Methods for evaluating biofilm formation

Abstract

The present invention provides a biofilm formation evaluation method that can evaluate the antibiofilm formation status under actual use conditions with general accuracy and in a short period of time. [Solution] The present invention provides a biofilm formation evaluation method that includes a sampling step of sampling water from a measurement environment, a placement step of placing a test piece in a mixed solution containing the water and culture medium, a cultivation step of culturing the mixed solution containing the test piece under test conditions, and an evaluation step of evaluating the biofilm formed on the test piece.

Description

Technical Field

[0005] , , ,

[0001] The present invention relates to a method for evaluating biofilm formation.

Background Art

[0002] A biofilm is a film that surrounds microorganisms formed by microorganisms such as those attached to a substance secreting extracellular polysaccharide (EPS, extracellar polysaccharide). EPS serves as a barrier to protect microorganisms, a transportation route for microorganisms to take in nutrients, etc., and protects the microorganisms inside the biofilm from environmental changes and chemical substances.

[0003] Biofilms are formed not only in nature such as ponds and rivers but also inside pipes of plants, etc. When the biofilm peels off, it causes problems such as foreign matter mixing into the product. Furthermore, when a biofilm is formed on water-related members used in bathrooms, kitchens, etc., it causes slipperiness and bad odors, resulting in hygienic problems. Also, when a biofilm is formed on the inner surface of an aquarium, it may have an adverse effect on the organisms in the aquarium. Therefore, under actual use, confirmation of the degree of biofilm formation is carried out as necessary. As a method for evaluating the formation state of a biofilm, ISO4768 stipulates an anti-biofilm test method.

Prior Art Documents

Non-Patent Documents

[0004]

Non-Patent Document 1

Summary of the Invention

Problems to be Solved by the Invention

[0005] However, the antibiofilm testing method specified in Non-Patent Document 1 involves culturing under predetermined specific conditions, which differ from the biofilm formation environment under actual use. Therefore, the antibiofilm activity evaluated by the testing method specified in Non-Patent Document 1 deviates from the antibiofilm activity under actual use, resulting in the problem that the biofilm formation status under actual use cannot be accurately evaluated.

[0006] Therefore, the present invention provides a method that can evaluate the formation status of antibiofilms under actual use conditions with general accuracy and in a short period of time. [Means for solving the problem]

[0007] The biofilm formation evaluation method of the present invention is A sampling process for collecting water samples from the measurement environment, A placement step involves placing a test specimen in a mixed solution containing the above-mentioned measurement water and culture medium, A culture step in which the mixed solution containing the above-mentioned test specimens is cultured under test conditions, The system is characterized by including an evaluation step of evaluating the biofilm formed on the above-mentioned test specimen. [Effects of the Invention]

[0008] The present invention's biofilm formation evaluation method allows for the cultivation of test specimens in a mixed solution containing measurement water and culture medium under test conditions similar to those actually used, enabling accurate and rapid evaluation of biofilm formation in the actual usage environment. Therefore, appropriate measures can be taken in advance to address biofilm formation in the actual usage environment, thereby suppressing biofilm growth. [Modes for carrying out the invention]

[0009] The present invention provides a method for evaluating biofilm formation, comprising: a sampling step of collecting water from a measurement environment; a placement step of placing a test piece in a mixed solution containing the water and culture medium; a cultivation step of culturing the mixed solution containing the test piece under test conditions; and an evaluation step of evaluating the biofilm formed on the test piece.

[0010] [Collection process] The biofilm formation evaluation method includes a sampling step of collecting water samples from the measurement environment.

[0011] In the sampling process, water samples are collected from the actual environment in which the sample is used (measurement environment). The measurement environment is not particularly limited as long as it is an environment in which the sample is actually used and where biofilm may form. Examples of measurement environments include sink drains, bath drains, washbasin drains, toilet drains, the inner surface of air conditioner drain pipes, inside water and sewage pipes, structures (e.g., bridge piers) or floating objects (e.g., buoys) installed in rivers or oceans.

