JP7436972B2 - Biofilm formation ability evaluation method, biofilm formation ability evaluation device, and multiwell plate lid member for use in biofilm formation ability evaluation - Google Patents

Description

The present invention relates to a novel method for evaluating biofilm formation ability, a biofilm formation ability evaluation device, and a lid member for a multiwell plate for use in evaluating biofilm formation ability.

A biofilm is a biological membrane formed by microorganisms attached to the surface of a solid or liquid, and is a structure in which colonies of microorganisms are formed inside a coating of extracellular polysaccharides (EPS) such as mucopolysaccharides produced by microorganisms. have. EPS protects internal microorganisms from environmental changes and chemicals, and also functions as a transport pathway for substances. Due to the action of such EPS, colonies with high population density are formed and dynamic equilibrium is maintained.

Biofilms can exist anywhere there is a solid or liquid base (such as a gas-liquid interface) and water, and are thought to be involved in material conversion and purification in the natural world. Applications to the agricultural field are also being considered through symbiosis between crops and rhizobia. On the other hand, biofilms are the cause of slime in kitchens and drains, clogging of filtration membranes in water treatment facilities, contamination of medical instruments such as catheters, and cavities and periodontal disease. In a wide range of fields such as dental materials, oral care, and sanitary, research and development is being conducted on materials, surface treatment techniques, etc. for suppressing biofilm formation (for example, see Non-Patent Document 1 and Patent Document 1). .

In the development of technologies to promote and inhibit biofilm formation, evaluation of biofilm formation ability plays an important role, and various evaluation methods have been proposed. Patent Document 2 discloses that raw water or reverse osmosis membrane concentrated water is passed through a water column having a biofilm-forming base material, and the biofilm-forming base material is periodically taken out from the water-passing column to remove the biofilms on the base material. A method for evaluating the amount of film based on the amount of ATP (adenosine triphosphate) is described. However, in order to accurately evaluate biofilm formation ability, these methods require a large amount of water, as well as ATP extraction operations and analysis equipment, so they require complicated operations and large-scale operations. There is a problem that a device is required, and in particular, it is difficult to evaluate multiple substances (substrates) in parallel.

In order to evaluate the biofilm formation ability of multiple substances in parallel on a laboratory scale, for example, put a bacterial suspension in each well of a multiwell plate and place a test piece on the bottom of the well. Alternatively, a method is used in which a biofilm is formed on the surface of the test piece by incubating the test piece while standing it upright in a well. However, with this method, the position of the test piece within the bacterial cell suspension is not stable, and furthermore, for example, in the case of a test piece made of a material with low specific gravity or a test piece with a water-repellent surface, the bacterial cell suspension is There is a risk of floating in the turbid liquid. Due to these factors, conventional methods that do not fix the test piece in a well have difficulty in forming a biofilm on the surface of the test piece with good reproducibility, and also require cleaning of the test piece on which a biofilm has been formed. In addition to requiring complicated operations such as picking up individual specimens with tweezers when staining, there were other problems such as the possibility that the biofilm would peel off during the operation.

Non-Patent Document 2 discloses that each well of a multi-well plate containing a bacterial cell suspension is formed into a disc shape so as to be approximately parallel to the liquid level, and a shaft for height adjustment is provided on the back side. A method has been proposed in which a test piece is loaded with the shaft protruding from a hole provided in the lid, and the plate is incubated while shaking, thereby evaluating the ability to form a biofilm.

Japanese Patent Application Publication No. 2008-13458 International Publication No. 2008/038575

Akihiko Terada et al., “Advances in materials that suppress biofilm formation in the field of water and wastewater treatment,” Journal of Environmental Biotechnology, Vol. 14, No. 2, p. 131-137 ( 2015) Shinobu Oda, “Exploration and production of pharmaceutical-related substances by interfacial bioprocessing”, 2018 New Technology Briefing Material, 15 pages, (URL: https://shingi.jst.go.jp/var/rev0/0000/ 7029/2018_kanazawa-it_3.pdf)

However, in the method described in Non-Patent Document 2, the preparation of a test piece having a complicated shape of a disk-like shape and a shaft for height adjustment is complicated, and the test piece in the well is difficult to prepare. There was a problem in that it was difficult to adjust the height of the pieces.

