Field

The disclosure relates to devices useful for the growing and detection of microorg including microbial growth nutrients. This disclosure also relates to methods of detecting enumerative microorganisms using the devices.

Background

A wide variety of culture devices have been developed. As one example, culture devices have been developed by 3M Company (hereafter "3M") of St. Paul, Minnesota. In particular, culture devices are sold by 3M under the trade name PETRIFILM plates. Culture devices 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, mold, Staphylococcus aureus, Listeria, Campylobacter, and the like. The use of

PETRIFILM plates, or other growth media, can simplify bacterial testing of food samples, for instance.

Culture devices can be used to enumerate or identify the presence of bacteria so that corrective measures can be performed (in the case of food testing) or proper diagnosis can be made (in the case of medical use). In other applications, culture devices may be used to rapidly grow microorganisms in laboratory samples, e.g., for experimental purposes.

Summary

Devices and methods for the propagation or storage of microorganisms are provided. In a first aspect, a device is provided. More particularly, a device is provided including a body member including a substrate having a first major surface and a second major surface. The device further includes a first adhesive composition adhered to a portion of the first major surface. A number of particles of a substantially dry, first microbial growth nutrient composition are distributed in the first adhesive composition, and a cold-water-soluble first hydrogel-forming composition is adhered to the first adhesive composition. The device also includes a cover sheet attached to the body member, where the cover sheet includes a first major surface facing the body member.

In a second aspect, another microbial detection device is provided. The microbial detection device includes a water-proof pouch including a first wall portion having an inner surface and an outer surface, a second wall portion having an inner surface and an outer surface, and a porous membrane filter disposed in the pouch between the inner surface of the first wall portion and the inner surface of the second wall portion. The membrane filter has a first major surface and a second major surface opposite the first major surface. The water-proof pouch further includes a first compartment defined in part by the inner surface of the first wall portion and defined in part by the first major surface of the membrane filter and a sealable sample port that provides access to deposit a liquid into the first compartment. Additionally, the water-proof pouch includes a second compartment defined in part by the inner surface of the second wall portion and defined in part by the second major surface of the membrane filter, and an absorbent pad disposed in the second compartment. The membrane filter permits passage of aqueous liquids from the first compartment to the second compartment and prevents passage of particles of a predetermined size from the first compartment to the second compartment. The water-proof pouch also includes an adhesive composition adhered to a portion of the pouch in the first compartment, a number of particles of a substantially dry, first microbial growth nutrient composition distributed in the adhesive composition, and a cold-water-soluble hydrogel-forming composition adhered to the adhesive composition.

In a third aspect, a method of detecting and enumerating at least one microorganism in a sample is provided. The method includes providing a device according to the first aspect, separating the first layer from the second layer, and adding a predetermined volume of a sample containing at least one microorganism onto the first hydrogel-forming composition to form an inoculated device. The method further includes contacting the first layer back to the second layer, incubating the inoculated device, and detecting the presence or an absence of a colony of the target microorganism in the device.

In a fourth aspect, another method for detecting and enumerating at least one

microorganism in a sample is provided. The method includes providing a device according to the second aspect, placing a predetermined volume of aqueous sample into the first compartment of the device, sealing the sample port, incubating the device, and detecting the presence or an absence of a colony of the target microorganism in the device.

The devices and methods allow for simple and rapid detection of microorganisms.

Brief Description of the Drawings

FIG. 1 A is a top perspective view, partially in section, of an exemplary embodiment of a microbiological growing device.

FIG. IB is a top perspective view, partially in section, of another exemplary embodiment of a microbiological growing device.

FIG. 2 is a cross sectional view of the device of FIG. 1A, including an optional spacer.

FIG. 3A is a scanning electron microscope (SEM) image of a surface of an adhesive composition layer according to an exemplary embodiment of the present disclosure. FIG. 3B is an SEM image of a surface of another adhesive composition layer according to an exemplary embodiment of the present disclosure.

FIG. 3C is an SEM image of a cross-section of an adhesive composition layer according to an exemplary embodiment of the present disclosure.

FIG. 3D is an SEM image of a cross-section of another adhesive composition layer according to an exemplary embodiment of the present disclosure.

FIG. 4 is a top view of the device of FIG. 2 showing a grid pattern printed on the substrate layer.

FIG. 5 is a perspective view of one embodiment of a device according to the present disclosure.

FIG. 6 is another perspective view, partially in section, of the device of FIG 5.

FIG. 7 is a cross-sectional view, taken along the line 7—7, of the device of FIG. 6.

FIG. 8 is an exploded cross-sectional view, of the device of FIG. 6.

FIG 9 is a plan view, partially in section, of an alternative embodiment of the device of FIG 5, showing an adhesive strip and a release liner releasably adhered thereto that form a sealable sample port.

FIG. 10 is a plan view of an alternative embodiment of a device according to the present disclosure, wherein the device comprises a sealable sample port with a screwcap.

FIG. 11 is an exploded view of another alternative embodiment of a device according to the present disclosure.

FIG. 12A is a first subassembly of the device of FIG. 11.

FIG. 12B is a second subassembly of the device of FIG. 11.

FIG. 13 is a plan view of the assembled device of FIG. 11.

FIG. 14 is a cross-sectional view, taken along the line 14—14, of the device of FIG. 13.

While the above-identified drawings, which may not be drawn to scale, set forth various embodiments of the present disclosure, other embodiments are also contemplated, as noted in the Detailed Description.

Detailed Description

Devices and methods of propagating or storing a microorganism are provided.

The recitation of any numerical range by endpoints is meant to include the endpoints of the range, all numbers within the range, and any narrower range within the stated range (e.g. 1 to 5 includes 1, 1.5, 2, 2.75, 3, 3.8, 4, and 5). Unless otherwise indicated, all numbers expressing quantities or ingredients, measurement of properties and so forth used in the specification and 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 attached listing of embodiments can vary depending upon the desired properties sought to be obtained by those skilled in the art utilizing the teachings of the present disclosure. At the very least, and not as an attempt to limit the application of the doctrine of equivalents to the scope of the claimed embodiments, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques.

For the following Glossary of defined terms, these definitions shall be applied for the entire application, unless a different definition is provided in the claims or elsewhere in the specification.

Glossary

Certain terms are used throughout the description and the claims that, while for the most part are well known, may require some explanation. It should be understood that, as used herein:

The term "a", "an", and "the" are used interchangeably with "at least one" to mean one or more of the elements being described.

The term "and/or" means either or both. For example, the expression "A and/or B" means

A, B, or a combination of A and B.

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

"Agglomerated" refers to a weak association of primary particles or aggregated particles usually held together by charge or polarity. Agglomerated particles can typically be broken down into smaller entities by, for example, shearing forces encountered during dispersion of the agglomerated particles in a liquid. The terms "aggregated" and "aggregates" refer to a strong association of primary particles often bound together by, for example, residual chemical treatment, covalent chemical bonds, or ionic chemical bonds. Further breakdown of the aggregates into smaller entities is very difficult to achieve.

"Cold-water-soluble" refers to material which forms a solution in water at room temperature (i.e., about 25°C).

"Hydrophobic" refers to a material that exhibits a water contact angle of 90° or larger on its surface.

"Opaque" refers to a substrate having at most 10% light transmission.

"Powder" refers to a finely divided particulate material having an average diameter in a range from 0.1 micrometer up to 400 micrometers.

"Reconstituted medium" refers to a solution or gel formed from the reconstitution of a cold-water-soluble powder with an aqueous liquid.

"Substantially impermeable to microorganisms and water vapor", as used herein, refers to a cover sheet that prevents undesired contamination and hydration of underlying layers of cold- water-soluble powder during shipping, storage, and use of thin film culture device(s), and avoids desiccation of the reconstituted medium, such that the reconstituted medium is suitable to support the growth of microorganisms during an incubation period.

"Substantially water-free", as used herein, designates a water content no greater than about the water content of the ambient environment.

"Test sample", as used herein, refers to a component or portion taken from a food product, a human or animal test subject, pharmaceutical or cosmetic commodity, soil, water, air or other environmental source, or any other source from which a presence and, optionally, an enumeration of aerobic and/or aerotolerant bacteria is to be determined. A test sample may be taken from a source using techniques known to one skilled in the art including, for example, pouring, pipetting, swabbing, filtering, and contacting. In addition, the test sample may be subjected to various sample preparation processes known in the art including, for example, blending, stomaching, homogenization, enrichment, selective enrichment, or dilution.

"Transparent" refers to a substrate having at least 90% light transmission.

Devices for the propagation and/or storage of microorganisms are provided having advantageous features such as controlling the amount of nutrients included in the device. Further, the nutrients are not directly exposed to samples during use, and thus are protected from potentially washing off when a fluid sample is introduced into the device.

In a first aspect, a device is provided. More particularly, a device is provided including a body member including a substrate having a first major surface and a second major surface. The device further includes a first adhesive composition adhered to a portion of the first major surface. A number of particles of a substantially dry, first microbial growth nutrient composition are distributed in the first adhesive composition, and a cold-water-soluble first hydrogel-forming composition is adhered to the first adhesive composition. The device also includes a cover sheet attached to the body member, where the cover sheet includes a first major surface facing the body member.

FIG. 1A illustrates an exemplary embodiment of device for growing microorganisms. The device 10 includes a body member 11 comprising a substrate 12 having a first major surface 12a (e.g., upper surface) and a second major surface 12b (e.g., lower surface). Moreover, the device 10 also includes a cover sheet 22 attached to the body member 11. The cover sheet 22 includes a first major surface 22a facing the body member 11.

