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) glucose18) and cultivate it with vigorous aeration at 37 &#176;C for 72 h or until &#62; 95% of the culture has sporulated. The spores of following strains are used: B. subtilis PY79 (wild type) B. subtilis PY79&#916;recA::neo (deficient of DNA repair protein RecA) B. subtilis PY79 recA-yfp::cat (RecA fused with yellow fluorescent protein [YFP]19).</li>
	<li>Harvest spores by centrifugation for 15 min at 3,000 x g in 50 mL tubes and purify the samples by repeated washing steps (up to 15 times) using sterile distilled H2O and check for purity and germination status by phase-contrast microscopy. Ensure that spore suspensions consist out of phase-bright spores (&#62; 99%) and are free of vegetative cells (rods), germinated spores (black/ grey appearance) and cell debris, otherwise further microscopy experiments can be disturbed. Wash the sample until desired purity is reached.</li>
	<li>Determinate the spore titer by plating out 50 &#181;L of 10-fold serial dilutions on LB-agar (i.e.: Use 30 &#181;L sample + 270 &#181;L sterile water for a 1:10 dilution. Take 30 &#181;L from the particular dilution to 270 &#181;L H2O for a 1:100 dilution and so forth) to calculate the CFU (colony forming units) and incubate the plates at 37 &#176;C overnight. After CFU determination, adjust the sample to 109 spores per mL by concentrating or diluting with sterile water.</li>
</ol>
2. Sample Preparation of Aerosol-deposited Bacillus subtilis Spores
NOTE: Accumulation and overlapping of spores might lead to shadowing effects during the treatment, ultimately resulting in falsified inactivation kinetics. To minimize this problem, prepare spore samples by an aerosol-deposition technique20. Briefly, control the high-precision two-substance nozzle with an electric timer that regulates the liquid throughput in concert with the flow of pressurized carrier gas (here N2). Disperse the injected liquid sample through the nozzle outlet using the nitrogen gas flow.
<ol>
	<li>Place a sample carrier in form of sterilized microscopic slides (for survival kinetics) or round 25 mm coverslips (for fluorescent tracking of DNA repair processes/cLSM; confocal laser scanning microscopy) inside the electrically operated aerosol spraying unit in alignment with the nozzle. The used spore concentration needs to correspond to a hundredfold of the desired final concentration.</li>
	<li>Transfer 1 mL of the spore culture to the nozzle fluid inlet and initiate the spraying process of 0.1 s at a pressure of 1.3&#160;bar. The sprayed spore suspension (1 x 107) forms a thin film on the microscopic slide that dries rapidly within seconds to form a uniformly distributed spore monolayer. Store the treated sample carriers in a sterile container at room temperature.</li>
</ol>
3. Low Pressure Plasma Treatment
<ol>
	<li>Prepare the plasma system for treatment of biological samples and operate the system at 5 Pa with argon plasma at 500 W for 5 min. By this, all surfaces in the system are cleaned and warmed up. This reduces sticking of molecules from ambient air, namely nitrogen, oxygen, and water, while venting the system. After the pretreatment of the system, vent the chamber and place the samples carefully in the center of the reactor vessel with the help of glass racks.</li>
	<li>Use at least three biological replicates. Close the chamber and evacuate below 2 Pa. Afterwards, fill the process gas into the chamber. Regulate the pressure in the system to 5 Pa.</li>
	<li>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 place the samples for the next parameter in the system. For non-plasma-treated controls expose samples to vacuum only (5 Pa) in presence of the process gas equivalent to the longest applied plasma time.</li>
</ol>
4. Recovery and Evaluation of Spore Survival
<ol>
	<li>Prepare a solution of autoclaved 10% polyvinyl acetate (PVA) and cover the sample carrier carefully with approximately 500 &#181;L and let them air-dry for 4 h. Strip off the dried PVA layer (now containing the spore sample) using sterile forceps and transfer it to a 2 mL reaction tube. Add 1 mL of sterile water to the tube and dissolve the PVA layer via vortexing. This procedure leads to &#62;95% recovery of spores and does not affect their germination capability21.</li>
	<li>Serially dilute the sample at 1:10 in sterile water in a 96-well plate (i.e. 270 &#181;L sterile H2O + 30 &#181;L sample/former dilution). Plate out 50 &#181;L of each dilution on Lysogeny broth nutrient agar (LB), incubate the plates at 37 &#176;C overnight and enumerate the number of grown colonies (CFU).</li>
</ol>
5. Live Cell Microscopy and Tracking of DNA Repair Processes in Germinating Spores
<ol>
