Journal of Applied Microbiology ISSN 1364-5072

ORIGINAL ARTICLE

Bacillus endospore resistance to gas dynamic heating

S.D. Gates1, A. Daniel McCartt1, P. Lappas1, J.B. Jeffries1, R.K. Hanson1, L.A. Hokama2 and K.E. Mortelmans2
1 Mechanical Engineering Department, Stanford University, Stanford, CA, USA 2 Microbiology Department, SRI International, Menlo Park, CA, USA

Keywords Bacillus spores, ow cytometry, heat resistance, sporeshock wave interaction. Correspondence Sean D. Gates, Stanford University, Building 520, 452 Escondido Mall, Stanford, CA 94305-3032, USA. E-mail: sdamien@stanford.edu

Abstract Aim: To develop a novel laboratory procedure for the study of shock waveinduced damage to Bacillus endospores. Methods and Results: Bacillus atrophaeus endospores are nebulized into an aerosol, loaded into the stanford aerosol shock tube and subjected to shock waves of controlled strength. Endospores experience uniform test temperatures between 500 and 1000 K and pressures ranging from 2 to 7 atm, for a relatively short time (23 ms). During this process, the bioaerosol is observed using in situ laser absorption and scattering diagnostics. Additionally, shock-treated samples are extracted for ex situ analysis including viability plating, ow cytometry and SEM imaging. Measurements indicate that endospores lose the ability to form colonies when heated to test temperatures above 500 K while signicant breakdown in morphology is observed at test temperatures above 750 K. Conclusion: These results demonstrate the disruption of essential biochemical pathways or biomolecules prior to the onset of signicant endospore morphological deterioration. Signicance and Impact of the Study: This novel laboratory approach to study the interaction of endospores with shock waves provides an experimental means to investigate the mechanisms of endospore resistance to rapid heating. In addition, this methodology allows for the direct simulation of a blast wave bioaerosol interaction in an atmospheric environment.

2010 0206: received 3 February 2010, revised 26 April 2010 and accepted 1 June 2010
doi:10.1111/j.1365-2672.2010.04785.x

Introduction The process of Bacillus sporulation forms extremely durable, metabolically dormant endospores, which have been shown to be highly resistant to a multitude of treatments. In the past, studies have focused upon the endospore response to stress mechanisms such as chemical treatments, moist and dry heat, and radiation (Nicholson et al. 2000; Setlow 2006; Coleman et al. 2007). In this study, we are exposing a Bacillus endospore laden aerosol to gas dynamic shock waves. During this process, the spore-laden aerosol encounters an effectively instantaneous increase in temperature and pressure as well as substantial acceleration forces produced by the passage of the shock wave. The acceleration rates and pressure changes seen in this study fall well below the lethal levels

cited in the literature for each effect (Lundbeck and Skoldberg 1963; Sislian et al. 2009). However, endospore response to very short time exposure to extreme temperatures coupled with minor pressure changes and acceleration forces is a heretofore unexplored subject. Furthermore, the ability to ne tune the shock conditions offers an empirical method to investigate theorized biological mechanisms of spore resistance to heat. Bacillus atrophaeus endospores of the strain (ATCC#9372) also referred to as Bacillus subtilis var. niger (American Type Culture Collection, Manassas, VA, USA) were used for this study. Aqueous B. atrophaeus-laden aerosols were loaded into the shock tube and subjected to shock waves of controlled strength. This process was monitored with in situ laser absorption and scattering diagnostics. Shock-treated samples were also collected for
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ex situ analysis. Samples were analysed for viability via plating, enumerated via ow cytometry, and the morphology observed via scanning electron microscopy (SEM). The new experimental method is rst described; including details concerning spore harvesting, aerosol production and the in situ and ex situ diagnostics. We then investigate the inuence of postshock temperature (T5) over the range from 450 to 1000 K. The results indicate three distinct stages of shock wave-induced endospore damage with inactivation occurring before morphological deterioration. Materials and methods Stanford aerosol shock tube The stanford aerosol shock tube (SAST) facility (Davidson et al. 2008) provides a laboratory environment to expose aerosols containing bacterial spores to shock waves of controlled strength with real-time in situ laser monitoring and sampling of the shock-treated spores for ex situ evaluation. The SAST consists of a driver section and a driven section separated with a diaphragm. To limit the diffusive transport of the bacterial aerosol away from the laser interrogation location, the driven section is divided with a sliding gate valve into two regions: an upstream section and a test section near the endwall. As illustrated in Fig. 1, the upstream section is lled with bath gas and the test section lled with a mixture of bath gas and aerosol produced by a pneumatic nebulizer from an aqueous spore suspension. After the two driven sections are loaded, the endwall poppet valves are closed, the gate valve is opened, and the driver section is pres-

