This work was supported by the French National Research Agency through the Laboratory of Excellence ARBRE (ANR-11-LABX-0002-01), the Plant-Microbe Interfaces Scientific Focus Area in the Genomic Science Program, and the Office of Biological and Environmental Research in the DOE Office of Science. Oak Ridge National Laboratory is managed by UT-Battelle, LLC, for the United States Department of Energy under contract DE-AC05-00OR22725.

Bakteriyel ve Fungal Kültürler 1. Hazırlık <ol><li> Mantar kültürleri hazırlanması <ol><li> Petri kapları ile aynı çapa sahip selofan membranlar hazırlayın ve 30 dakika boyunca EDTA (deiyonize su içinde 1 g / L) içinde kaynatın. deiyonize su ile yaprak durulayın ve iki kez otoklav. </li><li> mantar agar aşılayarak bir mantar öncesi kültür hazırlayın EDTA önceden tedavi selofan membran ile kaplı uygun agar ortamı üzerinde takar. koloniler çapı yaklaşık 1 cm elde etmek için uygun bir büyüme sıcaklığında inkübe edin. <ol><li> L. bicolor S238N için + diNH4 tartrat 0.5 g KH 2 PO4 1 g, MgSO 4 0.5 g .7H 2, O, Maltoz D 5 g yapılan Pachlewski P5 agar ortamı üzerinde 5 gün boyunca 25 ° C&#39;de inkübe , Glikoz D + 20 g,% 10 Tiamin-HCl 1 mi, 1 seyreltilmiş 1/10 mi mikro çözeltisi (6 g / L demir sülfat, 0.27 g / L molibden trioksit, 8.45 g / L, borik asit, 5 g / L adamganese sülfat; 5 g / L bakır sülfat; 2.27 g / l çinko sülfat), deiyonize su ile 1 L&#39;ye yapılan; ve agar 20 g; pH 5.5. </li></ol></li><li> Mantar ön kültür kaynaktan, kolonilerin radyal genişlemesini desteklemek için düşük karbonlu bir agar ortamı içeren EDTA önceden muamele edilmiş selofan zar ile kaplı yeni agar plaka inoküle. <ol><li> Petri kabı aşılamak için, hafifçe bir neşter ile ön kültürden bir mantar kolonisi (hücreleri aynı fizyolojik durumu var) ve yeni agar plaka hif aktarmak dış alanı çizebilir. </li><li> Yeni koloniler çapı yaklaşık 1 cm kadar optimum büyüme sıcaklığında inkübe edin. L. bicolor S238N için p20 agar ortamı (Dinh 4 tartrat, 0.25 g, 5 gün boyunca 25 ° C&#39;de inkübe; KH 2 PO 4 0.5 g, MgSO 4 .7H 2 O 0.25 g glikoz D + 1 g % 10 Tiamin-HCl, 0.5 mi, 1/10 seyreltilmiş mikro çözeltisi (CF 1.1.2 0.5 mi.1), deiyonize su ile 1 L&#39;ye yapılan; ve agar 20 g; pH 5.5). Not: nedeniyle selofan öncesi kültüre, ikinci kültür merkezi fişler agar ortamı içermez. Bu fişler agar ve analiz önyargıları oluşturabilir besin tanıtmak beri agar fişler, biyofilm daha fazla analiz için çıkarılmalıdır. Sadece mantar Bu aşaması, muhafaza ve agar plakaları üzerinde yayılır ve sporlar ya da donmuş stoktan yayılır mantarlar için gerekli değildir türlerin. </li></ol></li></ol></li><li> Bakteri kültürlerinin hazırlanması <ol><li> (Kullanılan gerginlik bağlı olarak ya da herhangi bir başka uygun bir ortamda), sıvı Luria-Bertani (LB) ortamı içinde 25 ml uygun bir agar ortamı üzerine bir kültürden 2 ila 3 bireysel bakteriyel koloniler, toplamak ve aşılanması için steril bir döngü kullanın. P. BBc6 fluorescens için hafifçe çalkalanarak 28 ° C (~ 150 rpm) bir gecede kültürü kuluçkaya yatmaktadır. Not: soyunun kullanılmasımikroskobik gözlemler önce çift boyama (mantar ve bakteri) yerine getirilmesinin önüne geçen, çünkü GFP gibi floresan protein, ile etiketlenmiş, tercih edilir. Zamanlama, karıştırma hızı, ve kuluçkalama sıcaklığı kullanılan soyu geç üstel büyüme bir kültür elde etmek için olan hedef bağlıdır. </li></ol></li></ol> Mantar Kolonisi Bakterilerin in vitro biyofilm N 2. Hazırlık <ol><li> Santrifüj bakteriyel 3 dakika boyunca 5000 x g&#39;de kültürü ve steril 0.1 M potasyum fosfat tampon maddesi (25 g / L KH 2 PO 4 ve 2.78 g / l K 2 HPO 4, pH 5.8), 25 ml pelet askıya. Bir kere yineleyin ve aynı tamponla 10 9 hücre / ml nihai bakteri konsantrasyonu ayarlanır. </li><li> Bakteriyel süspansiyon (veya negatif kontrol için, steril 0.1 M potasyum fosfat tamponu), 5 ml ile 6 mikroplakanın doldurun. </li><li> Fung selofan membran kestiSteril bir jilet ile al kültürü her membran üzerinde tek bir mantar kolonisi ile selofan kareler elde etmek. Dikkatle forseps kullanılarak katı ortamdan hif ihtiva selofan kareler kaldırmak ve bakteriyel süspansiyonunu içeren mikroplaka bir kuyu selofan ihtiva hif kare aktarın. </li><li> mantar kolonileri hala selofan bağlı iken yavaşça mikroplağı sallamak; Daha sonra, plaka fungal koloniler bırakarak, selofan sayfaları çıkarın. </li><li> çalışılan mikroorganizmalar için uyarlanmış bir ısıda hafif bir çalkalama (~ 60 dak) mikroplaka inkübe edin. Not: inkübasyon süresi, analiz edilecek biyofilmin ve sahne oluşturulması hızına bağlıdır. P. BBc6 / L fluorescens. bicolor, erken evre biyofilm almak için 30 dakika boyunca 20 ° C&#39;de inkübe edilmiş ve 16 st kadar biyofilm olgun alır. </li></ol> Biyofilm Format 3. Lazer Tarama Konfokal Mikroskop Analiziiyon <ol><li> örnek hazırlama <ol><li> Güçlü bir tuz çözeltisi (NaCI, 17 g / L) 17, 5 ml ile dolu yeni bir 6-çukurlu mikro-aktararak mantar durulama hif elektrostatik bağlı planktonik bakterilerin ve bakteri kaldırmak için; nazikçe 1 dakika sallayın. </li><li> yavaşça, 2 dakika için sallayın, steril 0.1 M potasyum fosfat tampon maddesi 5 ml ihtiva eden yeni bir 6-yuvalı mikroplakaya mantar transfer ve taze potasyum fosfat tamponuna mantar aktarın. </li><li> Elde edilen bölüm, mantar kolonisi yaklaşık yarısını içerecek şekilde, potasyum fosfat tamponu içinde tutarken mantar kolonisi kısmı kesilmiş bir neşter ile görüntülenmek üzere. </li><li> örnek leke için, uygun bir floresan boya (analizin amacı ile ilgili olarak) ihtiva eden steril su ile dolu bir Petri kabı transfer ve karanlıkta inkübe edilir. <ol><li> Mantar ağ görselleştirmek için, örneğin, kullanımı, Kongo Kırmızı (% 0.1 nihai eşncentration, inkübasyondan 5 dakika) ve Alexa Fluor 633 (10 ug / ml nihai konsantrasyonda konjüge, buğday tohumu agglutin (WGA) lektin, inkübasyon 10 dakika) ya da FUN1 (10 uM son konsantrasyon, kuluçkalama 10 dakika). </li><li> ticari olarak temin edilebilir bir çok hücreye nüfuz edici DNA, boyalar arasında bir DNA özgü flüoresan sonda ile bakteri hücreleri Counterstain olmayan bir floresan etiketli bakteri kullanarak. Örneğin, DAPI (0.25 ug / ml nihai konsantrasyon, kuluçkalama 10 dakika) kullanın. </li><li> Matris proteinleri görselleştirmek için, bir protein leke 21 kullanın. </li></ol></li><li> Boyamadan sonra, steril 0.1 M potasyum fosfat, 10 ml ihtiva eden bir Petri kabı kapağı aktararak örnek yıkayın ve hafifçe 1 dakika çalkalayın. </li><li> Yarım Petri kabı kapağı bir slayt batığın ve ince slayt üzerinde yüzer kesim bölümü getirmek. Sonra, yavaş yavaş numune nazikçe slayt üzerinde yerleşmek için izin tampon çözeltisinden slayt kaldırmak. <li> Son olarak, numune, anti-solma montaj orta 10 ul bir cam lamel ile örtün. Not: slayt hazırlandıktan sonra, mikroskobik analiz kısa sürede yapılmalıdır (en fazla 30 dakika içinde) biyofilm yapısı değişiklik önlemek için. </li></ol></li><li> Konfokal mikroskopik analiz <ol><li> görüntüleme yazılımı bağlanmış bir 10X veya 40X objektif lens ile konfokal lazer mikroskop altında slaytlar inceleyin. Not: Burada, bir çoklu ışın tarama konfokal mikroskop 405, 488, ve 561 nm eksitasyon lazerler, bir Gaasp PMT detektörü ve 10X 0.3 NA ve 40X 1.2 NA hedefleri ile donatılmış kullanılmıştır. <ol><li> Talep edilen veri tipine ve zaman kısıtlar uyarlanmış parametreleri kullanılarak görüntüleme gerçekleştirin. kullanılan leke ve üreticinin recommendati göre lazer uyarma / emisyon dalga boylarında kullanınons. mantar kolonisi ve biyofilm küresel 3D görüşlerini almak için kiremit tarama ve Z-yığın fonksiyonları bir arada kullanın. Not: Şekil 3 Görüntüler aşağıdaki parametreleri kullanılarak elde edildi: 8-bit / piksel, ortalama = 2 = 1.58 mikro-sn, fayans çevrimiçi dikiş (% 10 kapsama, eşik = 0.7) ile 5x5 görüntüleri taramak piksel bekleme; Z adımlı = 2 um. </li></ol></li><li> 3D veri görselleştirme için birçok ücretsiz veya ticari yazılımlarla (bu 3D rendering için birçok seçenek sunan) birini kullanın. <ol><li> Örneğin, parlak alan kanalını çıkarma, 2D maksimum yoğunluğu projeksiyonları olarak Z-yığın verilerini görüntülemek parlaklık ve kontrast ayarı ve kompozit bir görüntü oluşturmak için kanalları birleştirmek. Varsa, Z-yığın ekstremitelerde sinyali olmadan dilimleri ortadan kaldırır. Sonra, bir 2D maksimum yoğunluk projeksiyonu gerçekleştirmek ve RGB renk içine sonucu dönüştürmek. Görüntüler s elde etmek için ImageJ yazılım 22 &quot;Z projesi&quot; işlevini kullanın2B maksimum yoğunluğu projeksiyonları burada tepkiyle karşılanmıştır. </li></ol></li></ol></li></ol> 4. Elektron Mikroskobu Analizi <ol><li> Adım 3.1.2 hariç, LSCM için tarif edildiği gibi örnekleri hazırlamak durulama adımlarından sonra steril su yerine potasyum fosfat tamponu ile biyofilm aktarın. Bu SEM görüntü alma karıştırmayı olur dehidratasyon adımı sırasında tuz kristallerinin oluşumunu önler. </li><li> Bir örnek sahibine biyofilm aktarın ve aşırı su çıkarmak; Sadece örnek karşılamak için küçük bir su film tabakası sağlar. </li><li> değişken basınç taramalı elektron mikroskobu (VP-SEM) odasına örnek aktarın; Bir Peltier soğutma sahnede ° C -50 dondurma; ve yavaş dondurma-kurutma için, doğrudan SEM odası içinde, 15 saat süre ile belirlenen değişken basınç 100 Pa ile sağlar. </li><li> Dondurarak kurutma işlemi tamamlandıktan sonra, numune almak ve yüksek vakumlu film kaplama sistemi içine aktarın. Platinu 2 nm ile kaplayın örnekm argon plazmanın (2.5 x 10 -2 mbar, 35 mA) altında (kuvars ölçümü). </li><li> yüksek &quot;lens&quot; çözünürlüğü dedektörü ve 1 kV elektronik yüksek gerilim kullanarak bir alan emisyon tabanca (FEG) ile donatılmış bir SEM ile kaplanmış örnek gözlemleyin. </li></ol>

