The authors thank Andrea Schr&#246;der for her excellent technical assistance during parts of this work and Nikea J. Ulrich for her assistance during the video shoot.&#160;We would also like to thank Lyle A. Simmons for his generous donation of the Bacillus subtilis strains: LAS72 and LAS24. This work was supported in parts by grants from the German Research Foundation (DFG) Paketantrag (PlasmaDecon PAK 728) to PA (AW 7/3-1) and RM (MO 2023/2-1) and the DLR grant DLR-FuW-Projekt ISS LIFE, Programm RF-FuW, Teilprogramm 475 (to F.M.F, M.R. and R.M.). F.M.F. was supported by a PhD scholarship of the Helmholtz Space Life Sciences Research School (SpaceLife) at the German Aerospace Center (DLR) in Cologne, Germany, which was funded by the Helmholtz Association (Helmholtz-Gemeinschaft) over a period of six years (Grant No. VH-KO-300) and received additional funds from the DLR, including the Aerospace Executive Board and the Institute of Aerospace Medicine. The results of this study will be included in the Ph.D. thesis of Felix M. Fuchs.

1. Bacillus subtilis Spore Production and Purification
<ol>
	<li>For spore production, transfer a 5 mL overnight culture of the respective B. subtilis strain, supplemented with appropriate antibiotics, to 200 mL double-strength liquid Schaeffer sporulation medium (per liter 16 g nutrient broth, KCl 2 g, 0.5 g MgSO4*7 H2O, 2 mL 1 M Ca(NO3)2, 2 mL 0.1 M MnCl2 *&#8226; 4 H2O, 2 mL 1 mM FeSO4, 2 mL 50% (w/v) glucose<sup class="x...

