JP4521495B2 - Screening method for industrial disinfectant and its use - Google Patents

Description

  The present invention relates to a method for screening an industrial disinfectant necessary for accurately preventing or suppressing various obstacles derived from microorganisms in various industrial water, industrial products, etc., and a method for preventing microbial damage in a sterilization target system using the same. About.

  Conventionally, in industrial water systems such as pulp and paper manufacturing process water, various industrial cooling water, and seawater cooling water, slime derived from microorganisms such as bacteria and fungi has been generated, resulting in a decrease in product quality and heat exchange efficiency. A failure has occurred. In addition, many industrial products, such as metalworking fluids, textile fluids, paints, paper coating fluids, latexes, glues, etc., are rotted or contaminated by microorganisms, causing problems such as product contamination. .

  In order to prevent damage caused by these microorganisms, industrial disinfectants such as isothiazolones, dithiols, halogenated acetates, bromonitro compounds, bacteriostatic agents, preservatives, slime control agents, etc. (hereinafter referred to as “industrial disinfectants”) Is collectively used). Conventionally, these industrial disinfectants have been selected and used as a first indicator for reducing or suppressing the number of microorganisms (the number of bacteria) in a system in which microbial damage should be prevented.

  However, in actual manufacturing sites, for example, in the paper making process of the pulp and paper industry, despite the reduction in the number of microorganisms such as bacteria due to the addition of industrial disinfectants, troubles due to slime frequently occur, while the number of microorganisms Despite this, there have been many contradictions that slime does not cause trouble. Possible causes include the emergence of resistant bacteria and the difference in the ability to form slime depending on the type of microorganism present in the target water system.

  From this point of view, a slime generation prevention method (Patent No. 3073855: Patent Document) in which a chemical having growth inhibitory action or bactericidal action is added to both slime-causing microorganisms and latent slime-causing microorganisms detected from a specific process. 1), microorganisms isolated from microbial samples collected from industrial water systems are each cultured in a liquid medium containing cellulose fibers, the state of slime formation is observed, and microorganisms involved in slime formation based on the results Industrial water-based slime formation prevention method (see Japanese Patent Application Laid-Open No. 9-57275: Patent Document 2), and a type of microorganisms resistant to slime control agents and their generation Instead of the conventional viable count method, the microbial flora analysis by the base sequence of microbial DNA and its abundance ratio It identified from slime inhibiting method of adding a source treating agent chosen for generation sources previously accumulated drug search database: like (JP 2003-20598 JP Patent Document 3) are proposed.

  However, the techniques described in Patent Documents 1 and 3 have a problem that a complicated process or expensive equipment is required for the evaluation. In addition, in the technique described in Patent Document 2, since the slime is formed by adding cellulose fibers to the culture medium and shaking culture, the slime is easily peeled off due to the liquid flow caused by the shaking and the influence of the cellulose fibers. There is a problem that the results vary.

  Microorganisms, on the other hand, produce viscous polysaccharides (EPS) to protect themselves from external attacks and secrete them extracellularly to form higher order structures, so-called biofilms. It is known. EPS is also called “extracellular polymeric substances (EPS)”. This biofilm is generally formed from a plurality of types of microbial communities, and the microbial communities influence each other. For example, Pseudomonas aeruginosa that has formed a biofilm has been reported to have resistance to antibiotics several hundred times higher than that of floating cells. In addition, for the purpose of elucidating the mechanism for biofilm research and genetic research related to infectious microorganisms, pathogenic microorganisms are statically cultured using test tubes or multi-hole microplates, and the inner surface of the container is inoculated. A method for quantitatively determining the ability to form a biofilm by dyeing adherent cells with a dye and then measuring the absorbance after dissolving in a solvent has been reported (for example, Masaaki Morikawa, “Biofilm ”Chemistry and Biology, Japan Society for Agricultural Chemistry, 2003, Vol. 41, No. 1, p. 32-37: see Non-Patent Document 1).

  Biofilms are also known to cause microbial damage in the above industrial water systems and industrial products. Therefore, a method for directly and accurately preventing slime damage by evaluating the activity of an industrial fungicide against biofilm formation, that is, biofilm formation inhibitory activity has been proposed (for example, Japanese Patent Laid-Open No. Hei 6 (1994)). No. 327495 (see Patent Document 4).

