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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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. 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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 ...

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

Imaging Mismatch Repair and Cellular Responses to DNA Damage in Bacillus subtilis

Both prokaryotes and eukaryotes respond to DNA damage through a complex set of physiological changes. Alterations in gene expression, the redistribution of existing proteins, and the assembly of new protein complexes can be stimulated by a variety of DNA lesions and mismatched DNA base pairs. Fluorescence microscopy has been used as a powerful experimental tool for visualizing and quantifying these and other responses to DNA lesions and to monitor DNA replication status within the complex subcellular architecture of a living cell. Translational fusions between fluorescent reporter proteins and components of the DNA replication and repair machinery have been used to determine the cues that target DNA repair proteins to their cognate lesions in vivo and to understand how these proteins are organized within bacterial cells. In addition, transcriptional and translational fusions linked to DNA damage inducible promoters have revealed which cells within a population have activated genotoxic stress responses. In this review, we provide a detailed protocol for using fluorescence microscopy to image the assembly of DNA repair and DNA replication complexes in single bacterial cells. In particular, this work focuses on imaging mismatch repair proteins, homologous recombination, DNA replication and an SOS-inducible protein in Bacillus subtilis. All of the procedures described here are easily amenable for imaging protein complexes in a variety of bacterial species.

Imaging Mismatch Repair and Cellular Responses to DNA Damage in Bacillus subtilis

A detailed protocol is described for imaging the real time formation of DNA repair complexes in Bacillus subtilis cells.

Both prokaryotes and eukaryotes respond to DNA damage through a complex set of physiological changes. Alterations in gene expression, the redistribution ...

Identifying the Effects of BRCA1 Mutations on Homologous Recombination using Cells that Express Endogenous Wild-type BRCA1

The functional analysis of missense mutations can be complicated by the presence in the cell of the endogenous protein. Structure-function analyses of the BRCA1 have been complicated by the lack of a robust assay for the full length BRCA1 protein and the difficulties inherent in working with cell lines that express hypomorphic BRCA1 protein1,2,3,4,5. We developed a system whereby the endogenous BRCA1 protein in a cell was acutely depleted by RNAi targeting the 3'-UTR of the BRCA1 mRNA and replaced by co-transfecting a plasmid expressing a BRCA1 variant. One advantage of this procedure is that the acute silencing of BRCA1 and simultaneous replacement allow the cells to grow without secondary mutations or adaptations that might arise over time to compensate for the loss of BRCA1 function. This depletion and add-back procedure was done in a HeLa-derived cell line that was readily assayed for homologous recombination activity. The homologous recombination assay is based on a previously published method whereby a recombination substrate is integrated into the genome (Figure 1)6,7,8,9. This recombination substrate has the rare-cutting I-SceI restriction enzyme site inside an inactive GFP allele, and downstream is a second inactive GFP allele. Transfection of the plasmid that expresses I-SceI results in a double-stranded break, which may be repaired by homologous recombination, and if homologous recombination does repair the break it creates an active GFP allele that is readily scored by flow cytometry for GFP protein expression. Depletion of endogenous BRCA1 resulted in an 8-10-fold reduction in homologous recombination activity, and add-back of wild-type plasmid fully restored homologous recombination function. When specific point mutants of full length BRCA1 were expressed from co-transfected plasmids, the effect of the specific missense mutant could be scored. As an example, the expression of the BRCA1(M18T) protein, a variant of unknown clinical significance10, was expressed in these cells, it failed to restore BRCA1-dependent homologous recombination. By contrast, expression of another variant, also of unknown significance, BRCA1(I21V) fully restored BRCA1-dependent homologous recombination function. This strategy of testing the function of BRCA1 missense mutations has been applied to another biological system assaying for centrosome function (Kais et al, unpublished observations). Overall, this approach is suitable for the analysis of missense mutants in any gene that must be analyzed recessively.

We provide a method for testing BRCA1 variants in a tissue culture based assay for homologous recombination repair of DNA damage by depleting endogenous BRCA1 protein from a cell using RNAi and replacing it with a BRCA1 point mutant that contains a coding change.

The functional analysis of missense mutations can be complicated by the presence in the cell of the endogenous protein. Structure-function analyses of the ...

