Culture device capable of autonomously generating anaerobic environment and use method thereof

Abstract

A culture device for autonomously generating an anaerobic environment and a method of using the same. The invention provides a culture device capable of automatically generating an anaerobic environment. The culture device comprises a first substrate, wherein the first substrate is provided with an inner surface and an outer surface which are opposite to each other; the second substrate is provided with an inner surface and an outer surface which are opposite to each other; a growth region disposed between the inner surface of the first substrate and the inner surface of the second substrate; an effective amount of a substantially dry enzyme component in the enzyme-mediated oxygen consuming system; an effective amount of a substantially dry enzyme substrate component in the enzyme-mediated oxygen consumption system; and a cold water soluble dry gelling agent disposed in the growth region. The enzyme component and the enzyme substrate component are disposed within a coating in the growth zone. The first substrate and the second substrate are substantially impermeable to gaseous oxygen. Methods of making and using the culture device regions are also provided.

Description

[0001] This application is a divisional application of application number 201480058593.5 (International Application Number PCT / US2014 / 061351) filed on October 20, 2014, having the title "Culture Device for Autonomously Generating Anaerobic Environment and Methods of Use Thereof." Cross Reference to Related Applications

[0002] This application claims priority to U.S. Provisional Patent Application 61 / 895,170 filed on October 24, 2013, the disclosure of which is incorporated herein by reference in its entirety. BACKGROUND

[0003] Many bacteria are sensitive to oxygen and will not grow in the presence of oxygen. It can be useful to determine the viability of such anaerobic microorganisms in a variety of environments. For example, it can be important to determine whether anaerobic microorganisms are present in food processing and / or packaging equipment. It can also be important to determine whether anaerobic microorganisms are present in a medical environment, for example, to determine whether a pathogen is present using a diagnostic assay. As another example, water treatment facilities test water samples to determine whether such microorganisms are present.

[0004] A variety of devices are available for culturing microorganisms. For example, Petri dishes have been used for a long time to culture microorganisms. As is known in the art, a Petri dish is a shallow, flat-bottomed, circular dish that contains a suitable culture medium, such as agar and nutrients, to support the growth of microorganisms. However, the use of agar culture medium can be inconvenient and time consuming. For example, the agar culture medium must be sterilized, melted, and then cooled before a sample is added.

[0005] Additionally, it can be difficult to provide a suitable environment for culturing anaerobic microorganisms using a Petri dish. Because anaerobic microorganisms do not grow in large numbers in the presence of oxygen, it can be necessary to employ a cumbersome physico-chemical process to promote the growth of such microorganisms. Often, such devices must be modified, that is, shaped or constructed to provide a physical barrier that prevents the passage of oxygen.

[0006] Other techniques have been developed to remove oxygen using chemical reagents incorporated into anaerobic culture devices. Such devices typically include a reducing agent or pieces of bacterial-sterile membrane incorporated into a gel or nutrient medium. Additionally, U.S. Patent 3,387,794 describes an anaerobic bacterial culture device that is composed of an oxygen-impermeable membrane layer with a nutrient medium between the membranes, wherein the nutrient medium includes a reducing compound.

[0007] However, these devices and others can be expensive and can not be suitable for disposal after a single use. Assembly and / or use of these devices can also be cumbersome. Despite attempts in the art to produce simple devices for culturing anaerobic microorganisms in an aerobic environment, there remains a need for improved anaerobic culture devices. SUMMARY

[0008] The present invention relates generally to detecting microorganisms in a sample, optionally counting microorganisms in a sample. In particular, the present invention relates to culturing and detecting microaerophilic, microaerotolerant, or obligate anaerobic microorganisms. It is presently known that these microorganisms can be cultured and detected using culture devices that autonomously generate an anaerobic environment.

[0009] The culture devices and methods disclosed herein support the growth of microaerophilic, microaerotolerant, or obligate anaerobic microorganisms (e.g., bacteria, yeast), and the detection and differentiation of these microorganisms, even while the microorganisms are incubated in an oxygen-containing (e.g., normal atmospheric oxygen) environment. This advantageously eliminates the need for specialized incubation equipment and reagents (e.g., anaerobic jars, disposable anaerobic pouches, palladium catalyst, anaerobic glove boxes) that are typically required to culture anaerobic microorganisms. Additionally, the methods of the present invention allow for the detection of carbon dioxide gas produced by individual colonies, thereby supporting the differentiation of bacteria, eliminating the additional incubation time required to isolate pure cultures, and avoiding the use of fermentation tubes to detect gas production. Furthermore, the present invention relates to counting microaerophilic, microaerotolerant, or obligate anaerobic bacteria in a sample. Microaerophilic, microaerotolerant, and obligate anaerobic microorganisms share the common characteristic that they require low-oxygen environments for growth and propagation.

[0010] In one aspect, the present invention provides a method of detecting microorganisms in a sample. The method can include contacting a growth region of a culture device that autonomously generates an anaerobic environment with a predetermined volume of aqueous liquid in the culture device, the growth region comprising a cold water soluble dry gelling agent and an effective amount of substantially dry enzyme components of an enzyme-mediated oxygen consumption system prior to the step of contacting the growth region with the predetermined volume of aqueous liquid. The method can further include contacting the growth region with the sample; incubating the culture device for a period of time sufficient to allow microorganism colonies to form; and detecting the microorganism colonies.

[0011] In another aspect, the present invention provides a method of detecting microorganisms in a sample. The method can include contacting a growth region of a culture device that autonomously generates an anaerobic environment with a predetermined volume of aqueous liquid in the culture device, the growth region comprising a cold water soluble dry gelling agent prior to the step of contacting the growth region with the predetermined volume of aqueous liquid. The method can further include depositing an effective amount of enzyme components of an enzyme-mediated oxygen consumption system into the growth region; contacting the growth region with the sample; incubating the culture device for a period of time sufficient to allow microorganism colonies to form; and detecting the microorganism colonies. In any embodiment, the method can further include depositing an effective amount of enzyme substrate components of the enzyme-mediated oxygen consumption system into the growth region.

[0012] In yet another aspect, the present application provides a method of detecting microorganisms in a sample. The method can include contacting a growth area of a culture device that autonomously generates an anaerobic environment with a predetermined volume of aqueous liquid in the culture device, the growth area comprising a cold water soluble dry gelling agent prior to the step of contacting the growth area with the predetermined volume of aqueous liquid. The method can further include depositing an effective amount of an enzyme substrate component of an enzyme mediated oxygen depletion system into the growth area; contacting the growth area with the sample; incubating the culture device for a period of time sufficient to allow microorganism colonies to form; and then detecting the microorganism colonies. In any embodiment, the method can further include depositing an effective amount of an enzyme component of an enzyme mediated oxygen depletion system into the growth area.

[0013] In yet another aspect, the present application provides a culture device for counting microorganism colonies. The culture device can include a first substrate having opposing inner and outer surfaces; a second substrate having opposing inner and outer surfaces; a growth area disposed between the inner surface of the first substrate and the inner surface of the second substrate; a first effective amount of a substantially dry enzyme component of an enzyme mediated oxygen depletion system; a second effective amount of a substantially dry enzyme substrate component of the enzyme mediated oxygen depletion system; and a cold water soluble dry gelling agent disposed in the growth area. The first effective amount of a substantially dry enzyme component can be disposed in a first coating within the growth area. The second effective amount of a substantially dry enzyme substrate component can be disposed in a second coating within the growth area. The first substrate and the second substrate can be substantially impermeable to gaseous oxygen.

[0014] In yet another aspect, the present application provides a method of manufacturing a culture device for culturing anaerobic microorganisms. The method can include depositing a first coating onto a portion of a first substrate, the first coating formed using a liquid mixture comprising a liquid and an effective amount of an enzyme component of an enzyme mediated oxygen depletion system; drying the first coating; depositing a second coating onto a second substrate, the second coating comprising an enzyme substrate component of the enzyme mediated oxygen depletion system; positioning the first substrate proximate the second substrate with the first coating facing the second coating and with a growth area disposed between the first substrate and the second substrate overlapping a portion of the first coating and a portion of the second coating.

[0015] The words "preferred" and "preferably" do not mean "only this" or "only about this." Rather, the words mean that a certain embodiment is, for example, more advantageous than some other embodiments or is, for example, more useful in some circumstances than some other embodiments. The words "preferred" and "preferably" also mean that a certain embodiment is, for example, one of the best implementations.

[0016] The terms "comprise" and variations thereof herein, when appearing in the specification, do not have a limiting meaning. The terms "comprise", "comprising", "comprises" and "comprised of" as used herein are to be construed as meaning "including, but not limited to".

[0017] As used herein, "a," "an," "the," and "at least one" are used interchangeably and mean one or more than one. Thus, for example, a nutrient can be construed as meaning either one or more nutrients.

[0018] The terms "and / or," "and / or," and "and / or" mean one or all of the listed elements or a combination of any two or more of the listed elements.

[0019] Also, herein, ranges of values expressed in endpoints include all values included within that range (e.g., 1 to 5 includes 1, 1.5, 2, 2.75, 3, 3.80, 4, 5, etc.).

[0020] The above summary of the application is not intended to describe each disclosed embodiment or every implementation of the present application. The detailed description that follows more particularly exemplifies illustrative embodiments. Throughout the application the use of examples listing in several sections provides guidance as to how the examples in the list can be used in different ways. In each case the cited list is only used as a representative group and should not be interpreted as an exclusive list.

[0021] Further details of the above-described embodiments and other embodiments are described below with reference to the following drawings and detailed description. Other features, objects, and advantages will become apparent from the detailed description, drawings, and claims. BRIEF DESCRIPTION OF DRAWINGS

[0022] Figure 1 Top perspective view (partially cut away) of one embodiment of a culture device for autonomously generating an anaerobic environment according to the present application.

[0023] Figure 2 Top perspective view of an alternative embodiment of a culture device for autonomously generating an anaerobic environment according to the present application.

[0024] Figure 3 Top perspective view of an alternative embodiment of a culture device for autonomously generating an anaerobic environment according to the present application. Figure 2 Cross-sectional view of the culture device shown separated into the first base 12 and the second base 18 to facilitate depositing liquid and / or a sample into the growth area of the culture device.

[0025] Figure 4 Oxygen concentration versus time in a culture device is shown, some of the culture devices shown having received an enzyme-mediated oxygen-consuming system.

[0026] Figure 5 Oxygen concentration versus time in several culture devices for autonomously generating an anaerobic environment is shown, each of the culture devices shown having received an effective amount of an enzyme substrate. DETAILED DESCRIPTION

[0027] Before any embodiments of the application are explained in detail, it is to be understood that the application is not limited in its application to the details of construction and the arrangement of components set forth in the following description or illustrated in the following drawings. The application is capable of other embodiments and of being practiced or being carried out in various ways. Also, it is to be understood that the phraseology and terminology used herein is for the purpose of description and not of limitation. 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. Unless specified or limited otherwise, the terms "connected" and "coupled" and variations thereof are used broadly and encompass both direct and indirect connections, couplings, and attachments over both tangible and intangible means. Further, "connected" and "coupled" are not restricted to physical or mechanical connections or couplings. It is to be understood that other embodiments can be utilized, and structural or logical changes can be made without departing from the scope of the present application. Furthermore, embodiments can take on various other forms not specifically described herein. Also, terms such as "front," "back," "top," "bottom," and the like are used for description only and do not necessarily correspond to actual orientation of the device in use, or to indicate or imply necessary or required orientation of the device, or to specify how the application described herein is to be used, mounted, displayed, or positioned in use.

[0028] The present application generally relates to detecting microorganisms in a sample, optionally counting microorganisms in a sample. In particular, the present application relates to culturing and detecting microaerophilic, microaerobic, or obligate anaerobic microorganisms. It is presently known that these microorganisms can be cultured and detected using a culture device that autonomously generates an anaerobic environment.

[0029] It is also presently known that a dry, rehydratable culture device that autonomously generates an anaerobic environment can be manufactured. Such a culture device includes an effective amount of a substantially dry enzyme component and an effective amount of a substantially dry enzyme substrate component, which are disposed in a growth region of the culture device and are capable of being rehydrated in a predetermined volume of aqueous solution within the culture device, which, upon rehydration, are capable of participating in an oxygen-consuming reaction. Further, it is presently known that the oxygen-consuming reaction can consume a sufficient amount of oxygen to favor the growth of microaerophilic, microaerobic, or obligate anaerobic microorganisms. Further, the culture device can be maintained in an aerobic environment in which the culture device can maintain a low-oxygen environment for at least about 8 days to favor the growth of the aforementioned microorganisms.

[0030] Test samples that can be analyzed for the species of interest can originate from any source, such as physiological fluids (e.g., blood, saliva, lacrimal fluid, synovial fluid, cerebrospinal fluid, pus, sweat, exudate, urine, mucus), mucosal tissue (e.g., oral mucosa, gingival mucosa, nasal mucosa, ocular mucosa, tracheal mucosa, bronchial mucosa, gastrointestinal mucosa, rectal mucosa, urethral mucosa, ureteral mucosa, vaginal mucosa, cervical mucosa, uterine mucosa), breast milk during lactation, fecal matter, and the like. In addition, test samples can originate from a body site, such as a wound, skin, anterior nares, nasopharyngeal cavity, nasal cavity, nasal vestibule, scalp, fingernail / toenail, external ear, middle ear, mouth, rectum, vagina, axillary region, perineum, anus, or other similar site.

[0031] In addition to physiological fluids, other test samples can include other liquids as well as one or more solids dissolved or suspended in a liquid medium. Samples of interest can include process streams, water, food, food ingredients, beverages, soil, plant or other vegetation, air, surfaces (e.g., walls, floors, equipment, utensils in a manufacturing plant, hospital, clinic, or home), and the like.

[0032] Non-limiting examples of bacteria that require growth in an environment with reduced oxygen tension (i.e., microaerophilic bacteria) and / or bacteria that are tolerant of and can grow in an environment with reduced oxygen tension (i.e., anaerobic bacteria) include Helicobacter pylori, Campylobacter species (e.g., C. jejuni, C. coli, C. fetus), Streptococcus intermedius, Streptococcus sanguis, Streptococcus constellatus, Gemella morbillorum, Lactobacillus species, Streptococcus pyogenes.

[0033] Anaerobic bacteria are ubiquitous in nature. Anaerobic bacteria can be obligate anaerobes, or can be facultative anaerobes. Non-limiting examples of obligate anaerobes include Actinomyces species, Clostridium species (such as C. perfringens, C. tetani, C. sporogenes, C. botulinum, C. difficile, C. butyricum, C. acetobutylicum), lactic acid bacteria (such as Lactobacillus species, Leuconostoc species, Pediococcus species, Lactococcus species), Bacteroides species (such as B. fragilis), and Peptostreptococcus species (such as P. micros, P. magnus, P. asaccharolyticus, P. anaerobius, P. tetradius). Non-limiting examples of facultative anaerobes include Enterobacteria (such as E. coli), Salmonella species, Citrobacter freundii, Staphylococcus aureus, Listeria species (such as L. monocytogenes).

[0034] Because many yeast species are facultative anaerobes, and some yeast species are obligate anaerobes, it is expected that the culture device disclosed herein that autonomously generates an anaerobic environment can also be used to culture and detect yeast microorganisms.

[0035] In one aspect, the present application provides a culture device for culturing and detecting microorganisms that grow in a low-oxygen environment. See Figure 1The culture device 10 disclosed herein is configured to autonomously generate an anaerobic environment. The culture device 10 includes a first water-impermeable substrate 12, a second water-impermeable substrate 18, and a growth region 45 disposed between the first substrate 12 and the second substrate 18. The first substrate 12 has an inner surface 14 and an outer surface 16 opposite the inner surface 14. The second substrate 18 has an inner surface 20 and an outer surface 22 opposite the inner surface 20. In any embodiment, the inner surface 14 of the first substrate 12 is disposed facing the inner surface 20 of the second substrate 18.

[0036] A first dry coating 30 is disposed on the inner surface 14 of the first substrate 12. The first dry coating 30 is affixed to and covers at least the growth region 45 on the inner surface 14 of the first substrate 12. The first dry coating 30 includes a cold water-soluble dry gelling agent and a component of an enzyme-mediated oxygen-consuming system. In any embodiment, the component in the first coating includes a first effective amount of a substantially dry enzyme component of the enzyme-mediated oxygen-consuming system. Alternatively, in any embodiment, the component in the first coating includes a second effective amount of a substantially dry enzyme substrate capable of reacting with the enzyme component in a reaction that utilizes oxygen. The first dry coating 30 can cover the entire inner surface 14 of the first substrate 12, but preferably covers at least a portion of the inner surface 14 defining at least a first portion 40 of the growth region 45 in the culture device 10. The growth region 45 is located within the culture device 10 and is used to hold a sample during inoculation of the culture device with the sample and during incubation and detection of microorganisms, if any, in the sample.