surized. The resulting pressure differential between the driven and the driver section causes the diaphragm to rupture producing a planar shock wave that propagates into the driven section. The wave pattern generated by the shock event and the time evolution of the gas temperature and pressure observed at the laser diagnostic port 5 cm from the endwall are illustrated in Figs 2 and 3, respectively (Emanuel 2001). When the incident shock arrives at the measurement station (dened as t = 0), the preshock (T1, P1) gas aerosol mixture is abruptly heated and compressed (to T2, P2); when the shock wave reected by the endwall arrives a second time at the measurement port, the gas is once again heated and compressed (to T5, P5). For these experiments, the test conditions (T5 and P5) persist for 23 ms; the temperature and pressure then decay in c. 50 ms through a variety of expansion processes to room temperature and a resting pressure above atmospheric. Spore production, SAST loading and spore sampling To prepare the B. atrophaeus endospores used in our tests, we rst induced the sporulation of the bacterial strain by growth to exhaustion in Difco sporulation medium at 37C (Harwood and Cutting 1990). The spore suspension was puried through ddH2O washing. Subsequent incubation with 1 mg ml)1 lysozyme for 1 h at 37C and the addition of SDS (c. 1% nal concentration) for 20 min were implemented to eliminate vegetative cell debris (Harwood and Cutting 1990). The resulting suspension was once again washed with ddH2O until only phasebright spores were observed. Aqueous spore suspensions were subsequently concentrated to 1010 spores ml)1.

Wavelength-multiplexed NIR lasers (~14 m) for H2O absorption Diaphram Visible laser (660 nm) for aerosol scattering Ultraviolet laser (266 nm) for spore rupture products

Shockwave

High pressure driver gas

Bath gas

Bath gas + Aerosol

Endwall valves

Sliding gate valve Pump Impinger sampling Pump

BGI nebulizer with aqueous spore & SiO2 bead suspension

Wavelength dispersion with separate detectors for each laser

Figure 1 Schematic of the driven section of the stanford AST facility congured for shock-heating spore-laden aerosol. Three in situ laser diagnostics, 5 cm from the endwall, monitor the shock-heated gas aerosol, while pre- and postshock samples are taken with gas dynamic impingers for ex situ analysis. (Not drawn to scale).
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Contact surface
Time

Reflected shock wave

Reflected R expansion fan 3

R5 R2

Expansion fan

R4 Shock tube

Shock wave

R1 Position

High pressure region Diaphragm

Low pressure region Sliding gate valve Measurement location

Figure 2 Wave pattern generated by the diaphragm rupture in the stanford aerosol shock tube. Region 1 is the preshock arrival gas state. When the shock wave arrives, a step change in pressure and temperature occurs (Region 2). The passage of the reected shock wave once again compresses and heats the gas (Region 5) producing a stagnant ow region of high temperature and pressure.

Temperature (K)

400 5 200 0 0 1 2 3 Time (ms) 4 5 0 6

Figure 3 Theoretical time evolution of shock heating of gas observed at the measurement location of Fig. 1. The step change in gas temperature (solid line) and pressure (dashed line) from the incident shock (Region 12) and from the reected shock (Region 25) is observed. Uniform test conditions persist for 23 ms until a variety of processes result in the decay to room temperature and an elevated resting pressure (entire decay not shown here).

