KR102281121B1 - Self-Contained Biological Indicator for verification of room decontamination - Google Patents

Abstract

The present invention relates to a self-contained biological indicator for large space sterilization equipped with an intake and exhaust fan, and more particularly, when performing a sterilization or sterilization process, it is possible to check and verify whether sterilization was properly performed. It relates to a self-contained biological indicator for confirming sterilization of a large general closed space equipped with an intake and exhaust fan. The biological indicator of the present invention is self-contained, and is prepared in a separate medium from a separate sterilization process. It overcomes the difficulties of inoculation and aseptic handling of this process, and the advantage of maintaining uniformity from batch to batch by using possible, highly resistant and homogeneous spores, particularly for use in common closed-space sterilization. There is this.

Description

Self-Contained Biological Indicator for verification of room decontamination

The present invention relates to a self-contained biological indicator for confirming space bacteria decontamination, and more particularly, as a biological indicator that can confirm whether or not the normally blocked space is sterilized by vaporized hydrogen peroxide, etc., of a strain for separate culture It relates to a self-contained biological indicator for confirming decontamination of space bacteria that does not require inoculation.

The present invention uses endospores to sterilize a sterile room or a space for producing sterile products (eg, injections, eye drops, infusions, etc.) that must be maintained in a sterile state, for the purpose of verifying and verifying whether the sterilization has been performed normally. It relates to a biological indicator (indicator).

Biological indicators are used to verify or confirm that the sterilization or sterilization process has been performed effectively, that is, to evaluate and/or measure the effectiveness of the sterilization process. Specifically, common biological indicators include a population of calibrated microorganisms that are highly resistant to sterilization processes during the course of the study. After exposure to the sterilization process, the biological indicator is cultured in a culture medium to promote the growth of any viable remaining microorganisms.

Bacterial endospores are generally preferred as biological indicators because they are recognized to be more resistant to sterilization processes than most other types of microorganisms. That is, it is due to the fact that if a sterilization process to the extent of killing bacterial endospores is achieved, it is presumed to also kill any other microorganisms causing contamination.

Endospores are special objects that are produced by themselves to survive in an extreme environment where bacteria cannot survive, and they mainly occur in gram-positive bacteria. They are usually generated under conditions of lack of nutrients, and these endospores can withstand several years to several centuries, and when the surrounding environment is favorable for bacterial growth, they can be differentiated into vegetative cells again. In other words, endospores have strong resistance to heat, strong chemicals, and radiation, and have the function of a survival structure so that cells can overcome nutrient depletion or dryness, which is difficult for cells to survive. (Interphase) can be seen.

The selection of bacterial endospores depends on the sterilization modality, type of sterilization, and technique to be evaluated. For example, Geobacillus stearothermophilus spores are sterilized using moist heat, peracetic acid, hydrogen peroxide, and other peroxidizing compounds in both liquid and vapor states because these indicator spores are highly resistant to oxidative chemistries. Used to monitor the system. Similarly, Bacillus subtilis spores are used to monitor ethylene oxide sterilization, dry heat sterilization systems.

Microorganisms are generally supported on a carrier or substrate, such as a strip or disk. The substrate is made of materials compatible with the sterilization process and does not contain additives that may affect the sterilization evaluation. Materials such as filter paper, chromatography paper, blotter paper, fiberglass, polymer plastics, ceramics, stainless steel, and metaloxide articles are often used as substrates or carriers.

It is common to use chemical indicators as an auxiliary means. Chemical indicators have the advantage that they can be visually identified immediately after completion of the sterilization process, but strictly sterilization cannot be confirmed. Therefore, sterility must be confirmed using a biological indicator. G. stearothermophilus spores are used to verify and confirm moist heat sterilization or vaporized hydrogen peroxide sterilization process because spores of G. stearothermophilus show resistance to oxidizing substances or moist heat.

Self-contained biological indicators include culture medium within the indicator, typically in fragile vials. However, at present, these self-contained biological indicators are complete, such as chamber-type sterilizers (steam, EO gas, vaporized hydrogen peroxide: vH ₂ O ₂ ), etc. that can apply pressure by injecting steam, gas, or steam after applying a vacuum. It is used for sterilization verification only in a closed space.

