Name: monitoring extracellular ph in cross-kingdom biofilms using confocal microscopy
Uploaded: 2020-01-30T14:00:00
Description: Watch this Scientific Journal Video about Monitoring Extracellular pH in Cross-Kingdom Biofilms using Confocal Microscopy at JoVE.com

阿内特·阿克尔·汤姆森和哈维尔·加西亚因出色的技术支持而得到认可。作者感谢鲁本斯·斯宾-内托对图像分析进行了富有成果的讨论。

奥胡斯县道德委员会（M-20100032）审查并批准了唾液收集协议。
1. 跨王国生物膜的培育
<ol><li>在有氧条件下，在37°C的血琼脂板上生长S.mutans DSM 20523和C.白化菌NCPF 3179。</li>
	<li>将每个生物体的单个菌落转移到充满5 mL脑心脏输液（BHI）的试管中。在37°C的有氧条件下生长18小时。</li>
	<li>在1，200 x g下将过夜培养物离心5分钟。丢弃上清液，将细胞重新悬浮在?...

<ol>
	<li>Siqueira, J. F., Sen, B. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fungi+in+endodontic+infections.">Fungi in endodontic infections.</a> Oral Surgery, Oral Medicine, Oral Pathology, Oral Radiology, and Endodontics. 97 (5), 632-641 (2004).</li><li>Matic Petrovic, 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=Subgingival+areas+as+potential+reservoirs+of+different+Candida+spp+in+type+2+diabetes+patients+and+healthy+subjects.">Subgingival areas as potential reservoirs of different Candida spp in type 2 diabetes patients and healthy subjects.</a> PloS One. 14 (1), 0210527 (2019).</li><li>De-La-Torre, 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=Oral+Candida+colonization+in+patients+with+chronic+periodontitis.+Is+there+any+relationship.">Oral Candida colonization in patients with chronic periodontitis. Is there any relationship.</a> Revista Iberoamericana De Micologia. 35 (3), 134-139 (2018).</li><li>Xu, 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=Streptococcal+co-infection+augments+Candida+pathogenicity+by+amplifying+the+mucosal+inflammatory+response.">Streptococcal co-infection augments Candida pathogenicity by amplifying the mucosal inflammatory response.</a> Cellular Microbiology. 16 (2), 214-231 (2014).</li><li>Xu, H., Sobue, T., Bertolini, M., Thompson, A., Dongari-Bagtzoglou, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Streptococcus+oralis+and+Candida+albicans+Synergistically+Activate+&#956;-Calpain+to+Degrade+E-cadherin+From+Oral+Epithelial+Junctions.">Streptococcus oralis and Candida albicans Synergistically Activate &#956;-Calpain to Degrade E-cadherin From Oral Epithelial Junctions.</a> The Journal of Infectious Diseases. 214 (6), 925-934 (2016).</li><li>Dongari-Bagtzoglou, A., Kashleva, H., Dwivedi, P., Diaz, P., Vasilakos, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+mucosal+Candida+albicans+biofilms.">Characterization of mucosal Candida albicans biofilms.</a> PloS One. 4 (11), 7967 (2009).</li><li>Diaz, P. I., 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=Synergistic+interaction+between+Candida+albicans+and+commensal+oral+streptococci+in+a+novel+in+vitro+mucosal+model.">Synergistic interaction between Candida albicans and commensal oral streptococci in a novel in vitro mucosal model.</a> Infection and Immunity. 80 (2), 620-632 (2012).</li><li>Xiao, 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=Candida+albicans+and+Early+Childhood+Caries:+A+Systematic+Review+and+Meta-Analysis.">Candida albicans and Early Childhood Caries: A Systematic Review and Meta-Analysis.</a> Caries Research. 52 (1-2), 102-112 (2018).</li><li>Falsetta, M. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Symbiotic+relationship+between+Streptococcus+mutans+and+Candida+albicans+synergizes+virulence+of+plaque+biofilms+in+vivo.">Symbiotic relationship between Streptococcus mutans and Candida albicans synergizes virulence of plaque biofilms in vivo.</a> Infection and Immunity. 82 (5), 1968-1981 (2014).