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.

1.细菌和真菌培养的制备<ol><li> 真菌培养的制备 <ol><li>制备玻璃纸膜具有相同的直径的培养皿中并煮沸在EDTA中（1克/升用去离子水）30分钟。用去离子水冲洗的床单和高压灭菌他们两次。 </li><li>接种真菌琼脂制备的真菌预培养插头覆盖用EDTA预处理玻璃纸膜的适当的琼脂培养基上。孵育它在最佳生长温度以获得菌落直径约1厘米。 <ol><li>为L.双色 S238N，孵育在25℃下5天Pachlewski P5琼脂培养基上，制成0.5克diNH4酒石酸，1克KH 2 PO 4，0.5g的MgSO 4上的·7H 2 O，5克麦芽糖的D + 20克葡萄糖D +的1毫升10％盐酸硫胺素，将1ml的1/10稀释微量营养素溶液（6克/升硫酸亚铁0.27克/升钼trioxyde; 8.45克/升硼酸5克/大号男人ganese硫酸盐; 5g / L的硫酸铜; 2.27克/升的硫酸锌）补足至1升去离子水;和20g琼脂; pH值5.5。 </li></ol></li><li>从真菌预培养，接种覆盖有含有低碳琼脂培养基促进集落的径向膨胀的EDTA预处理玻璃纸膜新琼脂平板上。 <ol><li>接种的培养皿，轻轻刮擦外部区域（其中细胞具有相同的生理状态）从预培养用手术刀真菌菌落和菌丝转移到新的琼脂平板上。 </li><li>在最佳生长温度孵育，直到新的菌落的直径为约1厘米。对于二色补血草 S238N，培养他们在25℃，P20琼脂培养基（0.25克亭4酒石酸盐5天;0.5克KH 2 PO 4;0.25克硫酸镁4·7H 2 O的1克葡萄糖D +的0.5含10％盐酸硫胺素的0.5毫升1/10稀释微量营养素溶液（比照1.1.2。1）中，由向1升去离子水;和20g琼脂; pH值5.5）。 注:由于对玻璃纸预培养，第二个文化将不包含在中央插头琼脂培养基。琼脂塞应为生物膜的进一步分析被移除，因为这些插头引入琼脂和可以创建在分析偏差营养素。这一步骤只涉及真菌即保持和传播在琼脂平板上，并且这是没有必要对于从孢子或冷冻储存传播真菌物种。 </li></ol></li></ol></li><li> 细菌培养物的制备 <ol><li>用无菌环以收集从培养2至3个单独的细菌菌落的适当的琼脂培养基上并接种25毫升液体的Luria-BERTANI（LB）培养基（或任何其它适当的介质中，根据所用的菌株）。对于荧光假单胞菌 BBc6，在28℃轻轻摇动（〜150rpm）下孵育过夜培养。 注意:使用应变具有标记荧光蛋白，如绿色荧光蛋白，是优选的，因为这避免了前显微观察进行双重染色（真菌和细菌）。定时，搅拌速度，和孵育的温度取决于所用的菌株，其目标是获得在后期指数增长的培养。 </li></ol></li></ol> 2.对真菌菌落制备N 体外生物膜细菌<ol><li>离心机在5000×g离心3分钟的细菌培养和悬浮颗粒在25ml无菌0.1M磷酸钾缓冲液（25克/升KH 2 PO 4 2.78克/ LK 2 HPO 4，pH值5.8）的。重复此步骤一次，并调整最终细菌浓度到用相同缓冲液10 9细胞/ ml。 </li><li>填充6孔微量用5ml细菌悬浮液（或用于阴性对照的无菌的0.1M磷酸钾缓冲液）的。 </li><li>切烽的玻璃纸薄膜人文化用无菌刀片获得玻璃纸广场上每个膜单真菌菌落。小心地取出使用镊子从固体培养基含有菌丝玻璃纸正方形和含有玻璃纸菌丝的平方转移到含有细菌悬浮液的微板的孔中。 </li><li>轻轻摇动微孔板，而真菌菌落仍然附着于玻璃纸;那么，除去玻璃纸，留在板上的真菌菌落。 </li><li>孵育在适于所研究微生物的温度下温和搅拌（〜60转）微孔板。 注意:温育时间取决于建立生物膜和阶段的速度进行分析。对于荧光假单胞菌 BBc6 / L。双色，孵育在20℃下30分钟以获得早期生物膜和多达16个小时以获得成熟生物膜。 </li></ol> 3.