[0037] In any embodiment, the second dry coating 32 is disposed on the inner surface 20 of the second substrate 18. The second dry coating 32 is affixed to and covers at least the growth region 45 on the inner surface 20 of the second substrate 18. The second dry coating 32 includes a cold water-soluble dry gelling agent and a component of an enzyme-mediated oxygen-consuming system. In any embodiment, the component in the second coating includes a first effective amount of a substantially dry enzyme component of the enzyme-mediated oxygen-consuming system. Alternatively, in any embodiment, the component in the second coating includes a second effective amount of a substantially dry enzyme substrate capable of reacting with the enzyme component in a reaction that utilizes oxygen. The second dry coating 32 can cover the entire inner surface 20 of the second substrate 18, but preferably covers at least a portion of the inner surface 20 defining at least a second portion 42 of the growth region 45 in the culture device 10. The second portion 42 of the growth region 45 is located within the culture device 10 and is used to hold a sample during inoculation of the culture device with the sample and during incubation and detection of microorganisms, if any, in the sample. Figure 1 In the illustrated embodiment, the first dry coating 30 covers substantially the entire inner surface 14 of the first substrate 12. Accordingly, the growth region can be located anywhere within the culture device 10 between the first substrate 12 and the second substrate 18. Preferably, the growth region is located away from the peripheral edge 50.

[0038] The growth region 45 is defined as the space disposed between the inner surface 14 of the first substrate 12 and the inner surface 20 of the second substrate 18 that contains at least a portion of the first dry coating 30 and / or the second dry coating (discussed below). Accordingly, when an aqueous liquid is dispensed into the growth region, it comes into fluid contact with at least a portion of the first dry coating 30 and / or the second dry coating. The thickness of the growth region 45 can vary depending on a variety of factors, such as: the volume of aqueous liquid (not shown) deposited in the culture device, the presence of solids (e.g., suspended particulate matter and / or a membrane filter) associated with the sample (not shown), and / or the presence of a spacing member (if any) in the culture device 10 (discussed below).

[0039] The first substrate 12 is preferably a relatively rigid, water-resistant film made of a material that does not absorb water, or is otherwise not adversely affected by water (e.g., polyester, polypropylene, or polystyrene). The first substrate 12 is preferably made using a material that is substantially impermeable to gaseous oxygen. Non-limiting examples of suitable materials for making the first substrate 12 include polyester films of at least about 15 pm to at least about 180 pm, polypropylene films of at least about 100 pm to at least about 200 pm, and polystyrene films of at least about 300 pm to about 380 pm. Other suitable first substrates include ethylene-vinyl alcohol copolymer films, polyvinyl alcohol films, and polyvinylidene chloride films. If it is desired to view colonies through the first substrate 12, the first substrate 12 can be transparent.

[0040] The second substrate 18 overlies the inner surface 14 of the first substrate 12 to define a growth area 45, and optionally, to view the growth area during transport, storage, incubation, and / or colony counting. The second substrate 18 is preferably a relatively rigid, water-resistant film made of a material that does not absorb water, or is otherwise not adversely affected by water (e.g., polyester, polypropylene, or polystyrene). The second substrate 18 is preferably transparent to facilitate colony counting without opening the culture device 10, and is substantially impermeable to microorganisms and water vapor.

[0041] In general, the second substrate can be made of, for example, those materials used to make the first substrate 12. The second substrate 18 is preferably made using a material that is substantially impermeable to gaseous oxygen. Non-limiting examples of suitable materials for making the first substrate 12 include polyester films of at least about 15 pm to at least about 180 pm, polypropylene films of at least about 100 pm to at least about 200 pm, and polystyrene films of at least about 300 pm to about 380 pm. Other suitable first substrates include ethylene-vinyl alcohol copolymer films, polyvinyl alcohol films, and polyvinylidene chloride films. As Figure 1 As shown, the second substrate 18 can be attached in a flap-like manner (e.g., using double-sided tape) along one edge of each of the inner surfaces of the first substrate 12 and the second substrate 18.

[0042] One of ordinary skill in the art will recognize that the percentage of oxygen that penetrates a given type of polymer film can be reduced by increasing the thickness of the polymer film. In any embodiment, both the first substrate and the second substrate of the present disclosure are suitable thicknesses of polymer films, both of which are of a thickness that results in the film being substantially impermeable to gaseous oxygen.

[0043] If the first coating 30 is comprised primarily of dry powder or agglomerates of dry powder, it is preferred that the first coating 30 be disposed on an adhesive layer 32 disposed on at least a portion of the inner surface 14 of the first substrate 12. The first dry coating 30 can be deposited onto the first substrate 12, or optionally the adhesive layer 32, using, for example, the compounding processes, adhesive coating processes, liquid coating processes, and / or dry coating processes described in U.S. Patents 4,565,783; 5,089,413; and 5,232,838, all of which are incorporated herein by reference in their entirety.

[0044] With respect to the first dry coating 30, it optionally can include any nutrient or nutrient medium that can be rehydrated with cold water, that does not substantially interfere with the cold water gelling properties of the gelling agent, and that supports the growth of anaerobic microorganisms. The particular nutrient or nutrients suitable for use in the culture device depend on the microorganisms that will be grown in the device, and can be readily selected by one skilled in the art. In general, such nutrients are soluble in cold water. Suitable nutrients to support the growth of bacteria are known in the art, including but not limited to yeast extract, peptone, sugars, suitable salts, and the like. In any embodiment, the first dry coating can also include a selective agent (such as a nutrient, an antibiotic, and combinations thereof) that promotes the growth of a particular anaerobic microorganism or group of microorganisms to a greater extent than another microorganism or group of microorganisms. Those skilled in the art will recognize that a variety of other formulations can be used without detracting from the scope of the present invention.

[0045] Suitable gelling agents for use in the first dry coating 30 include cold water soluble natural gelling agents and synthetic gelling agents. Natural gelling agents (e.g., alginates, carboxymethylcellulose, tara gum, hydroxyethylcellulose, guar gum, locust bean gum, xanthan gum) and synthetic gelling agents (e.g., polyacrylamide, polyurethane, polyethylene oxide, polyvinyl alcohol) and mixtures thereof are generally suitable. Suitable gelling agents can be selected in accordance with the teachings of the present invention and the disclosures of U.S. Patents 4,565,783; 5,089,413; and 5,232,838. Preferred gelling agents include guar gum, locust bean gum, xanthan gum; these gelling agents can be used individually, and preferably in combination with one another.

[0046] The culture device of the present application includes an enzyme-mediated oxygen depletion system disposed therein, the system comprising at least one substantially dry component. The at least one dry component is hydrated with an aqueous liquid prior to, during, or after the introduction of sample material (e.g., inoculation) into the growth region of the culture device as described herein. Typically, the sample material and / or the aqueous liquid is introduced into the growth region of the culture device under ambient conditions (i.e., in the presence of an oxygenated gas environment). Thus, after inoculation of the growth region of the culture device with the sample under aerobic conditions, the aqueous liquid in the growth region has a first dissolved oxygen concentration. The enzyme-mediated oxygen depletion system in the culture device functions to reduce the first dissolved oxygen concentration of the aqueous liquid in the growth region to a second dissolved oxygen concentration that is substantially lower than the first dissolved oxygen concentration. As a result of the substantial reduction in the dissolved oxygen concentration in the inoculated growth region of the culture device, anaerobic or microaerophilic microorganisms are encouraged to grow in the culture device.

[0047] In any embodiment, the at least one dry component comprises an enzyme component. The enzyme component is capable of reacting with an enzyme substrate in a reaction that utilizes oxygen (e.g., dissolved oxygen in an aqueous medium). In use, the enzyme-mediated oxygen depletion system disclosed herein comprises a first effective amount of the enzyme component and a second effective amount of the enzyme substrate component. The first effective amount of the enzyme component and the second effective amount of the enzyme substrate component are capable of reacting to reduce a first dissolved oxygen concentration in an aqueous liquid in fluid communication with the growth region of the culture device according to the present application to a second dissolved oxygen concentration that is substantially lower than the first dissolved oxygen concentration, the second dissolved oxygen concentration being sufficiently low to encourage the growth of anaerobic or microaerophilic microorganisms.

[0048] In any embodiment, the first effective amount and the second effective amount are selected such that the first dissolved oxygen concentration is reduced to the second dissolved oxygen concentration within about 120 minutes after the enzyme component and the enzyme substrate component are brought into fluid contact with a predetermined volume of the aqueous liquid in the growth region of the culture device. In any embodiment, the first effective amount and the second effective amount are selected such that the first dissolved oxygen concentration is reduced to the second dissolved oxygen concentration within about 60 minutes after the enzyme component and the enzyme substrate component are brought into fluid contact with a predetermined volume of the aqueous liquid in the growth region of the culture device. In any embodiment, the first effective amount and the second effective amount are selected such that the first dissolved oxygen concentration is reduced to the second dissolved oxygen concentration within about 30 minutes after the enzyme component and the enzyme substrate component are brought into fluid contact with a predetermined volume of the aqueous liquid in the growth region of the culture device.

[0049] One of ordinary skill in the art will recognize that the rate of many enzyme-catalyzed reactions is generally related to the temperature of the reaction, within a range of temperatures that can be specific to a particular enzyme or a particular reaction. In any embodiment, the first dissolved oxygen concentration is reduced to the second dissolved oxygen concentration at a temperature between ambient temperature (e.g., about 23°C) and about 42°C, inclusive. Thus, in any embodiment of the method according to the present application, the first dissolved oxygen concentration is reduced to the second dissolved oxygen concentration within about 120 minutes, about 60 minutes, or about 30 minutes, without incubating the culture device at an elevated temperature (i.e., a temperature greater than ambient temperature), after the enzyme component and the enzyme substrate component are brought into fluid contact with the predetermined volume of aqueous liquid in the growth region of the culture device.

[0050] Many redox enzyme-mediated oxygen-consuming reactions are known, including, for example, reactions catalyzed by peroxidases, glucose oxidases, laccases, tyrosinases, ascorbate oxidases. However, some redox enzymes (e.g., peroxidases) react with enzyme substrates (e.g., hydrogen peroxide) that are less stable at lower concentrations in aqueous solution and are toxic to microorganisms (e.g., particularly some anaerobic microorganisms). Additionally, some redox enzymes (e.g., glucose oxidases) react with oxygen to produce reaction products (e.g., hydrogen peroxide) that are toxic to microorganisms. In any embodiment, catalase or other peroxide-consuming enzymes can be added to the reaction in order to reduce the level of accumulated hydrogen peroxide. Thus, a suitable enzyme-mediated oxygen-consuming system according to the present application utilizes an enzyme substrate that, when present in an effective amount in the growth region of the culture device, is in fluid communication with anaerobic microorganisms under conditions suitable for the growth of the anaerobic microorganisms, without significantly inhibiting the growth of the anaerobic microorganisms. Similarly, a suitable enzyme-mediated oxygen-consuming system according to the present application utilizes an enzyme and an enzyme substrate that, when each is present in an effective amount in the growth region of the culture device, and collectively consume sufficient oxygen to create a locally low-oxygen or anaerobic environment, generate reaction products of varying yield and type that are in fluid communication with anaerobic microorganisms under conditions suitable for the growth of the anaerobic microorganisms, without significantly inhibiting the growth of the anaerobic microorganisms.

[0051] One of ordinary skill in the art will recognize that the amount of oxygen removed from the growth region of the culture device disclosed herein, over a period of time suitable for culturing microorganisms, depends, inter alia, on the amount (i.e., activity) of the enzyme component and the enzyme substrate component. The experimental results presented herein show a relationship between the amount of enzyme substrate component available to participate in the oxygen-consuming reaction and the rate and extent of oxygen removal from the growth region. Thus, by adjusting the amount of each component in the enzyme-mediated oxygen-consuming system according to the present application, the culture device can be configured for culturing microaerophilic microorganisms, facultative anaerobic microorganisms, or obligate anaerobic microorganisms.

[0052] Non-limiting examples of suitable enzyme-mediated oxygen-consuming systems according to the invention include systems containing ascorbic acid oxidase and its corresponding enzyme substrate (e.g., L-ascorbic acid), and systems containing laccase and its corresponding enzyme substrate (e.g., hydroquinone or 2,6-dimethoxyphenol).

[0053] Referring again to the accompanying drawings, in any embodiment, the culture device 10 may optionally include a second dry coating (such as...). Figure 2 As shown, the second dry coating is disposed on the inner surface 20 of the second substrate 18. The second dry coating may comprise a cold water-soluble drying gelling agent and / or a component in an enzyme-mediated oxygen-consuming system. In any embodiment, the component in the second coating comprises a first effective amount of substantially dry enzyme component in an enzyme-mediated oxygen-consuming system. Alternatively, in any embodiment, the component in the second coating comprises a second effective amount of substantially dry enzyme substrate capable of reacting with the enzyme component in an oxygen-utilizing reaction. Suitable gelling agents used in the second dry coating include those described above applicable to the first dry coating.

[0054] Figure 2 Another embodiment of the invention is shown, namely, a culture device 10'. The culture device 10' and... Figure 1 The illustrated device 10 is similar, including a first waterproof substrate 12, a second waterproof substrate 18, and a growth region 45 disposed between the first substrate 12 and the second substrate 18. The first substrate 12 has an inner surface 14 and an outer surface 16 opposite to the inner surface 14. The second substrate 18 has an inner surface 20 and an outer surface 22 opposite to the inner surface 20. In any embodiment, the inner surface 14 of the first substrate 12 is disposed facing the inner surface 20 of the second substrate 18. Additionally, a first dry coating 30 is disposed on at least a portion 40 of the inner surface 14 of the first substrate 12. Optionally, an adhesive layer 32 is disposed between at least a portion of the first dry coating 30 and the first substrate 12.

[0055] The second substrate 18 may be without any coating, or may be as follows: Figure 2 As shown, a pressure-sensitive adhesive (e.g., adhesive 37) is applied to the inner surface 20 facing the first substrate 12. In any embodiment, a portion of the adhesive 37 layer may be used to cause the second substrate 18 to be sealed to at least a portion of the first substrate 12 or to a coating (such as a first dry coating 30) on the first substrate 12. Additionally or alternatively, a second dry coating 35 may be applied to a portion of the adhesive 37 layer as described herein.

[0056] The adhesive 37 disposed on the second substrate 18 may be the same as or different from the adhesive 32 disposed on the first substrate 12. Furthermore, the second dry coating 35 disposed on the second substrate 18 may be the same as or different from the first dry coating 30 disposed on the first substrate 12. The coating on the second substrate 18 may cover the entire surface facing the first substrate, but preferably at least a portion 41 of at least a portion of the growth region 45 defined in the culture device 10' on the inner surface 20. Such a coated second substrate is particularly preferred if it is desired that the device carries more gelling agent than can be incorporated into the single first dry composition.

[0057] If the second coating 35 consists primarily of dry powder or clumps of dry powder, it is preferable to arrange the second coating 30 on an adhesive layer 37, which is disposed on at least a portion of the inner surface 20 of the second substrate 18. The second dry coating 35 can be deposited onto the second substrate 18, or optionally the adhesive layer 37, using, for example, the compounding processes, adhesive coating processes, liquid coating processes, and / or dry coating processes described in U.S. Patents 4,565,783, 5,089,413, and 5,232,838.

[0058] The second dry coating 35 may optionally contain any nutrient or nutrient medium that can be rehydrated with cold water, substantially does not interfere with the cold water gelling properties of the gelling agent, and supports the growth of anaerobic microorganisms. One or more specific nutrients suitable for the culture device depend on the microorganisms to be grown in the device and can be readily selected by those skilled in the art. Generally, such nutrients are soluble in cold water. In any embodiment, the second dry coating 35 may also contain a selector (such as a nutrient, antibiotic, or a combination thereof) that promotes the growth of a particular anaerobic microorganism or microbial community to a greater extent than other microorganisms or microbial communities.