</li><li>Hwang, G., 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=Candida+albicans+mannans+mediate+Streptococcus+mutans+exoenzyme+GtfB+binding+to+modulate+cross-kingdom+biofilm+development+in+vivo.">Candida albicans mannans mediate Streptococcus mutans exoenzyme GtfB binding to modulate cross-kingdom biofilm development in vivo.</a> PLoS Pathogens. 13 (6), 1006407 (2017).</li><li>Koo, H., Falsetta, M. L., Klein, M. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+exopolysaccharide+matrix:+a+virulence+determinant+of+cariogenic+biofilm.">The exopolysaccharide matrix: a virulence determinant of cariogenic biofilm.</a> Journal of Dental Research. 92 (12), 1065-1073 (2013).</li><li>Schlafer, S., 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+Dental+Biofilms.">Ratiometric Imaging of Extracellular pH in Dental Biofilms.</a> Journal of Visualized Experiments. (109), 53622 (2016).</li><li>Schlafer, S., Kamp, A., Garcia, J. E. <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-based+method+to+monitor+extracellular+pH+in+fungal+biofilms.">A confocal microscopy-based method to monitor extracellular pH in fungal biofilms.</a> FEMS Yeast Research. 18 (5), (2018).</li><li>Schlafer, S., B&#230;lum, V., 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=Improved+pH-ratiometry+for+the+three-dimensional+mapping+of+pH+microenvironments+in+biofilms+under+flow+conditions.">Improved pH-ratiometry for the three-dimensional mapping of pH microenvironments in biofilms under flow conditions.</a> Journal of Microbiological Methods. 152, 194-200 (2018).</li><li>Hidalgo, G., 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=Functional+tomographic+fluorescence+imaging+of+pH+microenvironments+in+microbial+biofilms+by+use+of+silica+nanoparticle+sensors.">Functional tomographic fluorescence imaging of pH microenvironments in microbial biofilms by use of silica nanoparticle sensors.</a> Applied and Environmental Microbiology. 75 (23), 7426-7435 (2009).</li><li>Vroom, J. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Depth+Penetration+and+Detection+of+pH+Gradients+in+Biofilms+by+Two-Photon+Excitation+Microscopy.">Depth Penetration and Detection of pH Gradients in Biofilms by Two-Photon Excitation Microscopy.</a> Applied and Environmental Microbiology. 65, 3502-3511 (1999).</li><li>Lawrence, J. R., Swerhone, G. D. W., Kuhlicke, U., Neu, T. R. <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+evidence+for+metabolic+and+chemical+microdomains+in+the+structured+polymer+matrix+of+bacterial+microcolonies.">In situ evidence for metabolic and chemical microdomains in the structured polymer matrix of bacterial microcolonies.</a> FEMS Microbiology Ecology. 92 (11), (2016).</li><li>Franks, A. E., 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=Novel+strategy+for+three-dimensional+real-time+imaging+of+microbial+fuel+cell+communities:+monitoring+the+inhibitory+effects+of+proton+accumulation+within+the+anode+biofilm.">Novel strategy for three-dimensional real-time imaging of microbial fuel cell communities: monitoring the inhibitory effects of proton accumulation within the anode biofilm.</a> Energy Environmental Science. 2 (1), 113-119 (2009).</li><li>de Jong, M. H., van der Hoeven, J. S., van OS, J. H., Olijve, 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=Growth+of+oral+Streptococcus+species+and+Actinomyces+viscosus+in+human+saliva.">Growth of oral Streptococcus species and Actinomyces viscosus in human saliva.</a> Applied and Environmental Microbiology. 47 (5), 901-904 (1984).</li><li>Schneider, C. A., Rasband, W. S., Eliceiri, K. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NIH+Image+to+ImageJ:+25+years+of+image+analysis.">NIH Image to ImageJ: 25 years of image analysis.</a> Nature Methods. 9 (7), 671-675 (2012).