A method for verifying the effectiveness of a sterilization process based on the presence of volatile organic compounds

Abstract

According to the present disclosure, there is provided a method for determining whether spores in a self-contained biological indicator are in a germination stage, the method comprising the steps of subjecting the self-contained biological indicator to a sterilization process, exposing spores in the self-contained biological indicator to a growth medium, culturing the spores in the self-contained biological indicator, sampling the headspace within the self-contained biological indicator, and determining whether volatile organic compounds resulting from the germination stage of the spores in the self-contained biological indicator are released into the headspace upon contacting the spores with the growth medium, wherein the presence of volatile organic compounds resulting from the germination stage of the spores in the self-contained biological indicator indicates a failure of the sterilization process.

Description

[Technical Field]

[0001] (CROSS-REFERENCE TO RELATED APPLICATIONS) This application claims priority to U.S. Provisional Application No. 63 / 411,990, filed September 30, 2022, the disclosure of which is incorporated herein by reference.

[0002] (Technical field) The present invention generally relates to a method for verifying the Sterility Assurance Level of a sterilization process based on the presence of one or more volatile organic compounds as indicators of spore germination. [Background technology]

[0003] Biological indicators are used to monitor whether the conditions necessary for sterilization of medical devices, instruments, and other products requiring sterility are being met. Bacterial spores are the most resistant to the various sterilization processes currently in use. Therefore, most biological indicators (BIs) used to monitor sterilization processes are made from bacterial spores. Because the inactivation mechanisms of various sterilization processes differ, various bacterial strains have been selected to monitor the status of various sterilization processes. For example, biological indicators for steam or oxidation sterilization processes typically include spores of Geobacillus stearothermophilus (formerly known as Bacillus stearothermophilus), biological indicators for ethylene oxide sterilization processes typically include spores of Bacillus atrophaeus (formerly known as Bacillus subtilis), and biological indicators for radiation sterilization processes typically include spores of Bacillus pumilus.

[0004] Understanding the spore reproduction process is essential for developing methods to detect viable spores and determine whether a sterilization process has failed. Dormant spores sense their surrounding environment and germinate when conditions are favorable for growth. This occurs when the spore comes into contact with a growth medium and activates a biological indicator. The spore reproduction process is generally divided into two major stages: the first stage is the germination stage (germination and maturation), and the second stage is the growth stage. Depending on the bacterial strain, it can take up to two hours for a spore to revert to a vegetative cell capable of self-replication. Spore germination is triggered by exposure to a nutrient supplement, which occurs when germinant molecules, including low-molecular-weight amino acids, sugars, and purine nucleosides, are sensed by germinant receptors (GRs) present in the spore's inner membrane. Because germination of individual spores in a population of any species is heterogeneous, some spores require more time to germinate. In either case, the germination stage begins when the spores rehydrate, releasing dipicolinic acid (DPA) and partially hydrating the core. Approximately 50% of the spores lose their resistance to heat and chemicals within 15 minutes of the start of the incubation process (activation), indicating that they have undergone the germination stage.

[0005] The germination phase is followed by a maturation phase, which involves cortex hydrolysis, core hydration, core swelling, loss of dormancy, and loss of resistance. During the first 20–40 minutes of spore germination, up to 20% of dormant spore proteins are degraded to free amino acids, 90% of which are low-molecular-weight nonenzymatic glycoproteins (SAPs). These are used to synthesize small molecules such as nucleotides and new proteins. Proteins are synthesized during germination without the need for external nutrients. After the maturation phase, i.e., approximately 60 minutes after spore incubation (activation), the growth phase begins. This is the stage in which spores activate macromolecular synthesis to become vegetative cells and emerge from the decaying spore coat. Spores exposed to sublethal concentrations of sterilants (e.g., steam, H2O2, EtO) typically exhibit inhibition of the growth phase, rather than the germination phase. Even spores exposed to high concentrations of EtO are able to germinate freely under various conditions but are unable to grow. Therefore, detection of spore germination is more sensitive than detection of spore growth and replication.

[0006] Detection of the presence of viable spores after sterilization was initially based on the proliferation of spores to form vegetative cells and subsequent vegetative cell growth. Subsequently, to more quickly determine whether a sterilization process was effective, new methods were developed that determined the failure of the sterilization process under test if dormant spores entered the germination stage. However, these conventional methods required the implementation of fluorescence-based enzyme assays, which required additional steps, such as the addition of specific substrates, inducers, or sources of biological activity (e.g., microorganisms such as spores, enzymes, microbial metabolites, or ATP) or the modification of spores to incorporate target enzymes (genes and / or proteins). Furthermore, detection of enzyme activity could only indirectly detect spore germination. Thus, these conventional methods potentially introduced inaccuracies, additional steps, and other complications.

[0007] Therefore, there is a need for a method that can directly determine whether a sterilization process (e.g., a sterilization process using steam, hydrogen peroxide vapor, ethylene oxide (EtO), ozone, NO2, supercritical CO2, peracetic acid, or other liquids that meet the requirements to be defined as a sterilant, or any combination thereof) has entered the germination stage, which indicates failure, without modifying the spores or adding germination inhibitors, and that can provide rapid results in near real time. Summary of the Invention [Means for solving the problem]

[0008] Aspects and advantages of the invention will be set forth in part in the description that follows, or will be obvious from the description, or may be learned by practice of the invention.

[0009] In one embodiment of the present invention, a method for determining whether spores in a self-contained biological indicator are in a germination stage is provided, the disclosed method comprising the steps of subjecting the self-contained biological indicator to a sterilization process, exposing spores in the self-contained biological indicator to a growth medium, culturing the spores in the self-contained biological indicator, sampling the headspace within the self-contained biological indicator, and determining whether volatile organic compounds resulting from the germination stage of the spores in the self-contained biological indicator are released into the headspace upon contacting the spores with the growth medium, wherein the presence of volatile organic compounds resulting from the germination stage of the spores in the self-contained biological indicator indicates a failure of the sterilization process.

[0010] In one embodiment, the step of determining whether volatile organic compounds are released from the growth medium into the headspace includes collecting an air sample from the headspace for analysis of the volatile organic compounds.

[0011] In another embodiment, the sampling occurs within a self-contained biological indicator.

[0012] In another embodiment, sampling occurs external to the self-contained biological indicator.

[0013] In another embodiment, the air samples are collected and analyzed over a time frame of less than about 60 minutes.

[0014] In another embodiment, the sterilization process is a steam sterilization process.

[0015] In another embodiment, the sterilization process is a hydrogen peroxide gas sterilization process.

[0016] In another embodiment, the sterilization process is an ethylene oxide sterilization process.

[0017] In another embodiment, the self-contained biological indicator comprises Geobacillus stearothermophilus or Bacillus stearothermophilus spores.

[0018] In another embodiment, the growth medium comprises tryptic soy broth or modified soy casein digest broth.

[0019] In another embodiment, the volatile organic compound is polar.