<ol>
	<li>Nicholson, W. L., Schuerger, A. C., Race, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Migrating+microbes+and+planetary+protection.">Migrating microbes and planetary protection.</a> Trends Microbiol. 17, 389-392 (2009).</li><li>COSPAR. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=COSPAR+Planetery+Protection+Policy.">COSPAR Planetery Protection Policy.</a> Space Research Today, COSPAR's Information Bulletin. 193, 1-14 (2015).</li><li>De Geyter, N., Morent, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nonthermal+plasma+sterilization+of+living+and+nonliving+surfaces.">Nonthermal plasma sterilization of living and nonliving surfaces.</a> Annu Rev Biomed Eng. 14, 255-274 (2012).</li><li>Shimizu, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cold+atmospheric+plasma+-+A+new+technology+for+spacecraft+component+decontamination.">Cold atmospheric plasma - A new technology for spacecraft component decontamination.</a> Planet. Space Sci. 90, 60-71 (2014).</li><li>Lerouge, S., Fozza, A. C., Wertheimer, M. R., Marchand, R., Yahia, L. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sterilization+by+Low-Pressure+Plasma:+The+Role+of+Vacuum-Ultraviolet+Radiation.">Sterilization by Low-Pressure Plasma: The Role of Vacuum-Ultraviolet Radiation.</a> Plasma Polym. 5, 31-46 (2000).</li><li>Rossi, F., Kyli&#225;n, O., Rauscher, H., Gilliland, D., Sirghi, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+a+low-pressure+plasma+discharge+for+the+decontamination+and+sterilization+of+medical+devices.">Use of a low-pressure plasma discharge for the decontamination and sterilization of medical devices.</a> Pure Appl. Chem. 80, 1939-1951 (2008).</li><li>Halfmann, H., Hauser, J., Awakowicz, P., Koller, M., Esenwein, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+double+inductively+coupled+low-pressure+plasma+for+sterilization+of+medical+implant+materials.">A double inductively coupled low-pressure plasma for sterilization of medical implant materials.</a> Biomed Tech (Berl). 53, 199-203 (2008).</li><li>Halfmann, H., Denis, B., Bibinov, N., Wunderlich, J., Awakowicz, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+the+most+efficient+VUV/UV+radiation+for+plasma+based+inactivation+of+Bacillus+atrophaeus+spores.">Identification of the most efficient VUV/UV radiation for plasma based inactivation of Bacillus atrophaeus spores.</a> J. Phys. D: Appl. Phys. 40, 5907 (2007).</li><li>Vaishampayan, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bacillus+horneckiae+sp.+nov.,+isolated+from+a+spacecraft-assembly+clean+room.">Bacillus horneckiae sp. nov., isolated from a spacecraft-assembly clean room.</a> Int J Syst Evol Microbiol. 60, 1031-1037 (2010).</li><li>Mandic-Mulec, I., Stefanic, P., van Elsas, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ecology+of+Bacillaceae.">Ecology of Bacillaceae.</a> Microbiol Spectr. 3, (2015).</li><li>Alekhova, T. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diversity+of+bacteria+of+the+genus+Bacillus+on+board+of+international+space+station.">Diversity of bacteria of the genus Bacillus on board of international space station.</a> Dokl Biochem Biophys. 465, 347-350 (2015).</li><li>Claus, D., Bekerley, R. C. W., Sneath, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genus+Bacillus+Cohn+1872.">Genus Bacillus Cohn 1872.</a> Bergey's manual of systematic bacteriology. 2, 1105-1141 (1986).</li><li>Setlow, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spore+Resistance+Properties.">Spore Resistance Properties.</a> Microbiol Spectr. 2, (2014).</li><li>Setlow, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spores+of+Bacillus+subtilis:+their+resistance+to+and+killing+by+radiation,+heat+and+chemicals.">Spores of Bacillus subtilis: their resistance to and killing by radiation, heat and chemicals.