阅读说明：本技术 用于生长微生物的装置 (Device for growing microorganisms ) 是由 亚历克西斯·J·扬 库尔特·J·霍尔沃森 史蒂文·P·斯旺森 埃文·D·布鲁蒂内尔 卡尔布· 于 2020-05-29 设计创作，主要内容包括：本发明提供了一种用于生长微生物的装置。该装置包括主体构件,所述主体构件包括具有上表面和下表面的自支承防水基材；疏水间隔元件,所述疏水间隔元件粘附到形成侧壁的所述基材的所述上表面,以保持预定量的液体与所述基材接触,其中所述疏水间隔元件中具有孔；流体控制膜,所述流体控制膜处于所述疏水间隔元件的所述孔中；覆盖片,所述覆盖片具有面向内的表面和面向外的表面,所述覆盖片粘附到所述主体构件的至少一部分；以及设置在所述覆盖片的内表面的一部分上的基本上干燥的第一微生物生长营养物质组合物；粘附到所述第一微生物生长营养物质组合物的第一粘合剂组合物；以及粘附到所述第一粘合剂组合物的冷水可溶性第一水凝胶形成组合物。(The present invention provides an apparatus for growing microorganisms. The device includes a body member comprising a self-supporting waterproof substrate having an upper surface and a lower surface; a hydrophobic spacer element adhered to the upper surface of the substrate forming a sidewall to maintain a predetermined amount of liquid in contact with the substrate, wherein the hydrophobic spacer element has an aperture therein; a fluid control membrane in the pores of the hydrophobic spacer element; a cover sheet having an inwardly facing surface and an outwardly facing surface, the cover sheet adhered to at least a portion of the body member; and a substantially dry first microorganism growth nutrient composition disposed on a portion of the interior surface of the cover sheet; a first binder composition adhered to the first microorganism growth nutrient composition; and a cold water soluble first hydrogel-forming composition adhered to the first adhesive composition.)

1. An apparatus for growing microorganisms, the apparatus comprising:

a body member comprising a self-supporting waterproof substrate having an upper surface and a lower surface;

a fluid control membrane on the upper surface of the self-supporting waterproof substrate;

a cover sheet having an inwardly facing surface and an outwardly facing surface, the cover sheet adhered to at least a portion of the body member; and

a substantially dry first microorganism growth nutrient composition disposed on a portion of an interior surface of the cover sheet;

a first binder composition adhered to the first microorganism growth nutrient composition; and

a cold water soluble first hydrogel-forming composition adhered to the first adhesive composition.

2. The device of claim 1, wherein the fluid control membrane comprises a plurality of microreplicated structures.

3. The device of any one of claims 1-2, wherein the fluid control membrane comprises a plurality of fluid control channels extending along a channel longitudinal axis, each of the fluid control channels comprising a surface and configured to allow capillary movement of liquid in the channel.

4. A device according to any one of claims 1 to 3, wherein the fluid control membrane comprises a hydrophilic surface treatment covalently bonded to at least a portion of the surface of the fluid control channel.

5. The device of any one of claims 1 to 4, wherein the fluid control membrane comprises a non-covalent hydrophilic surface treatment disposed on at least a portion of the surface of the fluid control channel.

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

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

8. The device of any one of claims 1 to 7, wherein the spacer element comprises a sheet of hydrophobic foam.

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

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

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

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

13. The device of any one of claims 1 to 12, wherein the gelling agent is selected from the group consisting of a polysaccharide gum, guar gum, locust bean gum, carboxymethyl cellulose, hydroxyethyl cellulose, and algin.

14. The device of any one of claims 1 to 13, further comprising a hydrophobic spacer element adhered to the upper surface of the substrate forming the sidewall to maintain a predetermined amount of liquid in contact with the substrate, wherein the hydrophobic spacer element has an aperture therein, and wherein the fluid control membrane is in the aperture of the hydrophobic spacer element.

15. A method, the method comprising:

providing an apparatus according to any one of claims 1 to 14;

adding a predetermined volume of a sample containing at least one microorganism to the device to form an inoculated device;

contacting the cover sheet with the self-supporting water-repellent substrate;

incubating the inoculated device; and

detecting the presence or absence of colonies of the target microorganism in the device.

Background

Various culture devices have been developed. As one example, culture devices have been developed by 3M Company (3M Company, hereinafter "3M") of st paul, Minnesota. Specifically, the culture apparatus is sold by 3M under the trade name PETRIFILM plate. The culture device can be used to facilitate rapid growth and detection of microorganisms often associated with food contamination, including, for example, aerobic bacteria, e.coli, coliform, enterobacteria, yeast, mold, staphylococcus aureus, listeria, campylobacter, and the like. For example, the use of PETRIFILM plates or other growth media can simplify bacterial testing of food samples.

The culture device can be used to count or identify the presence of bacteria, thereby allowing improved assays (for food testing) or correct diagnosis (for medical use). In other applications, culture devices may be used to rapidly grow microorganisms in laboratory samples, e.g., for experimental purposes.