[0020] In another embodiment, the method of the present disclosure further comprises applying a correction factor to remove background volatile organic compounds that result from the growth medium rather than the germination stage of the spores in the self-contained biological indicator.

[0021] In another embodiment, the volatile organic compounds resulting from the germination stage of the spores of the self-contained biological indicator include an alkane, an alcohol, an ester, a ketone, a furan, or any combination thereof. For example, the volatile organic compounds resulting from the germination stage of the spores of the self-contained biological indicator include 2-pentanone, methyl isobutyl ketone, 4-methyl-2-heptanone, 2-methyl-2-propanol, amylene hydrate, 3-hydroxy-2,4,4-trimethylpentyl 2-methylpropanoate, 2,2,4-trimethyl-1,3-pentanediol diisobutyrate, 2-methyl-1,3-pentanediol, tetrahydro-2,2,5,5-tetramethylfuran, or any combination thereof.

[0022] In another embodiment, the self-contained biological indicator comprises spores in a cell number that will achieve a desired resistance in the indicator depending on the type of sterilization process in which the indicator is used.

[0023] In another embodiment, the presence of volatile organic compounds is determined directly without the use of enzymatic reagents.

[0024] In another embodiment, the presence of volatile organic compounds is determined directly without the use of fluorescent moieties.

[0025] In another embodiment, the step of determining whether volatile organic compounds resulting from the germination stage of spores in the self-contained biological indicator are released into the headspace when the spores are contacted with growth medium comprises comparing the measured concentration of the volatile organic compounds to a predetermined control concentration of the compounds.

[0026] In another embodiment, if the measured concentration of the volatile organic compound is greater than the control concentration of the compound, it is determined that the volatile organic compound resulting from the germination stage of the spores in the biological indicator was released into the headspace when the spores were contacted with the growth medium.

[0027] Another embodiment of the present invention provides a method for determining whether sterilization was successful, comprising the steps of subjecting a self-contained biological indicator to a sterilization process, exposing spores in the self-contained live biological indicator to a growth medium, culturing the spores in the self-contained biological indicator, sampling the headspace of the self-contained biological indicator, and measuring the concentration of volatile organic compounds released into the headspace when the spores are contacted with the growth medium.

[0028] In one embodiment, the step of measuring the concentration of volatile organic compounds comprises collecting an air sample from the headspace for analysis of the volatile organic compounds.

[0029] In another embodiment, the sampling occurs within a self-contained biological indicator.

[0030] In another embodiment, sampling occurs external to the self-contained biological indicator.

[0031] In another embodiment, the air samples are collected and analyzed over a time frame of less than about 60 minutes.

[0032] In another embodiment, the sterilization process is determined to be successful if the concentration of volatile organic compounds increases compared to a predetermined baseline concentration.

[0033] In another embodiment, the sterilization process is a steam sterilization process, a hydrogen peroxide gas sterilization process, or an ethylene oxide sterilization process.

[0034] In another embodiment, the self-contained biological indicator comprises Geobacillus stearothermophilus or Bacillus stearothermophilus spores.

[0035] In another embodiment, the growth medium comprises tryptic soy broth or modified soy casein digest broth.

[0036] In another embodiment, the volatile organic compound is non-polar.

[0037] In another embodiment, the volatile organic compounds include cyclododecanol; benzoic acid, 4-ethoxy-, ethyl ester; 3,4,4-trimethyl-3-pentanol; or any combination thereof.

[0038] In another embodiment, the presence of volatile organic compounds is determined directly without the use of enzymatic reagents.

[0039] In another embodiment, the presence of volatile organic compounds is determined directly without the use of fluorescent moieties.

[0040] These and other features, aspects, and advantages of the present invention will become better understood with reference to the following description and appended claims. The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and, together with the description, serve to explain the principles of the invention. [Brief explanation of the drawings]

[0041] A full and enabling disclosure of the present invention, including the best mode thereof, directed to one of ordinary skill in the art, is set forth in this specification, which makes reference to the accompanying drawings, in which:

