This project was supported by Midwestern University, IL Office of Research and Sponsored Programs (ORSP) and Midwestern University College of Dental Medicine-Illinois (CDMI).

1，HSV毒株和处理注:重组非扩频HSV-1（KOS）gL86和HSV-2（KOS）333gJ -与所使用的五Twiari 36,37分别提供β-半乳糖苷报告基因活性。 <ol><li>从单一的很多并储存在-80℃，使用的病毒以1:Dulbecco改良的Eagle培养基（DMEM），用20％胎牛血清（FBS）的比例为1和脱脂牛奶直到使用。病毒大量的存储之前，由邻 -硝基苯基β-D-吡喃半乳糖苷（ONPG）和5-溴-4-氯-3-吲哚基β-D-吡喃半乳糖苷（X-GAL）测定确定病毒浓度。 </li><li>确定通过X- gal染色病毒活力和感染复数（MOI）的多重性，使用报告病毒条目测定每个实验测定来说，如先前所描述（ 图1）14。 </li><li>稀释病毒（选件-MEM）到所需的MOI。修复单层（多聚甲醛0.5毫升/）染色前。放置病毒活力控制在一个单独的田鼠之三板与多种微生物检测板平行。 </li></ol> 2.海拉细胞299处理<ol><li>在37℃，5％CO 2在DMEM生长用4.5克/升葡萄糖，10％热灭活的胎牛血清（FBS），庆大霉素（50微克/ ml）和L-谷氨酰胺。传代细胞在含有胰蛋白酶溶液80％汇合（乙二胺四乙酸，0.53摩尔EDTA; 0.05％胰蛋白酶5毫升/瓶）。 </li></ol> 3. C.白色念珠菌处理注:C.从临床实验室来源获得白色念珠菌被储存在-80℃下在Remmel脱脂奶2倍介质。 <ol><li>培养冷冻库存到沙氏葡萄糖培养基（37℃）。 24小时后亚文化的C.白色念珠菌到Fungisel介质（37℃; 48小时），进行使用。 </li><li>产生芽管（GT）的形式（挑代表殖民地;3毫升FBS; 3小时; 37℃，绝对600，0.3）。孵育后，洗GT（HBSS; 2倍; 4000 XG）。洗GT加入回暖HBSS（37＃176;℃; 0.32绝对600）。 GT形式应是由血球计数来确定观察到的细胞的99％。 </li><li>通过选取有代表性Fungisel殖民地使酵母形式（YF）股票悬浮液（HBSS 3毫升，0.32绝对600）。算的YF形成数/ ml的显微镜使用血细胞计数器。 YF形式应是由血球计数来确定观察到的细胞的99％。 </li><li>让工作真菌的股票（250微升GT或YF股票在25毫升HBSS; 37℃; 10 5 CFU /毫升） </li></ol> 4. S.金黄色葡萄球菌处理<ol><li>商店S.金黄色葡萄球菌 ATCC 25923（-80℃; Remmel脱脂牛奶2倍）。文化上羊血琼脂（5％; 37℃; 24小时）。挑选有代表性的殖民地和转移到2天内中等甘露醇盐的股票（37℃; 18小时）。 </li><li>让S.金黄色葡萄球菌股票停牌（3毫升HBSS; 1.32阿布斯600 10 8 CFU /毫升） <ol><li>让工作S.金黄色葡萄球菌的股票（100微升在25毫升HBSS股票; 10 5 CFU / ml）中。 </li></ol></li></ol> 5. 念珠菌和S.金黄色葡萄球菌悬浮液<ol><li>使混合C.白色念珠菌和S.金黄色葡萄球菌悬液（250微升YF或GT的股票和25毫升HBSS 100微升金黄色葡萄球菌股票）。 </li></ol> 6.多种微生物生物膜分析<ol><li>种子的96孔板用200μl的2×10 5个 HeLa细胞/毫升（85％汇合的水平）。岩石板（30 - 45分钟; 37℃）温育前（37℃; 5％CO 2培养箱18小时）。洗涤单层（1X;选件-MEM），然后用HSV种子（HSV-1（KOS）gL86或HSV-2（KOS）33 GJ - 100微升选项-MEM; MOI 50和10）。孵育板（3小时; 37℃; 5％CO 2）。使用每天只有一个病毒株。 </li><li>洗感染单层（1×磷酸盐缓冲盐水（PBS）配的Mg +2和Ca +2; 100微升）。用温HBSS更换PBS留下25＆＃181;升的每个孔中。 <ol><li>添加YF，GT和/或S.金黄色葡萄球菌的工作悬浮液（100微升;靶细胞比= 5:1; N = 16）。如表1所示孵育板（静电; 30分钟; 37℃; 5％CO 2）。 </li></ol></li><li>温育后，在时间抽吸一列立即用300μl的PBS再填充用Mg +2和Ca +2。重复此步骤两次，然后添加无线免疫沉淀裂解液（RIPA;过滤消毒; 200微升1:50稀释）。 </li><li>迅速磨碎的HeLa细胞裂解物再放置50微升到甘露醇盐（MS）和/或Fungisel（F）的培养基（ 图2）。传播用玻璃棒弯曲溶解物以90°角。孵育所述板（在37℃下18小时）。手动计数每个平板菌落数。控制包括S的金黄色葡萄球菌和/或C.白色念珠菌坚持HSV未感染的HeLa细胞。 </li></ol> 7.影像学研究&lt;/ P&gt; <ol><li>洗每轮玻璃盖玻片（12毫米; 50ml丙酮在100毫升烧杯）。干燥和消毒盖玻片（的Kimwipes;玻璃培养皿）。放置干燥无菌盖玻片与醇火焰灭菌钳无菌24孔板的孔中。 </li><li> HeLa细胞（1毫升，用于96孔板5倍体积）加入含有洗过的无菌圆形玻璃盖玻片24孔板的孔中。根据在5倍用于96孔板的体积模板（表2）中添加的病毒，细菌和真菌，如在步骤6.2.1上述6.4中所述然后孵育和过程。 </li><li>最后一次洗涤后，通过将大量用甲醇滑动并使其蒸发定为显微镜的细胞。存储在RT板直至染色。 </li><li>对于亮视野显微镜（1000倍最终原放大）填充包含用去离子水甲醇固定盖玻片的井。立即吸水。盖上克结晶紫每个盖玻片。洗盖玻片免费的非绑定染色（去离子水）的。 原位干盖玻片，然后用硬组安装平台到一个标记滑动（图3）附着它们。 </li><li>对于荧光显微镜（100倍的目标;最后1000倍放大倍率原创）按步骤7.4中所述洗盖玻片免费甲醇基本上。干孔中的盖玻片。 <ol><li>干燥后，取出盖玻片，并把它们标记为幻灯片。然后加入1:20稀释的异硫氰酸荧光素（FITC）共轭型单纯疱疹病毒的足够量的1 + 2的gD抗体以覆盖盖玻片（1 - 5微升）。 </li><li>孵育在湿气室中的载玻片在37℃下30分钟。温育后，洗涤盖玻片在PBS中4变化。 </li><li>最后一次洗涤后，返回盖玻片标记的幻灯片。用4&#39;，6-二脒基-2-苯基（;湿气室; 37℃30分钟的DAPI）染色。孵育后洗盖玻片4瓒PBS的水电站，再贴上与硬组的安装介质中的标记幻灯片。 </li><li>允许安装介质以固化在室温在黑暗中24小时。检查既上一个明亮的视野显微镜或FITC和DAPI截止滤光片荧光显微镜下油浸物镜的盖玻片。检查至少50场（每生物体至少100个细胞）为共定位（ 图4）的照片。 </li></ol></li></ol>