Formulations for Freeze-drying of Bacteria and Their Influence on Cell Survival

Cellular water can be removed to reversibly inactivate microorganisms to facilitate storage. One such method of removal is freeze-drying, which is considered a gentle dehydration method. To facilitate cell survival during drying, the cells are often formulated beforehand. The formulation forms a matrix that embeds the cells and protects them from various harmful stresses imposed on the cells during freezing and drying. We present here a general method to evaluate the survival rate of cells after freeze-drying and we illustrate it by comparing the results obtained with four different formulations: the disaccharide sucrose, the sucrose derived polymer Ficoll PM400, and the respective polysaccharides hydroxyethyl cellulose (HEC) and hydroxypropyl methyl cellulose (HPMC), on two strains of bacteria, P. putida KT2440 and A. chlorophenolicus A6. In this work we illustrate how to prepare formulations for freeze-drying and how to investigate the mechanisms of cell survival after rehydration by characterizing the formulation using of differential scanning calorimetry (DSC), surface tension measurements, X-ray analysis, and electron microscopy and relating those data to survival rates. The polymers were chosen to get a monomeric structure of the respective polysaccharide resembling sucrose to a varying degrees. Using this method setup we showed that polymers can support cell survival as effectively as disaccharides if certain physical properties of the formulation are controlled1.

Freeze-drying is often an easy and convenient way to obtain dry products of viable bacterial cells. An issue of the process is cell survival. We detail here a procedure to investigate how cell survival during freeze-drying is influenced by the properties of the formulation used.

Cellular water can be removed to reversibly inactivate microorganisms to facilitate storage. One such method of removal is freeze-drying, which is ...

FRET Microscopy for Real-time Monitoring of Signaling Events in Live Cells Using Unimolecular Biosensors

F&ouml;rster resonance energy transfer (FRET) microscopy continues to gain increasing interest as a technique for real-time monitoring of biochemical and signaling events in live cells and tissues. Compared to classical biochemical methods, this novel technology is characterized by high temporal and spatial resolution. FRET experiments use various genetically-encoded biosensors which can be expressed and imaged over time in situ or in vivo1-2. Typical biosensors can either report protein-protein interactions by measuring FRET between a fluorophore-tagged pair of proteins or conformational changes in a single protein which harbors donor and acceptor fluorophores interconnected with a binding moiety for a molecule of interest3-4. Bimolecular biosensors for protein-protein interactions include, for example, constructs designed to monitor G-protein activation in cells5, while the unimolecular sensors measuring conformational changes are widely used to image second messengers such as calcium6, cAMP7-8, inositol phosphates9 and cGMP10-11. Here we describe how to build a customized epifluorescence FRET imaging system from single commercially available components and how to control the whole setup using the Micro-Manager freeware. This simple but powerful instrument is designed for routine or more sophisticated FRET measurements in live cells. Acquired images are processed using self-written plug-ins to visualize changes in FRET ratio in real-time during any experiments before being stored in a graphics format compatible with the build-in ImageJ freeware used for subsequent data analysis. This low-cost system is characterized by high flexibility and can be successfully used to monitor various biochemical events and signaling molecules by a plethora of available FRET biosensors in live cells and tissues. As an example, we demonstrate how to use this imaging system to perform real-time monitoring of cAMP in live 293A cells upon stimulation with a &beta;-adrenergic receptor agonist and blocker.

F&#246;rster resonance energy transfer (FRET) microscopy is a powerful technique for real-time monitoring of signaling events in live cells using various biosensors as reporters. Here we describe how to build a customized epifluorescence FRET imaging system from commercially available components and how to use it for FRET experiments.

F&ouml;rster resonance energy transfer (FRET) microscopy  continues to gain increasing interest as a technique for real-time monitoring  of biochemical ...

Live Imaging of Apoptotic Cell Clearance during Drosophila Embryogenesis

The proper elimination of unwanted or aberrant cells through apoptosis and subsequent phagocytosis (apoptotic cell clearance) is crucial for normal development in all metazoan organisms. Apoptotic cell clearance is a highly dynamic process intimately associated with cell death; unengulfed apoptotic cells are barely seen in vivo under normal conditions. In order to understand the different steps of apoptotic cell clearance and to compare 'professional' phagocytes - macrophages and dendritic cells to 'non-professional' - tissue-resident neighboring cells, in vivo live imaging of the process is extremely valuable. Here we describe a protocol for studying apoptotic cell clearance in live Drosophila embryos. To follow the dynamics of different steps in phagocytosis we use specific markers for apoptotic cells and phagocytes. In addition, we can monitor two phagocyte systems in parallel: 'professional' macrophages and 'semi-professional' glia in the developing central nervous system (CNS). The method described here employs the Drosophila embryo as an excellent model for real time studies of apoptotic cell clearance.

