Biological indicators for enhanced detection of volatile organic compounds

Abstract

A growth medium for a biological indicator is provided. The growth medium may include not only a base growth medium but also an additive. The additive may include, but is not limited to, a carbon source unit, such as glucose, and a volatile organic compound unit. Alternatively, the additive may include a carbohydrate source. The present invention also relates to a self-contained biological indicator (SCBI) comprising a container, spores disposed on a carrier, a growth medium, and an additive. The additive may include, but is not limited to, a carbon source unit, or a molecule containing a moiety that may form a VOC upon reduction or oxidation, and a volatile organic compound unit. Alternatively, the additive may include a carbohydrate source. It has been found that adding such additives to the growth medium facilitates efficient and accurate detection of failed sterilization processes.

Description

[Technical Field]

[0001] (CROSS-REFERENCE TO RELATED APPLICATIONS) This application claims priority to and the benefit of U.S. Provisional Patent Application No. 63 / 411,976, filed September 30, 2022, the entire disclosure of which is hereby incorporated by reference herein.

[0002] The present invention relates generally to growth media for biological indicators used to verify the effectiveness of a sterilization process. [Background technology]

[0003] Achieving and maintaining sterility is paramount in medical or pharmaceutical sterilization processes. In such cases, biological indicators (BIs) are often used to assess the lethality of a sterilization process. Biological indicators typically contain the most resistant spores, i.e., bacterial spores, from strains defined for a particular sterilization process. For example, Geobacillus stearothermophilus spores are typically used as biological indicators to assess process effectiveness during steam (gravity or dynamic air removal) or oxidative sterilization processes such as hydrogen peroxide, while Bacillus atrophaeus spores are typically used as biological indicators to assess process effectiveness during ethylene oxide sterilization cycles. Meanwhile, to avoid false negatives, it is important that the detection sensitivity of the measurement be accurate enough to identify cases where as few as one or two spores remain after a sterilization cycle. Currently available biological indicators rely on various techniques to identify whether biological activity of spores is still present after sterilization. In first-generation biological indicators, failure was measured by the turbidity of the growth medium. Second-generation biological indicators, on the other hand, are self-contained systems that contain the necessary spore strips and growth medium in a ready-to-use primary pack. These biological indicators rely on a pH indicator to measure the production of acidic metabolic products in the medium by germinated spores and replicating cells. Third-generation biological indicators, on the other hand, have a dual-readout system. The rapid portion detects active spore-associated α-glucosidase enzyme that survives the sterilization process within a specific time frame specified by the biological indicator and reader manufacturer. This enzyme is a common component of vegetative cells and spores of Geobacillus stearothermophilus and Bacillus atrophaeus. G. stearothermophilus spores have a large amount of associated α-glucosidase activity, resulting from at least two enzymes.This activity is due in part to enzymes present on the outer surface of the spore and in the initial dormant spore core, while the majority is due to enzymes synthesized during spore germination and growth. B. atrophaeus does not have such enzyme activity in its spores, and this activity is induced during germination. The activity of enzymes present in the outer membrane and cortex ceases 20 minutes after the onset of germination. After that, it is the inner membrane enzymes that become active.

[0004] The survival of spore-associated α-glucosidase enzyme after steam sterilization does not correlate well with spore survival. This is because normal (non-genetically modified) spores contain only small amounts of active spores that can be detected by fluorescence. If the microorganism is Geobacillus stearothermophilus or Bacillus atrophaeus, the number of microorganisms required to produce sufficient levels of the enzyme for detection by fluorescence is approximately 1 × 10 3 ~1×10 8 To increase sensitivity, it is possible to modify the genetic material of the spores or add additional enzyme extracts to the biological indicator. As part of the third generation, there are sometimes second readout systems that can include a pH indicator. This detects acidic metabolites produced by germinated spores or replicating cells, providing rapid confirmation of results within a specific time frame specified by the manufacturer of the biological indicator or reader. As generations of biological indicators increase, detection times decrease, but the need for response times as close to zero as possible remains.