	<li>For germination experiments, prepare a 1 mm thick 1.5% LB-agar pad, by boiling 700 &#181;L medium and pipet it into a sterile microscopy petri dish. After 10 min, cut out an 8 mm x 8 mm x 1 mm LB-agar pad with a sterile scalpel and transfer the agar carefully on top of the spore monolayers which are resting on 25 mm glass coverslips. 
	NOTE: This step is crucial for visualization of individual spores and allow following their reaction, towards the activation of germination induced by the nutrient agar. Thus, the LB-agar serves two purposes, (1) to fix the spores at the surface, which avoids relocalization along the surface and out of optical focus, and (2) to activate the spore for germination.</li>
	<li>After covering the sample with agar, transfer the glass coverslip quickly into an imaging chamber and microscope the samples with an automated confocal laser-scanning microscope with inverted optics using a 63X/1.3 plane apochromatic oil immersion objective.</li>
	<li>Perform imaging of fluorescence (YFP) with an excitation wavelength of 514 nm and emission can be detected between 520 and 560 nm.
	<ol>
		<li>Record bright-field images in scanning mode using one of the photo multipliers (transmitted-light path).</li>
		<li>Record time-lapse series with a laser power of 2.6%, and set the confocal aperture to 5 airy units and at a sample frequency of 1 frame per 30 s from 0 h to 5 h, depending on the experiment. It is of particular note that high doses of monochromatic laser illumination at 514 nm completely inhibit germination (Figure 1A, B).</li>
	</ol>
	</li>
	<li>Keep the samples at 37 &#176;C (ambient air humidity) in a heating stage during the entire imaging process. Use at least three biological replicates for each condition. In case of spore aggregation, multilayered spore distribution or contamination by dust particles, blocking of the plasma treatment (&#34;shadowing&#34;) might occur and enable germination of shadowed spores (Figure 1C, D).</li>
</ol>
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56666/56666fig1.jpg" /> 
Figure 1: Potential problems observed during confocal live cell fluorescence microscopy of plasma treated spores. (A, B) Inhibition of spore germination by high doses of monochromatic (514 nm) laser illumination. (A) Overview (3 x 3 stitched frames) of B. subtilis (LAS72, RecA-YFP) spores 180 min after initiation of germination. The frame in the middle was exposed at 30 s intervals to high doses of laser light (514 nm, 70% laser power), whereas the surrounding regions (= frames) were not illuminated (merged image of bright-field channel and RecA-YFP fluorescence; ordered structures were caused by using 35 mm imaging dishes with an imprinted 500 &#181;m grid). (B) demonstrates a 4X magnified view of the border between illuminated and non-illuminated region showing that spores, which were exposed to high doses of monochromatic laser illumination did not germinate and grow out, whereas spores in non-illuminated regions fully recover to vegetative bacteria expressing bright RecA-YFP fluorescence (green signal). (C, D) Spores covered by contaminating particles or multiple layers of spore (arrows) seems to protect underlying spores from inactivation by plasma treatment and allow their germination and outgrowth (&#34;shadowing effect&#34;). (C) Spores were plasma-treated for 60 s and imaged 180 min after initiation of germination or in (D) for 120 s and imaged after 240 min. <a href="//cloudfront.jove.com/files/ftp_upload/56666/56666fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
6. Scanning Electron Microscopy (SEM)
<ol>
	<li>Use scanning electron microscopy to provide ultrastructural information about the surface morphology of plasma treated spores in comparison to untreated controls. Coat dried spore monolayers on coverslips with gold-palladium (3 nm) by using a sputter-coater. Use a field-emission scanning electron microscope for imaging the samples, operated at 5 kV acceleration voltage including an in-lens secondary electron detector to reveal topography contrast.</li>
</ol>
7. Data Analysis
<ol>
	<li>Determine the spore survival from the quotient N/N0, where N is the average CFU of treated samples and N0 is the average CFU of untreated vacuum controls. Plot spore inactivation by argon plasma treatment as a function of time (in seconds). Express all data as averages and standard deviations (n = 3).</li>
	<li>Analyze images obtained by live cell imaging using the imaging software. Quantify the proportion of spore germination and outgrowing after plasma treatment, count spores in representative frames at the beginning of the experiment as well as after 4 h. For significance determination in spore survival assays, use one-way ANOVA-tests (analysis of variance) with statistical software). P values &#60;0.05 are considered as statistically significant.</li>
</ol>

