Method for detecting microorganisms in paints and coatings

Abstract

The present invention relates to an improved method for the detection of microorganisms in complex matrices, such as paints and coatings. The method utilizes a membrane-separated system with a microbial growth zone physically isolated from a detection zone. This configuration enhances performance by reducing interference from pigments, viscosity additives, and other matrix components in paints and coatings. The membrane can allow selective diffusion of microbial metabolites while preventing bulk transfer of the sample, which can allow for accurate detection.

Description

Attorney Docket No.: ARXPC-40Q-PCT (LP3745PC02)METHOD FOR DETECTING MICROORGANISMS IN PAINTS AND COATINGSCROSS-REFENCE TO RELATED APPLICATION

[0001] The present application is related and has right of priority to U.S. Provisional Application No. 63 / 679,872 fded on August 6, 2024, which is incorporated by reference in its entirety.BACKGROUND

[0002] The presence and enumeration of microorganisms in paint and coating samples have traditionally been determined by growing the microorganisms on an agar surface and counting the colonies. Such tests suffer drawbacks, including the significant time to result. For instance, conventional methods can require three to five (3-5) days to result, which can negatively affect productivity.

[0003] Systems and methods for more quickly determining the presence and enumeration of microorganisms in paint and coating samples would be useful.SUMMARY

[0004] In general, the present disclosure is directed to methods for detecting the presence and enumeration of microorganisms in paints and coatings, with improved time to result relative to conventional methods. For instance, the method of the present disclosure may advantageously have a time to result of less than forty -eight hours (48 hrs.), such as less than thirty -six hours (36 hrs.), such as about twenty-four hours (24 hrs.), such as less than twelve hours (12 hrs.).

[0005] In example embodiments, a method for detecting microorganisms in paints and coatings, includes: introducing a container with a test sample into a testing device, the test sample comprising a solvent and a polymer, the container comprising a growth zone, a detection zone, and a barrier layer, the growth zone disposed within the container and comprising a growth media for supporting growth of microorganisms, the detection zone adjacent a transparent section of the container containing an indicator substrate that changes optical properties due to growth of the microorganisms, the barrier layer disposed between the growth and detection zones and comprising porous material configured for allowing diffusion of metabolic by-products of the microorganisms between the growth and detection zones; incubating the microorganisms in the grow th media such that metabolic processes of the microorganisms generate the metabolic by-products in theAttorney Docket No.: ARXPC-40Q-PCT (LP3745PC02) grow th media in order to change the optical properties of the indicator substrate in the growth zone; diffusing the metabolic by-products into the detection zone through the barrier layer while blocking the microorganisms and particulate matter of the test sample from the detection zone; and detecting the change in the optical properties of the indicator substrate in the detection zone through the transparent section.

[0006] These and other features and aspects, embodiments and advantages of the present invention will become better understood with reference to the following description and appended claims.BRIEF DESCRIPTION OF THE DRAWINGS

[0007] A full and enabling disclosure of the present disclosure is set forth more particularly in the remainder of the specification, including reference to the accompanying figures, in which:FIG. 1 is a schematic view of a device for detecting microorganisms in paints and coatings according to an example embodiment of the present disclosure.

[0008] Repeat use of reference characters in the present specification and drawings is intended to represent the same or analogous features or elements of the present invention.DETAILED DESCRIPTION

[0009] Reference will now be made in detail to example embodiments of the disclosure. It is to be understood by one of ordinary skill in the art that the present disclosure is a description of exemplary embodiments only and is not intended as limiting the broader aspects of the present disclosure.

[0010] The present subject matter generally provides a method for detecting the presence and enumeration of microorganisms in paints and coatings, with improved time to result relative to conventional methods. For instance, the method of the present disclosure may advantageously have a time to result of less than sixty' hours (60 hrs.), such as about forty-eight hours (48 hrs.), such as about twenty-four hours (24 hrs.). The method may thus provide fast and reliable detection of microorganisms in paint and coating samples. Moreover, the method may include optical scanning of the paint and coating samples in media vials to provide quick, accurate detection of microorganisms in paint and coating samples. In example embodiments, the vials may be filled with a paint or coating sample, such as a small volume no greater than five millimeters (5 mm), such as no greater than one millimeter (1 mm), with the vials being preloaded with microbial growth medium that isAttorney Docket No.: ARXPC-400-PCT (LP3745PC02) kept separate from the paint or coating sample. Moreover, the vial may include an incubation zone and a reading zone that are separated from each other. The chemical characteristics of the indicator substrate may change color as metabolic processes occur within the vials filled with the paint or coating sample. Optical changes in the indicator substrate, e.g., within the reading zone, may be monitored over time to detect the presence and enumeration of microorganisms in the paint or coating sample. Moreover, changes in color and / or fluorescence in the microbial growth medium, e.g.. within the reading zone, may be detected by a photo detector, and the signal(s) from the photo detector may be recorded. In general, a higher number of microorganisms will result in faster detection times.

