The authors thank Christiaan Zeelenberg for his assistance during the SIM imaging. JW acknowledges the China Scholarship Council for a PhD fellowship and thanks Irene Stellingwerf for her help during the primary stage of imaging.

1. B. subtillis&#160;Sporulation (Timing: 7 Days Before Microscopic Observation)
<ol>
	<li>Day 1
	<ol>
		<li>Streak a bacterial culture on a Luria-Bertani Broth (LB) agar plate (1% tryptone, 0.5% yeast extract, 1% NaCl, 1% agar)13 and incubate overnight at 37 &#176;C to obtain single colonies. Use the B. subtilis KGB80 (PS4150 gerKA gerKC gerKB-mCherry cat, gerD-gfp kan) strain and its parent background strain B. subtilis PS4150 (PS832 &#916;gerE::spc, &#916;cotE::tet) as described previously7. 
		NOTE: The use of spores with the &#916;cotE&#916;gerE background is essential in germinosome visualization, in order to minimize the autofluorescence of the spore coat layers7.</li>
		<li>Sterilize all media, tubes, pipets and other culture materials to be used with appropriate methods.</li>
	</ol>
	</li>
	<li>Day 2
	<ol>
		<li>Inoculate a single colony into 5 mL of LB medium early in the morning and incubate the culture under continuous agitation in a screw cap tube at 200 rpm/min and 37 &#176;C until the OD600 reaches 0.3-0.4 (approximately 7 h).</li>
		<li>Make 500 mL of MOPS medium (pH 7.5) containing 3 nM (NH4)6Mo7O24&#183;4H2O, 0.4 &#956;M H3BO3, 30 nM CoCl2&#183;6H2O, 10 nM CuSO4&#183;5H2O, 10 nM ZnSO4&#183;7H2O, 1.32 mM K2HPO4, 0.4 mM MgCl2&#183;6H2O, 0.276 mM K2SO4, 0.01 mM FeSO4&#183;7H2O, 0.14 mM CaCl2&#183;2H2O, 80 mM MOPS, 4 mM Tricine, 0.1 mM MnCl2&#183;2H2O, 10 mM D-glucose-monohydrate, and 10 mM NH4Cl.</li>
		<li>Prepare in screw cap tubes 10-1- 10-7 serial dilutions of the LB culture in 5 mL MOPS medium each and incubate the cultures overnight under continuous agitation at 200 rpm/min and 37 &#176;C. 
		NOTE: The serial dilutions prepared here aim to obtain a dilution in early exponential phase in the next morning. This step and the next step are necessary to allow the cells to adapt to the MOPS buffered growth medium.</li>
	</ol>
	</li>
	<li>Day 3
	<ol>
		<li>Select one of the MOPS dilutions with an OD600 of 0.3-0.4. Inoculate 0.2 mL of the culture to pre-warmed (37 &#176;C) 20 mL of MOPS medium in a conical 250 mL flask and incubate under continuous agitation until the OD600 reaches 0.3-0.4 (~7 h).</li>
		<li>Inoculate the MOPS based sporulation medium (250 mL) with 1% (v/v) pre-culture from step 1.3.1 and incubate for 3 days at 37 &#176;C in a conical liter flask under continuous agitation. For FM4-64 staining of PS4150 spores, add 2 &#181;g/mL FM4-64 probe (see Table of Materials) to the sporulation medium 1 or 2 h after reaching the peak OD600 value of vegetative growth (generally approximately 2) and allow the culture to subsequently sporulate whilst protecting it from light5.</li>
	</ol>
	</li>
	<li>Day 7: Harvesting of spores
	<ol>
		<li>Determine the sporulation yield (spores vs vegetative cells) using phase contrast microscopy at 100X magnification to distinguish the phase bright spores from phase dark cells and possibly non-mature spores. A 90 % phase bright spore yield is expected.</li>
		<li>Pellet the spores at 4,270 x g for 15 min at 4 &#176;C in round bottom centrifuge tubes. Wash the spore pellet 2-3 times with 40 mL of sterile ultra-pure Type 1 demineralized water in 50 mL conical centrifuge tubes (see Table of Materials). Spin-down at each wash at 4,270 x g for 15 min (4 &#176;C).</li>
	</ol>
	</li>
	<li>Day 7: Spore purification
	<ol>
		<li>Spore purification: Suspend the spore pellet in 750 &#181;L of 20% nonionic density gradient medium (see Table of materials) and load onto 800 &#181;L of 50% nonionic density gradient medium in sterilized microcentrifuge tubes. Centrifuge for 60 min at 21,500 x g. The pellet obtained contains the free spores. Suspend the spore pellet in 200 &#181;L of 20% nonionic density gradient medium and load onto 1 mL of 50% nonionic density gradient medium in a microcentrifuge tube, and centrifuge for 15 min at 21,500 x g.</li>
		<li>Washing the final spore preparation and storage: The pellet obtained contains the purified dormant spores. Wash the spore pellet 2-3 times with 1.5 mL of sterile ultra-pure Type 1 water (see Table of Materials) and spin-down in between at 9,560 x g for 15 min at 4 &#176;C. Finally, suspend the pellet in sterile ultra-pure Type 1 water to a final OD600 of approximately 30. Aliquots of the spores can be stored at -80 &#176;C for 8 weeks14.</li>
	</ol>
	</li>
</ol>
2. Decoating
<ol>
	<li>Treat PS4150 spores with 0.1 M NaCl/0.1 M NaOH/1% sodium dodecyl sulfate (SDS)/0.1 M dithiothreitol (DTT) at 70 &#176;C for 1 h. Wash the spores 10 times with sterilized ultra-pure Type 1 water15,16. By doing so, any adsorbed FM4-64 probe in spore&#8217;s outer membrane and outer layers will be removed.</li>
</ol>
3. Coverslip and Slide Preparation11 (Timing: 1 H Before Observation)
<ol>
	<li>Pre-clean high precision coverslips (see Table of materials) with 1 M HCl for 30 min in a gently shaking water bath. Wash the coverslips twice in ultra-pure Type 1 water, and store them in 100% (vol/vol) EtOH. Let them dry and verify their clarity before use. Pre-clean the glass slides in 70% EtOH. Let them dry and verify their clarity before use.</li>
</ol>
4. Sampling Fluorescent Microspheres or Spores in the Gene Frame Slide10 (Timing 15 Min)
<ol>
	<li>Pre-warm two 70% EtOH cleaned and air dried glass slides (see Table of materials) for several seconds on a 70 &#176;C heating block, drop 65 &#956;L of sterilized 2% agarose held at 70 &#176;C on top of one glass slide and place the other glass slide on top to spread the agarose between the slides. The agarose patch will dry in approximately 5 min.</li>