Disclosure of Invention

Devices and methods for propagating or storing microorganisms are provided.

Accordingly, in one aspect, the present disclosure provides an apparatus for growing microorganisms. The device includes a body member comprising a self-supporting waterproof substrate having an upper surface and a lower surface; a hydrophobic spacer element adhered to the upper surface of the substrate forming a sidewall to maintain a predetermined amount of liquid in contact with the substrate, wherein the hydrophobic spacer element has an aperture therein; a fluid control membrane in the pores of the hydrophobic spacer element; a cover sheet having an inwardly facing surface and an outwardly facing surface, the cover sheet adhered to at least a portion of the body member; and a substantially dry first microorganism growth nutrient composition disposed on a portion of the interior surface of the cover sheet; a first binder composition adhered to the first microorganism growth nutrient composition; and a cold water soluble first hydrogel-forming composition adhered to the first adhesive composition.

In another aspect, the present disclosure provides a method. The method includes providing an apparatus of the present disclosure; adding a predetermined volume of a sample containing at least one microorganism to the device to form an inoculated device; contacting the cover sheet with the self-supporting water-repellent substrate; incubating the inoculated device; and detecting the presence or absence of colonies of the target microorganism in the device.

Various aspects and advantages of exemplary embodiments of the present disclosure have been summarized. The above summary is not intended to describe each illustrated embodiment or every implementation of the present disclosure. Additional features and advantages are disclosed in the following detailed description. The following drawings and detailed description more particularly exemplify certain embodiments using the principles disclosed herein.

Definition of

For the following defined terms, all definitions shall prevail throughout the specification, including the claims, unless a different definition is provided in the claims or elsewhere in the specification based on a specific reference to a modified form of the term as used in the following definition:

the terms "about" or "approximately" with respect to a numerical value or shape mean +/-5% of the numerical value or property or characteristic, but also expressly include any narrow range and exact numerical value within +/-5% of the numerical value or property or characteristic. For example, a temperature of "about" 100 ℃ refers to a temperature from 95 ℃ to 105 ℃, but also expressly includes any narrower temperature range or even a single temperature within that range, including, for example, a temperature of exactly 100 ℃. For example, a viscosity of "about" 1Pa-sec refers to a viscosity from 0.95Pa-sec to 1.05Pa-sec, but also expressly includes a viscosity of exactly 1 Pa-sec. Similarly, a perimeter that is "substantially square" is intended to describe a geometric shape having four lateral edges, wherein the length of each lateral edge is 95% to 105% of the length of any other lateral edge, but also encompasses geometric shapes wherein each lateral edge has exactly the same length.

The term "substantially" with respect to a property or characteristic means that the property or characteristic exhibits an extent greater than the opposite face of the property or characteristic. For example, a substrate that is "substantially" transparent refers to a substrate that transmits more radiation (e.g., visible light) than it does not. Thus, a substrate that transmits more than 50% of the visible light incident on its surface is substantially transparent, but a substrate that transmits 50% or less of the visible light incident on its surface is not substantially transparent.

The terms "a", "an" and "the" are used interchangeably, wherein "at least one" means one or more of the recited element(s).

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.

"agglomeration" refers to a weak association of primary particles or aggregated particles that are held together, typically by charge or polarity. For example, shear forces encountered when the agglomerate is dispersed in a liquid generally break the agglomerate down into smaller objects. The terms "aggregated" and "aggregate" refer to a strong association of primary particles that are typically bonded together by, for example, residual chemical treatment, covalent chemical bonding, or ionic chemical bonding. Further decomposition of aggregates into smaller entities is difficult to achieve.

"Cold water soluble" refers to materials that form aqueous solutions at room temperature (i.e., about 25℃.).

"hydrophobic" refers to a material that exhibits a water contact angle of 90 ° or greater on a surface.

By "opaque" is meant a substrate having a light transmission of at most 10%.

"powder" refers to finely divided particulate material having an average diameter in the range of 0.1 microns to 400 microns.

"reconstituted medium" refers to a solution or gel formed by reconstituting a cold water-soluble powder with an aqueous liquid.

As used herein, "substantially impermeable to microorganisms and water vapor" refers to a cover sheet that: it prevents undesirable contamination and hydration of the underlying layers of cold water soluble powder during shipping, storage and use of the thin film culture device(s) and avoids drying out of the reconstituted medium, making the reconstituted medium suitable for supporting growth of microorganisms during incubation.

As used herein, "substantially free of water" indicates that the water content is no greater than about the water content of the surrounding environment.

As used herein, "test sample" refers to a component or fraction taken from a food product, a human or animal test subject, a pharmaceutical or cosmetic, soil, water, air, other environmental source, or any other source from which the presence and optionally the count of aerobic and/or aerotolerant bacteria can be determined. The test sample can be taken from the source using techniques known to those skilled in the art, including, for example, pouring, pipetting, wiping, filtering, and contacting. In addition, the test sample may be subjected to various sample preparation processes known in the art, including, for example, blending, homogenization, enrichment, selective enrichment, or dilution.