[0011] FIG. 1 is a schematic view of a device 100 for detecting the presence and enumeration of microorganisms in paint and coating samples according to an example embodiment of the present subject matter. As shown in FIG. 1, the device 100 includes a container or vial 110. The vial 110 may contain a paint or coating test sample 102 and be configured for incubating the test sample 102 in liquid growth media. The vial 110 may be constructed of or with a plastic in some example embodiments, and the vial 110 or a portion of the vial 110 may be transparent to light. Moreover, at least a portion of the vial 110 may be transparent to visible light and / or ultraviolet radiation, such as a fraction of the long ultraviolet range. As an example, the vial 110 may include or be constructed of polycarbonate, which is transparent in the entire visible spectrum and to ultraviolet radiation greater than three hundred and fifty nanometers (350 nm). The vial 110 may be sealed with a cap 112, which may include or be constructed of polycarbonate in some example embodiments. The vial 110 and the cap 112 may be thermally sterilized, e.g., at a temperature greater than one hundred and tw enty-one degrees Celsius (121° C) prior to loading the vial 110 with the test sample 102.

[0012] A bottom portion of the vial 1 10 may include a window 1 16, which may be embedded in the vial 110. The window 116 may have parallel opposing surfaces and may be transparent to visible light and / or ultraviolet radiation, e.g., as described above. An internal volume 114 of the vial 110 may be configured for containing the test sample 102. In example embodiments, the internal volume 1 14 of the vial 110 may be no greater than fifty milliliters (50 mL), such as no greater than thirty milliliters (30 mL), such as no greater than ten milliliters (10 mL), such as about five milliliters (5 mL). The internal volume 114 of the vial 110 may include a growth zone 120 and a detection zone 130. A liquid mixture of reagents may be poured into the vial 110 to at least partially fill both theAttorney Docket No.: ARXPC-40Q-PCT (LP3745PC02) grow th and detection zones 120, 130. A barrier layer 140 may separate the grow th and detection zones 120, 130, and the barrier layer 140 may be configured for blocking particulate matter 104 (e.g., that may include sample particles and / or microorganisms) in the test sample 102 from flowing into the detection zone 130. The barrier layer 140 may also be configured to prevent microorganisms in the test sample 102 from entering the detection zone 130. The barrier layer 140 may be a porous material configured for diffusing liquid molecules and ions, e.g., with a selected pore size(s) designed to block the particulate matter 104 present in the growth zone 120 from entering the detection zone 130. The barrier layer 140 may be a porous membrane, such as Pall Supor®, which has an average pore size no less than a tenth of a micron (0.1 pm) and no greater than one and a half microns (1.5 pm), or other porous thick material, such as Porex 4897 sheet. In general, the pore size of the barrier layer 140 may be less than two microns (2 pm).

[0013] As may be seen from the above, the barrier layer 140 may provide continuity of the liquid phase in the growth and detection zones 120, 130 and allow liquid flow between the growth and detection zones 120, 130. Moreover, the barrier layer 140 may limit or prevent optically interfering substances, such as the particulate matter 104, from reaching the detection zone 130. The detection zones 130 separated from the growth zone 120 by the barrier layer 140 may thus allow visual observations and optical readings at the window' 116 that are unmasked by any interference resulting from the sample itself or turbidity of microorganisms therein.

[0014] The liquid phase in the vial 1 10 may be a mixture of growth media and at least one indicator substrate. The growth media enables efficient growth of microorganisms originating from the test sample 102. Selective media for the detection of molds and yeast, or selected groups of microorganisms can be used. The indicator substrate may be a chemical reagent capable of changing optical properties in the presence of metabolic byproducts, such as carbon dioxide, generated by the growing microorganisms. The amount and concentration of the indicator substrate may be chosen such that the mixture of the growth media and indicator significantly changes optical properties as a result of microbial growth. Suitable are several dyes that change colors due to either pH or oxygen reduction indicator (redox). Examples of indicators that can be used in the visible light range include pH indicators, such as Bromocresol Purple, Chlorophenol Red, Brothymol blue (Sigma- Aldrich, St. Louis, Mo., USA), and reduction indicators, such as Methylen Blue, Resazurin and Tetrazolium (Sigma-Aldrich, St. Louis, Mo., USA). Fluorescence compounds, with changes of properties in the UV range, can be used as indicator substrates, such as a varietyAttorney Docket No.: ARXPC-400-PCT (LP3745PC02) of Umbelliferones and Coumarins. 4-meth l-umbellifer l phosphate (MUP) (Sigma- Aldrich, St. Louis, Mo., USA) can be used as many organisms metabolize this compound to create fluorescence. There are many other chromomeric and enzymatic compounds that can be also utilized.