	<li>Cut the agarose patch into a 1 x 1cm section after removing one of the glass slides, add 0.4 &#956;L of sample (fluorescent microspheres or spores of ~108/mL), and transfer the patch onto the high precision coverslip by placing the coverslip onto the patch and sliding it off.</li>
	<li>Fix a Gene Frame (1.5 x 1.6 cm2, 65 &#181;L) onto the dried slide, onto which the coverslip is placed closing all corners of the frame, thus completing the slide for use in microscopy.</li>
</ol>
5. Imaging11,17(Timing: 1 H)
<ol>
	<li>Capture the transmission and fluorescence images of spores as well as a mixture of red and yellow-green carboxylate-modified fluorescent microspheres on a Structured Illumination Microscope (see Table of materials) equipped with a 100x oil objective (Numerical Aperture = 1.49), a CCD camera and image analysis software (see Table of materials). Generate all images at room temperature without the disturbance of ambient light. Make sure to always clean the 100x objective and the slide with 75% ethanol before imaging.</li>
	<li>Focus on 100 nm (diameter) fluorescent microspheres and optimize the point spread function (psf) by adjusting the correction ring on the 100x objective until a symmetric psf is obtained, thus minimizing blurring of the image. The psf is the impulse response or the response of an imaging system to a point source or point object.</li>
	<li>Select a field of view with approximately 10 round fluorescent microspheres. Apply a grating focus adjustment for both 561 nm and 488 nm excitation wave lengths as the guide for the image analysis software.</li>
	<li>Focus the spores with the transmission light and capture a transmission light image in the 16&#215; average mode with 200 ms exposures for each image.</li>
	<li>Capture 3D-SIM raw fluorescent images of the spores with the illumination mode &#8220;3D-SIM&#8221;, the camera settings to readout mode Electron Multiplying (EM) Gain 10MHz at 14-bit, and EM gain at 175. Excite the FM4-64 probe in PS4150 spores with 561 nm laser light at 20% laser power, and an illumination time of 400 ms.</li>
	<li>Excite the GerKB-mCherry and GerD-GFP in KGB80 spores with, respectively, 561 nm laser light at 30% laser power for 1 s, and 488 nm laser light at 60% laser power for 3 s. Z-Stack settings are in the top to bottom mode, 0.2 &#181;m/ step, 7 steps and 20 steps for germinosome and IM analysis, respectively. 
	NOTE: These laser parameters were applied in order to assure a maximum brightness value of the histogram window of around 4,000.</li>
</ol>
6. Reconstruct 3D-SIM Raw Images of FM4-64 Stained PS4150 Spores
<ol>
	<li>Perform the N-SIM Slice Reconstruction for the FM4-64 stained PS4150 spores&#8217; raw data. Click the Param button for Reconstruct Slice on the N-SIM pad tab sheet to open the N-SIM Slice Reconstruction window.</li>
	<li>Set the reconstruction parameters in the N-SIM Slice Reconstruction window. To get perfect reconstructed images, follow the suggestion of the N-SIM instructions and click on the appropriate controls in the N-SIM Slice Reconstruction window to set the Illumination Modulation Contrast (IMC) to Auto, High Resolution Noise Suppression (HRNS) to 1.00 and Out of the Focus Blur Suppression (OFBS) to 0.05 as starting points.</li>
	<li>Click the Reconstruct Slice button in the N-SIM Slice Reconstruction window to reconstruct the image. Evaluate the quality of the reconstructed images by the Fast Fourier Transformed (FFT) images and reconstruction score, which display after reconstruction12.</li>
	<li>Adjust the HRNS from 0.10 to 5.00 and OFBS from 0.01 to 0.50 by clicking on the appropriate controls in the N-SIM Slice Reconstruction window until the best parameter settings are obtained.</li>
	<li>Click the Apply button in the N-SIM Slice Reconstuction window to apply changed parameters. Click Close button to close the window.</li>
	<li>Make a FM4-64 stained PS4150 spores raw image active and click the Reconstruct Slice button on the N-SIM Pad tab sheet to execute Slice Reconstruction. Save the reconstructed image.</li>
</ol>
7. Image Analysis
<ol>
	<li>Convert Pseudo-Widefield images of the KGB80 germinosome
	<ol>
		<li>Convert 3D-SIM raw images of KGB80 into Pseudo-Widefield images by activating with a left click the ImageJ plugin SIMcheck utility Raw Data SI to Pseudo-Widefield12. Pseudo-Widefield averages images from raw SIM data and assembles, for comparison, an image equivalent to conventional widefield illumination12. For ImageJ itself see https://imagej.net/Welcome. Randomly select ~25 spores in each inverted transmission image for later germinosome analysis in the fluorescent Pseudo-Widefield images.</li>
		<li>Select in total approximately 350 (in this example, 346) KGB80 spores in 14 fields of view from two slides. The GerD-GFP and GerKB-mCherry fluorescent foci numbers in selected KGB80 spores should be counted independently by two researchers. The researcher can refer back to the 3D-SIM raw images whenever in doubt of the presence of separate fluorescent germinosome foci in spores.</li>
		<li>Assess the maximum intensities of each GerD-GFP and GerKB-mCherry focus and the integrated intensity of each KGB80 spore&#8217;s 3D image with ImageJ.</li>
	</ol>
	</li>
	<li>Analyze Pseudo-Widefield images of the KGB80 germinosome
	<ol>
		<li>Use the mean integrated intensity value of 7 stacks as the integrated signal intensity of the KGB80 spore. Determine background intensities by imaging the background strain PS4150 using identical settings. Regard fluorescent spots in individual KGB80 spores as germinosome foci when they are clearly distinguishable from the background.</li>
		<li>Apply one-way ANOVA-tests for significance determination with software Origin 9.0. considering P values &#60;0.05 as statistically significant. Use the Spearman&#8217;s rank correlation coefficient18 to evaluate the correlation of GerD-GFP and GerKB-mCherry foci number and the measurements of the integrated signal intensity.</li>
	</ol>
	</li>
</ol>