The authors have nothing to disclose.

目前暂无信息可到跨越多个域分类， 即原核，真核和病毒宿主微生物的半常任理事国，常任之间复杂的相互作用。因此，我们开发了一种新的体外模型系统来研究由S.生物膜启动金黄色葡萄球菌和C.白色念珠菌对HSV-1和HSV-2感染的HeLa 229（HeLa细胞）细胞38。 HeLa细胞模型系统呈现出独特的优势。这是由于它们缺乏表面纤连蛋白表达的，作为两个S的受体...

<html>人类微生物包括来自身体共享地理区域的多个分类王国不同的生物体。附着到细胞表面是在生物膜的形成，这是微生物定植过程的一部分重要的第一步。包括在微生物可以是引起慢性和持续感染病毒。慢性细胞感染这些病毒可以在推定受体可用性引起的改变。1,2-此外，通过细胞内病原体进入细胞也可能影响宿主细胞膜的流动性/疏水性这反过来又可以改变其他微生物部件的附着，包括细菌和真菌。为了理解可在这些多种病原体在人类宿主的同一地理区域该共定位之间发生的相互作用，我们必须能够学习的表示存在于粘膜表面生物分类王国的频谱病原体的相互作用。 t"的&gt;的疱疹病毒是存在于人类的微生物的永久成员100％的微生物的一个家族3,4-。此外，它们也可以持续在症状的存在和缺乏棚两者。具体地说，单纯疱疹病毒-1和单纯疱疹病毒-2（HSV-1和HSV-2，分别）是在oronasopharynx和生殖道的微生物的永久成员。在免疫能力的个体，无论是HSV-1和HSV-2引起龈，以及生殖器疱疹5-8。在这些站点，单纯疱疹病毒引起潜伏感染为特征的慢性持续性无症状病毒脱落的HSV 9。加入到在改变细胞的结果在nectins，硫酸乙酰肝素，脂筏和疱疹病毒条目介体的表面表达/肿瘤坏死因子受体（HVEM / TNFR）10-25。这些潜在代表共享受体某些细菌和真菌， 如金黄色葡萄球菌和白色念珠菌，这同时致病菌，也可以驻留作为oronasopharynx 26,27的粘膜微生物的成员。内oronasopharynx S.金黄色葡萄球菌和C.白色念珠菌定植占据两个不同的地点。与天然齿的主机，口腔粘膜用HSV-1和C共享白色念珠菌，而前鼻鼻孔都是由S.占用金黄色葡萄球菌 28。然而，尽管体外研究结果，即S。金黄色葡萄球菌坚持口上皮细胞，29,30 S.当正常组织存在金黄色葡萄球菌 29,30不经常口腔标本中分离。鲜为人知的是关于超越的临床研究结果，即生殖道共定植龛称为S.金黄色葡萄球菌与有氧阴道炎，其特点是生殖器炎症，分泌物和性交痛，而C.白色念珠菌产生粘膜病变类似于在口腔中31-35观察。因此，虽然口腔和生殖器microbi的这些部件青梅交叉分类王国小关于它们的相互作用是已知的，因为它影响其通过加入引发生物膜形成与宿主细胞表面5的能力。该协议得到了有效的应用，以确定共同定植/感染的功能性后果。

<table><tbody><tr><td>&lt;strong&gt;&lt;em&gt;C.albicans&lt;/em&gt;&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>BBL Sabouraud Dextrose</td><td>BD</td><td>211584</td><td></td></tr><tr><td>Fungisel Agar</td><td>Dot Scientific</td><td>7205A</td><td></td></tr><tr><td>&lt;strong&gt;&lt;em&gt;S.aureus&lt;/em&gt;&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Mannitol Salt Agar</td><td>Troy Biologicals</td><td>7143B</td><td></td></tr><tr><td>Sheep blood agar</td><td>Troy Biologicals</td><td>221239</td><td></td></tr><tr><td>&lt;strong&gt;Hela cells&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>1x DMEM (Dubelcco&#039;s Modified Eagle Medium, with 4.5 g/L glucose and L-glutamine, without sodium pyruvate</td><td>Corning</td><td>10-017-CM</td><td></td></tr><tr><td>Gentamicin 50 mg/ml</td><td>Sigma</td><td>1397</td><td>50 &amp;micro;g/ml final concentration in the complete DMEM</td></tr><tr><td>Trypsin EDTA (0.05% Trypsin, 0.53 M EDTA)Solution 1x</td><td>Corning</td><td>25-052-CI</td><td></td></tr><tr><td>Fetal Bovine Serum</td><td>Atlanta Biologicals</td><td>S11150</td><td>10% final concentration in the complete DMEM</td></tr><tr><td>&lt;strong&gt;Other medium and reagents&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>ONPG</td><td>Thermo Scientific</td><td>34055</td><td></td></tr><tr><td>Ultra-Pure X gal</td><td>Invitrogen</td><td>15520-018</td><td></td></tr><tr><td>1x HBSS (Hanks&#039; Balanced Salt Solution)</td><td>Corning</td><td>20-021-CV</td><td></td></tr><tr><td>1x PBS</td><td>Dot Scientific</td><td>30042-500</td><td></td></tr><tr><td>RIPA Lysis</td><td>Life Technologies</td><td>89901</td><td></td></tr><tr><td>&lt;strong&gt;Staining&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Methanol</td><td>Fisher Scientific</td><td>A433P-4</td><td></td></tr><tr><td>HSV 1&amp;amp;2, specific for gD</td><td>ViroStat</td><td>196</td><td></td></tr><tr><td>DAPI</td><td>SIGMA</td><td>D8417-5MG</td><td></td></tr><tr><td>Gram Crystal Violet</td><td>Troy Biologicals</td><td>212527</td><td></td></tr><tr><td>&lt;strong&gt;Supplies&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Petri dish 100 x 15</td><td>Dot Scientific</td><td>229693&amp;nbsp;</td><td></td></tr><tr><td>Petri dish 150 x 15</td><td>Kord Valmark</td><td>2902</td><td></td></tr><tr><td>96-Well plates</td><td>Evergreen Scientific</td><td>222-8030-01F</td><td></td></tr><tr><td>24-well plates</td><td>Evergreen Scientific</td><td>222-8044-01F</td><td></td></tr><tr><td>Culture tubes 100 x 13</td><td>Thomas Scientific</td><td>9187L61</td><td></td></tr><tr><td>Cover slip circles, 12 mm</td><td>Deckglaser</td><td>CB00120RA1</td><td></td></tr></tbody></table>