The substrate 12 is water-proof, and is optionally a self-supporting water-proof substrate. In some embodiments, the substrate 12 is a film of a material such as polyester, polypropylene, silicone, or polystyrene, which will not absorb or otherwise be affected by water. Polyester films and polypropylene films having a thickness from about 20 micrometers to about 250 micrometers, as well as polystyrene films having a thickness of about 380 micrometers, have each been found to be suitable for the substrate 12. Other suitable substrates include paper with a polyethylene or other water-proof coating. An example of a suitable polyethylene-coated paper substrate is "Schoeller Type MIL" photoprint paper (commercially available from Schoeller Pulaski, New York). The substrate 12 may be either transparent or opaque, depending on whether one wishes to view bacterial colonies through the substrate. In the exemplary embodiment shown in FIG. 4, the substrate 12 has a square grid pattern printed on the second major surface 12b to facilitate the counting of bacterial colonies.

The first major surface 12a comprises a first adhesive composition 14 adhered to a portion of the first major surface 12a. A plurality of particles of a substantially dry, first microbial growth nutrient composition is distributed in the first adhesive composition 14. This is in contrast to prior inclusion of substantially water-soluble microbial growth nutrients in adhesives using an aqueous emulsion. For instance, in co-owned PCT Publication No. WO 96/38533 (Nelson), an "adhesive composition comprises a water-insoluble adhesive, a non-inhibitory emulsifying agent, and at least one hydrophilic agent selected from the group consisting of a nutrient for growing

microorganisms, a selective agent, and combinations thereof (page 2, lines 8-11 of WO

96/38533). The nutrient is also described as being dissolved in an aqueous emulsion suspension in Example 2, not provided as particles dispersed and/or distributed in an adhesive composition. In some embodiments of the present disclosure, in contrast, the adhesive composition comprises a solvent based adhesive. Typically the adhesive composition is free of emulsifier.

The first adhesive composition 14 is (substantially) water-insoluble and non-inhibitory to the growth of microorganisms. In some embodiments, the first adhesive composition 14 is sufficiently transparent when wet to enable the viewing of bacterial colonies through the film coated with the adhesive. In some embodiments, the first adhesive composition 14 is a pressure- sensitive adhesive. In some other embodiments, heat-activated adhesives in which a lower melting substance is coated onto a higher melting substance may also be used. Water-activated adhesives such as mucilage may also be useful. A cold-water-soluble first hydrogel-forming composition 16 is adhered to the first adhesive composition 14. In many embodiments, the cold-water-soluble first hydrogel-forming composition is provided in the form of a powder.

Referring to FIG. 2, a cross sectional view is provided of the device 10 of FIG. 1A, plus further including an optional spacer 18 disposed on the cold-water-soluble first hydrogel-forming composition 16. In general, the spacer 18 comprises a water-insoluble substrate defining an aperture 20, the spacer 18 being positioned between the substrate sheet 12 and the cover sheet 22. Typically, the aperture 20 defines a peripheral boundary of a sample-receiving zone. The aperture 20 can be any shape. Non- limiting examples of useful shapes for the aperture 20 include a square, a rectangle, a circle, an oval, a polygon, a hexagon, and an octagon. The area of the sample- receiving zone (and aperture 20) may be selected based on, for example, the volume of sample (e.g., aqueous liquid) to be deposited in the zone. In any embodiment, for a 0.5-3 milliliter sample, the area of the sample-receiving zone is about 10 cm ² or about 15 cm ² . In any embodiment, for a 1-5 milliliter volume of sample, the area of the sample -receiving zone is about 20 cm ² , about 25 cm ² , about 30 cm ² , about 31 cm ² , or about 25-35 cm ² .

Referring to FIGS. 3A-3D, in some embodiments, the plurality of particles 4 of the substantially dry, microbial growth nutrient composition distributed in the adhesive composition 14 include one or more primary particles of the first microbial growth composition, a plurality of clusters of particles, or both. FIG. 3 A is an SEM image that shows a layer of an adhesive composition 14 prepared according to an embodiment of the present disclosure. Clusters of particles 4 (i.e., agglomerated and/or aggregated particles) can occur due to the microbial growth nutrient composition being incompatible with the adhesive composition, typically wherein the microbial growth nutrient composition is water soluble and the adhesive composition is water insoluble. Although the microbial growth nutrient composition can be distributed (e.g., dispersed) throughout the adhesive composition, such as by combining the two materials in a container and rolling the container, often the microbial growth nutrient composition gets distributed in discrete clumps. Referring again to FIG. 3A, a plurality of clusters of particles 4 of the microbial growth nutrient composition project above a plane of an upper surface 5 of the layer of the adhesive composition 14. Referring to FIG. 3B, a closer view of a layer of an adhesive composition 14 prepared according to the present disclosure is provided. Further, each of FIGS. 3C and 3D are SEM images of cross-sections of an adhesive composition 14 having particles 4 of the

substantially dry, microbial growth nutrient composition distributed therein. Each images show a cross-section of at least one cluster of particles 4 of the microbial growth nutrient composition. In certain embodiments, an average length of the plurality of clusters of particles of the first microbial growth composition is 250 micrometers or less, 200 micrometers or less, 150 micrometers or less, or 100 micrometers or less; and 10 micrometers or more, 25 micrometers or more, 50 micrometers or more, or 75 micrometers or more. In some embodiments, an average length of the plurality of clusters of the first microbial growth composition ranges from 10 micrometers to 100 micrometers, inclusive.

It has been unexpectedly discovered that despite the lack of compatibility between the materials of the microbial growth nutrient composition (e.g., being water soluble) and the adhesive composition (e.g., being water insoluble), a sufficient amount of nutrients are able to traverse through the adhesive composition to be available for microorganism consumption in the device. Advantageously, the amount of the microbial growth nutrient composition can be controlled by incorporating a desired amount into the adhesive composition. This is in contrast to coating a layer of microbial growth nutrient composition powder on the surface of an adhesive composition. In certain embodiments, the (first, second, or both) microbial growth nutrient composition is present in an adhesive composition in an amount of 1 weight percent (wt. %) or more, 2 wt. % or more, 5 wt. % or more, 7 wt. % or more, 10 wt. % or more, or 12.5 wt. % or more of the combined adhesive composition and the microbial growth nutrient composition; and 20 wt. % or less, 17 wt. % or less, 15 wt. % or less of the combined adhesive composition and the microbial growth nutrient composition. Moreover, due to being distributed in the first adhesive composition, the nutrients are not exposed on the surface of the film and thus are protected from washing off when a fluid sample is introduced into the device. This is particularly relevant for applications in which the sample must contact the powder layer and then pass through a filter (such as devices according to the second aspect of the present disclosure).

Referring again to FIG. 1A, the device further includes a cold-water-soluble first hydrogel- forming composition 16 adhered to the first adhesive composition 14. A uniform layer of powder of the hydrogel-forming composition 16 is desired with sufficient surface area exposed for hydration. Typically, an adhesive composition 14 layer in the thickness range from about 5 micrometers to about 150 micrometers is suitable.

Suitable adhesives are transparent when wetted with water. As noted 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, second adhesive composition often is a pressure sensitive adhesive. For instance, the adhesive may be a pressure- sensitive adhesive such as a water-insoluble adhesive comprising a copolymer of an alkyl acrylate monomer and an alkyl amide monomer. Preferably the weight ratio of alkyl acrylate monomer to alkyl amide monomer in these copolymers is from about 90: 10 to 99: 1, more preferably 94:6 to 98:2. The alkyl acrylate monomer comprises a lower alkyl (C2 to CIO) monomer of acrylic acid, including, for example, isooctyl acrylate (IOA), 2-ethylhexyl acrylate, butyl acrylate, ethyl acrylate, isoamyl acrylate, and mixtures thereof, while the alkyl amide monomer can comprise, without limitation, acrylamide (ACM), methacrylamide, N-vinylpyrrolidone (NVP), N - vinylcaprolactam (NVCL), N-vinyl-2-piperidine, N-(mono- or di -lower alkyl (C2 to

C5))(meth)acrylamides, N -methyl(meth)acrylamide, N ,N -dimethyl(meth) acrylamides, or mixtures thereof. Suitable adhesives may also 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, including for example those described in U.S. Patent Nos.

7,695,818 and 7,371,464. In the present disclosure, the cover sheet 22 is usually selected to be transparent, in order to facilitate counting of microbial colonies, and is typically also selected to be impermeable to bacteria and have low moisture vapor transmission rate (i.e., the cover sheet 22 prevents undesired contamination of the dehydrated medium during shipping, storage and use of the devices and provides an environment which will support the growth of microorganisms during the incubation period). In some embodiments, the cover sheet 22 has the same properties (e.g., being waterproof) as the substrate 12. The cover sheet 22 can be selected to provide the amount of oxygen transmission necessary for the type of microorganism desired to be grown. For example, some polyester films have low oxygen permeability (less than 5 g/645 cm ² /24 hours per 25 micrometers of thickness) and would be suitable for growing anaerobic bacteria. On the other hand, some polyethylenes have high oxygen permeability (e.g., approximately 500 g/645 cm ² /24 hours per 25 micrometers of thickness) and would be suitable for aerobic organisms. Suitable material for the cover sheet 22 includes 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.