  From this point of view, a method for reproducing a biofilm generated in an actual industrial water system at a laboratory level has been proposed. For example, a biofilm is produced using a biofilm-producing microorganism community isolated from a cooling tower by placing a medium and a synthetic cooling tower water in a stainless steel container having a 40-mesh 316 stainless steel wire mesh screen coupon rack. And a method for evaluating the activity of various enzymes using the same have been proposed (see JP-A-6-262165: Patent Document 5). This technique requires 7 to 10 days to produce a biofilm (see Patent Document 5, paragraph 0031).

  Bioreactors (continuous flow stirred tank reactors) and microorganisms commonly found in industrial process water systems (eg, Pseudomonas fluorescene) can be used for 72 hours on glass and stainless steel surfaces at room temperature. A method for forming a film and a method for evaluating a biodispersant used therefor have been proposed (see JP-A-10-80689: Patent Document 6).

  In addition, a method for culturing biofilms on sections (glass or stainless steel) placed on inoculated growth media such as agar and the biofilm sections are used to qualify the efficacy of bactericides against biofilms. Have been proposed (see Japanese Patent Application Laid-Open No. 2003-502063: Patent Document 7). Patent Document 7 (Paragraph No. 0012) describes that this method can be carried out at high speed (48 hours) simply using a laboratory basic apparatus and is reproducible. Yes. In this technique, it takes 2 weeks, preferably 2 to 3 days, to form a biofilm (see Patent Document 7, Paragraph 0020).

  As described above, in the above technique, the presence or absence of biofilm formation by each isolated bacterium is tested. However, isolation of bacteria requires at least about 2-3 days, and formation of a biofilm by the isolation bacteria requires about 1-3 days. In order to prevent slime damage occurring at actual production sites, it is necessary to predict the damage in a shorter time and select an effective industrial disinfectant.

Japanese Patent No. 3073855 JP-A-9-57275 JP 2003-20598 A JP-A-6-327495 JP-A-6-262165 Japanese Patent Laid-Open No. 10-80689 Japanese Patent Laid-Open No. 2003-502063 Masakawa Morikawa, “Biofilm”, Chemistry and Biology, Japan Agricultural Chemical Society, 2003, Vol. 41, No. 1, p. 32-37

  This invention makes it a subject to provide the evaluation method which screens the inhibitory effect with respect to the biofilm of an industrial disinfectant easily and in a short time, and the prevention method of the microbial disorder in the sterilization object system using the same.

  The inventors of the present invention, as a result of earnest research to solve such problems, have found that the formation of biofilms in sterilization target systems such as various industrial water and industrial products contaminated with microorganisms from the sterilization target system. Bacteria are not isolated from collected industrial water and slime such as slime, and this is cultured at a laboratory level, specifically in a microtube made of a hydrophobic material. It has been found that it can be reproduced in a very short period (within 24 hours) and that a biofilm can be formed with a certain degree of certainty from a very small amount of sample such as 1 to 100 μg, and the present invention has been completed.

Thus, according to the present invention, after adding a plurality of types of industrial disinfectants individually in known amounts to the object to be sterilized collected from the system to be sterilized or a dilution thereof, the obtained solutions are individually added together with the medium. Obtained after static culture under oligotrophic conditions set by using a dilution medium diluted 5 to 100 times with sterile water or distilled water, collected in a microtube made of a hydrophobic material of thermoplastic resin There is provided a screening method for industrial disinfectants characterized in that the effectiveness of an industrial disinfectant is determined by the presence or absence of a biofilm in each specimen.

  In addition, according to the present invention, based on the effectiveness of the industrial disinfectant determined by the industrial disinfectant screening method described above, an industrial disinfectant is added to the disinfectant system, and the microorganism in the disinfectant system There is provided a method for preventing a microbial disorder characterized by preventing the disorder.

According to the present invention, it is possible to provide an evaluation method for screening the inhibitory effect of an industrial disinfectant on a biofilm in a simple and shorter time and a method for preventing microbial damage in a sterilization target system using the same.
That is, according to the present invention, various industrial water contaminated with microorganisms, biofilm formation in the sterilization target system such as industrial products can be screened in a very short time, the effectiveness of the industrial disinfectant determined, That is, it is possible to accurately prevent or suppress various obstacles derived from microorganisms in the sterilization target system by adding the industrial sterilization system to the sterilization target system based on the type and amount of the preferred industrial sterilization agent. it can.