</li><li>Daims, H., L&#252;cker, S., Wagner, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Daime,+a+novel+image+analysis+program+for+microbial+ecology+and+biofilm+research.">Daime, a novel image analysis program for microbial ecology and biofilm research.</a> Environmental Microbiology. 8 (2), 200-213 (2006).</li><li>Barbosa, J. O., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Streptococcus+mutans+Can+Modulate+Biofilm+Formation+and+Attenuate+the+Virulence+of+Candida+albicans.">Streptococcus mutans Can Modulate Biofilm Formation and Attenuate the Virulence of Candida albicans.</a> PloS One. 11 (3), 0150457 (2016).</li><li>Thein, Z. M., Samaranayake, Y. H., Samaranayake, L. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+oral+bacteria+on+growth+and+survival+of+Candida+albicans+biofilms.">Effect of oral bacteria on growth and survival of Candida albicans biofilms.</a> Archives of Oral Biology. 51 (8), 672-680 (2006).</li><li>Krzy&#347;ciak, W., 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=Effect+of+a+Lactobacillus+Salivarius+Probiotic+on+a+Double-Species+Streptococcus+Mutans+and+Candida+Albicans+Caries+Biofilm.">Effect of a Lactobacillus Salivarius Probiotic on a Double-Species Streptococcus Mutans and Candida Albicans Caries Biofilm.</a> Nutrients. 9 (11), 1242 (2017).</li><li>Liu, 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=Nicotine+Enhances+Interspecies+Relationship+between+Streptococcus+mutans+and+Candida+albicans.">Nicotine Enhances Interspecies Relationship between Streptococcus mutans and Candida albicans.</a> BioMed Research International. 2017, 7953920 (2017).</li><li>Schlafer, S., Meyer, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Confocal+microscopy+imaging+of+the+biofilm+matrix.">Confocal microscopy imaging of the biofilm matrix.</a> Journal of Microbiological Methods. 138, 50-59 (2017).</li><li>Schlafer, 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=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> Applied and Environmental Microbiology. 81 (4), 1267-1273 (2015).</li><li>Ohle, 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=Real-time+microsensor+measurement+of+local+metabolic+activities+in+ex+vivo+dental+biofilms+exposed+to+sucrose+and+treated+with+chlorhexidine.">Real-time microsensor measurement of local metabolic activities in ex vivo dental biofilms exposed to sucrose and treated with chlorhexidine.</a> Applied and Environmental Microbiology. 76 (7), 2326-2334 (2010).</li><li>Schlafer, 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=pH+landscapes+in+a+novel+five-species+model+of+early+dental+biofilm.">pH landscapes in a novel five-species model of early dental biofilm.</a> PloS One. 6 (9), 25299 (2011).</li><li>Divaris, 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=The+Supragingival+Biofilm+in+Early+Childhood+Caries:+Clinical+and+Laboratory+Protocols+and+Bioinformatics+Pipelines+Supporting+Metagenomics,+Metatranscriptomics,+and+Metabolomics+Studies+of+the+Oral+Microbiome.">The Supragingival Biofilm in Early Childhood Caries: Clinical and Laboratory Protocols and Bioinformatics Pipelines Supporting Metagenomics, Metatranscriptomics, and Metabolomics Studies of the Oral Microbiome.</a> Methods in Molecular Biology. 1922, 525-548 (2019).</li><li>Stewart, P. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mini+review:+convection+around+biofilms.">Mini review: convection around biofilms.</a> Biofouling. 28 (2), 187-198 (2012).</li><li>Stoodley, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biofilms:+Flow+disrupts+communication.">Biofilms: Flow disrupts communication.</a> Nature Microbiology. 1, 15012 (2016).</li></ol>

