WO1999005310A1 - Spore germination media - Google Patents

Abstract

A spore germination medium that increases the rate of spore germination includes a germinant and an effector that interact synergistically. Said germinant and effector may comprise amino acids e.g. Ala, Val, Asn, Gln. Spore germination triggered by germination media provided by the invention can be used to measure and evaluate spore survival after exposing spores to a sterilant.

Description

SPORE GERMINATION MEDIA Field of the Invention The invention relates to microbial spore germination media. In particular, the invention relates to germination media that affect the rate of spore germination. The invention further relates to germination media that affect the relationship between spore germination rates and spore survival after exposing spores to a sterilant.

Background of the Invention In response to environmental factors, certain microorganisms, such as various Bacillus and Clostridium species, will form endospores . A spore is a rigid structure with no detectable metabolic activity. The general spore structure includes an outer exosporium, a spore coat, a spore cortex, an inner membrane and finally a spore core cell . Endospores are significantly more resistant to environmental fluctuations in pH, temperature, humidity and radiation than are vegetative cells. Foster & Johnstone, Molecular Microbiol., 4 (1) :137-41 (1990) .

Although microbial spores will remain dormant for extended periods of time, spores contain mechanisms that trigger germination under certain circumstances. The germination process consists of a series of degradative steps that break down the spore coat and spore cortex allowing water and nutrients to enter the spore core cell. Following germination, metabolic activity is reactivated and outgrowth of a new vegetative cell occurs .

Germination and subsequent outgrowth are distinct but related processes. That is, even though a spore germinates it might not complete the outgrowth process. Outgrowth of a new vegetative cell, however, cannot occur unless the spore germinates. The mechanisms that trigger spore germination are not fully understood. Depending on the species, germination can be triggered by exposing the spores to germinants or certain environmental stimuli or both. Johnstone, K. , J. APPI . Bac ■ Symposium Supp .. 76:17S-24S (1994) .

Early germination models suggested that Bacillus subtilis contained two germination pathways. In one pathway, -Ala alone was capable of triggering spore germination (GS-I) . Wax & Freese, J. Bac ■ , 95(2):433-38 (1968) . The other pathway required a combination of L- Asn or L-Gln plus glucose, fructose and potassium ions to trigger spore germination (GS-II) . Wax & Freese, J. Bac .. 95(2):433-38 (1968). Further research conducted with Bacillus subtilis has identified a third germination system. This third germination system is also triggered by a combination of L-Ala, glucose, fructose, and potassium ions (GS-III) , McCann et al., ^Letters Appl . Bact . , 23:290-93 (1996). Even with the identification of multiple germination systems, however, the identity of the receptors involved and the interactions between the receptors and germination active components remain unclear. See Foster and Johnstone, Molecular Microbiol . , 4:137 (1990); Johnstone, K, J. Appl . Bact_. Symposium Suppl. , 76:17S-24S (1994); Moir et al . , J. Appl. Bact . Symposium SUPPI .. 76:9S-16S (1994).

Summary of the Invention The present invention involves the discovery that when certain germinants and effectors are combined in a germination medium, they will act synergistically to alter microbial spore germination rates. Further, optimization of the germinant and effector concentrations alters the dose-response relationship between the rate of spore germination and sterilant exposure time, which facilitates rapid evaluation of sterilization efficacy. As used herein, a spore germination medium is any medium that induces a measurable amount of spore germination. An effector is a compound that, when added to a germination medium, acts synergistically with a germinant to produce an increase in the RV of spore germination and a decrease in the apparent K„ for the germinant triggering spore germination, compared to the same germination medium lacking the effector. The invention is useful for germinating spores including without limitation Bacillus subtilis, Bacillus circulans, Bacillus pu ilus, Clostridiu perfringe s, Clostridium sporogenes and Bacillus stearother ophilus spores.

In a first embodiment, the invention provides a spore germination medium containing a phosphate buffer at a concentration from about o.l molar to about 0.2 molar and synergistic amounts of a germinant and an effector. This spore germination medium can be optimized for the LRV response of microbial spores, that have been exposed to a sterilization process, by, for example, adjusting the phosphate buffer concentration to about 0.15 molar. The spore germination medium can also be optimized for particular germination systems including the GS-II system.

In another aspect, the invention provides a spore germination medium containing a phosphate buffer at a concentration from about 0.1 molar to about 0.2 molar, synergistic amounts of a germinant and an effector, sodium chloride, potassium chloride, glucose and fructose.

In another embodiment, the germination media disclosed herein can be useful for determining the effectiveness of a sterilization process by following the steps of exposing microbial spores to a sterilant, contacting the spores with a spore germination medium, and calculating the LRV of spore germination to determine the effectiveness of the sterilization process In another embodiment, the invention provides a biological indicator for indicating the effectiveness of a sterilization process. The biological indicator contains a germination medium having synergistic amounts of a germinant and an effector wherein the medium produces a sterilant exposure time/LRV response curve slope greater than about 0.016. The medium contained within the biological indicator may also contain a phosphate buffer at a concentration from about 0.1 molar to about 0.2 molar. These biological indicators can produce steepness indexes from about 10 to about 20.

In another embodiment, the invention provides a biological indicator that includes a first container containing dried microbial spores that is further adapted to exposing microbial spores to a sterilant and computing an LRV for germinating spores. Such a biological indicator also contains a second container adapted for storing a germination medium that contains a phosphate buffer at a concentration from about 0.1 molar to about 0.2 molar, and synergistic amounts of a germinant and an effector.

In another embodiment, the invention provides a method for identifying an effector by 1) contacting spores with a germination medium containing a compound with unknown effector properties and lacking a germinant, and then computing an LRV; 2) contacting spores with a germination medium containing the germinant and lacking the compound with unknown effector properties, and computing an LRV; 3) contacting spores with a germination medium containing the germinant and the compound with unknown effector properties, and computing an LRV; and 4) calculating an S value to determine the effectiveness of the compound with unknown effector properties.

The above-mentioned germination media and methods can be further modified by one or more of the following aspects of the invention. In one aspect, the germinant may include an L-amino acid or an operable derivative thereof at concentrations from about 0.0003 mg/ml to about 30 mg/ml, preferably from about 0.001 mg/ml to about 0.01 mg/ml or from about 0.1 mg/ml to about 1.0 mg/ml. Useful germinants include, but are not limited to, alanine, α-amino butyric acid, norvaline, cysteine, valine, isoleucine, β-alanine, serine, homoserine, leucine, methionine, allothreonine, and threonine . In another aspect, the effector may include a D-amino acid, an L-amino acid, or an operable derivative thereof at concentrations from about 0.1 mg/ml to about 20 mg/ml, preferably from about 0.1 mg/ml to about 10 mg/ml, more preferably from about 3 mg/ml to about 8 mg/ml . Useful effectors include, but are not limited to, asparagine, glutamine, homoserine, allothreonine, threonine, 4- acetamidobutyric acid, histidine, serine, 2- acetamidoacetic acid, and 6-acetamidohexanoic acid.

In another aspect, the invention is practical for use with a variety of sterilant sources including ethylene oxide, steam, radiation, heat, sodium hypochlorite, polyvinylpyrrolidone-iodine, sodium dichlorocyanurate, low temperature steam-formaldehyde, glutaraldehyde, hydrogen peroxide, hydrogen peroxide plasma, peracetic acid and mixtures thereof. Features and advantages of the invention include one or more of the following. The novel synergistic interactions facilitate rapid and accurate evaluation of a sterilization process. The germinants and effectors provide a consistent germination medium for triggering sporulation. The novel interactions discovered provide surprising results that are contrary to conventional thought in the field of bacterial spore germination. As a result, new and useful germination media and methods for using the media are provided. Unless otherwise defined, all technical and scientific terms and abbreviations, including three letter abbreviations commonly used for amino acids, used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. All publications, patent applications, patents and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. Other features and advantages of the invention will be apparent from the following detailed description and from the claims. >

Brief Description of the Drawings

FIG. 1 is a duplication of FIG. 1 of PCT International Publication WO 95/21936 and is a graph that depicts the absorbance at 480 nm versus time for a typical germinating suspension of Bacillus subtilis spores as described herein.

FIG. 2 is a duplication of FIG. 3 of PCT International Publication WO 95/21936 and is a graph that depicts the survival curve for Bacillus subtilis spores exposed to ethylene oxide having a D value of 2.7 minutes and an observed LRV curve for Bacillus subtilis spores.

Detailed Description of the Preferred Embodiments

The present invention involves the discovery that certain germinants and effectors, when combined in a germination medium (GM) , interact synergistically to alter microbial spore gemination rates. Further, optimization of germinant and effector concentrations alters the sterilant exposure time/germination response relationship, which facilitates rapid evaluation of a sterilization process.

