Biological indicator having a test microorganism encapsulated by a wax composition

Abstract

A self-contained sterilization process biological indicator is provided. The indicator includes a housing. Test microorganisms disposed in the housing are encapsulated by a wax composition. The wax composition includes a long chain (greater than C22) straight chain or branched chain alkyl or alkenyl alcohol, wherein the alkyl or alkenyl alcohol is not attached to a short chain alkyl or aryl (C1-C6) group by an ester or ether linkage, wherein the alkyl or alkenyl alcohol is not attached to a long chain (C8-C36) alkyl or alkenyl by an ester or ether linkage, wherein the alkyl or alkenyl alcohol is not attached to a straight chain or branched chain long chain (C8-C36) alkyl or alkenyl amine or acid by an amide linkage. The wax composition has a melting point between 78°C and 120°C. Also contained in the housing are a nutrient composition and an openable container containing a liquid medium.

Description

BACKGROUND

[0001] Sterilization of equipment, instruments, and other devices is critical in the healthcare industry. For example, hospitals and other medical facilities often sterilize medical instruments and equipment used in treating patients. The particular type of sterilization cycle used to sterilize such equipment can vary based on the particular equipment or device to be sterilized and based on the particular preferences of the entity performing the sterilization cycle. However, all such sterilization cycles or processes are generally designed to kill living organisms that can otherwise contaminate the equipment or device to be sterilized.

[0002] Various sterilization methods use different cycles or techniques to sterilize. For example, sterilization can include applying steam, dry heat, chemicals (e.g., ethylene oxide, hydrogen peroxide), or radiation to the equipment or device to be sterilized. Steam sterilization is generally recommended for metals, Teflon, and other high-melting point surgical instruments capable of holding temperatures in the range of 121-135 °C. The contact time in a sterilization cycle is temperature dependent. For example, the equipment or instrument to be sterilized is preferably exposed to steam sterilization at 132 °C for about three minutes. However, the contact period can be as long as 30-35 minutes at 121 °C.

[0003] Biological indicators are commonly used to evaluate and validate the effectiveness of sterilization processes in various environments. Generally, living but relatively more resistant spores of a thermophilic organism are subjected to the sterilization conditions along with any device or instrument to be sterilized. Generally, the test microorganism is more resistant to the sterilization process than most other organisms that would be present due to natural contamination. Applications have used spores of microorganisms that are capable of producing an enzyme that catalyzes a non-fluorescent substrate to a fluorescent product, which can be detected to indicate the presence of surviving spores.

[0004] Generally, after completion of the sterilization process, the test microorganism (e.g., spores) are incubated in a nutrient medium to determine whether any of the test organisms survived the sterilization procedure. In conventional biological indicators, the detection of growth of the number of organisms that can be detected can require 24 hours or more when using a pH indicator to detect growth.

[0005] The use of a rapid readout technique that detects enzyme activity of the test microorganism can reduce the time required to detect live test microorganisms. In some embodiments, analysis of the fluorescence intensity resulting from the fluorescent product of the enzyme reaction is used to determine whether the sterilization process was successful. SUMMARY

[0006] A complete sterilization process biological indicator is now provided, wherein everything needed to rapidly assess the effectiveness of a wide variety of steam sterilization processes is provided by the ability to detect the germination and / or outgrowth of live test microorganisms, if present, after contacting the complete biological indicator with a steam sterilization process. Advantageously, this discovery provides a user of the biological indicator with a test microorganism encapsulated by a wax composition that increases the resistance of the test microorganism to certain steam sterilization processes, thereby providing a biological indicator that can be used to assess the efficacy of a wide range of steam sterilization process conditions. Surprisingly, the wax composition encapsulated test microorganism of the present disclosure is suitable for both rapid detection (e.g., by detecting enzyme activity associated with the test microorganism) and detection based on traditional microbial growth.

[0007] In one aspect, the present disclosure provides a complete sterilization process biological indicator. The indicator can 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, wherein the test microorganisms are at least partially encapsulated by a wax composition; a liquid culture medium disposed in an openable container, the contents of which are in selective fluid communication with the compartment; and a nutrient composition that facilitates germination and / or outgrowth of the test microorganisms, wherein the nutrient composition is disposed in the container or the housing. The wax composition comprises a long chain (greater than C22) linear or branched alkyl or alkenyl alcohol, wherein the alkyl or alkenyl alcohol is not linked to a short chain alkyl or aryl (C1-C6) group by an ester or ether linkage, wherein the alkyl or alkenyl alcohol is not linked to a long chain (C8-C36) alkyl or alkenyl by an ester or ether linkage, wherein the alkyl or alkenyl alcohol is not linked to a linear or branched long chain (C8-C36) alkyl or alkenyl amine or acid by an amide linkage. The wax composition has a melting point between 78 °C and 120 °C. The housing includes an opening that allows a sterilant to enter the housing from outside the housing.

[0008] In certain embodiments of the complete sterilization process biological indicator, the test microorganisms can be immobilized to a carrier, wherein the test microorganisms are encapsulated by the wax composition and / or the carrier. In certain embodiments of the complete sterilization process biological indicator, the test microorganisms can be immobilized to a portion of a wall of the housing, wherein the test microorganisms are encapsulated by the wax composition and / or the portion of the wall. In any of the above embodiments, the complete biological indicator for a sterilization process can further include a wax composition wicking member. In certain embodiments, the wax composition wicking member can contact the wax composition.

[0009] In another aspect, the present disclosure provides methods for determining the effectiveness of a sterilization process. The methods can include positioning a sterilization process biological indicator in a sterilization chamber, wherein the biological indicator includes a plurality of test microorganisms at least partially encapsulated by a wax composition; contacting the biological indicator with moist heat at a temperature of at least 121 °C while the indicator is positioned in the sterilization chamber; contacting the test microorganisms with a detection medium after contacting the biological indicator with the moist heat; and incubating the indicator at a predetermined temperature for a period of time sufficient to detect the presence of one of the test microorganisms, if alive, after contacting the test microorganisms with the detection medium. The wax composition comprises a long chain (greater than C22) straight chain or branched chain alkyl or alkenyl alcohol, wherein the alkyl or alkenyl alcohol is not linked to a short chain alkyl or aryl (C1-C6) group by an ester or ether linkage, wherein the alkyl or alkenyl alcohol is not linked to a long chain (C8-C36) alkyl or alkenyl by an ester or ether linkage, wherein the alkyl or alkenyl alcohol is not linked to a straight chain or branched chain long chain (C8-C36) alkyl or alkenyl amine or acid by an amide linkage. The wax composition has a melting point between 78 °C and 120 °C.

[0010] In any of the above embodiments of the methods, positioning a sterilization process biological indicator in a sterilization chamber can include positioning a sterilization process biological indicator including a housing having test microorganisms therein, wherein contacting the test microorganisms with a detection medium can include contacting the test microorganisms with a detection medium inside the housing.

[0011] In yet another aspect, the present disclosure provides kits. The kits can include a plurality of test microorganisms, wherein the test microorganisms are at least partially encapsulated by a wax composition, and instructions for using the test microorganisms to evaluate the efficacy of a steam sterilization process. The wax composition comprises a long chain (greater than C22) straight chain or branched chain alkyl or alkenyl alcohol, wherein the alkyl or alkenyl alcohol is not linked to a short chain alkyl or aryl (C1-C6) group by an ester or ether linkage, wherein the alkyl or alkenyl alcohol is not linked to a long chain (C8-C36) alkyl or alkenyl by an ester or ether linkage, wherein the alkyl or alkenyl alcohol is not linked to a straight chain or branched chain long chain (C8-C36) alkyl or alkenyl amine or acid by an amide linkage. The wax composition has a melting point between 78 °C and 120 °C.

[0012] In any embodiment, the kit can further include a detection reagent selected from the group consisting of a nutrient, a pH indicator, a redox indicator, a fluorescent enzyme substrate, a chromogenic enzyme substrate, and a combination of any two or more of the foregoing detection reagents. In any of the above embodiments, the kit can further include a housing sized to contain the test microorganisms or the detection reagent. In certain embodiments of the kit, the test microorganisms and the detection reagent can be disposed in the housing.