[0015] The liquid in the growth zone 120 may be in molecular equilibrium with the liquid in the detection zone 130. Molecules of the growth media and the indicator substrate can rapidly diffuse back and forth. When the hydrostatic pressure of the growth and detection zones 120, 130 are equal, no flow of liquid may take place and the only transfer of material may be due to a diffusion process that takes place whenever the equilibrium is disturbed. During incubation of the sample in the grow th media in the growth zone 120, living organisms can grow and metabolize, generating metabolic by-products in the liquid. Since the metabolites are initially not present in the detection zone 130. the chemical equilibrium may be momentarily disturbed, and the metabolites can diffuse from the growth zone 120 into the detection zone 130 through the barrier layer 140. As a result, the indicator substance present in the detection zones 130 gradually changes optical properties as more metabolites are generated and diffused.

[0016] The vial 110 may be analyzed by optical instrumentation to detect the presence and enumeration of microorganisms in the test sample 102 within the vial 110. For example, a visible light source 150 and / or an ultraviolet light source 152 may be energized to illuminate the window 116. Thus, e.g., the visible light source 150 may be operable to direct visible light through the window 116 into the detection zone 130, and the ultraviolet light source 152 may be operable to direct ultraviolet radiation through the window' 116 into the detection zone 130. A photo detector 160 may be disposed for detecting the energy' generated by interaction of the visible light from the visible light source 150 and / or the ultraviolet radiation from the ultraviolet light source 152 with the indicator substrate in the detection zone 130. For instance, the photo detector 160 may be disposed opposite the visible light source 150 and / or the ultraviolet light source 152 about the detection zone 130. The photo detector 160 may include one or more of a photo diode, a photo transistor, a photon multiplying tube (PMT), or another light-sensitive device. A controller 170 may be configured for visible light source 150 and / or the ultraviolet light source 152 and for processing signal(s) from the photo detector 1 0 to detect the presence and enumeration of microorganisms in the test sample 102 w ithin the vial 110.

[0017] The test sample 102 may be placed in the vial 110, in which the growth and detection zones 120, 130 have been pre-filled with the liquid mixture of growth media andAttorney Docket No.: ARXPC-40Q-PCT (LP3745PC02) indicator substrates. The vial 110 may be closed with the cap 112. The vial 110 may then be engaged with the visible light source 150 and / or the ultraviolet light source 152, e.g., via fixture, such as a drawer. The fixture may be placed into an incubator, which is set to optimal temperature for growth of the target microorganisms. The controller 170 may periodically activate the visible light source 150 and / or the ultraviolet light source 152, e.g., for a fraction of a second. The photo detector 15 may detect the resulting visible light related data and / or ultraviolet light data.

[0018] The device 100 may be configured for detecting the presence and enumeration of various microorganisms in paints and coatings. Exemplary microorganisms can include one or more species from one or both of the following groups.

[0019] Bacteria: Alcaligenes, such as Alcaligenes faecalis. Acinetobacter , such as Acinetobacter calcoaceticus . Achromobacter, such as Achromobacter insolitus. Bacillus. such as Bacillus subtilis. Citrobacter , such as Citrobacter freundii. ('omomonas. such as Comamonas testosterone. Corynebacterium, such as Corynebacterium ammoniagenes . Enterobacter , such as Enterobacter aerogenes or Enterobacter cloacae, Enterococcus, such as Enterococcus hirae, Escherichia, such as Escherichia coli, Proteus, such as Proteus hauseri, Pseudomonas, such as Pseudomonas aeruginosa, Pseudomonas oloevorans, or Pseudomonas stutzeri, Salmonella, such as Salmonella enterica. Staphylococcus, such as Staphylococcus aureus.

[0020] Fungi: Acremonium, such as Acremonium strictum, Alternaria, such as Alternaria tenuis or Alternaria alternata, Aspergillus, such as Aspergillus niger or Aspergillus brasiliensis , Candida, such as Candida albicans, Chaetomium, such as Chaetomium globosum, Fusarium, such as Fusarium solani, Geotrichum, such as Geotrichum candidum. Lentinus, such as Lentinus tigrinus, Penicillium, such as Penicillium glaucum, Penicillium funiculosum or Penicillium pinophilum, Rhodotorula, such as Rhodotorula rubra or Rhodotorula mucilaginosa, Stachybotrys, such as Stachybotrys chartarum, Trichoderma, such as Trichoderma virens.