[0042] [Figure 1A] Figure 1A shows the total ion chromatogram (TIC) used to examine the headspace volatile organic compounds emitted from 48,000 viable G. stearothermophilus spores obtained from manufacturer A in a 10-fold dilution of soybean casein digest broth growth medium (upper graph) and a negative control of 48,000 steam-sterilized G. stearothermophilus spores in the same 10-fold dilution of growth medium (incubated at 60°C for 20 minutes to germinate) (lower graph). [Figure 1B]Figure 1B shows the total ion chromatogram (TIC) used to examine the headspace volatile organic compounds emitted from 24,000 viable G. stearothermophilus spores obtained from manufacturer B in a 1:10 dilution of soybean casein digest broth growth medium (upper graph) and a negative control of 24,000 steam-sterilized G. stearothermophilus spores obtained from manufacturer B in the same 1:10 dilution of growth medium (incubated at 60°C for 20 minutes to germinate) (lower graph). [Figure 2A] Figure 2A shows the total ion chromatogram (TIC) used to examine the headspace volatile organic compounds emitted from 42,000 viable G. stearothermophilus spores obtained from manufacturer C in a 1:10 dilution of soybean casein digest broth growth medium (upper graph) and a negative control of 42,000 steam-sterilized G. stearothermophilus spores obtained from manufacturer C in the same 1:10 dilution of growth medium (incubated at 60°C for 20 minutes to germinate) (lower graph). [Figure 2B] Figure 2B shows the total ion chromatogram (TIC) used to examine the headspace volatile organic compounds emitted from 14,000 viable B. atrophaeus spores obtained from manufacturer A in a 1:10 dilution of soybean casein digest broth growth medium (upper graph) and a negative control of 14,000 ethylene oxide-sterilized G. stearothermophilus spores obtained from manufacturer A in the same 1:10 dilution of growth medium (incubated at 60°C for 20 minutes to germinate) (lower graph). [Figure 3] FIG. 3 shows the differences in headspace VOC signatures in germinated suspensions and background growth media of different types of biological indicators as shown by principal component analysis (PCA). [Figure 4]FIG. 4 shows four total ion chromatograms (TICs) collected from the headspace VOCs of Manufacturer E growth medium, Manufacturer D growth medium, Manufacturer A growth medium, and Manufacturer C growth medium diluted 10-fold (DF) (all growth media are modified tryptic soy broth media). [Figure 5] FIG. 5 is a graph showing the relative peak intensities of various VOC groups emitted from the various spore growth media of FIG. [Figure 6] FIG. 6 shows three total ion chromatograms (TICs) collected from headspace VOCs in Manufacturer A's growth medium (background), 250 G. stearothermophilus spores cultured in Manufacturer A's growth medium, and 25,000 G. stearothermophilus spores cultured in Manufacturer A's growth medium. [Figure 7] FIG. 7 shows three total ion chromatograms (TICs) collected from headspace VOCs for 10-fold diluted (DF) Manufacturer C growth medium (background), 250 G. stearothermophilus spores cultured in 10-fold diluted (DF) Manufacturer C growth medium, and 25,000 G. stearothermophilus spores cultured in 10-fold diluted (DF) Manufacturer C growth medium. [Figure 8A] FIG. 8A shows the relationship between the number of spore cells and the peak intensity of the VOC methyl ethyl disulfide when G. stearothermophilus spores from Manufacturer A were cultured in Manufacturer A's growth medium. [Figure 8B] Figure 8B shows the relationship between spore cell number and peak intensity of the VOC 6-methyl-3-heptanone when G. stearothermophilus spores from manufacturer A were cultured in 10-fold diluted (DF) growth medium from manufacturer C. [Figure 8C] Figure 8C shows the relationship between spore cell number and peak intensity for the VOC benzaldehyde, 2,4-dimethyl-, when G. stearothermophilus spores from manufacturer A were cultured in manufacturer A's growth medium. [Figure 8D] Figure 8D shows the relationship between spore cell number and peak intensity for the VOC butanethioic acid, S-methyl ester, when G. stearothermophilus spores from manufacturer A were cultured in 10-fold diluted (DF) growth medium from manufacturer C. [Figure 9] FIG. 9 is a graph showing the relative peak intensities of various VOC groups emitted from spores when 250 G. stearothermophilus spores from Manufacturer A were cultured in background medium, Manufacturer A's growth medium, and when 25,000 G. stearothermophilus spores from Manufacturer A were cultured. [Figure 10] FIG. 10 is a graph showing the relative peak intensities of various VOC groups emitted from spores when 250 G. stearothermophilus spores from Manufacturer A were cultured in background medium, 10-fold diluted (DF) Manufacturer C growth medium, and when 25,000 G. stearothermophilus spores from Manufacturer A were cultured. [Figure 11] FIG. 11 is a graph showing the relative peak intensities of various VOC groups emitted by 250 G. stearothermophilus spores from Manufacturer A cultured in background medium, 10-fold diluted (DF) Manufacturer C growth medium, and 25,000 G. stearothermophilus spores from Manufacturer A cultured in background medium. [Figure 12] FIG. 12 is a graph showing the relative peak intensities of various VOC groups emitted by spores when 450 G. stearothermophilus spores from manufacturer B were cultured in background medium, 10-fold diluted (DF) growth medium from manufacturer C, and when 45,000 G. stearothermophilus spores from manufacturer B were cultured. [Figure 13] FIG. 13 is a graph showing the relative peak intensities of various VOC groups emitted from spores when 700 G. stearothermophilus spores from Manufacturer C were cultured in background medium, 10-fold diluted (DF) Manufacturer C growth medium, and when 42,000 G. stearothermophilus spores from Manufacturer C were cultured. [Figure 14] FIG. 14 shows three total ion chromatograms (TICs) collected from headspace VOCs released from Manufacturer A's G. stearothermophilus spores grown in ten-fold diluted (DF) Manufacturer C's growth medium with no steam sterilization, one cycle of steam sterilization (121° C. for 10 minutes), and two cycles of steam sterilization (121° C. for 10 minutes). [Figure 15] FIG. 15 shows three total ion chromatograms (TICs) collected from headspace VOCs released from Manufacturer B G. stearothermophilus spores grown in Manufacturer C growth medium at 10-fold dilution (DF) with no steam sterilization, one cycle of steam sterilization, and two cycles of steam sterilization. [Figure 16] FIG. 16 shows three total ion chromatograms (TICs) collected from headspace VOCs released from Manufacturer A's G. stearothermophilus spores grown in Manufacturer C's growth medium at 10-fold dilution (DF) with no steam sterilization, one cycle of steam sterilization, and two cycles of steam sterilization. [Figure 17] FIG. 17 shows three total ion chromatograms (TICs) collected from headspace VOCs released from Manufacturer B G. stearothermophilus spores grown in Manufacturer C growth medium at 10-fold dilution (DF) with no steam sterilization, one cycle of steam sterilization, and two cycles of steam sterilization. [Figure 18A] FIG. 18A shows the calibration curve for amylene hydrate, a VOC standard used to quantify the VOCs produced from the germ suspensions tested. [Figure 18B] FIG. 18B shows the calibration curve for 2-pentanone, a VOC standard used to quantify the VOCs produced from the germ suspensions tested. [Figure 18C]FIG. 18C shows the calibration curve for 2,3-dimethyl-2-butanol, a VOC standard used to quantify the VOCs produced from the spore suspensions tested. [Figure 18D] FIG. 18D shows the calibration curve for methyl isobutyl ketone, a VOC standard used to quantify the VOCs produced from the germ suspensions tested. [Figure 18E] FIG. 18E shows the calibration curve for 4-methyl-2-heptanone, a VOC standard used to quantify the VOCs produced from the germ suspensions tested. [Figure 19] FIG. 19 is a flow chart showing VOC production during fermentation. [Figure 20A] FIG. 20A is a graph showing the increase in cyclododecanol peak intensity with increasing number of sterilization cycles. [Figure 20B] FIG. 20B is a graph showing the increasing intensity of the benzoic acid, 4-ethoxy and ethyl ester peaks with increasing number of sterilization cycles. DETAILED DESCRIPTION OF THE INVENTION

[0043] Various embodiments of the present invention and one or more examples thereof are described in detail below. Each example is provided to explain the invention, not to limit it. For example, features illustrated or described as part of one embodiment can be used with another embodiment to yield a still further embodiment. Thus, it is intended that the present invention cover such modifications and variations insofar as they come within the scope of the appended claims and their equivalents.

[0044] As used herein, the terms "about," "approximately," or "approximately," when used to modify a value, indicate that values ​​within ±5% of that value are within the scope of the disclosed embodiments. Furthermore, when multiple ranges are provided, any combination of the minimum and maximum values ​​set forth in the ranges is also contemplated by the present invention. For example, when a range of "about 20% to about 80%" and a range of "about 30% to about 70%" are provided, the range of "about 20% to about 70%" or "about 30% to about 80%" is also contemplated by the present invention.

[0045] Generally, the present invention relates to a method for determining whether spores in a self-contained biological indicator are in the germination stage, even when very low concentrations of spores are activated and transition to the growth stage. The disclosed method includes subjecting the self-contained biological indicator to a sterilization process, exposing the spores in the self-contained biological indicator to a growth medium, culturing the spores in the self-contained biological indicator, sampling the headspace within the self-contained biological indicator, and determining whether volatile organic compounds resulting from the germination stage of the spores in the self-contained biological indicator are released into the headspace when the spores are contacted with the growth medium. The presence of volatile organic compounds resulting from the germination stage of the spores in the self-contained biological indicator indicates a failure of the sterilization process. The sterilization process can use steam, hydrogen peroxide gas, ethylene oxide ozone, NO, supercritical CO, peracetic acid, or any other liquid or gas that meets the requirements for being defined as a sterilant, or any combination thereof. The biological indicator is sterilized together with the item to be sterilized during the sterilization process, and after the sterilization process is completed, it is introduced into the growth medium. By collecting air samples above the growth medium and analyzing them for the presence of certain volatile organic compounds (VOCs), it is possible to verify or determine whether the sterilization process was successful. This is because when spores are in the germination stage, certain VOCs are released into the headspace, which means that the spores are active and have not been killed by the sterilization process.

