JP4806804B2 - Method for preventing microbial damage in sterilization target systems - Google Patents

Description

  The present invention relates to a method for preventing a microbial disorder, which accurately prevents and suppresses various disorders derived from microorganisms in various industrial water, industrial products and the like.

  Conventionally, slime derived from microorganisms such as bacteria and fungi has been generated in industrial water systems such as pulp and paper manufacturing process water, various industrial cooling water, seawater cooling water, etc., resulting in deterioration of product quality and heat exchange efficiency Disorders are known. In addition, many industrial products such as metalworking oils, textile oils, paints, paper coating fluids, latexes, glues, etc., cause rot or contamination by microorganisms, resulting in problems such as product fouling. ing.

In order to prevent damage by these microorganisms, industrial bactericides such as isothiazolones, dithiols, halogenated acetates, bromonitro compounds, bacteriostatic agents, preservatives and the like (hereinafter referred to as “industrial bactericides”). Is used. Conventionally, these industrial disinfectants have been selected and treated as a first indicator to reduce or suppress the number of microorganisms in the system in which microbial damage should be prevented.

However, in actual manufacturing sites, for example, the paper making process in the pulp and paper industry, the addition of industrial disinfectants caused many slime troubles despite the decrease in the number of microorganisms such as bacteria. Even though the price is high, there has been a lot of contradiction that troubles caused by slime do not occur. One possible reason is that the ability to form slime varies depending on the appearance of resistant bacteria and the type of microorganisms present in the water system.

From this point of view, a slime generation prevention method comprising adding a drug having a growth inhibitory action or a bactericidal action to both slime-causing microorganisms and latent slime-causing microorganisms detected from a specific process (Patent Document 1) (See below), cultivating bacteria isolated from microbial samples collected from industrial water systems in a liquid medium containing cellulose fibers, observing the state of slime formation, and effective against microorganisms involved in this slime formation An industrial water-based slime formation prevention method (see Patent Document 2) characterized by adding a bactericidal agent to the industrial water-based system, and the type of microorganism resistant to white water treatment agents in place of the conventional viable count method The source was identified from the microbiota analysis by the base sequence of microbial DNA and its abundance ratio, and selected by a drug search database accumulated in advance. Slime inhibiting method of adding Namagen treatment agent to the source (Patent Document 3) are proposed.

However, the inventions described in Patent Documents 1 and 3 have the problem that complicated processes and expensive equipment are required for the evaluation, and the invention described in Patent Document 2 adds pulp fibers to the medium. Since shaking culture is performed, the generated slime easily peels off due to the flow of liquid caused by shaking and the influence of pulp fibers, and there is a problem that the evaluation results vary.

On the other hand, in order to protect microorganisms from external attacks, it is known that so-called biofilms are formed by extracellular polymeric substances (EPS) that are secreted and produced extracellularly. This biofilm is generally formed from a multi-species microbial community. For example, it has been reported that the resistance to antibiotics is several hundred times higher in Pseudomonas aeruginosa that forms biofilms than in floating cells. Yes. 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-well microplates, and the inner surface of the container is cultured. A method for quantitatively determining the ability to form a biofilm by dyeing adherent cells with a dye, and then dissolving the resultant in a solvent and measuring the absorbance thereof has been introduced (for example, see Non-Patent Document 1).

Moreover, it is also known that a biofilm is a cause of microbial damage in the industrial water system and industrial products described above (see, for example, Patent Document 4). Therefore, it is preferable in that a microorganism that forms a biofilm is specified and an industrial disinfectant effective for the microorganism is added to the sterilization target system in order to prevent slime damage directly and accurately.

From this viewpoint, a method for reproducing a biofilm generated in an actual industrial water system or the like at the laboratory level has been proposed.
For example, a biofilm-producing microorganism community isolated from a cooling tower is placed in a stainless steel container with a 40-mesh 316 stainless steel wire mesh screen coupon rack and the biofilm-producing microorganism community isolated from the cooling tower. Producing a film and evaluating the activity of various enzymes using this method have been proposed (see Patent Document 5). Paragraph No. 0031 of this publication states that 7 to 10 days were required to produce a biofilm using this method.

In addition, using a bioreactor (continuous flow stirred tank reactor), Pseudomonas fluorescene , a microorganism usually found in industrial process water systems, requires 72 hours on glass and stainless steel surfaces at room temperature. A method for forming a biofilm and a method for evaluating a biodispersant using this method have been proposed (see Patent Document 6).

