Name: determination of biofilm initiation on virus-infected cells by bacteria and fungi
Uploaded: 2016-07-06T14:00:00.000+00:00
Duration: 12 min 33 s
Description: Watch this Scientific Journal Video about Determination of Biofilm Initiation on Virus-infected Cells by Bacteria and Fungi at JoVE.com

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的受体金黄色葡萄球菌和C.白色念珠菌 39-41。自粘膜上皮顶42面通常缺乏纤连蛋白，此系统更紧密，在自然感染和定植43-47观察模拟物。因此，我们可以通过微生物的其他成员更直接检查在随后的依从性作用的具体病毒进入受体营业额剧。 使用条目精通非扩频HSV-1和HSV-2，这些研究结果表明，HSV-1或HSV-2的细胞条目呈现难治超感染与S细胞金黄色葡萄球菌，同时增强C.白色念珠菌坚持在病毒浓度依赖性（ 图4）。有趣的是，HSV-1的在GT形式与YF的影响，是对于HSV-2，其可以具有在临床表现48-51上促进阴道念珠菌的显著影响的发现所测量的反向。从发病的角度来看，人们普遍认为C的GT表型白色念珠菌是致病的形式，与YF共生状态，51-54。 C.粘附白色念珠菌的GT该被共同孵育S.金黄色葡萄球菌显示出结合于HSV感染的HeLa细胞相比，对于YF测量的改变的图案- S.金黄色葡萄球菌具有约束力。 HSV-1增强GT的加入HeLa细胞，但是到了SIG着地较小的程度（P &lt;0.05）相比，对于YF粘附测定，如以上所讨论，对于YF粘附和GT粘附190％的控制，即 ，270％的控制。此外，虽然S。黄色葡萄球菌对HSV-1增强的GT结合没有影响，所述球菌否定由HSV-2的存在下介导的增强的附着性。观察到S的反转图案与病毒感染细胞的YF相互作用黄色葡萄球菌的效果。这种偏爱由HSV-1（YF）对HSV-2（GT）的不同真菌的形式在体内发挥的共生状态的作用，指导维修。至于S上对GT形式的效果金黄色葡萄球菌绑定，GT几乎完全取消了HSV-1抑制葡萄球菌坚持到HeLa细胞。相比之下，虽然燃气轮机的与HSV-2感染的细胞结合的能力相比，HSV-1，S的存在显著增加金黄色葡萄球菌挡在坚持了HSV-2介导的增加。 S.金黄色葡萄球菌 β细胞的相互作用，因为S.葡萄球菌仅仅粘附到未感染的HeLa细胞。此外，细胞的显微镜检查表明， 假丝酵母和HSV-1和HSV-2共定位。这里使用了来自该研究的结果平行殖民表明这款型号为多种微生物相互作用的研究报告的用途在体内的观测点的特异性。 有效地利用在此描述的协议的依赖于多种因素。首先，利用传播缺乏病毒。这使得清洁examina效果病毒进入和细胞信号传导的灰对后续微生物相互作用，没有可能发生由于包围采集离开细胞之前变化的并发症。此外，使用有缺陷的病毒允许对生物安全二班病原体的处理更安全的环境。其次，该协议允许使用这两种病毒和微生物粘附变种。变体的使​​用，只有结合于特异性受体允许微生物，或者，新的受体的备选的检测之间共享受体的划定。通过使用单克隆抗体的受体，存在用于微生物表面上粘附定位可视化的可能性，并提供了一​​种工具以研究改变粘附表达。这个协议是不适合用于无法鉴别培养基上被选择性地分离微生物的研究。该方法也依赖于单克隆抗体的可用性病毒特异性蛋白。虽然nalysis在这项研究是由酵母和细菌，利用差异荧光标记， 如得克萨斯州红色，微生物特定单克隆抗体之间的尺寸差提升将是一个有效的解决方法应该有问题的微生物在形态和大小相似。