</a> J Appl Microbiol. 101, 514-525 (2006).</li><li>Roth, S., Feichtinger, J., Hertel, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+Bacillus+subtilis+spore+inactivation+in+low-pressure,+low-temperature+gas+plasma+sterilization+processes.">Characterization of Bacillus subtilis spore inactivation in low-pressure, low-temperature gas plasma sterilization processes.</a> J Appl Microbiol. 108, 521-531 (2010).</li><li>Roth, S., Feichtinger, J., Hertel, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Response+of+Deinococcus+radiodurans+to+low-pressure+low-temperature+plasma+sterilization+processes.">Response of Deinococcus radiodurans to low-pressure low-temperature plasma sterilization processes.</a> J Appl Microbiol. 109, 1521-1530 (2010).</li><li>Setlow, B., Setlow, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+DNA+repair+in+Bacillus+subtilis+spore+resistance.">Role of DNA repair in Bacillus subtilis spore resistance.</a> J Bacteriol. 178, 3486-3495 (1996).</li><li>Schaeffer, P., Millet, J., Aubert, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Catabolic+repression+of+bacterial+sporulation.">Catabolic repression of bacterial sporulation.</a> Proc. Natl. Acad. Sci. 54, 704-711 (1965).</li><li>Simmons, L. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+responses+to+double-strand+breaks+between+Escherichia+coli+and+Bacillus+subtilis+reveals+different+requirements+for+SOS+induction.">Comparison of responses to double-strand breaks between Escherichia coli and Bacillus subtilis reveals different requirements for SOS induction.</a> J Bacteriol. 191, 1152-1161 (2009).</li><li>Raguse, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improvement+of+Biological+Indicators+by+Uniformly+Distributing+Bacillus+subtilis+Spores+in+Monolayers+To+Evaluate+Enhanced+Spore+Decontamination+Technologies.">Improvement of Biological Indicators by Uniformly Distributing Bacillus subtilis Spores in Monolayers To Evaluate Enhanced Spore Decontamination Technologies.</a> Appl Environ Microbiol. 82, 2031-2038 (2016).</li><li>Horneck, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protection+of+bacterial+spores+in+space,+a+contribution+to+the+discussion+on+Panspermia.">Protection of bacterial spores in space, a contribution to the discussion on Panspermia.</a> Orig Life Evol Biosph. 31, 527-547 (2001).</li><li>Opretzka, J., Benedikt, J., Awakowicz, P., Wunderlich, J., Keudell, A. v. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+chemical+sputtering+during+plasma+sterilization+of+Bacillus+atrophaeus.">The role of chemical sputtering during plasma sterilization of Bacillus atrophaeus.</a> J. Phys. D: Appl. Phys. 40, 2826 (2007).</li><li>Stapelmann, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Utilization+of+low-pressure+plasma+to+inactivate+bacterial+spores+on+stainless+steel+screws.">Utilization of low-pressure plasma to inactivate bacterial spores on stainless steel screws.</a> Int. J. Astrobiol. 13, 597-606 (2013).</li><li>Raguse, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Understanding+of+the+importance+of+the+spore+coat+structure+and+pigmentation+in+the+Bacillus+subtilis+spore+resistance+to+low-pressure+plasma+sterilization.">Understanding of the importance of the spore coat structure and pigmentation in the Bacillus subtilis spore resistance to low-pressure plasma sterilization.</a> J. Phys. D: Appl. Phys. 49, 285401 (2016).</li><li>Pandey, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Live+cell+imaging+of+germination+and+outgrowth+of+individual+Bacillus+subtilis+spores;+the+effect+of+heat+stress+quantitatively+analyzed+with+SporeTracker.">Live cell imaging of germination and outgrowth of individual Bacillus subtilis spores; the effect of heat stress quantitatively analyzed with SporeTracker.</a> PloS one. 8, e58972 (2013).</li></ol>

