CROSS-REFERENCE TO RELATED APPLICATIONS

[001] This application claims priority to U.S. Provisional Application No. 62/865,361, filed June 24, 2019, the subject matter of which is incorporated herein by reference in entirety.

DESCRIPTION

TECHNICAL FIELD

[002] The invention relates to the manufacturing of both biological indicators (Bis) and spore suspensions in which determining the ratio of normally dormant spores to superdormant spores is measured or manipulated.

BACKGROUND

[003] Bis consist of a population of spores on a carrier made of stainless steel, paper, Tyvek or other appropriate material. Bis are used for validating and monitoring processes for disinfection, decontamination and sterilization. Herein, all of these processes will be collectively referred to as sterilization, but it is understood that these methods apply equally to all such processes in which a reduction of microbial contamination is sought.

[004] In the healthcare industry, it is beneficial to assess the effectiveness of the sterilization of medical products and equipment used in manufacturing. Herein, all such products will be collectively referred to as medical products, but it is understood that this could apply equally to aseptic processing equipment, bioburden reductions on surfaces and the like.

[005] In determining sterility, bacterial spores are oftentimes used as Bis to monitor the efficacy of specific sterilization parameters by measuring spore lethality under the given sterilization conditions. Bis allow for the validation of sterilization parameters because if spores are inactivated during a sterilization process, then it is accepted that all microorganisms on a medical product will be inactivated, hence the validation of a sterile medical product (ref. 10). Therefore, it is important to manufacture predictable and reliable Bis to validate different sterilization conditions.

[006] It is known that the resistance of a BI increases as the population of the spores on the BI carrier increases. Furthermore, it is widely known that with sterilization processes that use gasses the predictable log-linear response is not always observed due to spore clumping and layering on the BI carrier. This layering is amplified as the spore population on the carrier increases. Therefore, to preserve predictable and log-linear BI response to a sterilization process, a BI should not have an excess of spores.

[007] As described herein, manufacturers of Bis must take into consideration the total spore particles on a BI carrier, which includes both normally dormant and superdormant spores. Normally dormant spores become enumerable during common germination processes. For the germination and enumeration process, spores are applied to the surface of a plate consisting of a nutritive agar media. After which, the spores grow, form colonies and can be counted. However, superdormant spores do not become enumerable with exposure to nutrients and instead remain dormant and are called superdormant. Due to the presence of superdormant spores in most crops of spores (and the spore suspensions made from each crop), the manufacturing of Bis can oftentimes incorrectly report the spore population in the spore suspension or on the BI due to the inability to enumerate superdormant spores.

[008] Superdormant spores can become enumerable during germination processes using treatments with triggering stimuli such as steam, chemicals, acids, and the like.

[009] Thus, a problem with existing manufactured Bis is that the number of spore particles is uncertain. If a manufacturer labels a BI carrier as having a certain number of spores, but there are superdormant spores that were not enumerated during germination processes, then an incorrect spore population is produced and the log-linear response of the BI is degraded. Consequently, an incorrectly manufactured BI (containing an excess of superdormant spores) could lead to an irreproducible sterilization process result. Furthermore, sterilization conditions can yield unexpected results due to inconsistency in determining the true number of spore particles on a BI carrier because the sterilization process can trigger superdormant spores to become germinating spores, becoming enumerable, thereby increasing the number of enumerable spores because of the sterilization process (when only a decrease in population is expected).

[010] BI performance consistency is critical for successfully monitoring and validating processes that rely on Bis. For example, with one lot used for initial process development and a new lot of Bis for validation, the non-log linear response can cause validation failure due to the rogue Bis. One cause has been identified as clumping of the spores on the BI carrier, and this clumping protects the spores that are covered by other spores. For example, this problem can be amplified when the BI population is determined by counting normally dormant spores and superdormant population is 300 % or more of the normally dormant population. Such a high superdormant population causes increased spore clumping.