Referring back to FIG. 1A, in certain embodiments, the device further comprises a second adhesive composition 24 adhered to a portion of the first major surface 22a of the cover sheet 22. For each of the substrate 12 and the cover sheet 22, a layer of a cold-water-soluble hydrogel- forming composition 16, 26 is typically adhered uniformly to the adhesive layer 14, 24, respectively. The layer of adhesive 24 is water-insoluble and non-inhibitory to the growth of microorganisms, and is sufficiently transparent when wet to enable the viewing of gas bubbles or microbial colonies through the film coated with the adhesive. In some embodiments, the layer of adhesive 24 comprises a pressure-sensitive adhesive. A uniform layer of the coating composition 26 is desired with sufficient surface area exposed for hydration.

Referring to FIG. IB, in any embodiment, the cover sheet 22 can be free of any coating.

Alternatively, the cover sheet 22 can be coated, e.g., on the surface facing the dehydrated medium with a layer of pressure-sensitive adhesive, in order to facilitate sealing of the cover means over the medium. Furthermore, the cover sheet 22 can optionally be coated on the surface facing the first cold-water-soluble hydrogel-forming composition 16 with layers of an adhesive composition 24 and a second cold-water-soluble hydrogel-forming composition 26, that are the same as or different from the first adhesive composition 14 and the first cold-water-soluble hydrogel-forming composition 16, respectively. Coatings on the cover sheet 22 can cover the entire surface, but preferably cover at least the part of the surface that is intended to cover the growth region of the culture device. Such coated coversheets are particularly preferred when it is desired to provide a device with more gelling agent than can be incorporated in the first cold-water-soluble hydrogel- forming composition alone.

In many embodiments, the device 10 further comprises a plurality of particles 4 of a substantially dry, second microbial growth nutrient composition distributed in the second adhesive composition. The second microbial growth nutrient composition may be the same as or different from the first microbial growth nutrient composition. In certain embodiments, the plurality of particles 4 of the second microbial growth composition comprise a plurality of clusters of particles of the second microbial growth composition, as discussed above with respect to the particles of the first microbial growth composition. Further, in some embodiments, the second adhesive composition also contains at least one indicator agent.

In certain embodiments, the cold-water-soluble hydrogel-forming composition (e.g., 16, 26) contains one or more organic cold-water-soluble agents, such as alginate, carboxymethyl cellulose, tara gum, hydroxyethyl cellulose, hydroxypropyl methylcellulose, guar gum, locust bean gum, xanthan gum, polyacrylamide, polyurethane, polyethylene oxides. Combinations of natural and/or synthetic gelling agents are contemplated. Preferred gelling agents include guar gum, xanthan gum, and locust bean gum, these gelling agents being useful individually or, in any embodiment, in combination with one another. A uniform monolayer of a cold-water-soluble hydrogel-forming composition is desired with sufficient surface area exposed for hydration. In any embodiment, the first and/or second cold-water-soluble hydrogel-forming composition comprises a mixture of gelling agents. Optionally, the first and/or second cold-water-soluble hydrogel-forming composition may further comprise an inducer, and indicator agent, or a combination of these.

Suitable microbial growth nutrient compositions typically comprise for instance and without limitation one or more nutrients including a meat peptone, a casein peptone, a gelatin peptone, a soy peptone, a beef extract, a yeast extract, lactose, glucose, dextrose, tryptose, galactose, tryptone, a fat, a mineral, or a vitamin. Further, non-limiting examples of nutrients, additional gelling agents, and mixtures thereof for supporting growth of microorganisms in a device of the present disclosure include those described in U.S. Patent Nos. 4,565,783; 5,089,413; 5,232,838; 5,364,766; 5,443,963; 5,462,860; 5,601,998; 5,635,367; and 5,681,712; these references also include non-limiting examples of indicator agents (e.g., detection reagents) and inducers.

Suitable indicator agents can include one or more indicator agents for detecting an alkyl esterase activity, a phosphatase enzyme activity, a glycosidase enzyme activity, a peptidase enzyme activity, a pH change, or a redox change. In any embodiment, the one or more indicator agents may be dissolved in an organic solvent (e.g., methanol) and blended with an adhesive composition (e.g., the first adhesive composition 14 and/or the second adhesive composition 24) before applying the composition to the substrate sheet 12 and/or cover sheet 22. In any embodiment, the first cold-water-soluble hydrogel-forming composition and/or the second cold- water-soluble hydrogel-forming composition may comprise one or more indicator agents, either the same or different.

Optionally, a plurality of indicator agents may be used for detecting a presence of an aerobic or aerotolerant bacterium. In any embodiment, suitable indicator agents comprise, for example, enzyme substrates. In any embodiment, each indicator agent can comprise a reporter group (e.g., a fluorogenic group or chromogenic group) that permits detection of a reaction between the indicator agent and a biological activity (i.e., an enzyme activity associated with an aerobic or aerotolerant bacterium) and the indicator agent. One suitable indicator agent is triphenyl tetrazolium chloride (TTC). Suitable redox indicators (e.g., triphenyl tetrazolium chloride) include a reporter group (e.g., a chromogenic and/or fluorogenic group) that is oxidized or reduced to form a detectable signal (e.g., a detectable color change or fluorescence change). For example, triphenyl tetrazolium chloride is reduced by bacteria to form a formazan product having a detectable color. Detecting the detectable reporter group (e.g., formazan) in a culture device of the present disclosure is indicative of a possible presence of an aerobic or aerotolerant bacterium.

Further, in a method wherein the culture medium comprises bromcresol purple as a pH indicator, the culture medium will have a purple or gray appearance at about a neutral pH. As the microorganisms grow and ferment a carbohydrate (e.g., glucose) in the culture medium, the bromcresol purple indicator will appear yellow adjacent the growing bacterial colonies. For example, in a method wherein the culture medium comprises chlorophenol red as a pH indicator, the culture medium will have a red or violet appearance at about a neutral pH. As the

microorganisms and ferment a carbohydrate in the culture medium, the chlorophenol red indicator will appear yellow adjacent the growing microbial colonies. Gas bubbles, if present in the growth compartment and associated with a colony of microorganisms (e.g., either touching the colony or within a distance of about 1 mm or less from the colony), can be detected visually and/or by the use of an imaging system. The gas bubbles may be associated with a visible colony and/or an acid zone detectable by a change in the color of a pH indicator in a region adjacent the colony of microorganisms. The gas bubble may comprise carbon dioxide generated by anaerobic fermentation of a carbohydrate, for example.

In a second aspect, another microbial detection device is provided. The microbial detection device includes a water-proof pouch including a first wall portion having an inner surface and an outer surface, a second wall portion having an inner surface and an outer surface, and a porous membrane filter disposed in the pouch between the inner surface of the first wall portion and the inner surface of the second wall portion. The membrane filter has a first major surface and a second major surface opposite the first major surface. The water-proof pouch further includes a first compartment defined in part by the inner surface of the first wall portion and defined in part by the first major surface of the membrane filter and a sealable sample port that provides access to deposit a liquid into the first compartment. Additionally, the water-proof pouch includes a second compartment defined in part by the inner surface of the second wall portion and defined in part by the second major surface of the membrane filter, and an absorbent pad disposed in the second compartment. The membrane filter permits passage of aqueous liquids from the first compartment to the second compartment and prevents passage of particles of a predetermined size from the first compartment to the second compartment. The water-proof pouch also includes an adhesive composition adhered to a portion of the pouch in the first compartment, a number of particles of a substantially dry, first microbial growth nutrient composition distributed in the adhesive composition, and a cold-water-soluble hydrogel-forming composition adhered to the adhesive composition.

For instance, a microbial detection device can comprise:

a water-proof pouch comprising:

a first wall portion having an inner surface and an outer surface; a second wall portion having an inner surface and an outer surface; a porous membrane filter disposed in the pouch between the inner surface of the first wall portion and the inner surface of the second wall portion, the membrane filter having a first major surface and a second major surface opposite the first major surface; a first compartment defined in part by the inner surface of the first wall portion and defined in part by the first major surface of the membrane filter;

a sealable sample port that provides access to deposit a liquid into the first compartment;

a second compartment defined in part by the inner surface of the second wall portion and defined in part by the second major surface of the membrane filter;

wherein the membrane filter permits passage of aqueous liquids from the first compartment to the second compartment and prevents passage of particles of a predetermined size from the first compartment to the second compartment;

an adhesive composition adhered to a portion of the pouch in the first

compartment;

a plurality of particles of a substantially dry, first microbial growth nutrient composition distributed in the adhesive composition; a cold-water-soluble hydrogel-forming composition adhered to the adhesive composition; and

an absorbent pad disposed in the second compartment.

Regarding the microbial detection device of the second aspect, FIGS. 5-8 show various views of one embodiment of a device 500 according to at least one embodiment of the present disclosure. The device 500 comprises a waterproof pouch 55 defined by at least one wall. The at least one wall comprises a first wall portion 510 and a second wall portion 520. The first wall portion 510 has an inner surface 512 and an outer surface 514. The second wall portion 520 has an inner surface 522 and an outer surface 524. Disposed in the pouch 55 between the inner surface 512 of the first wall portion 510 and the inner surface 522 of the second wall portion 520 is a membrane filter 540. The membrane filter has a first major surface 542 and a second major surface 544 opposite the first major surface.

Although the first wall portion 510 and second wall portion 520 may be distinct portions of a unitary pouch or bag, in any embodiment, the first wall portion and second wall portion alternatively may consist of separate sheets of polymeric film that are joined together (e.g., heat- sealed and/or adhesively sealed along the edges) to form the pouch, as shown in FIG. 9, for example, and described herein.