[0046] In another embodiment, the present invention relates to a method for determining whether sterilization has been successful. The disclosed method includes the steps of subjecting a self-contained biological indicator to a sterilization process, exposing spores within the self-contained live biological indicator to a growth medium, culturing the spores within the self-contained biological indicator, sampling the headspace of the self-contained biological indicator, and measuring the concentration of volatile organic compounds released into the headspace when the spores are contacted with the growth medium. The presence of volatile organic compounds resulting from the germination stage of the spores within the self-contained biological indicator indicates a failure of the sterilization process. The sterilization process can use steam, hydrogen peroxide gas, ethylene oxide ozone, NO, supercritical CO, peracetic acid, or any other liquid or gas that meets the requirements defined as a sterilant, or any combination thereof. The biological indicator is subjected to the sterilization process along with the item to be sterilized during the sterilization process and introduced into the growth medium after the sterilization process is completed. The success of the sterilization process can be verified or determined by collecting air samples above the growth medium and analyzing them for the presence of specific volatile organic compounds (VOCs). This is because when spores are in the germination stage, certain VOCs are released into the headspace, which means that the spores are active and not killed by the sterilization process.

[0047] The present inventors have discovered that the disclosed methods can improve detection of the growth stage or biological activity of hardy microorganisms (e.g., spores (endospores or bacterial spores)) by focusing on the detection and measurement of volatile organic compounds (VOCs). Examples of spores include, but are not limited to, Bacillus atrophaeus and Geobacillus stearothermophilus (e.g., Bacillus stearothermophilus, Bacillus megaterium, Bacillus coagulans, Clostridium sporogenes, Bacillus pumilus, or any combination thereof). If the sterilization process fails, specific volatile organic compounds (VOCs) are produced and rapidly detected in the headspace of the container containing the growth medium and biological indicator. For example, in some instances, a concentration of a specific VOC above a predetermined value can indicate a failed sterilization process or sterilization cycle. Meanwhile, in other instances, an increase in the concentration of a specific VOC over time can indicate a successful sterilization process or cycle. This reduces the time required to detect and report growth or biological activity after sterilization to less than about 30 minutes, e.g., less than about 15 minutes. This time is much faster than conventional methods that require culturing the growth medium for turbidity, which can take 2-7 days to confirm successful sterilization if turbidity is lacking. Furthermore, the disclosed method also eliminates the need for fluorescence measurements, which can take several hours depending on the measurement system. Fluorescence measurements, however, introduce complications and room for error due to the use of various reagents and enzymes in the multiple process steps that must be performed. In other words, the disclosed method directly measures the presence of volatile organic compounds (VOCs) without the use of extra steps or reagents.

[0048] The biological indicator system of the present disclosure contemplates the use of Geobacillus stearothermophilus or Bacillus atrophaeus spores, although other spores may also be used in the present invention. Additionally, the biological indicator may contain at least 100,000 spores for steam sterilization and at least 1 million spores for ethylene oxide and hydrogen peroxide sterilization.

[0049] On the other hand, the growth medium into which the biological indicator is introduced to determine whether spores are in the germination stage to verify or analyze the success of the sterilization process can be any suitable growth medium (e.g., growth medium from manufacturer A, B, C, D, or E), such as, but not limited to, tryptic soy broth or modified soybean casein digest broth. In some embodiments, the growth medium can be diluted (e.g., diluted 1x, 2x, 10x) to minimize the effect of background VOCs that may be present in the growth medium.

[0050] After the biological indicator is introduced into the growth medium, the growth medium / biological indicator combination is heated to a temperature in the range of about 50° C. to about 65° C., e.g., about 55° C. to about 60° C., for steam and hydrogen peroxide sterilization, or about 25° C. to about 40° C., e.g., about 30° C. to about 35° C., for ethylene oxide gas sterilization.

[0051] After contacting the biological indicator with the growth medium (activation), the biological indicator is incubated at a temperature range optimal for spore germination. The optimal temperature range for spore germination is approximately 50°C to approximately 65°C for Geobacillus stearothermophilus spores and approximately 25°C to approximately 40°C for Bacillus atrophaeus spores. The incubation is conducted for a time period of less than approximately 60 minutes, e.g., less than approximately 20 minutes, e.g., approximately 1 minute to approximately 20 minutes, e.g., approximately 3 minutes to approximately 17 minutes, e.g., approximately 5 minutes to approximately 15 minutes. During this incubation period, air samples are collected from the headspace above the growth medium for VOC analysis. During this incubation period, air samples from the headspace can be collected using any suitable sampling platform. Sampling platforms include, for example, absorbent films that generate a measurable electrical signal (e.g., to measure changes in resistance levels) and fibers for solid-phase microextraction / gas chromatography-mass spectrometry (SPME / GC-MS). The measured concentration of a particular VOC can then be compared to a control concentration of that VOC in growth medium without the presence of spores and / or active spores to determine whether the VOC is due to the germination phase of the biological indicator, i.e., whether it indicates a sterilization failure. For example, if the measured concentration of a particular VOC is higher than the control concentration of that VOC, it can be determined that the VOC was released from the growth medium into the headspace due to the germination phase of the biological indicator. It should be understood that in addition to the VOC concentration associated with the growth medium, the control concentration of the VOC can also include VOC concentrations due to other non-spore components of the biological indicator, such as, but not limited to, the cap, filter, sleeve, spore carrier, and VOC measurement board.

[0052] Specific VOCs detectable by the method of the present invention, which indicate sterilization failure due to the presence of spores in the growth medium, can be polar. These specific VOCs include ketones, alcohols, esters, and furans. In particular, VOCs detectable in the presence of spores, regardless of the type of growth medium used, include ketones such as 2-pentanone, methyl isobutyl ketone, and 4-methyl-2-heptanone; alcohols such as 2-methyl-2-propanol, amylene hydrate, and 2-methyl-1,3-pentanediol; esters such as 3-hydroxy-2,4,4-trimethylpentyl 2-methylpropanoate and 2,2,4-trimethyl-1,3-pentanediol diisobutyrate; and furans such as tetrahydro-2,2,5,5-tetramethylfuran. It should further be appreciated that the methods of the present invention may utilize a correction factor in the form of an algorithm to subtract the baseline concentration of a VOC from the concentration of the VOC released from the growth medium in order to distinguish between VOCs released from spores during germination and VOCs present only in the growth medium.

[0053] The present invention may be better understood with reference to the following examples.

[0054] Example

[0055] material

[0056] Growth media from manufacturers A, B, C, D, and E SPME fiber: A 50 / 30 μm divinylbenzene / carboxene (DVB / CAR) / PDMS (Sigma-Aldrich, catalog number: 57348-U) coated solid-phase microextraction (SPME) fiber was used for VOC extraction from the headspace. Three commercially available biological indicator (BI) products for steam-based sterilization, two for ethylene oxide-based sterilization, and one for hydrogen peroxide-based sterilization were obtained: spore suspensions from manufacturers A, B, and C were used as self-contained biological indicators. VOC standards and HPLC water: All VOC standards and HPLC-grade water were obtained from Sigma-Aldrich. They were used as is without further treatment.

