Name: investigating the detrimental effects of low pressure plasma sterilization on the survival of bacillus subtilis spores using live cell microscopy
Uploaded: 2017-11-30T14:00:00
Description: 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

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>
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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 ...

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