JPH0555105B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a novel blood bacteria isolation and incubator used for separating and culturing bacteria in blood.

Bacteria are present in the patient's blood in serious systemic infections such as sepsis and bacteremia, so a blood bacteriological test is performed to diagnose these infections. Bacteria testing of blood is also carried out to determine the efficacy of antibacterial agents through animal testing. In these bacterial tests, it is first necessary to culture and isolate bacteria from sample blood, and then drug susceptibility tests and bacterial identification are performed. The instrument of the present invention is used for specimen processing during such bacterial testing.

(Prior Art and Problems) Conventionally, blood bacteria are cultured and isolated by mixing collected blood with a liquid nutrient medium and culturing the culture until the medium becomes cloudy with the grown bacteria. This is carried out by transplanting to a medium such as an agar plate and further culturing.

In this way, it is difficult to directly isolate and culture small numbers of bacteria from blood using conventional methods, and it is complicated because it requires an additional operation called enrichment culture to increase the number of bacteria as a pretreatment for cultivating colonies. It requires operation and a long time. In particular, enrichment culture in a liquid as described above generally requires a long time. In addition, blood itself has the effect of suppressing the growth of bacteria,
When antibacterial agents are administered, it is more difficult for bacteria to grow in the blood, and if the number of bacteria in the sample is small, it may not be possible to detect them. Furthermore, when transferring bacteria from a growth medium to a separation medium, there is a risk that the test bacteria may contaminate the environment or contaminate the test surface, reducing test accuracy.

Purpose of the Invention Therefore, as a result of extensive research, the present inventors have found that the bacteria-contaminated blood is mixed with a solution consisting of at least a hemolytic agent and an anticoagulant, or a solution consisting of a hemolytic agent, an anticoagulant, and a liquid medium. An overculture in which a filter having holes large enough to prevent bacteria from passing through the mixture and a water absorbent adhered to the back side of the filter or a water absorbent impregnated with a liquid medium and dried are housed in a container with the filter facing upward. We have developed a method for isolating and culturing blood bacteria by directly culturing the bacteria on the filter.

According to this culture method, it is possible to directly collect blood bacteria without requiring any pretreatment operations such as enrichment culture of blood bacteria, and to culture them as they are without transplanting them to another medium. Therefore, in this culture method, it is essential to thoroughly mix the collected blood with a solution consisting of a hemolytic agent and an anticoagulant, or a solution consisting of a hemolytic agent, an anticoagulant, and a liquid medium. In order to prevent environmental contamination and sample contamination by test bacteria, it is required that the test be carried out in a closed container.

SUMMARY OF THE INVENTION Accordingly, an object of the invention is to provide an apparatus that allows mixing at least a solution of a hemolytic agent and an anticoagulant with blood, or a solution consisting of a hemolytic agent, an anticoagulant, and a liquid medium in a closed system. be.

The above object is achieved by the blood bacteria isolation and culture device of the present invention as described below.

(1)(a) A container containing a solution containing at least a hemolytic agent and an anticoagulant and having a blood storage space, and an elastic rubber stopper that is removably and sealably fitted into the opening of the container. (b) a container body containing a filter having holes large enough to prevent bacteria from passing through and a water absorbent adhered to the back side of the filter with the filter side facing up; and,
A blood bacteria isolation incubator consisting of a superincubator and a lid that removably covers the opening of a container body.

(2) The blood bacteria isolation and culturing device according to item 1, wherein the solution contained in the blood processing device contains a culture medium.

(3) The blood bacteria isolation and culturing device according to item 1 or 2, wherein the blood storage space of the blood processing device is under reduced pressure.

(4) Blood bacteria isolation and culture according to item 1 or 2, wherein the container of the blood processing device comprises a cylindrical body and a pusher arranged to slide on the inner wall of the cylindrical body in a liquid-tight manner. vessel.

By using the blood bacteria isolation incubator of the present invention having the above structure, blood can be sealed tightly with a solution consisting of a hemolytic agent and an anticoagulant, or a solution consisting of a hemolytic agent, an anticoagulant, and a liquid medium. The system allows sufficient mixing, and bacteria in the blood can be isolated and cultured without fear of the bacteria in the blood contaminating the environment or contaminating the blood sample with external bacteria.

DETAILED DESCRIPTION OF THE INVENTION The structure of the present invention will be described in detail below based on embodiments shown in the drawings.