Researchers have reported that L-Gln or L-Asn added to a base germination medium of fructose, glucose and potassium and phosphate buffer will trigger Bacillus subtilis spore germination. Wax & Freese, J. Bact . , 95:433-438 (1968); Johnstone, K. , J. Appl. Bact. Symposium Suppl . , 76X7S-24S (1994). As used herein, base germination medium (GM-base) is defined as a medium or solution that does not trigger spore germination by itself.

The present inventor has unexpectedly discovered that GM-base solutions further containing commercially available lots of L-Gln or L-Asn do not trigger Bacillus subtilis spore germination in a consistent manner. Some lots trigger germination (active lots) while other lots do not trigger germination (inactive lots) . The inventor has further discovered that substantially pure samples of L-Asn and L-Gln, when added to a GM-base solution, do not trigger spore germination to a significant extent.

The inventor has also discovered that L-Asn, L-Gln and certain other compounds interact synergistically with germinants to increase spore germination rates. In other words, it was discovered that L-Asn, L-Gln and certain other compounds function as "effectors" that promote spore germination triggered by L-Ala or other germinants through the GS-II mechanism. Such a discovery is useful in the general area of biological indicators. Germination media containing germinants and effectors and methods for using the media are disclosed and claimed herein.

Biological indicators and methods for producing biological indicators are known. Illustrative examples of biological indicators can be found in United States Patent NOS. 5,073,488, 3,661,717, 4,839,291, 4,741,437, and 4,416,984. Briefly, suitable biological indicators would allow access of a sterilant to the spores and would be adapted to contain a germination medium. Additionally, if the germination rate is determined spectrophotometrically, preferred biological indicators would be adapted to facilitate measuring the LRV of spore germination. A variety of containers made from quartz glass or polymeric materials such as poly (methylmethacrylate) or polystyrene, may be used to construct such a biological indicator.

Determining Spore Germination Rates .

As a bacterial spore germinates it absorbs water, altering the spore's spectrographic properties. For example, germinating spores lose their light scattering characteristics. As a result, spore germination occurring in solution can be followed spectrophotometrically. Dadd et al . , J . Appl . Bact . , 60:425-433 (1986) . Generally, any spore forming bacteria can be used. Useful spore forming bacteria include Bacillus subtilis, Bacillus circulans, Bacillus pumilus, Clostridium perfringens, Clostridium sporogenes, Bacillus cereus and Bacillus stearothermophilus . A preferred bacterium is Bacillus subtilis . By following the methods and examples disclosed herein, which were used to optimize preferred GMs for Bacillus subtilis spores, other preferred GMs optimized for other useful spore forming bacteria may be developed. Bacillus subtilis spores such as American Type Culture Collection (ATCC) accession no. 9372 can be used at concentrations ranging from about 0.5 x 10⁸ spores/ml to about 3 x 10⁸ spores/ml. Typically, spore suspensions used in germination reactions contained about 1.2 x 10⁸ viable spores per 1.2 ml of GM or GM-base solution. This concentration of Bacillus subtilis spores corresponds to an initial absorbance between about 0.45-0.50 absorbance units at 480 nm (Abs₄₈₀) measured in a 1 cm path length clear polymethacrylate cuvette.

Any spectrophotometer equipped with a temperature- controlled cuvette holder can be used to determine spore concentrations and/or spore germination kinetics. The experiments described herein were performed at 37°C in a Varian/Cary 1 UV/visible spectrophotometer equipped with a temperature control unit and a software package used to calculate slopes and other kinetic parameters (version 13) .

Methods to prepare spores for use in a GM are known. As is common practice, the spores used herein were heat activated. Spores can be heat activated by any acceptable method. An illustrative example of heat activating Bacillus subtilis spores is heating an aqueous suspension of spores at about 47°C for four hours. Another acceptable method for heat activating spores is to dry the spores onto a suitable carrier material at about 47°C for four hours. Heat activated spores are stable and will remain heat activated for an extended period of time, e.g., months. The spores can be unpurified or purified. Unpurified spores include spores harvested by conventional methods from bacterial cultures induced to sporulate by the appropriate stimuli. Unpurified spores often contain agar and/or cellular debris mixed in with the spores. Unpurified spores may be further purified by known methods. For example, to remove bits of agar, denatured nucleoproteins and other debris, spores can be resuspended in sterile deionized H₂0, which does not trigger germination. The suspension is then filtered through a Whatman GF/D glass fiber filter (2.7 μm retention) . After filtration, the spore suspension is considered purified.

Purified spores in aqueous solutions can be further separated into heavy and light spores by differential centrifugation. Heavy spores pellet after centrifugation for 12 to 15 minutes at 2,000 x g, whereas the light spores remain in suspension. Light spores can be pelleted by centrifuging the supernatant remaining after pelleting the heavy spores for 30 minutes at 2,000 x g. Purification does not alter spore viability.

Further, the level of purification does not affect the LRV when the GS-II mechanism is triggered. Purified spores can be stored as an aqueous suspension or dried.

Useful wavelengths of light for measuring changes in absorbance range from about 400 nm to about 700 nm, preferably about 460 nm to about 560 nm, and more preferably about 480 nm to about 520 nm. At these wavelengths, the absorbance per spore concentration is highest and the change in absorbance observed for germinating spores is still measurable. As the wavelength increases from 300 nm to 900 nm, the spore suspension's initial absorbance decreases asymptotically (more than 30% from 480 nm to 600 nm) , but the change in absorbance due to germination of the spores increases by less than 10%. Methods to observe spore germination are known and include methods disclosed in PCT International Publication WO 95/21936, which is hereby incorporated by reference .

Spore germination rates can be calculated from a plot of the absorbance of a germinating spore suspension versus time (germination kinetics curve) . The observed spore germination rate is a function of the number of spores that germinate per unit time, which is affected by both the number of spores germinating and the time needed for each spore to germinate.

FIG. 1 illustrates a typical germinating suspension of Bacillus subtilis spores observed at 480 nm (Abs₄₈₀) . As shown in FIG. 1, initially there is a lag period as spores begin to germinate. Then, as the spore germination process progresses, the Abs₄₈₀ of the spore suspension decreases. The decreasing absorbance is recorded until a majority of the spores that are competent to germinate do germinate. Typically, the decrease in Abs₄₈₀ following the lag period is linear for a period of time before approaching a final Abs₄₈₀.

The linear reaction velocity (LRV) is the maximum observed spore germination rate for a given suspension of spores. The LRV is represented by the absolute value of the slope of the descending linear portion of the germination kinetics curve following the lag period. As such, even though a descending line has a negative slope, the slope is reported as a positive number. Therefore, as used herein, germinants and effectors reference to an "increase" in the slope of the LRV actually refers to a slope that is more negative.

Under the conditions used herein, the LRV occurred within the first 15 minutes after initiating spore germination. Data points used to compute an LRV were typically taken between 2 and 10 minutes following the initiation of a spore germination reaction. The length of the lag period can vary depending on, for example, the condition of the spores and the germination medium used. Nevertheless, the linear portion of the kinetics curve will be apparent from the germination kinetics curve. For example, the LRV in FIG. 1 occurred between 5 and 10 minutes .

Identifying Synergistic Interactions

Germinants trigger germination by binding to specific protein receptors found on or in the dormant spore. In this way, germinants are activators of the proteins that initiate enzymatic reactions resulting in spore germination. It is generally understood, however, that germinants are neither consumed nor altered during the germination process. Even so, the LRV plotted against germinant concentration obeys Michaelis-Menten steady state kinetics equation. Accordingly, the LRV is directly proportional to the germinant concentration and obeys saturation kinetics principles with respect to the germinant concentration. See IVoese et al . , J. Bact. Vol. 761:578-588 (1958) . Thus, the K„ for a germinant can be determined. As used herein, the K„ is the germinant concentration that produces % maximal LRV. The K„ in such cases can be treated as an apparent dissociation or binding constant . The K„ values reported herein were computed using the computer program "Enzyme Kinetics" (Trinity Software Version 1.5) . The K„ values were derived from a Lineweaver-Burk (double reciprocal) plot of the LRV versus germinant concentration. The invention provides a three-step method for identifying useful germinants and/or effectors. First, the germination triggering characteristics of a compound having unknown germination properties (unknown compound) can be identified by computing an LRV for spores germinated in a GM-base solution containing the unknown compound and lacking any known germinants. This initial experiment is repeated at a series of concentrations for the unknown compound. If the unknown compound triggers germination, the data can be used to compute a Kψ, for the unknown compound. Compounds that trigger germination are classified as germinants. It is to be understood, however, that the compound can be further classified as a good or poor germinant. An illustrative example of a good germinant is L-Ala. Illustrative examples of poor germinants are L-Met and L-ornithine.