[011] Irreproducible BI results caused by layering and a large superdormant population is often blamed on ‘Rogue Bis’ (ref. 4), which is the observation that Bis unexpectedly survive sterilization (or microbial decontamination) processes. Practitioners try to compensate for the uncertainty of the process efficacy by placing three Bis in each location, rather than understand the deficiency of the BI.

[012] The population of superdormant spores varies from one spore crop to another. When used as a screening method for BI manufacturing, the inventor has found that between 30 % to 50 % of the spore crops screened have a significant population of superdormant spores.

[013] The disclosed methods provide a solution to manufacturing Bis and spore suspensions, wherein both normally dormant and superdormant spores are enumerated, allowing for predictable Bis for sterilization monitoring. Another aspect of the disclosed manufacturing methods triggers at least a portion of the superdormant spores to become germinating spores prior to applying these spores to the BI carrier.

SUMMARY

[014] A problem in the art of manufacturing Bis and spore suspensions is determining the number of spore particles on the BI carrier. The number of spore particles is defined as the number of both normally dormant and superdormant spores. Normally dormant spores are spores that can germinate and are therefore enumerable. Whereas normally dormant spores germinate and are enumerable with common microbiological methods, superdormant spores must be stimulated, or triggered, to germinate and become enumerable with the common enumeration methods.

[015] Commonly accepted practices in microbiology used for spore enumeration includes recovering the spores from the BI in a solution and germinating the spores on a nutritive surface (plate) so that each spore forms a visible colony. To avoid the confusion of a colony forming from multiple spores, proper dilution techniques yield sufficiently dilute solutions so that there are single spores sufficiently separated on the nutritive surface for accurate enumeration. These recovery, dilution and plate counting methods are common microbiological practices. However, as mentioned above, this method only germinates the normally dormant spores. Oftentimes, this method is not sufficient in determining the total number of spore particles on a BI carrier.

[016] Triggering mechanisms are often additionally necessary to determine the number of spore particles because triggering mechanisms allow for the conversion of superdormant spores to germinating spores, and in turn renders superdormant spores enumerable. The triggering mechanisms include heat-shocking, acid shocking, chemical exposure or other means. The triggering mechanism permits the heat labile, acid labile, protein labile to be counted. It is important to note that a portion of the superdormant spore population may convert to germinating spores during one triggering mechanism and a different cohort of superdormant spores can convert to germinating spores in response to a different triggering mechanism. Thus, it is useful to perform more than one triggering step to gain a full understanding of the triggerable spore particle population. A further aspect of this invention is the order of performing the triggering mechanism.

[017] Heat-shocking spores is a common microbiological practice and the method involves exposing a spore suspension to heat such as a water bath of 95°C or warmer for a pre-determined amount of time (e.g., 10 minutes). Heat-shocking spores can convert heat-labile superdormant spores to germinating spores. An estimate of the superdormant population in this case is the difference between the number of heat-labile spores and the number of normally dormant spores.

[018] Due to the nature of superdormant spores and the prevalence of manufacturing spore suspensions and Bis with a known amount of normally dormant and an unknown amount of superdormant spores, there is a need to manufacture Bis and spore suspensions with fewer superdormant spores. Therefore, the disclosed methods relate to the manufacturing of spore suspensions and Bis with superdormant spores accounting for less than thirty percent of spore particles on the BI carrier. In some embodiments, spore suspensions and Bis with known normally dormant and superdormant populations can be achieved by performing the three triggering mechanisms.

[019] An alternative to using triggering mechanisms is a method of counting all spore particles (normally dormant and superdormant) and comparing this number to the number of normally dormant, enumerated spores. For example, subtracting the number of normally dormant spores from the number of counted spore particles yields the total number of superdormant spores. Such counting methods can be flow cytometry, image cytometry, or other particle counting means. [020] The cytometric evaluation of a spore suspensions in Table 1 shows the total number of spore particles divided by the non-heat shock population of spores. Spore suspension 1 shows 136% more spores measured by image cytometry versus enumeration. While spore suspension 2 shows fewer spores found with image cytometry, this is likely just variation in the preparation of the samples. Spore suspension 3 and 4 have 166% and 451% more spores as measured with image cytometry. This is a typical distribution of normally dormant and superdormant spores.

