JPS5820262B2 - Collection equipment for anaerobic bacteria testing - Google Patents

Description

Detailed Description of the Invention ■Background Technical Field of the Invention The present invention relates to a sampling device for testing anaerobic bacteria, and in particular, a sampling device for testing anaerobic bacteria suitable for use as a reduced pressure blood collection tube (also referred to as a vacuum blood sampling tube). Regarding.

Prior Art and Problems In hospitals, etc., patients' blood is used as a liquid for testing, and the pathogenic bacteria contained in the blood are cultured to identify them and find appropriate drugs. .

As mentioned above, so-called reduced pressure blood collection tubes are known as instruments for collecting blood.

This vacuum blood collection tube consists of a container whose mouth is hermetically closed with a pierceable rubber stopper and whose interior is maintained at a predetermined degree of vacuum.

When blood is collected using this vacuum blood collection tube, one end of the piercing needle having needle tips at both ends is inserted into the patient's blood vessel, and the other end is inserted into the rubber stopper of the vacuum blood collection tube.

Then, an amount of blood commensurate with the degree of vacuum in the vacuum blood collection tube is collected from the patient's blood vessel into the vacuum blood collection tube.

However, even though a reduced pressure state is maintained in the reduced pressure blood collection tube as described above, some air and hence oxygen are present, which is not a favorable state for testing for anaerobic bacteria.

■Purpose of the Invention Therefore, an object of the present invention is to provide a testing instrument suitable for removing oxygen and testing for anaerobic bacteria.

According to the present invention, there is provided a mouthful container for containing a test liquid containing anaerobic bacteria, an airtight closing member for closing the mouth of the container, and a surface that allows gas to pass through but not liquid. A sampling device for testing anaerobic bacteria is provided with an oxygen scavenger capable of absorbing more oxygen than the amount of free oxygen in the container, which is housed in the container in a water-repellent packaging. provided.

It is most preferable that the sampling device for testing anaerobic bacteria of this invention takes the form of a vacuum blood collection tube.

In that case, the closure member is made of a pierceable material, such as rubber.

Further, the pressure inside the container is reduced to such an extent that a predetermined amount of test liquid can be aspirated.

The above-mentioned oxygen scavenger usually consists of powder of iron or an iron-based compound and a metal halide.

According to another aspect of the invention, an anaerobic bacterial culture medium is contained in the mouthed container.

①Detailed Description of the Invention Hereinafter, an embodiment in which the present invention is applied to a reduced pressure blood collection tube will be described with reference to the accompanying drawings.

As shown in FIG. 1, this vacuum blood collection tube includes a container 1 having a spout 3. As shown in FIG.

The container 1 may be made of any suitable material that can withstand reduced pressure, such as plastic or glass, but a transparent container, especially glass, is preferred because it allows the inside to be observed.

Further, the shape of the container is not limited to the tube shape as shown in the figure, but may also be a bottle shape.

As shown in the figure, it is advantageous, as will be described hereinafter, that an annular protrusion 3a is formed on the inner side of the end of the container mouth 3 by suitable means such as baking the end of the mouth.

The container 1 is hermetically closed in its recess 3 by a plug member 4.

This plug member 4 may be made of any material as long as it can maintain airtightness, but it should be made of a material that can be pierced, especially a rubber-like elastic material such as butyl rubber or styrene-butadiene. is preferred.

As clearly shown in FIG. 2, the plug member 4 has a concave portion 4a formed in its lower end surface, and these four portions 4a form a relatively thin peripheral wall 6 constituting a body portion.

This peripheral wall 6 is hermetically fitted to the mouth 3 of the container.

A head 5 having a slightly larger diameter than the body 6 is integrally formed therein, and a peripheral wall 5a of the head 5 is formed in a tapered shape that widens toward the body 6.

Further, a recess 5b is formed in the upper end surface of the head 5, and the wall thickness of the head 5 is made thin to facilitate piercing.

Preferably, the body 6 is formed with a through hole 7 that communicates with the internal atmosphere of the container 1.