[0005] Volatile organic compounds (VOCs) have been used as markers for endospore or bacterial spore germination and growth. Various detection methods are available for VOC measurement, as described in published literature and techniques. The concentration of captured VOCs directly corresponds to the spore germination activity. However, VOC detection sensitivity is crucial to identify the minimum threshold level of endospore- or bacterial spore-derived VOCs that indicates a sterilization failure. Therefore, to prevent false-negative results from biological indicators after sterilization, a combination of endospores or bacterial spores with a culture medium that produces detectable levels of VOCs is required. Summary of the Invention [Means for solving the problem]

[0006] The objects and advantages of the present invention will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned through practice of the present invention.

[0007] In one embodiment, a growth medium for a biological indicator is provided. The growth medium includes a base growth medium and an additive including a carbon source unit, such as, but not limited to, glucose, and a volatile organic compound unit. In another embodiment, a self-contained biological indicator (SCBI) may include a container, spores disposed on a carrier, the growth medium, and an additive. The additive may be contained within the growth medium, disposed on the carrier, or disposed on the spore itself. The additive may include a carbon source unit, such as, but not limited to, a carbohydrate or amino acid molecule, or a carbon source molecule containing a moiety that can form a VOC upon reduction or oxidization, such as, but not limited to, glucose, and a volatile organic compound unit.

[0008] In yet another embodiment, a growth medium for a biological indicator is provided. The growth medium includes a base growth medium and an additive containing a carbohydrate source that results in the formation of volatile organic compounds through the action of an enzyme or coenzyme active during the germination stage of spores introduced into the growth medium. For example, the additive may include an amino acid. For example, the additive may include an aliphatic amine. As a result of the germination stage of spores introduced into the growth medium, cytochrome c reductase is released, causing an oxidation reaction to produce volatile organic compounds. Furthermore, the volatile organic compounds may include aldehydes.

[0009] In yet another embodiment, a self-contained biological indicator (SCBI) is provided. The SCBI includes a container, spores disposed on a carrier, and a growth medium, and includes a carbohydrate source that results in the formation of volatile organic compounds through the action of an enzyme or coenzyme active during the germination stage of the spores introduced into the growth medium. The carbohydrate source may be included within the growth medium, disposed on the spore carrier, or disposed on the spore itself. During the germination stage of the spores introduced into the growth medium, cytochrome c reductase is released, causing an oxidation reaction that produces volatile organic compounds, including aldehydes.

[0010] 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.

[0011] 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. [Brief explanation of the drawings]

[0012] [Figure 1] FIG. 1 is a schematic diagram of one embodiment of a self-contained biological indicator contemplated by the present invention. DETAILED DESCRIPTION OF THE INVENTION

[0013] Reference will now be made in detail to one or more embodiments of the invention, examples of which are illustrated in the drawings. Each example and embodiment is provided to explain the invention and is not intended to limit the invention. For example, features illustrated or described as part of one embodiment may be used in combination with another embodiment to yield a still further embodiment. The present invention is intended to include these and other modifications and variations that are within the scope and spirit of the invention.

[0014] As used herein, the terms "about," "approximately," or "generally" used to modify a value indicate that the value may be increased or decreased by 5% and still fall 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 multiple ranges is contemplated by the present invention. For example, when the ranges "about 20% to about 80%" and "about 30% to about 70%" are provided, the ranges "about 20% to about 70%" and "about 30% to about 80%" are also contemplated by the present invention.

[0015] Generally speaking, the present invention relates to a growth medium for a biological indicator. The growth medium includes a base growth medium and an additive including, but not limited to, a carbon source unit, such as glucose, and a volatile organic compound unit. The present invention also relates to a self-contained biological indicator (SCBI) including a container, spores disposed on a carrier, the growth medium, and an additive. The additive may be contained within the growth medium, disposed on the spore carrier, or disposed on the spore itself. In some embodiments, the additive includes a carbon source unit, such as, but not limited to, a carbohydrate or amino acid molecule, or a carbon source molecule including a moiety that can form a VOC when reduced or oxidized, such as, but not limited to, glucose, and a volatile organic compound unit. Without being limited to a particular theory, the inventors have discovered that adding an additive to the growth medium facilitates efficient and accurate detection of a failed sterilization process. In particular, if the spores in the SCBI are not killed by the sterilization process, they proceed to a germination stage during which enzymes on the spore, such as, but not limited to, α-glucosidase, are activated and react with the additive to cleave the volatile organic compound unit from the carbon source unit, and the presence of the volatile organic compound, e.g., at a particular concentration, as measured by methods known to those skilled in the art, can indicate failure of the sterilization process in which the SCBI was utilized.

