Mechanisms of killing of spores of Bacillus subtilis by dimethyldioxirane

AIMS: To determine the mechanisms of Bacillus subtilis spore resistance to and killing by a novel sporicide, dimethyldioxirane (DMDO) that was generated in situ from acetone and potassium peroxymonosulfate at neutral pH. METHODS AND RESULTS: Spores of B. subtilis were effectively killed by DMDO. Rates of killing by DMDO of spores lacking most DNA protective alpha/beta-type small, acid-soluble spore proteins (alpha- beta- spores) or the major DNA repair protein, RecA, were very similar to that of wild-type spore killing. Survivors of wild-type and alpha- beta- spores treated with DMDO also exhibited no increase in mutations. Spores lacking much coat protein due either to mutation or chemical decoating were much more sensitive to DMDO than were wild-type spores, but were more resistant than growing cells. Wild-type spores killed with this reagent retained their large pool of dipicolinic acid (DPA), and the survivors of spores treated with DMDO were sensitized to wet heat. The DMDO-killed spores germinated with nutrients, albeit more slowly than untreated spores, but germinated faster than untreated spores with dodecylamine. The killed spores were also germinated by very high pressures and by lysozyme treatment in hypertonic medium, but many of these spores lysed shortly after their germination, and none of these treatments were able to revive the DMDO-killed spores. CONCLUSIONS: DMDO is an effective reagent for killing B. subtilis spores. The spore coat is a major factor in spore resistance to DMDO, which does not kill spores by DNA damage or by inactivating some component needed for spore germination. Rather, this reagent appears to kill spores by damaging the spore's inner membrane in some fashion. SIGNIFICANCE AND IMPACT OF THE STUDY: This work demonstrates that DMDO is an effective decontaminant for spores of Bacillus species that can work under mild conditions, and the killed spores cannot be revived. Evidence has also been obtained on the mechanisms of spore resistance to and killing by this reagent.     

Mechanisms of killing of Bacillus subtilis spores by Decon and Oxone, two general decontaminants for biological agents

AIMS: To determine the mechanisms of Bacillus subtilis spore killing by and resistance to the general biological decontamination agents, Decon and Oxone. METHODS AND RESULTS: Spores of B. subtilis treated with Decon or Oxone did not accumulate DNA damage and were not mutagenized. Spore killing by these agents was increased if spores were decoated. Spores prepared at higher temperatures were more resistant to these agents, consistent with a major role for spore coats in this resistance. Neither Decon nor Oxone released the spore core's depot of dipicolinic acid (DPA), but Decon- and Oxone-treated spores more readily released DPA upon a subsequent normally sublethal heat treatment. Decon- and Oxone-killed spores initiated germination with dodecylamine more rapidly than untreated spores, but could not complete germination triggered by nutrients or Ca(2+)-DPA and did not degrade their peptidoglycan cortex. However, lysozyme treatment did not recover these spores. CONCLUSIONS: Decon and Oxone do not kill B. subtilis spores by DNA damage, and a major factor in spore resistance to these agents is the spore coat. Spore killing by both agents renders spores defective in germination, possibly because of damage to the inner membrane of spore. SIGNIFICANCE AND IMPACT OF STUDY: These results provide information on the mechanisms of the killing of bacterial spores by Decon and Oxone.     

Mechanisms of Bacillus subtilis spore killing by and resistance to an acidic Fe-EDTA-iodide-ethanol formulation

AIMS: To determine the mechanisms of Bacillus subtilis spore killing by and resistance to an acidic solution containing Fe(3+), EDTA, KI and ethanol termed the KMT reagent. METHODS AND RESULTS: Wild-type B. subtilis spores were not mutagenized by the KMT reagent but the wild-type and recA spores were killed at the same rate. Spores (alpha(-)beta(-)) lacking most DNA-protective alpha/beta-type small, acid-soluble spore proteins were less resistant to the KMT reagent than wild-type spores but were also not mutagenized, and alpha(-)beta(-) and alpha(-)beta(-)recA spores exhibited nearly identical resistance. Spore resistance to the KMT reagent was greatly decreased if spores had defective coats. However, the level of unsaturated fatty acids in the inner membrane did not determine spore sensitivity to the KMT reagent. Survivors in spore populations killed by the KMT reagent were sensitized to killing by wet heat or nitrous acid and to high salt in plating medium. KMT reagent-killed spores had not released their dipicolinic acid (DPA), although these killed spores released their DPA more readily when germinated with dodecylamine than did untreated spores. However, KMT reagent-killed spores did not germinate with nutrients or Ca(2+)-DPA and were recovered only poorly by lysozyme treatment in a hypertonic medium. CONCLUSIONS: The KMT reagent does not kill spores by DNA damage and a major factor in spore resistance to this reagent is the spore coat. KMT reagent treatment damages the spore's ability to germinate, perhaps by damaging the spore's inner membrane. However, this damage is not oxidation of unsaturated fatty acids. SIGNIFICANCE AND IMPACT OF THE STUDY: These results provide information on the mechanism of spore resistance to and killing by the KMT reagent developed for killing Bacillus spores.     

