JP2001245962A - Biological indicator for ozone sterilization - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a novel biological indication for ozone sterilization having excellent flexibility and no influence on ozone sterilizing power. SOLUTION: An index germ 3 such as Bacillus subtilis ATCC9372 exhibiting resistance against ozone sterilizing power is stuck to the surface of a membrane filter 2 such as PVDF having acid resistance and flexibility. In this way, the ozone sterilizing biological indicator 1 having flexibility and no influence on ozone sterilizing power can be obtained, and consequently, biological sterilizing effect based on ozone to a container 4 such as an eye lotion container with a small aperture can be accurately determined.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a biological (biological) indicator used for ensuring the sterilization of a sterilizing device such as a surgical instrument or a medical container in the medical field, and particularly suitable for ozone sterilization. And a biological indicator for ozone sterilization.

[0002]

2. Description of the Related Art Conventionally, high-pressure steam sterilization, ethylene oxide gas sterilization, hydrogen peroxide water sterilization, and the like have been employed as sterilization methods for such medical instruments and pharmaceutical containers. Instead of the method, an ozone sterilization method using ozone gas or ozone water is being studied.

That is, this ozone (O ₃ ), of course, exhibits excellent sterilizing power due to its strong oxidizing power.
Since it does not require high-temperature and high-pressure conditions as in the high-pressure steam sterilization method, it is suitable for heat-sensitive objects such as plastic containers, and it decomposes into harmless oxygen in a short time. It has an excellent advantage that no harmful residue is left on the object to be sterilized as in a sterilization method. Furthermore, since this ozone can be easily obtained by causing silent discharge in dry air or oxygen, or irradiating ultraviolet light into the air, it can be obtained inexpensively and in large quantities as compared with the conventional method. It has the advantage of being able to work.

On the other hand, in an apparatus using a conventional sterilization method, a sterilization performance test is regularly performed to assure sterilization. Conventionally, as a method for inspecting the sterilization performance, a method of directly performing an aseptic test on an object to be sterilized after a sterilization treatment has been adopted. Recently, however, a simpler method is to use a biological sample at an arbitrary position in a sterilization apparatus. A method has been adopted in which a (biological) indicator is provided, and determination is made using the sterilization effect of the biological indicator after sterilization as an index.

[0005] For example, in the case of an apparatus for sterilizing a large number of pharmaceutical containers as an object to be sterilized, a maximum resistance value for the sterilization method is indicated in a dummy container having the same shape as the pharmaceutical container. After accommodating a sheet-shaped carrier coated with spores of possible bacteria, sterilization treatment was performed,
The biological sterilization effect is determined by injecting a culture solution into the dummy container and culturing it for a certain period of time.

[0006]

By the way, most of carriers conventionally used as such a biological indicator are made of a material having low flexibility such as stainless steel or glass fiber filter paper. Therefore, it has a drawback that it is difficult to put it into a small-diameter (several mm) container such as a container for eye drops.

For this reason, it is conceivable to form the carrier from a highly flexible material such as cellulose.
As described above, when ozone is used as a sterilization method, a part of the sterilizing power (oxidizing power) of ozone is used for the decomposition of cellulose, so that it is inappropriate to use it for an accurate sterilization assurance test. Was.

Further, the stainless steel carrier is not suitable as a biological indicator for ozone because the inoculated bacteria are easily condensed and the sterilization effect is greatly varied, and the glass fiber filter paper carrier generally has a coarse pore size, and the bacteria inside the glass carrier. It has a drawback that it is not suitable for sterilization of liquid systems such as ozone water because it sneaks down.

The present invention has been devised in order to effectively solve such a problem, and its main object is to provide a high flexibility and to affect the sterilizing power of ozone. The present invention provides a novel biological indicator for ozone sterilization without the use of a biological indicator.

[0010]

In order to solve the above-mentioned problems, according to the present invention, the surface of a flexible membrane filter having oxidation resistance has high ozone sterilization power. An indicator bacterium that exhibits resistance is attached.

That is, by using an oxidation-resistant flexible membrane filter as a carrier for bacteria, the membrane filter can be easily folded and stored in a small-diameter container such as a container for eye drops, etc. Thus, the contact with ozone water or ozone gas is improved, and the biological sterilization effect of ozone in the container can be accurately determined. In addition, the adhesion (application) of this membrane filter
Conventionally, it can be easily performed by a suction filtration method generally used as a microorganism test method.

As for ozone gas, as described in claim 2, if the membrane filter obtained in claim 1 is further wrapped with permeable sterilized paper, the biological indicator can be prevented from being contaminated or damaged during handling. In addition to being able to be effectively protected, it is also possible to simultaneously prevent secondary contamination of the sterilizing apparatus and the environment by surviving indicator bacteria.

