Methods, systems, and test kits for evaluating antimicrobial viscous liquids

Abstract

Methods, systems, devices, and kits are disclosed to assess or test antimicrobial and microbicidal characteristics and characteristics of viscous liquids, such as pigments and coatings, applied by a contact manner, such as smearing, brushing, or blade coating.

Description

Background Art

[0001] Many types of viscous liquids are formulated to have antimicrobial characteristics. Procedures and methods for evaluating and testing the antimicrobial characteristics and properties of materials (such as coatings and pigments) tested or applied in their liquid state have been reported. Such procedures and methods are included, for example, in protocol MRID 48482503 and protocol MRID 50025401. The method involves applying selected microorganisms to the surface of a carrier and then spraying the substance onto the carrier. However, this method is limited to using spray application and does not cover other common ways of applying viscous liquids to a substrate, such as brush application, scrape application, or roll application. Therefore, it also does not cover all formulations, as some formulations are developed specifically for application by non-spray methods. Compared to spray application, these other application methods require contact between the application device for applying the viscous liquid and the test bed containing the microorganisms. Existing methods for evaluating the antimicrobial characteristics of compositions applied by spraying cannot be used because the microorganisms involved in the test often spread, resulting in incorrect and inconsistent data.

[0002] Therefore, there is a need for a method and apparatus for evaluating and testing the antimicrobial characteristics of viscous liquid substances by direct application methods that will produce reliable and consistent data. Summary of the Invention

[0003] The present disclosure describes processes and procedures for evaluating or testing viscous liquids. The disclosed embodiments can determine the antimicrobial and germicidal characteristics of different types of materials, including but not limited to oils, mineral slurries, foods, jellies, latex polymers, personal care cream products, nail polishes, drugs, medicaments, pigments, coatings, and their precursors.

[0004] In one embodiment, the present disclosure describes a method for evaluating the microbicidal characteristics of a viscous liquid material or substance. The method may include the following steps: a) forming a microbial film on one or more carriers; b) exposing the microbially treated carriers to a uniform film of the viscous liquid using a platform including one or more pores; c) using a tool to lift the film-coated carriers out of the one or more pores to lift the carriers out of the pores; d) using a tool to remove the film-coated carriers from the platform to remove the carriers from the platform; e) incubating the film-coated carriers in a culture medium; and f) determining the viability of the microorganisms on the microbially treated carriers to evaluate the microbicidal characteristics of the viscous liquid. In an alternative embodiment, a uniform film of the viscous liquid is formed by smearing, roll coating, brush coating, or scraping the viscous liquid onto the microbially treated carriers. Those skilled in the art will understand that the described method can be used to evaluate the microbicidal properties of a liquid against various microorganisms, including but not limited to fungal spores, molds, bacteria, viruses, or selected combinations of these microorganisms. Those skilled in the art will also understand that the described method can be used to evaluate the microbicidal properties of a liquid against different cells, such as but not limited to skin cells, blood cells, cells from biopsy tissue, cultured cell samples, or combinations of such cells.

[0005] The present disclosure describes a method for depositing a thin film of a liquid for testing on a treated carrier, the method including the following steps: forming a microbial film or a material film on one or more carriers; exposing the carriers treated with the microbial film or the material film to a thin film of the liquid, the thin film being deposited by scraping the liquid on the platform; using a tool to lift the film-coated carriers out of the pores to lift the carriers out of the plurality of pores; and using a tool to remove the film-coated carriers from the platform to remove the carriers from the platform. In one embodiment, a uniform film of the viscous liquid is prepared by a scraping process that uses a doctor blade to uniformly apply a thin film of a liquid (such as a pigment or a coating) on the microbially treated carriers. However, the liquid film can also be deposited by smearing or brush coating the liquid film. Compared with existing methods, the present disclosure allows the evaluation of liquids that require contact between the application device and the carrier. However, existing methods do not support the evaluation of liquids applied in such a manner because the contact would dirty the microorganisms or the material film present on the carrier, but the method of the present invention supports application in such a manner and can distinguish the strength of the microbicidal activity provided between different liquid formulations against the microbial film or the material film present on the carrier.

[0006] The present disclosure also describes a system for preparing a uniform film on a carrier for further evaluation, processing, or testing. In some embodiments, the system can include: a well platform that includes wells for receiving carriers; a lifting tool configured to lift a microbially coated carrier from a well of the well platform; and a tool for removing the carrier from the well platform. The lifting tool can be configured to include a lifting structure, such as a tab, finger, protrusion, or fork, that will mate with a feature of the porous platform to remove the carrier from the well of the well platform.

[0007] Other embodiments described in the present disclosure include test kits for evaluating the resistance of a pigment or coating to mold or fungal growth. The features of the test kit can include a carrier, a well platform, a spreader bar, a lifting tool for lifting a microbially coated carrier from a well of the well platform and a removal tool for removing the microbially coated carrier from the well platform, as well as containers and tools suitable for preparing and processing the microbially treated carrier. Additional optional features of the kit can include microorganisms, growth medium reagents, and neutralizing reagents.

[0008] The foregoing embodiments can be combined with one or more optional features as further described herein. BRIEF DESCRIPTION OF THE DRAWINGS

[0009] Figure 1 is an isometric view of the porous platform.

