Name: paraffin embedding and thin sectioning of microbial colony biofilms for microscopic analysis
Uploaded: 2018-03-23T14:00:00
Description: Watch this Scientific Journal Video about Paraffin Embedding and Thin Sectioning of Microbial Colony Biofilms for Microscopic Analysis at JoVE.com

Dette arbejde blev støttet af NSF karriere AWARD 1553023 og NIH/NIAID award R01AI103369.

1. vækst af Pseudomonas aeruginosa koloni biofilm
<ol><li>Forberedelse af Medium-tolagede plader<ol>
<li>Forberede en 10 g/L trypton, 10 g/L agar (Se Tabel af materialer) løsning i ionbyttet vand.</li>
		<li>Autoklave i 20 min. afkøles til 50-60 ° C i et vandbad.</li>
		<li>Hældes 45 mL af opløsningen, agar-trypton i et 100 mm x 100 mm firkantet fad (Se Tabel af materialer) ved hjælp af en 50 mL konisk slange. Tillad agar størkne (~ 20-30 min). Hæld en anden, 15 mL lag ud over...

<ol>
	<li>Ray, V. A., Morris, A. R., Visick, K. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+semi-quantitative+approach+to+assess+biofilm+formation+using+wrinkled+colony+development.">A semi-quantitative approach to assess biofilm formation using wrinkled colony development.</a> J. Vis. Exp. (64), e4035 (2012).</li><li>Friedman, L., Kolter, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genes+involved+in+matrix+formation+in+Pseudomonas+aeruginosa+PA14+biofilms.">Genes involved in matrix formation in Pseudomonas aeruginosa PA14 biofilms.</a> Mol. Microbiol. 51 (3), 675-690 (2004).</li><li>Okegbe, C., 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=Electron-shuttling+antibiotics+structure+bacterial+communities+by+modulating+cellular+levels+of+c-di-GMP.">Electron-shuttling antibiotics structure bacterial communities by modulating cellular levels of c-di-GMP.</a> Proc. Natl. Acad. Sci. U. S. A. 114 (26), E5236-E5245 (2017).</li><li>Vlamakis, H., Aguilar, C., Losick, R., Kolter, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Control+of+cell+fate+by+the+formation+of+an+architecturally+complex+bacterial+community.">Control of cell fate by the formation of an architecturally complex bacterial community.</a> Genes Dev. 22 (7), 945-953 (2008).</li><li>Serra, D. O., Richter, A. M., Klauck, G., Mika, F., Hengge, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microanatomy+at+cellular+resolution+and+spatial+order+of+physiological+differentiation+in+a+bacterial+biofilm.">Microanatomy at cellular resolution and spatial order of physiological differentiation in a bacterial biofilm.</a> MBio. 4 (2), e00103-e00113 (2013).</li><li>Serra, D. O., Richter, A. M., Hengge, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellulose+as+an+architectural+element+in+spatially+structured+Escherichia+coli+biofilms.">Cellulose as an architectural element in spatially structured Escherichia coli biofilms.</a> J. Bacteriol. 195 (24), 5540-5554 (2013).</li><li>McGlinn, E., Mansfield, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+of+gene+expression+in+mouse+embryos+and+tissue+sections.">Detection of gene expression in mouse embryos and tissue sections.</a> Methods Mol. Biol. 770, 259-292 (2011).</li><li>Choi, Y. S., Kim, Y. C., Baek, K. J., Choi, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+Situ+Detection+of+Bacteria+within+Paraffin-embedded+Tissues+Using+a+Digoxin-labeled+DNA+Probe+Targeting+16S+rRNA.">In Situ Detection of Bacteria within Paraffin-embedded Tissues Using a Digoxin-labeled DNA Probe Targeting 16S rRNA.</a> J. Vis. Exp. (99), e52836 (2015).</li><li>James, G., Hunt, A. M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+Biofilms+in+Tissue+Specimens.">Imaging Biofilms in Tissue Specimens.</a> Antibiofilm Agents. , 31-44 (2014).</li><li>Madsen, J. S., 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=Facultative+control+of+matrix+production+optimizes+competitive+fitness+in+Pseudomonas+aeruginosa+PA14+biofilm+models.">Facultative control of matrix production optimizes competitive fitness in Pseudomonas aeruginosa PA14 biofilm models.