JP2001511356A - Spore germination medium - Google Patents

Abstract

(57) Abstract: A spore germination medium for increasing spore germination rate, comprising a germinating factor and an agent that interact synergistically. The germinating factor and the agent may include amino acids such as Ala, Val, Asn, Gln. The germination of spores caused by the germination medium provided by the invention can be used to measure and evaluate spore survival after exposing the spores to a sterilant.

Description

DETAILED DESCRIPTION OF THE INVENTION

FIELD OF THE INVENTION The invention relates to a microbial spore germination medium. In particular, the invention relates to germination media that affect spore germination rates. The invention further relates to a germination medium that affects the association between spore germination rate and spore survival after exposing the spores to a sterilant.

BACKGROUND OF THE INVENTION [0002] Depending on environmental factors, certain microorganisms, such as various Bacillus and Clostridium species, form endospores. Spores are rigid structures with no detectable metabolic activity. Common spore structures include the outer membrane, spore shell, spore cortex, inner membrane, and finally spore nucleus cells. Endospores are significantly more resistant than vegetative cells to environmental changes in pH, temperature, humidity and radiation. Foste
r & Johnstone Molecular Microbial. , 4
(1): 137-41 (1990).

[0003] While microbial spores remain dormant for extended periods of time, spores contain mechanisms that cause germination under certain circumstances. The germination process destroys the spore shell and spore cortex,
It consists of a series of degradation steps that allow water and nutrients to enter the spore nucleus cells.
Following germination, metabolic activity is reactivated and new vegetative cells undergo post-emergence growth.

[0004] Germination and subsequent post-emergence growth are different but related processes. That is, spores may germinate but not complete the post-emergence growth process. However, post-emergence growth of new vegetative cells cannot occur unless spores germinate. The mechanism that causes spore germination is not completely understood. In some species, spores can be germinated by exposing the spores to germinating factors and / or certain environmental stimuli. Johnstone, K .; , J. et al. Appl. Bact. Symposium
Suppl. , 76: 17S-24S (1994).

[0005] Early germination models suggest that Bacillus subtilis contains two germination pathways. In one pathway, L-Ala alone can cause spore germination (G
SI). Wax & Freese, J.M. Bact. , 95 (2): 433-
38 (1968). Another pathway requires a combination of glucose, fructose and potassium ions in addition to L-Asn or L-Gln to cause spore germination (GS-II). Wax & Freese, J.M. Bac
t. , 95 (2): 433-38 (1968). Further studies performed on Bacillus subtilis identified a third germination system. This third germination system was 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).
). However, despite the identification of multiple germination systems, the identity of the involved receptors and the interaction between the receptor and the germinatively active component remains unclear. Foster and Johnstone, Molecular
Microbiol. , 4: 137 (1990); Johnstone, K, J.
. Appl. Bact. Symposium Suppl. , 76: 17S-2
4S (1994); Moir et al. , J. et al. Appl. Bact. Sym
Posium Suppl. , 76: 9S-16S (1994).

SUMMARY OF THE INVENTION [0006] The present invention relates to the discovery that certain germinating agents and agents, when combined in a germination medium, act synergistically to alter microbial spore germination rates.
Furthermore, optimizing the concentrations of germinating agents and agents will alter the dose-response relationship between spore germination rate and sterilant exposure time, facilitating rapid assessment of bactericidal efficacy.
As used herein, a spore germination medium is any medium that induces a measurable amount of spore germination. The agent, when added to the germination medium, acts synergistically with the germinating element to increase LRV of spore germination compared to the same germination medium lacking the agent,
Is a compound that reduces the K _M apparent germination factors causing spore germination. The invention includes, but is not limited to, spores of Bacillus subtilis, Bacillus circulans, Bacillus plumis, Clostridium perfringens, Clostridium sporogenes and Bacillus stearothermophilus Useful for germinating non-spores.

[0007] In a first embodiment, the invention provides a spore germination medium comprising a phosphate buffer at a concentration of about 0.1 M to about 0.2 M, and a synergistic amount of germinal and agent.
The spore germination medium can be optimized for LRV reaction of microbial spores exposed to the sterilization process, for example, by adjusting the concentration of phosphate buffer to about 0.15M. Spore germination media can also be optimized for specific germination systems, including the GS-II system.

[0008] In another aspect, the invention comprises a phosphate buffer at a concentration of about 0.1 M to about 0.2 M, a synergistic amount of a germinant and agent, sodium chloride, potassium chloride, glucose and fructose. A spore germination medium is provided.

In another embodiment, the germination medium disclosed herein comprises the steps of exposing microbial spores to a sterilant, contacting the spores with the spore germination medium;
Calculating V and following the step of determining the effectiveness of the sterilization method is useful for determining the effectiveness of the sterilization method.

[0010] In another embodiment, the invention provides a biological indicator of the effectiveness of a sterilization method. Biological indicators include germination media having a synergistic amount of germinating agent and agent, resulting in a sterilant exposure time / LRV response curve slope of greater than about 0.016. The medium contained in the biological indicator may include a phosphate buffer at a concentration of about 0.1M to about 0.2M. These biological indicators can yield a slope index of about 10 to about 20.

[0011] In another embodiment, the invention relates to a biological method comprising exposing microbial spores to a sterilizing agent and comprising a first container containing dried microbial spores, further adapted to calculate LRV on germinated spores. Provide an indicator. Such biological indicators have concentrations of about 0.1 M to
A second container adapted to store a germination medium containing about 0.2 M phosphate buffer and a synergistic amount of germinating agent and agent is also included.

In another embodiment, the invention relates to 1) contacting a spore with a germination medium containing a compound having unknown agent properties and lacking a germinating factor, and then calculating the LRV; 2) germinating the spore LRV is calculated by contacting a germination medium containing a sex factor and lacking a compound with unknown agent properties, and 3) contacting the spores with a germination medium containing a germination factor and a compound having unknown agent properties. Calculating the LRV, and 4) calculating the S value to determine the efficacy of the compound with unknown agent properties,
A method for identifying an agent is provided.

[0013] The germination media and methods described above can be further modified by one or more of the following aspects of the invention. In one aspect, the germinating factor is present at a concentration of about 0.0003 mg / ml to about 3 mg / ml.
It may contain 0 mg / ml, preferably about 0.001 mg / ml to about 0.01 mg / ml or about 0.1 mg / ml to about 1.0 mg / ml of L-amino acid, or a functional derivative thereof. Useful germinating factors include, but are not limited to, alanine, α-aminobutyric acid, norvaline, cysteine, valine, isoleucine, β-alanine, serine, homoserine, leucine, methionine, allothreonine, and threonine. Not something. In another aspect, the agent has a concentration of about 0.1 mg / ml to about 20 mg / ml, preferably about 0.1 mg / ml to about 1 mg / ml.
It may contain 0 mg / ml, more preferably about 3 mg / ml to about 8 mg / ml of D-amino acids, L-amino acids, or functional derivatives thereof. Useful agents include, but are not limited to, asparagine, glutamine, homoserine, allothreonine, threonine, 4-acetamidobutyric acid, histidine, serine, 2-acetamidoacetic acid, and 6-acetamidohexanoic acid. Absent.

