WO2020136608A1

WO2020136608A1 - Instant read-out biological indicator

Info

Publication number: WO2020136608A1
Application number: PCT/IB2019/061382
Authority: WO
Inventors: Joshua D. Erickson; Francois AHIMOU
Original assignee: 3M Innovative Properties Company
Priority date: 2018-12-27
Filing date: 2019-12-26
Publication date: 2020-07-02

Abstract

A self-contained sterilization process biological indicator is provided. The indicator includes a cuvette having at least one liquid-impermeable wall that forms an opening into a compartment, a predetermined number of sterilization process-resistant spores disposed in the compartment, a liquid medium disposed in the compartment, and a nucleic acid-interacting dye disposed in the compartment, with the proviso that the self-contained sterilization process biological indicator does not include an effective amount of germination medium and/or microbial growth medium. The opening is part of a pathway that permits passage of a sterilant gas into the compartment. The nucleic acid-interacting dye is fluorescent when bound to DNA or RNA. A method of using the self-contained sterilization process biological indicator to determine the effectiveness of a sterilization process is also provided.

Description

INSTANT READ-OUT BIOLOGICAL INDICATOR

Background

[0001] Monitoring the effectiveness of processes used to sterilize equipment such as medical devices, instmments and other non-disposable articles is routinely carried out in health care as well as various industrial settings. An effective sterilization process is expected to completely destroy all viable microorganisms, including microorganism forms such as viruses and spores. Hospitals, as a standard practice to assay the lethality of a sterilization process, include a sterility indicator with a batch of articles to be sterilized. Both biological and chemical sterility indicators have been used.

[0002] A standard type of biological sterility indicator includes a known quantity of test microorganisms, for example Geobacillus stearothermophilus (formerly Bacillus stearothermophilus) or Bacillus atrophaeus (formerly Bacillus subtilis) spores, which are many times more resistant to a sterilization process than most contaminating organisms. After the indicator is exposed to the sterilization process, the spores are incubated in nutrient medium to determine whether any of the spores survived the sterilization process, with spore growth indicating that the sterilization process was insufficient to destroy all of the microorganisms. In another example, after being subjected to a sterilization process, the activity of an enzyme, which can be correlated with spore viability, is determined. Although advances have been made; the time required for determining with certainty whether the sterilization process was effective can be undesirably long.

[0003] Available chemical sterility indicators can be read immediately at the end of the sterilization process. However, the results indicate only that a particular condition was present, such as the presence of a particular chemical or a temperature for a certain period of time.

[0004] It is generally considered that the response of living organisms to all conditions actually present is a more direct and reliable test for how effective a sterilization process is in achieving sterilization. Accordingly, there is a continuing need for biological sterility indicators which can indicate the effectiveness of a sterilization process without an excessive delay after completion of the sterilization process.

Summary

[0005] It is now known that a self-contained sterilization process biological indicator, with everything therein needed to assess the effectiveness of a sterilization process for inactivating test microorganisms (e.g., spores) without the need for including an effective amount of germination medium and/or microbial growth medium in the self-contained sterilization process biological indicator. Advantageously, this discovery provides its user with the ability to assess the viability or capturability of the test microorganisms without the need for actually germinating and/or growing the test microorganisms. Thus, the aforementioned self-contained sterilization process biological indicator can be used in a method of determine effectiveness of a sterilization process. Because the method does not require any germination or growth of the test microorganism, the effectiveness of the sterilization process can be determined almost immediately after the sterilization process has been completed. Moreover, it is now known that a self-contained sterilization process biological indicator can be used in a method to assess the effectiveness of a sterilization process by measuring a parameter associated with killed spores, rather than a parameter associated with spores that have survived the sterilization process.

[0006] In one aspect, the present disclosure provides a self-contained sterilization process biological indicator. The biological indicator can comprise a cuvette having at least one liquid- impermeable wall that forms an opening into a compartment, a predetermined number of sterilization process-resistant spores disposed in the compartment, a liquid medium disposed in the compartment, and a nucleic acid-interacting dye disposed in the compartment, with the proviso that the self- contained sterilization process biological indicator does not include an effective amount of germination medium and/or microbial growth medium. The opening is part of a pathway that permits passage of a sterilant gas from outside the cuvette into the compartment. The nucleic acid-interacting dye is fluorescent when bound to DNA or RNA.

[0007] In certain of the above embodiments, the nucleic acid-interacting dye can be disposed in the aqueous liquid medium. In certain of the above embodiments, the liquid medium can be disposed in a container, wherein the container is disposed in the compartment. In certain of the above

embodiments, the spores can be disposed on a surface in a substantially water-free layer, wherein the surface is disposed in the compartment.

[0008] In any of the above embodiments, the biological indicator further can comprise an enhancer reagent, wherein the enhancer reagent contacts the spores. The enhancer reagent can be selected from the group consisting of glycerol, sucrose, trehalose, polyvinyl pyrrolidone, and a combination of any two or more of the foregoing enhancer reagents.

[0009] In certain of the above embodiments, the nucleic acid-interacting dye can be selected from the group consisting of Acridine Orange, SYTO 9, SYTO 16, DAPI, propidium iodide, and a combination of any two or more of the foregoing nucleic acid-interacting dyes. In any of the above embodiments, the spores can be produced by a species of microorganisms selected from the group consisting of Geobacillus stearothermophilus, Bacillus atrophaeus, Bacillus megaterium,

Clostridium sporogenes, Bacillus coagulans, and a combination of any two or more of the foregoing species. In any of the above embodiments, the biological indicator further can comprise a collisional quenching component disposed in the compartment.

[0010] In another aspect, the present disclosure provides a method. The method can be used to determine effectiveness of a sterilization process. The method can comprise positioning the self- contained sterilization process biological indicator of any one of the above embodiments of the self- contained sterilization process biological indicator in a sterilization chamber. The method further can comprise, while the indicator is positioned in the sterilization chamber, exposing the indicator to a sterilant gas; contacting the spores with the nucleic acid-interacting dye; contacting the spores with the liquid medium; after the exposing the indicator to the sterilant gas, measuring a first fluorescence intensity emitted by the nucleic acid-interacting dye in the indicator; and comparing the first fluorescence intensity to a reference fluorescence intensity to determine whether the exposing the indicator to the sterilant gas was effective to kill all of the spores; with the proviso that the method does not include, before measuring the first fluorescence intensity, incubating the spores in the indicator with a growth medium or germination medium for a sufficient period of time to cause germination or growth of the spores. In certain embodiments of the above method, contacting the spores with the nucleic acid-interacting dye can comprise contacting the spores with the nucleic acid interacting dye while exposing the indicator to the sterilant gas. In certain embodiments of the method, contacting the spores with the nucleic acid-interacting dye can comprise contacting the spores with the nucleic acid-interacting dye after the exposing the indicator to the sterilant gas.

[0011] In certain of the above embodiments of the method wherein the liquid medium is contained in an container that is disposed in the compartment and wherein the container is impermeable to the liquid medium, prior to the exposing the self-contained sterilization process biological indicator to the sterilant gas, the method further can comprise disintegrating the container to contact the spores with the liquid medium. In certain of the above embodiments of the method wherein the liquid medium is contained in an container that is disposed in the compartment and wherein the container is impermeable to the liquid medium, after the exposing the self-contained sterilization process biological indicator to the sterilant gas, the method further can compnse disintegrating the container to contact the spores with the liquid medium.

[0012] In any of the above embodiments, the method further can comprise measuring an amount of absorbance and/or scattering of electromagnetic radiation when the electromagnetic radiation is directed through the liquid medium in the cuvette. In certain embodiments, the method further can comprise calculating a ratio of the first fluorescence intensity to the measured amount of electromagnetic radiation absorbance and/or scattering.

[0013] In any of the above embodiments, the method further can comprise prior to the exposing the indicator to the sterilant gas, positioning an article to be sterilized in the sterilization chamber.

[0014] In yet another aspect, the present disclosure provides a composition. The composition can comprise a plurality of sterilization process-resistant microbial spores and a nucleic acid-interacting dye, wherein the composition is substantially water-free. In any embodiment of the composition, the plurality of sterilization process-resistant microbial spores can comprise a predefined number of spores.

[0015] In any embodiment of the above composition, the composition further can comprise an enhancer reagent that enables longer-term viability of the spores during storage, that facilitates resuspension of the spores into an aqueous medium, or that facilitates penetration of the nucleic acid interacting dye into the spores while the spores are contacted with a sterilant gas.

[0016] Additional details of these and other embodiments are set forth in the accompanying drawings and the description below. Other features, objects and advantages will become apparent from the description and drawings, and from the accompanying claims.

[0017] Herein, the terms " biological sterilization process indicators ",“sterilization process biological indicator”,“sterilization process indicator”,“biological indicator”,“BI”,“indicator”,“self- contained biological indicator”, and“SCBI” are used interchangeably.

[0018] Also herein, in the written description and the claims, the phrases "substantially dry", "substantially water- free" or the like refer to a composition or a coating which has a water content no greater than about the water content of the dehydrated coating once it has been permitted to equilibrate with the ambient environment.

[0019] The numbers, E5, E6, and E7 are used interchangeably herein with 10⁵, 10⁶, and 10⁷, respectively.

[0020] The term "comprising" and variations thereof (e.g., comprises, includes, etc.) do not have a limiting meaning where these terms appear in the description and claims.

[0021] As used herein, "a", "an", "the," "at least one, "and "one or more" are used interchangeably, unless the context clearly dictates otherwise.

[0022] Also herein, the recitations of numerical ranges by endpoints include all numbers subsumed within that range (e.g., 500 to 7000 nm includes 500, 530, 551, 575, 583, 592, 600, 620, 650, 700, etc ).