[0012] The measurement water should be collected from the measurement environment in such a way that the microorganisms that cause biofilm formation in the measurement environment are in a state as close as possible to that of the measurement environment. By using measurement water containing microorganisms that are in a state as close as possible to that of the measurement environment, it is possible to generate a biofilm in a state similar to that of the measurement environment during the culture process described later, and to accurately understand the state of biofilm formation in the measurement environment.

[0013] Examples of water samples taken from the measurement environment include contaminated water such as wastewater accumulated in the measurement environment. If the measurement environment is a structure or floating object, water in contact with the surface of the object or water present in its vicinity should be collected.

[0014] [Placement process] The biofilm formation evaluation method includes a placement step in which a test specimen is placed in a mixed solution containing the measurement water collected in the sampling step and the culture medium.

[0015] First, prepare a liquid medium containing a medium and purified water. The liquid medium can be prepared by uniformly mixing the medium in purified water. The content of the medium in the liquid medium is preferably 0.1 to 2.0% by mass, more preferably 0.3 to 1.5% by mass.

[0016] The medium contained in the liquid medium may be any medium that does not have an adverse effect on the growth of microorganisms that produce biofilms. The component composition of the liquid medium is not particularly limited, and it may contain at least one of components such as a carbon source, a nitrogen source, a sulfur source, a phosphorus source, a vitamin source, and a mineral source.

[0017] The liquid medium preferably contains glucose. When the liquid medium contains glucose, the content of glucose is preferably 0.1 to 2.0 g / L, more preferably 0.2 to 1.8 g / L.

[0018] The liquid medium preferably contains yeast extract. When the liquid medium contains yeast extract, the content of yeast extract is preferably 0.1 to 2.0 g / L, more preferably 0.2 to 1.8 g / L.

[0019] The liquid medium preferably contains peptone. When the liquid medium contains peptone, the content of peptone is preferably 0.1 to 2.0 g / L, more preferably 0.2 to 1.8 g / L.

[0020] The liquid medium preferably contains casamino acids. When the liquid medium contains casamino acids, the content of casamino acids is preferably 0.1 to 2.0 g / L, more preferably 0.2 to 1.8 g / L.

[0021] The liquid medium preferably contains active starch. When the liquid medium contains active starch, the content of active starch is preferably 0.1 to 2.0 g / L, more preferably 0.2 to 1.8 g / L.

[0022] The liquid medium preferably contains potassium hydrogen phosphate. When the liquid medium contains potassium hydrogen phosphate, the content of potassium hydrogen phosphate is preferably 0.02 to 1.50 g / L, more preferably 0.05 to 1.00 g / L.

[0023] The liquid medium preferably contains magnesium sulfate heptahydrate. When the liquid medium contains magnesium sulfate heptahydrate, the content of magnesium sulfate heptahydrate is preferably 0.01 to 1.00 g / L, more preferably 0.01 to 0.50 g / L.

[0024] The liquid medium preferably contains sodium pyruvate. When the liquid medium contains sodium pyruvate, the content of sodium pyruvate is preferably 0.02 to 1.50 g / L, more preferably 0.05 to 1.00 g / L.

[0025] The liquid medium is preferably heat-treated to kill the microorganisms contained in this liquid medium. By previously killing the microorganisms contained in the liquid medium before mixing the measurement water into the liquid medium, the evaluation of the formation of biofilm caused only by the measurement water can be performed more accurately.

[0026] The heat treatment of the liquid medium is not particularly limited as long as it can kill the microorganisms contained in the liquid medium. Examples of the heat treatment of the liquid medium include heating the liquid medium at 100 to 200 °C for a predetermined time (preferably 10 minutes or more) under pressure conditions (preferably 0.2 to 1.0 MPa).

[0027] Next, measurement water is uniformly mixed into the liquid medium to prepare a mixed solution. The amount of measurement water mixed into the liquid medium is appropriately changed according to the measurement environment and is not particularly limited, but it is preferably 3500 to 20000 parts by mass, more preferably 4000 to 15000 parts by mass, and even more preferably 4500 to 12500 parts by mass based on 100 parts by mass of the medium contained in the liquid medium.