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No conflicts of interest declared.

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 H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Sigma Aldrich</td><td>13152</td><td></td></tr><tr><td>Ca(NO&lt;sub&gt;3&lt;/sub&gt;)&lt;sub&gt;2&lt;/sub&gt;</td><td>Sigma Aldrich</td><td>202967</td><td></td></tr><tr><td>MnCl&lt;sub&gt;2&lt;/sub&gt; * 4 H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Sigma Aldrich</td><td>244589</td><td></td></tr><tr><td>FeSO&lt;sub&gt;4 * 7H2O&lt;/sub&gt;</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>&amp;micro;-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...

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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 ...

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

Research

JoVE Journal

Biology

9.4K Views.  German Aerospace Center (DLR e.V.). Dit protocol illustreert de belangrijke opeenvolgende stappen die nodig zijn voor de beoordeling van de relevantie van het toezicht op de parameter van de vitaliteit en DNA-herstelsynthese in de heropleving van sporen van Bacillus subtilis na behandeling met lage druk plasma door tracking fluorescentie-gelabelde DNA repair eiwitten via time-resolved confocale microscopie en elektronenmicroscopie scannen.

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

Live Cell Imaging of Bacillus subtilis and Streptococcus pneumoniae using Automated Time-lapse Microscopy

During the last few years scientists became increasingly aware that average data obtained from microbial population based experiments are not representative of the behavior, status or phenotype of single cells. Due to this new insight the number of single cell studies rises continuously (for recent reviews see 1,2,3). However, many of the single cell techniques applied do not allow monitoring the development and behavior of one specific single cell in time (e.g. flow cytometry or standard microscopy).
Here, we provide a detailed description of a microscopy method used in several recent studies 4, 5, 6, 7, which allows following and recording (fluorescence of) individual bacterial cells of Bacillus subtilis and Streptococcus pneumoniae through growth and division for many generations. The resulting movies can be used to construct phylogenetic lineage trees by tracing back the history of a single cell within a population that originated from one common ancestor. This time-lapse fluorescence microscopy method cannot only be used to investigate growth, division and differentiation of individual cells, but also to analyze the effect of cell history and ancestry on specific cellular behavior. Furthermore, time-lapse microscopy is ideally suited to examine gene expression dynamics and protein localization during the bacterial cell cycle. The method explains how to prepare the bacterial cells and construct the microscope slide to enable the outgrowth of single cells into a microcolony. In short, single cells are spotted on a semi-solid surface consisting of growth medium supplemented with agarose on which they grow and divide under a fluorescence microscope within a temperature controlled environmental chamber. Images are captured at specific intervals and are later analyzed using the open source software ImageJ.

Live Cell Imaging of Bacillus subtilis and Streptococcus pneumoniae using Automated Time-lapse Microscopy

This protocol provides a step-by-step procedure to monitor single cell behavior of different bacteria in time using automated fluorescence time-lapse microscopy. Furthermore, we provide guidelines how to analyze the microscopy images.

During the last few years scientists became increasingly aware that average data obtained from microbial population based experiments are not ...