By "transparent" is meant a substrate having a light transmission of at least 90%.

Drawings

The disclosure may be more completely understood in consideration of the following detailed description of various embodiments of the disclosure in connection with the accompanying drawings, in which:

fig. 1 is a top perspective view, partially in section, of another exemplary device according to the present disclosure.

Fig. 2 is a schematic view of a channeled microstructured surface of the present disclosure having an amount of fluid thereon.

While the above-identified drawing figures, which may not be drawn to scale, illustrate various embodiments of the disclosure, other embodiments are also contemplated, as noted in the detailed description. In all cases, this disclosure describes the presently disclosed invention by way of representation of exemplary embodiments and not by way of express limitations. It should be understood that numerous other modifications and embodiments can be devised by those skilled in the art that will fall within the scope and spirit of the principles of this disclosure.

Detailed Description

Before any embodiments of the disclosure are explained in detail, it is to be understood that the invention is not limited in its application to the details of the use, construction and arrangement of components set forth in the following description. The invention is capable of other embodiments and of being practiced or of being carried out in various ways that will become apparent to those skilled in the art upon reading this disclosure. Also, it is to be understood that the phraseology and terminology used herein is for the purpose of description and should not be regarded as limiting. The use of "including," "comprising," or "having" and variations thereof herein is meant to encompass the items listed thereafter and equivalents thereof as well as additional items. It is to be understood that other embodiments may be utilized and structural or logical changes may be made without departing from the scope of the present disclosure.

As used in this specification, the recitation of numerical ranges by endpoints includes all numbers subsumed within that range (e.g. 1 to 5 includes 1, 1.5, 2, 2.75, 3, 3.8, 4, and 5, etc.).

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 list 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.

Fig. 1 shows an exemplary embodiment of an apparatus for culturing microorganisms. The device 10 includes a body member 11 and a cover sheet 22 attached to at least a portion of the body member 11, the body member 11 including a substrate 12 having a first major surface 12a (e.g., an upper surface) and a second major surface 12b (e.g., a lower surface), wherein the cover sheet 22 includes a first major surface 22a (e.g., an inner surface) that faces the body member 11. The device 10 further includes a substantially dry first microorganism-growth nutrient composition 24 disposed on a portion of the first major surface 22a of the covering sheet 22, a first adhesive composition 26 adhered to the first microorganism-growth nutrient composition 24, and a cold-water soluble first hydrogel-forming composition 28 adhered to the first adhesive composition 26. Preferably, the device further comprises an optional hydrophobic spacer element 19 disposed on the first major surface 12a of the substrate 12. Generally, the spacer elements 19 comprise a water insoluble substrate defining pores or apertures 20. The spacer element 19 may be a sheet of hydrophobic foam, such as a sheet of polystyrene foam or a sheet of polyethylene foam. In use, the user separates the cover sheet 22 from the substrate 12 sufficiently to add a quantity of a sample containing at least one microorganism within the well or aperture 20 defined by the spacer 19, places the cover sheet 22 back in contact with the substrate 12 to form an inoculated device, and incubates the inoculated device. The area on the first major surface 12a of the substrate 12 defined by the aperture 20 may also be referred to as the sample-receiving zone 17. A fluid control membrane 18 may be disposed in the pores of the hydrophobic spacer member 19 and on the first major surface 12a of the substrate 12. The device 10 may further include a second adhesive composition 13 adhered to the upper surface 12a of the self-supporting, water-repellent substrate 12, with the second adhesive composition 13 being between the hydrophobic spacer element 19 and the substrate 12.

The apertures 20 may have any shape. Non-limiting examples of useful shapes for aperture 20 include square, rectangular, circular, elliptical, polygonal, hexagonal, and octagonal. The area of the sample-receiving zone (and the aperture 20) may be selected according to, for example, the volume of sample (e.g., aqueous liquid) to be deposited in that zone. In any embodiment, the sample receiving area is about 10cm in area for 0.5-3 ml of sample²Or about 15cm². In any embodiment, the sample receiving area is about 20cm in area for a sample volume of 1-5 ml²About 25cm, of²About 30cm, from the bottom²About 31cm²Or about 25-35cm²。

The substrate 12 is waterproof and optionally is a self-supporting waterproof substrate. In some embodiments, the substrate 12 is a film of a material such as polyester, polypropylene, silicone, or polystyrene that will not absorb or otherwise be affected by water. Polyester and polypropylene films having a thickness of about 20 microns to about 250 microns and polystyrene films having a thickness of about 380 microns have each been found to be suitable for use as the substrate 12. Other suitable substrates include papers with polyethylene coatings or other water resistant coatings. An example of a suitable polyethylene-coated paper substrate is "schoeler Type MIL" photographic paper (commercially available from schoeler pulski, New York). Substrate 12 may be transparent or opaque depending on whether it is desired to view bacterial colonies through the substrate. In some embodiments, the substrate 12 is printed with a square grid pattern on the second major surface 12b to facilitate counting of bacterial colonies.