The spore-laden aqueous aerosol is produced from the B. atrophaeus spore suspension with a commercial, gas dynamic nebulizer (6-jet Collison type; BGI Inc., Waltham, MA). Tests conducted in our laboratory have demonstrated that spore-laden aerosols generated using this nebulizer do not exhibit a signicant loss of viability or severe damage to the morphological structure. The spore-laden aerosol is subsequently introduced into the test section of the SAST through poppet valves in the endwall as shown in Fig. 1. The poppet valve conguration has been optimized to enhance turbulent mixing to maximize the uniformity of the aerosol mixture in the test section. Additionally, laser scattering and absorption data indicate that the spore density of the two-phase bioaerosol in the test section is on the order of 106 spores cm)3 with a H2O mole fraction of between 3 and 7%. Spore samples for ex situ analysis are vacuumextracted with a commercial (Ace Glass Inc., Vineland,

Pressure (atm)

600

10

NJ) gas dynamic liquid impinger from the sealed bioaerosol test section of the SAST before and after the shock event. The ow of aerosol through the impinger is directed onto a 10 ml pool of water. Any particulate in the ow impacts the water and is captured, while the gas ow turns and is extracted through the vacuum. Controlled experiments conrm that the collection with the impinger alone does not reduce the spore viability nor produce morphological damage to the B. atrophaeus spores. Two impingers are attached to the test region of the SAST as illustrated in Fig. 1. We collect a small preshock sample with the upstream impinger after the test region of the shock tube is lled, just prior to opening the sliding gate valve. The downstream impinger is used to collect a postshock sample from the resealed test section at the conclusion of the experiment. All sampling equipment is sterilized with an All American model 75 electric pressure steam autoclave between tests, and the shock tube is sterilized with high-temperature shocks in pure oxygen. The collected aqueous spore suspension in the impinger is then analysed with three different ex situ methods: nutrient agar (NA) plating, ow cytometry and scanning electron microscopy. Ex situ analysis One hundred microlitre samples from a series of dilutions of the preshock and postshock suspensions are plated on NA plates for viable counts. After overnight incubation at 29C, colonies are counted. The plates then continue to incubate at room temperature for another 48 h to insure the colony count has stabilized. For all experiments, an inoculum control was tested. In addition, each sample was plated ve times, at ve dilutions, to produce a statistical distribution of the viability results.
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Flow cytometry measurements serve to enumerate the particles in the pre- and postshock samples. A four-laser BD Biosciences, model LSR II with laser excitation at 405, 488, 532 and 635 nm, was employed to generate particle counts using forward and side scatter. Endospore resistance to shock waves is subsequently quantied by combining the results of the plating and ow cytometry analysis. Viable fraction (Vf) is dened as the fraction of the initially viable spores that remain viable after shock exposure. For the experiments with T5 < 750 K, the spore morphology remains largely unaltered, and spores in the collected samples can be directly counted by ow cytometry. For these shock treatments, the viable fraction Vf is given by: Vf Sp SFC Pv 1

Equation 1: Viable Fraction for low to moderate temperature shocks SP is the number of viable spores per ml in the postshock sample measured by plating. SFC is the number of intact spores per ml in the postshock sample determined by ow cytometer, and Pv is the fraction of viable spores in the preshock sample (typically 8095%). For T5 > 750 K, the spore morphology for a signicant number of spores is destroyed, and the collected spores cannot be fully counted by the ow cytometer. Thus, we add monodisperse one-micron SiO2 beads that are undamaged over the full range of T5 to the spore suspension at a known spore silica bead ratio (R). The viable fraction for these higher temperature shock experiments is given by: Vf Sp SiFC R Pv 2