The pouch 55 is divided into at least two compartments (first compartment 550 and second compartment 552, respectively). The first compartment 550 is defined in part by the inner surface 512 of the first wall portion 510 and also defined in part by the first major surface 542 of the membrane filter 540. The first compartment 550 has a sealable sample port 560. In the illustrated embodiment of FIGS. 5-7, the sealable sample port 560 is simply an opening 561 along a portion of the perimeter of the pouch 55. Nonlimiting exemplary means for closing the opening 561 are discussed herein. The second compartment 552 is defined in part by the inner surface 522 of the second wall portion 520 and defined in part by the second major surface 544 of the membrane filter 540.

The first compartment 550 is configured to receive a volume of liquid sample to be tested for presence of target microorganisms. The volume of liquid the first compartment 550 can receive will be influenced by several features of the device including, for example, the dimensions (e.g., the length "L" and width "W" shown in FIG. 7) of the first compartment and the flexibility of the materials (e.g., the first wall portion 510 and the membrane filter 540) that define the first compartment. The second compartment 552 is configured to receive a volume of liquid approximately equal to the volume of liquid sample to be tested. Thus, the pouch of a device of the present disclosure may be dimensioned to hold up to about twice the volume of the sample to be tested. In any embodiment, a device of the present disclosure is configured to test (i.e., configured to receive) at least about 25 milliliters of liquid sample. In any embodiment, a device of the present disclosure is configured to test at least about 50 milliliters of liquid sample. In any embodiment, a device of the present disclosure is configured to test at least about 75 milliliters of liquid sample. In any embodiment, a device of the present disclosure is configured to test at least about 100 milliliters of liquid sample. In any embodiment, a device of the present disclosure is configured to test at least about 125 milliliters of liquid sample. In any embodiment, a device of the present disclosure is configured to test at least about 150 milliliters of liquid sample. Thus, in any embodiment, the device according to the present disclosure is configured to receive at least about 25 mL, at least about 50 mL, at least about 75 mL, at least about 100 mL, at least about 125 mL, at least about 150 mL of liquid sample (e.g., aqueous liquid sample). Accordingly, in any embodiment, the first compartment of the device is configured to receive at least about 25 mL, at least about 50 mL, at least about 75 mL, at least about 100 mL, at least about 125 mL, at least about 150 mL of liquid sample (e.g., aqueous liquid sample).

The pouch 55 further comprises an adhesive composition adhered as a layer 530 to a portion of the pouch (e.g., the first wall portion 510 of the pouch) in the first compartment 550. In any embodiment, the adhesive composition comprises a pressure sensitive adhesive. As discussed above with respect to the first embodiment, the adhesive composition is often water insoluble. In certain embodiments, the adhesive composition comprises a solvent based adhesive. For instance, the adhesive may be a pressure-sensitive adhesive such as a water-insoluble adhesive comprising a copolymer of an alkyl acrylate monomer and an alkyl amide monomer.

A plurality of particles 535 of a substantially dry, first microbial growth nutrient composition is distributed in the adhesive composition. In any embodiment, a device of the present disclosure comprises an effective amount of one or more dry nutrient (e.g., a nutrient medium selected to support growth of the target microorganism). The microbial growth nutrient composition typically comprises at least one nutrient selected from the group consisting of a meat peptone, a casein peptone, a gelatin peptone, a soy peptone, a beef extract, a yeast extract, lactose, glucose, dextrose, tryptose, galactose, tryptone, a fat, a mineral, or a vitamin. As discussed above in detail with respect to the device of the first aspect, in some embodiments, the plurality of particles of the substantially dry, microbial growth nutrient composition distributed in the adhesive composition include one or more primary particles of the first microbial growth composition, a plurality of clusters of particles, or both. Similarly, in certain embodiments, an average length of the plurality of clusters of particles of the first microbial growth composition is 250 micrometers or less, 200 micrometers or less, 150 micrometers or less, or 100 micrometers or less; and 10 micrometers or more, 25 micrometers or more, 50 micrometers or more, or 75 micrometers or more. In some embodiments, an average length of the plurality of clusters of the first microbial growth composition ranges from 10 micrometers to 100 micrometers, inclusive.

Similar to the devices according to the first aspect, it has been unexpectedly discovered that despite the lack of compatibility between the materials of the microbial growth nutrient composition (e.g., being water soluble) and the adhesive composition (e.g., being water insoluble), a sufficient amount of nutrients are able to traverse through the adhesive composition to be available for microorganism consumption in the devices according to the third aspect.

In many embodiments, the microbial growth nutrient composition is present in an adhesive composition in an amount of 1 weight percent (wt. %) or more, 2 wt. % or more, 5 wt. % or more, 7 wt. % or more, 10 wt. % or more, or 12.5 wt. % or more of the combined adhesive composition and the microbial growth nutrient composition; and 20 wt. % or less, 17 wt. % or less, 15 wt. % or less of the combined adhesive composition and the microbial growth nutrient composition.

Moreover, due to being distributed in the first adhesive composition, the nutrients are not exposed on the surface of the film and thus are protected from washing off when a fluid sample is introduced into the device. This is particularly relevant for applications in which the sample must contact the powder layer and then pass through a filter (such as in the devices of this second aspect).

Further, a dry (i.e., substantially water-free) cold-water-soluble hydrogel-forming composition is adhered to the adhesive composition. For instance, FIG.7 shows the cold-water- soluble hydrogel-forming composition as a dry coating 532 disposed on the inner surface 512 of the first wall portion 510. The dry coating 532 is adhered to the first wall portion 510 via an adhesive layer 530. In addition, the pouch 55 has an absorbent pad 580 disposed in the second compartment 552. In any embodiment, the dry coating 532 may be adhered to a first substrate (e.g., adhered to an adhesive layer coated on the substrate) that is adhered to the first wall portion 510 of the pouch 55. This optional configuration is shown in FIG. 11 and described hereinbelow.

Whether the cold-water-soluble hydrogel-forming composition is adhered to the first wall portion of the pouch or to a first substrate that is adhered to the first wall portion, the area defined by the coating comprising the cold-water-soluble hydrogel-forming composition also defines the area in which microorganisms from the sample grow and are enumerated after a sample is deposited into the first compartment. Because the device comprises an absorbent pad (described below) that absorbs most of the liquid from the sample, the cold-water-soluble hydrogel-forming composition is hydrated by only a fraction of the liquid sample. Advantageously, these devices use a surprisingly smaller ratio of growth area: sample volume than previously-reported thin-film culture devices. For example, a device according to the second aspect of the present disclosure is configured to receive 100-150 mL of a liquid sample and has a growth area (that includes a cold- water-soluble hydrogel-forming composition) of about 80 cm ² . Thus, the microorganisms from the 150 mL sample volume is spread over a growth area that is equivalent to less than 1 cm ² per mL of sample.

The pouch 55 (i.e., at least one wall, and wall portions thereof) is fabricated of a water- proof, deformable material. In any embodiment, the deformable material may comprise a flexible, sheet-like material such as a polymeric film, for example. Suitable materials for use when fabricating the at least one wall include polyethylene, polypropylene, polyethylene terephthalate, polyamide, polyurethane, polyvinyl chloride, polyacrylate, polyurea, and combinations thereof. The at least one wall of the pouch can be relatively thin (e.g., approximately 25 microns thick) or relatively thicker (e.g., approximately 125 microns thick), provided at least a portion of the at least one wall (e.g., first wall portion 510, which is opposite the membrane filter 540 in the first compartment 550) can deform when the pouch 55 receives a liquid sample (not shown) and/or at least a portion of the at least one wall (e.g., second wall portion 520, which is proximate the absorbent pad described herein) can deform when at least a portion of the sample passes from the first compartment into the second compartment.

The membrane filter 540 permits passage of a liquid (an aqueous liquid, not shown) from the first compartment 550 to the second compartment 552 and prevents passage of particles of a predetermined size from the first compartment to the second compartment. Thus, when an aqueous liquid sample suspected of containing a target microorganism is placed into the first compartment 550, a first portion of the aqueous liquid passes (e.g., by gravity flow) through the membrane filter 540 into the second compartment 552 where it is absorbed by the absorbent pad 580. The target microorganism is trapped on or in the filter membrane 540 or is retained in a second portion of the aqueous liquid that remains in the first compartment 550.

The use of membrane filters to trap and retain microorganisms is well known in the art. Accordingly, there are a number of suitable membrane filters that can be used in a device according to the present disclosure. Nonlimiting examples of suitable membrane filters include fibrous membrane filters made of nylon, polyether sulfone, polytetrafluoroethylene, or cellulosic materials (e.g., mixed cellulose esters), microporous plastic films (e.g., laser-etched polycarbonate film), and ceramic membrane filters.

The porosity of the membrane filter generally is chosen so that the target microorganisms will not pass all the way though the pores from one side of the membrane filter to the other side, thereby insuring that substantially all target microorganisms in the sample are retained by the filter. Typical bacteria are about 0.5 to 5.0 μπι in length. Certain smaller bacteria, such as Mycoplasma spp., are approximately 0.3 μπι in diameter. Yeast cells are generally larger than bacteria. Typical yeast cells are approximately 3-4 μπι in diameter, although some are as large as about 40 μηι in diameter. Molds may exist as single cells, spores, or filamentous hyphae.

Although typically larger than bacteria, the average size of mold cells varies by species.