Table 1. Selected spore suspensions from different spore crops showing the ratio of cytometric population to the non-heat shock population.

BRIEF DESCRIPTION OT THE DRAWINGS

[021] Fig.l. A comparison of spore population recovered from Bis using heat shock, acid shock and chemical treatment. In this case, both acid shocking and chemical treatment (urea) triggered superdormant spores to become normally germinating.

[022] Fig. 2. The observation of population increase during a sterilization process that uses N02 as a component of the sterilization process.

PET ATT, ED DESCRIPTION

[023] Biological indicators that are used for monitoring sterilization and biodecontamination processes consist of spores applied to the surface of a carrier. The spores can be any organism that is appropriate for use as the indicating organism for a sterilant exposure process and the population of spores per carrier is usually 10,000 (104) to more than 1,000,000 (106) spores. The carrier may be made of stainless steel, paper, Tyvek, glass, quartz filter media, and the like. Often, these Bis are packaged in simple Tyvek pouches and in some cases the Bis are built into a more convenient, self-contained biological indicator package or used in a process challenge device. [024] All spores are dormant forms of organism. After germination, normally dormant spores become metabolizing and replicating organisms, referred to as vegetative organisms. The trigger for germination may be the presence of nutrients or other chemicals in the recovery and nutritive media.

[025] The method of testing viability of a spores on a BI is to use a recovery process. This recovery process can be either to test for any viable spores on a BI, or the recovery process may be to enumerate, or count the viable spores on a BI carrier. A non-quantitative test for any viable spores on a BI carrier consists of placing the entire BI in liquid growth medium, and after a period of time, evaluating the liquid growth medium for evidence of spores becoming vegetative organisms, shown by turbidity or the opaqueness of the liquid growth media. A quantitative method is an enumeration recovery method which counts the number of viable spores on a BI, using serial dilution and plating procedures. These are common microbiological techniques to those familiar with the testing Bis.

[026] In an embodiment, the method is applied to samples selected from a manufacturer batch to determine whether the batch meets production quality metrics and/or to characterize the batch so that it may be sold with a correct labeling. In an alternate embodiment, the process may be used to validate a batch of Bis received by a customer who would like to be certain that the labeled spore population is correct. In another embodiment, the process may be applied to trigger spores prior to applying a suspension of spores to a carrier to prepare the Bis.

[027] A sterilization process consists of exposing the load to be sterilized to the sterilizing agent (e.g., steam, sterilizing gas, etc.) for a specified time. After exposure to the sterilant exposure process, the spores can be recovered and evaluated for spore viability. When a population of spores are homogeneously presented to the sterilant exposure process, the rate at which exposed spores are inactivated (killed) will follow a log-linear response, where one log of the spore population is inactivated during each increment of the sterilant exposure process. Therefore, the time and exposure conditions required for the inactivation of the known population of spores is predictable and defines the resistance of the spores to the sterilant exposure process.

[028] Two principle factors that influence the measured resistance of a population of spores to the sterilant exposure process are the intrinsic resistance of the spores to the sterilization process and the impedance of the sterilant exposure conditions to homogeneously reach all the spores on the BI carrier. Excluding the variability of BI response due to load consideration, there are several factors that influence the impedance of the sterilization exposure conditions from reaching the spores equally, including cleanliness (e.g., no cellular debris) in the spore suspension, and the layering of the spores on the B1 carrier. While the degree of spore suspension cleanliness can be manipulated with cleaning of the spore suspension, the layering of the spores is a critical factor that is not so easily manipulated. Furthermore, the degree of layering will increase as the number of spores (normally dormant and superdormant) on the carrier increases.