Equation 2: Viable Fraction for all temperature shocks. We are able, therefore, to deduce the number of shocktreated spores from the ow cytometry count of the silica beads (SiFC) and the known spore silica bead ratio (R). The viable fraction can then be determined as per Eqn 2. For experiments with low and moderate values of T5, Eqn 2 can also be used, and the viable fraction determined is in excellent agreement with direct spore counting (Eqn 1). These redundant results give us condence that the SiO2 beads can be used to determine the viable fraction for the high-temperature shock experiments where the spore morphology is destroyed. Coupled with the aforementioned quantication techniques, we employ two additional qualitative methods that provide pertinent information regarding postshock
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spore morphological structure: SEM analysis of pre- and postshock samples and ow cytometer measurements of dye-induced spore uorescence. SEM micrographs are used to qualitatively characterize the morphology of shock-treated and untreated spores. The scanning electron microscope employed in this study, Hitachi model S-3400N VP, has a large analytical chamber with a ve-axis, computer eucentric stage and can be operated in the variable vacuum conditions needed for the analysis of aqueous spore suspensions. Portions (50 ll) of the impinger samples are ltered (022-lm cellulose; Millipore, Billerica, MA, USA), air-dried and mounted on double-sided carbon tape on aluminium stubs (Electron Microscopy Sciences, Hateld, PA, USA). Portions (1 ml) of some of the samples are centrifuged (2300 g for 5 min) to concentrate the collected material, and 50 ll subsamples from the tip of the Eppendorf tube are air-dried on carbon tape attached to aluminium stubs. All stubs were thereafter sputter-coated (100 A layer) with gold-palladium in a Denton Vacuum Desk II unit and visualized with secondary electron detection with the SEM, operated under high vacuum at 10 and 15 kV at a working distance of 89 mm. Images (2560 1920 pixels) were captured in TIF format using Quartz PCI software (Vancouver, BC, Canada). An example of a SEM image is shown in Fig. 4. Qualitative assessment of the SEM images provides signicant information regarding the onset of spore morphological breakdown. As Fig. 4a demonstrates, untreated spores are uniformly shaped and not ruptured. In the image of spores shock heated with T5 = 720 K (Fig. 4b), the endospore structure appears compromised and the presence of lysate material is apparent. The inclusion of propidium iodide (PI) uorescent dye in the spore samples allows for the interrogation of intact but morphologically damaged spores by ow cytometry. Generally, nucleic acid stains such as PI are weak binding agents for spores because of the connement of the nucleic acids to the spore core (Gould 1984; Setlow et al. 2002). It has been suggested, however, that reagents of this type will bind to the proteinaceous spore coat, the peptidoglycan cortex (Melly et al. 2002) and an insoluble shell below the proteinaceous coat termed a rind (Klobutcher et al. 2006). Because of the aforementioned considerations concerning the nonspecic adsorption of PI dye, conclusions regarding the integrity of the spore morphology drawn from the ow cytometry uorescence signals will be ambiguous. For the purposes of our analysis, however, we are interested in examining the relative change in uorescence signal levels between the pre- and postshock samples. Figure 5 illustrates ow cytometer results of a pre- and postshock sample for T5 $ 500 K. The image in Fig. 5a is from a preshock sample, with the

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(a)

(b)

examine the postshock sample in Fig. 5b, we note a substantial increase in the level of spore uorescence. In the light of the earlier considerations, this observed phenomenon implies two areas of potential spore morphological damage. Spore interaction with the shock wave may have induced the deterioration of the proteinaceous coat and subsequently increased the exposure of the rind or spore cortex to the dye molecules; additionally, the permeability of the inner membranes may have been compromised leading to the introduction of the PI dye directly into the spore core where substantial staining of the nucleic acids would occur. In all likelihood, both of these potential damage mechanisms are interrelated and exist to varying degrees in the postshock samples. While the nature of the biological components involved in the adsorption of the PI dye is an interesting consideration, what is more readily apparent, and important for our present purposes, is the relative change in uorescence signals from the preshock sample to the post. In the example illustrated in Fig. 5, it is clear that the passage of the shock wave through the bioaerosol-induced substantial deterioration of the morphological structure of the intact spore allowing increased adsorption of the PI dye, extending, perhaps, to the nucleic acid laden core. In situ analysis Three laser diagnostics (visible extinction, UV extinction and near-infrared absorption of water vapour) (Davidson and Hanson 2001) were used for in situ measurements of shock-heated spores as illustrated in Fig. 1. A separate paper, in preparation, will provide details on the in situ diagnostics; however, a brief summary is given here as the light scattering data are important