Accordingly, the selection of a membrane filter with a suitable pore size may depend upon the target microorganism. For example, a membrane filter with a nominal pore size of 1.0 μπι or less, 0.8 μπι or less, 0.6 μπι or less, 0.4 μπι or less, 0.2 μπι or less, 0.1 μπι or less, 0.05 μπι or less,

0.03 μπι or less, 0.02 μπι or less, or 0.01 μπι or less may be suitable to capture and detect target bacteria. For capturing and detecting target yeast or mold microorganisms, a membrane filter with a nominal pore size of 12 μπι or less, 8 μπι or less, 5 μπι or less, 3 μπι or less, 2 μπι or less, 1 μπι or less, 0.8 μπι or less, 0.6 μπι or less, 0.4 μπι or less, 0.2 μπι or less, or 0.1 μπι or less may be suitable.

Membrane filters may be prepared manually from suitable filtration media or,

alternatively, may be purchased in pre-cut sizes and shapes. The size and shape of the membrane filter can be chosen based upon the sample volume and the expected load of particulate material in the sample. In general, membrane filters with larger surface areas will allow for higher filtration rates than membrane filters with smaller surface areas. Membrane filters may be used in combination with other filtration media (e.g., a prefilter, to trap larger debris in the sample) or other membrane filters.

In any embodiment, the membrane filter may be supported (e.g., by a scrim, not shown) to provide physical stability for the membrane during use. In any embodiment, the support may be attached to the membrane filter (e.g., on the second major surface). In any embodiment, the membrane filter can comprise a wetting agent (e.g., a nonionic surfactant) to facilitate rapid and complete penetration of the liquid sample throughout the membrane filter. Preferably, the wetting agent is in an amount sufficient to facilitate wetting the membrane with an aqueous liquid, but in an amount that does not substantially inhibit growth of the target microorganism when using the device.

The dry, cold-water-soluble hydrogel-forming composition is hydrated and forms a hydrogel when an aqueous sample is placed into the first compartment 550 of the pouch 55. As the first portion of the aqueous liquid moves through the membrane filter 540 from the first compartment 550 to the second compartment 552, the hydrogel contacts the first surface of the membrane filter 540, thereby immobilizing any microorganisms retained on or in the membrane filter.

Cold-water-soluble gelling agents that are suitable for use in thin-film culture devices are known in the art and include, for example, cold-water-soluble natural and synthetic gelling agents. Natural gelling agents such as alginate, carboxymethyl cellulose, tara gum, hydroxyethyl cellulose, guar gum, locust bean gum, xanthan gum, and synthetic gelling agents such as polyacrylamide, polyurethane, polyethylene oxides, and mixtures thereof are generally suitable. Appropriate gelling agents can be selected according to the teaching of this disclosure and the disclosures of U.S. Patent Nos. 4,565,783; 5,089,413; and 5,232,838. Other preferred gelling agents include hydroxypropyl methylcellulose; these gelling agents being useful individually, or preferably, in combination with another gelling agent such as one of the aforementioned gelling agents.

In any embodiment, the dry, cold-water-soluble hydrogel-forming composition can be disposed in the pouch as a dry powder adhered to an adhesive layer, as described herein.

Processes and adhesives for coating a dry powder onto a flexible film for use in a thin-film culture device are described, for example, in U.S. Patent Nos. 4,565,783; 5,089,413; and 5,232,838. In any embodiment, the adhesive layer, if present may comprise an indicator for indicating microorganism growth. For example, the adhesive may comprise triphenyltetrazolium chloride as described in U.S. Patent No. 5,409,838, which is incorporated herein by reference in its entirety.

In any embodiment, the dry, cold-water-soluble hydrogel-forming composition can be deposited onto the first wall portion of the pouch as an aqueous composition and subsequently dried, as described in U.S. Patent Nos. 4,565,783; 5,089,413; and 5,232,838. Optionally, in any embodiment, the dried coating can be adhered to an adhesive layer coated onto the first wall portion of the pouch. In any embodiment, the adhesive layer may further comprise an indicator for indicating microorganism growth, as described above.

Before a liquid sample is deposited into the pouch, the absorbent pad 580 is preferably relatively thin (e.g., less than or equal to 5 mm thick, less than or equal to 4 mm thick, less than or equal to 3 mm thick, less than or equal to 2 mm thick, less than or equal to about 1 mm thick) and is configured to absorb a quantity of deionized water equal to many time its own weight (e.g., at least 100-times its own weight, at least 150-times its own weight, at least 200-times its own weight, at least 250-times its own weight, at least 300-times its own weight, at least 350-times its own weight, at least 400-times its own weight, at least 500-times its own weight). In any embodiment, the absorbent pad may comprise a plurality of materials such as, for example, a super-absorbent material (e.g., a superabsorbent polymer; "herein, "SAP") and a less-absorbent or nonabsorbent carrier (e.g., cellulosic fibers). A nonlimiting example of a suitable absorbent pad is a composite polyacrylate laminate structure comprising a superabsorbent polymer granule base disposed between two cellulose sheets. In any embodiment of the absorbent pad, the pad may comprise SAP granules disposed in an air-laid nonwoven material or SAP fibers blended with carrier fibers into a nonwoven material.

Optionally, in any embodiment (not shown), the absorbent pad may be coupled to a component of the pouch (e.g., the second wall portion) in the second compartment.

Advantageously, this can keep the pad from deforming (e.g., as it swells with liquid migrating from the first compartment) to an extent that it loses contact with a substantial portion of the membrane filter. The pad may be coupled to the pouch via an adhesive (e.g., a pressure-sensitive adhesive), a thermal weld or other suitable attachment means known in the art. In any embodiment, the absorbent pad may be releasably coupled to the pouch (e.g., by a water-soluble gum). This embodiment hold the pad in a proper position to receive liquid passing through the membrane filter, but permits lateral movement of the pad as it swells due to absorption of a large quantity of the liquid.

Referring back to the drawings, FIG. 9 shows one embodiment of a sealable sample port 560 of a device 501 according to the present disclosure. The device 501 comprises a pouch 55 having a first wall portion 510, a second wall portion 520, and a sealable sample port 560 consisting of an opening, each as described herein. The inner surface 512 of the first wall portion 510 comprises an adhesive strip 516 coated thereon along the edge of the inner surface proximate the opening. Adhered to the adhesive strip 516 is a release liner 518. After the sample is deposited (e.g., by pouring or pipetting) into the first compartment (not shown in FIG. 9) through the opening (sample port 560), the operator removes the release liner and contacts the adhesive strip 516 with the inner surface 522 of the second wall portion 520 proximate the opening in order to seal the opening. Optionally, the operator can expel (out of the opening) some or all of the air from the first compartment 550 when completing the sealing process.

FIG. 10 shows an alternative embodiment of a device 502 comprising a pouch 56 comprising a sealable sample port 560 with an opening 561. In this embodiment, the sealable sample port 560 is a screw-cap opening into which the liquid test sample can be poured or pipetted, for example. Alternatively, in any embodiment, the sealable sample port 560 can be a pierceable, elastically-deformable septum through which a needle or a pipet tip can be introduced to deliver the sample into the first compartment. After the needle or pipet is withdrawn from the septum, the elastically-deformable septum reseals the port. Advantageously, in these

embodiments, the introduction of air into the first compartment can be minimized.

In another alternative embodiment (not shown), the sealable sample port can comprise interlocking zipper components (e.g., similar to a ZIPLOK plastic storage bag) on each of the first wall portion and second wall portion and a zipper component that is used cooperatively with the interlocking components to open or seal the first compartment.

In another aspect, the present disclosure provides a method of assembling a large-volume, thin-film culture device. Devices of the present disclosure can be assembled entirely from sheetlike materials. Advantageously, this enables the use of roll-to-roll processes when assembling a plurality of devices. FIGS. 11-14 show various views of an alternative embodiment of a device 503 according to the present disclosure. FIG. 11 shows the sheet-like materials that are used to assemble one embodiment of a device according to the present disclosure. Each part of the device can be cut into appropriately- sized sheets and subsequently assembled into the device or, alternatively can be cut to the appropriate size using controlled-depth die cutting using a roll-to-roll process known in the art.

In any embodiment, a device of the present disclosure can be partially assembled into one or more subassembly, which is subsequently combined with other components to make the device. Referring to FIG. 11, the device 503 includes a first subassembly I that comprises a first part A, a second part B, and a third part C. Another view of the assembled first subassembly I is shown in FIG. 12A. The first part A consists of the first wall portion 510 with an adhesive layer 574 coated thereon as described herein. Second part B consists of a release liner 518 as described herein. Third part C consists of a first substrate 590 coated on one side with an adhesive layer 582.

Disposed on the adhesive layer 582 is a coating 584 that comprises the dry, cold-water-soluble hydrogel-forming composition described herein. The coating 584 can be deposited onto the adhesive layer 582 as a dry powder or as a liquid composition that is subsequently dried to a substantially water-free state, as described hereinabove. The first substrate 590 can comprise a sheet-like material similar to those used for the walls of the pouch as described above.

Alternatively, the first substrate can comprise a nonwoven fabric or a cellulosic material (e.g., paper). In any embodiment, the cellulosic material can be coated with a waterproof coating that is substantially noninhibitory to growth of microorganisms. The area defined by the coating 584 on third part C also defines the growth and colony-enumeration area in the assembled device.

