A device for growing microorganisms

Abstract

A device for growing microorganisms, the device including: a body member comprising a self-supporting, waterproof substrate having an upper surface and a lower surface; a hydrophobic spacer element adhered to the upper surface of the substrate and forming a sidewall for retaining a predetermined amount of liquid in contact with the substrate, the hydrophobic spacer element having pores therein; a fluid control film within the pores of the hydrophobic spacer element; a cover sheet having an inwardly facing surface and an outwardly facing surface and adhered to at least a portion of the body member; a substantially dry first microbial growth nutrient composition disposed on a portion of the inner surface of the cover sheet; a first adhesive composition adhered to the first microbial growth nutrient composition; and a cold-water soluble first hydrogel-forming composition adhered to the first adhesive composition.

Description

[Background technology]

[0001] A wide variety of culture devices have been developed. As an example, a culture device has been developed by 3M Company (hereinafter "3M") (St. Paul, Minnesota). In particular, a culture device is sold by 3M under the trade name PETRIFILM plates. The culture device can be utilized to facilitate the rapid growth and detection of microorganisms commonly associated with food contamination, including, for example, aerobic bacteria, E. coli., coliforms, enterobacteria, yeast, molds, Staphylococcus aureus, Listeria, Campylobacter, and the like. The use of PETRIFILM plates or other growth media can, for example, facilitate bacterial testing of food samples.

[0002] The culture device can be used to enumerate or identify the presence of bacteria so that corrective action can be taken (in the case of food testing) or an appropriate diagnosis can be made (in the case of medical use). In other applications, the culture device can be used to rapidly grow microorganisms in laboratory samples, for example for experimental purposes. Summary of the Invention

[0003] Devices and methods for the growth or storage of microorganisms are provided.

[0004] Thus, in one aspect, the disclosure provides a device for growing microorganisms, the device including a body member comprising a self-supporting waterproof substrate having an upper surface and a lower surface, a hydrophobic spacer element adhered to the upper surface of the substrate forming a sidewall for retaining a volume of liquid in contact with the substrate, the hydrophobic spacer element having pores therein, a fluid control film within the pores of the hydrophobic spacer element, a cover sheet having an inwardly facing surface and an outwardly facing surface, the cover sheet adhered to at least a portion of the body member, a substantially dry first microbial growth nutrient composition disposed on a portion of the inner surface of the cover sheet, a first adhesive composition adhered to the first microbial growth nutrient composition, and a cold water soluble first hydrogel forming composition adhered to the first adhesive composition.

[0005] In another aspect, the present disclosure provides a method, comprising providing a device according to the present disclosure, adding a volume of a sample containing at least one microorganism to the device to form an inoculated device, contacting a cover sheet with a self-supporting waterproof substrate, incubating the inoculated device, and detecting the presence or absence of colonies of the target microorganism in the device.

[0006] Various aspects and advantages of exemplary embodiments of the present disclosure are summarized. The above summary is not intended to describe each illustrated embodiment or every implementation of the present disclosure. Additional features and advantages are disclosed in the following embodiments. The figures and the detailed description that follow more particularly illustrate specific embodiments that utilize the principles disclosed herein.

[0007] definition For terms defined hereinafter, these definitions shall apply to the entire specification, including the claims, unless a different definition is presented in the claims or elsewhere in the specification based on specific reference to a change in the used terminology in the following definitions.

[0008] The term "about" or "approximately" with reference to a numerical value or shape means + / - 5 percent of the numerical value or property or characteristic, but is understood to also explicitly include any narrower range and exact numerical value within + / - 5 percent of the numerical value or property or characteristic. For example, a temperature of "about" 100°C refers to a temperature between 95°C and 105°C, but also explicitly includes any narrower range of temperatures or even a single temperature within that range, such as exactly 100°C. For example, a viscosity of "about" 1 Pa·sec refers to a viscosity between 0.95 Pa·sec and 1.05 Pa·sec, but also explicitly includes a viscosity of exactly 1 Pa·sec. Similarly, a "substantially square" perimeter is intended to describe a geometry having four lateral edges, each of which has a length between 95% and 105% of the length of any other lateral edge, but is also intended to include a geometry in which each lateral edge has the exact same length.

[0009] The term "substantially" with respect to a property or characteristic means that the property or characteristic is exhibited to a greater extent than the opposite of the property or characteristic is exhibited. For example, a "substantially" transparent substrate refers to a substrate that transmits more radiation (e.g., visible light) than it does not transmit (e.g., absorbs and reflects). Thus, a substrate that transmits more than 50% of the visible light incident on its surface is substantially transparent, while a substrate that transmits 50% or less of the visible light incident on its surface is not substantially transparent.

[0010] The terms "a," "an," and "the" are used interchangeably with "at least one" and mean one or more of the elements being described.

[0011] The term "and / or" means either or both. For example, the phrase "A and / or B" means A, B, or A in combination with B.

[0012] "Cluster" refers to a group of agglomerated and / or aggregated particles.

[0013] "Agglomerated" refers to a weak association of primary particles or aggregated particles that are usually held together by charge or polarity. Agglomerated particles can usually be broken down into smaller entities, for example, by shear forces encountered during dispersion of the agglomerated particles in a liquid. The terms "agglomerated" and "agglomerate" refer to a strong association of primary particles that are often bound together, for example, by chemical residue treatments, chemical covalent bonds, or chemical ionic bonds. Further breaking down of the aggregates into smaller units is very difficult to achieve.

