This study was financed in part by the Coordena&#231;&#227;o de Aperfei&#231;oamento de Pessoal de N&#237;vel Superior-Brasil (CAPES)-Finance Code 001; Governo do Estado do Amazonas with resources from Funda&#231;&#227;o de Amparo &#224; Pesquisa do Estado do Amazonas-FAPEAM; Conselho Nacional de Desenvolvimento Cient&#237;fico e Tecnol&#243;gico (CNPq).&#160;The authors thank the Program for Technological Development in Tools for Health PDTIS-FIOCRUZ for use of its facilities.

See the Table of Materials for details related to all materials, instruments, and software used in this protocol.
1. Flow cytometry setting
<ol>
	<li>Alignment of optical parameters of flow cytometer coupled to a computer
	<ol>
		<li>Log in to Cytometer software.</li>
		<li>From the software workspace, select Cytometer | Startup and wait a few minutes | Clean mode | Sit fluids. 
		&#8203;NOTE: Air bubbles and obstructions were removed during the cytometer initiation process, prior to sample acquisition.</li>
	</ol>
	</li>
	<li>Quality control
	<ol>
		<li>Use the quality control reagent to adjust the voltages of the photomultiplier tubes and evaluate their sensitivity.
		<ol>
			<li>Prepare the quality control reagent in a polystyrene tube.</li>
			<li>For defining the baseline, prepare the suspension beads by adding 0.5 mL of diluent (10 mM filtered PBS, pH 7) and 3 drops of beads to the labeled tube.</li>
			<li>Mix the bead vial by gentle inversion.</li>
		</ol>
		</li>
		<li>From the software workspace, select Cytometer | CST to connect to the CST interface. Wait a few minutes and observe the message cytometer disconnected.</li>
		<li>Attach the polystyrene tube containing the quality control reagent to the flow cytometer probe.</li>
	</ol>
	</li>
	<li>Verification of the configuration of the cytometer
	<ol>
		<li>In the System Summary window, check that the cytometer configuration is appropriate for the experiment.</li>
		<li>Select the setup beads batch ID corresponding to verify that the setup beads lot ID selected matches the current lot of CS&#38;T research beads.</li>
	</ol>
	</li>
	<li>Performance check
	<ol>
		<li>Select the option Check Performance and click Run.</li>
		<li>Upon completion of the performance check, the performance results will be displayed. View the Cytometer Performance Report. Either click Finish to complete the performance check or remove the tube from the cytometer and review the results in the System Summary window.</li>
		<li>Observe the final result of the morphometric and fluorescence sensitivity analysis that will appear in the | system summary| cytometer performance result| with the status: PASSED</li>
	</ol>
	</li>
</ol>
2. Preparation of the spores
<ol>
	<li>After sporulation, rinse the spores 3x with ultrapure ice-cold water by centrifugation at 17,949 &#215; g for 10 min.</li>
	<li>Resuspend the pellet obtained from the last centrifugation with 10 mL of ultrapure water.</li>
	<li>Autoclave the spores for 45 min at 121 &#186;C for inactivation of the vegetative cells. 
	NOTE: Previous tests carried out by our group comparing different inactivation methods demonstrated that the conditions mentioned in step 2.3 presented high efficiency, showing no colony formation in the plating analysis method on the LB agar medium.</li>
</ol>
3. Quantification of autoclaved spores using flow cytometry
<ol>
	<li>Take 50 &#181;L of autoclaved spores and incubate them protected from light with ethidium bromide (EtBr, 10 mg/mL diluted in water) at a dilution factor of 0.05% v/v for 30 min (Figure 1A).</li>
	<li>Wash the spores 3x with 1x phosphate-buffered saline (PBS) by centrifugation at 17,949 &#215; g for 10 min and resuspend in 1x PBS.</li>
	<li>Add 10 &#181;L of beads, following the manufacturer&#39;s recommendations for dilution.</li>
	<li>Analyze the sample using a flow cytometer as described in step 4. 
	&#8203;NOTE: It is important to emphasize that the beads should be pipetted as homogeneously as possible since disparities were observed in the calculations when this step was not well executed.</li>
</ol>