The present invention was made in view of the above circumstances, and it is possible to produce biofilms in parallel with multiple materials (test pieces, coating agents, etc.) with high precision without requiring complicated equipment, large amounts of sample water, or complicated operations. A method for evaluating the ability to form a biofilm, an apparatus for evaluating the ability to form a biofilm that can be suitably used to carry out the method, and a method for evaluating the ability to form a biofilm that can be suitably used in the above evaluation method and apparatus. The object of the present invention is to provide a lid member for a multi-well plate.

A first aspect of the present invention that meets the above objectives includes the steps of preparing a liquid containing microorganisms capable of forming a biofilm, and placing the liquid in a liquid container including a liquid container having one or more openings on the upper side. a step of accommodating one or more plate-shaped test pieces having a size and shape that can be accommodated inside the liquid accommodating section; fixing the test piece via a fixing means provided on a surface of a portion of the lid member that covers the opening of the liquid container that covers each of the openings of the liquid container; and fixing the test piece. The opening of the liquid container containing the liquid is covered with the lid member, and the liquid contained in the liquid container is placed in a state in which the surface of the test piece is perpendicular or substantially perpendicular to the surface of the liquid. The above-mentioned problem is solved by providing a method for evaluating biofilm formation ability, which includes the steps of: bringing the test piece into contact with the surface of the test piece to form a biofilm on the surface of the test piece; and evaluating the biofilm formation ability. This is to solve the problem.

In the biofilm formation ability evaluation method according to the first aspect of the present invention, it is preferable that the liquid container has a plurality of liquid storage parts, and the biofilm formation ability of a plurality of test pieces is evaluated simultaneously. .

In the biofilm formation ability evaluation method according to the first aspect of the present invention, the liquid container may be a multiwell plate.

In the biofilm forming ability evaluation method according to the first aspect of the present invention, in the step of evaluating the biofilm forming ability, the biofilm formed on the surface of the test piece is stained with a dye, and the biofilm forming ability is stained with a dye. The amount may be determined colorimetrically.

A second aspect of the present invention provides a liquid container including a liquid container having one or more openings on the upper side, and a lid that covers the opening of the liquid container while being positioned with respect to the liquid container. and one or more plate-shaped plates having a size and shape that can be accommodated inside the liquid container, on a surface of a portion of the lid member that covers each of the openings of the liquid container. Provided is a biofilm formation ability evaluation device having a fixing means for fixing a test piece so as to be able to contact the liquid in a state where the surface thereof is perpendicular or substantially perpendicular to the liquid level of the liquid contained in the liquid container. This solves the above problem.

In the biofilm formation ability evaluation device according to the second aspect of the present invention, the liquid container may be a multiwell plate.

In the biofilm formation ability evaluation device according to the second aspect of the present invention, the lid member is made of an elastic polymeric material, and the fixing means is arranged such that the tips thereof are positioned parallel to each other at predetermined intervals. Preferably, the pair of clamping pieces are formed integrally with the lid member and urged inward by the elasticity of the polymeric material so as to be able to clamp the test piece.

In the above case, it is more preferable that the interval between the pair of clamping pieces is formed so as to become narrower toward the distal end side.

In the biofilm formation ability evaluation device according to the second aspect of the present invention, it is preferable that the lid member has a sealing means for contacting the entire circumference of the edge of the opening and sealing the liquid storage section. .

In the biofilm formation ability evaluation device according to the second aspect of the present invention, it is preferable that the lid member is configured as a fitting lid for the liquid container.

In the biofilm formation ability evaluation device according to the second aspect of the present invention, a side skirt is provided around the lid member and extends in the same direction as one side of the clamping member formed on the lid member, and the lid member is It is preferable that the lower end of the side skirt is positioned above the bottom surface of the liquid storage part by a predetermined distance when the side skirt is fitted into the liquid storage container.

