WO2022172931A1

WO2022172931A1 - Method for evaluating biofilm formation ability, device for evaluating biofilm formation ability, and lid member for multi-well plate for use in evaluating biofilm formation ability

Info

Publication number: WO2022172931A1
Application number: PCT/JP2022/004984
Authority: WO
Inventors: 川島季晋; 塚谷忠之; 佐野善則
Original assignee: 株式会社同仁化学研究所; 福岡県; 有限会社佐野商会
Priority date: 2021-02-10
Filing date: 2022-02-08
Publication date: 2022-08-18
Also published as: JP7436972B2; JP2022122790A

Abstract

Disclosed is a method for evaluating biofilm formation ability having: a step for housing a cell suspension in a liquid-housing container provided with a liquid-housing portion having one or more openings on the upper side; a step for fixing, while positioned relative to the liquid-housing container, one or more plate-shaped test pieces having a size and shape that can be housed inside the liquid-housing portion; a step for bringing the test piece into contact with the liquid housed in the liquid-housing container with the surface of the test piece orthogonal or substantially orthogonal to the liquid surface of the liquid and forming a biofilm on the surface of the test piece; and a step for evaluating the biofilm formation ability. Also disclosed is a device for evaluating biofilm formation ability having: a liquid-housing container provided with a liquid-housing portion having one or more openings on the upper side; and a lid member for covering the openings of the liquid-housing portion while positioned relative to the liquid-housing container. The device for evaluating biofilm formation ability has, on the surface portions of the lid members that cover each of the openings of the liquid-housing container, a fixing means for fixing one or more plate-shaped test pieces having a size and shape that can be housed inside the liquid-housing portion in such a way as to be able to contact the liquid while the surface thereof is orthogonal or substantially orthogonal to the liquid surface of the liquid housed in the liquid-housing container. Also disclosed is a lid member for a multi-well plate for use in evaluating biofilm formation ability.

Description

METHOD FOR EVALUATING BIOFILM FORMABILITY, BIOFILM FORMABILITY EVALUATION DEVICE, AND LID MEMBER FOR MULTI-WELL PLATE USED TO EVALUATE BIOFILM FORMABILITY

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

Biofilm refers to a biofilm formed by microorganisms adhering to the surface of a solid or liquid, and is a structure in which colonies of microorganisms are formed inside a film of extracellular polysaccharides (EPS) such as mucopolysaccharides produced by microorganisms. have. EPS protects internal microorganisms from environmental changes and chemical substances, and also functions as a transport route for substances. Such EPS action forms colonies with high population densities and maintains dynamic equilibrium.

Biofilms can exist in any place where there is a solid or liquid (air-liquid interface, etc.) and water as a "base", and it is believed that biofilms are involved in material conversion and purification in the natural world. , Through the symbiosis of crops and root nodule bacteria, etc., application to the agricultural field is also being considered. On the other hand, biofilms also cause sliminess in kitchens and drains, clogging of filtration membranes in water treatment facilities, contamination of medical devices such as catheters, tooth decay and periodontal disease. , In a wide range of fields such as dental materials, oral care, and sanitary, research and development on materials, surface treatment technologies, etc. for suppressing biofilm formation are being conducted (see, for example, Non-Patent Document 1 and Patent Document 1). .

Evaluation of biofilm formation ability plays an important role in both the development of technologies to promote and suppress biofilm formation, and various evaluation methods have been proposed. In Patent Document 2, raw water or reverse osmosis membrane concentrated water is passed through a water-permeable column having a biofilm-forming substrate, and the biofilm-forming substrate is periodically removed from the water-permeable column. A method for evaluating film content based on ATP (adenosine triphosphate) content is described. However, in these methods, in order to accurately evaluate the biofilm formation ability, a large amount of water is required, and an ATP extraction operation and an analysis device are required. There is a problem that an apparatus is required, and it is particularly difficult to evaluate a plurality of substances (substrates) in parallel.

