JPS6233872B2 - - Google Patents

Description

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a novel blood processing device used to isolate and culture 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. Colonies are grown by transferring them onto a medium such as an agar plate and culturing them further.

In this way, with conventional methods, it is difficult to directly isolate and culture small numbers of bacteria from blood, and it is complicated because it requires an additional operation called enrichment culture as a pretreatment for cultivating colonies. This requires a lot of 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, and if antibacterial agents are administered, it is even more difficult for bacteria to grow in the blood, and if the number of bacteria in the sample is small, it can be detected. Sometimes it's not possible. moreover,
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 bacteria, reducing test accuracy.

Purpose of the Invention Therefore, as a result of intensive research, the inventors of the present invention have mixed bacteria-contaminated blood with a solution consisting of at least a hemolytic agent, an anticoagulant, and a liquid medium, and poured the mixture into holes large enough to prevent bacteria from passing through. A water absorbent adhered to the back surface of the filter or a water absorbent impregnated with a liquid medium and dried is filtered in a filtration incubator in which the filter is housed in a container with the filter facing upward. We developed a method to isolate and culture blood bacteria by culturing the bacteria as they are.

According to this culture method, it is possible to directly collect bacteria in blood without requiring pretreatment operations such as enrichment culture of blood bacteria, and to culture them as they are without transferring 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, an anticoagulant, and a liquid medium, and this operation prevents environmental contamination and specimen contamination by test bacteria. In order to prevent this, it is required that the process 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 thus achieved by the device of the invention as described below.

(1) A container that contains a solution containing at least a hemolytic agent, an anticoagulant, and a liquid medium, and has a blood storage space, and an elastic rubber stopper that is fitted into the opening of the container in a sealable manner. A blood processing device characterized by being sterilized.

(2) The blood processing device according to item 1, wherein the blood storage space is under reduced pressure.

(3) The blood processing device according to item 1, wherein the container comprises a cylindrical body and a pusher arranged to slide on the inner wall of the cylindrical body in a liquid-tight manner.

By using the blood processing device of the present invention having the above structure, blood can be sufficiently mixed with a solution consisting of a hemolytic agent, an anticoagulant, and a liquid medium in a closed system, and bacteria in the blood can be removed from the environment. There is no risk of contaminating the blood sample 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, an anticoagulant, and a liquid medium, and a rubber stopper 3 fitted in an opening of the container 1 in a sealing manner. is maintained at reduced pressure. Preferably, the rubber stopper 3 is removable. 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 is further mixed into these mixed solutions. Since the liquid medium is mixed in advance, there is no need to impregnate the water absorbing body of the separation culture vessel used in the next operation with the medium. 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, an anticoagulant, and a liquid medium similar to that of the previous embodiment. 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, anticoagulant, and liquid medium. 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 for the subsequent hemofiltration operation and culture of blood bacteria to be performed smoothly. Furthermore, since a liquid medium is mixed, mixing processing 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 separation incubator shown below. The filtration incubator is as shown in Figure 3.
The opening of the container body 8 is removably covered with 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 surface of the filter 6 with the filter 6 facing upward. It consists of a cover 9.
The container body 8 has a space 11 defined by the filter 6, the container body side wall 10, and the lid 9, and a space 13 defined by the water absorbent body 7, the container body side wall 10, and the container body bottom 12, and has a space 13 defined by the water absorbent body 7, the container body side wall 10, and the container body bottom 12. A ventilation hole 14 is provided in the.

The filter 6 is for filtering bacteria in the blood and has holes large enough to prevent bacteria from passing through. The size of the pores in the filter 6 is 0.75 microns or less, preferably about 0.45 microns. The material of the filter 6 is not particularly limited as long as it is inert to blood;
Typical examples include nitrocellulose, polycarbonate, polyamide, cellulose ester, etc. Commercially available products include Millipore (product of Millipore Corporation) and Metricel (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 absorbs and retains the filtered blood filtered by the filter 6, and provides 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 filtrate. Suitable materials include cellulose filter paper and nonwoven fabric. It is necessary that the water absorbent body 7 is 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 be tightly fixed to the back surface of the filter 6 after filtration. The bacteria on top are not sufficiently nourished.

As the adhesive used, heat sealing using low melting point polymer fibers that does not inhibit filtration is preferred. The integrated filter 6 and water absorber 7 are attached to the container body wall 1.
0 and is fixed without any gaps, thereby preventing 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 filtrate 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 filtrate, and is provided on the container body wall 10 or the container body bottom 12.
installed in such a way that it communicates with the atmosphere. Due to the presence of this vent 4, the entire amount of sample blood is quickly naturally filtered. It is desirable to fit a bacterial filter 16, such as a cotton stopper, into the vent hole 14 to prevent noise from entering. When the vent 14 is provided in the bottom 12 of the container body, a barrier 17 is provided around the vent 14 to prevent the stored filtrate from flowing out of the vent 14. It is also desirable to provide concentric barriers throughout the bottom 12 of the container body to prevent filtrate from collecting around the vent 14.

The blood bacteria isolation incubator having the above configuration improves the reliability of testing by performing sterilization treatment using gamma ray irradiation or ethylene oxide gas.

The shape of the separation culture vessel constructed in this way is not particularly limited, but 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 about 60 mm in order to accommodate this.

The blood sample thus treated with the blood processing device of the present invention is poured onto the filter 6 of the above-mentioned separation culture device, and the opening is covered with the lid 9. Blood is naturally filtered by 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 air compressed by the filtrate is discharged from the vent 14, filtration is smoothly performed by the weight of the sample blood. After the filtration is completed, bacterial colonies are formed on the filter 6 by maintaining the separation incubator at a constant temperature. Bacteria are collected from the colony and subjected to bacterial tests such as bacterial identification and drug susceptibility testing.

Specific Effects of the Invention According to the blood processing device of the present invention, a blood sample can be hemolyzed and anticoagulated in a sealed state. Therefore, it is possible to prevent environmental pollution caused by blood bacteria,
Furthermore, since it is possible to prevent bacteria from entering the sample from the outside, the reliability of the test can be increased.
Furthermore, by using the blood processing device of the present invention, hemolysis, anticoagulation, and mixing operations with culture media can be simplified, and environmental contamination caused by test bacteria caused by transplantation into an incubator after treatment can be reduced. The contamination of bacteria is also minimized. Furthermore, by using the blood processing device of the present invention, all bacteria in the collected blood sample are isolated and cultured as they are, so the detection rate of bacteria is high and it is also possible to measure the number of bacteria in the sample. be.

[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 a blood bacteria isolation and culturing device used in a blood bacteria isolation and culturing method together with the blood processing device of the present invention. DESCRIPTION OF SYMBOLS 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, 14...Vent.

Claims (1)

[Scope of Claims] 1. A container that contains a solution containing at least a hemolytic agent, an anticoagulant, and a liquid medium and has a blood storage space, and an elastic rubber stopper that is fitted into the opening of the container in a sealable manner. A blood processing device characterized in that it has a body and is sterilized. 2. The blood processing device according to claim 1, wherein the blood storage space is under reduced pressure. 3. The blood processing device according to claim 1, wherein the container comprises a cylindrical body and a pusher arranged to slide on the inner wall of the cylindrical body in a liquid-tight manner.