生物膜格式的激光扫描共聚焦显微镜分析离子<ol><li> 样品制备 <ol><li>以除去浮游的细菌和细菌静电附着到菌丝，通过将其转移到填充用5ml强烈盐溶液（氯化钠，17克/升）17的一个新的6孔微冲洗真菌;轻轻摇动1分钟。 </li><li>转移真菌含有5ml无菌0.1M磷酸钾缓冲液的新6孔微板，2分钟轻轻摇动，和真菌转移到新鲜的磷酸钾缓冲液中。 </li><li>切真菌菌落的一部分用手术刀，同时保持它在磷酸钾缓冲液，使得所获得的部分包含有关真菌菌落的一半进行成像。 </li><li>染色样品，将其转移到培养皿中填充有含有适当的荧光染料（取决于分析的目的）无菌水，并在黑暗中孵育它。 <ol><li>为了显现真菌网络，使用，例如，刚果红（0.1％最终共ncentration，5分钟温育），小麦胚芽凝集素（WGA）凝集素缀合的Alexa Fluor 633（10微克/毫升的最终浓度，孵育10分钟）或FUN1（10μM终浓度，孵育10分钟）。 </li><li>如果使用非荧光标记细菌，染液的细菌细胞与市售的许多细胞穿透物的DNA的染料中的特定DNA的荧光探针。例如，使用DAPI（0.25微克/毫升的最终浓度，孵育10分钟）。 </li><li>以可视化的基质蛋白，用蛋白质污渍21。 </li></ol></li><li>染色后，通过将其转移到含有10毫升无菌磷酸盐的0.1M钾的培养皿盖子冲洗样品和1分钟轻轻摇动。 </li><li>一半浸没在培养皿盖的滑动和微妙使切割部分浮在载玻片上方。然后，慢慢地取出从缓冲溶液中的滑动，使样品在幻灯片上轻轻沉降。 <li>最后，添加10微升抗褪色封固剂的样品中，用玻璃盖玻片覆盖。 注意:一旦滑动制备，必须尽快进行显微分析（至多30分钟内），以避免在生物膜结构修饰。 </li></ol></li><li> 共聚焦显微镜分析 <ol><li>检查具有耦合到成像软件10倍或40倍物镜的共聚焦激光显微镜下的幻灯片。 注意:在此，一个多光束扫描共聚焦显微镜配备405，488和561 nm激发激光器，一个的GaAsP PMT检测，和10X 0.3 NA和40X 1.2 NA目标使用。 <ol><li>使用适于所请求的数据的类型和时间约束参数来执行成像。根据所使用的染色剂和制造商的recommendati使用激光激发/发射波长附件。使用扫描瓷砖和Z堆栈功能的组合，以获得真菌菌落和生物膜的全球3D视图。 注意:使用下列参数被收购于图3图片:8位/像素，平均= 2，像素停留= 1.58微秒，地砖用扫描拼接在线（10％的覆盖率，阈值= 0.7）5x5的图像; Z-步骤= 2微米。 </li></ol></li><li>对于3D数据可视化，可以使用许多免费或商业软件（这些提供3D渲染许多选项）中的一个。 <ol><li>例如，要显示Z堆叠的数据作为二维最大强度投影，减亮场通道，调整亮度和对比度，并合并通道以创建合成图像。如果它们存在，消除片而不会在Z堆叠四肢信号。然后，执行一个二维最大强度投影和转换结果存入RGB颜色。使用的"Z项目"功能中的ImageJ软件22获取图像P通过二维最大强度的预测这​​里不满。 </li></ol></li></ol></li></ol> 4.电子显微镜分析<ol><li>制备如在步骤3.1.2 LSCM描述的，除了样品，漂洗步骤后生物膜转移到无菌水代替磷酸钾缓冲液。这避免了盐晶体在脱水步骤中，这会扰乱SEM成像的形成。 </li><li>转移生物膜到样品保持器和除去过量的水;只允许少量水防护膜，以覆盖样品。 </li><li>将样品转移到一个可变压力扫描电子显微镜（VP-SEM）的腔室;它冻至-50°在珀尔帖冷却阶段℃;并允许它慢慢冷冻干燥，直接在SEM室中，以100帕15小时设定可变压力。 </li><li>冷冻干燥结束后，检索样品并将其转移到一个高真空成膜装置。大衣platinu 2纳米样品米氩等离子体（2.5×10 -2毫巴，35个mA）的下（石英测量）。 </li><li>观察用SEM涂覆样品装有使用"镜头"高分辨率检测器和1千伏的电子高张力场发射枪（FEG）。 </li></ol>