In addition, a method of culturing biofilms on sections (glass or stainless steel) placed on inoculated growth media such as agar, and using such biofilm sections, qualitatively clarifies the efficacy of bactericides on biofilms. Has been proposed (see Patent Document 7). Paragraph No. 0012 of this publication describes that this method uses basic laboratory equipment, is fast (48 hours), simple, and reproducible. Paragraph No. 0020 describes that an appropriate time is required to form a biofilm, which is up to 2 weeks, and a preferred time is 2 to 3 days.

Japanese Patent No. 3073855 (Claims) JP-A-9-57275 (Claims, paragraph number 0007, Example 1) Japanese Patent Laying-Open No. 2003-20598 (Claims No. 0010) JP-A-6-327495 (see paragraphs 0002 and 0007) JP-A-6-262165 (Claims, Examples 1 to 15) JP 10-80689 A (paragraph number 0020, Examples 1 to 3) JP 2003-502063 A (claims, paragraph numbers 0007, 0012, 0019, 0020, 0030, 0033) Japan Agricultural Chemistry / Academic Society Center, Periodical Publication “Chemistry and Biology” Vol. 41 (2003) No. 1.32-36

In the conventional method, it takes 2 to 10 days for the biofilm formation of the isolated bacteria. If you can accurately identify the isolates that form biofilms that occur in the actual field in a shorter period of time, you can accurately prevent and control slime damage by applying an effective industrial disinfectant to the isolates. Therefore, it is preferable.
Therefore, the present invention can easily and accurately evaluate the biofilm forming ability of an isolated bacterium isolated from the sterilization target system of the sterilization target system in a shorter period of time, and is effective for the isolated bacterium. It is an object of the present invention to provide a method for preventing microbial damage by adding an antibacterial agent to a system to be sterilized.

  The inventors of the present invention, as a result of diligent research on a method that can easily and accurately reproduce the biofilm forming ability of various microorganisms at a laboratory level in a shorter time in order to solve such problems, By isolating bacteria from industrial water, slime, etc. collected from the system to be sterilized and cultivating the isolated bacteria in tubes made of hydrophobic materials under conditions of poor nutrition, extremely short period (within 24 hours) And found that a biofilm is formed with certain certainty on the inner wall of the tube. Furthermore, the present inventors have also found out that this method can be used to screen industrial disinfectants that are easy and reproducible in a short period of time. The present invention was completed by confirming the correlation between this fact and the effect of controlling slime damage in various industrial water and industrial products.

  Thus, according to the present invention, the suspension of bacteria isolated from the sterilization target system of the sterilization target system is collected in a microtube made of a hydrophobic material together with the culture medium, and after stationary culture under oligotrophic conditions, Based on the result of evaluating the presence or absence of a film, it is possible to identify an isolated bacterium that forms a biofilm and to add an industrial disinfectant effective against the isolated bacterium that forms a biofilm to the sterilization target system. A method for preventing microbial damage in a sterilization target system is provided.

  According to the present invention, the biofilm-forming ability of an isolated bacterium isolated from a sterilization target of a sterilization target system can be easily and accurately quantitatively evaluated in a shorter period of time, and an industrial that is effective for the isolated bacterium. By selecting the type, time of addition and amount of fungicide for use, and applying it to the system to be sterilized, various obstacles derived from microorganisms in various industrial water, industrial products, etc. can be accurately prevented. Has an effect.

The sterilization target system in this invention means a target system in which various obstacles occur due to slime derived from microorganisms, for example, industrial water systems such as paper pulp manufacturing process water, various industrial cooling water, seawater cooling water, Examples include various waste waters, metal processing oils, fiber oils, paints, paper coating liquids, latexes, pastes, and other industrial products and processes using these industrial products.
The sterilization target collected from these sterilization target systems refers to various industrial water or industrial products or microorganism-derived deposits.

In the present invention, the isolated bacterium is obtained by diluting these various industrial water or industrial products or microorganism-derived deposits, and then, first, a known medium commonly used for culturing various microorganisms, such as a normal agar medium. Inoculate asparagine / glucose agar, bouillon agar, Czapek agar, starch agar, egg agar, Waxmann agar, gelatin agar, etc. Then, after culturing the obtained colonies, each microorganism is identified from the colonies generated on the medium using a microscope or the like as necessary, and the microorganisms are collected separately. Thereafter, the isolated bacteria are suspended, for example, in a biolog suspension, and collected in a microtube made of a hydrophobic material together with the 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 bacterial species. In order to perform static culture under poor nutrition in the next step, the medium is a diluted medium diluted 5 to 100 times, preferably 10 to 50 times with sterilized water or distilled water. preferable. 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.
The total amount of the suspension of the isolated bacterium and the medium is about 50 to 200 μl, preferably about 50 to 100 μl, and the suspension is about 0.1 to 10% by weight, preferably 0.5 to 1% of the total amount. It is about wt%.