[0016] In other embodiments, the additive may be a carbohydrate source, such as an amino acid, such as an aliphatic amine. The additive may be a primary alkyl sulfate, or a primary alcohol may be converted by the action of a cytochrome (e.g., a coenzyme) to form a volatile organic compound. Cytochromes are present on the outer surface of the spore and in the early dormant spore nucleus. These are intermediates in the respiratory chain and generate the H+ needed to convert the abundant NAD and NADP molecules in the spore into NADH and NAD, which are used by enzymes within the spore to complete the germination and growth process. The outer layer of the Bacillus subtilis spore contains one-third of the cytochrome content of the entire spore and several enzymes of the electron transport chain (specifically, NADH oxidase, dehydrogenase, cytochrome c reductase, and NADPH dehydrogenase). In this embodiment, because many compounds can be converted to a single group of VOCs, such as aldehydes, detecting all compounds in a specific chemical group improves the sensitivity of detecting a small number of germinated spores.

[0017] In other embodiments, the additive may be converted to a second compound that is converted back to a VOC by one or more reactions. Carbon sources present in the growth medium enter glycolysis and / or the Entner-Doudoroff pathway to form pyruvate. Pyruvate can be fermented with alcohols, ketones, or acids such as 1-butanol, 2,3-butanediol (diacetyl), and propanoate (propanoic acid).

[0018] Upon spore germination, small acid-soluble spore proteins (SASPs) serve as a reservoir of amino acids to support protein synthesis. At this stage, utilization of SASPs is achieved through the activity of sequence-specific endoproteases called GPRs (germination proteases), which promote the degradation of SASPs.

[0019] Some amino acids, such as L-alanine, a germination initiator (germinant), are converted to pyruvate by the action of alanine dehydrogenase or L-glutamate-pyruvate transaminase. Other amino acids are converted by other pathways. Valine, leucine, threonine, and isoleucine are converted to keto acids such as α-ketoisocaproic acid (4-methyl-2-oxopentanoic acid). These products are converted to ethanol (VOC), isobutanol (VOC), 2-methyl-1-butanol (VOC), and 3-methyl-1-butanol (VOC).

[0020] Regardless of the composition, volatile organic compounds (VOCs) evaporate under average indoor temperature and pressure conditions. Several factors affect the volatility of VOCs, including molecular structure, molecular weight, polarity, and intermolecular forces. Volatility decreases with increasing chain length because longer chains provide more opportunity for interchain interactions via dispersion forces. Smaller molecules also have fewer intermolecular forces to overcome when transitioning from a liquid to a gaseous state, so chain length, aliphatic substitution, and the pathway / mechanism by which VOCs are produced must be considered. For example, shorter chain lengths or aliphatic substitutions, such as those found in the aliphatic amino acid subgroups of glycine, alanine, valine, leucine, and isoleucine, increase volatility. Threonine, which has a methyl hydroxyl group on one end and a carboxyl group on the other, can be converted to VOCs depending on the pathway (reduction / oxidation).

[0021] Anaerobic degradation of phenylalanine produces L-glutamic acid and phenylacetic acid. Phenylacetic acid can be fermented with toluene (VOC) and then with propanal (VOC). Some amino acids can be reduced by the Strickland reaction to produce 3-propanoate (3-propanoic acid) derivatives as a by-product.