The screening method for the industrial disinfectant of this invention,
(A) After individually adding a plurality of types of industrial disinfectants in known amounts to an object to be sterilized collected from the system to be sterilized or a dilution thereof, (B) the obtained solutions are individually hydrophobic together with the medium Collected in a microtube made of material, (C) after static culture under oligotrophic conditions, (D) the effectiveness of the industrial disinfectant is judged by the presence or absence of biofilm in each specimen obtained And
Each step will be specifically described.

(A) A plurality of types of industrial disinfectants are individually added in known amounts to an object to be sterilized collected from the system to be sterilized or a diluted product thereof.
In the present invention, the “sterilization target system” means a system in which slime derived from microorganisms is generated and various obstacles are generated thereby.
Examples of sterilization target systems include industrial water systems such as paper pulp manufacturing process water systems, industrial cooling water systems, seawater cooling water systems, and metal processing oils, textile oils, paints, paper coating liquids, latexes, and pastes. Industrial products such as agents and processes using these. The method of the present invention can be suitably used for paper pulp production process water systems and industrial cooling water systems.

An object to be sterilized is collected from the system to be sterilized by a known method.
In this invention, “object to be sterilized” means industrial water or industrial product, and “diluted object to be sterilized” refers to microorganism-derived deposits (slime) in the system to be sterilized, sterilized water, distilled water or sterilized object. Means diluted with industrial water or industrial products. The density | concentration of the deposit | attachment in a dilution is about 0.1 to 5 weight%, Preferably it is about 0.5 to 1 weight%.

Next, a plurality of types of industrial disinfectants are individually added in known amounts to the object to be sterilized or a diluted product thereof. When the object to be sterilized is industrial water or a liquid industrial product, the industrial bactericidal agent is added as it is. The addition amount varies depending on the state of microbial damage, for example, the number of microorganisms (the number of bacteria), but is usually an addition amount to be added to the actual sterilization target system, about 0.1 to 500 mg / liter with respect to the sample, Preferably it is about 1-100 mg / liter.
Specifically, for industrial disinfectants, select several types of industrial disinfectants used empirically depending on the system to be sterilized, the state of microbial damage, etc. You only have to set it.

  Examples of the industrial bactericides to be added include known bactericides such as organic nitrogen sulfur bactericides, organic bromine bactericides, organic nitrogen bactericides, and organic sulfur bactericides.

  Examples of organic nitrogen sulfur-based fungicides include alkylene bis thiocyanates such as methylene bis thiocyanate and ethylene bis thiocyanate; 5-chloro-2-methyl-4-isothiazolin-3-one, 2-methyl-4-isothiazolin-3-one, 3-isothiazolone compounds such as 4,5-dichloro-2-n-octyl-isothiazolin-3-one, 2-n-octyl-isothiazolin-3-one, 1,2-benzisothiazolin-3-one; Complex of isothiazolone compound and metal salt; Sulfonamide compound such as chloramine T, N, N-dimethyl-N ′-(fluorodichloromethylthio) -N′-phenylsulfamide; 2- (thiocyanomethylthio) benzo Such as thiazole, sodium 2-mercaptobenzothiazole Such azole compounds.

  Examples of organic bromides include 2-bromo-2-nitropropane-1,3-diol, 1,1-dibromo-1-nitro-2-propanol, and 2,2-dibromo-2-nitro-1-ethanol. 1,1-dibromo-1-nitro-2-acetoxyethane, 1,1-dibromo-1-nitro-2-acetoxypropane, 2-bromo-2-nitro-1,3-diacetoxy-propane, tribromonitromethane Organic bromonitro compounds such as 2,2-dibromo-3-nitrilopropionamide, organic bromocyano compounds such as 2-bromo-2-bromomethyl-glutaronitrile; 1,2-bis- (bromoacetoxy) -ethane, 1,2-bis- (bromoacetoxy) -propane, 1,4-bis- (bromoacetoxy) -2-butene, 1,2,3,- Organic bromoacetate esters or amides, such as squirrels bromoacetoxy propane; and organic bromo sulfone compounds such as bis tribromomethyl sulfone.