In another aspect, the invention provides ethylene oxide, steam, radiation, heat, sodium hypochlorite, polyvinylpyrrolidone-iodine, sodium dichlorocyanurate, low temperature steam-formaldehyde, glutaraldehyde, hydrogen peroxide, hydrogen peroxide plasma. It is practical to use with a variety of sterilant sources, including peracetic acid, and mixtures thereof.

[0015] Features and advantages of the invention include one or more of the following. New synergistic interactions facilitate quick and accurate evaluation of sterilization methods. The germinating factor and the agent are
Provide a consistent germination medium to cause sporulation. The novel interactions discovered provide surprising results contrary to conventional thinking in the field of bacterial spore germination. As a result, new and useful germination media and methods for using the media are provided.

Unless defined otherwise, all technical and scientific terms and abbreviations used herein, including the three-letter abbreviations commonly used for amino acids, are generally understood by those of ordinary skill in the art to which this invention belongs. Has the same meaning as 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.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS The present invention relates to the discovery that the combination of certain germinating agents and agents in a germination medium (GM) interacts synergistically to alter microbial spore germination rates. . In addition, optimizing the concentration of germinating agents and agents will alter the sterilant exposure time / germination response relationship and facilitate rapid evaluation of sterilization methods.

Researchers have reported that the addition of L-Gln or L-Asn to fructose, glucose, and potassium and phosphate buffered base germination media causes germination of Bacillus subtilis spores. I have. Wax & Fre
ese, J. et al. Bact. , 95: 433-438 (1968); Johnst.
one, K .; , J. et al. Appl. Bact. Symposium Suppl. ,
76: 17S-24S (1994). In this application, the base germination medium (GM
Base) is defined as a medium or solution that does not itself cause spore germination.

The present inventors have unexpectedly discovered that a GM-based solution further containing a commercial lot of L-Gln or L-Asn does not consistently cause spore germination of Bacillus subtilis. Some lots cause germination (active lots)
On the other hand, another lot does not cause germination (inactive lot). The inventors have further discovered that adding substantially pure samples of L-Asn and L-Gln to a GM-based solution does not cause spore germination to a significant extent.

[0020] The inventors have also discovered that L-Asn, L-Gln and certain other compounds interact synergistically with germinating factors to increase spore germination rates. In other words, L-Asn, L-Gln and certain other compounds are referred to as "agents" that promote spore germination triggered through the GS-II mechanism by L-Ala or other germinating factors. It was found to work. Such findings are useful in the general area of biological indicators. Here, a germination medium containing a germinating factor and an agent and a method of using the medium are disclosed and claimed.

[0021] Biological indicators and methods of obtaining biological indicators are known. Examples of biological indicators are described in U.S. Patent Nos. 5,073,488, 3,661,717.
No. 4,839,291, No. 4,741,437, and No. 4,416.
No. 984. Briefly, suitable biological indicators are adapted to allow contact of the sterilant with the spores to maintain the germination medium. When determining germination rates further spectrophotometrically, preferred biological indicators are adapted to facilitate measurement of LRV of spore germination. Various containers made of quartz glass or polymeric materials such as poly (methyl methacrylate) or polystyrene can be used to construct such biological indicators.

Determination of spore germination rate When bacterial spores germinate, they absorb water and change the spectral properties of the spores. Germinated spores, for example, lose their light scattering properties. As a result, spore germination occurring in solution can be tracked spectrophotometrically. Dadd et al. , J. et al. Appl. Bact. ,
60: 425-433 (1986).

Generally, any sporulating bacteria can be used. Useful spore-forming bacteria include Bacillus subtilis, Bacillus circulans, Bacillus plumis, Clostridium perfringens, Clostridium sporogenes, Bac
illus cereus and Bacillus stearothermophilus. A preferred bacterium is Bacillus subtilis. Preferred GM is Bacillus
By following the methods and examples disclosed herein, used to optimize against S. subtilis spores, optimized against other useful sporulating bacteria,
Other preferred GMs can be developed.

[0024] American Type Culture Collection (ATCC) Accession Number 9
Bacillus subtilis spores, such as 372, about 0.5x10 ⁸ cells / ml~ about 3x
It can be used at a concentration in the range of 10 ⁸ / ml. Typically, the spore suspension used in the germination reaction contains about 1.2 × 10 ⁸ viable spores per 1.2 ml of GM or GM base solution. This Bacillus subtilis spore concentration corresponds to an initial absorbance at ₄₈₀ nm (Abs ₄₈₀ ), measured in a 1 cm path clear polymethacrylate cuvette, of about 0.45 to 0.50 absorbance units.

For the determination of spore concentration and / or spore germination kinetics, any spectrophotometer equipped with a temperature controlled cuvette holder can be used. The experiments described here were performed at 37 ° C. in a Varian / Carry UV / visible spectrophotometer equipped with a temperature control unit and a software package (ver. 13) used to calculate gradients and other kinetic parameters. Carried out.

[0026] Methods for preparing spores for use in GM are known. According to practice, the spores used here were also heat activated. The spores can be heat activated in any acceptable way. An illustration of heat activation of Bacillus subtilis spores is to heat an aqueous suspension of spores at about 47 ° C for 4 hours. Another acceptable method of heat-activating spores is to dry the spores on a suitable carrier material at about 47 ° C for 4 hours. The heat-activated spores are stable and remain heat-activated for extended periods of, for example, several months.

The spores may or may not be purified. Unpurified spores include spores collected by conventional methods from bacterial cultures that have been induced to sporulate by appropriate stimulation. Crude spores often contain agar and / or cell debris mixed with spores. Unpurified spores can be further purified by known methods. For example, spores can be resuspended in sterile deionized water that does not cause germination to remove agar pieces, denatured nuclear proteins and other debris. The suspension is then filtered through Whatman GF / D glass fiber filters (2.7 μm hold). After filtration, the spore suspension is considered purified.

[0028] The purified spores in the aqueous solution can be further separated into heavy and light spores by differential centrifugation. Heavy spores pellet after centrifugation at 2,000 xg for 12-15 minutes, while light spores remain in suspension. Light spores can be pelleted by centrifuging the supernatant remaining after pelleting the heavy spores at 2,000 xg for 30 minutes. Purification does not alter spore viability. Furthermore, the level of purification does not affect LRV when the GS-II mechanism is triggered. The purified spores can be stored as an aqueous suspension or dried.

The wavelength of light useful for measuring absorbance changes is from about 400 nm to about 700 nm.
, Preferably about 460 nm to about 560 nm, and more preferably about 480 nm.
~ 520 nm. At these wavelengths, the absorbance per spore concentration is highest, and the change in absorbance observed for germinated spores is still measurable.
As the wavelength increases from 300 nm to 900 nm, the initial absorbance of the spore suspension decreases asymptotically (more than 30% from 480 nm to 600 nm), but the change in absorbance due to spore germination increases by less than 10%. Methods for observing spore germination are known, the entire contents of which are referred to in PCT International Publication WO95.
/ 21936, and the like.

The spore germination rate can be calculated from a plot of the absorbance of the germinated spore suspension against time (germination kinetic curve). The observed spore germination rate is a function of the number of spores that germinate per unit time and is affected by both the number of spores that germinate and the time required for each spore to germinate.