Therefore, in a general closed space (eg, a clean bench, a clean room, an isolator, a pass-box, a room, a hospital corridor, an entire laboratory, etc.), such strong pressure These self-contained biological indicators cannot be used because they are difficult to apply, and biological indicators in the form of spore strips are used. The biological indicator is combined with the culture medium contained in a separate container after a monitored sterilization process. Subsequent microbial growth, explained by color change or turbidity of the growth medium, is an indication that the sterilization process was not effective. At this time, as a sterilization method, a fumigation method for decontamination of space bacteria using vaporized hydrogen peroxide, chlorine dioxide vapor, glycel aldehyde vapor, etc. can be used.

Therefore, the present inventors are a biological indicator used for general closed space sterilization where pressure does not act as described above, but inoculation in a separate medium from a separate sterilization process and aseptically treating this process can be overcome. While contemplating a solution, we developed a structure that can be used for sterilization in a closed space that is in the same form as a self-contained biological indicator but does not apply pressure, and also has resistance in the biological indicator during space sterilization by vaporized hydrogen peroxide, etc. The present invention was completed by confirming whether or not endospores of Geobacillus stearomophilus, which are endospores having uniform characteristics and uniform characteristics, can be cultured.

It is an object of the present invention to provide a self-contained biological indicator for general closed space sterilization equipped with an intake and exhaust fan.

It is another object of the present invention to provide a self-contained biological indicator containing endospores of Geobacillus stearomophilus upon confirmation of sterilization of a common blockage by vaporized hydrogen peroxide.

In order to achieve the above object, the present invention is a case (3) and; a capsule 13 disposed inside the case 3 and storing the culture solution; a protective tube 11 disposed to surround the outside of the capsule 13 and having an outlet hole h through which the culture solution flows; And a cap portion 5 (Cap portion) coupled to the upper portion of the case (3); Intake and exhaust fans (7a, 7b) each disposed in the cap portion (5) for sucking or exhausting air; a pressure protrusion (9) disposed on the cap part (5) and descending when pressed to break the capsule (13) to drain the culture medium; a spore support unit 19 provided inside the cap unit 5 to pick up or drop spores (S) (Spore);

After the air disposed inside the case 3 and sucked in by the intake fan 7a flows along the first flow path a1, it flows along the second flow path a2 by the exhaust fans 7a and 7b and is discharged to the outside. an air circulation path 18 and;

a filter 14 disposed between the cap part 5 and the case 3 to prevent the inflow of bacteria or spores; And

It is formed between the first and second flow paths (a1, a2) on the air circulation path (18) and includes a culture unit (17) that promotes the culture of the discharged culture solution and the fallen spores (Spores), and the cap unit (5) ), the pressure protrusion 9 descends and breaks the capsule 13, so that the culture solution inside the capsule 13 flows out and falls through the outlet hole h of the protective tube 11 to the culture unit 17 It provides a biological indicator (1) for space sterilization in which spores (S) and culture are in progress by being supplied as

Hereinafter, the present invention will be described in detail.

In the biological indicator for space sterilization of the present invention (1), a plurality of micro-holes (h2) are formed on one outer peripheral surface of the protective tube (11), and the micro-holes (h2) are from the intake fan (7a) to the culture unit (17). By being formed in the flowing first flow path (a1) direction, the air introduced into the inside of the case 3 flows to the culture unit 17 along the first flow path (a1), and at the same time, some air passes through the plurality of microholes (h2). It is preferable to flow into the inside of the protective tube 11 through the.

In addition, in the biological indicator 1 for space sterilization of the present invention, the air circulation path 18 guides the air sucked by the intake fan 7a to the culture unit 17 along the first flow path a1 to circulate it. an intake guide surface 15; and an exhaust guide surface 16 guiding along the second flow path a2 so that the air that has passed through the culture unit 17 is discharged in the direction of the exhaust fan 7b, and includes an intake guide surface 15 and an exhaust guide surface 16 Preferably, the culture unit 17 is disposed between the intake and exhaust fans 7a and 7b are disposed on the intake guide surface 15 to suck the outside air and supply the inside of the case 3 to the intake fan 7a. class; It is more preferable to include an exhaust fan (7b) disposed on the exhaust guide surface (16) to discharge the internal air to the outside of the case (3).