determination of biofilm initiation on virus-infected cells by bacteria and fungi

整个分类王国，包括真菌，细菌，病毒等多种微生物相互作用的研究没有先前关于审查的微生物病毒的成员是如何影响这些病毒感染的宿主细胞随后微生物相互作用。与细菌和真菌病毒的同居主要是存在于口腔和生殖道的粘膜表面上。粘膜细胞，尤其是那些具有持久的慢性或持续性潜病毒感染，可以有一个显著对微生物的成员通过病毒改变数量和表达的受体的影响的类型。变形例在宿主细胞膜结构会导致正常菌群和机会致病菌的后续成员的以引发在生物膜的形成， 即 ，粘附在第一步骤改变的能力。这项研究描述了BIOFIL的启动为HSV的效果定量和视觉检查方法S. m的形成（粘附） 金黄色葡萄球菌和C.白色念珠菌 。

从这份报告中所描述的系统获得数据的稳健性的级别如图2所示AF 38。通过使用这种系统的与病毒感染的细胞和它们彼此的粘附效果葡萄球菌和真菌相互作用的调制可以划定。这些类型的研究需要的相互作用的显微镜检查中，以确定多种微生物相互作用是否在相同的细胞产生的示于图3和4 38。在这项研究中?...

Watch this Scientific Journal Video about Determination of Biofilm Initiation on Virus-infected Cells by Bacteria and Fungi at JoVE.com

Determination of Biofilm Initiation on Virus-infected Cells by Bacteria and Fungi

A method is described herein for the determination of inter-Kingdom association and competition (bacterial and fungal) for adherence to virus-infected HeLa cell monolayers. This protocol can be extended to multiple combinations of prokaryotes, eukaryotes, and viruses.

生物膜启动对病毒感染的细胞测定细菌和真菌

The study of polymicrobial interactions across the taxonomic kingdoms that include fungi, bacteria and virus have not been previously examined with ...

determination-biofilm-initiation-on-virus-infected-cells-bacteria

Research

JoVE Journal

Immunology and Infection

9.6K 次观看.  Midwestern University. A method is described herein for the determination of inter-Kingdom association and competition (bacterial and fungal) for adherence to virus-infected HeLa cell monolayers. This protocol can be extended to multiple combinations of prokaryotes, eukaryotes, and viruses.