The substantially dry first or second microorganism growth nutrient composition may comprise a microorganism growth nutrient composition coated at a weight of 2 milligrams per square inch (mg/in) or greater²)、5mg/in²Or greater, 10mg/in²Or greater, 12mg/in²Or greater, or 15mg/in²Or greater; and the coating weight was 50mg/in²Or less, 45mg/in²Or less, 40mg/in²Or less, 35mg/in²Or less, 30mg/in²Or less, 24mg/in²Or less, 22mg/in²Or less, 20mg/in²Or less, or 18mg/in²Or smaller. One suitable method for applying a microorganism growth nutrient composition to a substrate includes preparing an aqueous solution or suspension comprising at least a microorganism growth nutrient composition, disposing a coating of the solution or suspension on a surface of the substrate, and drying the coating to form a substantially dry microorganism growth nutrient composition. One skilled in the art can select a suitable coating method including, for example, but not limited to, knife coating, gravure coating, curtain coating, air knife coating, spray coating, die coating, draw bar coating, or curtain or roll coating. The coating is optionally dried at elevated temperature (e.g., in the range of 50 ℃ to 100 ℃) or at ambient conditions. In some embodiments, the microbial growth nutrient composition contains 75% or more by weight of microbial growth nutrients, or 80% or more by weight, or 85% or more by weight, or 90% or more by weight, or 95% or more by weight of microbial growth nutrients. Advantageously, in certain embodiments, a greater amount of microorganism growth nutrient may be included in the device as compared to a device in which the microorganism growth nutrient composition powder is applied to the adhesive layer and/or combined with a large amount of cold water soluble gelling agent.

The first adhesive composition or the second adhesive composition may be (substantially) water-insoluble and non-inhibitory to the growth of microorganisms. In some embodiments, the first adhesive composition 16 is sufficiently transparent when wet to allow for observation of bacterial colonies through the adhesive coated film. In some embodiments, the first adhesive composition or the second adhesive composition may be a pressure sensitive adhesive. In some other embodiments, a heat activated adhesive may also be used, wherein a lower melting point material is coated onto a higher melting point material. Water activated adhesives such as mucus may also be useful.

Suitable binders are transparent when wetted with water. As noted above, the adhesive composition is generally water insoluble. In certain embodiments, the adhesive composition comprises a solvent-based adhesive. The first adhesive composition and the second adhesive composition (if present) are typically pressure sensitive adhesives. For example, the adhesive may be a pressure sensitive adhesive, such as a water insoluble adhesive comprising a copolymer of an alkyl acrylate monomer and an alkylamide monomer or a copolymer of an alkyl acrylate monomer and acrylic acid. Preferably, the weight ratio of alkyl acrylate monomer to alkylamide monomer in these copolymers is from about 90:10 to 99:1, more preferably from 94:6 to 98: 2. The alkyl acrylate monomers comprise lower alkyl (C2 to C10) acrylate monomers including, for example, isooctyl acrylate (IOA), 2-ethylhexyl acrylate, butyl acrylate, ethyl acrylate, isoamyl acrylate, and mixtures thereof, while the alkyl amide monomers may include, but are not limited to, Acrylamide (ACM), methacrylamide, N-vinyl pyrrolidone (NVP), N-vinyl caprolactam (NVCL), N-vinyl-2-piperidine, N- (mono or di-lower alkyl (C2 to C5)) (meth) acrylamide, N-methyl (meth) acrylamide, N-dimethyl (meth) acrylamide, or mixtures thereof. Suitable adhesives may also include those described in U.S. Pat. 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. patents 7,695,818 and 7,371,464.

In the present disclosure, cover sheet 22 is typically selected to be transparent to facilitate enumeration of microbial colonies, and is also typically selected to be impermeable to bacteria and have a low moisture vapor transmission rate (i.e., cover sheet 22 prevents undesirable contamination of the dehydrated media during transport, storage, and use of the device, and provides an environment that supports growth of microorganisms during incubation). In some embodiments, the cover sheet 22 has the same characteristics as the substrate 12 (e.g., is waterproof). Cover sheet 22 may be selected to provide the amount of oxygen transmission required for the type of microorganism desired to be cultured. For example, some polyester films have low oxygen permeability (less than 5g/645cm per 25 micron thickness)²24 hours) and is suitable for the cultivation of anaerobic bacteria. On the other hand, some polyethylenes have high oxygen permeability (e.g., about 500g/645cm per 25 micron thickness²24 hours) and is suitable for aerobic organisms. Cover sheet 22Suitable materials include polypropylene, polyester, polyethylene, polystyrene or silicone. In certain embodiments, the cover sheet 22 comprises an oriented polypropylene, such as a biaxially oriented polypropylene, in some exemplary embodiments the oriented polypropylene has a thickness of about 40 microns.