Mechanisms of Bacillus subtilis spore resistance to and killing by aqueous ozone

AIMS: To determine the mechanisms of Bacillus subtilis spore killing by and resistance to aqueous ozone. METHODS AND RESULTS: Killing of B. subtilis spores by aqueous ozone was not due to damage to the spore's DNA, as wild-type spores were not mutagenized by ozone and wild-type and recA spores exhibited very similar ozone sensitivity. Spores (termed alpha-beta-) lacking the two major DNA protective alpha/beta-type small, acid-soluble spore proteins exhibited decreased ozone resistance but were also not mutagenized by ozone, and alpha-beta- and alpha-beta-recA spores exhibited identical ozone sensitivity. Killing of spores by ozone was greatly increased if spores were chemically decoated or carried a mutation in a gene encoding a protein essential for assembly of the spore coat. Ozone killing did not cause release of the spore core's large depot of dipicolinic acid (DPA), but these killed spores released all of their DPA after a subsequent normally sublethal heat treatment and also released DPA much more readily when germinated in dodecylamine than did untreated spores. However, ozone-killed spores did not germinate with either nutrients or Ca(2+)-DPA and could not be recovered by lysozyme treatment. CONCLUSIONS: Ozone does not kill spores by DNA damage, and the major factor in spore resistance to this agent appears to be the spore coat. Spore killing by ozone seems to render the spores defective in germination, perhaps because of damage to the spore's inner membrane. SIGNIFICANCE AND IMPACT OF THE STUDY: These results provide information on the mechanisms of spore killing by and resistance to ozone.     

Analysis of factors influencing the rate of germination of spores of Bacillus subtilis by very high pressure

AIMS: To elucidate the factors that determine the rate of germination of Bacillus subtilis spores with very high pressure (VHP) and the mechanism of VHP germination. METHODS AND RESULTS: Spores of B. subtilis were germinated rapidly with a VHP of 500 MPa at 50 degrees C. This VHP germination did not require the spore's nutrient-germinant receptors, as found previously, and did not require diacylglycerylation of membrane proteins. However, the spore's pool of dipicolinic acid (DPA) was essential. Either of the two redundant enzymes that degrade the spore's peptidoglycan cortex, and thus allow completion of spore germination, was essential for completion of VHP germination. However, neither of these enzymes was needed for DPA release triggered by VHP treatment. Completion of spore germination as well as DPA release with VHP had an optimum temperature of approx. 60 degrees C, in contrast to an optimum temperature of 40 degrees C for germination with the moderately high pressure of 150 MPa. The rate of spore germination by VHP decreased approx. fourfold when the sporulation temperature increased from 23 degrees C to 44 degrees C, and decreased twofold when 1 mol l(-1) salt was present in sporulation. However, large variations in levels of unsaturated fatty acids in the spore's inner membranes did not affect rates of VHP germination. Complete germination of spores by VHP was not inhibited significantly by killing of spores with several oxidizing agents, and was not inhibited by ethanol, octanol or o-chlorophenol at concentrations that abolish nutrient germination. Completion of spore germination by VHP was also inhibited by Hg(2+), but this ion did not inhibit DPA release caused by VHP. In contrast, dodecylamine, a surfactant that can trigger spore germination, strongly inhibited DPA release caused by VHP treatment. CONCLUSIONS: VHP does not cause spore germination by acting upon the spore's nutrient-germinant receptors, but by directly causing DPA release. This DPA release then leads to subsequent completion of germination. VHP likely acts on the spore's inner membrane to cause DPA release, targeting either a membrane protein or the membrane itself. However, the precise identity of this target is not yet clear. SIGNIFICANCE AND IMPACT OF THE STUDY: There is significant interest in the use of VHP to eliminate or reduce levels of bacterial spores in foods. As at least partial spore germination by pressure is almost certainly essential for subsequent spore killing, knowledge of factors involved and the mechanism of VHP germination are crucial to the understanding of spore killing by VHP. This work provides new insight into factors that can affect the rate of B. subtilis spore germination by VHP, and into the mechanism of VHP germination itself.     