[029] It is optimal for BI performance to prepare the BI so that the spores form a monolayer, without layering, on the carrier surface. The spores that lay under other spores are shielded from the full lethal effect of a gaseous sterilant exposure process, thereby increasing the measured resistance of the protected spores to the process. This uncontrollable increase in the measured resistance (due to the protected spores) results in irreproducibility of the BI response to the sterilant exposure process. Conversely, a monolayer of spores permits all of the spores to be homogeneously exposed to the sterilant exposure process.

[030] Since the spores are typically applied to the BI carrier as a liquid spore suspension, the volume of the liquid inoculum, the conditions for drying the liquid spore suspension, and other factors can affect the layering of the spores. However, even with these factors controlled, spore layering on the BI carrier is amplified by the presence of superdormant spores.

[031] Another factor that contributes to layering is the total population of the spores on the carrier. The total number of spore population on a carrier is known to change the apparent BI resistance, due to increasing the number of spores on the carrier increases the amount of layering may occur as shown in Table 2. (ref. 9). Applying more spores to the carrier surface than are needed to achieve the target population is detrimental to consistent and reproducible BI performance. Table 2 shows the response of Geobacillus stearothermophilus Bis with varying spore populations and exposed to increasing chlorine dioxide sterilant gas (mg-hr/L) at 65% RH to 75% RH. As shown in the table, 2 x 104 Bis are quickly inactivated, but when there are 2 x 106 spores on the carrier, sterilization with chlorine dioxide is not as effective and the amount of positive (non-sterile) Bis increases due to the layering of spores on the carrier. Table 2. The total number of spore population on a BI and BI Resistance

[032] Superdormant spores are described in the literature as spores that remain dormant and do not germinate during exposure to nutrients (Ref. 1, 10). Bacillus stearothermophilus spores specifically, are found to be persistently dormant to an unusual degree, relative to spores of other species (ref. 1). Each crop of spores and each spore suspension will have a unique fraction of normally dormant and superdormant spores. It is reported that using enumeration recovery methods, and without prior heat treatment, as few as 10% of the total number of spores might germinate, form colonies, and are able to be counted. However, after appropriate triggering mechanisms, such as heat activation, 50% or more of the spores might germinate and can be counted (ref. 1). Additionally, it has been reported that a percentage of these spores will germinate and can be counted after a triggering treatment with HC1 (ref. 2). Furthermore, in certain strains of Geobacillus stearothermophilus, dormancy may be induced, rather than alleviated, by prior heating at sublethal temperatures (ref. 2)

[033] With a significant population of superdormant spores on a BI carrier, superdormant spores, and especially the superdormant spores protected by layering, may remain viable after the sterilant exposure process is completed (ref. 4, 5, 6). Given these factors, the fraction of superdormant spores in a spore suspension or on a BI carrier can affect BI resistance due to increased layering during application of spore suspension to a BI carrier. [034] The fraction of the spores that germinate when presented with a trigger for germination can vary from nearly 100% of a spore population to less than 10% of the spore population. Superdormant spores can be converted to germinating spores when exposed to triggering mechanism such as heat shock, acid shock, or a chemical treatment. Such chemical for treatment include urea, cationic surfactants and proteins that can bind or react to germination binding sites on the spore.

[035] Fig. 1 shows an example of a spore suspension where 2 x 106 spores present germinate with the normal enumeration recovery techniques (the 0 minutes exposure time to 0.1 M HC1 datum on the graph), and where these recovery techniques included heat-shocking the spores. However, by using another trigger mechanism, exposing the spores to 0.1 M HC1 for varying times, the enumerated population exhibits a 400 % increase in the number of spores counted after 10 minutes of 0.1 M HC1 exposure. Fig. 1 shows that the common microbiological technique of heat- shocking spores is not sufficient in accounting for the total amount of spore particles. Heat- shocking the spores allowed for the conversion of normally dormant spores, but with twenty minutes of heat exposure at >95 °C, 2 x 106 spores were recovered, compared to the almost 5 x 106 spores recovered after 20 minutes of exposure to 0.1 M HC1, proving that the heat-shocking method is not sufficient in determining the spore particle population and additional triggering mechanisms are required.