This through hole 7 has a diameter of approximately 3 mm to 3 mm when viewed from the front.
It is a circular shape with a width of 0.3 mm to 2 mrrt and a length of 2 mm to 3 mm.

There may be a plurality of these through holes 7. Preferably diameter 9
．． Two circular through holes of 7 mm to 1.5 mm are symmetrically formed from the upper end of the body 6 to 37IL7IL to 5mtr.
Form at position t.

Further, it is convenient if an annular groove 8 is formed in the body portion 6 below the through hole 7.

Such a plug can be manufactured by pressing a heated, melted and pressurized rubber-like material into a mold and solidifying and molding it to obtain a plug without holes.

When making the through-hole part 7, it can be made by heating a needle having a predetermined shape and size and making a hole in the stopper, or by making a hole with something such as a punch.

After washing, the plug body thus obtained is dried naturally or by heating.

It is advantageous to coat this with silicone oil to improve sliding properties.

Returning to FIG. 1 again, the container 1 contains a medium 9 for culturing anaerobic bacteria.

The anaerobic bacterial culture medium 9 may be, for example, a plain vert infusion medium, a thiochelicolate medium, etc., but the composition of a particularly suitable medium is shown below.

Medium A and B are prepared by the following method.

Weigh out all of the medium components except for gelatin, and apply them to 500 mA! Dissolve in distilled water.

However, L-cystine hydrochloride and hemin are added after being dissolved in 0.01N hydrochloric acid and water, respectively.

Separately, weigh gelatin and add 500rfLl.
Dissolve completely while heating with distilled water.

After thoroughly mixing both, the mixture is cooled to room temperature, the pH is adjusted to a predetermined level with an alkaline solution such as 2N-NaOH, and the mixture is prefiltered using absorbent cotton.

The filtrate obtained from this is further filtered under reduced pressure through a glass filter, and the obtained filtrate is used as a culture medium.

Inside the container 1, an amount of oxygen scavenger 11 capable of absorbing more oxygen than the free oxygen in the container 1 is housed in a package 12, as shown in FIG.

As in the example shown in FIG.
is accommodated, the package 12 is located within the medium 9.

Examples of the oxygen scavenger 13 are listed below.

Metal powder such as iron powder coated with metal halide 0 Combination of at least one iron-based compound such as iron carbide, iron carbonyl, ferrous oxide, ferrous hydroxide, iron silicon, etc. and metal halide .

Iron and/or zinc and Na2 so4m 10 H
In combination with 2O or with the addition of metal halides.

Iron, copper, tin, zinc and/or nickel and Na
2 S 03 ・7 H20 or Na2CO3・
Combination of 10H20 and metal halide.

A combination of a transition metal from period 4 of the periodic table, tin and/or antimony, and water or a metal halide.

A combination of an alkaline earth metal sulfite and a ferrous oxide, a transition metal salt other than ferrous oxide, or an aluminum salt.

A combination of an alkaline earth metal sulfite, a transition metal salt or an aluminum salt, an alkaline compound containing an alkali metal or an alkaline earth metal, and an alkaline compound containing nitrogen.

Combinations of alkali metal or ammonium sulfites, bisulfites and/or pyrosulfites with transition metal salts or aluminum salts.

A combination of alkaline earth metal sulfite and ferrous salt compounds and ammonium salts.

Combination of dithionite and water-containing alkaline earth metal hydroxides and carbonates.

Combination of dithionite, activated carbon and water.

Combination of dithionite and a compound with water of crystallization.

Combination of dithionite, alkaline substance and alcohol compound.

The package 12 is made of a material that allows gases to pass through but not liquids, such as (3) RE-TEX (trade name manufactured by Junkosha). The surface is treated with a suitable water repellent to prevent premature deterioration of the oxygen scavenger.

The interior of the container 1 is maintained at a predetermined reduced pressure level, that is, a reduced pressure level that allows a predetermined amount of a liquid for testing anaerobic bacteria, such as patient's blood, to be aspirated.

Further, a cover 10 is installed so as to surround the outside of the stopper member 4 and the container mouth 3 with a slight gap from the lower tip 5b of the head 5 of the stopper member 4.