  Organic nitrogen-based fungicides include 1-bromo-3-chloro-5,5-dimethylhydantoin, 1,3-dibromo-5,5-diethylhydantoin, 1,3-dibromo-5-methyl-5-butylhydantoin Hydantoin compounds such as 1-bromo-3-chlorohydantoin; isophthalo such as 5-chloro-2,4,6-trifluoroisophthalonitrile, 5-chloro-2,4-difluoro-6-methoxyisophthalonitrile Nitrile compounds; s-triazine compounds such as hexahydro-1,3,5-triethyl-s-triazine, hexahydro-1,3,5-tris- (2-hydroxyethyl) -s-triazine; α-chlorobenzald Halogenated oxime compounds such as oxime and dichloroglyoxime; dichloroisocyanate and dichloro Chlorinated isocyanuric acid compounds such as sodium soocyanurate and trichloroisocyanuric acid; quaternary ammonium compounds such as benzalkonium chloride; N- (2-hydroxypropyl) -aminomethanol, 2- (hydroxymethylamino) ethanol And amino alcohol compounds such as 2-pyridinethiol sodium oxide.

Examples of the organic sulfur-based fungicide include 3,3,4,4-tetrachlorotetrahydrothiophene-1,1-dioxide, 4,5-dichloro-1,2-dithiol-3-one and the like.
Other known bactericides include glutardialdehyde, hydrogen peroxide, hypochlorite, hypobromite, and the like.

(B) Collect | recover the obtained several solution separately to the microtube which consists of hydrophobic materials with a culture medium.
Examples of the medium used in the present invention include known media such as bouillon media, zapek media, starch media, L media, and standard media, and are selected according to the sterilization target system and the bacterial species contained therein.
In order to perform stationary culture under oligotrophic conditions in the next step, the medium is preferably a diluted medium diluted 5 to 100 times, preferably 10 to 50 times with sterilized water or distilled water.
In order to form a biofilm in a short period of time, it is preferable to use a dilution of Czapek medium or L medium.

As the dilution medium, when the object to be sterilized is water and slime collected from industrial water systems such as paper pulp manufacturing process water systems, industrial cooling water systems, seawater cooling water systems, or waste water systems, and supernatants and filtered water A solution obtained by adding Czapek medium to the liquid may be used.
When the object to be sterilized is industrial products such as metalworking fluids, textile fluids, paints, paper coating fluids, latex, glues, or collected slime, water such as sterilized water or distilled water of the industrial products The dilution may be obtained by adding Czapek medium.
When the paste is starch paste, a water dilution such as sterilized water or distilled water of starch paste may be used without adding a medium.

  The total amount of the solution to which the industrial disinfectant is added and the medium is about 50 to 200 μl, preferably about 50 to 100 μl, and the solution is about 0.1 to 10% by weight, preferably 0.5 to 1% of the total amount. It is about wt%.

  “Hydrophobic” of the hydrophobic material constituting the microtube used in the present invention means that it is more hydrophobic than glass or stainless steel. Examples of such a material include thermoplastic resins selected from polyvinyl chloride, polystyrene, polyethylene, and polypropylene. In such a thermoplastic static resin, the mechanism is not clear, but it is possible to form a reproducible biofilm in a short period of time as compared with glass and stainless steel empirically.

This invention is characterized in that a biofilm is formed with a small amount of an object to be sterilized.
As the microtube, a well-known one can be used, and a multi-hole type microplate is particularly preferable in that a multi-sample sample can be collectively evaluated. A microtube or a microplate commercially available from Nippon Becton Dickinson Co., Ltd. or PerkinElmer Japan Co., Ltd. can be suitably used. Specifically, a 96-well microtiter plate (manufactured by Biolog, FALCON 353911: 96 well, U bottom, no lid, made of polyvinyl chloride) can be mentioned.

(C) Static culture under oligotrophic conditions.
In the present invention, the “oligotrophic condition” means that the medium is substantially diluted and is in an oligotrophic state as compared to the medium before dilution. As described in the description of the step (c), it is preferable to set the oligotrophic condition by using a diluted medium diluted 5 to 100 times with sterilized water or distilled water.
As a temperature condition of stationary culture, 20-50 degreeC is preferable at the point which can evaluate the presence or absence of a biofilm in a short time, and it is about 37 degreeC normally. You may set temperature conditions to 35-50 degreeC by the meaning matched with the conditions of the sterilization object type | system | group.
The culture time is about 12 to 72 hours, preferably about 24 hours.
Shaking culture is not preferable because the biofilm generated by the flow of the liquid may be peeled off and the evaluation results may vary.