Sterilization of surfaces using low-temperature, low-pressure plasma is a promising alternative to rather conventional sterilization procedures such as treatment with ionizing radiation, chemicals (e.g. gases like H2O2 or ethylene oxide) or dry and moist heat23. Ordinary sterilization methods mostly provide an effective sterilization, but they are known to influence the treated material and represent a potential risk for the operator. Low-pressure plasma offers a rap...

A major goal of space exploration is the search for signatures of life forms and biomolecules on other planetary bodies and moons in our solar system. The transfer of microorganisms or biomolecules of terrestrial origin to critical areas of exploration is of particular risk to impact the development and integrity of life-detection missions on planetary bodies such as Mars and Europa1. The international guidelines of planetary protection, established by the Committee of Space Research (COSPAR) in 1967, impose strict regulations on manned and robotic missions to other planets, their moons, asteroids, and other celestial bodies and regulate the cleaning and sterilization of a spacecraft and critical hardware components prior to launch in order to eliminate contaminating terrestrial microorganisms and prevent cross contamination of celestial bodies2. Over the last decade, the application of non-thermal plasmas has gained wide attention in biomedical and nutritional research, as well as in spaceflight applications3,4,5. Plasma sterilization is a promising alternative to conventional sterilization methods as it offers rapid and efficient microbial inactivation6, while being gentle to sensitive and heat labile materials. Plasma discharges contain a mixture of reactive agents such as free radicals, charged particles, neutral/excited atoms, photons in the ultraviolet (UV), and vacuum ultraviolet (VUV) spectrum which lead to rapid microbial inactivation3. In this study, we use low-pressure plasma generated by double inductively coupled low-pressure plasma (DICP) source7,8 to inactivate Bacillus subtilis endospores distributed on glass test surface.
Gram-positive bacteria of the family Bacillaceae are widely distributed in natural habitats of soil, sediments, and air as well as in unusual environments such as clean room facilities and the International Space Station9,10,11. The most distinct feature of the genus Bacillus is the ability to form highly resistant dormant endospores (hereafter referred to as spores) to survive unfavorable conditions, such as nutrient depletion12. Spores are generally much more resistant than their vegetative cell counterparts to a variety of treatments and environmental stresses, including heat, UV, gamma irradiation, desiccation, mechanical disruption, and toxic chemicals, such as strong oxidizers or pH-changing agents (reviewed in references13,14) and are therefore ideal objects for testing the efficiency of microbial inactivation methods. Since genomic DNA is a major target of the plasma treatment of bacteria15,16, the repair of plasma-induced DNA lesions (e.g. DNA double strand breaks) upon spore revival is crucial for survival of bacteria13,17.
Thus, we study the germination capacity of spores and the role of DNA repair during spore germination and outgrowth after treating the spores with low pressure argon plasma by following individual spores and their expression of fluorescence-labelled DNA repair protein RecA with time-resolved confocal fluorescence microscopy. We give a step by step instruction of the preparation of B. subtilis spores in monolayers for achieving reproducible test results, the treatment of spore monolayers with low pressure plasma for sterilization, the preparation of plasma treated spores for ultrastructural evaluation using scanning electron microscopy (SEM), and live cell microscopy analysis at the level of individual spores in concert with monitoring the active DNA repair processes occurring within the cell in response to plasma treatment.