[0023] The above summary of the present invention is not intended to describe each disclosed embodiment or every implementation of the present invention. The description that follows more particularly exemplifies illustrative embodiments.

Brief Description of Drawings

[0024] FIG. 1 is a partially-exploded, cross-sectional schematic view of one embodiment of a self- contained sterilization process biological indicator according to the present disclosure.

[0025] FIG. 2 is a top view of the self-contained sterilization process biological indicator of FIG. 1.

[0026] FIG. 3 is a partially-exploded, cross-sectional schematic view of an alternative embodiment of a self-contained sterilization process biological indicator according to the present disclosure.

[0027] FIG. 4 is a block diagram of one embodiment of a method of determining effectiveness of a sterilization process according to the present disclosure.

Detailed Description

[0028] As indicated above, biological sterilization process indicators are now provided which can detect an indication of viable spores almost immediately upon removal of the biological sterilization process indicators from a sterilization process. Advantageously, the near-immediate detection of an indication of viable spores (after removal of the BI from a sterilizer) eliminates undesirably-long waiting periods to determine whether a sterilization process was effective. In addition, the indication of viable spores present in the biological sterilization process indicators can be detected automatically, thereby eliminating a potential human error in deciding whether microbial growth or enzyme activity is present after the biological sterilization process indicators have been exposed to a sterilization process.

[0029] Methods of using the biological sterilization process indicators to determine the effectiveness of a sterilization process are also provided.

[0030] For certain embodiments, including any one of the biological indicator or method embodiments described herein, the at least one nucleic acid-interacting dye can interact with nucleic acids present in the spores, resulting in an increase in fluorescence intensity in the biological indicator. The interaction of the dye with the nucleic acids after the spores have been exposed to a sterilant gas provides an early indication of viable spores, if present, without a need for germination or growth of the spores.

[0031] For certain embodiments, including any one of the biological indicator or method embodiments described herein, the at least one nucleic acid-interacting dye can interact with nucleic acids present in the plurality of spores to produce a fluorescence intensity that corresponds to killed spores. Without being bound by theory, the investigators believe this is due to the ability of the dye to penetrate spores that have increased permeability (i.e., poor integrity of) the nonviable spores. This effect is not seen in vegetative cells. In contrast the SYT09 nucleic acid-interacting dye can penetrate both live and dead vegetative cells, as evidenced by the LIVE/DEAD® riacLight ^{1 M} Bacterial Viability Kit available from Molecular Probes, Inc.

(Eugene, OR). Selective interaction of the dye with nucleic acids present in the nonviable spores results in lower fluorescence when viable spores are present. Thus, an absence of viable spores in the biological indicator after exposure to a sterilant gas results in a correspondingly higher fluorescence intensity.

[0032] A number of sterilization process are presently known and in use, including, for example, exposure to steam, dry heat, gaseous or liquid agents such as ethylene oxide, hydrogen peroxide, peracetic acid, and ozone, and radiation. The plurality of sterilization process resistant spores may be selected according to the sterilization process to be used. Any spores may be used as long as they provide sufficient resistance to the sterilization process conditions, such that the spores are more resistant to the sterilization process conditions than most microorganisms encountered in natural contamination. For example, for a steam sterilization process, Gb.

stearothermophilus (formerly Bacillus stearothermophilus) may be used. In another example, for an ethylene oxide sterilization process, B. atrophaeus (formerly B subtilis) may be used. For certain embodiments, including any one of the above composition, indicator, and method embodiments, the plurality of sterilization process resistant spores is selected from the group consisting of Geobacillus stearothermophilus, Bacillus atrophaeus, Bacillus megaterium, Clostridium sporogenes, Bacillus coagulans, and a combination thereof. For certain of these embodiments, the plurality of sterilization process resistant spores is selected from the group consisting of Gb. stearothermophilus, B. atrophaeus, B. megaterium, and a combination thereof.

[0033] By way of example only, the present disclosure describes the microorganisms used in the biological sterilization indicator as being "spores;" however, it should be understood that the type of microorganism (e.g., spore) used in a particular embodiment of the biological sterilization indicator is selected for being highly resistant to the particular sterilization process contemplated. Accordingly, different embodiments of the present disclosure may use different microorganisms, depending on the sterilization process for which the particular embodiment is intended.

[0034] Using nucleic acid-interacting fluorescent dyes, the present biological indicators and methods can detect the presence of nucleic acids within the spores, without the need for germination and/or outgrowth. In any embodiment of the method disclosed herein, it is not necessary to use a sub-lethal concentration of nucleic acid-interacting dye because germination and/or outgrowth is not required to determine whether the microorganisms in the biological indicators of the present invention have been inactivated (e.g., killed).

[0035] In some, if not all, of the sterilization processes in use, an elevated temperature, for example, 50° C., 100° C., 121° C., 132° C., 134° C., or the like, is included or may be encountered in the process. Accordingly, for certain embodiments, including any one of the above biological indicator and method embodiments, the nucleic acid-interacting fluorescent dyes are stable at sterilization temperatures.

[0036] For certain embodiments, including any one of the above biological indicator and method embodiments, nucleic acid-interacting fluorescent dye is stable at a temperature up to at least 121° C. For certain of these embodiments, the nucleic acid-interacting fluorescent dye is stable at a temperature up to at least 132° C. For certain of these embodiments, the nucleic acid interacting fluorescent dye is stable at a temperature up to at least 134° C. For certain of these embodiments, the nucleic acid-interacting fluorescent dye is stable at a temperature up to at least 135° C. For certain of these embodiments, the nucleic acid-interacting fluorescent dye is stable when exposed to temperatures between 121-135° C for periods of time that are customary for sterilization processes.

[0037] For certain embodiments, including any one of the above biological indicator or method embodiments, the liquid medium is essentially free of any background fluorescence at emission and excitation wavelengths used to detect the fluorescence intensity of the nucleic acid interacting dye when it is interacting with nucleic acid in the biological indicator. This may provide an improved sensitivity because any background level of fluorescence occurring at the same wavelengths as the emission and excitation of the dye interacting with a nucleic acid, is minimized. [0038] For certain embodiments, including any one of the above biological indicator or method embodiments, the medium is essentially free of any nucleic acids other than nucleic acids present in the spores. This may provide an improved sensitivity because any baseline level of fluorescence resulting from the dye interacting with any nucleic acids not present in the spores is minimized.

[0039] The nucleic acid-interacting dye has a lower level of fluorescence at the emission wavelength (or wavelength range) when not interacting with a nucleic acid and a higher level fluorescence at this wavelength when interacting with a nucleic acid.

[0040] For certain embodiments, including any one of the above biological indicator or method embodiments, the nucleic acid-interacting fluorescent dye is a dye which interacts with DNA, RNA, or DNA and RNA. The interaction of the dye with DNA and RNA may be the same or different. The dye interacting with DNA may have a different excitation and/or emission maximum than the same dye interacting with RNA.

[0041] For certain embodiments, including any one of the above biological indicator or method embodiments, the nucleic acid-interacting fluorescent dye is a dye which interacts with the nucleic acids in a variety of ways know in the art, including intercalation, electrostatic attraction, charge interaction, hydrophilic-hydrophobic interaction, or a combination thereof. As indicated above, this interacting or binding of the dye with nucleic acids, which include total cellular nucleic acids, such as DNA, RNA (mRNA, rRNA, tRNA), and extrachromosomal nucleic acids, causes a relatively large increase in fluorescence from the dye. For certain of these embodiments, the nucleic acid-interacting fluorescent dye is selected from the group consisting of acridine orange, a substituted unsymmetrical cyanine dye, and salts thereof, and combinations thereof.

[0042] Acridine orange bound to a DNA has an excitation maximum at about 490 nm and an emission maximum at about 520 maximum, but when bound to an RNA about 530 nm and 620 nm, respectively. See Maclnnes, J. W and McClintock, M., Differences in Fluorescence Spectra of Acridine Orange-DNA Complexes Related to DNA Base Composition, Biopolymers.

Communications to the editor, Vol 9, Pages 1407-1411 (1970).

[0043] Suitable examples of substituted unsymmetrical cyanine dyes include dyes available under the trade name, SYTO (Invitrogen Corp., Carlsbad, Calif.). SYTO dyes may differ from each other, for example, in degree of permeability through intact spore membranes, amount of fluorescence intensity increase when bound to a nucleic acid, excitation and emission maxima, selectivity in binding to DNA and RNA, and binding affinity to DNA and RNA. See Tamok, Cytometry Part A, 73A, 477-479 (2008). Suitable nucleic acid-interacting dyes of the present disclosure include those nucleic acid-interacting dyes that are substantially excluded from viable spores (i.e., spores that are capable of germination to form vegetative cells that are capable of reproduction) but are not excluded from nonviable spores (e.g., spores that have been exposed to a lethal sterilization process. The determination of selective permeability of any given nucleic acid-interacting dye is easily performed by a person having ordinary skill in the art by separately contacting similar numbers of viable and nonviable spores of a given species with the nucleic acid-interacting dye in a microwell plate according to the method described in Example 2.

[0044] Suitable nucleic acid-interacting dyes include, but are not limited to Acridine Orange, SYT09, SYT016, DAPI, propidium iodide, and a combination of any two or more of the foregoing nucleic acid-interacting dyes. For certain embodiments, preferably the substituted unsymmetrical cyanine dye is SYT09 or SYTO 16.

[0045] Preferred nucleic acid-interacting dyes penetrate the spore coat and/or spore membrane substantially better after the spores have been exposed to a gas sterilant under conditions that result in the disruption and/or activation of substantially all of the spores. Without being bound by theory, it is believed the disruption of the spore coat and/or membrane facilitates penetration of the dye into the spore, thereby also facilitating interaction of the dye with nucleic acids disposed in and/or released from the disrupted spores.