A third aspect of the present invention is a lid member that covers the opening of each well while being positioned with respect to a multi-well plate having a plurality of wells, the lid member having a lid member that covers the opening of each well on a surface facing each of the wells. A biomedical device having a fixing means for fixing one or more plate-shaped test pieces having a size and shape that can be accommodated inside a well so as to be able to contact the liquid accommodated in the well in an orthogonal or substantially orthogonal state. The above-mentioned problem is solved by providing a lid member for a multi-well plate for use in evaluating film-forming ability.

In the multi-well plate lid member for use in evaluating biofilm forming ability according to the third aspect of the present invention, the lid member is made of an elastic polymeric material, and the fixing means has a predetermined tip. A pair of clamping pieces that are formed integrally with the lid member so as to be positioned parallel to each other at intervals, and are biased inwardly by the elasticity of the polymeric material so as to be able to clamp the test piece. is preferred.

In the above case, it is more preferable that the interval between the pair of clamping pieces is formed so as to become narrower toward the distal end side.

In the multi-well plate lid member for use in evaluating biofilm formation ability according to the third aspect of the present invention, the lid member contacts the entire circumference of the opening and seals the liquid storage portion. It is preferable to have a sealing means for this purpose.

In the multi-well plate lid member for use in evaluating biofilm formation ability according to the third aspect of the present invention, it is preferable that the lid member is configured as a fitting lid for the liquid container.

A lid member for a multi-well plate for use in evaluating biofilm formation ability according to a third aspect of the present invention, having a side skirt around the lid member extending in the same direction as one side of the clamping side formed on the lid member, It is preferable that the lower end of the side skirt be positioned above the bottom surface of the well by a predetermined distance when the lid member is fitted to the multi-well plate.

In the present invention, "to cover" means to cover and close, "to hold" means to hold a plate-shaped test piece in a state where it is sandwiched from both sides, and "to urge" is a direction in which the distance between the tips of the clamping pieces is narrowed by the elasticity of the material that makes up the clamping pieces, resisting the force of the test piece sandwiched between the clamping pieces to push the clamping pieces apart. This means that an elastic force acts on

In the biofilm formation ability evaluation method of the present invention, the test piece is fixed to a lid member and brought into contact with the liquid contained in the liquid storage part with its surface perpendicular or substantially perpendicular to the liquid surface. Since the position of the test piece in the liquid storage part can be kept constant, a biofilm can be formed with good reproducibility, and the ability to form a biofilm can be evaluated with high precision. Furthermore, by using a liquid container having a plurality of liquid storage parts in combination and a lid member having a fixing means for fixing a test piece on a surface facing each of the openings of the liquid storage part, The biofilm formation ability of multiple test pieces can be evaluated in parallel, and treatments such as washing the test pieces after biofilm formation can be performed at once with a simple operation.

3 is a flowchart showing each step of the biofilm formation ability evaluation method according to the first embodiment of the present invention. FIG. 2 is a schematic diagram of a biofilm formation ability evaluation device according to a second embodiment of the present invention. It is a schematic diagram of the lid member for multiwell plates concerning the 3rd embodiment of the present invention. It is a partial enlarged view of the clamping piece of the same lid member. FIG. 3 is a partially enlarged view showing a fitting structure between a multi-well plate and a lid member. FIG. 2 is a schematic diagram showing a state in which a test piece accommodated in a well is fixed by a clamping piece. 3 is a graph showing the results of Example 1. 3 is a graph showing the results of Example 2. 3 is a photograph showing the state of a test piece after staining in Example 2. 3 is a graph showing the results of Example 3.