FIG. 1 shows a typical germinating suspension of Bacillus subtilis spores observed at ₄₈₀ nm (Abs ₄₈₀ ). FIG. As shown by 1, there is a lag period when spores begin to germinate first. Next, as the spore germination process proceeds, the spore suspension of Abs _4
80 decreases. Decreasing absorbance is recorded until most of the germinable spores have germinated. A decrease in Abs ₄₈₀ , typically following a lag phase, is the final Abs _48
It is linear until it approaches _zero .

[0032] The linear reaction rate (LRV) is the maximum spore germination rate observed for a particular spore suspension. LRV is represented by the absolute value of the slope of the descending linear part of the germination kinetics curve following the lag phase. Thus, even though the descending line has a negative slope, the slope is shown as a positive number. Thus, as used herein, germinative and agent factors for the "increase" of the LRV gradient actually refer to the more negative gradient.

Under the conditions used herein, LRV occurs within the first 15 minutes after the start of spore germination. The data points used for LRV calculations are typically taken between 2 and 10 minutes following the start of the spore germination reaction. The length of the lag phase depends, for example, on the state of the spores and on the germination medium used. Still, the linear part of the dynamic curve
It is clear from the germination kinetics curve. For example, FIG. An LRV of 1 occurs between 5 and 10 minutes.

Identify synergistic interactions Germinating factors bind to and bind to specific protein receptors on or in dormant spores.
Causes buds. In this way, the germinating factor initiates the enzymatic reaction leading to spore germination
It is an activator of the protein that is However, germination factors are usually
The child is understood not to be consumed or altered. Still germinating factors
LRV plotted against mitochondrial concentration is the Michaelis-Menten steady state kinetic equation
Obey. Therefore, LRV is directly proportional to germinating factor concentration,
It follows the sum dynamic principle. Woese et al. , J. et al. Bact. Vol 761:
578-588 (1958). Thus, the germinating factor K_M
Is required. In this usage, K_MIs a germinant that produces half of the maximum LRV
Factor concentration. In such a case K_MAre treated as apparent dissociation or association constants.
I can. K reported here_MValues are based on the computer program “Enzyme Kinetics” (Tr
initi Software Ver. 1.5). K_M
Values are LRV Lineweaver-Burk (double
Reciprocal) obtained from the plot.

The invention provides a three-step method for identifying useful germinating and / or agent agents. First, by calculating the LRV for spores germinated in a GM-based solution containing an unknown compound and lacking any known germinating factors, the compound that has unknown germination properties (unknown compound) has the property of causing germination. Can be identified. This first experiment is repeated for unknown compounds at a range of concentrations. If the unknown compound causes germination, data can be calculated K _M for the unknown compound using. Compounds that cause germination are classified as germinating factors. However, the compounds shall be able to be further classified as good or bad germinating factors. An example of a good germinating factor is L-Ala. Examples of poor germinating factors are L-Met and L-ornithine.

Second, the agent properties of compounds having unknown agent properties can be evaluated. For spores that germinated in GM containing both unknown compounds and known germinating factors, LR
V is calculated. If the unknown compound is an agent for a known germinating factor, L
The RV reaction becomes synergistic. In Bacillus subtilis spores, the known germinating factor is typically L-Ala or L-Val. When assessing the agent properties of an unknown compound, in the absence of the unknown compound, the germinating agent concentration is sufficiently lower than the maximum LRV response to the germinating agent alone, and the LRV The germinating agent concentration must be sufficiently low so that an increase can be detected. As a control, the LRV for a known germinating factor in the absence of an unknown compound can also be determined. Typically, L-Ala was used at a concentration that yielded an LRV that was one half of the maximum LRV. Measuring the agent properties at multiple concentrations, as in step 1, facilitates identification of compounds that are agents only within a limited concentration range,
It facilitates the evaluation of the dose-response relationship between an agent and a germinating factor.

Formula 1 or 2 can be used to identify a synergy between an unknown compound and a known germinating factor.

Embedded image

Equation 1 requires three independent LRV calculations. LRV _EG is an LRV for GM containing both known germinating factors and unknown compounds. LRV
_G is the LRV for GM containing only known germinating factors at the same concentration used in the calculation of LRV _EG . LRV _E is the LRV for GM containing only unknown compounds at the same concentration used in the calculation of LRV _EG . Using Formula 1, known germinating factors and unknown compounds are synergistic when S ₁ > 1.1, additive when S ₁ = 1, and S ₁ <1 Competitive or competitive. Since true the agent (discussed below) to the K _M of known germination factors also cause concomitant decrease, the unknown compound in the case of S _1> 1.1, are considered agent "candidate". As is evident from Formula 1, antagonistic compounds may also produce negative _Sl values.

Synergy can also be identified using equation 2. Under the conditions disclosed herein, Equation 2 is preferred because the resulting S value is typically a positive number. In equation 2, LR
V _E , LRV _G , and LRV _EG are defined as in Equation 1.

Embedded image

If S ₂ > 1.1 using Equation 2, the unknown compound is a candidate agent It is considered a factor candidate. If S ₂ is less than about equal or 1 to 1, the unknown compounds that are not agent. In this usage, S ₁ corresponds to the S value calculated using Equation 1, and S ₂ corresponds to the S value calculated using Equation 2.

Using the third step, it can be determined whether the candidate agent is a true agent. This step can also typically be used to determine the degree of activity for an agent or borderline agent. A GM containing a saturating concentration of the unknown compound is prepared. The saturation concentration of the unknown compound can be calculated from step 2. The LRV is then calculated for spores that germinate in GM that contain a saturating concentration of the unknown compound and further contain increasing concentrations of known germinating factors. Use double reciprocal plot, it is possible to determine the K _M germination factors in the presence of the agent. By including candidate agent in GM, as compared to the K _M of germination factors causing germination in the absence of candidate agent, if K _M apparent germination factor decreases, candidate agent is true Is an agent.

The degree of synergy between any agent or unknown compound and a known germinating factor can be estimated by calculating the _KM ratio. K _M ratio, divided by the K _M calculated for germination factors in the presence of an agent or unknown compound is a calculated K _M for only germination factors. The compounds which act as an agent, a K _M ratio> 1.

Briefly, the LRV calculated for spores that germinated in a GM-based solution that also contains two or more test compounds gives the LRV calculated separately for each test compound.
A test compound that acts as a germinative and / or agent is synergistic if it exceeds the sum of the LRV calculated for spores that germinated in the M base solution. An LRV response is additive if the GM-based LRV additionally containing two or more test compounds together is equal to the sum of the LRVs measured for each test compound alone. If the GM-based LRV additionally containing two or more test compounds together is less than the sum of the LRVs measured for each test compound alone, the LRV response is antagonistic, ie, competitive.

Spore Susceptibility to Sterilizer Exposure Death of microorganisms or spores by exogenous sources such as ethylene oxide (EtO) is best described using first-order kinetics because the reduction in viable organism count is logarithmic. P
Flug and Holcomb, "Principles of th.
e thermal destruction of Microorgani
sms ", Disinfection, Sterilization, a
ndPreservation, 4th edition, S.M. S. Block ed., Lean an
d Febiger, (1991). The number of surviving organisms after exposure to sterilization or disinfection is determined using Equation 3.