<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。在这项研究中的差分细胞相互作用被观察为葡萄球菌和真菌粘附的HSV-调制即病毒物种特异性的结果。 金黄色葡萄球菌和C.白色念珠菌 （GT和YF）加入到同一HSV-未感染的HeLa对照细胞。在细胞上此共定位表明缺乏phy的<html>彼此的HeLa细胞粘附和所测量的差粘附的测量水平的iCal推断有可能HSV介导的（ 图3A中，A1）38。然而，在HSV-1或HSV-2感染的HeLa细胞葡萄球菌和C的无共定位白色念珠菌观察。 （ 图3B，B1，B2，B3，C1，C2）。使用荧光显微镜（FITC缀合的抗HSV-gD的单克隆抗体）进一步证实，S。葡萄球菌并未与C到共定位白色念珠菌也不HSV-1或HSV-2（ 图4A，A1，A2，A3，A4，B1，B2，B3）。 HSV和念珠菌之间的这种合作扩展到酵母和芽管形式C的（ 图2A-F）的 白色念珠菌 。微生物的黑社会之间的关联这种特殊性体现了殖民看到主机oronasopharynx黏膜更广泛的规模特异性。 <html> 1"&gt; <img alt="图1" src="/files/ftp_upload/54162/54162fig1.jpg" /> 在HeLa细胞用量依赖HSV-1感染的图1的X-gal染色图片。感染了HSV-1在不同的MOI和X-gal的染色HeLa细胞。 （A）HeLa细胞在96与X-gal的染色模拟感染的HeLa细胞控制孔板的;最初的20倍放大倍率; （B）中的HeLa细胞在96与感染HSV-1感染的多重性10（MOI）的X-gal染色的HeLa细胞孔板的;最初的20倍放大倍率; （C）的HeLa细胞在96与感染HSV-1感染的多重性50（MOI）的X-gal染色的HeLa细胞孔板的;最初的20倍放大倍率;比例尺适用于所有。 <a href="https://www.jove.com/files/ftp_upload/54162/54162fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> 文件/ ftp_upload / 54162 / 54162fig2.jpg"/&gt; 图2，HSV-1 的作用（板A，C，E）和HSV-2（图B，D，F）的感染多重性的50和10（MOI）S上粘附金黄色葡萄球菌和/或C.白色念珠菌到HeLa细胞 。 （A）S.金黄色葡萄球菌 （SA）结合的HSV-1感染的细胞中C的存在白色念珠菌芽管（GT）或酵母形式（YF）; （B）的S。金黄色葡萄球菌 （SA）结合的HSV-2感染的细胞中C的存在白色念珠菌芽管（GT）或酵母形式（YF）; （C）C.白色念珠菌芽管（GT）中S的存在下结合，HSV-1感染的细胞金黄色葡萄球菌 （SA）;（D）C.白色念珠菌芽管（GT）中S的存在下的结合对HSV-2感染的细胞金黄色葡萄球菌 （SA）; （E）C.白念珠菌酵母形式（YF）在S的存在下结合，HSV-1感染的细胞金黄色葡萄球菌 （SA）; （F）C.白念珠菌酵母形式（YF）在S的存在下结合到HSV-2感染的细胞金黄色葡萄球菌 （SA）。所有的数据点是平均值+/- SEM，每组16标准化为无病毒控制。* =显著不同未感染的HeLa细胞对照组（p &lt;0.05）。 ＃=显著不同于由括号表示成对点（P &lt;0.05）;比例尺适用于所有38 <a href="https://www.jove.com/files/ftp_upload/54162/54162fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图3" src="/files/ftp_upload/54162/54162fig3.jpg" /> 图3. S.缺乏金黄色葡萄球菌和C.白色念珠菌相互作用对HSV-1和HSV-2感染HeLa细胞，HSV-1和HSV-2感染（MOI 50）的HeLa细胞单层与S.金黄色葡萄球菌和C.白色念珠菌 （5:1靶细胞）。对于明场<html>显微镜，细胞单层用革兰氏结晶紫，然后通过光学显微镜检查染色。呈阳性的念珠菌或S细胞黄色葡萄球菌 （每微生物信号100个体细胞/每盖玻片）的二次扫描的附加 ​​的微生物共定位的信号的存在（1000倍初始倍率）。 （A＆A1）S.金黄色葡萄球菌 （SA）和C白色念珠菌酵母形式（YF）或芽管形式（GT）对未感染的HeLa细胞共定位; （A2，插入 ）。 HeLa细胞用共定位或单个微生物的百分比; （B - B3）S的缺乏。 金黄色葡萄球菌和C.白色念珠菌共定位在HSV-1的存在; （C - C2）S.缺乏金黄色葡萄球菌和C.白色念珠菌共定位在HSV-2的存在。平均值±SEM;比例尺适用于所有38rget ="_空白"&gt;点击此处查看该图的放大版本。 <img alt="图4" src="/files/ftp_upload/54162/54162fig4.jpg" /> 图4.缺乏S的共定位的黄色葡萄球菌 C.白色念珠菌对HSV-1和HSV-2感染HeLa细胞。HSV感染的HeLa细胞单层挑战与S.金黄色葡萄球菌和C.白色假丝酵母 （5:1靶细胞）染色（FITC缀合的抗HSV gD的抗体，和DAPI）。照片上有代表性的从细胞中筛选的是信号阳性HSV然后扫描念珠菌和S.结果金黄色葡萄球菌是随后二次扫描额外的微生物共定位信号的存在（每个盖玻片微生物信号100个体细胞）（1000倍初始放大倍数;尼康）。 （A - A4）C. 白色念珠菌 （A2，插入 ; DAPI染色）共定位与HSV-1; （B- B3）C.白色念珠菌共定位用HSV-2（B1，插入 ℃。 白色念珠菌 DAPI染色）。