<ol>
	<li>Frey-Klett, P., Burlinson, P., Deveau, A., Barret, M., Tarkka, M., Sarniguet, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bacterial-Fungal+Interactions:+Hyphens+between+Agricultural,+Clinical,+Environmental,+and+Food+Microbiologists.">Bacterial-Fungal Interactions: Hyphens between Agricultural, Clinical, Environmental, and Food Microbiologists.</a> Microbiol. Mol. Biol. Rev. 75 (4), 583-609 (2011).</li><li>Scherlach, K., Graupner, K., Hertweck, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+bacteria-fungi+interactions:+effects+on+environment,+food,+and+medicine.">Molecular bacteria-fungi interactions: effects on environment, food, and medicine.</a> Annu. Rev. Microbiol. 67, 375-397 (2013).</li><li>Schroeckh, V., Scherlach, 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=Intimate+bacterial-fungal+interaction+triggers+biosynthesis+of+archetypal+polyketides+in+Aspergillus+nidulans.">Intimate bacterial-fungal interaction triggers biosynthesis of archetypal polyketides in Aspergillus nidulans.</a> Proc. Natl. Acad. Sci. U. S. A. 106 (34), 14558-14563 (2009).</li><li>Hogan, D. A., Wargo, M. J., Beck, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bacterial+Biofilms+on+Fungal+Surfaces.">Bacterial Biofilms on Fungal Surfaces.</a> Biofilm mode of life: mechanisms and adaptations. 13, 235-245 (2007).</li><li>Hogan, D. A., 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=Pseudomonas+-+Candida+interactions:+an+ecological+role+for+virulence+factors.">Pseudomonas - Candida interactions: an ecological role for virulence factors.</a> Science. 296 (5576), 2229-2232 (2002).</li><li>Jayasinghearachchi, H. S., Seneviratne, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Can+mushrooms+fix+atmospheric+nitrogen?.">Can mushrooms fix atmospheric nitrogen?.</a> J. Biosci. 29 (3), 293-296 (2004).</li><li>Seneviratne, G., Zavahir, J. S., Weerasekara, W. M. M. S., Bandara, M. L. 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=Fungal-bacterial+biofilms+their+development+for+novel+biotechnological+applications.">Fungal-bacterial biofilms their development for novel biotechnological applications.</a> World J Microbiol Biotechnol. , 739-743 (2008).</li><li>Herath, H. M. L. I., Upamali, A., Vithanage, M., Seneviratne, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Developed+fungal+-+bacterial+biofilms+as+a+novel+tool+for+bioremoval+of+hexavelant+chromium+from+wastewater.">Developed fungal - bacterial biofilms as a novel tool for bioremoval of hexavelant chromium from wastewater.</a> Chem. Ecol. 00, 1-10 (2014).</li><li>Macedo, A. J., Kuhlicke, U., Neu, T. R., Timmis, K. N., Abraham, W. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three+stages+of+a+biofilm+community+developing+at+the+liquid-liquid+interface+between+polychlorinated+biphenyls+and+water.">Three stages of a biofilm community developing at the liquid-liquid interface between polychlorinated biphenyls and water.</a> Appl. Environ. Microbiol. 71 (11), 7301-7309 (2005).</li><li>Da Silva, W. J., Seneviratne, J., Samaranayake, L. P., Del Bel Cury, A. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bioactivity+and+architecture+of+Candida+albicans+biofilms+developed+on+poly(methyl+methacrylate)+resin+surface.">Bioactivity and architecture of Candida albicans biofilms developed on poly(methyl methacrylate) resin surface.</a> J. Biomed. Mater. Res. - Part B Appl. Biomater. 94 (1), 149-156 (2010).</li><li>Flemming, H., Wingender, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+biofilm+matrix.">The biofilm matrix.</a> Nat. Publ. Gr. 8 (9), 623-633 (2010).</li><li>Cerca, N., Gomes, F., Pereira, S., Teixeira, P., Oliveira, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Confocal+laser+scanning+microscopy+analysis+of+S.+epidermidis+biofilms+exposed+to+farnesol,+vancomycin+and+rifampicin.">Confocal laser scanning microscopy analysis of S. epidermidis biofilms exposed to farnesol, vancomycin and rifampicin.</a> BMC Res. Notes. 5, 244 (2012).</li><li>Lepanto, P., Lecumberry, F., Rossello, J., Kierbel, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+confocal+microscopy+image+analysis+method+to+measure+adhesion+and+internalization+of+Pseudomonas+aeruginosa+multicellular+structures+into+epithelial+cells.">A confocal microscopy image analysis method to measure adhesion and internalization of Pseudomonas aeruginosa multicellular structures into epithelial cells.</a> Mol. Cell. Probes. 28 (1), 1-5 (2014).</li><li>Mueller, L. N., de Brouwer, J. F. C., Almeida, J. S., Stal, L. J., Xavier, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+a+marine+phototrophic+biofilm+by+confocal+laser+scanning+microscopy+using+the+new+image+quantification+software+PHLIP.">Analysis of a marine phototrophic biofilm by confocal laser scanning microscopy using the new image quantification software PHLIP.</a> BMC Ecol. 6, (2006).</li><li>Murray, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methods+for+imaging+thick+specimens:+Confocal+microscopy,+deconvolution,+and+structured+illumination.">Methods for imaging thick specimens: Confocal microscopy, deconvolution, and structured illumination.</a> Cold Spring Harb. Protoc. 6 (12), 1399-1437 (2011).</li><li>Toljander, J. F., Artursson, V., Paul, L. R., Jansson, J. K., Finlay, R. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Attachment+of+different+soil+bacteria+to+arbuscular+mycorrhizal+fungal+extraradical+hyphae+is+determined+by+hyphal+vitality+and+fungal+species.">Attachment of different soil bacteria to arbuscular mycorrhizal fungal extraradical hyphae is determined by hyphal vitality and fungal species.</a> FEMS Microbiol. Ecol. 254, 34-40 (2006).</li><li>Brandl, M. T., Carter, M. Q., Parker, C. T., Chapman, M. R., Huynh, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Salmonella+Biofilm+Formation+on+Aspergillus+niger+Involves+Cellulose+-+Chitin+Interactions.">Salmonella Biofilm Formation on Aspergillus niger Involves Cellulose - Chitin Interactions.</a> PLoS One. 6 (10), (2011).</li><li>Balbont&#237;n, R., Vlamakis, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mutualistic+interaction+between+Salmonella+enterica+and+Aspergillus+niger+and+its+effects+on+Zea+mays+colonization.">Mutualistic interaction between Salmonella enterica and Aspergillus niger and its effects on Zea mays colonization.</a> Microb. Biotechnol. Publ. , (2014).</li><li>Frey-Klett, P., Garbaye, J., Tarkka, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+mycorrhiza+helper+bacteria+revisited.">The mycorrhiza helper bacteria revisited.</a> New Phytol. 176 (1), 22-36 (2007).</li><li>Deveau, A., Brul&#233;, 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=Role+of+fungal+trehalose+and+bacterial+thiamine+in+the+improved+survival+and+growth+of+the+ectomycorrhizal+fungus+Laccaria+bicolor+S238N+and+the+helper+bacterium+Pseudomonas+fluorescens+BBc6R8.">Role of fungal trehalose and bacterial thiamine in the improved survival and growth of the ectomycorrhizal fungus Laccaria bicolor S238N and the helper bacterium Pseudomonas fluorescens BBc6R8.