[0046] The nucleic acid-interacting dyes can be used in a self-contained biological sterilization process indicator at concentrations of about 0.05 mM to about 50 mM.

[0047] In certain preferred embodiments, the nucleic acid-interacting dye differentially penetrates (and interacts with the nucleic acid in) inactivated (killed) spores relative to viable spores. Thus, the higher the number of spores inactivated by the sterilant gas, the greater the interaction between the nucleic acid-interacting dye and the nucleic acid in the spores. Accordingly, the higher the number of spores inactivated by the sterilant gas, the greater the intensity of fluorescent signal emitted by the dye-bound nucleic acid from the spores in the sterilization process indicator.

[0048] For certain embodiments, including any one of the biological indicator or method embodiments described herein, the biological indicator further comprises a collisional quenching component disposed in the compartment (e.g., in the liquid medium. Such components reduce the background fluorescence signal of free (unbound to nucleic acid) nucleic acid-interacting dye through collisional quenching. Examples of species known to collisionally quench fluorescence include organic compounds such as purines, pyrimidines, aliphatic amines, and nitroxides, certain ions, for example, nitrate anions and dissolved metal ions. Other species known to collisionally quench fluorescence are described, for example, in Principles of Fluorescence Spectroscopy, Chapter 9, Joseph R. Lakowicz, Plenum Press, 1983.

[0049] For certain embodiments, including any one of the biological indicator or method embodiments described herein, the biological indicator further comprises at least one reference dye disposed in the compartment (e.g., in the liquid medium). The reference dye does not bind to nucleic acids but responds similarly to nucleic acid-interacting dyes to changes in temperature or changes in the medium induced by spore germination and outgrowth, for example an increase or decrease in pH, ionic strength, or concentration change in metabolic by-products that alter the fluorescent signal. By monitoring the reference dye, signal from the nucleic acid-interacting dye binding to nucleic acid can be distinguished from signal produced from a change in temperature or a change in the media. The reference dye preferably fluoresces at a different wavelength than the nucleic acid-interacting dye.

[0050] As indicated above, the sterilization process indicator provided herein comprises a predetermined number of sterilization process resistant viable spores disposed in a compartment of a cuvette. The predetermined number of spores can comprise at least 10, at least 10², at least 10³, at least 10⁴, at least 10⁵, at least 10⁶, or at least 10⁷ viable spores. In certain embodiments, of the present disclosure has a predetermined number of about 10⁶ viable spores disposed therein.

[0051] In certain embodiments, the spores may be disposed on a carrier. For certain embodiments, the carrier is a sheet material such as paper, woven cloth, nonwoven cloth, plastic, a polymeric material, a microporous polymeric material, metal foil, glass, porcelain, ceramic, or the like, or a combination thereof. For certain embodiments, the sheet material is water-absorbent or can be wetted to aid in quickly bringing the liquid medium in intimate contact with the spores at the appropriate time.

[0052] Known biological sterilization process indicator constructions such as those described, for example, in U.S. Pat. No. 5,073,488 (Matner et al.) and in U.S. Patent No. 10,047,334 (Chandrapaii et al.) may be adapted to construct a biological indicator of the present disclosure. FIGS. 1 and 2 show one embodiment of a sterilization process indicator 100 according to the present disclosure. The sterilization process indicator 100 includes a cuvette 10 having at least one liquid impermeable wall (e.g., side wall 12A and bottom wall 12B) that forms an opening 14 into a compartment 15. The cuvette 10 is shown as a circular tube, but other known

configurations can be used. The at least one wall is preferably transparent or translucent to the extent that electromagnetic radiation of a particular wavelength and intensity sufficient to cause a nucleic acid-interacting dye to fluoresce can be transmitted therethrough. In addition, the walls are preferably transparent or translucent to the extent that electromagnetic radiation at a particular wavelength emitted by the nucleic acid-interacting dye can be transmitted through the wall and be measured. Suitable materials for the walls may include glass, polycarbonate, polypropylene, polyester, and the like. For certain embodiments, the at least one wall of the cuvette transmits at least 90% of incident electromagnetic radiation within a wavelength range of at least 500 to 700 nm, preferably at least 500 to 675 nm.

[0053] In any embodiment, at least a portion of the cuvette can be configured (e.g., shaped and dimensioned) to be received into an instrument capable of detecting fluorescence emitted by the nucleic acid-interacting dye. That is, the instrument is configured to illuminate the portion of the cuvette with electromagnetic radiation of an appropriate first wavelength to cause fluorescence of the nucleic acid-interacting dye when the dye is bound to DNA or RNA. Moreover, the instmment is configured to detect and quantify electromagnetic radiation of a second wavelength emitted by fluorescence of the nucleic acid-bound nucleic acid-interacting dye. In certain embodiments, the instrument is also capable of measuring absorbance or scattering of electromagnetic radiation (e.g., at wavelengths typically absorbed or scattered by spores and/or microorganisms) passed through the portion of the cuvette. That is, the instrument is configured to pass the radiation through the portion of the cuvette and also detect an amount of radiation that passed through the cuvette. In certain alternative embodiments, at least a portion of the cuvette is configured (e g , shaped and dimensioned) to be received into a second instmment, the second instrument being capable of measuring the absorbance of electromagnetic radiation passed through the cuvette.

[0054] Disposed in the compartment 15 is a liquid medium 16, a nucleic acid-interacting dye (not shown), and a predetermined number of sterilization process-resistant spores (not shown). Suitable nucleic acid-interacting dyes are disclosed hereinabove. The liquid medium 16 can be a liquid (e.g., an aqueous liquid) in which the nucleic acid-interacting dye is suspended and/or dissolved. In certain embodiments, the nucleic acid-interacting dye is disposed in the liquid medium. In certain embodiments, the spores are disposed in the liquid medium. In certain embodiments, such as the illustrated embodiment of FIGS. 1 and 2, the spores and the nucleic acid-interacting dye are disposed in the liquid medium 16.

[0055] In use, the sterilization process indicator 100 is exposed to a sterilant gas (e.g., in the sterilization chamber of an automated sterilizer). The sterilant gas (not shown) passes through the closure member 22 into the compartment 15 where it contacts the liquid medium in which the spores and the nucleic acid-interacting dye are disposed. As the sterilant gas inactivates the spores, the nucleic acid-interacting dye penetrates the spore and interacts with the nucleic acid therein.

[0056] In certain embodiments, the liquid medium, whether present in the process indicator in the compartment or in a container as described herein, has a volume of not less than 10 microliters, not less than 20 microliters, not less than 50 microliters, not less than 100 microliters, or not less than 150 microliters. In certain embodiments, the liquid medium, whether present in the process indicator in the compartment or in a container as described herein, has a volume of not more than 1000 microliters, not more than 500 microliters, not more than 400 microliters, not more than 300 microliters, not more than 250 microliters, or not more than 200 microliters. In certain embodiments, the volume of the liquid medium is about 10 microliters to about 1000 microliters. In certain embodiments, the volume of the liquid medium is about 10 microliters to about 500 microliters. In certain embodiments, the volume of the liquid medium is about 100 microliters to about

250 microliters.

[0057] The opening 14 to compartment 15 is provided with a gas-transmissive,

microorganism -impermeable closure member 22, which may be adhered to cuvette 10 by an adhesive, a heat seal, or the like. Alternatively, closure member 22 may be held on to opening 14 with a cap 26 having an aperture 28. During exposure to a sterilant gas, the sterilant gas is directed through the closure member 22, enters the compartment 15, and contacts the spores. In certain preferred embodiments, the pathway that permits passage of the sterilant gas into the cuvette also resists or prevents passage into the container of bacteria from outside the sterilization process indicator. A nonlimiting example of suitable materials for closure members include microporous materials such as a filter membrane. Alternative embodiments for the above structures are shown in U S. Patent Publication No. 20150165082 (Chandrapati et al.), filed Oct. 17, 2008, entitled Biological Sterilization Indicator, System, and Methods of Using Same.

A skilled artisan will recognize alternative structures (e.g., a tortuous path) that may be suitable for providing passage of a sterilant gas into the compartment 15 while resisting passage of bacteria into the compartment.

[0058] A self-contained sterilization process indicator of the present disclosure does not include an effective amount of germination medium and/or microbial growth medium (e.g., either disposed in the cuvette or disposed in a container that is in selective fluid communication with the cuvette). Thus, the spores disposed in the cuvette 10 of the process indicator 100 do not have access to a source of germination medium and/or growth medium contained within the process indicator wherein the source would be present in an amount sufficient to support germination, growth, and detection of the germinated spores in 4 hours or less, more preferably in 2 hours or less, even more preferably in 1 hour or less, even more preferably in 30 minutes or less, and even more preferably in 15 minutes or less after the process indicator is exposed to a sterilization process.

[0059] In any embodiment of a sterilization process indicator of the present disclosure, the sterilant gasses include, but are not limited to, water vapor (i.e., steam), ethylene oxide, hydrogen peroxide, and ozone.

[0060] FIG. 3 shows an alternative embodiment of a stenlization process indicator 200 according to the present disclosure. The indicator compnses a cuvette 10 having at least one liquid impermeable wall (e.g., side wall 12A and bottom wall 12B) that forms an opening 14 into a compartment 15, each as described hereinabove. Disposed in the compartment 15 is a liquid medium 16, a nucleic acid-interacting dye and a predetermined number of sterilization process- resistant spores 20, each as described hereinabove. In the illustrated embodiment of FIG. 3, the liquid medium 16 is contained in a container 18. The sterilization process indicator 200 also includes a gas-transmissive, microorganism-impermeable closure member 22 and a cap 26, each as described hereinabove.