In certain embodiments, the cold water-soluble hydrogel-forming composition contains one or more organic cold water-soluble agents, such as alginates, carboxymethylcellulose, tara gum, hydroxyethylcellulose, hydroxypropylmethylcellulose, guar gum, locust bean gum, xanthan gum, polyacrylamide, polyurethane, polyethylene oxide. Combinations of natural and/or synthetic gellants are contemplated. Preferred gelling agents include guar gum, xanthan gum, and locust bean gum, which can be used alone or in combination with one another in any embodiment. 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 powdered cold water-soluble hydrogel-forming composition may further comprise an inducer, an indicator, or a combination of these.

Fluid control membranes may include those described in US 2017/0045284 a1(Meuler et al).

For example, the fluid control membrane includes a fluid control channel extending along a channel longitudinal axis. Each fluid control channel has a surface and is configured to allow capillary movement of liquid in the channel. In some embodiments, the fluid control membrane may further include a hydrophilic surface treatment covalently bonded to at least a portion of the surface of the fluid control channel. In some other embodiments, the fluid control membrane may have a non-covalent hydrophilic surface treatment, such as a surfactant treatment, disposed on at least a portion of the surface of the fluid control channels. The fluid control film exhibits a percent capillary rise recovery of at least 10%. Typically, the hydrophilic surface treatment comprises functional groups selected from non-zwitterionic sulfonates, non-zwitterionic carboxylates, zwitterionic sulfonates, zwitterionic carboxylates, zwitterionic phosphates, zwitterionic phosphonates, or combinations thereof.

A fluid control film according to the present disclosure includes a microstructured surface having a plurality of microreplicated structures. The fluid control film may have a variety of appearance characteristics. Exemplary fluid control membranes include channels having a V-shaped or rectangular cross-section, and combinations of these, as well as structures having channels, sub-channels (i.e., channels within a channel). Additionally, the features may include microstructured pillars and protrusions.

The channels on the microstructured surface have channel ends. In certain embodiments, the fluid control membrane may include a removal device. The removal device typically draws fluid from the channel adjacent to one channel end. In another embodiment, the removal device withdraws fluid from the channels adjacent to the ends of both channels. The removal device may include an absorbent material disposed in communication with the channel. In one embodiment, the removal device comprises a fluid droplet collector.

Generally, the channels in the microstructures are defined by generally parallel ridges, including a first set of ridges having a first height and a second set of ridges having a second, higher height. An upper portion of each ridge of the second set of ridges may have a lower melting temperature than a lower portion thereof. The channels have a pattern geometry selected from linear, curvilinear, radial, parallel, non-parallel, random, or intersecting.

In some embodiments, the fluid control film has a contact angle of less than 90 °. The contact angle Theta (θ) is the angle between the fluid bead on the surface at the point of contact with the surface and the tangent to the surface and the plane of the surface. A fluid bead with a tangent line perpendicular to the plane of the surface will have a contact angle of 90 deg.. Generally, a solid surface is considered to be wetted by a fluid if the contact angle is 45 ° or less. Surfaces on which a drop of water or an aqueous solution exhibits a contact angle of less than 45 ° are often referred to as "hydrophilic". As used herein, "hydrophilic" is used only to refer to the surface properties of a material, i.e., it is wetted by an aqueous solution, and does not express whether or not the material absorbs an aqueous solution. Thus, a material may be referred to as hydrophilic, regardless of whether the sheet of material is impermeable or permeable to aqueous solutions. Thus, the hydrophilic membrane used in the present patent application may be formed of a membrane prepared from an inherently hydrophilic resin material such as, for example, poly (vinyl alcohol). A fluid that produces a near-zero contact angle on a surface is considered to completely wet the surface. However, polyolefins are generally inherently hydrophobic, and polyolefin films (such as polyethylene or polypropylene) typically have a contact angle with water of greater than 90 °.

Generally, the channels in the microstructures are defined by generally parallel ridges, including a first set of ridges having a first height and a second set of ridges having a second, higher height. An upper portion of each ridge of the second set of ridges may have a lower melting temperature than a lower portion thereof. The channels have a pattern geometry selected from linear, curvilinear, radial, parallel, non-parallel, random, or intersecting.

Fig. 2 is a cross-section of a fluid control membrane 200 according to an example embodiment. The fluid control film 200 includes a fluid control film layer 201 having primary channels 230 and secondary channels 231 defined by primary ridges 220 and secondary ridges 221, wherein the channels 230, 231 and ridges 220, 221 extend along a channel axis (e.g., x-axis) that forms an angle θ with respect to a longitudinal axis of the fluid control film layer 201. Each primary channel 230 is defined by a set of primary ridges 220 (first and second) on either side of the primary channel 230. The main ridge 220 has a height h measured from the bottom surface 230a of the channel 230 to the top surface 220a of the ridge 220_p. In some embodiments, microstructures are disposed within primary channel 230. In some embodiments, the microstructure includes a secondary channel 231 disposed between the first and second major ridges 220 of the primary channel 230. Each secondary channel 231 is associated with at least one secondary ridge 221. The secondary channels 231 may be located between a set of secondary ridges 221 or between a secondary ridge 221 and a primary ridge 220.

