WO2022172931A1 - Procédé d'évaluation de la capacité de former un biofilm, dispositif d'évaluation de la capacité de former un biofilm, et élément formant couvercle pour plateau multi-puits destiné à être utilisé pour évaluer la capacité de former un biofilm - Google Patents

Procédé d'évaluation de la capacité de former un biofilm, dispositif d'évaluation de la capacité de former un biofilm, et élément formant couvercle pour plateau multi-puits destiné à être utilisé pour évaluer la capacité de former un biofilm Download PDF

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WO2022172931A1
WO2022172931A1 PCT/JP2022/004984 JP2022004984W WO2022172931A1 WO 2022172931 A1 WO2022172931 A1 WO 2022172931A1 JP 2022004984 W JP2022004984 W JP 2022004984W WO 2022172931 A1 WO2022172931 A1 WO 2022172931A1
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Prior art keywords
liquid
lid member
biofilm formation
biofilm
liquid container
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PCT/JP2022/004984
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English (en)
Japanese (ja)
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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
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Publication of WO2022172931A1 publication Critical patent/WO2022172931A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M1/00—Apparatus for enzymology or microbiology
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M1/00—Apparatus for enzymology or microbiology
    • C12M1/34—Measuring or testing with condition measuring or sensing means, e.g. colony counters
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
    • C12Q1/04—Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor
    • C12Q1/06—Quantitative determination

Definitions

  • 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.
  • EPS extracellular polysaccharides
  • 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). .
  • 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.
  • 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.
  • 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.
  • the biofilm may peel off during the operation.
  • 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.
  • 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.
  • 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. .
  • the liquid container may be a multiwell plate.
  • 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.
  • the liquid container may be a multiwell plate.
  • the lid member is made of an elastic polymer material
  • 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.
  • the gap between the pair of clamping pieces is formed so as to narrow toward the distal end side.
  • the lid member has sealing means for contacting the entire periphery of the edge of the opening and sealing the liquid storage portion. .
  • the lid member is configured as a fitting lid for the liquid container.
  • 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 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.
  • the gap between the pair of clamping pieces is formed so as to narrow toward the distal end side.
  • the lid member for use in evaluating biofilm formation ability according to the third aspect of the present invention, 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.
  • the 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 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.
  • to cover means to cover and block
  • to hold means to hold a plate-shaped test piece in a state of being sandwiched from both sides, and “to bias”.
  • 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.
  • 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.
  • 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.
  • cell suspension preparation a step of preparing a cell suspension (liquid containing microorganisms capable of forming a biofilm)
  • cell suspension preparation 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);
  • 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.
  • a cell suspension which is an example of a liquid containing microorganisms capable of forming biofilms.
  • 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 Porphy
  • 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 mm ⁇ 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.
  • 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.
  • the lid member 13 includes polypropylene, ethylene-propylene copolymer, AS resin, ABS resin, etc.
  • Polypropylene which has excellent elasticity and flexibility and is permeable, is preferred. is preferably used.
  • 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.
  • 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.
  • 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.
  • the lid member 13 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.
  • 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.
  • Side skirts 17 or end pins 19 may be used as vertical positioning means in retaining 18 .
  • 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.
  • the test piece 18 is attached to the clamping piece 14 of the lid member 13 and fixed.
  • 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.
  • 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 are not particularly limited, and can be appropriately selected according to the purpose.
  • 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.
  • 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.
  • the vertical positioning of the test piece 18 can be easily and reliably performed. can be done.
  • 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.
  • the test piece 18 on which the biofilm is formed 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.
  • 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.
  • 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.
  • 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.
  • 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).
  • 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
  • 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.
  • Example 2 Reproducibility of biofilm formation (comparison with conventional method)
  • a method hereinafter referred to as a conventional method
  • a biofilm formation test of Staphylococcus aureus 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.
  • 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 degree of biofilm staining was uneven, and biofilm formation varied widely.
  • 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.