<ol><li>Setlow, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Germination+of+spores+of+Bacillus+species%3A+what+we+know+and+do+not+know%5BTitle%5D)+AND+%22Journal+of+Bacteriology%22%5BJournal%5D)">Germination of spores of Bacillus species: what we know and do not know.</a> Journal of Bacteriology. 196 (7), 1297-1305 (2014).</li>
<li>Setlow, P., Wang, S., Li, Y. -Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Germination+of+spores+of+the+orders+Bacillales+and+Clostridiales%5BTitle%5D)+AND+%22Annual+Review+of+Microbiology%22%5BJournal%5D)">Germination of spores of the orders Bacillales and Clostridiales.</a> Annual Review of Microbiology. 71 (1), (2017).</li>
<li>Vepachedu, V. R., Setlow, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Analysis+of+interactions+between+nutrient+germinant+receptors+and+SpoVA+proteins+of+Bacillus+subtilis+spores%5BTitle%5D)+AND+%22FEMS+Microbiology+Letters%22%5BJournal%5D)">Analysis of interactions between nutrient germinant receptors and SpoVA proteins of Bacillus subtilis spores.</a> FEMS Microbiology Letters. 274 (1), 42-47 (2007).</li>
<li>Setlow, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Summer+meeting+2013%E2%80%93when+the+sleepers+wake%3A+the+germination+of+spores+of+Bacillus+species%5BTitle%5D)+AND+%22Journal+of+Applied+Microbiology%22%5BJournal%5D)">Summer meeting 2013–when the sleepers wake: the germination of spores of Bacillus species.</a> Journal of Applied Microbiology. 115 (6), 1251-1268 (2013).</li>
<li>Cowan, A. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Lipids+in+the+inner+membrane+of+dormant+spores+of+Bacillus+species+are+largely+immobile%5BTitle%5D)+AND+%22Proceedings+of+the+National+Academy+of+Sciences+of+the+United+States+of+America%22%5BJournal%5D)">Lipids in the inner membrane of dormant spores of Bacillus species are largely immobile.</a> Proceedings of the National Academy of Sciences of the United States of America. 101 (20), 7733-7738 (2004).</li>
<li>Loison, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Direct+investigation+of+viscosity+of+an+atypical+inner+membrane+of+Bacillus+spores%3A+a+molecular+rotor%2FFLIM+study%5BTitle%5D)+AND+%22Biochimica+et+Biophysica+Acta+%28BBA%29-Biomembranes%22%5BJournal%5D)">Direct investigation of viscosity of an atypical inner membrane of Bacillus spores: a molecular rotor/FLIM study.</a> Biochimica et Biophysica Acta (BBA)-Biomembranes. 1828 (11), 2436-2443 (2013).</li>
<li>Griffiths, K. K., Zhang, J., Cowan, A. E., Yu, J., Setlow, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Germination+proteins+in+the+inner+membrane+of+dormant+Bacillus+subtilis+spores+colocalize+in+a+discrete+cluster%5BTitle%5D)+AND+%22Molecular+Microbiology%22%5BJournal%5D)">Germination proteins in the inner membrane of dormant Bacillus subtilis spores colocalize in a discrete cluster.</a> Molecular Microbiology. 81 (4), 1061-1077 (2011).</li>
<li>Troiano, A. J., Zhang, J., Cowan, A. E., Yu, J., Setlow, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Analysis+of+the+dynamics+of+a+Bacillus+subtilis+spore+germination+protein+complex+during+spore+germination+and+outgrowth%5BTitle%5D)+AND+%22Journal+of+Bacteriology%22%5BJournal%5D)">Analysis of the dynamics of a Bacillus subtilis spore germination protein complex during spore germination and outgrowth.</a> Journal of Bacteriology. 197 (2), 252-261 (2015).</li>
<li>Wegel, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Imaging+cellular+structures+in+super-resolution+with+SIM%2C+STED+and+Localisation+Microscopy%3A+A+practical+comparison%5BTitle%5D)+AND+%22Scientific+Reports%22%5BJournal%5D)">Imaging cellular structures in super-resolution with SIM, STED and Localisation Microscopy: A practical comparison.</a> Scientific Reports. 6, 27290(2016).</li>
<li>Pandey, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Live+cell+imaging+of+germination+and+outgrowth+of+individual+Bacillus+subtilis+spores%3B+the+effect+of+heat+stress+quantitatively+analyzed+with+SporeTracker%5BTitle%5D)+AND+%22PloS+One%22%5BJournal%5D)">Live cell imaging of germination and outgrowth of individual Bacillus subtilis spores; the effect of heat stress quantitatively analyzed with SporeTracker.</a> PloS One. 8 (3), e58972(2013).</li>