Embedded image

In Equation 3, N ₀ is the initial number of viable spores or microorganisms, and N is the number of viable spores or microorganisms after exposure to a particular sterilant dose. U is the total sterilant exposure time, eg, total elapsed EtO exposure time, or sterilant dose. D is the D value, ie, the sterilant exposure required to kill one log of spores or cells. D is a set of specific conditions,
And constant for batches or crops of spores or cells. Thus, D is the negative reciprocal of the slope of the linear death curve. By identifying the conditions that change the D value, the slope of the survival curve can be manipulated.

A semilog plot of the LRV observed for microbial spore population versus sterilant exposure results in a negative slope (sterilant exposure time / LRV response curve). This curve is shown in FIG. Correlation with a linear mortality curve as shown in FIG. Sterilizer exposure time / LR
By adjusting the germination conditions so that the V response curve becomes flatter, the spore population L
The RV response is shown to be less sensitive to sterilant exposure, and vice versa.

For biological indicators, steeper gradients are preferred because of the increased sensitivity and accuracy of the biological indicator. As discussed above, "gradient" is the sterilant exposure time /
Absolute value of the slope of the LRV response curve. Under the conditions used here, the steeper slope is from about 0.014 to about 0.024. Preferably, the slope is from about 0.0185 to about 0.02. In the experiments described here, sterilant exposure time / y of the LRV response curve
The intercept, slope and coefficient of determination were calculated using the computer program SigmaPlot
(Jendel Scientific).

Instead of determining the actual slope of the sterilant exposure time / LRV response curve, a slope index can be calculated. It is particularly useful to use the slope index when EtO is a sterilant. The slope index is the value obtained by dividing the LRV response at 0 min. EtO exposure (y-intercept) by the LRV response calculated in a standard 60 min. EtO exposure cycle. useful 0.021). The germination media described herein may also be optimized for LRV reactions by adjusting the concentration of individual components to produce the steepest gradient, or maximum gradient index, achievable with different spore types and / or different sterilants. it can.

In a standard EtO sterilization cycle, conditions of EtO exposure of about 60% relative humidity and a temperature of about 55 ° C. with a concentration of 600 mg of EtO per liter of air are used. The ethylene oxide sterilization cycle used here is based on Joslyn Biologic.
cal Indicator Evaluator Resistor (B
. I. E. FIG. R. ) Performed in a gas sterilizer. The cycle was preheated at 55 ° C for 15 minutes, followed by a 30 minute dwell time at 60% relative humidity, followed by about 55 ° C.
Includes EtO sterilized dwell time at 600 mg EtO concentration per liter of air at a temperature of C, followed by three high vacuum cycles, and finally a low vacuum aeration at 55 ° C for 1 minute. As used herein, EtO exposure time refers to the length of only the EtO sterilization dwell time. As in conventional practice, preheating, humidity dwell, vacuum cycle, and low vacuum aeration are kept constant.

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

Generally, to expose spores to a sterilant, the spores must be dried on a suitable carrier material. The spores can be dried on any suitable carrier material, such as paper, plastic, glass, metal or other non-dispersible carriers. Suitable carriers include plastic or glass cuvettes. Spores
It can be dried in the presence of various additives that can change the spore sensitivity to sterilant exposure. Examples of additives and methods of applying the additives are described in US Patent Application Serial No. 09/06.
No. 1,293. Drying of spores in the presence of additives has been positioned as a test preparation for spores. The drying temperature is typically about 45-50 ° C, preferably about 47-48 ° C. For example, a suspension of treated spores can be dried at about 48 ° C for 4 hours. The spores dried in this way appear to be dry on the surface, but probably retain residual bound water within the spores. Alternatively, the spore suspension can be dried on the carrier at about 37 ° C. for about 12-20 hours. Typically,
Drying for 12 hours or overnight is sufficient.

To initiate spore germination, a brief vortex, low power sonication (
The dried spores can be resuspended in GM or GM based solution by 50W), stirring, or any other acceptable resuspension method. Typically, 10 of the dried spores
0% resuspension cannot be achieved. Normalizing the initial absorbance of the resuspended spores to the standard initial absorbance facilitates comparison of results between different experiments. Typically, the resuspended Bacillus subtilis spores were normalized to an initial Abs ₄₈₀ = 0.5.

[0053] In one aspect, the invention features a synthetic spore germination medium (GM) that increases LRV. Typically, a GM comprises a base solution (GM base), at least one germinating agent, and at least one agent. The GM can also include a GM-based solution that further contains a germinating agent, an agent, and / or a compound of unknown activity, or any combination thereof. The GM or GM base may be unfiltered or filtered through a 0.2 μm sterile filter. The filtered germination medium is indicated by "F" before the medium name, for example, FGM or FGM base.
It is represented with a.

While GM may or may not cause spore germination, the GM-based solution does not cause spore germination, ie, the measured LRV is about 0.0
008 Abs ₄₈₀ / min or less. As used herein, baseline germination, measured spectrophotometrically, or no germination

An optimal Bacillus subtilis spore GM-based solution can include the following components: (1) 0.01-0.2 M phosphate buffer (equivalent KH ₂ PO ₄ and Na
₂ HPO ₄ ), 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 embodiments disclosed herein use a GM-based solution within the above concentration range, the disclosed methods can also be applied to GM-based solutions lacking one or all of the listed components. A GM-based solution lacking one or more of the above components may be a non-GS
It may be useful to identify germinating and acting factors of the -II mediated germination mechanism and to optimize spore germination parameters other than Bacillus subtilis.

Varying the phosphate concentration is useful in accommodating the sensitivity of different types of spores or different spore crops for use in biological indicators. For Bacillus subtilis spores, the phosphate concentration in the FGM base solution was about 0.01
Increasing the phosphate concentration from about 0.2 M to about 0.2 M increases the slope of the EtO exposure time / LRV response curve from about 0.0118 to about 0.0205, respectively (ie, becomes more negative).

By increasing the phosphate concentration in the FGM base solution from about 0.01 to about 0.1 M phosphate, the y-intercept of the EtO exposure time / LRV response curve plot is about 0.1 M Up to phosphates. Increasing the phosphate concentration beyond about 0.15M causes a noticeable decrease in the y-intercept.

Preferred GM-based solutions of Bacillus subtilis spores are: (1) 0.1 M phosphate buffer (6.95 g / L KH ₂ PO ₄ +6.95)
g / L Na ₂ HPO ₄ ), pH 7.25 at 22 ° C., (2) 6.0 g / L NaCl (0.1 M), (3) 15 g / L KCl (0.201 M), (4) Contains 0.3 g / L glucose (1.67 mM), and (5) 0.3 g / L fructose (1.67 mM).