[0013] Additional details of these and other embodiments are set forth in the following drawings and description. Other features, objects, and advantages will become apparent from the description and drawings, and from the claims.

[0014] In this document, the terms "biological sterilization process indicator," "sterilization process biological indicator," "sterilization process indicator," "biological indicator," "BI," "indicator," "self-contained biological indicator," and "SCBI" can be used interchangeably.

[0015] Also in this document, in the written description and claims, the phrases "substantially dry," "substantially free of water," and the like mean a composition or coating having a water content no greater than the water content of the dehydrated coating once allowed to equilibrate with the ambient environment.

[0016] The numerals E5, E6, and E7 are used herein interchangeably with 10 5 , 10 6 , and 10 7 , respectively.

[0017] The terms "comprising," "including," "having," and the like, as used in the

[0018] As used herein, "a," "an," "the," and "at least one" are used interchangeably

[0019] Also in this document, a range expressed by an endpoint includes any number

[0020] The words "preferred" and "preferably" refer to embodiments of the invention that can provide certain benefits under certain circumstances. However, other embodiments can also be preferred under the same or other circumstances. Additionally, the use of certain of these terms in

[0021] As used herein, the term "subset" with respect to a given set S having n elements means a set having one or more elements of S. Power set"Power Set" refers to the mathematical definition of the power set of S and all possible subsets, including the empty set but not S itself, having 1 to n elements of each combination, and is denoted as P(S). Applicant notes that the mathematical definition of the power set includes the empty set (a set with no elements). However, the definition employed herein by Applicant excludes the empty set and includes all subsets having at least one element, including the full set of n elements (S). Generally, the power set includes all subsets having "i" elements, where i = 1 to n-1, as well as the subset having all n elements (n). For example, the power set of a subset S having elements a, b, and c (n = 3) includes the following 7 subsets: all possible subsets having one element: {(a), (b), (c)}; all possible subsets having any possible combination of two elements: {(a,b), (a,c), (b,c)}, and the subset having all 3 elements: (a,b,c).

[0022] As used herein, the term "container" Actuatable container "container" means a receptacle capable of being actuated to release its contents when desired. The container can be actuated, for example, by removing or taking out a stopper, actuating a valve to change it from a "closed" to an "open" state, or otherwise breaking at least a portion of the container.

[0023] The term "container" Frangible container "container" means any receptacle that can be acted upon, for example, by rupturing it, perforating it, breaking it, cutting it, and the like, to release its contents.

[0024] Unless defined otherwise, all scientific and technical terms used herein have the meanings commonly used in the art. The definitions given herein are intended to aid in understanding certain terminology frequently used in the present application and are not intended to limit reasonable interpretations of that terminology in the context of the present disclosure.

[0025] Unless otherwise indicated, all descriptions and claims contained in the specification and the claims are understood to be made using the terms "about" and "at or about" with respect to any quantitative expression of a physical property, amount or characteristic. Therefore, unless otherwise indicated, the numerical parameters listed in the specification and the claims are approximations. At the very least, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques. Numerical parameters in the specification and claims have, unless otherwise indicated, been determined by standard testing techniques.

[0026] The term "adjacent" as understood by its use in context refers to the relative position of two elements (such as, for example, two layers) that are near to each other and can or can not need to be in contact with each other or can have one or more layers separating the two elements.

[0027] The above summary of the application is not intended to describe each disclosed embodiment or every implementation of the present application. The following description more particularly exemplifies illustrative embodiments. BRIEF DESCRIPTION OF DRAWINGS

[0028] Figure 1 is a partially exploded cross-sectional view of the self-contained biological indicator of the present disclosure.

[0029] Figure 2 is a cross-sectional view of the self-contained biological indicator of Figure 1 along line 2-2.

[0030] Figure 3 is an exploded perspective view of the self-contained biological indicator of Figures 1-2

[0031] Figure 4A is a schematic plan view of one embodiment of a test microbe carrier according to the present disclosure, with test microbes encapsulated by a wax composition affixed to the test microbe carrier.

[0032] Figure 4B is a cross-sectional view of the test microbe carrier of Figure 4A along line 4B-4B.

[0033] Figure 5 ​is a cross-sectional view of an alternative embodiment of a housing of a self-contained biological indicator according to the present disclosure, wherein microorganisms encapsulated by a wax composition are affixed to at least one wall of the housing. DETAILED DESCRIPTION

[0034] Before any embodiments of the present disclosure 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 the terms "including," "comprising," or "having" and variations thereof herein is intended to be broad and encompass the recitation of steps, components, elements, or members that are either affirmatively recited or equivalently recited in a claim. 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 disclosure. Furthermore, as used herein, the terms "front," "rear," "top," "bottom," and the like, are merely used for description and do not indicate or imply that a particular orientation is required for the apparatus in use, installation, display, or positioning in use. It is to be understood that the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0035] The present disclosure relates generally to sterilization process indicators, kits containing test microorganisms for use as sterilization process indicators, and methods of use thereof. The self-contained biological indicators of the present disclosure include all components necessary to assess the survival of the test microorganisms contained therein and can be used to determine lethality of a sterilization process using moist heat as a sterilant.

[0036] As indicated above, there is now provided a self-contained biological sterilization process indicator that can be used to assess the efficacy of a variety of steam sterilization processes, including, for example, steam sterilization processes employing temperatures at 121 °C, 132 °C, 134 °C, or 135 °C. Advantageously, this means that an operator no longer has to use a different biological indicator for each of a plurality of different steam sterilization temperatures.

[0037] Housing

[0038] For non-limiting examples of housings suitable for use with 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 these is incorporated herein by reference in its entirety. Generally, a housing is a container, often an outer container, that designates a location for other components of a biological indicator, typically having walls that are impermeable to sterilants. The housing can be located within a 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 encompasses housings of any shape and size.

[0039] The housing contains at least one opening (sterilant path) that enables the flow of sterilant to the interior of the housing. In some embodiments, the housing can include a main body having an opening and a cover that closes the opening. In some embodiments, the cover can be capable of completely sealing the housing and eliminating any fluid communication between the interior of the housing and the surrounding environment (e.g., closing the sterilant path). Generally, the cover has an open position in which there is an opening (e.g., gap) between the cover and the main body of the container, enabling the flow of liquid or gas (e.g., sterilant) 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 can include vents that enable the passage of sterilant to the interior of the housing and form additional sterilant paths, even in the presence of the cover and when the cover is in the closed position. However, in other preferred embodiments, when the cover includes vents, placing the cover in the closed position simultaneously closes: (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.

[0040] In other embodiments, the cover can lack vents and the only sterilant path when the cover is in the open position can be through the space between the cover and the main body of the housing (or through another opening or vent, if present on the main body). In some embodiments, if vents are present on the housing, they are on the cover. In embodiments in which there are no openings other than the opening between the cover and the main body of the housing, placing the cover in the closed position completely seals 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 can be sealed when the cover is in the closed position.

[0041] Test microorganism

[0042] Generally, 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 a live culture of a known species of microorganism, typically in the form of spores of the microorganism. At least in part, spores (e.g., bacterial spores) are used instead of vegetative forms of microorganisms because vegetative microorganisms are known to be relatively easily killed by sterilization processes. Additionally, spores also have good storage properties and are able to remain in their dormant state for many years. Thus, sterilization of a seed culture of a standardized spore strain provides a higher degree of confidence that all microorganisms in the sterilization chamber have been inactivated.

[0043] The self-contained biological indicators of the present disclosure include a plurality of test microorganisms disposed therein (e.g., disposed in the interior of the housing). The test microorganisms can belong to one or more species. Typically, the biological indicators contain a predetermined amount of 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 test microorganisms.

[0044] By way of example only, the microorganisms used in the biological indicators are described as "spores"; however, it should be understood that the type of microorganism (e.g., spores) used in a particular embodiment of the biological indicator is selected for its ability to withstand the particular sterilization process contemplated (greater than the resistance of microorganisms typically present on the articles to be sterilized such that inactivation of the test microorganisms indicates successful sterilization). Thus, different embodiments of the present disclosure using different sterilants can use different microorganisms depending on the sterilization process intended to be employed by the particular embodiment.