Next, embodiments embodying the present invention will be described with reference to the attached drawings to provide an understanding of the present invention. The biofilm formation ability evaluation method according to the first embodiment of the present invention (hereinafter sometimes simply referred to as "biofilm formation ability evaluation method", "evaluation method", etc.) is shown in FIG. As shown, (1) preparing a bacterial cell suspension (liquid containing microorganisms capable of forming a biofilm) ("preparation of bacterial cell suspension"); and (2) preparing one or more openings on the upper side. (3) a step of seeding (accommodating) a bacterial suspension in a liquid container having a liquid storage part having a size ("cell suspension seeding"); and (3) a size that can be stored inside the liquid storage part and (4) A step of preparing one or more plate-shaped test pieces having a shape (not shown); and (4) a liquid container of a lid member that covers the opening of the liquid container while being positioned with respect to the liquid container. (5) step of attaching (fixing) the test piece to the fixing means provided on the surface of the part that covers each of the openings ("test piece attachment"); Cover the opening of the liquid container and immerse (contact) the test piece in the bacterial cell suspension contained in the liquid container with the surface of the test piece perpendicular or substantially perpendicular to the liquid surface. (“test piece immersion”), a step of culturing under predetermined conditions to form a biofilm on the surface of the test piece (“culturing”), and (6) for example, after washing the test piece, immersing it in a staining solution. After staining the biofilm and washing it to remove the dyes that do not adsorb to the biofilm, extract the dyes adsorbed to the biofilm, and measure the biofilm formation ability by colorimetric determination (measurement of absorbance) of the dye extract. It has evaluation steps ("washing", "staining", "washing", "extraction", "measurement"). Each step will be explained in detail below.

(1) Preparation of bacterial cell suspension First, a bacterial cell suspension, which is an example of a liquid containing microorganisms capable of forming a biofilm, is prepared. The liquid that is a dispersion medium for microbial cells is, for example, water or an aqueous solution, and may be environmental water collected from the natural environment or living environment, waste water, etc., and a culture solution (liquid medium) in which cultured microorganisms are dispersed. It may be. There is no particular limit to the number of microorganisms per unit volume, and if necessary, a solution diluted with any dispersion medium can be used as a microbial cell suspension.

The type of microorganism is not particularly limited as long as it has the ability to form a biofilm, but examples include Pseudomonas aeruginosa, Escherichia coli, Staphylococcus aureus, etc. Specific examples include Corynebacterium xerosis, Lactobacillus Lactobacillus brevis, Staphylococcus aureus, Staphylococcus epidermidis, Streptococcus mutans, Streptococcus oralis, Streptococcus oralis - Oral bacteria: Streptococcus salivarius, Acinetobacter baumannii, Cronobacter (formerly Enterobacter) - Cronobacter sakazakii, Escherichia coli, Porphyromonas gingivalis (periodontal disease bacteria), Examples include Serratia marcescens and Pseudomonas aeruginosa.

(2) Seeding of bacterial cell suspension Next, a predetermined volume of the bacterial cell suspension is stored (seeded) in the liquid container of the liquid container. The number of liquid storage parts may be one or more, and in that case, the biofilm formation ability of a plurality of test pieces can be evaluated simultaneously under the same conditions. An example of a liquid container having a plurality of liquid storage parts is a multiwell plate having wells of any number, size, and shape. Although the number, size, and shape of the wells are not particularly limited, they are, for example, cylindrical with an inner diameter of 15 mmφ and a height of 20 mm. For wells of this size, a few mL of bacterial cell suspension per well is sufficient to evaluate the ability to form a biofilm. The material constituting the multi-well plate is not particularly limited, and any material used for multi-well plates can be used without restriction.

A biofilm forming ability evaluation device 10 (hereinafter referred to as “ The evaluation device 10 (sometimes abbreviated as "evaluation device 10", etc.) has a structure as shown in FIG. The lid member for a multi-well plate for use in the evaluation of biofilm formation ability according to the third embodiment of the present invention, which is used in combination with the method, also has a similar structure. There is.