<table>
	<tbody>
		<tr>
			<td>Two substance nozzle (model 970-8)</td>
			<td>Schlick</td>
			<td>14,404</td>
			<td>230 V, 50 Hz, D 4.484/8, 0.8 mm bore diameter</td>
		</tr>
		<tr>
			<td>Luria Bertani Medium</td>
			<td>Sigma Aldrich</td>
			<td>70122-100G</td>
			<td></td>
		</tr>
		<tr>
			<td>Tube connectors</td>
			<td>Festo</td>
			<td>n/a</td>
			<td>G 1/8</td>
		</tr>
		<tr>
			<td>Magnetvalve DO35-3/2NC-G018-230AC</td>
			<td>Bosch Rexroth</td>
			<td>820005100</td>
			<td></td>
		</tr>
		<tr>
			<td>PLN Polyamid tube</td>
			<td>Festo</td>
			<td>558206</td>
			<td>d = 6 mm</td>
		</tr>
		<tr>
			<td>Glass slides</td>
			<td>VWR</td>
			<td>48300-026</td>
			<td></td>
		</tr>
		<tr>
			<td>Electric Timer 550-2-C</td>
			<td>Gefran</td>
			<td>F000074</td>
			<td>220 V</td>
		</tr>
		<tr>
			<td>attofluor cell chamber</td>
			<td>Menzel, Fisher Ref.</td>
			<td>3406816</td>
			<td>d=25 mm, round</td>
		</tr>
		<tr>
			<td>MgSO4*7 H2O</td>
			<td>Sigma Aldrich</td>
			<td>13152</td>
			<td></td>
		</tr>
		<tr>
			<td>Ca(NO3)2</td>
			<td>Sigma Aldrich</td>
			<td>202967</td>
			<td></td>
		</tr>
		<tr>
			<td>MnCl2 * 4 H2O</td>
			<td>Sigma Aldrich</td>
			<td>244589</td>
			<td></td>
		</tr>
		<tr>
			<td>FeSO4 * 7H2O</td>
			<td>AppliChem</td>
			<td>13446-34-9</td>
			<td></td>
		</tr>
		<tr>
			<td>Glucose</td>
			<td>Merck</td>
			<td>215422</td>
			<td></td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>Sigma Aldrich</td>
			<td>P9541-500G</td>
			<td></td>
		</tr>
		<tr>
			<td>Nutrient Broth (NB)</td>
			<td>Merck</td>
			<td>105443</td>
			<td></td>
		</tr>
		<tr>
			<td>Luria-Bertani (LB)</td>
			<td>Merck</td>
			<td>110283</td>
			<td></td>
		</tr>
		<tr>
			<td>96-wellplate</td>
			<td>ThermoFisher</td>
			<td>243656</td>
			<td></td>
		</tr>
		<tr>
			<td>Zeiss LSM 780, Axio Observer Z1</td>
			<td>Carl Zeiss Microscopy GmbH</td>
			<td>n/a</td>
			<td></td>
		</tr>
		<tr>
			<td>Leo 1530 Gemini</td>
			<td>Carl Zeiss Microscopy GmbH</td>
			<td>n/a</td>
			<td></td>
		</tr>
		<tr>
			<td>ZEN 2 and ZEN lite 2012 (Software)</td>
			<td>Carl Zeiss Microscopy GmbH</td>
			<td>n/a</td>
			<td></td>
		</tr>
		<tr>
			<td>SigmaPlot, version 13.0 (Statistic software)</td>
			<td>Systat GmbH, Erkrath, Germany</td>
			<td>n/a</td>
			<td></td>
		</tr>
		<tr>
			<td>Attofluor cell chamber</td>
			<td>Invitrogen</td>
			<td>A7816</td>
			<td></td>
		</tr>
		<tr>
			<td>&#181;-Dish 35 mm, high Grid-500 Glass Bottom</td>
			<td>ibidi</td>
			<td>81168</td>
			<td></td>
		</tr>
	</tbody>
</table>