The “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 such as polyvinyl chloride, polystyrene, polyethylene, and polypropylene. Although the mechanism is not clear, it is empirically possible to form a reproducible biofilm in a short period of time compared to glass or stainless steel.

This invention is characterized in that a biofilm is formed with a small amount of an object to be sterilized.
As the microtube, a publicly known one can be used, and it is particularly preferable to use a multi-hole type microplate from the viewpoint that multiple samples can be collectively evaluated. A microtube or a microplate commercially available from Nippon Becton Dickinson Co., Ltd. or Perkin Elmer Japan Co., Ltd. can be suitably used.

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 previous step, it is preferable to set the oligotrophic conditions by using a diluted medium diluted 5 to 100 times with sterilized water or distilled water.
The stationary culture condition is preferably 20 to 50 ° C. in that the presence or absence of a biofilm can be evaluated in a short period of time, and the temperature condition is set to the temperature condition of the sterilization target system in order to match the conditions of the sterilization target system. May be.

The culture time is about 12 to 72 hours, preferably about 24 hours. In the case of shaking culture, it is not preferable because the biofilm generated by the flow of the liquid may be peeled off and the evaluation result may vary.

In this invention, the biofilm may be evaluated by staining the biofilm using a staining solution after static culture and washing the microtube with pure water or the like, drying, and staining. It is preferable from the viewpoint that more objective quantitative evaluation can be performed by dissolving the biofilm in a solvent and measuring the absorbance of the obtained solution. The staining solution is appropriately selected depending on the bacterial species and the medium used, and basic dyes such as crystal violet and methylene blue can be suitably used. Further, the solvent for dissolving the dyed biofilm is not particularly limited as long as it is a solvent that dissolves a pigment such as dimethyl sulfoxide and alcohols. In the measurement of absorbance, the wavelength may be appropriately set depending on the type of 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 is simple and preferable.
As described above, after identifying an isolated bacterium that forms a biofilm, a microbial disorder in the sterilization target system can be prevented by adding an industrial disinfectant effective against the isolated bacterium.

In this invention, the selection of an effective bactericidal agent for sterilizing the isolated bacterium forming a biofilm is appropriately performed by measuring the number of viable bacteria after adding a known screening method, for example, an industrial bactericidal agent. You can choose. In order to select a more effective effective disinfectant, a plurality of industrial disinfectants individually added in known amounts to the suspension of isolated bacteria forming a biofilm, The suspension is individually collected in a microtube made of a hydrophobic material together with a medium, and after stationary culture under oligotrophic conditions, an effective industrial disinfectant is selected depending on the presence or absence of a biofilm.

It does not specifically limit as an industrial disinfectant to add, A well-known disinfectant can be used. For example, 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-isothiazoline-3- 3-isothiazolone compounds such as ON, 4,5-dichloro-2-n-octyl-isothiazolin-3-one, 2-n-octyl-isothiazolin-3-one, 1,2-benzisothiazolin-3-one, Complexes of 3-isothiazolone compounds and metal salts; Sulfonamide compounds such as chloramine T, N, N-dimethyl-N ′-(fluorodichloromethylthio) -N′-phenylsulfamide; 2- (thiocyano Thia such as methylthio) benzothiazole, sodium 2-mercaptobenzothiazole Examples thereof include sol 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, -trisbromoacetoxyprop Organic bromoacetate esters or amides and the like; bis tribromomethyl sulfone as the organic bromo sulfone compounds.

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, dichloroisocyanate Chlorinated isocyanuric acid compounds such as sodium oxalate and trichloroisocyanuric acid; quaternary ammonium compounds such as benzalkonium chloride; N- (2-hydroxypropyl) -aminomethanol, 2- (hydroxymethylamino) Examples include amino alcohol compounds such as ethanol; 2-pyridinethiol sodium oxide and the like.

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.
The addition amount of these industrial disinfectants is an addition amount to be added to the actual sterilization target system, and is usually selected from the range of 0.1 to 500 mg / liter.

  The present invention will be described in more detail with reference to the following test examples, but the present invention is not limited to these test examples.