4. Analysis using flow cytometry
<ol>
	<li>Log in to Cytometer software.</li>
	<li>From the software workspace, select Cytometer | Clean mode | Sit fluids.</li>
	<li>Adjust to the workspace to determine the analysis in flow cytometry.
	<ol>
		<li>Define the gates for the analyses (Figure 2).
		<ol>
			<li>Define the gating strategy based on the morphometric and fluorescence characteristics of the particles based on the negative control (unmarked spores).</li>
		</ol>
		</li>
		<li>To determine cell morphometry, choose Dotplot graphic for analyses of FSC-A parameters on the x-axis and SSC-A on the y-axis.</li>
		<li>To determine fluorescence, choose FL3 Dotplot plots on the y-axis using FL5 on the x-axis and create the gates in four quadrants.</li>
	</ol>
	</li>
	<li>Use a 12 x 75 mm stoppered polystyrene tube containing unlabeled samples, previously labeled with single-color EtBr, single-color APC, and multicolor EtBr+ APC.</li>
	<li>Mix the tube very gently containing the negative control and attach it to the flow cytometer probe.</li>
	<li>Click on Acquire.</li>
	<li>Set the power of the lasers by navigating to Cytometer | Parameters | FSC (375) and SSC (275).</li>
	<li>Set the threshold to (500) Cytometer | Threshold.</li>
	<li>To remove autofluorescence, analyze the dye-free sample containing only spores.</li>
	<li>Adjust the voltages for the filter detectors: FL3 (603) and FL5 (538) to discriminate negative and positive populations with respect to the chosen fluorescence in the controls. Cytometer | Parameters.</li>
	<li>Cytometer | Compensation | Set FL5/FL3 setup offset to 1.0.</li>
	<li>After morphometry and fluorescent analysis, configure the device for Acquisition 30,000 events | Experiment | Experiment layout | Acquisition |30,000 events&#8230;.</li>
	<li>After adjusting the parameters, acquire data for the samples that are labeled and contain beads in the flow cytometer.</li>
	<li>Calculate spore concentration as described by the manufacturer&#39;s instructions using equation (1). 
	<img alt="Equation 1" src="/files/ftp_upload/65141/65141eq01.jpg" style="margin: auto;" />&#160; &#160; (1) 
	NOTE: For this study, the bead quantification method was compared to the Petroff-Hausser counting chamber analysis. In addition, the labeling of autoclaved spores was compared to the labeling of non-autoclaved spores.</li>
</ol>
5. Estimation of the protein coupling index on a spore surface using flow cytometry
<ol>
	<li>Harvest by centrifugation of 50 &#181;L of spores (103/&#181;L) 17,949 &#215; g for 10 min and then, resuspend with 25 &#181;L of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) (300 mM)).</li>
	<li>Incubate for 15 min at room temperature.</li>
	<li>Add 25 &#181;L of N-hydroxysulfosuccinimide (NHS) (50 mM) to the spore suspension and incubate it at room temperature for 30 min.</li>
	<li>Wash the spores 3x with 1x PBS by centrifugation as described in step 5.1.</li>
	<li>Add fluorescent protein and leave the samples overnight at 15 &#176;C. 
	NOTE: The fluorescent protein used in this work was an APC-labeled anti-human IL-10 antibody. This antibody was used as a model for study, though the application of other fluorescent molecules is possible since EDC/NHS promote a covalent ligation between -COOH and -NH2 groups present in proteins on the spore surface.</li>
	<li>Wash the spores as per step 5.3.</li>
	<li>Add 10 mg/mL of ethidium bromide diluted at 1:50, then leave for 1 h on ice and protected from light (Figure 1B).</li>
	<li>Wash the spores as per step 5.3.</li>
	<li>Repeat step 4.</li>
	<li>To determine fluorescence, change the parameters FL3 on the X axis and FL5 on the Y axis of the dotplot.</li>
	<li>Analyze the sample using a flow cytometer with the blue laser (488 nm) at FL3 (670 LP filter) and the red laser (633 nm) in FL5 (660/20 filter), as described in step 4. 
	NOTE: The same samples were analyzed for immunofluorescence on slides under a microscope, at a magnification range of 1,000 x, and using the following filters: FITC 480/30 nm (green) and TRITC 540/25 nm (red).</li>
</ol>