[0010] Figure 2 is an isometric view of the lifting tool.

[0011] Figure 3 is an isometric view of the removal tool.

[0012] Figure 4 is a top view showing a film-coated carrier lifted from the porous tool by the lifting tool to allow the removal tool to transfer the film-coated carrier for further processing.

[0013] Figure 5 is Figure 4 a side view of, showing the carrier film lifted from the porous tool. DETAILED DESCRIPTION

[0014] The present disclosure describes processes and procedures for evaluating or testing the antimicrobial and microbicidal characteristics and properties of viscous liquids. The disclosed embodiments provide devices, tools, and methods for preparing samples coated with a liquid substance and processing the samples during subsequent testing procedures to provide consistent and reproducible results.

[0015] In the present disclosure, specific embodiments have been described above with reference to the disclosed embodiments and examples, but such embodiments are merely illustrative and do not limit the scope of the present disclosure.

[0016] All publications, patents, and patent documents are incorporated herein by reference as if each were incorporated by reference individually. It should be understood from such publications, patents, and patent documents that there are no limitations inconsistent with the present disclosure.

[0017] Method

[0018] The present disclosure describes contacting a microbially treated carrier or substrate with a liquid for a predetermined time, applying the liquid by contacting the carrier with an application device immersed in the liquid, removing the carrier via aseptic technique, and then evaluating the viability of the microorganisms on the carrier surface to grow under conditions providing quantifiable microbial activity. The present invention also discloses test devices and kits for performing the disclosed methods. Accordingly, the disclosed methods allow quantification of the microbicidal characteristics of viscous liquids.

[0019] In some embodiments, selected isolated microorganisms are applied to a suitable carrier, such as a slide, according to established protocols for assessing and testing microbial characteristics known to those of skill in the art. Prior to applying the isolated microorganisms, the carrier is sterilized to limit or eliminate contamination by external microorganisms. The carrier can be configured in a variety of shapes and sizes, but is preferably a thin slide. Additionally, the carrier can be made of a variety of materials such as glass, metal, plastic, or polymer, and is preferably non-porous. The carrier material can be selected for the microorganisms used for evaluation or testing. A suitable carrier material will support microbial film formation, allow processes using growth medium reagents, neutralizing agents, or other reagents, and will be used to provide a qualitative or quantitative determination of the microbicidal effect of the liquid being evaluated or tested according to the present disclosure.

[0020] The microorganisms used can broadly include one or more microorganism- or cell-based samples, which include, for example, bacteria, viruses, or fungi, as well as isolates or treated animal cells. Suitable animal cells include, for example, skin cells, blood cells, or cells from biopsy tissue. Additionally, the cell sample can be a cultured cell sample. A cultured cell sample can be used when known culturing processes are used to amplify cells or a small group of cells. When the cell culture reaches the desired cell density, it can be further processed, if needed, and then applied to a suitable carrier to form a microorganism-based film.

[0021] A viscous liquid is applied to a microbe-covered slide by way of contact between the viscous liquid (such as a coating) and an application device (such as a brush, roller, or squeegee). Different from existing methods that support testing only by spraying the viscous liquid, the present disclosure provides specific support for application by way of contact, which generally results in an undesired mess or spread of microbes on the carrier. The present disclosure also allows control of the amount of viscous liquid applied to each carrier, thereby allowing a volumetric evaluation of the microbicidal ability based on volume.

[0022] Thus, in an embodiment of the method of the present invention, an exact amount of viscous liquid can be dispensed in a small area without compromising the integrity of the microbes present on the surface of the carrier. The viscous liquid can be an oil, a mineral slurry, a foodstuff, a jelly, a latex polymer, a personal care cream product (e.g., a cream, a lotion), a nail polish, a drug (e.g., a tinea pedis cream), or any other viscous liquid that can be squeegeed onto the test carrier.

[0023] A suitable microbially-treated sample test carrier is placed in a well platform (the well platform can also be referred to as a squeegee template) having separate wells for the carriers, and the viscous liquid is applied to the carrier by using a straight-edge squeegee, brushing, or by roller. This allows the application of a viscous liquid (such as a pigment or a coating) that requires contact between the viscous liquid and the application device onto the microbe-covered slide without removing the microbes from the surface of the carrier by friction or fluid flow. The membrane-coated carrier in the template well can then be placed on a lifting tool to assist in lifting the membrane-coated test carrier out of the squeegee template well. Then, a slide remover is used to lift the sample test carrier off the lifting tool without direct human contact with the carrier, and the sample is transferred to a safe place to continue the test to evaluate the viability of the microbial growth present on the carrier. The use of the squeegee template in this method provides a consistent film build-up on multiple carriers in the wells of the squeegee template without compromising the microbes present on the surface of the carrier.