[0061] In certain embodiments of the process indicator 200, such as the illustrated embodiment of FIG. 3, the liquid medium 16 in the container 18 can have a portion or all of the nucleic acid interacting dye suspended and/or dissolved therein. In certain alternative embodiments (not shown), the nucleic acid-interacting dye can be disposed in a layer (e.g., as a powder or a coating) inside the compartment (e.g., on the bottom wall 12B; on a substrate such as a plastic film, for example) but not inside the container. [0062] The container 18, which holds the liquid medium 16, is shown within compartment 15. Container 18, which is sealed, can be a breakable (e.g., plastic or glass) ampoule, but could alternatively be a container equipped with a plug, or other mechanism which when activated (e.g., opened inside the compartment 15) allows the liquid medium 16 to contact the spores 20. Container 18 is shown as a frangible, elongated ampoule, but other known configurations can be used as well.

[0063] In any embodiment of a process indicator according to the present disclosure, the spores are disposed (e.g., in a substantially water-free layer or coating) on a surface in the compartment. For example, the spores may be suspended in a suitable liquid, which is then deposited onto the surface and subsequently dried (e.g., by evaporation). In certain embodiments (not shown), the surface can be a portion of the inner surface of one of the at least one walls (e.g., the bottom wall) of the cuvette.

[0064] Referring back to FIG. 3, the predetermined number of process-resistant spores 20 is disposed (e.g., as a layer or a substantially water-free coating) on an optional carrier 24. Optionally, in certain embodiments, the enhancer reagent (not shown) and/or the nucleic acid-interacting dye (not shown) can be disposed in the container 15 with the spores (e.g., in the layer or substantially water- free coating). The carrier 24 can be any suitable material onto which the spores 20 can be disposed (e.g., by a coating process) wherein the material does not substantially hinder or prevent 1) contact between the spores and a sterilant gas, 2) contact between the spores and the nucleic acid-interacting dye (e.g., by adsorbing and or sequestering the dye from contact with the spores), and/or 3) detection of fluorescence by the nucleic acid-interacting dye when it is bound to DNA or RNA (e.g., by substantially absorbing the electromagnetic radiation used to excite the fluorescent dye and/or by substantially absorbing the electromagnetic radiation emitted by the nucleic acid-bound dye).

Suitable materials for carriers 24 include, but are not limited to paper, glass, or a polymeric sheet or film.

[0065] In any embodiment, a self-contained sterilization process biological indicator can comprise an enhancer reagent disposed in the compartment. In certain embodiments, the enhancer reagent contacts the spores (e.g., it may be deposited on the inner surface of the wall or on the carrier with the spores). Additionally, or alternatively, in certain embodiments, the enhancer reagent is disposed in the liquid medium and contacts the spores when the liquid medium contacts the spores. The enhancer reagent, when present in the sterilization process indicator, improves the consistency of the sterilization of the spores and/or the detection of viable spores after the sterilization process indicator is exposed to the sterilant gas. Without being bound by theory, it is believed the enhancer reagent promotes stability of viable spores during storage and/or facilitates the resuspension of dried spores into the liquid medium and/or facilitates penetration of the nucleic acid-interacting dye into the spores.

[0066] Nonlimiting examples of suitable enhancer reagents include glycerol, sucrose, trehalose, polyvinyl pyrrolidone, and a combination of any two or more of the foregoing enhancer reagents. [0067] In any embodiment, a self-contained sterilization process biological indicator of the present disclosure can comprise a buffering agent disposed in the compartment. In certain embodiments, the buffering agent can be disposed in the liquid medium. Additionally, or alternatively, the buffering agent can be disposed with the spores (e.g., in a dried coating as discussed above). The buffering agent can serve to buffer the liquid medium at a pH that facilitates fluorescence of the nucleic acid interacting dye Suitable buffering agents include, but are not limited to HEPES buffer (e g., 50 mM), Tris-EDTA (e.g., 50 mM Tris, 10 mM EDTA) buffer, Tris buffer (e.g., 50 mM). The buffer can be selected for use at a neutral pH (e.g., about 6.0-8.0).

[0068] In certain embodiments, a self-contained sterilization process biological indicator of the present disclosure can comprise a collisional quenching component, as described hereinabove, disposed in the compartment. The collisional quenching component may be dissolved and/or suspended in the liquid medium, for example.

[0069] In certain embodiments, a self-contained biological indicator of the present disclosure can comprise a nucleic acid-interacting dye that, when bound to a nucleic acid can cause a change in the electrical conductance (or resistivity) of an aqueous mixture in which the dye and the nucleic acid are disposed. In some embodiments, this change can be due to a redox reaction in which the nucleic acid- bound dye can participate. Thus, the presence or absence of the dye bound to nucleic acid can be detected electrochemically (e.g., by measuring conductance of the aqueous medium. A non-limiting example of such a nucleic acid-interacting dye is Hoechst 33258 DNA binding dye (available from VWR International, Radnor PA).

[0070] The present disclosure additionally provides a method. The method can be used to determine the effectiveness of a stenlization process. FIG. 4 shows one embodiment of a method 500 of determining effectiveness of a sterilization process according to the present disclosure.

[0071] The method 500 includes the step 550 of positioning any embodiment of a self- contained sterilization process biological indicator according to the present invention in a sterilization chamber. The sterilization chamber can be a sterilization chamber of a sterilizer (e.g., a commercially-available automated sterilizer), the sterilization chamber being typically sized to contain a plurality of articles to be sterilized and equipped with a means of evacuating air and/or other gases from the chamber and adding a sterilant gas to the chamber. The biological sterilization indicator can be positioned in the most difficult location (e.g., above the drain) in the sterilizer to achieve proper sterilization conditions (e.g., temperature, pressure). Alternatively, the biological sterilization indicator can be positioned adjacent an article to be sterilized when placed in the sterilization chamber. Additionally, the biological sterilization indicator can be adapted into routinely used process challenge devices before positioning it in the sterilization chamber.

[0072] While the self-contained sterilization process biological indicator is positioned in the sterilization chamber, the method 500 includes the step 552 of exposing the sterilization process indicator to a sterilant gas during a sterilization process. The sterilization process indicator is exposed to the sterilant gas in the sterilization chamber. In certain embodiments, exposing the sterilization process indicator to a sterilant gas comprises exposing the sterilization process indicator to steam. In certain embodiments, exposing the sterilization process indicator to a sterilant gas comprises exposing the sterilization process indicator to ethylene oxide. In certain embodiments, exposing the sterilization process indicator to a sterilant gas comprises exposing the sterilization process indicator to a peroxide. In certain embodiments, exposing the sterilization process indicator to a sterilant gas comprises exposing the sterilization process indicator to ozone.

[0073] Because a sterilization process indicator according to the present disclosure comprises an opening that is part of a pathway that permits passage of a sterilant gas from outside the cuvette into the chamber of the indicator; exposing the sterilization process indicator to the sterilant gas comprises exposing the spores disposed in the compartment of the indicator to the sterilant gas. Typically, exposing the sterilization process indicator to the sterilant gas comprises exposing the spores to the sterilant gas under conditions (e.g., time, temperature, pressure, and concentration of sterilant gas) selected to be sufficient to inactivate (e.g., render nonviable and/or non- cultivable) all of the spores of the predetermined number of sterilization process-resistant spores disposed in the sterilization process indicator.

[0074] The sterilant gas can be added to the sterilization chamber after evacuating the chamber of at least a portion of any air or other gas present in the chamber. Alternatively, the sterilant gas may be added to the sterilization chamber without evacuating the chamber. A series of evacuation steps is often used to assure that the sterilant gas reaches all areas within the sterilization chamber and contacts all areas of the article(s) in the sterilization chamber to be sterilized. When the sterilant gas is added to the sterilization chamber, the sterilant gas also contacts the spores under conditions (e.g., temperature, pressure, concentration) where the sterilant gas reaches all areas within the sterilization chamber.

[0075] The method 500 further comprises the step 554 of contacting the spores with the nucleic acid-interacting dye. The spores are contacted with the nucleic acid-interacting dye in the compartment of the sterilization process indicator. In certain embodiments, contacting the spores with the nucleic acid-interacting dye comprises contacting the spores with the nucleic acid-interacting dye while of exposing the sterilization process indicator to the sterilant gas (see step 552). In these embodiments, if the nucleic acid-interacting dye is disposed in the sterilization process indicator in a container (e.g., in a liquid medium in the container as described hereinabove), the container is opened (e.g., by fracturing, crushing or otherwise disintegrating the container) prior to the exposing the indicator to the sterilant gas (e.g., before the indicator is positioned in the sterilization chamber). In certain alternative embodiments, contacting the spores with the nucleic acid-interacting dye comprises contacting the spores with the nucleic acid-interacting dye after the step 552 of exposing the sterilization process indicator to the sterilant gas. In these alternative embodiments, if the nucleic acid-interacting dye is disposed in the sterilization process indicator in a container (e g., in a liquid medium in the container as described hereinabove), the container is opened (e.g., by fracturing, crashing or otherwise disintegrating the container) after the exposing the indicator to the sterilant gas (e.g., after the indicator is removed from the sterilization chamber).

[0076] In certain embodiments of the method, the self-contained sterilization process biological indicator comprises a container disposed in the compartment, wherein the container contains the liquid medium. In these embodiments, prior to the exposing the self-contained sterilization process biological indicator to the sterilant gas, the method further may comprise disintegrating the container to contact the spores with the liquid medium. Thus, in these

embodiments, the spores are in contact with the liquid medium while the spores are exposed to the sterilant gas. In any of these embodiments, the nucleic acid-interacting dye may be dissolved and/or suspended in the liquid medium in the container.

