Name: paraffin embedding and thin sectioning of microbial colony biofilms for microscopic analysis
Uploaded: 2018-03-23T14:00:00.000+00:00
Duration: 8 min 52 s
Description: Watch this Scientific Journal Video about Paraffin Embedding and Thin Sectioning of Microbial Colony Biofilms for Microscopic Analysis at JoVE.com

这项工作得到了 NSF 职业奖1553023和 NIH/NIAID 奖 R01AI103369 的支持。

1. 铜绿假单胞菌菌落生物膜的生长
<ol><li>中双层板的制备<ol>
<li>准备一10克/升胰蛋白胨, 10 克/升琼脂 (参见材料表) 溶液在去离子水。</li>
		<li>20分钟的蒸压釜, 在水浴中冷却至50-60 摄氏度。</li>
		<li>将45毫升的琼脂-胰蛋白胨溶液倒入100毫米 x 100 毫米方盘 (参见材料表), 使用50毫升锥形管。允许琼脂凝固 (~ 20-30 分钟)。在第一层上倒入第二个, 15 毫升的层。让一夜之间凝固, 如果必要的话, 用不起毛的纸巾擦拭盖子。</li>
	</ol>
</li>
	<li>斑点菌落生物膜<ol>
<li>条纹的兴趣从冷藏库存到 LB 琼脂板13和孵化在黑暗中为12-16 小时37摄氏度和80-100% 相对湿度。</li>
		<li>对于每一个菌株或复制, 使用一个单一的殖民地接种2毫升 LB 和孵化在黑暗中12-16 小时37摄氏度与颤抖在 250 rpm。</li>
		<li>亚文化通过稀释1:100 入新鲜的磅和孵化在黑暗中为 2.5-3 h 在37°c 与震动在 250 rpm。</li>
		<li>使用分光光度计测量光学密度, 用无菌 LB 进行调整, 以达到500 nm的细胞悬浮, 直径为0.5。</li>
		<li>根据所需的菌落大小, 吸管 2.5-10 µL 的细胞悬浮在中双层板上 (在步骤1.1 中制备), 并允许斑点干20分钟. 离开培养盖半开靠近开放的火焰可以促进干燥。</li>
		<li>孵化点殖民地在25°c 和80-100% 相对湿度, 在黑暗中长达4天。</li>
	</ol>
</li>
</ol>2. 定影液的制备
<ol><li>在样品收获日准备固定液。稀释37% 甲醛 (FA) 在 1x PBS 到最后浓度为 4% FA。 注意: FA 是挥发性和毒性。在通风良好的油烟机上佩戴防护设备和工作。</li>
	<li>使用前立即将 l-赖氨酸盐酸盐溶于 4% FA (在步骤2.1 中制备) 室温下, 最终浓度为50毫米 l-赖氨酸盐酸。</li>
</ol>3. 将固定剂直接应用于菌落生物膜 [可选]
注: 我们发现在固定前添加琼脂覆盖物时, 生物膜形态最好保存。然而, 这一步骤也构成了环境条件的变化, 可能会影响基因表达。因此, 荧光记者的表达模式应使用单独的协议进行验证, 其中固定步骤是在添加琼脂叠加之前进行的, 如下所述。
<ol><li>在固定的日子里, 在通风良好的油烟机里工作, 将步骤2中准备好的定影液直接移到蚁群边缘周围的琼脂表面, 让它到达殖民地的外围而不淹没它。只应用尽可能多的固定, 以充分包围的殖民地, 大约500µL. 允许固定剂扩散到菌落和周围的琼脂。</li>
	<li>一旦固定剂被完全吸收到菌落和周围的介质中, 大约20-30 分钟, 剩余的 FA 就不再可见在盘子上, 重复步骤3.1。允许这个固定剂扩散到殖民地和周围的琼脂。</li>
	<li>一旦固定剂已完全吸收到菌落和周围介质重复步骤 3.1, 这一次将固定剂直接应用于菌落的表面。允许这个固定剂扩散到殖民地和周围的琼脂, 直到步骤 4, 只有在所有的固定剂已被吸收, 并不再可见的板块。</li>
</ol>4. 用琼脂覆盖菌落
<ol><li>在固定的日子, 准备一个10克/升琼脂溶液在去离子水和高压釜10-20 分钟溶解。在水浴冷却到50˚C。</li>
	<li>在生长培养基和菌落上轻轻倒入15毫升琼脂溶液。允许琼脂在室温下形成凝胶5分钟 (图 1B)。</li>
	<li>使用锋利的剃刀刀片切割一个正方形, 3 层的恰克与殖民地层间的前两层。如果准备多个样品, 确保每一个恰克被削减到一个可比的大小。</li>