涉及白化病和链球菌的跨王国生物膜的培养方案，在之前已经描述了9，22，23，24，25。然而，目前的设置侧重于简单的生长条件，与正常工作日兼容的时间表，平衡的物种组成，以及开发大量的生物膜基质。此外，96孔板涂有唾液溶液，在一定程度上模拟...

包括真菌和细菌物种的跨王国生物膜涉及口腔中的多种病理条件。念珠菌经常从内淋病感染1和牙周病变2，3分离。在粘膜感染中，米炎组链球菌物种已被证明能增强真菌生物膜的形成、组织入侵，并在体外和小鼠模型4、5、6、7中传播。最有趣的是，坎迪达spp的口服运输已被证明与儿童8的卡英的流行有关。如啮齿动物模型所示，粘膜链球菌和坎迪塔斯白化病之间的共生关系增加了细胞外多糖的产生，并导致形成更厚、更结血的生物膜9，10。
在上述所有条件下，特别是幼儿期，生物膜pH对疾病进展非常重要，生物膜基质在酸原微环境的发展中发挥突出作用11要求采用各种方法，允许研究跨王国生物膜内的pH变化。已开发出简单、准确的基于共聚焦显微镜的方法，用于监测细菌12和真菌13生物膜内的pH。通过比例染料C-SNARF-4和基于阈值的图像后处理，可以在生物膜14的所有三个维度中实时确定细胞外pH值。与生物膜中基于显微镜的pH监测技术相比，pH与C-SNARF-4的比值测量简单而便宜，因为它不需要合成颗粒或化合物，包括参考染料15或使用双光子激发16。仅使用一种染料可以防止探针条块分割、荧光透流和选择性漂白16、17、18的问题，同时仍然允许对细胞内和细胞外pH的可靠区分。最后，在生物膜生长后使用染料进行孵育，从而可以研究实验室和原位生长的生物膜。
本研究的目的是扩大pH比度测定的使用，并提供一种研究跨王国生物膜pH变化的方法。作为概念的证明，该方法用于监测暴露于葡萄糖的双种生物膜中的pH。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Blood agar plates</td>
			<td>Statens Serum Institut</td>
			<td>677</td>
			<td></td>
		</tr>
		<tr>
			<td>Brain heart infusion</td>
			<td>Oxoid</td>
			<td>CM1135</td>
			<td></td>
		</tr>
		<tr>
			<td>Brain heart infusion + 5 % sucrose</td>
			<td>BDH laboratory supplies</td>
			<td>10274</td>
			<td></td>
		</tr>
		<tr>
			<td>Candida albicans</td>
			<td>National Collection of Pathogenic Fungi</td>
			<td>NCPF 3179</td>
			<td></td>
		</tr>
		<tr>
			<td>D-(+)-Glucose</td>
			<td>Sigma-Aldrich</td>
			<td>G8270</td>
			<td></td>
		</tr>
		<tr>
			<td>daime: digital image analysis in microbial ecology</td>
			<td>Universit&#228;t Wien</td>
			<td>N/A</td>
			<td>Freeware; V2.1; <a href="https://dome.csb.univie.ac.at/daime" target="_blank">https://dome.csb.univie.ac.at/daime</a></td>
		</tr>
		<tr>
			<td>Dimethyl sulfoxide</td>
			<td>Life Technologies</td>
			<td>D12345</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum</td>
			<td>Gibco Life technologies</td>
			<td>10270</td>
			<td></td>
		</tr>
		<tr>
			<td>GS-6R refrigerated centrifuge</td>
			<td>Beckman</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>ImageJ</td>
			<td>National Institutes of Health</td>
			<td>N/A</td>
			<td>Freeware; V1.46r; <a href="https://imagej.nih.gov/ij" target="_blank">https://imagej.nih.gov/ij</a></td>
		</tr>
		<tr>
			<td>Java</td>
			<td>Oracle</td>
			<td>N/A</td>
			<td>Freeware necessary to run ImageJ; V8.0; <a href="https://java.com/en/download" target="_blank">https://java.com/en/download</a></td>
		</tr>
		<tr>
			<td>&#181;-Plate 96 Well Black</td>
			<td>Ibidi</td>
			<td>89626</td>
			<td></td>
		</tr>
		<tr>
			<td>MyCurveFit</td>
			<td>MyAssays Ltd.</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>2-(N-Morpholino)ethanesulfonic acid (MES) buffer</td>
			<td>Bioworld</td>
			<td>700728</td>
			<td></td>
		</tr>
		<tr>
			<td>PHM210 pH-meter</td>
			<td>Radiometer Analytical</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Plan-Apochromat 63x oil immersion objective</td>
			<td>Zeiss</td>
			<td>N/A</td>
			<td>NA=1.4</td>
		</tr>
		<tr>
			<td>SNARF&#174;-4F 5-(and-6)-Carboxylic Acid</td>
			<td>Life Technologies</td>
			<td>S23920</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile physiological saline</td>
			<td>VWR</td>
			<td>6404</td>
			<td></td>
		</tr>
		<tr>
			<td>Streptococcus mutans</td>
			<td>Deutsche Sammlung von Mikroorganismen und Zellkulturen</td>
			<td>DSM 20523</td>
			<td></td>
		</tr>
		<tr>
			<td>Vis-spectrophotometer V-3000PC</td>
			<td>VWR</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>XL Incubator</td>
			<td>PeCON</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Zeiss LSM 510 META</td>
			<td>Zeiss</td>
			<td>N/A</td>
			<td></td>
		</tr>
	</tbody>
</table>

