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

Fuchs, Felix M. and Raguse, Marina and Fiebrandt, Marcel and Madela, Kazimierz and Awakowicz, Peter and Laue, Michael and Stapelmann, Katharina and Moeller, Ralf (2017) Investigating the Detrimental Effects of Low Pressure Plasma Sterilization on the Survival of Bacillus subtilis Spores Using Live Cell Microscopy. Journal of Visualized Experiments (129), e56666. Journal of Visualized Experiments. DOI: 10.3791/56666 ISSN 1940-087X

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Official URL: http://dx.doi.org/10.3791/56666

Abstract

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.

Item URL in elib:https://elib.dlr.de/116598/
Document Type:Article
Title:Investigating the Detrimental Effects of Low Pressure Plasma Sterilization on the Survival of Bacillus subtilis Spores Using Live Cell Microscopy
Authors:
AuthorsInstitution or Email of AuthorsAuthors ORCID iD
Fuchs, Felix M.radiation biology department, institute of aerospace medicine, german aerospace center (dlr), cologne, germanyUNSPECIFIED
Raguse, Marinaradiation biology department, institute of aerospace medicine, german aerospace center (dlr), cologne, germany and Institute of Electrical Engineering and Plasma Technology, Ruhr-University BochumUNSPECIFIED
Fiebrandt, MarcelInstitute of Electrical Engineering and Plasma Technology, Faculty of Electrical Engineering and Information Technology, Ruhr-University BochumUNSPECIFIED
Madela, KazimierzAdvanced Light and Electron Microscopy (ZBS 4), Robert Koch InstituteUNSPECIFIED
Awakowicz, PeterInstitute of Electrical Engineering and Plasma Technology, Faculty of Electrical Engineering and Information Technology, Ruhr-University BochumUNSPECIFIED
Laue, MichaelAdvanced Light and Electron Microscopy (ZBS 4), Robert Koch InstituteUNSPECIFIED
Stapelmann, KatharinaInstitute of Electrical Engineering and Plasma Technology, Faculty of Electrical Engineering and Information Technology, Biomedical Applications of Plasma Technology, Ruhr-University BochumUNSPECIFIED
Moeller, Ralfradiation biology department, institute of aerospace medicine, german aerospace center (dlr), cologne, germany; ralf.moeller (at) dlr.dehttps://orcid.org/0000-0002-2371-0676
Date:2017
Journal or Publication Title:Journal of Visualized Experiments
Refereed publication:Yes
Open Access:No
Gold Open Access:No
In SCOPUS:Yes
In ISI Web of Science:Yes
DOI :10.3791/56666
Page Range:e56666
Publisher:Journal of Visualized Experiments
ISSN:1940-087X
Status:Published
Keywords:Plasma sterilization, Low pressure plasma (LPP), Bacillus subtilis spores, Live Cell Microscopy
HGF - Research field:Aeronautics, Space and Transport
HGF - Program:Space
HGF - Program Themes:Research under Space Conditions
DLR - Research area:Raumfahrt
DLR - Program:R FR - Forschung unter Weltraumbedingungen
DLR - Research theme (Project):R - Vorhaben Strahlenbiologie
Location: Köln-Porz
Institutes and Institutions:Institute of Aerospace Medicine > Radiation Biology
Deposited By: Kopp, Kerstin
Deposited On:07 Dec 2017 13:22
Last Modified:07 Dec 2017 13:22

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