平均值±SEM;比例尺适用于所有38 <a href="https://www.jove.com/files/ftp_upload/54162/54162fig4large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <table fo:keep-together.within-page="1"><tbody><tr><td></td><td> 1 </td><td> 2 </td><td> 3 </td><td> 4 </td><td> 6 </td><td> 7 </td><td> 8 </td><td> 9 </td><td> 10 </td><td> 11 </td><td> 12 </td></tr><tr><td>管</td><td> GT </td><td> YF </td><td> MSSA </td><td colspan="2"> GT / MSSA </td><td colspan="2"> YF / MSSA </td><td> GT </td><td> YF </td><td> MSSA </td><td colspan="2"> GT / MSSA </td><td colspan="2"> YF / MSSA </td><td></td><td></td></tr><tr><td>媒体</td><td> F </td><td> F </td><td>女士</td><td>女士</td><td> F </td><td>女士</td><td> F </td><td> F </td><td> F </td><td>女士</td><td>女士</td><td> F </td><td>女士</td><td> F </td><td></td><td></td></tr><tr><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>一个</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>乙</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td> C </td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td> ð </td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td> Ë </td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td> F </td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td> G </td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td> H </td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr></tbody></table> 表格1。 微生物在坚持多微生物相互作用的通用模板测定 GT = 白色念珠菌芽管型。 YF = C.白色念珠菌酵母型的形式; MSSA =甲氧西林敏感金黄色葡萄球菌 ; MS =甘露醇盐介质; F = Fungisel网上平台。 <a href="https://www.jove.com/files/ftp_upload/54162/54162table1.xlsx" target="_blank">请点击此处下载此文件。</a> <table fo:keep-together.within-page="1"><tbody><tr><td>板1 </td><td> 1 </td><td> 2 </td><td> 3 </td><td> 4 </td><td>五</td><td> 6 </td></tr><tr><tD&gt;控制</td><td>只有HeLa细胞</td><td>海拉+ GT </td><td>海拉+ YF </td><td>海拉+萨</td><td>海拉+ GT +萨</td><td>海拉+ YF +萨</td></tr><tr><td>一个</td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>乙</td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td> C </td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td> ð </td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>板2 </td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td> HSV </td><td> HeLa细胞+ HSV </td><td> HSV +海拉+ GT </td<html> &gt; <td> HSV +海拉+ YF </td><td> HSV +海拉+萨</td><td> HSV +海拉+ GT +萨</td><td> HSV +海拉+ YF +萨</td></tr><tr><td>一个</td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>乙</td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td> C </td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td> ð </td><td></td><td></td><td></td><td></td><td></td><td></td></tr></tbody></table> 表2中。 通用模板与HSV感染和未感染HeLa细胞相互作用的多种微生物的可视化分析 GT = 白色念珠菌芽管型。 YF = C.白色念珠菌酵母型的形式; SA =甲氧西林敏感金黄色葡萄球菌 ; HSV =单纯疱疹病毒。ve.com/files/ftp_upload/54162/54162table2.xlsx"目标="_空白"&gt;请点击这里下载该文件。

