Compositions Containing Scleroglucan, Articles Containing the Same, Production Methods, and Use Methods

Abstract

Compositions and articles containing scleroglucan that can be used for culturing and counting microorganisms even after exposure to a sterilizing dose of radiation. The compositions also include one or more water-swellable polymers other than scleroglucan, such as guar gum, locust bean gum, or xanthan gum. Also described are methods for producing and using the same.

Description

Technical Field

[0001] [Cross - Reference to Related Applications] This application claims priority to U.S. Patent Application No. 63 / 367,956, filed on July 8, 2022, the content of which is incorporated herein by reference in its entirety.

[0002] The field of the present invention generally relates to polymer films and their manufacture and use.

Background Art

[0003] Thin - film culture devices have become quite popular in recent decades. Generally, thin - film culture devices include a film disposed on a substrate. This film contains a culture medium capable of growing microorganisms or functions as a culture medium. This film has a wide variety of uses, one of which is to evaluate the level of microbial contamination in a test sample.

[0004] Patent Document 1 discloses a thin - film culture plate having culture medium particles containing a mixture of nutrients and a gelling agent. The gelling agent is a substance such as carbohydrates, proteins, and minerals, and specifically is a mixture of xanthan gum, locust bean gum, and guar gum.

[0005] Patent Document 2 discloses a dry - powder cell culture medium containing components embedded in a polymer.

[0006] Patent Document 3 discloses a device for separately counting colonies of coliform bacteria and Escherichia coli microorganisms. This device includes a first sheet containing a first cold - water - soluble gelling agent adhered to a first sheet and a second sheet containing a second cold - water - soluble gelling agent adhered to a second sheet. Examples of the gelling agent include guar gum, polyacrylamide, locust bean gum, and agar, where guar gum and xanthan gum alone or in combination are preferred, and guar gum is exemplified.

[0007] Patent Document 4 teaches a method for producing a fluid agglomerated nutrient medium using a fluidized bed agglomeration chamber. A gelling agent may be included. A binder is optionally included. Examples of the binder include PEG, polyvinylpyrrolidone, polyvinyl alcohol, polysaccharides, dextran, dextrin, maltodextrin, microcrystalline cellulose, HPMC, methylcellulose, starch, and saccharides.

[0008] Patent Document 5 provides a culture device for counting colonies of microorganisms. A cold water-soluble gelling agent, a buffer system in a dry state, a carbon dioxide generation system in a dry state, and a deoxygenation reagent in a dry state are arranged in the growth section. The gelling agents mentioned include algin, carboxymethyl cellulose, tara gum, hydroxyethyl cellulose, guar gum, locust bean gum, xanthan gum, polyacrylamide, polyurethane, and polyethylene oxide. Guar gum, locust bean gum, and xanthan gum are preferred either individually or in combination.

[0009] Feng et al. disclose in Non-Patent Document 1 an experiment of "examining the potential enzymatic degradation of guar gum, a gelling agent used in Petrifilm (trademark) plates". This paper concludes that since liquefier organisms can hydrolyze guar gum, "(i) in the liquefied region, motile organisms may move and amplify,... two or more colonies may arise from one cell, resulting in an overestimation of the microbial load, and (ii) in the blurred region, other colonies become difficult to see, which may lead to an underestimation of the potential (counting) accuracy" in two aspects of the accuracy.

[0010] Scleroglucan is available under the trademark ACTIGUM from Cargill, Inc. (Minnetonka, Minnesota, USA), and according to its manufacturer, it is stated to provide a stable viscosity over a pH range of 2.5 to 12 and temperatures between 10 degrees Celsius and 120 degrees Celsius for use in surfactants for construction, paints, household products, and applications in drilling, crude oil recovery, and asphalt emulsions.

Prior Art Documents

Patent Documents

[0011]

Patent Document 1

Patent Document 2

Patent Document 3

Patent Document 4

Patent Document 5

Non-Patent Documents

[0012]

Non-Patent Document 1

Summary of the Invention

[0013] The present invention relates to compositions, articles, devices, and methods related to thin films comprising scleroglucan and one or more water-swellable polymers.

[0014] In some embodiments, the present invention includes a composition comprising scleroglucan and one or more water-swellable polymers other than scleroglucan.

[0015] In some embodiments, the present invention includes a culture device comprising a base member, a cover sheet connected to the base member, and a growth compartment disposed between the cover sheet and the base member. In some embodiments, the scleroglucan layer is located within or adjacent to the growth compartment. In some embodiments, the scleroglucan layer further comprises one or more water-swellable polymers other than scleroglucan.

[0016] In some embodiments, the present invention includes a method for culturing a microorganism. The culturing method may include contacting the microorganism with a composition according to the present invention described herein to form an inoculated composition, and replicating the microorganism at least once.

[0017] In some embodiments, the present invention includes a method for culturing a microorganism, the method including contacting the microorganism with an article according to the present invention described herein (e.g., a culture device described herein) to form an inoculated article, and replicating the microorganism at least once.

[0018] In some embodiments, the present invention includes a method for sterilizing a culture device. The sterilization method may include providing an article according to the present invention described herein (e.g., a culture device described herein) that includes one of the compositions according to the present invention described herein. The sterilization method may further include directing radiation sufficient to sterilize the culture device towards the article.

[0019] In some embodiments, the present invention includes a film comprising one of the compositions according to the present invention described herein.

[0020] In some embodiments, the present invention includes an article comprising a substrate and a film according to the present invention described herein. BRIEF DESCRIPTION OF THE DRAWINGS

[0021]

Figure 1

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Figure 8

Figure 9

Best Mode for Carrying Out the Invention

[0022] In this application, terms such as "a", "an", and "the" are intended to refer not only to a single entity but also to include general categories for which specific examples may be used in the description. The terms "a", "an", and "the" are used interchangeably with the phrases "at least one" and "one or more". The phrases "at least one of" and "comprises at least one of" following a list refer to any one of the items in the list and any combination of two or more of the items in the list.

[0023] Terms such as "common", "commonly", "often", "frequent", and "frequently" are used to refer to features typically used in the present invention, but are not to be construed as meaning that the features so described were known or common prior to the present disclosure, unless otherwise indicated.

[0024] The use of "or" means "and / or" unless specifically stated otherwise.