Immunology and Infection

Basic Research in Plasma Medicine - A Throughput Approach from Liquids to Cells

In plasma medicine, ionized gases with temperatures close to that of vertebrate systems are applied to cells and tissues. Cold plasmas generate reactive species known to redox regulate biological processes in health and disease. Pre-clinical and clinical evidence points to beneficial effects of plasma treatment in the healing of chronic ulcer of the skin. Other emerging topics, such as plasma cancer treatment, are receiving increasing attention. Plasma medical research requires interdisciplinary expertise in physics, chemistry, and biomedicine. One goal of plasma research is to characterize plasma-treated cells in a variety of specific applications. This includes, for example, cell count and viability, cellular oxidation, mitochondrial activity, cytotoxicity and mode of cell death, cell cycle analysis, cell surface marker expression, and cytokine release. This study describes the essential equipment and workflows required for such research in plasma biomedicine. It describes the proper operation of an atmospheric pressure argon plasma jet, specifically monitoring its basic emission spectra and feed gas settings to modulate reactive species output. Using a high-precision xyz-table and computer software, the jet is hovered in millisecond-precision over the cavities of 96-well plates in micrometer-precision for maximal reproducibility. Downstream assays for liquid analysis of redox-active molecules are shown, and target cells are plasma-treated. Specifically, melanoma cells are analyzed in an efficient sequence of different consecutive assays but using the same cells: measurement of metabolic activity, total cell area, and surface marker expression of calreticulin, a molecule important for the immunogenic cell death of cancer cells. These assays retrieve content-rich biological information about plasma effects from a single plate. Altogether, this study describes the essential steps and protocols for plasma medical research.

A high-throughput cold physical plasma treatment protocol of liquids and cells is shown. It involves setting up different feed gas compositions for plasma ignition, measuring the plasma&#39;s emission spectra, and the subsequent analysis of liquids and cellular activity after plasma treatment.

In plasma medicine, ionized gases with temperatures close to that of vertebrate systems are applied to cells and tissues. Cold plasmas generate reactive ...

Environment

Single-cell Analysis of Bacillus subtilis Biofilms Using Fluorescence Microscopy and Flow Cytometry

Biofilm formation is a general attribute to almost all bacteria 1-6. When bacteria form biofilms, cells are encased in extracellular matrix that is mostly constituted by proteins and exopolysaccharides, among other factors 7-10. The microbial community encased within the biofilm often shows the differentiation of distinct subpopulation of specialized cells 11-17. These subpopulations coexist and often show spatial and temporal organization within the biofilm 18-21.
Biofilm formation in the model organism Bacillus subtilis requires the differentiation of distinct subpopulations of specialized cells. Among them, the subpopulation of matrix producers, responsible to produce and secrete the extracellular matrix of the biofilm is essential for biofilm formation 11,19. Hence, differentiation of matrix producers is a hallmark of biofilm formation in B. subtilis.
We have used fluorescent reporters to visualize and quantify the subpopulation of matrix producers in biofilms of B. subtilis 15,19,22-24. Concretely, we have observed that the subpopulation of matrix producers differentiates in response to the presence of self-produced extracellular signal surfactin 25. Interestingly, surfactin is produced by a subpopulation of specialized cells different from the subpopulation of matrix producers 15.
We have detailed in this report the technical approach necessary to visualize and quantify the subpopulation of matrix producers and surfactin producers within the biofilms of B. subtilis. To do this, fluorescent reporters of genes required for matrix production and surfactin production are inserted into the chromosome of B. subtilis. Reporters are expressed only in a subpopulation of specialized cells. Then, the subpopulations can be monitored using fluorescence microscopy and flow cytometry (See Fig 1).
The fact that different subpopulations of specialized cells coexist within multicellular communities of bacteria gives us a different perspective about the regulation of gene expression in prokaryotes. This protocol addresses this phenomenon experimentally and it can be easily adapted to any other working model, to elucidate the molecular mechanisms underlying phenotypic heterogeneity within a microbial community.

Single-cell Analysis of Bacillus subtilis Biofilms Using Fluorescence Microscopy and Flow Cytometry

Microbial biofilms are generally constituted by distinct subpopulations of specialized cells. Single-cell analysis of these subpopulations requires the use of fluorescent reporters. Here we describe a protocol to visualize and monitor several subpopulationswithin B. subtilis biofilms using fluorescence microscopy and flow cytometry.

