<meta charset="utf8"></head><body>פרוטוקול מקיף לזיהוי תאים וניתוח מטבולי באמצעות OPTIR-FISH

<ol>
	<li>Evers, T. M. J., 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=Deciphering+metabolic+heterogeneity+by+single-cell+analysis.">Deciphering metabolic heterogeneity by single-cell analysis.</a> Anal Chem. 91 (21), 13314-13323 (2019).</li><li>Overmann, J., Abt, B., Sikorski, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Present+and+future+of+culturing+bacteria.">Present and future of culturing bacteria.</a> Annu Rev Microbiol. 71, 711-730 (2017).</li><li>Epstein, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+phenomenon+of+microbial+uncultivability.">The phenomenon of microbial uncultivability.</a> Curr Opin Microbiol. 16 (5), 636-642 (2013).</li><li>Su, C., Lei, L., Duan, Y., Zhang, K. -. 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C., Wagner, M., Cheng, J. -. X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mid-infrared+photothermal-fluorescence+in+situ+hybridization+for+functional+analysis+and+genetic+identification+of+single+cells.">Mid-infrared photothermal-fluorescence in situ hybridization for functional analysis and genetic identification of single cells.</a> Anal Chem. 95 (4), 2398-2405 (2023).</li><li>Manz, W., Amann, R., Ludwig, W., Wagner, M., Schleifer, K. -. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phylogenetic+oligodeoxynucleotide+probes+for+the+major+subclasses+of+proteobacteria:+problems+and+solutions.">Phylogenetic oligodeoxynucleotide probes for the major subclasses of proteobacteria: problems and solutions.</a> System Appl Microbiol. 15 (4), 593-600 (1992).</li><li>Manz, W., Amann, R., Ludwig, W., Vancanneyt, M., Schleifer, K. -. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Application+of+a+suite+of+16S+rRNA-specific+oligonucleotide+probes+designed+to+investigate+bacteria+of+the+phylum+cytophaga-flavobacter-bacteroides+in+the+natural+environment.">Application of a suite of 16S rRNA-specific oligonucleotide probes designed to investigate bacteria of the phylum cytophaga-flavobacter-bacteroides in the natural environment.</a> Microbiology. 142, 1097-1106 (1996).</li><li>Daims, H., Stoecker, K., Wagner, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescence+In+Situ+Hybridization+for+the+Detection+of+Prokaryotes.">Fluorescence In Situ Hybridization for the Detection of Prokaryotes.</a> Molecular Microbial Ecology. , (2004).</li><li>Bridger, J. M., Volpi, E. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Fluorescence In Situ Hybridization (FISH): Protocols and Applications. , (2010).</li><li>Wang, D. O., 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=A+quick+and+simple+FISH+protocol+with+hybridization-sensitive+fluorescent+linear+oligodeoxynucleotide+probes.">A quick and simple FISH protocol with hybridization-sensitive fluorescent linear oligodeoxynucleotide probes.</a> RNA. 18 (1), 166-175 (2012).</li><li>Greuter, D., Loy, A., Horn, M., Rattei, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=probeBase-an+online+resource+for+rRNA-targeted+oligonucleotide+probes+and+primers:+new+features+2016.">probeBase-an online resource for rRNA-targeted oligonucleotide probes and primers: new features 2016.</a> Nucleic Acids Res. 44, D586-D589 (2016).