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作者什么都没有透露。

细菌生物膜在许多环境中检索并自1950年代以来已被研究，导致了一些方法的发展，对它们进行分析24。经典方法来量化和显示器生物膜包括微量滴定测定法和，最广泛使用的方法中，结晶紫（CV）染色。这些方法是速度快，成本低，并且容易处理25和是特别有用的量化总生物膜的生物量或执行的生存能力和矩阵量化测定。上的另一方面，"组学"的方法，可以在生物膜研究是有用的，允许生物膜26，27的数量和功能的分析。尽管微滴定板的优点和"组学"的方法，生物膜的几个基本功能不能与这些技术捕获，阻碍该过程的一个完整的理解。这些功能包括矩阵结构S，细菌菌落架构，细胞/细胞的相互作用，以及定植模式，这对于理解生物膜两者的功能和它们的形成的动态密钥数据。尽管显微镜捕捉到这些功能的能力，对丝状真菌细菌生物膜的显微镜分析仍然很少。这主要是由于丝状真菌的生长，其经常形成厚的，复杂的，三维网络的菌落。对真菌细菌生物膜的形成是常见的在不同环境中，并显著涉及各个领域4（ 例如 ，医学，农业，和环境。）;因此，关键是开发新的方法，以促进他们的调查。为此目的，我们结合的方法，以生成与细菌生物膜的显微成像非常薄的真菌菌落。此外，我们提出了一套显微镜工具来定性分析这些生物膜。该方法的成功依赖于产生非常薄菌丝殖民地和应用适当的染料的能力。这些点在下面讨论。 由于生物膜的结构复杂，了解他们的功能需要的多尺度方法28,29。生物膜，细菌菌落结构和矩阵结构和组成的分布格局不同尺度（ 即中尺度和微观尺度）进行了分析。此外，纳米级分辨率允许访问的小区/细胞物理相互作用和基质的纳米结构。因此，所建立的方法很容易地使形成在真菌菌落的细菌生物膜的一个多尺度分析。 在大多数研究中，LSCM生物膜的分析仅限于微米级，中尺度通常由光学相干断层30,31 进行 <sup类="外部参照"&gt; 32。这里介绍的方法使双方微观和中观尺度由激光共聚焦显微镜分析。它表明结合的实用性使用新一代共焦显微镜具有高分辨率（ 图3）中的样品的相同区域，甚至在相同的图像既分析。因此，挂钩汇编的数据收集问题，在不同的尺度有不同的方法在这里避免。 分析这种组合给访问伴随生物膜重新分区上的真菌菌落，沿显影生物膜的细菌菌落的体系结构，和基质结构。中尺度分析显示细菌生物膜的真菌菌落（ 图2和3）一个异种分布。这个观察不会已经可能的协议仅允许真菌菌落，这不一定是代表整个群体的一小部分的摄像。因此，尽管往往被忽视，中尺度分析可以提供有关生物膜分布格局宝贵的信息。 最后，该建立的方法可以用来分析不同显微镜技术，包括扫描电子显微镜的样品。这里，扫描电镜是用来达到纳米级，并且获得生物膜内的细菌空间组织。它与薄真菌菌落表现非常出色，而SEM只允许表面成像。在对比LSCM，扫描电镜，然而，所需的样本脱水，最经常，涂覆有导电金属。此脱水过程可能改变生物结构时，不正确地执行它，并可能需要优化。在这里，用慢速冷冻干燥脱水样品用33。然而，同时应用的LSCM和SEM的样品将允许相关显微镜在样品的相同位置的性能。 尽管优势如上所述，存在一定的局限性。首先，它可能并不适用于所有种类的真菌。事实上，这种培养方法是真菌固体介质的表面上的沿径向扩展的发展。这种方法可能不用于形成主要的气生菌丝（ 例如，镰孢属 ）的真菌或微需氧真菌主要扩频内琼脂是合适的。此外，真菌降解玻璃纸可能会有问题，以及（ 例如 ， 木霉属）。其次，需要注意的是染色的策略是一个关键点和污点的选择必须精心制作，如污渍必须不干扰生物膜是非常重要的。例如，我们注意到，Calcofluor白由于高pH值这个污渍引起的局部生物膜分裂（数据未示出），可能的。而且，一些染料产生异质染色（ 例如 ，刚果红），而其他生产均匀染色（ 例如 ，与WGA凝集素的细胞壁染色），得到非均相的图像质量。此外，要知道，某些染料可能不是充分具体是很重要的。例如，WGA污渍不仅真菌细胞壁但在期间生物膜形成34，35由革兰氏阳性和阴性细菌产生的革兰氏阳性细菌细胞壁和黏附也N-乙酰神经氨酸。因此，在使用荧光蛋白标记的细菌和/或建议真菌避免多次染色。如果使用多个染料，它们必须不化学干扰，并且其发射光谱不应当重叠。 尺度分析需要大的扫描区域，因此，LSCM可以是耗时的（40分钟至1小时，这取决于样品的厚度）和瓶颈大量样品的分析。尽管如此，调整可以根据所需的数据的类型进行。有可能通过改变图像质量降低采集时间和图像尺寸。例如，高清晰度的不需要分析生物膜一般再分配。 最后，一些局限性需要选择显示Z-堆栈的数据作为二维或三维凸起时加以考虑。二维预测是汇总数据的好方法，但深度信息丢失，重叠的结构变得隐藏。另一方面，3D突起允许从不同的观点，可视化，但它们往往在空间复杂的情况下，差的渲染。 总之，我们已经报道对菌丝细菌生物膜在结构水平上表征的方法。该方法可以扩展到其他的应用程序。事实上，此方法允许形成在真菌菌丝细菌生物膜的功能性或化学特性的性能。由于各种各样的现有荧光报道系统，LSCM分析可以用于多种用途29。例如，荧光麦克风roscopy可以用来监测pH梯度36或分子扩散在生物膜37。另外，该方法允许在多物种生物膜群落分析。例如， 在原位杂交靶向特定细菌群体荧光是特别有用的研究特定细菌再分配在多物种生物膜38,39。最后，大量的荧光染料可用于表征生物膜21的基质组合物。这里，蛋白用SYPRO，其染色的大范围的蛋白，其中基质蛋白之间（ 图4）定位，但其他的染料允许其他重要基质成分，如胞外多糖或胞外DNA的可视化。有趣的是，所有这些分析，可以在中尺度使用所描述的方法进行。由于激光共聚焦显微镜可在活体标本进行，也有可能使用例如，coverwell腔室，特别适合于薄真菌菌落实现延时成像。该选项是特别有趣，因为生物膜形成是一个复杂的，动态的过程。最后，对于一个定量目的，所报道的方法可以通过使这种量化可能在尺度图像提高自动定量分析的准确度。这可能克服生物膜的异质性和统计问题29。