[0028] In the above description, we explained the case in which a liquid culture medium is prepared in advance and the measurement water is mixed with this liquid culture medium to make a mixed solution. However, the order in which the purified water, culture medium, and measurement water are mixed is not particularly limited. For example, the purified water, culture medium, and measurement water may be mixed all at once to make a mixed solution, or the culture medium may be uniformly mixed into the mixed water prepared by mixing purified water and measurement water to make a mixed solution. It is preferable to heat-treat the purified water in the same manner as the heat treatment of the liquid culture medium before mixing it with the culture medium and / or measurement water.

[0029] Alternatively, instead of using purified water, a mixed solution may be prepared by directly mixing the powdered culture medium with the water used for measurement.

[0030] The test specimen is placed in the mixed solution prepared as described above. The shape of the test specimen is not particularly limited and can be, for example, a sheet or a block. The arrangement of the test specimen in the mixed solution should preferably be as close as possible to the biofilm formation environment in the measurement environment. By arranging it in this way, it becomes possible to accurately evaluate the biofilm formation status in the actual measurement environment. For example, in the case of a sink drain, the area where biofilm is formed is generally submerged in contaminated water, so the test specimen is placed so that its entire body is immersed in the mixed solution. In addition, for example, when evaluating the biofilm formation status on the surface of the piers of a suspension bridge constructed over a river, only the lower end of the pier is placed in water, and the rest of the pier is placed above the water surface. In this case, the test specimen is placed so that its lower half is immersed in the mixed solution and its upper half is above the water surface, thus simulating a condition close to the actual measurement environment.

[0031] Furthermore, the arrangement of the test specimens in the mixed solution does not need to be kept constant throughout the culture process described later. If the measurement environment changes over time, it is preferable to change the arrangement of the test specimens in the mixed solution over time to approximate the changes in the measurement environment over time.

[0032] The test specimen is preferably made of the same material as the component on which the biofilm is formed in the measurement environment, as this allows for accurate evaluation of the biofilm formation status in the measurement environment. However, it may also be made of a different material from the component on which the biofilm is formed in the measurement environment, as long as the biofilm formation status can be evaluated similarly.

[0033] [Culture process] The biofilm formation evaluation method includes a culture step in which a mixed solution containing test specimens is cultured under test conditions.

[0034] In the culture process, a biofilm is formed on the surface of the test specimen by microorganisms contained in the mixed solution due to the water used for measurement. The culture conditions for forming the biofilm by microorganisms in the culture process are not particularly limited. The mixed solution should be maintained at a temperature that is generally conducive to the growth of the microorganisms contained in it (for example, 20-40°C). The culture time should be set appropriately depending on the type of microorganism and the concentration of microorganisms in the mixed solution. The culture time is preferably 0.5-40 hours, more preferably 1-35 hours, and even more preferably 12-30 hours.

[0035] The microorganisms mentioned above are not particularly limited as long as they form biofilms, and may be prokaryotes such as bacteria, or eukaryotes such as yeasts and fungi. The bacteria may be either Gram-positive or Gram-negative.

[0036] Gram-positive bacteria include bacteria of the genus Bacillus (e.g., Bacillus coagulans, Bacillus anthracis, Bacillus atrophaeus, Bacillus cereus, Bacillus megaterium, Bacillus pumilus, Bacillus subtilis, etc.), bacteria of the genus Clostridium (e.g., Clostridium botulinum, Clostridium difficile, Clostridium perfringens, Clostridium sporogenes, Clostridium tetani, etc.), bacteria of the genus Enterococcus (e.g., Enterococcus faecalis, Enterococcus faecium, etc.), and bacteria of the genus Lactobacillus (e.g., Lactobacillus Bacteria of the Mycobacterium genus (e.g., Mycobacterium bovis, Mycobacterium leprae, Mycobacterium terrae, Mycobacterium tuberculosis, etc.), Bacteria of the Propionibacterium genus (e.g., Propionibacterium acnes, etc.), Bacteria of the Staphylococcus genus (e.g., Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus lugdunensis, Staphylococcus saprophyticus, etc.), Bacteria of the Streptococcus genus (e.g., Streptococcus mitis, Streptococcus mutans, StreptococcusExamples include *Streptococcus oralis*, *Streptococcus pneumoniae*, and *Streptococcus pyogenes*.