</li><li>Yilmaz, L. S., Parnerkar, S., Noguera, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=mathFISH,+a+web+tool+that+uses+thermodynamics-based+mathematical+models+for+in+silico+evaluation+of+oligonucleotide+probes+for+fluorescence+in+situ+hybridization.">mathFISH, a web tool that uses thermodynamics-based mathematical models for in silico evaluation of oligonucleotide probes for fluorescence in situ hybridization.</a> Appl Environ Microbiol. 77 (3), 1118-1122 (2011).</li><li>Lima, C., Muhamadali, H., Xu, Y., Kansiz, M., Goodacre, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+isotopically+labeled+bacteria+at+the+single-cell+level+using+high-resolution+optical+infrared+photothermal+spectroscopy.">Imaging isotopically labeled bacteria at the single-cell level using high-resolution optical infrared photothermal spectroscopy.</a> Anal Chem. 93 (6), 3082-3088 (2021).</li><li>Yin, J., 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=Nanosecond-resolution+photothermal+dynamic+imaging+via+MHZ+digitization+and+match+filtering.">Nanosecond-resolution photothermal dynamic imaging via MHZ digitization and match filtering.</a> Nat Commun. 12 (1), 7097 (2021).</li><li>Bai, Y., Yin, J., Cheng, J. -. X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bond-selective+imaging+by+optically+sensing+the+mid-infrared+photothermal+effect.">Bond-selective imaging by optically sensing the mid-infrared photothermal effect.</a> Sci Adv. 7 (20), (2021).</li><li>Xia, Q., Yin, J., Guo, Z., Cheng, J. -. X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mid-infrared+photothermal+microscopy:+principle,+instrumentation,+and+applications.">Mid-infrared photothermal microscopy: principle, instrumentation, and applications.</a> J Phys Chemi B. 126 (43), 8597-8613 (2022).</li><li>Du, J., Wang, H., Wei, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bringing+vibrational+imaging+to+chemical+biology+with+molecular+probes.">Bringing vibrational imaging to chemical biology with molecular probes.</a> ACS Chem Biol. 17 (7), 1621-1637 (2022).</li><li>Miao, Y., Shi, L., Hu, F., Min, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Probe+design+for+super-multiplexed+vibrational+imaging.">Probe design for super-multiplexed vibrational imaging.</a> Phys Biol. 16 (4), 041003 (2019).</li><li>Shi, L., 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=Mid-infrared+metabolic+imaging+with+vibrational+probes.">Mid-infrared metabolic imaging with vibrational probes.</a> Nat Methods. 17 (8), 844-851 (2020).</li><li>Bai, Y., 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=Single-cell+mapping+of+lipid+metabolites+using+an+infrared+probe+in+human-derived+model+systems.">Single-cell mapping of lipid metabolites using an infrared probe in human-derived model systems.</a> Nat Commun. 15 (1), 350 (2024).</li><li>Zhang, L., 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=Spectral+tracing+of+deuterium+for+imaging+glucose+metabolism.">Spectral tracing of deuterium for imaging glucose metabolism.</a> Nat Biomed Eng. 3 (5), 402-413 (2019).</li><li>Ge, X., 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=SRS-FISH:+A+high-throughput+platform+linking+microbiome+metabolism+to+identity+at+the+single-cell+level.">SRS-FISH: A high-throughput platform linking microbiome metabolism to identity at the single-cell level.