视频: Determination of Biofilm Initiation on Virus-infected Cells by Bacteria and Fungi

Assay for Adhesion and Agar Invasion in S. cerevisiae

Yeasts are found in natural biofilms, where many microorganisms colonize surfaces. In artificial environments, such as surfaces of man-made objects, biofilms can reduce industrial productivity, destroy structures, and threaten human life. 1-3 On the other hand, harnessing the power of biofilms can help clean the environment and generate sustainable energy. 4-8
The ability of S. cerevisiae to colonize surfaces and participate in complex biofilms was mostly ignored until the rediscovery of the differentiation programs triggered by various signaling pathways and environmental cues in this organism. 9, 10 The continuing interest in using S. cerevisiae as a model organism to understand the interaction and convergence of signaling pathways, such as the Ras-PKA, Kss1 MAPK, and Hog1 osmolarity pathways, quickly placed S. cerevisiae in the junction of biofilm biology and signal transduction research. 11-20 To this end, differentiation of yeast cells into long, adhesive, pseudohyphal filaments became a convenient readout for the activation of signal transduction pathways upon various environmental changes. However, filamentation is a complex collection of phenotypes, which makes assaying for it as if it were a simple phenotype misleading. In the past decade, several assays were successfully adopted from bacterial biofilm studies to yeast research, such as MAT formation assays to measure colony spread on soft agar and crystal violet staining to quantitatively measure cell-surface adherence. 12, 21 However, there has been some confusion in assays developed to qualitatively assess the adhesive and invasive phenotypes of yeast in agar.
Here, we present a simple and reliable method for assessing the adhesive and invasive quality of yeast strains with easy-to-understand steps to isolate the adhesion assessment from invasion assessment. Our method, adopted from previous studies, 10, 16 involves growing cells in liquid media and plating on differential nutrient conditions for growth of large spots, which we then wash with water to assess adhesion and rub cells completely off the agar surface to assess invasion into the agar. We eliminate the need for streaking cells onto agar, which affects the invasion of cells into the agar. In general, we observed that haploid strains that invade agar are always adhesive, yet not all adhesive strains can invade agar medium. Our approach can be used in conjunction with other assays to carefully dissect the differentiation steps and requirements of yeast signal transduction, differentiation, quorum sensing, and biofilm formation.

We describe a qualitative assay for yeast adhesion and agar invasion as a measure of invasive and pseudohyphal differentiation. This simple assay can be used to assess the invasive phenotype of various mutants as well as the effects environmental cues and signaling pathways on yeast differentiation.

化验粘附和酿酒酵母琼脂入侵

Yeasts are found in natural biofilms, where many microorganisms colonize surfaces. In artificial environments, such as surfaces of man-made objects, ...

科研

生物学

Invasion of Human Cells by a Bacterial Pathogen

Here we will describe how we study the invasion of human endothelial cells by bacterial pathogen Staphylococcus aureus . The general protocol can be applied to the study of cell invasion by virtually any culturable bacterium. The stages at which specific aspects of invasion can be studied, such as the role of actin rearrangement or caveolae, will be highlighted. Host cells are grown in flasks and when ready for use are seeded into 24-well plates containing Thermanox coverslips. Using coverslips allows subsequent removal of the cells from the wells to reduce interference from serum proteins deposited onto the sides of the wells (to which S. aureus would attach). Bacteria are grown to the required density and washed to remove any secreted proteins (e.g. toxins). Coverslips with confluent layers of endothelial cells are transferred to new 24-well plates containing fresh culture medium before the addition of bacteria. Bacteria and cells are then incubated together for the required amount of time in 5% CO2 at 37&deg;C. For S. aureus this is typically between 15-90 minutes. Thermanox coverslips are removed from each well and dip-washed in PBS to remove unattached bacteria. If total associated bacteria (adherent and internalised) are to be quantified, coverslips are then placed in a fresh well containing 0.5% Triton X-100 in PBS. Gentle pipetting leads to complete cell lysis and bacteria are enumerated by serial dilution and plating onto agar. If the number of bacteria that have invaded the cells is needed, coverslips are added to wells containing 500 &mu;l tissue culture medium supplemented with gentamicin and incubation continued for 1 h, which will kill all external bacteria. Coverslips can then be washed, cells lysed and bacteria enumerated by plating onto agar as described above. If the experiment requires direct visualisation, coverslips can be fixed and stained for light, fluorescence or confocal microscopy or prepared for electron microscopy.

A general protocol for the study of invasion of host cells by a bacterial pathogen, focusing on Staphylococcus aureus and human endothelial cells.

由细菌病原体入侵人体细胞

Here we will describe how we study the invasion of human endothelial cells by bacterial pathogen Staphylococcus aureus . The general protocol can be ...

免疫与感染

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.

