Fixed pH indicator for growth indication of biological indicators

Abstract

The present invention provides a self-contained biological indicator. The biological indicator includes a housing having at least one liquid-impermeable wall forming an opening to a compartment; a plurality of test microorganisms disposed in the housing; an aqueous liquid culture medium disposed in an openable container; and a pH indicator dye substrate having a pH indicator dye bound to the pH indicator dye substrate. The pH indicator is not disposed in the aqueous liquid culture medium or in liquid contact with the aqueous liquid culture medium. The biological indicator also includes a nutrient composition that promotes germination and / or growth of the test microorganism if the test microorganism is viable; the nutrient composition may be disposed in the housing or in the openable container, the openable container being in selective fluid communication with the compartment.

Description

Technical Field

[0001] The present disclosure relates generally to sterilization indicators and, in particular, to biological sterilization indicators and methods of evaluating the efficacy of a sterilization process. Background Art

[0002] In many industries, such as the healthcare industry, and in other industrial applications, it may be necessary to monitor the effectiveness of processes used to sterilize equipment such as medical devices, instruments, and other disposable and non-disposable products. In these cases, sterilization is generally defined as a process that completely destroys all viable sources of biological activity, such as microorganisms (including structures such as viruses and spores). As a standard practice, hospitals have sterilization indicators that are placed with a batch of products to test the lethality of the sterilization process. Both biological and chemical sterilization indicators have been used.

[0003] One standard type of biological sterilization indicator includes a known amount of test microorganisms, such as Geobacillus stearothermophilus (formerly known as Bacillus stearothermophilus) or Bacillus atrophaeus (formerly known as Bacillus subtilis) spores, which can be many times more resistant to a particular sterilization process than other contaminating organisms. After subjecting the indicator to the sterilization process, the sources of biological activity (e.g., spores) can be cultured in a nutrient medium to determine whether any of the sources of biological activity survive the sterilization process, wherein source metabolism and / or growth indicates that the sterilization process was insufficient to destroy all of the sources of biological activity.

[0004] Available chemical sterilization indicators can be read immediately at the end of the sterilization process. However, the results only indicate that specific conditions, such as a specific chemical or temperature, existed during the sterilization process and potentially that those conditions were achieved for a period of time. In contrast, the response of a bioactive source to all conditions present can be a more direct and reliable test of how effective the sterilization process was in achieving sterilization. Summary of the Invention

[0005] The present disclosure generally relates to self-contained biological indicators and methods for evaluating the efficacy of a sterilization process. The self-contained biological indicator includes a pH indicator dye substrate disposed therein, the pH indicator dye substrate having a pH indicator dye bonded to the pH indicator dye substrate. The disclosed method can be used to detect pH changes as an indicator of the survival of at least one test microorganism undergoing a sterilization process. Advantageously, the substrate-bound pH indicator dye of the disclosed articles and methods allows the use of a pH indicator dye that, if present in a solution with the test microorganism, could negatively impact the germination and / or growth of the test microorganism in the biological indicator. Alternatively or in addition, the use of a fixed pH indicator in the biological indicator allows the use of a higher (or lower) amount of pH indicator dye than is typically used in biological indicators. Even more advantageously, the use of a substrate-bound pH indicator dye allows the use of a dry form of the pH indicator dye in the biological indicator, thereby providing stability (e.g., hydrolysis resistance) over a longer period of time during storage. Furthermore, when the pH indicator dye substrate-bound pH indicator dye is used in methods involving detection of an enzyme catalysis product of a fluorogenic or chromogenic enzyme substrate, it provides for more rapid detection of the product than was previously possible.

[0006] In one aspect, the present disclosure provides a self-contained biological indicator. The self-contained biological indicator may include a housing having at least one liquid-impermeable wall forming an opening to a compartment; a plurality of test microorganisms disposed in the housing; an aqueous liquid culture medium disposed in an openable container disposed in the housing; a nutrient composition that promotes germination and / or growth of the test microorganisms if the test microorganisms are alive; and a pH indicator dye substrate disposed in the housing, the pH indicator dye substrate having a pH indicator dye bound to the pH indicator dye substrate, wherein the pH indicator is not disposed in the aqueous liquid culture medium or is not in liquid contact with the aqueous liquid culture medium. The nutrient composition may be disposed in the housing or in the openable container.

[0007] In any embodiment in the above-mentioned embodiment, the pH indicator dye substrate can be substantially free of water. In any embodiment in the above-mentioned embodiment, the pH indicator dye substrate can be substantially free of water. In any embodiment in the above-mentioned embodiment, the pH indicator dye substrate can comprise a cationic polymer. In any embodiment in the above-mentioned embodiment, the pH indicator dye substrate can be selected from thymol blue, nasturtium orange 00, methyl yellow, methyl orange, bromophenol blue, bromocresol green, methyl red, bromothymol blue, phenol red, chlorophenol red, neutral red, naphtholphthalein, phenolphthalein, thymolphthalein, alizarin yellow, nasturtium orange 0, nitroamine, trinitrobenzoic acid, thymol blue, bromophenol blue, tetrabromophenol blue, bromocresol green, bromocresol purple, methyl red, bromothymol blue, Congo red and cresol red. In any embodiment in the above-mentioned embodiment, described multiple test microorganisms can be arranged on a carrier. In certain embodiments, the pH indicator dye substrate can be a carrier. In any embodiment in the above-mentioned embodiment, the aqueous liquid nutrient medium can comprise the germination and / or the nutrients that promote the growth of test microorganisms. In any of the above embodiments, the self-contained biological indicator may further include a fluorescent enzyme substrate or a chromogenic enzyme substrate disposed in the housing.

[0008] In another aspect, the present disclosure provides a method for evaluating the efficacy of a sterilization process. The method may include positioning a biological indicator in a sterilization chamber, wherein the self-contained biological indicator includes: a housing having at least one liquid-impermeable wall forming an opening to a compartment, a plurality of test microorganisms disposed in the compartment; and a substantially non-aqueous pH indicator dye substrate disposed in the compartment, the pH indicator dye substrate having a substantially non-aqueous pH indicator dye bound to the pH indicator dye substrate. The pH indicator dye is substantially present on the pH indicator dye substrate in a first state, and the pH indicator dye is convertible into a second state that is optically distinguishable from the first state. The method may further include subjecting a biological indicator in a sterilization chamber to a sterilization process; after subjecting the biological indicator in the sterilization chamber to the sterilization process, contacting the test microorganism and the pH indicator dye substrate with an aqueous liquid culture medium and nutrients in the housing of the biological indicator, the nutrients promoting germination and / or growth of the test microorganism; incubating the biological indicator for a certain period of time; and after incubating the biological indicator for the period of time, observing the pH indicator dye to detect a second state, wherein detected presence of the second state of the pH indicator dye on the pH indicator dye substrate indicates a lack of efficacy of the sterilization process, and wherein detected absence of the second state of the pH indicator dye on the pH indicator dye substrate indicates efficacy of the sterilization process.

[0009] Unless otherwise indicated, all scientific and technical terms used herein have the meanings commonly used in the art. The definitions given herein are intended to facilitate understanding of certain terms frequently used in this application and are not intended to exclude reasonable interpretations of those terms within the context of this disclosure. Unless otherwise indicated, all numerical values used in the specification and claims that express feature sizes, quantities, and physical properties should be understood to be modified by the term "about" in all cases. Therefore, unless otherwise indicated, the numerical parameters listed in the above specification and the appended claims are approximate values, which may vary based on the desired properties sought to be obtained by a person skilled in the art using the teachings disclosed herein. At the very least, and without attempting to limit the application of the doctrine of equivalents to the scope of the claims, each numerical parameter should at least be interpreted in accordance with the number of reported significant digits and by applying customary rounding techniques. Although the numerical ranges and parameters shown within the broad scope of the present invention are approximate, the numerical values shown in the specific examples are reported as accurately as possible. However, any numerical value inherently contains certain errors, which are necessarily caused by the standard deviation present in their corresponding experimental measurements.