In order to evaluate the biofilm formation ability of multiple substances in parallel on a laboratory scale, for example, a bacterial cell suspension is placed in each well of a multiwell plate, and a test piece is placed on the bottom of the well. Alternatively, a method is used in which incubation is performed while the specimen is leaned against a well to form a biofilm on the surface of the specimen. However, with this method, the position of the test piece in the cell suspension is not stable. It may float in the turbid liquid. Due to these factors, it is difficult to form a biofilm on the surface of the test piece with good reproducibility in conventional methods that do not fix the test piece in the well. In addition to the need for complicated operations such as picking up individual specimens with tweezers when staining, there is a problem that the biofilm may peel off during the operation.

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

Japanese Patent Application Laid-Open No. 2008-13458 WO2008/038575

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

The present invention has been made in view of such circumstances, and does not require a complicated device, a large amount of sample water, or a complicated operation. A method for evaluating the ability to form biofilms, a biofilm-forming ability evaluation device that can be suitably used in the implementation of the method, and a biofilm-forming ability evaluation device that can be suitably used in the above-described evaluation method and evaluation device It is an object of the present invention to provide a lid member for a multiwell plate.

A first aspect of the present invention in accordance with the above object includes a step of preparing a liquid containing microorganisms having biofilm-forming ability, and storing the liquid in a liquid container having one or more liquid container portions having openings on the upper side. preparing one or a plurality of plate-shaped test pieces having a size and shape that can be accommodated in the liquid container; a step of fixing the test piece via a fixing means provided on a surface of a portion of a lid member covering the opening of the container portion covering each of the openings of the liquid container; and fixing the test piece. The lid member covers the opening of the liquid container containing the liquid, and the liquid contained in the liquid container in a state in which the surface of the test piece is orthogonal or substantially orthogonal to the liquid surface of the liquid. By providing a biofilm formation ability evaluation method comprising the step of contacting the test piece to form a biofilm on the surface of the test piece, and the step of evaluating the biofilm formation ability is to solve

In the biofilm-forming ability evaluation method according to the first aspect of the present invention, it is preferable that the liquid container has a plurality of liquid-containing portions, and that the biofilm-forming ability is evaluated simultaneously for a plurality of test pieces. .

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

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

A second aspect of the present invention is a liquid container comprising one or more liquid containing portions having openings on the upper side, and a lid that covers the opening of the liquid containing portion while being positioned with respect to the liquid container. and one or more plates having a size and shape that can be accommodated inside the liquid container on the surface of the portion of the lid member that covers each of the openings of the liquid container. is fixed so that the test piece can come into contact with the liquid in such a state that the surface of the test piece is orthogonal or substantially orthogonal to the liquid surface of the liquid contained in the liquid container. This solves the above problems.

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 polymer material, and the fixing means is arranged such that the tips thereof are positioned in parallel at a predetermined interval. It is preferable that the clamping pieces are formed integrally with the cover member and biased inward by the elasticity of the polymer material so that the test piece can be clamped.

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

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

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 forming ability evaluation device according to the second aspect of the present invention, a side skirt extending in the same direction as the clamping piece formed on the lid member is provided around the lid member, and the lid member It is preferable that the lower end of the side skirt is located above the bottom surface of the liquid container by a predetermined distance when fitted to the liquid 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 multiwell plate having a plurality of wells, wherein the surface facing each of the wells has the A biotechnology device having fixing means for fixing one or more plate-shaped test strips having a size and shape that can be accommodated inside the well so as to be in contact with the liquid contained in the well in an orthogonal or substantially orthogonal state. The above problem is solved by providing a multiwell plate lid member for use in evaluating film-forming ability.

In the multiwell plate lid member for use in evaluating biofilm formation ability according to the third aspect of the present invention, the lid member is made of an elastic polymer material, and the fixing means has a predetermined tip end. A pair of clamping pieces integrally formed with the lid member so as to be positioned parallel to each other with a space therebetween and biased inward by the elasticity of the polymer material so as to be able to clamp the test piece. is preferred.