[0022] Various embodiments of the present invention will now be described in more detail with reference to FIG. 1 . FIG. 1 illustrates a self-contained biological indicator 100 including a container 101. The container 101 is sealed by a cap 118 and holds a growth medium 110 in an ampoule 108 and a growth chamber 104 housing a spore carrier 106 containing spores 102. The growth medium 110 may include an additive 112, although it should be understood that the additive 112 may alternatively or additionally be present on the spores 102 themselves and / or on the spore carrier 106. The additive 112 includes a glucose unit 114 and a volatile organic compound unit 115. The ampoule 108 and the growth chamber 104 are separated by an ampoule crusher 116, which, when activated, is used to introduce the growth medium 110 to the spores 102 after a sterilization cycle has been performed. If the sterilization cycle is successful, the spores 102 will not germinate. On the other hand, if the sterilization cycle is unsuccessful, the spores 102 enter a germination stage, during which they release a target enzyme or coenzyme. The target enzyme or coenzyme reacts with the additive 112 to cleave the volatile organic compound unit 115 from the carbon unit 114. The volatile organic compound unit 115 is then released into the headspace 120 within the ampoule 108. The presence of the volatile organic compound unit 115 in the headspace 120 indicates a sterilization cycle failure and can be collected and analyzed by any suitable method, including, but not limited to, solid phase microextraction (SPME) and gas chromatography-mass spectrometry (GC-MS).

[0023] In some embodiments, the growth medium 110 may include tryptic soy broth, modified soybean casein digest broth, or AGFK (L-asparagine, D-glucose, D-fructose, and K+) germination medium. Additionally, the SCBI may include spores of Geobacillus stearothermophilus and / or Bacillus atrophaeus.

[0024] Further, in some embodiments, the carbon unit 114 may be an α-glucopyranoside. Further, in some embodiments, the volatile organic compound unit 115 may be a functional alkyl, where the alkyl may be methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, undecyl, or dodecyl. Furthermore, the additive 112 comprising the glucose unit 114 and the volatile organic compound unit 115 may be present in the growth medium at a concentration ranging from about 1 millimolar to about 20 millimolar, e.g., from about 2.5 millimolar to about 15 millimolar, e.g., from about 5 millimolar to about 10 millimolar.

[0025] In some embodiments, the additive 112 may have the following structure:

[0026] [ka]

[0027] In some embodiments, the additive 112 may have the following structure:

[0028] [ka]

[0029] In some embodiments, the additive may have the following structure:

[0030] [ka]

[0031] In some embodiments, the additive may have the following structure:

[0032] [ka]

[0033] In some embodiments, the additive may have the following structure:

[0034] [ka]

[0035] In some embodiments, the additive may have the following structure:

[0036] [ka]

[0037] In some embodiments, the additive may have the following structure:

[0038] [ka]

[0039] The present invention can be better understood with reference to the following examples.

[0040] Example 1

[0041] the purpose

[0042] We demonstrate that the addition of specific compounds to the base growth medium increases VOC production during the first 20 minutes of germination of Geobacillus stearothermophilus spores.

[0043] methodology

[0044] Geobacillus stearothermophilus spore suspension (1 × 10 6 The suspension (CFU / 0.1 mL) was heat shocked at 95-99°C for 15 minutes to kill any vegetative cells that may be present in the suspension, and then diluted to a concentration of 500-800 CFU / 30-50 μL.

[0045] The modified soybean casein digest broth was diluted (10x) with HPLC-grade water and then sterilized by 0.45 μm filtration to limit background VOC levels. It was used with or without additives. The number of samples used and the combinations investigated are summarized in Table 1 below.

[0046] Table 1. Number of test samples per combination

[0047] [Table 1]

[0048] For VOC testing, 0.5 mL of sterile diluted growth medium was transferred to each VOC collection vial. The vials were preheated to at least 60°C for 10 minutes. Before VOC sampling began, additives were added to the vials, followed by the addition of spores according to Table 1, depending on the test being performed. Immediately after spore addition, an SPME fiber was inserted into the vial to sample the VOCs, and the vial was returned to the temperature-controlled system. The SPME fiber used was a divinylbenzene / carboxin / polydimethylsiloxane (DVB / CAR / PDMS) assembly (Sigma Aldrich, product number 57328-U, manufactured by Supelco) that had been conditioned at 270°C for 30 minutes prior to use. The SPME fiber was removed 20 minutes after spore addition and analyzed using GC-MS according to the device's experimental protocol.