investigating the detrimental effects of low pressure plasma sterilization on the survival of bacillus subtilis spores using live cell microscopy

Plasma sterilization is a promising alternative to conventional sterilization methods for industrial, clinical, and spaceflight purposes. Low pressure plasma (LPP) discharges contain a broad spectrum of active species, which lead to rapid microbial inactivation. To study the efficiency and mechanisms of sterilization by LPP, we use spores of the test organism Bacillus subtilis because of their extraordinary resistance against conventional sterilization procedures. We describe the production of B. subtilis spore monolayers, the sterilization process by low pressure plasma in a double inductively coupled plasma reactor, the characterization of spore morphology using scanning electron microscopy (SEM), and the analysis of germination and outgrowth of spores by live cell microscopy. A major target of plasma species is genomic material (DNA) and repair of plasma-induced DNA lesions upon spore revival is crucial for survival of the organism. Here, we study the germination capacity of spores and the role of DNA repair during spore germination and outgrowth after treatment with LPP by tracking fluorescently-labelled DNA repair proteins (RecA) with time-resolved confocal fluorescence microscopy. Treated and untreated spore monolayers are activated for germination and visualized with an inverted confocal live cell microscope over time to follow the reaction of individual spores. Our observations reveal that the fraction of germinating and outgrowing spores is dependent on the duration of LPP-treatment reaching a minimum after 120 s. RecA-YFP (yellow fluorescence protein) fluorescence was detected only in few spores and developed in all outgrowing cells with a slight elevation in LPP-treated spores. Moreover, some of the vegetative bacteria derived from LPP-treated spores showed an increase in cytoplasm and tended to lyse. The described methods for analysis of individual spores could be exemplary for the study of other aspects of spore germination and outgrowth.

Survival of plasma-treated B. subtilis spores
Plasma treatment of the B. subtilis spores used in this study show a decrease in survival with increasing duration of the plasma treatment (Figure 2). Spores of the strain expressing the recA-gene fused to YFP showed survival curves similar to spores of the wild type strain, indicating that the genetic modification has no signific...

Watch this Scientific Journal Video about Investigating the Detrimental Effects of Low Pressure Plasma Sterilization on the Survival of Bacillus subtilis Spores Using Live Cell Microscopy at JoVE.com

Investigating the Detrimental Effects of Low Pressure Plasma Sterilization on the Survival of Bacillus subtilis Spores Using Live Cell Microscopy

This protocol illustrates the important consecutive steps required to assess the relevance of monitoring vitality parameter and DNA repair processes in reviving Bacillus subtilis spores after treatment with low pressure plasma by tracking fluorescence-labelled DNA repair proteins via time-resolved confocal microscopy and scanning electron microscopy.