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	<li>McKenney, P. T., Driks, A., Eichenberger, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Bacillus+subtilis+endospore:+assembly+and+functions+of+the+multilayered+coat.">The Bacillus subtilis endospore: assembly and functions of the multilayered coat.</a> Nature Reviews. Microbiology. 11 (1), 33-44 (2013).</li><li>Cutting, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bacillus+probiotics.">Bacillus probiotics.</a> Food Microbiology. 28 (2), 214-220 (2011).</li><li>Ricca, E., Baccigalupi, L., Cangiano, G., De Felice, M., Isticato, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mucosal+vaccine+delivery+by+non-recombinant+spores+of+Bacillus+subtilis.">Mucosal vaccine delivery by non-recombinant spores of Bacillus subtilis.</a> Microbial Cell Factories. 13, 115 (2014).</li><li>Falahati-Pour, S. K., Lotfi, A. S., Ahmadian, G., Baghizadeh, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Covalent+immobilization+of+recombinant+organophosphorus+hydrolase+on+spores+of+Bacillus+subtilis.">Covalent immobilization of recombinant organophosphorus hydrolase on spores of Bacillus subtilis.</a> Journal of Applied Microbiology. 118 (4), 976-988 (2015).</li><li>Harrold, Z., Hertel, M., Gorman-Lewis, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimizing+Bacillus+subtilis+spore+isolation+and+quantifying+spore+harvest+purity.">Optimizing Bacillus subtilis spore isolation and quantifying spore harvest purity.</a> Journal of Microbiological Methods. 87 (3), 325-329 (2011).</li><li>Nicholson, W. L., Setlow, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sporulation,+germination+and+outgrowth.">Sporulation, germination and outgrowth.</a> Molecular biological methods for Bacillus. , (1990).</li><li>Mora-Uribe, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+the+adherence+of+Clostridium+difficile+spores:+the+integrity+of+the+outermost+layer+affects+adherence+properties+of+spores+of+the+epidemic+strain+R20291+to+components+of+the+intestinal+mucosa.">Characterization of the adherence of Clostridium difficile spores: the integrity of the outermost layer affects adherence properties of spores of the epidemic strain R20291 to components of the intestinal mucosa.</a> Frontiers in Cellular and Infection Microbiology. 6, 99 (2016).</li><li>Paidhungat, M., Setlow, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+ger+proteins+in+nutrient+and+nonnutrient+triggering+of+spore+germination+in+Bacillus+subtilis.">Role of ger proteins in nutrient and nonnutrient triggering of spore germination in Bacillus subtilis.</a> Journal of Bacteriology. 182 (9), 2513-2519 (2000).</li><li>Schnizlein-Bick, C., Spritzler, J., Wilkening, C., Nicholson, J., O'Gorman, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+TruCount+absolute-count+tubes+for+determining+CD4+and+CD8+cell+numbers+in+human+immunodeficiency+virus-positive+adults.+Site+Investigators+and+The+NIAID+DAIDS+New+Technologies+Evaluation+Group.">Evaluation of TruCount absolute-count tubes for determining CD4 and CD8 cell numbers in human immunodeficiency virus-positive adults. Site Investigators and The NIAID DAIDS New Technologies Evaluation Group.</a> Clinical and Diagnostic Laboratory Immunology. 7 (3), 336-343 (2000).</li><li>Godfrey, A., Alsharif, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+enumeration+of+viable+spores+by+flow+cytometry.">Rapid enumeration of viable spores by flow cytometry.</a> US Patent. , (2003).</li><li>Karava, M., Bracharz, F., Kabisch, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantification+and+isolation+of+Bacillus+subtilis+spores+using+cell+sorting+and+automated+gating.">Quantification and isolation of Bacillus subtilis spores using cell sorting and automated gating.</a> PLoS ONE. 14 (7), e021989 (2019).</li><li>Genovese, M., Poulain, E., Doppler, F., Toussaint, R., Boyer, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bacillus+spore+enumeration+using+flow+cytometry:+A+proof+of+concept+for+probiotic+application.">Bacillus spore enumeration using flow cytometry: A proof of concept for probiotic application.</a> Journal of Microbiological Methods. 190, 106336 (2021).</li><li>Trunet, C., Ngo, H., Coroller, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantifying+permeabilization+and+activity+recovery+of+Bacillus+spores+in+adverse+conditions+for+growth.">Quantifying permeabilization and activity recovery of Bacillus spores in adverse conditions for growth.</a> Food Microbiology. 81, 115-120 (2019).</li><li>Tehri, N., Kumar, N., Raghu, H., Vashishth, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biomarkers+of+bacterial+spore+germination.">Biomarkers of bacterial spore germination.</a> Annals of Microbiology. 68, 513-523 (2018).</li><li>Isticato, R., Ricca, E., Baccigalupi, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spore+adsorption+as+a+nonrecombinant+display+system+for+enzymes+and+antigens.">Spore adsorption as a nonrecombinant display system for enzymes and antigens.</a> Journal of Visualized Experiments. 145, e59102 (2019).</li><li>Song, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Killed+Bacillus+subtilis+spores+as+a+mucosal+adjuvant+for+an+H5N1+vaccine.">Killed Bacillus subtilis spores as a mucosal adjuvant for an H5N1 vaccine.</a> Vaccine. 30 (22), 3266-3277 (2012).</li></ol>