[0024] After applying a uniform film on a carrier, the carrier is lifted out of the well platform and transferred to a suitable growth or nutrient broth for further processing. A nutrient broth suitable for the microorganism in the evaluation or test method is selected. In some methods, the nutrient broth can be Sabouraud dextrose medium, potato dextrose medium, malt medium, glucose medium, basal salt medium, trypticase soy medium, nutrient medium, brain heart infusion medium, plate count medium, R2A medium, blood medium, or charcoal medium, or another suitable nutrient medium. When in the nutrient broth, the membrane-coated carrier can be maintained at a desired temperature that allows the transferred microorganism to multiply or grow. It is well known that there are microorganisms that grow optimally at elevated temperatures (such as 37 °C). In some embodiments, the incubated membrane-coated carrier is stirred at an elevated temperature for a predetermined period within the range of several hours to several days.

[0025] When the incubation period ends, many types of qualitative or quantitative methods can be used to determine the results of the evaluation or test. In some embodiments, the incubated sample is contacted with a neutralizing reagent solution, and the turbidity of the solution can be visually analyzed to evaluate the microbicidal effect of the viscous liquid. The neutralizing agent can be, for example, the neutralizing and inactivating agents in ASTM E1054-08(2013) entitled "Standard Test Method for Inactivators of Antimicrobial Agents for Evaluation". Alternatively, an aliquot from the nutrient broth can be added to the growth medium agar in a Petri dish or agar-coated membrane (such as a PETRIFILM count plate (commercially available from 3M, Saint Paul, MN)), and established methods can be used to determine the microbial viability.

[0026] In some embodiments, after the contact time of the viscous liquid with the treated carrier, the carrier is immediately removed from the test device using a lifting tool and added to a sterile master test container. A sterile lecithin / dextrose-based neutralizing broth medium (e.g., 20 mL) is added to the master container, and the carrier is dropped into the broth medium. A sterile cell scraper can be used to scrape any test substance remaining on the carrier in the master neutralizing container. Then, using the cell scraper as a spatula for transferring the carrier, the carrier is placed in a secondary container containing unused neutralizing medium (e.g., 20 mL).

[0027] All of the primary and secondary containers are then incubated for a predetermined time necessary to evaluate microbial growth. In some methods, this may involve, for example, incubating at 30 °C + / - 3 °C for 10 days. After the incubation period, 200 μL aliquots are removed from each primary container, inoculated onto a suitable agar, and incubated at 30 °C + / - 3 °C for 5 days. Additional spread plates may be used to confirm whether the fungal growth observed in the primary and secondary containers has the characteristic color of the microbial colonies being tested.

[0028] The criterion for a passing result can be zero recovery of the test organism from the primary and secondary neutralization containers, or from any spread plates taken from the primary and / or secondary containers from all parallel determinations. In some methods, the criterion for a passing result can be adjusted such that a passing result is a low number of test organisms recovered from the primary or secondary neutralization container. In this way, the criterion can be adjusted to allow for comparative testing between samples.

[0029] In a selected embodiment, the present disclosure describes a method for evaluating the resistance of a pigment or coating to mold or fungal growth, the method comprising preparing a microbially treated carrier; placing the microbially treated carrier in a platform comprising one or more wells; forming a uniform film of the pigment or coating sample on the microbially treated carrier using the platform with wells and a spreader bar to provide a film-coated carrier; exposing the carrier to the pigment or coating sample; using a tool to lift the film-coated carrier out of the well to lift the carrier out of the plurality of wells; using a tool to remove the film-coated carrier from the one or more wells to remove the carrier from the platform; incubating the film-coated carrier in a culture medium at a temperature above ambient temperature for a predetermined incubation time; neutralizing the culture medium with a neutralizing reagent solution; and determining the viability of the microorganisms in the neutralizing solution by qualitative macroscopic measurement of the turbidity of the neutralizing solution and quantitative evaluation of mold or fungal growth on a spread plate of the neutralizing solution to evaluate the resistance of the pigment or coating to mold or fungal growth.

[0030] Platforms and Tools

[0031] Suitable preparation and processing equipment and tools that can be used in the disclosed method include single-well or multi-well platforms, spreading tools, lifting tools, and transfer tools. The various features of these equipment and tools are described in the section below that details these equipment and tools.

[0032] Figure 1is an embodiment of a porous platform or tool. In this embodiment, the porous platform 10 includes ten holes 12 that accommodate ten rectangular carriers 14. The holes are sized to accommodate carriers containing selected microorganisms. The holes have a depth greater than the thickness of the carriers. The depth of the holes is selected to allow a film of a desired depth of a viscous liquid material to form on the exposed upper surface of the carriers. As part of this procedure, the viscous liquid material is spread over the carriers using a doctor blade application. However, in some embodiments, the viscous liquid can be spread by troweling, brushing, or another contact application. In embodiments using a doctor blade application, the viscous liquid material is brought into contact with the upper surface of the hole platform, and a sterilized straight-edge tool is pulled horizontally across the holes to form a uniform liquid film in each hole. Additionally, each hole has an orifice or opening to provide access to a carrier lifting tool. The number of holes can be one, two, three, four, or more. The shape or depth of the holes can be selected, for example, from a plurality of numbers, shapes, or depths in order to optimize these features for the type of microorganism to be evaluated or tested and for the characteristics and properties of the viscous liquid that provides the film-coated carriers.

