JPH11319040A - Sterilization of culture medium - Google Patents

Abstract

PROBLEM TO BE SOLVED: To surely sterilize a culture medium without exerting an influence on a culture medium compsn. by irradiating the culture medium with a specific range of electron beam as a substantial irradiation dose. SOLUTION: When various bacteria or microorganisms present in a container made of a synthetic resin such as a polyolefine resin, an acrylic resin or a vinyl chloride resin or a container made of glass and a culture medium are little, the container and the culture medium are irradiated with electron beam of 5-40 kGy as a substantial irradiation dose. When the synthetic resin or glass container is housed in a corrugated cardboard box or the like to be sterilized by electron beam, the substantial irradiation dose received by the housed container is set to 10-40 kGy. By this constitution, the powder culture medium, granular culture medium, agar culture medium or liquid culture medium housed in the container can be sterilized within a short time.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a method for sterilizing a culture medium, and more particularly to a method for performing a sterilization treatment by irradiating an electron beam.

[0002]

2. Description of the Related Art Conventionally, as methods for sterilizing a medium, EOG sterilization using ethylene oxide gas (hereinafter referred to as "EOG"), γ-ray sterilization, and the like are well known.

[0003] However, since EOG sterilization cannot sterilize the container in a sealed state, there is a possibility that various bacteria may be mixed in the sealing operation performed later. Further, since the sterilizing gas is toxic, it is necessary to perform post-treatment such as aeration, and even if the sterilizing gas remains in the medium container at all, there is a possibility that the sterilizing gas may adversely affect the human body. Furthermore, the treatment time generally requires several tens of hours, which causes problems such as solidification and discoloration of the medium over time and deterioration of the performance as a medium.

On the other hand, γ-ray sterilization has a large effect on the physical properties of the container itself, causing coloring of the container itself, and troublesome management and maintenance of the radiation source. Also, similar to EOG sterilization, the time required for sterilization is long. For example, it may take as long as 25 hours to irradiate 25 kGy of γ-rays. Therefore, there are problems such as a high running cost.

The present invention has been made to solve the above-mentioned problems in the prior art, and has as its object to surely sterilize a medium without affecting the composition of the medium, and to improve the time-dependent change and performance of the medium over time. It is an object of the present invention to provide a method for sterilizing a medium that can be performed at a low cost while preventing or suppressing the decrease.

[0006]

Means for Solving the Problems As a result of intensive studies, the present inventors have solved the above-mentioned problems by irradiating a medium with an electron beam of 3 kGy-40 kGy as a substantial irradiation dose, and at the same time, the above-mentioned objects have been achieved. Achieved.

[0007] That is, if an electron beam is used for sterilizing the medium, the medium can be surely sterilized without affecting the composition of the medium, and the composition change of the medium over time and the performance deterioration accompanying the medium can be prevented. It has been found that sterilization can be performed at low cost and in a short time.

The irradiation of the electron beam in the present invention is performed using an electron beam accelerator. The electron beam accelerator is not particularly limited as long as it emits high-energy electron beams. For example, "Dynamitron (mark)" manufactured by Sumitomo Heavy Industries, Ltd. is used. This electron beam accelerator
Acceleration energy can be set steplessly from 0.5MeV to 5MeV,
The object to be processed conveyed by the cart conveyor is irradiated with an electron beam having the above energy.

[0009] The medium used in the sterilization method according to the present invention may be not only a commonly used powder medium or granule medium but also a liquid medium or a gel medium.
However, when electron beam sterilization is performed on agar in a powder state or a powder medium containing agar, the three-dimensional structure of the agar changes,
It is not suitable as a medium component used in the present invention because it does not gel. When the agar medium is subjected to electron beam sterilization, if the agar is gelled and then subjected to electron beam sterilization, the sterilization treatment can be performed in the same manner as the other medium without changing the three-dimensional structure of the agar.

As the medium container used in the method for sterilizing a medium according to the present invention, a container made of glass or synthetic resin can be used. There is no particular limitation on the material of the synthetic resin, for example, polyolefin resin, AS resin, A
Containers made of BS resin, polystyrene, polyethylene, polypropylene, acrylic resin, polyethylene terephthalate, polycarbonate, methylpentene polymer, polysulfone, polyamide, polyacetal, polyester, and vinyl chloride resin can be used.

According to the electron beam sterilization method of the present invention, a container containing a culture medium can be sterilized in large quantities at a time, and can be sterilized while being stored in a cardboard box or the like. For example, the above glass or synthetic resin round bottle (body diameter: 18 mm, height: 100 mm, wall thickness: 1.5 mm)
Sterilization can be performed even in a state in which the cardboard boxes are housed in a cardboard box that can be accommodated, and the cardboard boxes are further housed in a packaging container that can house seven boxes.