Center-to-center distance d between major ridges_prMay range from about 25 microns to about 3000 microns; center-to-center distance d between major ridge and nearest minor ridge_psMay range from about 5 microns to about 350 microns; center-to-center distance d between two minor ridges_ssAnd may range from about 5 microns to about 350 microns. In some cases, the major ridges and/or minor ridges may taper with distance from the base. Distance d between outer surfaces of the main ridge at the base_pbCan range from about 15 microns to about 250 microns, and can taper to a smaller distance d ranging from about 1 micron to about 25 microns_pt. The distance d between the outer surfaces of the minor ridges at the base_sbCan range from about 15 microns to about 250 microns, and can taper to a smaller distance d ranging from about 1 micron to about 25 microns_st. In one example, d_pr0.00898 inches (228 microns), d_ps0.00264 inch (67 microns), d_ss0.00185 inches (47 microns), d_pb0.00251 inches (64 microns), d_pt0.00100 inch (25 microns), d_sb0.00131 inch (33 microns), d_st0.00100 inch (25 microns), h_p0.00784 inches (199 microns), and h_s0.00160 inches (41 microns).

The minor ridge has a height h measured from the bottom surface 230a of the channel 230 to the top surface 221a of the minor ridge 221_s. Height h of main ridge 220_pGenerally greater than the height h of the minor ridge 221_s. In some embodiments, the height of the major ridges is between about 25 microns to about 3000 microns, and the height of the minor ridges is between about 5 microns to about 350 microns. In some embodiments, the minor ridge 221 height h_sHeight h from main ridge 220_pThe ratio of about 1: 5. The major ridges 220 may be designed to provide durability to the fluid control film layer 200 and protection to the minor channels 231, minor ridges, and/or other microstructures disposed between the major ridges 220.

The fluid control film 200 optionally has an adhesive layer 205 disposed on the bottom surface 201a of the fluid control film layer 201. The adhesive layer 205 may allow the fluid control film layer 200 to be attached to some of the outer surfaces 202 to help manage liquid dispersion across the outer surfaces. The combination of adhesive layer 205 and fluid control film layer 201 forms a fluid control strip. The adhesive layer 205 may be continuous or discontinuous.

The fluid control film layer 201 is configured to distribute fluid over the entire surface of the fluid control film layer 201 to facilitate evaporation of the fluid. In some embodiments, the adhesive layer 205 may be or include a hydrophobic material that repels liquid at the interface 202a between the adhesive layer 205 and the outer surface 202, thereby reducing liquid collection at the interface 202 a.

Adhesive layer 205 has a thickness t_aAnd the fluid control membrane layer 201 has a thickness t from the bottom surface 230a of the channels 230, 231 to the bottom surface 201a of the fluid control membrane layer 201_v. In some embodiments, the total thickness t between the bottom surface 230a of the channels 230, 231 and the bottom surface 205a of the adhesive layer 205_v+t_aAnd may be less than about 300 microns, for example, about 225 microns. The total thickness t_v+t_aMay be selected to be small enough to allow liquid to be effectively wicked from the outer surface 202 and into the channels 230, 231 through the channel openings at the edges of the fluid control film layer 201.

A method of detecting and enumerating at least one microorganism in a sample is provided. The method comprises the following steps: providing a device according to the present disclosure, adding a predetermined volume of a sample containing at least one microorganism to the pores 20 of the spacer element 19 to form an inoculated device, contacting the cover sheet with the substrate, incubating the inoculated device, and detecting the presence or absence of colonies of the target microorganism in the device. The cold water-soluble hydrogel-forming composition on the cover sheet hydrates and forms a hydrogel when the aqueous sample is placed in the device, and the hydrogel can self-spread and fill the channels of the flow control membrane. It has been surprisingly found that colonies will form such punctate colonies and do not grow along the channel longitudinal axis of the channel of the fluid control membrane.

The method further includes the step of incubating the device for a period of time at a temperature that facilitates growth and detection of the target microorganism. One of ordinary skill in the art will recognize that the temperature and time of incubation will depend on a variety of factors (e.g., the target microorganism, nutrients present in the sample, nutrients present in the device, inhibitors present in the sample and/or the device), and that the incubation time and temperature will be adjusted accordingly.

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

The following embodiments are intended to illustrate the disclosure, but not to limit it.

Detailed description of the preferred embodiments

Embodiment 1 is an apparatus for growing microorganisms, the apparatus comprising: a body member comprising a self-supporting waterproof substrate having an upper surface and a lower surface; a hydrophobic spacer element adhered to the upper surface of the substrate forming a sidewall to maintain a predetermined amount of liquid in contact with the substrate, wherein the hydrophobic spacer element has an aperture therein; a fluid control membrane in the pores of the hydrophobic spacer element;

a cover sheet having an inwardly facing surface and an outwardly facing surface, the cover sheet adhered to at least a portion of the body member; and a substantially dry first microorganism growth nutrient composition disposed on a portion of the interior surface of the cover sheet; a first binder composition adhered to the first microorganism growth nutrient composition; and a cold water soluble first hydrogel-forming composition adhered to the first adhesive composition.

