices are stacked one above the other. It is now possible to do so.

Next, a procedure for testing live yeast in a beer bottle using the testing device of this embodiment will be described.

In the state shown in FIG. 1, the automatic inspection machine removes the lid member 36 from the second member 34. In this case, the lid member 36 can be easily removed by inserting an arm (not shown) into the ring groove 84. After being removed, the lid member 36 is held at another location by this automatic inspection machine.

In the state where the second member 34 and the first member 32 are combined, the fifth
As shown, the test device is inverted into an inverted position, and the bonding wall 72 is bonded to the spout outer layer of the beer bottle 74 after the stopper has been removed. The crown frame portion of the beer bottle 74 has been sterilized in advance using a chemical, flame, or the like.

As shown in FIG. 6, this inspection device is turned upside down again together with the beer bottle 74, and the bottom ring 42 is placed in the royal state.
The lower end portion of is supported on a pedestal 86 via an O-ring 87. This pedestal 86 has a liquid medicine inner hole 88 formed at its axial center and communicates with a pipe 92 . For this reason beer bottle 74
The test liquid in the beer bottle is discharged through the test liquid inlet chamber 70 and the check valve 58 due to the internal pressure caused by the carbonic acid in the beer liquid. When the negative pressure source starts suctioning, outside air enters the beer bottle 74 through the air filter unit 78 and creates a back pressure.As a result, all the sample liquid in the beer bottle 74 is discharged through the filter 48.

After filtration by the filter 48 is completed, the pressure reduction is stopped, and after the pressure returns to normal pressure, both the beer bottle 74 and the second member 34 are removed from the first member 32.The upper part of the first member 32 is thereby opened. After injecting a medium (not shown) from above, the lid member 36 held elsewhere is placed over the upper end of the intermediate ring 44, as shown in FIG.

In this case, since the gap between the lid member 36 and the intermediate ring 44 is made liquid-tight by the O-ring 62, when the lid member 36 is pushed down from the state shown in FIG. 2 to the state shown in FIG. The test liquid remaining in the absorbent pad 50 is removed to avoid dilution of the medium, and the medium is permeated into the absorption pad 50.

In this state, culture is performed at a predetermined temperature for a predetermined period of time, and the filter 4
The test is performed by counting the number of colonies on 8. In this case, the inspection is performed on the transparent lid plate portion 36B of the lid member 36.
Alternatively, the lid member 36 may be removed for direct observation.

In order to prevent the first member 32, second member 34 and intermediate ring 44 from accidentally separating from each other or leaking liquid during operation, the ring-shaped protrusions on the outer periphery are held by an automatic inspection machine,
Special retaining means may be considered, such as sealing the outer periphery of the joint with adhesive tape. In addition, the first and second members 32
．． 34 and the intermediate ring 44 are also prevented from disengaging by axially compressing them together.

As described above, in the microbial test according to this embodiment, the apparatus can be handled entirely automatically, and the test liquid can be directly filtered from a container such as a beer bottle, eliminating the need for pouring the test liquid into a funnel or the like.

Therefore, since the filtration operation itself is a closed system, there is no need to worry about cross-contamination. Furthermore, there is no need to use sterilized funnels to administer doses for the number of tests.

Note that the shape of the connecting wall 72 can be changed in various ways in order to connect it with other glass containers, metal containers, etc. instead of the beer bottle 74. Further, instead of the O-ring 76 used in the above embodiment, an elastomer such as a packing may be used.

〔Effect of the invention〕

Since the present invention has the above-mentioned configuration, it has excellent effects in that microbial testing is reliable and simple, and there is no risk of cross-contamination.

[Brief explanation of the drawing]

FIG. 1 is a vertical cross-sectional view along the axial direction showing a test container according to an embodiment of the present invention, and FIG. 2 is a cross-sectional view showing a state in which the lid member starts to be connected to the intermediate ring after the second member is removed. Figure 3 is a sectional view showing the state in which the lid member is securely connected to the intermediate ring, Figure 4 is an exploded perspective view showing each part of this embodiment, and Figure 5 is a beer bottle used for inspection of this embodiment. FIG. 6 is a side view showing a state in which the device is connected; FIG. 6 is a side view showing a state in which membrane filtration is being performed to remove a test liquid from a beer bottle; FIGS. 7 and 8 are conventional testing means and methods. FIG. 32... First member, 34... Second member, 36... Lid member, 42... Bottom ring, 44... Intermediate ring, 48... Filter, 64.66... -Ring groove, 70...Test liquid inflow chamber, 74...Beer bottle, 78...Air filter unit, 84...Ring groove.

Claims (3)

[Claims] (1) a first member that holds a microorganism-trapping filter; a second member that is separably coupled to the first member and forms a test liquid inflow chamber together with the first member and the filter; A test liquid container connecting portion provided on the second member and communicating with the test liquid inflow chamber, and an air filter provided on the first member or the second member to guide outside air to the test liquid inflow chamber. A method for detecting liquid in a liquid, characterized by having an air inlet, and a lid member that is connectable to the second member and the first member and that closes the test liquid inflow chamber when the second member is removed. Microbiological testing equipment. (2) The device for testing microorganisms in a liquid according to claim (1), wherein the first member is provided with a sealing member that makes the joint part liquid-tight when combined with the second member and the lid member. . (3) The device for testing microorganisms in a liquid according to claim (1), wherein a ring groove is formed in the outer periphery between the first member and the second member, the ring groove having a narrow width toward the groove bottom. .