When assembling subassembly I, the release liner 518 is releasably adhered to the adhesive layer 574 along the edge (edge 511) of the first wall portion 510 that forms the opening of the assembled device. In addition, the third part C is positioned centrally over part A with the coating 584 facing away from the adhesive layer 574. Part C is then contacted with adhesive 574 to affix part C to part A with the coating 584 exposed., as shown in FIG. 12A.

Referring back to FIG. 11, a second subassembly II includes a fourth part D and a fifth part E. The fourth part D comprises a second substrate 591. The second substrate 591 forms a frame comprising an aperture 592. The second substrate 591 is coated on one side with an adhesive layer 593. The second substrate 591 can comprise a sheet-like material (e.g., a flexible film) similar to those used for the walls of the pouch as described above. Alternatively, the second substrate can comprise a nonwoven fabric or a cellulosic material (e.g., paper). In any

embodiment, the cellulosic material can be coated with a waterproof coating that is substantially noninhibitory to growth of microorganisms. Optionally, the absorbent pad can be coupled to the second substrate in the second compartment. The second subassembly II also includes the fifth part E (i.e., membrane filter 540, as described herein). The membrane filter 540 is dimensioned so that it completely covers the area defined by the aperture 592. When assembling subassembly II, the membrane filter 540 is adhered to the adhesive layer 593 so that it completely covers the aperture 592 of the second substrate 591, as shown in FIG. 12B. In use, liquid passes through the aperture from the first compartment to the second compartment of the device as the liquid passes through the membrane filter. In any embodiment, the aperture 592 defines a first area and the coating 584 defines a second area.

Preferably, the second area is greater than or equal to the first area. More preferably, the second area is shaped and dimensioned to completely overlap the area of the aperture.

Optionally, when assembling the device 503 of FIG. 11, the subassembly I can be coupled to subassembly II to form a subassembly III. This can be done by placing the back side (i.e., the side that does not include adhesive layer 593) of subassembly II in overlaying contact with the adhesive-coated side of subassembly I. In addition, the aperture 592 of subassembly II is aligned with subassembly I so that it overlaps the third part C of subassembly I.

To complete the construction of the device 503, the sixth part F (i.e., absorbent pad 580, as described herein) is placed in overlaying contact with the membrane filter 540 of the subassembly III and the seventh part (i.e., second wall portion 520, as described herein) is placed in overlaying contact with the first part A such that the seventh part G is adhesively coupled to the portion of the adhesive layer 574 at the periphery of the first part A. FIG. 13 shows a plan view and FIG. 14 shows a cross-sectional view of the assembled device 503 of FIG. 11.

In any embodiment, a device of the present disclosure further comprises an indicator reagent for indicating a presence of a viable microorganism. The indicator reagent is disposed in the pouch. In any embodiment, the indicator reagent may be disposed as a dry powder or dried coating in the first compartment and/or the second compartment of the pouch. In any embodiment, the indicator reagent can be disposed in an adhesive layer as described herein. Alternatively, or additionally, the indicator reagent may be a dry reagent coated onto an adhesive layer (e.g., with the cold-water-soluble hydrogel-forming composition as described herein).

In any embodiment, the indicator reagent may be a general indicator (e.g., a redox indicator such as triphenyltetrazolium chloride, for example) of viable microorganisms or an indicator of a large class of target microorganisms (e.g., total aerobic microorganisms).

Alternatively, the indicator reagent can be an indicator (e.g., a chromogenic or fluorogenic enzyme substrate) that reacts with a smaller group of target microorganisms. A person having ordinary skill in the will recognize an appropriate indicator reagent for a particular target microorganism.

In any embodiment of a device according to the present disclosure, the device further comprises a stand-off layer (not shown) disposed in the second compartment between the membrane filter and the absorbent pad. The stand-off layer is a relatively-thin (e.g., about 0.1 mm to 2 mm thick) sheet-like material. In any embodiment, the stand-off layer is shaped and dimensioned to be at least coextensive with the membrane filter. In any embodiment, the stand-off layer is substantially less absorbent than the absorbent pad. In any embodiment, the absorbency of the stand-off layer is less than or equal to the absorbency of the membrane filter. The stand-off layer may comprise or consist essentially of a hydrophobic material (e.g., unmodified

polypropylene).

The stand-off layer functions to permit the passage of aqueous liquid from the membrane filter to the absorbent layer during the initial period in which over half of the aqueous liquid deposited into the first compartment passes into the second compartment, while restricting diffusion of nutrient from the first compartment to the second compartment while the device is being incubated to facilitate microbial colony growth.

Suitable materials for use as the stand-off layer include, for example nonwoven fabrics comprising polypropylene; polyethylene; polyethylene terephthalate; a blend of polyethylene terephthalate and cellulose; a blend of polyethylene terephthalate and rayon; and mixtures thereof. Advantageously, devices comprising the stand-off layer can include dry nutrients coated on the first wall portion of the pouch and can retain enough nutrients in the hydrated cold water- soluble gelling agent to support growth of the target microorganisms in the hydrated nutrient gel.

In a third aspect, a method of detecting and enumerating at least one microorganism in a sample is provided. The method includes providing a device according to the first aspect, separating the first layer from the second layer, adding a predetermined volume of a sample containing at least one microorganism onto the first hydrogel-forming composition to form an inoculated device, contacting the first layer back to the second layer, incubating the inoculated device, and detecting the presence or an absence of a colony of the target microorganism in the device. Any of the devices described in detail above with respect to the first aspect are suitable for use in the methods of the third aspect.

For instance, the use of the devices of the present invention for detecting and enumerating microorganisms can be discussed with specific reference to the device of FIGS. 1 and 3. In certain embodiments, the second layer 22 acts as a cover sheet and is pulled back, and then a

predetermined quantity of water or an aqueous test sample is placed on the first layer 12 of the body member 11. The particles of the gelling agent 16 adhered to the first layer 12 by the adhesive 14 are quickly hydrated or dissolved and a gel is formed. The second layer 22 is then replaced over the first layer 12 carefully, in order to minimize entrapment of air bubbles. The device is then incubated for a predetermined period of time. The inoculated device typically includes incubating for up to about 48 hours, or up to about 24 hours, or even up to about 14 hours. Any bacterial colonies growing in the medium can be detected and optionally enumerated (e.g., counted) through the transparent cover film. In some embodiments, the microorganisms can be counted using an automated system, such as an automated colony counter.

In a fourth aspect another method of detecting and enumerating at least one microorganism in a sample is provided. The method includes providing a device according to the second aspect, placing a predetermined volume of aqueous sample into the first compartment of the device, sealing the sample port, incubating the device, and detecting the presence or an absence of a colony of the target microorganism in the device. Any of the devices described in detail above with respect to the second aspect are suitable for use in the methods of the fourth aspect.

The method of the fourth aspect comprises a step of placing a predetermined volume of aqueous sample into the first compartment of the device of any one of the embodiments of the present disclosure. The aqueous sample can be any filterable liquid sample to be tested for presence of a target microorganism. The method is particularly useful for water samples that are suspected of containing relatively low concentrations (e.g., less than or equal to 10

microorganisms per milliliter, less than or equal to 1 microorganism per milliliter, less than or equal to 0.1 microorganisms per milliliter, less than or equal to 0.01 microorganism per milliliter,) of target microorganisms. Placing a predetermined volume of aqueous sample into the first compartment of the device comprises placing the predetermined volume into the device (e.g., via pipetting, pouring, injecting, or the like) through the sealable sample port.

The method further comprises a step of sealing the sample port. The procedure for sealing the sample port will depend upon the particular sealable sample port that is present in the device used in the method. For example, if the device 503 of FIGS. 11-13 is used in the method, sealing the sample port comprises removing the release liner 518 to expose an adhesive disposed on the first wall portion 510 and then contacting the adhesive on the first wall portion with the second wall portion to form a waterproof seal that closes the opening of the pouch.

For example, if the device 502 of FIG. 10 is used in the method, sealing the sample port comprises screwing the cap back onto the sample port, thereby forming a waterproof seal.

For example, if a device comprising an elastically-deformable pierceable septum (not shown) is used in the method, sealing the sample port will spontaneously occur as the pipet or needle used to introduce the sample into the device is withdrawn from the septum.

In any embodiment of the method, air may be expelled (e.g., manually, by squeezing) from the pouch via the sealable sample port before and or during the process of forming the waterproof seal.

The method further comprises a step of incubating the device for a period of time at a temperature that facilitates growth and detection of a target microorganism. A person having ordinary skill in the art will recognize the incubation temperature and period of time will depend upon a number of factors (e.g., the target microorganism, nutrients present in the sample, nutrients present in the device, inhibitory agents present in the sample and/or the device) and will adjust the incubation time and temperature accordingly.

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

In any embodiment, after sealing the sample port, the method further comprises laying the outer surface of the first wall portion of the device or laying the outer surface of the second wall portion of the device onto a surface that is substantially perpendicular to gravitational force.

Advantageously, laying the outer surface of its second wall portion of the device onto a surface that is substantially perpendicular to the force of gravity facilitates flow of the sample liquid through the membrane filter by force of gravity. In addition, laying the outer surface of its second wall portion of the device onto a surface that is substantially perpendicular to the force of gravity facilitates contact between the hydrated cold-water-soluble hydrogel-forming composition adhered to the first wall portion and the membrane filter as the liquid passes through the membrane filter from the first compartment to the second compartment.

In any embodiment, the method further comprises passing at least 90%, at least 92%, at least 95%, at least 97% or at least 98% of the predetermined volume from the first compartment to the second compartment. The portion of the predetermined volume that remains in the first compartment is substantially present as part of the gel formed by hydrating the cold-water-soluble first hydrogel-forming composition.