	<li>轻轻地从包含的卡盘中取出多余的琼脂。</li>
	<li>要将琼脂 (包含菌落) 的两个上层层从恰克的底层 (图 1C) 中抬起, 请在 1x PBS 或水中将刮刀的扁头湿润, 并在顶层和底部之间轻轻插入。层合群应与底层分离。小心不要扭曲或弯曲的层压的殖民地时, 从它的基础上解除。</li>
</ol>5. 固定
<ol><li>在通风良好的油烟机中工作时, 立即将层压的菌落 (即2层卡盘) 转移到嵌入盒中 (请参阅材料表), 并用抗化学标记笔标记。将嵌入的卡带放入玻璃滑动邮件 (见材料表) 中, 其中包含步骤 2.1-2.2 中准备的固定剂。确保嵌入盒内没有气泡。</li>
	<li>在室温下, 在黑暗中在夜间固定固定的样品。</li>
</ol>6. 样品处理: 缓冲洗涤、脱水、清除和渗透
<ol><li>夜间固定后, 每两次在 1x PBS 中清洗样品1小时。为取得最佳效果, 使用自动组织处理器的自旋功能 (见材料表) 自动将试剂均匀化, 设置为低设置。如果没有可用的处理器, 则可以手动运行处理。</li>
	<li>通过在 1x PBS (25%, 50%, 70%, 95%) 的梯度系列乙醇 (乙醇) 稀释在室温下的1小时, 通过脱水来洗涤缓冲液。</li>
	<li>脱水在三洗涤100% 乙醇为1小时在室温下。</li>
	<li>清除脱水样品在三洗涤100% 橙色油基清洁剂 (参见材料表) 为 1 h 每在室温。</li>
	<li>两次渗透清除样品与熔融石蜡 (参见材料表), 加热到 55˚C, 每2小时。</li>
</ol>7. 样品嵌入
<ol><li>填充蜡模与熔融石蜡加热到55˚C。使用一个加热, 平铲, 迅速将渗入的恰克从嵌入的卡带到蜡模, 确保恰克与模具底座平行。避免在卡盘上施加过多的压力。蜡模可以是定制的3维打印, 或者是商用的 (请参阅材料表)。</li>
	<li>允许蜡在4˚C 一夜之间凝固。在固体蜡模压的样品可以无限期储存在4˚C。</li>
	<li>一旦蜡固化, 从模具中的样品和修剪多余的蜡从样品周围的刀片。将多余的蜡从试样的一端延伸, 平行于切片的平面, 可用于将其夹紧到切片 (参见材料表)。也留下一鞘蜡, 大约1毫米厚, 在样品附近和光滑它的面孔。</li>
</ol>8. 切片
<ol><li>把水浴加热到42˚C。将样品夹在切片上, 使其表面垂直定向到刀片的边缘 (参见材料表)。</li>
	<li>将样品修剪成50µm 的间隔, 直到达到所需的菌落平面, 从中收集剖面。</li>
	<li>使用切片设置为 75-80 rpm 的切片速度和6-10˚的间隙角, 将所需的 10-µm 厚截面的带状带剪切。使用细尖的画笔从刀片式服务器分离功能区。</li>
	<li>使用一滴水在巴斯德吸管 (首选) 或镊子的尖端, 轻轻地把丝带转移到水浴。小心避免在剖面下捕捉气泡。</li>
	<li>立即在水浴中插入一张幻灯片, 并在45˚的角度将其放置在丝带下面。</li>
	<li>触摸丝带的窄边到幻灯片, 就在磨砂标签下面。丝带应该坚持。</li>
	<li>将滑块从水浴中拉出, 调整角度, 使其与水面垂直, 并允许丝带沿其长度平放在滑轨上。避免在丝带下诱捕多余的水。</li>
	<li>轻轻地将滑块放在一个无起毛的无绒组织上, 从切片中吸出多余的水分。</li>
	<li>把滑梯放在纸巾上, 让它在黑暗中过夜, 在室温下晾干。幻灯片可以无限期存储在4˚C 在黑暗中。</li>
</ol>9. 热固、补液和安装
<ol><li>加热一个平整的热板到45˚C。将幻灯片加热30-60 分钟。蜡将变成半熔化和平压在幻灯片上。</li>
	<li>轻轻地从热板上提起滑块, 将其平放到平滑、平整、室温的表面, 使用小心, 以确保熔融蜡不会拉到任何一侧的幻灯片, 直到蜡固化 (~ 1 分钟)。</li>
	<li>使用玻璃滑动邮件, 脱蜡幻灯片在四洗涤清洁剂每5分钟, 使用 Büchner 吸引器, 以消除清洗之间的解决方案。</li>
	<li>在100% 乙醇中三次清洗幻灯片, 每张1分钟。</li>
	<li>水化在连续的95%、70%、50% 和25% 的加工顺序中, 依次为1分钟, 然后在 1x PBS 中进行两次1分钟的洗涤。</li>
	<li>立即装入三缓冲安装介质中的截面 (请参阅材料表) 并应用盖玻片, 避免引入气泡。允许安装介质在室温下聚合过夜。</li>
	<li>一旦聚合, 使用明确的指甲油密封盖玻片到幻灯片。密封的幻灯片可以无限期地存储在黑暗中4˚C。</li>
</ol>