The multi-well plate has a plurality of wells 12 (an example of a liquid storage part) each having an opening on the upper side. The lid member 13 is a member for covering the opening of each well 12 while positioned with respect to the multi-well plate 11, and a plate-shaped test piece is provided on the surface of the portion covering the opening of each well 12. It has a clamping piece 14 (an example of a fixing means) for fixing the piece so that the piece can come into contact with the liquid in a state where the surface thereof is perpendicular or substantially perpendicular to the liquid level of the liquid contained in the liquid container. . The lid member 13 is made of an elastic polymer material. Specific examples of polymeric materials that can be used for the lid member 13 include polypropylene, ethylene-propylene copolymer, AS resin, ABS resin, etc. Polypropylene, which has excellent elasticity and flexibility and is transparent, is preferably used. As shown in FIG. 3, the clamping piece 14 is formed integrally with the lid member 13 so that its tips are positioned parallel to each other at predetermined intervals, and the clamping piece 14 is made of the polymeric material so as to be able to clamp the test piece (18). They are a pair of plate-shaped members that are urged inward by their elasticity.

As shown in FIG. 4, it is preferable that the clamping pieces 14 are formed so that the intervals become narrower from the root side to the tip side so that they are bilaterally symmetrical when viewed from the side. The distance between the tips of the pair of clamping pieces 14 is preferably narrower than the thickness of the test piece. By configuring the pair of clamping pieces 14 in this way, the test piece 18 is fixed almost perpendicularly to the lid member 13, so that when it is later immersed in a bacterial cell suspension, it is placed almost against the liquid surface. Can be kept vertical. Further, as shown in FIGS. 2, 3, and 4, the lid member 13 includes a seal member 15 (an example of a sealing means) that contacts the entire circumference of the opening edge of each well 12 and seals the well 12. may be formed.

As shown in FIGS. 2 and 5, the lid member 13 is formed as a fitting lid for the multi-well plate 11 so that it can cover each well 12 while positioned relative to the multi-well plate 11. Good too. More specifically, a plate-shaped peripheral wall 16 is formed around the multi-well plate 11, and around the lid member 13, the side on which the clamping piece 14 is provided faces downward. The side skirt 17 is formed so as to hang down toward the surface on which the holding piece 14 is formed when the side skirt 17 is arranged as shown (corresponding to the arrangement during use). As shown in FIG. 2, end pins 19 having a predetermined length may be formed at the four distal corners of the side skirt 17. By fitting both the peripheral wall 16 and the side skirt 17, the lid member 13 fits into the multiwell plate 11 while being positioned in the horizontal direction. Furthermore, since the sealing member 15 is configured to cover each well 12 while sealing the opening of each well 12, the lid member 13 is fitted in a state in which it is positioned perpendicularly to the multi-well plate 11. do. With such a configuration, it is possible to prevent the volatilization of the bacterial cell suspension contained in each well 12 and contamination by microorganisms in the atmosphere, and, as will be described later, the test piece can be attached to the clamping piece 14. When holding 18, side skirts 17 or end pins 19 can be used as vertical positioning means. Further, as shown in FIG. 2, the side skirt 17 is provided with a notch to make it easier to hold only the lid member 13 or the lid member 13 and the multiwell plate 11 at the same time. Good too.

(3) Preparation and Fixation of Test Piece Next, the test piece 18 is attached to the clamping piece 14 of the lid member 13 and fixed. As shown in FIG. 6, the test piece 18 is a plate-like member that can be accommodated inside the well 12 and has a size and shape that allows part of its surface to come into contact with the bacterial cell suspension. The test piece is preferably rectangular, and the thickness and size are appropriately selected depending on the size and shape of the well 12, the accumulation of the bacterial suspension to be accommodated, etc. Preferably, if the diameter is about 10 to 20 mm, the ability to form a biofilm can be evaluated with sufficient accuracy. The material and surface properties (surface processing mode, presence or absence of coating agent, etc.) of the test piece 18 are not particularly limited and can be appropriately selected depending on the purpose. To fix the test piece 18 to the clamping piece 14, place the lid member 13 on a horizontal table with the clamping piece 14 facing upward, pinch the test piece 18 with tweezers, etc., and pinch it between the clamping pieces 14. This can be done by any method such as. At this time, the test piece 18 is slightly sandwiched between the clamping pieces 14, and then the upper limit of the lid member 13 is reversed, placed on a horizontal table with the clamping pieces 14 facing downward, and pressed lightly. By aligning the lower end of the side skirt 17 or end pin 19 with the lower end of the test piece 18 (the position indicated by the dashed line in FIG. 6), the test piece 18 can be easily and reliably positioned in the vertical direction. I can do it.