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

Nanyang Technological University

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

Microtiter Dish Biofilm Formation Assay

Biofilms are communities of microbes attached to surfaces, which can be found in medical, industrial and natural settings. In fact, life in a biofilm probably represents the predominate mode of growth for microbes in most environments. Mature biofilms have a few distinct characteristics. Biofilm microbes are typically surrounded by an extracellular matrix that provides structure and protection to the community. Microbes growing in a biofilm also have a characteristic architecture generally comprised of macrocolonies (containing thousands of cells) surrounded by fluid-filled channels. Biofilm-grown microbes are also notorious for their resistance to a range of antimicrobial agents including clinically relevant antibiotics.
The microtiter dish assay is an important tool for the study of the early stages in biofilm formation, and has been applied primarily for the study of bacterial biofilms, although this assay has also been used to study fungal biofilm formation. Because this assay uses static, batch-growth conditions, it does not allow for the formation of the mature biofilms typically associated with flow cell systems. However, the assay has been effective at identifying many factors required for initiation of biofilm formation (i.e, flagella, pili, adhesins, enzymes involved in cyclic-di-GMP binding and metabolism) and well as genes involved in extracellular polysaccharide production. Furthermore, published work indicates that biofilms grown in microtiter dishes do develop some properties of mature biofilms, such a antibiotic tolerance and resistance to immune system effectors.
This simple microtiter dish assay allows for the formation of a biofilm on the wall and/or bottom of a microtiter dish. The high throughput nature of the assay makes it useful for genetic screens, as well as testing biofilm formation by multiple strains under various growth conditions. Variants of this assay have been used to assess early biofilm formation for a wide variety of microbes, including but not limited to, pseudomonads, Vibrio cholerae, Escherichia coli, staphylocci, enterococci, mycobacteria and fungi.
In the protocol described here, we will focus on the use of this assay to study biofilm formation by the model organism Pseudomonas aeruginosa. In this assay, the extent of biofilm formation is measured using the dye crystal violet (CV). However, a number of other colorimetric and metabolic stains have been reported for the quantification of biofilm formation using the microtiter plate assay. The ease, low cost and flexibility of the microtiter plate assay has made it a critical tool for the study of biofilms.

The assay describes a rapid means to measure early biofilm formation in bacteria and fungi. This method uses a microtiter plate as the substratum for microbial biofilm formation, and the biofilm is visualized using crystal violet strain. The assay provides either a qualitative or quantitative assay for early biofilm formation.

酶标菜生物膜形成实验

Biofilms are communities of microbes attached to surfaces, which can be found in medical, industrial and natural settings.  In fact, life in a biofilm ...

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In vitro Biofilm Formation in an 8-well Chamber Slide

The chronic nature of many diseases is attributed to the formation of bacterial biofilms which are recalcitrant to traditional antibiotic therapy. Biofilms are community-associated bacteria attached to a surface and encased in a matrix. The role of the extracellular matrix is multifaceted, including facilitating nutrient acquisition, and offers significant protection against environmental stresses (e.g. host immune responses). In an effort to acquire a better understanding as to how the bacteria within a biofilm respond to environmental stresses we have used a protocol wherein we visualize bacterial biofilms which have formed in an 8-well chamber slide. The biofilms were stained with the BacLight Live/Dead stain and examined using a confocal microscope to characterize the relative biofilm size, and structure under varying incubation conditions. Z-stack images were collected via confocal microscopy and analyzed by COMSTAT. This protocol can be used to help elucidate the mechanism and kinetics by which biofilms form, as well as identify components that are important to biofilm structure and stability.