真菌和细菌有很多机会，因为他们在大多数陆地环境同居彼此交互。由于它们的多样性及其普及，这些相互作用在许多生物领域包括生物技术，农业，食品加工，以及医药1,2重要。分子间的相互作用需要一定程度的接近，以允许伙伴之间的交流，并在某些情况下，合伙物理缔合是必要的功能性相互作用3。细菌和真菌之间的共同物理缔是细菌生物膜的真菌面4的形成。细菌细胞和真菌菌丝之间的这种直接接触允许所涉及的各种生物过程亲密相互作用。例如，在医学上，对机会的所在铜绿假单胞菌的生物膜形成的研究tunistic真菌病原体白色念珠菌能提供深入了解生物膜的形成和毒力5之间的链路。在农业方面，研究表明当与在混合生物膜真菌相关联的植物生长促进根际细菌和生物防治菌具有增加的效率。例如，当与平菇在混合生物膜6关联Bradyrhizobium属elkanii具有增强的 N 2 -fixing活性。最后，在生物修复，细菌，真菌混合生物膜已用于污染场地7的整治，8。 LSCM是特别合适的，研究的生物膜，因为它允许对居住与最小预处理水合生物膜，从而保持生物膜的结构和组织的三维观察。因此，通过激光共聚焦显微镜生物膜分析是非常丰富，特别是DET貂生物膜形成的时间过程和特征阶段9中 ，10的检测，从粘合步骤以一个成熟生物膜的发展。它也特别适合于可视化生物膜结构和矩阵11,12或量化生物膜尺寸13,14。虽然这种方法是合适的，研究了非生物或薄生物表面生物膜上的真菌菌落丝状研究细菌生物膜仍然是非常具有挑战性的。事实上，大多数丝状真菌打造厚重的文化底蕴，复杂的，立体的网络。即使厚目的可以通过共焦显微镜成像，激光穿透衰减和荧光发射常常降低最终图像的质量超过50微米15的深度。此外，由于真菌菌落不是刚性的，我t是难以处理的微生物而不干扰生物膜。由于样品的厚度，对真菌菌丝细菌生物膜的少数微观分析通常仅在真菌菌落的一小部分进行的，因此，只含有少数菌丝16，17，18。这一切都限制了我们来描述在真菌菌落生物膜分配能力，因此可以将偏压入分析中生物膜的真菌菌落内的异种分布的情况下。 为了克服这些困难，我们报告的生长和细菌生物膜的真菌菌丝的分析的方法。应用此方法研究生物膜形成的荧光假单胞菌的外生菌根担子菌双色蜡蘑 S238N的菌丝BBc6。这两个森林土微生物先前描述以形成混合生物膜状结构19,20。这种方法可以很容易地进一步适于其它丝状真菌/细菌系统中。这里介绍的方法是基于真菌培养方法的结合，从而允许非常薄的真菌菌落的生长，具有LSCM和SEM成像。这允许我们获得微（微米范围），内消旋 - （毫米范围）缩放视图的两种微生物之间的相互作用，允许生物膜的定性鉴定。我们还表明，样品可通过SEM观察到的，在纳米尺度水平（纳米范围）使生物膜的结构分析。