[0049] result

[0050] α-Glucosidase activity converts additives into methanol and glucose. Methanol, being a VOC, can be analyzed. Glucose may be used by spores to generate additional VOCs via other enzymes or pathways. In addition to VOC identification, their detection intensity was also analyzed. Because the presence of spores produces more VOCs, the intensity may be reduced even if the same amount of VOC is produced. On the other hand, an increase in the concentration of a specific VOC in the presence of spores compared to the growth medium, regardless of whether an additive is present, indicates an increase in its concentration. Analysis of the VOCs generated is complicated by the fact that not all spores germinate simultaneously. Furthermore, some water-soluble compounds may be available to spores more quickly during germination. Therefore, some VOCs may only be detected in a few samples. While some pathways to VOCs may have been identified (Table 2), the pathways for other VOCs detected only in the presence of additives, such as 1-nonene, nonane, 1-octene, octane, and heptane, remain unknown.

[0051] The detected VOCs indicate that they may be generated by cytochrome activity (aldehyde VOCs - formaldehyde, nonanal, octanal, pentanal, heptanal, hexanal, 2-propenal), which may be generated by pyruvate fermentation using additives (glucose or methanol released by α-glucosidase activity) (1-butanol, 2,3-butanedione, propionic acid), methanol-derived VOCs (formaldehyde), and other pathways such as amino acid degradation (formaldehyde, propionic acid).

[0052] Table 2. Number of samples testing positive for specific VOCs for which metabolic pathways could be identified.

[0053] [Table 2]

[0054] Examples of possible pathways to VOCs are given below. Propene apparently originates from germinated spores, and its concentration is not increased by the presence of additives. Propene can be produced by the reaction of fatty acids with hydrogen peroxide or acyl-[acyl-carrier-protein] with malonyl-CoA+ reduced electron carrier + H+, as described in equation (VIII).

[0055] [ka]

[0056] [ka]

[0057] Formaldehyde is produced from methanol (IX) or as another aldehyde (X) by the action of cytochromes.

[0058] [ka]

[0059] [ka]

[0060] 2,3-Butanedione can be produced by fermentation of pyruvate to acetoin (XI), which can accumulate in spores and in the medium because the conversion to acetoin requires NADH. The spontaneous reaction occurs under aerobic conditions.

[0061] [ka]

[0062] It can also occur via the general reaction (XII).

[0063] [ka]

[0064] Propionate may be produced by the fermentation of pyruvate, the breakdown of L-threonine (XIII), or from other amino acids such as L-valine.

[0065] [ka]

[0066] Aldehydes such as nonanal, octanal, hexanal, and heptanal can be produced by the conversion of aliphatic amines by cytochrome (XIV) and primary alkyl sulfates (XV).

[0067] [ka]

[0068] [ka]

[0069] Example 2

[0070] the purpose

[0071] We will examine whether the addition of specific compounds to the germination medium (which allows germination but limits exogenous growth) increases VOC production during the first 15 minutes of germination of Geobacillus stearothermophilus spores.

[0072] methodology

[0073] Geobacillus stearothermophilus spore suspension (1 × 10 6 CFU / 0.1 mL) were heated at 99–100°C for 30 min to kill any vegetative cells that may be present in the suspension.

[0074] The growth medium was a 200-fold dilution of modified soybean casein digest broth mixed with sterilized germination medium (1.0 mM L-valine in 10 mM sodium phosphate buffer, pH 8.0). This medium was based on the method described in "Kinetics of Germination of Individual Spores of Geobacillus stearothermophilus as Measured by Raman Spectroscopy and Differential Interference Contrast Microscopy, PLoS ONE 8(9):e74987" by Zhou T, Dong Z, Setlow P, Li Yq et al. (2013). Germination medium was tested with or without 5 mM L-threonine or glucose. Experiments were also performed with diluted modified soybean casein digest broth (200-fold) and with germination medium alone without any additives. Controls included SPME fiber alone, in the presence of 2500 μg methanol, and growth medium with or without L-threonine or glucose.