Thus, the container accommodating the culture medium can be sealed and the sterilization process can be performed even in the packed state, so that the operation can be greatly simplified. In addition, since sterilization can be performed after the packing operation is completed, sterilization can be surely performed without fear of contamination by germs during the operation.

When there are few germs or microorganisms present in a synthetic resin container or a glass container or a medium, a substantial irradiation dose of about 3 kGy can be used for sterilization. Effective irradiation dose is 5kGy-
Desirably 40 kGy. However, with substantial irradiation dose
When the irradiation dose is set to 40 kGy or more, not only the medium components are denatured or discolored, but also the container may be colored, which is not preferable.

When the electron beam sterilization is performed in the state of being housed in a cardboard box or the like as described above, an obstacle (a cardboard box or the like) is present before the electron beam reaches the surface of the object (container and culture medium) to be sterilized. Therefore, the irradiation dose (substantial irradiation dose) actually reaching the inner surface of the target object is slightly smaller than the irradiation dose emitted from the electron beam accelerator. Therefore, when irradiating an electron beam, it is necessary to set the irradiation dose of the electron beam in consideration of the substantial irradiation dose. That is, when sterilizing the container containing the culture medium at 5 kGy, set the irradiation dose so that the dose received by the container (substantial irradiation dose) becomes 5 kGy or more. Therefore, the irradiation dose in this case is preferably from 10 kGy to 40 kGy.

However, since the substantial irradiation dose cannot be actually measured, the “substantial irradiation dose” in the present invention refers to the irradiation dose applied to the surface of the object to be sterilized (surface irradiation dose). Is measured by a dosimeter, and the value is estimated and applied as a substantial irradiation dose for convenience. However, this is a case where the electron beam sterilization is performed in the package state, and when directly irradiating the container, the surface irradiation dose can be considered to be substantially the same as the substantial irradiation dose.

According to the method for sterilizing a medium according to the present invention, the medium can be sterilized in only a few seconds. Therefore, unlike EOG sterilization and γ-ray sterilization, which require several hours for sterilization, deterioration of the culture medium and container can be minimized.

When a container containing a medium is sealed and sterilized in a packaged state, continuous sterilization can be performed by using a cart conveyor. Sterilization can be performed by electron beam irradiation for 3 seconds.

[0018]

Now, the present invention will be described in further detail with reference to Comparative Test Examples and Examples.

Comparative Test Example 1 (Study on Storage Stability of Medium) (1) A medium sterilized by the method of the present invention and a conventional medium
To compare and examine the storage stability of the medium sterilized by EOG sterilization and γ-ray sterilization, sterilization was performed by the following method.
In this comparative test example, peptone (5.
0g), sodium chloride (5.0g), potassium dihydrogen phosphate
(1.0 g), sodium lauryl sulfate (0.1 g), sodium pyruvate (1.0 g), potassium nitrate (1.0 g), isopropyl-β-D-thiogalactopyranoside (IPTG, 0.1 g), 5-bromo-4 -Chloro-3-indolyl-β-D-galactopyranoside (X-GAL, 0.1 g), 4-methylumbelliferyl-β-D-
The case where a water quality test medium (powder) composed of glucuronide (MUG, 0.1 g) is used is described.

Sterilization of Medium by Electron Beam A medium for water quality test is placed in a glass round bottle (body diameter 16 m).
m, height 100mm, wall thickness 1.5mm. The same shall apply hereinafter), and the medium-containing container is charged with an electron beam accelerator (“Dynamitron” mark)
Using 4.8 MeV voltage, 5 mA current,
The planned irradiation dose was set to 5 kGy, the speed of the cart conveyor was set to 6.44 m / min. Under the beam, and the round bottle was laid on its side to irradiate an electron beam from above. After irradiation, the surface irradiation dose of the container was measured with a dosimeter, and it was 6.6 kGy in the upper part of the glass container, 7.4 kGy in the middle part of the glass container, and 8.7 kGy in the lower part of the glass container.

Sterilization of Medium by EOG A medium for water quality test is put in a glass round bottle in the same manner as the electron beam sterilization medium, and a mixed gas of 20% of EOG and 80% of carbon dioxide is added.
After treating at a temperature of 50 ° C. for 5 hours under a pressure condition of 0.4 kg / cm ^2, the gas remaining in the chamber was replaced nine times with air passed through a hepar filter.