[0045] In some embodiments, the test microorganisms can include, but are not limited to, at least one of Geobacillus stearothermophilus, Bacillus stearothermophilus, Bacillus subtilis, Bacillus atrophaeus, Bacillus megaterium, Bacillus coagulans, Clostridium sporogenes, Bacillus pumilus, or combinations thereof.

[0046] Enzyme and enzyme substrate

[0047] The self-contained biological indicators of the present disclosure include a detection reagent (e.g., a nutrient that facilitates germination and / or outgrowth of a test microorganism). In some embodiments, the biological indicator can include a detection reagent (e.g., an enzyme substrate) capable of detecting an enzyme present in and / or on the test microorganism, or the test microorganism is capable of producing such an enzyme, or both. Enzymes that can be used in the biological indicators of the present disclosure include extracellular and intracellular enzymes whose activity correlates with the viability of at least one of the microorganisms typically used to monitor sterilization efficacy ("test" microorganisms or "test spores"). In this context, "correlates" means enzyme activity above background that can be used to indicate the survival of at least one of the test microorganisms. The enzyme should be one that retains sufficient activity to react with an enzyme substrate of the enzyme within twenty-four hours, and in preferred embodiments, within one hour or less, after a sublethal sterilization cycle to the test microorganism, while being inactivated or significantly less active after a lethal sterilization cycle to the test microorganism.

[0048] Examples of suitable enzymes include alpha-glucosidase, alpha-galactosidase, lipase, esterase, acid phosphatase, alkaline phosphatase, protease, aminopeptidase, chymotrypsin, beta-glucosidase, beta-galactosidase, alpha-glucuronidase, beta-glucuronidase, phospho-hydrolase, alpha-mannosidase, beta-mannosidase, a-L-fucosidase, leucine aminopeptidase, a-L-arabinofuranosidase, cysteine aminopeptidase, valine aminopeptidase, beta-xylosidase, alpha-L-iduronidase, dextranase, cellobiohydrolase, cellulase, alpha-arabinosidase, poly-saccharase, sulfatase, butyrate esterase, glycosidase, arabinosidase, and combinations of any two or more of the foregoing enzymes. In certain embodiments of the articles, kits, systems, and methods of the present disclosure, the source of biological activity used includes an isolated or otherwise purified form of any of the above suitable enzymes.

[0049] In the context of the present application, an enzyme substrate includes a substance or mixture of substances that is converted to an enzyme-modified product when acted upon by an enzyme. While the preferred substrate produces a compound that is capable of fluorescent 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 consist of a compound that, when reacted with an enzyme, will produce a product that will react with an additional compound or composition to produce a luminescent, fluorescent, or colored material. Preferably, if the substrate is to be included in the indicator device during sterilization, the substrate should not spontaneously decompose or convert to a detectable product during sterilization or incubation. For example, in a device for monitoring steam and dry heat sterilization, the substrate must be stable at temperatures between about 20°C and 180°C. Also preferably, where the enzyme substrate is to be included with a conventional growth medium, the enzyme substrate must be stable in the growth medium, e.g., not spontaneously fluorescent in the growth medium.

[0050] In general, there are two basic types of enzyme substrates that can be used in the biological indicators of the present disclosure. The first type of substrate can be fluorescent (or chromogenic), and can be given a chemical formula such as AB. When acted upon by an enzyme, AB is broken down into products A and B. B, for example, can be fluorescent or colored. A specific example of this type of fluorescent substrate is a salt of 4-methylumbelliferyl. Other fluorescent substrates of this type include derivatives of 4-methylumbelliferyl, 7-amido-4-methylcoumarin (7-AMC), indoxyl, and fluorescein. An example of a chromogenic substrate of this type is 5-bromo-4-chloro-3-indolyl phosphate. In the presence of a phosphatase, the substrate will break down into indigo blue and phosphate. Other chromogenic substrates of this type include derivatives of 5-bromo-4-chloro-3-indolyl, nitrophenol, and phenolphthalein.

[0051] The second type of substrate can be given a chemical formula such as CD, which will be converted to C and D by a specific enzyme. However, in this case, neither C nor D will be fluorescent or colored, but either C or D is capable of further reacting with a 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 remaining portion of lysine is called cadaverine, which is strongly basic. A basic indicator such as 4-methylumbelliferone can be incorporated, and this basic indicator will fluoresce in the presence of a strong base. A chromogenic substrate of this type will be 2-naphthyl phosphate. Phosphatase reacts with this substrate to produce β-naphthol. The released β-naphthol reacts with a chromogenic reagent comprising 1-diazo-4-benzamido-2,5-diethoxybenzene (commercially available as "Fast Blue BB salt" from Sigma Chemical) to produce a violet color.

[0052] As noted above, preferred enzyme substrates are in some embodiments fluorescent substrates, which are defined herein as compounds that are capable of being modified by an enzyme (e.g., by hydrolysis or other enzymatic action) to give a derivative fluorophore that has significantly altered or enhanced fluorescence.

[0053] It will be appreciated by those of ordinary skill in the art that suitable fluorescent compounds are themselves non-fluorescent or meta-fluorescent (i.e., fluoresce in a manner that is significantly different (e.g., in color or intensity) from the corresponding enzyme-modified product). In this regard, appropriate excitation and detection wavelengths are used in a manner known to users of fluorescence technology to separate the fluorescent signal formed by enzyme modification from any other fluorescence that can be present.

[0054] 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: 4-methylumbelliferyl a-D-glucopyranoside, 4-methylumbelliferyl a-D- galactopyranoside, heptanoic acid 4-methylumbelliferyl ester, palmitic acid-4-methylumbelliferyl ester, oleic acid 4-methylumbelliferyl ester, acetic acid 4-methylumbelliferyl ester, nonanoic acid 4-methylumbelliferyl ester, octanoic acid 4-methylumbelliferyl ester, butyric acid 4-methylumbelliferyl ester, 4-methylumbelliferyl-P-D-cellobioside, acetic acid 4-methylumbelliferyl ester, phosphoric acid 4-methylumbelliferyl ester, sulfuric acid 4-methylumbelliferyl ester, cinnamic acid 4-methylumbelliferyl-P-trimethylammonium chloride, 4-methylumbelliferyl-P-D-N,N',N"-triacetylchitotriose, 4-methylumbelliferyl-P-D- xyloside, 4-methylumbelliferyl-N-acetyl-P-D-glucosaminide, 4-methylumbelliferyl-N-acetyl-a-D- glucosaminide, propionic acid 4-methylumbelliferyl ester, stearic acid 4-methylumbelliferyl ester, 4-methylumbelliferyl-a-L-arabinofuranoside, 4-methylumbelliferyl a-L-arabinoside;methylumbelliferyl-β-D-N,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, 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 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-p-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-glutamic acid 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-cyanophthalimide) 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-methylphthalimide, and 3-(4-imidazolyl)phthalimide; 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)-carboxynaphthofluorescein, rhodamine, 2',7'-dichlorofluorescein diacetate, 5(6)-carboxyfluorescein, 5(6)-carboxyfluorescein diacetate, 5-(bromomethyl)fluorescein, 5-(iodoacetamido)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, anthrofluorescein, rhodols, halofluoresceins; derivatives of rhodamines, including: tetramethyl rhodamine, carboxytetramethyl rhodamine, carboxy-X-rhodamines, sulfonyl rhodamine 101, and rhodamine B; fluoroglycosamine derivatives; derivatives of benzanthracene dyes, including: heminaphthofluorone, carboxyheminaphthofluorone, heminaphthofluorescein, seminaphthorhodafluors; derivatives of cyanines, including sulfonated pentamethine cyanine and septamethine cyanine.

[0055] In some embodiments, the enzyme whose activity is to be detected can be selected from a-D-glucosidase, chymotrypsin, or fatty acid esterase. In the case of B. stearothermophilus, the fluorescent enzyme substrate is preferably 4-methylumbelliferyl-a-D-glucopyranoside, 7- glutarylphenylalanine-7-amido-4-methylcoumarin, or heptanoic acid 4-methylumbelliferyl ester. In certain preferred embodiments, 4-methylumbelliferyl a-D-glucopyranoside is the enzyme substrate used to generate metabolic activity, and the enzyme is a glucosidase, such as b-D-glucosidase.