[0010] The words "preferred" and "preferably" refer to embodiments of the invention that may provide certain benefits under certain circumstances. However, other embodiments may also be preferred, under the same or other circumstances. Furthermore, the recitation of one or more preferred embodiments does not imply that other embodiments are unusable and is not intended to exclude other embodiments from the scope of the invention.

[0011] The terms "include" and variations thereof do not have a limiting meaning where these terms appear in the description and claims.

[0012] As used herein, "a," "an," "the," "at least one," and "one or more" are used interchangeably. Thus, for example, a substrate can be interpreted as meaning "one or more" substrates.

[0013] The term "and / or" means one or all of the listed elements, or a combination of any two or more of the listed elements.

[0014] Also herein, the recitations of numerical ranges by endpoints include all numbers subsumed within that range (eg, 1 to 5 includes 1, 1.5, 2, 2.75, 3, 3.80, 4, 5, etc.).

[0015] The above summary of the invention is not intended to describe every disclosed embodiment or every implementation of the present invention. The following description more specifically illustrates exemplary embodiments. In several places throughout this application, guidance is provided by lists of examples, which can be used in various combinations. In each case, the cited lists serve only as representative groups and should not be construed as exclusive lists. DETAILED DESCRIPTION

[0016] Before explaining any embodiment of the present disclosure in detail, it should be understood that the present invention is not limited in its application to the construction details and component arrangements mentioned in the following description or shown in the following drawings. The present invention is capable of other embodiments and can be practiced or implemented in various ways. Moreover, it should be understood that the words and terms used herein are for illustrative purposes and should not be considered restrictive. The use of "including", "comprising" or "having" and their variations herein is intended to cover the items listed thereafter and their equivalents as well as additional items. It should be understood that other embodiments may be adopted and structural or logical changes may be made without departing from the scope of the present disclosure. In addition, terms such as "front", "back", "top", "bottom" are only used to describe elements that are related to each other and are by no means meant to describe a specific orientation of a device, to indicate or imply a necessary or desired orientation of the device, or to specify how the invention described herein is to be used, installed, displayed or positioned in use.

[0017] The present disclosure relates generally to self-contained sterilization indicators and, in particular, to self-contained biological sterilization indicators. Self-contained biological indicators include all components necessary to assess the viability of test microorganisms contained therein and can be used to determine the lethality of a sterilization process. The present disclosure generally relates to constructions of biological sterilization indicators that achieve at least one or more of the following functions: provide a pH indicator dye that binds to a pH indicator dye substrate (e.g., with high affinity); contain a liquid (e.g., an aqueous liquid culture medium) separately from the pH indicator dye and one or more test microorganisms during sterilization, and allow the liquid and test microorganisms to combine with the pH indicator dye (i.e., bind to the pH indicator dye substrate) after sterilization; facilitate movement of sterilant to a location (e.g., a closed end) in the biological sterilization indicator where a source of one or more test microorganisms is contained; secure an openable container (e.g., a frangible ampoule, such as a glass ampoule) containing the liquid in a position separated from the test microorganisms in the biological sterilization indicator during sterilization; release the liquid from the openable container during activation of the biological sterilization indicator (e.g., by fracturing the container); provide a substantially constant sterilant path; and integrally control and / or assist in the flow of fluid within the biological sterilization indicator (e.g., by using one or more internal vents).

[0018] Pressurized steam or other common sterilants can be used to sterilize equipment and supplies used in healthcare settings. Small, self-contained indicators, such as biological sterilization indicators, can be used to verify the effectiveness of the sterilization process. These indicators can be biological indicators and can contain a source of biological activity (e.g., a test microorganism).

[0019] case

[0020] For non-limiting examples of housings suitable for self-contained biological indicators, see U.S. Patents 3,661,717; 5,223,401; and 6,623,955; and U.S. Patent Application Publications 2013 / 0302849 and 2014 / 0349335; each of which is incorporated herein by reference in its entirety. Generally speaking, a housing refers to a container, typically an outer container, having at least one wall that is impermeable to the sterilant. The at least one wall forms a reservoir in which the other components of the biological indicator are located. The housing can be disposed within the process challenge device or can be the process challenge device itself. In some embodiments, the housing can have dimensions that can be used to produce a flat or substantially planar biological indicator. The present disclosure contemplates housings of any shape and size.

[0021] The housing includes at least one opening (sterilant path) that enables the sterilant to flow into the interior of the housing. In some embodiments, the housing may include a main body with an opening and a cover that closes the opening. In some embodiments, the cover may be able to completely seal the housing and eliminate any fluid communication between the interior of the housing and the surrounding environment (e.g., closing the sterilant path). Generally speaking, the cover has an open position, in which there is an opening (e.g., a gap) between the cover and the main body of the container, so that liquid or gas (e.g., sterilant) can flow into and out of the interior of the housing. The cover also has a closed position, in which the opening is sealed and any fluid flow through the gap is eliminated. In other embodiments, the cover may include vents, which allow the sterilant to be transferred to the interior of the housing and form an additional sterilant path even if the cover is present and the cover is in the closed position. However, in other preferred embodiments, when the cover includes vents, placing the cover in the closed position will simultaneously close: (a) the gap between the cover and the main body of the container and (b) the vents present on the cover, thereby substantially closing the sterilant path.

[0022] In other embodiments, the cover may lack a vent, and when the cover is in the open position, the only sterilant path may be through the space between the cover and the body of the housing (or through another opening or vent, if present on the body). In some embodiments, if vents are present on the housing, they are located on the cover. In embodiments where no openings exist other than the opening between the cover and the body of the housing, placing the cover in the closed position completely seals off the interior of the housing, thereby preventing fluid communication between the interior of the housing and the surrounding environment. In those embodiments, the sterilant path may be sealed when the cover is in the closed position.

[0023] pH indicator dye substrates

[0024] The self-contained biological indicator of the present disclosure includes a pH indicator dye substrate disposed therein (e.g., disposed within a housing). The pH indicator dye substrate has a pH indicator dye bonded to the pH indicator dye substrate. The pH indicator dye can be bonded to the pH indicator dye substrate by any means, including but not limited to covalent bonding, ionic bonding, hydrophobic interaction, and a combination of any two or more of the aforementioned bonding means.

[0025] Preferably, the pH indicator dye binds to the pH indicator dye substrate with high affinity (i.e., after 10 mg of the indicator dye-bound substrate is rinsed with deionized water and then placed in 1 mL of deionized water; less than 10%, preferably less than 5%, and more preferably less than 1% of the indicator dye is released into deionized water within 1 hour at 23°C).

[0026] In some embodiments, the pH indicator dye substrate can be formed by various materials, and precondition is that it is with high-affinity in conjunction with the pH indicator dye.If necessary, can modify the pH indicator dye substrate material and / or its surface in conjunction with the pH indicator dye.The example of pH indicator dye substrate material includes but not limited to cotton, glass wool, cloth, non-woven polypropylene, non-woven rayon, non-woven polypropylene / rayon blend, non-woven nylon, non-woven glass fiber or other non-woven fibers, filter paper, microporous hydrophobicity and hydrophilic membrane, glass fiber, open-cell polymer foam and semi-permeable plastic membrane (for example, particle-filled film, thermally induced phase separation (TIPS) film etc.) and their combination.

[0027] In some embodiments, the pH indicator dye substrate material comprises a cationic charged substance, such as an anionic charged substance, which is (for example, with high affinity) in conjunction with the pH indicator dye with a cationically charged solvent having a neutral pH (for example, approximately 6.5 to approximately 7.5 pH). Alternatively, the pH indicator dye substrate material comprises a cationic charged substance, which is (for example, with high affinity) in conjunction with the pH indicator dye with a cationically charged solvent having a neutral pH (for example, approximately 6.5 to approximately 7.5 pH). In certain embodiments, the pH indicator dye substrate material comprises a cationic polymer, which is selected from any two or more combinations of polyamide polymers, polyethyleneimine polymers, polyvinylidene fluoride polymers and aforementioned cationic polymers.