Biofilm formation is a general attribute to almost all bacteria 1-6. When bacteria form biofilms, cells are encased in extracellular matrix that is mostly ...

Establishing the Minimal Bactericidal Concentration of an Antimicrobial Agent for Planktonic Cells (MBC-P) and Biofilm Cells (MBC-B)

This protocol allows for a direct comparison between planktonic and biofilm resistance for a bacterial strain that can form a biofilm in vitro. Bacteria are inoculated into the wells of a 96-well microtiter plate. In the case of the planktonic assay, serial dilutions of the antimicrobial agent of choice are added to the bacterial suspensions. In the biofilm assay, once inoculated, the bacteria are left to form a biofilm over a set period of time. Unattached cells are removed from the wells, the media is replenished and serial dilutions of the antimicrobial agent of choice are added. After exposure to the antimicrobial agent, the planktonic cells are assayed for growth. For the biofilm assay, the media is refreshed with fresh media lacking the antimicrobial agent and the biofilm cells are left to recover. Biofilm cell viability is assayed after the recovery period. The MBC-P for the antimicrobial agent is defined as the lowest concentration of drug that kills the cells in the planktonic culture.&#160; In contrast, the MBC-B for a strain is determined by exposing preformed biofilms to increasing concentrations of antimicrobial agent for 24 hr. The MBC-B is defined as the lowest concentration of antimicrobial agent that kills the cells in the biofilm.

Establishing the Minimal Bactericidal Concentration of an Antimicrobial Agent for Planktonic Cells MBC-P and Biofilm Cells MBC-B

This protocol allows for a direct comparison between planktonic and biofilm resistance for a bacterial strain that can form a biofilm in vitro using a 96-well microtiter plate. Planktonic or biofilm bacteria are exposed to serial dilutions of the antimicrobial agent of choice. Viability is assayed by growth on agar plates.

This protocol allows for a direct comparison between planktonic and biofilm resistance for a bacterial strain that can form a biofilm in vitro. Bacteria ...

Light-Induced In Situ Transmission Electron Microscopy for Observation of the Liquid-Soft Matter Interaction

The current protocol describes the modifications of the transmission electron microscope (TEM) setup for in situ light-induced observations. A glass optical fiber inserted into the electron column above the objective lens polepiece, and a laser, an adjustable light source, was used to fabricate the device. After the illuminator has been calibrated using an external measuring system, it allows one to adjust the intensity of the lighting to the needs of the observed process. This lighting system was utilized to image antimicrobial photodynamic therapy phenomena, which are currently the subject of intense research. The sample was prepared by spotting a suspension of bacteria on a carbon, graphene, or silicon nitride substrate, blotting the excess solution, spotting the photosensitizer solution, blotting the excess liquid again, and then assembling the liquid cell with a second substrate or graphene film. The process of the imaging experiment itself includes choosing the right place for observation with the use of low magnification and a minimum dose of electrons, and then cyclical activation of the light source to capture subsequent images at specified intervals with the minimum amount of electrons necessary. The electron dose of each exposure and the time and intensity of lighting used need to be carefully recorded due to the complexity of the observed phenomena as, at the same time, the process is both light- and electron-driven. After the actual experiment is performed, additional control observations must be made, in which the same doses of electrons are used but without additional light influence and smaller doses of electrons are used for higher doses of light. This makes it possible to distinguish light-induced microstructural effects from those caused by electrons in both the fields of life and materials science.

Light-Induced In Situ Transmission Electron Microscopy for Observation of the Liquid-Soft Matter Interaction

The present protocol describes transmission electron microscope (TEM) modifications with a light illumination system, the fabrication of liquid cells, and in situ TEM observations of light-induced interactions between bacterial cells and a photosensitizer. The sample preparation methods, electron beam damage, and imaging are also discussed.

The current protocol describes the modifications of the transmission electron microscope (TEM) setup for in situ light-induced observations. A glass ...