[0056] The concentration of enzyme substrate present in the biological indicator (e.g., when dissolved and / or suspended in the aqueous liquid medium of the biological indicator) depends on the identity of the particular enzyme substrate and enzyme, the amount of enzyme product that must be generated to be detectable visually or by instrument, and the amount of time one is willing to wait in order 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, such that at least 10 -8Molarzyme modification product. Where the enzyme substrate is a 4-methylumbelliferyl derivative, the inventors have found that its concentration in the aqueous liquid medium disclosed herein is preferably between about 10 -5 and 10 -3 moles. In some embodiments, 4-methylumbelliferyl-a-D-glucoside can be used in the aqueous mixture, for example, at a concentration of about 0.05 g / L to about 0.5 g / L (such as 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).

[0057] pH indicator dye

[0058] In any embodiment, the self-contained, packaged biological indicator of the present disclosure can include a pH indicating dye disposed in the housing (e.g., disposed in the compartment). In certain embodiments, the pH indicating dye can be bound (e.g., with high affinity) to a pH indicating dye substrate material, as described in U.S. Provisional Patent Application No. 62 / 990,483; filed March 17, 2020, and entitled “IMMOBILIZED PH INDICATOR FOR BIOLOGICAL INDICATOR GROWTH INDICATION,” which is incorporated herein by reference in its entirety. In any embodiment, the indicating dye can be a pH indicator suitable for detecting biological activity (e.g., fermentation of a carbohydrate nutrient). The indicating dye can be selected according to standard known in the art, e.g., pH range, compatibility with biological activity, and solubility. In some embodiments, a salt form of the pH indicator can be used, e.g., to increase solubility of the pH indicator in the aqueous mixture. Non-limiting examples of suitable pH indicator dyes include, e.g., thymol blue, chrysamine G, methyl yellow, methyl orange, bromophenol blue, bromocresol green, methyl red, bromothymol blue, phenol red, chlorophenol red, neutral red, naphthol phthalein, phenolphthalein, thymolphthalein, alizarin yellow, chrysamine O, 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 an anionic dye in a solution having a pH of about neutral.

[0059] In some embodiments, the pH indicator dye produces a color change upon a decrease in pH, thereby indicating growth of the test microorganism. In some embodiments, the pH indicator dye is bromocresol purple. The pH indicator can be used to detect biological activity, such as fermentation of a carbohydrate to an acidic end product (indicative of survival of the test microorganism). For example, these activities can indicate the presence or absence of viable spores after a biological indicator has been processed through a sterilization process. For example, bromocresol purple can be used in the aqueous mixture at a concentration of about 0.03 g / L.

[0060] The combination of bromocresol purple and 4-methylumbelliferyl-a-D-glucoside represents a preferred combination of enzyme substrate and pH indicator dye in an article or method according to the present disclosure, but other combinations can be contemplated within the scope of the present disclosure.

[0061] Self-contained biological indicator

[0062] The various test microorganisms encapsulated by the wax compositions described herein can be used as a modification to various biological indicators known in the art to produce a biological indicator or a self-contained biological indicator according to the present disclosure. The resulting biological indicator or self-contained biological indicator is particularly useful for evaluating the effectiveness of a steam sterilization process. Further, the various test microorganisms encapsulated by the wax compositions described herein can be used as a modification to various methods for evaluating the effectiveness of a sterilization process.

[0063] For example, the self-contained biological indicator of U.S. Patent No. 3,661,717, the entirety of which is incorporated herein by reference, can be modified to provide test microorganisms encapsulated by a wax composition as described herein. In certain embodiments, the test microorganisms encapsulated by the wax composition can be provided on a carrier substrate or on an interior surface of a housing of the biological indicator.

[0064] Additionally, the self-contained biological indicators of U.S. Patent Nos. 5,223,401 and 6,623,955, the entireties of both patents are incorporated herein by reference, can be modified to provide test microorganisms encapsulated by a wax composition as described herein. In certain embodiments, the test microorganisms encapsulated by the wax composition can be provided on a carrier substrate or on an interior surface of a housing of the biological indicator.

[0065] Further, the self-contained biological indicator of U.S. Patent Application Publication No. US 2013 / 0302849, the entirety of which is incorporated herein by reference, can be modified to provide test microorganisms encapsulated by a wax composition as described herein. In certain embodiments, the test microorganisms encapsulated by the wax composition can be provided on a carrier substrate or on an interior surface of a housing of the biological indicator.

[0066] Those skilled in the art will recognize how other existing biological indicators can be modified using test microorganisms encapsulated by the wax composition of this disclosure to obtain the articles and methods of this disclosure.

[0067] In this disclosure, the process of bringing together spores and culture medium is referred to as “activation” of a bioindicator. That is, the term “activation” and its variations, when used in relation to a bioindicator, generally refer to bringing one or more test microorganisms (e.g., spores) into fluid communication with an aqueous liquid culture medium (e.g., a liquid culture medium containing nutrients and / or enzyme substrates). For example, a bioindicator may be described as being “activated” when an openable container within a bioindicator 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 microorganism.

[0068] Turn to the attached diagram. Figures 1-3 Various views of one embodiment of the complete set of bio-indicators 100 according to this disclosure are shown.

[0069] The complete biometric indicator 100 is shown with a housing 10, which includes a compartment 11 and a cover 28. The compartment 11 has at least one wall 12 forming an opening 14. For example, at least one wall may be made of a moisture-impermeable, non-absorbent material (such as glass or plastic). In some preferred embodiments, the compartment is formed of an optically transparent or translucent material.

[0070] The bioindicator 100 contains a variety of test microorganisms (e.g., bacterial spores) 17 disposed within the housing 10. For example, the test microorganisms 17 may optionally be disposed on a carrier 16 (e.g., a sheet material, such as filter paper strips or polymer membranes) as a substantially anhydrous coating. In some embodiments, the carrier 16 is made of a water-impermeable material; therefore, in those embodiments, the test microorganisms 17 are composed of a wax composition as described herein (…). Figure 1 Encapsulation with (not shown in the image) and optional waterproof substrate, such as Figure 4A -B is shown and described below.

[0071] The complete biological indicator 100 includes a liquid culture medium 20 (e.g., an aqueous liquid culture medium) disposed in an openable container 18. The contents of the openable container 18 (e.g., the liquid culture medium 20) are selectively in communication with a compartment 11 of the housing 10. Figures 1-3In the illustrated embodiments, the openable container 18 is a normally sealed, pressure-openable container, such as a fragile glass ampoule. Those skilled in the art will recognize other suitable openable containers 18 (including some embodiments in which the container is disposed outside the housing 10) and components (e.g., valves, protruding seals) that provide selective fluid communication between the openable container and the compartment.

[0072] The complete set of bioindicators 100 includes a nutrient composition that promotes the germination and / or growth of test microorganisms. The nutrient composition ( Figures 1-3 (Not shown) is disposed within a container or housing. Figures 1-3 In one embodiment of the illustration, the nutrient composition is dissolved and / or suspended in a liquid culture medium 20 disposed in an openable container 18 disposed within a housing 10. In some alternative embodiments, the nutrient composition may be disposed in a dry form (e.g., a dry coating, powder, tablet) within the housing, which may dissolve and / or suspend in the liquid culture medium upon actuation of the openable container. Additionally, upon actuation of the openable container, the liquid culture medium and the nutrient composition may come into contact with the test microorganisms, thereby promoting the growth (and detection) of any live test microorganisms (if present) in the bioindicator.

[0073] exist Figures 1-3 In the illustrated embodiment, container 18 is retained close to the compartment, such that little volume of compartment 10 is unoccupied. Container 18 is separated from the wall 12 of compartment 10 by a carrier 16, thereby providing cavities 24 and 26 between the wall 12 and container 18. The open end 14 of compartment 10 is provided with a breathable, bacteria-impermeable sealing member, shown as sheet 22. Sheet 22 can be sealed, for example, by heat or adhesive or by means of cap 28 (in... Figure 2 The cover 28 is shown as being removed from the opening 14 of the compartment 10, and has an orifice 29 passing through it at an adjacent sheet 22. The orifice 29 allows sterilizing agent to enter the housing from the outside. The cover 28 can be formed from various materials (e.g., metals, plastics) using methods well known in the art. During steam sterilization, steam permeates the sheet 22 and through cavities 24 and 26 to contact the test microorganisms 17 disposed on the carrier 16.