[0013] More specifically, it is most preferable to use either Bacillus subtilis ATCC9372 or Bacillus stearothermophilus ATCC12980 as the indicator bacterium. That is, as will be described later, as a result of examining the ozone sterilization resistance of bacteria belonging to the genus Bacillus, which is said to have high resistance to ozone sterilization power among known indicator bacteria, Bacillus stearothermophi
lus was found to exhibit the best resistance to ozone sterilization, and Ba is often detected as a biobargain.
This is because cillus subtilis was found to be usable as well.

As the membrane filter, specifically, hydrophilic PVDF (polyvinylidene difluoride) is suitable as described in claim 4, and an eye dropper having a small diameter as described above is used. When Bacillus subtilis ATCC9372 or the like is used as the target bacterium and as an indicator bacterium, it is desirable to use a membrane filter having a diameter of 47 mm or less and a pore diameter of 0.45 μm or less. That is, if the diameter is 47 mm or less, it can be easily placed in an eye drop container or the like having a small diameter, and at the same time, it can be restored to almost its original shape after being placed in a small container. Also, the hole diameter 0.4
If it is 5 μm or less, the indicator bacteria, such as Bacillus subtilis ATCC9372, will only bite into the eyes of the filter and will not enter the inside of the filter, so that an accurate sterilization effect can be obtained.

[0015]

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS Next, a preferred embodiment of the present invention will be described with reference to the accompanying drawings.

FIG. 1 shows an embodiment of a biological indicator (hereinafter abbreviated as BI) 1 for ozone sterilization according to the present invention.

As shown in the figure, this BI is formed on one surface of an oxidation-resistant flexible membrane filter 2 having a diameter of 25 mm, a film thickness of 125 μm and a pore diameter of 0.22 μm, for example.
The indicator bacteria 3 exhibiting high resistance to ozone sterilization power are attached (applied) by suction filtration or the like.

The membrane filter 2 has excellent oxidation resistance and flexibility, that is, oxidation resistance that is inactive against ozone and does not affect the sterilizing power of ozone, and can be easily folded and stored. A material having flexibility and restoring properties such that the material can be easily restored to the original shape after it has been stored (in a container) is used. In addition, it is desirable that the surface has appropriate hydrophilicity for adhering indicator bacteria in the liquid by suction filtration or the like.

As a known material satisfying such conditions, hydrophilic PVDF (Polyvinylidenedifluoride) is most suitable, and in addition, hydrophilic PTFE (Polytetrafluid) is used.
oroethilene) can also be used.

On the other hand, the indicator bacteria 3 attached to the membrane filter 2 are of the genus Bacillus, which is said to have the highest resistance to the sterilizing power of ozone water.
Bacillus stearothermophilus ATCC12980, or Bacill
It is desirable to use us subtilis ATCC9372. That is, when the present inventors preliminarily selected the most resistant bacteria to ozone water sterilization before reaching the present invention,
Kinds of bacteria exhibit high resistance to ozone water, especially
This is because Bacillus stearothermophilus ATCC12980 exhibited the most resistance, and thus was found to be optimal as an indicator bacterium applied to the BI of the present invention. In particular,
Spore suspensions and Aspergillus of 11 major Bacillus species
Using Nigar spore suspension, water temperature about 20 ℃, concentration about 5
As a result of conducting a resistance test to 0 mg / L ozone water, the initial number of bacteria was about 10 ⁴ CFU (Colony Forming Unit,
Bacillus stearothe per colony forming unit) / ml
rmophilus ATCC12980 only 244C even with a retention time of 45 seconds
This is because FU / ml survived, but the remaining 10 species of Bacillus and Aspergillus Nigar almost all died in a retention time of 45 seconds. However, this Bacillus stearothermophi
lus ATCC12980 rarely exists as bioburden bacteria in plants, but rather Bacillus subtilis ATCC
9372 is better detected, so actually this Bacill
It is considered that us subtilisATCC9372 is more frequently used.