[0075] For VOC testing, 0.5 mL of control solution (methanol) or sterile growth medium was transferred to each VOC collection vial. The vials were preheated to at least 65°C for 10 minutes. Before VOC sampling began, 5 mM L-threonine or glucose was added to the vial, followed by 5,000–10,000 spores (except for the control). Immediately after spore addition, an SPME fiber was inserted into the vial to sample the VOCs, and the vial was then returned to the temperature-controlled system. The SPME fiber used was a divinylbenzene / carboxin / polydimethylsiloxane (DVB / CAR / PDMS) assembly (Sigma-Aldrich, product number 57328-U, manufactured by Supelco) that was treated at 270°C for 30 minutes before use. The SPME fiber was removed 15 minutes after spore addition and analyzed using GC-MS according to the device's experimental protocol.

[0076] result

[0077] L-threonine led to the formation of carbon dioxide, propionic acid, acetic acid, and other volatile fatty acids, while glucose led to acetate (anaerobic respiration) or pyruvate, lactate, or butanediol (fermentation).

[0078] In this example, VOCs emanating from the growth medium itself were less dominant than in Example 1. It also demonstrates that not only can VOCs be linked to glucose molecules, but also that VOCs produced using glucose itself can be detected.

[0079] Addition of L-threonine or glucose produces common VOCs such as 1-nonene, but also other VOCs, as shown in Table 3.

[0080] Table 3. VOCs (qualitative amounts) produced in the presence of L-threonine and / or glucose in the presence of spores.

[0081] [Table 3]

[0082] Some examples of possible pathways leading to VOCs are given below: Propene was detected in Example 1, which indicates that propene is associated with the use of carbohydrate compounds such as glucose that are present in or added to the growth medium.

[0083] Glucose can be fermented to 1-butanal by Clostridium (Biocycle Pathway PWY-6594, www.biocyc.org). Bacillus species possess some of the same enzymes as Clostridium when grown under anaerobic conditions (fermentation).

[0084] Threonine can be degraded to acetaldehyde by Bacillus subtilis or Clostridium pasteurianum via L-threonine aldolase (XVI). See www.Biocyc.org for details (Threonine Degradation Pathway 4). Threonine can also be fermented to 1-butanol via pyruvate (XVI).

[0085] [ka]

[0086] Other VOCs, such as benzaldehyde, 3-ethyl-, are known to be produced by Clostridium species (C.A. Rees, A. Shen, J.E. Hill et al. 2016. Comprehensive two-dimensional gas chromatography-to-flight mass spectrometry characterization of the Clostridium difficile volatile metabolome. Journal of Chromatography B, Vol. 1039, pp. 8–16).

[0087] This 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 the devices or systems, or performing the incorporated methods. The patentable scope of the invention is defined by the claims, and may include other examples that occur to those skilled in the art. Such other examples are intended to be within the scope of the claims if they include elements that do not depart from the literal language of the claims, or equivalent elements that do not differ substantially from the literal language of the claims.

Claims

1. 1. A growth medium for a biological indicator, comprising: a base growth medium; A growth medium comprising a carbon source unit and an additive comprising a volatile organic compound unit.

2. 10. The growth medium of claim 1, wherein the base growth medium comprises tryptic soy broth, modified soy casein digest broth, or AGFK (L-asparagine, D-glucose, D-fructose, and K+) germination medium.

3. 2. The growth medium of claim 1, wherein the carbon source unit comprises an α-glucopyranoside and the volatile organic compound unit comprises a functional alkyl.