In addition, in the biological indicator for space sterilization of the present invention (1), the spore support portion 19 is an upper coupling piece 21 connected to the cap portion (5) and; The upper side is male and female coupled to the upper coupling piece 21, and the lower side is a lower coupling piece 23 to which the spores (S) are connected; It includes a support protrusion 25 for supporting the bottom surface of the upper coupling piece 21, and when the cap part 5 is pressed, the upper coupling piece 21 descends so that the bottom surface 21b is in contact with the supporting protrusion 25 By doing so, it proceeds to the outside, and the spore (S) falls by releasing the coupling state of the first engaging projection 21a of the upper engaging piece 21 and the second engaging projection 23a of the lower engaging piece 23. desirable.

In addition, in the biological indicator for space sterilization of the present invention (1), the spores (S) may include a device that can be placed in a place close to the outside or where air flow can be easily and subsequently dropped. In addition, the filter does not pass bacteria or spores between the case and the cap, but allows gas or vaporized hydrogen peroxide to pass therethrough.

In addition, in the biological indicator for space sterilization of the present invention (1), the air is preferably used for space sterilization by containing at least one vapor selected from the group consisting of vaporized hydrogen peroxide, chlorine dioxide vapor and glycel aldehyde vapor. In this case, the spore (Spore; S) is preferably an endospore, and the endospore is Bacillus subtilis ( Bacillus subtilis ), Bacillus subtilis globigii ( Bacillus subtilis globigii ), Clostridium sporogenes ( Clostridium sporogenes ), Bacillus cereus ( Bacillus cereus ), Bacillus circulans ( Bacillus circulans ), Bacillus atrophaeus ( Bacillus atrophaeus ) and Geobacillus stearothermophilus ( Geobacillus stearothermophilus ) consisting of at least one selected from the group More preferably, in this case, the endospores are Bacillus atropaeus, Geobacillus stearomophilus, or a combination thereof, even more preferably.

In addition, in the biological indicator for space sterilization (1) of the present invention, the endospores are cultured in a medium to which hydrogen peroxide is added at a high temperature, and the resulting endospores are subjected to density gradient centrifugation (density gradient centrifugation). It is more preferable to purely separate only resistant endospores from vegetative cells, common spores and other impurities using gradient centrifugation, wherein the density gradient centrifugation is a group consisting of sugar, glycerol, linogriffin and percol. It is even more preferable to use at least one selected from among as a medium, and for the medium to use a continuous density gradient of 50% to 100%.

In addition, in the biological indicator for space sterilization (1) of the present invention, in order to facilitate attachment of the indicator to a wall or any surface, it is directly attached to the surface of the iron material with a magnetic adhesive, or a magnetic adhesive is one side for attachment of a non-iron surface. Adhesive is applied to the surface and can be protected with a protective film. After the protective film is removed, it can be attached to a wall or surface. In addition, a CI (chemical indicator) sticker or CI ink can be printed on the outside of the indicator so that it can be checked immediately after removing (sterilization) of bacterial contamination.

The biological indicator (1) used in the present invention is also called a biological indicator, and when performing sterilization using a living organism, sterilization was successful if the organism dies, and if the organism survives, it is used for the purpose of determining that sterilization is incomplete. do. A typical biological indicator is sterilized by attaching the biological indicator (indicator) to the wall in the space to be sterilized during sterilization, collecting the indicator, and culturing in a culture medium in which the spores can grow to determine whether the spores survive. Check it.

In contrast, the present invention is a biological indicator for space sterilization, and as described above, it is a self-contained biological indicator, and the configuration is the same as the detailed description of the above and the following drawings.

Organisms used for biological indicators include microorganisms, microbial spores, and thermostable enzymes. Among them, endospores are the most used. Since resistant spores have very strong viability, it can be inferred that when the spores die, all other common microorganisms and pathogenic bacteria are killed. The present invention particularly relates to the improvement of productivity with respect to the conditions for the production of resistant endospores and the formation of spores.