</a> Environ. Microbiol. Rep. 2 (4), 560-568 (2010).</li><li>Neu, T. R., Lawrence, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advanced+Techniques+for+In+Situ+Analysis+of+the+Biofilm+Matrix+(Structure,+Composition,+Dynamics)+by+Means+of+Laser+Scanning+Microscopy.">Advanced Techniques for In Situ Analysis of the Biofilm Matrix (Structure, Composition, Dynamics) by Means of Laser Scanning Microscopy.</a> Methods Mol. Biol. 1147, 43-64 (2014).</li><li>Berggren, K. N., Schulenberg, B., 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=An+improved+formulation+of+SYPRO+Ruby+protein+gel+stain:+Comparison+with+the+original+formulation+and+with+a+ruthenium+II+tris+(bathophenanthroline+disulfonate)+formulation.">An improved formulation of SYPRO Ruby protein gel stain: Comparison with the original formulation and with a ruthenium II tris (bathophenanthroline disulfonate) formulation.</a> Proteomics. 2 (5), 486-498 (2002).</li><li>Pantanella, F., Valenti, P., Natalizi, T., Passeri, D., Berlutti, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analytical+techniques+to+study+microbial+biofilm+on+abiotic+surfaces:+pros+and+cons+of+the+main+techniques+currently+in+use.">Analytical techniques to study microbial biofilm on abiotic surfaces: pros and cons of the main techniques currently in use.</a> Ann. di Ig. 25 (1), 31-42 (2013).</li><li>Toole, G. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microtiter+Dish+Biofilm+Formation+Assay.">Microtiter Dish Biofilm Formation Assay.</a> J. Vis. Exp. (47), (2011).</li><li>Azevedo, N. F., Lopes, S. P., Keevil, C. W., Pereira, M. O., Vieira, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Time+to+&amp;#34;go+large&amp;#34;+on+biofilm+research:+Advantages+of+an+omics+approach.">Time to &amp;#34;go large&amp;#34; on biofilm research: Advantages of an omics approach.</a> Biotechnol. Lett. 31 (4), 477-485 (2009).</li><li>Koerdt, A., Orell, A., 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=Macromolecular+fingerprinting+of+Sulfolobus+species+in+biofilm:+A+transcriptomic+and+proteomic+approach+combined+with+spectroscopic+analysis.">Macromolecular fingerprinting of Sulfolobus species in biofilm: A transcriptomic and proteomic approach combined with spectroscopic analysis.</a> J. Proteome Res. 10 (9), 4105-4119 (2011).</li><li>Morgenroth, E., Milferstedt, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biofilm+engineering:+Linking+biofilm+development+at+different+length+and+time+scales.">Biofilm engineering: Linking biofilm development at different length and time scales.</a> Rev. Environ. Sci. Biotechnol. 8 (3), 203-208 (2009).</li><li>Neu, T. R., Lawrence, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Innovative+techniques,+sensors,+and+approaches+for+imaging+biofilms+at+different+scales.">Innovative techniques, sensors, and approaches for imaging biofilms at different scales.</a> Trends Microbiol. 23 (4), 233-242 (2015).</li><li>Xi, C., Marks, D., Schlachter, S., Luo, W., Boppart, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-resolution+three-dimensional+imaging+of+biofilm+development+using+optical+coherence+tomography.">High-resolution three-dimensional imaging of biofilm development using optical coherence tomography.</a> J. Biomed. Opt. 11 (3), 34001 (2006).</li><li>Janjaroen, D., Ling, F., 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=Roles+of+ionic+strength+and+biofilm+roughness+on+adhesion+kinetics+of+Escherichia+coli+onto+groundwater+biofilm+grown+on+PVC+surfaces.">Roles of ionic strength and biofilm roughness on adhesion kinetics of Escherichia coli onto groundwater biofilm grown on PVC surfaces.</a> Water Res. 47 (7), 2531-2542 (2013).</li><li>Derlon, N., Koch, N., 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=Activity+of+metazoa+governs+biofilm+structure+formation+and+enhances+permeate+flux+during+Gravity-Driven+Membrane+(GDM)+filtration.">Activity of metazoa governs biofilm structure formation and enhances permeate flux during Gravity-Driven Membrane (GDM) filtration.</a> Water Res. 47 (6), 2085-2095 (2013).</li><li>Karcz, J., Bernas, T., Nowak, A., Talik, E., Woznica, A. <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+lyophilization+to+prepare+the+nitrifying+bacterial+biofilm+for+imaging+with+scanning+electron+microscopy.">Application of lyophilization to prepare the nitrifying bacterial biofilm for imaging with scanning electron microscopy.</a> Scanning. 34 (1), 26-36 (2012).</li><li>Burton, E., Yakandawala, N., LoVetri, K., Madhyastha, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+microplate+spectrofluorometric+assay+for+bacterial+biofilms.">A microplate spectrofluorometric assay for bacterial biofilms.</a> J. Ind. Microbiol. Biotechnol. 34 (1), 1-4 (2007).</li><li>Berne, C., Ducret, A., Hardy, G. G., Brun, Y. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adhesins+Involved+in+Attachment+to+Abiotic+Surfaces+by+Gram-Negative+Bacteria.">Adhesins Involved in Attachment to Abiotic Surfaces by Gram-Negative Bacteria.</a> Microbiol. Spectr. 3 (4), 1-45 (2015).</li><li>Schlafer, S., Garcia, J. E., Greve, M., Raarup, M. K., Nyvad, B., Dige, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ratiometric+imaging+of+extracellular+pH+in+bacterial+biofilms+with+C-SNARF-4.">Ratiometric imaging of extracellular pH in bacterial biofilms with C-SNARF-4.</a> Appl. Environ. Microbiol. 81 (4), 1267-1273 (2015).</li><li>Daddi Oubekka, S., Briandet, R., Fontaine-Aupart, M. P., Steenkeste, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Correlative+time-resolved+fluorescence+microscopy+to+assess+antibiotic+diffusion-reaction+in+biofilms.">Correlative time-resolved fluorescence microscopy to assess antibiotic diffusion-reaction in biofilms.</a> Antimicrob. Agents Chemother. 56 (6), 3349-3358 (2012).</li><li>Almeida, C., Azevedo, N. F., Santos, S., Keevil, C. W., Vieira, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Discriminating+multi-species+populations+in+biofilms+with+peptide+nucleic+acid+fluorescence+in+situ+hybridization+(PNA+FISH).">Discriminating multi-species populations in biofilms with peptide nucleic acid fluorescence in situ hybridization (PNA FISH).</a> PLoS One. 6 (3), (2011).</li><li>Malic, S., Hill, K. E., Hayes, A., Percival, S. L., Thomas, D. W., Williams, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+and+identification+of+specific+bacteria+in+wound+biofilms+using+peptide+nucleic+acid+fluorescent+in+situ+hybridization+(PNA+FISH).">Detection and identification of specific bacteria in wound biofilms using peptide nucleic acid fluorescent in situ hybridization (PNA FISH).</a> Microbiology. 155 (8), 2603-2611 (2009).</li></ol>