[0033] Figure 2 is an embodiment of a carrier lifting tool 20. The carrier lifting tool includes structures 22 that extend vertically from the surface of the carrier lifting tool. Suitable structures can include, for example, tabs, fingers, protrusions, or forks. These structures fit into and pass through the orifices or openings of each hole and have a vertical height that allows the lower surface of the carrier to be lifted above the upper surface of the porous platform. In one embodiment, the Figure 3 carrier removal tool shown is inserted between the upper surface of the porous platform and the bottom surface of the carrier to remove the carrier for subsequent processing during an evaluation or testing procedure.

[0034] Figure 3 is an embodiment of a carrier removal tool 30. The carrier removal tool includes features for removing the carrier from the porous platform. A handle 32 on the removal tool allows for contact and removal of the carrier during a sanitation procedure, thus avoiding undesired contamination of the carrier. When suspended on the lifting structures of a carrier lifting tool above the upper surface of the porous platform, a lifting fork 34 on the removal tool provides a structure for contacting and lifting the carrier. The fork fits over the outside of the lifting structure to allow for a stable procedure for removing and transporting the carrier. In some aspects, the lifting fork can include an orifice for receiving the carrier.

[0035] Figure 4 is an embodiment of a step of an evaluation or testing process or procedure of the present disclosure. Figure 4An exemplary step is an embodiment in which the vertical structure of the carrier lifting tool 20 is assembled into the orifices of the orifice platform 10 and extends through these orifices, thereby suspending the carrier 14 above the upper surface of the porous platform. The carrier removal tool 30 is assembled between the upper surface of the orifice platform and the lower surface of the carrier, and the fork 34 of the carrier removal tool is assembled around the outside of the lifting structure. The two forks of the carrier removal tool will allow the carrier to be easily removed and transported in subsequent procedures.

[0036] Figure 5 is Figure 4 A side view of the illustrated procedural step. This side view shows the position of the components when the carrier 14 is suspended above the upper surface of the orifice platform 10. In this figure, the structure 22 of the lifting tool 20 is assembled into the orifices of the porous platform and extends through these orifices, thereby suspending the carrier above the upper surface of the orifice platform. Additionally, the fork 34 of the carrier removal tool is assembled into the area between the upper surface of the orifice platform and the bottom surface of the carrier.

[0037] The orifice platform, the lifting tool, and the removal tool can be made of any material that can be sterilized. Suitable materials include, for example, glass, metal, or plastic. Combinations of suitable materials can also be used. Additionally, the orifice platform, the lifting tool, and the removal tool can be disposable disposal materials. Alternatively, the porous platform, the lifting tool, and the removal tool can be reused as long as the equipment is cleaned and sterilized before each use.

[0038] Kits and Systems

[0039] The features of a test kit or test system can include components such as carriers, porous platforms, spreaders, a lifting tool for lifting a microorganism-coated carrier from the orifices of the orifice platform, and a removal tool for removing a membrane-coated carrier from the orifice platform, as well as containers and tools suitable for preparing and processing the microorganism-treated carriers. Additional features of the kit can include microorganisms, growth medium reagents, and neutralizing reagents. The combination of all components in kit form makes the method easy to use and avoids unwanted variability in assessment or testing when the components used in the method are sourced from multiple suppliers or vendors.

[0040] As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless expressly and unambiguously limited to one referent. Thus, for example, reference to "an additive" includes two or more different additives. As used herein, the term "comprising" and its grammatical variants are intended to be non-limiting, such that the recitation of items in a list does not preclude other similar items that may be substituted or added to the listed items.

[0041] A numerical range expressed by endpoints includes all the values included within that range (e.g., 1 to 5 includes 1, 1.5, 2, 2.75, 3, 3.80, 4, 5, etc.). In addition, the disclosure of a range is intended as a specific disclosure of all sub-ranges included within a broader range (e.g., 1 to 5 discloses 1 to 4, 1.5 to 4.5, 1 to 2, etc.).

[0042] The terms "preferred" and "preferably" refer to embodiments of the present invention that can provide certain beneficial effects in certain circumstances. However, in the same or other circumstances, other embodiments may also be preferred. In addition, the recitation of one or more preferred embodiments does not imply that other embodiments are not available and is not intended to exclude other embodiments from the scope of the present invention.

[0043] When the term "comprising" and its variants appear in the specification and claims, they do not have a limiting meaning.

[0044] It should be understood that each component, compound, substituent, or parameter disclosed herein is to be interpreted as being disclosed for use alone or in combination with one or more of each and every other component, compound, substituent, or parameter disclosed herein.

[0045] It should also be understood that each range disclosed herein will be interpreted as a disclosure of each specific value having the same number of significant digits within the disclosed range. Thus, for example, the range of 1 to 4 will be interpreted as a specific disclosure of the values 1, 2, 3, and 4 and any range of such values.

[0046] Those skilled in the art will understand that the description of the exemplary embodiments in this disclosure is not intended to limit the disclosure to the specific forms disclosed, but is intended to cover all modifications, equivalents, and alternatives falling within the spirit and scope of the disclosure as defined by the embodiments and the following claims. The scope of the disclosure should be interpreted with reference to the embodiments and the claims.

[0047] The following examples are intended to illustrate the above disclosure and should not be construed as narrowing its scope. Those skilled in the art will recognize that the examples present many other ways in which the disclosure can be practiced. Many variations and modifications can be made while remaining within the scope of the disclosure.