<li>Demmerle, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Strategic+and+practical+guidelines+for+successful+structured+illumination+microscopy%5BTitle%5D)+AND+%22Nature+Protocols%22%5BJournal%5D)">Strategic and practical guidelines for successful structured illumination microscopy.</a> Nature Protocols. 12 (5), 988-1010 (2017).</li>
<li>Ball, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((SIMcheck%3A+a+toolbox+for+successful+super-resolution+structured+illumination+microscopy%5BTitle%5D)+AND+%22Scientific+Reports%22%5BJournal%5D)">SIMcheck: a toolbox for successful super-resolution structured illumination microscopy.</a> Scientific Reports. 5, 15915(2015).</li>
<li>Bertani, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((STUDIES+ON+LYSOGENESIS+I.%3A+The+Mode+of+Phage+Liberation+by+Lysogenic+Escherichia+coli1%5BTitle%5D)+AND+%22Journal+of+Bacteriology%22%5BJournal%5D)">STUDIES ON LYSOGENESIS I.: The Mode of Phage Liberation by Lysogenic Escherichia coli1.</a> Journal of Bacteriology. 62 (3), 293(1951).</li>
<li>Pandey, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Quantitative+analysis+of+the+effect+of+specific+tea+compounds+on+germination+and+outgrowth+of+Bacillus+subtilis+spores+at+single+cell+resolution%5BTitle%5D)+AND+%22Food+Microbiology%22%5BJournal%5D)">Quantitative analysis of the effect of specific tea compounds on germination and outgrowth of Bacillus subtilis spores at single cell resolution.</a> Food Microbiology. 45, 63-70 (2015).</li>
<li>Zheng, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Bacillus+subtilis+spore+inner+membrane+proteome%5BTitle%5D)+AND+%22Journal+of+Proteome+Research%22%5BJournal%5D)">Bacillus subtilis spore inner membrane proteome.</a> Journal of Proteome Research. 15 (2), 585-594 (2016).</li>
<li>Vepachedu, V. R., Setlow, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Localization+of+SpoVAD+to+the+inner+membrane+of+spores+of+Bacillus+subtilis%5BTitle%5D)+AND+%22Journal+of+Bacteriology%22%5BJournal%5D)">Localization of SpoVAD to the inner membrane of spores of Bacillus subtilis.</a> Journal of Bacteriology. 187 (16), 5677-5682 (2005).</li>
<li>Schouten, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Imaging+dendritic+spines+of+rat+primary+hippocampal+neurons+using+structured+illumination+microscopy%5BTitle%5D)+AND+%22Journal+of+Visualized+Experiments%3A+JoVE%22%5BJournal%5D)">Imaging dendritic spines of rat primary hippocampal neurons using structured illumination microscopy.</a> Journal of Visualized Experiments: JoVE. (87), (2014).</li>
<li>Mukaka, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+guide+to+appropriate+use+of+correlation+coefficient+in+medical+research%5BTitle%5D)+AND+%22Malawi+Medical+Journal%22%5BJournal%5D)">A guide to appropriate use of correlation coefficient in medical research.</a> Malawi Medical Journal. 24 (3), 69-71 (2012).</li>
<li>Stewart, K. A., Setlow, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Numbers+of+individual+nutrient+germinant+receptors+and+other+germination+proteins+in+spores+of+Bacillus+subtilis%5BTitle%5D)+AND+%22Journal+of+Bacteriology%22%5BJournal%5D)">Numbers of individual nutrient germinant receptors and other germination proteins in spores of Bacillus subtilis.</a> Journal of Bacteriology. 195 (16), 3575-3582 (2013).</li>
<li>Laflamme, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Flow+cytometry+analysis+of+germinating+Bacillus+spores%2C+using+membrane+potential+dye%5BTitle%5D)+AND+%22Archives+of+Microbiology%22%5BJournal%5D)">Flow cytometry analysis of germinating Bacillus spores, using membrane potential dye.</a> Archives of Microbiology. 183 (2), 107-112 (2005).</li>