[0014] "Cold water soluble" is a substance that forms an aqueous solution at room temperature (ie, about 25°C).

[0015] "Hydrophobic" refers to a material that exhibits a water contact angle of 90° or greater on its surface.

[0016] "Opaque" refers to a substrate having a maximum light transmission of 10%.

[0017] "Powder" refers to finely divided particles having an average diameter ranging from 0.1 micrometers up to 400 micrometers.

[0018] "Reconstituted medium" refers to the solution or gel formed by reconstitution of a cold water soluble powder with an aqueous liquid.

[0019] "Substantially impermeable to microorganisms and water vapor," as used herein, refers to a cover sheet that prevents undesired contamination and hydration of the underlying layer of cold water soluble powder during transportation, storage, and use of the thin film culture device, as well as avoids drying out of the reconstituted medium so that the reconstituted medium is suitable for supporting microbial growth during the incubation period.

[0020] "Substantially free of water," as used herein, indicates that the water content is about equal to or less than the water content of the surrounding environment.

[0021] "Test sample", as used herein, refers to a component or portion taken from a food product, human or animal test subject, pharmaceutical or cosmetic product, soil, water, air or other environmental source, or any other source in which the presence and, optionally, enumeration of aerobic and / or aerobic bacteria is measured. Test samples can be taken from sources using techniques known to those skilled in the art, including, for example, pouring, pipetting, wiping, filtering, and contacting. In addition, test samples may be subjected to various sample preparation methods known in the art, including, for example, blending, digestion, homogenization, enrichment, selective enrichment, or dilution.

[0022] "Transparent" refers to a substrate that has a light transmittance of at least 90%. [Brief description of the drawings]

[0023] The present disclosure may be more fully understood from the following detailed description of various embodiments of the disclosure when considered in conjunction with the accompanying drawings, in which: [Figure 1] FIG. 2 is a top perspective view, partially in cross section, of yet another exemplary device according to the present disclosure. [Diagram 2] FIG. 2 is a schematic diagram of a channeled microstructured surface of the present disclosure having an amount of fluid thereon.

[0024] While the above-identified drawings, which may not be drawn to scale, reveal various embodiments of the present disclosure, other embodiments are contemplated, as pointed out in the detailed description. In all cases, the present disclosure describes the invention of the present disclosure by way of illustration of exemplary embodiments, and not by way of express limitation. It should be understood that numerous other modifications and embodiments may be devised by those skilled in the art that are within the scope and spirit of the present disclosure. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0025] Before any embodiment of the present disclosure is described in detail, it is understood that the present invention is not limited in its application to the details of use, structure, and composition of matter set forth in the following description. The present invention is capable of other embodiments and can be implemented or carried out in various ways that will become apparent to those skilled in the art upon reading this disclosure. It is also understood that the terminology and terminology used herein is for descriptive purposes and should not be considered limiting. The use of "including," "comprising," or "having" and variations thereof herein means the inclusion of the items listed thereafter and equivalents thereof, as well as additional items. It is understood that other embodiments may be utilized and structural or logical changes may be made without departing from the scope of the present disclosure.

[0026] As used herein, the recitations of numerical ranges by endpoints include all numbers subsumed within that range (eg, 1 to 5 includes 1, 1.5, 2, 2.75, 3, 3.8, 4, and 5, etc.).

[0027] Unless otherwise indicated, all numbers expressing quantities or ingredients, measurements of properties, and the like used in the specification and the embodiments are to be understood as being modified in all instances by the term "about." Accordingly, unless indicated to the contrary, the numerical parameters set forth in the foregoing specification and accompanying listing of embodiments may vary depending upon the desired properties one of ordinary skill in the art would desire to obtain utilizing the teachings of the present disclosure. At the very least, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques, which is not intended to limit the application of the doctrine of equivalents to the scope of the claimed embodiments.

[0028] FIG. 1 shows an exemplary embodiment of a device for growing microorganisms. The device 10 includes a body member 11 comprising a substrate 12 having a first major surface 12a (e.g., an upper surface) and a second major surface 12b (e.g., a lower surface), and a cover sheet 22 attached to at least a portion of the body member 11, the cover sheet 22 comprising a first major surface 22a (e.g., an inner surface) facing the body member 11. The device 10 further includes a substantially dry first microbial growth nutrient composition 24 disposed on a portion of the first major surface 22a of the cover sheet 22, a first adhesive composition 26 adhered to the first microbial growth nutrient composition 24, and a cold water soluble first hydrogel forming composition 28 adhered to the first adhesive composition 26. Preferably, the device also includes an optional hydrophobic spacer element 19 disposed on the first major surface 12a of the substrate 12. Generally, the spacer element 19 comprises a water-insoluble substrate defining holes or openings 20. The spacer element 19 may be a hydrophobic foam sheet, such as a polystyrene foam sheet or a polyethylene foam sheet. In use, the user sufficiently separates the cover sheet 22 from the substrate 12, adds an amount of sample containing at least one microorganism within the holes or openings 20 defined by the spacer 19, places the cover sheet 22 back in contact with the substrate 12 to form an inoculated device, and incubates the inoculated device. The area on the first major surface 12a of the substrate 12 defined by the openings 20 may also be referred to as the sample receiving zone 17. A fluid control film 18 may be disposed within the holes of the hydrophobic spacer element 19 and on the first major surface 12a of the substrate 12. The device 10 further includes a second adhesive composition 13 adhered to the top surface 12a of the self-supporting waterproof substrate 12, the second adhesive composition 13 being between the hydrophobic spacer element 19 and the substrate 12.