Engineering

Methodologies for Studying B. subtilis Biofilms as a Model for Characterizing Small Molecule Biofilm Inhibitors

This work assesses different methodologies to study the impact of small molecule biofilm inhibitors, such as D-amino acids, on the development and resilience of Bacillus subtilis biofilms. First, methods are presented that select for small molecule inhibitors with biofilm-specific targets in order to separate the effect of the small molecule inhibitors on planktonic growth from their effect on biofilm formation. Next, we focus on how inoculation conditions affect the sensitivity of multicellular, floating B. subtilis cultures to small molecule inhibitors. The results suggest that discrepancies in the reported effects of such inhibitors such as D-amino acids are due to inconsistent pre-culture conditions. Furthermore, a recently developed protocol is described for evaluating the contribution of small molecule treatments towards biofilm resistance to antibacterial substances. Lastly, scanning electron microscopy (SEM) techniques are presented to analyze the three-dimensional spatial arrangement of cells and their surrounding extracellular matrix in a B. subtilis biofilm. SEM facilitates insight into the three-dimensional biofilm architecture and the matrix texture. A combination of the methods described here can greatly assist the study of biofilm development in the presence and absence of biofilm inhibitors, and shed light on the mechanism of action of these inhibitors.

Methodologies for Studying B. subtilis Biofilms as a Model for Characterizing Small Molecule Biofilm Inhibitors

This study presents the development of reproducible methodologies to study biofilm inhibitors and their effects on Bacillus subtilis multicellularity.

This work assesses different methodologies to study the impact of small molecule biofilm inhibitors, such as D-amino acids, on the development and ...

Improvement of Bacillus subtilis Spore Enumeration and Label Analysis in Flow Cytometry

The spores of Bacillus subtilis have already been proposed for different biotechnological and immunological applications; however, there is an increasing need for the development of methodologies that improve the detection of antigens immobilized on the surface of spores together with their quantification. Flow cytometry-based analyses have been previously proposed as fast, reliable, and specific approaches for detecting labeled cells of B. subtilis. Herein, we propose the use of flow cytometry to evaluate the display efficiency of a fluorescent antibody (FA) on the surface of the spore and quantify the number of spores using counting beads. 
For this, we used ethidium bromide as a DNA marker and an allophycocyanin (APC)-labeled antibody, which was coupled to the spores, as a surface marker. The quantification of spores was performed using counting beads since this technique demonstrates high accuracy in the detection of cells. The labeled spores were analyzed using a flow cytometer, which confirmed the coupling. As a result, it was demonstrated that DNA labeling improved the accuracy of quantification by flow cytometry, for the detection of germinated spores. It was observed that ethidium bromide was not able to label dormant spores; however, this technique provides a more precise determination of the number of spores with fluorescent protein coupled to their surface, thus helping in the development of studies that focus on the use of spores as a biotechnological platform in different applications.

Improvement of Bacillus subtilis Spore Enumeration and Label Analysis in Flow Cytometry

This protocol focuses on the use of flow cytometry and counting beads to quantify bacterial spores labeled with ethidium bromide. The method is also efficient for analyzing the covalent coupling of proteins on the surface of intact spores.

The spores of Bacillus subtilis have already been proposed for different biotechnological and immunological applications; however, there is an increasing ...

Biochemistry

Visualization of Germinosomes and the Inner Membrane in Bacillus subtilis Spores

The small size of spores and the relatively low abundance of germination proteins, cause difficulties in their microscopic analyses using epifluorescence microscopy. Super-resolution three-dimensional Structured Illumination Microscopy (3D-SIM) is a promising tool to overcome this hurdle and reveal the molecular details of the process of germination of Bacillus subtilis (B. subtilis) spores. Here, we describe the use of a modified SIMcheck (ImageJ)-assistant 3D imaging process and fluorescent reporter proteins for SIM microscopy of B. subtilis spores&#8217; germinosomes, cluster(s) of germination proteins. We also present a (standard)3D-SIM imaging procedure for FM4-64 staining of B. subtilis spore membranes. By using these procedures, we obtained unsurpassed resolution for germinosome localization and show that &#62;80% of B. subtilis KGB80 dormant spores obtained after sporulation on defined minimal MOPS medium have one or two GerD-GFP and GerKB-mCherry foci. Bright foci were also observed in FM4-64 stained spores&#8217; 3D-SIM images suggesting that inner membrane lipid domains of different fluidity likely exist. Further studies that use double labeling procedures with membrane dyes and germinosome reporter proteins to assess co-localization and thus get an optimal overview of the organization of Bacillus germination proteins in the inner spore membrane are possible.