[0074] like Figure 3 As shown, the complete set of bioindicators 100 can be easily assembled by sequentially inserting the carrier 16 (on which the test microorganism 17 is disposed) and the container 18 into the opening end 14 of the compartment 10 and sealing the opening end 14 of the tube with the sheet 22. Before assembly, the test microorganism 17 can be deposited on the carrier 16 as a liquid suspension, and then dried before placing the carrier 16 into the container 10.

[0075] Optionally, the biological indicator 100 may also include detector material (e.g., a chromogenic or fluorescent enzyme substrate and / or a pH indicator; not shown) that is capable of undergoing a visible color change in response to spore growth. The detector material may be present in a liquid culture medium and / or may be present in a dry form (e.g., powder or tablet) disposed in the housing 10.

[0076] Figure 4A -B shows Figure 1 and Figure 3 Various views of the carrier 16 of the all-in-one bioindicator 100 are shown. The carrier 16 is provided with a variety of test microorganisms 17 and a wax composition 30, both as described herein. When producing the bioindicator of this disclosure, the test microorganisms 17 may be applied to the carrier 16 suspended in a liquid solvent (e.g., sterile water, not shown). The solvent is then removed (e.g., by evaporation), leaving a dry coating of the test microorganisms on the carrier. The wax composition 30 may then be applied to the carrier 16, either encapsulating individual test microorganisms within the wax composition or encapsulating test microorganisms between the wax composition and the carrier material.

[0077] Figure 5 Alternative methods for providing test microorganisms within the housing (including compartment 11 and cover 28) of a fully assembled bioindicator according to this disclosure are shown. In this embodiment, instead of placing the test microorganisms in a carrier ( Figure 5 Instead of providing the test microorganism 17 on the inner surface of at least one wall 12 within the housing (not shown), the test microorganism 17 is placed in a compartment. The test microorganism 17 and the wax composition 30 can be delivered onto the wall 12 as described above for placing the test microorganism and the wax composition onto a carrier. After depositing the test microorganism 17 and the wax composition 30 into the housing, a container containing a liquid culture medium nutrient composition (e.g., a fragile glass ampoule, not shown) can be positioned within the housing to produce a fully-equipped bioindicator.

[0078] Sterilization process

[0079] The biological indicator disclosed herein can be used to monitor the effectiveness of one or more types of sterilization procedures, including sterilization procedures that use steam (e.g., pressurized steam) as a sterilizing agent.

[0080] At least some steps in the sterilization process include or may involve high temperatures, such as 121°C, 132°C, 134°C, 135°C, etc. Additionally, high pressure and / or vacuum, such as 15 psi (1 × 10⁻⁶), may be encountered at different stages within a single given sterilization cycle or in different sterilization cycles. 5 Pa).

[0081] In the case of steam as the sterilant, the sterilization temperature can include 121 °C, 132 °C, 134 °C, 135 °C. The rapid readout biological indicator is suitable for steam sterilization cycles at each of the above temperatures, and for each temperature, the cycle can have a different air removal process selected from gravity, pre-vacuum ("pre-vac"), and steam flash pressure pulse (SFPP). Each of these cycles can have a different contact time, depending on the type of instrument / device to be sterilized. In the present disclosure, pre-vacuum and SFPP are also labeled as dynamic air removal (DAR) cycles.

[0082] A tabular representation of exemplary steam sterilization cycles in which the biological indicators of the present invention can be used is shown below:

[0083]

[0084] In general, the sterilization process includes placing the biological indicators of the present disclosure in a sterilizer. In some embodiments, the sterilizer includes a sterilization chamber that can be sized to hold a plurality of articles to be sterilized, and can be equipped with components to exhaust air and / or other gases from the chamber and components to add steam to the chamber. The self-contained biological indicators can be positioned in the most difficult to sterilize area of the sterilizer. Alternatively, the biological indicators can be positioned in a process challenge device to simulate the sterilization conditions, where steam can not be delivered as directly as in a more favorable sterilization situation.

[0085] Steam sterilant can be added to the sterilization chamber after at least a portion of any air or other gases present in the chamber is exhausted from the chamber. Alternatively, steam can also be added to the chamber without evacuating the chamber. A series of evacuation steps can be used to ensure that the steam sterilant reaches all desired areas within the chamber and contacts all desired articles to be sterilized, including the biological indicators.

[0086] The self-contained biological indicators are capable of determining the efficacy of one or more steam sterilization cycles selected from the power set of the following eleven cycles: 121 C gravity, 121 C pre-vacuum, 121 C SFPP, 132 C gravity, 132 C pre-vacuum, 132 C SFPP, 134 C pre-vacuum, 134 C SFPP, 135 C gravity, 135 C pre-vacuum, and 135 C SFPP, preferably in less than 1 hour.

[0087] Liquid medium

[0088] The self-contained biological indicators of the present disclosure include a liquid medium disposed in an openable container, the contents of which are in selective fluid communication with a compartment. Suitable containers include, for example, glass ampoules 18 described in U.S. Patent No. 3,661,717; inner containers 48 described in U.S. Patent No. 5,223,401; inner compartments 18 as well as inner containers 48 and 78 described in U.S. Patent No. 6,623,955; and frangible containers 120 described in U.S. Patent Application Publication No. 2013 / 0302849.

[0089] The liquid medium can contain one or more enzyme substrates mentioned herein. In certain embodiments, the enzyme substrate is 4-methylumbelliferyl-a-D-glucoside (MUG). In some embodiments, the liquid medium can also optionally include a nutrient composition that facilitates germination and / or outgrowth of the test microorganism. In some preferred embodiments, the solvent of the liquid medium is water.

[0090] Suitable nutrients can be initially provided in the housing in a dry form (e.g., a powder form, a tablet form, a caplet form, a capsule form, a film or coating, entrapped in a bead or other carrier, another suitable shape or configuration, or combinations thereof). When combined with the liquid medium (e.g., when the biological indicator is actuated), the nutrients can contact the test microorganism and facilitate growth of any live test microorganisms that remain in the housing after the biological indicator is exposed to a sterilization process.

[0091] The nutrients can include one or more sugars, including but not limited to glucose, fructose, dextrose, maltose, trehalose, cellobiose, and the like or combinations thereof. Alternatively, the nutrients can include complex media such as peptone, tryptone, vegetable peptone, yeast extract, soybean casein digest, other extracts, hydrolysates, and the like or combinations thereof. In other embodiments, the nutrients comprise a combination of one or more complex media components and other specific nutrients. The nutrients can also include salts, including but not limited to sodium chloride, potassium chloride, calcium chloride, and the like or combinations thereof. In some embodiments, the nutrients can also include at least one amino acid, including but not limited to at least one of methionine, phenylalanine, alanine, tyrosine, and tryptophan.

[0092] A liquid medium, as part of a self-contained, self-contained biological indicator; optionally containing nutrients, enzyme substrates, and other components; is typically present throughout the sterilization process, but remains segregated in an actuatable container and inaccessible to the test microorganisms until the biological indicator is actuated. After the sterilization process is complete and the biological indicator is used to determine the efficacy of the sterilization, the liquid medium is placed in contact with the test microorganisms and nutrients, creating a mixture. In the present disclosure, placing the liquid medium in contact with the test microorganisms includes activating the actuatable container so that the liquid medium is released and contacts the test microorganisms. This process can include mixing the liquid medium with the test microorganisms, such as manually or mechanically shaking the housing of the biological indicator to thoroughly mix the liquid culture with the test microorganisms.