Figure 4 (a) SEM image of untreated spore suspension. (b) SEM image of shock-heated spores with T5 = 720 K, where morphological damage and lysate material are apparent. Silica beads remain undamaged.

two regions representing viable spores labelled I and II. The presence of two fairly distinct regions in the untreated spore sample may be the result of the nonspecic adsorption of dye previously addressed. If we then

(a) 105
Forward scatter

Pre shock

(b) 105

Post shock T5 ~ 500 K

II

103 BA Spores 102 101 101 102 103 104 Fluorescence 105

Forward scatter

104

104 103 102 101 101 102 103 104 Fluorescence 105

Figure 5 Flow cytometry data. Image a is of a preshock sample clearly showing two distinct spore regions with differential adsorption of the PI dye. Image b is of a postshock sample tested at 520 K. The spore region has shifted to the right indicating increased dye adsorption and morphological damage.
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for our interpretation of the morphological breakdown of the shock-treated endospores. Time-resolved optical extinction measurements of the shock-heated bioaerosols at 266 and 665 nm are taken. A double-pass optical set-up was used for the measurements to optimize the signal-to-noise ratio. In addition to scattering by the aerosol, the UV light at 266 nm is strongly absorbed by UV active endospore biochemicals (e.g. dipicolinic acid (DPA) and nucleic acids) (Nudelman et al. 2000). The visible light at 665 nm is only scattered by the bioaerosol. Thus, the postshock decay of 665-nm extinction provides a time history for the destruction of the spore morphology, while the UV light at 266 nm indicates the release of spore lysate. For low postshock temperatures (T5), where the spores remain intact, both extinction signals undergo step changes in density as the shock waves pass through the optical beam path. For hightemperature cases, decay in the visible optical extinction data from the shock-heated spores shows evidence of spore morphology destruction, while the maintained level of optical extinction at 266 nm indicates the presence of spore lysate. Water vapour is monitored in the SAST using two lasers tuned to different absorption transitions near 13433 and 13917 nm (Baer et al. 1996). Each of the lasers is scanned in wavelength over the absorption feature, and the two NIR wavelengths are separated by a diffraction grating onto separate detectors. The two transitions have different lower state energies, and thus their absorption has different temperature dependence. The gas temperature is calculated from the absorption ratio. Rapid modulation of the lasers provides 50-ls time resolution of the dynamic processes. Water absorption measurements from region 2 and the measured shock speed are used to iteratively solve for the temperature and gas composition in all three regions (1, 2 and 5). Results Quantitative analysis of shock-heated spores was performed using the procedures and protocols described previously. Pre- and postshock samples were taken for each test and subjected to viability analysis and ow cytometry quantication. The results indicate a dramatic decrease in the postshock viable fraction as a function of the postshock temperature. As is apparent from Fig. 6, the viable fraction decreases with temperature in a roughly linear manner on a semilog plot. Good agreement is noted between the measurements taken with the silica beads and those taken with direct spore counts. In addition, a test conducted with a postshock temperature of 1018 K yielded a viable fraction
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101 100 Viable fraction 101 102 103 104 105 106

400

500

600 700 800 900 Post shock temperature (K)

1000

Figure 6 Viable fraction as a function of postshock temperature. The circles are data points calculated using direct ow cytometer spore counts (Eqn 1). Triangles are data points calculated using silica bead ow cytometer counts (Eqn 2).