[0028] For example, in embodiments where a pH indicator dye substrate can be used to bind bromocresol purple (BCP), the substrate can be formed from a charged nylon, such as a heavy, charged transfer membrane available from GE Water & Process Technologies, Trevose, Pa., under the trade designation "MAGNAPROBE" (e.g., 0.45 micron pore size, 30 cm x 3 m roll, catalog number NPOHY00010, material number 1226566). In certain embodiments, the pH indicator dye substrate material can be a hydrophobic material that binds the pH indicator dye with high affinity, for example, through hydrophobic interactions. Other suitable combinations of pH indicator dye substrate materials and pH indicator dyes will be apparent to those of ordinary skill in the art.

[0029] In some embodiments, it is contemplated that a particular pH indicator dye substrate material may be treated (e.g., by subjecting it to a plasma, corona treatment, or electron beam process to form a surface charge on the substrate material) to promote binding of the pH indicator dye to the pH indicator dye substrate. Alternatively or in addition, binding of the pH indicator dye to the pH indicator dye substrate may be carried out in a solvent having a non-neutral pH to promote ionic conditions that are favorable for binding of the dye to the substrate.

[0030] In any embodiment, the pH indicator dye substrate may be substantially free of water. In any embodiment, the pH indicator dye in combination with the pH indicator dye substrate may be substantially free of water. As used herein, "substantially free of water" refers to a substrate or pH indicator dye having a water content no greater than about the water content of the dehydrated substrate or dye after being allowed to equilibrate with the surrounding environment after excess water has been removed by drying.

[0031] The shape and size of the pH indicator dye substrate are designed to be adapted to the housing interior of the biological indicator in a position that can contact (for example, when the biological indicator is activated) with an aqueous culture medium and a nutrient substance for cultivating a test microorganism, and the aqueous culture medium is in fluid contact with the test microorganism, each as disclosed herein. In certain embodiments, the pH indicator dye substrate can be substantially planar (for example, as a membrane filter). In some embodiments, for example, the shape and size of the pH indicator dye substrate can be designed to be similar to the filter paper 16 in (relative to other components in the biological indicator) in U.S. Patent No. 3,661,717; the carrier 46 or wicking strip 76 in U.S. Patent No. 5,223,401; or the substrate 119 in U.S. Patent Application Publication No. 2013 / 03022849. In some embodiments, the pH indicator dye substrate can be a fiber substrate (for example, as yarn). In some embodiments, the pH indicator dye substrate can be a granular substrate (for example, as porous or non-porous beads).

[0032] Test microorganisms (e.g., spores)

[0033] Generally speaking, test microorganisms that are particularly resistant to a given sterilization process are selected for use in biological indicators. In certain embodiments, the biological indicators of the present disclosure include living cultures of known species of microorganisms, typically in the form of microbial spores. Spores (e.g., bacterial spores) are used, at least in part, rather than vegetative forms of the microorganisms, because vegetative microorganisms are known to be killed relatively easily by sterilization processes. In contrast, spores are relatively more resistant to sterilization processes. In addition, spores also have good storage characteristics and can maintain their dormant state for many years. Therefore, sterilization of an inoculum of a standardized spore strain provides a higher degree of confidence that all microorganisms in the sterilization chamber have been inactivated.

[0034] The self-contained biological indicator of the present disclosure includes a plurality of test microorganisms disposed therein (e.g., disposed within the interior of a housing). The test microorganisms may belong to one or more species. Typically, the biological indicator contains at least 10 3 , at least 10 4 , at least 10 5 , at least 10 6 , at least 10 7 or at least 10 8 A test microorganism.

[0035] By way of example only, the present disclosure describes the microorganisms used in biological indicators as "spores"; however, it should be understood that the type of microorganism (e.g., spores) used in a particular embodiment of a biological indicator is selected to be resistant to the particular sterilization process envisioned (more resistant than microorganisms typically present on the article to be sterilized so that inactivation of the test microorganism indicates successful sterilization). Thus, different embodiments of the present disclosure using different sterilizing agents may use different microorganisms, depending on the sterilization process intended for the particular embodiment.

[0036] Generally speaking, the test microorganisms used in a particular system are selected based on the sterilization process at hand. For example, for a steam sterilization process, Bacillus stearothermophilus or Bacillus stearothermophilus spores can be used. As another example, for an ethylene oxide sterilization process, Bacillus atrophaeus (formerly known as Bacillus subtilis) spores can be used. In some embodiments, the spores may include, but are not limited to, at least one of the following: Bacillus stearothermophilus, Bacillus stearothermophilus, Bacillus subtilis, Bacillus atrophaeus, Bacillus megaterium, Bacillus coagulans, Clostridium sporogenes, Bacillus pumilus, or a combination thereof.

[0037] Aqueous liquid culture medium

[0038] Aqueous liquid culture medium is arranged in an openable container, and this openable container is arranged in the inside of the shell formed by described at least one wall of shell.In certain embodiments, the solvent of aqueous liquid culture medium is water.In certain embodiments, aqueous liquid culture medium may have dissolved and / or suspended nutrient composition (as described below) therein, and this nutrient composition promotes the germination and / or growth of living test microorganisms.In certain embodiments, aqueous liquid culture medium may have dissolved and / or suspended one or more enzyme substrates as disclosed herein therein.In certain embodiments, aqueous liquid culture medium may have dissolved and / or suspended salt therein, and this salt includes but is not limited to sodium chloride, potassium chloride, calcium chloride etc. or their combination.In certain embodiments, salt may include the buffer (such as sodium phosphate, potassium phosphate) for buffering aqueous liquid culture medium at the pH that is applicable to the germination and / or growth of test microorganisms.In certain embodiments, aqueous liquid culture medium may have dissolved and / or suspended the following component therein: nutrient composition as disclosed herein, enzyme substrate as disclosed herein, salt as disclosed herein, buffer as disclosed herein and any two or more combination of aforementioned components.

[0039] Nutritional composition

[0040] The self-contained biological indicator of the present disclosure includes a nutrient composition that promotes the germination and / or growth of a test microorganism in one of the multiple test microorganisms if the test microorganism is alive. Initially, the nutrients applicable to the nutrient composition can be provided in the interior of the biological indicator in a dry form (e.g., powder form, tablet form, caplet form, capsule form, film or coating, trapped in beads or other carriers, another suitable shape or configuration, or a combination thereof). Alternatively or in addition, suitable nutrients can be provided in an aqueous liquid culture medium.

[0041] The nutritional composition may comprise, for example, one or more sugars including, but not limited to, glucose, fructose, dextrose, maltose, trehalose, cellobiose, and the like, or a combination thereof. Alternatively or in addition thereto, the nutritional composition may comprise a complex culture medium such as peptone, tryptone, phytone, yeast extract, soy casein digest, other extracts, hydrolysates, and the like, or a combination thereof. In other embodiments, the nutritional composition may comprise a combination of one or more complex culture medium components and other specific nutrients. In some embodiments, the nutritional composition may further comprise at least one amino acid including, but not limited to, at least one of methionine, phenylalanine, alanine, tyrosine, and tryptophan.