</a> Appl. Environ. Microbiol. 81 (24), 8414-8426 (2015).</li><li>Jo, J., Cortez, K. L., Cornell, W. -. C., Price-Whelan, A., Dietrich, L. E. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+orphan+cbb3-type+cytochrome+oxidase+subunit+supports+Pseudomonas+aeruginosa+biofilm+growth+and+virulence.">An orphan cbb3-type cytochrome oxidase subunit supports Pseudomonas aeruginosa biofilm growth and virulence.</a> bioRxiv. , 171538 (2017).</li><li>Fong, J. C., 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=Structural+dynamics+of+RbmA+governs+plasticity+of+Vibrio+cholerae+biofilms.">Structural dynamics of RbmA governs plasticity of Vibrio cholerae biofilms.</a> Elife. 6, (2017).</li><li>Bertani, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lysogeny+at+mid-twentieth+century:+P1,+P2,+and+other+experimental+systems.">Lysogeny at mid-twentieth century: P1, P2, and other experimental systems.</a> J. Bacteriol. 186 (3), 595-600 (2004).</li><li>Schindelin, 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=Fiji:+an+open-source+platform+for+biological-image+analysis.">Fiji: an open-source platform for biological-image analysis.</a> Nat. Methods. 9 (7), 676-682 (2012).</li><li>Dietrich, L. E. P., Teal, T. K., Price-Whelan, A., Newman, D. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Redox-active+antibiotics+control+gene+expression+and+community+behavior+in+divergent+bacteria.">Redox-active antibiotics control gene expression and community behavior in divergent bacteria.</a> Science. 321 (5893), 1203-1206 (2008).</li><li>Zupan&#269;i&#269;, D., Ter&#269;elj, M., &#352;trus, B., Verani&#269;, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How+to+obtain+good+morphology+and+antigen+detection+in+the+same+tissue+section?.">How to obtain good morphology and antigen detection in the same tissue section?.</a> Protoplasma. , (2017).</li><li>Priester, J. 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=Enhanced+visualization+of+microbial+biofilms+by+staining+and+environmental+scanning+electron+microscopy.">Enhanced visualization of microbial biofilms by staining and environmental scanning electron microscopy.</a> J. Microbiol. Methods. 68 (3), 577-587 (2007).</li><li>Boyles, J., Anderson, L., Hutcherson, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+fixative+for+the+preservation+of+actin+filaments:+fixation+of+pure+actin+filament+pellets.">A new fixative for the preservation of actin filaments: fixation of pure actin filament pellets.</a> J. Histochem. Cytochem. 33 (11), 1116-1128 (1985).</li><li>Blackburn, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Examination+of+normal+and+abnormal+placentation+in+the+mouse.">Examination of normal and abnormal placentation in the mouse.</a> Methods Mol. Biol. 136, 185-193 (2000).</li><li>Hoffman, E. A., Frey, B. L., Smith, L. M., Auble, D. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Formaldehyde+crosslinking:+a+tool+for+the+study+of+chromatin+complexes.">Formaldehyde crosslinking: a tool for the study of chromatin complexes.</a> J. Biol. Chem. 290 (44), 26404-26411 (2015).</li><li>Jennings, L. K., 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=Pel+is+a+cationic+exopolysaccharide+that+cross-links+extracellular+DNA+in+the+Pseudomonas+aeruginosa+biofilm+matrix.">Pel is a cationic exopolysaccharide that cross-links extracellular DNA in the Pseudomonas aeruginosa biofilm matrix.</a> Proc. Natl. Acad. Sci. U. S. A. 112 (36), 11353-11358 (2015).</li></ol>

Integrering af paraffin og tynd-skæring vævsprøver er en klassisk histologisk teknik, der gør det muligt for billeddannelse af mikro-morfologiske strukturer og er almindeligt anvendt på eukaryote væv, og er blevet anvendt med en vis succes på mikrobiel prøver8 ,9. Mens cryoembedding giver mulighed for stærk fastholdelse af endogene og immunfluorescent signal, er paraffin indlejring generelt at foretrække, da det giver bedre bevarelse af morfologi<sup c...