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作者没有什么可透露的。

石蜡嵌入和薄切片组织标本是一种经典的组织学技术, 使显微形态学结构的成像和通常用于真核组织, 并已成功地应用于微生物样本8 ,9。虽然 cryoembedding 允许强保留内源和免疫荧光信号, 石蜡嵌入通常是可取的, 因为它提供更好的保存形态学16。在将这种方法应用于微生物生物膜时, 我们关注的是我们系统特有的特性。组织的细胞和基质?...

大多数微生物都有能力形成生物膜, 细胞群落由自制的基质聚集在一起。生物膜可以生长在许多类型的物理设置, 与各种制度的营养和基质供应。特定的生物膜形成方法往往产生可重复的多细胞结构, 并观察到在群落或宏观层面上 phylogenetically 不同物种的共同结构。当微生物在大气层中作为固体介质的菌落生长时, 宏观形态传达有关矩阵生产能力的信息, 并且经常与其他特性相关, 1,2,3。微生物菌落的内部结构也可以提供有关生物膜特异化学和生理学的线索, 但很难描述。最近, cryoembedding 和 cryosectioning 技术在细菌菌落中的应用已使特定功能的成像和可视化达到空前的分辨率4,5,6。然而, 与动物组织的研究表明, 石蜡嵌入提供了卓越的保存形态, 当与 cryoembedding 7相比, 并已用于可视化的细菌在组织8,9。因此, 我们制定了一个固定, 石蜡嵌入和薄切片微生物菌落生物膜的协议。在这里, 我们将描述铜绿假单胞菌 PA14 菌落-生物膜薄部分10,11的制备, 但我们也成功地将此技术应用于细菌形成的生物膜铜绿假单胞菌 synxantha, 枯草芽孢杆菌,和霍乱弧菌12。
石蜡嵌入和薄切片生物膜的过程遵循一个简单的逻辑。首先, 生物膜被包裹在一层琼脂中, 以保持加工过程中的形态学。第二, 包裹生物膜被浸没在一个固定剂中, 以交联大分子并保存形貌。然后用酒精脱水, 用更非极性溶剂清除, 然后用液体石蜡渗透。一旦渗入, 样品被嵌入到蜡块中切片。节被剪切, 装在幻灯片上, 然后水化以返回到更纯的状态。从这一点上, 它们可以被染色或覆盖在安装介质中进行微观分析。
本协议生产适合于组织学分析的微生物生物膜薄切片。当使用这种方法制备的薄切片被光镜成像时, 菌落生物膜子结构可见。生物膜也可以在含有特定于个别特征的荧光污渍的介质上生长, 或者在安装前立即在补液步骤 (步骤 9.5-9.6) 上染色。最后, 微生物可以被设计成以一种本构或调控的方式生产荧光蛋白, 允许就地报告这些社区内的细胞分布或基因表达。利用这些方法确定了菌落的生物膜深度、细胞分布、基质分布、生长形态和时空基因表达。