方法学研究<em&gt;乙。枯草</em&gt;生物膜作为模型的表征小分子抑制剂生物膜

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

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

Bacterial biofilms frequently form on fungal surfaces and can be involved in numerous bacterial-fungal interaction processes, such as metabolic cooperation, competition, or predation. The study of biofilms is important in many biological fields, including environmental science, food production, and medicine. However, few studies have focused on such bacterial biofilms, partially due to the difficulty of investigating them. Most of the methods for qualitative and quantitative biofilm analyses described in the literature are only suitable for biofilms forming on abiotic surfaces or on homogeneous and thin biotic surfaces, such as a monolayer of epithelial cells.
While laser scanning confocal microscopy (LSCM) is often used to analyze in situ and in vivo biofilms, this technology becomes very challenging when applied to bacterial biofilms on fungal hyphae, due to the thickness and the three dimensions of the hyphal networks. To overcome this shortcoming, we developed a protocol combining microscopy with a method to limit the accumulation of hyphal layers in fungal colonies. Using this method, we were able to investigate the development of bacterial biofilms on fungal hyphae at multiple scales using both LSCM and scanning electron microscopy (SEM). This report describes the protocol, including microorganism cultures, bacterial biofilm formation conditions, biofilm staining, and LSCM and SEM visualizations.

This protocol describes a new method to grow and qualitatively analyze bacterial biofilms on fungal hyphae by confocal and electron microscopy.

一种用于丝状真菌菌落细菌生物膜的定性多尺度分析的新方法利用共聚焦和电子显微镜

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

Visualization of Biofilm Formation in Candida albicans Using an Automated Microfluidic Device

Candida albicans is the most common fungal pathogen of humans, causing about 15% of hospital-acquired sepsis cases. A major virulence attribute of C. albicans is its ability to form biofilms, structured communities of cells attached to biotic and abiotic surfaces. C. albicans biofilms can form on host tissues, such as mucosal layers, and on medical devices, such as catheters, pacemakers, dentures, and joint prostheses. Biofilms pose significant clinical challenges because they are highly resistant to physical and chemical perturbations, and can act as reservoirs to seed disseminated infections. Various in vitro assays have been utilized to study C. albicans biofilm formation, such as microtiter plate assays, dry weight measurements, cell viability assays, and confocal scanning laser microscopy. All of these assays are single end-point assays, where biofilm formation is assessed at a specific time point. Here, we describe a protocol to study biofilm formation in real-time using an automated microfluidic device under laminar flow conditions. This method allows for the observation of biofilm formation as the biofilm develops over time, using customizable conditions that mimic those of the host, such as those encountered in vascular catheters. This protocol can be used to assess the biofilm defects of genetic mutants as well as the inhibitory effects of antimicrobial agents on biofilm development in real-time.

Visualization of Biofilm Formation in Candida albicans Using an Automated Microfluidic Device

This protocol describes the use of a customizable automated microfluidic device to visualize biofilm formation in Candida albicans under host physiological conditions.

全自动微流控装置在白色念珠菌生物膜形成中的可视化

Candida albicans is the most common fungal pathogen of humans, causing about 15% of hospital-acquired sepsis cases. A major virulence attribute of C. ...

Microfluidic Tools for Probing Fungal-Microbial Interactions at the Cellular Level

Filamentous fungi are successful inhabitants of soil and play a major role in soil ecosystems, such as in the decomposition of organic and inorganic matter, as well as regulation of nutrient levels. There they also find numerous opportunities to interact with a variety of other microbes such as bacteria or other fungi. Studying fungal interactions at the cellular level, however, can be challenging owing to the black box-like nature of soil. New microfluidic tools are being developed for the study of fungal interactions; two platforms designed to study bacterial-fungal and fungal-fungal interactions are highlighted. Within these microchannels, fungal-microbial interactions can be monitored in controlled physico-chemical environments at higher temporal and spatial resolution than previously possible. Application of these tools have yielded numerous novel biological insights, such as the observation of bacterial polar attachment to hyphae or revealing uncharacterised fungal-fungal antagonisms. A key feature of these methodologies regards the ease of use of this tool by non-experts, yielding highly translatable technologies for use in microbiology labs.

Owing to the opacity of soil, interactions between its constituent microbes cannot easily be visualised with cellular resolution. Here, two microfluidic tools are presented, which offer new opportunities for investigating fungal-microbial interactions. The devices are versatile and simple to use, enabling high spatiotemporal control and high-resolution imaging at the cellular level.