(4) Formation of biofilm (culture)
The surface of the test piece 18 is cultured at a predetermined temperature for a predetermined time in the evaluation device 10, which is equipped with a lid member 13 that holds a bacterial culture solution in each well 12 and fixes the test piece 18 to the clamping piece 14. to form a biofilm. Shaking etc. may be performed as necessary. The culture temperature and culture time can be appropriately selected depending on the type of bacteria in the bacterial suspension. During culture, the medium in the bacterial cell suspension may be replaced with a fresh medium as appropriate (for example, at predetermined intervals).

(5) Evaluation of biofilm formation ability Biofilm formation ability was evaluated by measuring the mass change of each specimen before and after biofilm formation, and by evaluating the colony formation ability of microorganisms in the biofilm detached by ultrasonic irradiation. Any method such as evaluation can be used, but in the evaluation method according to the first embodiment of the present invention, a biofilm is stained with a dye, and the amount of dye in the stained biofilm (the mass of the biofilm is The biofilm forming ability is evaluated by a method of quantifying (proportional to). More specifically, the test piece 18 on which a biofilm has been formed is washed and stained with a dye, the stained test piece 18 is washed, and after removing the dye that has not been adsorbed to the biofilm, the biofilm is removed. The ability to form biofilms is evaluated by extracting pigments and performing colorimetric determination of the pigments.

As for dyes used for biofilm staining, the amount of adsorption to the biofilm is a function of the amount of biofilm formed, and the amount of biofilm formed can be quantitatively evaluated from the results of colorimetric analysis by creating a calibration curve, etc. Any possible dye can be used. The dye preferably has a high molecular extinction coefficient and is soluble in organic solvents such as alcohols. Specific examples of such dyes include crystal violet and the like. An aqueous solution such as water or physiological saline is used to wash the test piece 18 after biofilm formation and after staining. For dyeing and extraction of the dye after dyeing, a solvent with high solubility of the dye is appropriately selected depending on the type of dye. Preferred examples of dyeing and extraction solvents include alcoholic solvents such as ethanol and methanol, and water-soluble organic solvents such as acetone.

Any known method can be used for each of the cleaning, staining, and extraction steps, but when using the evaluation method, evaluation device, or lid member according to each embodiment of the present invention, cleaning liquid, staining liquid, and extraction liquid The biofilm is damaged with a simple operation by preparing a multi-well plate 11 containing a sample and replacing the multi-well plate 11 one after another with the test piece 18 fixed to the lid member 13 by the clamping piece 14. It is possible to implement it without

The purpose and application of biofilm formation ability evaluation include evaluation of materials and surface treatment technologies for preventing environmental pollution in water treatment facilities, housing equipment, and sanitary fields, and evaluation of microorganisms in fields such as medical equipment, dental materials, and oral care. evaluation of anti-proliferation materials and technologies.

Next, examples performed to confirm the effects of the present invention will be described.

Example 1: Formation of biofilm on test piece A culture solution of Staphylococcus aureus (NBRC13276) was diluted and prepared with a medium (Mueller-Hinton broth) to a concentration of about 10 ⁷ cfu/mL, and this bacterial cell suspension was prepared. 2 mL of the solution was dispensed into each well of a 24-well microplate. A polystyrene test piece (thickness 2 mm x width 10 mm x length 20 mm) is fixed on a lid member for a multiwell plate having the structure shown in Figure 3, and placed over the 24-well microplate into which the bacterial cell suspension has been dispensed. The test piece was placed so as to be perpendicular to the cell suspension (10 mm of the test piece in the longitudinal direction was immersed in the cell suspension). Furthermore, a biofilm was formed on the test piece by static culture at 37°C for a certain period of time. Culture conditions were set as follows.
・Test group 1: Culture for 24 hours ・Test group 2: Culture for 48 hours ・Test group 3: After 24 hours of culture, replace with fresh medium and culture for another 24 hours