Another preferred GM-based solution comprises the above components 1-5, wherein the phosphate buffer is at a concentration of about 0.15M. Other buffers, including zwitterionic buffers, such as ACES, HEPES, MOPS, PIPES, and TRIZMA buffers can be used in the GM-based solution. However, the LRV response measured on spores germinated in GM containing other buffers other than phosphate is less reactive, so phosphate is preferred for GM used for biological indicators. Further, MgSO ₄ , CaC
l ₂ and the effect of urea were evaluated. Other than urea, which reduced the observed lag time of spore germination by up to 1 minute, there appeared to be no advantage in adding such compounds.

[0061] As used herein, germination factors when added to GM-based solution, cause spore germination significant extent (i.e. LRV> 0.008Abs ₄₈₀ / min). The agent, when added to GM containing germination factors, cause synergistic increase in LRV, causes concomitant decrease in K _M of germination factors. Germinating agents and agents include test compounds and their functional derivatives. As used herein, a functional derivative is a derivative of a germinating agent or agent that continues to function as a germinating agent, an agent, or both when tested using the methods disclosed herein. Further, the compound can be both a germinating agent and an agent, for example, L-Thr.

It is important to use germinating agents and agents that are used here and that are substantially free of other germinating contaminants because of their low concentrations. Methods for identifying contaminants are known. These methods include the known D ₂ O nuclear magnetic resonance (“NMR”) technique. Usually, the detection limit of these methods is about 0.01% weight / weight (w / w). Substantially free of other germinating active contaminants is defined as contaminants having a concentration of less than about 0.01% w / w.

Since L-Ala is significantly more active than other known germination active compounds,
L-Ala is probably the most common detectable contaminant. In commercial lots of L-Gln or L-Asn, most of the contaminants are <0.1%,
Concentrations of other contaminating amino acids do not appear to have a significant effect on LRV.

Useful germinating factors include the compounds listed in Table 1. The concentration ranges listed in Table 1 are (Normalize Abs ₄₈₀ to 0.5) The range that caused Bacillus subtilis spore germination from a value slightly above to the maximum observed for each germinating factor.

[Table 1]

As used herein, the preferred agent is the steepest EtO exposure time / LR in combination with an identified germinant factor such as L-Ala or L-Val.
A compound that gives a V response curve. Preferred agents include about 0.3 g / L
L-Gln and L-Asn used at a concentration of 約 10 g / L.
Synergy can be measured with L-Asn or L-Gln as low as 0.1 mg / ml, but at concentrations less than about 1 mg / ml, LRV or EtO exposure time / LRV
Neither of the reactions is useful in biological indicators.

Illustrative examples of useful GMs containing both a germinating agent and an agent include germinating factor L-Val at a concentration of about 0.1 g / L to about 1.0 g / L, or a concentration of about 1.0 g / L.
mg / L to about 10 mg / L of germinating factor L-Ala at a concentration of about 1 g / L to
Mention may be made of about 10 g / L of the agent L-Gln, or of a GM further comprising a concentration of about 1 g / L to about 10 g / L of the agent L-Asn.

Preferred GMs include 0.15 M phosphate buffer (pH 7 to 7.4), L-Val at a concentration of about 0.1 g / L to about 0.3 g / L, and a concentration of about 3 g / L. L to about 8
Preferred FGM-based solutions further containing g / L of L-Gln.
More preferable GM is L-Val having a concentration of about 0.1 g / L (0.85 mM).
Preferred FG further containing L-Gln at a concentration of about 5 g / L (34.2 mM)
M base solution. Concentration of about 1 mg / L to about 4 mg / L, preferably 1 m
g / L of L-Ala replaces L-Val. However, at 1 mg / L, F
It is difficult to maintain the stability of L-Ala concentration in GM. Concentration about 3g /
L-Asn of L replaces L-Gln, but the slope of the EtO exposure time / LRV response curve is smaller.

Tested for germinative or agent activity according to the methods described herein,
Additional examples of compounds such as known amino acids and their derivatives include the compounds listed in Table 2 (active agents), Table 3 (boundary agents), and Table 4 (inactive agents).

[Table 2]

[Table 3]

[Table 4]

[Table 5]

[0069] Tables 5 and 6, in the presence or absence of the agent or border agents respectively Tables 2 and 3 show a K _M calculated for germination factors L-Ala.

[Table 6]

[Table 7]

In another aspect of the invention, GS-II is shown to be more sensitive to EtO than either GS-I or GS-III. Because GS-II is sensitive to EtO, for biological indicators used in EtO sterilants,
It is preferred to cause a GS-II mechanism over a GS-I or GS-III mechanism. In addition, the discovery of a synergistic effect between the agent molecule and the germinating agent has led to the manipulation of germinating agent and agent concentrations in GM to alter the correspondence between spore germination rate and sterilant exposure time. It's easier to do.

The invention is further understood with reference to the following examples, which are purely intended to be illustrative and do not limit the true scope of the invention, which is set forth in the following claims. Unless otherwise noted, in each Example, L-Asn or L-Asn substantially free from other germination active contaminants
A commercial lot of Gln was used.

Example 1 Synergistic Interaction between L-Asn or L-Gln and L-Ala In this example, the difference in germination activity levels observed for commercial lots of L-Gln and L-Asn was determined. Illustrate. Commercial lots of L-Gln and L-Asn are available from Sigma Chemical Company (lot number 1 respectively).
5F-0680 and 89F-0398) or Aldrich Chemical
al Company (lot numbers 06710EV and 03220PN).

20 μl of heat-activated Bacillus subtilis spore suspension (approximately 1.2 × 10⁸
Individual spores) were placed in a polymethacrylate semi-micro cuvette. 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 KH
₂PO₄/L+6.95g of Na₂HPO₄/ L, pH 7.25 at 22 ° C))
1.2 ml containing the appropriate concentration of germinating and / or agent
FGM was placed in each cuvette to initiate germination. L-Ala (Aldric
h lot number AF0631PZ), L-Gln, and / or L-Asn
Suitable concentrations are shown in Table 7. FGM containing L-Asn or L-Gln
Germination of Bacillus subtilis spores in the presence or absence of L-Ala
Caused. L-Asn (lot number 06710EV) or L-Gln (
F, which further contains an active lot of lot number 15F-0680) in increasing concentrations.
From the first experiment performed with the GM base solution, L-Asn showed that the LRV reaction
Saturated at 10 mg / ml and L-Gln saturated the LRV reaction at 6 mg / ml.
Was shown. The L-Ala concentration was below the saturation concentration.

Temperature control Varian / Cary 1 UV / visible light spectrophotometer at 37 ° C.
Changes in Abs ₄₈₀ in were recorded and spore germination was followed. LRV was calculated from the germination kinetic curve obtained. The average LRV calculated for each reaction and the associated% standard deviation (% SD) are shown in Table 7.

[Table 8]

FGM containing inert L-Asn (Lot No. 03220PN) or F
When L-Ala was added to the GM base, LRV increased in a concentration-dependent manner. Active L
LRV for Asn (lot no. 06710EV) is 0.001 mg / m
It was approximately equal to the LRV calculated for inactive L-Asn formulated with L-Ala at a concentration of l. For GM 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. From the results, the active lot of L-Asn is determined by the chemical detection limit of D ₂ O NMR (
That is, approximately 0.001 mg / ml LA which is 0.01% w / w or less.
It is suggested that la was contained.