Endospores used for biological indicators have strong resistance to chemicals or heat, and representative bacteria that produce these spores are Bacillus subtilis , Bacillus atrophaeus , Geobacillus stea Thermophilus ( Geobacillus stearothermophilus ) and the like are mainly used. In order to efficiently produce endospores from these bacteria, the nutritional conditions, pH, and culture temperature of the sporulation medium are reviewed to produce a large number of spores with high resistance. In addition, the endospores used in the biological indicator of the present invention are induced to have resistance by adding a small amount of hydrogen peroxide to the medium to form highly resistant spores.

The generated spores also contain cell debris (debis) and feeder cells, so there is a problem in using it as it is. To solve this problem, it is necessary to separate cell debris or feeder cells from spores. In addition, by separating spores that are resistant to spores and spores that are not, only clean and highly resistant spores are separated and used for indicators to produce a consistent product every time it is produced.

Density gradient centrifugation is used to separate common spores, debis, and feeder cells from resistant spores. As a density gradient medium, sugar (sucrose), glycerol (glycerol), rinograffin, and Percoll are used. The concentration of the medium is between 50% and 100%. The spores thus separated are checked for purity with an optical microscope or an electron microscope.

As described above, the biological indicator of the present invention has the following effects.

First, by having self-contained, it is possible to overcome the difficulty of inoculating a separate sterilization process and inoculating a separate medium and treating this process aseptically, especially for general closed space sterilization. , and also has the advantage of maintaining uniformity for each production batch by using highly resistant and uniform spores.

Second, the interior of the indicator is divided into two areas on the left and right, an intake fan is arranged in one area to suck outside air, and an exhaust fan is arranged in the other area to discharge the internal air to the outside, thereby forming an air flow path inside the indicator Thus, there is an advantage that culture can proceed smoothly by exposing the culture medium.

Third, a plurality of micro-holes are formed on the outer circumferential surface of the protective tube surrounding the outside of the capsule in which the culture medium is stored, but the micro-holes are formed in the direction of the intake fan so that the sucked air flows to the inside of the protective tube through the micro-holes. Because the surface of the can also be sterilized, it is possible to prevent false positives during culture after sterilization.

Fourth, by printing a sticker-type chemical indicator or ink on the outside of the indicator, it is possible to easily check whether sterilization is achieved by color change.

1 is a perspective view showing the appearance of a biological indicator for space sterilization according to an embodiment of the present invention.
FIG. 2 is an exploded perspective view of FIG. 1 ;
FIG. 3 is a front view showing the internal structure of the biological indicator shown in FIG. 1 .
FIG. 4 is a view showing a state of use of the biological indicator shown in FIG. 1 .
FIG. 5 is a partially enlarged perspective view illustrating a structure in which an inclined surface is formed at a corner and a fan is disposed on the inclined surface as another embodiment of the cap part of the indicator shown in FIG. 1 .

Hereinafter, a biological indicator for space sterilization according to an embodiment of the present invention will be described in detail with reference to the accompanying drawings.

As shown in Figures 1 to 4, the biological indicator (1) for space sterilization proposed by the present invention (hereinafter, biological indicator) is completed by sterilizing the space to be sterilized, by pressing the biological indicator (1) The glass ampoule inside is broken to leak the culture solution, and after culturing for a predetermined time, the sterilization is determined by checking the color change.

This biological indicator (SCBI; Self-Contained Biological Indicator; 1) is,

Case (Case; 3) and; It is disposed inside the case (3) and the culture medium is stored in the capsule (Capsule; 13) and;

a protective tube 11 disposed to surround the outside of the capsule 13 and having an outlet hole h through which the culture solution flows;

And a cap portion (Cap portion; 5) coupled to the upper portion of the case (3);

Intake and exhaust fans (7a, 7b) each disposed in the cap portion (5) for sucking or exhausting air;

a pressure protrusion (9) disposed on the cap part (5) and descending when pressed to break the capsule (13) to drain the culture medium;

a spore support part 19 provided inside the cap part 5 to pick up or drop spores (Spore);

an air circulation path 18 disposed inside the case 3 to circulate the air supplied by the intake and exhaust fans 7a and 7b;

a filter (14) disposed between the cap part (5) and the case (3) to prevent the inflow of bacteria or spores; And

It is formed on the air circulation path 18 and includes a culture unit 17 that promotes the culture of the spilled culture solution and the fallen spores (Spores).