[0042] As part of a self-contained biological indicator, the aqueous liquid culture medium is typically present in or within the biological indicator throughout the sterilization process, but is separated from the test microorganism (e.g., in an openable container) to the desired extent. Other components in the biological indicator (e.g., nutrients and / or enzyme substrates and / or other components (e.g., neutralizers, buffer components, salts)) may also be present in or within the biological indicator throughout the sterilization process, but are separated from the test microorganism (e.g., in an openable container) to the desired extent. After the sterilization process is completed and the biological indicator is used to determine the efficacy of the sterilization, the aqueous liquid culture medium is placed in contact with the test microorganism, thereby producing a mixture. In the present disclosure, placing the aqueous liquid culture medium in contact with the test microorganism includes activating an openable container so that the aqueous liquid culture medium is released and contacts the test microorganism. The process may include mixing the aqueous liquid culture medium with the spores, such as manually or mechanically shaking the housing of the biological indicator so that the aqueous liquid culture medium is fully mixed with the test microorganism, nutrients, and other components (if present).

[0043] Enzymes and enzyme substrates

[0044] The self-contained supporting biological indicator of the present disclosure optionally includes an enzyme that can catalyze the cracking of an enzyme substrate to produce a fluorescent detectable compound. In certain embodiments, the enzyme may be present in and / or on the test microorganism, or the test microorganism can produce such an enzyme, or both. The enzyme that can be used for the biological indicator of the present disclosure includes extracellular enzymes and intracellular enzymes, and the activity of these enzymes is associated with at least one of the vigor of the microorganisms ("test" microorganisms or "test spores") that are typically used to monitor sterilization efficacy. In this context, "association" means the enzymatic activity that exceeds background, which can be used to indicate the survival of at least one test microorganism in the test microorganism. The enzyme should be an enzyme that, after a sterilization cycle that is sublethal to the test microorganism, maintains enough activity to react with the enzyme substrate of the enzyme within 24 hours and in a preferred embodiment within one hour or less, and is inactivated or has significantly reduced activity after a sterilization cycle that is lethal to the test microorganism.

[0045] Examples of suitable enzymes include α-glucosidase, α-galactosidase, lipase, esterase, acid phosphatase, alkaline phosphatase, protease, aminopeptidase, β-glucosidase, β-galactosidase, α-glucuronidase, β-glucuronidase, phosphohydrolase, α-mannosidase, β-mannosidase, α-L-fucosidase, leucine aminopeptidase, α-L-arabinofuranosidase, cysteine aminopeptidase, valine aminopeptidase, β-xylosidase, α-L-iduronidase, glucanase, cellobiosidase, cellulase, α-arabinosidase, glycanase, sulfatase, butyrate, glycosidase, arabinosidase, and combinations of any two or more of the foregoing enzymes.

[0046] In the context of the present application, an enzyme substrate includes a substance or mixture of substances that is converted into an enzyme-modified product when the enzyme acts on it. Although preferred enzyme substrates produce compounds capable of fluorescence detection, in other embodiments, the product of the enzymatic action can be a luminescent or colored material. However, in other embodiments, the enzyme substrate can be composed of a certain compound that will produce a product when reacting with the enzyme, and the product will react with additional compounds or compositions to produce a luminescent, fluorescent or colored material. Preferably, if the enzyme substrate is to be included in an indicator device during sterilization, the enzyme substrate should not spontaneously decompose or be converted into a detectable product during sterilization or incubation. For example, in devices for monitoring steam and dry heat sterilization, the enzyme substrate must be stable at a temperature between about 20°C and 180°C. Also preferably, when the enzyme substrate is to be included with a conventional growth medium, the enzyme substrate must be stable in the growth medium, for example, it will not spontaneously fluoresce in the growth medium. Advantageously, whether the enzyme substrate is a fluorescent substrate or a chromogenic substrate, the pH indicator binds (eg, with high affinity) to the pH indicator dye substrate such that the pH indicator dye is significantly less likely to interfere with detection of the enzyme reaction product in the aqueous broth.

[0047] Generally speaking, there are two basic types of enzyme substrates that can be used for the biological indicators of the present disclosure. The first type of enzyme substrate can be fluorescent (or chromogenic) and can be given with a chemical formula such as AB. When acted upon by an enzyme, AB decomposes into products A and B. B can, for example, be fluorescent or colored. A specific example of this type of fluorescent enzyme substrate is a salt containing a 4-methylumbelliferyl component. Other fluorescent enzyme substrates of this type include derivatives of 7-amido-4-methylcoumarin (7-AMC), indoxyl, and fluorescein. An example of a chromogenic enzyme substrate of this type is 5-bromo-4-chloro-3-indolyl phosphate. In the presence of a phosphatase, the enzyme substrate will decompose to form indigo and phosphate. Other chromogenic enzyme substrates of this type include derivatives of the following 5-bromo-4-chloro-3-indolyl, nitrophenol, and phenolphthalein.

[0048] The second type of enzyme substrate can be given, for example, by the chemical formula CD, which will be converted into C and D by a specific enzyme. However, in this case, neither C nor D will fluoresce or be colored, but either C or D can further react with compound Z to produce a fluorescent or colored compound, thereby indicating enzyme activity. A specific fluorescent example of this type is the amino acid lysine. In the presence of lysine decarboxylase, lysine loses a CO2 molecule. The product produced by the enzymatic decarboxylation of lysine is cadaverine, which is a strong base. An alkaline indicator such as 4-methylumbelliferone can be incorporated, and this alkaline indicator will fluoresce in the presence of a strong base. A chromogenic enzyme substrate of this type would be 2-naphthyl phosphate. Phosphatase reacts with this enzyme substrate to produce β-naphthol. The released β-naphthol reacts with a chromogenic reagent containing 1-diazo-4-benzamido-2,5-diethoxybenzene (commercially available as "Fast Blue BB salt" from Sigma Chemical) to produce a violet color.

[0049] As noted above, preferred enzyme substrates are in some embodiments fluorogenic enzyme substrates, which are defined herein as compounds capable of being modified by an enzyme (e.g., by hydrolysis or other enzymatic action) to yield a derivative fluorophore having significantly altered or enhanced fluorescence.

[0050] Those of ordinary skill in the art will appreciate that suitable fluorescent compounds are themselves non-fluorescent or meta-fluorescent (i.e., fluoresce in a manner that is distinctly different (e.g., in color or intensity) from the corresponding enzyme modification product). In this regard, the fluorescent signal formed by the enzyme modification is separated from any other fluorescence that may be present using appropriate excitation and detection wavelengths in a manner known to users of fluorescence techniques.