[0048] Examples

[0049] The following examples describe a test method for evaluating the microbicidal ability of a test liquid (in this embodiment, a wet paint composition). Briefly, fungal cells and a dry film of the carrier on the glass carrier surface are exposed to the test substance applied by scraping or spraying for a 10-minute exposure time. After exposure, the carrier is transferred to a container containing a neutralizing medium, and the surviving organisms are determined by the growth of the test organisms. The examples show that this test can distinguish the microbicidal properties of paint compositions applied by spraying or by direct contact application (such as scraping).

[0050] Example 1

[0051] Inoculum preparation: Trichophyton interdigitale (previously called Trichophyton mentagrophytes) ATCC 9533 (VWR P / N 89504-368) obtained from Microbiologics was grown on Sabouraud dextrose agar (SDA, VWR P / N 90000-038) at 25 °C for 12 days.

[0052]

[0053] The fungal growth was harvested by scraping into a 50 mL sterile centrifuge tube (VWR #93000-032) containing 8 sterile 3 mm glass beads (VWR P / N 26396-508) and 20 mL of a solution of 0.85% saline and 0.05% Triton X-100 (VWR P / N EM-TX1568-1).

[0054] The fungal growth was vortexed at maximum speed for 5 minutes and then filtered through a sterile Falcon 40 μm pore size cell strainer (VWR P / N 21008-949) into a second sterile 50 mL centrifuge tube.

[0055] The fungal filtrate was serially diluted 1:10 in sterile DI water, and then 100 μL aliquots were inoculated on SDA plates to determine the initial population and then adjusted to 4.0×10 7 CFU / mL in sterile DI water. The adjusted inoculum was stored refrigerated at 4 °C to 10 °C until used for testing.

[0056] Sample Preparation

[0057] The glass carriers used are hemocytometer slides (Muhwa Scientific P / N MH-800G1) that are 26 mm long × 22 mm wide × 0.5 mm thick. They are prepared by cleaning with 95% ethanol, rinsing with DI water, allowing to air dry, and then autoclaving for 15 minutes using a dry goods sterilization cycle at 121 °C.

[0058] Place the sterile slides on an in-house produced plastic frame that is sterilized by immersion in 10% bleach, rinsed with water, and allowed to air dry.

[0059] Mix the conditioned inoculum to resuspend the fungal component.

[0060] Pipette a 10 μL aliquot of the fungal inoculum onto the middle of the glass carrier and spread with the pipette tip to cover the surface of the glass carrier without exceeding the edges.

[0061] Allow the inoculated glass carriers to dry in a 35 °C incubator for 30 minutes.

[0062] After the 30-minute drying period, coat the inoculated carriers with the test substance (primer).

[0063] After coating, remove the carriers from the test frame and place them on the inner (unlined) lid (VWRP / N 16087-264) of a sterile 125 mL container (VWRP / N 89045-874) with a 60 mm opening. To minimize contamination, place an empty container on top of the carriers on the lid and expose at room temperature for the duration of the exposure time.

[0064] Before recovery, expose each inoculated carrier to the test substance for a 10-minute contact time. Evaluate two different coating application methods in separate rounds of testing.

[0065] Doctor Blade Application

[0066] Pull 4 mL of the coating test sample horizontally across the carrier in the frame hole with a sterilized straight edge, where the depth of the hole is the thickness of the glass carrier plus the film thickness of the 4 mil test substance, resulting in a 4 mil coating thickness.

[0067] Spray Application

[0068] Spray the test substance vertically (at approximately a 60° angle) with a Wagner Spraytech 0417005HVLP Control Spray Stain sprayer until all carrier surfaces are covered, which typically requires 1 - 4 passes depending on the viscosity of the test substance. Clean the sprayer according to the manufacturer's instructions and allow to air dry between samples.

[0069] Bioburden Recovery

[0070] After 10 minutes of contact time, immediately pipette 20 mL of sterile neutralizing broth medium 1 into a container and drip the carrier from the cap into the medium. Fix the cap to the container and then gently shake the container for 5 seconds.

[0071] Use a sterile cell scraper (VWR P / N 76036-006) to scrape the test substance remaining on the glass carrier in the main neutralization container. Record any growth.

[0072] Then use the cell scraper as a spatula to transfer the glass carrier to a secondary neutralization container containing 20 mL of neutralization medium. Record any growth.

[0073] Incubate all recovery containers at 30 °C + / - 3 °C for 10 days.

[0074] Take 200 μL aliquots from each main container on day 7 and inoculate onto SDA 2 Then incubate at 30 °C + / - 3 °C for 5 days to evaluate the test microorganisms. Use additional SDA plates to confirm whether the fungal growth observed in the recovery containers is characteristic white colonies with a light orange substrate of the test microorganisms.

[0075] The acceptance criterion is zero recovery of the test organisms from the main neutralization container, the secondary neutralization container, or any spread plates taken from the main containers and / or secondary containers from all parallel determinations.

[0076] The summary results of the pigment samples tested according to the method of the present disclosure are listed in Table 1, where the viscous test liquid is applied by scraping and by spraying.