[0025] The terms "comprise", "comprises", "comprising", "include", "includes", and "including" can be used interchangeably and are not intended to be limiting. Further, when the term "comprising" is used in the description of one or more embodiments, those skilled in the art will understand that in some specific examples, the embodiment(s) may alternatively be described using the terms "consisting essentially of" and / or "consisting of".

[0026] As used herein, the term "about" refers to a variation of ±10% from a set value. Such variation is understood to always be included in any value provided herein, whether or not it is specifically recited.

[0027] Ranges provided by absolute or approximate language are intended to include both, and any definitions used herein are intended to clarify, not to limit. Numerical ranges and parameters that indicate the broad scope of the invention are approximate, but the numerical values set forth in specific examples are reported as precisely as possible. Further, all ranges disclosed herein are to be understood to encompass all sub-ranges subsumed therein (including all fractional and integral values).

[0028] As used herein, the word "sterile" is used only as an adjective and never as a verb. It may be used with or without reference to ISO 11137 or ISO 11137-2:2013. Whether or not it is used with reference to ISO 11137 or ISO 11137-2:2013, "sterile" means that the noun it modifies, such as a composition, film, article, or culture device, is "negative in the sterility test" as defined in ISO 11137-2:2013. That the sterility test is negative can be established by either Method 1 or Method 2 of ISO 11137-2:2013. Thus, an article that is negative in the sterility test when tested according to Method 1 of ISO 11137-2:2013 and not negative or vice versa when tested according to Method 2 of ISO 11137-2:2013, or an article that is negative in the sterility test when tested according to both Method 1 and Method 2 of ISO 11137-2:2013, is considered "sterile" as the term is used herein.

[0029] As used herein, the words "sterilize", "sterilizes", "sterilizing", and "sterilized" are used only as verbs and are never used as adjectives. (i) The act of sterilizing a thing (such as a composition, film, article, or culture device), (ii) the act of sterilizing a thing (such as a composition, film, article, and culture device), (iii) a sterilized thing (such as a composition, film, article, or culture device), or (iv) the act of sterilizing a thing (such as a composition, film, article, or culture device) all have the same meaning and differ only in the form of the verb "sterilize" due to grammatical requirements. In any case, the verb (either "sterilize", "sterilizes", "sterilizing", or "sterilized") means to bring its object into a sterilized state (the term "sterile" is defined above). References to ISO 11137 or ISO 11137-2:2013 are to the English version of ISO 111347-2:2013.

[0030] For example, thin film culture devices such as those disclosed in Patent Document 1, Patent Document 2, and US Patent Application Publication No. 202001943 include a substrate provided with a film that may contain a water-swellable polymer such as gum (e.g., guar gum, xanthan gum, or locust bean gum). These water-swellable polymers can provide a matrix in which target microorganisms can replicate and form colonies.

[0031] An exemplary thin film culture device is available under the trade name PETRIFILM from Neogen Corporation in Lansing, Michigan, USA. According to the instruction for use ("IFU") of the culture device of the PETRIFILM brand, these are decontaminated but not sterilized.

[0032] The present disclosure recognizes that it has not been possible to provide, in a sterile state, prior art culture media and culture devices, such as those containing one or more water-swellable polymers, such as one or more of guar gum, xanthan gum, and locust bean gum, particularly thin film culture devices containing such polymers. The inventors have attempted to expose such conventional culture devices to a radiation dose sufficient to sterilize the device, for example at least 25 kGy, or at least about 30 kGy, or about 25 kGy to about 30 kGy. However, when using prior art culture devices exposed to a sterilizing dose of radiation, the inventors have found that the microorganisms to be cultured are not visualized as distinct colonies, but rather spots can form. As a result, it has been difficult or impossible to count the resulting colonies and accurately count the number of colony forming units (“cfu”) on the culture device.

[0033] Non-sterile culture devices with reduced bioburden after decontamination, such as those sold under the trade name PETRIFILM™, are acceptable for many applications, but in some applications, such as medical diagnostics, cleanroom monitoring, and biopharmaceutical purification, it may be necessary to culture microorganisms under sterile conditions.

[0034] Accordingly, the problem can be stated as how to form a water-swellable polymer composition that is suitable for use as a component of a culture medium, such as a culture device, and that does not exhibit an inappropriate increase in the mobility of microorganisms after sterilization, particularly after exposure to radiation of at least about 25 kGy or at least about 30 kGy, or about 25 kGy to about 30 kGy. Another problem can be stated as how to form a sterilized composition of a water-swellable polymer composition that is suitable for use in a culture device and that does not exhibit an inappropriate increase in the mobility of microorganisms.

[0035] Yet another problem is how to produce a sterilizing film for use in microbial cultures that contains a water-swellable polymer composition and, after sterilization, does not exhibit an inappropriate increase in microbial mobility, especially after exposure to radiation of at least about 25 kGy or at least about 30 kGy, or from about 25 kGy to about 30 kGy.

[0036] Another problem is how to produce a sterilized culture device, especially a thin film culture device, that has been exposed to radiation of at least about 25 kGy or at least about 30 kGy, or from about 25 kGy to about 30 kGy, and does not exhibit an inappropriate increase in microbial mobility.

[0037] In connection with these problems, if microorganisms form spots or patches after incubation on a film or culture device, this can make it impossible to identify cfu or count the number of colonies with reasonable accuracy.

[0038] Briefly stated, a solution to one or more of the above problems, and potentially several other problems, involves using scleroglucan alone or with other water-swellable polymers in compositions, films, or articles for cell culture.

[0039] Scleroglucan is a polysaccharide commercially available under the trademark ACTIGUM from Cargill, Inc. (Minnetonka, Minnesota, USA). Scleroglucan has a chemical structure similar to that of polysaccharides such as guar gum, xanthan gum, and locust bean gum. However, unexpectedly and surprisingly, while polysaccharides generally cannot withstand radiation, scleroglucan has been found to be radiation-tolerant without the above problems.

[0040] Composition In a culture device such as a thin film culture device, the composition that can be used as a culture medium can have scleroglucan alone as its matrix, or can have scleroglucan in combination with one or more additional water-swellable polymers other than scleroglucan. Examples of the one or more additional water-swellable polymers can include polymers that are at least partially soluble in water. For example, the water-soluble polymers that can be used in the compositions disclosed herein include those disclosed in Patent Document 1, Patent Document 2, or Patent Document 3, which are used in water-swellable compositions or hydrogel-forming compositions. In some embodiments, when one or more additional water-swellable polymers are used, these can include one or more of guar gum, xanthan gum, or locust bean gum. In a further embodiment, a combination of locust bean gum and xanthan gum can be used.