<table><tbody><tr><td>5 3/4&quot; Pasteur pipette</td><td>Fisher Scientific</td><td>13-678-6A</td><td>Purchased from univeristy biostores&amp;nbsp;</td></tr><tr><td>Agar&amp;nbsp;</td><td>Teknova&amp;nbsp;</td><td>A7777</td><td></td></tr><tr><td>Buchner Aspirator (Vacuum) Flask&amp;nbsp;</td><td>Pyrex</td><td>5340</td><td>Purchased from univeristy biostores&amp;nbsp;</td></tr><tr><td>Chemically-resistant Marking Pen</td><td>VWR</td><td>103051-182</td><td>Manufacturer: Leica</td></tr><tr><td>Clear Fingernail Polish&amp;nbsp;</td><td>********</td><td>********</td><td>Store bought</td></tr><tr><td>Congo Red Indicator Grade</td><td>VWR</td><td>AAAB24310-14</td><td>Manufacturer: Alfa Aesar</td></tr><tr><td>Coomassie Blue&amp;nbsp;</td><td>VWR</td><td>EM-3340</td><td>Manufacturer: EMD Millipore</td></tr><tr><td>TRIS-buffered Mounting Medium (w/ DAPI)&amp;nbsp;</td><td>Fisher Scientific</td><td>50 247 04</td><td>Manufacturer: Electron Microscopy Sciences</td></tr><tr><td>Embedding Mold&amp;nbsp;</td><td>********</td><td>********</td><td>3D printed in-house</td></tr><tr><td>Embedding Mold (commercial)&amp;nbsp;</td><td>Electron Microscopy Sciences</td><td>70182</td><td></td></tr><tr><td>Ethanol 200P</td><td>Decon Labs, Inc.&amp;nbsp;</td><td>2701</td><td>Purchased from univeristy biostores&amp;nbsp;</td></tr><tr><td>Fine-tipped Brush</td><td>********</td><td>********</td><td>Store bought, paint brush</td></tr><tr><td>Glass Coverslips 60x22mm</td><td>Fisher Scientific</td><td>12-519-21C</td><td></td></tr><tr><td>Glass Rehydration Mailer&amp;nbsp;</td><td>Ted Pella</td><td>21043</td><td>20 slide mailer&amp;nbsp;</td></tr><tr><td>Histoclear-II, orange oil-based clearing agent&amp;nbsp;</td><td>Fisher Scientific</td><td>50 899 90150</td><td>Manufacturer: National Diagnostics&amp;nbsp;</td></tr><tr><td>Histosette, Embedding Casette</td><td>Fisher Scientific</td><td>15 182 701A</td><td></td></tr><tr><td>L-lysine hydrochloride&amp;nbsp;</td><td>Fisher Scientific</td><td>BP386 100</td><td></td></tr><tr><td>Low Profile Microtome Blades</td><td>Fisher Scientific</td><td>22 210 048</td><td>Manufacturer: Sturkey&amp;nbsp;</td></tr><tr><td>Micropipette&amp;nbsp;</td><td>VWR</td><td>89080-004</td><td>Promo-pack</td></tr><tr><td>Micropipette Tips&amp;nbsp;</td><td>See comments section</td><td>See comments section</td><td>p10 (Fisher Scientific, 02 707 469), p200 (VWR, 89079-474), p1250 (VWR, 89079-486)</td></tr><tr><td>Microtome&amp;nbsp;</td><td>Fisher Scientific</td><td>905200U/00016050</td><td>Model: HM355S, Manufacturer: Microm, NON-CATALOG, Vendor Catalog # 905200U/00016050</td></tr><tr><td>Formaldehyde, 37% Aqueous (Formalin)</td><td>Ricca Chemical</td><td>RSOF0010-500A</td><td></td></tr><tr><td>Paraplast Xtra (paraffin wax)</td><td>VWR</td><td>15159-486</td><td>Manufacturer: McCormick Scientific&amp;nbsp;</td></tr><tr><td>Petri Dishes Square 100x100x15mm</td><td>Laboratory Disposable Products&amp;nbsp;</td><td>D210-16</td><td></td></tr><tr><td>Potassium chloride&amp;nbsp;</td><td>EMD Chemicals&amp;nbsp;</td><td>PX1405-1</td><td>Component of phosphate buffered saline, prepared in-house&amp;nbsp;</td></tr><tr><td>Potassium phosphate&amp;nbsp;</td><td>Fisher Scientific</td><td>P380-500</td><td>Component of phosphate buffered saline, prepared in-house&amp;nbsp;</td></tr><tr><td>Razor Blades&amp;nbsp;</td><td>VWR</td><td>55411-050</td><td>Purchased from univeristy biostores&amp;nbsp;</td></tr><tr><td>Slide Warmer&amp;nbsp;</td><td>Fisher Scientific</td><td>NC0865259</td><td>NON-CATALOG, Vendor Catalog # 12857D</td></tr><tr><td>Sodium chloride&amp;nbsp;</td><td>VWR</td><td>0241-1KG</td><td>Component of phosphate buffered saline, prepared in-house&amp;nbsp;</td></tr><tr><td>Sodium phosphate&amp;nbsp;</td><td>VWR</td><td>BDH9296.500</td><td>,Component of phosphate buffered saline, prepared in-house&amp;nbsp;</td></tr><tr><td>Suprafrost Histology Slides&amp;nbsp;</td><td>Fisher Scientific</td><td>12-544-2</td><td></td></tr><tr><td>Tissue Flotation Water Bath&amp;nbsp;</td><td>Fisher Scientific</td><td>NC0815797</td><td>Manufacturer: Ted Pella, Vendor Catalog # 28156-B</td></tr><tr><td>Automatic Tissue Processor&amp;nbsp;</td><td>Fisher Scientific</td><td>813160U/Q#00009061</td><td>Model: STP120 Tissue Processor</td></tr><tr><td>Tryptone&amp;nbsp;</td><td>Teknova&amp;nbsp;</td><td>T9012</td><td></td></tr><tr><td>Yeast extract</td><td>Teknova&amp;nbsp;</td><td>Y9010</td><td></td></tr></tbody></table>