用于在细胞水平上探测真菌 - 微生物相互作用的微流体工具

Filamentous fungi are successful inhabitants of soil and play a major role in soil ecosystems, such as in the decomposition of organic and inorganic ...

生物工程

Measuring the Migration and Biofilm Formation of Various Bacteria

As microbes that thrive in the host body primarily have adaptive abilities that facilitate their survival, methods for classifying and identifying their nature would be beneficial in facilitating their characterization. Currently, most studies focus only on one specific characterization method; however, the isolation and identification of microorganisms from the host is a continuous process and usually requires several combinatorial characterization methods. Herein, we describe methods of identifying the microbial biofilm-forming ability, the microbial respiration state, and their chemotaxis behavior. The methods are used to identify five microbes, three of which were isolated from the bone tissue of Sprague-Dawley (SD) rats (Corynebacterium stationis, Staphylococcus cohnii subsp. urealyticus, and Enterococcus faecalis) and two from the American Type Culture Collection (ATCC)-Staphylococcus aureus ATCC 25923&#160;and Enterococcus faecalis V583. The microbes isolated from the SD rat bone tissue include the gram-positive microbes. These microbes have adapted to thrive under stressful and nutrient-limiting environments within the bone matrix. This article aims to provide the readers with the specific know-how of determining the nature and behavior of the isolated microbes within a laboratory setting.

<meta charset="utf8"></head><body>测量各种细菌的迁移和生物膜形成

Here, we present a practical method for the isolation and identification of microorganisms within the host. In this way, the physicochemical properties of microorganisms and possible ways of living in the host are clearly described.

测量各种细菌的迁移和生物膜形成

As microbes that thrive in the host body primarily have adaptive abilities that facilitate their survival, methods for classifying and identifying their ...

Candida albicans Biofilm Formation in an In Vivo Mouse Model

Source: Kuchar&#237;kov&#225;, S., et al.&#160; <a href="https://app.jove.com/v/52239">Candida albicans Biofilm Development on Medically-relevant Foreign Bodies in a Mouse Subcutaneous Model Followed by Bioluminescence Imaging.</a> J. Vis. Exp. (2015).
This video demonstrates the development of an in vivo mouse model to study the biofilm formation of Candida albicans in subcutaneous tissue. The experiment involves implanting Candida-infected devices in the animal&#39;s back region for biofilm development.

白假丝酵母体内小鼠模型中的生物膜形成

1. C. albicans Growth

	Twenty-four hours before the initiation of the animal experiment, prepare yeast extract peptone dextrose, YPD plates by adding 10 ...

JoVE Encyclopedia of Experiments

Immunology

Immunodiagnostics

In Vitro and In Vivo Models

A Method for Sample Preparation to Visualize Biofilm Forming Bacteria on Fungal Hyphae

Source: Guennoc, Cora Miquel, et al, <a href="https://app.jove.com/v/54771">A New Method for Qualitative Multi-scale Analysis of Bacterial Biofilms on Filamentous Fungal Colonies Using Confocal and Electron Microscopy.</a> J. Vis. Exp. (2017).
This video describes a method to grow and qualitatively analyze bacterial biofilms on fungal hyphae using microscopy. Bacterial biofilms frequently form on fungal surfaces and involve numerous bacterial-fungal interaction processes, such as metabolic cooperation, competition, or predation.

一种在真菌菌丝上可视化生物膜形成细菌的样品制备方法

1. Preparation of an In Vitro Biofilm of Bacteria on the Fungal Colony

	Centrifuge the bacterial culture at 5,000 x g for 3 min and suspend the pellet in ...

生物膜启动对病毒感染的细胞测定细菌和真菌

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化验粘附和酿酒酵母琼脂入侵

由细菌病原体入侵人体细胞

方法学研究

一种用于丝状真菌菌落细菌生物膜的定性多尺度分析的新方法利用共聚焦和电子显微镜

全自动微流控装置在白色念珠菌生物膜形成中的可视化

用于在细胞水平上探测真菌 - 微生物相互作用的微流体工具

测量各种细菌的迁移和生物膜形成

测量各种细菌的迁移和生物膜形成

白假丝酵母体内小鼠模型中的生物膜形成

一种在真菌菌丝上可视化生物膜形成细菌的样品制备方法