Various embodiments are described that are devices, kits, methods of making the devices, or methods of detecting and enumerating microorganisms.

Embodiment 1 is a microbial detection device. The device includes a body member including a substrate having a first major surface and a second major surface. The device further includes a first adhesive composition adhered to a portion of the first major surface. A number of particles of a substantially dry, first microbial growth nutrient composition are distributed in the first adhesive composition, and a cold-water-soluble first hydrogel-forming composition is adhered to the first adhesive composition. The device also includes a cover sheet attached to the body member, where the cover sheet includes a first major surface facing the body member.

Embodiment 2 is the device of embodiment 1, further including a second adhesive composition adhered to a portion of the first major surface of the cover sheet.

Embodiment 3 is the device of embodiment 2, further including a plurality of particles of a substantially dry, second microbial growth nutrient composition distributed in the second adhesive composition.

Embodiment 4 is the device of embodiment 3, wherein the second microbial growth nutrient composition is present in the second adhesive composition in an amount of 1 weight percent or more of the total second adhesive composition.

Embodiment 5 is the device of embodiment 3 or embodiment 4, wherein the plurality of particles of the second microbial growth composition include a plurality of clusters of particles of the second microbial growth composition.

Embodiment 6 is the device of embodiment 5, wherein an average length of the plurality of clusters of particles of the second microbial growth composition is 250 micrometers or less.

Embodiment 7 is the device of embodiment 5 or embodiment 6, wherein an average length of the plurality of clusters of particles of the second microbial growth composition ranges from 10 micrometers to 100 micrometers, inclusive.

Embodiment 8 is the device of any of embodiments 2 to 7, wherein the second adhesive composition includes at least one indicator agent.

Embodiment 9 is the device of any of embodiments 2 to 8, wherein the second adhesive composition includes a pressure sensitive adhesive.

Embodiment 10 is the device of any of embodiments 2 to 9, wherein the second adhesive composition includes a solvent based adhesive.

Embodiment 11 is the device of any of embodiments 2 to 10, wherein the second adhesive composition includes an alkyl acrylate copolymer.

Embodiment 12 is the device of any of embodiments 1 to 11, wherein the first microbial growth nutrient composition is present in the first adhesive composition in an amount of 1 weight percent or more of the combined first adhesive composition and the first microbial growth nutrient composition.

Embodiment 13 is the device of any of embodiments 1 to 12, wherein the plurality of particles of the first microbial growth composition include a plurality of clusters of particles of the first microbial growth composition Embodiment 14 is the device of embodiment 13, wherein an average length of the plurality of clusters of particles of the first microbial growth composition is 250 micrometers or less.

Embodiment 15 is the device of embodiment 13 or embodiment 14, wherein an average length of the plurality of clusters of the first microbial growth composition ranges from 10 micrometers to 100 micrometers, inclusive.

Embodiment 16 is the device of any of embodiments 1 to 15, wherein the first adhesive composition includes at least one indicator agent.

Embodiment 17 is the device of any of embodiments 1 to 16, wherein the first adhesive composition includes a pressure sensitive adhesive.

Embodiment 18 is the device of any of embodiments 1 to 17, wherein the first adhesive composition includes a solvent based adhesive.

Embodiment 19 is the device of any of embodiments 1 to 18, wherein the first adhesive composition includes an alkyl acrylate copolymer.

Embodiment 20 is the device of any of embodiments 1 to 17, wherein the first microbial growth nutrient composition is present in the first adhesive composition in an amount of 1 weight percent or more of the total first adhesive composition.

Embodiment 21 is the device of any of embodiments 1 to 18, wherein the first microbial growth nutrient composition includes at least one nutrient selected from the group consisting of a meat peptone, a casein peptone, a gelatin peptone, a soy peptone, a beef extract, a yeast extract, lactose, glucose, dextrose, tryptose, galactose, tryptone, a fat, a mineral, or a vitamin.

Embodiment 22 is the device of any of embodiments 1 to 21, wherein the cold-water- soluble first hydrogel-forming composition includes a cold-water-soluble gelling agent selected from the group consisting of alginate, carboxymethyl cellulose, tara gum, hydroxyethyl cellulose, hydroxypropyl methylcellulose, guar gum, locust bean gum, xanthan gum, polyacrylamide, polyurethane, polyethylene oxides, and combinations thereof.

Embodiment 23 is a microbial detection device. The microbial detection device includes a water-proof pouch including a first wall portion having an inner surface and an outer surface, a second wall portion having an inner surface and an outer surface, and a porous membrane filter disposed in the pouch between the inner surface of the first wall portion and the inner surface of the second wall portion. The membrane filter has a first major surface and a second major surface opposite the first major surface. The water-proof pouch further includes a first compartment defined in part by the inner surface of the first wall portion and defined in part by the first major surface of the membrane filter and a sealable sample port that provides access to deposit a liquid into the first compartment. Additionally, the water-proof pouch includes a second compartment defined in part by the inner surface of the second wall portion and defined in part by the second major surface of the membrane filter, and an absorbent pad disposed in the second compartment. The membrane filter permits passage of aqueous liquids from the first compartment to the second compartment and prevents passage of particles of a predetermined size from the first compartment to the second compartment. The water-proof pouch also includes an adhesive composition adhered to a portion of the pouch in the first compartment, a number of particles of a substantially dry, first microbial growth nutrient composition distributed in the adhesive composition, and a cold-water-soluble hydrogel-forming composition adhered to the adhesive composition.

Embodiment 24 is the device of embodiment 23, wherein the pouch includes a deformable first wall portion disposed opposite the membrane filter in the first compartment and the adhesive composition is disposed on the first wall portion.

Embodiment 25 is the device of embodiment 23, wherein the membrane filter is coupled to a frame, wherein the frame comprises an aperture through which liquid passes from the first compartment into the membrane filter, wherein the aperture defines a first area, and wherein the adhesive composition disposed on the pouch defines a second area that is greater than or equal to the first area.

Embodiment 26 is the device of any of embodiments 23 to 25, wherein the device is dimensioned to receive a liquid sample having a volume between 25 mL and 150 mL, inclusive.

Embodiment 27 is the device of any of embodiments 23 to 26, wherein the adhesive composition includes at least one indicator agent.

Embodiment 28 is the device of any of embodiments 23 to 27, wherein the adhesive composition includes a pressure sensitive adhesive.

Embodiment 29 is the device of any of embodiments 23 to 28, wherein the adhesive composition includes a solvent based adhesive.

Embodiment 30 is the device of any of embodiments 23 to 29, wherein the adhesive composition includes an alkyl acrylate copolymer.

Embodiment 31 is the device of any of embodiments 23 to 30, wherein the plurality of particles of the first microbial growth composition include a plurality of clusters of particles of the first microbial growth composition

Embodiment 32 is the device of embodiment 31, wherein an average length of the plurality of clusters of particles of the microbial growth composition is 250 micrometers or less.

Embodiment 33 is the device of claim 31 or claim 32, wherein an average length of the plurality of clusters of the microbial growth composition ranges from 10 micrometers to 100 micrometers, inclusive. Embodiment 34 is the device of any of embodiments 23 to 33, wherein the microbial growth nutrient composition is present in the adhesive composition in an amount of 1 weight percent or more of the total adhesive composition.

Embodiment 35 is the device of any of embodiments 23 to 34, wherein the microbial growth nutrient composition includes at least one nutrient selected from the group consisting of a meat peptone, a casein peptone, a gelatin peptone, a soy peptone, a beef extract, a yeast extract, lactose, glucose, dextrose, tryptose, galactose, tryptone, a fat, a mineral, or a vitamin.

Embodiment 36 is the device of any of embodiments 23 to 35, wherein the cold- water- soluble hydrogel-forming composition includes a cold-water-soluble gelling agent selected from the group consisting of alginate, carboxymethyl cellulose, tara gum, hydroxyethyl cellulose, hydroxypropyl methylcellulose, guar gum, locust bean gum, xanthan gum, polyacrylamide, polyurethane, polyethylene oxides, and combinations thereof.

Embodiment 37 is a method for detecting and enumerating at least one microorganism in a sample. The method includes providing a device according to any of embodiments 1 to 22, separating the first layer from the second layer, and adding a predetermined volume of a sample containing at least one microorganism onto the first hydrogel-forming composition to form an inoculated device. The method further includes placing the first layer back in contact with the second layer, incubating the inoculated device, and detecting the presence or an absence of a colony of the target microorganism in the device.

Embodiment 38 is a method for detecting and enumerating at least one microorganism in a sample. The method includes providing a device according to any of embodiments 23 to 36, placing a predetermined volume of aqueous sample into the first compartment of the device, sealing the sample port, incubating the device, and detecting the presence or an absence of a colony of the target microorganism in the device.

Examples

Objects and advantages of this invention are further illustrated by the following examples, but the particular materials and amounts thereof recited in these examples, as well as other conditions and details, should not be construed to unduly limit this invention. These examples are for illustrative purposes only and are not meant to be limiting on the scope of the appended claims.

Materials

Unless otherwise noted, all parts, percentages, ratios, etc., in the examples and in the remainder of the specification are by weight. R2a broth powder was obtained from HIMEDIA Laboratories (Mumbai, India). The manufacturer reported the composition of the R2a broth powder to be: casein acid hydroxylase (16 weight%); yeast extract (16 weight%); proteose peptone (16 weight%); dextrose (16 weight%); starch, soluble (16 weight%); dipotassium phosphate (9.6 weight%); magnesium sulphate (0.8 weight%); sodium pyruvate (9.6 weight%).