The authors have no conflicts of interest to disclose.

Traditional methods, such as plate counting of colonies, are not only time-consuming, but also need viable cells and do not allow for quantification of inactivated spores5. The Petroff-Hausser chamber is an alternative methodology, but it requires an experienced microscopist to perform it. Flow cytometry has proven to be a useful alternative for this purpose.
Genovese et al.12 described the use of flow cytometry for the quantification of viable c...

Bacillus subtilis is a rod-shaped, gram-positive bacterium that is able to produce quiescent spores when environmental conditions do not allow cell growth1. Spores are extremely stable cell forms and those of several species, including B. subtilis,&#160;are widely used as probiotics for human and animal use2. Due to its resistance and safety properties, the spore of B. subtilis, which displays heterologous proteins, has been proposed as a mucosal adjuvant, a vaccine delivery system, and an enzyme-immobilization platform3,4.
To obtain spores from B. subtilis, it is necessary to expose it to nutrient deprivation using a special culture medium. After obtaining and purifying these spores, one must quantify them to improve test efficiency5,6. Thus, certain methods are applied to analyze the concentration of the spores obtained. Plate counting and a Petroff-Hausser chamber, also known as a counting chamber, can be used. The latter was originally developed to determine the concentration of blood cells; however, it is possible to use it in the field of microbiology for spore counting7,8. Despite being the standard method used for cell counting, reading is laborious since this method is completely manual and its accuracy depends on the experience of the operator.
Flow cytometry-based (FC) analyses have been previously proposed as fast, reliable, and specific approaches for detecting labeled cells of Bacillus spp. The use of flow cytometry counting beads has guaranteed reproducibility in cell counting in routine examinations (absolute count of CD4 and CD8 T lymphocytes) and in the development of research involving particles capable of being detected and counted using flow cytometry9. Godjafrey and Alsharif suggested the use of counting beads for FC quantification of unlabeled spores10. The use of flow cytometry was described for the monitoring of sporulation in Bacillus spp. via&#160;labeling the spore DNA10,11,12,13. Yet another study used FC to evaluate the amount of fluorescently labeled proteins on the spore surface15.
This study sought to use commercial counting beads to ensure a standard of reproducibility with respect to event counting using flow cytometry. Herein, we suggest the use of counting beads for cell counting in FC to refine spore enumeration and evaluate the coupling efficiency of fluorescently labeled antibodies on the spore surface.