In the multiwell plate lid member for use in evaluating biofilm formation ability according to the third aspect of the present invention, the lid member is in contact with the entire circumference of the edge of the opening and seals the liquid container. It is preferred to have a sealing means for.

In the multiwell 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.

In the multiwell plate lid member for use in evaluating biofilm formation ability according to the third aspect of the present invention, a side skirt extending in the same direction as the clamping piece side formed on the lid member is provided around the periphery, It is preferable that the lower ends of the side skirts are positioned above the bottom surfaces of the wells by a predetermined distance when the lid member is fitted to the multiwell plate.

In the present invention, “to cover” means to cover and block, and “to hold” means to hold a plate-shaped test piece in a state of being sandwiched from both sides, and “to bias”. A direction in which the gap between the tips of the clamping pieces is narrowed against the force of the test piece sandwiched between the clamping pieces that pushes the clamping pieces apart due to the elasticity of the material that constitutes the clamping pieces. It means that elastic force acts on

In the biofilm-forming ability evaluation method of the present invention, the test piece is fixed to the lid member and brought into contact with the liquid contained in the liquid container in a state where the surface of the test piece is orthogonal or substantially orthogonal to the liquid surface of the liquid. Since the position of the test piece in the liquid container can be kept constant, the biofilm can be formed with good reproducibility, and the biofilm formation ability can be evaluated with high accuracy. Further, by using a combination of a liquid container having a plurality of liquid containing portions and a lid member having fixing means for fixing the test piece on the surface facing each of the openings of the liquid containing portion, The ability to form biofilms can be evaluated in parallel for a plurality of test pieces, and the treatment such as washing of the test pieces after biofilm formation can be performed at once with a simple operation.

BRIEF DESCRIPTION OF THE DRAWINGS It is a flowchart which shows each process of the formation ability evaluation method of the biofilm which concerns on the 1st Embodiment of this invention. FIG. 2 is a schematic diagram of a biofilm formation ability evaluation device according to a second embodiment of the present invention. FIG. 4 is a schematic diagram of a multiwell plate lid member according to a third embodiment of the present invention. It is the elements on larger scale of the clamping piece of the same cover member. FIG. 4 is a partially enlarged view showing a fitting structure between a multiwell plate and a lid member; FIG. 4 is a schematic diagram showing a state in which a test strip accommodated in a well is fixed by clamping pieces. 4 is a graph showing the results of Example 1. FIG. 4 is a graph showing the results of Example 2. FIG. 4 is a photograph showing the state of a test piece after dyeing in Example 2. FIG. 4 is a graph showing the results of Example 3. FIG.

Next, with reference to the attached drawings, specific embodiments of the present invention will be described 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) a step of preparing a cell suspension (liquid containing microorganisms capable of forming a biofilm) ("cell suspension preparation"), and (2) one or more openings on the upper side (3) a step of seeding (accommodating) a cell suspension in a liquid container having a liquid container having a (4) a step of preparing one or more plate-shaped test pieces having a shape (not shown); (5) a step of attaching (fixing) the test piece to the fixing means provided on the surface of the portion that covers each of the openings ("test piece mounting"); The opening of the liquid container containing the liquid container is covered, and the test piece is immersed (brought into contact) with the bacterial cell suspension contained in the liquid container in a state where the surface of the test piece is perpendicular or substantially perpendicular to the surface of the liquid. ("test piece immersion"), culturing under predetermined conditions to form a biofilm on the surface of the test piece ("culturing"); After staining the biofilm and washing to remove the dye that does not adsorb to the biofilm, the dye adsorbed to the biofilm is extracted, and the biofilm formation ability is determined by colorimetric determination (measurement of absorbance) of the dye extract. evaluation steps (“washing”, “staining”, “washing”, “extraction”, “measurement”). Each step will be described in detail below.