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

细菌生物膜经常形成于真菌表面，并且可以参与许多细菌真菌交互过程，如代谢合作，竞争，或捕食。生物膜的研究是在许多生物领域，包括环境科学，食品生产和药品的重要。然而，很少研究都集中在这样的细菌生物膜，部分由于调查它们的困难。大部分为定性和定量生物膜的方法在文献中分析描述是仅适用于形成在非生物表面或上均匀且薄的生物表面，如上皮细胞的一个单层生物膜。 而激光扫描共聚焦显微镜（LSCM）经常被用来在原位 和体内生物膜来分析，当施加到上真菌菌丝细菌生物膜此技术变得非常具有挑战性的，由于厚度和菌丝NETW的三维兽人。为了克服这个缺点，我们开发了一种协议与方法结合显微镜来限制在真菌菌落菌丝层的积累。使用这种方法，我们能够调查细菌生物膜的真菌菌丝在同时使用的LSCM和扫描电子显微镜（SEM）多尺度的发展。这份报告介绍了协议，其中包括微生物培养，细菌生物膜形成的条件，生物膜染色，激光共聚焦显微镜和扫描电镜和可视化。

真菌培养和生物膜制剂的整体概略过程在图1中给出。培养方法使我们能够获得真菌菌落20-50微米厚的含菌丝几层，允许微型和中型规模使用的LSCM水合生活生物膜分析。该方法的应用允许的P的高品质的图像采集荧光上L.双色菌丝BBc6生物膜沿生物膜（ 图2 - 4）的形成。 生物膜的尺度分析显示P的生物膜的异种分布荧光 BBc6上L.双色 S238N的菌丝（ 图2和3）。尺度分析还允许生物膜形成随时间变化的跟踪（ 图3）从早期步骤，其中只有一些细菌附着到菌丝（ 图3a），一个厚的，成熟生物膜（ 图3b的形成）。中尺度的图像的高分辨率允许我们以获得菌落架构（ 图3c - d）关于同一图像执行微观尺度分析。 微观尺度分析与特定标签相结合，使我们能够更进一步生物膜的表征。这里，SYPRO Ruby的23，其标签的蛋白质的大多数类，施加到样品（ 图4），并显示蛋白质在基质中的存在。 最后，通过脱水和涂层（ 图5）后的相同样品的SEM成像获得的生物膜结构的进一步细节。 SEM成像提供获得纳米级，从而使获得的基质结构。 <img alt="图1" src="/files/ftp_upload/54771/54771fig1.jpg" /> 图1:真菌培养和生物膜编制总体原理的过程。此图描述该方法的主要步骤，从微生物培养物样品的分析。更多细节在协议给出。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54771/54771fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图2" src="/files/ftp_upload/54771/54771fig2.jpg" /> 图2:在真菌菌落BBc6生物膜再分配。将样品之后在10倍放大倍率的相互作用的18小时进行成像。通过三维共焦显微镜图像的2D最大强度投影获得的图像。在figu灰色网格重新描绘了马赛克位置。 二色补血草菌丝沾满刚果红（红色）和细菌GFP标记（绿色）。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54771/54771fig2large.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/54771/54771fig3.jpg" /> 图3:对二色补血草菌丝BBc6生物膜形成早期和晚期。通过3D共聚焦显微镜图像的二维最大强度投影获得此图片。放大40倍进行成像。 （ 一 ）后相互作用的2小时生物膜再分配。 （b）在相互作用的14小时生物膜再分配。 （c）在该白色矩形的放大（a）中。 （ 四 ）在白色矩形的放大（B）。真菌菌丝沾满刚果红（红色）和细菌GFP-TAgged（绿色）。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54771/54771fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图4" src="/files/ftp_upload/54771/54771fig4.jpg" /> 图 4: 二色补血草 菌丝 BBc6生物膜矩阵染色 。 （ 一 ）后相互作用的16小时生物膜。 （ 二 ）在白色的矩形的放大（a）中。在40X放大率下进行成像，并通过三维共焦显微镜图像的2D最大强度投影获得这些图像。细菌是GFP标记（绿色），木耳沾有FUN1（深绿色），和蛋白与S.红宝石（红色）染色。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54771/54771fig4large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> 图5: 二色补血草菌丝BBc6生物膜的SEM图像。 SEM成像在2360X进行，EHT:千伏。成像前，将试样缓慢地在SEM室中冷冻干燥，并涂覆有铂。绿色箭头指向细菌生物膜和黄色箭头指向真菌菌丝。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54771/54771fig5large.jpg" target="_blank">请点击此处查看该图的放大版本。</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

这个协议描述成长和定性通过共聚焦和电镜分析关于真菌菌丝细菌生物膜的新方法。

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 ...