[0059] The culture device 10' also includes a spacer member disposed between the first substrate 12 and the second substrate 18 to create a well that defines both the position and thickness of the growth region 45 of the culture device 10' and to confine the aqueous sample within the growth region 45. The spacer member... Figure 2A spacer 46 is shown defining a circular aperture 48. The walls of the circular aperture 48 form a well of predetermined size and shape and define the thickness of the growth region 45 of the culture device 10'. The spacer 46 should be thick enough to form a well of the desired volume, e.g., 1 ml, 2 ml, or 3 ml, depending on the size of the growth region and the size of the sample to be placed in the culture device. In any embodiment, the spacer 46 is made of closed-cell polyethylene foam; however, any material that is hydrophobic (not wetted), inert to microorganisms, and sterilizable can be used. In any embodiment, the spacer member can be directly coupled to the first substrate or the second substrate (e.g., via a pressure sensitive adhesive). Additionally or alternatively, the spacer member can be indirectly coupled to the first substrate or the second substrate (e.g., via a pressure sensitive adhesive) (e.g., the spacer member can be coupled to a first dry coating or a second dry coating that is separately coated onto the first substrate or the second substrate, respectively).

[0060] In Figure 2 In the illustrated embodiment, the culture device 10' includes a first dry coating 30 disposed on the inner surface 14 of the first substrate 12 in the growth region 45, and the culture device 10' includes a second dry coating 35 disposed on the inner surface 20 of the second substrate 18 in the growth region. The first dry coating 30 contains a first effective amount of a component (e.g., an enzyme component) of an enzyme-mediated oxygen-consuming system, and the second dry coating 35 contains a second effective amount of another component (e.g., a corresponding enzyme substrate of the enzyme component) of the enzyme-mediated oxygen-consuming system. Thus, when an aqueous liquid (e.g., water, an aqueous buffer, an aqueous nutrient medium, an aqueous sample) is introduced into the growth region of the culture device, the first effective amount of the component and the second effective amount of the other component are in fluid communication, and the system reacts with oxygen present in the coating and the aqueous liquid, thereby consuming oxygen in the growth region of the culture device to create an environment conducive to the growth of microaerophilic or anaerobic microorganisms.

[0061] In Figure 2 and Figure 3 In the illustrated embodiment, the first dry coating 30 covers substantially the entire inner surface 14 of the first substrate 12, and only the portion 40 exposed by the circular aperture 48 in the spacer 46 is accessible. Thus, when the first substrate 12 and the second substrate 18 are positioned adjacent to each other, the growth region 45 of the culture device 10' is defined by the area of the circular aperture 48 and the portions (41 and 40, respectively) of the first dry coating 30 and the second dry coating 35 that overlap the circular aperture 48. In this embodiment, the growth region is located away from the peripheral edge 50, and communication between the growth region 45 and the external environment (e.g., air) is substantially blocked by the spacer 46.

[0062] The culture apparatus of the present invention optionally further includes means for indicating the presence of oxygen in the culture apparatus. This means is preferably capable of indicating the amount of oxygen present in the apparatus (e.g., a predetermined threshold or relative amount). Advantageously, the means can indicate whether the enzyme-mediated oxygen-consuming system has adequately consumed oxygen in the growth region of the culture apparatus to a concentration favorable to the growth of microaerophilic or anaerobic microorganisms, or can indicate the moment when the enzyme-mediated oxygen-consuming system has adequately consumed oxygen in the growth region of the culture apparatus to a concentration favorable to the growth of microaerophilic or anaerobic microorganisms. Components for detecting the means in the culture apparatus are known in the art, including, for example, redox dyes (e.g., methylene blue) and oxygen-quenched fluorescent dyes.

[0063] Such a device may be a luminescent compound indicating the presence of oxygen within the device. Suitable oxygen indicators are disclosed in U.S. Patent 6,689,438 (Kennedy et al.), the entire contents of which are incorporated herein by reference. The fluorescence displayed by luminescent compounds suitable for use as indicators in the culture apparatus disclosed herein is quenched by oxygen. More specifically, these indicators emit light upon exposure to an excitation frequency, with the intensity of the light inversely proportional to the oxygen concentration. Such an indicator may be coated, laminated, or extruded onto another layer within the device, or onto a portion of another layer. This layer may be disposed within the growth region, optionally separated from the growth region by one or more additional oxygen-permeable layers. Suitable compounds for indicating oxygen include octaethylporphyrin, tetraphenylporphyrin, tetrabenzoporphyrin, dihydroporphyrin, or metal derivatives of bacterial porphyrin. Other suitable compounds include coprophyllite palladium (PdCPP), octaethylporphyllite platinum and octaethylporphyllite palladium (PtOEP, PdOEP), tetraphenylporphyllite platinum and tetraphenylporphyllite palladium (PtTPP, PdTPP), camphorquinone (CQ), and xanthracene dyes (such as erythrosine B (EB)). Other suitable compounds include complexes of ruthenium, osmium, and iridium with ligands such as 2,2'-dipyridine, 1,10-o-phenanthroline, 4,7-diphenyl-1,10-o-phenanthroline, etc. Suitable examples of these complexes include tris(4,7-diphenyl-1,10-o-phenanthroline)ruthenium(II) perchlorate, tris(2,2'-dipyridine)ruthenium(II) perchlorate, tris(1,10-o-phenanthroline)ruthenium(II) perchlorate, etc.

[0064] Those skilled in the art will recognize that the rate of an enzyme-catalyzed reaction can be affected by the pH of the reaction environment. Furthermore, those skilled in the art will recognize that the growth of microaerophilic or anaerobic microorganisms in the culture apparatus of the present invention can also be affected by the pH of the microbial culture environment. It is known that the pH of the growth zone can be pre-selected to promote both aerobic enzyme reactions and microbial growth. In any embodiment, a buffer can be incorporated into at least one dry coating and / or the plate can be inoculated with a buffered aqueous solution to control the pH of the growth zone.

[0065] In use, the growth region of the culture apparatus disclosed in this invention comprises a first effective amount of one component (e.g., an enzyme component) in an enzyme-mediated oxygen-consuming system, and a second effective amount of another component (e.g., the corresponding enzyme substrate of the enzyme component) in an enzyme-mediated oxygen-consuming system, the two components being fluidly connected to each other. The maximum oxygen-consuming capacity of the system is generally determined by the amount of enzyme substrate in the growth region. Preferably, the aqueous growth region contains sufficient enzyme substrate to react with substantially all dissolved oxygen in the region. More preferably, the aqueous growth region contains sufficient enzyme substrate to react with more dissolved oxygen than is normally present in the region.

[0066] In any embodiment, the growth region can be sized to hydrate with 1 ml of aqueous liquid. Each milliliter of water contains approximately 0.54 micromoles of dissolved oxygen. Therefore, the first and / or second drying coating preferably contains at least a sufficient amount of enzyme substrate, which can consume 0.54 micromoles of oxygen over a period of 120 minutes or less at a temperature of approximately 22°C to approximately 42°C. More preferably, the first and / or second drying coating preferably contains at least a sufficient amount of enzyme substrate, which can consume more than 0.54 micromoles of oxygen over a period of 120 minutes or less at a temperature of approximately 22°C to approximately 42°C.

[0067] In any embodiment, the first and / or second dry coating may contain any number of other components, such as dyes (e.g., pH indicators), crosslinking agents, reagents (e.g., selection reagents or indicator reagents, such as chromogenic enzyme substrates or luciferase substrates), or combinations of any two or more of the foregoing components. For example, for some applications, it is necessary to incorporate microbial growth indicators (e.g., pH indicators, chromogenic enzyme substrates, redox dyes) into the first and / or second dry coatings, or into the adhesive to which the dry coatings adhere. Suitable dyes include those that are metabolized by growing microorganisms, or those that otherwise react with growing microorganisms, thereby causing colony staining or fluorescence (making colonies more visible). Such dyes include triphenyltetrazolium chloride, p-tolyltetrazole red, tetrazolium violet, veratrolityltetrazole blue, and related dyes, as well as disodium 5-bromo-4-chloroindole phosphate. Other suitable dyes include those that are sensitive to pH changes during microbial growth, such as neutral red.

[0068] For some applications, it is necessary to form a dry coating that, upon rehydration with an aqueous liquid, forms a hydrogel with sufficient rigidity to allow the operator to inoculate using the streak method. To form a streakable culture medium, an effective amount of a suitable crosslinking agent can be incorporated into one or more dry coatings containing a gelling agent. A suitable crosslinking agent substantially does not affect the growth of the target microorganism. Those skilled in the art can readily select suitable types of crosslinking agents and their appropriate amounts. For example, for guar gum, suitable crosslinking agents are (such as) potassium tetraborate, aluminum salts, or calcium salts, and an effective amount (e.g., less than about 1.0% by weight of the dry coating) of these suitable crosslinking agents can be added to the dry coating.

[0069] At least one dry coating may optionally contain reagents necessary for performing certain microbiological tests. For example, it may contain antibiotics for performing antibiotic susceptibility testing. For microbial identification, it may contain a differential reagent that changes color in the presence of a specific type of microorganism.

[0070] The culture apparatus of the present invention can be prepared using a variety of techniques. Generally, the culture apparatus of the present invention can be manufactured by hand or using common laboratory equipment described herein and, for example, in U.S. Patents 4,565,783, 5,089,413 and 5,232,838.

[0071] The first and / or second dry coatings in the culture apparatus of the present invention may contain adhered powdered culture medium. The steps for preparing and fixing the adhered powdered culture medium are as follows: First, an adhesive layer (layer 32 or layer 37, respectively) is formed on at least a portion of the inner surface of the first and / or second substrates in the growth region. This adhesive is preferably a water-insoluble pressure-sensitive adhesive that substantially does not inhibit the growth of microorganisms to be cultured in the culture apparatus. Preferably, the adhesive layer 32 or layer 37 remains sufficiently transparent when wetted, allowing an observer to observe microbial colonies through it.

[0072] Non-limiting examples of suitable pressure-sensitive adhesives are copolymers of 2-methylbutyl acrylate and acrylic acid in a 90 / 10 molar ratio. Other preferred pressure-sensitive adhesives that can be used include copolymers of isooctyl acrylate and acrylic acid in a 95 / 5 or 94 / 6 molar ratio, and silicone rubber. Adhesives that become emulsified (e.g., opaque) upon exposure to water are less preferred but can be used in conjunction with an opaque first substrate or where colony visibility is not required. Heat-activated adhesives and / or water-activated adhesives (such as adhesives) are also known, in which a lower-melting-point substance in the heat-activated adhesive is coated onto a higher-melting-point substance; both types of adhesives can be used in this invention. When incorporating the aforementioned indicator reagents to facilitate colony visibility, it is generally preferred to incorporate the indicator reagents into the adhesive or broth coating mixture rather than into the powder.

[0073] An adhesive is applied (e.g., using a doctor blade coater) to the top surface of a first or second substrate to form an adhesive layer, the thickness of which is preferably less than the average particle size of the dry powder or agglomerated powder to be adhered to the adhesive. Generally, the amount of adhesive applied is sufficient to adhere the powder particles to the substrate (e.g., the first or second substrate described herein) without being excessive and causing the particles to become completely embedded in the adhesive. Generally, an adhesive layer of about 5 µm to about 12 µm thick is suitable.

[0074] To form an adhesive powder culture medium, a layer of cold water-soluble powder (e.g., including a cold water-soluble gelling agent), optionally a nutrient, a selective reagent, a component of an enzyme-mediated oxygen-consuming system, or a combination of any two or more of the aforementioned powders is then substantially uniformly adhered to an adhesive layer to be arranged in at least a portion of the growth area of ​​the culture device.

[0075] Preferably, when the first and / or second dry coatings contain a gelling agent, the amount of gelling agent used is such that a predetermined amount of water or an aqueous sample (e.g., 1 to 3 ml) placed in the growth zone will form a hydrogel of suitable viscosity, for example, approximately 1500 cps or greater when measured at 60 rpm using a Brookfield LVF viscometer at 25°C. Because of this viscosity, the operator can move and stack the culture apparatus during incubation, and this viscosity of hydrogel supports the formation of clear colonies in the culture medium. For example, 0.025 g to 0.050 g of powdered guar gum in a 20.3 cm² medium... 2 When spread substantially evenly over a surface area, these powdered guar gums will form a sufficiently viscous culture medium upon rehydration with 1 to 3 ml of aqueous sample. The coating weight per unit area can be controlled by adjusting the particle size. For example, a 100-mesh guar gum coating would yield approximately 0.05 g / 20.3 cm². 2 At this weight, the 400-mesh guar gum coating reaches approximately 0.025g / 20.3cm. 2 The weight.

[0076] In any embodiment, the first or second dry coating may contain one or more nutrients to promote microbial growth. If the coating consists substantially of powder or powder clumps, the preferred ratio of gelling agent to nutrients in the adhered powder culture medium is determined by the specific microorganisms to be cultured in the culture device. However, for general purposes, a ratio of about 4:1 to about 5:1 (total gelling agent to total nutrients, by weight) is preferred. The powder in the adhered powder culture medium can be applied to the adhesive layer (e.g., adhesive layer 32 and / or adhesive layer 37) using any method suitable for substantially uniformly applying the powder layer. Examples of suitable methods for applying the powder layer include the use of a vibratory apparatus or a powder coating machine.

[0077] Another preferred method for forming a dry coating (i.e., a first dry coating and / or a second dry coating) is a liquid coating method. In this method, a liquid mixture (e.g., an aqueous liquid mixture) is first prepared, containing a gelling agent, a component of an enzyme-mediated oxygen-consuming system (e.g., an enzyme component), and optionally nutrients, a selector, and / or an indicator. This liquid mixture is then (e.g., using a doctor blade coater) coated onto a first or second substrate, and subsequently (e.g., by flash evaporation) substantially all of the liquid is removed, resulting in a substantially dry (e.g., substantially anhydrous) coating. The gelling agent can be used to increase the consistency of the liquid mixture to facilitate its coating onto the substrate. From a practical standpoint, the amount of gelling agent is preferably insufficient to increase the consistency of the mixture to a degree unsuitable for coating the culture medium onto the substrate. These coated mixtures are generally self-adhesive to the substrate, thus eliminating the need for an adhesive layer between the substrate and the culture medium.

[0078] In either embodiment, the first and second dry coatings can be prepared using similar processes (i.e., both coatings are prepared using either a powder coating process or a liquid coating process). Alternatively, in either embodiment, one dry coating (e.g., the first dry coating) is applied using a liquid coating process, and the other dry coating (e.g., the second dry coating) is applied using a powder coating process. Furthermore, the first and second dry coatings may contain the same or different components.

[0079] In any embodiment, the culture apparatus of the present invention includes a substrate (e.g., a first substrate) having a dry coating (e.g., a first dry coating) comprising a gelling agent and an enzyme component in an enzyme-mediated oxygen-consuming system, and another substrate (e.g., a second substrate) having a dry coating (e.g., a second dry coating) comprising a gelling agent and / or an enzyme substrate component in an enzyme-mediated oxygen-consuming system. Preferably, the first dry coating is applied using a liquid coating process, and the second dry coating is applied using a powder coating process.

[0080] When using the culture apparatus of the present invention, it may be desirable to accurately count the colonies of present microorganisms. Therefore, in any embodiment, the culture apparatus of the present invention may have a grid pattern on a first substrate or a second substrate. This grid pattern may include a square grid pattern, such as the square grid pattern disclosed in U.S. Patent 4,565,783. This grid pattern may be generated on the first substrate or the second substrate by any suitable process, such as printing.

[0081] We also envision that the enzyme-mediated oxygen-consuming system can be used in a culture medium similar to that described in U.S. Patent 6,331,429, which is incorporated herein by reference. This culture medium, when arranged in a culture apparatus as disclosed herein, or arranged between a suitable first substrate and a second substrate as described herein, can be used for methods of culturing microaerophilic or obligate anaerobic microorganisms.

[0082] In another aspect, the present invention provides a method for preparing a culture device that autonomously generates an anaerobic environment. The method includes: depositing a first coating onto a portion of a first substrate, the first coating being formed using a liquid mixture comprising a liquid and an effective amount of an enzyme component in an enzyme-mediated oxygen-consuming system; drying the first coating; positioning the first substrate near a second substrate such that the first coating faces the second coating, and such that a growth region disposed between the first and second substrates overlaps with a portion of the first coating and a portion of the second coating.

[0083] The enzyme component in the first coating is an enzyme capable of reacting with an enzyme substrate and oxygen to produce a product. As described herein, this reaction occurs in an aqueous mixture, resulting in localized oxygen depletion within the aqueous mixture. Optionally, in any embodiment of the method, an adhesive layer may be disposed on a first substrate at a location between the first coating and the first substrate. Optionally, in any embodiment of the method, the first coating may also contain nutrients or nutrient culture media, a selector, an indicator, a crosslinking agent, a dye, or any combination of two or more of the foregoing substances, as described herein. The composition of the first coating is adjusted such that an effective amount of the enzyme component (e.g., ascorbic acid oxidase) is dispersed (e.g., uniformly dispersed) within a first portion of the growth region defined by the first substrate.