paraffin embedding and thin sectioning of microbial colony biofilms for microscopic analysis

石蜡切片是一种在真核细胞中广泛建立的技术。在这里, 我们提供了一种方法, 以固定, 嵌入和切片的完整的微生物群生物膜使用灌注石蜡。为了适应这种方法在菌落生物膜上的使用, 我们开发了技术, 以保持每个样品在其生长基质和层压它与琼脂层叠, 并添加赖氨酸的固定液。这些优化改进了果皮微形态特性的样本保留和保存。以这种方式制备的样品可以通过光、荧光和透射电镜进行薄切片和成像。我们已将此技术应用于铜绿假单胞菌、假单胞菌 synxantha、枯草芽孢杆菌和霍乱弧菌的菌落生物膜。该方法所产生的样品中可见的高度细节, 加上报告应变工程或特定染料的使用, 可为微生物群落的生理和发育提供令人振奋的见解。

该方法产生的生物膜薄切片, 其中明显的形态学特征和区域的基因表达可以成像的 DIC, 荧光显微镜, 和 TEM。虽然 DIC 成像使用一个40X 的油浸泡目标可以足以显示一些形态学特征 (图 2E), 我们发现, 荧光显微镜的菌株设计的组成性快速荧光蛋白提供了增强示例中单元格分布的可视化 (图 2D)。可以将单个剖面的图像缝合在一起, 生成?...

Watch this Scientific Journal Video about Paraffin Embedding and Thin Sectioning of Microbial Colony Biofilms for Microscopic Analysis at JoVE.com

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

微生物菌落生物膜的石蜡嵌入和薄切片显微分析

我们描述了微生物菌落生物膜的固定、石蜡嵌入和薄切片技术。在制备的样品中, 生物膜子结构和记者的表达模式可以通过显微技术进行可视化。

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

paraffin-embedding-thin-sectioning-microbial-colony-biofilms-for

Research

JoVE Journal

Immunology and Infection

14.3K 次观看.  Columbia University. 我们描述了微生物菌落生物膜的固定、石蜡嵌入和薄切片技术。在制备的样品中, 生物膜子结构和记者的表达模式可以通过显微技术进行可视化。

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

A Method for Obtaining Serial Ultrathin Sections of Microorganisms in Transmission Electron Microscopy