When describing the biological indicator 1 having this structure in more detail, the capsule 13 is disposed in the vertical direction inside the case 3 , and the culture solution is stored in the inner bottom. In this case, the capsule 13 has various shapes, for example, a cylindrical shape, a polygonal shape such as a triangular shape, a quadrilateral shape, and the like.

In addition, the capsule 13 is formed of a material that can be broken, for example, is formed of glass, synthetic resin, or the like. Therefore, as will be described later, when the pressing protrusion 9 of the cap part 5 is pressed, the pressing protrusion 9 is damaged by pressing the upper portion of the capsule 13 . And, when the capsule 13 is broken, the culture solution flows out into the protective tube 11 .

The protective tube 11 has a cylindrical shape, and the capsule 13 is disposed therein so as to surround the outside of the capsule 13 .

And, a plurality of micro-holes (h2) are formed on the outer peripheral surface of one side of the protective tube (11). These fine holes h2 are formed in the direction of one side of the case 3 , that is, in the direction of the first flow path a1 flowing from the intake fan 7a to the culture unit 17 . That is, it is not formed in the direction of the second flow path a2 flowing from the culture unit 17 to the exhaust fan 7b.

Therefore, the air introduced into the inside of the case 3 through the intake fan 7a flows to the culture unit 17 along the first flow path a1, and at the same time, some air flows through the plurality of fine holes h2 through the protective tube ( 11) is introduced into the

And, the outlet hole (h) is formed in the lower portion of the protective pipe (11). Therefore, when the culture solution is leaked due to the breakage of the capsule 13 , the culture solution leaked falls down through the outlet hole h of the protective tube 11 and is stored in the culture unit 17 .

At this time, the culture unit 17 is a space formed concavely on the air circulation path 18, and the culture solution and the spores (S) come into contact.

The air circulation path 18 includes an intake guide surface 15 for circulating the air sucked by the intake fan 7a by guiding it to the culture unit 17 along the first flow path a1; and an exhaust guide surface 16 guiding the air passing through the culture unit 17 to be discharged in the direction of the exhaust fan 7b along the second flow path a2.

Therefore, the external air sucked by the intake fan 7a is supplied to the culture unit 17 through the intake guide surface 15 of the culture unit 17 along the first flow path a1, or a plurality of fine holes h2. After flowing into the inside of the protective tube 11 through the , it is supplied to the culture unit 17 to promote culture, and then passes through the exhaust guide surface 16 by the suction force of the exhaust fan 7b to pass the second flow path a2. is discharged to the outside.

On the other hand, the intake and exhaust fans (7a, 7b) are detachably disposed on the cap portion (5), the intake fan (7a) is disposed on the upper portion of the intake guide surface (15) to suck outside air and supply it to the inside of the case (3) and ; and an exhaust fan 7b disposed on the exhaust guide surface 16 to discharge internal air to the outside of the case 3 .

At this time, the cap part 5 may be divided into left and right sides by forming a partition wall W in the middle of the inner side. Accordingly, it is possible to prevent the air sucked in by the intake fan 7a from flowing directly into the exhaust fan 7b region without flowing into the case 3 .

In addition, the cap part 5 may have a hexahedral shape as shown in FIG. 2 , but as shown in FIG. 5 , an inclined surface is formed at the corner, and the intake and exhaust fans 7a and 7b are disposed on the inclined surface. is also possible

The intake/exhaust fans 7a and 7b are rotated by the driving of a motor, and may include various types of fans, for example, a micro fan or the like.

At this time, since the intake fan 7a and the exhaust fan 7b have opposite fan structures, the air flows in opposite directions during rotation. That is, the intake fan 7a sucks in the outside air of the cap part 5 and flows it inwardly to form a first flow path a1, and the exhaust fan 7b causes the air inside the case 3 to flow outward. A second flow path a2 is formed.

Therefore, after the external air is sucked into the inside of the case 3 by the driving of the intake fan 7a and the exhaust fan 7b, it flows along the first flow path a1, passes through the culture unit 17, and then goes back to the second flow path. According to (a2), a circulation structure discharged to the outside is formed.