[036] The measured population on a BI carrier is shown in Figure 2, where the measured spore population initially increases due to exposure to the sterilization process. In this case, the sterilization process makes some amount of HN03 acid that triggers the population of superdormant spores to germinate with enumeration methods. This aligns with the reported stimulus provided by dipicolinic acid (DP A), as is reported in the literature (ref. 1). Furthermore, given that DP A, HC1 and HN03 are able to trigger acid labile spores, it is obvious that peracetic acid and acetic acid could cause a similar triggering of superdormant spores.

[037] After chemical, protein or acid triggering treatment of a spore suspension, the suspension may need to be washed to neutralize triggering treatment and the remove unwanted residuals of the triggering treatment. This is done by diluting the spore suspension, centrifuging the diluted spore suspension, removing the supernatant, and resuspending the spores in suspension. The washing may need to be repeated to result in a clean spore suspension. [038] Another embodiment of this invention is the order of the triggering steps. For example, the inventor found that performing acid shocking of the spore suspension prior to heat shocking of the spore suspension results in Bis with the greatest germinating spore population and the fewest superdormant spores.

[039] It is another embodiment of this disclosure to provide a method for manufacturing Bis that do not have an excess of superdormant spores. The term“excess” relates to BI carriers that do not have a superdormant population exceeding 30% of the total amount of spore particles. This allows for controlling the spore population on BI indicators and any BI carriers that have an excess of superdormant spores will not be used for manufacturing purposes.

[040] This disclosure also includes methods of manufacturing spore suspension for Bis in which the population of superdormant spores is determined. Heretofore, spore suspension and Bis are not tested to determine the fraction of superdormant spores. By using a method to determine the total number of spore particles, the BI manufacturer can either choose to not use that spore suspension, or the superdormant spores can be converted to normally geminating spores using heat, acid, or protein or treatments.

[041] Further, embodiments include determining the superdormant spore population by spore particle counting via cytometric methods such as flow cytometry or image cytometry and comparing the particle count with the enumeration recovery results. This might include using at least two techniques of counting the spores applied to a biological indicator carrier and using the difference from counting techniques to characterize the quality of the biological indicator.

[042] Identifying the presence of a significant fraction of super dormant spores can be used as one criterion for screening spore crops that might be used for manufacturing Bis. Alternatively, triggering can be used for converting superdormant spores to germinating spores prior to applying the spores to the BI carrier.

[043] Embodiments include the following examples:

[044] 1. A method of manufacturing a biological indicator (BI) comprising: enumerating spores with at least two different methods, comprising: germinating untreated spores to determine a quantity of normally dormant spores on the BI; and performing a triggering mechanism to determine the presence of superdormant spores on the BI. [045] 2. The method of example 1 wherein the total amount of superdormant spores are determined by a combination of heat-labile, acid labile, protein labile spores, non-germinating, and non-viable spores.

[046] 3. The method of example 1 wherein the number of normally dormant spores is determined by the germination method, which includes enumerating growing colonies of the BI organism, wherein growing colonies are a group of reproducing organisms that when diluted arise from one germinating spore.

[047] 4. The method of example 3wherein a 10-fold serial dilution of the liquid recovery medium is performed prior to enumerating growing colonies of the BI organism.

[048] 5. The method of example 3 wherein the germination method is performed by resuspending spores from a BI carrier into a recovery tube with a liquid recovery medium prior to enumerating growing colonies of the BI organism.

[049] 6. The method of example 5 wherein the resuspended spores in the liquid-filled recovery tube are plated on a nutritive agar media and viable spores are determined by counting colonies on the agar surface and normally dormant spores are determined by counting the number of spore particles using a cytometric method.