Killing of spores of Bacillus subtilis by tert-butyl hydroperoxide plus a TAML activator

AIMS: To determine the effectiveness of tert-butyl hydroperoxide (tBHP) plus the cationic surfactant cetyltrimethyl ammonium bromide (CTAB) and a tetra-amido macrocyclic ligand (TAML) activator in killing spores of Bacillus subtilis and the mechanisms of spore resistance to and killing by this reagent. METHODS AND RESULTS: Killing of spores of B. subtilis by tBHP was greatly stimulated by the optimum ratio of concentrations of a TAML activator (1.7 micromol l(-1)) to tBHP (4.4%, vol/vol) plus a low level (270 mg l(-1)) of CTAB. Rates of killing of spores lacking most DNA protective alpha/beta-type small, acid-soluble spore proteins (alpha(-)beta(-) spores) or the major DNA repair protein, RecA, by tBHP plus CTAB and a TAML activator were essentially identical to that of wild-type spore killing. Survivors of wild-type and alpha(-)beta(-) spores treated with tBHP plus CTAB and a TAML activator also exhibited no increase in mutations. Spores lacking much coat protein either because of mutation or chemical decoating were much more sensitive to this reagent than were wild-type spores, but were more resistant than growing cells. Wild-type spores killed with this reagent retained their large pool of dipicolinic acid (DPA), and the survivors of spores treated with this reagent were sensitized to wet heat. The tBHP plus CTAB and TAML activator-killed spores germinated with nutrients, albeit more slowly than untreated spores, but germinated faster than untreated spores with dodecylamine. The killed spores were also germinated by application of 150 and 500 megaPascals of pressure for 15 min and by lysozyme treatment in hypertonic medium, but these spores lysed shortly after their germination. CONCLUSIONS: The combination of tBHP plus CTAB and a TAML activator is effective in killing B. subtilis spores. The spore coat is a major factor in spore resistance to this reagent system, which does not kill spores by DNA damage or by inactivating some component needed for spore germination. Rather, this reagent system appears to kill spores by damaging the spore's inner membrane in some fashion. SIGNIFICANCE AND IMPACT OF THE STUDY: This work demonstrates that tBHP plus CTAB and a TAML activator is an effective and mild decontaminant for spores of Bacillus species. Evidence has also been obtained on the mechanisms of spore resistance to and killing by this reagent system.     

Germination of spores of Bacillus subtilis with dodecylamine

AIMS: To determine the properties of Bacillus subtilis spores germinated with the alkylamine dodecylamine, and the mechanism of dodecylamine-induced spore germination. METHODS AND RESULTS: Spores of B. subtilis prepared in liquid medium were germinated efficiently by dodecylamine, while spores prepared on solid medium germinated more poorly with this agent. Dodecylamine germination of spores was accompanied by release of almost all spore dipicolinic acid (DPA), degradation of the spore's peptidoglycan cortex, release of the spore's pool of free adenine nucleotides and the killing of the spores. The dodecylamine-germinated spores did not initiate metabolism, did not degrade their pool of small, acid-soluble spore proteins efficiently and had a significantly lower level of core water than did spores germinated by nutrients. As measured by DPA release, dodecylamine readily induced germination of B. subtilis spores that: (a) were decoated, (b) lacked all the receptors for nutrient germinants, (c) lacked both the lytic enzymes either of which is essential for cortex degradation, or (d) had a cortex that could not be attacked by the spore's cortex-lytic enzymes. The DNA in dodecylamine-germinated wild-type spores was readily stained, while the DNA in dodecylamine-germinated spores of strains that were incapable of spore cortex degradation was not. These latter germinated spores also did not release their pool of free adenine nucleotides. CONCLUSIONS: These results indicate that: (a) the spore preparation method is very important in determining the rate of spore germination with dodecylamine, (b) wild-type spores germinated by dodecylamine progress only part way through the germination process, (c) dodecylamine may trigger spore germination by a novel mechanism involving the activation of neither the spore's nutrient germinant receptors nor the cortex-lytic enzymes, and (d) dodecylamine may trigger spore germination by directly or indirectly activating release of DPA from the spore core, through the opening of channels for DPA in the spore's inner membrane. SIGNIFICANCE AND IMPACT OF THE STUDY: These results provide new insight into the mechanism of spore germination with the cationic surfactant dodecylamine, and also into the mechanism of spore germination in general. New knowledge of mechanisms to stimulate spore germination may have applied utility, as germinated spores are much more sensitive to processing treatments than are dormant spores.     