[0084] In any embodiment of the method, the method optionally further includes forming a second coating on a second substrate. This second coating contains an enzyme substrate component in an enzyme-mediated oxygen-consuming system, capable of reacting with the enzyme component and oxygen in an aqueous medium to generate a product. Optionally, in any embodiment of the method, an adhesive layer may be disposed on the second substrate at a location between the second coating and the second substrate. In any embodiment, the second coating comprises a substantially dry powder or powder agglomerate disposed on the adhesive layer adhered to the second substrate. The composition of the second coating is adjusted such that an effective amount of the enzyme substrate component (e.g., sodium ascorbate) is dispersed (e.g., uniformly dispersed) within a second portion of the growth region defined by the second substrate.

[0085] In any embodiment of the method, the liquid used to form the liquid mixture comprises an aqueous liquid. In any embodiment, the liquid used to form the liquid mixture comprises an organic liquid. In any embodiment of the method, the liquid mixture used to form the first coating further comprises a cold water-soluble gelling agent.

[0086] In any embodiment of the method, depositing the first coating onto a portion of the first substrate comprises: mixing an enzyme component and a gelling agent in a predetermined volume of aqueous liquid to form a coating mixture, and then applying the coating mixture onto the first substrate. In any embodiment, applying the coating mixture onto the first substrate may comprise: applying the coating mixture onto an adhesive layer disposed on the first substrate as described herein.

[0087] The first coating can be dried using a variety of processes known in the art. For example, the first coating can be dried in an oven (e.g., a gravity oven, a convection oven) according to the process described in U.S. Patent 5,601,998, the entire contents of which are incorporated herein by reference. Preferably, the first coating is dried until it is substantially free of water. As used herein, the phrases “substantially dry,” “substantially free of water,” etc., refer to a coating whose moisture content, once allowed to equilibrate with its surroundings, is substantially no greater than that of the dehydrated coating.

[0088] Various methods can be used to position the first substrate near the second substrate, so that the first coating faces the second coating, and the growth area arranged between the first substrate and the second substrate overlaps with a portion of the first coating and a portion of the second coating. Figures 1 to 3 Representative examples of applicable methods are shown, suitable for positioning a first substrate and a second substrate close to each other such that a portion of the first coating overlaps a portion of the second coating. As can be seen from the figures, this overlapping configuration allows an operator to deposit an aqueous liquid between the first and second substrates, thereby enabling fluid communication between the first and second coatings (if present).

[0089] In another aspect, the present invention provides a culture apparatus for counting Clostridium difficile colony-forming units in a sample. The culture apparatus includes, as described above, a first substrate having opposing inner and outer surfaces; a second substrate having opposing inner and outer surfaces; and a growth region disposed between the first and second substrates. The culture apparatus further includes a first effective amount of substantially dry enzyme component in an enzyme-mediated oxygen-consuming system, the first effective amount of substantially dry enzyme component disposed within a first coating in the growth region; a second effective amount of substantially dry enzyme substrate component in an enzyme-mediated oxygen-consuming system, the second effective amount of substantially dry enzyme substrate component disposed within a second coating in the growth region; and an effective amount of substantially dry nutrient composition disposed within the growth region. In any embodiment, the nutrient composition comprises a nutrient mixture (such as brain heart extract and / or yeast extract) for promoting the growth of nutritionally demanding microorganisms. In any embodiment, the culture device further includes an effective amount of at least one substantially dry selector (e.g., bile salts, such as sodium taurocholate; one or more antibiotics, such as cefoxitin, cycloserine), which, when hydrated with a predetermined volume of aqueous liquid, substantially allows the growth of *Clostridium difficile* and substantially inhibits the growth of non-*Clostridium difficile* microorganisms, such as *Escherichia coli*, *S. aureus*, *C. sporogenes*, *C. perfringens*, *Bacteroides fragilis*, *Prevotella melaninogencia*, *Fusobacterium* species, and *Peptostreptococcus anaerobius*. The culture device optionally also includes an effective amount of a substantially dry reducing agent (e.g., sodium thioglycolate and / or L-cysteine) arranged within the growth zone.

[0090] In another aspect, the present invention provides a method for detecting microorganisms. In any embodiment, the method utilizes a culture device that autonomously generates an anaerobic environment as disclosed in the present invention. The culture device includes: a first substrate having opposing inner and outer surfaces; a second substrate having opposing inner and outer surfaces; a growth region disposed between the inner surfaces of the first and second substrates; a first effective amount of substantially dry enzyme components in an enzyme-mediated oxygen-consuming system disposed within the growth region; and a cold water-soluble substantially dry gelling agent disposed within the growth region; wherein the first and second substrates are substantially impermeable to gaseous oxygen. Optionally, the culture device further includes a second effective amount of substantially dry enzyme substrate components in an enzyme-mediated oxygen-consuming system disposed within the growth region. In a preferred embodiment, the culture device further includes spacers defining the perimeter of the growth region.

[0091] In any embodiment of the method, a cold water-soluble, essentially dry gelling agent arranged within the growth region of a culture device that autonomously generates an anaerobic environment is hydrated with a predetermined volume of aqueous liquid to: i) promote the reaction of several components of the enzyme-mediated oxygen-consuming system, and ii) provide an aqueous environment to promote microbial growth. As discussed herein, the cold water-soluble gelling agent in the culture device can be hydrated before, during, and / or after sample deposition into the culture device.

[0092] In any embodiment, the predetermined volume of the aqueous liquid used for hydration and / or inoculation culture is about 1 ml. In any embodiment, the predetermined volume of the aqueous liquid used for hydration and / or inoculation culture is about 2 ml. In any embodiment, the predetermined volume of the aqueous liquid used for hydration and / or inoculation culture is about 3 ml. In any embodiment, the predetermined volume of the aqueous liquid used for hydration and / or inoculation culture is about 4 ml. In any embodiment, the predetermined volume of the aqueous liquid used for hydration and / or inoculation culture is about 5 ml. In any embodiment, the predetermined volume of the aqueous liquid used for hydration and / or inoculation culture is about 10 ml. In any embodiment, the aqueous liquid used for the growth region of the hydration culture is dispersed within the growth region at 20.3 cm intervals. 2 There is approximately 1 ml of liquid in the growth area.

[0093] In any embodiment, the effective amount of enzyme substrate component (such as L-ascorbic acid or its salt) in the growth region of the culture device can be selected based on a predetermined volume of the aqueous liquid to be deposited into the growth region. For example, if the predetermined volume is about 1 ml, the effective amount of enzyme substrate component (such as L-ascorbic acid or its salt) arranged or deposited in the growth region can be about 3 μmol to about 4.5 μmol (to create an environment for culturing microaerophilic or microaerophilic microorganisms), or about 7 μmol to about 30 μmol (to create an environment for culturing obligate anaerobic microorganisms). For example, if the predetermined volume is about 2 ml, the effective amount of enzyme substrate component (such as L-ascorbic acid or its salt) arranged or deposited in the growth region can be about 6 μmol to about 9 μmol (to create an environment for culturing microaerophilic or microaerophilic microorganisms), or about 14 μmol to about 60 μmol (to create an environment for culturing obligate anaerobic microorganisms). Those skilled in the art will recognize that the number of micromoles of enzyme substrate in a given enzyme-mediated oxygen-consuming system can vary depending on the number of micromoles of oxygen consumed per micromole of enzyme substrate component in a reaction catalyzed by a particular enzyme component.

[0094] In any embodiment, a predetermined volume of aqueous solution is dispersed in a space having a predetermined area (e.g., by a...). Figure 2 The growth region (defined by the holes 48 in the spacers 46 of the culture apparatus 10') is thus a predetermined area. Therefore, when coating the enzyme substrate component onto the first or second substrate, the effective amount of the enzyme substrate component (e.g., L-ascorbic acid or its salt) in the growth region of the culture apparatus can be selected based on the predetermined area of ​​the growth region. For example, if the predetermined area of ​​the growth region is defined by a circle with a diameter of about 2 inches (about 5.1 cm), the effective amount of the enzyme substrate component (e.g., L-ascorbic acid or its salt) disposed in or deposited in the growth region can be from about 0.15 μmol / cm² to about 0.22 μmol / cm² (to create an environment for culturing microaerophilic or microaerophilic microorganisms when the growth region is hydrated with 1 mL of liquid), or from about 0.35 μmol / cm² to about 1.5 μmol / cm² (to create an environment for culturing obligate anaerobic microorganisms when the growth region is hydrated with 1 mL of liquid).

[0095] The culture device is typically placed on a generally horizontal surface, separating the first and second substrates (e.g., raising the upper substrate) to allow access to the growth region while hydrating and / or inoculating it. It is advantageous to hydrate and / or inoculate the culture device in an aerobic environment (i.e., in air). Typically, an aqueous solution (which may contain the sample material to be tested) is transferred to the growth region between the first and second substrates for hydration. After a predetermined volume of aqueous solution has deposited into the growth region, the culture device is closed. Optionally, a planar or concave applicator (similar to those used for inoculating PETRIFILM culture devices) can be used to spread the aqueous solution over a predefined area within the culture device.

[0096] After hydrating the culture device, if the enzyme component and enzyme substrate component in the enzyme-mediated oxygen-consuming system are present in the growth region (e.g., the enzyme component is present in a substantially dry first composition located on one of the substrates in the growth region, while the enzyme substrate component is present in a substantially dry second composition located on another substrate in the growth region), then hydrating the growth region includes fluidly communicating the enzyme component and the enzyme substrate component with an aqueous fluid.

[0097] When the first composition, the second composition, and the aqueous liquid are fluidly connected, a mixture having a first dissolved oxygen concentration is formed. In any embodiment, the first dissolved oxygen concentration substantially inhibits the growth of obligate anaerobic microorganisms, microaerophilic microorganisms, and / or microaerophilic microorganisms. In these embodiments, fluidly connecting several components with an aqueous fluid initiates a reaction between the enzyme and its substrate and any oxygen dissolved in the coating and / or the aqueous fluid, thereby reducing the first dissolved oxygen concentration in the growth region to a second concentration lower than the first concentration (e.g., at least about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 98%, or about 99% lower than the first concentration).

[0098] In any embodiment, reducing the first dissolved oxygen concentration to the second dissolved oxygen concentration may include reducing the dissolved oxygen to a sufficiently low second concentration to support the growth of microaerophilic microorganisms. In any embodiment, reducing the first dissolved oxygen concentration to the second dissolved oxygen concentration may include reducing the dissolved oxygen to a sufficiently low second concentration to support the growth of microaerophilic microorganisms. In any embodiment, reducing the first dissolved oxygen concentration to the second dissolved oxygen concentration may include reducing the dissolved oxygen to a sufficiently low second concentration to support the growth of obligate anaerobic microorganisms.

[0099] In any embodiment, reducing the first dissolved oxygen concentration to the second dissolved oxygen concentration includes reducing the dissolved oxygen to the second concentration within approximately 120 minutes after the mixture is formed. In any embodiment, reducing the first dissolved oxygen concentration to the second dissolved oxygen concentration includes reducing the dissolved oxygen to the second concentration within approximately 90 minutes after the mixture is formed. In any embodiment, reducing the first dissolved oxygen concentration to the second dissolved oxygen concentration includes reducing the dissolved oxygen to the second concentration within approximately 60 minutes after the mixture is formed. In any embodiment, reducing the first dissolved oxygen concentration to the second dissolved oxygen concentration includes reducing the dissolved oxygen to the second concentration within approximately 45 minutes after the mixture is formed. In any embodiment, reducing the first dissolved oxygen concentration to the second dissolved oxygen concentration includes reducing the dissolved oxygen to the second concentration within approximately 30 minutes after the mixture is formed.

[0100] In any embodiment, the aqueous liquid used to hydrate the culture device includes water (e.g., sterile deionized water, biological buffer, sterile nutrient medium), and optionally may include one or more additives. These additives can function in various ways in the method. For example, in any embodiment, the additives may include nutrients or nutrient media to support the growth of anaerobic, microaerophilic, or microaerophilic microorganisms to be cultured in the culture device. Such nutrients or nutrient media are well known in the art and can be selected based on the specific microorganism to be cultured. These nutrients or nutrient media should substantially not interfere with enzyme-mediated oxygen-consuming systems. This can be easily detected using an oxygen sensor as described herein.

[0101] Additionally or alternatively, in any embodiment, the additive comprises at least a portion (or all) of a substantially dry enzyme substrate component (e.g., L-ascorbic acid, a metal salt of L-ascorbic acid, hydroquinones such as 2,5-dichlorohydroquinone, D-red ascorbic acid, chlorohydroquinone) in an effective amount in the enzyme-mediated oxygen-consuming system. Therefore, the culture device may contain an effective amount of a substantially dry enzyme component in the enzyme-mediated oxygen-consuming system, or may not contain an effective amount of the enzyme substrate component in the enzyme-mediated oxygen-consuming system. In these embodiments, the enzyme substrate component is an additive in the aqueous liquid used to hydrate the culture device. Therefore, the operation of depositing the aqueous liquid into the growth region fluidly connects the substantially dry enzyme component present in the culture device with the enzyme substrate component present in the aqueous liquid via the aqueous fluid, thereby initiating a reaction between the enzyme and the enzyme substrate and any oxygen dissolved in the coating and / or the aqueous fluid. This reaction depletes oxygen in the growth region of the culture device.

[0102] Additionally or alternatively, in any embodiment, the additive includes one or more selectors (e.g., antibiotics, salts) that promote the growth of a microorganism to a greater extent than at least one other microorganism. In one embodiment, the selector promotes the growth of *Clostridium difficile*. Additionally or alternatively, in any embodiment, the additive includes an indicator (e.g., a pH indicator, a redox indicator, a chromogenic enzyme substrate, a luciferase substrate) for detecting the presence of a certain microorganism. Those skilled in the art will recognize that both selectors and indicators can be used to detect microorganisms. The selector and / or indicator should substantially not interfere with enzyme-mediated oxygen-consuming systems. This can be easily detected using oxygen sensors as described herein. Advantageously, by adding one or more selectors and / or indicators to a solution for a hydration culture device, a wide variety of specific microorganisms or microbial communities can be cultured using a specific culture device containing a specific nutrient medium (e.g., a general nutrient medium for supporting the growth of a wide variety of microorganisms).

[0103] If the culture device is hydrated before the sample material is placed in it, the device is reopened so that, optionally, a cold water-soluble gelling agent (e.g., at room temperature) can be allowed to hydrate for several minutes to about 30 minutes or longer before inoculation with the culture material, thereby forming a gel. As described herein, during the period when the gelling agent is allowed to hydrate, thereby forming a gel, the enzyme-mediated components in the oxygen-consuming system (if all are present) reduce the dissolved oxygen concentration in the hydrated gelling agent from a first concentration to a second concentration that favors the growth of microaerophilic, microaerophilic, facultative anaerobic, or obligate anaerobic microorganisms.

[0104] Before or after the growth region of the culture device is hydrated, sample material can be brought into contact with the growth region using various methods known in the art. In any embodiment, sample material is brought into contact with the growth region by depositing sample material onto the growth region. Deposition of sample material into the growth region can be achieved, for example, by: removing sample material with a pipette; bringing the growth region into contact with a swab used to acquire the sample material (e.g., wiping the surface with a swab); bringing the growth region into contact with an inoculation loop or inoculation needle (e.g., using a streak plate technique); or indirectly placing a sample capture device (such as a swab, sponge, or membrane filter) into the growth region, followed by reclosing the culture device. After the sample has settled and the culture device is reclosed (care should be taken not to allow visible air bubbles to become trapped in the culture device during reclosing), the enzyme-mediated oxygen-consuming system resumes consuming dissolved oxygen within the growth region.

[0105] In any embodiment, after the growth region of the culture device becomes hydrated and the gelling agent forms a gel, the culture device can be used as a contact plate (such as a RODAC plate). Therefore, after gel formation, the first and second substrates of the culture device are separated to expose the hydrated gel within the growth region. The hydrated gel is brought into contact with the sample surface, and the culture device is resealed, taking care not to allow visible air bubbles to become trapped within the culture device. Advantageously, the contact plate process can be performed in an aerobic environment, and after resealing the culture device, a hypoxic environment can be regenerated within the culture device using the enzyme-mediated oxygen-consuming system.