And, between the intake and exhaust fans (7a, 7b), the above-described pressing protrusion (9) is disposed to pressurize the capsule (13) to break it. That is, the pressing protrusion 9 is disposed in the vertical direction on the bottom surface of the cap part 5, and when the user presses the upper surface of the cap part 5, the pressing protrusion 9 descends by a certain distance to the lower side of the capsule 13. The upper surface is pressed, and the capsule 13 is damaged when the pressure is greater than a certain force.

Therefore, the culture solution stored in the capsule 13 may be leaked to the outside due to the breakage of the capsule 13 .

On the other hand, the spores (S) are supported by the spore support unit 19, and when the cap unit 5 is pressed, the spores S may fall and be supplied to the culture unit 17.

The spore support portion 19 includes an upper coupling piece 21 connected to the cap portion 5; The upper side is male and female coupled to the upper coupling piece 21, and the lower side is a lower coupling piece 23 to which the spores (S) are connected; It includes a support protrusion 25 for supporting the bottom surface of the upper coupling piece (21).

More specifically, the upper coupling piece 21 has an upper side connected to the cap part 5, a first locking protrusion 21a is formed on the lower side, and the bottom surface 21b is inclined to the support protrusion 25 by being inclined. contact

In addition, the second locking protrusion 23a is formed on the upper side of the lower coupling piece 23 , and the spores S are connected to the lower side by the connecting member 27 . At this time, the connection member 27 includes various types, for example, includes a wire (Wire), a thread, and the like.

Therefore, when the process of separating and dropping the spores S from the spore support 19 having this structure is described, first pressing the upper side of the cap part 5, the upper coupling piece 21 is descended a certain distance, at this time , the lower bottom surface 21b of the upper coupling piece 21 is in contact with the support protrusion 25, thereby proceeding outward along the arrow direction (a1).

In this way, when the lower side of the upper coupling side advances to the outside, the first locking jaw 21a of the upper coupling piece 21 also proceeds outward, so that the second locking jaw 23a of the lower coupling piece 23 and The bonding state is canceled.

Therefore, since the lower coupling piece 23 is dropped, the spores (S) connected thereto also fall to the culture unit 17 .

In this way, the spores (S) that have fallen into the culture unit 17 are in contact with the culture solution, and the culture proceeds.

On the other hand, since an attachment member M is provided on one side of the biological indicator 1, the indicator 1 can be stably fixed to a wall or the like.

The attachment member M includes various types for fixing the indicator 1 to the wall, and includes, for example, a magnetic method, a double-sided adhesive tape method, a Velcro method, and the like.

In addition, by attaching a sticker-type chemical indicator (CI) to the outside of the indicator 1, it is possible to check whether the sterilization has been removed from bacterial contamination.

In addition to sticker-type indicators, chemical indicator ink (CI Ink) can be printed on the surface.

Hereinafter, the method of using the biological indicator for space sterilization (1) proposed by the present invention will be described in more detail with reference to the accompanying drawings.

1 to 5, the user first lowers the pressing protrusion 9 by pressing the upper portion of the cap portion (5). The pressing protrusion 9 descends and comes into contact with the upper portion of the capsule 13 , and when a force exceeding a certain standard is applied, the capsule 13 is damaged. When the capsule 13 is damaged, the culture solution stored therein flows out, and the culture solution leaks fall through the outlet hole h of the protective tube 11 .

Then, the fallen culture solution is supplied to and stored in the lower culture unit 17 .

As such, when the capsule 13 is pressed and broken and the culture solution flows out, the upper coupling piece 21 also descends a certain distance.

At this time, the lower bottom surface 21b of the upper coupling piece 21 comes into contact with the support protrusion 25 to proceed outward along the arrow direction a1.

And, when the lower side of the upper coupling side advances to the outside, the first locking projection 21a of the upper coupling piece 21 also proceeds to the outside, so that the second locking jaw 23a of the lower coupling piece 23 and The bonding state is canceled. Accordingly, the lower coupling piece 23 is dropped, and the spores (S) connected thereto also fall to the culture unit 17 .

Therefore, the spore (S) that has fallen into the culture unit 17 is cultured by contacting the culture solution.

At this time, by turning on the switch, the intake fan 7a and the exhaust fan 7b are driven.

As the intake fan 7a is driven, external air is sucked in, passes through the filter 14 and then sucked into the case 3 .