[0077] In certain alternative embodiments of the method, the self-contained sterilization process biological indicator comprises a container disposed in the compartment, wherein the container contains the liquid medium. In these alternative embodiments, after the exposing the self-contained sterilization process biological indicator to the sterilant gas, the method further comprises disintegrating the container to contact the spores with the liquid medium. Thus, in these embodiments, the spores are not in contact with the liquid medium while the spores are exposed to the sterilant gas. In any of these alternative embodiments, the nucleic acid-interacting dye may be dissolved and/or suspended in the liquid medium in the container.

[0078] After exposing the sterilization process indicator to the sterilant gas, the method 500 comprises the step 556 of measuring a first fluorescence intensity emitted by the nucleic acid interacting dye in the self-contained sterilization process biological indicator. Prior to measuring the first fluorescence intensity, the spores are contacted with the liquid medium. Measuring the first fluorescence intensity can comprise illuminating at least a portion of the cuvette with electromagnetic radiation of a first (excitation) wavelength and intensity sufficient to cause the nucleic acid interacting dye that is bound to nucleic acid to fluoresce at a second (emission) wavelength. The first fluorescence intensity is measured (e.g., in a fluorometer adapted to receive the cuvette) at the second wavelength and is an indication of the effectiveness of the inactivation of the spores in the sterilization process indicator, as discussed hereinabove.

[0079] After measuring the first fluorescence intensity, the method 500 comprises the step 558 of comparing the first fluorescence intensity to a reference fluorescence intensity to determine whether the sterilization process was effective. In certain embodiments, the reference fluorescence intensity is a measured intensity of fluorescence (i.e., a second fluorescence intensity) emitted by a“control” sterilization process indicator (e.g., of a“positive control”, such as an identical sterilization process indicator that has not been exposed to a sterilant gas and, thus, the spores are substantially all viable; or a“negative control”, such as an identical sterilization process indicator that has been treated (e.g., in an“overkill” sterilization process) so that substantially all of the spores are nonviable). In certain embodiments, the reference fluorescence intensity is a measured intensity (i.e., a second fluorescence intensity) emitted by a“fluorescence control” (e.g., a solution or a solid state material that emits a predetermined fluorescence intensity used to set a threshold intensity value to which the first fluorescence intensity can be compared to determine whether or not all of the spores in the sterilization process monitor that was exposed to the sterilant gas have been inactivated. In certain embodiments, the reference fluorescence intensity can be an arbitrary fluorescence intensity value, for example, in a printed publication such as instructions for use that can be used by the operator to make a comparison with the first fluorescence intensity, or in an electronic data set that can be used (e.g., by a fluorimeter or a microprocessor) to make a comparison with the first fluorescence intensity.

[0080] Comparing the first fluorescence intensity to a reference fluorescence intensity comprises determining whether the first fluorescence intensity is less than, equal to, or greater than the reference fluorescence intensity. In certain embodiments, a first fluorescence intensity that is greater than the reference intensity indicates the sterilization process was effective to inactivate all of the

predetermined number of spores. In certain embodiments, a first fluorescence intensity that is greater than or equal to the reference intensity indicates the sterilization process was effective to inactivate all of the predetermined number of spores. In certain embodiments, a first fluorescence intensity that is less than the reference intensity indicates the sterilization process was not effective to inactivate all of the predetermined number of spores. In certain embodiments, a first fluorescence intensity that is less than or equal to the reference intensity indicates the sterilization process was not effective to inactivate all of the predetermined number of spores.

[0081] In any embodiment of a method of determining effectiveness of a sterilization process according to the present disclosure, before measuring the first fluorescence intensity, the method does not include a step of incubating the spores in the self-contained sterilization process biological indicator with a growth medium or germination medium for a sufficient period of time to cause germination or growth of the spores. Thus, a method according to the present disclosure does not require germination of viable spores and/or growth of germinated spores in order to determine whether the sterilization process was effective.

[0082] The present method can, therefore, be sufficiently sensitive to the presence of viable spores to provide an indication thereof immediately upon removal of the sterilization process indicator from the sterilization chamber or, if necessary, immediately after sufficient cooling of the process indicator to a temperature that permits safe handling of the process indicator. In addition, the indication can be provided even when the number of viable spores present is relatively low.

[0083] For certain embodiments, including any one of the above method embodiments, the method further comprises placing an article to be sterilized along with the sterilization process indicator in the sterilization chamber. For certain of these embodiments, the method further comprises determining whether or not the sterilization process was effective for sterilizing the article. An indication of no viable spores may be used to determine that the sterilization process was effective for sterilizing the article, whereas an indication of viable spores may be used to determine that the process was not effective. Thus, an assessment of the sterility of an article subjected to a sterilization process may be made in a relatively short time using the method and self-contained sterilization process biological indicators described herein.

[0084] In any of the above embodiments, a method according to the present disclosure further can comprise measuring an amount of absorbance and/or scattering of electromagnetic radiation as the electromagnetic radiation is directed through the liquid medium in the cuvette. Preferably, the absorbance and/or scattering is measured after the exposing the sterilization process indicator to the sterilant gas. The wavelength of electromagnetic radiation used to measure the absorbance and/or scattering can be, for example, a wavelength used in the art to assess the optical density of spores suspended in a liquid medium. Nonlimiting examples of suitable wavelengths for measuring the absorbance and/or scattering are those wavelengths in the range of about 500nm to about 600nm. The amount of absorbance and/or scattering of the electromagnetic radiation by the liquid medium is a further indication of the inactivation (e.g., disintegration) of the spores by the sterilization process. In certain embodiments, the method further includes a step of calculating a ratio of the first fluorescence intensity to the measured amount of electromagnetic radiation absorbance and/or scattering.

[0085] The ratio of the first fluorescence intensity to the measured amount of electromagnetic radiation absorbance and/or scattering can be used to calculate and adjusted first fluorescent intensity (IA) according to the following formula:

I_A = I/O.D.,

wherein I = the first fluorescent intensity and O.D. is the measured absorbance and/or lights scattering (i.e., optical density) of the spores in the liquid medium after the sterilization process indicator has been exposed to the sterilant gas.

[0086] In any embodiment of a method according to the present disclosure, the method further can comprise agitating the self-contained sterilization process biological indicator for a period of time. Agitation can be used to mix the contents present in the chamber of the indicator. The indicator can be agitated, for example, to suspend the spores in the liquid medium (e.g., after a container has been actuated to release the liquid medium into the chamber). Additionally, or alternatively, the indicator can be agitated before measuring the first fluorescence intensity and/or before measuring the amount of absorbance and/or scattering of electromagnetic radiation as the electromagnetic radiation is directed through the liquid medium in the cuvette. The indicator can be agitated manually (e.g., by swirling, tapping, or shaking) or it can be agitated using a sample-vortexing machine. [0087] In certain alternative embodiments of a method according to the present disclosure, detection of the nucleic acid-interacting dye with a nucleic acid after a sterilization process can be effected by measuring the electrical conductance of a liquid medium in which the nucleic acid interacting dye with the nucleic acid, as described, for example, in Examples 7 and 8 herein.

[0088] In yet another aspect, the present disclosure provides a composition. The composition can be used in a method of manufacturing a self-contained biological sterilization process indicator. The composition comprises a plurality (e.g., a predefined number) of sterilization process-resistant microbial spores and a nucleic acid-interacting dye, wherein the composition is substantially water- free.

[0089] The plurality of process-resistant microbial spores can be any of the spores described herein for use in a sterilization process indicator. In any embodiment, the plurality of spores can be a predefined number of spores. The predefined number can be an approximate number (e g., about 10, about 10², about 10³, about 10⁴, about 10⁵, about 10⁶, or about 10⁷ viable spores). Alternatively, the predefined number of spores can be a threshold number of spores (e.g., a lower threshold of at least 10, at least 10², at least 10³, at least 10⁴, at least 10^s, at least 10⁶, or at least 10⁷; and/or an upper threshold of not more than 10², not more than 10³, not more than 10⁴, not more than 10⁵. not more than 10⁶, not more than 10⁷, or not more than 10⁸ viable spores).

[0090] The composition can comprise any suitable nucleic acid-interacting dye as described herein, and suitable alternative nucleic acid-interacting dyes recognized by a person having ordinary skill in the art in view of the present disclosure.

[0091] The composition can be made, for example, by suspending the spores in a suspending solution (e.g. sterile water, a sterile buffer solution) comprising the nucleic acid-mteracting dye. An appropriate volume of the suspended spores (e.g., a volume containing the predefined number of spores) is deposited onto a surface (e.g., a surface of a earner or an inner surface of a cuvette as described herein) and subsequently dried to produce the substantially water-free composition of the present disclosure. The concentration of the nucleic acid-interacting dye in the suspending solution is selected so that, when the resulting composition is rehydrated with a predetermined volume of liquid medium (e.g., a predetermined volume of about 10 microliters to about 1000 microliters of an aqueous liquid), the concentration of the nucleic acid-interacting dye in the resulting rehydrated mixture is about 0.05 mM to about 20 mM.

Examples

[0092] Materials and Equipment

[0093] Table 1. Materials

[0094] Table 2. Equipment

[0095] Reference Example 1. Contacting spores with a Nucleic Acid-Interacting Dye after Exposing the Spores to a Steam Sterilization Process.