Yazarlar ifşa hiçbir şey yok.

Bacterial biofilms are retrieved in many environments and have been studied since the 1950s, leading to the development of a number of methods to analyze them24. Classical methods to quantify and monitor biofilms include micro-titer assays and, the most widely-used method, crystal violet (CV) staining. These methods are fast, low cost, and easy to handle25 and are particularly useful to quantify total biofilm biomass or to perform viability and matrix quantification assays. On an other hand, &#34;omics&#34; methods are also useful in biofilm studies, allowing for quantitative and functional analyses of biofilms26,27. Despite the advantages of micro-titer plate and &#34;omics&#34; methods, several essential features of biofilms cannot be captured with these techniques, hindering a complete understanding of this process. Such features include matrix structures, bacterial colony architectures, cell/cell interactions, and colonization patterns, which are key data for understanding both the functioning of biofilms and the dynamics of their formation. Despite the capacity of microscopy to capture these features, microscopy analysis of bacterial biofilms on filamentous fungi are still scarce. This is mainly due to the growth of filamentous fungi, which often forms colonies of thick, complex, tridimensional networks. Formation of bacterial biofilms on fungi is common in diverse environments and is significantly involved in various fields4 (e.g., medicine, agriculture, and environment); hence, it is critical to develop new methods to facilitate their investigation. To this end, we combined a method to generate very thin fungal colonies with microscopic imaging of the bacterial biofilms. In addition, we proposed a set of microscopy tools to qualitatively analyze those biofilms. The success of the method relies upon the ability to produce very thin hyphal colonies and to apply the appropriate dyes. These points are discussed below.
Due to the complex structures of the biofilms, understanding their function requires a multi-scale approach28,29. Distribution patterns of the biofilms, bacterial colony architecture, and matrix structure and composition are analyzed at different scales (i.e., meso-scale and micro-scale). Moreover, nanoscale resolution allows access to the cell/cell physical interactions and the nano-structure of the matrix. Thus, the developed method easily enables a multi-scale analysis of the bacterial biofilms formed on the fungal colony.
In most studies, LSCM analyses of biofilms are limited to the micro-scale, the meso-scale usually being performed by optical coherence tomography30,31,32. The method presented here enables both micro- and meso-scale analyses by LSCM. It demonstrates the utility of combining both analyses in the same region of the sample and even on the same image using new-generation confocal microscopes with high resolution (Figure 3). Thus, issues linked to compiled data gathered at different scales with different methods are here avoided.
This combination of analyses gave access concomitantly to the biofilm repartition on the fungal colony, the bacterial colony architecture along the developing biofilm, and the matrix structure. The meso-scale analysis showed a heterogenic distribution of the bacterial biofilms on the fungal colonies (Figures 2 and 3). This observation would not have been possible with protocols that only permit imaging of a small portion of the fungal colony, which is not necessarily representative of the entire colony. Thus, while often neglected, the meso-scale analysis can give precious information about biofilm distribution patterns.
Finally, the developed method can be used to analyze samples with different microscopy techniques, including scanning electron microscopy. Here, SEM was used to reach the nano-scale and to obtain the bacterial spatial organization within the biofilm. It performed very well with the thin fungal colonies, while SEM only permitted surface imaging. In contrast to LSCM, SEM, however, required sample dehydration and, most often, coating with a conductive metal. This dehydration process might alter biological structures when it is not properly executed and may require optimization. Here, sample dehydration using slow lyophilization was used33. Nevertheless, applying both LSCM and SEM to the samples will allow the performance of correlative microscopy at the same location of the sample.
Despite the advantages described above, some limitations exist. Firstly, it may not be applicable to all kind of fungi. Indeed, this culturing method is developed for fungi spreading radially on the surface of solid media. This method may not be suitable for fungi forming mainly aerial hyphae (e.g., Fusarium sp.) or for micro-aerobic fungi spreading mainly inside agar. Moreover, fungi degrading cellophane may be problematic as well (e.g., Trichoderma sp.). Secondly, it is important to note that the staining strategy is a critical point and the choice of the stain must be made carefully, as the stain must not disturb the biofilm. For example, we noticed that Calcofluor White caused partial biofilm disruption (data not shown), likely due to the high pH of this stain. Also, some dyes produced heterogeneous staining (e.g., Congo Red), while others produced homogeneous staining (e.g., cell wall staining with WGA lectin), giving a heterogeneous image quality. Moreover, it is important to be aware that some dyes might not be fully specific. For example, WGA stains not only fungal cell walls but also N-acetylneuraminic acid in gram-positive bacterial cell walls and adhesins produced by gram-positive and -negative bacteria during biofilm formation34,35. Therefore, using fluorescent protein-tagged bacteria and/or fungi is recommended to avoid multiple staining. If multiple dyes are used, they must not chemically interfere, and their emission spectra should not overlap.
Meso-scale analyses require a large scanned area, and therefore, LSCM may be time-consuming (40 min to 1 hr, depending on the sample thickness) and bottleneck the analysis of a large number of samples. Nonetheless, adjustments can be made depending on the type of data required. It is possible to decrease acquisition time and image size by altering the image quality. For example, high resolution is not necessary to analyze the biofilm general repartition.
Finally, some limitations need to be considered when choosing to display Z- stack data as 2D or 3D projections. Two-dimensional projections are a good way to summarize data, but depth information is lost, and overlapped structures become hidden. On the other hand, 3D projections allow the visualization from different points of view, but they often render poorly in case of spatial complexity.
In conclusion, we have reported a method for the characterization of bacterial biofilms on hyphae at the structural level. The methodology can be extended to other applications. Indeed, this method allows the performance of functional or chemical characterization of bacterial biofilms forming on fungal hyphae. Due to the great variety of existing fluorescent reporter systems, LSCM analysis can be used for multiple purposes29. For example, the fluorescence microscopy could be used to monitor pH gradient36 or molecule diffusion in biofilms37. Additionally, the method allows for community analysis in multispecies biofilms. For example, fluorescence in situ hybridization targeting specific bacterial groups is particularly useful to study specific bacterial repartition in multispecies biofilms38,39. Last, numerous fluorescent dyes can be used to characterize the matrix composition of the biofilms21. Here, proteins were targeted using Sypro, which stains a large range of proteins, among them matrix proteins (Figure 4), but other dyes allow for the visualization of other important matrix constituents, such as exopolysaccharides or extracellular DNA. Interestingly, all these analyses could be performed at the meso-scale using the described method. Since LSCM can be performed on living samples, it is also possible to achieve time-lapse imaging using, for example, coverwell chambers, particularly suitable for thin fungal colonies. This option is particularly interesting, as biofilm formation is a complex, dynamic process. Finally, for a quantitative purpose, the reported method may improve the accuracy of automatic quantitative analysis by making this quantification possible on meso-scale images. This may overcome biofilm heterogeneity and statistical issues29.