monitoring extracellular ph in cross-kingdom biofilms using confocal microscopy

由真菌和细菌细胞组成的跨王国生物膜涉及各种口腔疾病，如内膜感染、牙周炎、粘膜感染，以及最值得注意的是儿童早期幼细胞。在所有这些条件下，生物膜基质中的pH影响微生物-宿主相互作用，从而影响疾病进展。本协议描述了一种基于共聚焦显微镜的方法，用于监测包括念珠菌和链球菌在内的跨王国生物膜内的pH动力学。利用pH依赖双发射光谱和比例探头C-SNARF-4的染色特性，确定生物膜细胞外区域pH的下降。在探头中使用 pH 比度测量需要仔细选择成像参数、彻底校准染料以及仔细、基于阈值的图像数据后处理。正确使用后，该技术可在生物膜的不同区域快速评估细胞外 pH 值，从而监测水平和垂直 pH 梯度随时间推后。虽然共聚焦显微镜的使用将Z型探型限制在75μm或以下的薄生物膜，但pH比度测定法的使用非常适合对跨王国生物膜中一个重要的毒力因子进行非侵入性研究。

24小时和48小时后，在井板中开发出了强大的跨王国生物膜。C. 白化菌表现出不同程度的丝状生长，而S.mutans在高度形成高达35μm的密集簇。单细胞和S.mutan的链群在真菌的hyphae周围，和较大的细胞间空间表明存在一个大量的矩阵（图S1）。
比例染料的校准产生不对称的sigmoidal曲线13，14。在接触葡萄糖?...

Watch this Scientific Journal Video about Monitoring Extracellular pH in Cross-Kingdom Biofilms using Confocal Microscopy at JoVE.com

Monitoring Extracellular pH in Cross-Kingdom Biofilms using Confocal Microscopy

该协议描述了由念珠菌和链球菌组成的跨王国生物膜的培养，并提出了一种基于共焦点显微镜的方法，用于监测这些生物膜内的细胞外pH值。

使用共聚焦显微镜监测跨王国生物膜中的细胞外pH值

Cross-kingdom biofilms consisting of both fungal and bacterial cells are involved in a variety of oral diseases, such as endodontic infections, ...