Second, the effector characteristics of the compound with unknown effector properties can be evaluated. LRVs are computed for spores germinated in a GM containing both the unknown compound and a known germinant. If the unknown compound is an effector for the known germinant, the LRV response will be synergistic. For Bacillus subtilis spores, the known germinant is typically L-Ala or L-Val . When evaluating the effector characteristics of an unknown compound, the germinant concentration should be sufficiently low such that, in the absence of the unknown compound, the germinant concentration should be sufficiently below the maximum LRV response for the germinant alone so as to allow detection of an increase in LRV due to the presence of the effector molecule. As a control, the LRV for the known germinant in the absence of the unknown compound may be determined. Typically, L-Ala was used at a concentration that produced an LRV of about 1/2 max LRV. As in step one, the effector characteristics are measured at multiple concentrations, which facilitates identifying compounds that are effectors over only a limited concentration range and facilitates evaluating the dose- response relationship between an effector and germinant. Equations 1 or 2 can be used to identify synergy between the unknown compound and the known germinant .

LRV_EΓ-LRV_{C Ώ}

S. = ≤2 . Equa_tti.on (,,1_λ)

LRV_E Equation 1 requires computing three independent LRVs . LRV_EG is the LRV for a GM containing both the known germinant and the unknown compound. LRV_G is the LRV for the GM containing only the known germinant at the same concentration used to compute the LRV_EG. LRV_E is the LRV for the GM containing only the unknown compound at the same concentration used to compute the LRV_EG. Using equation 1, the known germinant and the unknown compound are synergistic when S_t > 1.1, additive when S_x = 1, and antagonistic or competitive when S_x < 1. The unknown compound is considered an effector "candidate" when S_x > 1.1 because a true effector will also cause a concomitant decrease in the K„ of the known germinant (discussed below) . As is apparent from equation 1, compounds that are antagonistic may produce negative S_x values.

Synergy can also be identified using equation 2. Equation 2 is preferred because under the conditions disclosed herein the resulting S values are typically positive. In equation 2, the LRV_E, LRV_G, and LRV_EG are defined the same as in equation 1.

Equation (2)

Using equation 2, the unknown compound is considered an effector candidate if S₂ > 1.1. The unknown is considered a marginal effector candidate if 1 ≤ S₂ ≤ 1.15. The unknown is not an effector if S₂ is about equal to or less than 1. As used herein, S_x corresponds to an S value computed using equation 1 and S₂ corresponds to an S value computed using equation 2.

A third step can be used to determine if an effector candidate is a true effector. This step is typically also used to determine the degree of activity for an effector or marginal effector. GMs containing a saturating concentration of the unknown compound are prepared. Saturating concentrations of the unknown compound can be computed from step two . LRVs are then computed for spores germinated in GMs containing a saturating concentration of the unknown compound and further containing increasing concentrations of the known germinant. Using a double reciprocal plot, the K„ for the germinant in the presence of the effector can be determined. Effector candidates are true effectors when the inclusion of the effector candidate in a GM causes a decrease in the apparent K„ for the germinant compared to the K„ for the germinant triggering germination in the absence of the effector candidate.

The degree of synergy between any effector or unknown compound and the known germinant can be estimated by computing a K„ ratio. The K„ ratio is the K„ computed for the germinant alone divided by the K^ computed for the germinant in the presence of the effector or unknown compound. A compound that acts as an effector will result in a K„ ratio > 1.

Simply put, tested compounds that act as germinants and/or effectors are synergistic when the LRV computed for spores germinated in a GM-base solution further containing two or more of the tested compounds is greater than the sum of the LRVs computed for spores germinated in the GM-base solution further containing each of the tested compounds individually. When the LRV for the GM-base further containing two or more tested compounds together is equal to the sum of the LRVs measured for each tested compound alone, the LRV response is additive. When the LRV for the GM-base further containing two or more tested compounds together is less than the sum of the LRVs for each tested compound alone, the LRV response is antagonistic, i.e., competitive. Spore Sensitivity to Sterilant Exposure

Microorganism or spore death caused by an external factor such as ethylene oxide (EtO) is described best using first order kinetics since the decrease in the number of surviving organisms is logarithmic. Pflug and Holcomb, "Principles of the thermal destruction of microorganisms", Disinfection, Sterilization, and Preservation, 4th edition, S.S. Block ed. , Lea and Febiger, (1991) . The number of organisms surviving after exposure to a sterilization or killing treatment can be determined using equation 3.

LogiN)=-— +Log(N₀) Equation (3)

In equation 3, N₀ is the initial number of viable spores or microorganisms and N is the number of surviving spores or microorganisms after exposure to a given sterilant dosage. U is the total sterilant exposure time, e.g., total elapsed EtO exposure time or sterilant dosage. D is a decimal reduction time, i.e., sterilant exposure required to kill one log of spores or cells. D is a constant for a given set of conditions and batch or crop of spores or cells. Thus, D is the negative reciprocal of the slope of a straight-line death curve. By identifying conditions that change the D value, the slope of the survival curve can be manipulated. A semilog plot of the observed LRV for a population of microbial spores versus sterilant exposure results in a negative-sloping line (sterilant exposure time/LRV response curve) . This curve correlates to the straight-line death curve as shown in FIG. 2. Adjusting the germination conditions such that the sterilant exposure time/LRV response curve is flatter indicates that the LRV response of a population of spores is less sensitive to sterilant exposure and visa versa. A steep slope is preferred for biological indicators because it increases the sensitivity and accuracy of the biological indicator. As discussed above, the "slope" is the absolute value of the slope of the sterilant exposure time/LRV response curve. For the conditions used herein, a steep slope is from about 0.014 to about 0.024. Preferably, the slope is from about 0.0185 to about 0.02. For the experiments described herein, the y- intercept, slope and coefficient of determination for sterilant exposure time/LRV response curves were computed using the computer program SigmaPlot (Jendel Scientific) .

A steepness index may be computed instead of determining the actual slope of the sterilant exposure time/LRV response curve. Using a steepness index is particularly useful when EtO is the sterilant. The steepness index is the value obtained when the LRV response at 0 minutes of EtO exposure (y-intercept) is divided by the LRV response computed at 60 minutes of a standard EtO exposure cycle. Useful steepness indexes are from about 7 (slope = 0.014) to about 30 (slope = 0.03-0.035), preferably from about 10 (slope « 0.016) to about 20 (slope = 0.021) . The germination media described herein may be optimized for the LRV response by adjusting the concentration of the individual ingredients so as to produce the steepest slope or largest slope index attainable for different spore types and/or different sterilants.

A standard EtO sterilization cycle employs conditions of about 60% relative humidity, EtO exposure at a concentration of 600 mg EtO/L of air at a temperature of about 55°C. The ethylene oxide sterilization cycles used herein were conducted in a Joslyn Biological Indicator Evaluator Resistomer (B.I.E.R.) gas sterilizer. The cycles included a 15 minute preheat at 55°C followed by a 30 minute dwell time at 60% relative humidity followed by an EtO sterilization dwell time at a concentration of 600 mg EtO/L of air at a temperature of about 55°C followed by 3 deep vacuum cycles, and a final 1 minute low vacuum aeration at 55 °C. As used herein, EtO exposure times refer to the length of the EtO sterilization dwell time only. As is conventional practice, the preheat, humidity dwell, vacuum cycles, and low vacuum aeration were kept constant.

As used herein, sterilant refers to a sterilizing agent used in any method of sterilization and includes, for example, steam, ethylene oxide, radiation, heat, sodium hypochlorite, polyvinylpyrrolidone- iodine, sodium dichlorocyanurate, low temperature steam-formaldehyde, glutaraldehyde, hydrogen peroxide, hydrogen peroxide plasma, peracetic acid and mixtures thereof.

Generally, spores must be dried onto a suitable carrier material in order to expose the spores to a sterilant. The spores can be dried on any suitable carrier material such as paper, plastic, glass, metal or other nondispersible carrier. A suitable carrier includes plastic or glass cuvettes. The spores can be dried down in the presence of various additives that can alter a spore's sensitivity to sterilant exposure. Illustrative examples of additives and methods for applying additives are disclosed in United States Patent Application Serial No. 09/061,293. Drying the spores in the presence of additives is defined as pre-conditioning the spores. The drying temperature is typically between about 45-50°C, preferably, between about 47-48°C. For example, a suspension of treated spores can be dried down in four hours at about 48°C. Spores dried in this manner appear dry on the surface, but likely retain residual amounts of bound water within the spore. Alternatively, the spore suspension can be dried onto a carrier for about 12-20 hours at about 37°C. Typically, 12 hours or overnight drying is sufficient.