Mantarlar ve bakteriler en çok karasal ortamlarda yaşayacakları çünkü birbirleri ile etkileşim için birçok fırsat var. Çeşitliliği ve bunların aynı anda her yerde nedeniyle, bu etkileşimler biyoteknoloji, tarım, gıda işleme ve tıp 1, 2 gibi birçok biyolojik alanlarda önemlidir. Moleküler etkileşimler ortaklar arasındaki alışverişi sağlamak yakınlığı belli bir derecede gerektirir ve bazı durumlarda, ortak bir fiziksel ilişki fonksiyonel etkileşim 3 için gereklidir. Bakteri ve mantar arasındaki ortak bir fiziksel ilişki mantar yüzeyleri 4 üzerinde bakteri biyofilmlerin oluşumudur. Bakteri hücreleri ve hyphe&#39;nin arasındaki bu doğrudan temas, çeşitli biyolojik süreçlerde katılan samimi etkileşimleri izin verir. Örneğin, ilaç, Fırsatlar başarıyla Pseudomonas aeruginosa biyofilm çalışmadatunistic mantar patojen Candida albicans biyofilm oluşumu ve virülans 5 arasındaki bağlantı içgörüler sağlayabilir. Tarımda, çalışmalar karışık biyofilm bir mantar ile ilişkili o zaman bitki büyüme destekleyici Bu bakteriler ve biyo bakteriler bir verimlilik artışı olduğu ileri sürüldü. Karışık bir biyofilm 6 kavak mantarı ile birlikte, örneğin, Bradyrhizobium elkanii N2 -fixing aktivitesi arttırmıştır. Son olarak, bio-temizlenmesi için, bakteri, mantar karışık biyofilm kirli sitelerinin 7 iyileştirme, 8 kullanılmıştır. LSCM bu minimum ön işlemlerin ile sulu biyofilm oturma, böylece, biyofilm yapısını ve organizasyonunu korumanın bir üç boyutlu gözlem izin verdiği için biyofilm çalışma özellikle uygundur. Böylece, LSCM biyofilm analizi, özellikle det, çok bilgilendiriciolgun biyofilm gelişmesine yapıştırma aşamasından, biyofilm zaman sürecini ve karakteristik aşamaları 9, 10 saptanmasını ermin. Aynı zamanda, özellikle biyofilm yapısı ve matris 11, 12 görselleştirmek veya biyofilm boyutu 13, 14 ölçmek için adapte edilir. Bu yöntem, bir mantar ipliksi kolonisi bakteri biyofilm okuyan, abiyotik veya ince biyotik yüzeylerde biyofilm incelemek için uygun olmasına rağmen hala çok zordur. Gerçekten de, çoğu ipliksi mantarlar kültür kalın, karmaşık, üç boyutlu ağlar kurmak. Kalın nesneler konfokal mikroskobu ile görüntülü olabilir olsa bile, lazer penetrasyon zayıflama ve floresan emisyon genellikle 50 mikron 15 derinliğe üzerinde nihai görüntülerin kalitesini düşürür. Ayrıca, mantar kolonileri sert değildir çünkü, benT biyofilm bozmadan mikroorganizmaları ele almak zordur. Ancak örnekler kalınlığı, mantar hyphae&#39;nin bakteri biyofilmlerin birkaç mikroskopik analizleri genellikle bu nedenle sadece az sayıda hif 16, 17, 18 ihtiva eden, mantar kolonisi küçük bir bölümü üzerinde gerçekleştirilir. Bütün bu mantar koloni içinde biyofilm heterojen dağılımı durumunda analiz içine önyargıları getirebilir ve böylece mantar kolonisi üzerinde biyofilm dağılımını açıklamak için yeteneğimizi sınırlar ve. Bu zorlukların üstesinden gelmek için, büyüme ve mantar hyphae&#39;nin bakteri biyofilm analizi için bir yöntem sunulmaktadır. Bu yöntem, Pseudomonas ectomycorrhizal basidiomiset grubu mantarın Laccaria bicolor S238N bölgesinin hifler üzerinde BBc6 fluorescens biyofilm oluşumunu çalışmak için uygulanmıştır. Bu iki orman-toprak mikroorganizmaları daha önce karma oluşturmak için tarif edilmiştirbiyofilm benzeri yapılar 19, 20. Bu yöntem, kolaylıkla daha başka bir filamentli mantar / bakteri sistemlere adapte edilebilir. Burada sunulan yöntem LSCM ve SEM görüntüleme ile çok ince mantar kolonilerinin büyümesi sağlayan, bir mantar kültürü yöntemi kombinasyonuna dayanmaktadır. Bu biyofilm nitel karakterizasyonu için izin iki mikroorganizma arasındaki etkileşimin görüşlerini ölçek mikro (mikron aralığında) almak ve (mm aralığı) mezo bize izin verdi. Biz de numuneler nano ölçekli düzeyinde (nm aralığında) de biyofilm yapısal analizini izin, SEM ile görülebilir olduğunu gösterdi.

<table border="0" cellpadding="0" cellspacing="0" width="1069">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">6 well Falcon Tissue Culture Plates</td>
			<td style="width:381px;">Fisher Scientific</td>
			<td style="width:136px;">08-772-33</td>
			<td style="width:141px;">Used in 2.2 &#38; 3.1</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">Congo Red</td>
			<td style="width:381px;">Fisher Scientific</td>
			<td style="width:136px;">C580-25</td>
			<td style="width:141px;">Used in 3.1.4.1</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">FUN 1 Cell Stain</td>
			<td style="width:381px;">Thermo Fisher Scientific</td>
			<td style="width:136px;">F7030</td>
			<td style="width:141px;">Used in 3.1.4.1</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">Wheat Germ Agglutinin, Alexa Fluor 633 Conjugate</td>
			<td style="width:381px;">Thermo Fisher Scientific</td>
			<td style="width:136px;">W21404</td>
			<td style="width:141px;">Used in 3.1.4.1</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">DAPI solution</td>
			<td style="width:381px;">Thermo Fisher Scientific</td>
			<td style="width:136px;">62248</td>
			<td style="width:141px;">Used in 3.1.4.2</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">Propidium iodide</td>
			<td style="width:381px;">Thermo Fisher Scientific</td>
			<td style="width:136px;">P3566</td>
			<td style="width:141px;">Used in 3.1.4.3</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">FilmTracer SYPRO Ruby Biofilm Matrix protein Stain</td>
			<td style="width:381px;">Thermo Fisher Scientific</td>
			<td style="width:136px;">F10318</td>
			<td style="width:141px;">Used in 3.1.4.4</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">Fluoromount-G Slide Mounting Medium</td>
			<td style="width:381px;">Fisher Scientific</td>
			<td style="width:136px;">OB100-01</td>
			<td style="width:141px;">Used in 3.1.7</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">LSM780 Axio Observer Z1</td>
			<td style="width:381px;">Zeiss</td>
			<td style="width:136px;"></td>
			<td style="width:141px;">Used in 3.2.1</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">ZEN 2.1 lite black software&#160;</td>
			<td style="width:381px;">Zeiss</td>
			<td style="width:136px;"></td>
			<td style="width:141px;">Used in 3.2.1</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">High Vacuum Coater Leica EM ACE600</td>
			<td style="width:381px;">Leica</td>
			<td style="width:136px;"></td>
			<td style="width:141px;">Used in 4</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">GeminiSEM-FEG</td>
			<td style="width:381px;">Zeiss</td>
			<td style="width:136px;"></td>
			<td style="width:141px;">Used in 4</td>
		</tr>
	</tbody>
</table>

a new method for qualitative multi-scale analysis of bacterial biofilms on filamentous fungal colonies using confocal and electron microscopy