Test Example 1 (Biofilm formation ability test by stationary culture under oligotrophic conditions)
By stationary culture using normal L medium and L medium diluted 10-fold with pure water, using bacteria isolated from slime adhering to the primary screen of circulating white water in the papermaking process of paper manufacturer A The biofilm forming ability was tested.

(Test method)
The collected slime was diluted and cultured in a standard medium. Four colonies obtained were randomly picked and isolated in BUG medium (BiOLOG UNIVERSAL GROUTH AGAR: preculture medium). The isolated and grown bacteria were further planted in a BUG medium. Bacteria were suspended in a biolog suspension from a colony cultured overnight.

100 μl each of normal L medium and L medium (sterilized) diluted 10-fold with pure water was poured into each well of a 96-well microtiter plate (FALCON 353911: 96 well, U bottom, without lid, made of polyvinyl chloride) Thereto, 1 μl of the cell suspension 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 dried in a high-temperature bath adjusted to 70 ° C. for 5 to 10 minutes. Next, 100 μl of a 1% crystal violet solution (solvent: ethyl alcohol) was added to each well and allowed to stand at room temperature for 20 minutes to stain the biofilm. 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). Meanwhile, 100 μl of dimethyl sulfoxide (DMSO) as a solvent was added to each well and left at room temperature for 5 minutes to dissolve the biofilm stained with 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 for each isolated bacterium.

The composition of the L medium is as follows.
Composition of L medium (Lennox medium): Bacto-Tryptone 10g
Bacto-Yeast extract 5g
NaCl 5g
Glucose 1g
1000ml distilled water
pH 7.2

Note) 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)

(Discussion)
From Table 1 and the above criteria, a biofilm can be formed in a short period of time with a small amount of sample by stationary culture of the isolated bacterium under oligotrophic conditions of L medium diluted 10-fold with pure water. I understand.

Example 1 (Anti-rotation test for microbial damage in sterilization target system)
2,2-Dibromo-2-nitroethanol was added at 7.5 mg / liter for 15 minutes 6 times a day in the externally added size liquid circulation system of B Paper Company. It occurred and became an obstacle. Therefore, after obtaining and isolating normal and abnormal externally added sizing solutions, and investigating the slime-forming ability of each, the drug effect on representative bacteria was examined. Based on the result, a mixture of 5-chloro-2-methyl-4-isothiazolin-3-one and 2-methyl-4-isothiazolin-3-one at a weight ratio of 3: 1 was 2.5 mg / liter for 15 minutes. Was added six times a day to the externally added size liquid circulation system, discoloration and precipitates due to microbial decay disappeared, and microbial decay could be prevented. Test methods and test results are shown below.

(Test method)
A normal product and an abnormal product (discolored and precipitated) of the externally added sizing solution were obtained, and 10 colonies that had grown using the standard medium were randomly collected and isolated on the BUG medium. Table 2 shows each bacterial species and the ratio.
Among isolated bacteria, L medium (sterilized) diluted 10-fold with pure water in the same manner as in Test Example 1 using Roseomonas fauriae as a typical bacterial species and Vibrio proteolyticus and Chryseobacterium balustinum as abnormal species, respectively. ) Was used to perform a biofilm-forming test by static culture under oligotrophic conditions. The results are shown in Table 3. It was found that Roseomonas fauriae detected from normal products had almost no biofilm forming ability, whereas Vibrio proteolyticus and Chryseobacterium balustinum detected from abnormal products had high biofilm forming ability.

  Therefore, to the preparation prepared by adding boric acid, potassium chloride and sodium hydroxide to pure water to pH 8.9, Roseomonas fauriae detected from normal products, Vibrio proteolyticus and Chryseobacterium balustinum detected from abnormal products, Were collected separately. Then, various industrial disinfectants were added, and cultured with shaking at 30 ° C. for 2 hours and 24 hours, and the number of viable bacteria at each time was measured. Test results are shown in Tables 4-6. Table 4 shows the drug effect on Roseomonas fauriae detected from normal products, Table 5 shows the drug effect on Vibrio proteolyticus detected from abnormal products, and Table 6 shows the drug effect on Chryseobacterium balustinum detected from abnormal products. Show.