Embodiment 2 is the device of embodiment 1, wherein the fluid control film comprises a plurality of microreplicated structures.

Embodiment 3 is the device of any one of embodiments 1-2, wherein the fluid control membrane comprises a plurality of fluid control channels extending along a channel longitudinal axis, each of the fluid control channels comprising a surface and configured to allow capillary movement of liquid in the channel.

Embodiment 4 is the device of any of embodiments 1-3, wherein the fluid control membrane includes a hydrophilic surface treatment covalently bonded to at least a portion of the surface of the fluid control channel.

Embodiment 5 is the device of any of embodiments 1-4, wherein the fluid control membrane comprises a non-covalent hydrophilic surface treatment disposed on at least a portion of the surface of the fluid control channel.

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

Embodiment 7 is the device of any one of embodiments 1 to 6, further comprising a second adhesive composition adhered to the upper surface of the self-supporting waterproof substrate, wherein the second adhesive composition is between the hydrophobic spacer elements and the substrate.

Embodiment 8 is the device of any one of embodiments 1 to 7, wherein the spacer element comprises a hydrophobic foam sheet.

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

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

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

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

Embodiment 13 is the device of any one of embodiments 1 to 12, wherein the gelling agent is selected from the group consisting of a polysaccharide gum, guar gum, locust bean gum, carboxymethyl cellulose, hydroxyethyl cellulose, and algin.

Embodiment 14 is a method comprising: providing a device according to any one of embodiments 1 to 13;

adding a predetermined volume of a sample containing at least one microorganism to the device to form an inoculated device; contacting the cover sheet with the self-supporting water-repellent substrate; incubating the inoculated device; and detecting the presence or absence of colonies of the target microorganism in the device.

The following working examples are intended to illustrate the disclosure and are not intended to be limiting.

Examples

Table 1: material

Incubation and inoculation

The bacterial strain escherichia coli (ATCC 25922) was obtained from microbiology Incorporated, st. cloud, MN, santa clara, and was incubated in an inova 44 incubator (New Brunswick Scientific, infield, CT) in pancreatin soybean broth (TSB) overnight at 37 ℃ and 200 rpm. The inoculum was prepared by serial dilution of culture samples with Butterfield buffer (3M Corporation, st. paul, MN), a 3M company of st paul, minnesota. Culture samples were diluted to yield a final concentration of about 50-250 colony forming unit (cfu) counts per 1mL of inoculum.

Preparation example 1 fluid control Membrane manufacture

The fluid control film of fig. 2 was prepared according to the extrusion stamping procedure described in U.S. patent application 20017/0045284(Meuler), which is incorporated by reference in its entirety. Using the indicators of fig. 2, the fluid control films of the examples had the following dimensions: dpr 0.00898 inches (228 microns), dps 0.00264 inches (67 microns), dss 0.00185 inches (47 microns), dpb 0.00251 inches (64 microns), dpt 0.00100 inches (25 microns), dsb 0.00131 inches (33 microns), dst 0.00100 inches (25 microns), hp 0.00784 inches (199 microns), and hs 0.00160 inches (41 microns). The film was made from a low density polyethylene polymer (available from DOW Chemical Company, Midland, MI) under the trade designation "DOW LDPE 9551" from DOW Chemical Company of Midland, michigan).

Preparation example 2 plasma treatment of fluid control film

A silicon-containing film layer [ the formation methods of which are described in U.S. patent nos. 6696157(David) and 8664323(Iyer) and U.S. patent application No. 2013/0229378(Iyer) ] was applied to the fluid control film of preparation example 1 using a Plasma-Therm 3032 batch Plasma reactor, available from Plasma-Therm LLC of st. The instrument was configured for reactive ion etching with a 26 inch low power electrode and central gas pumping. The chamber was aspirated with a roots-type blower (model EH1200, obtained from Edwards Engineering, Burgess Hill, UK) supported by a dry mechanical pump (model iQDP80, obtained from Edwards Engineering) to aspirate the chamber. RF power was delivered by a 3kW, 13.56Mhz solid state generator (RFPP model RF30S, available from Advanced Energy Industries, Fort Collins, CO, coringberg, colorado). The system has a nominal base pressure of 5 mTorr. The flow rate of the gas was controlled by an MKS flow controller (available from MKS Instruments, Andover, MA).

A sample of the fluid control film is secured to a power electrode of the plasma reactor. After pumping to base pressure, gases Tetramethylsilane (TMS) and oxygen (O) were introduced at different flow rates₂) (see Table 2). Once the gas flow was stabilized in the reactor, rf power (1000 watts) was applied to the electrode to generate a plasma. The plasma exposure time also varied (see table 2). After the plasma treatment is completed, the chamber is connected to the chamberThe gas is communicated and the treated fluid control film is removed from the chamber.

Table 2: plasma treatment conditions for making fluid control films A-D

PREPARATION EXAMPLE 3 SURFACE-ACTIVE AGENT-CONTAINING FLUID CONTROL FILM

A fluid control film was prepared as described in preparative example 1, except that 0.5 wt% of the nonionic surfactant TRITON-X100 was incorporated into the low density polyethylene polymer used in the extrusion embossing procedure. The resulting fluid control membrane was designated fluid control membrane E.