[0106] In any embodiment of the method, after the growth area comes into contact with the sample (e.g., inoculates) and is closed, the incubation device is incubated for a period of time (e.g., a predetermined time period). As is well known to those skilled in the art, incubation conditions (e.g., incubation temperature) can affect the growth rate of microaerophilic bacteria, microaerophilic bacteria, facultative anaerobic bacteria, or obligate anaerobic bacteria, and can also affect the species of bacteria being detected. For example, incubation at lower temperatures (e.g., about 25°C) can enable the detection of psychrophilic bacteria. Incubation at higher temperatures (e.g., about 30°C, about 32°C, about 35°C, about 37°C) can favor the faster growth of certain microaerophilic, microaerophilic, facultative anaerobic, or obligate anaerobic microorganisms.

[0107] In some embodiments, the culture device may be incubated for at least about 16 hours, at least about 18 hours, at least about 24 hours, or at least about 48 hours. In some embodiments, the culture device may be incubated for no more than about 24 hours, no more than about 48 hours, or no more than about 72 hours. In some preferred embodiments, the culture device is incubated for about 24 hours to about 48 hours. In any embodiment, the culture device may be incubated and maintained in a low-oxygen environment for about 72 hours, about 96 hours, about 120 hours, about 7 days, or about 8 days before detecting or counting anaerobic microbial colonies growing in the growth area. In any embodiment, incubating the culture device for a sufficiently long period to allow for the formation of microbial colonies includes incubating the culture device in an oxygen-containing gas environment for a period of time.

[0108] Following incubation in the culture device, the method further includes detecting microbial colonies. Microbial colonies can be detected in the culture device using a variety of techniques known in the art. After a suitable incubation period, the absence of microorganisms in the culture device can be detected by the observable absence of colonies, no change in growth indicators (e.g., pH indicators, chromogenic enzyme substrates, redox indicators such as TTC, luciferase substrates), and the absence of bubbles in the growth medium associated with the metabolism of fermentable carbohydrates.

[0109] Acidic regions associated with microbial colonies can be detected visually and / or using an imaging system. For example, in a method where the culture medium contains bromocresol purple as a pH indicator, the medium will have a purple or gray appearance at approximately neutral pH. As microorganisms grow in the medium and ferment carbohydrates (e.g., glucose), the bromocresol purple indicator will turn yellow at adjacent bacterial colonies. Similarly, in a method where the culture medium contains chlorophenol red as a pH indicator, the medium will have a red or violet appearance at approximately neutral pH. As microorganisms grow in the medium and ferment carbohydrates, the chlorophenol red indicator will turn yellow at adjacent microbial colonies.

[0110] Bubbles associated with microbial colonies (if present in the growth area) can be detected visually and / or using an imaging system. Bubbles can be associated with visible colonies and / or acidic areas detectable by color changes in pH indicators in areas adjacent to microbial colonies. For example, bubbles may contain carbon dioxide produced by the anaerobic fermentation of carbohydrates. Some microorganisms can generate hydrogen sulfide (H2S) gas through the reduction of sulfate ions. Hydrogen sulfide can react with metal ions (e.g., iron ions) present in the culture device to generate an insoluble black precipitate (e.g., FeS) adjacent to sulfate-reducing bacterial colonies. Therefore, in any embodiment, the first or second coating of the present invention may contain a metal salt capable of reacting with hydrogen sulfide to generate an insoluble colored precipitate.

[0111] In any of the above embodiments of the method, the culture device may have a growth region that does not contain a substantially dry amount of the enzyme component effective in the enzyme-mediated oxygen-consuming system prior to the step of contacting the growth region with the predetermined volume of aqueous liquid. In these embodiments, the method further includes the step of depositing a substantially dry amount of the enzyme component effective in the enzyme-mediated oxygen-consuming system into the growth region. The enzyme component may be dispensed in dry or liquid form. In any embodiment, the enzyme component may be mixed with a liquid sample before depositing the sample into the growth region. In any embodiment, the method further includes depositing a substantially dry amount of the enzyme substrate component effective in the enzyme-mediated oxygen-consuming system into the growth region.

[0112] In any of the above embodiments, the method may further include acquiring images of the culture apparatus. In these embodiments, detecting the presence or absence of microorganisms includes displaying, printing, or analyzing images of the culture apparatus. The imaging system includes an imaging device and may include a processor. In some embodiments, the imaging device may include a line scanner or a surface scanner (e.g., a camera). The imaging device may include a monochrome (e.g., black and white) or multicolor (e.g., color) scanner. Advantageously, a monochrome imaging system can provide higher resolution images, which can improve the accuracy of the results and / or reduce the time required to detect the presence of microorganisms in the culture apparatus.

[0113] In some embodiments, the imaging system further includes an illumination system. The illumination system may include at least one broad-spectrum visible light (e.g., "white" light) source. In some embodiments, the illumination system may include at least one narrow-spectrum visible light source (e.g., a light-emitting diode emitting visible light with a relatively narrow bandwidth, such as red, green, or blue light). In some embodiments, the illumination system may include a narrow-spectrum visible light source (e.g., a light-emitting diode) with a light emission peak at a preselected wavelength (e.g., about 525 nm).

[0114] Images can be obtained from light reflected by components in the growth region of the culture device (e.g., microbial colonies, growth medium, and indicators), or from light transmitted through components in the growth region of the culture device. Suitable imaging systems and corresponding illumination systems are described, for example, in International Patent Publication No. WO 2005 / 024047 and U.S. Patent Application Publications Nos. US 2004 / 0101954 and US 2004 / 0102903, each of which is incorporated herein by reference in its entirety. Non-limiting examples of suitable imaging systems include the PETRIFILM plate reader (PPR) from 3M Company, St. Paul, MN, USA; the Petriscan colony counter from Spiral Biotech, Norwood, MA, USA; and the PROTOCOL and ACOLYTE plate scanners from Synbiosis, Cambridge, UK.

[0115] In some implementations, image acquisition includes acquiring wavelength-biased images. For example, the imaging system may include a bias filter that biases the light acquired by the imaging device. Filter elements are known in the art and include "cutoff" filters (i.e., filters that allow light wavelengths above or below a specified wavelength to pass through) and "bandpass" filters (i.e., filters that allow light wavelengths between a specified upper and lower limit to pass through). The bias filter may be positioned between the illumination source and the culture device. Alternatively, or otherwise, the bias filter may be positioned between the culture device and the imaging device. Exemplary Implementation

[0116] Implementation scheme A is a method for detecting microorganisms in a sample, the method comprising: The growth region of the culture device that autonomously generates an anaerobic environment is brought into contact with a predetermined volume of aqueous liquid in the culture device. Prior to the step of bringing the growth region into contact with the predetermined volume of aqueous liquid, the growth region contains a cold water-soluble drying gelling agent. This allows an effective amount of enzyme components in an enzyme-mediated oxygen-consuming system to be deposited into the growth region. Make the growth area contact the sample; The incubation device should be used for a sufficient period of time to allow for microbial colony formation; and Detect microbial colonies.

[0117] Implementation scheme B is the method described in implementation scheme A, and further includes depositing an effective amount of enzyme substrate component in the enzyme-mediated oxygen-consuming system into the growth region.

[0118] Implementation scheme C is a method for detecting microorganisms in a sample, the method comprising: The growth region of the culture device that autonomously generates an anaerobic environment is brought into contact with a predetermined volume of aqueous liquid in the culture device. Prior to the step of bringing the growth region into contact with the predetermined volume of aqueous liquid, the growth region contains a cold water-soluble drying gelling agent and an effective amount of a basic dry enzyme component in an enzyme-mediated oxygen-consuming system. Make the growth area contact the sample; The incubation device should be used for a sufficient period of time to allow for microbial colony formation; and Detect microbial colonies.

[0119] Implementation scheme D is a method for detecting microorganisms in a sample, the method comprising: The growth region of the culture device that autonomously generates an anaerobic environment is brought into contact with a predetermined volume of aqueous liquid in the culture device. Prior to the step of bringing the growth region into contact with the predetermined volume of aqueous liquid, the growth region contains a cold water-soluble drying gelling agent. This allows an effective amount of enzyme substrate components in an enzyme-mediated oxygen-consuming system to be deposited into the growth region. Make the growth area contact the sample; The incubation device should be used for a sufficient period of time to allow for microbial colony formation; and Detect microbial colonies.

[0120] Implementation scheme E is the method described in implementation scheme D, and further includes depositing an effective amount of enzyme components in the enzyme-mediated oxygen-consuming system into the growth region.

[0121] Implementation scheme F is the method of any one of implementation schemes A to C, wherein the growth region of the culture device further contains an effective amount of essentially dried enzyme substrate components in an enzyme-mediated oxygen-consuming system.

[0122] Implementation scheme G is the method of any one of the foregoing implementation schemes, wherein contacting the growth region with the aqueous liquid includes contacting the growth region with the sample.

[0123] Implementation scheme H is the method of any one of the foregoing implementation schemes, wherein the step of contacting the growth region with the sample is not synchronized with the step of contacting the growth region with a predetermined volume of aqueous liquid.

[0124] Implementation scheme I is the method described in implementation scheme H, wherein the step of contacting the growth region with the sample occurs after the step of contacting the growth region with a predetermined volume of aqueous liquid.

[0125] Implementation scheme J is the method described in implementation scheme H, wherein the step of contacting the growth region with the sample occurs before the step of contacting the growth region with a predetermined volume of aqueous liquid.

[0126] Implementation scheme K is the method of any one of the foregoing implementation schemes, wherein contacting the growth region with the sample or contacting the growth region with a predetermined volume of aqueous liquid includes providing an additive in the sample or the predetermined volume of aqueous liquid.

[0127] Implementation scheme L is the method described in implementation scheme K, wherein the additive includes an effective amount of a selector or indicator for detecting microbial growth.

[0128] Implementation scheme M is the method described in implementation scheme L, wherein an effective amount of the selector substantially allows the germination and growth of Clostridium difficile microorganisms in the culture device, and an effective amount of the selector substantially inhibits the growth of Escherichia coli, Staphylococcus aureus, Clostridium sporogenes, Clostridium perfringens, Bacteroides fragilis, Prevotella melaninogen, Fusobacterium species and / or anaerobic Streptococcus.

[0129] Implementation scheme N is the method of any one of the foregoing implementation schemes, wherein the incubation device is incubated for a sufficient period of time to allow the formation of microbial colonies, including incubating the incubation device for a period of time in an oxygen-containing gas environment.

[0130] Implementation scheme O is the method of any one of the foregoing implementation schemes, wherein a first drying composition is coated on a first substrate of the culture device, and a second drying composition is coated on a second substrate of the culture device.

[0131] Implementation scheme P is the method of any one of implementation schemes A to O: When the first composition, the second composition, and the aqueous liquid are arranged in fluid communication in the growth region of the culture device, they form an aqueous mixture containing a first dissolved oxygen concentration. In this process, after the liquid mixture is formed, when the device is kept in an oxygen-containing environment for less than or equal to about 120 minutes, the first dissolved oxygen concentration is reduced to a second dissolved oxygen concentration that does not substantially inhibit the growth of microaerophilic microorganisms through an enzyme-mediated oxygen-consuming system.

[0132] Implementation scheme Q is the method of any one of implementation schemes A to O: When the first composition, the second composition, and the aqueous liquid are arranged in fluid communication in the growth region of the culture device, they form an aqueous mixture containing a first dissolved oxygen concentration. In this process, after the liquid mixture is formed, when the device is kept in an oxygen-containing environment for less than or equal to about 120 minutes, the first dissolved oxygen concentration is reduced to a second dissolved oxygen concentration that does not substantially inhibit the growth of microaerophilic microorganisms through an enzyme-mediated oxygen-consuming system.

[0133] Implementation scheme R is the method of any one of implementation schemes A to O: When the first composition, the second composition, and the aqueous liquid are arranged in fluid communication in the growth region of the culture device, they form an aqueous mixture containing a first dissolved oxygen concentration. In this process, after the liquid mixture is formed, when the device is kept in an oxygen-containing environment for less than or equal to about 120 minutes, the first dissolved oxygen concentration is reduced to a second dissolved oxygen concentration that does not substantially inhibit the growth of obligate anaerobic microorganisms through an enzyme-mediated oxygen-consuming system.

[0134] Implementation S is the method described in any one of the foregoing implementation schemes, wherein detecting microbial colonies includes detecting Campylobacter colonies.

[0135] Implementation scheme T is the method of any one of implementation schemes A to R, wherein detecting microbial colonies includes detecting microbial colonies that ferment glucose into lactic acid.

[0136] Implementation scheme U is a culture device for counting microbial colonies, the device comprising: A first substrate having opposing inner and outer surfaces; A second substrate having opposing inner and outer surfaces; The growth region is located between the inner surface of the first substrate and the inner surface of the second substrate. A first effective amount of substantially dry enzyme component in an enzyme-mediated oxygen-consuming system is arranged in a first coating within a growth region. A second effective amount of substantially dry enzyme substrate component in an enzyme-mediated oxygen-consuming system, wherein the second effective amount of substantially dry enzyme substrate component is disposed in a second coating within a growth region; and A cold water-soluble drying gelling agent placed in the growth zone; Among them, the first substrate and the second substrate are basically impermeable to gaseous oxygen.

[0137] Implementation scheme V is the culture device described in implementation scheme U, wherein when an effective amount of enzyme component and enzyme substrate component are fluidly connected in the growth region by a predetermined volume of aqueous liquid, the enzyme component and enzyme substrate component can react to reduce the first dissolved oxygen concentration in the aqueous liquid to a second dissolved oxygen concentration that is significantly lower than the first dissolved oxygen concentration.

[0138] Implementation scheme W is the culture apparatus described in implementation scheme V, wherein effective amounts of enzyme component and enzyme substrate component are selected such that the first dissolved oxygen concentration is reduced to the second dissolved oxygen concentration within 60 minutes of fluid communication between the first effective amount of enzyme component and the second effective amount of enzyme substrate component in a predetermined volume of aqueous liquid.

[0139] Implementation X is a culture device according to any one of implementations S to U, the device further comprising a spacer disposed between a first substrate and a second substrate, wherein the spacer defines the perimeter of a growth region in the culture device.

[0140] Implementation scheme Y is the culture device described in any one of implementation schemes U to X, and the culture device further contains nutrients that are conducive to the growth of microorganisms.

[0141] Implementation scheme Z is the culture device described in implementation scheme Y, wherein nutrients are conducive to the growth of Clostridium difficile microorganisms.

[0142] Implementation scheme AA is the culture device described in implementation scheme Z, wherein the culture device further comprises an effective amount of selective agent, wherein when the growth area is rehydrated with a predetermined volume of aqueous liquid, the effective amount of selective agent substantially allows the germination and growth of Clostridium difficile microorganisms in the culture device, and the effective amount of selective agent substantially inhibits the growth of Escherichia coli, Staphylococcus aureus, Clostridium sporogenes, Clostridium perfringens, Bacteroides fragilis, Prevotella melaninogen, Fusobacterium species and / or anaerobic Streptococcus.

[0143] Implementation scheme AB is a culture device according to any one of implementation schemes U to Y, wherein nutrients are conducive to the growth of Campylobacter microorganisms.

[0144] Implementation scheme AC is the culture device described in implementation scheme AA, the culture device further comprising an effective amount of selective agent, wherein, when the growth area is rehydrated with a predetermined volume of aqueous liquid, the effective amount of selective agent substantially allows the germination and growth of Campylobacter microorganisms in the culture device, and the effective amount of selective agent substantially inhibits the growth of non-Campylobacter microorganisms.

[0145] Implementation scheme AD is a culture device according to any one of implementation schemes U to Y, wherein the nutrients are conducive to the growth of lactic acid producing microorganisms.

[0146] Implementation scheme AE is a culture device according to any one of implementation schemes U to AD, the culture device further comprising an indicator.

[0147] Implementation scheme AF is the culture device described in any one of implementation schemes U to AE, wherein the culture device further comprises an effective amount of reducing agent.

[0148] Implementation scheme AG is the culture device described in any one of implementation schemes U to AF, wherein the enzyme component includes ascorbic acid oxidase.

[0149] Implementation scheme AH is the culture device described in implementation scheme AG, wherein the enzyme substrate component includes L-ascorbic acid or a salt thereof.

[0150] Implementation scheme AI is the culture device described in implementation scheme AH, wherein the second effective amount of L-ascorbic acid or its salt arranged in the first coating within the growth region is approximately 1.5 μmol / 10 cm. 2 Approximately 15 micromoles / 10cm 2 .

[0151] Implementation scheme AJ is a method for preparing a culture device for culturing anaerobic microorganisms, the method comprising: The first coating is deposited on a portion of the first substrate, and the first coating is formed by using a liquid mixture containing enzyme components of an enzyme-mediated oxygen-consuming system; Dry the first coating; A second coating is deposited on a second substrate, the second coating comprising an enzyme substrate component of an enzyme-mediated oxygen-consuming system; and The first substrate is positioned near the second substrate, with the first coating facing the second coating, and the growth region disposed between the first substrate and the second substrate overlaps with a portion of the first coating and a portion of the second coating.