(Discussion)
From the test results in Tables 4 to 6, it is clear that Vibrio proteolyticus and Chryseobacterium balustinum detected from abnormal products are clearly the same concentration compared to Roseomonas fauriae detected from normal products in 2,2-dibromo-2-nitroethanol. It turns out that the bactericidal effect with respect to a chemical | medical agent changes with microbial species, and it becomes difficult to be effective with respect to microbe with high biofilm formation ability.
On the other hand, in a 3: 1 weight ratio mixture of 5-chloro-2-methyl-4-isothiazolin-3-one and 2-methyl-4-isothiazolin-3-one, Roseomonas fauriae (detected from normal product), Vibrio Both proteolyticus and Chryseobacterium balustinum (detected from abnormal products) have almost the same bactericidal effect against drugs, indicating that they are also effective against bacteria with high biofilm-forming ability.

Example 2 (Test for confirming prevention of microbial damage in sterilization target system)
In the white water circulation system of the papermaking process of C Paper Company, 1,4-bis- (bromoacetoxy) -2-butene was added at 60 mg / liter for 20 minutes four times a day. There was a failure. When the properties of white water were investigated, the pH was 5.35 and the sulfite ion concentration was 40 ppm. The slime-forming ability was investigated using bacteria isolated from this slime, drug selection for highly-forming bacteria was carried out, and 2-bromo-2-nitro-1,3-diacetoxy-propane was selected based on the results. When 45 mg / liter, 20 minutes, was added four times a day in the white water circulation system, no slime adhered to the papermaking tool even after 40 days of operation. Test methods and test results are shown below.

(Test method)
Dilute slime adhering to the papermaking tool, use standard medium, and randomly collect 10 colonies from the grown colonies and isolate them on BUG medium. The biofilm-forming ability test was carried out by stationary culture under oligotrophic conditions using L medium (sterilized) diluted 10-fold. The results are shown in Table 7. The results revealed that Rhizobium radiobacter and Burkholderia vietnamiensis have the ability to form biofilms.
Therefore, various industrial fungicides were screened for Rhizobium radiobacter and Burkholderia vietnamiensis.

  That is, after adding to a preparation prepared by adding disodium hydrogen phosphate and citric acid to pure water so as to have a pH of 5.4, sodium sulfite was added to a sulfite ion concentration of 40 ppm, and then isolated bacteria Rhizobium radiobacter and Burkholderia Vietnamiensis was collected separately. Thereafter, various industrial disinfectants were added, and cultured with shaking at 40 ° C. for 20 minutes, and the number of viable bacteria at each time was measured.

At the same time, 100 μl each of L medium (sterilized) diluted 10 times with pure water was poured into each well of a 96-well microtiter plate (FALCON 353911: 96 well, U bottom, without lid, made of polyvinyl chloride) 1 μl of sample after counting 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 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 100 μl of dimethyl sulfoxide (DMSO) as a solvent was added to each well and left at room temperature for 5 minutes to dissolve the biofilm stained with 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. Biofilm formation ability was determined according to the same criteria as in Test Example 1. The test results are also shown in Table 8 and Table 9. Table 8 shows the drug effect on Rhizobium radiobacter, and Table 9 shows the drug effect on Burkholderia vietnamiensis.

(Discussion)
From the test results in Table 8 and Table 9, it was found that 2-bromo-2-nitro-1,3-diacetoxy-propane was formed at a low concentration compared to 1,4-bis- (bromoacetoxy) -2-butene. You can see that it is inhibiting.

Claims (3)

The suspension of bacteria isolated from the sterilization target system is collected in a microtube made of a thermoplastic resin selected from polyvinyl chloride, polystyrene, polyethylene, and polypropylene together with the culture medium, and then sterilized or distilled water. Based on the result of evaluating the presence or absence of a biofilm after static culture in a diluted medium diluted to 100 to 100 times, an isolated bacterium forming a biofilm is identified, and an effective bactericidal agent for the isolated bacterium A method for preventing microbial damage in a system to be sterilized , comprising selecting and adding the bactericide to the system to be sterilized. After the biofilm is dyed with a staining solution, the microtube is washed and dried, the dyed biofilm is dissolved in a solvent, and the absorbance of the obtained solution is measured. The method for preventing microbial damage according to claim 1, wherein The selection of an effective fungicide is the addition of several industrial fungicides individually in known amounts to the suspension of isolates forming the biofilm, and the resulting suspensions individually In addition, it is collected in a microtube made of a thermoplastic resin selected from polyvinyl chloride, polystyrene, polyethylene, and polypropylene, statically cultured in a diluted medium diluted 5 to 100 times with sterilized water or distilled water, and then biofilm The method for preventing microbial damage according to claim 1 or 2, wherein the method is carried out by evaluating the presence or absence of the microorganism.