<li>Magge, A., Setlow, B., Cowan, A. E., Setlow, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Analysis+of+dye+binding+by+and+membrane+potential+in+spores+of+Bacillus+species%5BTitle%5D)+AND+%22Journal+of+Applied+Microbiology%22%5BJournal%5D)">Analysis of dye binding by and membrane potential in spores of Bacillus species.</a> Journal of Applied Microbiology. 106 (3), 814-824 (2009).</li>
<li>Ghosh, S., Scotland, M., Setlow, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Levels+of+germination+proteins+in+dormant+and+superdormant+spores+of+Bacillus+subtilis%5BTitle%5D)+AND+%22Journal+of+Bacteriology%22%5BJournal%5D)">Levels of germination proteins in dormant and superdormant spores of Bacillus subtilis.</a> Journal of Bacteriology. 194 (9), 2221-2227 (2012).</li>
<li>Abhyankar, W. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+influence+of+sporulation+conditions+on+the+spore+coat+protein+composition+of+Bacillus+subtilis+Spores%5BTitle%5D)+AND+%22Frontiers+in+microbiology%22%5BJournal%5D)">The influence of sporulation conditions on the spore coat protein composition of Bacillus subtilis Spores.</a> Frontiers in microbiology. 7, 1636(2016).</li>
<li>Rose, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Comparison+of+the+properties+of+Bacillus+subtilis+spores+made+in+liquid+or+on+agar+plates%5BTitle%5D)+AND+%22Journal+of+Applied+Microbiology%22%5BJournal%5D)">Comparison of the properties of Bacillus subtilis spores made in liquid or on agar plates.</a> Journal of Applied Microbiology. 103 (3), 691-699 (2007).</li>
<li>Stewart, K. A., Yi, X., Ghosh, S., Setlow, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Germination+protein+levels+and+rates+of+germination+of+spores+of+Bacillus+subtilis+with+overexpressed+or+deleted+genes+encoding+germination+proteins%5BTitle%5D)+AND+%22J+Bacteriol%22%5BJournal%5D)">Germination protein levels and rates of germination of spores of Bacillus subtilis with overexpressed or deleted genes encoding germination proteins.</a> J Bacteriol. 194 (12), 3156-3164 (2012).</li>
<li>Ramirez-Peralta, A., Zhang, P., Li, Y. -q, Setlow, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Effects+of+sporulation+conditions+on+the+germination+and+germination+protein+levels+of+Bacillus+subtilis+spores%5BTitle%5D)+AND+%22Applied+and+Environmental+Microbiology%22%5BJournal%5D)">Effects of sporulation conditions on the germination and germination protein levels of Bacillus subtilis spores.</a> Applied and Environmental Microbiology. 78 (8), 2689-2697 (2012).</li>
<li>Laue, M., Han, H. -M., Dittmann, C., Setlow, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Intracellular+membranes+of+bacterial+endospores+are+reservoirs+for+spore+core+membrane+expansion+during+spore+germination%5BTitle%5D)+AND+%22Scientific+Reports%22%5BJournal%5D)">Intracellular membranes of bacterial endospores are reservoirs for spore core membrane expansion during spore germination.</a> Scientific Reports. 8, 11388(2018).</li>
<li>Strahl, H., Bürmann, F., Hamoen, L. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+actin+homologue+MreB+organizes+the+bacterial+cell+membrane%5BTitle%5D)+AND+%22Nature+Communications%22%5BJournal%5D)">The actin homologue MreB organizes the bacterial cell membrane.</a> Nature Communications. 5, (2014).</li>
<li>Strahl, H., Hamoen, L. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Membrane+potential+is+important+for+bacterial+cell+division%5BTitle%5D)+AND+%22Proceedings+of+the+National+Academy+of+Sciences+of+the+United+States+of+America%22%5BJournal%5D)">Membrane potential is important for bacterial cell division.</a> Proceedings of the National Academy of Sciences of the United States of America. 107 (27), 12281-12286 (2010).</li>
<li>Swarge, B. N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((%22One-Pot%22+sample+processing+methodfor+proteome-wide+analysis+of+microbial+cells+and+spores%5BTitle%5D)+AND+%22Proteomics+Clinical+Applications%22%5BJournal%5D)">"One-Pot" sample processing methodfor proteome-wide analysis of microbial cells and spores.</a> Proteomics Clinical Applications. (5), 1700169(2018).</li>
</ol>