[0057] Sample preparation

[0058] Stock spore suspensions were prepared by suspending the target spores in HPLC water. Depending on the form factor of the biological indicator (BI) product, this could be done by suspending the BI carrier in HPLC water or by adding HPLC water to the BI container to remove any adhering spores. Spore suspensions prepared from BIs for steam sterilization and BIs for hydrogen peroxide vapor were heat-shocked at 95°C for 15 minutes. The 15-minute heat shock treatment began when the spore suspension reached 85°C. Spore suspensions prepared from BIs for ethylene oxide sterilization were heat-shocked at 80°C for 10 minutes. The 10-minute heat shock treatment began when the spore suspension reached 70°C. After the heat shock treatment, the mixture was cooled to room temperature. The cooled mixture was then serially diluted six times in 10-fold steps. To confirm the target spore concentration, 20 μl of each dilution was plated (in triplicate) on a TSA plate. TSA plates were incubated at 60°C or 37°C (depending on spore type) for 24 hours, after which the cells were counted. All dilutions were labeled with the appropriate concentration and stored at 4°C until use. All spore suspensions used for GC-MS analysis were examined to confirm the number of spores in the GC-MS samples.

[0059] To establish a baseline for VOCs in sterilized BIs, each type of spore was sterilized according to the manufacturer's protocol, and the processed spores were recovered by HPLC water. For baseline testing, sterilized spores (plated to confirm death / lack of growth) were inoculated into growth medium. This allows for detection of VOCs generated by unsterilized spores (plated to confirm cell concentration) within 15 minutes of the germination stage. Tests were also conducted to determine whether BIs sterilized with one or two cycles produced different VOCs.

[0060] All spore samples for GCMS were prepared in airtight 10 ml glass vials containing 500 μl to 1 ml of growth medium. 40–50 μl of spore suspension was added to the vials in real time, 2–3 min before the start of a 15-min incubation / fiber VOC collection. The sample spore concentrations inoculated into the growth medium were targeted at two ranges: a low concentration range (100–900 spores / vial) and a high concentration range (10,000–90,000 spores / vial). The spore suspension concentration was confirmed after heat shock treatment and again before sample storage. The spore suspension was spread onto TSA plates and incubated (in triplicate) at 60°C or 37°C for 24 h, depending on the spore type.

[0061] Headspace VOCs

[0062] Headspace VOC analysis was performed using an SPME / GC-MS method using an Agilent 8890GC coupled to an Agilent 7250 Q-TOF system (Agilent, Santa Clara, CA, USA) equipped with a Gerstel TDU / CIS autosampler (Gerstel GmbH & Co., Germany). This system was coupled to a sample preparation system (Gerstel GmbH, Germany) for incubation and sample mixing. At the start of the incubation, an SPME fiber was inserted into the headspace of the sample vial. The sample vial was stirred at 60 °C or 37 °C to extract the VOCs. All fibers were conditioned in the GC injection port before use according to the manufacturer's guidelines.

[0063] The fiber collected VOCs from the headspace over a 15-minute period and desorbed them into the GC injector at 250 °C for 3 minutes. Chromatographic separation was performed using a DB-5 GC column (L × ID 30 m × 0.25 mm, film thickness 0.25 μm, Agilent Technologies, Palo Alto, CA, USA). The GC oven temperature was programmed to increase from 40 °C to 280 °C. Helium was used as the carrier gas, and the column flow rate was constant at 1.2 ml / min. The transfer line temperature was maintained constant at 220 °C. Compounds exiting the column were ionized by 70 eV electron impact and detected with a time-of-flight mass spectrometer with mass-to-charge ratios (m / z) ranging from 45 to 300 thomsons. The mass-to-charge ratios were chosen to avoid background from N2 and CO2. To detect additional potential compounds of interest in the VOCs collected from the headspace, data were processed using Agilent MassHunter Unknowns Analysis Version 10.0 software with the SureMass algorithm and NIST17 EI library search. Agilent Mass Profiler Professional (MPP) Version 15.1 software was used to compare and extract significantly different VOCs between different groups. MPP was also used to perform statistical analyses, such as ANOVA, PCA, fold analysis, volcano plots, hierarchical trees, self-organizing maps, and various methods for class prediction.

[0064] result:

[0065] SPME / GC-MS for Microbial VOC Testing

[0066] Monitoring bacterial volatile emissions has been facilitated by the use of SPME / GC-MS. SPME is an extraction method that combines analyte sampling, extraction, and concentration. SPME-based extraction of VOCs from headspace is a passive sampling method that does not interfere with the sample. In addition, this method offers the advantage of automation, improving sensitivity and consistency. The polarity of the adsorbent on the fiber, ranging from nonpolar PDMS to polar polyacrylate coatings, can be adjusted based on the application. Based on the fact that VOCs of various polarities are produced by microorganisms, a PDMS / DVB / CAR fiber with a moderate polarity was selected. Furthermore, this type of fiber has been reported to have the inherent ability to extract a wide range of microbial-derived VOCs, including low-molecular-weight compounds.

[0067] VOCs of germinating spores

[0068] Spore suspensions were prepared from steam sterilization BIs obtained from manufacturers A, B, and C, ethylene oxide (ETO) sterilization BIs obtained from manufacturer A, and vaporized hydrogen peroxide (VHP) sterilization BIs obtained from manufacturers A and B. To examine the release of spore-derived VOCs during germination, the heat-activated spore suspensions were incubated at their optimal temperatures (60°C for steam sterilization and VHP sterilization BIs, 37°C for ETO sterilization BIs), and VOCs were collected from the headspace for 15 min using an SPME fiber. Because the spore suspensions were heat-activated, the spore germination rate and extent increased. Because spore germination typically occurs between 0 and 15 min, VOC collection from the headspace was limited to 15 min. After 15 min of headspace VOC extraction, the SPME fiber was heated at 250°C for 3 min at the GC inlet to desorb attached VOCs. The desorbed VOCs were introduced into the GC column by the carrier gas at a rate of 1.2 ml / min. For details of GC-MS data collection and analysis, see the Experimental Section.

[0069] In this study, 40 μl of a known concentration of heat-activated spore suspension was added to 1 ml of 10x diluted medium from Manufacturer C in a 10 ml glass vial. The average number of spores observed in the high concentration range per sample vial was 48,000 for Manufacturer A (for steam sterilization), 24,000 for Manufacturer B (for steam sterilization), 42,000 for Manufacturer C (for steam sterilization), and 14,000 for Manufacturer A (for ethylene oxide sterilization). The average number of spores observed in the low concentration range was 100-fold lower than the corresponding number of spores in the high concentration range. Negative control samples were prepared in the same manner by adding the high concentration range to the sterilized spore suspension. Total ion chromatograms (TICs) for the background (negative control) and viable spore-containing (positive) samples are shown in Figures 1A-1B and 2A-2B.