(D) The effectiveness of an industrial disinfectant is determined based on the presence or absence of a biofilm in each specimen obtained.
This evaluation may be carried out by staining the biofilm with a staining solution after static culture, and washing and drying the inside of the microtube with pure water or the like, and using the stained biofilm as a solvent. It is preferable to carry out by dissolving and measuring the absorbance of the obtained solution from the viewpoint of more objective quantitative evaluation.

The staining solution is appropriately selected depending on the bacterial species and the type of medium used, and basic dyes such as crystal violet and methylene blue can be preferably used.
The solvent for dissolving the dyed biofilm is not particularly limited as long as it is a solvent that dissolves a dye such as dimethyl sulfoxide and alcohols.

  In the measurement of absorbance, the wavelength may be appropriately set depending on the type of the staining solution. For example, when crystal violet is used as the staining solution, the wavelength may be set to 590 nm. The measuring instrument to be used is not particularly limited, but measurement with a microplate reader or the like is simple and preferable.

  As described above, according to the screening method of the present invention, it is possible to easily and quickly select the type of industrial bactericides effective for preventing microbial damage in the sterilization target system and the amount of addition thereof. That is, the present invention accurately selects an effective industrial disinfectant and its addition amount against the environmental change of the microbial community involved in biofilm formation that can occur in the actual sterilization target system and the emergence of resistant bacteria. Can prevent the occurrence of microbial damage that may occur in the future.

  Therefore, based on the effectiveness of the industrial disinfectant determined by the above industrial disinfectant screening method, the industrial disinfectant can be added to the disinfectant system to prevent microbial damage in the disinfectant system. it can. That is, the timely implementation of the screening method of the present invention makes it possible to manage the addition of industrial fungicides more accurately. Here, “control” means selection of the type of industrial disinfectant, setting of addition time and addition amount, and application. Specifically, it is preferable to periodically perform screening at a rate of, for example, once every 2-3 days, and add an industrial disinfectant to the sterilization target system based on the result.

  The present invention will be specifically described with reference to test examples and examples, but the present invention is not limited to these examples.

Test Example 1 (Biofilm formation ability test by stationary culture under oligotrophic conditions)
Static culture using slime adhering to the primary screen of circulating white water and PDF (Polydisc filter) collected in the papermaking process of A paper company using L medium diluted 10 times with normal L medium and pure water. And their biofilm-forming ability was tested.

Each well of a 96-well microtiter plate (Biolog, FALCON 353911: 96 wells, U bottom, without lid, made of polyvinyl chloride) 10 times with normal L medium (Lennox medium, composition below) and pure water 100 μl of each diluted L medium (sterilized) was poured, and the collected slime was dispersed in pure water so as to be 0.3% by weight, and 1 μl thereof was added to the medium in each well. Thereafter, in order to prevent drying, the microtiter plate was placed in a glass petri dish and statically cultured in a high-temperature bath adjusted to 37 ° C. After 24 hours, the medium in each well was removed, washed with pure water, and then dried in a high-temperature bath adjusted to 70 ° C. for 5 to 10 minutes. Next, 100 μl of 1% crystal violet solution (solvent: ethyl alcohol) was added to each well and left at room temperature for 20 minutes. Thereafter, the crystal violet solution in each well was removed, and the stained state of the inner wall of each well was visually observed to determine the presence or absence of a biofilm (BF). On the other hand, 100 μl of dimethyl sulfoxide (DMSO) as a solvent was added to each well and allowed to stand at room temperature for 5 minutes to dissolve crystal violet. Thereafter, the absorbance (abs) at 590 nm of the obtained solution was measured. When the absorbance exceeded 1.0 abs, the solution was arbitrarily diluted with DMSO and the absorbance was measured again.
The obtained results are shown in Table 1 together with the place where the slime was collected.