[0041] When one or more additional water-swellable polymers are used, the weight ratio of scleroglucan to the one or more additional water-swellable polymers can vary depending on the requirements of the final product. In some embodiments, the ratio of the total weight of the one or more water-swellable polymers to the weight of scleroglucan can be from about 1:99 to about 99:1, optionally from about 1:10 to about 10:1, and further optionally from about 3:1 to about 1:3. In a further embodiment, the ratio of the total weight of the one or more water-swellable polymers to the weight of scleroglucan can be from about 10:90 to about 90:10, from about 20:80 to about 80:20, from about 25:75 to about 75:25, from about 30:70 to about 70:30, from about 40:60 to about 60:40, or from about 55:45 to about 45:55.

[0042] The amount of one or more additional water-swellable polymers can be characterized as a percentage of the total weight of the one or more additional water-swellable polymers and the total weight of scleroglucan. In some embodiments, the weight percentage of the one or more additional water-swellable polymers relative to the total weight of the one or more additional water-swellable polymers and the total weight of scleroglucan is about 1 wt% or more, optionally about 5 wt% or more, optionally about 10 wt% or more, optionally about 20 wt% or more, optionally about 25 wt% or more, optionally about 30 wt% or more, optionally about 33 wt% or more, optionally about 40 wt% or more, optionally about 50 wt% or more, optionally about 60 wt% or more, optionally about 67 wt% or more, optionally about 70 wt% or more, optionally about 75 wt% or more, optionally about 80 wt% or more, optionally about 90 wt% or more, or optionally about 95 wt% or more, or about 1 wt% to about 95 wt%, or about 5 wt% to about 75 wt%, or about 10 wt% to about 50 wt%, or about 5 wt% to about 30 wt%.

[0043] The amount of scleroglucan can also be characterized as a percentage of the total weight of the one or more additional water-swellable polymers and the total weight of scleroglucan. In some embodiments, the weight percentage of scleroglucan relative to the total weight of the one or more additional water-swellable polymers and the total weight of scleroglucan is about 1 wt% or more, optionally about 5 wt% or more, optionally about 10 wt% or more, optionally about 20 wt% or more, optionally about 25 wt% or more, optionally about 30 wt% or more, optionally about 33 wt% or more, optionally about 40 wt% or more, optionally about 50 wt% or more, optionally about 60 wt% or more, optionally about 67 wt% or more, optionally about 70 wt% or more, optionally about 75 wt% or more, optionally about 80 wt% or more, optionally about 90 wt% or more, or optionally about 95 wt% or more, 100 wt%, or about 1 wt% to 100 wt%, or about 25 wt% to about 100 wt%, or about 10 wt% to about 70 wt%.

[0044] In the compositions disclosed herein, additional components may be used. Examples of such additional components include one or more nutrients that promote the growth of one or more microorganisms. In some embodiments, the nutrients include one or more saccharides such as one or more of glucose, lactose, fructose, maltose, and dextrose, soluble starch, tryptone, proteose peptone, soy tone, yeast extract, casamino acids, casein, pancreatic digest of casein, casein acid hydroxylase, papain digest of soybeans, sodium pyruvate, sodium chloride, magnesium chloride, magnesium sulfate, or dipotassium phosphate. In further embodiments, glucose may be used as an additional component.

[0045] Other additional components that may be used include selection agents (e.g., antibiotics), detection agents (e.g., dyes such as redox dyes), carbon dioxide generators, and oxygen reducing agents.

[0046] In some embodiments, the composition can be a sterilized composition, such as an irradiated composition, for example, a composition exposed to radiation of about 25 kGy or more, or even more preferably about 30 kGy or more.

[0047] The composition may preferably meet the ISO 11737-1:2013 standard. In some embodiments, the composition may be placed within a package such as a sterile package, which can meet this standard while it is within the package, but may become contaminated, for example, immediately after being removed from the package, and thus may not meet the standard when removed from the package.

[0048] Article Another aspect of the present invention relates to an article comprising any of the compositions discussed herein. Layers such as coatings and / or dried films comprising any of the compositions discussed herein are also contemplated. The article may comprise a layer such as a coating or a dried film, and the layer or film may likewise comprise any of the compositions discussed herein. In some embodiments, the article may also comprise a substrate that can be in contact with the layer or film. In further embodiments, the article may be sterilized. The sterilized article can be an irradiated article that has been exposed to radiation of 25 kGy or more, optionally 30 kGy or more.

[0049] The article can be in the form of a culture device such as a culture plate. However, other forms may also be possible. An example of another form may be a bag in which the composition is contained.

[0050] In some embodiments, when the article can be a culture device, particularly a culture plate, the article can comprise a base member, a cover sheet (which may also be referred to as a "cover slip") on the base member, and a growth compartment between the cover sheet and the base member. The growth compartment can comprise any of the compositions described herein, for example, scleroglucan that does not contain another water-swellable polymer, or a combination of scleroglucan and one or more other water-swellable polymers. Additional components such as those discussed above with respect to the composition can also be included. In some embodiments, the cover sheet is movable and optionally removable to expose the growth compartment and enable inoculation of the growth compartment.

[0051] The composition comprising scleroglucan may be disposed on the base member, in which case the cover sheet can be disposed to cover the composition. In an alternative configuration, the composition or film can be disposed on the cover sheet. In another alternative configuration, the composition or film can be disposed on both the substrate and the cover sheet.

[0052] In some embodiments, a spacing member may be disposed between the base member and the cover sheet. When present, the spacing member may provide a space for the growth zone, such as a scleroglucan-containing composition that may also include one or more additional components discussed herein.

[0053] In some embodiments, an adhesive may be used. The adhesive may be disposed on the base layer, the cover sheet, or both, depending on the desired configuration of the device. Suitable adhesives include those known for use in thin film culture devices, such as acrylates (e.g., isooctyl acrylate-based adhesives), or mixtures of one or more acrylates and acrylamides.