Sterilization of culture medium by γ-ray As in the case of the electron-beam sterilization medium, a culture medium for water quality inspection is placed in a glass round bottle, and irradiated with γ-rays by a ⁶⁰ Co source raising / lowering device for 6 hours, thereby performing γ-ray sterilization treatment equivalent to 6 kGy. Was done.

(2) Using an electron beam sterilized medium, an EOG sterilized medium, and a γ-ray sterilized medium treated by the above sterilization method, an appearance test, an absorbance test, a pH test, and a culture test were performed as follows. As a control, a test was similarly performed on a medium that was not sterilized (unsterilized medium).

Appearance test The medium obtained by the above sterilization method was subjected to 4 ° C., 10 ° C., 30 ° C.
The medium was stored under different temperature conditions of 40 ° C., and changes in the color, shape, etc. of the medium were observed over time for 12 months. In addition, 4 ℃, 10
Five culture media were prepared under the condition of ℃ and observed until 12 months. On the other hand, six media were prepared at 30 ° C. and 40 ° C., respectively, and observed until 6 months had elapsed.

The results are shown in Table 1. The medium after sterilization is
Although it was slightly browned by the sterilization treatment, the electron beam sterilization medium maintained a powdered state after sterilization, like the γ-ray sterilization medium and the non-sterilization medium, whereas the EOG sterilization medium solidified. It was confirmed that. In addition, it was found that under the storage conditions of 4 ° C and 40 ° C, the mediums of the electron beam sterilization medium and the γ-ray sterilization medium began to solidify after 3 months.

From the above results, if the medium is irradiated with an electron beam and sterilized, the medium can be stored with higher quality than EOG sterilization, and the storage stability equivalent to that of γ-ray sterilization can be obtained. Became clear.

[0027]

[Table 1]

Absorbance test After dissolving the medium subjected to the appearance test in sterile water (10 ml), the wavelength was measured using a photoelectric photometer (ANA-7S, manufactured by Tokyo Koden).
The absorbance was measured at 420 nm. The results are shown in Table 2.
The absorbance of the non-sterile medium was the lowest, followed by the electron beam sterilized medium, the γ-ray sterilized medium, and the EOG sterilized medium. From the above results, it was found that the discoloration of the medium after the sterilization treatment was the least in electron beam sterilization.

[0029]

[Table 2]

PH Test The pH of the medium subjected to the absorbance test was measured using a pH meter (HM-60V, manufactured by Toa Corporation). The results are as shown in Table 3, and the pH immediately after the sterilization treatment was
Although the pH of the medium sterilized by EOG slightly increased, the medium sterilized by γ-rays and electron beams showed almost the same value as that of the non-sterile medium. Has not been found to give a large fluctuation. Thereafter, the medium sterilized by the sterilization method according to the present invention did not show a large change in pH.

[0031]

[Table 3]

Culture test The sterilized purified water (10%) was added to the electron beam sterilized medium, EOG sterilized medium, γ-ray sterilized medium and non-sterile medium obtained by the sterilization method described above.
After adding Escherichia coli (ATCC
11775) and X-GAL (5-Bromo-4-chloro-3-indoxyl)
-β-D-galactopyranoside, BIOSYNTH) and M
UG (4-Methylumbelliferyl-β-D-glucuronic acid dihy
_{drate, C 16 H 16 O 9} · 2H 2 O, it was observed color development reaction in manufactured BIOTHYNTH Co.).

Table 4 shows the results. If the medium components have deteriorated due to sterilization or due to changes over time, the color reaction is weakened or no color reaction is observed.However, the medium treated by the sterilization method of the present invention can be used for the X-GAL reaction and MUG reaction. A color reaction was observed due to the reaction, and it was clarified that the medium components did not change with an electron beam with a substantial irradiation dose of about 7 kGy, suggesting that the storage stability was maintained for at least 12 months. On the other hand, it was observed that the X-GAL reaction of EOG sterilized medium and γ-ray sterilized medium weakened over time,
It was presumed that the medium components had deteriorated with time.

[0034]

[Table 4] X: X-GAL reaction, M: MUG reaction, +: reaction, W: weak reaction, NT: not tested

Comparative Test Example 2 (Examination of Irradiation Dose) (1) Next, in the sterilization method according to the present invention, sterilization was performed by changing the electron beam irradiation conditions to 6 kGy to 60 kGy in the following manner. The influence of the difference on the culture medium and the container was examined.