[0096] Spores of Geobacillus stearothermophilus were produced in liquid sporulation medium. The spores were washed in sterile deionized water and were resuspended in sterile deionized water to achieve a concentration of about 10⁸ spores/mL. This spore crop was then diluted 1: 100 in sterile DI water, and aliquoted (2 mL volumes) into glass test tubes to yield 3.4* 10⁶ spores/mL. [0097] Table 3 provides the six different exposure times at 121°C in a dynamic-air removal steam sterilization cycle. Commercial 1492V biological indicators (3M Company, St. Paul, MN) were used as a control. This experiment was performed in duplicate.

[0098] The tubes containing the spores were placed in a Midmark M9 steam sterilizer and were held in the sterilization chamber at 121° C for the times listed in Table 3.

[0099] After the tubes were removed from the sterilizer, the tubes were agitated to obtain a uniform suspension and 0.1 mL of each suspension was transferred to individual wells of a 96-well plate, each well containing 100 pL of 3 mM SYT09 dye. The fluorescence intensity of each well was measured in a BIOTEK 96-well plate reader at room temperature. The excitation wavelength was 480 nm. The detection (emission) wavelength was 510 nm. In addition, the optical density (600 nm) of each well was measured in the BIOTEK plate reader. A 0.1 mL portion from each spore suspension was spread onto Tryptic Soy Agar and incubated at 60° C for 18-24 to determine the number of viable spores (colony-forming units) in the suspension.

[00100] The data are shown in Table 4. The values shown in Table 4 are the average of 2 replicates per treatment time. The RFU/ODeoo calculation provided the greatest differentiation between the samples that had viable spores left after exposure to steam the and samples in which there were no viable spores after exposure to steam, as shown in Table 4.

[00101] Table 4.

¹ - Positive Control samples were not exposed to steam.

[00102] Reference Example 2. Contacting spores with a Nucleic Acid-Interacting Dye during or after Exposing the Spores to a Steam Sterilization Process.

[00103] Geobacillus stearothermophilus ATCC 7953 spores were produced and washed in sterile water as described in Reference Example 1. After the final wash, the spores were suspended in a trehalose solution (25% w/v). A concentrated solution of SYT09 dye was added to a first portion (“pre-sterilization”) of the resuspended spores to a final concentration of 10 mM SYT09. A second portion (“post-sterilization”) of the resuspended spores did not contain SYT09 dye. Twenty- microliter aliquots of each portion of the resuspended spores were spotted onto polypropylene film carriers (¼” diameter discs) to yield >10⁶ spores/carrier. The coated carriers were dried at 60°C for 15 minutes.

[00104] The coated carriers were placed into individual tubes that were then placed into a Midmark M9 steam sterilizer and were held in the sterilization chamber at 12G C for six minutes. No-exposure controls were prepared identically but were not subjected to steam sterilization. After removing the tubes from the sterilizer, 0.2 mL of sterile water was added to each tube containing a“presterilization” carrier and the tubes were agitated to resuspend the spores. In addition, 0.2 mL of 1 pm SYT09 dye in sterile water was added to each of the tubes containing a“post-sterilization” carrier and the tubes were agitated to resuspend the spores.

[00105] Each of the tubes was analyzed for fluorescence and optical density as described in

Reference Example 1. The results are shown in Tables 5 and 6. The“Exposure Time denotes the number of minutes the carriers were exposed to steam at 12 G C.

[00106] Table 5. Results from Pre-sterilization Addition of SYT09 Dye to the Spores.

¹ - Controls were prepared like the other samples except the spores in the trehalose solution were omitted from the dye solution that was spotted onto the carriers. Thus, the RFU/ODgoo calculation was not applicable because there were no spores to contribute to the ODgnn.

[00107] Table 6. Results from Post-sterilization Addition of SYT09 Dye to the Spores.

[00108] The data indicate the signal is higher when the dye contacts the spores during exposure to the sterilant when the spores are coated with trehalose on a carrier. Without being bound by theory, it is thought this effect may be because the steam facilitates penetration of the nucleic acid-interacting dye into the spores.

[00109] Example 1. Preparation of Self-contained Sterilization Process Biological Indicators having Dried Spores Coated on a Cuvette Wall. [00110] Geobacillus stearothermophilus spores can be produced as described in Reference Example 1. The washed spores can be resuspended in a trehalose solution (25% w/v) containing 10 mM SYT09, as described in Reference Example 2.

[00111] Assembly of cuvettes

[00112] Biological indicators (part number 1292) can be obtained from 3M Company (St. Paul, MN). Caps on the biological indicators can be removed and the contents (glass ampule, spore strip, and nonwoven packing adjacent the spore strip in the bottom of each tube) can be removed. The nonwoven sheet in the cap of can be left in place. The caps (with the nonwoven sheets therein) can be replaced on the tubes and each assembled tube to form the assembled cuvette. The cuvettes can be sterilized in a steam sterilizer.

[00113] Process indicators can be made by spotting (20 mΐ) of the spore suspension onto the inner surface of a wall of the cuvettes to yield >10⁶ spores/indicator. The spore-coated cuvettes can be dried at 56°C for approximately 1 hour. After drying the spores, a cylindrical glass ampule (approximately 25 mm long and 4 mm in diameter) containing approximately 0.2 mL sterile water can be placed into the cuvettes and the caps can be replaced on the cuvettes to complete the assembly of the process indicators.

[00114] The assembled process indicators can then be exposed to steam at 121°C in a dynamic-air- removal benchtop commercial vessel at 2 different exposure points (To exposure and 6 minutes exposure), as described in Reference Example 1.

[00115] Sample analysis

[00116] After being exposed to the stenlant in the sterilizer, the process indicators of Example 1 can be removed from the sterilizer and the glass ampule can be crushed (e.g., by carefully pinching the flexible plastic cuvette with pliers to break the glass ampule. The process indicators can be agitated for about 10-20 seconds. Subsequently, 100 mΐ samples from each process indicator can be transferred to a 96-well plate and the fluorescence and optical density of each sample can be measured as described in Reference Example 1.

[00117] Projected Results

[00118] The projected estimated results for the process indicators of Example 1 are shown in Table 7. The values in Table 7 are intended to represent approximate relative differences between the optical density and relative fluorescence that would be observed in process indicators that would be expected to contain a relatively large number of viable spores (e.g., the 0 minute exposure) and process indicators that would be expected to contain a relatively small number of viable spores (e.g., the 6 minute exposure). The exposure time (to the steam sterilant) can indicate the exposure time at 121°C in a Midmark M9 commercial steam sterilization vessel.

[00119] Table 8.

¹ - Projected Estimate

² - Projected Estimate

³ - Projected Estimate

[00120] Reference Example 3 - Spore Suspension

[00121] Sample Preparation.

[00122] Geobacillus stearothermophilus ATCC 7953 spores were suspended in sterile TbO (3.4xl0⁶ spores/mL) and aliquoted (2 mL volumes) into six glass test tubes. Table 9 shows the six different steam -exposure times at 121°C in a dynamic-air removal steam sterilization process in a MidMark M9 sterilizer. Commercial 1492V biological indicators (3M Company, St. Paul, MN) were used as growth controls as described below.

[00123] Table 9. Samples and steam SFPP exposure times

[00124] Sample Analysis:

[00125] After the tubes containing the spore suspensions were exposed to the sterilization processes, Syto9 (3.0 mM) nucleic acid binding dye (100 pL) was then added to aliquots of each test sample (100 pL) and fluorescence intensity was immediately measured in a BIOTEK 96-well plate reader at room temperature. The excitation wavelength used in the plate reader was 480 nm and the emission wavelength measured was 510 nm. In addition, the optical density (600 nm) was also measured in the BIOTEK plate reader. The colony forming units for each test sample was determined by diluting the spore suspensions and surface-plating the dilutions onto Tryptic Soy Agar followed by incubation of the plates at 60°C.

[00126] Results

[00127] The data for this example are shown in Table 10. The RFU/OD600 value shown in Table 10 correlated well with the length of exposure of the spores to the steam sterilization process. 1492V commercial biological indicator controls were all growth negative at exposure times greater than 2 minutes of exposure in this experiment.

[00128] Table 10. Reference Example 3 Data. The OD600, Syto9 (RFU), and Syto9 (RFU)/OD600 reported in this table are the average of the triplicate technical replicates for each condition. The CFU/mL is the average of duplicate technical replicates for each condition.

[00129] Example #2 - Dried spores - DNA dye added to spore coating solution

[00130] Sample preparation:

[00131] Geobacillus stearothermophilus ATCC 7953 spores were suspended in an aqueous trehalose solution (25% w/v) with and without syto9 dye (10mM) and spotted (20pL) onto circular polypropylene film carriers to yield approximately >10⁶ spores/carrier. The coated carriers were then dried at 60°C for 15 minutes. The spore-coated and dried carriers were then exposed to a 6-minute steam sterilization process at 121°C in a dynamic-air removal steam sterilization cycle using a MidMark M9 sterilizer. Samples were prepared and tested in duplicate.

[00132] Sample analysis:

[00133] Following stenlization of the samples, 200 pL of FFO was added to the samples that already had syto9 dried with the spores prior to sterilization. For samples that had syto9 added post sterilization, 20 pL of syto9 (10 pM) was added to the wells followed by 180 pL of H₂0. Therefore, all samples had a final concentration of 1 pM syto9 at the time of the reading. The samples were pipetted up and down approximately 5 times and fluorescence intensity and optical density of each well was immediately measured in a BIOTEK 96-well plate reader at room temperature as described in Reference Example 3.

[00134] Results:

[00135] The data for this example are shown in Table 11, 12 and 13. The exposure time indicates the length of exposure to steam at 121C in a Midmark M9 commercial sterilizer. Syto9 pre/post sterilization indicates whether the sample had syto9 dye added to the coating solution (Pre sterilization), or after the sterilization process (Post). The RFU/OD600 values shown in Table 11 correlated well with the length of exposure of the spores to the steam sterilization process. The observed fluorescent signal in the steam-exposed samples was higher when the dye is added prior to sterilization.