[0077] Table 1. Test Samples with Summary Results

[0078]

[0079]

[0080]

[0081] *Growth of organisms other than the test organisms (Trichophyton interdigitale, previously known as Trichophyton mentagrophytes) is shown in more detail in Table 2 for samples applied by spraying according to the method of Example 1.

[0082] Table 2: Detailed Results of Samples Applied and Evaluated by Spray Application via the Method of the Invention

[0083]

[0084]

[0085]

[0086] *Indicates the recovery of organisms other than the test organism (Trichophyton interdigitale, previously known as Trichophyton mentagrophytes, ATCC 9533).

[0087] More detailed results of samples applied and evaluated by scraping according to the methods of the present disclosure are shown in Table 3.

[0088] Table 3: Samples Applied by Doctor Blade and Evaluated by Doctor Blade Application and Evaluation via the Method of the Invention Detailed Results 。

[0089]

[0090]

[0091] *Indicates the recovery of organisms other than the test organism (Trichophyton interdigitale, previously known as Trichophyton mentagrophytes, ATCC 9533).

[0092] Based on the results of the tests completed, the methods of the present disclosure are capable of showing differences in the samples submitted. Based on 1 CFU in 5 parallel determinations of the test organism recovered from the spread plates of the main neutralization container, scraping application showed 3 minor failures, and based on growth in the secondary neutralization container and the spread plates from the main neutralization container, one obvious failure (Sample 7, Zinsser mold killing primer).

[0093] Based on growth in the secondary neutralization container and the spread plates from the main neutralization container, spraying application showed two minor failures, with 1 CFU in five parallel determinations. All samples from spraying application showed contamination on the spread plates from the main neutralization container, which may be due to the sprayer not being able to be disinfected. Nevertheless, the methods of the present disclosure are capable of differentiating samples showing growth of the test organism from samples not showing growth of the test organism.

[0094] Equivalent results for these two application methods were shown only in three of the five samples showing failures, indicating differences in the antimicrobial quality based on the application method (spraying versus scraping). It is hypothesized that highly variable film thickness applied by the sprayer due to different viscosities of the samples may result in differences in the results.

[0095] The foregoing examples demonstrate that the methods of the present disclosure are capable of evaluating the antimicrobial characteristics of viscous liquid compositions (such as paints) applied by means that require direct contact between the coating composition and the application device.

[0096] The foregoing includes examples of this specification. Of course, for the purposes of describing this specification, it is not possible to describe every conceivable combination of components or methods, but one of ordinary skill in the art will recognize that many additional combinations and permutations of this specification are possible. Each of the systems, components, and / or methods described above can be combined in any permutation or added together. Accordingly, this specification is intended to cover all such changes, modifications, and variations that fall within the spirit and scope of the appended claims. Additionally, insofar as the term "comprising" is used in the detailed description or claims, such term is intended to be inclusive in a manner similar to the term "including" as interpreted when used as a transitional word in a claim to mean "including but not limited to".

[0097] List of Embodiments

[0098] Embodiment 1: A method for evaluating the microbicidal characteristics of a viscous liquid, the method comprising: a) forming a microbial film on one or more carriers; b) exposing the microbially treated carriers to a uniform film of the viscous liquid while fixing the carriers on a platform comprising one or more holes; c) using a lifting tool to lift the film-coated carriers out of the one or more holes of the platform to remove the carriers from the one or more holes; d) using a removal tool to remove the film-coated carriers from the platform; e) incubating the film-coated carriers in a culture medium; and f) determining the viability of the microorganisms on the microbially treated carriers to evaluate the microbicidal characteristics of the viscous liquid.

[0099] Embodiment 2: The method according to Embodiment 1, wherein the uniform film is formed by smearing, roll coating, brush coating, or scraping the viscous liquid on the microbially treated carriers.

[0100] Embodiment 3: The method according to Embodiment 1, wherein the uniform film is prepared by a scraping process of uniformly applying the viscous liquid on the microbially treated carriers using a rod.

[0101] Embodiment 4: The method according to any one of Embodiments 1 to 3, wherein the platform comprises a single hole.

[0102] Embodiment 5: The method according to any one of Embodiments 1 to 3, wherein the platform comprises two or more holes.

[0103] Embodiment 6: The method according to any one of Embodiments 1 to 5, wherein at least one carrier is a glass slide.

[0104] Embodiment 7: The method according to any one of Embodiments 1 to 5, wherein at least one carrier is metal.

[0105] Embodiment 8: The method according to any one of claims 1 to 5, wherein at least one carrier comprises a polymer.

[0106] Embodiment 9: The method according to any one of the foregoing embodiments, wherein the carrier is sterile before forming the microbial film.

[0107] Embodiment 10: The method according to any one of the foregoing embodiments, wherein the viscous liquid is a pigment or a paint.

[0108] Embodiment 11: The method according to any one of the foregoing embodiments, wherein the film-coated carrier is incubated at a temperature above the ambient temperature for a predetermined incubation time.

[0109] Embodiment 12: The method according to any one of the foregoing embodiments, wherein after the predetermined incubation time, the culture medium is further neutralized with a neutralizing reagent.

[0110] Embodiment 13: The method according to any one of the foregoing embodiments, wherein the determination of viability is a qualitative or quantitative measurement.