[0021] The device 100 may be configured for detecting the presence and enumeration of microorganisms in various paints and coatings. For instance, the device 100 may be configured for detecting the presence and enumeration of microorganisms in latex paints. In example embodiments, the paint may include a solvent, such as water, and a latex binder (e.g., a polymer including one or more acrylate, vinyl acetate, vinyl chloride, and / or styrene butadiene monomers). Optionally, the paint may further include a dispersant and / or surfactant to improve distribution of the latex binder throughout the paint. In this manner,Attomey Docket No.: ARXPC-40Q-PCT (LP3745PC02) the dispersant and / or surfactant may be used to produce a more homogenous mixture that can provide a more even coating of the paint. Optionally, the paint may include a thickening agent to adjust the viscosity of the paint to improve adhesion of the wet paint to an applicator (e.g., a brush or roller). Optionally, the paint may include one or more pigments (e.g., TiO2) for providing a color to the paint. Optionally, the paint may include a cosolvent (e.g., ethylene glycol) that can improve solubility of components of the paint.

[0022] As another example, the device 100 may be configured for detecting the presence and enumeration of microorganisms in antifouling coatings. The antifouling coatings may include a polymer and / or copolymer allowing controlled release of one or more biocidal agents included therein, e.g., by releasing these agents from an antifouling coating overtime as is the case with self-polishing or ablative coatings. Suitable polymers and / or copolymers for that purpose are known to the person skilled in the art. For example, the polymers and / or copolymers that are used as binders in “self-polishing antifouling coatings” allowing the controlled release of the one or more biocidal agents may be hydrolysable acry late polymers such as (meth)acrylate based polymers and / or copolymers, such as those described in U.S. Patent Publication No. 2022 / 0095625, which is incorporated by reference in its entirety. The (meth)acrylate polymers and / or copolymers may also include a metal salt moiety of acry lic or methacrylic acid, referred to herein as a “metal salt (meth)acrylate”. The metal may be any suitable metal known to the skilled artisan, e g., zinc, calcium, magnesium, lithium, iron, zirconium, aluminum, cobalt, zirconium, barium and bismuth. The polymer and / or copolymer allowing the controlled release of one or more biocidal agents may also be a VAGH copolymer. The VAGH copolymer may be dissolved in 2:3 xylene: MIBK.

[0023] The paints and coatings analyzed by the device 100 may also include one or more biocides. In example embodiments, the biocides may include at least one 4- isothiazolin-3-one biocidal active compound, which may include any 4-isothiazolin-3-one derivative known in the art that exhibits biocidal activity. For example, the at least one 4- isothiazolin-3-one biocidal active compound may be one or more of 1,2- benzisothiazolin- 3-one (BIT), N-(w-butyl)-l,2-benzisothiazolin-3-one (BBIT), 5- chloro-2-methyl-2H- isothiazolin-3-one (CMIT), 2-methyl-2H-isothiazolin-3-one (MIT), 2- methyl-4,5- trimethylene-4-isothiazolin-3-one (MTI), 2-octyl-3(2H)-isothiazolone (OIT), dichloro-n- octyl-2H-isothiazolin-3-one (DCOIT), N-methyl-l,2-benzisothiazolin-3-one (nMBIT), and 2,2-dithiobis(N-methylbenzamide) (DTMB). Other examples of biocidal active compounds are bronopol. benzyl hemiformal, tetramethylazodi carboxamide (TMAD), 1,3-Attorney Docket No.: ARXPC-400-PCT (LP3745PC02) bis(hydroxymethyl)-5,5-dimethylimidazolidine-2, 4-dione (DMDMH), tris-hydroxymethyl nitromethane (THNM), dimethyl oxazolidine (DMO). p-chloro-m-cresol, dimethylol urea, l,2-dibromo-2.4-dicyanobutane. 2, 2-dibromo-3 -nitrilopropionic acid amide, glutaric dialdehyde, ethylene glycol hemiformal, ethylenglykol-bis-hemiformal, N-methylol urea, bis(tetrakis hydroxymethyl) phosphonium sulfate (THPS), thiabendazol, carbendazim, zinc pyrithione, sodium pyrithione, potassium pyrithione, lithium pyrithione, ammonium pyrithione, calcium pyrithione, magnesium pyrithione, an organic amine pyrithione, barium pyrithione, strontium pyrithione, copper pyrithione, cadmium pyrithione, 2- phenoxyethanol, phenoxypropanol, o-phenyl-phenol, benzyl alcohol, sodium dithiocarbamates, chlorophen, quarternary ammonium salts, such as N-alkyl-N,N- dimethyl-benzyl-ammonium chloride, or tri-n-butyl tetradecyl phosponium chloride, C8- C16 alkyl amines, and organic acids such as benzoic acid, dehydroacetic acid, sorbic acid, salicylic acid, and combinations thereof. Further examples of biocidal active compounds are cuprous oxide (Cu20), zinc oxide (ZnO), 4-bromo-2-(4-chlorophenyl)-5- (trifluoromethyl)-lH-pyrrole-3-carbonitrile (tralopyril), zinc ethane-1,2- diylbis(dithiocarbamate) (zineb). zinc N,N-dimethylcarbamodithioate (ziram), 3-(3,4- dichlorophenyl)- 1,1 -dimethylurea (diuron), copper(I) thiocyanate (CuSCN), 4-[l -(2,3- dimethylphenyl)ethyl]-lH-imidazole (medetomidine), triazines, fluanids and 2, 4,5,6- tetrachloroisophthalonitrile (chlorothalonil), and combinations thereof.