To initiate spore germination, dried spores can be resuspended in a GM or GM-base solution by briefly vortexing, low power sonication (50 watt) , stirring, or any other acceptable method of resuspension. Typically, 100% resuspension of the dried spores is unattainable. Normalizing the initial absorbance of the resuspended spores to a standard initial absorbance can facilitate comparing results between different experiments. Typically, resuspended Bacillus subtilis spores were normalized to an initial Abs₄₈₀ = 0.5.

In one aspect, the invention features a defined spore germination medium (GM) that increases the LRV.

Typically, the GM includes a base solution (GM-base) , at least one germinant and at least one effector. The GM can also include a GM-base solution further containing a germinant, an effector, and/or a compound of unknown activity or any combination thereof. The GM or GM-base can be unfiltered or filtered through a 0.2 μm sterile filter. A filtered germination medium is designated by an "F" preceding the media designation, e.g., FGM or FGM- base . A GM may or may not trigger spore germination whereas the GM-base solution will not trigger spore germination, i.e., the measured LRV is less than or equal to about 0.0008 Abs₄₈₀/min. As used herein, baseline germination or no germination measured spectrophotometrically is an LRV ≤ 0.0005 ± 0.0003 Abs₄₈₀/min.

Optimal Bacillus subtilis spore GM-base solutions can include the following constituents: (1) 0.01-0.2 M phosphate buffer (equal parts KH₂P0₄ and Na₂HP0₄) , pH 6.8-7.8 at 23°C;

(2) 1-10 g/L NaCl; (3) 5-15 g/L KCl ;

(4) 0.03-1.5 g/L glucose; and

(5) 0.03-1.5 g/L fructose.

Although the examples disclosed herein used GM- base solutions that were within the concentration ranges recited above, the disclosed methods are applicable to GM-base solutions lacking one or all of the constituents listed. GM-base solutions lacking one or more of the described constituents can be useful for identifying germinants and effectors of non-GS-II mediated germination mechanisms and for optimizing the germination parameters of spores other than Bacillus subtilis .

Altering the phosphate concentration is useful for accommodating the sensitivity of different types of spores or different spore crops used in a biological indicator. For Bacillus subtilis spores, increasing the phosphate concentration in a FGM-base solution from about 0.01 to about 0.2 M phosphate concentration increased the slope (i.e., more negative) of the EtO exposure time/LRV response curve from about 0.0118 to about 0.0205, respectively. Increasing the phosphate concentration in a FGM-base solution from about 0.01 to about 0.1 M phosphate increases in the y-intercept for a plot of the EtO exposure time/LRV response curve up to about 0.1 M phosphate. Increasing the phosphate concentration above about 0.15 M causes an appreciable decrease in the y- intercept .

A preferred Bacillus subtilis spore GM-base solution includes: (1) 0.1 M phosphate buffer (6.95 g/L KH₂P0₄ + 6.95 g/L Na₂HP0₄) , pH 7.25 at 22 °C;

(2) 6.0 g/L NaCl (0.1 M) ; (3) 15 g/L KC1 (0.201 M) ;

(4) 0.3 g/L glucose (1.67 mM) ; and

(5) 0.3 g/L fructose (1.67 mM) .

Another preferred GM-base solution includes constituents 1-5 from above wherein the phosphate buffer is at a concentration of about 0.15 M. Other buffers including the zwitterion buffers such as ACES, HEPES , MOPS, PIPES and TRIZMA buffers can be used in GM-base solutions. However, the LRV response measured for spores germinated in GMs containing buffers other than phosphate is less responsive and thus phosphate is preferred for GMs used in biological indicators. Additionally, the effects of MgS0₄, CaCl₂ and urea have been evaluated. Other than urea, which reduced the observed lag period for spore germination by up to 1 minute, there does not appear to be any benefit to adding such compounds.

As used herein, germinants trigger spore germination to a significant extent when added to a GM- base solution, i.e., LRV > 0.008 Abs₄₈₀/min. Effectors cause a synergistic increase in the LRV when added to a GM containing a germinant and cause a concomitant decrease in the K^ of the germinant. Germinants and effectors include the tested compounds and operable derivatives thereof. As used herein, an operable derivative is a derivative of a germinant or effector that when tested using the methods disclosed herein continues to function as a germinant, effector or both. Moreover, a compound can be both a germinant and an effector, e.g., L-Thr.

Due to the low concentrations of germinants and effectors used herein, it is important to use germinants and effectors that are substantially free of other germination active contaminants. Methods to identify contaminants are known. These methods include known D₂0 nuclear magnetic resonance ("NMR") techniques. Generally, the detection limits of these methods are on the order of 0.01 % weight/weight (w/w) . Substantially free of other germination active contaminants is defined as a contaminant at a concentration below about 0.01% w/w. L-Ala is likely to be the most prevalent detectable contaminant because L-Ala is orders of magnitude more active than other known germination active compounds. Since most contaminants are expected to be < 0.1% in commercial lots of L-Gln or L-Asn, the concentrations of other contaminating amino acids would not be expected to have a significant effect on the LRV.

Useful germinants include the compounds listed in Table 1. The concentration ranges listed in Table 1 are the ranges that triggered Bacillus subtilis spore (initial Abs₄₈₀ normalized to 0.5) germination in FGM from barely above the background (LRV « 0.001 Abs₄₈₀/min) to about the observed maximum for each germinant .

Table 1 Germinant Concentration Ranges GERMINANT CONCENTRATION RANGE

L-alanine (L-Ala) 0.0003-1 mg/ml L-o.-amino butyric acid (L-α..Abu) 0.001-1 mg/ml

L-norvaline (L-nVal) 0.01-30 mg/ml

L-cysteine (L-Cys) 0.03-3 mg/ml L-valine (L-Val) 0.03-30 mg/ml

L-isoleucine (L-Ile) 0.1-10 mg/ml β-alanine (β-Ala) 0.2-10 mg/ml

L-serine (L-Ser) 0.3-10 mg/ml

L-homoserine (L-hSer) 0.3-30 mg/ml L-leucine (L-Leu) 1-30 mg/ml

L-methionine (L-Met) 1-30 mg/ml

L-allothreonine (L-aThr) 1-30 mg/ml

L-threonine (L-Thr) 3-30 mg/ml As used herein, preferred effectors are those compounds that, in combination with identified germinants such as L-Ala or L-Val, gave the steepest EtO exposure time/LRV response curves. Preferred effectors include L- Gin and L-Asn used at concentrations from about 0.3 g/L to about 10 g/L. Synergy can be measured at L-Asn or L- Gln concentrations as low as 0.1 mg/ml, but neither the LRV nor the EtO exposure time/LRV response is useful in biological indicators at concentrations below about 1 mg/ml .

Illustrative examples of useful GMs containing both germinants and effectors include GMs containing the germinant L-Val at a concentration from about 0.1 g/L to about 1.0 g/L or the germinant L-Ala from about 1.0 mg/L to about 10 mg/L and further containing the effector L- Gln at a concentration from about 1 g/L to about 10 g/L or the effector L-Asn at a concentration from about 1 g/L to about 10 g/L.

A preferred GM includes a preferred FGM-base solution containing 0.15 M phosphate buffer (pH 7-7.4) additionally containing L-Val at a concentration from about 0.1 g/L to about 0.3 g/L and L-Gln at a concentration from about 3 g/L to about 8 g/L. A more preferred GM includes the preferred FGM-base solution additionally containing L-Val at a concentration of about 0.1 g/L (0.85 mM) and L-Gln at a concentration of about 5 g/L (34.2 mM) . L-Ala at a concentration from about 1 mg/L to about 4 mg/L, preferably 1 mg/L, can be substituted for L-Val. At 1 mg/L, however, the stability of the L-Ala concentration in the FGM is difficult to maintain. L-Asn at a concentration of about 3 g/L can be substituted for L-Gln, but the slope of the EtO exposure time/LRV response curve is not as steep.

Additional examples of compounds, such as known amino acids and derivatives thereof, tested for their germinant or effector activity according to the methods described herein include the compounds listed in Tables 2 (active effectors) , 3 (borderline effectors) , and 4 (inactive effectors).

LRV measured for GM containing L-Ala at a concentration of 0.006 mg/ml,

RV measured for GM containing L-Ala at a concentration of 0.006 mg/ml.

germinant . Tables 5 and 6 depict the K„ computed for the germinant L-Ala in the presence or absence of effectors or borderline effectors in Tables 2 and 3, respectively.