<table><tbody><tr><td>(N-hydroxysuccinimide) (NHS)</td><td>Sigma</td><td>130672</td><td></td></tr><tr><td>Anti-human fluorescent antibody</td><td>BioLegend</td><td>501410</td><td>APC anti-human IL-10</td></tr><tr><td>Anti-mouse fluorescent antibody</td><td>Thermo Scientific</td><td>A32723</td><td>Alexa Fluor Plus 488</td></tr><tr><td>BD FACSCanto II</td><td>&amp;nbsp;BD</td><td></td><td>Flow cytometer</td></tr><tr><td>BD FACSDiva Cytometer Setup &amp;amp; Tracking Beads Kit (use with BD FACSDiva software v 6.x)</td><td>BD</td><td>642412</td><td>Quality control reagent</td></tr><tr><td>BD FACSDiva Software v. 6.1.3</td><td>BD</td><td>643629</td><td>Software</td></tr><tr><td>Centrifuge MegaFuge 8R</td><td>Thermo Scientific</td><td>75007213</td><td></td></tr><tr><td>Counting Beads</td><td>BD</td><td>340334</td><td>TruCount Tubes</td></tr><tr><td>Eclipse 80i</td><td>Nikon</td><td></td><td>Fluorescent Microsope</td></tr><tr><td>Ethidium Bromide</td><td>Ludwig Biotec</td><td></td><td></td></tr><tr><td>Phosphate buffered saline</td><td>Sigma-Aldrich</td><td>A4503</td><td></td></tr><tr><td>Plastic Microtubes</td><td>Eppendorf</td><td></td><td></td></tr><tr><td>Polystyrene tube</td><td>Falcon</td><td>352008</td><td>5 mL polystyrene tube, 12 x 75 mm, without lid, non-sterile</td></tr></tbody></table>

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.

In autoclaved spore (AS) samples, 2 &#215; 103 spores/&#181;l and 1 &#215; 103 spores/&#181;l were detected by using counting beads and the Petroff-Hausser method, respectively (Figure 2).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/65141/65141fig01.jpg" /> 
Figure 1: General scheme of quantification of spores. (A) Spores labeled with...

Watch this Scientific Journal Video about Improvement of Bacillus subtilis Spore Enumeration and Label Analysis in Flow Cytometry at JoVE.com

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.

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 ...

improvement-bacillus-subtilis-spore-enumeration-label-analysis-flow

Research

JoVE Journal

Biochemistry

1.0K Views.  Instituto Leônidas e Maria Deane - Fiocruz Amazônia. Het verzamelen van sporen van Bacillus subtilis met behulp van traditionele methoden kan een arbeidsintensieve taak zijn, aangezien deze methoden volledig handmatig kunnen zijn en hun nauwkeurigheid afhankelijk is van de ervaring van de operator. Dit protocol maakt het mogelijk om slapende en ontkiemende sporen te inventariseren. Bovendien maakt de techniek het ook mogelijk om het percentage coping van fluorescerende eiwitten op het oppervlak te bepalen.Gezien de installatiestap die in...