Effect of mechanical abrasion on the viability, disruption and germination of spores of Bacillus subtilis

AIMS: To elucidate the factors influencing the sensitivity of Bacillus subtilis spores in killing and disrupting by mechanical abrasion, and the mechanism of stimulation of spore germination by abrasion. METHODS AND RESULTS: Spores of B. subtilis strains were abraded by shaking with glass beads in liquid or the dry state, and spore killing, disruption and germination were determined. Dormant spores were more resistant to killing and disruption by abrasion than were growing cells or germinated spores. However, dormant spores of the wild-type strain with or without most coat proteins removed, spores of strains with mutations causing spore coat defects, spores lacking their large depot of dipicolinic acid (DPA) and spores with defects in the germination process exhibited essentially identical rates of killing and disruption by abrasion. When spores lacking all nutrient germinant receptors were enumerated by plating directly on nutrient medium, abrasion increased the plating efficiency of these spores before killing them. Spores lacking all nutrient receptors and either of the two redundant cortex-lytic enzymes behaved similarly in this regard, but the plating efficiency of spores lacking both cortex-lytic enzymes was not stimulated by abrasion. CONCLUSIONS: Dormant spores are more resistant to killing and disruption by abrasion than are growing cells or germinated spores, and neither the complete coats nor DPA are important in spore resistance to such treatments. Germination is not essential for spore killing by abrasion, although abrasion can trigger spore germination by activation of either of the spore's cortex-lytic enzymes. SIGNIFICANCE AND IMPACT OF THE STUDY: This work provides new insight into the mechanisms of the killing, disruption and germination of spores by abrasion and makes the surprising finding that at least much of the spore coat is not important in spore resistance to abrasion.     

Comparison of the inactivation of Bacillus subtilis spores and MS2 bacteriophage by MIOX, ClorTec and hypochlorite

AIMS: To compare the disinfection ability of two widely used electrolytic generation systems (ClorTec and MIOX) and the conventional chlorine disinfectant (sodium hypochlorite) using three strains of Bacillus subtilis spores and MS2 bacteriophage. METHODS AND RESULTS: Three B. subtilis aerobic spore strains (ATCC1A1, 35021 and 35946) and the bacteriophage MS2 (ATCC 15597-B1) were propagated and sporulated. Four indicator organisms were exposed to four disinfectant treatments for comparing the effectiveness of inactivation: hypochlorite, ClorTec, MIOX and MIOX-anode. The results indicated that the two electrolytic generation systems were as effective as the conventional chlorination for the inactivation of micro-organisms used. Some data points showed the variation using anova analysis, in which the inactivation of MIOX and ClorTec was higher than that of hypochlorite. CONCLUSIONS: The ClorTec and MIOX systems are quite similar to hypochlorite in the inactivation-effectiveness for aerobic spores and bacteriophage in drinking water. SIGNIFICANCE AND IMPACT OF THE STUDY: Laboratory-scale investigation proved that gaseous chlorine could be replaced by either ClorTec or MIOX systems for the drinking water treatment utilities, which still could maintain the same disinfection efficiency.     