Table 5 !£„ (mM) Ratios of Active Effectors

K„ L-Ala i , L-Ala EFFECTOR met /ml (alone) (+ef f ector) Ratio

L-asparagine 3 0.0157 0.0024 6.54

L-glutamine 3 0.0157 0.0033 4.76

L-homoserine 3 0.014 0.009 1.55

L-threonine 5 0.015 0.008 1.88 4-acetamidobutyric 6 0.015 0.006 2.5 acid Table 6 K_JJ (mM) Ratios of Borderline Effectors

K„ L-Ala I, L-Ala

EFFECTOR mq/ml (alone) (+effector) Ratio

L-histidine 10 0.015 0.017 0.88

L-serine 0.6 0.014 0.015 0.93

2 -acetamidoacetic 3 0.014 0.014 1.0 acid

6- 6 0.015 0.014 1.0 acetamidohexanoic acid

In another aspect of the invention, GS-II is shown to be more sensitive to EtO than either GS-I or GS-III. Since GS-II is sensitive to EtO, it is preferable to trigger the GS-II mechanism as opposed to the GS-I or GS- III mechanism for biological indicators used in EtO sterilizers. Further, the discovery of cooperativity between effector molecules and germinants facilitates the manipulation of the germinant and effector concentrations in a GM in order to alter the correspondence between spore germination rates and sterilant exposure time. The invention will be further understood with reference to the following illustrative embodiments, which are purely exemplary, and should not be taken as limiting the true scope of the present invention as described in the claims. Unless otherwise indicated, each example used commercial lots of L-Asn or L-Gln that were substantially free of other germination active contaminants .

Example 1 Synergistic Interactions between L-Asn or L-Gln and L-Ala

This example illustrates the difference in germination activity levels observed for commercial lots of L-Gln and L-Asn. Commercial lots of L-Gln and L-Asn were purchased from Sigma Chemical Company (Lot# 15F-0680 & 89F-0398) or Aldrich Chemical Company (Lot# 06710EV & 03220PN) , respectively. Twenty microliters of heat activated Bacillus subtilis spore suspensions, approximately 1.2 x 10⁸ spores, were added to polymethacrylate semimicro cuvettes. 1.2 ml of FGM containing FGM-base solution (1.67 mM fructose, 1.67 mM glucose, 0.2 M KCl, 0.1 M NaCl and 0.1 M phosphate buffer (6.95 g KH₂P0₄/L + 6.95 g Na₂HP0₄/L, pH 7.25 at 22°C) ) and the appropriate concentration of germinant and/or effector were added to each cuvette to initiate germination. The appropriate concentrations of L-Ala (Aldrich Lot# AF06311PZ) , L-Gln, and/or L-Asn are indicated in Table 7. FGMs containing L-Asn or L-Gln were used to trigger Bacillus subtilis spore germination in the presence or absence of L-Ala. Initial experiments conducted with FGM-base solutions further containing increasing concentrations of active lots of L-Asn (lot# 06710EV) or L-Gln (Lot# 15F-0680) indicated that at L-Asn would saturate the LRV response at 10 mg/ml and L-Gln would saturate the LRV response at 6 mg/ml . The L-Ala concentrations were below saturating concentrations.

Spore germination was followed by recording the change in Abs₄₈₀ at 37 °C in a temperature controlled Varian/Cary 1 UV/visible spectrophotometer. LRVs were calculated from the resulting germination kinetics curves . The mean LRV and accompanying percent standard deviation (%SD) computed for each reaction are shown in Table 7.

TABLE 7 LRVs Computed for L-Ala, L-Asn, or L-Gln

Compound 1 Compound 2 Cmpd#2 Mean LRV (mcr/ml) (mq/ml) Lot

%SD

L-Ala (0.001) 0.008 10.2 L-Ala (0.003) 0.017 2.9 L-Ala (0.01) 0.026 3.0 L-Ala (0.03) 0.035 2.8

L-Asn (10) 06710EV 0.028 0.2 L-Asn (10) 03220PN 0.006 7.8

L-Ala (0.001) L-Asn (10) 03220PN 0.027 3.6 L-Ala (0.003) L-Asn (10) 03220PN 0.037 2.3 L-Ala (0.01) L-Asn (10) 03220PN 0.041 1.1 L-Ala (0.03) L-Asn (10) 03220PN 0.042 5.6 L-Ala (0.001) 0.007 8.3 L-Ala (0.003) 0.017 2.2 L-Ala (0.01) 0.026 4.0 L-Ala (0.03) 0.034 1.8

L-Gln (6) 15F-0680 0.044 3.0 L-Gln (6) 89F-0398 0.003 9.8

L-Ala (0.001) L-Gln (6) 89F-0398 0.031 1.1 L-Ala (0.003) L-Gln (6) 89F-0398 0.040 1.0 L-Ala (0.01) L-Gln (6) 89F-0398 0.042 1.5 L-Ala (0.03) L-Gln (6) 89F-0398 0.044 3.5 L-Ala added to FGMs containing inactive L-Asn (lot#

0322 OPN) or FGM-base increased the LRV in a concentration dependent manner. The LRV for active L-Asn (lot# 06710EV) was approximately equal to the LRV computed for inactive L-Asn combined with L-Ala at a concentration of 0.001 mg/ml. For GMs containing inactive L-Asn, increasing the L-Ala concentration increased the LRV to a maximum of 0.042 at an L-Ala concentration of 0.03 mg/ml. The results suggested that the active lot of L-Asn contained approximately 0.001 mg/ml of L-Ala, which was at or below the chemical detection limits of D₂0 NMR (i.e. , 0.01% w/w) .

The LRV computed for the active lot of L-Gln (lot# 15F- 0680) was equivalent to the LRV computed for 0.01 mg/ml L-Ala added to the inactive lot of L-Gln. When inactive L-Gln (lot# 89F-0398) was combined with L-Ala, the LRV reached a plateau at an L-Ala concentration between 0.003-0.006 mg/ml, which suggested that the active lot of L-Gln contained at least 0.06% L-Ala.

It is known that commercial lots of amino acids are not 100% pure. Thus, the LRV activity of "inactive" L-Gln or L-Asn may have been due to trace contaminations of a germination active compound. Samples of active and inactive L-Gln were analyzed using D₂0 NMR analysis, a minimum of 0.06% L-Ala was detected in the active lot of L-Gln and no L-Ala could be detected in the inactive lot of L-Gln. These results indicated that GS-II was not triggered by L-Asn or L-Gln in a FGM containing glucose, fructose, and potassium, as is described in the literature. See Wax & Freese, J. Bact. , 95:433-437 (1968) ; Johnstone, J. Appl. Bact. Symposium Suppl., 76:17S-24S (1994) . The S values computed for L-Asn in the presence of 0.001 mg/ml of L-Ala {S₁ = 3.2; S₂ = 2.6) indicated that L-Asn was synergistic with L-Ala. The S values computed for L-Gln in the presence of 0.001 mg/ml of L-Ala (S₁ = 8.0; S₂ = 4.0) indicated that L-Gln was synergistic with L-Ala.

Example 2 Identifying Synergy

This example illustrates a method for identifying additional germinants and/or effector candidates . LRVs were computed in FGMs prepared according to the procedures described in Example 1. Each unknown compound was used to compute an LRV alone, combined with L-Ala, and combined with L-Gln. The results are shown in Table 8. The LRV computed for the FGM-base solution was < 0.001 ABS₄₈₀/min. TABLE 8 Identifying Synergy Between Compounds of Unknown Spore

Germination Activity

The L-Ala concentrat on was fixed in the FGM at 0.006 mg/ml .

² LRV_G = L-Ala at a concentration of 0.006 mg/ml (LRV 0.0232 Abs₄₈₀/min) (see row 2 of this table).

³ The L-Gln concentration was fixed in the FGM at 4.0 mg/ml . ⁴ LRV_G = L-Gln at a concentration of 4.0 mg/ml (LRV 0.0037 Abs₄₈₀/min) (see row 18 of this table).

Compounds that produced an LRV_E greater than about 0.01 .Abs₄₈₀/min were considered germinants. Compounds that were synergistic with L-Ala were considered effector candidates . Compounds that had an LRV less than or equal to L-Gln or L-Asn alone and an S value greater than 1 when tested in combination with L-Ala were also considered effector candidates. Some compounds were classified as both a germinant and an effector candidate. For example, L-Thr had an LRV_E of 0.0135 at 7.5 mg/ml. In addition, L-Thr had an S_x > 1 with both L-Ala and L-Gln. As is apparent from Table 8, some compounds (e.g., L- Asn, L-Gln) are weaker germinants than others (e.g., L- Ala and L-Val) . Further, the LRV of the L-Gln or L-Asn may have been due to trace contaminations of a germination active compound, most likely L-Ala at a concentration below about 0.3 μg/ml. Using this method any compound or an operable derivative thereof can be identified as a germinant or an effector candidate.

Example 3 Concentration Dependence of Synergistic Interactions This example illustrates a method for computing changes in the K„ for germinants in the presence of effector compounds or effector candidate compounds. FGMs were prepared according to the procedures described in Example 1. The concentration of the germinant L-Ala or L-Val was increased as indicated in Table 9 to create a saturation kinetics curve. LRVs including standard deviations (%SD) were computed.