[0111] Embodiment 14: The method according to any one of embodiments 1 to 12, wherein the determination of viability is a qualitative measurement of turbidity.

[0112] Embodiment 15: The method according to any one of embodiments 1 to 12, wherein the determination of viability is a qualitative measurement of the turbidity of the solution after neutralization.

[0113] Embodiment 16: The method according to any one of embodiments 1 to 12, wherein the determination of viability is a quantitative measurement of the spreading position of the solution after neutralization.

[0114] Embodiment 17: The method according to any one of the foregoing embodiments, wherein the measuring step evaluates the microbicidal ability of the viscous liquid against the microbial film.

[0115] Embodiment 18: A method for depositing a thin film of a liquid for testing on a treated carrier, the method comprising: a) forming a microbial film on one or more carriers; b) exposing the carrier treated with the microbial film to a thin film of the liquid while fixing the carrier on a platform comprising one or more holes, the thin film being deposited by scraping the liquid on the platform; c) lifting the film-coated carrier from the holes using a lifting tool; and d) removing the film-coated carrier from the platform using a removal tool.

[0116] Embodiment 19: The method according to any one of the preceding embodiments, wherein the microbial film comprises fungal spores.

[0117] Embodiment 20: The method according to any one of the preceding embodiments, wherein the microbial film comprises mold.

[0118] Embodiment 21: The method according to any one of the preceding embodiments, wherein the microbial film comprises bacteria.

[0119] Embodiment 22: The method according to any one of the preceding embodiments, wherein the microbial film comprises viruses.

[0120] Embodiment 23: The method according to any one of the preceding embodiments, wherein the microbial film comprises cells.

[0121] Embodiment 24: The method according to any one of the preceding embodiments, wherein the viscous liquid comprises a pharmaceutically active compound.

[0122] Embodiment 25: A method for evaluating the resistance of a pigment or a coating to mold or fungal growth, the method comprising: a) preparing a microbially treated carrier; b) placing the microbially treated carrier in a platform comprising one or more holes; c) forming a uniform film of the pigment or the coating on the microbially treated carrier using a doctor blade and the platform comprising a plurality of holes to expose the microbially treated coated carrier to the pigment or the coating; d) lifting the film-coated carrier from the one or more holes using a lifting tool; e) removing the film-coated carrier from the one or more holes using a removal tool; f) incubating the film-coated carrier in a culture medium at a temperature higher than the ambient temperature for a predetermined incubation time; g) neutralizing the culture medium with a neutralizing reagent solution; and h) determining the viability of the microorganisms in the neutralizing reagent solution by qualitative measurement of the turbidity of the neutralizing solution to evaluate the resistance of the pigment or the coating to mold or fungal growth.

[0123] Embodiment 26: A system for preparing a uniform film on a carrier, the system comprising: a platform comprising one or more holes for receiving the carrier; a lifting tool for lifting the microbially coated carrier from the holes of the platform; and a removal tool for removing the carrier from the platform.

[0124] Embodiment 27: The system according to Embodiment 26, wherein the holes comprise orifices for receiving the lifting tool.

[0125] Embodiment 28: The system according to Embodiment 27, wherein the removal tool comprises a fork head for removing the carrier from the holes of the platform.

[0126] Embodiment 29: A test kit for evaluating the resistance of a pigment or a coating to the growth of mould or fungi, the test kit comprising a carrier; a platform comprising one or more wells; a lifting tool for lifting one or more microorganism-coated carriers from the wells of the platform; and a removal tool for removing the microorganism-coated carriers from the multi-well platform.

[0127] Embodiment 30: The test kit according to Embodiment 29, further comprising a microorganism and a growth medium reagent.

[0128] Embodiment 31: The test kit according to any one of Embodiments 28 and 29, further comprising a spreading rod.

[0129] Embodiment 32: The test kit according to any one of claims 28 to 30, further comprising a neutralizing reagent.

[0130] Embodiment 33: The test kit according to any one of claims 28 to 31, further comprising an incubation and neutralization container.

[0131] Embodiment 34: The test kit according to any one of claims 28 to 32, wherein the microorganism is a mould or a fungal spore.

[0132] Embodiment 35: A method for evaluating the resistance of a pigment sample to the growth of mould or fungi, the method comprising using the test kit according to any one of the preceding claims.

[0133] Embodiment 36: A method for evaluating the microbicidal property of a pigment sample, the method comprising using the test kit according to any one of the preceding claims.

[0134] Exemplary embodiments have been described above. It will be apparent to those skilled in the art that, without departing from the general scope of the present disclosure, the above-described compositions and methods may include variations and modifications. The general scope of the present disclosure is intended to cover all such variations and alterations.

Claims

1. A method for evaluating the microbicidal characteristics of a viscous liquid, the method comprising a. forming a microbial film on one or more carriers; b. exposing the microbially treated carriers to a uniform film of the viscous liquid while fixing the carriers on a platform comprising one or more holes; c. using a lifting tool to lift the film-coated carriers out of the one or more holes of the platform to lift the carriers out of the one or more holes; d. using a removal tool to remove the film-coated carriers from the platform; e. incubating the film-coated carriers in a culture medium; and f. determining the viability of the microorganisms on the microbially treated carriers to evaluate the microbicidal characteristics of the viscous liquid.