[0029] The aperture 20 can be any shape. Non-limiting examples of useful shapes for the aperture 20 include square, rectangular, circular, oval, polygonal, hexagonal, and octagonal. The area of ​​the sample receiving zone (and aperture 20) can be selected based on, for example, the volume of sample (e.g., aqueous liquid) to be injected into the zone. In any embodiment, for a sample of 0.5 milliliters to 3 milliliters, the area of ​​the sample receiving zone is about 10 cm. 2 Or about 15cm 2 In any embodiment, for a sample volume between 1 milliliter and 5 milliliters, the area of ​​the sample receiving zone is about 20 cm 2 , about 25cm 2 , about 30cm 2 , about 31cm 2 , or about 25 cm 2 ~35cm 2 It is.

[0030] Substrate 12 is waterproof, and optionally is a self-supporting waterproof substrate. In some embodiments, substrate 12 is a film of a material such as polyester, polypropylene, silicone, or polystyrene that does not adsorb or otherwise be affected by water. Polyester and polypropylene films having a thickness of about 20 micrometers to about 250 micrometers, and polystyrene films having a thickness of about 380 micrometers have each been found to be suitable for substrate 12. Other suitable substrates include paper with a polyethylene or other waterproof coating. An example of a suitable polyethylene-coated paper substrate is "Schoeller Type MIL" photographic paper (commercially available from Schoeller Pulaski, New York). Substrate 12 can be either transparent or opaque, depending on whether a user desires to view the bacterial colonies through the substrate. In some embodiments, substrate 12 has a square grid pattern printed on second major surface 12b to facilitate counting of bacterial colonies.

[0031] The substantially dry first or second microbial growth nutrient composition has a density of 2 milligrams per square inch (mg / in 2 ) or more, 5mg / in 2 More than 10mg / in 2 Above 12 mg / in 2 or more than 15 mg / in 2 At coating weights of 50 mg / in or more 2 Below, 45mg / in 2 Below, 40mg / in 2 Below, 35mg / in 2 Below, 30mg / in 2 Below, 24mg / in 2 Below, 22mg / in 2 Below, 20mg / in 2 or less than 18 mg / in 2 The following coating weights of the microbial growth nutrient composition may be included. One suitable method for applying the microbial growth nutrient composition onto a substrate includes preparing an aqueous solution or suspension including at least the microbial growth nutrient composition, disposing a coating of the solution or suspension on the substrate surface, and drying the coating to form a substantially dry microbial growth nutrient composition. Those skilled in the art can select suitable coating methods including, for example, but not limited to, knife coating, gravure coating, curtain coating, air knife coating, spray coating, die coating, draw bar coating, or curtain coating or roll coating. The coating is optionally dried at elevated temperatures (e.g., in the range of 50°C to 100°C) or at ambient conditions. In some embodiments, the microbial growth nutrient composition contains 75% or more by weight of microbial growth nutrients, or 80% or more by weight, or 85% or more by weight, or 90% or more by weight, or 95% or more by weight of microbial growth nutrients. Advantageously, in certain embodiments, the device can include a greater amount of microbial growth nutrients than in devices where the microbial growth nutrient composition is powder coated onto an adhesive layer and / or combined with a significant amount of a cold water soluble gelling agent.

[0032] The first or second adhesive composition is (substantially) water insoluble and does not inhibit microbial growth. In some embodiments, the first adhesive composition 16 is sufficiently transparent when wet that bacterial colonies can be seen through the adhesive coated film. In some embodiments, the first or second adhesive composition is a pressure sensitive adhesive. In some other embodiments, a heat activated adhesive may be used in which a lower melting material is coated onto a higher melting substrate. Water activated adhesives such as rubber cement may also be useful.

[0033] A suitable adhesive is transparent when wetted with water. As mentioned above, the adhesive composition is often water-insoluble. In certain embodiments, the adhesive composition comprises a solvent-based adhesive. The first adhesive composition and, if present, the second adhesive composition are often pressure-sensitive adhesives. For example, the adhesive can be a pressure-sensitive adhesive, such as a water-insoluble adhesive comprising a copolymer of an alkyl acrylate monomer and an alkyl amide monomer, or a copolymer of an alkyl acrylate monomer and an acrylic acid. Preferably, the weight ratio of alkyl acrylate monomer to alkyl amide monomer in these copolymers is about 90:10 to 99:1, more preferably 94:6 to 98:2. Alkyl acrylate monomers include lower alkyl (C2-C10) monomers of acrylic acid, including, for example, isooctyl acrylate (IOA), 2-ethylhexyl acrylate, butyl acrylate, ethyl acrylate, isoamyl acrylate, and mixtures thereof, and alkylamide monomers include, but are not limited to, acrylamide (ACM), methacrylamide, N-vinylpyrrolidone (NVP), N-vinylcaprolactam (NVCL), N-vinyl-2-piperidine, N-(mono- or di-lower alkyl (C2-C5))(meth)acrylamide, N-methyl(meth)acrylamide, N,N-dimethyl(meth)acrylamide, or mixtures thereof. Suitable adhesives include those described in U.S. Patent Nos. 4,565,783, 5,089,413, 5,681,712, and 5,232,838. In some embodiments, silicone pressure sensitive adhesives may be used, such as those described in US Pat. Nos. 7,695,818 and 7,371,464.