[0152] Implementation scheme AK is the method described in implementation scheme AJ, wherein the liquid includes water.

[0153] Implementation scheme AL is the method described in implementation scheme AJ, wherein the liquid includes an organic liquid.

[0154] Implementation scheme AM is the method of any one of implementation schemes AJ to AL, wherein the liquid mixture further comprises a cold water-soluble gelling agent.

[0155] Implementation scheme AN ​​is a method of implementation scheme AM which is subordinate to implementation scheme AK, wherein depositing the first coating on a portion of the first substrate includes: The enzyme components and gelling agent are mixed in a predetermined volume of liquid to form a coating mixture; and The coating mixture is applied to the first substrate.

[0156] The following embodiments further illustrate the purpose and advantages of the invention, but the specific materials and quantities listed in these embodiments, as well as other conditions and details, should not be construed as undue limitation of the invention. Example

[0157] Table 1. List of Materials

[0158] Example 1. Preparation and use of a cultivation device that autonomously generates an anaerobic environment.

[0159] Construction and Figure 2 and Figure 3 The culture apparatus shown, 10', is a self-generating anaerobic environment culture apparatus. The first substrate consists of a 5-mil (0.127 mm) thick polyester film (MELINEX grade 377 biaxially oriented polyester (PET) film, purchased from DuPont Teijin, Chester, VA). The second substrate consists of a transparent polyester (PET) film (0.073 mm thick). Powders containing nutrients (as listed in Table 2), polyvinyl alcohol (PVA), and guar gum were stirred in deionized water to obtain a substantially homogeneous mixture containing 3% (w / v) nutrients, 10% (w / v) PVA, and 0.3% (w / v) guar gum, respectively. As described in U.S. Patent 4,565,783, this mixture was spread onto the first substrate using a spatula and dried in a convection oven at 210℉ (98.9°C) for 7–8 minutes. The nutrient layer was applied by scraping until the target coating weight (after drying) was 0.150 g / 24 in. 2 (1.0mg / cm 2 The thickness of the polystyrene foam spacers (approximately 0.038 cm thick, with a density of approximately 19.3 kg / m³) after drying. 3 A dry coating is adhered to a first substrate via a thin layer of pressure-sensitive adhesive (a copolymer of 98 wt% isooctyl acrylate and 2 wt% acrylic acid). The spacer comprises a circular opening with a diameter of 2 inches (5.1 cm), which defines... Figure 2 The hole in the spacer is shown. The second substrate is attached to the first substrate along one edge using double-sided tape, and the device is cut to fit the... Figure 2 The rectangles shown are similar to those in the rectangles, approximately 3 inches (7.6 cm) x 4 inches (10.1 cm).

[0160] Table 2. Composition of nutrient powders used to prepare coating mixtures.

[0161] Tetraazole salt (2,3,5-triphenyltetrazolium chloride (TTC); final concentration = 20 µg / mL), L-ascorbic acid (final concentration = 1 mg / mL), and ascorbic acid oxidase (final concentration = 4 units / mL) were added to an aqueous diluent (Butterfield buffer). The resulting solution was filtered and sterilized. A stockpile of Clostridium sporogenes ATCC#3584 spores in water was serially diluted in the aqueous diluent to obtain final suspensions of approximately 10, 100, and 1000 bacteria per milliliter. One milliliter of each suspension was used to inoculate a 3M PETRIFILM aerobic microbial counting (PAC) plate (3M Company, St. Paul, MN, USA) and the culture apparatus prepared according to this example. The PAC plates were incubated in an anaerobic culture chamber, where an anaerobic atmosphere was generated using an air bag (GASPAK EZ Anaerobe Container System Sachet, purchased from BD, Dickinson and Company, Franklin Lakes, NJ). The culture apparatus from Example 1 was then incubated in an incubator with an aerobic (environmental) atmosphere.

[0162] The PAC plates and culture devices from Example 1 were incubated at 37°C for 24 hours, and bacterial colony indicators were observed. Red colonies were observed in all PAC plates and all culture devices prepared according to Example 1. The number of colonies observed in each plate and culture device was approximately consistent with the number of bacteria in the corresponding suspension inoculated into each plate.

[0163] Examples 2-3. Preparation and use of a culture device that autonomously generates an anaerobic environment, including an oxygen indicator.

[0164] 3M PETRIFILM Aerobic Count plates were purchased from 3M Company (St. Paul, MN), Minnesota, USA. Following the manufacturer's instructions for inoculation, the plates were opened, and 1 mL of sterile Butterfield's buffer solution containing the components listed in Table 3 was deposited into the growth area of ​​each plate. Before closing the plates, at least one oxygen sensor (a plate-type oxygen sensor, 5 mm in diameter, part number 200000023; purchased from Presents Precision Sensing GmbH (Regensburg, DE), Germany) was placed on the hydrated growth area. The top membrane (i.e., the second substrate) was lowered onto the bottom membrane (i.e., the first substrate), and the solution was spread evenly across the circular area of ​​the device using the concave side of the applicator provided by the PAC plate manufacturer.

[0165] Table 3. Components of the liquid used to hydrate the culture device.

[0166] After shutting down the incubation apparatus, fluorescence from the sensor was monitored using an OXY-MINI fiber optic microsensor (Presens Precision Sensing) equipped with supplier-provided software for oxygen measurement. The incubation apparatus was kept at room temperature (approximately 22°C). Data were collected at 30-second intervals after the incubation apparatus was shut down. The results are shown below. Figure 4 middle.

[0167] Figure 4 As shown, after the device was shut down, the plates receiving the enzyme-mediated oxygen-consuming system (e.g., sodium ascorbate and ascorbate oxidase) reduced the oxygen concentration in the growth region of the device to at least about 25% or less of that of the air-saturated control within 50 minutes. Unbound by theory, it is believed that the enzyme-mediated oxygen-consuming system first removes oxygen from the liquid used to inoculate the PETRIFILM plates at a rate faster than the rate at which oxygen re-enters the culture devices of Examples 2 and 3. However, over time, the rate at which oxygen (e.g., via diffusion through an oxygen-permeable cover of the PETRIFILM plate) re-enters eventually exceeds the rate at which it is consumed by the enzymatic reaction, resulting in a relatively high concentration of oxygen returning to the device. Unbound by theory, PVA appears to slow the rate at which oxygen re-enters the device of Example 3. However, these examples demonstrate that oxygen can be removed from the culture device for a period of time that allows for detectable growth of anaerobic and / or microaerophilic microorganisms.

[0168] Examples 4-6. Preparation and use of a culture device for autonomously generating an anaerobic environment for enzymes, including a PET second substrate and a basically dried substrate.

[0169] The cultivation device for autonomously generating an anaerobic environment is constructed as described in Example 1, except for the following: 1) a layer of adhesive is first applied to the inner surface of the second substrate, and then guar gum is powder-coated on the adhesive layer, essentially as described in Example 1 of U.S. Patent 4,565,783; 2) the liquid coating mixture consists of nutrients (30 g / L), powdered guar gum (12 g / L), and 4000 U / L ascorbic acid oxidase as described in Example 1.

[0170] The resulting culture devices were hydrated with 1 mL of Butterfield's buffer containing sodium ascorbate at the corresponding concentrations shown in Table 4. After hydration, oxygen sensors were placed in the culture devices, the devices were turned off, and the liquid was spread evenly across the circular growth area defined by spacers using the flat side of the applicator provided by the PAC plate manufacturer. Oxygen sensors were placed in the growth area of ​​each culture device as described in Examples 2-3. Oxygen saturation in the growth areas of these culture devices was measured and recorded as described in Examples 2-3.

[0171] Table 4. Concentration of sodium ascorbate in the aqueous liquid used to prepare the culture devices described in Examples 4-6.

[0172] Figure 5 The oxygen concentrations recorded in Examples 4-6 are shown during the first 50 minutes after hydration of the culture apparatus. Although not shown in Figure 5 As shown, but data collection took about 8 days. The representative culture apparatus of each of Examples 4-6 was kept anaerobic (e.g., no detectable oxygen in the growth area) until at least about 8 days after hydration.

[0173] Examples 7-12. Preparation and use of a culture device for autonomously generating an anaerobic environment, comprising substantially dried enzymes and various amounts of substantially dried enzyme substrates.

[0174] The culture apparatus was prepared as described in Examples 4-6, except that the powder used to coat the adhesive layer on the second substrate contained various amounts of sodium ascorbate powder. The concentration of sodium ascorbate in each powder mixture is shown in Table 5.

[0175] Table 5. Concentration of sodium ascorbate in the powder mixture used for coating the second substrate of the culture apparatus of Examples 7-12.

[0176] As described in Examples 4-6, the culture devices were opened, and 1 mL of sterile Butterfield's buffer solution was deposited into the growth region of each culture device. In these examples, the aqueous liquid did not contain sodium ascorbate or ascorbate oxidase. After the liquid was deposited into the growth region of the device, an oxygen sensor was placed in the growth region of each culture device as described in Examples 2-3. As described in Examples 2-3, the oxygen saturation of the growth regions of these culture devices was measured and recorded. The initial rate of oxygen consumption in the growth regions of the hydrated culture devices (i.e., during the first 1-15 minutes after hydration of the culture devices) was calculated, and the data are shown in Table 6.

[0177] Table 6. Oxygen consumption rate after hydration in culture devices that autonomously generate anaerobic environments. The reported data are the average and standard deviation of five replicates for each culture device. Oxygen consumption rate data are reported as the percentage of oxygen removed per minute from the growth region of the culture device. 1 Within 24 hours of hydration, the culture devices failed to reach -0% O2. Although oxygen could not be completely removed within 24 hours, these devices achieved a stable low oxygen concentration sufficient to support the growth of microaerophilic bacteria or fungi within the growth area.

[0178] The data in Table 6 show that in culture devices comprising a second substrate coated with up to 6% (w / w) sodium ascorbate powder, there is an approximately linear relationship between the amount of enzyme substrate (sodium ascorbate) and the rate of oxygen consumption in the hydrated culture device. The data also indicate that the O2 concentrations in Examples 7 and 8 appear to have stabilized at approximately 50% and 75% of their initial concentrations, respectively.

[0179] Example 13. Effect of gelling agent on the observed oxygen consumption rate in a culture device that autonomously generates an anaerobic environment.

[0180] The culture device was prepared according to Example 11 (above), except that the gelling agent used to prepare the liquid coating mixture (applied to the first substrate) was changed, as shown in Table 7. Additionally, the control culture device was prepared using 12 g / L powdered guar gum, but the liquid coating mixture did not contain any ascorbic acid oxidase (enzyme).

[0181] Table 7. Gel composition of the culture device.

[0182] As described in Examples 4-6, the culture devices were opened, and 1 mL of sterile Butterfield's buffer solution was deposited into the growth region of each culture device. In this example, the aqueous liquid did not contain sodium ascorbate or ascorbate oxidase. After the liquid was deposited into the growth region of the device, an oxygen sensor was placed in the growth region of each culture device, as described in Examples 2-3. As described in Examples 2-3, the oxygen saturation of the growth regions of these culture devices was measured and recorded, and as described in Examples 7-12, the initial rate of oxygen consumption in the growth regions of the hydrated culture devices was calculated. The calculated oxygen consumption rates are shown in Table 8.

[0183] Table 8. Oxygen consumption rate after hydration in culture devices that autonomously generate anaerobic environments. The reported data are the mean and standard deviation (where applicable) for each culture device.

[0184] The data in Table 8 show that although the culture apparatus without ascorbic acid oxidase appears to have a relatively low oxygen consumption rate, it is still four times higher than that of the culture apparatus in which guar gum is the only cold water-soluble gelling agent. The oxygen consumption rate of the culture apparatus containing PVA-56 and PVA-47 is slightly higher than that of the control apparatus lacking ascorbic acid oxidase.

[0185] Example 14. A comparison of the oxygen consumption rate in a culture device in which the enzyme is coated and dried with oxygen consumption rate in a culture device in which the enzyme is added as a component of the hydration medium.

[0186] A group of culture devices (“Group E”) was prepared as described in Example 11 above. Another group of culture devices (“Group F”) was prepared in a similar manner, except that the liquid coating mixture (used for coating the first substrate) did not contain ascorbic acid oxidase.

[0187] As described in Examples 4-6, the culture devices were opened, and 1 mL of sterile Butterfield's buffer solution was deposited into the growth region of each culture device. In this example, the aqueous solution used to inoculate the "Group E" culture devices did not contain sodium ascorbate or ascorbate oxidase. However, the aqueous solution used to inoculate the "Group F" culture devices contained ascorbate oxidase (at a concentration of 4 units / mL). After the solution was deposited into the growth region of the device, an oxygen sensor was placed in the growth region of each culture device as described in Examples 2-3. As described in Examples 2-3, the oxygen saturation of the growth regions of these culture devices was measured and recorded, and the initial rate of oxygen consumption in the growth regions of the hydrated culture devices was calculated as described in Examples 7-12. The calculated oxygen consumption rates are shown in Table 9.

[0188] Table 9. Oxygen consumption rate after hydration in culture devices that autonomously generate anaerobic environments. The reported data are the mean and standard deviation of three replicates for each culture device formulation.

[0189] The data in Table 9 show that the enzyme (ascorbic acid oxidase) and enzyme substrate (sodium ascorbate) can be coated onto the device in a substantially dry form and can be rehydrated and participate in oxygen-consuming reactions when in contact with a suitable liquid.

[0190] Examples 15-16. Cultivation devices that autonomously generate anaerobic environments and their usage methods.

[0191] The independent anaerobic culture device was prepared according to Example 12 above, with the following differences: 1) In Examples 15 and 16, the liquid coating mixture contained the components shown in Table 10. 2) In Example 16, the adhesive used for coating the second substrate (reaching 0.230 g / 24 in) 2 (1.49mg / cm 2 The dried coating weight contains 0.04% (w / w) of ALDOL 515 acetic acid; and in Examples 15 and 16, the coating weight (after drying) of the liquid coating mixture applied to the first substrate is approximately 0.257 g / 24 in. 2 (1.66mg / cm 2 ).

[0192] A spore stock of Clostridium spore-forming bacteria ATCC#3584 was serially diluted in Butterfield buffer to obtain final suspensions of approximately 10 and 100 bacteria per milliliter. One milliliter aliquot of each suspension was placed in the growth zone of a single culture device and spread using the flat side of a spreader provided by the PAC plate manufacturer. Another one milliliter aliquot of each suspension was inoculated into a separate PAC plate as a growth control. The suspension was spread into a circle of approximately 5 cm in the growth zone using the concave side of the spreader provided by the PAC plate manufacturer. The culture devices of Examples 15 and 16 were incubated in ambient air at 37°C for approximately 24 hours. The inoculated PAC plates were incubated in an anaerobic chamber at 37°C for approximately 24 hours.

[0193] Table 10.

[0194] After incubation, the culture device containing ALDOL 515 acetic acid (Example 16) was observed under ultraviolet light. Bright fluorescent Clostridium spore-forming colonies were observed in the growth area of ​​the device.

[0195] After incubation, all inoculated culture devices containing Na2SO3 (Example 16) exhibited characteristic gray-black colonies in their growth areas, surrounded by gray precipitate, indicating the growth of spore-forming Clostridium difficile and the production of hydrogen sulfide. Furthermore, bubbles were observed near the gray-black colonies, further indicating the production of gaseous hydrogen sulfide.

[0196] After incubation, all inoculated growth controls showed characteristic red spore-forming Clostridium colonies in the growth areas of the PAC plates. The colony counts observed in the culture devices of Examples 15 and 16 were within approximately 0.5 log CFU of the colony counts observed in anaerobic PAC plates.

[0197] Example 17. Reconstruction of the anaerobic environment after the culture device that autonomously generates an anaerobic environment is turned on.

[0198] The culture device for autonomously generating an anaerobic environment was prepared according to Example 12, with the following differences: 1) Dehydrated Lactobacillus MRS nutrients (110 g / L) were used instead of the powdered nutrient mixture (as described in Example 1) to prepare the liquid coating mixture; 2) The adhesive used for coating the second substrate (reaching 0.210 g / 24 in) 2 (1.36mg / cm 2 The dry coating weight contains 0.1375% (w / w) of 2,3,5-triphenyltetrazole chloride; and the concentration of guar gum in the liquid coating mixture is 14 g / L instead of 12 g / L.