No conflicts of interest declared.

&#160;The protocol presented contains a standard 3D-SIM procedure for analysis of FM4-64 stained B. subtilis spores that includes sporulation, slide preparation and imaging processes. In addition, the protocol describes a modified SIMcheck (ImageJ)-assisted 3D imaging process for SIM microscopy of B. subtilis spore germinosomes labeled with fluorescent reporters. The latter procedure allowed us to observe this dim substructure with enhanced contrast. By coupling two imaging procedures, it is possible to...

Spores of the orders Bacillales and Clostridiales are metabolically dormant and extraordinarily resistant to harsh decontamination regimes, but unless they germinate, cannot cause deleterious effects in humans1. In nutrient germinant triggered germination of Bacillus subtilis (B. subtilis) spores, the initiation event is germinant binding to germinant receptors (GRs) located in the spore&#8217;s inner membrane (IM). Subsequently, the GRs transduce signals to the SpoVA channel protein also located in the IM. This results in the onset of the exchange of spore core pyridine-2,6-dicarboxylic acid (dipicolinic acid; DPA; comprising 20% of spore core dry wt) for water via the SpoVA channel. Subsequently, the DPA release triggers the activation of cortex peptidoglycan hydrolysis, and additional water uptake follows2,3,4. These events lead to mechanical stress on the coat layers, its subsequent rupture, the onset of outgrowth and, finally, vegetative growth. However, the exact molecular details of the germination process are still far from resolved.
A major question about spore germination concerns the biophysical properties of the lipids surrounding the IM germination proteins as well as the IM SpoVA channel proteins. This largely immobile IM lipid bilayer is the main permeability barrier for many small molecules, including toxic chemical preservatives, some of which exert their action in the spore core or vegetative cell cytoplasm5,6. The IM lipid bilayer is likely in a gel state, although there is a significant fraction of mobile lipids in the IM5. The spore&#8217;s IM also has the potential for significant expansion5. Thus, the surface area of the IM increases 1.6-fold upon germination without additional membrane synthesis and is accompanied by the loss of this membrane&#8217;s characteristic low permeability and lipid immobility5,6.
While the molecular details of the activation of germination proteins and organization of IM lipids in spores are attractive topics for study, the small size of B. subtilis spores and the relatively low abundance of germination proteins, pose a challenge to microscopic analyses. Griffiths et al. compelling epifluorescence microscope evidence, using fluorescent reporters fused to germination proteins, suggests that in B. subtilis spores the scaffold protein GerD organizes three GR subunits (A, B and C) for the GerA, B and K GRs, in a cluster7. They coined the term &#8216;germinosome&#8217; for this cluster of germination proteins and described the structures as ~300 nm large IM protein foci8. Upon initiation of spore germination, fluorescent germinosome foci ultimately change into larger disperse fluorescent patterns, with &#62;75% of spore populations displaying this pattern in spores germinated for 1 h with L-valine8. Note that the paper mentioned above used averaged images from dozens of consecutive fluorescent pictures, to gain statistical power and overcome the hurdle of low fluorescent signals observed during imaging. This visualization of these structures in bacterial spores was at the edge of what is technically feasible with classical microscopic tools and neither an evaluation of the amount of foci in a single spore nor their more detailed subcellular localization was possible with this approach.
Here, we demonstrate the use of Structured Illumination Microscopy (SIM) to obtain a detailed visualization and quantification of the germinosome(s) in spores of B. subtilis, as well as of their IM lipid domains9. The protocol also contains instructions for the sporulation, slide preparation and image analysis by SIMcheck (v1.0, an imageJ plugin) as well as ImageJ10,11,12.

<table><tbody><tr><td>Air dried glass slides</td><td>Menzel Gl&amp;auml;ser</td><td>630-2870</td><td></td></tr><tr><td>APO TIRF N20R8 100&amp;times; oil objective (NA=1.49)</td><td></td><td></td><td></td></tr><tr><td>&lt;em&gt;B. subtilis&lt;/em&gt; KGB80 (PS4150 &lt;em&gt;gerKA gerKC gerKB-mCherry cat, gerD-gfp kan&lt;/em&gt;)</td><td></td><td></td><td></td></tr><tr><td>&lt;em&gt;B. subtilis &lt;/em&gt;PS4150 (PS832&lt;em&gt; &amp;Delta;gerE::spc, &amp;Delta;cotE::tet&lt;/em&gt;)</td><td></td><td></td><td></td></tr><tr><td>Erlenmeyer flasks 1 L</td><td>Sigma-Aldrich</td><td>Z567868</td><td></td></tr><tr><td>Erlenmeyer flasks 250 mL</td><td>Sigma-Aldrich</td><td>Z723088</td><td></td></tr><tr><td>FluoSpheres carboxylate-modified microspheres</td><td>Invitrogen, 0.1 &amp;mu;m</td><td>F8803</td><td></td></tr><tr><td>FM4-64</td><td>Thermo Fisher Scientific</td><td>F34653</td><td></td></tr><tr><td>Histodenz nonionic density gradient medium</td><td>Sigma-Aldrich</td><td>D2158</td><td></td></tr><tr><td>ImageJ</td><td></td><td></td><td></td></tr><tr><td>iXON3 DU-897 X-6515 CCD camera</td><td>Andor Technology</td><td></td><td>https://imagej.net/Welcome</td></tr><tr><td>LB Agar</td><td>Sigma-Aldrich</td><td>L2897</td><td></td></tr><tr><td>Microfuge tubes 1.5 mL</td><td>Thermo Fisher Scientific</td><td>3451PK</td><td></td></tr><tr><td>Microscope imaging software</td><td>Nikon, Japan</td><td>NIS-Element AR 4.51.01</td><td></td></tr><tr><td>MilliQ Ultrapure Deminerilzed Water</td><td>Millipore</td><td>Milli-Q IQ 7003</td><td></td></tr><tr><td>Nikon Eclipse Ti microscope</td><td></td><td></td><td></td></tr><tr><td>Polypropylene Screw Cap Bottle 180 mL</td><td>Thermo Fisher Scientific</td><td>75003800</td><td></td></tr><tr><td>Precision Coverslips</td><td>Paul Marienfeld</td><td>117650</td><td></td></tr><tr><td>Round Bottom tubes 15 mL</td><td>Thermo Fisher Scientific</td><td></td><td>Nunc TM</td></tr><tr><td>Screw cap tubes 50 mL</td><td>Thermo Fisher Scientific</td><td></td><td>Nunc TM</td></tr></tbody></table>

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.

The current protocol presents a SIM microscope imaging procedure for bacterial spores. The sporulation and slide preparation procedures were carried out as shown in Figure 1 before imaging. Later, the imaging and analysis procedures were applied both for dim (fluorescent protein labeled germination proteins) and bright (lipophilic probe stained IM) spore samples as shown in the following text.
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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.