[0034] In the present disclosure, the cover sheet 22 is generally selected to be transparent to facilitate counting of microbial colonies, and is typically also selected to be impermeable to bacteria and to have a low water vapor transmission rate (i.e., the cover sheet 22 prevents undesired contamination of the dehydration medium during shipping, storage, and use of the device, and provides an environment that supports microbial growth during the incubation period). In some embodiments, the cover sheet 22 has the same properties as the substrate 12 (e.g., waterproof). The cover sheet 22 can be selected to provide the oxygen transmission rate required for the type of microorganism desired to grow. For example, some polyester films have low oxygen permeability (5 g / 645 cm per 25 micrometers of thickness). 2 / 24 hours) and is suitable for the growth of anaerobic bacteria. On the other hand, some polyethylenes have high oxygen permeability (e.g., about 500 g / 645 cm per 25 micrometers of thickness). 2 / 24 hours), suitable for aerobic organisms. Suitable materials for the cover sheet 22 include polypropylene, polyester, polyethylene, polystyrene, or silicone. In certain embodiments, the cover sheet 22 comprises oriented polypropylene, such as biaxially oriented polypropylene, which in some exemplary embodiments has a thickness of about 40 micrometers.

[0035] In certain embodiments, the cold water soluble hydrogel-forming composition contains one or more organic cold water soluble agents, such as alginate, carboxymethylcellulose, tara gum, hydroxyethylcellulose, hydroxypropylmethylcellulose, guar gum, locust bean gum, xanthan gum, polyacrylamide, polyurethane, polyethylene oxide, etc. Combinations of natural and / or synthetic gelling agents are contemplated. Preferred gelling agents include guar gum, xanthan gum, and locust bean gum, which are useful individually or in any embodiment in combination with each other. It is desirable for a uniform layer of the cold water soluble hydrogel-forming composition to have sufficient exposed surface area for hydration. In any embodiment, the cold water soluble first and / or second hydrogel-forming compositions comprise a mixture of gelling agents. Optionally, the powdered cold water soluble hydrogel-forming composition may further comprise an inducer, an indicator, or a combination thereof.

[0036] Fluid control films can include those described in U.S. Patent Application No. 2017 / 0045284(A1) (Meuler et al.).

[0037] For example, the fluid control film includes fluid control channels extending along a channel longitudinal axis. Each of the fluid control channels has a surface and is configured to allow capillary movement of liquid within the channel. In some embodiments, the fluid control film can further include a hydrophilic surface treatment covalently bonded to at least a portion of the surface of the fluid control channels. In some other embodiments, the fluid control film can have a non-covalently bonded hydrophilic surface treatment, e.g., a surfactant treatment, disposed on at least a portion of the surface of the fluid control channels. The fluid control film exhibits a capillary rise recovery of at least 10%. Typically, the hydrophilic surface treatment includes a functional group selected from a non-zwitterionic sulfonate, a non-zwitterionic carboxylate, a zwitterionic sulfonate, a zwitterionic carboxylate, a zwitterionic phosphate, a zwitterionic phosphonic acid, a zwitterionic phosphonate, or a combination thereof.

[0038] The fluid control film according to the present disclosure includes a microstructured surface having a plurality of microreplicated structures. The fluid control film may have a variety of topographies. An exemplary fluid control film is composed of structures having a plurality of channels with V-shaped or rectangular cross sections, and combinations thereof, as well as secondary channels, i.e., channels within channels. In addition, the topography may include microstructured posts and protrusions.

[0039] The channels on the microstructured surface have channel ends. In certain embodiments, the fluid control film may include a removal means. The removal means generally draws fluid from the channels adjacent one of the channel ends. In another embodiment, the removal means draws fluid from the channels adjacent both channel ends. The removal means may include an absorbent material disposed in communication with the channels. In one embodiment, the removal means includes a fluid drip collector.

[0040] Generally, the channels in the microstructure are defined by generally parallel ridges including a first set of ridges having a first height and a second set of ridges having a second, higher height. An upper portion of each ridge in the second set of ridges may have a lower melting temperature than a lower portion. The channels have a pattern shape selected from the group consisting of straight, curvilinear, radial, parallel, non-parallel, random, or crossing.

[0041] In some embodiments, the fluid control film has a contact angle of less than 90 degrees. The contact angle theta (θ) is the angle between a line tangent to the surface of a bead of fluid on a surface at its point of contact with the surface and the plane of the surface. A bead of fluid with a tangent perpendicular to the plane of the surface will have a contact angle of 90 degrees. Typically, a solid surface is considered wetted by a fluid if the contact angle is 45 degrees or less. Surfaces on which a drop of water or an aqueous solution exhibits a contact angle of less than 45 degrees are generally referred to as "hydrophilic." As used herein, "hydrophilic" refers only to the surface properties of a material, i.e., that it is wetted by aqueous solutions, and does not describe whether the material absorbs or does not absorb aqueous solutions. Thus, a material can be referred to as hydrophilic regardless of whether a sheet of material is impermeable or permeable to aqueous solutions. Thus, hydrophilic films as used in this application can be formed from films prepared from resin materials that are inherently hydrophilic, such as poly(vinyl alcohol). A fluid that produces a contact angle of nearly zero on a surface is considered to have completely wetted out the surface. However, polyolefins are typically hydrophobic in nature, and the contact angle with water of polyolefin films such as polyethylene or polypropylene is typically greater than 90 degrees.