[0051] Non-limiting examples of suitable enzyme substrates can include, for example, derivatives of coumarin (including 7-hydroxycoumarin (also known as umbelliferone or 7-hydroxy-2H-benzopyran-2-one) derivatives) and 4-methylumbelliferone (7-hydroxy-4-methylcoumarin) derivatives, including, for example: 4-methylumbelliferyl α-D-glucopyranoside, 4-methylumbelliferyl α-D-galactopyranoside, 4-methylumbelliferyl heptanoate, 4-methylumbelliferyl palmitate, 4-methylumbelliferyl oleate, 4-methylumbelliferyl acetate, 4-methylumbelliferyl nonanoate, 4-methylumbelliferyl octanoate, 4-methylumbelliferyl butyrate, 4-methylumbelliferyl- β-D-cellobioside, 4-methylumbelliferyl acetate, 4-methylumbelliferyl phosphate, 4-methylumbelliferyl sulfate, 4-methylumbelliferyl-β-trimethylammonium chloride cinnamate, 4-methylumbelliferyl-β-DN,N′,N″-triacetylchitotriose, 4-methylumbelliferyl-β-D-xyloside, 4-methylumbelliferyl-N-acetyl-β-D-glucosaminide, 4-methylumbelliferyl-N-acetyl-α-D-glucosaminide, 4-methylumbelliferyl propionate, 4-methylumbelliferyl stearate, 4-methylumbelliferyl-α-L-arabinofuranoside, 4-methylumbelliferyl α-L-arabinoside;4-Methylumbelliferyl-β-DN,N′-diacetylchitobioside, 4-methylumbelliferyl elaidate, 4-methylumbelliferyl-α-D-mannopyranoside, 4-methylumbelliferyl-β-D-mannopyranoside, 4-methylumbelliferyl-β-D-fucoside, 4-methylumbelliferyl-α-L-fucoside, 4-methylumbelliferyl-β-L-fucoside, 4-methylumbelliferyl-α-D-galactoside, 4-methylumbelliferyl-β-D-galactoside, 4-trifluoromethylumbelliferyl-β-D-galactoside Glycosides, 4-methylumbelliferyl-α-D-glucoside, 4-methylumbelliferyl-β-D-glucoside, 4-methylumbelliferyl-7,6-sulfo-2-acetamido-2-deoxy-β-D-glucoside, 4-methylumbelliferyl-β-D-glucuronide, 6,8-difluoro-4-methylumbelliferyl-β-D-glucuronide, 6,8-difluoro-4-methylumbelliferyl-β-D-galactoside, 6,8-difluoro-4-methylumbelliferyl phosphate, 6,8-difluoro-4-methylumbelliferyl-β-D-xylobioside. The second enzyme substrate can also be a derivative of 7-amido-4-methylcoumarin, including: Ala-Ala-Phe-7-amido-4-methylcoumarin, Boc-Gln-Ala-Arg-7-amido-4-methylcoumarin hydrochloride, Boc-Leu-Ser-Thr-Arg-7-amido-4-methylcoumarin, Boc-Val-Pro-Arg-7-amido-4-methylcoumarin hydrochloride, D-Ala-Leu-Lys-7-amido-4-methylcoumarin, L-alanine-7-amido-4-methylcoumarin trifluoroacetate, L-methionine-7-amido-4-methylcoumarin trifluoroacetate, L-tyrosine-7-amido-4-methylcoumarin, Lys-Ala-7-amido-4-methylcoumarin dihydrochloride, N-toluenesulfonyl-Gly -Pro-Arg-7-amido-4-methylcoumarin hydrochloride, N-succinyl-Ala-Ala-Phe-7-amido-4-methylcoumarin, N-succinyl-Ala-Ala-Pro-Phe-7-amido-4-methylcoumarin, N-succinyl-Ala-Phe-Lys-7-amido-4-methylcoumarin acetate, N-succinyl-Leu-Leu-Val-Tyr-7-amido-4-methylcoumarin, D-Val-Leu-Lys-7-amido-4-methylcoumarin, Fmoc-L-glutamate 1-(7-amido-4-methylcoumarin), Gly-Pro-7-amido-4-methylcoumarin hydrobromide, L-leucine-7-amido-4-methylcoumarin hydrochloride, L-proline-7-amido-4-methylcoumarin hydrobromide;Other 7-hydroxycoumarin derivatives include 3-cyano-7-hydroxycoumarin (3-cyanumbelliferone) and 7-hydroxycoumarin-3-carboxylic acid esters such as 7-hydroxycoumarin-3-carboxylic acid ethyl ester, 7-hydroxycoumarin-3-carboxylic acid methyl ester, 3-cyano-4-methylumbelliferone and 3-(4-imidazolyl)umbelliferone; derivatives of fluorescein including: 2′,7′-bis-(2-carboxyethyl)-5-(and-6-)carboxyfluorescein, 2′,7′-bis-(2-carboxypropyl)-5-(and-6-)-carboxyfluorescein, 5-(and 6)-carboxynaphthylfluorescein, fluorescein, 2′,7′-dichlorofluorescein diacetate, 5(6)-carboxyfluorescein, 5(6)-carboxyfluorescein diacetate, 5-(bromomethyl)fluorescein, 5-(iodoacetamido)fluorescein. Fluorescein, 5-([4,6-dichlorotriazin-2-yl]amino)fluorescein hydrochloride, 6-carboxyfluorescein, eosin Y, fluorescein diacetate 5-maleimide, fluorescein-O'-acetic acid, O'-(carboxymethyl)fluorescein amide, anthrafluorescein, rhodols, halogenated fluoresceins; rhodamine derivatives, including tetramethylrhodamine, carboxytetramethylrhodamine, carboxy-X-rhodamine, sulforhodamine 101, and rhodamine B; fluoroglucosamine derivatives; benzanthracene dye derivatives, including seminaphthorhodafluorone, carboxyseminaphthorhodafluorone, seminaphthorhodafluors; cyanine derivatives, including sulfonated pentamethine cyanine and septamethine cyanine.

[0052] In some embodiments, the enzyme to be detected can be selected from α-D-glucosidase, chymotrypsin or fatty acid esterase. With regard to Bacillus stearothermophilus, the luciferase substrate is preferably 4-methylumbelliferyl-α-D-glucoside, 7-glutarylphenylalanine-7-amido-4-methylcoumarin or heptanoic acid 4-methylumbelliferyl ester. If the enzyme to be detected is α-L-arabinofuranosidase (e.g., derived from Bacillus atrophaeus), the preferred luciferase substrate is 4-methylumbelliferyl-α-L-arabinofuranoside. In a preferred embodiment, 4-methylumbelliferyl α-D-glucopyranoside is an enzyme substrate for generating metabolic activity, and the enzyme is a glucosidase, such as β-D-glucosidase.

[0053] The concentration of enzyme substrate present in the aqueous liquid medium depends on the specific enzyme substrate and enzyme species, the amount of enzyme product that must be generated to be detectable visually or instrumentally, and the amount of time one is willing to wait to determine whether active enzyme is present in the reaction mixture. Preferably, the amount of enzyme substrate is sufficient to react with any residual active enzyme present within a period of about eight hours after the sterilization cycle to produce at least 10 -8In the case where the enzyme substrate is a 4-methylumbelliferyl derivative, the present inventors have found that its concentration in the aqueous liquid culture medium disclosed herein is preferably about 10 -5 with 10 -3 In some embodiments, 4-methylumbelliferyl-α-D-glucoside can be used in the aqueous mixture at a concentration of, for example, about 0.05 g / L to about 0.5 g / L (e.g., about 0.05 g / L, about 0.06 g / L, about 0.07 g / L, about 0.08 g / L, about 0.09 g / L, about 0.1 g / L, about 0.15 g / L, about 0.2 g / L, about 0.25 g / L, about 0.3 g / L, about 0.35 g / L, about 0.4 g / L, about 0.45 g / L, about 0.5 g / L).

[0054] Optional components

[0055] In some embodiments, the aqueous liquid culture medium may include a buffer solution. The ionic condition of the buffer solution should be such that the enzyme and enzyme substrate are unaffected. In some embodiments, the buffer solution is used as a part for the aqueous liquid culture medium, such as phosphate buffer (for example, phosphate buffered saline solution, potassium phosphate or dipotassium hydrogen phosphate), tris (hydroxymethyl) aminomethane-HCl solution or acetate buffer or any other buffer known in the art that is applicable to sterilization. The buffer solution that is applicable to biological indicators of the present invention should be compatible with the fluorescent enzyme substrate and the colorimetric enzyme substrate used as a part for the aqueous liquid culture medium. Another consideration when selecting the buffer solution is their impact on enzyme activity. For example, phosphate buffered saline comprises a relatively high concentration of inorganic phosphate, which is a competitive inhibitor of alkaline phosphatase. Therefore, for this enzyme, it is recommended to use Tris-HCl buffer. The intensity of the buffer solution can be 0.05M to 0.5M, preferably 0.05M to 0.25M, more preferably 0.05M to 0.15M, even more preferably about 0.1M.