[0093] In the present disclosure, the process of bringing the test microorganisms and liquid medium together is referred to as "activation" of the biological indicator. That is, the term "activation" and variations thereof, when used in relation to a biological indicator, generally refers to bringing one or more test microorganisms (e.g., spores) together with a liquid medium (including, for example, a nutrient medium and enzyme substrate for the test microorganisms of interest). For example, a biological indicator containing a liquid medium can be described as having been "activated" when an actuatable container within the biological indicator is at least partially fractured, perforated, punctured, crushed, broken, ruptured, etc., such that the medium has been placed in fluid communication with the test microorganisms. In other words, a biological indicator has been activated when the test microorganisms have contacted a liquid medium that was previously housed separately from the test microorganisms.

[0094] In some preferred embodiments, the mixture created by the mixing of the liquid medium with the test microorganisms after activation remains segregated within the housing of the biological indicator after the sterilization cycle has been completed, and no additional reagents or components are added to it during or after activation. If at least one of the test microorganisms is alive and grows, the enzymes produced by the test microorganisms can catalyze the cleavage of the enzyme substrate, which can produce a compound that is capable of fluorescent detection. This means that the same solution in the same container (housing) is used for three separate events: (a) test microorganism germination / growth (if the test microorganism is alive), (b) enzymatic cleavage of the enzyme substrate, producing a compound that is capable of fluorescent detection, and (c) fluorescent detection of the compound that is capable of fluorescent detection.

[0095] In some embodiments, the liquid medium can include a buffered solution, such as the buffered solutions described in U.S. Patent Application No. 62 / 964,369, entitled “SELF-CONTAINED BIOLOGICAL INDICATOR WITH SALT COMPOUND,” filed January 22, 2020; which is incorporated herein by reference in its entirety. The ionic conditions of the buffered solution should be such that the enzyme and enzyme substrate, if present in the biological indicator, are not affected. In some embodiments, the buffered solution is used as part of the liquid medium, such as a phosphate buffer (e.g., a 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 to be suitable for sterilization. If a fluorescent enzyme substrate and a chromogenic enzyme substrate are used as part of the biological indicator, the buffer suitable for use in the present biological indicator should be compatible with the enzyme substrates.

[0096] The concentration of the enzyme substrate, if present in the liquid medium prior to or after activation of the biological indicator, depends on the properties of the particular substrate and enzyme, the amount of enzyme product that must be generated for visual or instrumental detection, and the amount of time one is willing to wait in order 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 for a period of about eight hours after the sterilization cycle, such that at least 10 -8 moles of enzyme modification product are produced. In the case where the enzyme substrate is a 4-methylumbelliferyl derivative, the present inventors have found that its concentration in the aqueous buffered solution is preferably between about 10 -5 and 10 -3 moles.

[0097] In some embodiments, the biological indicator can include an additional indicator compound (in addition to the enzyme substrate that can produce a fluorescently detectable compound) that can facilitate detection of another metabolic activity of the test microorganism (e.g., spores). This additional metabolic activity can also be an enzyme activity. Non-limiting examples of indicator compounds include chromogenic enzyme substrates (e.g., observable in the visible spectrum), pH indicators, redox indicators, chemiluminescent enzyme substrates, dyes, and combinations of any two or more of the foregoing indicator compounds.

[0098] In some embodiments, the additional indicator is a pH indicator that produces a color change upon a decrease in pH, thereby indicating growth of the test microorganism. In some embodiments, the pH indicator is bromocresol purple. The pH indicator can be used to detect a second biological activity such as fermentation of a carbohydrate to an acidic end product (indicative of survival of the test microorganism), and an enzymatic biological activity such as alpha-D-glucoside enzyme activity. For example, these activities can be indicative of the presence or absence of viable test microorganisms after the biological indicator has been exposed to a sterilization process. For example, bromocresol purple can be used in the aqueous mixture at a concentration of about 0.03 g / L. 4-methylumbelliferyl-alpha-D-glucoside can be used in the aqueous mixture, for example, at a concentration of about 0.05 to about 0.5 g / L (such as 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).

[0099] Wax composition wicking member

[0100] The self-contained biological indicator of the present disclosure optionally includes a wax composition wicking member. The function of the wax composition wicking member is to absorb the molten wax composition and / or wick the wax composition away from the test microorganism when the biological indicator is exposed to a steam sterilization process, and to isolate the wax from the test microorganism when the test microorganism is contacted with the liquid medium and nutrient composition to detect viable test microorganism in the biological indicator, if present, after the biological indicator has been exposed to a sterilization process. In certain embodiments in which the test microorganism encapsulated in a wax composition is affixed to a substrate (e.g., at least one wall of a carrier or housing as described herein), the wax composition wicking member can be positioned in contact with the carrier or wall, respectively, so as to wick the wax away when molten. Additionally or alternatively, the wax composition wicking member can be positioned in contact with the wax so that the wax can flow into or onto the wicking member when molten.

[0101] Materials for wicking (e.g., by capillary action) molten wax composition away from a source (e.g., a fibrous material such as filter paper) are known in the art of time-temperature dosimeters (see, e.g., U.S. Patent No. 9,301,258; which is incorporated by reference herein in its entirety. Materials suitable for use as the wax composition wicking member of the present disclosure include, for example, paper (e.g., a filter paper strip).

[0102] Method of evaluating efficacy of a sterilization process

[0103] In another aspect, the present disclosure provides a method for determining efficacy of a sterilization process. The method includes positioning a sterilization process biological indicator in a sterilization chamber. The biological indicator includes a plurality of test microorganisms at least partially encapsulated by a wax composition. As used with reference to the methods of the present disclosure, "sterilization process biological indicator" is used broadly to include an article that contains or comprises a plurality of test microorganisms and is intended to validate the efficacy of a moist heat sterilization process. The sterilization process biological indicator includes, for example, a container that houses a substrate (e.g., a strip, test sample, bead, or thread made of a material known in the art of biological indicators for carrying test microorganisms in a sterilization process) having sterilization process test microorganisms affixed thereto, or an article (e.g., a bead) comprising the foregoing wax composition and test microorganisms encapsulated thereby.

[0104] The wax composition in the sterilization process biological indicator used in the methods of the present disclosure comprises a long chain (greater than C22) straight chain or branched chain alkyl or alkenyl alcohol, wherein the alkyl or alkenyl alcohol is not linked to a short chain alkyl or aryl (C1-C6) group by an ester or ether linkage, wherein the alkyl or alkenyl alcohol is not linked to a long chain (C8-C36) alkyl or alkenyl by an ester or ether linkage, wherein the alkyl or alkenyl alcohol is not linked to a straight chain or branched chain long chain (C8-C36) alkyl or alkenyl amine or acid by an amide linkage. The wax composition has a melting point of 78 °C to 120 °C.

[0105] In certain embodiments, positioning the sterilization process biological indicator in the sterilization chamber includes positioning the biological indicator such that the wax composition will flow away from the test microorganisms when it reaches its melting point. For example, if the test microorganisms are disposed on a carrier (such as Figure 4A As shown, if a first edge of the carrier is positioned higher in the biological indicator than a second edge, then when the wax composition melts, gravity will tend to draw the wax composition to the second edge (and away from at least some of the test microorganisms).

[0106] The method further includes contacting the biological indicator with moist heat at a temperature of at least 121 °C while the indicator is positioned in the sterilization chamber. In certain embodiments, contacting the biological indicator with moist heat at a temperature of at least 121 °C can include processing the biological indicator in an automatic steam sterilizer using a preprogrammed sterilization cycle selected from the power set of the following eleven cycles: 121 C gravity, 121 C pre-vacuum, 121 C SFPP, 132 C gravity, 132 C pre-vacuum, 132 C SFPP, 134 C pre-vacuum, 134 C SFPP, 135 C gravity, 135 C pre-vacuum, and 135 C SFPP.

[0107] After contacting the biological indicator with moist heat, the method includes contacting the test microorganism with a detection medium. The detection medium typically comprises an aqueous liquid comprising reagents that facilitate detection of the live test microorganism. The reagents can be any reagents known in the art that are in effective amounts to propagate the test microorganism to a detectable extent (e.g., by turbidity) or to perform one or more reactions (e.g., enzyme catalyzed reactions) to a detectable extent (e.g., by colorimetric, fluorescent, or chemiluminescent detection methods).