Bakteriyel biyofilm sık sık mantar yüzeylerde oluşturacak ve metabolik işbirliği, rekabet veya avlanma gibi çok sayıda bakteri-mantar etkileşim süreçleri, dahil edilebilir. biyofilm çalışma çevre bilimleri, gıda üretimi ve tıp gibi birçok biyolojik alanlarda önemlidir. Ancak az sayıda çalışma Kısmen onları soruşturma zorluğu, bu tür bakteriyel biyofilmlerin odaklanmıştır. literatürde tarif analizleri nitel ve nicel biyofilm yöntemlerin çoğu abiyotik yüzeyleri üzerinde ya da epitelyal hücrelerin tek bir tabakası homojen ince biyotik yüzeyler üzerinde oluşturan biyofilm için uygundur. Lazer tarama konfokal mikroskopi (LSCM) sıklıkla in situ ve in vivo biyofilmlerinde analiz etmek için kullanılır iken hyphe&#39;nin bakteri biyofilm uygulandığında, bu teknoloji sayesinde kalınlığı ve hifal şebkd üç boyutları, çok zor olurorks. Bu eksikliği gidermek için, biz mantar kolonileri içinde hifal katmanların birikimini sınırlamak için bir yöntem ile mikroskopi birleştiren bir protokol geliştirmiştir. Bu yöntemi kullanarak, biz de LSCM ve taramalı elektron mikroskobu (SEM) kullanarak birden fazla ölçeklerde hyphe&#39;nin bakteriyel biyofilm gelişimini araştırmak için başardık. Bu rapor mikroorganizma kültürleri, bakteri biyofilm oluşumu koşulları, biyofilm boyama ve LSCM ve SEM görselleştirme dahil olmak üzere protokol açıklanmaktadır.

Fungal kültür ve biyofilm hazırlık genel şematik prosedürü, Şekil 1 &#39;de verilmiştir. Kültür yöntemi bize kalın mikro ve mezo ölçekli izin hif birkaç katman içeren 20 ila 50 mikron LSCM kullanarak sulandırılmış yaşam biyofilm analizleri mantar koloniler elde etmek için izin verdi. Yöntemin uygulama P. yüksek kaliteli görüntülerin elde edilmesi biyofilm (- 4 Şekil 2) oluşumu boyunca L. bicolor hifler üzerinde BBc6 biyofilm fluorescens izin verdi. Biyofilm mezo ölçekli analizi P. biyofilm heterojen dağılımı L. bicolor S238N ve hif (Şekil 2 ve 3) üzerine BBc6 fluorescens gösterdi. Mezo-ölçekli analizi de zamanla biyofilm oluşumunun takibi (Şekil izin3) Sadece bazı bakteri hifler (Şekil 3a bağlı edildiği erken safhalarında) ile ilgili), kalın, olgun bir biyofilm (Şekil 3b oluşumuna. Mezo ölçekli görüntüleri yüksek çözünürlüklü bizi koloni mimarileri (- d Şekil 3c) elde etmek amacıyla aynı görüntülerde mikro ölçekli analizi gerçekleştirmek için izin verdi. Belirli etiketleme ile kombine mikro ölçekli analiz biyofilm karakterizasyonu bir adım daha ileri gitmek sağladı. Burada, proteinlerin çoğu sınıfları etiketler SYPRO Ruby 23, numune (Şekil 4) uygulanır ve matriks proteinlerinin mevcudiyetini göstermektedir edildi. Son olarak, biyofilm yapısının daha detaylı bilgi için dehidrasyon ve kaplamanın (Şekil 5) sonra aynı numune SEM görüntüleme ile elde edilmiştir. SEM görüntüleme Resimnanoölçekli seviyesine erişim, böylece matris yapısına erişim sağlayarak. <img alt="Şekil 1" src="/files/ftp_upload/54771/54771fig1.jpg" /> Şekil 1: mantar kültürü ve biyofilm hazırlık Genel şematik prosedür. Bu rakam analizleri örnek mikroorganizma kültürleri, yöntemin temel adımları açıklar. Daha fazla detay protokolde verilmiştir. <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54771/54771fig1large.jpg" target="_blank">Bu rakamın büyük halini görmek için lütfen buraya tıklayınız.</a> <img alt="şekil 2" src="/files/ftp_upload/54771/54771fig2.jpg" /> Şekil 2: mantar kolonisi üzerinde BBc6 biyofilm yeniden bölme. Numune 10X büyütmede etkileşim 18 saat sonra görüntülendi. Görüntü 3D konfokal mikroskopi görüntüleri 2D maksimum intensite projeksiyon yoluyla elde edilmiştir. figu gri ızgaraYeniden mozaik pozisyonları göstermektedir. L. bicolor hifler Kongo Kırmızısı (kırmızı) ve bakteri ile GFP etiketli (yeşil) vardır boyandı. <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54771/54771fig2large.jpg" target="_blank">Bu rakamın büyük halini görmek için lütfen buraya tıklayınız.</a> <img alt="Şekil 3," src="/files/ftp_upload/54771/54771fig3.jpg" /> Şekil 3: L. bicolor hifler üzerinde BBc6 biyofilm oluşumunun erken ve geç evreleri. Bu görüntü 3D konfokal mikroskopi görüntüleri 2D maksimum intensite projeksiyon yoluyla elde edilmiştir. Görüntüleme 40X büyütmede yapılmıştır. (A) etkileşim 2 saat sonra yeniden bölmek biyofilm. Etkileşim 14 saat sonra (b) Biyofilm yeniden bölme. (C), beyaz bir dikdörtgen genişleme (a) dır. (D) &#39;de beyaz bir dikdörtgenin Büyütme (B). GFP-ta mantar hyphae&#39;nin Kongo Kırmızısı (kırmızı) ve bakteriler ile boyanır olangged (yeşil). <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54771/54771fig3large.jpg" target="_blank">Bu rakamın büyük halini görmek için lütfen buraya tıklayınız.</a> <img alt="Şekil 4," src="/files/ftp_upload/54771/54771fig4.jpg" /> Şekil 4: L. bicolor hifler üzerinde BBc6 biyofilm Matris boyama. (A) etkileşim 16 saat sonra Biyofilmlererde. (B) de beyaz bir dikdörtgenin Büyütme (a) dır. Görüntüleme 40X büyütmede yapılmıştır ve bu görüntülerin 3D konfokal mikroskopi görüntü 2D maksimum yoğunluk projeksiyon ile elde edildi. Bakteri GFP etiketli (yeşil), mantar FUN1 (koyu yeşil) ile boyandı, ve proteinler S. Ruby (kırmızı) ile boyandı. <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54771/54771fig4large.jpg" target="_blank">Bu rakamın büyük halini görmek için lütfen buraya tıklayınız.</a> Şekil 5: L. bicolor hifler üzerinde BBc6 biyofilm SEM görüntüleme. SEM görüntü alma EHT, 2360X gerçekleştirildi: 1kV. görüntüleme önce, numune yavaşça SEM bölmesine dondurarak kurutuldu ve platin kaplanmış. Yeşil oklar bakteriyel biyofilmlerin işaret ve sarı oklar hyphe&#39;nin işaret etmektedir. <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54771/54771fig5large.jpg" target="_blank">Bu rakamın büyük halini görmek için lütfen buraya tıklayınız.</a>

Watch this Scientific Journal Video about A New Method for Qualitative Multi-scale Analysis of Bacterial Biofilms on Filamentous Fungal Colonies Using Confocal and Electron Microscopy at JoVE.com

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

Bu protokol büyümek ve niteliksel konfokal ve elektron mikroskobu ile mantar hif bakteri biyofilm analiz etmek için yeni bir yöntem açıklanır.

Yeni Bir Yöntem Filamentli Mantar kolonileri ile ilgili Bakteriyel Biyofilmler Niteliksel Çok ölçekli Analizi için kullanılması Eşodaklı ve Elektron Mikroskobu

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

a-new-method-for-qualitative-multi-scale-analysis-bacterial-biofilms

Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

19.4K Görüntüleme.  INRA Université de Lorraine.  Bu protokol büyümek ve niteliksel konfokal ve elektron mikroskobu ile mantar hif bakteri biyofilm analiz etmek için yeni bir yöntem açıklanır. 