This cover 10 does not inhibit the movement of the plug member 4, and may be made of any material such as plastic or aluminum.

Molton stoppers are preferred. The vacuum blood collection tube having the above configuration is prepared as follows.

20m1 tube (diameter 15.5mm length 165mm
) or a suitable flask-like or bottle-like container, dispense the specified amount of the culture medium (18 ml for a 20 ml tube, 45 ml for a 50 ral flask-like or bottle-like container).

Next, the package 12 containing the oxygen absorber is stored, and the plug member is pushed in to seal the container.

Then, the temperature is 115°C to 12°C in a high-temperature autoclave sterilizer.
1℃, pressure 1.5-2 kgA! r? Autoclave the blood collection tube for 15-20 minutes at i.

The pressure inside the container 1 becomes more positive than the atmospheric pressure by the amount that the plug member 4 is pushed in, but as the oxygen absorber 11 absorbs oxygen, the pressure as a whole becomes lower than the atmospheric pressure and the above-mentioned degree of reduced pressure is achieved. can.

That is, when medium 9 is not used, approximately 1 of the volume of container 1
Blood can be collected by a volume equal to /, and even when the medium 9 is used, blood can be collected by a volume corresponding to about - the space inside the container 1 above the medium 9.

The degree of pressure relief can be arbitrarily adjusted by reducing the pressure before closing the container with the stopper member.

■Specific functions and effects of the invention Blood is collected using the vacuum blood collection tube described above, and anaerobic bacteria in the blood are cultured as follows.

First, a liquid sample containing microorganisms, such as patient's blood, is collected in a container. At this time, it is convenient to use a vacuum blood collection tube holder (manufactured by Terumo Corporation).

This holder consists of a hollow body capable of accommodating a blood collection tube and a sample collection needle supported at the tip of the body and having needle tips formed at both ends.

After disinfecting the stopper of the blood collection tube with alcohol, iodine tincture, etc., insert the culture device into a holder with a sample injection needle.

Then, after puncturing one end of the sample collection needle into the patient's vein,
When the blood collection tube is inserted sufficiently deeply into the holder and the other end of the collection needle is inserted into the container through the stopper member, the pressure inside the blood collection tube is reduced, so a predetermined amount of sample is injected into the inside of the blood collection tube.

In addition, the present invention can also be practiced using a syringe.

After collecting a sample with a syringe, insert the syringe needle into the center of the stopper member of the blood collection tube whose stopper member has been sterilized with alcohol, iodine tincture, etc.

At this time, since the pressure inside the blood collection tube is reduced, a predetermined amount of sample is automatically aspirated from the syringe and injected.

After collecting the sample in this manner, it is cultured at 27° to 37°C for 1 to 14 days.

Cultivation can be continued further if necessary.

Note that if the stopper member is rotated once just before the start of culture, degassing will be performed well.

In addition, when the medium 9 is not stored in the container 1 in advance, the medium is filled after blood collection and the above-mentioned culture is performed.

In this anaerobic culture, anaerobic bacteria produce gas during culture, and the pressure inside the container 1 increases.

When the pressure inside the container 1 increases and exceeds a certain point, the plug member 4 is pushed up by the pressure. Or, the stopper member 3 will stop after reaching a slightly higher point.

Then, the gas generated inside the container 1 is discharged to the outside of the container 1 through the through hole 7.

Note that even if the stopper member 4 is pushed up further by the generated gas, the annular groove 8 formed in the body 6 will engage with the protrusion 3a of the container mouth, preventing further movement of the stopper member 4. is prevented.

Then, after degassing, the inside of the culture container 1 can be maintained in an airtight state again by pushing down by hand so that the through hole 7 is closed by the inner wall of the mouth 3 of the container 1.

Therefore, gas generated inside the culture container 1 can be vented while maintaining an airtight state inside the culture container 1, and the plug member 4 will not fly away due to internal pressure or the culture solution will not overflow during or after culture. It is extremely safe.

