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

Summary

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.

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.

Introduction

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 cl....

Protocol

1. Bacillus subtilis Spore Production and Purification

1. 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 *• 4 H₂O, 2 mL 1 mM FeSO₄, 2 mL 50% (w/v) glucose¹⁸) and cultivate it with vigorous aeration at 37 °C for 72 h or until > 95% of the culture has sporulated. The spores of following strains are u....

Results

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.......

Discussion

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.......

Materials

List of materials used in this article

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