[0042] Generally, the channels in the microstructure are defined by generally parallel ridges including a first set of ridges having a first height and a second set of ridges having a second, higher height. An upper portion of each ridge in the second set of ridges may have a lower melting temperature than a lower portion. The channels have a pattern shape selected from the group consisting of straight, curvilinear, radial, parallel, non-parallel, random, or crossing.

[0043] 2 is a cross-sectional view of a fluid control film 200, according to an exemplary embodiment. The fluid control film 200 comprises a fluid control film layer 201 having primary and secondary channels 230, 231 defined by primary and secondary ridges 220, 221, the channels 230, 231 and ridges 220, 221 running along a channel axis that is at an angle θ with respect to a longitudinal axis of the fluid control film layer 201, e.g., the x-axis. Each primary channel 230 is defined by a pair of (first and second) primary ridges 220 on either side of the primary channel 230. The primary ridges 220 have a height h 2 , measured from a bottom surface 230a of the channel 230 to a top surface 220a of the ridge 220. p In some embodiments, the microstructures are disposed within the primary channel 230. In some embodiments, the microstructures comprise secondary channels 231 disposed between the first and second primary ridges 220 of the primary channel 230. Each secondary channel 231 is associated with at least one secondary ridge 221. The secondary channel 231 may be located between a pair of secondary ridges 221 or between a secondary ridge 221 and a primary ridge 220.

[0044] Center-to-center distance between primary ridges d pr may range from about 25 micrometers to about 3000 micrometers, and the center-to-center distance d between the primary ridge and the nearest secondary ridge ps may range from about 5 micrometers to about 350 micrometers, and the center-to-center distance d between the two secondary ridges may range from about 5 micrometers to about 350 micrometers. ss may range from about 5 micrometers to about 350 micrometers. In some cases, the primary and / or secondary ridges may taper with distance from the base. The distance d between the outer surfaces of the primary ridges at the base pb may range from about 15 micrometers to about 250 micrometers, with a smaller distance d ranging from about 1 micrometer to about 25 micrometers. pt The distance d between the outer surfaces of the secondary ridges at their bases sbmay range from about 15 micrometers to about 250 micrometers, with a smaller distance d ranging from about 1 micrometer to about 25 micrometers. st In one example, the taper may be d pr = 0.00898 inches (228 micrometers), d ps = 0.00264 inches (67 micrometers), d ss = 0.00185 inches (47 micrometers), d pb = 0.00251 inches (64 micrometers), d pt = 0.00100 inches (25 micrometers), d sb = 0.00131 inches (33 micrometers), d st = 0.00100 inches (25 micrometers), h p = 0.00784 inches (199 micrometers), and h s =0.00160 inches (41 micrometers).

[0045] The secondary ridge has a height h measured from the bottom surface 230a of the channel 230 to the top surface 221a of the secondary ridge 221. s The height of the primary ridge 220 is h p is the height h of the secondary ridge 221 s In some embodiments, the height of the primary ridges is between about 25 micrometers and about 3000 micrometers, and the height of the secondary ridges is between about 5 micrometers and about 350 micrometers. In some embodiments, the height h of the secondary ridges 221 is between about 25 micrometers and about 3000 micrometers. s and the height h of the primary ridge 220 p The ratio of is about 1:5. The primary ridges 220 may be designed to provide durability to the fluid control film 200 and protection to the secondary channels 231, secondary ridges, and / or other microstructures disposed between the primary ridges 220.

[0046] The fluid control film 200 optionally has an adhesive layer 205 disposed on the bottom surface 201a of the fluid control film layer 201. The adhesive layer 205 may enable attachment of the fluid control film 200 to a portion of the exterior surface 202 to aid in managing liquid distribution across the exterior surface. The combination of the adhesive layer 205 and the fluid control film layer 201 forms a fluid control tape. The adhesive layer 205 may be continuous or non-continuous.

[0047] The fluid control film layer 201 is configured to distribute fluid across a surface of the fluid control film layer 201 to promote evaporation of the fluid. In some embodiments, the adhesive layer 205 may be or include a hydrophobic material that repels liquid at the interface 202a between the adhesive layer 205 and the exterior surface 202, reducing collection of liquid at the interface 202a.

[0048] The adhesive layer 205 has a thickness t a and the fluid control film layer 201 has a thickness t v In some embodiments, the total thickness between the bottom surface 230a of the channels 230, 231 and the bottom surface 205a of the adhesive layer 205, t v +t a The total thickness t may be less than about 300 micrometers, for example, about 225 micrometers. v +t a may be selected to be small enough to efficiently wick liquid from the exterior surface 202 through the channel openings at the edges of the fluid control film layer 201 and into the channels 230 , 231 .