FIG. 1 shows an embodiment of the blood processing device of the present invention. This blood processing device consists of a container 1 containing at least a mixed solution 2 of a hemolytic agent and an anticoagulant, and a rubber stopper 3 that is removably and sealably fitted into the opening of the container 1. The interior is maintained at reduced pressure. To process blood using this blood processing device, the needle of a syringe (not shown) used to collect blood is pierced through the rubber stopper 3, and the blood is introduced into the container 1 using the reduced pressure inside the container 1. After the blood has been introduced, the needle is removed and the container is inverted to mix the blood with the solution in the container. Saponin is preferably used as the hemolytic agent, and sodium amylosulfate, sodium polyanethole sulfate, etc. are preferably used as the anticoagulant. A liquid medium can also be mixed into these mixed solutions as desired. In this case, there is no need to impregnate the water absorbing body of the separation culture vessel with the medium to be used in the next operation. Also, bacteria multiply faster.

FIG. 2 shows another embodiment of the blood processing device of the present invention. This blood processing device consists of a cylindrical body 4 and a pusher 5 arranged to slide on the inner wall of the cylindrical body in a fluid-tight manner.A rubber stopper 3 is removably attached to the opening of the cylindrical body 4. The space formed by the cylindrical body 4 and the pusher 5, which are fitted in a sealing manner, contains a liquid mixture 2 consisting of a hemolytic agent and an anticoagulant similar to that of the previous embodiment. This mixed solution may also be mixed with a liquid medium. To process blood using this blood processor, the needle of the syringe used to collect blood is inserted through the rubber stopper 3, and when the blood is forced in with the syringe, the pusher 5 of the blood processor slides downward. Blood is introduced into the container. After the blood has been introduced, the container is inverted to thoroughly mix the blood with the hemolytic agent and anticoagulant. These blood processing devices are sterilized by γ-ray irradiation or autoclaving to prevent contamination of sample blood with bacteria.

By treating blood with the blood processor of the present invention, red blood cells are destroyed and blood coagulation is prevented. These operations are essential to perform the following blood overoperation and culture of blood bacteria. Furthermore, when a liquid medium is mixed, mixing treatment with the culture solution can be performed at the same time.

After treating the blood with the blood processor of the present invention, the rubber stopper is removed and the sample blood is poured onto the filter of the overincubator shown below. The superincubator is as shown in Figure 3.
A container body 8 containing a filter 6 having holes large enough to prevent bacteria from passing through and a water absorbent 7 bonded to the back side of the filter 6 with the filter 6 side facing up, and an opening of the container body 8 can be freely attached and detached. It consists of a covering lid 9. The container body 8 includes a filter 6 and a side wall 10 of the container body.
and a space 11 defined by the lid 9, the water absorbent body 7, the container body side wall 10, and the container body bottom 12.
It has a space 13 defined by, and a ventilation hole 14 is provided below the water absorbent body 7.

The filter 6 is for passing bacteria in the blood, and has holes large enough to prevent bacteria from passing through. The hole size of the filter 6 is preferably 0.75 microns or less.
It is about 0.45 microns. The material of the filter 6 is not particularly limited as long as it is inert to blood, but representative examples include nitrocellulose, polycarbonate, polyamide, cellulose ester, etc. Commercially available materials include Millipore (Millipora Corporation), etc. products) and Metricel (a product of Gelman Instrument Company). It is preferable that the filter 6 is subjected to hydrophilic treatment by a method known per se so that blood is easily absorbed therein.

The water absorbent body 7 is for absorbing and retaining the excess blood passed through the filter 6 and providing nutrients to the bacteria captured on the filter 6. It is desirable that the water absorbent body 7 has the ability to absorb almost the entire amount of liquid. Suitable materials include cellulose paper and nonwoven fabric. The water absorbent body 7 must be tightly fixed to the back surface of the filter 6 with an adhesive or the like. If the degree of adhesion is insufficient, filtration will not be carried out smoothly, and the water absorbent body 7 will not be able to pass through the filter 6 after filtration.
The bacteria on top are not sufficiently nourished.