Live Imaging of Apoptotic Cell Clearance during Drosophila Embryogenesis

Here we describe an effective method for studying dynamics of apoptotic cell clearance in vivo. This method employs live Drosophila embryos as a powerful model for monitoring phagocytosis of apoptotic cells using specific labeling of apoptotic cells and phagocytes.

The  proper elimination of unwanted or aberrant cells through apoptosis and  subsequent phagocytosis (apoptotic cell clearance) is crucial for normal  ...

Analyzing Craniofacial Morphogenesis in Zebrafish Using 4D Confocal Microscopy

Time-lapse imaging is a technique that allows for the direct observation of the process of morphogenesis, or the generation of shape. Due to their optical clarity and amenability to genetic manipulation, the zebrafish embryo has become a popular model organism with which to perform time-lapse analysis of morphogenesis in living embryos. Confocal imaging of a live zebrafish embryo requires that a tissue of interest is persistently labeled with a fluorescent marker, such as a transgene or injected dye. The process demands that the embryo is anesthetized and held in place in such a way that healthy development proceeds normally. Parameters for imaging must be set to account for three-dimensional growth and to balance the demands of resolving individual cells while getting quick snapshots of development. Our results demonstrate the ability to perform long-term in vivo imaging of fluorescence-labeled zebrafish embryos and to detect varied tissue behaviors in the cranial neural crest that cause craniofacial abnormalities. Developmental delays caused by anesthesia and mounting are minimal, and embryos are unharmed by the process. Time-lapse imaged embryos can be returned to liquid medium and subsequently imaged or fixed at later points in development. With an increasing abundance of transgenic zebrafish lines and well-characterized fate mapping and transplantation techniques, imaging any desired tissue is possible. As such, time-lapse in vivo imaging combines powerfully with zebrafish genetic methods, including analyses of mutant and microinjected embryos.

Time-lapse confocal imaging is a powerful technique useful for characterizing embryonic development. Here, we describe the methodology and characterize craniofacial morphogenesis in wild-type, as well as pdgfra, smad5, and smo mutant embryos.

Time-lapse imaging is a technique that allows for the direct observation of the process of morphogenesis, or the generation of shape. Due to their optical ...

Standardized Method for High-throughput Sterilization of Arabidopsis Seeds

Arabidopsis thaliana (Arabidopsis) seedlings often need to be grown on sterile media. This requires prior seed sterilization to prevent the growth of microbial contaminants present on the seed surface. Currently, Arabidopsis seeds are sterilized using two distinct sterilization techniques in conditions that differ slightly between labs and have not been standardized, often resulting in only partially effective sterilization or in excessive seed mortality. Most of these methods are also not easily scalable to a large number of seed lines of diverse genotypes. As technologies for high-throughput analysis of Arabidopsis continue to proliferate, standardized techniques for sterilizing large numbers of seeds of different genotypes are becoming essential for conducting these types of experiments. The response of a number of Arabidopsis lines to two different sterilization techniques was evaluated based on seed germination rate and the level of seed contamination with microbes and other pathogens. The treatments included different concentrations of sterilizing agents and times of exposure, combined to determine optimal conditions for Arabidopsis seed sterilization. Optimized protocols have been developed for two different sterilization methods: bleach (liquid-phase) and chlorine (Cl2) gas (vapor-phase), both resulting in high seed germination rates and minimal microbial contamination. The utility of these protocols was illustrated through the testing of both wild type and mutant seeds with a range of germination potentials. Our results show that seeds can be effectively sterilized using either method without excessive seed mortality, although detrimental effects of sterilization were observed for seeds with lower than optimal germination potential. In addition, an equation was developed to enable researchers to apply the standardized chlorine gas sterilization conditions to airtight containers of different sizes. The protocols described here allow easy, efficient, and inexpensive seed sterilization for a large number of Arabidopsis lines.

The objective of this study was to determine the effects of bleach and chlorine gas sterilization on seed germination of a range of Arabidopsis genotypes grown on sterile media. Optimized sterilization protocols have been developed to prevent the growth of microbial contaminants while providing satisfactory seed survival.

Arabidopsis thaliana (Arabidopsis) seedlings often need to be grown on sterile media. This requires prior seed sterilization to prevent the growth of ...