[0024] The paints and coatings analyzed by the device 100 may also include a pigment. Representative pigments that can be used include, but are not limited to, rutile and anatase TiO2, clays such as kaolin clay, asbestos, calcium carbonate, zinc oxide, chromium oxide, barium sulfate, iron oxide, tin oxide, calcium sulfate, talc, mica, silicas, dolomite, zinc sulfide, antimony oxide, zirconium dioxide, silicon dioxide, cadmium sulfide, cadmium selenide, lead chromate, zinc chromate, nickel titanate, diatomaceous earth, glass fibers, glass powders, glass spheres, MONASTAL Blue G (C. I. Pigment Blue 15), molybdate Orange (C. I. Pigment Red 104), Toluidine Red YW (C I. Pigment 3)-process aggregated crystals, Phthalo Blue (C I. Pigment Blue 15)-cellulose acetate dispersion, Toluidine Red (C. I. Pigment Red 3), Watchung Red BW (C. I. Pigment Red 48), Toluidine Yellow GW (C. I. Pigment Yellow 1), MONASTRAL Blue BW (C. I. Pigment Blue 15), MONASTRAL Green BW (C. I. Pigment Green 7), Pigment Scarlet (C. I. Pigment Red 60), Auric Brown (C. I. Pigment Brown 6), MONASTRAL Green G (C I. Pigment Green 7), MONASTRAL Maroon B, MONASTRAL Orange, and Phthalo Green GW 951.Attorney Docket No.: ARXPC-40Q-PCT (LP3745PC02)

[0025] The device 100 may surprisingly detect the presence and enumeration of various microorganisms in paints and coatings despite the presence of latex binder(s), polymer(s), copolymer(s), biocide(s), and / or pigment(s).

[0026] The present disclosure may be better understood with reference to the following examples.Example 1: Detection of High Level of Bacterial Contamination in Paint Using Detection Method with Barrier Layer Membrane Versus Detection Method without Barrier Layer Membrane

[0027] Interior latex paint samples were inoculated with bacterial cultures representing a high contamination level and analyzed using both a detection method employing a membrane between the growth media and indicator substrate and a detection method without a membrane between the growth media and indicator substrate.Experimental Method Test Procedure1) Bacterial cultures were grown on tryptic soy agar (Difco, Becton Dickenson and Company, Franklin Lakes, NJ. USA) for 18-24 hours at 35°C.2) Bacterial suspensions were made in Butterfield’s phosphate buffer (Neogen Corporation, Lansing, MI, USA) and adjusted to ~1 x 108CFU (colony forming units) / mL.3) A sample of experimental interior latex paint of a volume of 4.95 mL was added to sterile 25 mL tubes.4) The bacterial suspension (0.05 mL) was added to the paint sample followed by vortexing, resulting in a bacterial contamination level of ~1 x 106CFU / mL.5) Following vortexing, 1 mL samples of paint were transferred to Soleris® NF-TVC vials (Neogen Corporation, Lansing. MI, USA) and mixed via inverting the vial and sample. The Soleris® NF-TVC vials represent a detection method with a barrier layer membrane between the growth media and indicator substrate. In parallel, 1 mL samples of paint were transferred to CertaBlue CB-TVC vials (CertaBlue, Sw-almen, Netherlands) and mixed via inverting the vial and sample. The CertaBlue CB-TVC vials represent a detection method without a barrier layer membrane between the growth media and indicator substrate.6) The Soleris® NF-TVC vials with sample were placed into a Soleris® Next Generation instrument (Neogen Corporation, Lansing. MI, USA) and analyzed for contamination detection with the included Neogen Fusion software. Detection time was set to 24 hrsAttorney Docket No.: ARXPC-40Q-PCT (LP3745PC02) and incubation temperature was set to 35°C. The CertaBlue CB-TVC vials with sample were placed into a CertaBlue AutoScanner System (CertaBlue, Swalmen, Netherlands) and analyzed for contamination detection with the included CertaSoft Professional X software. Detection time was set to 24 hrs and incubation temperature was set to 35°C.

[0028] Results for high level contamination detection are shown in Table 1.