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

Microwave Assisted Rapid Diagnosis of Plant Virus Diseases by Transmission Electron Microscopy

Investigations of ultrastructural changes induced by viruses are often necessary to clearly identify viral diseases in plants. With conventional sample preparation for transmission electron microscopy (TEM) such investigations can take several days1,2 and are therefore not suited for a rapid diagnosis of plant virus diseases. Microwave fixation can be used to drastically reduce sample preparation time for TEM investigations with similar ultrastructural results as observed after conventionally sample preparation3-5. Many different custom made microwave devices are currently available which can be used for the successful fixation and embedding of biological samples for TEM investigations5-8. In this study we demonstrate on Tobacco Mosaic Virus (TMV) infected Nicotiana tabacum plants that it is possible to diagnose ultrastructural alterations in leaves in about half a day by using microwave assisted sample preparation for TEM. We have chosen to perform this study with a commercially available microwave device as it performs sample preparation almost fully automatically5 in contrast to the other available devices where many steps still have to be performed manually6-8 and are therefore more time and labor consuming. As sample preparation is performed fully automatically negative staining of viral particles in the sap of the remaining TMV-infected leaves and the following examination of ultrastructure and size can be performed during fixation and embedding.

This study describes a method that allows the rapid and clear diagnosis of plant virus diseases in about half a day by using a combination of microwave assisted plant sample preparation for transmission electron microscopy and negative staining methods.

Mikrodalga Transmisyon Elektron Mikroskobu ile Bitki Virüs Hastalıkları Hızlı Tanı Destekli

Investigations of ultrastructural changes induced by viruses are often necessary to clearly identify viral diseases in plants. With conventional sample ...

İmmünoloji ve Enfeksiyon

Intravital Microscopy of the Spleen: Quantitative Analysis of Parasite Mobility and Blood Flow

The advent of intravital microscopy in experimental rodent malaria models has allowed major advances to the knowledge of parasite-host interactions 1,2. Thus, in vivo imaging of malaria parasites during pre-erythrocytic stages have revealed the active entrance of parasites into skin lymph nodes 3, the complete development of the parasite in the skin 4, and the formation of a hepatocyte-derived merosome to assure migration and release of merozoites into the blood stream 5. Moreover, the development of individual parasites in erythrocytes has been recently documented using 4D imaging and challenged our current view on protein export in malaria 6. Thus, intravital imaging has radically changed our view on key events in Plasmodium development. Unfortunately, studies of the dynamic passage of malaria parasites through the spleen, a major lymphoid organ exquisitely adapted to clear infected red blood cells are lacking due to technical constraints.
Using the murine model of malaria Plasmodium yoelii in Balb/c mice, we have implemented intravital imaging of the spleen and reported a differential remodeling of it and adherence of parasitized red blood cells (pRBCs) to barrier cells of fibroblastic origin in the red pulp during infection with the non-lethal parasite line P.yoelii 17X as opposed to infections with the P.yoelii 17XL lethal parasite line 7. To reach these conclusions, a specific methodology using ImageJ free software was developed to enable characterization of the fast three-dimensional movement of single-pRBCs. Results obtained with this protocol allow determining velocity, directionality and residence time of parasites in the spleen, all parameters addressing adherence in vivo. In addition, we report the methodology for blood flow quantification using intravital microscopy and the use of different colouring agents to gain insight into the complex microcirculatory structure of the spleen. 
Ethics statement
All the animal studies were performed at the animal facilities of University of Barcelona in accordance with guidelines and protocols approved by the Ethics Committee for Animal Experimentation of the University of Barcelona CEEA-UB (Protocol No DMAH: 5429). Female Balb/c mice of 6-8 weeks of age were obtained from Charles River Laboratories.

We show the method for performing intravital microscopy of the spleen using GFP transgenic malaria parasites and the quantification of parasite mobility and blood flow within this organ.

Dalak intravital Mikroskopi: Parazit Mobilite ve Kan Akımının Kantitatif Analiz

The advent of intravital microscopy in experimental rodent malaria models has allowed major advances to the knowledge of parasite-host interactions 1,2. ...

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.

Tek hücreli Analizi Bacillus subtilis</emFloresan Mikroskobu ve Akım Sitometri Kullanma> Biyofilmler

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 ...

Visualization of Bacterial Toxin Induced Responses Using Live Cell Fluorescence Microscopy

Bacterial toxins bind to cholesterol in membranes, forming pores that allow for leakage of cellular contents and influx of materials from the external environment. The cell can either recover from this insult, which requires active membrane repair processes, or else die depending on the amount of toxin exposure and cell type1. In addition, these toxins induce strong inflammatory responses in infected hosts through activation of immune cells, including macrophages, which produce an array of pro-inflammatory cytokines2. Many Gram positive bacteria produce cholesterol binding toxins which have been shown to contribute to their virulence through largely uncharacterized mechanisms.
Morphologic changes in the plasma membrane of cells exposed to these toxins include their sequestration into cholesterol-enriched surface protrusions, which can be shed into the extracellular space, suggesting an intrinsic cellular defense mechanism3,4. This process occurs on all cells in the absence of metabolic activity, and can be visualized using EM after chemical fixation4. In immune cells such as macrophages that mediate inflammation in response to toxin exposure, induced membrane vesicles are suggested to contain cytokines of the IL-1 family and may be responsible both for shedding toxin and disseminating these pro-inflammatory cytokines5,6,7. A link between IL-1&beta; release and a specific type of cell death, termed pyroptosis has been suggested, as both are caspase-1 dependent processes8. To sort out the complexities of this macrophage response, which includes toxin binding, shedding of membrane vesicles, cytokine release, and potentially cell death, we have developed labeling techniques and fluorescence microscopy methods that allow for real time visualization of toxin-cell interactions, including measurements of dysfunction and death (Figure 1). Use of live cell imaging is necessary due to limitations in other techniques. Biochemical approaches cannot resolve effects occurring in individual cells, while flow cytometry does not offer high resolution, real-time visualization of individual cells. The methods described here can be applied to kinetic analysis of responses induced by other stimuli involving complex phenotypic changes in cells.

Methods for purifying the cholesterol binding toxin streptolysin O from recombinant E. coli and visualization of toxin binding to live eukaryotic cells are described. Localized delivery of toxin induces rapid and complex changes in targeted cells revealing novel aspects of toxin biology.

Canlı Hücre Floresan Mikroskopi kullanma Bakteriyel Toksin İsteyerek Yanıtların Görselleştirme

Bacterial toxins bind to cholesterol in membranes, forming pores that allow for leakage of cellular contents and influx of materials from the external ...

Live-cell Video Microscopy of Fungal Pathogen Phagocytosis

Phagocytic clearance of fungal pathogens, and microorganisms more generally, may be considered to consist of four distinct stages: (i) migration of phagocytes to the site where pathogens are located; (ii) recognition of pathogen-associated molecular patterns (PAMPs) through pattern recognition receptors (PRRs); (iii) engulfment of microorganisms bound to the phagocyte cell membrane, and (iv) processing of engulfed cells within maturing phagosomes and digestion of the ingested particle. Studies that assess phagocytosis in its entirety are informative1, 2, 3, 4, 5 but are limited in that they do not normally break the process down into migration, engulfment and phagosome maturation, which may be affected differentially. Furthermore, such studies assess uptake as a single event, rather than as a continuous dynamic process. We have recently developed advanced live-cell imaging technologies, and have combined these with genetic functional analysis of both pathogen and host cells to create a cross-disciplinary platform for the analysis of innate immune cell function and fungal pathogenesis. These studies have revealed novel aspects of phagocytosis that could only be observed using systematic temporal analysis of the molecular and cellular interactions between human phagocytes and fungal pathogens and infectious microorganisms more generally. For example, we have begun to define the following: (a) the components of the cell surface required for each stage of the process of recognition, engulfment and killing of fungal cells1, 6, 7, 8; (b) how surface geometry influences the efficiency of macrophage uptake and killing of yeast and hyphal cells7; and (c) how engulfment leads to alteration of the cell cycle and behavior of macrophages 9, 10.
In contrast to single time point snapshots, live-cell video microscopy enables a wide variety of host cells and pathogens to be studied as continuous sequences over lengthy time periods, providing spatial and temporal information on a broad range of dynamic processes, including cell migration, replication and vesicular trafficking. Here we describe in detail how to prepare host and fungal cells, and to conduct the video microscopy experiments. These methods can provide a user-guide for future studies with other phagocytes and microorganisms.

We describe methods for live-cell video microscopy of Candida albicans phagocytosis by macrophages. These methods enable stage-specific analysis of macrophage migration, recognition, engulfment and phagosome maturation and reveal novel aspects of phagocytosis.

Fungal Patojen Fagositoz Canlı hücre video Mikroskopi

Phagocytic clearance of fungal pathogens, and microorganisms more generally, may be considered to consist of four distinct stages: (i) migration of ...