The BI of the present invention having such a structure
Although the method for producing is not particularly limited, it is necessary to prevent the indicator bacteria 3 attached to the membrane filter 2 from easily detaching from the membrane filter 2. That is, when the indicator bacteria 3 flow out to the ozone water side when immersed in the ozone water or are blown off by the flow of the ozone gas, it is impossible to accurately determine the sterilization effect to be performed later. B that satisfies such conditions
When the membrane filter 2 made of PVDF as described above is used as the method for producing I, suction filtration is optimal, and specifically, about 1.5 ×
After preparing a bacterial solution diluted to 10 ³ / ml and counting the initial number of bacteria, a 25-fold diluted water of the bacterial solution was prepared, and 2 times diluted water was prepared on the membrane filter 2 installed in the filter trap.
5 ml was poured, and this was sucked with a vacuum pump at a pressure of about 300 mmHg so that the indicator bacterium 3 was brought into close contact with the membrane filter 2, and then the membrane filter 2 was transferred onto a sterile petri dish with sterile tweezers.
It can be easily obtained by natural drying on a clean bench.

The thus obtained BI of the present invention is folded into a small size, for example, a PET (polyethylene terephthalate) or the like having a small diameter of several mm as shown in FIG. The membrane filter 2 can be easily stored in the eye drop container 4 and returns to the substantially original shape by the restoring force of the membrane filter 2 itself after the storage. Therefore, at the time of ozone sterilization, the indicator bacteria 3 on the surface and the ozone water or ozone gas flowing into the container 4 come into reliable contact with each other, so that the sterilization effect by ozone can be accurately determined. Becomes

The BI for ozone gas is as follows:
As shown in FIG. 3, it is possible to apply the BI as it is, but it is preferable to use the BI by first wrapping it in a permeable sterile paper 5. That is, this BI
Is protected by air permeable sterilized paper 5 to prevent damage and contamination of BI during handling, and at the same time, prevent secondary contamination of the production environment such as clean room at the time of equipment and transportation due to surviving indicator bacteria. This is because it can be prevented. In addition, it is necessary to use an air-permeable sterilizing paper that does not absorb ozone gas and that allows good permeation of the ozone gas. For example, it has been used as a sterilizing paper for the primary packaging of hydrogen peroxide sterilizing BI. Tyvek wrapping paper or the like is suitable.

[0024]

EXAMPLES Next, specific examples relating to the evaluation of the sterilizing power of ozone using the BI for ozone sterilization according to the present invention will be described.

(Example 1: Ozone water sterilization) The medical container obtained by the above-mentioned method is put into a medical container, filled with ozone water, and after the elapse of the holding time, a sodium thiosulfate solution is dropped. After removing the ozone, the treated water is sprinkled out of the container, and the treated water and BI are
The cells were cultured in a CD medium (Soybean Casein Digest, soybean casein digest medium) for one week to check for survival of the indicator bacterium, and the fraction negative method of Stumbo-Murphy
The D value was calculated by the Cochran Procedure (SMCP).

When sterilized with ozone water at a concentration of 40 mg / L and a water temperature of 20 ° C., the initial number of bacteria is 1.0 × 10
The D value of ³ CFU in Bacillus stearothermophilus ATCC 12980 is about 47 seconds, and the same initial number of Bacillus subtilis
The D value at ATCC 9372 was 6.8 seconds.

As shown in Table 1 below, the concentration was 3
When sterilized with ozone water at a water temperature of 20 ° C even when the concentration is reduced to 0 mg / L, the initial number of bacteria is 1.0 × 10 ³ CFU of Bacillus s.
The D value for tearothermophilus ATCC12980 is about 57.2
Seconds, D in Bacillus subtilis ATCC9372 with the same initial bacterial count
The value was 7.5 seconds, and it was found that the sterilizing power was not so different from that at a concentration of 40 mg / L.

[0028]

[Table 1]

As shown in Table 2 below, the concentration was 3
Bacillus subtilis ATCC with an initial cell count of 1.0 × 10 ³ CFU, fixed at 0 mg / L, and the water temperature was divided into 23 ° C. and 17 ° C.
When the same sterilization test was performed on 9372, the higher the water temperature, the lower the D
It was confirmed that the value was small and the sterilizing power was high.

[0030]

[Table 2]

(Example 2: Ozone gas sterilization) The same BI as used in Example 1 was prepared by tyvek packaging, and the BI was set in a thermo-hygrostat at a set temperature of 23 ° C, and ozone gas having a concentration of 6000 ppm was used. At a flow rate of 2.5 L / min, purged of oxygen gas after the elapse of the holding time to remove ozone, and then cultivated BI treated with ozone gas in an SCD medium for 1 week to survive the survival of indicator bacteria. And the D value was calculated by the SMCP method.

As a result, as shown in Table 3 below, 1.
When 0 × 10 ³ CFU of Bacillus subtilis ATCC9372 was treated with dry ozone gas, the D value was 18 minutes. On the other hand, when the same BI was treated with ozone gas having a relative humidity of 60%, the D value was 10.7 minutes. Furthermore, when the same BI was treated with ozone gas at a relative humidity of 80%, it was presumed that only 10 treatments resulted in the death of all 20 specimens, and the D value was within 2 minutes. In addition, it was calculated that the D value was also within 2 minutes even when the temperature was lowered from 23 ° C. to 18 ° C. at the same relative humidity of 80%.