The inhaled external air is supplied to the culture unit 17 through the intake guide surface 15 of the culture unit 17 along the first flow path (a1), or through a plurality of micro-holes (h2) inside the protective tube 11 . After being introduced into the, it is supplied to the culture unit (17).

And, after promoting the culture between the spores (S) and the culture medium in the culture unit 17, it is discharged to the outside along the second flow path (a2) through the exhaust guide surface (16) by the suction force of the exhaust fan (7b).

Through this process, it is possible to check whether the space is sterilized.

There are various ways to check whether sterilization is possible, but as described above, it can be confirmed through a chemical indicator attached to the outside of the case 3 in the form of a sticker, or through a color change of the chemical indicator ink.

Hereinafter, the present invention will be described in more detail through examples. These examples are only for illustrating the present invention in more detail, and it will be apparent to those of ordinary skill in the art that the scope of the present invention is not limited by these examples according to the gist of the present invention. .

<Example 1> Preparation of Geobacillus Stearothermophilus Endospores

Geobacillus sterarothermophilus standard strain (ATCC 12980) capable of forming endospores with high heat resistance was selected as the target bacteria and inoculated into Tryptic soy broth (TSB, Difco, Detroit, MI, USA) After that, the primary culture was performed at 55°C for 48 hours.

After the culture, hydrogen peroxide was added from 10 uM to 10 mM in the same medium, and secondary culture was performed for 7 days. After sufficiently washing the culture solution with distilled water, it was transferred to a 15 ml plastic tube (Becton Diskinson, Franklin Lakes, NJ, USA) and centrifuged at 3,000 × g for 15 minutes. After removing the supernatant by decanting, endospores were generated by heat shock incubating at 100° C. for 15 minutes. The formation of endospores was confirmed by observing non-refractive endospores under a relative-ratio microscope.

<Example 2> Isolation of endospores

The endospores obtained in Example 1 were separated from the feeder cells by repeated centrifugation (5,000×g, 10 minutes), and then washed 4 to 5 times with deionized water to isolate the endospores. The washed cells were examined under a microscope to confirm that they contained 95% or more of spores.

<Example 3> Isolation of endospores by density gradient centrifugation

The endospores isolated in Example 2 were redissolved in sucrose-containing Tris buffer (hereinafter referred to as 'buffer') and subjected to glycerol density gradient centrifugation. Glycerol was made from 50 (w/v)% to 100% with TS buffer, and the suspension containing endospores of Example 2 was placed on a glycerol gradient and centrifuged at 3,000×g for 30 minutes. After centrifugation, the endospore layer (density 1.13 to 1.15) formed below was separated, and then washed 5 times with TS buffer to remove glycerol. The content of the purified endospores was measured using the absorbance of a spectrophotometer, and the purity was confirmed with an electron microscope.

Then, the purified endospores were placed in a stainless steel disk, dried, and packaged aseptically.

<Example 4 and Example 5> Check the function of the biological indicator

The function was confirmed using the endospores prepared in Example 3 inside the biological indicator prepared according to FIG. 1 . The room was sterilized with vaporized hydrogen peroxide at a concentration of 300 ppm or higher (Example 4) and 50 ppm or lower (Example 5) for 15 minutes.

Then, after completion of sterilization, the indicator was incubated for 48 hours under a sterile, normal atmosphere. As a result, in Example 5, colonies of the strain were formed, and it could be confirmed that Geobacillus steathermophilus was proliferated into feeder cells, whereas in the strain of Example 4, no colonies were formed.

As mentioned above, although preferred embodiments of the present invention have been described in detail, the present invention is not limited to the above embodiments, and various modifications can be made by those skilled in the art within the scope of the technical spirit of the present invention. This is possible.