The L calculated for the active lot of L-Gln (lot number 15F-0680)
RV was 0.01 mg / ml L-Ala added to the inactive lot of L-Gln.
Was equal to the LRV calculated for Inactive L-Gln (Lot No. 89F-
0398) to L-Ala, the LRV is 0.003-0.006 mg /
A plateau was reached at L-Ala concentrations between ml, suggesting 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 be due to contamination with trace amounts of germinatively active compounds. Using D ₂ O NMR analysis, L-
Analysis of the Gln active and inactive samples showed that at least 0.06% of L-Ala was detected in the L-Gln active lot and L-Ala was not detectable in the L-Gln inactive lot. . These results suggest that GS-II was not caused by L-Asn or L-Gln in FGM containing glucose, fructose, and potassium as the literature states. Wax & Freese, J.M. Bact. , 95: 433-437 (19
68); Johnstone, J .; Appl. Bact. Symposium
Suppl. , 76: 17S-24S (1994).

S calculated for L-Asn in the presence of 0.001 mg / ml L-Ala
The values (S ₁ = 3.2, S ₂ = 2.6) suggested that L-Asn was synergistic with L-Ala. L-Gln in the presence of 0.001 mg / ml L-Ala
From the S values (S ₁ = 8.0, S ₂ = 4.0) calculated for, L-Gln is L
-It was suggested to be synergistic with Ala.

Example 2 Identifying Synergism This example illustrates a method for identifying additional germinating factors and / or candidate agents. The LRV in FGM prepared according to the procedure described in Example 1 was calculated. Using each unknown compound, alone, in combination with L-Ala, and L
LRV in the formulation with Gln was calculated. Table 8 shows the results. The LRV calculated for the FGM base solution was <0.001 ABS ₄₈₀ / min.

[Table 9]

[0080] Compounds causing LRV _E of greater than about 0.01Abs ₄₈₀ / min, was regarded as germination factors. Compounds synergistic with L-Ala were considered as potential agent. L
Has -Gln or L-Asn single value below LRV, was also considered a candidate agent compound having a _{S 1} value exceeding 1 when tested by blending a L-Ala.

Some compounds have been classified as both germinating factors and candidate agents. For example L-Thr had LRV _E of 0.0135 at 7.5 mg / ml.
Further, L-Thr was S ₁ > 1 in the combination with both L-Ala and L-Gln.

As apparent from Table 8, some compounds (eg, L-Asn, L-Gln
) Are even weaker germinating factors than others (eg, L-Ala and L-Val). Furthermore, the LRV of L-Gln or L-Asn is probably about 0.3
This may be due to trace contamination of the germinating active compound, which is less than μg / ml L-Ala. Using this method, any compound or functional derivative thereof can be identified as a germinant or candidate agent.

[0083] In the concentration-dependent this example of Example 3 Synergistic interaction, germination factors in the presence of the agent the compound or agent candidate compound, illustrates a method of calculating the change in K _M. FGM was prepared according to the procedure described in Example 1. As shown in Table 9, saturation kinetic curves were generated by increasing the concentration of the germinating factors L-Ala or L-Val. Standard deviation (% SD)
And LRV was calculated.

[Table 10]

[0084] The LRV response of FGM containing both germinating factors and agents is L-Al
a saturates at about 0.01 mg / ml (0.112 mM) and L-Val at about 1.0 mg / ml (8.54 mM), which is the saturation of both germinating factors in the absence of the agent. Well below the concentration.

A double reciprocal plot was used to evaluate the synergistic interaction between the agent (L-Gln and L-Asn) and the germinant (L-Ala and L-Val). Using the data from Table 9, a commercially available enzyme kinetics software package (ver.
. 5, Trinity Software) using the, child _K M (mM of germination factor
) Was calculated. Table 10 shows the results. Correlation coefficient, i.e. quantitative coefficient or ^{R 2} with respect to _{K M} Quantification was between 0.983 and 1.0.

[Table 11]

The sprouting factor concentrations for the LRV response of L-Ala are biphasic, suggesting that L-Ala binds to one or more receptors with different affinities. Therefore, L-Ala (L) (0.001-0.01 mg / ml), and L-Al
Two _{K M} values of a (H) (0.01~03mg / ml ) was calculated.

The sprouting factor concentration for the LRV response of L-Val was negative. In addition L-Val, in concentrations as low as L-Ala so did not cause spore germination, _{K M} quantification of L-Val less than 0.1 mg / ml was prevented.

[0088] Both L-Asn and L-Gln as shown in Table 10, germination causing the L-Ala and L-Val, substantially reduced the _{K M.} L-Ala
The synergistic effect observed for (L) was approximately twice that observed for L-Val. The synergistic LRV response observed for L-Val did not show biphasic kinetics, so it was not clear if synergy occurred at low affinity sites.

Example 4 Assessing EtO Exposure Time / LRV Response 20 μl of an aqueous Bacillus subtilis spore suspension (approximately 1.2 × 10 ⁸ spores) is placed in a polymethacrylate semi-micro cuvette and then at 47 ° C. For 4 hours. Next, Joslyn B. I. E. FIG. Dried spores were exposed to various EtO exposure times in an R gas sterilizer. Each complete cycle proceeded as follows. 1) Preheating at 55 ° C for 15 minutes. 2) 30 minute dwell time at 60% relative humidity. 3) EtO sterilized dwell time at a temperature of about 55 ° C. and 600 mg of EtO per liter of air (total time varies as shown in Table 11). 4) Three advanced vacuum cycles. 5) Low vacuum aeration at 55 ° C for 1 minute.

After EtO exposure, 1.67 mM fructose, 1.67 mM glucose, 0
. 2 M KCl, 0.1 M NaCl, and 0.1 M phosphate buffer (6.9
5 g KH ₂ PO ₄ /L+6.95 g Na ₂ HPO ₄ / L, pH 7 at 22 ° C.
. 1.2) FGM containing 25) was placed in each cuvette. The FGM also included appropriate concentrations of germinal and / or agent as shown in Table 11.

The spores were resuspended by vortexing for 10 seconds. Temperature control Varian / Car
Changes in Abs ₄₈₀ were recorded at 37 ° C. in a y1 UV / visible spectrophotometer to observe spore germination. Table 11 shows L calculated from germination kinetics curves following EtO exposure.
Indicates RV.

[Table 12]

A plot of log (LRV) against EtO exposure time provided a straight line.
Line slopes were calculated using Sigmaplot (Jendel Scientific). In the presence of 3.0 mg / ml L-Asn, a concentration of 0.001, 0
. Et for spore germination caused by L-Ala at 0.1 and 0.1 mg / ml
The slope value of the O exposure time / LRV response curve was 0.0157, 0.011,
And 0.0095. In the presence of L-Gln, a concentration of 0.001, 0.01
And the slope values of the EtO exposure time / LRV response curves for spore germination caused by 0.1 mg / ml L-Ala were 0.0177, 0.014, and 0.0087, respectively. F containing either L-Asn or L-Gln
Increasing L-Ala concentration in GM decreased the slope of the EtO exposure time / LRV response curve. Increasing L-Ala concentration decreased the sensitivity of the LRV response, presumably because L-Ala causes a non-GSII spore germination mechanism.