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

Nanyang Technological University

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JoVE Journal

Immunology and Infection

19.4K 次观看.  INRA Université de Lorraine. 这个协议描述成长和定性通过共聚焦和电镜分析关于真菌菌丝细菌生物膜的新方法。 

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

Oral Biofilm Analysis of Palatal Expanders by Fluorescence In-Situ Hybridization and Confocal Laser Scanning Microscopy

Confocal laser scanning microscopy (CLSM) of natural heterogeneous biofilm is today facilitated by a comprehensive range of staining techniques, one of them being fluorescence in situ hybridization (FISH).1,2 We performed a pilot study in which oral biofilm samples collected from fixed orthodontic appliances (palatal expanders) were stained by FISH, the objective being to assess the three-dimensional organization of natural biofilm and plaque accumulation.3,4 FISH creates an opportunity to stain cells in their native biofilm environment by the use of fluorescently labeled 16S rRNA-targeting probes.4-7,19 Compared to alternative techniques like immunofluorescent labeling, this is an inexpensive, precise and straightforward labeling technique to investigate different bacterial groups in mixed biofilm consortia.18,20 General probes were used that bind to Eubacteria (EUB338 + EUB338II + EUB338III; hereafter EUBmix),8-10 Firmicutes (LGC354 A-C; hereafter LGCmix),9,10 and Bacteroidetes (Bac303).11 In addition, specific probes binding to Streptococcus mutans (MUT590)12,13 and Porphyromonas gingivalis (POGI)13,14 were used. The extreme hardness of the surface materials involved (stainless steel and acrylic resin) compelled us to find new ways of preparing the biofilm. As these surface materials could not be readily cut with a cryotome, various sampling methods were explored to obtain intact oral biofilm. The most workable of these approaches is presented in this communication. Small flakes of the biofilm-carrying acrylic resin were scraped off with a sterile scalpel, taking care not to damage the biofilm structure. Forceps were used to collect biofilm from the steel surfaces. Once collected, the samples were fixed and placed directly on polysine coated glass slides. FISH was performed directly on these slides with the probes mentioned above. Various FISH protocols were combined and modified to create a new protocol that was easy to handle.5,10,14,15 Subsequently the samples were analyzed by confocal laser scanning microscopy. Well-known configurations3,4,16,17 could be visualized, including mushroom-style formations and clusters of coccoid bacteria pervaded by channels. In addition, the bacterial composition of these typical biofilm structures were analyzed and 2D and 3D images created.

We present a protocol for structural and compositional analysis of natural oral biofilm from orthodontic appliances with in situ hybridization (FISH) and confocal laser scanning microscopy (CLSM). Oral biofilm samples were collected from palatal expanders, scraping acrylic-resin flakes off their surface and referring them for molecular processing.

腭扩展口腔生物膜分析，荧光原位杂交和激光共聚焦显微镜

Confocal laser scanning microscopy (CLSM) of natural heterogeneous biofilm is today facilitated by a comprehensive range of staining techniques, one of ...

科研

医学

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.

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 ...

免疫与感染

Characterization of Aquatic Biofilms with Flow Cytometry

Biofilms are dynamic consortia of microorganism that play a key role in freshwater ecosystems. By changing their community structure, biofilms respond quickly to environmental changes and can be thus used as indicators of water quality. Currently, biofilm assessment is mostly based on integrative and functional endpoints, such as photosynthetic or respiratory activity, which do not provide information on the biofilm community structure. Flow cytometry and computational visualization offer an alternative, sensitive, and easy-to-use method for assessment of the community composition, particularly of the photoautotrophic part of freshwater biofilms. It requires only basic sample preparation, after which the entire sample is run through the flow cytometer. The single-cell optical and fluorescent information is used for computational visualization and biological interpretation. Its main advantages over other methods are the speed of analysis and the high-information-content nature. Flow cytometry provides information on several cellular and biofilm traits in a single measurement: particle size, density, pigment content, abiotic content in the biofilm, and coarse taxonomic information. However, it does not provide information on biofilm composition on the species level. We see high potential in the use of the method for environmental monitoring of aquatic ecosystems and as an initial biofilm evaluation step that informs downstream detailed investigations by complementary and more detailed methods.

Flow cytometry in combination with visual clustering offers an easy-to-use and fast method for studying aquatic biofilms. It can be used for biofilm characterization, detection of changes in biofilm community structure, and detection of abiotic particles embedded in the biofilm.

流式细胞仪对水生生物膜的表征

Biofilms are dynamic consortia of microorganism that play a key role in freshwater ecosystems. By changing their community structure, biofilms respond ...