Guar gum (Meyprogat 150) was obtained from Danisco (Copenhagen, Denmerk).

BACTO tryptic soy broth (TSB) was obtained from the Becton, Dickinson Company (Franklin Lakes, NJ).

2,3,5-triphenyl tetrazolium chloride (TTC) was obtained from the Sigma-Aldrich

Corporation (St. Louis, MO).

Butterfield's buffer was obtained from the 3M Corporation (St. Paul, MN).

MELINEX 454 PET film (3 mil (0.076 mm) thick) was obtained from Tekra (A Division of EIS) Incorporated (New Berlin, WI).

Incubation and Inoculation

The bacterial strains listed in Table 1 were obtained from MICROBIOLOGICS,

Incorporated (St. Cloud, MN). The bacterial strains used in Example 1 were individually cultured by incubating overnight in tryptic soy broth (TSB) at 30 °C without shaking. For Examples 2-6, the Escherichia coli (ATCC 25922) and the Pseudomonas aeruginosa (ATCC 15442) samples were individually cultured by incubating overnight in tryptic soy broth (TSB) at 37 °C with shaking at 250 rpm (New Brunswick™ INNOVA 44 Incubator Shaker, Eppendorf AG, Germany). For all Examples, inoculums were prepared by serially diluting each culture sample in

Butterfield's Buffer (10-fold dilutions) to yield final concentrations that provided colony forming unit (cfu) counts of about 10-300 cfu per ImL of inoculum (this was typically the 10 ^"7 dilution).

Table 1. Bacterial Strains

Staphylococcus aureus subsp. aureus (ATCC 6538)

Lactococcus lactis subsp. cremoris (ATCC 19257)

Kocuria rhizophila (ATCC 51820)

Microbacterium esteraromaticum (ATCC 51822)

Bacillus subtilis subsp. spizizenii (ATCC 6633)

Staphylococcus aureus subsp. aureus (ATCC 25923)

Escherichia coli (ATCC 51813)

Streptococcus agalactiae (ATCC 27956)

Escherichia coli (ATCC 25922)

Chryseobacterium shigense (ATCC 51823)

Acinetobacter sp. (ATCC 51819)

Pseudomonas sp. (ATCC 51821)

Pseudomonas aeruginosa (ATCC 15442)

Example 1.

Microbial detection devices according to the device of FIG. 1A were constructed. A nutrient coating formulation was prepared by adding R2a broth powder to an

isoctylacrylate/acrylamide (96/4 weight ratio) pressure sensitive adhesive formulation (about 22- 25% solids in a heptane and ethyl acetate solution) at a powder concentration of 10.28 % by weight. The bottle containing the coating formulation was mixed at 40 rpm for 4 hours using a bottle roller. The formulation was then notch bar coated by hand onto one side of a PET film substrate (3 mil (0.076 mm) thick) using a 6 mil (0.15 mm) gap setting. The coated film was dried in an oven at 68 °C for 10 minutes. The adhesive coated side of the substrate was then was powder coated with guar gum. The powder was evenly applied and excess powder was removed from the adhesive layer by hand shaking. The resulting powder coated substrate was next and cut into 76 mm wide by 102 mm long sections that were used as the body member of the device.

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 coated on one side with an isooctyl acrylate/acrylic acid (98/2 weight ratio) pressure-sensitive adhesive coating formulation containing TTC as described in Example 4 of U.S. Patent No. 5,409,838. The adhesive layer was subsequently powder coated with guar gum. The powder was evenly applied and excess powder was removed from the adhesive layer. The cover sheet was cut to match the dimensions of the body member and then attached to the body member in a hinge-like fashion along one edge (76 mm edge) using double sided adhesive tape. For each device, the cover sheet and the body member were oriented so that the coated surface of the cover sheet faced the coated surface of the body member.

The detection devices were inoculated with a single microbial sample selected from Table 1. The cover sheet of the device was lifted and 1 mL of the inoculum (i.e. final dilution as described above) was added by pipet to the exposed coated surface of the body member. The cover sheet was replaced and the sample was uniformly spread by applying downward pressure with a 3M PETRIFILM Flat Spreader (3M Corporation, St. Paul, MN). The inoculated devices were incubated at 30 °C for 40 hours. The red-colored colonies were counted by visual examination at the end of the incubation period. As comparative examples, commercially available PETRIFILM Aerobic Count (AC) Plates (3M Corporation) were individually inoculated, incubated, and counted in the same manner as with the detection devices. The results are presented in Table 2.

Example 2.

Microbial detection devices according to the device of FIG. 1A were constructed. A nutrient coating formulation was prepared by adding tryptic soy broth (TSB) powder to an isoctylacrylate/acrylamide (96/4 weight ratio) pressure sensitive adhesive formulation (about 22- 25% solids in a heptane and ethyl acetate solution) at a powder concentration of 5.14 % by weight. The bottle containing the coating formulation was mixed at 40 rpm for 4 hours using a bottle roller. The formulation was then notch bar coated by hand onto one side of a PET film substrate (3 mil (0.076 mm) thick) using a 12 mil (0.30 mm) gap setting. The coated film was dried in an oven at 68 °C for 10 minutes. The adhesive coated side of the substrate was then was powder coated with guar gum. The powder was evenly applied and excess powder was removed from the adhesive layer by hand shaking. The resulting powder coated substrate was next and cut into 76 mm wide by 102 mm long sections that were used as the body member of the device.

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 coated on one side with an isooctyl acrylate/acrylic acid (98/2 weight ratio) pressure-sensitive adhesive coating formulation containing TTC as described in Example 4 of U.S. Patent No. 5,409,838. The adhesive layer was subsequently powder coated with guar gum. The powder was evenly applied and excess powder was removed from the adhesive layer. The cover sheet was cut to match the dimensions of the body member and then attached to the body member in a hinge-like fashion along one edge (76 mm edge) using double sided adhesive tape. For each device, the cover sheet and the body member were oriented so that the coated surface of the cover sheet faced the coated surface of the body member.

A finished detection device was inoculated with Escherichia coli (ATCC 25922) (Table 1). The cover sheet of the device was lifted and 1 mL of the inoculum (i.e. final dilution as described above) was added by pipet to the exposed coated surface of the body member. The cover sheet was replaced and the sample was uniformly spread by applying downward pressure with a 3M PETRIFILM Flat Spreader (3M Corporation, St. Paul, MN). The inoculated devices were incubated at 37 °C for 18 hours. The red-colored colonies were counted by visual examination at the end of the incubation period. As comparative examples, commercially available PETRIFILM Aerobic Count (AC) Plates were individually inoculated, incubated, and counted in the same manner as with the detection devices. The results are presented in Table 3.

Example 3.

A Microbial detection device was constructed according to the procedure described in

Example 2 with the exception that gap setting for the coating step was 6 mil (0.15 mm) instead of 12 mil (0.30 mm). The device was inoculated (with Escherichia coli (ATCC 25922), incubated, and counted according to the procedure described in Example 2. The results are reported in Table 3. Example 4.

A Microbial detection device was constructed according to the procedure described in Example 2 with the exception that the TSB in the nutrient coating formulation was 10.28% by weight, instead of 5.14% by weight. The device was inoculated (with Escherichia coli (ATCC 25922), incubated, and counted according to the procedure described in Example 2. The results are reported in Table 3.

Example 5.

A Microbial detection device was constructed according to the procedure described in Example 2 with the exception that 1) the TSB in the nutrient coating formulation was 10.28% by weight, instead of 5.14% by weight, and 2) the gap setting for the coating step was 6 mil (0.15 mm) instead of 12 mil (0.30 mm). The device was inoculated (with Escherichia coli (ATCC 25922), incubated, and counted according to the procedure described in Example 2. The results are reported in Table 3.

Table 3. Colony (cfu) Counts for Escherichia coli (ATCC 25922) Using the Devices of Examples 2-5

Example 6.

A Microbial detection device was constructed according to the procedure described in

Example 2 with the exception that the bottle containing the coating formulation was mixed at 40 rpm for 18 hours using a bottle roller.

A finished detection device was inoculated with Pseudomonas aeruginosa (ATCC 15442) (Table 1). The cover sheet of the device was lifted and 1 mL of the inoculum (i.e. final dilution as described above) was added by pipet to the exposed coated surface of the body member. The cover sheet was replaced and the sample was uniformly spread by applying downward pressure with a 3M PETRIFILM Flat Spreader (3M Corporation, St. Paul, MN). The inoculated device was incubated at 37 °C for 40 hours. The red-colored colonies were counted by visual examination at the end of the incubation period. As a comparative example, a commercially available

PETRIFILM Aerobic Count (AC) Plate was inoculated, incubated, and counted in the same manner as with the detection device. The results are reported in Table 4. Table 4. Colony (cfu) Counts for Pseudomonas aeruginosa (ATCC 15442) Using the Device of Example 6.

While the specification has described in detail certain exemplary embodiments, it will be appreciated that those skilled in the art, upon attaining an understanding of the foregoing, may readily conceive of alterations to, variations of, and equivalents to these embodiments.

Furthermore, all publications and patents referenced herein are incorporated by reference in their entirety to the same extent as if each individual publication or patent was specifically and individually indicated to be incorporated by reference. Various exemplary embodiments have been described. These and other embodiments are within the scope of the following claims.