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 ...

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9.8K Views.  University of Amsterdam. Super-resolutie gestructureerde verlichting microscopie, 3D-SIM, is een innovatieve techniek voor de visualisatie van fluorescerende reporter eiwitten van kiemreceptor clusters in sporenmembranen. Met behulp van deze procedures kan een onovertroffen oplossing van germinosome lokalisatie en sporen binnenmembraan lipide domeinen worden bereikt. De techniek wordt gedemonstreerd door promovendus Juan Wen en Master of Science student Raymond Pasman van ons laboratorium.Voordat u met de proc...

Video: Visualization of Germinosomes and the Inner Membrane in Bacillus subtilis Spores

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

Visualization of Cortex Organization and Dynamics in Microorganisms, using Total Internal Reflection Fluorescence Microscopy

TIRF microscopy has emerged as a powerful imaging technology to study spatio-temporal dynamics of fluorescent molecules in vitro and in living cells1. The optical phenomenon of total internal reflection occurs when light passes from a medium with high refractive index into a medium with low refractive index at an angle larger than a characteristic critical angle (i.e. closer to being parallel with the boundary). Although all light is reflected back under such conditions, an evanescent wave is created that propagates across and along the boundary, which decays exponentially with distance, and only penetrates sample areas that are 100-200 nm near the interface2. In addition to providing superior axial resolution, the reduced excitation of out of focus fluorophores creates a very high signal to noise ratios and minimizes damaging effects of photobleaching2,3. Being a widefield technique, TIRF also allows faster image acquisition than most scanning based confocal setups.
At first glance, the low penetration depth of TIRF seems to be incompatible with imaging of bacterial and fungal cells, which are often surrounded by thick cell walls. On the contrary, we have found that the cell walls of yeast and bacterial cells actually improve the usability of TIRF and increase the range of observable structures4-8. Many cellular processes can therefore be directly accessed by TIRF in small, single-cell microorganisms, which often offer powerful genetic manipulation techniques. This allows us to perform in vivo biochemistry experiments, where kinetics of protein interactions and activities can be directly assessed in living cells.
We describe here the individual steps required to obtain high quality TIRF images for Saccharomyces cerevisiae or Bacillus subtilis cells. We point out various problems that can affect TIRF visualization of fluorescent probes in cells and illustrate the procedure with several application examples. Finally, we demonstrate how TIRF images can be further improved using established image restoration techniques.

Total Internal Reflection Fluorescence (TIRF) microscopy is a powerful approach to observe structures close to the cell surface at high contrast and temporal resolution. We demonstrate how TIRF can be employed to study protein dynamics at the cortex of cell wall-enclosed bacterial and fungal cells.

TIRF microscopy has emerged as  a powerful imaging technology to study spatio-temporal dynamics of fluorescent molecules  in vitro and in living cells1. ...

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 ...

Biology

Detecting Cortex Fragments During Bacterial Spore Germination

The process of endospore germination in Clostridium difficile, and other Clostridia, increasingly is being found to differ from the model spore-forming bacterium, Bacillus subtilis. Germination is triggered by small molecule germinants and occurs without the need for macromolecular synthesis. Though differences exist between the mechanisms of spore germination in species of Bacillus and Clostridium, a common requirement is the hydrolysis of the peptidoglycan-like cortex which allows the spore core to swell and rehydrate. After rehydration, metabolism can begin and this, eventually, leads to outgrowth of a vegetative cell. The detection of hydrolyzed cortex fragments during spore germination can be difficult and the modifications to the previously described assays can be confusing or difficult to reproduce. Thus, based on our recent report using this assay, we detail a step-by-step protocol for the colorimetric detection of cortex fragments during bacterial spore germination.

Herein, we describe a colorimetric assay to detect the presence of reducing sugars during bacterial spore germination.

The process of endospore germination in Clostridium difficile, and other Clostridia, increasingly is being found to differ from the model spore-forming ...

Visualizing Yeast Organelles with Fluorescent Protein Markers

The budding yeast, Saccharomyces cerevisiae, is a classic model system in studying organelle function and dynamics. In our previous works, we have constructed fluorescent protein-based markers for major organelles and endomembrane structures, including the nucleus, endoplasmic reticulum (ER), Golgi apparatus, endosomes, vacuoles, mitochondria, peroxisomes, lipid droplets, and autophagosomes. The protocol presented here describes the procedures for using these markers in yeast, including DNA preparation for yeast transformation, selection and evaluation of transformants, fluorescent microscopic observation, and the expected outcomes. The text is geared toward researchers who are entering the field of yeast organelle study from other backgrounds. Essential steps are covered, as well as technical notes about microscope hardware considerations and several common pitfalls. It provides a starting point for people to observe yeast subcellular entities by live-cell fluorescent microscopy. These tools and methods can be used to identify protein subcellular localization and track organelles of interest in time-lapse imaging.

Here we describe the use of a set of fluorescent protein-based organelle markers in live-cell imaging of the budding yeast, Saccharomyces cerevisiae.

The budding yeast, Saccharomyces cerevisiae, is a classic model system in studying organelle function and dynamics. In our previous works, we have ...

Biochemistry

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 ...