[0108] In certain embodiments, contacting the test microorganism with the detection medium includes adding (e.g., by pipette) the detection medium to the housing or other container in which the test microorganism is disposed. In certain embodiments, contacting the test microorganism with the detection medium includes activating the biological indicator (e.g., by opening a frangible container containing the medium in the biological indicator) to cause contact between the medium, detection reagents, and test microorganism. Optionally, after placing the test microorganism in liquid contact with the detection medium, the components can be mixed (e.g., by manual or mechanical agitation or vortexing).

[0109] After contacting the test microorganism with the detection medium, the method includes incubating the indicator at a predetermined temperature for a period of time sufficient to detect the presence of one of the test microorganisms, if alive. The predetermined temperature can be any suitable incubation temperature described herein for the test microorganism and / or its enzyme activity. The period of time can be any suitable incubation period known for detection of the microorganism or its enzyme activity. In certain embodiments, the specified period of time is less than 8 hours, in some embodiments, less than 1 hour, in some embodiments, less than 30 minutes, in some embodiments, less than 15 minutes, in some embodiments, less than 5 minutes, and in some embodiments, less than 1 minute. In other embodiments, suitable incubation times for the biological indicators of the present disclosure are 10 minutes to 4 hours, or 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.

[0110] During and / or after incubating the mixture for the period of time, the viability of the at least one test microorganism in the biological indicator can be detected using procedures known in the art, including visual detection and / or automated detection. Detecting a fluorescent product of an enzyme reaction, for example, includes directing electromagnetic radiation (e.g., radiation within the ultraviolet spectrum of electromagnetic energy) into the mixture and detecting electromagnetic radiation (e.g., radiation within the ultraviolet spectrum or visible spectrum of electromagnetic energy) emitted by the fluorescent product in the mixture, as described herein. In certain embodiments, detecting electromagnetic radiation emitted by the fluorescent product includes using an automated detector (e.g., an automated reader as described herein) to detect the electromagnetic radiation.

[0111] In any of the above embodiments, the method can further include positioning the article to be sterilized in the sterilization chamber prior to contacting the indicator with the steam sterilization process.

[0112] In any of the embodiments of the method, detecting the fluorescent product includes detecting an amount of fluorescence emitted by the fluorescent product. In any embodiment, the amount of fluorescence detected can be compared to a threshold amount. In any embodiment, a first amount of fluorescence detected after a first specified period of time can be compared to a second amount of fluorescence detected after a second specified period of time. In certain embodiments, detecting at least a threshold amount of the fluorescent product or detecting a threshold amount of the test microorganism (e.g., by turbidity) indicates a lack of efficacy of the sterilization process.

[0113] In any of the embodiments of the method, positioning the sterilization process biological indicator in the sterilization chamber can include positioning a sterilization process biological indicator including a housing having the test microorganism therein. In these embodiments, contacting the test microorganism with the detection medium includes contacting the test microorganism with the detection medium inside the housing. In these embodiments, wherein contacting the test microorganism with the detection medium can include contacting the test microorganism with an aqueous medium including a detection reagent. The detection reagent can be selected from the group consisting of a nutrient, a pH indicator, a redox indicator, a fluorescent enzyme substrate, a chromogenic enzyme substrate, and a combination of any two or more of the foregoing detection reagents.

[0114] To detect a detectable change caused by a live test microorganism, the biological indicator can be measured immediately after the liquid medium and test microorganism have been combined to obtain a baseline reading. Thereafter, any detectable change from the baseline reading can be detected. The biological indicator can be monitored and measured continuously or intermittently. In some embodiments, a portion or all of an incubation step can be performed prior to measuring the detectable change. In some embodiments, the incubation can be performed at one temperature (e.g., at 37°C, at 50-60°C, etc.) and the measurement of the detectable change can be performed at another, different temperature (e.g., at room temperature, 25°C, or 37°C). In other embodiments, the incubation and fluorescence measurement can be performed at the same temperature.

[0115] The readout time of the biological indicator (i.e., the time used to determine the effectiveness of the sterilization process) can be less than 8 hours in some embodiments, less than 1 hour in some embodiments, less than 30 minutes in some embodiments, less than 15 minutes in some embodiments, less than 5 minutes in some embodiments, and less than 1 minute in some embodiments. In other embodiments, the readout time of the 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. The detection of fluorescence above the baseline reading that would indicate the presence of live spores (i.e., that the sterilization process failed) can be performed according to any method known in the art, including area under the curve (in a plot of time versus fluorescence intensity), monitoring the slope change of the curve, using a threshold value of fluorescence, etc., or a combination of two or more of these techniques.

[0116] One of the advantages of the biological indicators of the present disclosure is that a single type can be used for a variety of steam sterilization conditions. The following working examples show that a single type of biological indicator can be used for all of the following steam sterilization cycles: 121 °C gravity, 121 °C prevac, 121 °C SFPP, 132 °C gravity, 132 °C prevac, 132 °C SFPP, 134 °C prevac, 134 °C SFPP, 135 °C gravity, 135 °C prevac, and 135 °C SFPP. To this end, the biological indicator can be used for any subset of cycles selected from the above set. That is, a single biological indicator is capable of determining the efficacy of one or more sterilization cycles selected from the power set of 121 °C gravity, 121 °C prevac, 121 °C SFPP, 132 °C gravity, 132 °C prevac, 132 °C SFPP, 134 °C prevac, 134 °C SFPP, 135 °C gravity, 135 °C prevac, and 135 °C SFPP.

[0117] Kit

[0118] In another aspect, the present disclosure provides a kit useful for determining the efficacy of a sterilization process. The kit can include a plurality of test microorganisms, wherein the test microorganisms are at least partially encapsulated by a wax composition, and instructions for using the test microorganisms to assess the efficacy of a steam sterilization process. The wax composition comprises a long chain (greater than C22) straight chain or branched alkyl or alkenyl alcohol, wherein the alkyl or alkenyl alcohol is not linked to a short chain alkyl or aryl (C1-C6) group by an ester or ether linkage, wherein the alkyl or alkenyl alcohol is not linked to a long chain (C8-C36) alkyl or alkenyl by an ester or ether linkage, wherein the alkyl or alkenyl alcohol is not linked to a straight chain or branched long chain (C8-C36) alkyl or alkenyl amine or acid by an amide linkage. The wax composition has a melting point between 78 °C and 120 °C.

[0119] In any embodiment, the kit can further include a detection reagent selected from the group consisting of a nutrient, a pH indicator, a redox indicator, a fluorescent enzyme substrate, a chromogenic enzyme substrate, and a combination of any two or more of the foregoing detection reagents. In any of the above embodiments, the kit can further include a housing sized to contain the test microorganisms or the detection reagent. In certain embodiments of the kit, the test microorganisms and the detection reagent can be disposed in the housing. In certain embodiments, the wax composition has a melting point of at least about 78 °C, at least about 91 °C, at least about 99 °C, or at least about 105 °C.

[0120] In any embodiment of the kit, the test microorganisms are completely encapsulated by the wax composition. In any embodiment, the kit can include any embodiment of the self-contained, packaged biological indicator of the present disclosure.

[0121] Examples

[0122] Example 1-4. Assembly of self-contained sterilization process biological indicators .

[0123] The wax compositions used in the examples are listed in Table 1. The wax compositions were obtained from Baker Hughes (Houston, TX). The wax compositions were heated just above the melting point, after which they were deposited on the spore strips as described below.

[0124] Table 1

[0125] Example number Wax name Melting point (°C) 1 UNILIN TM 350 alcohol 78 2 UNILIN TM 425 alcohol 91 3 UNILIN TM 550 alcohol 99 4 UNILIN TM 700 alcohol 105 Control None N / A

[0126] 3M TM ATTEST TMUltrafast readout biological indicators (part number 1492V) were obtained from 3M Company (St. Paul, MN). Those self-contained biological indicators were assembled as shown in Figures 4-6 of U.S. Patent Application Publication No. US 2014 / 0349335, the entirety of which is incorporated herein by reference. The cap of the biological indicator was removed and the contents (glass ampoule, insert, and spore reservoir) were removed. The interior of the tube and the exterior of the glass ampoule and insert were rinsed with sterile deionized water. 100 microliters of molten wax composition was added to the spore reservoir through a micropipette, completely covering the dried spores on the concave surface of the spore reservoir. The biological indicator was reassembled. Five commercially available ATTEST 1492V biological indicators were used as controls.