(1) Preparation of Cell Suspension First, a cell suspension, which is an example of a liquid containing microorganisms capable of forming biofilms, is prepared. The liquid that is the dispersion medium for the microorganisms is, for example, water or an aqueous solution, and may be environmental water, waste water, etc. collected from the natural environment or living environment, and a culture solution (liquid medium) in which cultured microorganisms are dispersed. may be There is no particular limitation on the number of microorganisms per unit volume, and if necessary, the suspension can be used after being appropriately diluted with an arbitrary dispersion medium.

The type of microorganism is not particularly limited as long as it has biofilm-forming ability, and examples thereof include Pseudomonas aeruginosa, Escherichia coli, and Staphylococcus aureus. Specific examples include Corynebacterium xerosis, Lactobacillus xerosis, Brevis (Lactobacillus brevis), Staphylococcus aureus: Staphylococcus epidermidis, Streptococcus mutans (dental caries): Streptococcus mutans, Streptococcus oralis: Streptococcus oralis, Streptococcus・Salivarius (oral indigenous bacteria): Streptococcus salivarius, Acinetobacter baumannii: Acinetobacter baumannii, Cronobacter (former Enterobacter) ・Sakazaki: Cronobacter sakazakii, Escherichia coli: Escherichia coli Porphyromonas gingivalis (periodontal bacteria): Porphyromonas gingivalis, Serratia marcescens: Serratia marcescens, Pseudomonas aeruginosa: Pseudomonas aeruginosa, etc. are mentioned.

(2) Seeding of Cell Suspension Next, a predetermined volume of the cell suspension is accommodated (inoculated) in the liquid storage portion of the liquid container. The number of liquid storage units may be only one, but may be multiple, and in this case, biofilm formation ability can be evaluated simultaneously for multiple test pieces under the same conditions. An example of a liquid container having a plurality of liquid containing portions is a multi-well plate having wells of any number, size and shape. The number, size, and shape of the wells are not particularly limited, but they are, for example, cylindrical with an inner diameter of 15 mmφ and a height of 20 mm. With wells of this size, several mL of cell suspension per well is enough to evaluate the biofilm formation ability. The material constituting the multiwell plate is not particularly limited, and any material used for multiwell plates can be used without limitation.

A biofilm-forming ability-evaluating apparatus 10 according to the second embodiment of the present invention, which is an example of a biofilm-forming ability-evaluating apparatus that can be suitably used to implement the evaluation method according to the present embodiment (hereinafter referred to as " The evaluation device 10" or the like may be abbreviated) has a structure as shown in FIG. The multi-well plate lid member for use in evaluating biofilm formation ability according to the third embodiment of the present invention for use in combination with also has a similar structure.) there is

A multi-well plate has a plurality of wells 12 (an example of a liquid container) with openings on the upper side. The lid member 13 is a member for covering the opening of each well 12 while being positioned with respect to the multiwell plate 11, and a plate-shaped test plate is provided on the surface of the portion covering the opening of each well 12. It has a clamping piece 14 (an example of fixing means) for fixing the piece so that it can come into contact with the liquid in a state where the surface of the piece is perpendicular or substantially perpendicular to the liquid surface of the liquid contained in the liquid container. . The lid member 13 is made of an elastic polymeric 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 permeable, is preferred. is preferably used. As shown in FIG. 3, the clamping piece 14 is formed integrally with the cover member 13 so that the ends thereof are positioned parallel to each other at a predetermined interval, and is made of the polymer material so as to be able to clamp the test piece (18). They are a pair of plate-like members that are biased inward by their elasticity.

As shown in FIG. 4, it is preferable that the clamping pieces 14 are formed so that the interval between them becomes 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 substantially perpendicularly to the lid member 13, so that when it is immersed in the bacterial cell suspension later, it is almost perpendicular to the liquid surface. It can be kept vertical. As shown in FIGS. 2, 3 and 4, the lid member 13 is provided with a sealing member 15 (an example of sealing means) for contacting the entire circumference of the edge of the opening of each well 12 and sealing the well 12. may be formed.