Example 1 microbial detection device

A microorganism detection apparatus of the apparatus according to fig. 1 was constructed. For each device, the base material of the body member was a transparent biaxially oriented polypropylene (BOPP) film (1.6 mils (0.04mm) thick and corona treated on both sides) cut into sections 76mm wide by 102mm long. The body member was completed by adhesively laminating a 76mm wide by 102mm long polyethylene film spacer (Optimum Plastics, Bloomer, W) to one side of the substrate. The septum was approximately 20 mils (0.51 mm) thick and contained a circular hole (5.1cm diameter) located near the center of the septum. The circular aperture defines the perimeter of the sample receiving zone of the device. The circular portion of the fluid control film (selected from the fluid control films designated a-E in table 2) was cut and sized to fit in the aperture (5.1cm diameter) and oriented such that the non-microreplicated surface of the film was adhesively laminated to the exposed substrate surface defined by the aperture.

The cover sheet of the device was a clear biaxially oriented polypropylene (BOPP) film (1.6 mil (0.04mm) thick and corona treated on both sides) coated on one side in sequence with a microbial growth nutrient composition, a binder composition, and a guar gum (e.g., cold water soluble hydrogel-forming) composition according to the following procedure.

30g of pancreatin soy broth (TSB) and 500mL of purified water [ obtained from MILLI-Q gradient water purification system (model # ZMDS 6V00Y, Merck Millipore Corporation, Billerica, MA) ] by vigorous mixing (using an air-driven overhead mixer with JIFFY-type mixing impeller)]Until the TSB is completely dissolved to prepare the microbial growth nutrient coating composition. The pH of the resulting solution was 7.3(Mettler-Toledo FE20 FIVEEASY pH meter, Mettler-Toledo LLC, Columbus, OH) from Columbus, Ohio). Guar gum (10g) was added to the nutrient solution and vigorous stirring was continued for about 10 minutes. The resulting solution was knife coated onto one side of a BOPP cover sheet film with a 14 mil (0.35mm) gap setting. The nutrient coated film was dried in an oven at 85 ℃ for 12 minutes to give about 360mg/24in²(2.3mg/cm²) Dry coating weight of (c).

A isooctyl acrylate/acrylic acid (98/2 weight ratio) Pressure Sensitive Adhesive (PSA) coating formulation containing a TTC (2, 3, 5-triphenyltetrazolium chloride) indicator as described in example 4 of U.S. patent 5,409,838, which is incorporated herein by reference, was drawn down on the exposed nutrient coating at a 2 mil (0.05mm) gap setting. The resulting coated film was dried in an oven at 65 ℃ for 6 minutes to give a dry coating weight of about 180mg/24in²(1.15mg/cm²) The PSA coating of (1). The adhesive coated side of the coverlay film is then coated with guar powder. The powder was applied uniformly and excess powder was removed from the adhesive layer by manually shaking the film and then gently wiping the surface with a paper towel. The final coat weight of guar gum was about 400mg/24in²(2.6mg/cm²)。

The coated coverlay film is then cut to match the dimensions of the body member. The finished device was assembled by attaching the cover sheet to the body member (in a hinge-like manner) along one edge of the spacer (76mm edge) using a double-sided adhesive tape. For each device, the cover sheet and the body member are oriented such that the coated surface of the cover sheet faces the septum side of the body member.

The finished product testing device was inoculated with the E.coli inoculum. The cover sheet of the device was lifted and 1mL of inoculum (i.e., the final dilution as described above) was added by pipette across the fluid control membrane so that the channel was filled with liquid. The cover sheet is gently returned to its original position. All devices demonstrated self-spreading of water in the channels, allowing the guar to be uniformly wetted and form a hydrogel filling the channels of the fluid control membrane. The device was incubated at 37 ℃ for 24 hours. At the end of the incubation period, red colonies were counted by visual inspection. For all devices, it was observed that spot-like colonies were disposed on the entire surface of the hydrogel. The results are presented in table 3.

TABLE 3：

An apparatus comprising	Colony (cfu) count	Self-spreading of hydrogels	Punctate bacterial colony
				Fluid control membrane A	202	Is that	Is that
Fluid control membrane B	144	Is that	Is that
				Fluid control membrane C	214	Is that	Is that
Fluid control membrane D	154	Is that	Is that
				Fluid control membrane E	180	Is that	Is that

All references and publications cited herein are expressly incorporated by reference into this disclosure in their entirety. Illustrative embodiments of the invention are discussed herein and reference is made to possible variations within the scope of the invention. For example, features depicted in connection with one exemplary embodiment may be used in connection with other embodiments of the invention. These and other variations and modifications in the invention will be apparent to those skilled in the art without departing from the scope of the invention, and it should be understood that this invention is not limited to the illustrative embodiments set forth herein. Accordingly, the invention is to be limited only by the claims provided below and equivalents thereof.