After culturing, the lid member for the multiwell plate on which the test piece was fixed was transferred to a 24-well microplate filled with physiological saline and washed to remove floating microbial cells. Furthermore, the biofilm was transferred to a 24-well microplate containing 0.1% crystal violet and stained for 30 minutes. After staining, the test piece was washed with physiological saline, transferred to a 24-well microplate containing 99.5% ethanol, and extracted for 15 minutes. A 24-well microplate containing the extract was placed in a plate reader and subjected to absorbance measurement at 595 nm. The results are shown in Table 1 and FIG.

Compared to Test Groups 1 and 2, more biofilms could be formed on the test pieces in Test Group 3. Therefore, in Examples 2 and 3 below, biofilm forming ability was evaluated according to the procedure of Test Group 3.

Example 2: Reproducibility of biofilm formation (comparison with conventional method)
Currently, a method of placing a test piece at the bottom of each well of a microplate to form a biofilm (hereinafter referred to as the conventional method) is widely used. Therefore, we conducted a biofilm formation test on Staphylococcus aureus (S. aureus NBRC13276) using a method using a multi-well plate lid member having the structure shown in Figures 2 and 3 (hereinafter referred to as this method) and a conventional method. The reproducibility of biofilm formation was compared.

This method was carried out according to the conditions of Test Group 3 of Example 1. On the other hand, in the conventional method, a test piece (2 mm thick x 10 mm wide x 10 mm long) was placed at the bottom of each well of a 24-well microplate, and 2 mL of the bacterial cell suspension was dispensed into the test group 3 of Example 1. Medium exchange, washing, staining, extraction, and measurement were performed according to the procedure. All movements of the test piece in the conventional method were performed using tweezers. The results are shown in Table 2 and FIG.

The coefficient of variation (CV) (n=8) was 5.6% for the present method, whereas it was 42.3% for the conventional method, indicating that the reproducibility of the present method was significantly superior. As shown in FIG. 9, in the conventional method, the degree of staining of the biofilm was uneven, and the biofilm formation was highly variable. On the other hand, with this method, a reproducible biofilm could be formed.

Example 3: Effect of positioning the test piece in the vertical direction using the multi-well plate lid member When using the multi-well plate lid member having the structure shown in FIG. However, in this example, the biofilm formation ability was compared with and without vertical positioning to verify its effectiveness. A biofilm formation test of S. aureus NBRC13276 was performed according to the procedure of test group 3 in Example 1, with and without vertical positioning ("with height adjustment"). (“No height adjustment”). The results are shown in Table 3 and FIG.

The coefficient of variation (CV) (n = 6) was 2.1% with the height adjustment feature compared to 11.7% without it; The reproducibility of the cases was excellent.

10: Biofilm formation ability evaluation device 11: Multi-well plate 12: Well 13: Lid member 14: Clamping piece 15: Seal member 16: Peripheral wall 17: Side skirt 18: Test piece 19: End pin

Claims (15)