[0029] The detection method with a barrier layer membrane outperformed, that is, detected contamination in a shorter time, the method without a barrier for even’ bacterium tested in a high level contamination experiment. It was unexpected that the barrier layer membrane method was able to detect contamination significantly faster than the method without a barrier layer membrane. Detection times with the barrier layer membrane method were between 1.7 and 10.4 hours faster than the method without a barrier layer membrane.TABLE 1Time to Detection in Hours for High Level Bacterial ContaminationSoleris® CertaBlueOrganism NF-TVC CB-TVCPseudomonas aeruginosa 5.0 15.4Pseudomonas oleovorans 9.3 19.6Enterobacter cloacae 3.7 5.5Citrobacter freundii 4.0 5.8Bacillus subtilis 10.1 1 1.8Example 2: Detection of Moderate Level of Bacterial Contamination in Paint Using Detection Method with Barrier Layer Membrane Versus Detection Method without Barrier Layer Membrane

[0030] Interior latex paint samples were inoculated with bacterial cultures representing a moderate contamination level and analyzed using both a detection method employing a membrane between the grow th media and indicator substrate and a detection method without a membrane between the growth media and indicator substrate.Experimental Method Test Procedure1) Bacterial cultures were grown on tryptic soy agar (Difco, Becton Dickenson and Company, Franklin Lakes. NJ. USA) for 18-24 hours at 35°C.2) Bacterial suspensions were made in Butterfield’s phosphate buffer (Neogen Corporation, Lansing, MI, USA) and adjusted to ~1 x 106CFU (colony forming units) / mL.Attorney Docket No.: ARXPC-40Q-PCT (LP3745PC02)3) A sample of experimental interior latex paint of a volume of 4.95 mL was added to sterile 25 mL tubes.4) The bacterial suspension (0.05 mL) was added to the paint sample followed by vortexing, resulting in a bacterial contamination level of ~1 x 104CFU / mL.5) Follow ing vortexing, 1 mL samples of paint were transferred to Soleris® NF-TVC vials (Neogen Corporation, Lansing, MI, USA) and mixed via inverting the vial and sample. The Soleris® NF-TVC vials represent a detection method with a barrier layer membrane between the growth media and indicator substrate. In parallel, 1 mL samples of paint were transferred to CertaBlue CB-TVC vials (CertaBlue, Swalmen, Netherlands) and mixed via inverting the vial and sample. The CertaBlue CB-TVC vials represent a detection method without a barrier layer membrane between the growth media and indicator substrate.6) The Soleris® NF-TVC vials with sample were placed into a Soleris® Next Generation instrument (Neogen Corporation, Lansing, MI, USA) and analyzed for contamination detection with the included Neogen Fusion software. Detection time was set to 24 hrs and incubation temperature was set to 35°C. The CertaBlue CB-TVC vials with sample were placed into a CertaBlue AutoScanner System (CertaBlue, Swalmen, Netherlands) and analyzed for contamination detection with the included CertaSoft Professional X software. Detection time was set to 24 hrs and incubation temperature was set to 35°C.

[0031] Results for moderate level contamination detection are shown in Table 2.

[0032] The detection method with a barrier layer membrane outperformed, that is, detected contamination in a shorter time, the method without a barrier for every bacterium tested in a moderate level contamination experiment. It w as unexpected that the barrier layer membrane method was able to detect contamination significantly faster than the method without a barrier layer membrane. Detection times with the barrier layer membrane method were between 1 .0 and 3.5 hours faster than the method without a barrier layer membrane.TABLE 2Time to Detection in Hours for Moderate Level Bacterial ContaminationSoleris® CertaBlueOrganism NF-TVC CB-TVCPseudomonas aeruginosa 8.2 11.7Pseudomonas oleovorans 16.3 18.8Enterobacter cloacae 6.5 7.5Citrobacter freundii 6.2 8.1Attorney Docket No.: ARXPC-40Q-PCT (LP3745PC02)Bacillus subtilis 15,5 17,7Example 3: Detection of Low Level of Bacterial Contamination in Paint Using Detection Method with Barrier Layer Membrane Versus Detection Method without Barrier Layer Membrane

[0033] Interior latex paint samples were inoculated with bacterial cultures representing a low contamination level and analyzed using both a detection method employing a membrane between the growth media and indicator substrate and a detection method without a membrane between the growth media and indicator substrate.Experimental Method Test Procedure1) Bacterial cultures were grown on tryptic soy agar (Difco, Becton Dickenson and Company, Franklin Lakes, NJ, USA) for 18-24 hours at 35°C.2) Bacterial suspensions were made in Butterfield’s phosphate buffer (Neogen Corporation, Lansing, MI, USA) and adjusted to ~1 x 104CFU (colony forming units) / mL.3) A sample of experimental interior latex paint of a volume of 4.95 mL w as added to sterile 25 mL tubes.4) The bacterial suspension (0.05 mL) was added to the paint sample followed by vortexing, resulting in a bacterial contamination level of ~1 x 102CFU / mL.5) Following vortexing, 1 mL samples of paint w ere transferred to Soleris® NF-TVC vials (Neogen Corporation, Lansing, MI, USA) and mixed via inverting the vial and sample. The Soleris® NF-TVC vials represent a detection method with a barrier layer membrane between the grow th media and indicator substrate. In parallel, 1 mL samples of paint w ere transferred to CertaBlue CB-TVC vials (CertaBlue, Swalmen, Netherlands) and mixed via inverting the vial and sample. The CertaBlue CB-TVC vials represent a detection method without a barrier layer membrane between the growth media and indicator substrate.6) The Soleris® NF-TVC vials with sample were placed into a Soleris® Next Generation instrument (Neogen Corporation, Lansing, MI, USA) and analyzed for contamination detection with the included Neogen Fusion software. Detection time was set to 24 hrs and incubation temperature was set to 35°C. The CertaBlue CB-TVC vials with sample were placed into a CertaBlue AutoScanner System (CertaBlue, Swalmen, Netherlands) and analyzed for contamination detection with the included CertaSoft Professional X software. Detection time was set to 24 hrs and incubation temperature was set to 35°C.Attorney Docket No.: ARXPC-400-PCT (LP3745PC02)