[0054] FIG. 7 shows one embodiment of an article according to the present invention in the form of a culture device 700. The culture device 700 includes a base member 702 and a cover sheet 704. The cover sheet 704 covers one side of the base member 702. FIG. 7 shows two enlarged views of different portions of the device 700. Enlarged view 706 shows a cross-sectional view of the cover sheet 704, while enlarged view 708 shows a cross-sectional view of the base member 702.

[0055] As shown in enlarged view 706, the cover sheet 704 includes at least three different layers, such as a plastic film layer 710, an adhesive layer 712, and a composition layer 714 according to the present invention. The plastic film layer 710 is above both the adhesive layer 712 and the composition layer 714 according to the present invention. The plastic film layer 710 is water-impermeable and flexible, allowing the cover sheet 704 to be peeled from the base member 702. The adhesive layer 712 is disposed below the plastic film layer 710 and above the composition layer 714 according to the present invention, providing improved adhesion between layer 710 and layer 714. The composition layer 714 according to the present invention is below both layer 710 and layer 712 and includes one of the scleroglucan-containing compositions according to the present invention described herein.

[0056] As shown in the enlarged view 708, the base member 702 comprises at least three different layers such as a plastic-coated paper layer 720, an adhesive layer 718, and a composition layer 716 according to the present invention. The plastic-coated paper layer 720 is below both the adhesive layer 718 and the composition layer 716 according to the present invention. The plastic-coated paper layer 720 is impermeable to water and provides a relatively rigid substrate for the base member 702, from which the cover sheet 704 can be peeled or pressed against. The adhesive layer 718 is disposed above the plastic-coated paper layer 720 and below the composition layer 716 according to the present invention, resulting in an improved adhesiveness between layer 720 and layer 716. The composition layer 716 according to the present invention is above both layer 718 and layer 720 and contains one of the scleroglucan-containing compositions according to the present invention described herein. The plastic-coated paper layer 720 is printed with a grid that enables the user to more easily count the cfu during use of the device 700.

[0057] The device 700 comprises a spacing member 722 which is a layer of plastic or plastic-coated paper disposed between the lower surface of the cover sheet 704 and the upper surface of the base member 702. The cover sheet 704 functions as a spacer between the cover sheet 704 and the base member 702, defining a circular or oval space in the form of a growth compartment 724 therebetween. The growth compartment 724 is essentially a void defined at the top by the lower surface of the composition layer 714 of the cover sheet 704 according to the present invention, at the bottom by the upper surface of the composition layer 716 of the base member 702 according to the present invention, and laterally by the spacing member 722. In some embodiments, one of the compositions according to the present invention described herein is disposed within the growth compartment of the culture device according to the present invention.

[0058] Manufacturing method The compositions and articles described herein can be made by any suitable method. Figure 8 shows a flowchart depicting an embodiment of a method of the invention that can be used to produce or make the compositions and articles of the invention in the form of method 800.

[0059] Step 802 of method 800 includes forming one of the compositions according to the invention described herein.

[0060] In some embodiments, the compositions according to the invention are made by mixing their components. For example, if the composition comprises scleroglucan and one or more nutrients that promote the growth of one or more microorganisms, these components may be mixed as powders, or in some embodiments, mixed in the presence of a liquid to form a dispersion. Similarly, if the composition comprises scleroglucan and one or more additional water-swellable polymers, these components may be mixed as powders, or mixed in combination with a liquid to form a dispersion.

[0061] In some embodiments, when a liquid is used, this may be water or a combination of water and an organic solvent. When an organic solvent is used, the organic solvent may preferably be selected such that a mixture of water and the organic solvent can form a single phase. This can be achieved, for example, by selecting a solvent that is miscible with water, such as a short-chain alcohol like methanol, ethanol, or propanol, or an ether like ethyl acetate, or some esters like tetrahydrofuran, or by using an organic solvent at a sufficiently low concentration such that a mixture of water and the organic solvent can form only a single phase.

[0062] When the components are mixed as a liquid dispersion, the concentration of the components in the liquid will depend on the exact nature of the components used and the type of article intended. If a film is to be formed from the liquid, the concentration of scleroglucan and, if used, one or more additional water-swellable polymers should be such that the viscosity of the dispersion is low enough to be handled and coated onto a substrate. If the concentration is too high, a gel will form, making it impossible to coat the solid components of the dispersion. However, if a film is not intended to be formed, but instead the solid components and liquid are to be used as a gel in an article, for example as a gel in a culture bag, then a concentration that forms a gel is acceptable.

[0063] Step 804 of method 800 includes forming a film or layer from the composition according to the invention formed in step 802. If a film or layer can be formed, the liquid can be coated onto the desired substrate in any suitable manner. Examples include knife coating, die coating, spray coating, spin coating, etc. After coating, the film can be partially or completely dried to remove all or part of the liquid. This can be achieved by any known method, heating, exposure to reduced pressure, or air drying the film or article under ambient conditions.

[0064] In some embodiments, when a spray can be formed, such as when the components are mixed as a powder and the powder is sprayed onto a base member, a cover sheet, or both, any known powder spraying device or powder coating device can be used. In further embodiments, an adhesive can be applied to the cover sheet, the base layer, or both to adhere the powder composition to one or more appropriate components of the device.

[0065] Step 806 of method 800 is optional and includes packaging the composition, film, or article of the present invention. In some embodiments, the packaging comprises a barrier layer that seals the contents, including the film, article, or composition, from the external environment. Any packaging known for this purpose can be used, for example, packaging known for use in medical devices or thin film culture plates of the PETRIFILM™ brand.

[0066] Step 808 of method 800 includes sterilizing the composition, film, or article according to the present invention. If any packaging step is performed, the sterilization step can be performed before, simultaneously with, or after any packaging step. In further embodiments, since the sterilization step can be performed simultaneously with or after any packaging step, the composition, film, or article can be protected by the packaging, for example, until the packaging is opened, and can maintain a sterile state over a certain period of time.

[0067] A sterilization step can be used to render the composition, film, or article sterile. This can be achieved by exposing the composition, film, or article to ionizing radiation such as radiation. For this purpose, a wide variety of types of radiation can be used, such as gamma rays, electron beams (eBeam), and X-rays. In some embodiments, the composition, film, or article can be irradiated with radiation to render them sterile. Typically, at least 25 kGy of radiation is used. In some cases, at least 30 kGy of radiation is used.