[0033]

[Table 3]

As can be seen from the above examples, Bacillus stearothermophil was added to the membrane filter.
With the BI for ozone sterilization of the present invention to which us ATCC12980 or Bacillus subtilis ATCC9372 is adhered (applied), it can be confirmed that a stable D value can be obtained in an ozone water sterilization validation test. By packaging, it was confirmed that it can be used as a BI for ozone gas sterilization.

[0035]

In summary, according to the present invention, the following excellent effects can be exerted.

(1) Since PVDF or the like having a hydrophilic property is used as a carrier for bacteria by using a membrane filter, it can be easily folded and stored in a small-diameter container such as a container for eye drops or the like. Thus, the contact with ozone water or ozone gas is improved, and the biological sterilization effect of ozone in the container can be accurately determined.

(2) The membrane filter is further wrapped in air-permeable sterilized paper to prevent contamination of a clean room or the like by surviving indicator bacteria and to aseptically transport BI after sterilization. It can be used for sterilization of equipment and manufacturing environment.

(3) Since either Bacillus subtilis ATCC9372 or Bacillus stearothermophilus ATCC12980, which has high resistance to ozone sterilization, is used as an indicator bacterium, the sterilization effect of ozone can be accurately determined. .

(4) A membrane filter having a diameter of 47
By using a membrane filter of a thin film having a diameter of 0.4 mm or less and a pore diameter of 0.45 μm or less, it can be easily placed in a small-diameter eye dropping container, etc. In addition, since the indicator bacteria do not enter the inside thereof, an accurate sterilization effect can be obtained.

[Brief description of the drawings]

FIG. 1 is an enlarged perspective view showing an embodiment of a biological indicator for ozone sterilization according to the present invention.

FIG. 2 is an explanatory view showing an example of use of the biological indicator for ozone sterilization according to the present invention.

FIG. 3 is an enlarged perspective view showing another embodiment of the biological indicator for ozone sterilization according to the present invention.

[Explanation of symbols]

 1 Biological Indicator for Ozone Sterilization 2 Membrane Filter 3 Indicator Bacteria 4 Small Diameter Container 5 Sterile Paper

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) G01N 33/50 G01N 33/50 Z // (C12Q 1/02 (C12Q 1/02 C12R 1: 125) C12R 1: 125) (C12Q 1/02 (C12Q 1/02 C12R 1:07) C12R 1:07) (72) Inventor Tomio Mizuno 3-1-1-15 Toyosu, Koto-ku, Tokyo Ishikawajima Harima Heavy Industries, Ltd. Tokyo Engineering Center (72) Inventor Hiroshi Takahashi 3-2-1-16 Toyosu, Koto-ku, Tokyo Ishikawajima-Harima Heavy Industries, Ltd.Tokyo Engineering Center (72) Inventor Sakae Fukunaga 1 Shinnakaharacho, Isogo-ku, Yokohama, Kanagawa Prefecture 2G045 AA28 BB19 CB21 FA07 FB07 FB15 HA10 4B029 AA07 BB02 CC02 CC11 FA01 FA15 4B033 NA01 NA12 NB34 NB70 ND05 ND11 NF06 4B063 QA01 QA20 QQ06 QQ89 QQ98 QR50 QR75 QR84 QS24 QS39 4C058 AA12 AA14 AA16 BB07 DD15 JJ14

Claims (5)

[Claims] 1. A biological indicator for ozone sterilization, comprising an indicator bacterium exhibiting high resistance to ozone sterilization attached to the surface of a flexible membrane filter having oxidation resistance. . 2. An indicator bacterium exhibiting high resistance to ozone sterilization force is attached to the surface of a flexible membrane filter having oxidation resistance, and this is wrapped with ozone-permeable sterilized paper. A biological indicator for ozone sterilization, comprising: 3. The method according to claim 2, wherein the indicator bacterium is Bacillus subtilis ATCC9.
The biological indicator for ozone sterilization according to claim 1 or 2, which is any one of 372 and Bacillusstearothermophilus ATCC12980. 4. The method according to claim 1, wherein the membrane filter is a hydrophilic P.
The biological indicator for ozone sterilization according to any one of claims 1 to 3, comprising VDF (polyvinylidene difluoride). 5. The diameter of the membrane filter is 47 m.
5. The biological indicator for ozone sterilization according to claim 1, wherein the pore size is 0.45 μm or less.