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Abstract

La présente invention concerne un procédé d'évaluation de l'aptitude à la formation d'un biofilm comprenant les étapes suivantes : une étape pour loger une suspension cellulaire dans un récipient de logement de liquide muni d'une partie de logement de liquide ayant une ou plusieurs ouvertures sur le côté supérieur ; une étape pour fixer, tout en étant positionné par rapport au récipient de logement de liquide, une ou plusieurs pièces d'essai en forme de plateau ayant une taille et une forme permettant de les loger à l'intérieur de la partie de logement de liquide ; une étape pour mettre l'éprouvette en contact avec le liquide logé dans le récipient de logement de liquide, la surface de l'éprouvette étant orthogonale ou sensiblement orthogonale à la surface du liquide et formant un biofilm sur la surface de l'éprouvette ; et une étape pour évaluer la capacité de formation du biofilm. La présente invention concerne également un dispositif d'évaluation de la capacité de formation de biofilm comportant les éléments suivants : un récipient de logement de liquide muni d'une partie de logement de liquide ayant une ou plusieurs ouvertures sur le côté supérieur ; et un élément de couvercle destiné à couvrir les ouvertures de la partie de logement de liquide tout en étantpositionné par rapport au récipient de logement de liquide. La présente invention concerne également un dispositif d'évaluation de la capacité de formation d'un biofilm, comportant les éléments suivants : un récipient de logement de liquide muni d'une partie de logement de liquide ayant une ou plusieurs ouvertures sur le côté supérieur ; et un élément formant couvercle pour couvrir les ouvertures de la partie de logement de liquide lorsqu'il est positionné par rapport au récipient de logement de liquide. La présente invention concerne également un élément formant couvercle pour un plateau multi-puits destiné à être utilisé pour évaluer la capacité de formation de biofilm.
PCT/JP2022/004984 2021-02-10 2022-02-08 Procédé d'évaluation de la capacité de former un biofilm, dispositif d'évaluation de la capacité de former un biofilm, et élément formant couvercle pour plateau multi-puits destiné à être utilisé pour évaluer la capacité de former un biofilm WO2022172931A1 (fr)

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JP2021020261A JP7436972B2 (ja) 2021-02-10 2021-02-10 バイオフィルムの形成能評価方法、バイオフィルムの形成能評価装置及びバイオフィルム形成能の評価に用いるためのマルチウェルプレート用蓋部材

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TSUKATANI TADAYUKI, KAWAGUCHI TOMOAKI, SUENAGA HIKARU, SHIGA MASANOBU, IKEGAMI TAKASHI: "RAPID AND SIMPLE DETERMINATION OF MINIMUM BIOFILM ERADICATION CONCENTRATION BY A COLORIMETRIC MICROBIAL VIABILITY ASSAY BASED ON REDUCTION OF A WATER-TETRAZOLIUM SALT AND COMBINATED EFFECT OF ANTIBIOTICS AGAINST MICROBIAL BIOFILM", JOURNAL OF MICROBIOLOGY, BIOTECHNOLOGY AND FOOD SCIENCES, vol. 6, no. 1, 1 January 2016 (2016-01-01), pages 677 - 680, XP055958933, DOI: 10.15414/jmbfs.2016.6.1.677-680 *
TSUKATANI TADAYUKI; SAKATA FUMIHIKO; KURODA RIEKO: "A rapid and simple measurement method for biofilm formation inhibitory activity using 96-pin microtiter plate lids", WORLD JOURNAL OF MICROBIOLOGY AND BIOTECHNOLOGY, vol. 36, no. 12, 1 January 2020 (2020-01-01), Dordrecht, XP037305804, ISSN: 0959-3993, DOI: 10.1007/s11274-020-02964-6 *
TSUKATANI TADAYUKI; SAKATA FUMIHIKO; KURODA RIEKO; AKAO TETSUYUKI: "Biofilm Eradication Activity of Herb and Spice Extracts Alone and in Combination Against Oral and Food-Borne Pathogenic Bacteria", CURRENT MICROBIOLOGY, vol. 77, no. 9, 11 May 2020 (2020-05-11), New York , pages 2486 - 2495, XP037213649, ISSN: 0343-8651, DOI: 10.1007/s00284-020-02017-z *

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