[0056] In some cases, one or more components of the biological indicator (e.g., gaps in the housing, pH indicator dye substrates and / or carriers for spores, walls of the container, etc.) may retain residual oxidizing sterilants. This can occur with, for example, hydrogen peroxide vapor and other vapor sterilants (such as ozone and peracetic acid). For example, certain carrier materials (e.g., hydrophilic ones, such as glass fiber and cellulose materials) may retain residual oxidizing sterilants, particularly hydrogen peroxide. In this context, "residual" refers to the amount of sterilant retained that inhibits the growth of a small number of viable spores. Typically, this means that more than 10 micrograms of sterilant are retained per microgram of carrier. In some cases, the amount of residual sterilant can be greater than 40 micrograms of sterilant per milliliter of growth medium. In comparison, if the carrier material has a contact angle greater than 90°, it is hydrophobic and typically retains no more than 10 micrograms of sterilant per microgram of carrier.

[0057] Thus, in some embodiments, the biological indicator includes one or more neutralizing agents that are neither enzymes nor metal catalysts disposed within the biological indicator. Neutralizing agents are compounds or materials that react with residual sterilant (e.g., hydrogen peroxide) to neutralize its effects, wherein the neutralizing agent is neither an enzyme nor a metal catalyst. Enzyme neutralizers are generally unstable at high temperatures and are therefore undesirable.

[0058] Suitable examples of neutralizing agents include sulfur-containing materials (e.g., methionine, L-cysteine, D-ethionine, S-methyl-L-cysteine, S-benzyl-L-cysteine, sodium thiosulfate, glutathione, L-cystathionine, N-acetyl-L-cysteine, carboxymethylcysteine, D,L-homocysteine, D,L-homocysteine-thiolactone, and thiodipropionic acid) and non-sulfur-containing materials (e.g., isoascorbic acid, potassium ferricyanide, and sodium pyruvate). Various combinations of these neutralizing agents can be used. Preferred neutralizing agents include methionine, L-cysteine, D-ethionine, S-methyl-L-cysteine, S-benzyl-L-cysteine, sodium thiosulfate, thiodipropionic acid, isoascorbic acid, potassium ferricyanide, sodium pyruvate, and combinations thereof.

[0059] pH indicator dyes

[0060] The self-contained supporting biological indicator of the present disclosure includes (for example, with high affinity) the pH indicator dye that is combined with the pH indicator dye substrate material. In any one of the embodiments of the method, the indicator dye can be a pH indicator suitable for detecting biological activity. The indicator dye can be selected according to standards known in the art, for example, pH range, compatibility with biological activity and solubility. In certain embodiments, the salt form of the pH indicator can be used, for example, to increase the solubility of the pH indicator in aqueous mixtures. The non-limiting example of suitable pH indicator dyes includes for example thymol blue, tropaeolum orange 00, methyl yellow, methyl orange, bromophenol blue, bromocresol green, methyl red, bromothymol blue, phenol red, chlorophenol red, neutral red, naphtholphthalein, phenolphthalein, thymolphthalein, alizarin yellow, tropaeolum orange 0, nitroamine, trinitrobenzoic acid, thymol blue, bromophenol blue, tetrabromophenol blue, bromocresol green, bromocresol purple, methyl red, bromothymol blue, Congo red and cresol red. In certain embodiments, the pH indicator dye is anionic in solution at a pH of about neutral.

[0061] In some embodiments, the pH indicator dye produces a color change when the pH decreases, thereby indicating the growth of the test microorganism. In some embodiments, the pH indicator dye is bromocresol purple. For example, pH indicators can be used to detect biological activities such as fermentation of carbohydrates to acidic end products (indicating the survival of the test microorganism) and enzyme biological activities such as α-D-glucosidase enzyme activity. For example, these activities can indicate the presence or absence of live spores after the biological indicator is treated by a sterilization process. For example, bromocresol purple can be used in an aqueous mixture at a concentration of about 0.03 g / L.

[0062] The combination of bromocresol purple and 4-methylumbelliferyl-α-D-glucoside represents a preferred combination of enzyme substrate and pH indicator dye in articles or methods according to the present disclosure, but other combinations are contemplated within the scope of the present disclosure.

[0063] Complete biological indicator

[0064] The pH indicator dye-conjugated pH indicator dye substrates described herein can be used as modifications to various biological indicators known in the art to produce self-contained biological indicators according to the present disclosure. In addition, the pH indicator dye-conjugated pH indicator dye substrates described herein can be used as modifications to various methods for evaluating the effectiveness of a sterilization process.

[0065] For example, the self-contained biological indicator of U.S. Pat. No. 3,661,717 (incorporated herein by reference in its entirety) can be modified to provide a pH-sensitive dye indicator in combination with a pH indicator dye substrate as described herein, rather than providing it in an aqueous nutrient medium disposed in an inner container (e.g., a frangible ampoule). Optionally, a portion of the pH indicator dye substrate can be used as a carrier for the test microorganisms (e.g., spores).

[0066] In addition, the self-contained biological indicators of U.S. Pat. Nos. 5,223,401 and 6,623,955 (both of which are incorporated herein by reference in their entirety) can be modified to provide a pH-sensitive dye indicator in combination with a pH indicator dye substrate as described herein, rather than providing it in an aqueous nutrient medium disposed in an inner container (e.g., a frangible ampoule). Optionally, a portion of the pH indicator dye substrate can be used as a carrier for the test microorganisms (e.g., spores).

[0067] In addition, the self-contained biological indicator of U.S. Patent Application Publication No. US 2013 / 0302849 (incorporated herein by reference in its entirety) can be modified to provide a pH-sensitive dye indicator in combination with a pH indicator dye substrate as described herein, rather than providing it in an aqueous nutrient medium disposed in an internal container (e.g., a frangible ampoule). For example, substrate 119 disclosed in U.S. Patent Application Publication No. US 2013 / 0302849 can be used as a pH indicator dye substrate for a self-contained biological indicator according to the present disclosure.

[0068] One of ordinary skill in the art will recognize how to modify other existing biological indicators with the disclosed pH indicator dye-bound substrates to yield the disclosed articles and methods.

[0069] In the present disclosure, the process of bringing spores and culture medium together is referred to as "activation" of a biological indicator. That is, the term "activation" and variations thereof, when used with respect to a biological indicator, generally refers to placing one or more test microorganisms (e.g., spores) in fluid communication with an aqueous liquid culture medium (e.g., a liquid culture medium containing nutrients and / or enzyme substrates). For example, a biological indicator can be described as having been "activated" when an openable container within a biological indicator containing an aqueous liquid culture medium is at least partially opened (e.g., broken, perforated, punctured, crushed, fractured, ruptured, etc.) such that the culture medium has been placed in fluid communication with the test microorganisms. In other words, a biological indicator is activated when the test microorganisms have been exposed to an aqueous liquid culture medium that was previously separated from the test microorganisms.

[0070] Methods for evaluating the efficacy of sterilization processes

[0071] In another aspect, the present disclosure provides a method for evaluating the efficacy of a sterilization process. In certain embodiments, the sterilization process can be a process intended to sterilize an article or a group of articles. In certain embodiments, the method is performed using a sterilizer. In certain embodiments, the method is performed using an automated sterilizer comprising a sterilization chamber and programmed to subject articles disposed within the sterilization chamber to predetermined conditions or a set of conditions intended to render the articles sterile.

[0072] The method includes positioning a biological indicator in a sterilization chamber. The biological indicator includes: a housing having at least one liquid-impermeable wall forming an opening to a compartment as disclosed herein; a plurality of test microorganisms disposed in the housing; and a pH indicator dye substrate disposed in the housing.

[0073] The pH indicator dye substrate is substantially free of water and has a pH indicator dye as disclosed herein that is bound to the pH indicator dye substrate. The pH indicator dye is substantially present on the pH indicator dye substrate in a first state (e.g., when the dye is primarily deprotonated, it has a color associated with the pH indicator dye). The pH indicator dye can be converted (e.g., by protonation in an aqueous liquid culture medium) to a second state that is optically distinguishable from the first state (e.g., when the dye is primarily protonated, it has a color associated with the pH indicator dye). When the biological indicator is positioned in the sterilization chamber (i.e., before the biological indicator is subjected to a sterilization process), the pH indicator dye is not disposed in the aqueous liquid culture medium or in liquid contact with the aqueous liquid culture medium.