环境科学

Concurrent Quantification of Cellular and Extracellular Components of Biofilms

Confocal laser scanning microscopy (CLSM) is a powerful tool for investigation of biofilms. Very few investigations have successfully quantified concurrent distribution of more than two components within biofilms because: 1)&#160;selection of fluorescent dyes having minimal spectral overlap is complicated, and 2)&#160;quantification of multiple fluorochromes poses a multifactorial problem. Objectives: Report a methodology to quantify and compare concurrent 3-dimensional distributions of three cellular/extracellular components of biofilms grown on relevant substrates. Methods: The method consists of distinct, interconnected steps involving biofilm growth, staining, CLSM imaging, biofilm structural analysis and visualization, and statistical analysis of structural parameters. Biofilms of Streptococcus mutans (strain UA159) were grown for 48 hr on sterile specimens of Point 4 and TPH3 resin composites. Specimens were subsequently immersed for 60 sec in either Biot&#232;ne PBF (BIO) or Listerine Total Care (LTO) mouthwashes, or water (control group; n=5/group). Biofilms were stained with fluorochromes for extracellular polymeric substances, proteins and nucleic acids before imaging with CLSM. Biofilm structural parameters calculated using ISA3D image analysis software were biovolume and mean biofilm thickness. Mixed models statistical analyses compared structural parameters between mouthwash and control groups (SAS software; &#945;=0.05). Volocity software permitted visualization of 3D distributions of overlaid biofilm components (fluorochromes). Results: Mouthwash BIO produced biofilm structures that differed significantly from the control (p&#60;0.05) on both resin composites, whereas LTO did not produce differences (p&#62;0.05) on either product. Conclusions: This methodology efficiently and successfully quantified and compared concurrent 3D distributions of three major components within S. mutans biofilms on relevant substrates, thus overcoming two challenges to simultaneous assessment of biofilm components. This method can also be used to determine the efficacy of antibacterial/antifouling agents against multiple biofilm components, as shown using mouthwashes. Furthermore, this method has broad application because it facilitates comparison of 3D structures/architecture of biofilms in a variety of disciplines.

A protocol for the concurrent quantification and comparison of three cellular and extracellular components within biofilms is presented. The methodology involves the use of confocal laser scanning microscopy, biofilm structural analysis and visualization software, and statistical analysis software.

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 ...

Methods for Characterizing the Co-development of Biofilm and Habitat Heterogeneity

Biofilms are surface-attached microbial communities that have complex structures and produce significant spatial heterogeneities. Biofilm development is strongly regulated by the surrounding flow and nutritional environment. Biofilm growth also increases the heterogeneity of the local microenvironment by generating complex flow fields and solute transport patterns. To investigate the development of heterogeneity in biofilms and interactions between biofilms and their local micro-habitat, we grew mono-species biofilms of Pseudomonas aeruginosa and dual-species biofilms of P. aeruginosa and Escherichia coli under nutritional gradients in a microfluidic flow cell. We provide detailed protocols for creating nutrient gradients within the flow cell and for growing and visualizing biofilm development under these conditions. We also present protocols for a series of optical methods to quantify spatial patterns in biofilm structure, flow distributions over biofilms, and mass transport around and within biofilm colonies. These methods support comprehensive investigations of the co-development of biofilm and habitat heterogeneity.

Biofilms have complex interactions with their surrounding environment. To comprehensively investigate biofilm-environment interactions, we present here a series of methods to create heterogeneous chemical environment for biofilm development, to quantify local flow velocity, and to analyze mass transport in and around biofilm colonies.

方法表征的共同发展生物膜和生境异质性

Biofilms are surface-attached microbial communities that have complex structures and produce significant spatial heterogeneities. Biofilm development is ...

生物工程

Paraffin Embedding and Thin Sectioning of Microbial Colony Biofilms for Microscopic Analysis

Sectioning via paraffin embedding is a broadly established technique in eukaryotic systems. Here we provide a method for the fixation, embedding, and sectioning of intact microbial colony biofilms using perfused paraffin wax. To adapt this method for use on colony biofilms, we developed techniques for maintaining each sample on its growth substrate and laminating it with an agar overlayer, and added lysine to the fixative solution. These optimizations improve sample retention and preservation of micromorphological features. Samples prepared in this manner are amenable to thin sectioning and imaging by light, fluorescence, and transmission electron microscopy. We have applied this technique to colony biofilms of Pseudomonas aeruginosa, Pseudomonas synxantha, Bacillus subtilis, and Vibrio cholerae. The high level of detail visible in samples generated by this method, combined with reporter strain engineering or the use of specific dyes, can provide exciting insights into the physiology and development of microbial communities.