[050] 7. The method of example 6 wherein the at least a portion of the superdormant spores is determined by taking the difference between the counted growing colonies and normally dormant spores.

[051] 8. The method of example 2 wherein a heat-shock step is used as the triggering mechanism to convert superdormant spores to germinating spores and after the spores are heat-shocked, they are enumerated using the germination method to determine the number of heat labile superdormant spores.

[052] 9. The method of example 8 wherein at least a portion of the superdormant population is estimated as the difference between the number of heat-shock enumerated spores and the normally dormant spores.

[053] 10. The method of example 2 wherein an acid-shock step is used as a trigger to convert superdormant spores to germinating spores and following acid-shocking the spores, the spores are enumerated using germination of the acid-shock treated spores by enumerating growing colonies of the BI organism to determine the number of acid labile superdormant spores. [054] 11. The method of example 10 wherein at least a portion of the superdormant population is estimated as the difference between the number of acid-shock enumerated spores and the normally dormant spores.

[055] 12. The method of example 2 wherein a protein exposure is used as a trigger to convert superdormant spores to germinating spores and following the protein exposure, the spores are enumerated using germination of the protein-exposed spores by enumerating growing colonies of the BI organism to determine the amount of protein labile superdormant spores.

[056] 13. The method of example 12 wherein at least a portion of the superdormant population is estimated as the difference between the number of protein-exposed spores and the normally dormant spores.

[057] 14. A method of manufacturing a biological indicator (BI) wherein the spores are enumerated with at least two distinct methods, wherein one method involves germination of untreated spores to determine the quantity of normally dormant spores on the BI and the second method involves determining the presence of superdormant spores on the BI, wherein the total number of spore particles are counted.

[058] 15. The method of example 14 wherein the spore particles are counted using flow cytometry.

[059] 16. The method of example 14 wherein the spore particles are counted using image cytometry.

[060] 17. The method of example 14 wherein the number of normally dormant spores is determined using the germination method.

[061] 18. The method of example 14 wherein the total amount of superdormant spores are determined by the combination of heat-labile, acid labile, protein labile spores, non-germinating, and non-viable spores.

[062] 19. The method of example 14 wherein the number of normally dormant spores is determined by the germination method, which includes enumerating growing colonies of the BI organism, wherein growing colonies are a group of reproducing organisms that when diluted arise from one germinating spore.

[063] 20. The method of example 19 wherein a 10-fold serial dilution of the liquid recovery medium is performed prior to enumerating growing colonies of the BI organism. [064] 21. The method of example 19 wherein the germination method is performed by resuspending spores from a BI carrier into a recovery tube with a liquid recovery medium prior to enumerating growing colonies of the BI organism.

[065] 22. The method of example 21 wherein the resuspended spores in the liquid-filled recovery tube are plated on a nutritive agar media and viable spores are determined by counting colonies on the agar surface and normally dormant spores are determined by counting the number of spore particles using a cytometric method.

[066] 23. The method of example 22 wherein the at least a portion of the superdormant spores is determined by taking the difference between the counted growing colonies and normally dormant spores.

[067] 24. The method of example 18 wherein a heat-shock step is used as the triggering mechanism to convert superdormant spores to germinating spores and after the spores are heat- shocked, they are enumerated using the germination method to determine the number of heat labile superdormant spores.

[068] 25. The method of example 24 wherein at least a portion of the superdormant population is estimated as the difference between the number of heat-shock enumerated spores and the normally dormant spores.

[069] 26. The method of example 18 wherein an acid-shock step is used as a trigger to convert superdormant spores to germinating spores and following acid-shocking the spores, the spores are enumerated using germination of the acid-shock treated spores by enumerating growing colonies of the BI organism to determine the number of acid labile superdormant spores.

[070] 27. The method of example 26 wherein at least a portion of the superdormant population is estimated as the difference between the number of acid-shock enumerated spores and the normally dormant spores.