As mentioned above, in a vacuum blood collection tube, the oxygen in the air remaining in the container 11 is absorbed by the oxygen scavenger 13 in a relatively short period of time.
On the other hand, the oxygen scavenger 13 absorbs oxygen so that, for example, the iron(1) compound that constitutes it is absorbed and removed by iron(l).
[)・It is oxidized to a compound.

In this way, no oxygen exists within the container 11.

That is, the above-mentioned blood collection and culture will be performed in such a state, and the anaerobic bacteria contained in the blood will survive and be cultured in the anaerobic state.

An example in which anaerobic bacteria were cultured using a vacuum blood collection tube as shown in FIG. 1 will be described below.

Example 1 In anaerobic culture, various gas-producing bacteria shown in Tables 1 and 2 were suspended in appropriate amounts of sterile distilled water, and 101 to 10
2 CC of a suspension prepared to have a bacterial concentration of 2 cells/1 rLl were each collected in container 1, and then cultured at 37°C.

This was observed every day and the state of growth of the bacteria within the culture device and the movement of the stopper due to gas pressure were investigated, and the results were as shown in Tables 1 and 2.

The movement of the stopper is expressed as the number of days required for part or all of the hole in the stopper to move to the tip of the mouth of the culture device after the start of culture.

Even if the culture was continued from the day when the plug body moved to this point, no further movement of the plug body was observed.

Example 3 Various bacterial strains were suspended in an appropriate amount of sterile distilled water, and Habo I
The bacteria repellency was adjusted to 101 to 5 x 102 cells/rule before use in the experiment.

Cultivation was started at 37° C., and the number of viable cells was measured every day according to a conventional method, and the number of days required for the number of viable cells to reach approximately 5×10 7 cells/ml or more was determined.

[Brief explanation of the drawing]

FIG. 1 is a partially cross-sectional side view of a reduced pressure blood collection tube according to an embodiment of the present invention, FIG. 2 is a partially cross-sectional side view of a stopper member used in the reduced pressure blood collection tube shown in FIG. 1, and FIG. 3 is a side view of the present invention. FIG. 2 is a perspective view showing an oxygen absorber used in a package. 1... Container, 3... Container mouth, 4.
...Plug member, 7...Through hole, 9...
・Medium, 11...Oxygen absorber, 12...
packaging.

Claims (1)

[Scope of Claims] 1. A container with a mouth for containing a liquid for testing anaerobic bacteria, an airtight closing member that closes the mouth of the container, and a surface that is repellent to allow gas to pass through but not to allow liquid to pass through. 1. A sampling device for anaerobic bacteria testing, comprising an oxygen scavenger that can absorb more oxygen than the amount of free oxygen in the container, which is housed in the container in the form of a water-treated package. 2. The collection instrument according to claim 1, wherein the closing member is pierceable. 3. The sampling device according to claim 2, wherein the closing member is made of rubber. 4. The sampling device according to claim 1 or 2, wherein the inside of the container is reduced in pressure to the extent that a predetermined amount of the test liquid can be aspirated. 5. The collection device according to any one of claims 1 to 4, wherein the oxygen scavenger comprises powder of iron or an iron-based compound and a metal halide. 6. A mouthed container for containing a liquid for anaerobic bacteria testing, an airtight closing member that closes the mouth of this container, which allows the anaerobic bacteria culture medium and gas contained in the container to pass through, but not the liquid. The container is housed in the container in the form of a package whose surface has been treated with water so as not to damage the container, and the container is sterilized in an autoclave and is equipped with an oxygen scavenger capable of absorbing more oxygen than the amount of free oxygen in the container. Collection equipment for anaerobic bacteria testing. 7. The collection instrument according to claim 6, wherein the closing member is pierceable. 8. The collection instrument according to claim 7, wherein the closing member is made of rubber. 9. The sampling device according to claim 6 or 7, wherein the inside of the container is reduced in pressure to the extent that a predetermined amount of test liquid can be aspirated. 10. The collection device according to any one of claims 6 to 9, wherein the oxygen scavenger comprises iron or iron-based compound powder and a metal halide.