[0068] Gamma irradiation can be generated using commercially available sources such as those available from Atomic Energy of Canada, Inc. Some of such sources use cobalt 60 high energy sources. Electron beam irradiation sources such as the electron beam system CB-300 available from ESI are also commercially available. X-ray devices are also well known and are available to those skilled in the art.

[0069] Usage The present invention further includes a method of using the compositions, layers, films, or articles disclosed herein for culturing one or more microorganisms. FIG. 9 shows a flowchart illustrating an embodiment of a method of using the compositions, layers, films, or articles disclosed herein in the form of method 900.

[0070] In step 902 of method 900, a sample (e.g., an aqueous sample) containing one or more microorganisms can be contacted with the composition, layer, film, or article to form an inoculated composition, layer, film, or article.

[0071] Next, in step 904 of method 900, the inoculated composition, layer, film, or article can be incubated at an elevated temperature for a time sufficient to allow at least one replication of the one or more microorganisms. Exemplary elevated temperatures include from about 30°C to about 80°C such as about 30°C, about 32°C, about 37°C, about 40°C, about 42°C, about 45°C, about 50°C, about 60°C, or about 70°C. Other temperatures may be used depending on the exact nature of the composition, film, or article, the nature of the microorganisms being cultured, and other factors known to those of skill in the relevant art. The required time can be from about 30 minutes to about 72 hours, from about 1 hour to about 60 hours, from about 2 hours to about 50 hours, from about 5 hours to about 40 hours, from about 6 hours to about 30 hours, from about 7 hours to about 25 hours, or from about 8 hours to 24 hours. The required time can be from about 8 hours to about 10 hours, from about 10 hours to 12 hours, from about 12 hours to 14 hours, from about 14 hours to 16 hours, from about 16 hours to about 18 hours, from about 18 hours to about 20 hours, from about 20 hours to about 22 hours, or from about 22 hours to about 24 hours.

[0072] In any step 906 of method 900, the composition, film, or article can optionally be counted to determine the number of cfu of the one or more microorganisms that may have formed. This can be done manually by human observation or by using commercially available equipment such as a PETRIFILM™ Plate Reader Advanced (3M Company, St. Paul, Minnesota, USA) designed for this purpose.

Example

[0073] Materials BACTO Tryptic Soy Broth (TSB) powder was obtained from Becton, Dickinson and Company in Franklin Lakes, New Jersey. According to the manufacturer, the composition of the TSB powder was reported to be tryptone (57% by weight), soy tone (10% by weight), glucose (8% by weight), sodium chloride (17% by weight), dipotassium phosphate (8% by weight).

[0074] R2A broth powder was obtained from HIMEDIA Laboratories (Mumbai, India). According to the manufacturer, the composition of the R2A broth powder was reported to be casein acid hydroxylase (16% by weight), yeast extract (16% by weight), proteose peptone (16% by weight), dextrose (16% by weight), soluble starch (16% by weight), dipotassium phosphate (9.6% by weight), magnesium sulfate (0.8% by weight), sodium pyruvate (9.6% by weight).

[0075] Scleroglucan was obtained from Cargill, Incorporated under the trade name ACTIGUM CS-6 scleroglucan.

[0076] 2,3,5-Triphenyltetrazolium chloride (TTC) and sodium pyruvate were obtained from MilliporeSigma Company in St. Louis, Missouri.

[0077] 3M PETRIFILM Aerobic Count Plates were obtained from 3M Company in Maplewood, Minnesota and designated as Comparative Example A (CE-A).

[0078] Butterfield's Buffer was obtained from 3M Company.

[0079] Xanthan gum and locust bean gum were obtained from CP Kelco Company in Atlanta, Georgia.

[0080] Guar gum (Meyprogat 150) was obtained from Danisco in Copenhagen, Denmark.

[0081] The bacterial strains Escherichia coli (ATCC 25922) and Bacillus subtilis (ATCC 6633) were obtained from Microbiologics in St. Cloud, Minnesota.

[0082] The bacterial strain Proteus mirabilis (ATCC 14153) was obtained from ATCC (American Type Culture Collection) in Manassas, Virginia.

[0083] Unless otherwise stated, water was obtained from a MILLI-Q water purification system (EMD Millipore, Billerica, Massachusetts).

[0084] Preparation Example 1. Preparation of inoculum Sterile TSB liquid medium was prepared according to the manufacturer's instructions (30 g of TSB powder per liter of purified water (pH 7.3 ± 0.2)) and sterilized using an autoclave. Cultures of each bacterial strain were prepared individually in sterile test tubes containing 9 mL of sterile TSB and incubated overnight at 30 °C and 225 rpm (revolutions per minute) in an orbital shaking incubator. Each inoculum was prepared by serially diluting a single culture sample with Butterfield's buffer. The inocula of Escherichia coli (ATCC 25922) and Proteus mirabilis (ATCC 14153) were serially diluted (10-fold dilution) to the final 10 -8 dilution sample. The inoculum of Bacillus subtilis (ATCC 6633) was serially diluted to the final 10 -7Serial dilutions were performed up to the diluted sample. When used for inoculating thin film culture devices AA to DD, the inoculation sample of Bacillus subtilis (ATCC 6633) was supplemented with sodium pyruvate (2 g / L).

[0085] Example 1. Production of the cover sheet of the thin film device Powder compositions A to D were prepared from scleroglucan powder, xanthan gum powder, and locust bean gum powder. Powder composition A contained only scleroglucan powder. Powder compositions B to D were powder mixtures containing scleroglucan powder, xanthan gum powder, and locust bean gum powder at various weight percent (wt%) concentrations. For each powder mixture, the components were combined and shaken in a plastic bag for 30 seconds to form a homogeneous mixture. The ratios of the powder components in the compositions are summarized in Table 1.

[0086] The cover sheet components of each thin film device were produced starting from a biaxially oriented polypropylene (BOPP) film (1.6 mil thickness) having a pressure-sensitive adhesive (isooctyl acrylate:acrylamide in a weight ratio of 96:4) at a coating amount of 1.3 mg / cm 2 The adhesive contained 0.1 wt% of TTC based on the dry weight of the adhesive. The adhesive-coated surface of the film was powder-coated with a single homogeneous powder mixture selected from powder compositions A to D. Each powder composition was uniformly applied to the adhesive surface, and excess powder was removed from the adhesive layer by shaking the film by hand. The resulting powder-coated film was cut into 76 mm wide × 100 mm long sections to form the cover sheet of the thin film device.