Composition of L medium: Bacto-Tryptone 10g
Bacto-Yeast extract 5g
NaCl 5g
Glucose 1g
1000ml distilled water
pH: 7.2

Biofilm-forming ability was determined from the obtained absorbance according to the following criteria.
Absorbance: less than 0.67 abs-(no ability to form)
0.67 or more and less than 1.33 abs ±
1.33 abs or more + (formation potential)

  From Table 1 and the above criteria, it can be seen that slime can be statically cultured under oligotrophic conditions of L medium diluted 10-fold with pure water. Therefore, it can be seen that a biofilm is formed by a small amount of sample (collected slime dispersion).

Example 1 (Screening of industrial fungicides)
Circulating white water (pH 7.0, sulfite ion concentration 27 ppm, bacterial species: Acidovorax sp., Acinetobacter sp., Enterococcus sp., Viable cell count 2.2 × 10 ⁷ CFU / ml collected in the paper making process of B Paper Company ) And slime slightly adhering to the primary screen (bacteria species: Enpedobacter sp., Stenotrophomonas sp., Enterobacter sp.) Were screened for industrial fungicides.
In this papermaking process, 2-bromo-2-nitro-1,3-diacetoxy-propane was added at an addition amount of 15 mg / liter, an addition time of 20 minutes per time, three times a day, and slime. There is almost no adhesion and stable operation is possible.

Similarly, the collected slime was added to 200 ml of the collected circulated white water so as to be 0.3% by weight, and 10 ml of this circulated white water was collected in an L-shaped tube. The industrial disinfectant shown in Table 2 was added to the circulating white water in the specified amount and cultured with shaking at 30 ° C. for 20 minutes. Thereafter, the viable count (CFU / ml) in the circulating white water was measured.
On the other hand, 100 μl each of a mixed medium (sterilized) of 9 ml of circulating white water and 1 ml of Czapek medium was poured into each well of a 96-well microtiter plate (see Test Example 1), and then the above-mentioned circulation in which the number of viable bacteria was measured. 1 μl of system white water was added to each medium. Thereafter, in order to prevent drying, the microtiter plate was placed in a glass petri dish and statically cultured in a high-temperature bath adjusted to 37 ° C. After 24 hours, the medium in each well was removed, washed with pure water, and then dried in a high-temperature bath adjusted to 70 ° C. for 5 to 10 minutes. Next, 100 μl of 1% crystal violet solution (solvent: ethyl alcohol) was added to each well and left at room temperature for 20 minutes. Thereafter, the crystal violet solution in each well was removed, and the stained state of the inner wall of each well was visually observed to determine the presence or absence of a biofilm (BF). On the other hand, 100 μl of dimethyl sulfoxide (DMSO) as a solvent was added to each well and allowed to stand at room temperature for 5 minutes to dissolve crystal violet. Thereafter, the absorbance (abs) at 590 nm of the obtained solution was measured. When the absorbance exceeded 1.0 abs, the solution was arbitrarily diluted with DMSO and the absorbance was measured again.
Based on the same standards as in Test Example 1, biofilm-forming ability was determined from the obtained absorbance.
The obtained results are shown in Table 2 together with the industrial disinfectant used and the amount added.

The abbreviations for industrial fungicides in Table 2 represent the following compounds.
DBNPA: 2,2-dibromo-3-nitrilopropionamide BBAB: 1,4-bis- (bromoacetoxy) -2-butene MAC: 2-bromo-2-nitro-1,3-diacetoxy-propane CL-BA: α-Chlorobenzaldoxime

From the results in Table 2, under the conditions of this example, 2-bromo-2-nitro-1,3-diacetoxy-propane was added at a lower concentration (15 mg / liter) among the industrial fungicides used. It can be seen that biofilm formation can be inhibited. Moreover, it turns out that the formation inhibitory effect of biofilm has no correlation with the effect of reducing the number of viable bacteria.
The above results are consistent with the actual state of the addition of industrial disinfectant in the paper making process of the B paper company that collected circulating white water and slime.
Therefore, according to this invention, it turns out that selection of the industrial disinfectant which inhibits biofilm formation effectively, and the setting of the addition amount can be performed.