[0074] Optionally, in any embodiment of the method, the biological indicator can further include an aqueous liquid culture medium disposed in an openable container disposed in the housing, as disclosed above.

[0075] Optionally, in any embodiment of the method, the biological indicator can further include a nutrient composition as described herein that promotes germination and / or growth of a test microorganism in the plurality of test microorganisms.

[0076] The method also includes subjecting the biological indicator in the sterilization chamber to a sterilization process. The sterilization process can be any sterilization process that renders the test microorganism inviable. Non-limiting examples of sterilization processes include sterilization processes in which the test microorganism is placed in fluid communication with a sterilant such as steam, ethylene oxide, vapor hydrogen peroxide, ozone, dry heat, ionizing radiation, or a combination thereof.

[0077] After the biological indicator in the sterilizing chamber is subjected to the processing of sterilization process, the method also includes contacting test microorganisms and pH indicator dye with aqueous liquid culture medium and nutrients in the housing of the biological indicator, and this nutrients promotes germination and / or growth of test microorganisms. In some specific implementations, nutrients can be arranged in the housing before the biological indicator is positioned in the sterilizing chamber. In some specific implementations, after the biological indicator in the sterilizing chamber is subjected to the processing of sterilization process, nutrients can be added to the housing (for example, as liquid or dry composition). In some specific implementations, nutrients can be present in the aqueous liquid culture medium. In some specific implementations where the biological indicator does not include a container for holding aqueous liquid culture medium, aqueous liquid culture medium (for example, sterile water) can (for example, by pipette) be added to the housing.

[0078] In some embodiments, the aqueous liquid culture medium may be present in (or adjacent to) the biological indicator in an openable (e.g., breakable) container. In these embodiments, contacting the test microorganism and the pH indicator dye with the aqueous liquid culture medium in the housing of the biological indicator comprises opening the openable container (e.g., by crushing the breakable container). In any embodiment, when the biological indicator is positioned in the sterilization chamber, the nutrient may be present in the housing of the biological indicator (e.g., as a dry powder or capsule that is dissolved and / or suspended in the aqueous liquid culture medium, or dissolved and / or suspended in the aqueous liquid culture medium contained in the openable container). Alternatively or in addition, the nutrient may be present in the aqueous liquid culture medium that may be added to the housing (e.g., by pipette) after the biological indicator in the sterilization chamber has been subjected to the sterilization process.

[0079] After contacting the test microorganism and the pH indicator dye substrate with an aqueous liquid culture medium containing nutrients in the housing of the biological indicator, the method also includes incubating the biological indicator for a period of time. Incubating the biological indicator for a period of time includes incubating the biological indicator at a specified temperature while the aqueous liquid culture medium is in contact with the test microorganism and the pH indicator dye substrate. The specified temperature can be any suitable incubation temperature for testing microorganisms and / or enzyme activity as described herein. In some specific implementations, the incubation temperature is about 30°C and 40°C. In some specific implementations, the incubation temperature is about 52°C to 65°C.

[0080] Incubation time can be any suitable incubation time period that is suitable for the indication of live test microorganism in the detection biological indicator.The variation of pH indicator dyestuff from the first state to the second state is a kind of indication of live test microorganism.The existence of the enzyme reaction product with fluorogenic enzyme substrate or chromogenic enzyme substrate is another kind of indication of live test microorganism.In certain embodiments, the specified time period is less than 8 hours, and in some embodiments, is less than 1 hour, and in some embodiments, is less than 30 minutes, and in some embodiments, is less than 15 minutes, and in some embodiments, is less than 5 minutes, and in some embodiments, is less than 1 minute.In other embodiments, the suitable incubation time that is used for biological indicator of the present disclosure is 10 minutes to 1 hour or 10 minutes to 50 minutes or 10 minutes to 30 minutes or 10 minutes to 20 minutes or 10 minutes to 25 minutes or 15 minutes to 30 minutes or 15 minutes to 25 minutes or 15 minutes to 20 minutes.

[0081] After the biological indicator is incubated for the described time period, the method also comprises observing pH indicator dye to detect the second state.Can visual observation or by using automated detector (for example, colorimeter) to observe pH indicator dye.Observing pH indicator dye comprises observing the pH indicator dye that is combined with pH indicator dye substrate to detect the second state.Detecting the existence indication sterilization process lacks efficacy of the second state of pH indicator dye on the pH indicator dye substrate.Detecting the efficacy of the non-existence indication sterilization process of the second state of pH indicator dye on the pH indicator dye substrate.

[0082] system

[0083] In another aspect, the present disclosure provides a system that can be used to determine the efficacy of a sterilization process. The system includes any embodiment of a self-contained biological indicator according to the present invention and an automatic reader. The automatic reader is constructed to i) receive at least a portion of the biological indicator, ii) direct electromagnetic radiation of a first wavelength into an aqueous liquid culture medium in a housing, and iii) detect or measure the amount of electromagnetic radiation of a second wavelength emitted by a fluorescent product. Therefore, it should be recognized by those skilled in the art that the automatic reader includes, among other things, a site (e.g., a chamber) sized to receive the biological indicator, a source of ultraviolet electromagnetic radiation, a light detector for detecting and measuring fluorescence emitted from the biological indicator, and at least one microprocessor for controlling components of the automatic reader. Optionally, the automatic reader also includes software or firmware having an algorithm for identifying biological indicators that exhibit fluorescence, which indicates that the source of biological activity is completely inactivated or at least a portion of the source of biological activity survives after being treated by the sterilization process.

[0084] The present invention is illustrated by the following examples. It is to be understood that the specific examples, materials, amounts and procedures are to be interpreted broadly in accordance with the scope and spirit of the invention as set forth herein.

[0085] Example

[0086] Example 1: Preparation of pH indicator-bound pH indicator dye substrate .

[0087] At room temperature, 3M TM ATTEST TM The 1492V biological indicator has a 2 The charged nylon substrate (GE MAGNAPROBE charged nylon) of 1000 square meters is immersed in the ampoule culture medium in the 3M ATTEST 1492V biological indicator for one minute, during which time substantially all of the bromocresol purple is concentrated from the culture medium onto the pH indicator dye substrate. The pH indicator dye substrate is taken out from the culture medium, rinsed with sterile deionized water, and dried at room temperature. The substrate bound by the pH indicator dye presents a dark purple color. After the pH indicator dye substrate is dried, they are placed on the housing of the biological indicator, as shown in " substrate 119 " in Fig. 1-Fig. 4 of U.S. Patent Application Publication No. 2013 / 0302849.

[0088] Example 2: Preparation of biological indicators .

[0089] The components used in this example were from the 3M ATTEST 1492V biological indicator (described in U.S. Patent Application Publication No. 2013 / 0302849 and obtained from 3M Company, St. Paul, MN). In contrast to the commercially available 1492V biological indicator, the fragile ampoule containing the nutrient medium and pH indicator was removed from the biological indicator used in this example. A pH indicator dye substrate incorporating the pH indicator was prepared as described in Example 1 and inserted into the biological indicator (ampoule lacking the nutrient medium) at the position indicated by substrate 119 of U.S. Patent Application Publication No. 2013 / 0302849. These biological indicators were used to evaluate the efficacy of a steam sterilization process, as described below.

[0090] Although the biological indicator of this embodiment does not include nutrients to promote germination and / or growth of test microorganisms or an openable container for holding an aqueous liquid culture medium, the inventors recognize that these components can be added to the biological indicator of Example 2 to produce a self-contained biological indicator.

[0091] Example 3: Evaluating the efficacy of a sterilization process .