[0087]

Table 1

[0088] Example 2. Production of the base member components of the thin film culture device containing TSB powder as a nutrient The base member components of each thin-film device were manufactured starting from a clear polyethylene-coated paper sheet (1 cm × 1 cm in size, 0.15 mm thick, coated on both sides). The paper sheet component is white and has yellow grid lines (1 cm × 1 cm). On one side of the polyethylene-coated paper sheet, a pressure-sensitive adhesive (isooctyl acrylate: acrylic acid in a weight ratio of 98:2) was coated at a coating amount of 1.3 mg / cm 2 Next, the adhesive-coated surface was powder-coated with a single powder mixture selected from powder compositions E to H (Table 2). For each powder mixture, the components were combined and shaken in a plastic bag to form a homogeneous mixture. Each powder composition was uniformly applied to the adhesive surface, and excess powder was removed from the adhesive layer by hand-shaking the film. The resulting powder-coated film was cut into sections 76 mm wide × 100 mm long to form the base member components of the thin-film device.

[0089]

Table 2

[0090] Example 3. Production of the base member components of a thin-film culture device containing R2A broth powder as a nutrient The base member components were individually manufactured from powder compositions I to L (Table 3) using the same procedure as described in Example 2, except that TSB powder was replaced with R2A broth powder in the mixture used.

[0091]

Table 3

[0092] Example 4. Production of a thin-film device A thin-film culture device was assembled by attaching a cover sheet selected from Example 1 to a base member component selected from Example 2 and Example 3. Each cover sheet was attached (like a hinge) to the base member along one edge (the 76 mm edge) using a single-sided double-sided adhesive tape (about 9.5 mm wide). For each device, the cover sheet and the base member were oriented such that their edges were aligned and the powder-coated surface of the cover sheet faced the powder-coated surface of the base member. The configurations of thin-film culture devices AA to HH are summarized in Table 4.

[0093]

Table 4

[0094] Example 5. Inoculation, Incubation, and Colony Observation of Thin-Film Devices Thin film culture devices AA to HH were exposed to gamma radiation of 30 kGy. The irradiated thin film culture devices AA to HH of Example 4 were inoculated with a single inoculum of any one of Escherichia coli (ATCC 25922), Bacillus subtilis (ATCC 6633), or Proteus mirabilis (ATCC 14153). The device was placed on a flat horizontal surface. The cover sheet of each device was lifted, and 1 mL of the single inoculum (i.e., the final dilution described in Preparation Example 1) was carefully added by pipetting as a single application to the dense area in the center of the coated base member. The cover sheet was gently returned to its original position. When a 3M PETRIFILM Yeast and Mold Spreader (obtained from 3M Company) was applied with hand pressure to the outer surface of the cover sheet to spread the inoculum, a circular area having a diameter of about 2 and 3 / 8 inches (6.03 cm) was formed. Then, the spreader was removed, and the plate was left as it was for 1 to 5 minutes to form a gel. Inoculated devices were produced in triplicate (n = 3 for each of devices AA to HH). Then, each device was incubated at 32 °C for 48 hours. The same inoculation and incubation procedures were also carried out on 3M PETRIFILM aerobic count plates (CE-A) exposed to a gamma radiation dose of 30 kGy and non-irradiated 3M PETRIFILM aerobic count plates (CE-A).

[0095] Images of the devices were taken using a 3M PETRIFILM Advanced Plate Reader (obtained from 3M Company and operated according to the manufacturer's instructions) and exported as JPEG image files. The area of the red-colored colonies present within the devices was measured using ImageJ software (National Institutes of Health, Bethesda, Maryland, USA). Once the image file was opened, the "Global" settings were selected and the scale of the image was set using the internal 10 mm standard. For each image, the scale of the image was set using the distance between the yellow grid lines in the base member. Next, the "Image-Adjust-Color Balance" setting was selected, and subsequently the "Yellow" setting was selected from the drop-down menu. The minimum and maximum sliders were used to enhance the contrast at the edges of the colonies. The image was then converted to 8-bit and the threshold was set so that only the pixels representing the colonies were selected. In the "Analyze Particles" menu (within the "Analyze" menu of the software), the smallest bacterial colonies within the image were visually identified and small non-colony particles were excluded from the analysis by setting the area of that colony as the minimum size for colony identification by the software. The "OK" button was selected and the area of the colonies (mm 2 ) was measured by the software program. The results are reported in Tables 5 and 6. For samples of Escherichia coli (ATCC 25922) and Proteus mirabilis (ATCC 14153), the average colony size was determined based on the analysis of 25 colony-forming units (cfu) to 100 cfu per device. For samples of Bacillus subtilis (ATCC 6633), the average colony size was determined based on the analysis of 5 colony-forming units (cfu) to 10 cfu per device.

[0096] The indication "ND" in Tables 5 and 6 indicates that large, spreading, and / or irregularly shaped spots have occurred where the boundaries of multiple colonies have fused together and cannot be counted for the purpose of colony counting.

[0097]

Table 5

[0098]

Table 6

[0099] List of Embodiments The following is a non-limiting list of embodiments.

[0100] 1. A composition comprising: a. One or more water-swellable polymers other than scleroglucan, optionally including one or more of guar gum, locust bean gum, or xanthan gum; and b. Scleroglucan (which may be present in an amount of about 1% by weight or more, optionally about 5% by weight or more, optionally about 10% by weight or more, optionally about 20% by weight or more, optionally about 25% by weight or more, optionally about 30% by weight or more, optionally about 33% by weight or more, optionally about 40% by weight or more, optionally about 50% by weight or more, optionally about 60% by weight or more, optionally about 67% by weight or more, optionally about 70% by weight or more, optionally about 75% by weight or more, optionally about 80% by weight or more, optionally about 90% by weight or more, or optionally about 95% by weight or more, 100% by weight, or about 1% by weight to 100% by weight, or about 25% by weight to about 100% by weight, or about 10% by weight to about 70% by weight, based on the total weight of one or more water-swellable polymers other than scleroglucan and the total weight of scleroglucan). The composition as described above.