[00136] Table 11. Pre-sterilization addition of Syto9 dye.

[00137] Table 12. Post-sterilization addition of Syto9 dye

[00138] Table 13. Pre-sterilization addition of Syto9 dye - dye only control (no spores)

[00139] Example #3 - Dried spores in biological indicator

[00140] Sample Preparation

[00141] Geobacillus stearothermophilus ATCC 7953 spores were suspended in a trehalose solution (25% w/v) with syto9 dye (IOmM) and spotted (20pL) on the inside of the wall of polycarbonate housings from 3M™ ATTEST™ 1295 biological indicators. Each resulting biological indicator contained approximately >10⁶ spores coated on inside of the wall of the housing. The coated BI sleeves were then dried at 56°C for approximately 1 hour. SRBI caps were then placed on the coated BI sleeves. The Samples were then exposed to steam at 121°C in a dynamic-air-removal benchtop commercial sterilization vessel at 2 different exposure points (TO exposure and 6 minutes exposure). Samples were prepared in duplicate.

[00142] Sample analysis:

[00143] EbO (220 pL) was added to each BE The Bis were then vortexed for 3 seconds. Aliquots were then pulled (200 pL) from each BI and the fluorescence and optical density were immediately measured in a BIOTEK 96-well plate reader at room temperature as described in Reference Example 3.

[00144] Results: [00145] The data for example #3 are shown in Table 14. Note that the Bis that were aborted at TO of exposure were exposed to the pre-conditioning phase on the commercial sterilization cycle. The data show higher values of fluorescence in the BTs exposed to steam for 6 minutes.

[00146] Table 14. Example #3 Data.

[00147] Example #4 - Creation of Reference Standard Curve

[00148] Sample Preparation

[00149] Spores were produced in liquid cultures. The spores were harvested by centrifugation, washed several times in sterile water, and resuspended in a sufficient volume of sterile water to achieve a concentration of 2.0xl0⁸ CFU/mL). 150uL of the resulting suspension was placed in a 1.5 mL micro-centrifuge tube and centrifuged at 3400 RPM for 5 minutes in a micro-centrifuge. The supernatant was removed.

[00150] Preparation of coating solution (12% PYP W/V, and 2.5% Trehalose W/V in H2O): Mix 3 mL of H2O with Polyvinyl Pyrrolidone (0.36 g) and Trehalose (0.075 g).

[00151] The spore pellet in the microcentrifuge tube was then resuspended with the coating solution (1 mL). Four mΐ of Syto9 (5000 mM stock) was then added to the suspension to yield a 20 mM concentration. The suspension was then coated onto polypropylene film carriers. Carriers were dried for 25 minutes at 56°C. The biological indicators were then placed in an H&W 105 resistometer, where they were exposed to an ISO 132°C Pre-vacuum sterilization cycle for various periods of 132°C exposure ( 30 seconds, 1.5 minutes, 2 minutes, 2.5 minutes, and 3.5 minutes).

[00152] Sample Analysis

[00153] After exposure to the sterilization process, sterile FLO (220 pL) was added to each Biological indicator and the indicators were vortexed briefly to resuspend spores. The resuspended spores were then transferred to a clear bottom, black walled 96-well plate. Plates were read at room temperature in the BioTek plate reader at 480nm excitation/520nm emission, and the OD600 was measured. Readings were performed as quickly as possible from the time of resuspension to reading in the plate reader. Following the fluorescence reading, nutrient media (containing bromocresol purple) that supports spore germination and outgrowth was added to the wells. The plate was then placed in a 60°C incubator for a minimum of 24 hours, after which the media was examined for indications (e.g., turbidity, pH change) of spore germination and growth. [00154] Results

[00155] Table 15 and 16 show the fluorescence and growth data respectively for this example. The values shown in Table 15 are relative fluorescence units (RFU) for five biological indicator replicates across 5 exposure times. The RFU values shown in Table 15 are with the background (from unexposed BTs) subtracted. Table 16 shows the growth result with bromocresol purple and media that supports spore germination and outgrowth.

[00156] These data indicate that a threshold value could be set that indicates where positive and negative fluorescence could be established based on how it correlates to spore growth. For example anything below 8000 RFU would be assigned a fluorescent positive value (Growth positive) and anything above 8000 RFU would be assigned a fluorescent negative value (Growth negative). This data set indicates an example reference standard curve data set that could be used to assign fluorescent designation for near immediate monitoring of sterilization cycles.

[00157] Table 15. Example #4 Fluorescence Data following exposure in ISO 132°C cycle.

[00158] Table 16. Example #4 Growth Data (P = growth positive, N = Growth negative) following exposure in ISO 132°C cycle.

[00159] Example #5 - Hydrogen Peroxide Monitoring

[00160] Sample Preparation

[00161] Six 3M™ Attest™1295 biological indicators were placed into a Tyvek™ polyethylene fiber pouch that was exposed to H2O2 vapor for 90 seconds of exposure in an otherwise empty Sterrad® 100NX sterilizer. Preliminary experiments showed that a 90-second exposure in the sterilizer was sufficient to kill all of the spores in the biological indicators. [00162] The carriers were then removed from 3 of the HaCE-exposed biological indicators and 3 unexposed (control) biological indicators and the carriers were placed in separate borosilicate glass vials with 1 mL of Butterfields buffer. The vials were then vortexed briefly and sonicated for 15 minutes in a water bath sonicator.

[00163] Sample Analysis

[00164] Duplicate samples (100 pL) of the buffer from each sonicated tube were then placed into separate wells of a clear bottom 96-well plate. 100pL of Propidium iodide (PI) only (30 mM) or Syto9 (10pM)+PI(30pM) was added to individual wells as shown in Table 9. Samples were mixed and immediately transferred to a 96-well plate for analysis using a BIOTEK synergy4 plate reader. The plate was sealed using an adhesive seal (Bio-Rad Life Sciences; Hercules, CA) The Optical density of each sample was measured at 600 nm. The Syto9 fluorescence was measured according to Reference Example 3. Propidium iodide fluorescence was measured using an excitation wavelength of 490 nm and an emission wavelength of 635 nm.

[00165] Results

[00166] The Optical Density (600 nm) and fluorescence (at 520nm and/or at 635 nm as indicated in

Table 17) were analyzed using the BioTek plate reader described above. The results for are shown in Table 17. Note the increase in signal from live (unexposed) to dead (¾0₂-exposed) for the syto9+PI samples. In addition, the response can be measured by taking the ratio of Syto9+PI/OD600 (using the 490ext/635em values for syto9+PI).

[00167] Table 17. Example #5 Data for hydrogen peroxide monitoring.

^A - Unexposed to sterilant

B - Exposed to sterilant

[00168] Example #6 - Detection using Electrochemical Methods - DNA Solutions Mimicking Different Levels of Spore DNA Available for Detection after Sterilization

[00169] Sample preparation: [00170] A sample stock solution of DNA (Lambda DNA available from Invitrogen, Waltham, MA) is prepared in a 50 mM phosphate buffer (40 mM K2HPO4 and 10 mM KH2PO4, pH 7.0) to a DNA concentration of 100 pg/mL. The stock solution is diluted with additional buffer to make a range of sample solutions with DNA concentrations of: 10, 1 and 0.1 pg/mL. A control sample solution containing no DNA is also prepared. The DNA sample solutions simulate samples of spore DNA that is released as a result of a sterilization process. The varying concentrations of DNA in the sample solutions simulate exposures to the sterilant for different exposure times. Hoechst 33258 DNA binding dye (available from VWR International, Radnor PA) is added to the DNA sample solutions as well as the control solution, to give a concentration of 1 pM for the Hoechst binding dye. The Hoechst binding dye concentration is constant (1 pM) for all DNA sample solutions as well as the control solution.

[00171] Sample analysis:

[00172] Because Hoechst 33258 is irreversibly oxidized, an oxidative linear sweep voltammetry (LSV) with scan is used for the electrochemical analysis of the DNA sample solutions. LSV is performed using a Metrohm AUTOLAB potentiostat (available from Metrohm, Riverview, FL) in the voltage range from 0 to 1.0 V at a sweep rate of 100 mV/s. The electrochemical cell consists of a Metrohm DropSense screen printed three electrode cell (DropSense 220AT available from Metrohm, Riverview, FL). The electrochemical cell has gold working and auxiliary electrodes and a silver reference electrode. The working electrode is 4mm in diameter. The DNA sample solutions are tested by placing a 100 pL drop of the sample solution onto the screen-printed electrochemical cell covering all three electrodes. After a five-minute equilibration at 0 volts, the LSV described above is initiated. The LSV analysis is repeated for the control solution containing no DNA. For each LSV analysis a new electrochemical cell is used.

[00173] Results:

[00174] The LSV curves will show oxidation of the Hoechst binding dye at 0.6V. The peak current measured in pA at this oxidation potential is always lower for the control solution in comparison to the DNA sample solutions. The ratio of the peak current at the oxidation potential for the series of DNA sample solution divided by the peak current of the control solution at that same potential increases proportionally with increasing DNA concentration in the sample solution series.