Claims (11)

a case (3);
a capsule 13 disposed inside the case 3 and storing the culture solution;
a protective tube 11 disposed to surround the outside of the capsule 13 and having an outlet hole h through which the culture solution flows;
A cap portion coupled to the upper portion of the case (3) (Cap portion; 5) and;
Intake and exhaust fans (7a, 7b) each disposed in the cap portion (5) for sucking or exhausting air;
a pressure protrusion (9) disposed on the cap part (5) and descending when pressed to break the capsule (13) to drain the culture medium;
a spore support unit 19 provided inside the cap unit 5 to pick up or drop the spores (S) (Spore);
After the air disposed inside the case 3 and sucked in by the intake fan 7a flows along the first flow path a1, it flows along the second flow path a2 by the exhaust fans 7a and 7b and is discharged to the outside. an air circulation path 18 being;
a filter 14 disposed between the cap part 5 and the case 3 to prevent the inflow of bacteria or spores; And
It is formed between the first and second flow paths (a1, a2) on the air circulation path (18) and includes a culture unit (17) that promotes the culture of the spilled culture solution and the fallen spores (Spores);
When the cap part 5 is pressurized, the pressure protrusion 9 descends and breaks the capsule 13, so that the culture solution inside the capsule 13 is leaked and falls through the outlet hole h of the protective tube 11 to the culture part. Biological indicator (1) for space sterilization in which culture with spores (S) is progressed by being supplied to (17). The method of claim 1,
A plurality of micro-holes h2 are formed on the outer peripheral surface of one side of the protective tube 11, and the micro-holes h2 are formed in the direction of the first flow path a1 flowing from the intake fan 7a to the culture unit 17, whereby the case 3 ) flows into the culture unit 17 along the first flow path (a1), and at the same time, some air is introduced into the inside of the protective tube 11 through a plurality of micro-holes h2. indicator (1). The method of claim 1,
The air circulation path 18 includes an intake guide surface 15 for circulating the air sucked by the intake fan 7a by guiding it to the culture unit 17 along the first flow path a1; and an exhaust guide surface 16 guiding along the second flow path a2 so that the air that has passed through the culture unit 17 is discharged in the direction of the exhaust fan 7b, and includes an intake guide surface 15 and an exhaust guide surface 16 A biological indicator (1) for space sterilization between which the culture section (17) is disposed. 4. The method of claim 3,
The intake and exhaust fans (7a, 7b) are disposed on the intake guide surface (15), the intake fan (7a) for supplying external air to the inside of the intake (3); A biological indicator (1) for space sterilization including an exhaust fan (7b) disposed on the exhaust guide surface (16) to exhaust the internal air to the outside of the case (3). The method of claim 1,
The spore support 19 includes an upper coupling piece 21 connected to the cap 5; The upper side is male and female coupled to the upper coupling piece 21, and the lower side is a lower coupling piece 23 to which the spores (S) are connected; It includes a support protrusion 25 for supporting the bottom surface of the upper coupling piece 21,
When the cap part 5 is pressed, the upper coupling piece 21 descends and the bottom surface 21b comes into contact with the support protrusion 25 to advance outwardly, and the first engaging protrusion 21a of the upper coupling piece 21 is lowered. ) and a biological indicator (1) for space sterilization in which the spores (S) fall by releasing the coupling state of the second locking jaw (23a) of the lower coupling piece (23). The biological indicator (1) for space sterilization according to claim 1, wherein the air is used for space sterilization by containing at least one vapor selected from the group consisting of vaporized hydrogen peroxide, chlorine dioxide vapor and glycel aldehyde vapor. . The biological indicator (1) for space sterilization according to claim 1, wherein the spore (Spore;S) is an endospore. According to claim 7, wherein the endospore is Bacillus subtilis ( Bacillus subtilis ), Bacillus subtilis globigii ( Bacillus subtilis globigii ), Clostridium sporogenes , Bacillus cereus ) , Bacillus circulans ( Bacillus circulans ), Bacillus atrophaeus ( Bacillus atrophaeus ) and Geobacillus stearothermophilus ) Biological indicator for space sterilization, characterized in that at least one selected from the group consisting of (1). The biological indicator for space sterilization (1) according to claim 8, wherein the endospores are Bacillus atropaeus, Geobacillus stearomophilus, or a combination thereof. The method of claim 7, wherein the endospores are vegetative cells (vegetative) using the endospores produced by culturing the strain producing endospores in a medium to which hydrogen peroxide is added at 55 ° C. cell), a biological indicator for space sterilization (1), characterized in that only resistant endospores are purely separated from normal spores and other impurities. 11. The method of claim 10, wherein the density gradient centrifugation uses one or more selected from the group consisting of sugar, glycerol, linogriffin and percol as a medium, and the medium uses a continuous density gradient of 50% to 100%. Biological indicator for space sterilization with (1).

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