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

Analyzing Cellular Internalization of Nanoparticles and Bacteria by Multi-spectral Imaging Flow Cytometry

Nanoparticulate systems have emerged as valuable tools in vaccine delivery through their ability to efficiently deliver cargo, including proteins, to antigen presenting cells1-5. Internalization of nanoparticles (NP) by antigen presenting cells is a critical step in generating an effective immune response to the encapsulated antigen. To determine how changes in nanoparticle formulation impact function, we sought to develop a high throughput, quantitative experimental protocol that was compatible with detecting internalized nanoparticles as well as bacteria. To date, two independent techniques, microscopy and flow cytometry, have been the methods used to study the phagocytosis of nanoparticles. The high throughput nature of flow cytometry generates robust statistical data. However, due to low resolution, it fails to accurately quantify internalized versus cell bound nanoparticles. Microscopy generates images with high spatial resolution; however, it is time consuming and involves small sample sizes6-8. Multi-spectral imaging flow cytometry (MIFC) is a new technology that incorporates aspects of both microscopy and flow cytometry that performs multi-color spectral fluorescence and bright field imaging simultaneously through a laminar core. This capability provides an accurate analysis of fluorescent signal intensities and spatial relationships between different structures and cellular features at high speed.
	Herein, we describe a method utilizing MIFC to characterize the cell populations that have internalized polyanhydride nanoparticles or Salmonella enterica serovar Typhimurium. We also describe the preparation of nanoparticle suspensions, cell labeling, acquisition on an ImageStreamX system and analysis of the data using the IDEAS application. We also demonstrate the application of a technique that can be used to differentiate the internalization pathways for nanoparticles and bacteria by using cytochalasin-D as an inhibitor of actin-mediated phagocytosis.

In this article, we describe a method utilizing multi-spectral imaging flow cytometry to quantify the internalization of polyanhydride nanoparticles or bacteria by RAW 264.7 cells.

Nanoparticulate  systems have emerged as valuable tools in vaccine delivery through their  ability to efficiently deliver cargo, including proteins, to ...

Bioengineering

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 ...

Immunology and Infection

Characterizing Microbiome Dynamics &#8211; Flow Cytometry Based Workflows from Pure Cultures to Natural Communities

The investigation of pure cultures and monitoring of microbial community dynamics is vital to understand and control natural ecosystems and technical applications driven by microorganisms. Next generation sequencing methods are widely utilized to resolve microbiomes, but they are generally resource and time intensive and deliver mostly qualitative information. Flow cytometric microbiome analysis does not suffer from those disadvantages and can provide relative subcommunity abundances and absolute cell numbers at-line. Although it does not deliver direct phylogenetic information, it can enhance the analysis depth and resolution of sequencing approaches. In sharp contrast to medical applications in both research and routine settings, flow cytometry is still not widely used for microbiome analysis. Missing information on sample preparation and data analysis pipelines may create an entry barrier for the researchers facing microbiome analysis challenges that would often be textbook flow cytometry applications. Here, we present three comprehensive workflows for pure cultures, complex communities in clear medium and complex communities in challenging matrices, respectively. We describe individual sampling and fixation procedures and optimized staining protocols for the respective sample sets. We elaborate the cytometric analysis with a complex research centered and an application focused bench top device, describe the cell sorting procedure and suggest data analysis packages. We furthermore propose important experimental controls and apply the presented workflows to the respective sample sets.

Flow cytometric analysis has proven valuable for investigating pure cultures and monitoring microbial community dynamics. We present three comprehensive workflows, from sampling to data analysis, for pure cultures and complex communities in clear medium as well as in challenging matrices, respectively.

The investigation of pure cultures and monitoring of microbial community dynamics is vital to understand and control natural ecosystems and technical ...

Environment

Visualization of Bacterial Resistance using Fluorescent Antibiotic Probes

Fluorescent antibiotics are multipurpose research tools that are readily used for the study of antimicrobial resistance, due to their significant advantage over other methods. To prepare these probes, azide derivatives of antibiotics are synthesized, then coupled with alkyne-fluorophores using azide-alkyne dipolar cycloaddition by click chemistry. Following purification, the antibiotic activity of the fluorescent antibiotic is tested by minimum inhibitory concentration assessment. In order to study bacterial accumulation, either spectrophotometry or flow cytometry may be used, allowing for much simpler analysis than methods relying on radioactive antibiotic derivatives. Furthermore, confocal microscopy can be used to examine localization within the bacteria, affording valuable information about mode of action and changes that occur in resistant species. The use of fluorescent antibiotic probes in the study of antimicrobial resistance is a powerful method with much potential for future expansion.