[00175] Example #7 - Detection using Electrochemical Methods - Spore Suspension

[00176] Sample Preparation:

[00177] Geobacillus stearothermophilus ATCC 7953 spores are suspended in sterile H2O (3.4xl0⁶ spores/mL) and aliquoted (2 mL volumes) into multiple glass test tubes. This experiment is performed in duplicate. Table 18 provides the six different exposure times at 121°C in a dynamic-air removal steam sterilization cycle (MidMark M9 available from MidMark, Traverse City, MI). Commercial 1492V biological indicators (available from 3M, St. Paul, MN) are used as a control. [00178] Table 18. Samples and steam SFPP exposure times

[00179] Sample Analysis:

[00180] Hoechst 33258 (available from VWR International, Radnor, PA) nucleic acid binding dye is added to aliquots of each test sample and LSV is immediately conducted. LSV is performed using a Metrohm AUTOLAB potentiostat (available from Metrohm, Riverview, FL) in the voltage range from 0 to 1.0 V at a sweep rate of 100 mV/s. The electrochemical cell consists of a Metrohm

DropSens screen printed three electrode cell (DropSense 220AT available from Metrohm, Riverview, FL). The electrochemical cell has gold working and auxiliary electrodes and a silver reference electrode. The working electrode is 4mm in diameter. The DNA sample solutions are tested by placing a 100pL drop of the test solution onto the screen-printed electrochemical cell covering all three electrodes. After a five-minute equilibration at 0 volts, the LSV described above is initiated.

The colony forming units for each test sample was determined by diluting the spore suspensions and surface-plating the dilutions onto Tryptic Soy Agar followed by incubation of the plates at 60°C.

[00181] Results

[00182] The LSV curves show oxidation of the Hoechst binding dye at 0.6V. Sample 6 results the lowest peak current at the oxidation potential. Sample 1 results in a peak current greater than sample 6 at the oxidation potential. This is because, even though the sterilization exposure is TO (meaning the cycle is stopped at the very beginning of the sterilization exposure phase of the cycle) the preconditioning portion of the cycle results in a very small amount of released DNA available for detection. The peak current for samples 2-5 is significantly larger than for sample 6, indicating greater amounts of DNA detected electrochemically. The peak current for samples 4 and 5 is comparable, indicating that for exposure times greater than 6 minutes all the possible DNA generated as a result of the sterilization cycle is detected electrochemically. 1492V commercial biological indicator controls are all growth negative at 2 minutes of exposure in this experiment.

[00183] Example #8 - Detection using Electrochemical Methods - Hydrogen Peroxide Monitoring

[00184] Sample Preparation [00185] Commercial 3M™ Attest™1295 biological indicators (available from 3M, St. Paul, MN) are exposed to VH202 in an incomplete Standard Cycle for the Sterrad 100NX (available from ASP, Irvine, CA) sterilizer providing a total of 90 seconds of exposure. Six of the BI's from this lot are packaged in a TYVEK® polypropylene fiber pouch and placed in an otherwise empty chamber. This cycle is run in order to kill the BI's.

[00186] The carriers are then removed from 3 of the exposed (Dead) and unexposed (Live) BI's and placed in separate borosilicate glass vials with 1 mL of Butterfields buffer. The vials are vortexed briefly and sonicated for 15 minutes in a water bath sonicator.

[00187] Sample Analysis

[00188] 100pL of each sample is placed in a microcentrifuge tube. To each sample, 100pL of a

200uM Hoechts 33258 DNA binding dye solution is added. In addition, a control sample with no Hoechts dye added is prepared for both Dead and Live samples. Samples are mixed and immediately tested using LSY. LSV is performed using a Metrohm AUTOLAB potentiostat (available from Metrohm, Riverview, FL) in the voltage range from 0 to 1.0 V at a sweep rate of 100 mV/s. The electrochemical cell consists of a Metrohm DropSens screen printed three electrode cell (DropSense 220AT available from Metrohm, Riverview, FL). The electrochemical cell has gold working and auxiliary electrodes and a silver reference electrode. The working electrode is 4mm in diameter. The DNA sample solutions are tested by placing a 100pL drop of the test solution onto the screen-printed electrochemical cell covering all three electrodes. After a five-minute equilibration at 0 volts, the LSV described above is initiated.

[00189] Results

[00190] Except for the control samples, the LSV curves show oxidation of the Hoechst binding dye at 0.6V. The control samples do not have an oxidation peak at 0.6V and result in negligible current flow at that potential. The peak current for the Dead samples is significantly larger than for Live samples, indicating greater amounts of DNA detected electrochemically.

[00191] Various modifications and alterations to this invention will become apparent to those skilled in the art without departing from the scope and spirit of this invention. It should be understood that this invention is not intended to be unduly limited by the illustrative embodiments and examples set forth herein and that such examples and embodiments are presented by way of example only with the scope of the invention intended to be limited only by the claims set forth herein.

Claims

What is claimed is:

1. A self-contained sterilization process biological indicator, comprising:

a cuvette having at least one liquid-impermeable wall that forms an opening into a compartment;

a predetermined number of sterilization process-resistant spores disposed in the compartment;

a liquid medium disposed in the compartment; and

a nucleic acid-interacting dye disposed in the compartment;

with the proviso that the self-contained sterilization process biological indicator does not include an effective amount of germination medium and/or microbial growth medium; wherein the opening is part of a pathway that permits passage of a sterilant gas from outside the cuvette into the compartment;

wherein the nucleic acid-interacting dye is fluorescent when bound to DNA or RNA.

2. The self-contained sterilization process biological indicator of claim 1, wherein the nucleic acid-interacting dye is disposed in the liquid medium.

3. The self-contained sterilization process biological indicator of claim 1 or claim 2, wherein the spores are disposed in the liquid medium.

4. The self-contained sterilization process biological indicator of claim 1 or claim 2, wherein the liquid medium is disposed in a container, wherein the container is disposed in the compartment.

5. The self-contained sterilization process biological indicator of claim 4, wherein the spores are disposed on a first surface in a substantially water-free first coating, wherein the first surface is disposed inside the compartment.

6. The self-contained sterilization process biological indicator of claim 5, wherein the nucleic acid-interacting dye is disposed in the first coating.

7. The self-contained sterilization process biological indicator of any one of the preceding claims, further comprising an enhancer reagent, wherein the enhancer reagent contacts the spores.

8. A method of determining effectiveness of a sterilization process, the method comprising:

positioning the self-contained sterilization process biological indicator of any one of claims 1 through 7 in a sterilization chamber;

while the indicator is positioned in the sterilization chamber, exposing the indicator to a sterilant gas;

contacting the spores with the nucleic acid-interacting dye;

after the exposing the indicator to the sterilant gas, measuring a first fluorescence intensity emitted by the nucleic acid-interacting dye in the indicator; and

comparing the first fluorescence intensity to a reference fluorescence intensity to determine whether the exposing the indicator to the sterilant gas was effective to kill all of the spores;

with the proviso that the method does not include, before measuring the first fluorescence intensity, incubating the spores in the indicator with a growth medium or germination medium for a sufficient period of time to cause germination or growth of the spores.

9. The method of claim 8, wherein the exposing the sterilization process indicator to a sterilant gas comprises exposing the sterilization process indicator to steam.

10. The method of claim 8 or claim 9, wherein contacting the spores with the nucleic acid-interacting dye comprises contacting the spores with the nucleic acid-interacting dye while exposing the stenlization process indicator to the sterilant gas.

11. The method of claim 8 or claim 9, wherein contacting the spores with the nucleic acid-interacting dye comprises contacting the spores with the nucleic acid-interacting dye after the exposing the sterilization process indicator to the sterilant gas.

12. The method claim 8 or claim 9,

wherein the liquid medium is contained in a container that is disposed in the compartment;

wherein the container is impermeable to the liquid medium;

wherein, prior to the exposing the self-contained sterilization process biological indicator to the sterilant gas, the method further comprises disintegrating the container to contact the spores with the liquid medium.

13. The method of claim 8 or claim 9, wherein the liquid medium is contained in a container that is disposed in the compartment;

wherein the container is impermeable to the liquid medium;

wherein, after exposing the self-contained sterilization process biological indicator to a sterilant gas during a sterilization process, the method further comprises disintegrating the container to contact the spores with the liquid medium.

14. The method of any one of claims 8 through 13, further comprising a step of measuring an amount of absorbance and/or scattering of an electromagnetic radiation when the electromagnetic radiation is directed through the liquid medium in the cuvette.

15. The method of any one of claims 8 through 14, wherein the reference fluorescence intensity is obtained by measuring a second fluorescence intensity.

16. The method of any one of claims 8 through 15, further comprising agitating the sterilization process indicator for a period of time.

17. The method of any one of claims 8 through 16, further comprising:

prior to the exposing the sterilization process indicator to the sterilant gas, positioning an article to be sterilized in the stenlization chamber.

18. A composition comprising a plurality of stenlization process-resistant microbial spores and a nucleic acid-interacting dye, wherein the composition is substantially water-free.

19. The composition of claim 18, wherein the plurality of sterilization process-resistant microbial spores comprises a predefined number of spores.

20. A method of determining effectiveness of a sterilization process, the method comprising:

positioning the self-contained sterilization process biological indicator of any one of the preceding claims in a sterilization chamber;

contacting the spores with the liquid medium;

contacting the spores with the nutrient composition; after contacting the spores with the nutrient composition, incubating the indicator at a predetermined temperature for a period of time sufficient to permit germination and at least one cell division of a germinated spore;

after the exposing the sterilization process biological indicator to the sterilant gas and after the contacting the spores with the liquid medium and before the incubating the indicator at the predetermined temperature, measuring a first electrical conductance of the liquid medium;

comparing the first electrical conductance to a reference electrical conductance to determine whether the exposing the sterilization process biological indicator to the sterilant gas was effective to kill all of the spores; and

after the incubating the sterilization process indicator at the predetermined temperature, detecting a presence, an absence, or a quantity of a parameter associated with a germinated spore.