As the adhesive to be used, heat sealing using low melting point polymer fibers that does not inhibit adhesive properties is preferred. The water absorbent body 7 can also be impregnated with a liquid medium and dried if desired. In this case, the dry medium is dissolved in the liquid that is absorbed and retained by the water absorbent body 7, and then filtered through the filter 6.
Provide nutrients and moisture to the bacteria on top. The integrated filter 6 and water absorber 7 are attached to the container body wall 10.
It is quickly fitted and fixed, and prevents the sample blood from leaking out without passing through the filter 6. The filter 6 and the water absorbent body 7 are fixed to the wall of the container body using an adhesive or by crimping with a caulking tool 15 as shown in the figure. In this case, a step S is formed on the side wall 10 of the container body 8, and the integrated filter 6 and water absorbent 7 are placed on the step S, and then the caulking tool 15 is press-fitted. A space 11 above the filter 6 is for storing sample blood, and a space 13 below the water absorbent body 7 is for receiving liquid that has not been completely absorbed by the water absorbent body 7. The vent hole 14 is for discharging the air in the space 13 compressed by the liquid, and is provided in the container body wall 10 or the container body bottom 12 so as to communicate with the atmosphere. Due to the presence of this vent 14, the entire amount of sample blood is quickly allowed to pass through naturally. It is desirable to fit a bacterial filter 16, such as a cotton stopper, into the vent hole 14 to prevent the intrusion of germs. When the vent hole 14 is provided in the container body bottom 12, a barrier 17 is provided around the vent hole 14 to prevent the stored liquid from flowing out through the vent hole 14. It is also desirable to provide concentric barriers throughout the bottom 12 of the container body to prevent liquid from collecting around the vent 14.

The over-incubator having the above configuration is sterilized by γ-ray irradiation or ethylene oxide gas to improve the reliability of testing.

Although the shape of the superincubator configured in this way is not particularly limited, it is generally desirable to have a circular shape. Although its size is not particularly limited, for example, when 2 ml of blood is used as a specimen, the diameter of the container body is preferably approximately 60 mm in order to accommodate this.

The blood specimen thus treated with the blood processing device of the present invention is poured onto the filter 6 of the above-mentioned superincubator, and the opening is covered with the lid 9. Blood drains naturally under its own weight. Bacteria in the blood remain on the filter 6, blood passes through the filter 6 and is absorbed by the water absorbent 7, and blood in excess of the saturated amount drips and accumulates at the bottom 12 of the container body.
Since the compressed air is discharged from the vent 14, the evaporation is carried out smoothly under the weight of the sample blood. After the over-incubation is completed, bacterial colonies are formed on the filter 6 by maintaining the over-incubator at a constant temperature. Bacteria are collected from the colony and subjected to bacterial tests such as bacterial identification and drug susceptibility testing. Note that if the water absorbent body 7 is impregnated with a liquid medium and dried, it is not necessary to add the liquid medium in the blood processing operation.

Specific Effects of the Invention According to the blood bacteria isolation and culturing device of the present invention, a blood sample can be subjected to hemolytic and anticoagulant treatment in a sealed state. Therefore, it is possible to prevent environmental contamination due to blood bacteria, and it is also possible to prevent contamination of external bacteria into the specimen, thereby increasing the reliability of the test. Furthermore, by using the blood bacteria isolation incubator of the present invention, hemolysis, anticoagulation, and mixing operations with the culture medium can be simplified, and environmental contamination by test bacteria associated with transplantation into the incubator after treatment can be prevented. The contamination of bacteria with bacteria is also minimized. Furthermore, by using the blood bacteria isolation and incubator of the present invention, all the bacteria in the collected sample blood can be isolated and cultured as is, resulting in a high detection rate of bacteria and making it possible to measure the number of bacteria in the sample. It is possible.

[Brief explanation of the drawing]

FIG. 1 is a sectional view showing one embodiment of the blood processing device of the present invention, and FIG. 2 is a sectional view of another embodiment. FIG. 3 is a sectional view of the overincubator of the present invention. 1... Container, 2... Mixed solution of hemolytic agent and anticoagulant, 3... Rubber stopper, 4... Cylindrical body, 5... Pusher, 6... Filter, 7... Water absorbent, 8... Container body, 9... Lid, 1
4...Vent.

Claims (1)

[Scope of Claims] 1 (a) A container containing a solution containing at least a hemolytic agent and an anticoagulant and having a blood accommodation space, and an elastic member that is removably and sealably fitted into the opening of the container. (b) A filter having holes large enough to prevent bacteria from passing through, and a water absorbent adhered to the back of the filter, with the filter side facing up. A blood bacteria isolation and incubator consisting of a container body containing bacteria, and a superincubator comprising a lid that removably covers the opening of the container body. 2. The blood bacteria isolation and culturing device according to claim 1, wherein the solution contained in the blood processing device contains a culture medium. 3. The blood bacteria isolation and culturing device according to claim 1 or 2, wherein the blood storage space of the blood processing device is under reduced pressure. 4. Blood bacteria according to claim 1 or 2, wherein the container of the blood processing device comprises a cylindrical body and a pusher arranged so as to be able to slide liquid-tightly on the inner wall of the cylindrical body. Separation incubator.