[0070] The TICs collected from positive samples showed significantly higher peak counts and peak intensities than the negative controls. To extract VOCs from the TICs, the SureMass algorithm was applied, and features were extracted by spectral analysis. The number of features extracted from positive and negative spore samples is shown in Table 1. The number of features for negative samples was approximately 180. Meanwhile, the number of features for positive samples was highly dependent on the type of BI.

[0071] The MPP was used to compare the characteristics obtained from the positive and negative samples. To extract VOCs released from spores, the following two selection criteria were used: (a) the peak intensity of the spore-containing sample differs by at least two-fold compared to the negative sample (either an increase or decrease in intensity); and (b) the VOC is present in more than 80% of the positive samples. Based on these selection criteria, the number of VOCs associated with viable spores is shown in Table 1.

[0072] Common examples of VOCs released by spores include 2-methyl-2-propanol, acetic acid, pentyl esters, amylene hydrate, methyl isobutyl ketone, and tetrahydro-2,2,5,5-tetramethylfuran. The results of the above tests clearly demonstrate the release of VOCs from spores during germination. As demonstrated by the principal component analysis (PCA) shown in Figure 3, VOC signatures can be used to identify viable BIs from sterilized samples and differentiate between the types of BIs used in the sterilization process.

[0073] [Table 1]

[0074] Effect of growth medium and cell number

[0075] Growth media play an important role in volatile production and function by influencing metabolism and growth rate. The growth media itself emits significant amounts of background VOCs, such as pyrazine VOCs, which are by-products of sterilization of the growth media by autoclaving amino acids and reducing sugars. We investigated the profile of background VOCs emitted from various growth media from manufacturers A, C, D, and E. In this study, 500 μl of growth media was placed in a 10 ml glass vial, and the same VOC extraction method as for headspace VOC analysis was used.

[0076] The number of background VOCs is shown in Table 2, and the corresponding TICs are shown in Figure 4. It can be clearly seen that the growth media from manufacturers D and E have both higher peak intensities and higher peak counts compared to the media from manufacturer A and the 10x diluted media from manufacturer C. As shown in Figure 5, the growth media from manufacturers A and E were observed to contain more S-containing compounds. The growth media from manufacturer D contained very high relative levels of pyrazine-related compounds. The growth media from manufacturers A and C contained higher relative levels of ester- and N-containing compounds compared to the media from manufacturers D and E.

[0077] To minimize background interference, Manufacturer C's 10x diluted medium was selected for subsequent spore VOC testing. The germination and growth rates of spores in both Manufacturer C's 10x diluted medium and Manufacturer A's medium were compared and confirmed using Manufacturer A's spores for both ethylene oxide-based and steam-based sterilization.

[0078] [Table 2]

[0079] In the following examples, two concentration ranges of Geobacillus stearothermophilus spores (250 cells and 25,000 cells) from Manufacturer A (for steam sterilization) were spiked into Manufacturer A's growth medium and Manufacturer C's 10x diluted medium, respectively. Total ion chromatograms (TICs) for the background and spore-containing samples are shown in Figures 6 and 7. It can be seen that the number of features and peak intensities are much higher in Manufacturer A's growth medium compared to Manufacturer C's 10x diluted medium. The number of features detected in the growth medium and spore-containing samples is shown in Table 3. To extract VOCs released from spores, the following two selection criteria were used: (a) The peak intensity of the spore-containing sample is at least two-fold higher than that of the negative sample (either an increase or decrease in intensity). (b) VOCs are present in more than 80% of the positive samples.

[0080] For samples with high cell counts (25,000 cells), 47 unique spore VOCs were detected in Manufacturer A's growth medium, while 26 VOCs were detected in Manufacturer C's 10x diluted medium. For samples with low cell counts (250 cells), 53 unique spore VOCs were detected in Manufacturer A's growth medium, while 32 VOCs were detected in Manufacturer C's 10x diluted medium. Some VOCs showed higher peak intensities in samples with low cell counts. Additionally, some VOCs showed lower peak intensities in samples with high cell counts. Comparative examples of spore VOC peak intensities in samples with various cell counts are shown in Figures 8A-8D. This study also demonstrated that spore germination could be detected even at concentrations as low as 100 cells.

[0081] The composition of VOCs released by spores also varied significantly depending on the type of growth medium, as shown in Figures 9 and 10. The positive samples from Manufacturer A's growth medium produced higher amounts of both nitrogen- and sulfur-containing compounds compared to the positive samples from Manufacturer C's 10x diluted medium. This is consistent with the increased relative peak intensities for both sulfur- and nitrogen-containing compounds detected in Manufacturer A's growth medium. These results highlight the potential for target spore VOCs to be generated by modifying the growth medium composition.

[0082] The results showed an increased number of ketones, aldehydes, and alcohols in the positive samples prepared with 10x diluted medium from manufacturer C. Surprisingly, as shown in Figures 8A-8D, an increase in the peak intensity of ester-, aldehyde-, and alcohol-based VOCs was observed in the spore samples in the low concentration range but not in the high concentration range.

[0083] [Table 3]

[0084] Common spore-associated VOCs were detected in both growth media, including ketones such as 3-hexanone, 4-methyl-2-heptanone, and phorone; aldehydes such as 3-ethylbenzaldehyde and 2,4-dimethylbenzaldehyde; esters such as 2,2,4-trimethyl-1,3-pentanediol diisobutyrate; and alkanes such as 3,6,6-trimethyl-bicyclo[3.1.1]hept-2-ene and 2,2,4,6,6-pentamethyl-heptane.

[0085] Effects of various spores

[0086] In this study, spore samples prepared for steam sterilization from manufacturers A, B, and C were spiked into 10x diluted GM from manufacturer C. Headspace VOCs from the positive samples were collected and compared with those from the negative sample (headspace VOCs from manufacturer C's 10x diluted GM). Table 4 shows the number of compounds detected in each sample group. Figures 11–13 show a comparison of the relative peak intensities of various VOC groups emitted from the three spore sample groups and background medium. The number of compounds increased in the positive samples, and more VOCs were obtained from spore samples with higher cell counts. Based on the selection criteria for extracting spore VOCs, approximately 26, 80, and 26 spore-associated VOCs were detected in the samples from manufacturers A, B, and C, which contained high spore counts, respectively. The composition and peak intensities of VOCs generated from the three spore sample groups are shown in Figures 11–13.

[0087] [Table 4]

[0088] Common VOCs found in all three types of spore-containing samples include ketones (2-pentanone, methyl isobutyl ketone, 3-hexanone, cyclohexanone, 4-methyl-2-heptanone, and 6-methyl-3-heptanone); alcohols (2-methyl-2-propanol, amylene hydrate, and 2-methyl-1,3-pentanediol); esters (acetic acid, pentyl ester, benzoic acid, and methyl ester); aldehydes (2,4-dimethylbenzaldehyde); and furans (tetrahydro-2,2,5,5-tetramethylfuran). Table 5 summarizes common VOCs detected in association with spore germination.