[0101] 2. A device comprising: a. A base member; and b. A cover sheet connected to the base member. c. A growth compartment disposed between the cover sheet and the base member, and comprising d. The cover sheet can be moved to expose the growth compartment, and e. Further, the growth compartment contains the composition of Embodiment 1, said device.

[0102] 3. The composition or device according to any of the above embodiments, further comprising one or more nutrients that promote the growth of one or more microorganisms.

[0103] 4. The composition or device according to any of the above embodiments, wherein the ratio of (i) the weight of one or more water-swellable polymers to (ii) the weight of scleroglucan is from 1:99 to 99:1, optionally from 1:10 to 10:1, and further optionally from 3:1 to 1:3.

[0104] 5. The weight percentage of the water-swellable polymer with respect to the total weight of the water-swellable polymer and the total weight of scleroglucan is 1 or more, optionally 5 or more, optionally 10 or more, optionally 20 or more, optionally 25 or more, optionally 30 or more, optionally 33 or more, optionally 40 or more, optionally 50 or more, optionally 60 or more, optionally 67 or more, optionally 70 or more, optionally 75 or more, optionally 80 or more, optionally 90 or more, or optionally 95 or more, the composition or device according to any of the above embodiments.

[0105] 6. The weight percentage of scleroglucan with respect to the total weight of the water-swellable polymer and the total weight of scleroglucan is 95 or less, optionally 90 or less, optionally 80 or less, optionally 75 or less, optionally 70 or less, optionally 67 or less, optionally 60 or less, optionally 50 or less, optionally 40 or less, optionally 33 or less, optionally 30 or less, optionally 25 or less, optionally 20 or less, optionally 10 or less, optionally 5 or less, or optionally 1 or less, the composition or device according to any of the above embodiments.

[0106] 7. The composition or device according to any of the above embodiments, comprising scleroglucan and one or more nutrients that promote the growth of one or more microorganisms.

[0107] 8. The composition or device according to any of the above embodiments, wherein one or more nutrients contain one or more saccharides, and the saccharides optionally contain at least one of glucose, lactose, fructose, maltose, or sucrose, soluble starch, tryptone, soy tone, yeast extract, casamino acids, sodium pyruvate, sodium chloride, magnesium chloride, magnesium sulfate, or dipotassium phosphate.

[0108] 9. The composition or device according to any of the above embodiments, wherein one or more nutrients contain glucose.

[0109] 10. The composition or device according to any of the above embodiments, wherein the composition or device is an irradiated composition or device that has been exposed to radiation of 25 kGy or more, optionally 30 kGy or more.

[0110] 11. The composition or device according to any of the above embodiments, which is sterilized.

[0111] 12. The composition or device according to any of the above embodiments, which is disposed inside a package, and optionally the package provides a sterilization barrier between the inside of the package and the external environment.

[0112] 13. An article comprising the composition or device according to any of the above embodiments, optionally a culture device, optionally a thin-film culture device.

[0113] 14. The article according to embodiment 13, wherein the article is an irradiated article that has been exposed to radiation of 25 kGy or more, optionally 30 kGy or more, or radiation of about 25 kGy to about 35 kGy.

[0114] 15. The article according to any one of embodiments 13 to 14, which is sterilized.

[0115] 16. An article according to any one of embodiments 13 to 15, disposed inside the package, optionally providing a sterilization barrier between the inside of the package and the external environment.

[0116] 17. A film comprising the composition according to any one of embodiments 1 to 12.

[0117] 18. An article comprising a substrate and the film of embodiment 17.

[0118] 19. A culture device comprising the film according to any one of embodiments 17 to 18, the culture device comprising a growth compartment defined by a backing material and a cover member covering the growth compartment, and the film being disposed on the backing material, the cover member, or both.

[0119] 20. The composition according to any one of embodiments 1 to 13, used in the culture of one or more microorganisms.

[0120] 21. The film according to embodiment 17, used in the culture of one or more microorganisms.

[0121] 22. The article according to any one of embodiments 14 to 16 or embodiments 18 to 20, used in the culture of one or more microorganisms.

[0122] 23. A method for culturing microorganisms, a. contacting the microorganism with the composition according to any one of embodiments 1 to 13 to form an inoculated composition; b. replicating the microorganism at least once; comprising the above method.

[0123] 24. A method for culturing microorganisms, a. contacting the microorganism with the device according to any one of embodiments 2 to 12 or the article according to any one of embodiments 13 to 15 to form an inoculated article; [[ID=4l]] b. replicating the microorganism at least once; The method as described above, including

[0124] 25. A method for culturing microorganisms, comprising: a. Contacting the microorganism with the film described in Embodiment 17 or the article described in any one of Embodiments 18 to 22 to form an inoculated article; b. Replicating the microorganism at least once; including c. The microorganism is optionally a microorganism of the genus Bacillus, and further optionally a species of the genus Bacillus. The method as described above.

[0125] 26. The method according to any one of Embodiments 24 to 25, further comprising optionally incubating the inoculated composition or article at a temperature of 25°C to 80°C for 30 minutes to 72 hours.

[0126] 27. The method according to any one of Embodiments 24 to 26, further comprising a step of forming one or more microbial colonies on the microorganism and a step of counting the number of microbial colonies.

[0127] 28. A method for preparing the composition according to Embodiment 7, comprising mixing scleroglucan with one or more nutrients that promote the growth of one or more microorganisms and optionally a water-swellable polymer other than scleroglucan. The method as described above.

[0128] 29. A method for sterilizing the composition or device according to any one of Embodiments 1 to 14, comprising exposing the composition or device to radiation sufficient to sterilize the composition. The method as described above.

[0129] 30. The method according to Embodiment 28, further comprising exposing the composition to radiation sufficient to sterilize the composition.

[0130] 31. The method according to any one of Embodiments 28 to 30, further comprising exposing the composition to radiation of at least 25 kGy, optionally at least 30 kGy.

[0131] 32. The method according to any one of embodiments 28 to 31, wherein the water-swellable polymer other than scleroglucan comprises one or more of guar gum, locust bean gum, and xanthan gum, and optionally locust bean gum, xanthan gum, or a mixture of locust bean gum and xanthan gum.