2. The method according to claim 1, wherein the uniform film is formed by smearing, roll-coating, brush-coating or scraping the viscous liquid onto the microbially treated carriers.

3. The method according to claim 1, wherein the uniform film is prepared by a scraping process of uniformly applying the viscous liquid onto the microbially treated carriers using a bar.

4. The method according to any one of claims 1 to 3, wherein the platform comprises a single hole.

5. The method according to any one of claims 1 to 3, wherein the platform comprises two or more holes.

6. The method according to any one of claims 1 to 5, wherein at least one carrier is a glass slide.

7. The method according to any one of claims 1 to 5, wherein at least one carrier is metal.

8. The method according to any one of claims 1 to 5, wherein at least one carrier comprises a polymer.

9. The method according to any one of the preceding claims, wherein the carriers are sterile before forming the microbial film.

10. The method according to any one of the preceding claims, wherein the viscous liquid is a pigment or a paint.

11. The method according to any one of the preceding claims, wherein the film-coated carriers are incubated at a temperature higher than the ambient temperature for a predetermined incubation time.

12. The method according to any one of the preceding claims, wherein after the predetermined incubation time, the culture medium is further neutralized with a neutralizing reagent.

13. The method according to any one of the preceding claims, wherein the determination of viability is a qualitative or quantitative measurement.

14. The method according to any one of claims 1 to 12, wherein the determination of viability is a qualitative measurement of turbidity.

15. The method according to any one of claims 1 to 12, wherein the determination of viability is a qualitative measurement of the turbidity of the solution after neutralization.

16. The method according to any one of the preceding claims, wherein the determination step evaluates the microbicidal ability of the viscous liquid against the microbial film.

17. A method for depositing a thin film of a liquid for testing on a treated carrier, the method comprising: a. forming a microbial film on one or more carriers; b. Exposing the carrier treated with the microbial film to a thin film of liquid while fixing the carrier on a platform including one or more pores, and depositing the thin film by doctor - blading the liquid on the platform; c. Using a lifting tool to lift the film - coated carrier from the pores; and d. Using a removal tool to remove the film - coated carrier from the platform.

18. The method according to any one of the preceding claims, wherein the microbial film comprises fungal spores.

19. The method according to any one of the preceding claims, wherein the microbial film comprises molds.

20. The method according to any one of the preceding claims, wherein the microbial film comprises bacteria.

21. The method according to any one of the preceding claims, wherein the microbial film comprises viruses.

22. The method according to any one of the preceding claims, wherein the microbial film comprises cells.

23. The method according to any one of the preceding claims, wherein the viscous liquid comprises a pharmaceutically active compound.

24. A method for evaluating the resistance of a pigment or a coating to the growth of molds or fungi, the method comprising: a. Preparing a microbially - treated carrier; b. Placing the microbially - treated carrier in a platform including one or more pores; c. Using a doctor - blading bar and the platform including a plurality of pores to form a uniform film of the pigment or the coating on the microbially - treated carrier to expose the microbially - treated coated carrier to the pigment or the coating; d. Using a lifting tool to lift the film - coated carrier from the one or more pores; and e. Using a removal tool to remove the film - coated carrier from the one or more pores; f. Incubating the film - coated carrier in a culture medium at a temperature higher than the ambient temperature for a predetermined incubation time; g. Neutralizing the culture medium with a neutralizing reagent solution; and h. Determining the viability of the microorganisms in the neutralizing reagent solution by qualitative measurement of the turbidity of the neutralized solution to evaluate the resistance of the pigment or the coating to the growth of molds or fungi.

25. A system for preparing a uniform film on a carrier, the system comprising: A platform, the platform including one or more pores for accommodating the carrier; A lifting tool, the lifting tool for lifting the microbially - coated carrier from the pores of the platform; and a removal tool, the removal tool for removing the carrier from the platform.

26. The system according to claim 25, wherein the pores include orifices for receiving the lifting tool.

27. The system according to claim 25, wherein the removal tool includes a fork head for removing the carrier from the pores of the platform.

28. A test kit for evaluating the resistance of a pigment or coating to the growth of mold or fungus, the test kit comprising a carrier; a platform, the platform comprising one or more wells; A lifting tool, the lifting tool for lifting one or more microbially - coated carriers from the pores of the platform; and a removal tool, the removal tool for removing the microbially - coated carrier from the porous platform.

29. The test kit according to claim 28, the test kit further comprising microorganisms and growth medium reagents.

30. The test kit according to any one of claims 28 and 29, the test kit further comprising a doctor - blading bar.

31. The test kit according to any one of claims 28 to 30, wherein the test kit further comprises a neutralizing reagent.

32. The test kit according to any one of claims 28 to 31, wherein the test kit further comprises an incubation and neutralization container.

33. The test kit according to any one of claims 28 to 32, wherein the microorganism is a mold or fungal spore.

34. A method for evaluating the resistance of a pigment sample to mold or fungal growth, the method comprising using the test kit according to any one of the preceding claims.

35. A method for evaluating the microbicidal properties of a pigment sample, the method comprising using the test kit according to any one of the preceding claims.

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