Visual and Microscopic Evaluation of Streptomyces Developmental Mutants

Streptomycetes are filamentous soil bacteria belonging to the phylum Actinobacteria that are found throughout the world and produce a wide array of antibiotics and other secondary metabolites. Streptomyces coelicolor is a well-characterized, non-pathogenic species that is amenable to a variety of analyses in the lab. The phenotyping methods described here use S. coelicolor as a model streptomycete; however, the methods are applicable to all members of this large genus as well as some closely related actinomycetes. Phenotyping is necessary to characterize new species of Streptomyces identified in the environment, and it is also a vital first step in characterizing newly isolated mutant strains of Streptomyces. Proficiency in phenotyping is important for the many new researchers who are entering the field of Streptomyces research, which includes the study of bacterial development, cell division, chromosome segregation, and second messenger signaling. The recent crowdsourcing of antibiotic discovery through the isolation of new soil microbes has resulted in an increased need for training in phenotyping for instructors new to the field of Streptomyces research and their college or high school students. This manuscript describes methods for bacterial strain propagation, storage, and characterization through visual and microscopic examination. After reading this article, new researchers (microbiology education laboratories and citizen scientists) should be able to manipulate Streptomyces strains and begin visual characterization experiments.

Visual and Microscopic Evaluation of Streptomyces Developmental Mutants

Here we present protocols for novice researchers to initiate phenotyping for the pharmacologically important bacterial genus Streptomyces.

Streptomycetes are filamentous soil bacteria belonging to the phylum Actinobacteria that are found throughout the world and produce a wide array of ...

Applying Live Cell Imaging and Cryo-Electron Tomography to Resolve Spatiotemporal Features of the Legionella pneumophila Dot/Icm Secretion System

The Dot/Icm secretion system of Legionella pneumophila is a complex type IV secretion system (T4SS) nanomachine that localizes at the bacterial pole and mediates the delivery of protein and DNA substrates to target cells, a process generally requiring direct cell-to-cell contact. We have recently solved the structure of the Dot/Icm apparatus by cryo-electron tomography (cryo-ET) and showed that it forms a cell envelope-spanning channel that connects to a cytoplasmic complex. Applying two complementary approaches that preserve the native structure of the specimen, fluorescent microscopy in living cells and cryo-ET, allows in situ visualization of proteins and assimilation of the stoichiometry and timing of production of each machine component relative to other Dot/Icm subunits. To investigate the requirements for polar positioning and to characterize dynamic features associated with T4SS machine biogenesis, we have fused a gene encoding superfolder green fluorescent protein to Dot/Icm ATPase genes at their native positions on the chromosome. The following method integrates quantitative fluorescence microscopy of living cells and cryo-ET to quantify polar localization, dynamics, and structure of these proteins in intact bacterial cells. Applying these approaches for studying the Legionella pneumophila T4SS is useful for characterizing the function of the Dot/Icm system and can be adapted to study a wide variety of bacterial pathogens that utilize the T4SS or other types of bacterial secretion complexes.

Applying Live Cell Imaging and Cryo-Electron Tomography to Resolve Spatiotemporal Features of the Legionella pneumophila Dot/Icm Secretion System

Imaging of bacterial cells is an emerging systems biology approach focused on defining static and dynamic processes that dictate the function of large macromolecular machines. Here, integration of quantitative live cell imaging and cryo-electron tomography is used to study Legionella pneumophila type IV secretion system architecture and functions.

The Dot/Icm secretion system of Legionella pneumophila is a complex type IV secretion system (T4SS) nanomachine that localizes at the bacterial pole and ...

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.

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.

Plasma sterilization is a promising alternative to conventional sterilization methods for industrial, clinical, and spaceflight purposes. Low pressure ...

Application of Membrane and Cell Wall Selective Fluorescent Dyes for Live-Cell Imaging of Filamentous Fungi

The application of membrane and cell wall selective fluorescent dyes for live-cell imaging analyses of organelle dynamics in fungal cells started two decades ago and since then continues to contribute greatly to our understanding of the filamentous fungal lifestyle. This paper provides a practical guide for the utilization of the two membrane dyes FM 1-43 and FM 4-64 and the four cell wall stains Calcofluor White M2R, Solophenyl Flavine 7GFE 500, Pontamine Fast Scarlet 48 and Congo Red. The focus is on their low-dose application to ascertain artefact-free staining, their co-imaging properties, and their quantitative evaluation. The presented methods are applicable to all filamentous fungal samples that can be prepared in the described ways. The fundamental staining approaches can serve as starting points for adaptations to species that might require different cultivation conditions. First, biophysical and biochemical properties are reviewed as their understanding is essential for using these dyes as truly vital fluorescent stains. Secondly, step-by-step protocols are presented that detail the preparation of various fungal sample types for fluorescent live-cell imaging. Finally, example experiments illustrate different approaches to: (1) identify defects in the spatio-temporal organization of endocytosis in genetic mutants, (2) comparatively characterize shared and distinct co-localization of GFP-labeled target proteins in the endocytic pathway, (3) identify morphogenetic cell wall defects in a genetic mutant, and (4) monitor cell wall biogenesis in real time.

Vital fluorescent dyes are essential tools for live-cell imaging analyses in modern fungal cell biology. This paper details the application of established and lesser-known fluorescent dyes for tracking plasma membrane dynamics, endo-/exocytosis and cell wall morphogenesis in filamentous fungi.

The application of membrane and cell wall selective fluorescent dyes for live-cell imaging analyses of organelle dynamics in fungal cells started two ...

Visualization of Germinosomes and the Inner Membrane in Bacillus subtilis Spores

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