[0049] A method for detecting and enumerating at least one microorganism in a sample is provided. The method includes providing a device according to the present disclosure, adding a volume of a sample containing at least one microorganism to the opening 20 of the spacer element 19 to form an inoculated device, contacting a cover sheet with a substrate, incubating the inoculated device, and detecting the presence or absence of colonies of the target microorganism in the device. The cold water soluble hydrogel-forming composition on the cover sheet is hydrated to form a hydrogel when an aqueous sample is placed in the device, and the hydrogel can self-spread to fill the channels of the flow control film. It has been unexpectedly discovered that the colonies form such punctate colonies and do not grow along the longitudinal axis of the channels of the flow control film.

[0050] The method further includes incubating the device for a period of time at a temperature that promotes growth and detection of the target microorganism. One skilled in the art will recognize that the temperature and duration of incubation will depend on a number of factors (e.g., the target microorganism, nutrients present in the sample, nutrients present in the device, inhibitors present in the sample and / or device) and will adjust the incubation time and temperature accordingly.

[0051] The method further includes detecting the presence or absence of a colony of the target microorganism in the device. In any embodiment, detecting the presence or absence of a colony of the target microorganism in the device may include detecting a colony in a first compartment of the device (e.g., visually or using machine vision). In any embodiment, detecting the presence or absence of a colony of the target microorganism in the device may include detecting a change associated with an indicator reagent. The indicator reagent may change from a first state (e.g., substantially colorless or non-fluorescent) to a second state (e.g., colored or fluorescent) in and / or around the colony of the target microorganism. In any embodiment, the colonies may be counted and, optionally, the number of colonies of the target microorganism may be recorded. In some embodiments, the microorganisms may be counted using an automated system, such as an automated colony counter.

[0052] The following embodiments are intended to illustrate, but not limit, the present disclosure.

[0053] Embodiment Embodiment 1 is a device for growing microorganisms comprising: a body member comprising a self-supporting waterproof substrate having an upper surface and a lower surface; a hydrophobic spacer element adhered to the upper surface of the substrate forming a sidewall for retaining a predetermined amount of liquid in contact with the substrate, the hydrophobic spacer element having pores therein; a fluid control film within the pores of the hydrophobic spacer element; a cover sheet having an inwardly facing surface and an outwardly facing surface, the cover sheet adhered to at least a portion of the body member; a substantially dry first microbial growth nutrient composition disposed on a portion of the inner surface of the cover sheet; a first adhesive composition adhered to the first microbial growth nutrient composition; and a cold water soluble first hydrogel forming composition adhered to the first adhesive composition.

[0054] Embodiment 2 is a device of embodiment 1, wherein the fluid control film comprises a plurality of high-definition structures.

[0055] Embodiment 3 is a device described in embodiment 1 or 2, wherein the fluid control film has a plurality of fluid control channels extending along the channel longitudinal axis, each of the fluid control channels having a surface and configured to enable capillary movement of liquid within the channel.

[0056] Example 4 is a device according to any one of Examples 1-3, wherein the fluid control film comprises a hydrophilic surface treatment covalently bonded to at least a portion of the surface of the fluid control channel.

[0057] Example 5 is a device according to any one of Examples 1 to 4, wherein the fluid control film comprises a non-covalent hydrophilic surface treatment disposed on at least a portion of the surface of the fluid control channel.

[0058] Embodiment 6 is a device according to any one of embodiments 1 to 5, wherein the fluid control film has a contact angle of less than 90 degrees.

[0059] Embodiment 7 is a device described in any one of embodiments 1 to 6, further comprising a second adhesive composition adhered to the top surface of the self-supporting waterproof substrate, the second adhesive composition being between the hydrophobic spacer element and the substrate.

[0060] Example 8 is a device according to any one of Examples 1 to 7, wherein the spacer element comprises a hydrophobic foam sheet.

[0061] Embodiment 9 is the device of embodiment 8, wherein the hydrophobic foam is a polystyrene foam or a polyethylene foam.

[0062] Embodiment 10 is the device of any one of embodiments 1-9, wherein the cover sheet comprises a transparent film.

[0063] Embodiment 11 is the device of embodiment 10, wherein the film is selected from the group consisting of polyester, polyethylene, polypropylene, polystyrene, and silicone.

[0064] Embodiment 12 is the device according to any one of embodiments 1 to 11, wherein the substrate is a film selected from the group consisting of polyester, polypropylene, polyethylene, and polystyrene.

[0065] Embodiment 13 is a device according to any one of embodiments 1 to 12, wherein the gelling agent is selected from the group consisting of xanthan gum, guar gum, locust bean gum, carboxymethylcellulose, hydroxyethylcellulose, and algin.

[0066] Embodiment 14 is a method comprising providing a device according to any one of embodiments 1 to 13, adding a volume of a sample containing at least one microorganism to the device to form an inoculated device, contacting a cover sheet with a self-supporting waterproof substrate, incubating the inoculated device, and detecting the presence or absence of colonies of the target microorganism in the device.

[0067] The following examples are intended to illustrate, but not limit, the disclosure. EXAMPLES

[0068] [Table 1]

[0069] Incubation and inoculation The bacterial strain Escherichia coli (ATCC25922) was obtained from Microbiologics Incorporated (St. Cloud, MN) and incubated overnight in tryptic soy broth (TSB) at 37°C and 200 rpm in an INNOVA44 incubator (New Brunswick Scientific, Enfield, CT). Inocula were prepared by serially diluting each culture sample in Butterfield buffer (3M Corporation, St. Paul, MN). Culture samples were diluted to obtain a final concentration of approximately 50 to 250 colony forming units (cfu) per mL of inoculum.