Visualization of Germinosomes and the Inner Membrane in Bacillus subtilis Spores

Germinant receptor proteins cluster in &#8216;germinosomes&#8217; in the inner membrane of Bacillus subtilis spores. We describe a protocol using super resolution microscopy and fluorescent reporter proteins to visualize germinosomes. The protocol also identifies spore inner membrane domains that are preferentially stained with the membrane dye FM4-64.

The small size of spores and the relatively low abundance of germination proteins, cause difficulties in their microscopic analyses using epifluorescence ...

Bioengineering

Kinetic Visualization of Single-Cell Interspecies Bacterial Interactions

Polymicrobial communities are ubiquitous in nature, yet studying their interactions at the single-cell level is difficult. Thus, a microscopy-based method has been developed for observing interspecies interactions between two bacterial pathogens. The use of this method to interrogate interactions between a motile Gram-negative pathogen, Pseudomonas aeruginosa and a non-motile Gram-positive pathogen, Staphylococcus aureus is demonstrated here. This protocol consists of co-inoculating each species between a coverslip and an agarose pad, which maintains the cells in a single plane and allows for visualization of bacterial behaviors in both space and time.
Furthermore, the time-lapse microscopy demonstrated here is ideal for visualizing the early interactions that take place between two or more bacterial species, including changes in bacterial species motility in monoculture and in coculture with other species. Due to the nature of the limited sample space in the microscopy setup, this protocol is less applicable for studying later interactions between bacterial species once cell populations are too high. However, there are several different applications of the protocol which include the use of staining for imaging live and dead bacterial cells, quantification of gene or protein expression through fluorescent reporters, and tracking bacterial cell movement in both single species and multispecies experiments.

This live-bacterial cell imaging protocol allows for visualization of interactions between multiple bacterial species at the single-cell level over time. Time-lapse imaging allows for observation of each bacterial species in monoculture or coculture to interrogate interspecies interactions in multispecies bacterial communities, including individual cell motility and viability.

Polymicrobial communities are ubiquitous in nature, yet studying their interactions at the single-cell level is difficult. Thus, a microscopy-based method ...

Plasmid Stability Analysis with Open-Source Droplet Microfluidics

Plasmids play a vital role in synthetic biology by enabling the introduction and expression of foreign genes in various organisms, thereby facilitating the construction of biological circuits and pathways within and between cell populations. For many applications, maintaining functional plasmids without antibiotic selection is critical. This study introduces an open-hardware-based microfluidic workflow for analyzing plasmid retention by culturing single cells in gel microdroplets and quantifying microcolonies using fluorescence microscopy. This approach allows for the parallel analysis of numerous droplets and microcolonies, providing greater statistical power compared to traditional plate counting and enabling the integration of the assay into other droplet microfluidic workflows. By using plasmids expressing fluorescent proteins alongside a non-specific fluorescent DNA stain, single colonies can be identified and differentiated based on plasmid loss or fluorescent marker expression. Notably, this advanced workflow, implemented with open-source hardware, offers precise flow control and temperature management of both the sample and the microfluidic chip. These features enhance the workflow&#39;s ease of use, robustness, and accessibility. While the study focuses on Escherichia coli as the experimental model, the method&#39;s true potential lies in its versatility. It can be adapted for various studies requiring fluorescence signal quantification from plasmids or stains, as well as for other applications. The adoption of open-source hardware broadens the potential for conducting high-throughput bioanalyses using accessible technology in diverse research settings.

An accessible, open-source microfluidic workflow is presented for the parallelized analysis of plasmid retention in bacteria. By employing fluorescence microscopy to quantify plasmid presence in single-cell microcolonies within gel microdroplets, this method provides a precise, accessible, and scalable alternative to traditional plate counting.

Plasmids play a vital role in synthetic biology by enabling the introduction and expression of foreign genes in various organisms, thereby facilitating ...

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

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