Fluorescently tagged antibiotics are powerful tools that can be used to study multiple aspects of antimicrobial resistance. This article describes the preparation of fluorescently tagged antibiotics and their application to studying antibiotic resistance in bacteria. Probes can be used to study mechanisms of bacterial resistance (e.g., efflux) by spectrophotometry, flow cytometry, and microscopy.

Fluorescent antibiotics are multipurpose research tools that are readily used for the study of antimicrobial resistance, due to their significant ...

Quantifying Human Norovirus Virus-like Particles Binding to Commensal Bacteria Using Flow Cytometry

Commensal bacteria are well established to impact infection of eukaryotic viruses. Direct binding between the pathogen and the host microbiome is responsible for altering infection for many of these viruses. Thus, characterizing the nature of virus-bacteria binding is a foundational step needed for elucidating the mechanism(s) by which bacteria alter viral infection. For human norovirus, commensal bacteria enhance B cell infection. The virus directly binds to these bacteria, indicating that this direct interaction is involved in the mechanism of infection enhancement. A variety of techniques can be used to quantify interactions between bacteria and viruses including scintillation counting of radiolabeled viruses and polymerase chain reaction (PCR). Both methods require the use of live virus, which may need to be generated in the laboratory. Currently, none of the established in vitro culture systems available for human norovirus are robust enough to allow for generation of highly concentrated viral stocks. In lieu of live virus, virus-like particles (VLPs) have been used to characterize the interactions between norovirus and bacteria. Herein a flow cytometry method is described with uses virus specific antibodies to quantify VLP binding to gram-negative and gram-positive bacteria. Inclusion of both bacteria only and isotype controls allowed for optimization of the assay to reduce background antibody binding and accurate quantification of VLP attachment to the bacteria tested. High VLP:bacterium ratios result in VLPs binding to large percentages of the bacterial population. However, when VLP quantities are decreased, the percent of bacteria bound also decreases. Ultimately, this method can be employed in future experiments elucidating the specific conditions and structural components that regulate norovirus:bacterial interactions.

The goal of this protocol is to quantify binding of the eukaryotic pathogen human norovirus to bacteria. After performing an initial virus-bacterium attachment assay, flow cytometry is used to detect virally-bound bacteria within the population.

Commensal bacteria are well established to impact infection of eukaryotic viruses. Direct binding between the pathogen and the host microbiome is ...

Flow Cytometric Analysis of Bimolecular Fluorescence Complementation: A High Throughput Quantitative Method to Study Protein-protein Interaction

Among methods to study protein-protein interaction inside cells, Bimolecular Fluorescence Complementation (BiFC) is relatively simple and sensitive. BiFC is based on the production of fluorescence using two non-fluorescent fragments of a fluorescent protein (Venus, a Yellow Fluorescent Protein variant, is used here). Non-fluorescent Venus fragments (VN and VC) are fused to two interacting proteins (in this case, AKAP-Lbc and PDE4D3), yielding fluorescence due to VN-AKAP-Lbc-VC-PDE4D3 interaction and the formation of a functional fluorescent protein inside cells.
BiFC provides information on the subcellular localization of protein complexes and the strength of protein interactions based on fluorescence intensity. However, BiFC analysis using microscopy to quantify the strength of protein-protein interaction is time-consuming and somewhat subjective due to heterogeneity in protein expression and interaction. By coupling flow cytometric analysis with BiFC methodology, the fluorescent BiFC protein-protein interaction signal can be accurately measured for a large quantity of cells in a short time. Here, we demonstrate an application of this methodology to map regions in PDE4D3 that are required for the interaction with AKAP-Lbc. This high throughput methodology can be applied to screening factors that regulate protein-protein interaction.