[0089] [Table 5]

[0090] Spores for ethylene oxide-based sterilization and spores for vapor hydrogen peroxide-based sterilization were obtained from Manufacturers A and B, respectively. Spore suspensions were prepared in 10-fold diluted GM from Manufacturer C, and their headspace VOCs were examined as shown in Table 6. An increased number of VOCs was detected in the spore-containing samples.

[0091] [Table 6]

[0092] Effect of sterilization on spore-forming VOCs

[0093] Geobacillus stearothermophilus spore samples (for steam sterilization) from both Manufacturer A and Manufacturer B were spiked into 10x diluted GM from Manufacturer C. The stock spore suspensions were divided into three groups. Each group was subjected to 0, 1, and 2 10-minute sterilizations at 121°C. Headspace VOCs from the sterilized spore samples were collected and compared with those from unsterilized spore samples. TICs for spore samples from Manufacturer A and Manufacturer B, which were sterilized using different types of sterilization, are shown in Figures 14 and 15, respectively. The number of detected compounds is shown in Table 7. Increased VOC emissions were observed in the sterilized samples. As shown in Figures 20A and 20B, the peak intensities of certain VOCs, namely, cyclododecanol, benzoic acid, 4-ethoxybenzoate, and ethyl ester, increased with increasing sterilization cycles. Therefore, certain VOCs such as cyclododecanol; benzoic acid, 4-ethoxy-, ethyl ester; and 3,4,4-trimethyl-3-pentanol can be used to confirm sterilization.

[0094] [Table 7]

[0095] Verification and quantification of spore-forming VOCs

[0096] Due to the availability of VOC standards, only a few common VOCs were verified. Stock VOCs were prepared by adding approximately 20 mg of standard to 10 ml of HPLC-grade methanol in a volumetric flask. Serial dilutions were performed in 10x Manufacturer C GM to obtain VOC standards at concentrations of 0, 5, 10, 15, 25, and 50 ppb. Identical SPME VOC extraction and GC-MS parameters were used to measure the VOC standards. Both retention times and mass spectra were used to verify the VOCs. The verified spore VOCs are listed in Table 8. Calibration curves for example VOC standards are shown in Figures 18A-18E. These were used to quantify the amount of VOCs generated from spore suspensions containing 48,000 Geobacillus stearothermophilus spores (for steam sterilization) from Manufacturer A, 24,000 Geobacillus stearothermophilus spores (for steam sterilization) from Manufacturer C in 10x diluted GM from Manufacturer B, 42,000 Geobacillus stearothermophilus spores (for steam sterilization) from Manufacturer C, and 14,000 Bacillus atrophaeus spores (for ethylene oxide sterilization) from Manufacturer A. The quantitative results of spore VOCs released from the various spore samples are shown in Table 9.

[0097] [Table 8]

[0098] [Table 9]

[0099] Consideration

[0100] Microorganisms produce various small molecules, such as volatile compounds, as secondary metabolic by-products throughout their growth cycle. Volatile organic compounds (VOCs) are carbon-based, low-molecular-weight compounds that generally have high vapor pressures and therefore readily diffuse into the environment. The higher the volatility (lower the boiling point), the more likely the compound is to be released into the atmosphere from products, media, or surfaces. VOCs have been reported to be involved in microbial interactions. The chemical composition of bacterial volatilomes is defined by genetic determinants and can be used as a chemical taxonomic marker under standardized conditions. Microorganisms can also emit volatiles induced by biological interactions or environmental factors. Fundamental biosynthetic pathways that generate VOCs include heterotrophic carbon metabolism, fermentation, amino acid catabolism, fatty acid degradation, sulfur reduction, and terpenoid biosynthesis. Some VOCs are regularly produced by various microorganisms, while others are only produced by specific strains.

[0101] Potential metabolic pathways for VOC production

[0102] Metabolism of exogenous and endogenous compounds begins immediately after spore germination, and much of the spore's energy requirement during the first 10-15 minutes of germination is met by catabolism of molecules stored in the dormant spore.

[0103] Dormant spores retain large amounts of NAD and NADP, but no NADH or NADPH, and very little other common energy compounds such as ATP. During the first minute of spore germination, the activated metabolic pathways primarily involve ATP production and nicotinamide nucleotide reduction (e.g., conversion of NAD and NADP to NADH and NADPH). These pathways are largely anaerobic during the first 5 minutes of the germination process, after which aerobic pathways begin to be used.

[0104] Germinating spores appear to utilize internal energy and carbon sources. Glutamate, malate, arginine, sulfolactate, and 3-phosphoglycerate are some of the organic compounds detected inside spores. Sporulation conditions (sporulation medium, temperature, etc.) affect spore content and sterilization resistance.

[0105] In addition, pyridine-2,6-dicarboxylic acid and peptidoglycan fragments (N-acetylmuramic acid and N-acetyl-D-glucosamine) are released into the growth medium during early germination, as are amino acids generated by proteolysis of acid-soluble microspore proteins.

[0106] Additionally, the germination process can be activated by the reaction of enzymes activated in the spore exine or exosporium with external compounds (e.g., glucose or alanine-based compounds). The pH of the growth medium also influences the VOCs produced. The effect of pH can be limited by maintaining the BI growth medium at a pH of approximately 6.8–7.2.

[0107] Studies of microbial VOC production demonstrated that esters were detected during anaerobic metabolism in the presence of glucose or alcohol. Organosulfur volatiles were detected when peptone was added. The VOCs produced also depended on the electron donor and acceptor.

[0108] Spore-forming bacteria produce the following compounds under anaerobic conditions: Acids: formic acid (PubChem #284), acetic acid (#176), caproic acid (#8892), isocaproic acid (#12587), valeric acid (#7991), isovaleric acid (#10430), acrylic acid (#6581), butyric acid (#264), isobutyric acid (#6590), propionic acid (#1032), crotonic acid (#637090); Alcohols: 2,3-butanediol (#262), methanol (#887), ethanol (#702), isobutanol (#6560), isopentanol (#31260); Other: Acetoin (#179).

[0109] Fatty acids and their derivatives, such as alkanes, alkenes, aldehydes, ketones, alcohols, ethers, and esters, are likely products of incomplete oxidation of primary metabolites. Common secondary metabolites are found in the groups of terpenes, aromatic compounds, furans, sulfur-containing compounds, and nitrogen-containing compounds.

[0110] The importance of growth media

[0111] As shown in this example, the growth medium influences the types of VOCs observed. It has been observed that the addition of peptone to the growth medium increases the detection of sulfur organic volatiles. Based on the amount of sulfur compounds detected in Manufacturer A's growth medium, the presence of bacteriological peptone in the medium can be confirmed. In contrast, Manufacturer C's growth medium is a modified soybean casein digest broth with a pH indicator. The predominance of ester and aldehyde VOCs suggests that this medium does not contain many accessible sulfur compounds.