Single-cell Microfluidic Analysis of Bacillus subtilis

Microfluidic technology overcomes many of the limitations to traditional analytical methods in microbiology. Unlike bulk-culture methods, it offers single-cell resolution and long observation times spanning hundreds of generations; unlike agarose pad-based microscopy, it has uniform growth conditions that can be tightly controlled. Because the continuous flow of growth medium isolates the cells in a microfluidic device from unpredictable variations in the local chemical environment caused by cell growth and metabolism, authentic changes in gene expression and cell growth in response to specific stimuli can be more confidently observed. Bacillus subtilis is used here as a model bacterial species to demonstrate a &#34;mother machine&#34;-type method for cellular analysis. We show how to construct and plumb a microfluidic device, load it with cells, initiate microscopic imaging, and expose cells to a stimulus by switching from one growth medium to another. A stress-responsive reporter is used as an example to reveal the type of data that may be obtained by this method. We also briefly discuss further applications of this method for other types of experiments, such as analysis of bacterial sporulation.

Single-cell Microfluidic Analysis of Bacillus subtilis

We present a method for the microfluidic analysis of individual bacterial cell lineages using Bacillus subtilis as an example. The method overcomes shortcomings of traditional analytical methods in microbiology by allowing observation of hundreds of cell generations under tightly controllable and uniform growth conditions.

Microfluidic technology overcomes many of the limitations to traditional analytical methods in microbiology. Unlike bulk-culture methods, it offers ...

Biological Sample Preparation by High-pressure Freezing, Microwave-assisted Contrast Enhancement, and Minimal Resin Embedding for Volume Imaging

The described sample preparation technique is designed to combine the best quality of ultrastructural preservation with the most suitable contrast for the imaging modality in a focused ion beam scanning electron microscope (FIB-SEM), which is used to obtain stacks of sequential images for 3D reconstruction and modelling. High-pressure freezing (HPF) allows close to native structural preservation, but the subsequent freeze substitution often does not provide sufficient contrast, especially for a bigger specimen, which is needed for high-quality imaging in the SEM required for 3D reconstruction. Therefore, in this protocol, after the freeze substitution, additional contrasting steps are carried out at room temperature. Although these steps are performed in a microwave, it is also possible to follow traditional bench processing, which requires longer incubation times.The subsequent embedding in minimal amounts of resin allows for faster and more precise targeting and preparation inside the FIB-SEM. This protocol is especially useful for samples that require preparation by high-pressure freezing for a reliable ultrastructural preservation but do not gain enough contrast during the freeze substitution for volume imaging using FIB-SEM. In combination with the minimal resin embedding, this protocol provides an efficient workflow for the acquisition of high-quality volume data.

Here we present a protocol for combining two sample processing techniques, high-pressure freezing and microwave-assisted sample processing, followed by minimal resin embedding for acquiring data with a focused ion beam scanning electron microscope (FIB-SEM). This is demonstrated using a mouse tibial nerve sample and Caenorhabditis elegans.

The described sample preparation technique is designed to combine the best quality of ultrastructural preservation with the most suitable contrast for the ...

Live-Cell Imaging of the Life Cycle of Bacterial Predator Bdellovibrio bacteriovorus using Time-Lapse Fluorescence Microscopy

Bdellovibrio bacteriovorus is a small gram-negative, obligate predatory bacterium that kills other gram-negative bacteria, including harmful pathogens. Therefore, it is considered a living antibiotic. To apply B. bacteriovorus as a living antibiotic, it is first necessary to understand the major stages of its complex life cycle, particularly its proliferation inside prey. So far, it has been challenging to monitor successive stages of the predatory life cycle in real-time. Presented here is a comprehensive protocol for real-time imaging of the complete life cycle of B. bacteriovorus, especially during its growth inside the host. For this purpose, a system consisting of an agarose pad is used in combination with cell-imaging dishes, in which the predatory cells can move freely beneath the agarose pad while immobilized prey cells are able to form bdelloplasts. The application of a strain producing a fluorescently tagged &#946;-subunit of DNA polymerase III further allows chromosome replication to be monitored during the reproduction phase of the B. bacteriovorus life cycle.

Live-Cell Imaging of the Life Cycle of Bacterial Predator Bdellovibrio bacteriovorus using Time-Lapse Fluorescence Microscopy

Presented here is a protocol that describes monitoring of the complete life cycle of predatory bacterium Bdellovibrio bacteriovorus using time-lapse fluorescence microscopy in combination with an agarose pad and cell-imaging dishes.

Bdellovibrio bacteriovorus is a small gram-negative, obligate predatory bacterium that kills other gram-negative bacteria, including harmful pathogens. ...