Table 9 Concentration Dependence of Synergy

Germinant Effector Mean LRV % SD

(mq/ml) (mg/ml) ABS,,_R„/min

L-Ala ( 0.001) 0.012 1.0

L-Ala ( 0.003) 0.023 1.3

L-Ala ( 0.01) 0.030 1.3

L-Ala ( 0.03) 0.035 1.0

L-Ala ( 0.1) 0.040 0.9

L-Ala 0.3) 0.042 1.0

L-Ala 0.001) L-Gln (4) 0.038 0.9

L-Ala 0.003) L-Gln (4) 0.043 0.7

L-Ala 0.01) L-Gln (4) 0.046 1.1

L-Ala 0.03) L-Gln (4) 0.046 1.5

L-Ala 0.1) L-Gln (4) 0.046 1.2

L-Ala 0.3) L-Gln (4) 0.046 1.3

L-Ala (0.001) L-Asn (3) 0.038 0.6

L-Ala (0.003) L-Asn (3) 0.043 1.3

L-Ala (0.01) L-Asn (3) 0.045 1.5

L-Ala (0.03) L-Asn (3) 0.046 0.9

L-Ala (0.1) L-Asn (3) 0.046 3.2

L-Ala (0.3) L-Asn (3) 0.045 0.6

L-Val (0.03) 0.012 0.8

L-Val (0.1) 0.030 0.8

L-Val (1.0) 0.038 0.6

L-Val (3.0) 0.042 1.2

L-Val (10.0) 0.045 1.4

L-Val (30.0) 0.044 0.7

L-Val (0.03) L-Gln (4) 0.014 6.6

L-Val (0.1) L-Gln (4) 0.036 1.1

L-Val (0.3) L-Gln (4) 0.045 0.6

L-Val (1.0) L-Gln (4) 0.048 1.2

L-Val (3.0) L-Gln (4) 0.048 0.5

L-Val (10.0) L-Gln (4) 0.048 1.3

L-Val (0.03) L-Asn (3) 0.016 0.6

L-Val (0.1) L-Asn (3) 0.037 0.7

L-Val (0.3) L-Asn (3) 0.046 1.6

L-Val (1.0) L-Asn (3) 0.048 1.1

L-Val (3.0) L-Asn (3) 0.049 0.8

L-Val (10.0) L-Asn (3) 0.048 1.9

The LRV response for FGMs containing both germinant and effector was saturated at about 0.01 mg/ml for L-Ala (0.112 mM) and at about 1.0 mg/ml for L-Val (8.54 mM) , well below the saturating concentration for either of the germinants in the absence of the effectors.

Synergistic interactions between the effectors (L-Gln and L-Asn) and the germinants (L-Ala and L-Val) were assessed using a double reciprocal plot . A commercial enzyme kinetics software package (version 1.5, Trinity Software) was used to compute a 1^ (mM) for the germinants using the data from Table 9. The results are shown in Table 10. The correlation coefficients, i.e., coefficient of determination or R², for the K„ determinations fell between 0.983 and 1.0.

Table 10 ]£„ (mM) Ratios

Germinant Alone +L-Asn Ratio -t-L-Gln Ratio L-Ala (L) 0.02 0.002 10 0.002 10 L-Ala (H) 0.088 0.002 44 0.002 44 L-Val 1.342 0.255 5.3 0.292 4.6

The germinant concentration versus LRV response for L-

Ala was biphasic indicating that L-Ala bound to more than one receptor with different affinities. Therefore, two K^ values were computed: L-Ala (L) (0.001-0.01 mg/ml) and L-

Ala (H) (0.01-0.3 mg/ml).

The germinant concentration versus LRV response for L-

Val was monophasic. In addition, L-Val did not trigger spore germination at concentrations as low as L-Ala preventing K„ determinations for L-Val below 0.1 mg/ml. As shown in Table 10, both L-Asn and L-Gln substantially reduced the K„ for L-Ala and L-Val triggered germination. The synergistic effect observed for L-Ala (L) was approximately twice that observed for L-Val.

Since the synergistic LRV response observed for L-Val did not show biphasic kinetics, it was not clear whether or not synergy occurred at low affinity sites. Example 4 Evaluating the EtO Exposure time/LRV response

Twenty microliters of an aqueous Bacillus subtilis spore suspension, approximately 1.2 x 10⁸ spores, was added to polymethacrylate semimicro cuvettes and then dried for 4 hours at 47°C. The dried spores were then exposed to various EtO exposure times in a Joslyn B.I.E.R gas sterilizer. Each complete cycle proceeded as follows: 1) 15 minute preheat at 55°C; 2) 30 minute dwell time at 60% relative humidity; 3) EtO sterilization dwell time at a concentration of 600 mg EtO/L of air at a temperature of about 55°C (total time varied as indicated in Table 11) ; 4) 3 deep vacuum cycles; and 5) a 1 minute low vacuum aeration at 55°C. After EtO exposure, 1.2 ml of FGM containing 1.67 mM fructose, 1.67 mM glucose, 0.2 M KCl, 0.1 M NaCl and 0.1 M phosphate buffer (6.95 g KH₂P0₄/L + 6.95 g Na₂HP0₄/L, pH 7.25 at 22°C) was added to each cuvette. The FGM also included the appropriate concentration of a germinant and/or effector as indicated in Table 11.

The spores were resuspended by vortexing for 10 seconds. Spore germination was observed by recording the change in Abs₄₈₀ at 37°C in a temperature controlled Varian/Cary 1 UV/visible spectrophotometer. Table 11 depicts the LRVs calculated from the germination kinetics curves following EtO exposure.

Table 11 EtO Exposure Time/LRV Response

Germinant Effector EtO Exp. Mean LRV

(mg /ml) (mq/m. (Min) (Min) %SD

L-Ala (0.001) L-Asn ( 3.0) 5 0.020 3.1

L-Ala (0.001) L-Asn ( 3.0) 20 0.010 5.8

L-Ala (0.001) L-Asn ( 3.0) 40 0.005 10.9

L-Ala (0.001) L-Asn ( 3.0) 60 0.003 15.2

L-Ala (0.01) L-Asn ( 3.0) 5 0.025 3.2

L-Ala (0.01) L-Asn ( 3.0) 20 0.016 7.4

L-Ala (0.01) L-Asn ( 3.0) 40 0.009 7.8

L-Ala (0.01) L-Asn ( 3.0) 60 0.006 5.6

L-Ala (0.1) L-Asn ( 3.0) 5 0.025 1.1

L-Ala (0.1) L-Asn ( 3.0) 20 0.015 3.0

L-Ala (0.1) L-Asn ( 3.0) 40 0.010 1.5

L-Ala (0.1) L-Asn 3.0) 60 0.007 15.4

L-Ala (0.001) L-Gln 3.0) 5 0.013 13.1

L-Ala (0.001) L-Gln 3.0) 20 0.006 12.4

L-Ala (0.001) L-Gln (3.0) 40 0.002 8.2

L-Ala (0.001) L-Gln (3.0) 60 0.001 22.9

L-Ala (0.01) L-Gln (3.0) 5 0.020 7.7

L-Ala (0.01) L-Gln (3.0) 20 0.013 8.9

L-Ala (0.01) L-Gln (3.0) 40 0.006 9.0

L-Ala (0.01) L-Gln (3.0) 60 0.003 13.4

L-Ala (0.1) L-Gln (3.0) 5 0.020 14.2

L-Ala (0.1) L-Gln (3.0) 20 0.015 15.7

L-Ala (0.1) L-Gln (3.0) 40 0.007 11.0

L-Ala (0.1) L-Gln (3.0) 60 0.007 14.9

A plot of the EtO exposure time versus log (LRV) resulted in a straight line. Slopes of the lines were computed using Sigmaplot (Jendel Scientific) . The slope values for the EtO exposure time/LRV response curves for spore germination triggered with L-Ala at a concentration of 0.001, 0.01, and 0.1 mg/ml in the presence of 3.0 mg/ml L-Asn were 0.0157, 0.011, and 0.0095, respectively. The slope values for the EtO exposure time/LRV response curves for spore germination triggered with L-Ala at a concentration of 0.001, 0.01, and 0.1 mg/ml in the presence of L-Gln were 0.0177, 0.014, and 0.0087, respectively . Increasing the L-Ala concentration in the FGM containing either L-Asn or L-Gln decreased the slope of the EtO exposure time/LRV response curve. Increasing the L-Ala concentration lowered the sensitivity of the LRV response, which is likely due to L-Ala triggering a non-GSII spore germination mechanism.

Example 5 Synergistic interactions between L-Thr, L-aThr and L-Ala

Synergistic interactions between L-Thr, L-aThr and L- Ala were determined by computing LRVs using the experimental procedures indicated in Examples 1 and 2. The results are shown in Table 12.