Investigating the Detrimental Effects of Low Pressure Plasma Sterilization on the Survival of Bacillus subtilis Spores Using Live Cell Microscopy

Plasma sterilization is a promising alternative to conventional sterilization methods for industrial, clinical, and spaceflight purposes. Low pressure ...

The overall goal of this set of consecutive methods is to visualize and monitor vitality parameters in DNA repair in reviving Bacillus subtilis spores after Low Pressure Plasma Treatment using time resolved confocal fluorescence microscopy and scanning electron microscopy. Our method can help answer key questions in the field of Microbial Inactivation and Sterilization. It helps us to analyze the detrimental effect of low pressure plasma treatment on biological indicators such as Bacillus subtilis spores.The main advantage of our technique is that we combine several methods view determination, scanning electron microscopy and monitoring the DNA repair using time result fluorescence microscopy in order to verify the detrimental effect of low pressure plasma treatment. To carry out spore production, transfer a five milliliter overnight culture of the respective B.subtilis strain supplemented with appropriate antibiotics to 200 milliliters of double strength liquid shafer sporulation medium and cultivate it with vigorous aeration at 37 degrees Celsius for at least 72 hours or longer until greater than 95%of the culture has been sporulated. Harvest spores in 15-milliliter tubes by centrifugation at 3000 G for 15 minutes and purify the samples using sterile distilled H2O in repeated washing steps.Check for purity and germination status by face contrast miscroscopy and ensure that spore suspensions consist of greater than 99%of phase bright spores and are free of vegetative cells, germinated spores and cell debris, otherwise further microscopy experiments can be disturbed. Determinate the spore titer by plating 50 microliters of ten-fold serial dilutions on LB agar to calculate the CFUs and incubate the plates at 37 degrees Celsius overnight. Following CFU determination, adjust the sample to 10 to the 9th spores per milliliter by concentrating or using sterile water to dilute the samples.Place a sample carrier in the form of sterilized microscopic slides or round 25-millimeter cover slips inside the electrically operated aerosol spraying unit in alignment with the nozzle. Transfer the spore culture to the nozzle fluid inlet and initiate the spraying at 0.15 seconds with a pressure of 1.3 bar. The sprayed spore suspension forms a thin film on the microscopic slide that dries rapidly within seconds to form a uniformly distributed spore mono layer.Store the treated sample carriers in a sterile container at room temperature. To clean and warm up all surfaces in the plasma system, initiate the system at five Pascals with Argon plasma at 500 watts for five minutes. The pretreatment reduces the sticking of molecules from ambient air such as nitrogen, oxygen, and water during venting of the chamber.After venting the chamber, carefully place the samples on glass racks in the center of the reactor vessel. Close the chamber and evacuate it. Afterwards, use the desired process gas to fill the chamber and regulate the pressure in the system to five Pascals.Then, start the plasma process. After the defined process time, turn off the power and gas supply and carefully vent the system to prevent blowing the samples from the sample holder. After ventilation, remove the samples and store them in a sterile container.For non-plasma treated controls, expose samples to vacuum only in the presence of the process gas equivalent to the longest applied plasma time. Prepare a solution of autoclaved 10%polyvinyl acetate or PVA and use 500 microliters of it to carefully cover the sample carriers. After letting them air dry for four hours, use sterile forceps to strip off the dried PVA layer that now contains the spore sample and transfer it to a 2-milliliter reaction tube.Add one milliliter of sterile water to the tube and vortex it to dissolve the PVA layer to recover greater than 95%of the spores capable of germination. Use sterile water to serially dilute the sample at one to 10 in a 96-well plate. After plating 50 microliters of each dilution on LB agar, and incubating at 37 degrees Celsius overnight, count the number of colonies and determine the CFUs per milliliter.For germination experiments, prepare a one-millimeter thick 1.5 LB agar pad by boiling 700 microliters of medium and pipette it into a sterile microscopy petridish. After 10 minutes, use a sterile scalpel to cut out an eight millimeter by eight millimeter by one millimeter LB agar pad, and carefully transfer the agar on top of the spore monolayers which are resting on 25-millimeter glass cover slips already mounted in an imaging chamber. The agar put method combines two specific functions.It stabilizes the samples in the optic plane during laser microscopy and it restricts lateral cell movement. Apart from using it for germination essays, this method can be applied to other microorganisms as well as to eukaryotic cells. After covering the sample with agar, quickly transfer the glass cover slip into an imaging chamber.Keep the sample at 37 degrees Celsius on a heated stage during the entire imaging process. Use at least three biological replicates for each condition. Following the imaging of the samples by automated confocal laser scanning and bright field microscopy, record time lapse series with a laser power of 2.6%and set the confocal aperture to five aer units and a sample frequency of one frame per 30 seconds from zero to five hours depending on the experiment.Applying high doses of monochromatic light, may completely inhibit spore germination during laser microscopy. Therefore, use the laser intensity and just longer intervals obtaining single frames. In case of spore aggregation, multi-layered spore distribution or contamination by dust particles, blocking of the plasma treatment or shadowing might occur and enable germination of shadowed spores.To carry out scanning electron microscopy, once the spore monolayers have been sputter coded with gold palladium, image the samples with the field emission SEM operated at five kilovolts acceleration voltage including an inlan secondary electron detector to reveal topography contrast. As shown in this graph, plasma treatment of the B.subtilis spores results in a decrease in survival with increasing duration of the plasma treatment. These SEM images reveal characteristic longitudinal ridge-like structures, which are constantly visible in all treated and untreated spores.Plasma treatment up to 30 seconds, induces no significant changes of the spore surface morphology in comparison to controls. Increasing duration of plasma treatment leads to a more granular surface and small cracks and fissures can be observed in spores treated for 120 or 240 seconds. In these time resolved confocal laser scanning microscopy experiments, almost all spores treated with vacuum as a control germinate and develop a rod-shape cell morphology with a rather uniform length of individual cells.In contrast, spores treated for 15 seconds with plasma already show a reduced germination capacity of less than 75 plus or minus four percent. In addition, cells grow either into extensively long rods or remain small and stick in the spore coat. After 30 seconds of plasma treatment, only 25 plus or minus six percent of the spores germinate and vegetative cells are notably delayed in growth and vary in length.While attempting this procedure, it's important to confirm the quality of the spore monolayers using phase contrast microscopy in order to ensure that there are no overlapping or germinating spores. After its development, the agar pad method for image stabilization, paved the way for researchers in plasma sterilization and live cell microscopy. After watching this video, you should have a better understanding of how to prepare spore monolayers on glass slides, determinacy of use using plasma treatments for spore inactivation and conduct live cell and scanning electron microscopy.In contrast to other decontamination methods, for example ethylene oxide or gamma radiation, plasma sterilization is an efficient and safe approach to reduce a number of unwanted microorganisms on almost any kind of surface.

investigating-detrimental-effects-low-pressure-plasma-sterilization

Research

JoVE Journal

Biology

9.3K Views.  German Aerospace Center (DLR e.V.). The overall goal of this set of consecutive methods is to visualize and monitor vitality parameters in DNA repair in reviving Bacillus subtilis spores after Low Pressure Plasma Treatment using time resolved confocal fluorescence microscopy and scanning electron microscopy. Our method can help answer key questions in the field of Microbial Inactivation and Sterilization. It helps us to analyze the detrimental effect of low pressure plasma treatment on biological indicators such as Bacillus sub...

Video: Investigating the Detrimental Effects of Low Pressure Plasma Sterilization on the Survival of Bacillus subtilis Spores Using Live Cell Microscopy