Electron beam sterilization A culture medium for water quality inspection is placed in a polyolefin resin round bottle (diameter of main body: 18 mm, height: 100 mm, wall thickness: 1.5 mm).
The container was sealed with a lid made of polypropylene, and the container was irradiated with an electron beam using an electron beam accelerator ("Dynamitron"). The voltage was set to 4.8 MeV, and the current set value and the cart conveyor speed under the beam were set to the values shown in Table 5 below so that the surface irradiation dose was 6 kGy, 10 kGy, 20 kGy, 40 kGy, and 60 kGy, respectively.

[0037]

[Table 5]

Here, 40 kGy irradiation is obtained by repeating 20 kGy irradiation twice, and 60 kGy irradiation is 20 kGy irradiation.
Repeated three times. The irradiation of the electron beam was performed from the upper part with the container standing. Table 6 shows the surface irradiation dose measured with a dosimeter.

[0039]

[Table 6]

EOG sterilization In the same manner as in the case of electron beam sterilization, a granular medium for water quality inspection is placed in a polyolefin resin round bottle, and EOG 20% and carbon dioxide gas are added.
80% mixed gas at a pressure of 0.4 kg / cm ² at a temperature of 5
After treatment at 0 ° C for 5 hours, the gas in the chamber was replaced nine times with air passed through a hepar filter.

Γ-ray sterilization In the same manner as the electron beam sterilization medium, a granular medium for water quality inspection was placed in a polyolefin resin round bottle, and γ-rays were irradiated at 6 kGy, 10 kGy, 20 kGy, 40 kGy, and 60 kGy by a ⁶⁰ Co source raising / lowering device.

(2) Test method Using an electron beam sterilized medium, an EOG sterilized medium, and a γ-ray sterilized medium treated by the sterilization method described above, the following container appearance test, medium appearance test, pH test, absorbance test, A sterility test and a performance test were performed. In addition, the same test was performed on a non-sterile medium as a control.

Container appearance test As a result of observation of the container obtained by the above sterilization method, no coloration was observed in the appearance of the container after irradiation with an electron beam even when the container was irradiated with an electron beam as strong as 60 kGy. From this, it became clear that the sterilization method according to the present invention is suitable for sterilization of a culture medium for which a test result is determined based on color development or luminescence. On the other hand, in EOG sterilization, peeling of the inner lid of the container was observed, and it was found that the container colored brown when irradiated with γ-rays at 40 kGy or 60 kGy (see Table 7 “Appearance of container”). .

Culture Medium Appearance Test As a result of observing the culture medium obtained by the above sterilization method, no change was observed in the culture medium that had been subjected to electron beam sterilization even under the irradiation conditions of 40 kGy. On the other hand, the medium, which should have been milky white in the past, turned yellow after EOG sterilization, and it was observed that the medium adhered to the wall of the container.
On the other hand, when the γ-ray and electron beam irradiation conditions were 60 kGy, the color changed to gray (see the column in Table 7, “Appearance of culture medium”).

PH Test The medium tested in the above appearance test was dissolved in sterilized water (10 ml), and measured with a pH meter (HM-60V, manufactured by Toa Corporation). As a result, in the medium treated by the sterilization method according to the present invention, no significant pH fluctuation was observed under any of the sterilization conditions, as in the other sterilization methods (Table 7).
PH column).

Absorbance Test The medium tested in the above pH test was measured for absorbance at a wavelength of 420 nm using a photoelectric photometer (ANA-7S, manufactured by Tokyo Koden Co., Ltd.). As a result, the absorbance tended to increase slightly as the irradiation amount of the electron beam increased, but the change in the absorbance was smaller than that of the γ-ray sterilized medium or the EOG sterilized medium as a whole. From this, it was presumed that the discoloration of the medium in the sterilization was the least in the case of electron beam sterilization (see the column of "absorbance" in Table 7).

Sterility test The medium obtained by each sterilization method was dissolved in sterilized tryptosome bouillon (manufactured by Nissui Pharmaceutical Co., Ltd.),
After culturing at 35 ° C for 1 week and confirming the sterility, the medium treated by the sterilization method of the present invention was confirmed to be sterile under all irradiation conditions, and the sterilizing effect of the electron beam according to the present invention was confirmed. Proven. However, the growth of Gram-positive staphylococci was observed only in the non-sterile medium, and it was confirmed that such a medium for water quality test also needs to be sterilized (see Table 7 “Sterility Test”).

[0048]

[Table 7] * 1: Medium adheres to the wall, * 2: Gram-positive staphylococci grow, NG: No growth

Performance test Sterilized purified water (10 ml) was added to the electron beam sterilized medium, EOG sterilized medium, γ-ray sterilized medium and non-sterile medium obtained by the sterilization method described above.
, And the medium is sufficiently stirred, and then Escherichia coli (ATC
C 11775), Citrobacter freundii (ATCC 8090) and St
aphylococcusaureus (ATCC 6538)
-The reaction of GAL and MUG and the presence or absence of bacterial growth were observed. The medium used for the water quality test was E. coli.
And C. freundii can grow, but S. aureus , a gram-positive bacterium, has the ability to grow. In addition, C. freundii cannot secrete an enzyme for producing a MUG reaction even when proliferated, so that the MUG reaction does not occur if the medium components are normal.