TABLE 12

L-Thr and L-aThr Evaluations

Germinant Effector Mean LRV (mg/ml) (mg/ml) (Min) %SD s₂ L-Ala (0.0006) 0.024 2. ,0 - L-Thr (5.0) 0.005 6. .3 - L-Thr (7.5) 0.014 2. .3 - L-Thr (10) 0.024 5, .4 - L-Thr (15) 0.038 0, .7 - L-Thr (20) 0.042 1. .3 -

L-Ala (0.0006) L-Thr (5.0) 0.041 2 .6 1.50

L-Ala (0.0006) L-Thr (7.5) 0.044 1 .1 1.25

L-Ala (0.0006) L-Thr (10) 0.045 0 .6 0.87

L-Ala (0.0006) L-Thr (15) 0.046 2 .4 0.33 L-Ala (0.0006) L-Thr (20) 0.049 2 .4 0.29

L-Ala (0.0006) 0.036 L-aThr (1.0) 0.013 L-aThr (2.0) 0.027 L-aThr (4.0) 0.040 L-aThr (7.0) 0.045 L-aThr (10) 0.047

L-Ala (0.0006) L-aThr (1.0) 0.039 0.72

L-Ala (0.0006) L-aThr (2.0) 0.041 0.39

L-Ala (0.0006) L-aThr (4.0) 0.044 0.11 L-Ala (0.0006) L-aThr (7.0) 0.048 0.08

L-Ala (0.0006) L-aThr (10) 0.049 0.06

S₂ values greater than 1.1, indicated that L-Thr was synergistic with L-Ala at concentrations below 7.5 mg/ml. Additionally, unlike L-Asn and L-Gln, L-Thr itself triggered spore germination. L-Thr may constitute a compound that is capable of homotropic positive cooperativity .

The experimental procedures set forth in Example 3 were used to compute the K„ for L-Ala in the presence of L-Thr. FGMs containing L-Ala between 0.001 and 0.01 mg/ml with or without L-Thr at 10 mg/ml resulted in a ^ ratio of 5.5 (K„ L-Ala = 0.033 mM, K„ L-Ala+L-Thr = 0.006 mM) . The correlation coefficients for the transformations were between 0.941 and 0.992. L-Thr increased the binding efficiency for L-Ala and indicated that L-Thr was an effector of GS-II germination triggered by L-Ala.

L-aThr triggered spore germination. S₂ values << 1, however, indicated that L-aThr was not synergistic with L-Ala. Additional experiments using the procedures described in Example 3 were conducted to determine if L- aThr altered the binding efficiency of L-Ala. In the additional experiments, L-Ala was varied from 0.001 mg/ml to 0.009 mg/ml in the presence or absence of L-aThr at a concentration of 5.0 mg/ml. The K,,, ratio for L-Ala in the presence or absence of L-aThr was 12.7 (K„ (L-Ala) = 0.014 mM, K„ (L-Ala+L-aThr) = 0.0011 mM) . Thus, L-aThr was anomalous in that its S₂ values indicated that L-aThr was not synergistic with L-Ala and yet the K„ ratio indicated a significant synergistic interaction. It is possible that L-aThr is anomalous because its germinant characteristics dominated over its effector characteristics .

Example 6 Synergistic Interactions Between L-Cvs and L-Ala Experimental procedures as described in Examples 1 and 2 were used to compute LRVs for L-Cys and L-Ser with L- Ala. The results are shown in Table 13.

Table 13 Synergistic Interactions of L-Cys or L-Ser with L-Ala

Germinant Effector Mean LRV s₂

L-Ala (0.006) 0.028 -

L-Cys (0.03) 0.006 -

L-Cys (0.1) 0.012 -

L-Cys (0.3) 0.029 -

L-Cys (1.0) 0.037 -

L-Ala (0.006) L-Cys (0.03) 0.027 0.75

L-Ala (0.006) L-Cys (0.1) 0.028 0.57

L-Ala (0.006) L-Cys (0.3) 0.031 0.07

L-Ala (0.006) L-Cys (1.0) 0.036 -0.04

L-Ala (0.006) 0.026 -

L-Ser (0.3) 0.002 -

L-Ser (1.0) 0.009 -

L-Ser (3.0) 0.016 -

L-Ser (10.0) 0.021 -

L-Ala (0.006) L-Ser (0.3) 0.031 1.15

L-Ala (0.006) L-Ser (1.0) 0.036 1.0

L-Ala (0.006) L-Ser (3.0) 0.030 0.54

L-Ala (0.006) L-Ser (10.0) 0.026 0.19

L-Cys was not synerg:istic wi ,th L-Ala. An S₂ << 1 indicated an antagonistic effect similar to the effects observed for other germinants that were also not synergistic with L-Ala (see Table 8) . L-Cys did trigger spore germination with relatively high germination rates.

Thus, it is expected that L-Gln and L-Asn would be synergistic with L-Cys. At lower concentrations of L-Ser, L-Ser was synergistic with L-Ala. At higher concentrations the L-Ser and L-Ala became antagonistic, similar to the response observed for L-Thr (Example 5) . Thus, L-Ser was a borderline effector candidate .

Example 7 Effect of GM Composition on the LRV Response This example illustrates the superiority of the additional ingredients contained in a preferred FGM-base solution compared to an FPO solution containing only filtered 50 mM phosphate buffer (equal parts Na₂HP0₄ and KH₂P0₄) . FPO is the standard medium used for activating only GS-I mediated spore germination in the presence of a germinant. GS-I and GS-III mediated germination are activated when an FGM contains a germinant. GS-II mediated germination is activated when the FGM contains both a germinant and an effector. Only the GS-II mediated germination, however, is sensitive enough to EtO sterilization to be useful for monitoring the effectiveness of an EtO sterilization process.

FGM-base solution containing 1.67 mM fructose, 1.67 mM glucose, 0.2 M KCl, 0.1 M NaCl and 0.1 M phosphate buffer (6.95 g KH₂P0₄/L + 6.95 g Na₂HP0₄/L, pH 7.25 at 22°C) was combined with L-Ala at concentrations indicated in Table 14. Each germination reaction was repeated in the presence or absence of the effector L-Gln at a concentration of 3 mg/ml . The experimental procedures were repeated using an FGM-base solution containing only filtered 50 mM phosphate (FPO-base) . The results are shown in Table 14

Table 14 Buffer Effects on LRV Response

FGM-base [L-Ala] L-Gln LRV %SD

FPO 0.001 0.0169 2.3

FPO 0.003 - 0.0171 3.5

FPO 0.01 - 0.0250 1.3

FPO 0.03 - 0.0288 0.4

FPO 0.3 - 0.0295 1.9

FPO 0.001 + 0.0164 6.3

FPO 0.003 + 0.0264 2.2

FPO 0.01 + 0.0313 2.0

FPO 0.03 + 0.0342 1.6

FPO 0.3 + 0.0345 3.7

FGM 0.001 - 0.0182 4.3

FGM 0.003 - 0.0300 2.3

FGM 0.006 - 0.0359 2.5

FGM 0.009 - 0.0377 0.8

FGM 0.001 + 0.0417 1.8

FGM 0.003 + 0.0485 1.7

FGM 0.006 + 0.0511 2.6

FGM 0.009 + 0.0527 1.7

The Kψ, computed for L-Ala in the presence or absence of L-Gln in the FPO-base solution was 0.0257 mM and 0.0119 mM (K„ Ratio = 2.2) , respectively. The K„ computed for L- Ala in the presence or absence of L-Gln in the preferred FGM-base solution was 0.0157 mM and 0.0033 mM (K„ ratio = 4.8), respectively. Thus, even though synergy was observed in the FPO-base media, it was apparent that the additional ingredients contained in the FGM enhanced the synergistic interaction of the effector and the germinant . Example 8

Differentiating the Buffer Effects that

Contribute to the EtO Exposure Time/LRV Response

Experimental procedures described in Example 4 were followed in order to compare the y-intercept and slope of the EtO exposure time/LRV response curve for the combination of L-Gln and L-Val in either an FGM-base solution containing 1.67 mM fructose, 1.67 mM glucose, 0.2 M KCl, 0.1 M NaCl and 0.1 M phosphate buffer (6.95 g KH₂P0₄/L + 6.95 g Na₂HP0₄/L, pH 7.25 at 22°C) or in filtered 50 mM phosphate buffer (FPO) consisting of equal parts Na₂HP0₄ and KH₂P0₄.