Observing cells and cell components in three dimensions at high magnification in transmission electron microscopy requires preparing serial ultrathin sections of the specimen. Although preparing serial ultrathin sections is considered to be very difficult, it is rather easy if the proper method is used. In this paper, we show a step-by-step procedure for safely obtaining serial ultrathin sections of microorganisms. The key points of this method are: 1) to use the large part of the specimen and adjust the specimen surface and knife edge so that they are parallel to each other; 2) to cut serial sections in groups and avoid difficulty in separating sections using a pair of hair strands when retrieving a group of serial sections onto the slit grids; 3) to use a 'Section-holding loop' and avoid mixing up the order of the section groups; 4) to use a 'Water-surface-raising loop' and make sure the sections are positioned on the apex of the water and that they touch the grid first, in order to place them in the desired position on the grids; 5) to use the support film on an aluminum rack and make it easier to recover the sections on the grids and to avoid wrinkling of the support film; and 6) to use a staining tube and avoid accidentally breaking the support films with tweezers. This new method enables obtaining serial ultrathin sections without difficulty. The method makes it possible to analyze cell structures of microorganisms at high resolution in 3D, which cannot be achieved by using the automatic tape-collecting ultramicrotome method and serial block-face or focused ion beam scanning electron microscopy.

This study presents reliable and easy procedures for obtaining serial ultrathin sections of a microorganism without expensive equipment in transmission electron microscopy.

一种在透射电镜中获取微生物连续超薄切片的方法

Observing cells and cell components in three dimensions at high magnification in transmission electron microscopy requires preparing serial ultrathin ...

科研

工程学

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 ...

免疫与感染

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 ...

Yeast Colony Embedding Method

Patterning of different cell types in embryos is a key mechanism in metazoan development. Communities of microorganisms, such as colonies and biofilms also display patterns of cell types. For example, in the yeast S. cerevisiae, sporulated cells and pseudohyphal cells are not uniformly distributed in colonies. The functional importance of patterning and the molecular mechanisms that underlie these patterns are still poorly understood.
One challenge with respect to investigating patterns of cell types in fungal colonies is that unlike metazoan tissue, cells in colonies are relatively weakly attached to one another. In particular, fungal colonies do not contain the same extensive level of extracellular matrix found in most tissues . Here we report on a method for embedding and sectioning yeast colonies that reveals the interior patterns of cell types in these colonies. The method can be used to prepare thick sections (0.5 &mu;) useful for light microscopy and thin sections (0.1 &mu;) suitable for transmission electron microscopy. Asci and pseudohyphal cells can easily be distinguished from ovoid yeast cells by light microscopy , while the interior structure of these cells can be visualized by EM.
The method is based on surrounding colonies with agar, infiltrating them with Spurr's medium, and then sectioning. Colonies with a diameter in the range of 1-2 mm are suitable for this protocol. In addition to visualizing the interior of colonies, the method allows visualization of the region of the colony that invades the underlying agar.

A method for embedding yeast colonies allowing sectioning for light and electron microscopy. This protocol allows determination of the distribution of sporulated cells and pseudohyphal cells within colonies providing a new tool toward understanding the organization of cell types within a fungal community.

酵母殖民地嵌入方法

Patterning of  different cell types in embryos is a key mechanism in metazoan development. Communities of microorganisms, such as colonies and biofilms ...

生物学

Rigid Embedding of Fixed and Stained, Whole, Millimeter-Scale Specimens for Section-free 3D Histology by Micro-Computed Tomography

For over a hundred years, the histological study of tissues has been the gold standard for medical diagnosis because histology allows all cell types in every tissue to be identified and characterized. Our laboratory is actively working to make technological advances in X-ray micro-computed tomography (micro-CT) that will bring the diagnostic power of histology to the study of full tissue volumes at cellular resolution (i.e., an X-ray Histo-tomography modality). Toward this end, we have made targeted improvements to the sample preparation pipeline. One key optimization, and the focus of the present work, is a straightforward method for rigid embedding of fixed and stained millimeter-scale samples. Many of the published methods for sample immobilization and correlative micro-CT imaging rely on placing the samples in paraffin wax, agarose, or liquids such as alcohol. Our approach extends this work with custom procedures and the design of a 3-dimensional printable apparatus to embed the samples in an acrylic resin directly into polyimide tubing, which is relatively transparent to X-rays. Herein, sample preparation procedures are described for the samples from 0.5 to 10 mm in diameter, which would be suitable for whole zebrafish larvae and juveniles, or other animals and tissue samples of similar dimensions. As proof of concept, we have embedded the specimens from Danio, Drosophila, Daphnia, and a mouse embryo; representative images from 3-dimensional scans for three of these samples are shown. Importantly, our methodology leads to multiple benefits including rigid immobilization, long-term preservation of laboriously-created resources, and the ability to re-interrogate samples.