We describe fixation, paraffin embedding, and thin sectioning techniques for microbial colony biofilms. In prepared samples, biofilm substructure and reporter expression patterns can be visualized by microscopy.

微生物菌落生物膜的石蜡嵌入和薄切片显微分析

Sectioning via paraffin embedding is a broadly established technique in eukaryotic systems. Here we provide a method for the fixation, embedding, and ...

Real-time Imaging and Quantification of Fungal Biofilm Development Using a Two-Phase Recirculating Flow System

In oropharyngeal candidiasis, members of the genus Candida must adhere to and grow on the oral mucosal surface while under the effects of salivary flow. While models for the growth under flow have been developed, many of these systems are expensive, or do not allow imaging while the cells are under flow. We have developed a novel apparatus that allows us to image the growth and development of Candida albicans cells under flow and in real-time. Here, we detail the protocol for the assembly and use of this flow apparatus, as well as the quantification of data that are generated. We are able to quantify the rates that the cells attach to and detach from the slide, as well as to determine a measure of the biomass on the slide over time. This system is both economical and versatile, working with many types of light microscopes, including inexpensive benchtop microscopes, and is capable of extended imaging times compared to other flow systems. Overall, this is a low-throughput system that can provide highly detailed real-time information on the biofilm growth of fungal species under flow.

We describe the assembly, operation, and cleaning of a flow apparatus designed to image fungal biofilm formation in real time while under flow. We also provide and discuss quantitative algorithms to be used on the acquired images.

用两相循环流系统实时成像和定量真菌生物膜的研究

In oropharyngeal candidiasis, members of the genus Candida must adhere to and grow on the oral mucosal surface while under the effects of salivary flow. ...

A Semi-quantitative Approach to Assess Biofilm Formation Using Wrinkled Colony Development

Biofilms, or surface-attached communities of cells encapsulated in an extracellular matrix, represent a common lifestyle for many bacteria. Within a biofilm, bacterial cells often exhibit altered physiology, including enhanced resistance to antibiotics and other environmental stresses 1. Additionally, biofilms can play important roles in host-microbe interactions. Biofilms develop when bacteria transition from individual, planktonic cells to form complex, multi-cellular communities 2. In the laboratory, biofilms are studied by assessing the development of specific biofilm phenotypes. A common biofilm phenotype involves the formation of wrinkled or rugose bacterial colonies on solid agar media 3. Wrinkled colony formation provides a particularly simple and useful means to identify and characterize bacterial strains exhibiting altered biofilm phenotypes, and to investigate environmental conditions that impact biofilm formation. Wrinkled colony formation serves as an indicator of biofilm formation in a variety of bacteria, including both Gram-positive bacteria, such as Bacillus subtilis 4, and Gram-negative bacteria, such as Vibrio cholerae 5, Vibrio parahaemolyticus 6, Pseudomonas aeruginosa 7, and Vibrio fischeri 8.
The marine bacterium V. fischeri has become a model for biofilm formation due to the critical role of biofilms during host colonization: biofilms produced by V. fischeri promote its colonization of the Hawaiian bobtail squid Euprymna scolopes 8-10. Importantly, biofilm phenotypes observed in vitro correlate with the ability of V. fischeri cells to effectively colonize host animals: strains impaired for biofilm formation in vitro possess a colonization defect 9,11, while strains exhibiting increased biofilm phenotypes are enhanced for colonization 8,12. V. fischeri therefore provides a simple model system to assess the mechanisms by which bacteria regulate biofilm formation and how biofilms impact host colonization.
In this report, we describe a semi-quantitative method to assess biofilm formation using V. fischeri as a model system. This method involves the careful spotting of bacterial cultures at defined concentrations and volumes onto solid agar media; a spotted culture is synonymous to a single bacterial colony. This 'spotted culture' technique can be utilized to compare gross biofilm phenotypes at single, specified time-points (end-point assays), or to identify and characterize subtle biofilm phenotypes through time-course assays of biofilm development and measurements of the colony diameter, which is influenced by biofilm formation. Thus, this technique provides a semi-quantitative analysis of biofilm formation, permitting evaluation of the timing and patterning of wrinkled colony development and the relative size of the developing structure, characteristics that extend beyond the simple overall morphology.

We provide a simple, semi-quantitative method to investigate biofilm formation in vitro. This method takes advantage of the Zeiss stemi 2000-C Dissecting Microscope (with camera attachment) to monitor both the timing and pattern of biofilm formation, as assessed by the development of wrinkled colonies.

一个半定量的方法来评估生物膜形成皱殖民地的发展

Biofilms, or surface-attached communities of cells encapsulated in an extracellular matrix, represent a common lifestyle for many bacteria. Within a ...

生物学

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.

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, ...

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.remotexs.ntu.edu.sg/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 ...