of zero, providing an upper temperature bound to spore viability in these harsh conditions. While Fig. 6 provides interesting results regarding endospore response to shock heating, it fails to elucidate some of the more subtle interactions that we believe are occurring. In particular, with the use of the laser diagnostic and ow cytometry data, we are able to investigate the endospore morphological response to shock-heated ows. In Fig. 7, the triangles represent the viable fraction data seen on the semi-log plot in Fig. 6. The circles represent the percentage of spores that retain their morphological structure as determined by the in situ laser diagnostics. As demonstrated previously, viable fraction levels are near

100 80 Increased PI dye uptake 60 % 40 20 0 300 400 500 600 700 800 Post shock temperature (K) 900 1000 01% 001% 0001%
Figure 7 Viable fraction and intact spore structure percentage as a function of postshock temperature. Circles represent the percentage of intact spores, while the triangles indicate the viable fraction.

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zero at postshock temperatures above 500 K. However, the visible laser diagnostic maintained a high level of extinction indicating little to no endospore break-up below 750 K. These data suggest that for temperature treatment between 500 and 750 K, the majority of spores remain intact but are no longer viable. This indicates a mechanism of spore inactivation that does not signicantly alter the endospores overall morphological structure. Temperatures above this regime lead to total spore morphology breakdown, as determined with ow cytometry and conrmed by both visible laser scattering and UV laser absorption of lysate material. While the overall morphological structure of the spore may be largely undamaged in the postshock temperature range of 500750 K, the increased PI dye uptake demonstrated at a T5 value of c. 520 K indicates the possible onset of spore coat deterioration. Discussion Considering the body of results summarized in Figs 6 and 7, we propose a three-stage framework for the response of endospores exposed to shock-heated ows: i Weak shocks (T5 < 500 K) cause minimal morphological damage to the spores and a signicant fraction of the collected postshock spores remain viable. ii Moderate shocks (T5 between 500 and 750 K) cause a rapid rate of decline in viability and a corresponding but signicantly slower rate of severe spore morphological breakdown. iii Strong shocks (T5 > 750 K) cause a near complete loss of viability and the majority of spores experience complete morphological deterioration coupled with the rapid release of lysate. This qualitative trend has been observed in all of our tests. As detailed in the second stage of the endospore response framework, the existence of a rate differential between the loss of viability and morphological deterioration has led to the conclusion that essential biological mechanisms or biomolecules are being disrupted prior to major endospore structural damage. Possible mechanisms include the damaging of essential proteins, nucleic acids and the endospore coat. In an attempt to further elucidate the biological pathways being disrupted, we plan on extending our studies to the strains B. subtilis PS533 and Bacillus thuringiensis. Not only would this enable us to examine the effect of differing exosporiums on the ability of spores to retain viability but it would also create the opportunity to perform measurements on genetically modied spores to aid in the identication of proteins that may be critical to shock wave spore resistance. Additionally, specic mutants, such as ones decient in DPA, may be employed to aid in the

determination of the specic components of the UV absorbing lysate. The methodology and experiments described in this paper illustrate a new laboratory protocol for the study of interactions of endospores with shock blast waves. Acknowledgements This work is supported by the Defense Threat Reduction Agency via grant AB07TAS014, administered by the Army Research Ofce under grant 51532CHCBB with Dr Jennifer Becker of the Chemical Sciences Division as contract monitor. We would also like to acknowledge the assistance of Dr Lydia Joubert of the Cell Sciences Imaging Facility at Stanford University, Drs David Parks and Marty Bigos of the Stanford Shared FACS Facility and Drs Steve Biller and Bill Bunkholder of Stanford University. References
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Setlow, B., Loshon, C.A., Genest, P.C., Cowan, A.E., Setlow, C. and Setlow, P. (2002) Mechanisms of killing of spores of Bacillus subtilis by acid, alkali and ethanol. J Appl Microbiol 92, 362375. Sislian, P.R., Zhang, X., Li, M., Pham, D., Madler, L. and Christodes, P.D. (2009) Bacterial aerosol neutralization by aerodynamic shocks using a novel impactor system: design and computation. Chem Eng Sci 64, 19531967.

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