Example 5 Synergistic Interaction Between L-Thr, L-aThr and L-Ala LRV was calculated using the experimental procedures set forth in Examples 1 and 2, and L-Thr,
A synergistic interaction between L-aThr and L-Ala was determined. Table 12 shows the results.

[Table 13]

An S ₂ value greater than 1.1 indicates that L-Thr at concentrations below 7.5 mg / ml LA
It was shown to be synergistic with la. Furthermore, unlike L-Asn and L-Gln,
-Thr itself causes spore germination. L-Thr may be a compound capable of homotropic positive cooperation.

Using the experimental procedure described in Example 3, K of L-Ala in the presence of L-Thr
_M was calculated. 0.001 in the presence or absence of 10 mg / ml L-Thr
From the FGM containing L-Ala of ~0.01mg / ml, _{K M} ratio of 5.5 was obtained _{(K M L-Ala = 0.033mM} , K M L-Ala + L-Thr
= 0.006 mM). The conversion correlation coefficients were 0.941 to 0.992. L-
Thr increased binding efficiency for L-Ala, suggesting that L-Thr is an agent of L-Ala-induced GS-II germination.

L-aThr caused spore germination. But _{S 2} value << 1, L-aT
hr was not synergistic with L-Ala. Additional experiments using the procedure described in Example 3 were performed to determine whether L-aThr alters the binding efficiency of L-Ala. In additional experiments, L-Ala was added in the presence or absence of 5.0 mg / ml L-aThr from 0.001 mg / ml to 0.009 mg / ml.
Was changed to. L-aThr _{K M} ratio in the presence or absence L-Ala is 12
. _{7 (K M (L-Ala} ) = 0.014mM, K M (L-Ala + L-aThr
) = 0.0011 mM). Therefore _{S 2} value L-ATHR is L-Al
While indicating that no synergistic with a, K _M ratio in terms indicating significant synergistic interaction, L-ATHR was unusual. L-aThr may be unusual because germinant factor properties predominate over agonist properties.

Example 6 Synergistic Interaction between L-Cys and L-Ala Using the experimental procedure described in Examples 1 and 2, LC in the presence of L-Ala
The LRV of ys and L-Ser was calculated. Table 13 shows the results.

[Table 14]

[0098] L-Cys was not synergistic with L-Ala. From S ₂ << 1, L-
A similar antagonistic effect was suggested, as was the effect observed for other germinating factors that were not synergistic with Ala (see Table 8). L-Cys caused spore germination at a relatively high germination rate. Therefore, L-Gln and L-Asn are converted to L-Cy
Expected to be synergistic with s.

[0099] At lower concentrations of L-Ser, L-Ser was synergistic with L-Ala. At higher concentrations, the L-Sr was similar to the reaction observed for L-Thr (Example 5).
er and L-Ala became antagonistic. Thus, L-Ser was a candidate for a borderline factor.

Example 7 Effect of GM Composition on LRV Reaction In this example, the additional components contained in the preferred FGM base solution were filtered 50 mM phosphate buffer (equal Na ₂ HPO ₄ and KH It illustrates the advantage over FPO solutions containing only ₂ PO ₄ ). FPO is a standard medium used to activate only GS-I-mediated spore germination in the presence of germinating factors. GS-I and GS-III-mediated germination are activated when FGM contains a germinating factor. GS-II-mediated germination is activated when FGM contains both germinal and agent. However, only GS-II mediated germination was to an extent that was useful to monitor the effectiveness of the EtO sterilization method.
Sufficiently sensitive to tO sterilization.

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 KH ₂ PO
FGM based solution containing pH 7.25) at ₄ /Ltasu6.95G of _{Na 2 HPO 4 / L, 22} ° C, was mixed with L-Ala concentration shown in Table 14. Concentration 3mg
Each germination reaction was repeated in the presence or absence of an L-Gln / ml agent. The experimental procedure was repeated using an FGM-based solution containing only filtered 50 mM phosphate (FPO-based). Table 14 shows the results.

[Table 15]

[0102] FPO-based solution, the presence L-Gln or absence, _{K M} calculated for L-Ala, respectively 0.0257mM and 0.0119mM _{(K M} ratio =
2.2). In a preferred FGM-based solution, the KM calculated for L-Ala in the presence or absence of L-Gln is 0.0157 mM and 0.1 _M , respectively.
It was 0033mM _{(K M} ratio 4.8). Thus, even though synergy is observed in FPO-based media, it is clear that the additional components contained in FGM enhanced the synergistic interaction between the agent and the germinating factor.

Example 8 Identifying the Effect of Buffer Contributed to EtO Exposure Time / LRV Response 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 KH ₂ P
Filtered 50 mM phosphoric acid in FGM base solution containing O ₄ /L+6.95 g Na ₂ HPO ₄ / L, pH 7.25 at 22 ° C. or consisting of equal parts Na ₂ HPO ₄ and KH ₂ PO ₄ L-Gln in any of the buffer solutions (FPO)
The experimental procedure described in Example 4 was followed to compare the y-intercept and slope of the EtO exposure time / LRV response curve for the combination of and L-Val.

[Table 16]

Both the initial LRV expressed in y-sections and the slope of the EtO exposure time / LRV response indicated that the FGM-induced germination mechanism was superior to FPO. Therefore, when monitoring the EtO sterilization method, it is desirable to trigger the GS-II germination mechanism. In addition, this example suggests that excessive germinal factor concentrations decrease the slope of the EtO exposure time / LRV response (ie, to a lesser degree), which was also observed in Example 4.

Example 9 Effect of D-Isomer The D-isomer of the germinating active amino acid is known to competitively inhibit spore germination. Woese et al. , J. et al. Bact. , 76: 578-88 (
1958). Using the experimental procedures described in Examples 1 and 2, D-Gln, D-
Asn and D-Thr were evaluated as agonist compounds. The results are shown in Tables 16 and 17.

[Table 17]

[Table 18]

D-Asn and D-Gln are synergistic with L-Ala, whereas DT
hr is antagonistic. From these results, it was confirmed that L-Thr was both an acting factor and a germinating factor. Further, from these results, it can be seen that the D-isomer is L-
Since germination caused by Ala was not inhibited and germination was not caused even when used alone, it was confirmed that L-Asn and L-Gln were merely acting factor molecules and did not act as germinating factors. Normally, D-amino acids are thought to act as competitive inhibitors against known germinating factors, so D-Asn and DG
The results for ln were very unexpected. However, the observed lack of germination inhibition by D-Asn and D-Gln suggests that the binding sites for the agonist and germinant are distinct. The results also supported the conclusion that germinant factor activity in commercial lots of L-Asn and L-Gln was indeed due to trace amounts of contaminants such as L-Ala. See Example 1.

Other aspects, advantages, and modifications are within the scope of the following claims.

[Brief description of the drawings]

FIG. 1: PCT International Publication WO 95/21936, FIG. 1 is a graph showing absorbance at 480 nm versus time for a typical germinated suspension of Bacillus subtilis spores as described herein.

FIG. 2: PCT International Publication WO 95/21936, FIG. 3 is a copy of 2.
Figure 4 is a graph depicting the survival curve of Bacillus subtilis spores exposed to ethylene oxide having a D value of 7 minutes, and the LRV curve of Bacillus subtilis spores observed.