[0070] Preparation Example 1. Production of fluid control film The fluid control film of Figure 2 was prepared according to the extrusion embossing procedure described in U.S. Patent Application Publication No. 20017 / 0045284 (Meuler), which is incorporated herein by reference in its entirety. Using the indicators from Figure 2, the example fluid control film had the following diameters: dpr = 0.00898 inches (228 microns), dps = 0.00264 inches (67 microns), dss = 0.00185 inches (47 microns), dpb = 0.00251 inches (64 microns), dpt = 0.00100 inches (25 microns), dsb = 0.00131 inches (33 microns), dst = 0.00100 inches (25 microns), hp = 0.00784 inches (199 microns), hs = 0.00160 inches (41 microns). The films were made from a low density polyethylene polymer (obtained under the trade designation "DOW LDPE9551" from Dow Chemical Company, Midland, Mich.).

[0071] Preparation Example 2. Plasma treatment of fluid control film A silicon-containing film layer (formation method described in US Pat. Nos. 6,696,157 (David) and 8,664,323 (Iyer) and US Patent Application Publication No. 2013 / 0229378 (Iyer)) was applied to the fluid control film of Preparation Example 1 using a Plasma-Therm 3032 batch plasma reactor (obtained from Plasma-Therm LLC, St. Petersburg, FL). The tool was configured for reactive ion etching with a 26-inch low-power electrode and central gas pumping. The chamber was pumped with a root-type blower (model EH1200 obtained from Edwards Engineering, Burgess Hill, UK) backed by a dry mechanical pump (model iQDP80 obtained from Edwards Engineering). RF power was provided by a 3 kW, 13.56 Mhz solid-state generator (RFPP model RF30S obtained from Advanced Energy Industries, Fort Collins, CO). The system had a nominal base pressure of 5 mTorr. Gas flow rates were controlled by MKS flow regulators (obtained from MKS Instruments, Andover, Mass.).

[0072] The fluid control film samples were fixed onto the powered electrodes of a plasma reactor. After pumping down to base pressure, the gases tetramethylsilane (TMS) and oxygen (O2) were introduced at various flow rates (see Table 2). Once the gas flows in the reactor were stabilized, rf power (1000 watts) was applied to the electrodes to generate the plasma. The plasma exposure time was also varied (see Table 2). After completion of the plasma treatment, the chamber was vented to atmosphere and the treated fluid control film was removed from the chamber.

[0073] [Table 2]

[0074] Preparation Example 3. Fluid control film containing surfactant A fluid control film was prepared as described in Preparative Example 1, except that 0.5 wt % of the non-ionic surfactant TRITON-X100 was incorporated into the low density polyethylene polymer used in the extrusion embossing process. The resulting fluid control film was designated as Fluid Control Film E.

[0075] Example 1. Microorganism detection device Microbial detection devices according to the device of FIG. 1 were fabricated. For each device, the substrate for the body member was a clear biaxially oriented polypropylene (BOPP) film (1.6 mil (0.04 mm) thick and corona treated on both sides) cut into a 76 mm wide by 102 mm long piece. The body member was completed by adhesively laminating a 76 mm wide by 102 mm long polyethylene film spacer (Optimum Plastics, Bloomer, WI) to one side of the substrate. The spacer was approximately 20 mil (0.51 mm) thick with a circular hole (5.1 cm diameter) positioned near the center of the spacer. The circular hole defined the periphery of the sample receiving zone of the device. A circular section of fluid control film (selected from the fluid control films labeled A through E in Table 2) was cut and sized to fit the hole (5.1 cm diameter) and oriented such that the non-fine-grained surface of the film was adhesively laminated to the exposed substrate surface defined by the hole.

[0076] The cover sheet of the device was a clear biaxially oriented polypropylene (BOPP) film (1.6 mil (0.04 mm) thick and corona treated on both sides) that was sequentially coated on one side with a microbial growth nutrient composition, an adhesive composition, and a guar gum (e.g., cold water soluble hydrogel forming) composition by the following procedure.

[0077] A microbial growth nutrient coating composition was prepared by vigorously mixing (using an air-driven overhead mixer with a JIFFY-type mixing impeller) 30 g of tryptic soy broth (TSB) and 500 mL of purified water (MILLI-Q Gradient Water Purification System, model number: ZMQS6V00Y, obtained from Merck Millipore Corporation, Billerica, MA) until the TSB was completely dissolved. The resulting solution had a pH of 7.3 (Mettler-Toledo FE20 FIVEEASY pH meter, Mettler-Toledo LLC, Columbus, OH). Guar gum (10 g) was added to the nutrient solution and vigorous mixing was continued for approximately 10 minutes. The resulting solution was knife coated onto one side of a BOPP cover sheet film with a gap setting of 14 mil (0.35 mm). The nutrient-coated film was dried in an oven at 85° C. for 12 minutes to yield a nutrient content of approximately 360 mg / 24 in 2 (2.3 mg / cm 2 ) dry coating weight was obtained.