</a> Proc Natl Acad Sci U S A. 119 (26), e2203519119 (2022).</li><li>Bai, Y., 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=Ultrafast+chemical+imaging+by+widefield+photothermal+sensing+of+infrared+absorption.">Ultrafast chemical imaging by widefield photothermal sensing of infrared absorption.</a> Sci Adv. 5 (7), 7127 (2019).</li><li>Zhang, J., Zhao, J., Lin, H., Tan, Y., Cheng, J. -. X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-speed+chemical+imaging+by+dense-net+learning+of+femtosecond+stimulated+Raman+scattering.">High-speed chemical imaging by dense-net learning of femtosecond stimulated Raman scattering.</a> J Phys Chem Lett. 11 (20), 8573-8578 (2020).</li><li>Lin, H., 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=Microsecond+fingerprint+stimulated+Raman+spectroscopic+imaging+by+ultrafast+tuning+and+spatial-spectral+learning.">Microsecond fingerprint stimulated Raman spectroscopic imaging by ultrafast tuning and spatial-spectral learning.</a> Nat Commun. 12 (1), 3052 (2021).</li><li>Fu, 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=Super-resolution+imaging+of+non-fluorescent+molecules+by+photothermal+relaxation+localization+microscopy.">Super-resolution imaging of non-fluorescent molecules by photothermal relaxation localization microscopy.</a> Nat Photon. 17, 330-337 (2023).</li><li>Tamamitsu, 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=Mid-infrared+widefield+nanoscopy.">Mid-infrared widefield nanoscopy.</a> arXiv. , (2023).</li></ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>96% Ethanol</td>
			<td>ThermoScientific</td>
			<td>T032021000</td>
			<td></td>
		</tr>
		<tr>
			<td>Calcium Fluoride</td>
			<td>Crystran</td>
			<td>CAFP10-0.35</td>
			<td></td>
		</tr>
		<tr>
			<td>D-(+)-Glucose</td>
			<td>Sigma-Aldrich</td>
			<td>G7021-1KG</td>
			<td></td>
		</tr>
		<tr>
			<td>D-Gluocose (U-13C6, 99%)</td>
			<td>Cambridge Isotopic Laboratories</td>
			<td>CLM-1396-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethylenediaminetetraacetic acid</td>
			<td>Sigma-Aldrich</td>
			<td>E9884-100G</td>
			<td></td>
		</tr>
		<tr>
			<td>formamide</td>
			<td>ThermoScientific</td>
			<td>17899</td>
			<td></td>
		</tr>
		<tr>
			<td>Luria-Bertani broth&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>L3522-250G</td>
			<td></td>
		</tr>
		<tr>
			<td>M9 Minimal Salts 5x</td>
			<td>SIGMA</td>
			<td>M6030-1KG</td>
			<td></td>
		</tr>
		<tr>
			<td>OPTIR instrument</td>
			<td>Photothermal Spectroscopy Corp.</td>
			<td>mIRage LS</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraforaldehyde Solution, 4% in PBS</td>
			<td>ThermoScientific</td>
			<td>J19943-K2</td>
			<td></td>
		</tr>
		<tr>
			<td>poly-L-lysine solution 0.1% (w/v)&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>P8920-500ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Chloride</td>
			<td>Sigma-Aldrich</td>
			<td>S9888-25G</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium dodecyl sulfate</td>
			<td>Sigma-Aldrich</td>
			<td>L3771-25G</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris(hydroxymethyl)aminomethane hydrochloride, 99+%</td>
			<td>ThermoScientific</td>
			<td>A11379.18</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypic Soy Broth</td>
			<td>Sigma-Aldrich</td>
			<td>22092-500G</td>
			<td></td>
		</tr>
	</tbody>
</table>