[0037] Gram-negative bacteria include those of the genus Bordetella (e.g., Bordetella pertussis), Campylobacter (e.g., Campylobacter jejuni), Enterobacter (e.g., Enterobacter cloacae), Escherichia (e.g., Escherichia coli), Fusobacterium (e.g., Fusobacterium nucleatum), Helicobacter (e.g., Helicobacter pylori), Klebsiella (e.g., Klebsiella pneumoniae), and Neisseria (e.g., Neisseria gonorrhoeae). Examples include bacteria of the genus Pseudomonas (e.g., Meningococcus meningitidis), Pseudomonas (e.g., Pseudomonas aeruginosa, Pseudomonas putida), Salmonella (e.g., Salmonella enterica serovar Typhi, Salmonella enterica serovar Paratyphi A, Salmonella enterica serovar Typhimurium, Salmonella enterica serovar Enteritidis), Serratia (e.g., Serratia marcescens), and Vibrio (e.g., Vibrio cholerae, Vibrio parahaemolyticus).

[0038] [Evaluation Process] The biofilm formation evaluation method includes an evaluation step of evaluating the biofilm formed on a test specimen.

[0039] The biofilm formed on the surface of the test specimen during the culture process is evaluated. The biofilm formed on the surface of the test specimen is cultured in an environment similar to the measurement environment. Therefore, by evaluating the biofilm formed on the surface of the test specimen, it is possible to easily predict how the biofilm will form in the measurement environment, and to prepare in advance for the biofilm under the measurement environment. By evaluating not only the quantification of the biofilm formed on the test specimen but also its distribution, the biofilm formation morphology under the measurement environment can be understood more accurately, and biofilm suppression can be implemented more effectively.

[0040] Any known method that can confirm the degree of biofilm formation can be used to evaluate the biofilm formed on the test specimen.

[0041] For example, one method involves observing the surface of a test specimen using a microscope (e.g., a laser microscope) and evaluating the biofilm formed on the specimen. Methods for evaluating the biofilm include, for example, photographing the biofilm on the specimen and calculating the total area of ​​the biofilm visible in the resulting magnified photograph to objectively evaluate the degree of biofilm formation numerically. The biofilm may also be stained using known methods.

[0042] Alternatively, biofilms may be quantified by measuring the amount of adenosine triphosphate (ATP) contained in them. Furthermore, biofilms may be quantified by measuring not only the amount of adenosine triphosphate (ATP) but also the amounts of ADP (adenosine diphosphate) and AMP (adenosine monophosphate). An adenosine triphosphate measuring device is commercially available from Todenpa Kogyo Co., Ltd. under the product name "ATP Tester". A device for measuring the total amount of adenosine triphosphate (ATP), ADP (adenosine diphosphate), and AMP (adenosine monophosphate) is commercially available from Kikkoman Bio-Chemifa Co., Ltd. under the product name "Lumitester smart".

[0043] Alternatively, the biofilm may be quantified by staining it with a crystal violet solution and measuring its absorbance. For example, the biofilm may be stained with a crystal violet solution, the surface may be washed with purified water, the stained biofilm may be wiped off with a water-soluble nonwoven fabric, and the resulting water-soluble nonwoven fabric may be added to 5 mL of a 1% by mass sodium dodecyl sulfate aqueous solution and shaken to dissolve it and prepare a solution. The absorbance of the resulting solution at 590 nm may be measured using a microplate reader.

[0044] The biofilm evaluation may be performed only once after the completion of the culture process, or it may be performed once or multiple times during the culture process, not just at the end of the culture process. By evaluating the biofilm once or multiple times during the culture process, it is possible to understand the changes in the biofilm over time, implement measures to suppress biofilm growth more effectively, and suppress the occurrence of biofilm in the measurement environment more easily.

Claims

[Claim 1] A sampling process for collecting water samples from the measurement environment, A placement step involves placing a test specimen in a mixed solution containing the above-mentioned measurement water and culture medium, A culture step in which the mixed solution on which the above test specimens are placed is cultured under test conditions, A method for evaluating biofilm formation, characterized by including an evaluation step of evaluating the biofilm formed on the above-mentioned test piece.