[0078] A pressure sensitive adhesive (PSA) coating formulation of isooctyl acrylate / acrylic acid (98 / 2 weight ratio) containing TTC (2,3,5-triphenyltetrazolium chloride) indicator as described in Example 4 of U.S. Patent No. 5,409,838 (incorporated herein by reference) was knife coated onto the exposed nutrient coating at a gap setting of 2 mil (0.05 mm). The resulting coated film was dried in a 65° C. oven for 6 minutes to yield a mass of approximately 180 mg / 24 in 2 (1.15 mg / cm 2 A PSA coating with a dry coating weight of about 400 mg / 24 in was then obtained. The adhesive coated side of the cover sheet film was then powder coated with guar gum. The powder was applied evenly and excess powder was removed from the adhesive layer by shaking the film by hand, followed by lightly polishing the surface with a paper towel. The final coating weight of guar gum was about 400 mg / 24 in 2(2.6 mg / cm 2 ) was.

[0079] The coated cover sheet film was then cut to match the dimensions of the body member. The finished devices were assembled by attaching the cover sheet to the body member (in a hinge fashion) along one edge of the spacer (the 76 mm edge) using double-sided adhesive tape. For each device, the cover sheet and body member were oriented so that the coated surface of the cover sheet faced the spacer side of the body member.

[0080] The completed detection devices were inoculated with E. coli inoculum. The cover sheet of the device was lifted and 1 mL of the inoculum (i.e., the final dilution above) was added by pipette across the fluid control film, thereby filling the channels with liquid. The cover sheet was gently replaced in its original position. All devices showed self-diffusion of water within the channels such that the guar gum was uniformly wetted and formed a hydrogel that filled the channels of the fluid control film. The devices were incubated at 37° C. for 24 hours. At the end of the incubation period, red-stained colonies were counted by visual inspection. For all devices, dot-like colonies were observed, distributed across the surface of the hydrogel. The results are reported in Table 3.

[0081] [Table 3]

[0082] All references and publications cited herein are expressly incorporated by reference in their entirety into this disclosure. Exemplary embodiments of the invention have been discussed and reference has been made to possible variations within the scope of the invention. For example, a feature described in the context of one exemplary embodiment may be used in the context of other embodiments of the invention. These and other variations and modifications of the invention will be apparent to those skilled in the art without departing from the scope of the invention, and it should be understood that the invention is not limited to the exemplary embodiments described herein. Therefore, the present invention should be limited only by the claims provided below and their equivalents.

Claims

1. 1. A device for growing microorganisms, comprising: a body member comprising a self-supporting waterproof substrate having an upper surface and a lower surface; a fluid control film overlying an upper surface of the self-supporting waterproof substrate; a cover sheet having an inwardly facing surface and an outwardly facing surface, the cover sheet being adhered to at least a portion of the body member; a first microbial growth nutrient composition substantially free of water disposed on a portion of an inner surface of the cover sheet; a first adhesive composition adhered to the first microbial growth nutrient composition; a cold water soluble first hydrogel-forming composition adhered to the first adhesive composition; A device comprising:

2. The device of claim 1 , wherein the fluid control film comprises a plurality of high definition structures.

3. 3. The device of claim 1 or 2, wherein the fluid control film comprises a plurality of fluid control channels extending along a longitudinal channel axis, each of the fluid control channels having a surface configured to allow capillary movement of liquid within the channel.

4. The device of claim 3 , wherein the fluid control film comprises a hydrophilic surface treatment covalently bonded to at least a portion of a surface of the fluid control channel.

5. 5. The device of claim 3 or 4, wherein the fluid control film comprises a non-covalent hydrophilic surface treatment disposed on at least a portion of the surface of the fluid control channel.

6. The device of any one of claims 1 to 5, wherein the fluid control film has a contact angle of less than 90 degrees.

7. The device of any one of claims 1 to 6, further comprising a second adhesive composition adhered to an upper surface of the self-supporting waterproof substrate, the second adhesive composition being between a hydrophobic spacer element and the substrate.

8. The device of claim 7 , wherein the spacer elements comprise hydrophobic foam sheets.

9. The device of claim 8 , wherein the hydrophobic foam is a polystyrene foam or a polyethylene foam.

10. The device of any one of claims 1 to 9, wherein the cover sheet comprises a transparent film.

11. The device of claim 10 , wherein the film is selected from the group consisting of polyester, polyethylene, polypropylene, polystyrene, and silicone.

12. The device according to any one of claims 1 to 11, wherein the substrate is a film selected from the group consisting of polyester, polypropylene, polyethylene and polystyrene.

13. 13. The device of any one of claims 1 to 12, wherein the cold water soluble first hydrogel-forming composition is selected from the group consisting of xanthan gum, guar gum, locust bean gum, carboxymethylcellulose, hydroxyethylcellulose, and alginate.

14. The device described in claim 7, wherein the hydrophobic spacer element is adhered to an upper surface of the substrate to form a sidewall for retaining a predetermined amount of liquid in contact with the substrate, the hydrophobic spacer element having holes therein, and the fluid control film is within the holes of the hydrophobic spacer element.

15. Providing a device according to any one of claims 1 to 14; adding a volume of a sample containing at least one microorganism to the device to form an inoculated device; contacting the cover sheet with the self-supporting waterproof substrate; incubating the inoculated device; and detecting the presence or absence of colonies of a target microorganism in the device; A method comprising:

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