optical photothermal infrared-fluorescence in situ hybridization (optir-fish)

Cellular metabolism stands as a foundational pillar in cell biology, steering many processes that determine cell health, function, and interaction with the environment. Analyzing metabolism at the individual cell level, particularly within the native environments, provides invaluable insights to reveal the heterogeneous and complex activities in biological systems1. This is especially crucial in the study of microorganisms, as many microbes exhibit unique growth requirements or environmental dependencies that challenge traditional cultivation methods2. For instance, some species may require specific nutrient compositions or symbiotic relationships not easily replicated in a laboratory setting, thus rendering them non-culturable by standard techniques3. Furthermore, the extended periods needed for the cultivation of certain species pose significant challenges for microbiological research, often extending beyond practical time frames for study and analysis. To circumvent these limitations, alternative methods such as polymerase chain reaction and fluorescence in situ hybridization (FISH) allow for the identification of microbial species without necessitating cultivation4, which can achieve a more accurate and holistic view of microbial ecosystems. However, these analytical technologies lack the ability to elucidate cellular metabolism. This gap highlights the ongoing challenges in the microbiology field: the concurrent task of differentiating cellular identity and elucidating metabolism at the single-cell level. Advancements in techniques such as imaging mass spectrometry (IMS) coupled with stable isotopes have emerged as powerful tools for single-cell metabolic analysis5. In these experiments, cells were incubated with substrates containing isotopes such as 13C or 15N. The newly anabolized biomolecules carry these isotopes, making them distinguishable on the m/z spectra. However, IMS suffers from expensive instrumentation, complicated sample preparation, relatively low throughput, and expertise required for analyzing the m/z spectra. When combined with molecular marker-specific fluorescence imaging, advancements have been made in elucidating cellular metabolism with increased specificity6. Nevertheless, challenges persist in bridging these two modalities. The difference in resolution between IMS and fluorescence imaging, combined with different operational setups, makes it difficult to align and correlate findings7.
The integration of vibrational spectroscopic imaging with stable isotope labeling offers a novel solution for the study of single-cell metabolism. The incorporation of heavier isotopes slows down the vibration of chemical bonds, leading to red-shifted peaks in the vibrational spectra8. Notably, vibrational imaging provides a spatial resolution comparable to fluorescence imaging, and both metabolic quantification and cell identification can be performed in a single setup, simplifying the image registration and correlation. Our recent work has demonstrated the combination of an advanced vibrational imaging platform: optical photothermal infrared (OPTIR) with fluorescence in situ hybridization (FISH) to probe glucose metabolism in bacterial communities9 (Figure 1). OPTIR is a vibrational spectroscopic microscopy system that harnesses the photothermal effect of mid-IR absorption by detecting the visible light change, which provides sub-micrometer resolution as in optical microscopy but with the additional vibrational spectroscopic information originating from the mid-IR absorption. FISH is a commonly used technique to determine the microorganism identity at the single-cell level. The sequence of oligonucleotides could be designed to target specific 16S sequences of different taxa, and different fluorophores could be attached. Specific hybridization of the designed oligonucleotide probes with the target rRNA leads to strong fluorescence signals of target species within individual cells, and multi-channel fluorescence imaging could be performed to identify multiple species within the population. As both OPTIR and fluorescence imaging are based on optical detection, combining OPTIR and FISH fluorescence is straightforward to implement. The two modalities share the same optical resolution for single-cell analysis and can be switched conveniently without requiring additional alignment or co-registration.
This protocol presents a detailed guide to leveraging the OPTIR-FISH platform for advanced single-cell structure-function analysis. We used the bacterial samples grown in 13C-glucose-containing media as a test bed and quantified de novo protein synthesis from 13C-glucose. In order to demonstrate the ability of the platform to identify cells, we used bacterial mixtures in which each species was labeled using rRNA-targeted FISH probes. This approach facilitated precise single-cell identification of specific bacterial strains, such as Escherichia coli (E. coli) and Bacteroides thetaiotaomicron (B. theta), and their metabolism. This protocol offers researchers a powerful tool for simultaneous metabolic profiling and species identification at the single-cell level, promising to advance our understanding of cellular interactions, physiology, and their roles in complex environments.

<meta charset="utf8"></head><body>מחבר זרקור: OPTIR-FISH משולב לניתוח מטבולי וזהות של תא בודד בסביבות מורכבות

הכלאה פוטותרמית אופטית אינפרא אדום-פלואורסצנטי באתרו (OPTIR-FISH)

The scope of our research is to develop and apply OPTIR-FISH to identify individual cells, and analyze their metabolism at the single-cell level. We aim to understand how individual cells interact within a complex environment, and understand their physiology and roads. Analyzing metabolism at the individual cell level, particularly within the native environment, could provide invaluable insights into revealing the heterogeneous complex activities in biological systems.However, the concurrent task of differentiating cellular identity, and elucidating metabolism at the single-cell level remains challenging. Our advanced vibrational imaging platform, Optical Photothermal Infrared offers the study of single cell metabolism. At the same time, it is naturally highly compatible with the well established forests in situ hybridization for the identification of microbial species.Therefore, straightforward integration offers metabolism and identification at the same imaging resolution simultaneously.

Read this Scientific Article Protocol about Optical Photothermal Infrared - Fluorescence In Situ Hybridization OPTIR-FISH at JoVE.com

Understanding the metabolic activities of individual cells within complex communities is critical for unraveling their role in human disease. Here, we ...

optical-photothermal-infrared-fluorescence-situ-hybridization-optir

Research

JoVE Journal

Bioengineering

Optical Photothermal Infrared-Fluorescence In Situ Hybridization (OPTIR-FISH)

701 Views.  Boston University. היקף המחקר שלנו הוא לפתח וליישם OPTIR-FISH כדי לזהות תאים בודדים, ולנתח את חילוף החומרים שלהם ברמת התא היחיד. אנו שואפים להבין כיצד תאים בודדים מתקשרים בסביבה מורכבת, ולהבין את הפיזיולוגיה והדרכים שלהם. ניתוח חילוף החומרים ברמת התא הבודד, במיוחד בסביבה הטבעית,...