De fleste mikrober har kapacitet til form biofilm, holdt Fællesskaber af celler sammen af egen-producerede matricer. Biofilm kan dyrkes i mange typer af fysiske opsætninger, med forskellige regimer af næringsstof og substrat bestemmelse. Specifikke assays til Biofilmdannelse tendens til at give reproducerbare flercellede strukturer, og udbredte arkitekturer er observeret for Fylogenetisk forskelligartede arter på fællesskabsplan eller makroskopisk plan. Når mikrober er vokset som kolonier på solid medium under en atmosfære, makroskopisk morfologi formidler oplysninger om kapacitet til produktion af matrix og ofte korrelerer med andre træk 1,2,3. Den interne arkitektur af mikrobielle kolonier kan også give et fingerpeg om biofilm-specifikke kemi og fysiologi, men har været svært at karakterisere. Seneste programmer af cryoembedding og cryosectioning teknikker til bakteriel kolonier har aktiveret imaging og visualisering af specifikke funktioner i hidtil uset opløsning 4,5,6. Undersøgelser med animalsk væv har dog vist at paraffin indlejring giver overlegen bevarelse af morfologi i forhold til cryoembedding 7 og har været brugt til at visualisere bakterier i væv 8,9. Vi har derfor udviklet en protokol for fiksering, paraffin indlejring og tynde skæring af mikrobielle koloni biofilm. Her vil vi beskrive forberedelse af Pseudomonas aeruginosa PA14 koloni-biofilm tyndslib 10,11, men vi har også med succes anvendt denne teknik til biofilm dannes af bakterier Pseudomonas synxantha, Bacillus subtilis, og Vibrio cholerae12.
Processen med integrering af paraffin og tynd-skæring biofilm følger en simpel logik. Først, biofilm er indkapslet i et lag af agar at bevare morfologi under behandlingen. For det andet indkapslet biofilm er neddykket i fiksativ til bitmapgenkendelse makromolekyler og bevare micromorphology. Disse er derefter dehydreret med alkohol, ryddet med en mere ikke-polære opløsningsmidler og derefter infiltreret med flydende paraffin voks. Når infiltreret, er prøverne indlejret i voks blokke for skæring. Sektioner er skåret, monteret på dias og derefter rehydreret for at returnere dem til en mere naturlig tilstand. Fra dette punkt, kan de farves eller omfattet af montering medium til mikroskopisk analyse.
Denne protokol producerer tynde sektioner af mikrobielle biofilm egnet til histologisk analyse. Koloni biofilm delstrukturer er synlige, når tyndslib udarbejdet ved hjælp af denne metode er afbildet ved lysmikroskopi. Biofilm kan også dyrkes på medier indeholdende fluorescerende pletter specifikke for individuelle funktioner eller plettet på trinnet rehydrering umiddelbart inden montering (trin 9,5-9,6). Endelig, mikrober kan blive manipuleret til at producere fluorescerende proteiner i en konstitutiv eller regulerede mode tillader i situ rapportering af celle distribution eller gen udtryk inden for disse samfund. Vi har brugt disse metoder til at bestemme koloni biofilm dybde, celle distribution, matrix distribution, vækstmønstre og spatiotemporelle genekspression.

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			<td height="21" style="height:21px;width:276px;">Yeast extract</td>
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			<td style="width:161px;">Y9010</td>
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paraffin embedding and thin sectioning of microbial colony biofilms for microscopic analysis

Skæring via paraffin indlejring er en bredt etablerede teknik i eukaryote systemer. Her giver vi en metode til fiksering, indlejring, og skæring af intakt mikrobielle koloni biofilm ved hjælp af perfunderet paraffinvoks. For at tilpasse denne metode til brug på koloni biofilm, vi udviklet teknikker til vedligeholdelse af hver prøve på substratet vækst og laminering det med en agar overlayer, og tilføjet lysin til Fikseringsvæske løsningen. Disse optimeringer forbedre prøve opbevaring og bevarelse af micromorphological funktioner. Prøver forberedt på denne måde er indstillet til tynde skæring og imaging af lys, fluorescens og transmissions Elektron Mikroskopi. Vi har anvendt denne teknik til kolonien biofilm af Pseudomonas aeruginosa, Pseudomonas synxantha, Bacillus subtilisog Vibrio cholerae. Det høje niveau af detailrigdommen i prøver genereret ved denne metode, kombineret med reporter stamme engineering eller brug af specifikke farvestoffer, kan give spændende indsigt i fysiologi og udvikling af mikrobielle samfund.

Denne metode genererer biofilm tynde sektioner hvori forskellige morfologiske funktioner og zoner af genekspression kan være afbildet af DIC, Fluorescens mikroskopi og TEM. Mens DIC imaging ved hjælp af en 40 X oliebestandighedsobjektet kan være tilstrækkeligt til at vise nogle morfologiske træk (figur 2E), har vi fundet at Fluorescens mikroskopi af stammer, der er udviklet til constitutively express fluorescerende protein giver forbedret visualisering a...