As shown in FIGS. 2 and 5, lid member 13 is formed as a mating lid for multiwell plate 11 so that it can cover each well 12 while positioned relative to multiwell plate 11 . good too. More specifically, a plate-like peripheral wall 16 is formed around the multiwell plate 11, and around the lid member 13, the surface on which the clamping pieces 14 are provided faces downward. The side skirts 17 are formed so as to hang down toward the side on which the clamping pieces 14 are formed when 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 corners of the side skirt 17 on the distal end side. By fitting both the peripheral wall 16 and the side skirts 17, the lid member 13 is fitted to the multi-well plate 11 in a horizontally positioned state. In addition, 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 to the multiwell plate 11 while being positioned vertically. do. By adopting such a configuration, volatilization of the cell suspension contained in each well 12 and contamination by microorganisms in the air can be prevented. Side skirts 17 or end pins 19 may be used as vertical positioning means in retaining 18 . In addition, as shown in FIG. 2, the side skirt 17 is provided with a notch for making it easier to hold only the lid member 13 or both the lid member 13 and the multiwell plate 11 at the same time, if necessary. good too.

(3) Preparation and Fixing 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-shaped member that can be accommodated inside the well 12 and has a size and shape that allows a portion of its surface to come into contact with the cell suspension. The test piece is preferably rectangular, and the thickness and size are appropriately selected according to the size and shape of the well 12, the deposition of the bacterial cell suspension to be accommodated, and the like. Preferably, if the distance is about 10 to 20 mm, the biofilm formation ability can be evaluated with sufficient accuracy. The material of the test piece 18 and the properties of the surface (the mode of surface processing, the presence or absence of a coating agent, etc.) are not particularly limited, and can be appropriately selected according to the purpose. To fix the test piece 18 to the clamping piece 14, the cover member 13 is placed on a horizontal table so that the clamping piece 14 faces upward, and the test piece 18 is pinched with tweezers or the like to be sandwiched between the clamping pieces 14. It can be performed by any method such as. At that time, the test piece 18 is slightly sandwiched between the clamping pieces 14, then the upper limit of the lid member 13 is turned over, placed on a horizontal table with the clamping pieces 14 facing downward, and lightly pressed. By aligning the lower end of the side skirt 17 or the end pin 19 with the lower end of the test piece 18 (the position indicated by the dashed line in FIG. 6), the vertical positioning of the test piece 18 can be easily and reliably performed. can be done.

(4) Formation of biofilm (culture)
Cell culture solution is accommodated in each well 12, and cultured at a predetermined temperature for a predetermined time in the evaluation device 10 equipped with a lid member 13 in which the test piece 18 is fixed to the clamping piece 14, so that the surface of the test piece 18 is to form a biofilm. Shaking or the like may be performed as necessary. The culture temperature and culture time can be appropriately selected according to the type of cells in the cell suspension. During culturing, the medium in the cell suspension may be replaced with fresh medium as appropriate (for example, at predetermined intervals).

(5) Evaluation of biofilm formation ability As a method for evaluating biofilm formation ability, measurement of the mass change of each test piece before and after biofilm formation, colony formation ability of microorganisms in the biofilm peeled off 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, the biofilm is dyed with a dye, and the amount of dye in the dyed biofilm (the mass of the biofilm (proportional to ) is evaluated for biofilm formation ability. More specifically, after washing the test piece 18 on which the biofilm is formed, it is stained with a dye, the test piece 18 after staining is washed, and after removing the dye that has not been adsorbed to the biofilm, the biofilm Biofilm-forming ability is evaluated by extracting the pigment from the plant and performing colorimetric determination of the pigment.