[0034] Results for low level contamination detection are shown in Table 3.

[0035] The detection method with a barrier layer membrane outperformed, that is, detected contamination in a shorter time, the method without a barrier for three bacteria tested in a moderate level contamination experiment. In the case of two of the bacteria tested, Pseudomonas oleovorans, and Bacillus subtilis, the method without a barrier layer membrane was unable to detect the low level of contamination present, whereas the method with a barrier layer membrane was able to detect the contamination. It was unexpected that the barrier layer membrane method was able to detect contamination significantly faster than the method without a barrier layer membrane. It was also unexpected that the barrier layer membrane method was able to detect bacterial contamination successfully when the method without a barrier layer membrane was unable to detect the contaminating organisms. Detection times with the barrier layer membrane method were between 1.1 and 4.0 hours faster than the method without a barrier layer membrane.TABLE 3 Time to Detection in Hours for Low Level Bacterial ContaminationSoleris® CertaBlueOrganism NF-TVC CB-TVCPseudomonas aeruginosa 12.0 16.0Pseudomonas oleovorans 22.8 NDEnterobacter cloacae 9.0 10.1Citrobacter freundii 9.3 10.7Bacillus subtilis 21.2 NDND = no detectionAdditional Disclosure

[0036] The preceding description is exemplary in nature and is not intended to limit the scope, applicability or configuration of the disclosure in any way. Various changes to the described embodiments may be made in the function and arrangement of the elements described herein without departing from the scope of the disclosure.

[0037] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention is related.

[0038] As used in this application and in the claims, the singular forms “a”, "an", and ■‘the” include the plural forms unless the context clearly dictates otherwise. Additionally,Attorney Docket No.: ARXPC-40Q-PCT (LP3745PC02) the term “includes” means “comprises”. The methods and compositions of the present disclosure, including components thereof, can comprise, consist of, or consist essentially of the essential elements and limitations of the embodiments described herein, as well as any additional or optional ingredients, components or limitations described herein or otherwise useful in biocidal compositions.

[0039] Unless otherwise indicated, all numbers expressing quantities of ingredients, properties such as molecular weight, percentages, and so forth, as used in the specification or claims are to be understood as being modified by the term “about”. Accordingly, unless otherwise indicated, implicitly or explicitly, the numerical parameters set forth are approximations that may depend on the desired properties sought and / or limits of detection under standard test conditions / methods. When directly and explicitly distinguishing embodiments from discussed prior art. the embodiment numbers are not approximates unless the word “about” is recited.

[0040] As used herein, “optional” or “optionally” means that the subsequently described material, event or circumstance may or may not be present or occur, and that the description includes instances where the material, event or circumstance is present or occurs and instances in which it does not. As used herein, “w / w%” and “wt%” mean by weight as relative to another component or a percentage of the total weight in the composition.

[0041] The term “about” is intended to mean approximately, in the region of. roughly, or around. When the term “about” is used in conjunction with a numerical range, it modifies that range by extending the boundaries above and below the numerical values set forth. Unless otherwise indicated, it should be understood that the numerical parameters set forth in the following specification and attached claims are approximations. At the very least, and not as an attempt to limit the application of the doctrine of equivalents to the scope of the claims, numerical parameters should be read in light of the number of reported significant digits and the application of ordinary' rounding techniques.

[0042] The term “substantially free of’ when used to describe the amount of substance in a material is not to be limited to entirely or completely free of and may correspond to a lack of any appreciable or detectable amount of the recited substance in the material. Thus, e.g., a material is “substantially free of’ a substance when the amount of the substance in the material is less than the precision of an industry -accepted instrument or test for measuring the amount of the substance in the material. In certain example embodiments, a material may be “substantially free of’ a substance when the amount of the substance in theAttorney Docket No.: ARXPC-40Q-PCT (LP3745PC02) material is less than 10%, less than 9%, less than 8%, less than 7%, less than 6%, less than 5%, less than 4%, less than 3%, less than 2%, less than 1%, less than 0.5%, or less than 0.1% by weight of the material.