该协议提出了一种易于执行的方法，用于培育由链球菌组成的跨王国生物膜。穆坦人和坎迪达阿尔比坎人。随后的基于共分显微镜的pH比率测量允许准确监测这些生物膜的细胞外基质内的pH发展。仔细选择图像采集参数对于获取具有足够对比度的图像数据进行后续分析至关重要。pH 比率测量是一种快速、精确、廉价的实时研究跨王国生物膜中水平和垂直 pH 梯度的方法。pH 比率也可以在纯真菌和细菌生物膜中进行。该方法有助于增进我们对生物膜中微生物代谢的理解。展示生物膜生长过程将是阿内特·阿贾尔·汤姆森，一个技术员从我的实验室。在有氧条件下，在37摄氏度的血脂盘上生长S.mutans和C.albicans。然后将每个生物体的单菌体转移到充满5毫升脑心输液的试管中，再生长18个小时。第二天，将培养物在1200次g下离心5分钟，然后丢弃上一代。在生理盐水中重新消耗细胞，并调整OD 550到0.5为C.albicans和S.mutans。然后稀释S.mutans悬浮液1至10，以达到等值浓度。将50微升无菌唾液滴入光学底部96孔板的孔中进行显微镜检查。在37摄氏度下孵育板30分钟，然后用100微升无菌生理盐水洗三次，并排空井。在每个井中加入100微升的C.albicans悬浮液，在37摄氏度下孵育板90分钟，然后用盐水洗井三次。接下来，在每一个井中加入100微升热灭活的胎儿牛血清。孵育盘子两小时，然后用盐水洗三次。清空油井，但留下一个20微升的储层，以避免过度的剪切力。在每个井中加入 100 微升的 S.mutans 悬浮液和 150 微升的 BHI，每井加 5% 蔗糖。然后在37摄氏度下孵育板24小时或更长时间。培养较老的生物膜时，每天更换介质。在跨王国生长阶段结束时，用无菌生理盐水洗盘子五次。对于比位 pH 成像，请使用带 63X 油或水浸透镜、543 纳米激光线和光谱成像系统的倒置共声激光扫描显微镜。使用培养箱将显微镜加热到 35 摄氏度。设置检测器，用于同时检测绿色荧光从 576 到 608 纳米，红色荧光从 629 到 661 纳米。然后选择适当的激光功率和增益，以避免过度和曝光不足。将针孔大小设置为一个空气单元或约 0.8 微米的光学片。然后将图像大小设置为 512 x 512 像素，将扫描速度设置为 2。使用均值选项选择 2 的线平均值。准备100微升无菌生理盐水，0.4%葡萄糖滴定到pH7。然后制作C-SNARF-4的库存溶液，并将染料加入30微摩尔的最终浓度。用跨王国生物膜空空一口，留下一个20微升的储液罐，然后加入葡萄糖和比例染料的盐水。将板放在显微镜舞台上并开始成像。在 35 摄氏度下，以 0.2 pH 单位的增量准备一系列 50 毫摩尔 MES 缓冲液滴定到 pH 4 至 7.8。将每个缓冲液的150微升移液插入光学底部96孔板的孔中。将染料以30微摩尔浓度加入充满缓冲的井中，并平衡5分钟。将显微镜阶段加热到 35 摄氏度，并选择与前面描述的比数 pH 成像相同的设置。将 96 孔板放在显微镜阶段，并聚焦在井底。获取所有缓冲解决方案的绿色和红色通道图像。定期拍摄激光关闭的图像，以校正探测器偏移。在三次迭代中执行校准实验，并导出所有图像作为 TIFF 文件。将绿色和红色图像存储在单独的文件夹中，并重命名两个包含序号的文件系列。将图像导入专用图像分析软件（如 ImageJ）。在用激光关闭拍摄的图像中，单击分析和直方图以确定平均荧光强度。通过单击过程、数学和减法从生物膜图像中减去此值。然后将两个图像系列导入 daime，然后通过单击分段、自动分段和自定义阈值对红色通道图像执行基于阈值的分段。设置高于真菌细胞质荧光强度的低阈值，将高阈值设置为真菌细胞壁和细菌的强度以下。然后通过单击分段和传输对象图层将分段图像系列的对象图层传输到绿色通道图像系列。删除红色和绿色通道系列中的非对象像素。现在，生物膜图像从细菌和真菌细胞中清除，可以导出为TIFF文件。将图像系列导入 ImageJ，并自行划分红色图像系列。然后将结果乘以原始图像序列。生成的图像系列与原始图像系列相同，但删除所有零强度像素除外。使用绿色图像系列重复此过程。接下来，使用两个图像系列上的均值滤波器来补偿探测器噪声。将绿色除以红色图像系列，然后生成所有对象像素的绿与红比。24 小时和 48 小时后，在井板中研制出坚固的跨王国生物膜。围绕真菌催眠和大细胞空间的S.mutans的单细胞和链表明存在大量的基质。使用查找表可视化了细胞外空间中的 pH 值。在接触葡萄糖后头五分钟内，生物膜中的细胞外pHH迅速下降。此后，酸化速度减慢，通常在15分钟后达到5.5至5.8的值。由于局部pH的变化，生物膜中的荧光强度随时间而变化。在图像分割过程中，选择高阈值和低阈值以充分消除细菌和真菌细胞覆盖的所有区域。蓝色和绿色区域分别被低阈值和高阈值消除。仔细选择图像采集参数对于在细菌细胞、真菌细胞和生物膜基质之间获得良好的对比度至关重要。如果跨王国生物膜生长在流动细胞中，pH的发展可以在流动条件下监测，模仿口腔中的细胞。pH 比率测量已用于识别牙齿生物膜中pH值特别低的局部区域，即所谓的酸性热点。