Example 2 (Method for preventing microbial damage in simulated sterilization target system)
Similarly, the slime used in Example 1 was added to the circulated white water used in Example 1 so as to be 0.3% by weight to prepare a test solution. A polyethylene plastic wire (90 mm × 270 mm) was attached to the inner side of the side of a 500 ml beaker, 500 ml of the prepared test solution was added thereto, and the test solution was stirred at a temperature of 30 ° C. and a rotation number of 60 rpm with a jar tester with a thermostatic bath. Immediately, the industrial fungicide shown in Table 3 was added to the test water in its defined amount of stock solution. 20 minutes after the addition, the test water in the beaker was gently discarded.
Newly prepared test solution not containing industrial disinfectant was placed in a 500 ml beaker. The test liquid was stirred for 12 hours at a temperature of 30 ° C. and a rotation speed of 60 rpm with a jar tester with a thermostatic bath. Immediately, the industrial fungicide shown in Table 3 was added to the test water in its defined amount of stock solution. 20 minutes after the addition, the test water in the beaker was gently discarded.
Newly prepared test solution not containing industrial disinfectant was placed in a 500 ml beaker. The test liquid was stirred for 12 hours at a temperature of 30 ° C. and a rotation speed of 60 rpm with a jar tester with a thermostatic bath.
The above operation was repeated twice a day, and the test was conducted for 10 days.
Ten days later, the test water was gently discarded from the beaker, and then the plastic wire attached to the beaker was removed and dried to determine the surface area of the adhering slime.
The obtained results are shown in Table 3 together with the industrial disinfectant used and the amount added.

The ratio of the surface area of the slime adhering to the plastic wire (amount of dirt) was determined according to the following criteria.
0% or more and less than 25% of the total: 1
25% or more and less than 50% of the total: 2
50% or more and less than 75% of the total: 3
75% or more and less than 100% of the total: 4

From the results in Table 3, under the conditions of this example, 2-bromo-2-nitro-1,3-diacetoxy-propane was added at a lower concentration (15 mg / liter) among the industrial fungicides used. It can be seen that biofilm formation can be inhibited.
Similar to the result of Example 1, the above results are in agreement with the actual state of addition of industrial disinfectant in the paper making process of the B paper company that collected the circulating white water and slime.
Therefore, it can be seen that the method of the present invention has a correlation with the current state of the sterilization target system and can be applied to this.

Claims (10)

After individually adding several kinds of industrial disinfectants in known amounts to the object to be sterilized collected from the system to be sterilized or its dilution, the obtained solutions are individually separated from the thermoplastic resin hydrophobic material together with the culture medium. After being placed in a microtube and statically cultured under oligotrophic conditions set by using a diluted medium diluted 5 to 100 times with sterile water or distilled water, and then depending on the presence or absence of a biofilm in each specimen obtained A screening method for an industrial fungicide characterized by judging the effectiveness of an industrial fungicide. The sterilizing object, an industrial water or industrial products, dilutions of the sterilizing object, sterile water as the deposits derived from microorganisms becomes 0.1 to 1 wt%, distilled water or sterilized system The method for screening an industrial disinfectant according to claim 1, which is diluted with industrial water or an industrial product. The method for screening an industrial disinfectant according to claim 1 or 2, wherein 0.1 to 500 mg / liter of the industrial disinfectant is added to the object to be sterilized or a diluted product thereof. It said solution and a total amount of the medium 50-200, a screening method of an industrial disinfectant according to any one of claims 1-3 wherein the solution is a 0.1 to 10% by weight of the total amount. The method for screening an industrial disinfectant according to any one of claims 1 to 4 , wherein the hydrophobic material is a thermoplastic resin selected from polyvinyl chloride, polystyrene, polyethylene, and polypropylene. The screening method of the industrial disinfectant according to any one of claims 1 to 5 , wherein the culture is statically cultured at a temperature of 25 to 50 ° C for 24 hours. After the static culture, to stain the biofilm using a dyeing solution by washing and drying the inside of the microtube, the dyed biofilm is dissolved in a solvent, measuring the absorbance of the resulting solution, The screening method of the industrial disinfectant according to any one of claims 1 to 6 , wherein the presence or absence of a biofilm is evaluated. The method for screening an industrial disinfectant according to any one of claims 1 to 7 , wherein the sterilization target system is a paper pulp manufacturing process water system or an industrial cooling water system. Based on the effectiveness of the industrial disinfectant determined by the industrial disinfectant screening method according to any one of claims 1 to 8, an industrial disinfectant is added to the disinfectant system, A method for preventing microbial damage characterized by preventing microbial damage in a system. The method for preventing microbial damage according to claim 9 , wherein screening of industrial disinfectant is conducted once every 2-3 days.