Watch this Scientific Journal Video about Paraffin Embedding and Thin Sectioning of Microbial Colony Biofilms for Microscopic Analysis at JoVE.com

Paraffin Embedding and Thin Sectioning of Microbial Colony Biofilms for Microscopic Analysis

Paraffin indlejring og tynde skæring af mikrobielle koloni biofilm til mikroskopisk analyse

Vi beskriver fiksering, paraffin indlejring og tynde skære teknikker for kimtal biofilm. I rede prøver, kan biofilm underkonstruktion og reporter udtryk mønstre visualiseres ved mikroskopi.

Sectioning via paraffin embedding is a broadly established technique in eukaryotic systems. Here we provide a method for the fixation, embedding, and ...

Research

JoVE Journal

Immunology and Infection

Colony Forming Cell (CFC) Assay for Human Hematopoietic Cells

Colony Forming Cell CFC Assay for Human Hematopoietic Cells

Human hematopoietic stem/progenitor cells are usually obtained from bone marrow, cord blood, or peripheral blood and are used to study hematopoiesis and ...

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

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

Encellede Analyse af Bacillus subtilis Biofilm Brug fluorescensmikroskopi og Flowcytometri

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 ...

Growth of Mycobacterium tuberculosis Biofilms

Growth of Mycobacterium tuberculosis Biofilms

Vækst af Mycobacterium tuberculosis Biofilm

Mycobacterium tuberculosis, the etiologic agent of human tuberculosis, has an extraordinary ability to survive against environmental stresses including ...

Isolation and Genome Analysis of Single Virions using 'Single Virus Genomics'

Isolering og genom analyse af enkelte Virioner benytter &quot;enkelt virus Genomics &#39;

Whole genome amplification and sequencing of single microbial cells enables genomic characterization without the need of cultivation 1-3. Viruses, which ...

A New Method for Qualitative Multi-scale Analysis of Bacterial Biofilms on Filamentous Fungal Colonies Using Confocal and Electron Microscopy

En ny metode til Kvalitativ Multi-skala Analyse af Bakteriel biofilm på Filamentøs Kolonier Brug Konfokal og Electron Microscopy

Bacterial biofilms frequently form on fungal surfaces and can be involved in numerous bacterial-fungal interaction processes, such as metabolic ...

Extraction of Hemocytes from Drosophila melanogaster Larvae for Microbial Infection and Analysis

Extraction of Hemocytes from Drosophila melanogaster Larvae for Microbial Infection and Analysis

Ekstraktion af Hemocytes fra Drosophila melanogaster larver for mikrobiel infektion og analyse

During the pathogenic infection of Drosophila melanogaster, hemocytes play an important role in the immune response throughout the infection. Thus, the ...

Complementary Use of Microscopic Techniques and Fluorescence Reading in Studying Cryptococcus-Amoeba Interactions

Complementary Use of Microscopic Techniques and Fluorescence Reading in Studying Cryptococcus-Amoeba Interactions

Supplerende anvendelse af mikroskopiske teknikker og fluorescens aflæsning i studiet af Cryptococcus-Amoeba interaktioner

To simulate Cryptococcus infection, amoeba, which is the natural predator of cryptococcal cells in the environment, can be used as a model for ...

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

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

Anvendelse af Live Cell Imaging og Cryo-Electron Tomography at løse Spatiotemporal Funktioner i Legionella pneumophila Dot / Icm Sekresion 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 ...

Analysis of the Lipid Composition of Mycobacteria by Thin Layer Chromatography

Analyse af Lipid sammensætning af mycobacteria af tyndt lag kromatografi

Mycobacteria species can differ from one another in the rate of growth, presence of pigmentation, the colony morphology displayed on solid media, as well ...

Standardized In vitro Assays to Visualize and Quantify Interactions between Human Neutrophils and Staphylococcus aureus Biofilms

Standardized In vitro Assays to Visualize and Quantify Interactions between Human Neutrophils and Staphylococcus aureus Biofilms

Standardiserede in vitro-assays til visualisering og kvantificering af interaktioner mellem humane neutrofiler og Staphylococcus aureus biofilm

Neutrophils are the first line of defense deployed by the immune system during microbial infection. In vivo, neutrophils are recruited to the site of ...