[0199] Two mL of Butterfield buffer was added to the growth regions of both culture devices. Four oxygen sensors were placed in the growth region of the first culture device (“G”), both culture devices were shut down, and liquid was spread in the growth regions of both culture devices as described in Example 2. Both culture devices were placed in an environment with ambient oxygen levels at room temperature (approximately 22°C), and the oxygen concentration in the growth region of culture device G was measured and recorded as described in Example 2. After 45 minutes, the second culture device (“H”) was turned on for less than 1 minute, and the four oxygen sensors were placed in the growth region. Culture device H was shut down again (i.e., the first and second substrates were brought back into contact with each other, and the hydrated growth region was sandwiched between the first and second substrates), and the oxygen concentration in the growth region was measured and recorded as described in Example 2. When the oxygen sensors were placed in culture device H and then shut down again, care should be taken to avoid introducing air bubbles. Representative portions of the oxygen concentration data for each sensor in the two culture devices are shown in Table 11.

[0200] Table 11. Removal of oxygen from the culture apparatus after activation of the enzyme-mediated oxygen consumption system. The data shown here are the average values ​​of four oxygen sensors placed in the growth zones of each culture apparatus. 1. Time “0” for culture device H begins immediately after the oxygen sensor is placed therein and the device is shut down again. Without being bound by theory, it can be expected that when the plate is opened (40 min reference time point for culture device G), culture device H contains less than 40% oxygen, and the oxygen concentration may briefly increase before both culture devices are shut down again.

[0201] Data shows that more than half of the oxygen was removed from the culture device within 40 minutes of hydration with buffer, and all oxygen was removed within approximately 100 minutes (see data from sensors in device G). The data also shows that when the culture device was turned on 45 minutes after hydration, the oxygen concentration in the growth zone rose back to approximately 75% of the initial oxygen content in the device. However, the data further shows that after the culture device was turned off again, the enzyme-mediated oxygen-consuming system was able to continue removing oxygen from the growth zone and re-establish an anaerobic environment in the culture device. Therefore, after activating the oxygen-consuming system, the culture device can be turned on in the presence of oxygen (e.g., for streaking inoculum onto the culture medium, or for placing a membrane filter containing sample material onto the culture medium), and an anaerobic environment can be re-established in the culture device.

[0202] Example 18. Use of a culture device that autonomously generates an anaerobic environment in streak plate culture technology.

[0203] A culture device capable of generating its own anaerobic environment was prepared according to Example 17. A control device with the same nutrient medium composition but lacking the oxygen-consuming components was also prepared. Cultures of anaerobic microorganisms were prepared by individually growing *Lactobacillus brevis*, *L. plantarum*, *L. paracasei*, *Pediococcus acidilactici*, *P. damnosus*, and *P. dextrinicus* in *Lactobacillus MRS* fermentation broth for 24-48 hours under anaerobic conditions. As described in Example 17, the culture devices were hydrated with 2 mL of Butterfield buffer. After hydration, each hydrated culture device was opened for approximately 30 minutes, and a full loop of one culture was streaked onto the medium in the growth area using an inoculation loop. The culture devices were then closed again, taking care not to introduce air bubbles, and incubated aerobically at 37°C for up to 7 days. The control device was hydrated and streaked with a full loop of culture in a similar manner. The control apparatus was incubated in an anaerobic chamber at 37°C. Colonies were typically observed in the culture apparatus after 24-48 hours of incubation.

[0204] After incubation, colonies of each microorganism were observed in typical streak plates of the growth areas in all culture devices and all control devices.

[0205] Example 19. Use of a culture device that autonomously generates an anaerobic environment in membrane filter culture technology.

[0206] A culture device capable of generating its own anaerobic environment was prepared according to Example 17. A control device with the same nutrient medium composition but lacking the oxygen-consuming components was also prepared. The microbial culture of the anaerobic microorganisms prepared in Example 18 was serially diluted in sterile buffer to approximately 10 culture-forming units (CFU) per milliliter. The culture device was hydrated with 2 milliliters of Butterfield buffer as described in Example 17.

[0207] Approximately 10 mL of the diluted culture was filtered through a single sterile membrane filter (0.2 µm SUPORE membrane filter, obtained from Pall Corporation; Port Washington, NY). After hydration, each hydrated culture unit was turned on for approximately 30 minutes, and a filter was placed on the culture medium in the growth zone, through which the diluted culture would pass. Care should be taken to avoid introducing air bubbles between the membrane filter and the culture medium in the culture unit. The culture units were then turned off again, taking care not to introduce air bubbles, and incubated aerobically at 37°C for up to 7 days. The control units were hydrated in a similar manner and inoculated using one of the filters through which the diluted culture had passed. The control units were incubated in an anaerobic chamber at 37°C. Colonies were typically observed in the culture units after 24–48 hours of incubation.

[0208] After incubation, colonies of each microorganism were observed on membrane filters placed in the growth areas of all culture devices and all control devices. Compared with the control devices, the number of colonies of each organism on the filters was similar to the number of colonies in the culture devices that autonomously generated anaerobic environments.

[0209] Example 20. Preparation and use of a culture device containing a selective culture medium for culturing Clostridium difficile that autonomously generates an anaerobic environment.

[0210] The cultivation device for autonomously generating an anaerobic environment is constructed as described in Example 1, except for the following: 1) substantially as described in Example 1 of U.S. Patent 4,565,783, an adhesive layer is first applied to the inner surface of the second substrate, and then a mixture comprising 90% (w / w) guar gum and 10% (w / w) sodium ascorbate is powder-coated onto the adhesive layer; 2) the adhesive used for coating the second substrate contains 0.4 mg of ALDOL 515 acetic acid per gram, and the adhesive is applied to the second substrate with a coating weight of 1.84 mg / cm². 2 (The coating weight was measured after drying at 115℉ (46℃) for 5 minutes); 3) The liquid coating mixture consisted of the components listed in Table 12; and 4) The liquid coating mixture was applied to the first substrate using a doctor blade, and the coating weight was 2.00 mg / cm³. 2 (The coating weight was measured after the coating was dried at 210℉ (98.9℃) for 8 minutes.)

[0211] The "control" device was prepared in a similar manner, except that the liquid coating mixture did not contain cefoxitin or cycloserine. All devices were assembled with foam spacers as described in Example 1.

[0212] Table 12. Composition of the selective culture medium for culturing Clostridium difficile. Mix all components in deionized water at the listed concentrations.

[0213] Suspensions of *Clostridium difficile* ATCC 43598, *Escherichia coli* ATCC 25922, *Staphylococcus aureus* ATCC 43598, and spore-forming *Clostridium* were prepared in Butterfield buffer. These suspensions were diluted with Butterfield buffer to obtain final suspensions containing approximately 10 colony-forming units (CFU) and 100 CFU per milliliter (for each organism). One milliliter of each suspension was used to inoculate the respective culture apparatus prepared according to this embodiment. The culture apparatus was inoculated by the following steps: lifting the second substrate to expose the growth area, transferring 1 milliliter of suspension onto the growth area of ​​the first substrate, gently lowering the second substrate until it contacts the suspension and foam spacer, and then gently pressing a plate plastic applicator onto the closed apparatus to apply the liquid suspension into the openings of the foam spacer.

[0214] All culture devices were placed in an incubator at 37°C and incubated in an aerobic atmosphere for approximately 48 hours. The devices were removed from the incubator and examined while being illuminated with green light (i.e., from an LED with a peak emission wavelength of approximately 530 nm) to determine the presence of microbial colonies (e.g., fluorescent halos around colonies indicating ALDOL 515 acetic acid hydrolysis by microorganisms within the colonies). Fluorescent colonies were observed and / or photographed using a high-pass red filter with a cutoff value ≥590 nm. Qualitative results are shown in Table 13. These results show that all tested microorganisms could grow in the control device, indicating that the device supports the growth of both facultative anaerobes (e.g., *Escherichia coli* and *Staphylococcus aureus*) and obligate anaerobes (e.g., spore-forming *Clostridium difficile* and *Clostridium difficile*). However, only *Clostridium difficile* microorganisms were able to grow in the device containing cefoxitin and cycloserine, indicating that these devices are selective for *Clostridium difficile* microorganisms.

[0215] Table 13. Growth of various microorganisms in culture devices that autonomously generate anaerobic environments. "+" indicates colonies were detected after 48 hours of incubation. "-" indicates no colonies were detected after 48 hours of incubation. The number of *Clostridium difficile* colonies observed in the antibiotic-containing culture device was similar to that observed in the control plate.

[0216] Example 21. Quantitative comparison of the growth of Clostridium difficile in three different culture environments.

[0217] As described in Example 20, a culture device containing cefoxitin and cyclic serine was constructed to autonomously generate an anaerobic environment.

[0218] Two types of agar media were purchased from Remel Microbiology Products (Lenexa, KS) in Lenexa, Kansas, USA: anaerobic blood agar plate (catalog number R01040) and cyclic serine-cefoxitin-fructose agar plate containing horse blood (catalog number R01266).

[0219] A suspension of spores (Clostridium difficile ATCC 43598) was prepared in Butterfield buffer. The first portion of the suspension (i.e., the heat-treated (HT) portion) was kept at 80°C for 20 minutes, while the second portion (i.e., the untreated (NT) portion) was kept at ambient temperature.

[0220] The HT and NT suspensions were diluted with Butterfield buffer, and 100 μL aliquots of the diluted suspensions were inoculated onto the surface of each type of agar plate using a sterile applicator. After inoculation, the agar plates were placed in an anaerobic container containing a bag that, when activated, creates an anaerobic environment. The anaerobic container was sealed and placed in an incubator with an aerobic atmosphere at 37°C.

[0221] Using a dilution micropipette, a 100 μL aliquot of the suspension was diluted to a 1 mL volume and inoculated into a culture device containing cefoxitin and cycloserine, which generates its own anaerobic environment. After inoculation, the culture device was placed in an aerobic atmosphere in an incubator at 37°C.

[0222] All inoculated agar plates and all inoculated culture devices that generate their own anaerobic environment were incubated for 48 hours. After incubation, bacterial colonies in each plate or device were counted. The number of colonies was multiplied by the dilution factor to obtain the number of spores in the original suspension. The calculated data are shown in Table 14.

[0223] Table 14. CFU per milliliter of spore suspension, calculated based on the number of colonies observed on agar plates and in culture devices that generate their own anaerobic environments. 1. The results reported for culture devices that autonomously generate anaerobic environments are the average colony counts obtained from two identical culture devices.

[0224] These results indicate that the recovery and growth of *Clostridium difficile* colonies in culture devices that autonomously generate anaerobic environments are similar to those in cycloserine-cefoxitin-fructose agar plates incubated under anaerobic conditions. Furthermore, these results suggest that the recovery and growth of *Clostridium difficile* colonies in culture devices that autonomously generate anaerobic environments are superior to those in anaerobic blood agar plates incubated under anaerobic conditions.

[0225] Example 22. Preparation and use of a culture device for cultivating lactic acid-producing bacteria to autonomously generate an anaerobic environment.

[0226] Construction and Figure 2 and Figure 3 The culture apparatus shown in Figure 10' is a self-generating anaerobic environment culture apparatus. The first substrate consists of a 5-mil (0.127 mm) thick polyester film (MELINEX grade 377, biaxially oriented polyester (PET) film, obtained from DuPont Teijin, Chester, VA, USA). The components listed in Table 15 were added to deionized water (1 L) and the mixture was stirred. Then, sodium hydroxide (1 N) was added to the stirred mixture to adjust the pH to approximately 6.5. Guar gum (14 g) was then added and stirring continued to obtain a substantially homogeneous mixture. As described in U.S. Patent 4,565,783, the mixture was spread onto the first substrate with a spatula and placed in a convection oven to dry at 210℉ (98.9 °C) for 7–8 minutes. The coating weight (after drying) was 0.4 g / 24 in. 2 (2.6mg / cm 2 After drying, the polystyrene foam spacers (approximately 0.038 cm thick, with a density of approximately 19.3 kg / m³) 3 A pressure-sensitive adhesive thin layer (composed of 98% isooctyl acrylate and 2% acrylic acid copolymer) is adhered to the dried coating on the first substrate. Figure 2 As shown, the spacer has a circular opening with a diameter of 2 inches (5.1 cm). The circular opening defines the growth area of ​​the device.

[0227] The second substrate consists of a transparent polyester (PET) film (0.073 mm thick). The inner surface of the second substrate is coated with a first coating formulation containing TTC in the same adhesive as the second substrate adhesives described in Examples 4 to 6. The dry coating weight in this configuration is 0.2 g / 24 in. 2 (1.3mg / cm 2 The concentration of TTC in the dried coating was approximately 0.02 mg / in. 2 (0.003mg / cm 2Then, a homogeneous mixture of guar gum (pre-mixed with 0.01% CAB-O-SIL fused silica) (90% w / w) and sodium ascorbate (10% w / w) is powder-coated onto the adhesive layer of the second substrate.

[0228] Use double-sided tape to adhere the second substrate to the first substrate along one edge, and cut the assembled device into a rectangle of approximately 3" (7.6cm) × 4" (10.1cm), so that the circular opening is roughly positioned with... Figure 2 The center of those similar devices shown. The second substrate serves as a cover for the culture device that autonomously generates an anaerobic environment.

[0229] Single microbial samples selected from bacterial strain set A (Table 16) were inoculated into culture devices that autonomously generate an anaerobic environment. The suspension of each sample was diluted with Butterfield buffer to obtain final suspensions (for each organism) containing approximately 100 CFU / mL (diluted sample A), 1000 CFU / mL (diluted sample B), and 10000 CFU / mL (diluted sample C). One mL of each suspension was used to inoculate the respective culture devices prepared according to this embodiment. The culture devices were inoculated by the following steps: lifting the second substrate to expose the growth area, transferring 1 mL of suspension onto the growth area of ​​the first substrate, gently lowering the second substrate until it contacts the suspension and foam spacers, and then gently pressing a flat plastic applicator onto the closed device to apply the liquid suspension into the openings of the foam spacers.

[0230] After inoculation, the culture devices that autonomously generate an anaerobic environment were incubated in an aerobic incubator at 32°C for 60 hours. Red bacterial colonies in each culture device were counted visually at 24 and 60 hours. The results are shown in Tables 17 and 18.

[0231] Table 15. Compositions used to prepare coating mixtures for the first substrate of Example 22

[0232] Table 16. Bacterial strain collection A (used in Example 22)

[0233] Table 17. Example 22 (24-hour incubation) *TNTC = "Too many to count"

[0234] Table 18. Example 22 (60-hour incubation) *TNTC = "Too many to count"

[0235] Example 23. Preparation and use of a culture device for cultivating lactic acid-producing bacteria to autonomously generate an anaerobic environment.

[0236] The preparation of the culture device for autonomously generating an anaerobic environment as described in Example 22 differs in that the components listed in Table 15 are added to deionized water (1L) and the mixture is stirred. Sodium hydroxide (1N) is then added to the stirred mixture to adjust the pH value to approximately 5.4 (instead of pH 6.5 in Example 22).

[0237] Single microbial samples selected from bacterial strain set A (Table 16) were inoculated into culture devices that autonomously generate an anaerobic environment. The suspension of each sample was diluted with Butterfield buffer to obtain final suspensions (for each organism) containing approximately 100 CFU / mL (diluted sample A), 10 CFU / mL (diluted sample D), and 1 CFU / mL (diluted sample E). One mL of each suspension was used to inoculate the respective culture devices prepared according to this embodiment. The culture devices were inoculated by the following steps: lifting the second substrate to expose the growth area, transferring 1 mL of suspension onto the growth area of ​​the first substrate, gently lowering the second substrate until it contacts the suspension and foam spacers, and then gently pressing a flat plastic applicator onto the closed device to apply the liquid suspension into the openings of the foam spacers.

[0238] After inoculation, the culture devices that autonomously generate an anaerobic environment were incubated at 32°C for 72 hours in an aerobic incubator. At 72 hours, the presence or absence of red bacterial colonies (positive) or negative was assessed in the culture devices. For devices inoculated with *Leuconostoc mesenteroides* subsp. *dextrose* and *Leuconostoc mesenteroides* subsp. *mesenteroides*, related colony bubbles were also observed. The results are shown in Table 19.

[0239] Table 19. Colony detection using the culture device for autonomously generating an anaerobic environment as described in Example 23.

[0240] Example 24. Preparation and use of a culture device for autonomously generating an anaerobic environment for culturing lactic acid-producing bacteria.

