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Immunologia e infezioni

使用共聚焦显微镜监测跨王国生物膜中的细胞外pH值

Published: January 30, 2020
doi:
10.3791/60270
,
1Department of Dentistry and Oral Health,Aarhus University

Summary

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。

Abstract

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。

Introduction

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。

Protocol

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 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。 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。 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?…

Representative Results

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?…

Discussion

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…

Divulgazioni

The authors have nothing to disclose.

Acknowledgements

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。

Materials

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

Cross-kingdom BiofilmsStreptococcus MutansCandida AlbicansConfocal MicroscopyPH RatiometryExtracellular MatrixMicrobial MetabolismBiofilm GrowthSalivary SolutionFetal Bovine SerumBrain Heart InfusionSucrose
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Schlafer, S., Frost Kristensen, M. Monitoring Extracellular pH in Cross-Kingdom Biofilms using Confocal Microscopy. J. Vis. Exp. (155), e60270, doi:10.3791/60270 (2020).
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