Video: מחבר זרקור: OPTIR-FISH משולב לניתוח מטבולי וזהות של תא בודד בסביבות מורכבות

Understanding the metabolic activities of individual cells within complex communities is critical for unraveling their role in human disease. Here, we present a comprehensive protocol for simultaneous cell identification and metabolic analysis with the OPTIR-FISH platform by combining rRNA-tagged FISH probes and isotope-labeled substrates. Fluorescence imaging provides cell identification by the specific binding of rRNA-tagged FISH probes, while OPTIR imaging provides metabolic activities within single cells by isotope-induced red shift on OPTIR spectra. Using bacteria cultured with 13C-glucose as a test bed, the protocol outlines microbial culture with isotopic labeling, fluorescence in situ hybridization (FISH), sample preparation, optimization of the OPTIR-FISH imaging setup, and data acquisition. We also demonstrate how to perform image analysis and interpret spectral data at the single-cell level with high throughput. This protocol&#39;s standardized and detailed nature will greatly facilitate its adoption by researchers from diverse backgrounds and disciplines within the broad single-cell metabolism research community.

Here, we present a protocol using optical photothermal infrared-fluorescence in situ hybridization (OPTIR-FISH), also known as mid-infrared photothermal-FISH (MIP-FISH), to identify individual cells and understand their metabolism. This methodology can be applied broadly for diverse applications, including mapping cellular metabolism with single-cell resolution.

Hybridizing Bacterial Cells with an Oligonucleotide Probe Labeled with a Fluorophore Using Fluorescence In Situ Hybridization (FISH)

To begin, take bacterial cells fixed in paraformaldehyde. Centrifuge the cells at 14, 000 x g for 10 minutes. Then remove the supernatant.Resuspend the pellet in 100 microliters of 96%analytical grade ethanol and incubate for one minute at room temperature. Repeat the centrifugation for five minutes, then remove the ethanol and air dry the cell pellet. Now hybridize the cells in a solution containing fluorescently labeled oligonucleotide for three hours at 46 to degrees Celsius.Immediately after hybridization, centrifuge the samples in a rotor preheated at 46 degrees Celsius for 15 minutes at the maximum allowed temperature. Then wash the samples in a buffer with suitable stringency for 15 minutes at 48 degrees Celsius. After centrifuging the sample, once again, wash the cells with 500 microliters of ice cold PBS.Resuspend them in 20 microliters of PBS and store them at four degrees Celsius until further use.

הכלאה של תאי חיידקים עם בדיקת אוליגונוקלאוטיד המסומנת בפלואורופור באמצעות הכלאה פלואורסצנטית באתרו (FISH)

Sample Preparation for OPTIR-FISH to Probe Glucose Metabolism in Bacterial Communities

To begin, take standardized calcium fluoride slides. Immerse them in 70%ethanol for 15 minutes, and rinse them two times with deionized water. Transfer the clean slides into 0.1%poly L lysine solution and incubate them overnight at four degrees Celsius.Then rinse the slides with deionized water and let them dry. Next, thaw the prepared cell samples at room temperature while keeping them protected from light. To avoid excessive salt crystals from PBS during sample drying, wash the thawed cells twice with deionized water.Centrifuge at 14, 000 G for 10 minutes, and remove the supernatant. Reintroduce the original volume of deionized water to the cells and gently vortex them. After washing centrifuge the cells at 14, 000 G for 10 minutes to concentrate the bacterial cell samples and remove the supernatant.Gently vortex the remaining solution for approximately 30 seconds and apply one to two microliters of this solution onto the prepared calcium fluoride slide.