[071] 28. The method of example 18 wherein a protein exposure is used as a trigger to convert superdormant spores to germinating spores and following the protein exposure, the spores are enumerated using germination of the protein-exposed spores by enumerating growing colonies of the BI organism to determine the amount of protein labile superdormant spores.

[072] 29. The method of example 28 wherein at least a portion of the superdormant population is estimated as the difference between the number of protein-exposed spores and the normally dormant spores. [073] 30. A method of manufacturing a spore suspension used for a biological indicator (BI) wherein spore particles are enumerated with at least two distinct methods, wherein one method involves germination of untreated spores to determine the quantity of normally dormant spores on the BI and the second method involves performing a triggering mechanism to determine the presence of superdormant spores on the BI.

[074] 31. The method of example 30 wherein the total amount of superdormant spores are determined by the combination of heat-labile, acid labile, protein labile spores, non-germinating, and non-viable spores.

[075] 32. The method of example 30 wherein the number of normally dormant spores is determined by the germination method, which includes enumerating growing colonies of the BI organism, wherein growing colonies are a group of reproducing organisms that when diluted arise from one germinating spore.

[076] 33. The method of example 32 wherein a 10-fold serial dilution of the liquid recovery medium is performed prior to enumerating growing colonies of the BI organism.

[077] 34. The method of example 32 wherein the germination method is performed by resuspending spores from a BI carrier into a recovery tube with a liquid recovery medium prior to enumerating growing colonies of the BI organism.

[078] 35. The method of example 34 wherein the resuspended spores in the liquid-filled recovery tube are plated on a nutritive agar media and viable spores are determined by counting colonies on the agar surface and normally dormant spores are determined by counting the number of spore particles using a cytometric method.

[079] 36. The method of example 35 wherein the at least a portion of the superdormant spores is determined by taking the difference between the counted growing colonies and normally dormant spores.

[080] 37. The method of example 31 wherein a heat-shock step is used as the triggering mechanism to convert superdormant spores to germinating spores and after the spores are heat- shocked, they are enumerated using the germination method to determine the number of heat labile superdormant spores.

[081] 38. The method of example 37 wherein at least a portion of the superdormant population is estimated as the difference between the number of heat-shock enumerated spores and the normally dormant spores. [082] 39. The method of example 31 wherein an acid-shock step is used as a trigger to convert superdormant spores to germinating spores and following acid-shocking the spores, the spores are enumerated using germination of the acid-shock treated spores by enumerating growing colonies of the BI organism to determine the number of acid labile superdormant spores.

[083] 40. The method of example 39 wherein at least a portion of the superdormant population is estimated as the difference between the number of acid-shock enumerated spores and the normally dormant spores.

[084] 41. The method of example 31 wherein a protein exposure is used as a trigger to convert superdormant spores to germinating spores and following the protein exposure, the spores are enumerated using germination of the protein-exposed spores by enumerating growing colonies of the BI organism to determine the amount of protein labile superdormant spores.

[085] 42. The method of example 41 wherein at least a portion of the superdormant population is estimated as the difference between the number of protein-exposed spores and the normally dormant spores.

[086] 43. A method of manufacturing a spore suspension used for a biological indicator (BI) wherein the spore particles are enumerated with at least two distinct methods, wherein one method involves germination of untreated spores to determine the quantity of normally dormant spores on the in the spore suspension, and the second method involves performing a triggering mechanism to determine the quantity of superdormant spores in the spore suspension, and spore particles are counted.

[087] 44. The method of example 43 wherein the spore particles are counted using flow cytometry.

[088] 45. The method of example 43 wherein the spore particles are counted using image cytometry.

[089] 46. The method of example 43 wherein the number of normally dormant spores is determined using the germination method.

[090] 47. The method of example 43 wherein the total amount of superdormant spores are determined by the combination of heat-labile, acid labile, protein labile spores, non-germinating, and non-viable spores.