Research

JoVE Journal

Immunology and Infection

Neutrophil Extracellular Traps: How to Generate and Visualize Them

嗜中性粒细胞胞外的陷阱：如何生成和可视化

Neutrophil granulocytes are the most abundant group of leukocytes in the peripheral blood. As professional phagocytes, they engulf bacteria and kill them ...

科研

免疫与感染

Visualizing Cell-to-cell Transfer of HIV using Fluorescent Clones of HIV and Live Confocal Microscopy

可视化的细胞与细胞转移的艾滋病毒，艾滋病毒和现场共聚焦显微镜的荧光克隆

By fusing the green fluorescent protein to their favorite proteins, biologists now have the ability to study living complex cellular processes using ...

A 96 Well Microtiter Plate-based Method for Monitoring Formation and Antifungal Susceptibility Testing of Candida albicans Biofilms

A 96 Well Microtiter Plate-based Method for Monitoring Formation and Antifungal Susceptibility Testing of Candida albicans Biofilms

一个96孔微孔板为基础的方法监测的形成和抗真菌的药敏试验白色念珠菌生物膜

Candida albicans remains the most frequent cause of fungal infections in an expanding population of compromised patients and candidiasis is now the third ...

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

活体显微镜的脾：寄生虫的流动性和血流量的定量分析

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

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

单细胞分析枯草芽孢杆菌生物膜，用荧光显微镜和流式细胞仪

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 ...

Concurrent Quantification of Cellular and Extracellular Components of Biofilms

细胞和生物膜细胞外成分的定量并发

Confocal laser scanning microscopy (CLSM) is a powerful tool for investigation of biofilms. Very few investigations have successfully quantified ...

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

评估隔离肺泡巨噬细胞的激光共聚焦显微镜抗真菌活性

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

Measuring Phagosome pH by Ratiometric Fluorescence Microscopy

由成比例的荧光显微镜测量吞噬体pH值

Phagocytosis is a fundamental process through which innate immune cells engulf bacteria, apoptotic cells or other foreign particles in order to kill or ...

Ratiometric Imaging of Extracellular pH in Dental Biofilms

在口腔生物膜细胞外pH值成比例成像

The pH in bacterial biofilms on teeth is of central importance for dental caries, a disease with a high worldwide prevalence. Nutrients and metabolites ...