We developed protocols and designed a custom apparatus to enable embedding of millimeter-scale specimens. We present sample preparation procedures with an emphasis on embedding in acrylic resin and polyimide tubing to achieve rigid immobilization and long-term storage of specimens for the interrogation of tissue architecture and cell morphology by micro-CT.

用微计算机断层扫描法对无节段3D 组织学进行固定染色、全尺寸、毫米度试样的刚性嵌入

For over a hundred years, the histological study of tissues has been the gold standard for medical diagnosis because histology allows all cell types in ...

生物工程

Microscopy Techniques for Interpreting Fungal Colonization in Mycoheterotrophic Plants Tissues and Symbiotic Germination of Seeds

Structural botany is an indispensable perspective to fully understand the ecology, physiology, development, and evolution of plants. When researching mycoheterotrophic plants (i.e., plants that obtain carbon from fungi), remarkable aspects of their structural adaptations, the patterns of tissue colonization by fungi, and the morphoanatomy of subterranean organs can enlighten their developmental strategies and their relationships with hyphae, the source of nutrients. Another important role of symbiotic fungi is related to the germination of orchid seeds; all Orchidaceae species are mycoheterotrophic during germination and seedling stage (initial mycoheterotrophy), even the ones that photosynthesize in adult stages. Due to the lack of nutritional reserves in orchid seeds, fungal symbionts are essential to provide substrates and enable germination. Analyzing germination stages by structural perspectives can also answer important questions regarding the fungi interaction with the seeds. Different imaging techniques can be applied to unveil fungi endophytes in plant tissues, as are proposed in this article. Freehand and thin sections of plant organs can be stained and then observed using light microscopy. A fluorochrome conjugated to wheat germ agglutinin can be applied to the fungi and co-incubated with Calcofluor White to highlight plant cell walls in confocal microscopy. In addition, the methodologies of scanning and transmission electron microscopy are detailed for mycoheterotrophic orchids, and the possibilities of applying such protocols in related plants is explored. Symbiotic germination of orchid seeds (i.e., in the presence of mycorrhizal fungi) is described in the protocol in detail, along with possibilities of preparing the structures obtained from different stages of germination for analyses with light, confocal, and electron microscopy.

This protocol aims to provide detailed procedures for collecting, fixing, and maintaining mycoheterotrophic plant samples, applying different microscopy techniques such as scanning and transmission electron microscopy, light, confocal, and fluorescence microscopy to study fungal colonization in plants tissues and seeds germinated with mycorrhizal fungi.

用于解释霉菌异养植物组织中真菌定植和种子共生萌发的显微镜技术

Structural botany is an indispensable perspective to fully understand the ecology, physiology, development, and evolution of plants. When researching ...

Histological Sample Preparation for Light Microscopy

Histology is the study of cells and tissues, which is typically aided by the use of a light microscope. The preparation of histological samples can vary greatly based on the inherent properties of the samples such as size and hardness as well as expected post-processing which includes planned staining techniques or other down-stream applications. As described in this video, specimen preparation typically begins with a fixation procedure to prevent degradation of the sample by naturally occurring enzymes that are released by the cells upon death. Once fixed, samples are placed into an embedding medium that is able to sufficiently support the sample. Most commonly this is paraffin wax, but other materials such as a glycerin based freezing medium and agars are also used to surround the sample during sectioning. Sectioning then takes place on a microtome or other cutting device that allows the user to shave the sample into thin slices ranging from a few microns to a few millimeters in thickness. Once cut, sections are mounted on a glass slide and stained to bring out specific features of the sample before being imaged on a microscope.

Histological Sample Prep for a Light Microscope

制备组织学样品用于光学显微镜技术

Histology is the study of cells and tissues, which is typically aided by the use of a light microscope.  The preparation of histological samples can vary ...

教育

JoVE Science Education

基础生物学

常用的实验室技术

Formalin-Fixed Paraffin Embedded (FFPE) Tissue Preservation: A Method for Studying Tissue Morphology

Source: Nagaraj A.S., et al., <a href="https://www.jove.com/v/58569/establishment-analysis-tumor-slice-explants-as-prerequisite-for">Establishment and Analysis of Tumor Slice Explants As a Prerequisite for Diagnostic Testing.</a> J. Vis. Exp. (2018)
The following video describes how to preserve morphology of a tissue with formalin fixation and paraffin embedding.