Treatment with oxidizing agents damages the inner membrane of spores of Bacillus subtilis and sensitizes spores to subsequent stress

AIMS: To determine if treatment of Bacillus subtilis spores with a variety of oxidizing agents causes damage to the spore's inner membrane. METHODS AND RESULTS: Spores of B. subtilis were killed 80-99% with wet heat or a variety of oxidizing agents, including betadine, chlorine dioxide, cumene hydroperoxide, hydrogen peroxide, Oxone, ozone, sodium hypochlorite and t-butylhydroperoxide, and the agents neutralized and/or removed. Survivors of spores pretreated with oxidizing agents exhibited increased sensitivity to killing by a normally minimal lethal heat treatment, while spores pretreated with wet heat did not. In addition, spores treated with wet heat or the oxidizing agents, except sodium hypochlorite, were more sensitive to high NaCl in plating media than were untreated spores. The core region of spores treated with at least two oxidizing agents was also penetrated much more readily by methylamine than was the core of untreated spores, and spores treated with oxidizing agents but not wet heat germinated faster with dodecylamine than did untreated spores. Spores of strains with very different levels of unsaturated fatty acids in their inner membrane exhibited essentially identical resistance to oxidizing agents. CONCLUSIONS: Treatment of spores with oxidizing agents has been suggested to cause damage to the spore's inner membrane, a membrane whose integrity is essential for spore viability. The sensitization of spores to killing by heat and to high salt after pretreatment with oxidizing agents is consistent with and supports this suggestion. Presumably mild pretreatment with oxidizing agents causes some damage to the spore's inner membrane. While this damage may not be lethal under normal conditions, the damaged inner membrane may be less able to maintain its integrity, when dormant spores are exposed to high temperature or when germinated spores are faced with osmotic stress. Triggering of spore germination by dodecylamine likely involves action by this agent on the spore's inner membrane allowing release of the spore core's depot of dipicolinic acid. Presumably dodecylamine more readily alters the permeability of a damaged inner membrane and thus more readily triggers germination of spores pretreated with oxidizing agents. Damage to the inner spore membrane by oxidizing agents is also consistent with the more rapid penetration of methylamine into the core of treated spores, as the inner membrane is likely the crucial permeability barrier to methylamine entry into the spore core. As spores of strains with very different levels of unsaturated fatty acids in their inner membrane exhibited essentially identical resistance to oxidizing agents, it is not through oxidation of unsaturated fatty acids that oxidizing agents kill and/or damage spores. Perhaps these agents work by causing oxidative damage to key proteins in the spore's inner membrane. SIGNIFICANCE AND IMPACT OF THE STUDY: The more rapid heat killing and germination with dodecylamine, the greater permeability of the spore core and the osmotic stress sensitivity in outgrowth of spores pretreated with oxidizing agents is consistent with such agents causing damage to the spore's inner membrane, even if this damage is not lethal under normal conditions. It may be possible to take advantage of this phenomenon to devise improved, less costly regimens for spore inactivation.     

Analysis of damage due to moist heat treatment of spores of Bacillus subtilis

Aims:  To determine conditions for generation and recovery of Bacillus subtilis spore populations heavily damaged by moist heat treatment.
Methods and Results:  Bacillus subtilis spores were treated with moist heat and spore viability was assessed on different media. A rich medium and several minimal media gave similar spore recoveries after moist heat treatment, but lack of glucose in minimal media greatly decreased spore recovery. High NaCl levels also greatly decreased the recovery of moist heat-treated spores on minimal media, and addition of good osmoprotectants reversed this effect. Moist heat treatment did not decrease spore recovery on minimal media with high salt through DNA damage or by eliminating spore germination, but by affecting spore outgrowth.
Conclusions:  Conditions for generating B. subtilis spore populations with high levels of conditional moist heat damage have been determined. The major conditional damage appears to be in spore outgrowth, perhaps because of damage to one or more important metabolic enzymes.
Significance and Impact of the Study:  This work has provided new insight into the mechanism of B. subtilis spore killing by moist heat.     

Disinfection of Bacillus subtilis spore-contaminated surface materials with a sodium hypochlorite and a hydrogen peroxide-based sanitizer

Aims: To evaluate a sodium hypochlorite and hydrogen peroxide solution (Ox‐B7) as a potential decontaminant of Bacillus subtilis spore‐contaminated surface materials (porous and nonporous). Methods and Results: Test materials were contaminated with B. subtilis spores to a final concentration in the range of 5·7–6·6 log CFU cm^?2. Ox‐B7 reduced spore counts by 99·999% (5 log) for both porous and nonporous surfaces within a 5‐min contact. Treatment with equivalent concentrations of only sodium hypochlorite reduced spore counts by 99% (2 log) on porous materials and by 99·99% (4 log) on nonporous materials. Hydrogen peroxide treatments reduced spores by less than 90% (<1 log) on both porous and nonporous materials when compared with untreated samples. Conclusions: A combination of sodium hypochlorite and hydrogen peroxide (Ox‐B7) effectively killed B. subtilis spores on both porous and nonporous surface materials. Significance and Impact of the Study: The combination of sodium hypochlorite and hydrogen peroxide can be used as an alternative disinfectant of spore‐contaminated surface materials, as it is more effective than when hydrogen peroxide or sodium hypochlorite are used separately.     