──────────────────────────────────────────────────続 き Continuation of front page (81) Designated country EP (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE ), OA (BF, BJ, CF, CG, CI, CM, GA, GN, GW, ML, MR, NE, SN, TD, TG), AP (GH, GM, KE, LS, MW, SD, SZ, UG, ZW), EA (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), AL, AM, AT, AU, AZ, BA, BB, BG, BR, BY, CA, CH, CN, CU, CZ, DE, DK, EE, ES, FI, GB, GE, GH, GM, HR, HU, ID, IL, IS, JP, KE, KG, KP , KR, KZ, LC, LK, LR, LS, LT, LU, LV, MD, MG, MK, MN, MW, MX, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, SL, TJ, TM, TR, TT, UA, UG, UZ, VN, YU, ZWF Term (reference) 4B063 QA01 QQ05 QR44 QR49 QR50 QR69 4B065 AA15X AA19X AA23X BB02 BB12 BB15 CA46

Claims (33)

[Claims] 1. A spore germination medium comprising: a) a phosphate buffer at a concentration of about 0.1 M to about 0.2 M; and b) a synergistic amount of a germinating agent and an agent. 2. The spore germination medium according to claim 1, wherein the medium is optimized for microbial spore LRV reaction. 3. The method of claim 2, wherein the phosphate buffer is at a concentration of about 0.15M.
A spore germination medium according to item 1. 4. The spore germination medium according to claim 1, wherein the at least one germinating factor comprises an amino acid or a functional derivative thereof. 5. The spore germination medium according to claim 4, wherein at least one of the germinating factors comprises an L-amino acid. 6. The at least one germinating factor is selected from the group consisting of alanine, α-aminobutyric acid, norvaline, cysteine, valine, isoleucine, β-alanine, serine, homoserine, leucine, methionine, allothreonine, and threonine. The spore germination medium according to claim 1, which is selected. 7. The spore germination medium according to claim 6, wherein the at least one germinating factor comprises L-valine. 8. The spore germination medium according to claim 6, wherein the at least one germinating factor comprises L-alanine. 9. The spore germination medium according to claim 1, wherein the at least one agent comprises an amino acid or a functional derivative thereof. 10. The spore germination medium according to claim 9, wherein the at least one agent comprises an L-amino acid. 11. The spore germination medium according to claim 9, wherein the at least one agent comprises a D-amino acid. 12. The at least one agent is selected from the group consisting of asparagine, glutamine, homoserine, allothreonine, threonine, 4-acetamidobutyric acid, histidine, serine, 2-acetamidoacetic acid, and 6-acetamidohexanoic acid. The spore germination medium according to claim 9. 13. The spore germination medium according to claim 12, wherein the at least one agent includes glutamine. 14. The spore germination medium according to claim 12, wherein the at least one agent comprises asparagine. 15. The method of claim 15, wherein the at least one germinating factor is alanine, L-α.
-Aminobutyric acid, norvaline, cysteine, valine, isoleucine, β-alanine, serine, homoserine, leucine, methionine, allothreonine, and threonine, at least one of the above-mentioned agents is asparagine, glutamine, homoserine, The spore germination medium according to claim 1, which is selected from the group consisting of allothreonine, threonine, 4-acetamidobutyric acid, histidine, serine, 2-acetamidoacetic acid, and 6-acetamidohexanoic acid. 16. The spore germination medium may be Bacillus subtilis (Baci).
llus subtilis), Bacillus circulans (Bacillu)
s circulans), Bacillus pumils
us), Clostridium perfringens (Clostridium p.
erfringens), Clostridium sporogenes (Clostri)
dium sporogenes) or Bacillus stearothermophilus (
16. The spore germination medium according to claim 15, which is optimized for spores of Bacillus stearothermophilus. 17. The spore germination medium according to claim 16, wherein the spore germination medium is optimized for Bacillus subtilis. 18. The method according to claim 17, wherein the concentration of the germinating factor is from about 0.1 mg / ml to about 1 m.
The spore germination medium according to claim 15, which is g / ml. 19. The method according to claim 18, wherein the concentration of the agent is about 0.3 mg / ml to about 10 mg.
The spore germination medium according to claim 15, which is / ml. 20. The method according to claim 11, wherein the concentration of the agent is about 3 mg / ml to about 8 mg / ml.
The spore germination medium according to claim 15, which is: 21. The spore germination medium according to claim 15, wherein the germinating factor concentration is about 0.1 mg / ml, and the agent concentration is about 3 mg / ml to about 8 mg / ml. 22. The germinating factor comprises L-valine and the agent is L-valine.
The spore germination medium according to claim 21, comprising -Gln or L-Asn. 23. The germinating factor has a concentration of about 0.001 mg / ml to about 0 mg / ml.
. 01 mg / ml and the concentration of the agent is about 3 mg / ml to about 8 mg / m
The spore germination medium according to claim 15, which is 1. 24. The spore germination medium according to claim 23, wherein the germinating factor includes L-alanine, and the agent includes L-Gln or L-Asn. 25. a) a phosphate buffer at a concentration of about 0.1 M to about 0.2 M, b) a synergistic amount of germinating and agent, and c) sodium chloride, potassium chloride, glucose and fructose. Essentially spore germination medium. 26. (a) exposing microbial spores to a sterilant; (b) a phosphate buffer at a concentration of about 0.1 M to about 0.2 M, and a synergistic amount of germinating factor. And (c) calculating the LRV of the spore germination to determine the effectiveness of the sterilization method. 27. A composition comprising a synergistic amount of a germinating agent and an agent, wherein said agent comprises about 0.
A biological indicator of the effectiveness of a sterilization method that includes a germination medium that produces a sterilant exposure time / LRV response curve gradient greater than 016. 28. The biological indicator of claim 27, wherein said medium further comprises a phosphate buffer at a concentration of about 0.1M to about 0.2M. 29. The biological indicator of claim 28, wherein said medium produces a slope index of about 10 to about 20. 30. The sterilizing agent used in the sterilizing method includes steam, ethylene oxide, radiation, heat, sodium hypochlorite, polyvinylpyrrolidone-iodine,
30. The biological indicator of claim 29, wherein the biological indicator is selected from the group consisting of 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) a first container adapted to expose microbial spores to a sterilizing agent and to calculate LRV for germinated spores; b) a second container adapted to store germination medium. C) a germination medium stored in said second container, comprising a phosphate buffer at a concentration of about 0.1 M to about 0.2 M, and a synergistic amount of germinating agent and agent;
And d) a biological indicator of the effectiveness of the sterilization method comprising the dried microbial spores. 33. a) the method of claim 1, wherein the spores comprise a compound having unknown agent properties;
Contacting a germination medium lacking a germinative factor to calculate LRV; b) calculating a LRV by contacting a spore with a germination medium lacking said compound containing said germinative factor and having unknown agent properties C) contacting a spore with a germination medium containing said germinating agent and said compound having unknown agent properties to calculate an LRV; and d) calculating an S value to obtain an unknown agent. A method of identifying an agent comprising the step of determining the efficacy of said compound having properties.