Table 8 shows the results. It has been found that the medium treated by the sterilization method according to the present invention exhibits normal medium performance under any irradiation conditions and does not affect the change of the medium components due to electron beam irradiation. On the other hand, in the EOG sterilized medium, the X-GAL reaction in E. coli was weak, and it was inferred that the medium components slightly changed.

[0051]

[Table 8] Increase: bacterial growth, X: X-GAL reaction, M: MUG reaction, G: bacterial growth, NG: no bacterial growth, +: reactive,-: no reactive, W: weak response

Example 1 (Electron Beam Sterilization for Different Medium Compositions) Various media prepared with different compositions were sterilized using the sterilization method according to the present invention. That is, a standard agar medium, a desoxycholate medium, a blood agar medium, a tryptosome agar medium, a TSI medium, and a wipe were sterilized at a surface irradiation dose of 10 kGy and 40 kGy, respectively, and the effects on the coloring and shape of the medium and container were observed. did. The medium composition of the medium used is as shown in Table 9 below. The wiping solution was liquid, and the other media were all agar media. Prior to the sterilization treatment, the medium containing agar was heated and dissolved by adding water, and then cooled and gelled.

[0053]

[Table 9]

Table 10 shows the results. No change was observed in the medium under the electron beam irradiation condition of 10 kGy, and it was revealed that agar medium and liquid medium can be used in the sterilization method according to the present invention. Regarding the effect on the container, no significant change was observed in the TSI medium glass container, which was slightly brownish. On the other hand, under relatively intense irradiation conditions of 40 kGy, there were sporadic changes in the medium, such as formation of bubbles in the medium and fading of the color of the medium, and some of the containers were colored brown. It was concluded that it is appropriate to perform the electronic sterilization of the medium at 40 kGy or less.

[0055]

[Table 10] -: No change

[0056]

According to the method for sterilizing a medium using an electron beam according to the present invention, a powder medium, a granule medium contained in a container,
Agar medium and liquid medium can be sterilized in a short time,
Changes over time (color tone change or deterioration) are also extremely small. Therefore, when using a culture medium, a microorganism can be tested by directly adding a specimen without sterilization again.

Further, since the present invention uses an electron beam, it is possible to control the irradiation dose by setting the voltage and current values, so that sterilization control is easy and the irradiated electron beam remains. It is safe because there is nothing to do.
The sterilization time is greatly reduced as compared with the conventional γ-ray sterilization, and sterilization in a packaged state is also possible, so that a large amount of sterilization can be performed at one time.

Further, when the running cost is roughly estimated, the maintenance cost of the equipment is about one-tenth of that of γ-ray sterilization, and γ-rays use imported radioactive materials as the source, whereas Wire is excellent in cost performance because it uses electricity as a source.

 ──────────────────────────────────────────────────続 き Continuing from the front page (72) Inventor Shingo Mizuochi 2-11-1, Sugamo, Toshima-ku, Tokyo Nissui Pharmaceutical Co., Ltd. (72) Inventor Hiroshi Sengoku 2-1-1, Sugamo, Toshima-ku, Tokyo Nissui Pharmaceutical Co., Ltd. (72) Inventor Kunitachi Tachibana 2-1-1, Sugamo, Toshima-ku, Tokyo Nissui Pharmaceutical Co., Ltd. (72) Inventor Yutaka Yamase 4-16 Midorigahara, Tsukuba, Ibaraki Prefecture Japan Irradiation Service Co., Ltd. Tsukuba Electron Irradiation Center

Claims (4)

[Claims] 1. A method for sterilizing a medium, which comprises irradiating an electron beam at a substantial irradiation dose of 3 kGy to 40 kGy. 2. The method for sterilizing a medium according to claim 1, wherein the medium is in the form of a film, a powder, a granule, a gel, or a liquid. 3. The method for sterilizing a medium according to claim 1, wherein the medium is a medium for testing water quality or a medium for growing microorganisms. 4. An irradiation dose of 10k is applied to a packaged culture medium contained in a container and a predetermined number of said containers contained in a packaging container.
4. The method for sterilizing a medium according to claim 1, wherein the electron beam of Gy-40 kGy is irradiated from outside the packaging container.