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 obstructio...

<ol>
	<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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>((1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride) (EDC)</td>
			<td>Sigma</td>
			<td>341006</td>
			<td></td>
		</tr>
		<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>&#160;BD</td>
			<td></td>
			<td>Flow cytometer</td>
		</tr>
		<tr>
			<td>BD FACSDiva Cytometer Setup &#38; 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 ...

Cell counter of Bacillus subtilis spores using traditional methods can be a laborious task, as these methods can be completely manual and their accuracies depending on the operator's experience. This protocol makes the enumeration of dormant and germinating spores possible. In addition, the technique also makes it possible to determine the percentage of coping of fluorescent proteins on its surface.Given the setup step present in this protocol the vision demonstrates with facilities and the technical understanding and the detail of care in this execution. Begin by logging into the cytometer software. In the software workspace, select Cytometer, followed by Fluidic Startup.Then choose Cleaning Modes. And finally, initiate SIT Flush. Take 50 microliters of autoclave spores and incubate them with ethidium bromide at a dilution factor of 0.05%volume by volume for 30 minutes protected from light.Wash the spores three times with PBS by centrifuging at 17, 949 G for 10 minutes, and re-suspending in PBS. Next, add 10 microliters of beads following the manufacturer's recommendations for dilution and analyze the sample using a flow cytometer. Define the gating strategy based on the morpho-metric and fluorescence characteristics of the particles using the negative control as a reference.Then mix the tube gently. Attach it to the flow cytometer probe and click Acquire. To set the laser power, go to the parameters tab in the cytometer window, adjust forward scatter to 375 and side scatter to 275.Then select the threshold tab and set it to 500. Next, analyze the dye-free sample containing only spores to eliminate autofluorescence. In the parameter tab, adjust the filter detector three's voltages to 603 and filter detector five's voltages to 538 to differentiate between negative and positive populations using fluorescence in the controls.Then click Compensation, and set filter detector 5 by 3 setup offset to one. To configure the device for acquisition, select Experiment, choose the experiment layout and set the acquisition to 30, 000 events. After adjusting the parameters, acquire data for the samples that are labeled and contain beads in the flow cytometer.Centrifuge 50 microliters of spores at 17, 949 G for 10 minutes. Next, re-suspend the spores using 25 microliters of 1-ethyl-3-3-dimethylaminopropyl carbodiimide and incubate for 15 minutes. Afterward, add 25 microliters of N-hydroxysulfosuccinimide at a concentration of 50 millimolar to the spore suspension.Incubate the spore suspension at room temperature for 30 minutes. Wash the spores three times with PBS by centrifuging as shown earlier. Add fluorescent protein to the samples and incubate overnight at 15 degrees Celsius.Add 10 milligrams per milliliter of ethidium bromide diluted at 1 to 50, then leave the samples on ice for one hour, protected from light. After washing the spores and defining the gates as shown earlier, change the parameters for filter detector three on the x axis, and filter detector five on the Y axis of the dot plot. The counting beads method detected two times 10 to the third spores per microliter in autoclave spore samples.A ethidium bromide staining of autoclave spore samples showed higher mean fluorescence intensity than the staining of non-autoclaved spores, indicating greater staining of genetic material. The autoclave spore surface, coupled with APC labeled, anti-human interleukin 10 antibody showed greater coupling efficiency compared to the non-autoclaved spores. The presence of spores permeable to ethidium bromide indicated the possible presence of germinated spores in the total population.Based on the flow cytometry analyses, it was observed that the fluorescent antibody showed a higher percentage of coupling with the dormant spores in comparison to the germinated ones. Moreover, as the concentration of fluorescent antibodies increased, an increase in the percentage of coupled spores and mean fluorescence intensity was observed. It is important to perform a thorough homogenization of the current beads to ensure precise readings through flow cytometry and accurate enumeration of spores.Standardizing the concentration of antigens attached to spore, Canadian studies analyze the use of spore as vaccine antibodies.

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Research

JoVE Journal

Biochemistry

723 Görüntüleme.  Instituto Leônidas e Maria Deane - Fiocruz Amazônia. Geleneksel yöntemler kullanılarak Bacillus subtilis sporlarının hücre sayacı zahmetli bir iş olabilir, çünkü bu yöntemler tamamen manuel olabilir ve doğrulukları operatörün deneyimine bağlı olabilir. Bu protokol, hareketsiz ve çimlenen sporların sayımını mümkün kılar. Ek olarak, teknik, yüzeyindeki floresan proteinlerin başa çıkma yüzdesini belirlemeyi de mümkün kılar.Bu protokolde yer alan kurulum adımı göz önüne alındığında, vizyon, tesisler ...