Assessing Anti-fungal Activity of Isolated Alveolar Macrophages by Confocal Microscopy

The lung is an interface where host cells are routinely exposed to microbes and microbial products. Alveolar macrophages are the first-line phagocytic cells that encounter inhaled fungi and other microbes. Macrophages and other immune cells recognize Aspergillus motifs by pathogen recognition receptors and initiate downstream inflammatory responses. The phagocyte NADPH oxidase generates reactive oxygen intermediates (ROIs) and is critical for host defense. Although NADPH oxidase is critical for neutrophil-mediated host defense1-3, the importance of NADPH oxidase in macrophages is not well defined. The goal of this study was to delineate the specific role of NADPH oxidase in macrophages in mediating host defense against A. fumigatus. We found that NADPH oxidase in alveolar macrophages controls the growth of phagocytosed A. fumigatus spores4. Here, we describe a method for assessing the ability of mouse alveolar macrophages (AMs) to control the growth of phagocytosed Aspergillus spores (conidia). Alveolar macrophages are stained in vivo and ten days later isolated from mice by bronchoalveolar lavage (BAL). Macrophages are plated onto glass coverslips, then seeded with green fluorescent protein (GFP)-expressing A. fumigatus spores. At specified times, cells are fixed and the number of intact macrophages with phagocytosed spores is assessed by confocal microscopy.

A method to evaluate the ability of isolated mouse alveolar macrophages to control the growth of phagocytosed Aspergillus spores by confocal microscopy.

Eşodaklı Mikroskopi İzole Alveoler Makrofagların anti-fungal Hareketi değerlendirilmesi

The lung is an interface where host cells are routinely exposed to microbes and microbial products. Alveolar macrophages are the first-line phagocytic ...

Methodologies for Studying B. subtilis Biofilms as a Model for Characterizing Small Molecule Biofilm Inhibitors

This work assesses different methodologies to study the impact of small molecule biofilm inhibitors, such as D-amino acids, on the development and resilience of Bacillus subtilis biofilms. First, methods are presented that select for small molecule inhibitors with biofilm-specific targets in order to separate the effect of the small molecule inhibitors on planktonic growth from their effect on biofilm formation. Next, we focus on how inoculation conditions affect the sensitivity of multicellular, floating B. subtilis cultures to small molecule inhibitors. The results suggest that discrepancies in the reported effects of such inhibitors such as D-amino acids are due to inconsistent pre-culture conditions. Furthermore, a recently developed protocol is described for evaluating the contribution of small molecule treatments towards biofilm resistance to antibacterial substances. Lastly, scanning electron microscopy (SEM) techniques are presented to analyze the three-dimensional spatial arrangement of cells and their surrounding extracellular matrix in a B. subtilis biofilm. SEM facilitates insight into the three-dimensional biofilm architecture and the matrix texture. A combination of the methods described here can greatly assist the study of biofilm development in the presence and absence of biofilm inhibitors, and shed light on the mechanism of action of these inhibitors.

Methodologies for Studying B. subtilis Biofilms as a Model for Characterizing Small Molecule Biofilm Inhibitors

This study presents the development of reproducible methodologies to study biofilm inhibitors and their effects on Bacillus subtilis multicellularity.

Araştırma yöntemleri<em&gt; B. subtilis</emKüçük Molekül Biyofilm İnhibitörleri Karakterizasyonu için Bir Model Olarak&gt; Biyofilmler

This work assesses different methodologies to study the impact of small molecule biofilm inhibitors, such as D-amino acids, on the development and ...

Live Imaging of Antifungal Activity by Human Primary Neutrophils and Monocytes in Response to A. fumigatus

Aspergillus fumigatus is an opportunistic fungal pathogen causing invasive infections in immunocompromised hosts with a high case-fatality rate. Research investigating immunological responses against A. fumigatus has been limited by the lack of consistent and reliable assays for measuring the antifungal activity of specific immune cells in vitro. A new method is described to assess the antifungal activity of primary monocytes and neutrophils from human donors against A. fumigatus using FLuorescent Aspergillus REporter (FLARE) conidia. These conidia contain a genetically encoded dsRed reporter, which is constitutively expressed by live FLARE conidia, and are externally labeled with Alexa Fluor 633, which is resistant to degradation within the phagolysosome, thus allowing a distinction between live and dead A. fumigatus conidia. Video microscopy and flow cytometry are subsequently used to visualize the interaction between conidia and innate immune cells, assessing fungicidal activity whilst also providing a wealth of information on phagocyte migration, phagocytosis and the inhibition of fungal growth. This novel technique has already provided exciting new insights into the host-pathogen interaction of primary immune cells against A. fumigatus. It is important to note the laboratory setup required to perform this assay, including the necessary microscopy and flow cytometry facilities, and the capacity to work with human donor blood and genetically manipulated fungi. However, this assay is capable of generating large amounts of data and can reveal detailed insights into the antifungal response. This protocol has successfully been used to study the host-pathogen interaction of primary immune cells against A. fumigatus.
It is important to note the laboratory setup required to perform this assay, including the necessary microscopy and flow cytometry facilities, and the capacity to work with human donor blood and genetically manipulated fungi. However, this assay is capable of generating large amounts of data and can reveal detailed insights into the antifungal response. This protocol has successfully been used to study the host-pathogen interaction of primary immune cells against A. fumigatus.

Live Imaging of Antifungal Activity by Human Primary Neutrophils and Monocytes in Response to A. fumigatus

Here, we describe a protocol to assess antifungal activity of primary human immune cells in real-time using fluorescent Aspergillus reporter conidia in conjunction with live-cell video microscopy and flow cytometry. Generated data provide insight into host cell-Aspergillus interactions such as fungicidal activity, phagocytosis, cell migration and inhibition of fungal growth.

Cevabı İnsan İlköğretim nötrofiller ve monositler tarafından Antifungal Faaliyet Canlı Görüntüleme<em&gt; A. fumigatus</em

Aspergillus fumigatus is an opportunistic fungal pathogen causing invasive infections in immunocompromised hosts with a high case-fatality rate. Research ...

Visualizing Macrophage Extracellular Traps Using Confocal Microscopy

A primary method used to define the presence of neutrophil extracellular traps (NETs) is confocal microscopy. We have modified established confocal microscopy methods to visualize macrophage extracellular traps (METs). These extracellular traps are defined by the presence of extracellular chromatin with co-expression of other components such as granule proteases, citrullinated histones, and peptidyl arginase deiminase (PAD). The expression of METs is generally measured after exposure to a stimulus and compared to un-stimulated samples. Samples are also included for background and isotype control. Cells are analyzed using well-defined image analysis software. Confocal microscopy may be used to define the presence of METs both in vitro and in vivo in lung tissue.

Macrophage extracellular traps are a newly described entity. This article will concentrate on confocal microscopy methods and how they are visualized in vitro and in vivo from lung samples.

Makrofaj hücre dışı tuzaklar Confocal mikroskobu kullanılarak görüntülenmesi

A primary method used to define the presence of neutrophil extracellular traps (NETs) is confocal microscopy. We have modified established confocal ...

Monitoring Extracellular pH in Cross-Kingdom Biofilms using Confocal Microscopy

Cross-kingdom biofilms consisting of both fungal and bacterial cells are involved in a variety of oral diseases, such as endodontic infections, periodontitis, mucosal infections and, most notably, early childhood caries. In all of these conditions, the pH in the biofilm matrix impacts microbe-host interactions and thus the disease progression. The present protocol describes a confocal microscopy-based method to monitor pH dynamics inside cross-kingdom biofilms comprising Candida albicans and Streptococcus mutans. The pH-dependent dual-emission spectrum and the staining properties of the ratiometric probe C-SNARF-4 are exploited to determine drops in pH in extracellular areas of the biofilms. Use of pH ratiometry with the probe requires a meticulous choice of imaging parameters, a thorough calibration of the dye, and careful, threshold-based post-processing of the image data. When used correctly, the technique allows for the rapid assessment of extracellular pH in different areas of a biofilm and thus the monitoring of both horizontal and vertical pH gradients over time. While the use of confocal microscopy limits Z-profiling to thin biofilms of 75 &#181;m or less, the use of pH ratiometry is ideally suited for the noninvasive study of an important virulence factor in cross-kingdom biofilms.

The protocol describes the cultivation of cross-kingdom biofilms consisting of Candida albicans and Streptococcus mutans and presents a confocal microscopy-based method for the monitoring of extracellular pH inside these biofilms.