[0241] The same self-generating anaerobic culture apparatus as described in Example 22 was prepared, with the difference that the bromocresol green pH indicator listed in Table 15 was replaced with chlorophenol red (0.5 g, Sigma-Aldrich). Single microbial samples of *Leuconostoc mesenteroides* subsp. *dextrose* or *Leuconostoc mesenteroides* subsp. *mesenteroides* were inoculated into the self-generating anaerobic culture apparatus. The suspension of each sample was diluted with Butterfield buffer to obtain a final suspension containing approximately 100 CFU / mL (for each organism). Inoculation was performed as described in Example 22, and the apparatus was incubated in an aerobic incubator at 32°C for 48 hours. After incubation, bacterial colonies in the culture apparatus were visually evaluated. Colonies with associated air bubbles (red) were observed in all culture apparatuses.

[0242] Example 25. Preparation and use of a culture device for cultivating lactic acid-producing bacteria that autonomously generates an anaerobic environment.

[0243] The same self-generating anaerobic culture apparatus as described in Example 24 was prepared, except that the bromocresol green pH indicator listed in Table 15 was replaced with chlorophenol red (0.25 g). Single microbial samples of *Leuconostoc mesenteroides* subsp. *dextrose* or *Leuconostoc mesenteroides* subsp. *mesenteroides* were inoculated into the self-generating anaerobic culture apparatus. The suspension of each sample was diluted with Butterfield buffer to obtain a final suspension containing approximately 100 CFU / mL (for each organism). Inoculation was performed as described in Example 22, and the apparatus was incubated in an aerobic incubator at 32°C for 48 hours. After incubation, bacterial colonies in the culture apparatus were visually evaluated. Colonies with associated air bubbles (red) were observed in all culture apparatuses.

[0244] Example 26. Preparation and use of a culture device for cultivating lactic acid-producing bacteria that autonomously generates an anaerobic environment.

[0245] A culture device for autonomously generating an anaerobic environment was prepared as described in Example 22, but with two differences. First, the bromocresol green pH indicator listed in Table 15 was replaced with chlorophenol red (0.5 g). Second, the components listed in Table 15 were added to deionized water (1 L) and the mixture was stirred. Sodium hydroxide (1 N) was then added to the stirred mixture to adjust the pH to approximately 5.4 (instead of pH 6.5 in Example 22). Single microbial samples of *Leuconostoc mesenteroides* subsp. *dextrose* or *Leuconostoc mesenteroides* subsp. *mesenteroides* were inoculated into the culture device for autonomously generating an anaerobic environment. The suspension of each sample was diluted with Butterfield buffer to obtain a final suspension containing approximately 100 CFU / mL (for each organism). Inoculation was performed as described in Example 22, and the device was then incubated in an aerobic incubator at 32°C for 48 hours. After incubation, bacterial colonies in the culture devices were visually evaluated. Colonies with associated air bubbles (red) were observed in all culture devices.

[0246] Example 27. Preparation and use of a culture device for autonomously generating an anaerobic environment for culturing lactic acid-producing bacteria.

[0247] A culture device for autonomously generating an anaerobic environment was prepared as described in Example 22, but with two differences. First, the bromocresol green pH indicator listed in Table 15 was replaced with chlorophenol red (0.25 g). Second, the components listed in Table 15 were added to deionized water (1 L) and the mixture was stirred. Sodium hydroxide (1 N) was then added to the stirred mixture to adjust the pH to approximately 5.4 (instead of pH 6.5 in Example 22). Single microbial samples of *Leuconostoc mesenteroides* subsp. *dextrose* or *Leuconostoc mesenteroides* subsp. *mesenteroides* were inoculated into the culture device for autonomously generating an anaerobic environment. The suspension of each sample was diluted with Butterfield buffer to obtain a final suspension containing approximately 100 CFU / mL (for each organism). Inoculation was performed as described in Example 22, and the device was incubated in an aerobic incubator at 32°C for 48 hours. After incubation, bacterial colonies in the culture devices were visually evaluated. Colonies with associated air bubbles (red) were observed in all culture devices.

[0248] Example 28. Preparation and use of a culture device for cultivating Campylobacter bacteria that autonomously generates a microaerophilic environment.

[0249] Construction and Figure 2 and Figure 3The culture apparatus shown in Figure 10' is a culture device that autonomously generates a microaerophilic environment. The first substrate consists of a 5-mil (0.127 mm) thick polyester film (MELINEX grade 377, biaxially oriented polyester (PET) film, obtained from DuPont Teijin, Chester, VA, USA). The components listed in Table 20 were added to deionized water (1 L) and the mixture was stirred. Guar gum (14 g) was then added and stirring was continued to obtain a substantially homogeneous mixture. As described in U.S. Patent 4,565,783, the mixture was spread onto the first substrate with a spatula and placed in a convection oven to dry at 210℉ (98.9 °C) for 7–8 minutes. The coating weight (after drying) was 0.25 g / 24 in. 2 (1.6mg / cm 2 After drying, the polystyrene foam spacers (approximately 0.038 cm thick, with a density of approximately 19.3 kg / m³) 3 A pressure-sensitive adhesive thin layer (composed of 98% isooctyl acrylate and 2% acrylic acid copolymer) is adhered to the dried coating on the first substrate. Figure 2 As shown, the spacer has a circular opening with a diameter of 2 inches (5.1 cm). The circular opening defines the growth area of ​​the device.

[0250] The second substrate consists of a transparent polyester (PET) film (0.073 mm thick). The inner surface of the second substrate is coated with a first coating formulation containing TTC in the same adhesive as the second substrate adhesives described in Examples 4 to 6. The dry coating weight in this configuration is 0.2 g / 24 in. 2 (1.3mg / cm 2 The concentration of TTC in the dried coating was approximately 0.02 mg / in. 2 (0.003mg / cm 2 Then, a homogeneous mixture of guar gum (pre-mixed with 0.01% CAB-O-SIL fused silica) (97.5% w / w) and sodium ascorbate (2.5% w / w) is powder-coated onto the adhesive layer of the second substrate.

[0251] Use double-sided tape to adhere the second substrate to the first substrate along one edge, and cut the assembled device into a rectangle of approximately 3" (7.6cm) × 4" (10.1cm), so that the circular opening is roughly positioned with... Figure 2 The center of those similar devices shown. The second substrate serves as a cover plate for the culture device that autonomously generates a microaerophilic environment.

[0252] Suspensions of Campylobacter jejuni ATCC 29428 and Campylobacter jejuni ATCC 33291 were prepared in Butterfield buffer. These suspensions were serially diluted with Butterfield buffer to obtain a final suspension with a concentration providing approximately 10-100 CFU (for each organism). One mL of each suspension was used to inoculate the respective culture apparatus prepared according to this example. The culture apparatus was inoculated by the following steps: lifting the second substrate to expose the growth area, transferring 1 mL of suspension onto the growth area of ​​the first substrate, gently lowering the second substrate until it contacts the suspension and the foam spacer, and then gently pressing a plate plastic applicator onto the closed apparatus to apply the liquid suspension into the openings of the foam spacer.

[0253] After inoculation, the culture devices that autonomously generate an anaerobic environment were incubated in an aerobic incubator at 41.5°C for 48 hours. After incubation, the number of red bacterial colonies in each culture device was counted by visual inspection. The cell count in the original suspension (CFU / mL) was calculated by multiplying the counted colony count (the average of two replicate samples) by the dilution factor. The calculated data are shown in Table 21.

[0254] As a control, suspensions of Campylobacter jejuni ATCC 29428 and Campylobacter jejuni ATCC 33291 were inoculated onto agar plates. Agar plates were prepared using Campylobacter bloodless selective medium (CBFSM, catalog number C03-102, Alpha Biosciences, Baltimore, MD, USA). CBFSM (44.5 g) was sequentially suspended in 1 L of purified water; the medium was dissolved by boiling for 1 min; autoclaving was performed at 121 °C for 15 min; and the medium was cooled to 45–50 °C. The cooled medium (15–20 mL) was then poured into sterile Petri dishes. 100 μL aliquots of serially diluted samples were spread onto the agar plates. The inoculated plates were then placed in an anaerobic chamber along with gas-generating bags (GASPAK EZ Campylobacter container system bags, obtained from BD (Becton, Dickinson and Company, Franklin Lakes, NJ)) that provided a microaerophilic atmosphere during incubation. The agar plates were incubated at 41.5°C for 48 hours under the above conditions. The cell count (CFU / mL) in the original suspension was calculated by multiplying the colony count (average of two replicates) on each agar plate by the dilution factor. The calculated data are shown in Table 21.

[0255] Table 20. Compositions used to prepare coating mixtures for the first substrate of Example 28.

[0256] Table 21. CFU / mL of cells in the original suspension, calculated based on the number of colonies observed on the agar plates and in the culture device that autonomously generates a microaerophilic environment in Example 28.

[0257] Example 29. Preparation and use of a culture device for cultivating Campylobacter bacteria that autonomously generates a microaerophilic environment.

[0258] The same microaerophilic environment-generating culture device as described in Example 28 was prepared, with one difference: the coating on the first substrate was prepared using the components listed in Table 22 (instead of those in Table 20), and the dried coating weight was 0.26 g / 24 in. 2 (1.7mg / cm 2 ).

[0259] Suspensions of Campylobacter jejuni ATCC 29428, Campylobacter jejuni ATCC 33291, Campylobacter coli ATCC 33559, and Campylobacter coli ATCC 43476 were prepared in Butterfield buffer. These suspensions were serially diluted with Butterfield buffer to obtain a final suspension with a concentration providing approximately 10-100 CFU (for each organism). One mL of each suspension was inoculated into the respective culture apparatus prepared according to Example 28. The culture apparatus was inoculated by the following steps: lifting the second substrate to expose the growth area, transferring 1 mL of suspension onto the growth area of ​​the first substrate, gently lowering the second substrate until it contacts the suspension and the foam spacer, and then gently pressing a flat plastic applicator onto the closed apparatus to apply the liquid suspension into the openings of the foam spacer.

[0260] After inoculation, the culture devices that autonomously generate a microaerophilic environment were incubated in an aerobic incubator at 41.5°C for 48 hours. After incubation, the number of red bacterial colonies in each culture device was counted by visual inspection. The cell count in the original suspension (CFU / mL) was calculated by multiplying the counted colony count (the average of two replicate samples) by the dilution factor. The calculated data are shown in Table 23.

[0261] As a comparative reference, suspensions of Campylobacter jejuni ATCC 29428, Campylobacter jejuni ATCC 33291, Campylobacter coli ATCC 33559, and Campylobacter coli ATCC 43476 (one organism per plate) were inoculated onto CBFSM agar plates (as described above) and treated according to the method described in Example 28. The cell count (CFU / mL) in the original suspension was calculated by multiplying the number of colonies counted on each agar plate (the average of two replicates) by the dilution factor. The calculated data are shown in Table 23.

[0262] Table 22. Compositions used to prepare coating mixtures for the first substrate of Example 29.

[0263] Table 23. CFU / mL of cells in the original suspension, calculated based on the number of colonies observed on the agar plates and in the culture device that autonomously generates a microaerophilic environment in Example 29.

[0264] All patents, patent applications, and patent publications cited herein, as well as available electronic materials, are incorporated herein by reference. In the event of any conflict between the disclosure of this patent application and the disclosure of any document incorporated herein by reference, the disclosure of this patent application shall prevail. The specific embodiments and examples described above are provided only for the purpose of clearly understanding the invention and should not be construed as unnecessary limitations. The invention is not limited to the specific details shown and described, and variations that will be obvious to those skilled in the art are also included in the invention as defined by the claims.

[0265] All headings are for the reader's convenience and should not be used to limit the meaning of the text that follows the heading, unless explicitly stated otherwise.

[0266] Various modifications may be made without departing from the spirit and scope of the invention. These and other embodiments are all within the scope of the following claims.

Claims

1. A method of detecting anaerobic or microaerophilic microorganisms in a sample, the method comprising: contacting a growth area of a culture device that autonomously generates an anaerobic environment with a predetermined volume of aqueous liquid in the culture device, the growth area comprising a cold water soluble dry gelling agent prior to the step of contacting the growth area with the predetermined volume of aqueous liquid; depositing an effective amount of an enzyme component of an enzyme mediated oxygen depletion system into the growth area; contacting the growth area with a sample; incubating the culture device in an oxygen containing environment for a period of time sufficient to allow for microorganism colony formation; and detecting the microorganism colony; wherein contacting the growth area with the aqueous liquid comprises contacting the growth area with the sample; and wherein incubating the culture device in an oxygen containing environment for a period of time sufficient to allow for microorganism colony formation comprises incubating the culture device in an oxygenated gaseous environment for the period of time; wherein the culture device comprises: a first substrate having opposing inner and outer surfaces; a second substrate having opposing inner and outer surfaces; wherein the second substrate is used to cover the inner surface of the first substrate, and wherein the second substrate is attached along one edge of the respective inner surfaces of the first and second substrates.

2. The method of claim 1, further comprising depositing an effective amount of an enzyme substrate component of the enzyme mediated oxygen depletion system into the growth area.

3. The method of claim 1, wherein contacting the growth area with the sample or contacting the growth area with a predetermined volume of aqueous liquid comprises providing an additive in the sample or the predetermined volume of aqueous liquid, wherein the additive comprises an effective amount of a selective agent or an indicator for detecting microorganism growth.

4. The method of claim 3, wherein the effective amount of selective agent substantially allows for Clostridium difficile microorganisms to germinate and grow in the culture device and substantially inhibits growth of Escherichia coli, Staphylococcus aureus, Clostridium sporogenes, Clostridium perfringens, Bacteroides fragilis, Prevotella melaninogenica, Fusobacterium species, and / or Anaerobic digistis.

5. The method of claim 1, wherein a first dry composition is coated on a first substrate of the culture device and a second dry composition is coated on a second substrate of the culture device; wherein when the first composition, the second composition, and the aqueous liquid are arranged in fluid communication in the growth area of the culture device, the first composition, the second composition, and the aqueous liquid form an aqueous mixture having a first dissolved oxygen concentration; wherein the first dissolved oxygen concentration is reduced to a second dissolved oxygen concentration by the enzyme mediated oxygen depletion system when the device is maintained in an oxygen containing environment for less than or equal to about 120 minutes after the liquid mixture is formed, the second dissolved oxygen concentration substantially not inhibiting growth of microaerophilic microorganisms.

6. The method of claim 1: wherein a first dry composition is coated on a first substrate of the culture device and a second dry composition is coated on a second substrate of the culture device; wherein, wherein the first composition, the second composition, and the aqueous liquid form an aqueous mixture having a first dissolved oxygen concentration when arranged in fluid communication in the growth region of the culture device; wherein the first dissolved oxygen concentration is reduced to a second dissolved oxygen concentration by the enzyme-mediated oxygen-consuming system when the device is maintained in an oxygen-containing environment for less than or equal to about 120 minutes after formation of the liquid mixture, the second dissolved oxygen concentration being substantially non-inhibitory to growth of the microaerophilic microorganism.

7. The method of any one of claims 1 to 4: wherein a first dry composition is coated on a first substrate of the culture device and a second dry composition is coated on a second substrate of the culture device; wherein, wherein the first composition, the second composition, and the aqueous liquid form an aqueous mixture having a first dissolved oxygen concentration when arranged in fluid communication in the growth region of the culture device; wherein the first dissolved oxygen concentration is reduced to a second dissolved oxygen concentration by the enzyme-mediated oxygen-consuming system when the device is maintained in an oxygen-containing environment for less than or equal to about 120 minutes after formation of the liquid mixture, the second dissolved oxygen concentration being substantially non-inhibitory to growth of the obligate anaerobic microorganism.

8. A culture device for growing and enumerating anaerobic or microaerophilic microorganism colonies in an oxygen-containing environment, the device comprising: a first substrate having opposing inner and outer surfaces; a second substrate having opposing inner and outer surfaces; a growth region disposed between the inner surface of the first substrate and the inner surface of the second substrate; an effective amount of a substantially dry enzyme component of an enzyme-mediated oxygen-consuming system, a first effective amount of the substantially dry enzyme component being arranged in a first coating in the growth region; an effective amount of a substantially dry enzyme substrate component of the enzyme-mediated oxygen-consuming system, a second effective amount of the substantially dry enzyme substrate component being arranged in a second coating in the growth region; a cold water soluble dry gelling agent arranged within the growth region; wherein the first substrate and the second substrate are substantially impermeable to gaseous oxygen.

9. The culture device of claim 8, wherein the enzyme component and the enzyme substrate component are capable of reacting when placed in fluid communication in the growth region using a predetermined volume of aqueous liquid, thereby reducing a first dissolved oxygen concentration in the aqueous liquid to a second dissolved oxygen concentration that is substantially less than the first dissolved oxygen concentration.

10. The culture device of claim 8, further comprising a nutrient that is beneficial to growth of a microorganism.

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