Permeability of dormant spores of Bacillus subtilis to gramicidin S

Abstract Gramicidin S, dissolved in ethanol, penetrated into the inside of the dormant spores of Bacillus subtilis , had a partial inhibitory effect on l-alanine-initiated germination and completely inhibited their outgrowth and vegetative growth. The activity of particulate NADH oxidase of the antibiotic-treated dormant spores was also influenced significantly. Abnormal morphological changes were observed in germinated spores from gramicidin S-treated dormant spores. An immunoelectron microscopy method with colloidal gold-IgG complex showed that the penetration site of gramicidin S inside dormant spores was mainly the core region. These facts suggest that gramicidin S induces the damage of not only the outer membrane-spore coat complex but also the inner membrane surrounding the spore protoplast, and is able to penetrate into the core region of B. subtilis dormant spores.     

Germination and inactivation of Bacillus subtilis spores induced by moderate hydrostatic pressure

In this study, we investigated the mechanisms of spore inactivation by high pressure at moderate temperatures to optimize the sterilization efficiency of high‐pressure treatments. Bacillus subtilis spores were first subjected to different pressure treatments ranging from 90 to 550 MPa at 40°C, with holding times from 10 min to 4 h. These treatments alone caused slight inactivation, which was related to the pressure‐induced germination of the spores. After these pressures treatments, the sensitivity of these processed spores to heat (80°C/10 min) or to high pressure (350 MPa/40°C/10 min) was tested to determine the pressure‐induced germination rate and the advancement of the spores in the germination process. The subsequent heat or pressure treatments were applied immediately after decompression from the first pressure treatment or after a holding time at atmospheric pressure. As already known, the spore germination is more efficient at low pressure level than at high pressure level. Our results show that this low germination efficiency at high pressure seemed not to be related either to a lower induction or a difference in the induction mechanisms but rather to an inhibition of enzyme activities which are involved in germination process. In fact, high pressure was necessary and very efficient in inducing spore germination. However, it seemed to slow the enzymatic digestion of the cortex, which is required for germinated spores to be inactivated by pressure. Although these results indicate that high‐pressure treatments are more efficient when the two treatments are combined, a small spore population still remained dormant and was not inactivated with any holding time or pressure level. Biotechnol. Bioeng. 2010;107: 876–883. © 2010 Wiley Periodicals, Inc.     

Analysis of the slow germination of multiple individual superdormant Bacillus subtilis spores using multifocus Raman microspectroscopy and differential interference contrast microscopy

Aim: To analyse the dynamic germination of hundreds of individual superdormant (SD) Bacillus subtilis spores. Methods and Results: Germination of hundreds of individual SD B. subtilis spores with various germinants and under different conditions was followed by multifocus Raman microspectroscopy and differential interference contrast microscopy for 12 h and with temporal resolutions of ≤30 s. SD spores germinated poorly with the nutrient germinant used to isolate them and with alternate germinants targeting the germinant receptor (GR) used originally. The mean times following mixing of spores and nutrient germinants to initiate and complete fast release of Ca‐dipicolinic acid (CaDPA) (T_lag and T_release times, respectively) of SD spores were much longer than those of dormant spores. However, the ΔT_release times (T_release?T_lag) of SD spores were essentially identical to those of dormant spores. SD spores germinated almost as well as dormant spores with nutrient germinants targeting GRs different from the one used to isolate the SD spores and with CaDPA that does not trigger spore germination via GRs. Conclusions: Since (i) ΔT_release times were essentially identical in GR‐dependent germination of SD and dormant spores; (ii) rates of GR‐independent germination of SD and dormant spores were identical; (iii) large increases in T_lag times were the major difference in the GR‐dependent germination of SD as compared with spores; and (iv) higher GR levels are correlated with shorter T_lag times, these results are consistent with the hypothesis that low levels of a GR are the major reason that some spores in a population are SD with germinants targeting this same GR. Significance and Impact of the Study: This study provides information on the dynamic germination of individual SD spores and improves the understanding of spore superdormancy.