A lid member made of an elastic polymer material and covering the opening of each well while positioned with respect to a multi-well plate having a plurality of wells,
One or more plate-shaped test pieces having a size and shape that can be accommodated inside the well are placed on the surface of the portion covering each of the wells in a state that is perpendicular or substantially perpendicular to the well. has a fixing means for fixing it so that it can come into contact with the liquid to be
The fixing means is formed integrally with the lid member so that the tips are positioned parallel to each other at predetermined intervals, and is biased inward by the elasticity of the polymeric material so as to be able to clamp the test piece. A lid member for a multi-well plate for use in evaluating biofilm formation ability , which is a pair of clamping pieces. The lid member for a multiwell plate for use in evaluating biofilm forming ability according to claim 1 , wherein the distance between the pair of clamping pieces is formed so as to become narrower toward the tip side. Used for the evaluation of biofilm formation ability according to claim 1 or 2, characterized in that it has a sealing means for contacting the entire periphery of the opening edge of the well and sealing each well of the multiwell plate. Lid member for multi-well plate. The lid member for a multi-well plate for use in evaluating biofilm formation ability according to any one of claims 1 to 3 , characterized in that it is configured as a fitting lid for the multi-well plate. A side skirt is provided around the lid member and extends in the same direction as one side of the clamping side formed on the lid member, and when the lid member is fitted to the multi-well plate, the lower end of the side skirt is lower than the bottom surface of the well. 5. The lid member for a multi-well plate for use in evaluating biofilm forming ability according to any one of claims 1 to 4 , wherein the lid member is configured to be positioned above by a predetermined distance. a liquid container comprising one or more liquid storage portions having an opening on the upper side;
a lid member made of an elastic polymeric material and covering an opening of the liquid storage portion while positioned relative to the liquid storage container;
On the surface of the portion of the lid member that covers each of the openings of the liquid container, one or more plate-shaped test pieces having a size and shape that can be accommodated inside the liquid container are placed. having a fixing means for fixing the liquid in a state where the surface thereof is perpendicular or substantially perpendicular to the liquid level of the liquid contained in the liquid container so as to be able to contact the liquid;
The fixing means is formed integrally with the lid member so that the tips are positioned parallel to each other at predetermined intervals, and is biased inward by the elasticity of the polymeric material so as to be able to clamp the test piece. A device for evaluating biofilm formation ability , characterized in that it is a pair of clamping pieces . 7. The biofilm formation ability evaluation device according to claim 6 , wherein the liquid container is a multiwell plate. The biofilm forming ability evaluation device according to claim 6 or 7, wherein the interval between the pair of clamping pieces is formed so as to become narrower toward the tip side. Biofilm formation according to any one of claims 6 to 8, characterized in that the lid member has a sealing means for contacting the entire circumference of the edge of the opening and sealing the liquid storage part. performance evaluation device. The biofilm forming ability evaluation device according to any one of claims 6 to 9, wherein the lid member is configured as a fitting lid for the liquid container. A side skirt is provided around the lid member and extends in the same direction as the clamping piece formed on the lid member, and when the lid member is fitted into the liquid container, the lower end of the side skirt is The biofilm forming ability evaluation device according to any one of claims 6 to 10, wherein the device is configured to be located a predetermined distance above the bottom surface of the liquid storage section. preparing a liquid containing microorganisms capable of forming a biofilm;
accommodating the liquid in a liquid container including one or more liquid accommodating portions having an opening on the upper side;
preparing one or more plate-shaped test pieces having a size and shape that can be accommodated inside the liquid storage section;
a lid member made of an elastic polymer material that covers the openings of the liquid storage section when positioned with respect to the liquid storage container , and a surface of a portion that covers each of the openings of the liquid storage container; At the top , the lid member was formed integrally with the lid member so that the tips were positioned parallel to each other at predetermined intervals, and was urged inward by the elasticity of the polymer material so as to be able to clamp the test piece. This is a step of fixing the test piece to the lid member that is provided with fixing means that are a pair of clamping pieces, and fixing the test piece by sandwiching the test piece between the pair of clamping pieces of the lid member. process and
The opening of the liquid container containing the liquid is covered with the lid member to which the test piece is fixed, and the liquid is contained in a state in which the surface of the test piece is perpendicular or substantially perpendicular to the surface of the liquid. bringing the test piece into contact with the liquid contained in a container to form a biofilm on the surface of the test piece;
A method for evaluating biofilm formation ability, comprising the step of evaluating the biofilm formation ability. 13. The method for evaluating biofilm formation ability according to claim 12 , wherein the liquid container has a plurality of liquid storage sections, and the biofilm formation ability of a plurality of test pieces is evaluated simultaneously. The biofilm formation ability evaluation method according to claim 12 or 13, wherein the liquid container is a multiwell plate. Any one of claims 12 to 14 , characterized in that in the step of evaluating the ability to form a biofilm, the biofilm formed on the surface of the test piece is stained with a dye, and the amount of the dye is quantified colorimetrically. The method for evaluating biofilm formation ability according to item 1.

Images