4. 2. The growth medium of claim 1, wherein the additive has the following structure: 【Chemical 1】

5. 2. The growth medium of claim 1, wherein the additive has the following structure: 【Chemistry 2】

6. 2. The growth medium of claim 1, wherein the additive has the following structure: 【Chemistry 3】

7. 2. The growth medium of claim 1, wherein the additive has the following structure: 【Chemistry 4】

8. 2. The growth medium of claim 1, wherein the additive has the following structure: 【Chemistry 5】

9. 2. The growth medium of claim 1, wherein the additive has the following structure: 【Chemistry 6】

10. 2. The growth medium of claim 1, wherein the additive has the following structure: 【Chemistry 7】

11. 10. The growth medium of claim 1, wherein the additive is present at a concentration ranging from about 1 millimolar to about 20 millimolar.

12. 1. A self-contained biological indicator comprising: A container and spores disposed on a carrier; a growth medium; and an additive, The additive is a self-contained biological indicator comprising: (1) a carbon source unit, or (2) a carbon source molecule comprising a moiety that forms a volatile organic compound when oxidized, and (3) a volatile organic compound unit.

13. 13. The self-contained biological indicator of claim 12, comprising spores of Geobacillus stearothermophilus or Bacillus atrophaeus.

14. 13. The self-contained biological indicator of claim 12, wherein the growth medium comprises tryptic soy broth or modified soybean casein digest broth.

15. 13. The self-contained biological indicator of claim 12, wherein the carbon source unit comprises an α-glucopyranoside, the carbon source molecule comprises glucose, and the volatile organic compound unit comprises a functional alkyl.

16. 13. The self-contained biological indicator of claim 12, wherein the additive has the following structure: 【Chemistry 8】

17. 13. The self-contained biological indicator of claim 12, wherein the additive has the following structure: 【Chemistry 9】

18. 13. The self-contained biological indicator of claim 12, wherein the additive has the following structure: 【Chemistry 10】

19. 13. The self-contained biological indicator of claim 12, wherein the additive has the following structure: 【Chemistry 11】

20. 13. The self-contained biological indicator of claim 12, wherein the additive has the following structure: 【Chemistry 12】

21. 13. The self-contained biological indicator of claim 12, wherein the additive has the following structure: 【Chemistry 13】

22. 13. The self-contained biological indicator of claim 12, wherein the additive has the following structure: 【Chemistry 14】

23. 13. The self-contained biological indicator of claim 12, wherein the additive is included in the growth medium at a concentration ranging from about 1 millimolar to about 20 millimolar.

24. 13. The self-contained biological indicator of claim 12, further comprising a growth medium ampoule and an ampoule crusher.

25. 13. The self-contained biological indicator of claim 12, wherein the resulting release of α-glucosidase during the germination stage of the spore causes cleavage of the volatile organic compound unit.

26. 1. A growth medium for a biological indicator, comprising: a base growth medium; and an additive comprising a carbohydrate source that causes the formation of volatile organic compounds by the action of an enzyme or coenzyme active during the germination stage of spores introduced into the growth medium.

27. 27. The growth medium of claim 26, wherein the spores introduced into the growth medium are in a germination stage, resulting in the release of cytochrome c reductase, followed by an oxidation reaction to produce volatile organic compounds.

28. 28. The growth medium of claim 27, wherein the volatile organic compounds comprise aldehydes.

29. 1. A self-contained biological indicator comprising: A container and spores disposed on a carrier; a growth medium; A self-contained biological indicator comprising a carbohydrate source that causes the formation of volatile organic compounds through the action of an enzyme or coenzyme active during the germination stage of spores introduced into the growth medium.

30. 30. The self-contained biological indicator of claim 29, wherein the carbohydrate source comprises an amino acid.

31. 30. The self-contained biological indicator of claim 29, wherein the carbohydrate source comprises an aliphatic amine.

32. 30. The self-contained biological indicator of claim 29, wherein the carbohydrate source is disposed within the growth medium, on the carrier, or on the spores.

33. 30. The self-contained biological indicator of claim 29, wherein the spores introduced into the growth medium are in a germination stage resulting in the release of cytochrome c reductase, which then undergoes an oxidation reaction to yield volatile organic compounds.

34. 34. The self-contained biological indicator of claim 33, wherein the volatile organic compound comprises an aldehyde.

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