[0092] The biological indicator prepared as described in Example 2 was placed in the sterilization chamber of an automatic steam sterilizer and The bioindicators were steam sterilized at 121°C for 15 minutes in a Lab 110 steam sterilizer (STERIS, Mentor, OH). A control biological indicator from Example 2 was not subjected to the steam sterilization process. After the indicators were removed from the sterilizer, approximately 0.5 mL of nutrient medium (containing fermentable carbohydrates) for culturing Geobacillus stearothermophilus was pipetted into the bioindicators that had been steam sterilized, and the control and all bioindicators were incubated at 60°C to allow spore growth. After incubation, the pH indicator-bound pH indicator dye substrate in the bioindicators that had been subjected to the steam sterilization process remained a dark purple color. In contrast, the pH indicator-bound pH indicator dye substrate in the bioindicators that had not been subjected to the steam sterilization process exhibited a yellow color.

[0093] Reference Example 1: Concentration of pH Indicator Dye onto pH Indicator Dye Substrate .

[0094] 3M TM ATTEST TM The charged nylon substrate in the 1492V biological indicator is combined with the 3M TM ATTEST TM Approximately 0.5 mL of nutrient medium from the ampoule of the 1492V biological indicator was placed into a colorimetric tube. The colorimetric tube with the medium and substrate was incubated at 58° C. The time intervals ( exist After contacting the substrate with the culture medium, the TM GENESYS TM The absorbance (450 nm) of the nutrient medium was measured using a BIOL® 20 spectrophotometer (Fisher Scientific, Waltham, MA).

[0095] The data in Table 1 indicate that less than half of the pH indicator dye was removed from the nutrient medium during the first 25 minutes of contact between the substrate and the nutrient medium. This suggests that attempts to detect the fluorescent enzyme product in the nutrient medium may be hampered by residual pH indicator dye remaining in the medium. In contrast, when the pH indicator dye is bound to the pH indicator dye substrate, the pH indicator dye will experience little or no interference in detecting the weak fluorescent signal in the nutrient medium.

[0096] Table 1: Removal of Bromoform from Nutrient Medium (at 58°C) by Placement of Charged Nylon Substrates into the Medium Time course of phenol violet pH indicator dye .

[0097] Time (min) <![CDATA[A 450 ]]> 0.5 0.465±0.007 5 0.429±0.003 19 0.402±0.010 15 0.387±0.015 20 0.359±0.016 25 0.345±0.007

[0098] Although the numerical ranges and parameters setting forth the broad scope of the invention are approximations, the numerical values set forth in the specific examples are reported as precisely as possible. Nevertheless, all numerical values inherently encompass a range inevitably resulting from the standard deviation found in their respective testing procedures.

[0099] All headings are for the convenience of the reader and should not be used to limit the meaning of the text that follows the heading unless so specified.

[0100] The complete disclosures of all patents, patent applications, publications, and nucleic acid and protein database entries cited herein are hereby incorporated by reference in their entirety, as if each were individually incorporated. Various modifications and alterations of the present invention will become apparent to those skilled in the art without departing from the scope and spirit of the invention, and it should be understood that this invention is not to be unduly limited to the illustrative embodiments set forth herein.

Claims

1. A self-contained biological indicator for a sterilization process, comprising: a housing having at least one liquid-impermeable wall forming an opening to the compartment; a plurality of test microorganisms disposed in the housing; an aqueous liquid culture medium, wherein the aqueous liquid culture medium is disposed in an openable container, and the openable container is disposed in the housing; a nutrient that promotes germination and / or growth of the test microorganism if the test microorganism is viable; wherein the nutrient is disposed in the housing or in the container; a pH indicator dye substrate disposed in the housing, the pH indicator dye substrate having a pH indicator dye associated with the pH indicator dye substrate, wherein the pH indicator is not disposed in or in liquid contact with the aqueous liquid culture medium; and A fluorescent enzyme substrate or a chromogenic enzyme substrate is disposed in the shell.

2. The self-contained biological indicator for a sterilization process according to claim 1, wherein the pH indicator dye substrate is substantially free of water.

3. The self-contained sterilization process biological indicator of claim 1, wherein the pH indicator dye is substantially free of water.

4. The self-contained sterilization process biological indicator of claim 1, wherein the pH indicator dye substrate comprises a cationic polymer. 5 . The self-contained biological indicator for a sterilization process according to claim 4 , wherein the cationic polymer comprises a polyamide polymer, a polyethyleneimine polymer, a polyvinylidene fluoride polymer, or a combination of any two or more of the foregoing cationic polymers.

6. The self-contained biological indicator for a sterilization process according to claim 1, wherein the pH indicator dye has an anionic charge in a solvent having a neutral pH.

7. The self-contained biological indicator for sterilization process according to claim 1, wherein the pH indicator is selected from the group consisting of thymol blue, tropaeolum orange 00, methyl yellow, methyl orange, bromophenol blue, bromocresol green, methyl red, bromothymol blue, phenol red, chlorophenol red, neutral red, naphtholphthalein, phenolphthalein, thymolphthalein, alizarin yellow, tropaeolum orange 0, nitroamine, trinitrobenzoic acid, thymol blue, bromophenol blue, tetrabromophenol blue, bromocresol green, bromocresol purple, methyl red, bromothymol blue, Congo red and cresol red.

8. The self-contained biological indicator for a sterilization process according to claim 1, wherein the plurality of test microorganisms are disposed on a carrier.

9. The self-contained biological indicator for a sterilization process according to claim 8, wherein the pH indicator dye substrate is the carrier.

10. The self-contained biological indicator for a sterilization process according to claim 1, wherein the test microorganism is a microorganism selected from the group consisting of: Bacillus stearothermophilus, Bacillus stearothermophilus, Bacillus subtilis, Bacillus atrophaeus, Bacillus megaterium, Bacillus coagulans, Clostridium sporogenes, Bacillus pumilus, and a combination of any two or more of the foregoing microorganisms.

11. The self-contained biological indicator for a sterilization process according to claim 1, wherein the aqueous liquid culture medium comprises the nutrient substances that promote the germination and / or growth of the test microorganisms. 12 . The self-contained biological indicator for a sterilization process according to claim 1 , wherein the fluorescent enzyme substrate or the chromogenic enzyme substrate is disposed in the openable container.

13. A method of evaluating the efficacy of a sterilization process, the method comprising: Positioning a biological indicator in the sterilization chamber, wherein the self-contained biological indicator comprises: a housing having at least one liquid-impermeable wall forming an opening to the compartment; a plurality of test microorganisms disposed in the compartment; a substantially non-aqueous pH indicator dye substrate disposed in the compartment, the pH indicator dye substrate having a substantially non-aqueous pH indicator dye associated with the pH indicator dye substrate; and a fluorescent enzyme substrate or a chromogenic enzyme substrate disposed in the shell; wherein the pH indicator dye is substantially present in a first state on the pH indicator dye substrate, and wherein the pH indicator dye is convertible to a second state optically distinguishable from the first state; subjecting the biological indicator in the sterilization chamber to the sterilization process; after subjecting the biological indicator in the sterilization chamber to the sterilization process, contacting the test microorganism and the pH indicator dye substrate with an aqueous liquid culture medium and nutrients in the housing of the biological indicator, the nutrients promoting germination and / or growth of the test microorganism; incubating the biological indicator for a period of time; and after incubating the biological indicator for the period of time, observing the pH indicator dye to detect the second state; wherein detecting the presence of the second state of the pH indicator dye on the pH indicator dye substrate indicates a lack of efficacy of the sterilization process; Wherein detecting the absence of the second state of the pH indicator dye on the pH indicator dye substrate indicates efficacy of the sterilization process.

14. The method of claim 13, wherein the biological indicator further comprises an aqueous liquid culture medium disposed in an openable container disposed in the housing, wherein contacting the test microorganism and the pH indicator dye substrate with the aqueous liquid culture medium in the housing of the biological indicator comprises opening the openable container.