[0127] Example 5: Detection of viable spores after exposure to a 135°C steam sterilization process .

[0128] Each of the biological indicators of Examples 1-4 and five of the five control biological indicators were placed into the sterilization chamber of a steam resistance meter (H&W 101 Steam Resistance Meter, available from H&W Technology LLC, Rochester, NY) and exposed to a dynamic air removal steam sterilization cycle (135°C for 3.5 minutes). After the exposure cycle, all of the biological indicators were activated by breaking the glass ampoule containing the detection media in the biological indicator. After the biological indicators were activated, they were incubated in a biological indicator auto-reader available from 3M Company (St. Paul, MN). The auto-reader indicated that a positive fluorescence was detected in each of the biological indicators within 24 minutes of incubation. After incubation at 60°C for 7 days, the detection media visually observed a color change due to acid production in the biological indicator. A pH change (as observed by a change in pH indicator from purple to yellow) was observed in each of the biological indicators. The data showed that the biological indicators with the wax composition covering the spores had the same response (fluorescence positive, growth positive) to the 135°C steam sterilization cycle as the control biological indicators.

[0129] Example 6: Detection of viable spores after exposure to a 121°C steam sterilization process .

[0130] Each of the biological indicators of Examples 1-4 and five of the five control biological indicators were placed into the sterilization chamber of a steam resistance tester (H&W 101 Steam Resistance Tester, available from H&W Technologies, LLC, Rochester, NY) and exposed to a dynamic air removal steam sterilization cycle (121 °C for 10 minutes). The biological indicators were placed on a shelf with the cover on top. In this position, the spore reservoir was tilted relative to the vertical axis of the biological indicator. Thus, it was noted after the biological indicators were processed in the steam resistance tester that at least some of the wax composition had dripped from the spore reservoir and into the bottom of the housing.

[0131] After the exposure cycle, all of the biological indicators were activated by breaking the glass ampoule containing the detection media in the biological indicator. After activation, the biological indicators were incubated in a biological indicator auto reader available from 3M Company (St. Paul, MN) and tested for fluorescence (enzyme-based early readout) and growth (spore survival) parameters as described in Example 5. The results are shown in Table 2. The data indicate that the resistance of the spores (as indicated by both fluorescence measurements and growth measurements) generally increased in the biological indicators including the wax compositions having a melting point greater than or equal to 91 °C. The biological indicators having the wax composition with a melting point of 78 °C showed higher resistance to spore viability for the 121 °C process, but not higher resistance to spore-related enzyme activity.

[0132] Table 2. Effect of a 121 °C steam sterilization cycle on the rapid (fluorescence) readout and 7-day growth readout in biological indicators including spores encapsulated by a wax composition. Five biological indicators were tested for each condition.

[0133] Biological indicator Number of fluorescence positives Number of growth positives Control 0 / 5 0 / 5 Example 1 0 / 5 4 / 5 Example 2 3 / 5 3 / 5 Example 3 4 / 5 5 / 5 Example 4 5 / 5 5 / 5

[0134] Various modifications and alterations to this application will become apparent to those skilled in the art from the disclosure thereof. It is intended that all such alterations and modifications be considered equivalents thereof. It should be understood that this application is not to be unduly limited to illustrative embodiments and examples that are presented herein and / or that have been set forth in the foregoing description / It is intended that the scope of the application should only be limited by the claims.

Claims

1. A complete set of biological indicators for sterilization processes, comprising: A housing having at least one liquid-impermeable wall forming an opening to a compartment; Multiple test microorganisms are disposed in the housing, wherein the test microorganisms are encapsulated, and at least a portion of the test microorganisms are encapsulated by a wax composition; The wax composition comprises long-chain straight-chain or branched alkyl or alkenyl alcohols with a C22 or greater; The alkyl or alkenyl alcohol is not connected to a short-chain alkyl or aryl group of C1-C6 via an ester bond or an ether bond; The alkyl or alkenyl alcohols thereon are not connected to long-chain alkyl or alkenyl groups of C8-C36 via ester or ether bonds; The alkyl or alkenyl alcohol is not connected to a straight-chain or branched C8-C36 long-chain alkyl or alkenyl amine or acid via an amide bond; The wax composition is configured to increase the resistance of the test microorganisms to the steam sterilization process and has a melting point of at least 91°C; A liquid culture medium is disposed in an openable container, the contents of which are in selective fluid communication with the compartment; and A nutrient composition that promotes the germination and / or growth of the test microorganism, wherein the nutrient composition is disposed in the container or the shell; The housing includes an opening that allows sterilizing agents to enter the housing from the outside.

2. The complete sterilization process biological indicator according to claim 1, wherein the container is disposed in the housing.

3. The complete sterilization process biological indicator according to claim 1, wherein the nutrient composition is disposed in the container.

4. The complete sterilization process biological indicator according to claim 1 further includes a detection reagent disposed in the housing.

5. The complete sterilization process biological indicator according to claim 4, wherein the detection reagent is disposed in the container.

6. The complete sterilization process biological indicator according to claim 1, wherein the test microorganism is immobilized to a carrier, and wherein the test microorganism is encapsulated by the wax composition and the carrier.

7. The complete sterilization process biological indicator according to claim 6, wherein the carrier is moisture-proof.

8. The complete sterilization process biological indicator according to claim 1, wherein the test microorganism is fixed to a portion of the wall of the housing, wherein the test microorganism is encapsulated by the wax composition and a portion of the wall of the housing.

9. The complete sterilization process biological indicator according to claim 1, wherein the melting point of the wax composition is at least 99°C.

10. The complete sterilization process biological indicator according to claim 9, wherein the melting point of the wax composition is at least 105°C.

11. The complete sterilization process biological indicator according to claim 1, further comprising a wax composition wicking member disposed in the housing.

12. The complete sterilization process biological indicator according to claim 11, wherein the wax composition wicking member contacts the carrier.

13. The complete sterilization process biological indicator according to claim 11, wherein the wax composition wicking member contacts the wall of the housing.

14. The complete sterilization process biological indicator according to claim 11, wherein the wax composition wicking member contacts the wax composition.

15. The complete sterilization process biological indicator according to claim 11, wherein the wax composition wicking member comprises a fibrous material.

16. A method for determining the effectiveness of a sterilization process, the method comprising: The biological indicator for the sterilization process is positioned in the sterilization chamber; The bioindicator includes a plurality of encapsulated test microorganisms, at least a portion of which is encapsulated by a wax composition; The wax composition comprises long-chain straight-chain or branched alkyl or alkenyl alcohols with a C22 or greater; The alkyl or alkenyl alcohol is not connected to a short-chain alkyl or aryl group of C1-C6 via an ester bond or an ether bond; The alkyl or alkenyl alcohols thereon are not connected to long-chain alkyl or alkenyl groups of C8-C36 via ester or ether bonds; The alkyl or alkenyl alcohol is not connected to a straight-chain or branched C8-C36 long-chain alkyl or alkenyl amine or acid via an amide bond; The wax composition is configured to increase the resistance of the test microorganisms to the steam sterilization process and has a melting point of at least 91°C; When the indicator is positioned in the sterilization chamber, the biological indicator is exposed to moist heat at a temperature of at least 121°C. After the bioindicator is exposed to the humid heat, the test microorganism is brought into contact with the detection medium; After the test microorganism is brought into contact with the detection medium, the indicator is incubated at a predetermined temperature for a period of time sufficient to detect the presence of one of the test microorganisms, if the test microorganism is alive.

17. The method of claim 16, wherein positioning the sterilization process bioindicator in the sterilization chamber comprises positioning the sterilization process bioindicator comprising a housing having the test microorganism therein, wherein contacting the test microorganism with the detection medium comprises contacting the test microorganism with the detection medium inside the housing.

18. The method of claim 17, wherein contacting the test microorganism with the detection medium comprises contacting the test microorganism with an aqueous medium containing a detection reagent selected from: nutrients, pH indicators, redox indicators, luciferase substrates, chromogenic enzyme substrates, and any two or more of the aforementioned detection reagents.

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