Table 15 Buffer Effects

Germinant (mg/ml)/

Media Effector (mq/ml) Y-Int . Slope Rj;

FPO L-Ala (0.01) 0. .0149 0. ,0066 0. .99 FPO L-Ala (0.10) 0. .0171 0. .0056 0. .98 FPO L-Val (0.1) 0. .0003 0. .0000 -- FPO L-Ala (0.01) /L-Gln (3.0) 00.. .00119922 0, .0079 1. .00 FGM L-Val (0.1) 0, .0060 0, .0106 1. .00 FGM L-Val (0.1) /L-Gln (3.0) 0, .0349 0, .0177 1. .00 FGM L-Val (1.0) /L-Gln (3.0) 0 .0410 0 .0094 1 .00 FGM L-Val (10.0) /L-Gln (3.0] 0 .0406 0 .0082 1, .00 Both the initial LRV, represented by the y- intercept, and the slope of the EtO exposure time/LRV response demonstrate that the germination mechanism triggered by the FGM is superior to FPO. Thus, it is desirable to trigger the GS-II germination mechanism when monitoring an EtO sterilization process. Further, this example indicates that excessive germinant concentrations will decrease (i.e., less negative) the slope of the EtO exposure time/LRV response, which was also observed in Example 4. Example 9 Effect of D-isomers

It is known that D-isomers of germination active amino acids competitively inhibit spore germination. Woese et al., J. Bact. , 76:578-88 (1958). D-Gln, D-Asn and D-Thr were evaluated as effector compounds using the experimental procedures set forth in Examples 1 and 2. The results are shown in Tables 16 and 17.

Table 16 D- Amino Acids

LRV_G = 0.0360 A s₄₈₀ m n L-A a 0.006 mg m . ² LRV_G = 0.0370 Abs₄₈₀/min (L-Ala 0.006 mg/ml) . ³ LRV_G = 0.0381 Abs₄₈₀/min (L-Ala 0.006 mg/ml) ; Table 17 K„ Ratios

i , L-Ala K_M L-Ala + K„ Ratio

Effector mg/ml Alone (a) Effector (b) a/b D-Asn 10.0 0.0129 0.0035 3.7

D-Gln 10.0 0.0128 0.0097 1.3

D-Thr 0.3 0.0153 0.0958 0.2

D-Asn and D-Gln were synergistic with L-Ala while D-Thr was antagonistic. These results confirmed that L-Thr is both an effector and a germinant. Further, these results confirm that L-Asn and L-Gln are only effector molecules and do not act as germinants because the D-isomers did not inhibit L-Ala triggered germination and did not trigger germination when used alone. The results for D- Asn and D-Gln were very unexpected because it is generally thought that the D-amino acids act as competitive inhibitors for known germinants. The observed lack of germination inhibition by D-Asn and D- Gln, however, suggests that the binding sites for effectors and germinants are distinct. The results also support the conclusion that the germinant activity attributed to commercial lots of L-Asn and L-Gln is indeed caused by a contaminant such as trace amounts of L-Ala . See Example 1. Other aspects, advantages, and modifications are within the scope of the following claims.

Claims

What is claimed is:

1. A spore germination medium comprising: a) a phosphate buffer at a concentration from about 0.1 molar to about 0.2 molar; and b) synergistic amounts of a germinant and an effector.

2. The spore germination medium of claim 1 wherein said medium is optimized for the LRV response of microbial spores .

3. The spore germination medium of claim 2 wherein said phosphate buffer is at a concentration of about 0.15 molar.

4. The spore germination medium of claim 1 wherein at least one said germinant comprises an amino acid or an operable derivative thereof.

5. The spore germination medium of claim 4 wherein at least one said germinant comprises an L-amino acid.

6. The spore germination medium of claim 1 wherein at least one said germinant is selected from the group consisting of alanine, ╬▒-amino butyric acid, norvaline, cysteine, valine, isoleucine, ╬▓- alanine, serine, homoserine, leucine, methionine, allothreonine, and threonine.

7. The spore germination medium of claim 6 wherein at least one said germinant comprises L-valine.

8. The spore germination medium of claim 6 wherein at least one said germinant comprises L-alanine.

9. The spore germination medium of claim 1 wherein at least one said effector comprises an amino acid or an operable derivative thereof.

10. The spore germination medium of claim 9 wherein at least one said effector comprises an L-amino acid.

11. The spore germination medium of claim 9 wherein at least one said effector comprises a D-amino acid.

12. The spore germination medium of claim 9 wherein at least one said effector is selected from the group consisting of asparagine, glutamine, homoserine, allothreonine, threonine, 4-acetamidobutyric acid, histidine, serine, 2-acetamidoacetic acid, and 6- acetamidohexanoic acid.

13. The spore germination medium of claim 12 wherein at least one said effector comprises glutamine.

14. The spore germination medium of claim 12 wherein at least one said effector comprises asparagine.

15. The spore germination medium of claim 1 wherein at least one said germinant is selected from the group consisting of alanine, L-╬▒-amino butyric acid, norvaline, cysteine, valine, isoleucine, ╬▓- alanine, serine, homoserine, leucine, methionine, allothreonine, and threonine and at least one said effector is selected from the group consisting of asparagine, glutamine, homoserine, allothreonine, threonine, 4-acetamidobutyric acid, histidine, serine, 2-acetamidoacetic acid, and 6- acetamidohexanoic acid.

16. The spore germination medium of claim 15 wherein said spore germination medium is optimized for Bacillus subtilis, Bacillus circulans, Bacillus pumilus, Clostridium perfringens, Clostridium sporogenes or Bacillus stearothermophilus spores.

17. The spore germination medium of claim 16 wherein said spore germination medium is optimized for Bacillus subtilis .

18. The spore germination medium of claim 15 wherein said germinant concentration is from about 0.1 mg/ml to about 1 mg/ml .

19. The spore germination medium of claim 15 wherein said effector concentration is from about 0.3 mg/ml to about 10 mg/ml.

20. The spore germination medium of claim 15 wherein said effector concentration is from about 3 mg/ml to about 8 mg/ml .

21. The spore germination medium of claim 15 wherein said germinant concentration is about 0.1 mg/ml and said effector concentration is from about 3 mg/ml to about 8 mg/ml .

22. The spore germination medium of claim 21 wherein said germinant comprises L-valine and said effector comprises L-Gln or L-Asn.

23. The spore germination medium of claim 15 wherein said germinant concentration is from about 0.001 mg/ml to about 0.01 mg/ml and said effector concentration is from about 3 mg/ml to about 8 mg/ml .

24. The spore germination medium of claim 23 wherein said germinant comprises L-alanine and said effector comprises L-Gln or L-Asn.

25. A spore germination medium consisting essentially of: a) a phosphate buffer at a concentration from about 0.1 molar to about 0.2 molar; b) synergistic amounts of a germinant and an effector; and c) sodium chloride, potassium chloride, glucose and fructose.

26. A method for determining the effectiveness of a sterilization process comprising the steps of:

(a) exposing microbial spores to a sterilant;

(b) contacting said spores with a spore germination medium comprising a phosphate buffer at a concentration from about 0.1 molar to about 0.2 molar, and synergistic amounts of a germinant and an effector; and (c) calculating the LRV of spore germination to determine the eff ctiveness of the sterilization process.

27. A biological indicator for indicating the effectiveness of a sterilization process comprising a germination medium comprising synergistic amounts of a germinant and an effector wherein said medium produces a sterilant exposure time/LRV response curve slope greater than about 0.016.

28. The biological indicator of claim 27 wherein said medium further comprises a phosphate buffer at a concentration from about 0.1 molar to about 0.2 molar.

29. The biological indicator of claim 28 wherein said medium produces a steepness index from about 10 to about 20.

30. The biological indicator of claim 29 wherein a sterilant used in said sterilization process is selected from the group consisting of steam, ethylene oxide, radiation, heat, sodium hypochlorite, polyvinylpyrrolidone-iodine, sodium dichlorocyanurate, low temperature steam- formaldehyde, glutaraldehyde, hydrogen peroxide, hydrogen peroxide plasma, peracetic acid and mixtures thereof.

31. The biological indicator of claim 30 wherein said sterilant comprises ethylene oxide.

32. A biological indicator for indicating the effectiveness of a sterilization process comprising: a) a first container adapted for exposing microbial spores to a sterilant and computing an LRV for germinating spores; and b) a second container adapted for storing a germination medium; c) a germination medium stored in said second container said medium comprising a phosphate buffer at a concentration from about 0.1 molar to about 0.2 molar, and synergistic amounts of a germinant and an effector; and d) dried microbial spores.

33. A method for identifying an effector comprising the steps of : a) contacting spores with a germination medium comprising a compound with unknown effector properties and lacking a germinant, and computing an LRV; b) contacting spores with a germination medium comprising said germinant and lacking said compound with unknown effector properties, and computing an LRV; c) contacting spores with a germination medium comprising said germinant and said compound with unknown effector properties, and computing an LRV; and d) calculating an S value to determine the effectiveness of said compound with unknown effector properties.