As a dye used for staining biofilms, the amount of adsorption to biofilms is a function of the amount of biofilm formation, and the amount of biofilm formation is 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 alcohol. Specific examples of such dyes include crystal violet and the like. An aqueous solution such as water or physiological saline is used for washing the test piece 18 after biofilm formation and dyeing. For dyeing and extraction of dye after dyeing, a solvent having high dye solubility is appropriately selected according to 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 step of washing, staining, and extraction. is prepared, and while the test piece 18 is fixed to the lid member 13 by the clamping piece 14, the multiwell plate 11 is sequentially replaced to damage the biofilm with a simple operation. can be implemented without

Purposes and applications of biofilm formation ability evaluation include evaluation of materials and surface treatment technology for environmental pollution prevention in water treatment facilities, housing equipment and sanitary fields, microorganisms in fields such as medical equipment, dental materials and oral care. and evaluation of anti-proliferation materials and techniques.

Next, an example conducted to confirm the effects of the present invention will be described.

Example 1: Biofilm Formation on Test Piece A culture solution of Staphylococcus aureus NBRC13276 was diluted and prepared with a medium (Muller-Hinton Broth) to about 10 ⁷ cfu/mL, and this cell suspension was prepared. 2 mL of the liquid 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) was fixed to a multiwell plate cover member having the structure shown in Fig. 3, and covered with a 24-well microplate in which a cell suspension was dispensed. , the test piece was placed perpendicular to the cell suspension (10 mm in the longitudinal direction of the test piece was immersed in the cell suspension). Furthermore, static culture was carried out at 37° C. for a certain period of time to form a biofilm on the test piece. Culture conditions were set as follows.
Test group 1: culture for 24 hours Test group 2: culture for 48 hours Test group 3: After culturing for 24 hours, replace with fresh medium and culture for an additional 24 hours

After culturing, the multi-well plate lid member to which the test piece was fixed was transferred to a 24-well microplate filled with physiological saline and washed to remove floating cells. Furthermore, 0.1% crystal violet was dispensed into a 24-well microplate to stain the biofilm for 30 minutes. After staining, the specimen was washed with physiological saline, transferred to a 24-well microplate dispensed with 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 piece in test group 3. Therefore, in Examples 2 and 3 below, the biofilm formation ability was evaluated according to the procedure of test group 3.

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

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

The coefficient of variation (CV) (n = 8) was 5.6% for this method, while it was 42.3% for the conventional method, indicating that the reproducibility of this method was remarkably excellent. As shown in FIG. 9, in the conventional method, the degree of biofilm staining was uneven, and biofilm formation varied widely. On the other hand, this method was able to form reproducible biofilms.

Example 3: Effect of Vertical Positioning of Test Piece by Multiwell Plate Lid Member Using a multiwell plate lid member having the structure shown in FIG. However, in this example, the biofilm formation ability was compared between the case where vertical positioning was performed and the case where vertical positioning was not performed, and the effect was verified. S. aureus NBRC13276 biofilm formation test with and without vertical positioning ("with height adjustment") according to the procedure of Test Group 3 of Example 1. (“without height adjustment”) were compared for reproducibility. The results are shown in Table 3 and FIG.

The coefficient of variation (CV) (n = 6) was 2.1% with height adjustability vs. 11.7% without height adjustability. The reproducibility was remarkably excellent.

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

It should be noted that the present invention allows for various embodiments and modifications without departing from the broad spirit and scope of the present invention. Moreover, the above-described embodiments are for explaining the present invention, and do not limit the scope of the present invention. In other words, the scope of the present invention is indicated by the claims rather than the embodiments. Various modifications made within the scope of the claims and within the meaning of the invention equivalent thereto are considered to be within the scope of the present invention.

This application is based on Japanese Patent Application No. 2021-20261 filed on February 10, 2021, and includes the specification, claims, drawings and abstract thereof. The disclosure in the above Japanese patent application is incorporated herein by reference in its entirety.