[0043] As used herein, the terms “first”, “second”, and “third” may be used interchangeably to distinguish one component from another and are not intended to signify location or importance of the individual components.

[0044] Here and throughout the specification and claims, range limitations are combined and interchanged, such ranges are identified and include all the sub-ranges contained therein unless context or language indicates otherwise. For example, all ranges disclosed herein are inclusive of the endpoints, and the endpoints are independently combinable with each other.

[0045] This written description uses examples to disclose the present disclosure, including the best mode, and also to enable any person skilled in the art to practice the disclosure, including making and using any devices or systems and performing any incorporated methods. The patentable scope of the disclosure is defined by the claims, and may include other examples that occur to those skilled in the art. Such other examples are intended to be within the scope of the claims if they include structural elements that do not differ from the literal language of the claims, or if they include equivalent structural elements with insubstantial differences from the literal languages of the claims.

[0046] These and other modifications and variations to the present invention may be practiced by those of ordinary skill in the art, without departing from the spirit and scope of the present invention, which is more particularly set forth in the appended claims. In addition, it should be understood that aspects of the various embodiments may be interchanged both in whole or in part. Furthermore, those of ordinary skill in the art will appreciate that the foregoing description is by way of example only, and is not intended to limit the invention so further described in such appended claims.

Claims

Attorney Docket No.: ARXPC-40Q-PCT (LP3745PC02)What Is Claimed;1. A method for detecting microorganisms in paints and coatings, comprising: introducing a container with a test sample into a testing device, the test sample comprising a solvent and a polymer, the container comprising a growth zone, a detection zone, and a barrier layer, the growth zone disposed within the container and comprising a growth media for supporting growth of microorganisms, the detection zone adjacent a transparent section of the container, the barrier layer disposed between the growth and detection zones and comprising porous material configured for allowing diffusion of the growth media, an indicator substrate that changes optical properties due to growth of the microorganisms, and metabolic by-products of the microorganisms between the growth and detection zones; incubating the microorganisms in the growth media such that metabolic processes of the microorganisms generate the metabolic by-products in the growth media in the growth zone in order to change the optical properties of the indicator substrate in the growth zone; diffusing the metabolic by-products into the detection zone through the barrier layer while blocking the microorganisms and particulate matter of the test sample from the detection zone; and detecting the change in the optical properties of the indicator substrate in the detection zone through the transparent section.

2. The method of claim 1, w herein the test sample further comprises one or both of a biocide and a pigment.

3. The method of claim 1, wherein the barrier layer comprises a membrane.

4. The method of claim 3. wherein the membrane is hydrophilic.

5. The method of claim 4, wherein the membrane is configured to allow a flow of liquids between through the membrane when a hydrostatic pressure gradient is present between opposing surfaces of the membrane.Attorney Docket No.: ARXPC-40Q-PCT (LP3745PC02)6. The method of claim 3. wherein a maximal pore size of the membrane is less than one micrometer.

7. The method of claim 1, wherein the porous material is a polymer.

8. The method of claim 1. wherein the indicator substrate comprises one or both of: a visible dye configured to change color due to the metabolic processes of the microorganisms; and fluorescence dye configured to change fluorescent properties due to the metabolic processes of the microorganisms.

9. The method of claim 1 , further comprising: directing electromagnetic energy from an electromagnetic energy source at the transparent section; and detecting reactive electromagnetic energy resulting from interaction of the electromagnetic energy with the indicator substrate in the detection zone.

10. The method of claim 9, wherein the electromagnetic energy7source comprises a visible light source, and the indicator substrate comprises a visible dye.1 1 . The method of claim 10, wherein the electromagnetic energy source is a lightemitting diode.

12. The method of claim 9. wherein the electromagnetic energy source comprises an ultraviolet light source, and the indicator substrate comprises a fluorescence dye generating visible reactive energy.

13. The method of claim 12, wherein said electromagnetic energy source comprises one or both of an ultraviolet light-emitting diode and a gas discharge tube.

14. The method of claim 9, wherein a photo detector is utilized for the detection of the reactive electromagnetic energy, and the photo detector comprises one or more of a photo diode, a photon multiplying tube (PMT), and a photo transistor.Attorney Docket No.: ARXPC-40Q-PCT (LP3745PC02)15. The method of claim 1. further comprising activating an optical instrumentation comprising a light source and a photodetector, the light source disposed exterior to the container and producing light that passes through the detection zone prior to detection by the photodetector, wherein detecting the change in the optical properties of the indicator substrate comprises detecting the change in the optical properties of the indicator substrate based on one or more signals from the photodetector.

Images