[0112] Potential pathways for VOC generation

[0113] It is difficult to determine the metabolic pathways that produce the detected VOCs. The pathways shown in Figure 19 are known. However, because spores seek the reduction of nucleosides such as NAD+ in NADH early in the germination process, products of the fermentation process such as butanol are oxidized to butanal. This may also occur with other fermentation by-products.

[0114] Some enzymes are only active during the first few minutes of spore germination and are therefore difficult to detect; only metabolic by-products are detected.

[0115] One of the VOCs detected in Table 9 of the GC-MS study, 2-propanol 2-methyl, is produced by the tert-butyl methyl ether (MTBE) pathway, which has been identified in many bacterial species. For the other VOCs, no direct metabolic pathway has been identified.

[0116] Generally, sulfur-containing VOCs are formed by the metabolism of sulfur-containing amino acids, for example, via transamination, demethylation, or recombination pathways.

[0117] This written description uses examples to disclose the invention, including the best mode, and also to enable any person skilled in the art to practice the invention, including making and using any devices or systems, and practicing any methods incorporated herein. The patentable scope of the invention is defined by the claims, and may include other embodiments that occur to those skilled in the art. Such other embodiments are within the scope of the claims if they contain elements that do not differ from the literal language of the claims, or if they contain equivalent elements that do not differ substantially from the literal language of the claims.

Claims

1. 1. A method for determining whether spores in a self-contained biological indicator are in the germination stage, comprising: subjecting the self-contained biological indicator to a sterilization process; exposing spores in the self-contained live biological indicator to a growth medium; culturing the spores in the self-contained biological indicator; sampling the headspace within the self-contained biological indicator; and determining whether volatile organic compounds resulting from the germination stage of the spores within the self-contained biological indicator are released into the headspace upon contacting the spores with the growth medium; The method of claim 1, wherein the presence of the volatile organic compounds resulting from the germination stage of the spores in the self-contained biological indicator indicates a failure of the sterilization process.

2. 10. The method of claim 1, The method, wherein the step of determining whether the volatile organic compounds are released from the growth medium into the headspace comprises collecting an air sample from the headspace for analysis of the volatile organic compounds.

3. 10. The method of claim 1, The method, wherein the sampling occurs within the self-contained biological indicator.

4. 10. The method of claim 1, A method wherein the sampling occurs external to the self-contained biological indicator.

5. 3. The method of claim 2, The method, wherein the air sample is collected and analyzed in a time frame of less than about 60 minutes.

6. 10. The method of claim 1, The method, wherein the sterilization process is a steam sterilization process.

7. 10. The method of claim 1, The method, wherein the sterilization process is a hydrogen peroxide gas sterilization process.

8. 10. The method of claim 1, The method wherein the sterilization process is an ethylene oxide sterilization process.

9. 10. The method of claim 1, The method, wherein the self-contained biological indicator comprises a spore of Geobacillus stearothermophilus or Bacillus stearothermophilus.

10. 10. The method of claim 1, The method, wherein the growth medium comprises tryptic soy broth or modified soy casein digest broth.

11. 10. The method of claim 1, The method wherein the volatile organic compound is polar.

12. 10. The method of claim 1, The method further comprising applying a correction factor to remove background volatile organic compounds resulting from the growth medium but not from the germination stage of the spores in the self-contained biological indicator.

13. 10. The method of claim 1, The method, wherein the volatile organic compounds resulting from the germination stage of the spores of the self-contained biological indicator comprise alkanes, alcohols, esters, ketones, furans, or any combination thereof.

14. 14. The method of claim 13, 10. The method of claim 1, wherein the volatile organic compounds resulting from the germination stage of the spores of the self-contained biological indicator comprise 2-pentanone, methyl isobutyl ketone, 4-methyl-2-heptanone, 2-methyl-2-propanol, amylene hydrate, 3-hydroxy-2,4,4-trimethylpentyl 2-methylpropanoate, 2,2,4-trimethyl-1,3-pentanediol diisobutyrate, 2-methyl-1,3-pentanediol, tetrahydro-2,2,5,5-tetramethylfuran, or any combination thereof.

15. 10. The method of claim 1, The method, wherein the self-contained biological indicator comprises spores in a cell number that allows the indicator to achieve a desired resistance depending on the type of sterilization process in which the indicator is used.

16. 10. The method of claim 1, A method wherein the presence of said volatile organic compounds is determined directly without the use of enzymatic reagents.

17. 10. The method of claim 1, A method wherein the presence of said volatile organic compounds is determined directly without the use of fluorescent moieties.

18. 10. The method of claim 1, The method, wherein the step of determining whether the volatile organic compounds resulting from the germination stage of the spores in the self-contained biological indicator are released into the headspace when the spores are contacted with the growth medium comprises the step of comparing the measured concentration of the volatile organic compounds with a predetermined control concentration of the compounds.

19. 20. The method of claim 18, If the measured concentration of the volatile organic compound is higher than the control concentration of the compound, it is determined that the volatile organic compound was released into the headspace due to the germination stage of the spores in the biological indicator when the spores were contacted with the growth medium.

20. 1. A method for determining whether sterilization has been successful, comprising: subjecting the self-contained biological indicator to a sterilization process; exposing spores in the self-contained live biological indicator to a growth medium; culturing the spores in the self-contained biological indicator; sampling the headspace of the self-contained biological indicator; and measuring the concentration of volatile organic compounds released into the headspace when the spores are contacted with the growth medium.

21. 21. The method of claim 20, The method, wherein the step of measuring the concentration of the volatile organic compounds comprises collecting an air sample from the headspace for analysis of the volatile organic compounds.

22. 21. The method of claim 20, The method, wherein the sampling occurs within the self-contained biological indicator.

23. 21. The method of claim 20, A method wherein the sampling occurs external to the self-contained biological indicator.

24. 22. The method of claim 21, The method, wherein the air sample is collected and analyzed in a time frame of less than about 60 minutes.

25. 21. The method of claim 20, The method of claim 1, wherein the sterilization process is determined to be successful if the concentration of the volatile organic compounds increases compared to a predetermined baseline concentration.

26. 21. The method of claim 20, The method, wherein the sterilization process is steam sterilization, hydrogen peroxide gas sterilization, or ethylene oxide sterilization.

27. 21. The method of claim 20, The method, wherein the self-contained biological indicator comprises a spore of Geobacillus stearothermophilus or Bacillus stearothermophilus.

28. 21. The method of claim 20, The method, wherein the growth medium comprises tryptic soy broth or modified soy casein digest broth.

29. 21. The method of claim 20, The method wherein the volatile organic compound is non-polar.

30. 21. The method of claim 20, The method, wherein the volatile organic compound comprises cyclododecanol; benzoic acid, 4-ethoxy-, ethyl ester; 3,4,4-trimethyl-3-pentanol; or any combination thereof.

31. 21. The method of claim 20, A method wherein the presence of said volatile organic compounds is determined directly without the use of enzymatic reagents.

32. 21. The method of claim 20, A method wherein the presence of said volatile organic compounds is determined directly without the use of fluorescent moieties.

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