Claims

preparing a liquid containing microorganisms capable of forming a biofilm;
a step of storing the liquid in a liquid container having one or more liquid containing portions having openings on the upper side;
a step of preparing one or more plate-shaped test pieces having a size and shape that can be accommodated inside the liquid storage portion;
Via the fixing means provided on the surface of the portion of the lid member that covers the opening of the liquid container while being positioned with respect to the liquid container and covers each of the openings of the liquid container. fixing the test piece;
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 liquid surface of the liquid. contacting the test strip with the liquid contained in the vessel to form a biofilm on the surface of the test strip;
A biofilm-forming ability evaluation method comprising the step of evaluating the biofilm-forming ability.
The biofilm-forming ability evaluation method according to claim 1, wherein the liquid container has a plurality of liquid-containing parts, and the biofilm-forming ability is evaluated for a plurality of test pieces at the same time.
The biofilm formation ability evaluation method according to claim 2, wherein the liquid container is a multiwell plate.
4. Any one of claims 1 to 3, wherein 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 colorimetrically determined. 1. The biofilm formation ability evaluation method according to claim 1.
a liquid container comprising one or more liquid containing portions having openings on the upper side;
a lid member that covers the opening of the liquid container while being positioned with respect to 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 on the surface of the portion of the lid member that covers each of the openings of the liquid container. A biofilm formation ability evaluation device having fixing means for fixing the liquid in such a manner that the surface of the liquid is perpendicular or substantially perpendicular to the surface of the liquid contained in the liquid container so as to be in contact with the liquid.
The biofilm formation ability evaluation device according to claim 5, wherein the liquid container is a multiwell plate.
The lid member is made of an elastic polymeric material, and the fixing means is integrally formed with the lid member so that the ends thereof are positioned parallel to each other at a predetermined interval so that the test piece can be clamped, 7. The biofilm formation ability evaluation device according to claim 5, wherein the pair of clamping pieces are biased inward by the elasticity of the polymer material.
The biofilm formation ability evaluation device according to claim 7, characterized in that the gap between the pair of clamping pieces is formed so as to narrow toward the distal end side.
Biofilm formation according to any one of claims 5 to 8, characterized in that the lid member has sealing means for contacting the entire perimeter of the rim of the opening and sealing the liquid container. ability evaluation device.
The biofilm formation ability evaluation device according to any one of claims 5 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. When the lid member is fitted to the liquid container, the lower end of the side skirt extends to the above-described side skirt. The biofilm formation ability evaluation device according to any one of claims 7 to 10, wherein the biofilm formation ability evaluation device is configured to be positioned above the bottom surface of the liquid container by a predetermined distance.
A lid member that covers the opening of each well while being positioned with respect to a multiwell plate having a plurality of wells,
One or a plurality of plate-shaped test strips having a size and shape that can be accommodated inside the wells are accommodated in the wells in an orthogonal or substantially orthogonal state on the surface of the portion that covers each of the wells. A lid member for a multiwell plate for use in evaluating biofilm formation ability, having fixing means for fixing in contact with a liquid to be applied.
The fixing means is made of an elastic polymeric material, and the fixing means is formed integrally with the lid member so that the ends thereof are positioned parallel to each other at a predetermined interval, and is made of the polymeric material so as to be able to hold the test piece. 13. The multi-well plate lid member for use in biofilm-forming ability evaluation according to claim 12, wherein the lid member is a pair of clamping pieces that are biased inward by their elasticity.
The lid member for a multiwell plate for use in evaluating biofilm formation ability according to claim 13, characterized in that the distance between the pair of clamping pieces is formed so as to narrow toward the distal end side.
15. Biofilm formation according to any one of claims 12 to 14, characterized in that it comprises sealing means for sealing each well of the multiwell plate in contact with the entire perimeter of the well opening rim. Lid member for multi-well plate for use in evaluation of ability.
The lid member for a multiwell plate for use in evaluating biofilm formation ability according to any one of claims 12 to 15, characterized by being configured as a fitting lid for the multiwell plate.
A side skirt extending in the same direction as the clamping piece formed on the lid member is provided on the periphery, and when the lid member is fitted to the multiwell plate, the lower end of the side skirt extends from the bottom surface of the well. 17. The lid member for a multiwell plate for use in evaluating biofilm formation ability according to any one of claims 13 to 16, characterized in that the lid member for a multiwell plate is configured to be positioned above by a predetermined distance.