[091] 48. The method of example 43 wherein the number of normally dormant spores is determined by the germination method, which includes enumerating growing colonies of the BI organism, wherein growing colonies are a group of reproducing organisms that when diluted arise from one germinating spore.

[092] 49. The method of example 48 wherein a 10-fold serial dilution of the liquid recovery medium is performed prior to enumerating growing colonies of the BI organism.

[093] 50. The method of example 48 wherein the germination method is performed by resuspending spores from a BI carrier into a recovery tube with a liquid recovery medium prior to enumerating growing colonies of the BI organism.

[094] 51. The method of example 50 wherein the resuspended spores in the liquid-filled recovery tube are plated on a nutritive agar media and viable spores are determined by counting colonies on the agar surface and normally dormant spores are determined by counting the number of spore particles using a cytometric method.

[095] 52. The method of example 51 wherein the at least a portion of the superdormant spores is determined by taking the difference between the counted growing colonies and normally dormant spores.

[096] 53. The method of example 47 wherein a heat-shock step is used as the triggering mechanism to convert superdormant spores to germinating spores and after the spores are heat- shocked, they are enumerated using the germination method to determine the number of heat labile superdormant spores.

[097] 54. The method of example 53 wherein at least a portion of the superdormant population is estimated as the difference between the number of heat-shock enumerated spores and the normally dormant spores.

[098] 55. The method of example 47 wherein an acid-shock step is used as a trigger to convert superdormant spores to germinating spores and following acid-shocking the spores, the spores are enumerated using germination of the acid-shock treated spores by enumerating growing colonies of the BI organism to determine the number of acid labile superdormant spores.

[099] 56. The method of example 55 wherein at least a portion of the superdormant population is estimated as the difference between the number of acid-shock enumerated spores and the normally dormant spores.

[0100] 57. The method of example 47 wherein a protein exposure is used as a trigger to convert superdormant spores to germinating spores and following the protein exposure, the spores are enumerated using germination of the protein-exposed spores by enumerating growing colonies of the BI organism to determine the amount of protein labile superdormant spores.

[0101] 58. The method of example 57 wherein at least a portion of the superdormant population is estimated as the difference between the number of protein-exposed spores and the normally dormant spores.

[0102] 59. A method of manufacturing biological indicator (BI) wherein triggering spores is performed on a spore suspension prior to applying the spores to the BI carrier.

[0103] 60. The method of example 59 wherein the spore suspension is cleaned after triggering.

[0104] 61. The method of example 60 wherein the cleaning step includes diluting the spore suspension.

[0105] 62. The method of example 61 wherein the cleaning includes a centrifuging step to create a stratified solution with one strata containing spores.

[0106] 63. The method of example 62 includes removing supernatant from the centrifuged and stratified solution.

[0107] 64. The method of example 63 wherein the cleaning step that includes resuspending the spores from the strata to form a homogeneous spore suspension.

[0108] 65. The method of example 59 wherein the triggering step involves heat-shocking the spores.

[0109] 66. The method of example 59 wherein the triggering step involves acid-shocking the spores.

[0110] 67. The method of example 59 wherein the triggering step involves exposing the spores to a protein.

[0111] 68. The method of example 66 wherein a cleaning step neutralizes the spore suspension pH after the acid-shock step.

[0112] 69. The method of example 59 wherein the spore suspension is evaluated for the ratio of normally dormant spores to superdormant spores after triggering.

[0113] 70. The method of example 61 wherein the spore suspension is evaluated for the ratio of normally dormant spores to superdormant spores after cleaning.

[0114] Although the invention has been described in detail for the purpose of illustration based on what is currently considered to be the most practical and preferred embodiments, it is to be understood that such detail is solely for that purpose and that the invention is not limited to the disclosed embodiments, but, on the contrary, is intended to cover modifications and equivalent arrangements that are within the spirit and scope of the appended claims. For example, it is to be understood that the present invention contemplates that, to the extent possible, one or more features of any embodiment can be combined with one or more features of any other embodiment.

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