In vitro Biofilm Formation in an 8-well Chamber Slide

This article describes the procedure for the formation and visualization of a bacterial biofilm grown within an 8-well chamber slide

在体外生物膜的形成以及8商会幻灯片

The chronic nature of many diseases is attributed to the formation of bacterial biofilms which are recalcitrant to traditional antibiotic therapy. ...

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. ...

Bile Salt-induced Biofilm Formation in Enteric Pathogens: Techniques for Identification and Quantification

Biofilm formation is a dynamic, multistage process that occurs in bacteria under harsh environmental conditions or times of stress. For enteric pathogens, a significant stress response is induced during gastrointestinal transit and upon bile exposure, a normal component of human digestion. To overcome the bactericidal effects of bile, many enteric pathogens form a biofilm hypothesized to permit survival when transiting through the small intestine. Here we present methodologies to define biofilm formation through solid-phase adherence assays as well as extracellular polymeric substance (EPS) matrix detection and visualization. Furthermore, biofilm dispersion assessment is presented to mimic the analysis of events triggering release of bacteria during the infection process. Crystal violet staining is used to detect adherent bacteria in a high-throughput 96-well plate adherence assay. EPS production assessment is determined by two assays, namely microscopy staining of the EPS matrix and semi-quantitative analysis with a fluorescently-conjugated polysaccharide binding lectin. Finally, biofilm dispersion is measured through colony counts and plating. Positive data from multiple assays support the characterization of biofilms and can be utilized to identify bile salt-induced biofilm formation in other bacterial strains.

This protocol enables the reader to analyze bile salt-induced biofilm formation in enteric pathogens using a multifaceted approach to capture the dynamic nature of bacterial biofilms by assessing adherence, extracellular polymeric substance matrix formation, and dispersion.

胆汁盐诱导肠道病原菌生物膜形成的鉴别和定量技术

Biofilm formation is a dynamic, multistage process that occurs in bacteria under harsh environmental conditions or times of stress. For enteric pathogens, ...

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.

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.

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

生物学

不动杆菌生物膜形成的评估和生物膜的表征

Quantification, Viability Assessment, and Visualization Strategies for Acinetobacter Biofilms

Acinetobacter causes nosocomial infections and its biofilm formation can contribute to the survival on dry surfaces such as hospital environments. Thus, biofilm quantification and visualization are important methods to assess the potential of Acinetobacter strains to cause nosocomial infections. The biofilms forming on the surface of the microplate can be quantified in terms of volume and cell numbers. Biofilm volumes can be quantified by staining using crystal violet, washing, destaining using ethanol, then measuring the solubilized dye using a microplate reader. To quantify the number of cells embedded in the biofilms, the biofilms are scrapped off using cell scrapers, harvested in the saline, vigorously agitated in the presence of glass beads, and spread on Acinetobacter agar. Then, the plates are incubated at 30 &#176;C for 24-42 h. After incubation, the red colonies are enumerated to estimate the number of cells in biofilms. This viable count method can also be useful for counting Acinetobacter cells in mixed-species biofilms. Acinetobacter biofilms can be visualized using fluorescent dyes. A commercially available microplate designed for microscopic analysis is employed to form biofilms. Then, the bottom-surface attached biofilms are stained with SYTO9 and propidium iodide dyes, washed, then visualized with confocal laser scanning microscopy.

作者聚焦：不动杆菌生物膜定量、评估和可视化的综合方案 

This protocol describes the preparation of the inoculum, the biofilm quantification on microtiter plates using crystal violet dye, the viable count in biofilms, and the visualization of biofilms of Acinetobacter.

不动杆菌生物膜的定量、活力评估和可视化策略

Acinetobacter causes nosocomial infections and its biofilm formation can contribute to the survival on dry surfaces such as hospital environments. Thus, ...

生物膜启动对病毒感染的细胞测定细菌和真菌

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Measuring the Migration and Biofilm Formation of Various Bacteria

不动杆菌生物膜的定量、活力评估和可视化策略

不动杆菌生物膜的定量、活力评估和可视化策略

不动杆菌生物膜的定量、活力评估和可视化策略