[0132] 33. The method according to any one of embodiments 28 to 31, wherein the ratio of the weight of (i) one or more water-swellable polymers to (ii) the weight of scleroglucan is from 1:99 to 99:1, optionally from 1:10 to 10:1, and further optionally from 3:1 to 1:3.

[0133] 34. The weight percentage of the water-swellable polymer relative to the total weight of the water-swellable polymer and the total weight of scleroglucan is 1 or more, optionally 5 or more, optionally 10 or more, optionally 20 or more, optionally 25 or more, optionally 30 or more, optionally 33 or more, optionally 40 or more, optionally 50 or more, optionally 60 or more, optionally 67 or more, optionally 70 or more, optionally 75 or more, optionally 80 or more, optionally 90 or more, or optionally 95 or more. The method according to any one of embodiments 28 to 32.

[0134] 35. The weight percentage of the water-swellable polymer relative to the total weight of the water-swellable polymer and the total weight of scleroglucan is 95 or less, optionally 90 or less, optionally 80 or less, optionally 75 or less, optionally 70 or less, optionally 67 or less, optionally 60 or less, optionally 50 or less, optionally 40 or less, optionally 33 or less, optionally 30 or less, optionally 25 or less, optionally 20 or less, optionally 10 or less, optionally 5 or less, or optionally 1 or less. The method according to any one of embodiments 28 to 31.

[0135] 36. A method for producing a sterilized culture device, a. fixing the composition according to any one of embodiments 1 to 13 on a substrate to form a culture device; b. exposing the culture device to radiation sufficient to sterilize the composition. The method as described above.

[0136] 37. The method according to embodiment 36, comprising exposing the composition to radiation of at least 25 kGy, optionally at least 30 kGy, or radiation of about 25 kGy to about 35 kGy.

[0137] 38. The method according to any one of embodiments 36 - 37, further comprising placing the culture device in a package.

[0138] 39. The method according to embodiment 38, wherein the step of exposing the culture device to radiation is performed while the culture device is disposed within the package.

[0139] 40. The method according to any one of embodiments 38 - 39, wherein the step of exposing the culture device to radiation is performed while the culture device is sealed within the package.

[0140] 41. The composition or device according to any of the above embodiments, wherein scleroglucan is present in a weight percentage of scleroglucan relative to the total weight of one or more additional water - swellable polymers and the total weight of scleroglucan, and scleroglucan can be about 1 wt% or more, optionally about 5 wt% or more, optionally about 10 wt% or more, optionally about 20 wt% or more, optionally about 25 wt% or more, optionally about 30 wt% or more, optionally about 33 wt% or more, optionally about 40 wt% or more, optionally about 50 wt% or more, optionally about 60 wt% or more, optionally about 67 wt% or more, optionally about 70 wt% or more, optionally about 75 wt% or more, optionally about 80 wt% or more, optionally about 90 wt% or more, or optionally about 95 wt% or more, 100 wt%, or about 1 wt% - 100 wt%, or about 25 wt% - about 100 wt%, or about 10 wt% - about 70 wt%.

[0141] References In order to more fully explain and disclose the present invention and the state of the art to which the present invention pertains, a number of patents and publications have been cited above. The complete citations for these references are shown below. Each of these references is incorporated by reference into the present disclosure in its entirety to the same extent as if each individual reference had been specifically and individually indicated to be incorporated by reference.

[0142] All publications mentioned in this specification are incorporated by reference to the extent they support the present invention.

Claims

1. (i) One or more water-swellable polymers other than scleroglucan, (ii) Sclerogulcan and, A composition containing the following:

2. The composition according to claim 1, wherein the one or more water-swellable polymers include one or more selected from guar gum, locust bean gum, or xanthan gum.

3. The composition according to claim 1 or 2, wherein the weight ratio of the one or more water-swellable polymers to the scleroglucan is 1:99 to 99:

1.

4. The composition according to claim 1, wherein the weight percentage of the one or more water-swellable polymers relative to the sum of the total weight of the one or more water-swellable polymers and the total weight of the scleroglucan is 1 weight percent or more.

5. The composition according to claim 1, wherein the weight percentage of the scleroglucan relative to the sum of the total weight of the water-swellable polymer and the total weight of the scleroglucan is 95% by weight or less.

6. The composition according to claim 1, further comprising one or more nutrients that promote the growth of one or more microorganisms.

7. The composition according to claim 1, wherein the composition is sterilized by exposure to radiation of 25 kGy or more.

8. Base member and A cover sheet connected to the base member, A growth section is placed between the cover sheet and the base member, A scleroglucan layer containing scleroglucan located within or adjacent to the aforementioned growth area, A culture device equipped with the following features.

9. The culture device according to claim 8, wherein the scleroglucan layer further comprises one or more water-swellable polymers other than scleroglucan.

10. The culture device according to claim 8, wherein the one or more water-swellable polymers include one or more selected from guar gum, locust bean gum, or xanthan gum.

11. The culture device according to claim 9 or 10, wherein the weight ratio of the one or more water-swellable polymers in the scleroglucan layer to the scleroglucan is 1:99 to 99:

1.

12. The culture device according to claim 9, wherein the weight percentage of the one or more water-swellable polymers relative to the sum of the total weight of the one or more water-swellable polymers and the total weight of the scleroglucan is 1 weight percent or more.

13. The culture device according to claim 9, wherein the weight percentage of the scleroglucan relative to the sum of the total weight of the water-swellable polymer and the total weight of the scleroglucan is 95% by weight or less.

14. The culture device according to claim 8, wherein the scleroglucan layer further comprises one or more nutrients that promote the growth of one or more microorganisms.

15. The culture device according to claim 8, wherein the culture device is sterilized by exposure to radiation of 25 kGy or more.

16. The culture device according to claim 8, further comprising packaging, wherein the culture device is located inside the packaging, and the packaging provides a sterile barrier between the culture device and the external environment.

17. A method for culturing microorganisms, The process involves contacting a microorganism with the composition described in claim 1 to form an inoculated composition, The microorganisms are to be replicated at least once, The method, including the method described above.

18. A method for sterilizing culture devices, To prepare a culture device containing the composition described in claim 1, The culture device is exposed to radiation sufficient to sterilize it, The method, including the method described above.

19. A film comprising the composition described in claim 1.

20. An article comprising a base material and the film described in claim 19.

Images