福尔马林固定石蜡包埋 （FFPE） 组织保存:一种研究组织形态的方法

Source: Nagaraj A.S., et al., Establishment and Analysis of Tumor Slice Explants As a Prerequisite for Diagnostic Testing. J. Vis. Exp. (2018)
The ...

JoVE Encyclopedia of Experiments

癌症研究

肺癌

Ex vivo study

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 ...

Immunology

Immunodiagnostics

In Vitro and In Vivo Models

Fixation and Sectioning

Two basic types of preparation are used to visualize specimens with a light microscope: wet mounts and fixed specimens.
The simplest type of preparation is the wet mount, in which the specimen is placed in a drop of liquid on the slide. A liquid specimen can be directly deposited on the slide using a dropper. Solid specimens, such as skin scraping, can be placed on the slide before adding a drop of liquid to prepare the wet mount. Sometimes the liquid is simply water, but stains are often added to enhance contrast. Once the liquid has been added to the slide, a coverslip is placed on top, and the specimen is ready for examination under the microscope.
The second method of preparing specimens for light microscopy is fixation. The &#8220;fixing&#8221; of a sample refers to the process of attaching cells to a slide. Fixation is often achieved by heating (heat fixing) or chemically treating the specimen. In addition to attaching the specimen to the slide, fixation also kills microorganisms in the specimen, stopping their movement and metabolism while preserving the integrity of their cellular components for observation.
To heat-fix a sample, a thin layer of the specimen is spread on the slide (called a smear), and the slide is then briefly heated over a heat source. Chemical fixatives are often preferable to heat for tissue specimens. Chemical agents such as acetic acid, ethanol, methanol, formaldehyde (formalin), and glutaraldehyde can denature proteins, stop biochemical reactions, and stabilize cell structures in tissue samples.
In addition to fixation, staining is almost always applied to color certain features of a specimen before examining it under a light microscope. Stains, or dyes, contain salts made up of a positive ion and a negative ion. Depending on the type of dye, the positive or the negative ion may be the chromophore (the colored ion); and the other uncolored ion is called the counterion. If the chromophore is the positively charged ion, the stain is classified as a basic dye; if the negative ion is the chromophore, the stain is considered an acidic dye.
Dyes are selected for staining based on the chemical properties of the dye and the specimen being observed, which determine how the dye will interact with the specimen. In most cases, it is preferable to use a positive stain, a dye that will be absorbed by the cells or organisms being observed, adding color to objects of interest to make them stand out against the background. However, there are scenarios where it is advantageous to use a negative stain absorbed by the background but not by the cells or organisms in the specimen. Negative staining produces an outline or silhouette of the organisms against a colorful background.
Because cells typically have negatively charged cell walls, the positive chromophores in basic dyes tend to stick to the cell walls, making them positive stains. Thus, basic dyes such as basic fuchsin, crystal violet, malachite green, methylene blue, and safranin typically serve as positive stains. On the other hand, the negatively charged chromophores in acidic dyes are repelled by negatively charged cell walls, making them negative stains. Commonly used acidic dyes include acid fuchsin, eosin, and rose bengal.
Some staining techniques involve the application of only one dye to the sample; others require more than one dye. In simple staining, a single dye is used to emphasize particular structures in the specimen. A simple stain will generally make all of the organisms in a sample appear the same color, even if the sample contains more than one type of organism. In contrast, differential staining distinguishes organisms based on their interactions with multiple stains. In other words, two organisms in a differentially stained sample may appear different colors. Differential staining techniques commonly used in clinical settings include Gram staining, acid-fast staining, endospore staining, flagella staining, and capsule staining.
This text is adapted from <a href="https://openstax.org/books/microbiology/pages/2-4-staining-microscopic-specimens">Openstax, Microbiology 2e, Section 2.4: </a><a href="https://openstax.org/books/microbiology/pages/2-4-staining-microscopic-specimens">Staining microscopic specimens</a>

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Two basic types of preparation are used to visualize specimens with a light microscope: wet mounts and fixed specimens.
The simplest type of preparation ...

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