Flow cytometric analysis of Bimolecular Fluorescence Complementation provides a high throughput quantitative method to study protein-protein interaction. This methodology can be applied to mapping protein binding sites and for screening factors that regulate protein-protein interaction.

Among methods to study protein-protein interaction inside cells, Bimolecular Fluorescence Complementation (BiFC) is relatively simple and sensitive. BiFC ...

Biology

Monitoring Intraspecies Competition in a Bacterial Cell Population by Cocultivation of Fluorescently Labelled Strains

Many microorganisms such as bacteria proliferate extremely fast and the populations may reach high cell densities. Small fractions of cells in a population always have accumulated mutations that are either detrimental or beneficial for the cell. If the fitness effect of a mutation provides the subpopulation with a strong selective growth advantage, the individuals of this subpopulation may rapidly outcompete and even completely eliminate their immediate fellows. Thus, small genetic changes and selection-driven accumulation of cells that have acquired beneficial mutations may lead to a complete shift of the genotype of a cell population. Here we present a procedure to monitor the rapid clonal expansion and elimination of beneficial and detrimental mutations, respectively, in a bacterial cell population over time by cocultivation of fluorescently labeled individuals of the Gram-positive model bacterium Bacillus subtilis. The method is easy to perform and very illustrative to display intraspecies competition among the individuals in a bacterial cell population.

Bacteria may accumulate either detrimental or beneficial mutations during their lifetime. In a population of cells individuals that have accumulated beneficial mutations may rapidly outcompete their fellows. Here we present a simple procedure to visualize intraspecies competition in a bacterial cell population over time using fluorescently labeled individuals.&#160;

Many microorganisms such as bacteria proliferate extremely fast and the populations may reach high cell densities. Small fractions of cells in a ...

Protocol for Co-Culturing Sucrose-Secreting Cyanobacteria with Fungal Partners

Assembly and Quantification of Co-Cultures Combining Heterotrophic Yeast with Phototrophic Sugar-Secreting Cyanobacteria

With the increasing demand for sustainable biotechnologies, mixed consortia containing a phototrophic microbe and heterotrophic partner species are being explored as a method for solar-driven bioproduction. One approach involves the use of CO2-fixing cyanobacteria that secrete organic carbon to support the metabolism of a co-cultivated heterotroph, which in turn transforms the carbon into higher-value goods or services. In this protocol, a technical description to assist the experimentalist in the establishment of a co-culture combining a sucrose-secreting cyanobacterial strain with a fungal partner(s), as represented by model yeast species, is provided. The protocol describes the key prerequisites for co-culture establishment: Defining the media composition, monitoring the growth characteristics of individual partners, and the analysis of mixed cultures with multiple species combined in the same growth vessel. Basic laboratory techniques for co-culture monitoring, including microscopy, cell counter, and single-cell flow cytometry, are summarized, and examples of nonproprietary software to use for data analysis of raw flow cytometry standard (FCS) files in line with FAIR (Findable, Accessible, Interoperable, Reusable) principles are provided. Finally, commentary on the bottlenecks and pitfalls frequently encountered when attempting to establish a co-culture with sugar-secreting cyanobacteria and a novel heterotrophic partner is included. This protocol provides a resource for researchers attempting to establish a new pair of co-cultured microbes that includes a cyanobacterium and a heterotrophic microbe.

This protocol provides a comprehensive guideline for the setup and quantitative monitoring of co-cultures, including photoautotrophic sugar-secreting cyanobacteria and heterotrophic yeasts.

With the increasing demand for sustainable biotechnologies, mixed consortia containing a phototrophic microbe and heterotrophic partner species are being ...

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 ...

Single-cell Microfluidic Analysis of Bacillus subtilis

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

Single-cell Microfluidic Analysis of Bacillus subtilis

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

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

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

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