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。

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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

14.2K 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

Colony Forming Cell (CFC) Assay for Human Hematopoietic Cells

Human hematopoietic stem/progenitor cells are usually obtained from bone marrow, cord blood, or peripheral blood and are used to study hematopoiesis and leukemogenesis. They have the capacity to differentiate into lymphoid and myeloid lineages. The colony forming cell (CFC) assay is used to study the proliferation and differentiation pattern of hematopoietic progenitors by their ability to form colonies in a semisolid medium. The number and the morphology of the colonies formed by a fixed number of input cells provide preliminary information about the ability of progenitors to differentiate and proliferate. Cells can be harvested from individual colonies or from the whole plate to further assess their numbers and differentiation states using flow cytometry and morphologic evaluation of Giemsa-stained slides. This assay is useful for assessing myeloid but not lymphoid differentiation. The term myeloid in this context is used in its wider sense to encompass granulocytic, monocytic, erythroid, and megakaryocytic lineages.

We have used this assay to assess the effects of oncogenes on the differentiation of primary human CD34+ cells derived from peripheral blood. For this purpose cells are transduced with either control retroviral construct or a construct expressing the oncogene of interest, in this case NUP98-HOXA9. We employ a commonly used retroviral vector, MSCV-IRES-GFP, that expresses a bicistronic mRNA that produces the gene of interest and a GFP marker. Cells are pre-activated by growing in the presence of cytokines for two days prior to retroviral transduction. After another two days, GFP+ cells are isolated by fluorescence-activated cell sorting (FACS) and mixed with a methylcellulose-containing semisolid medium supplemented with cytokines and incubated till colonies appear on the surface, typically 14 days. The number and morphology of the colonies are documented. Cells are then removed from the plates, washed, counted, and subjected to flow cytometry and morphologic examination. Flow cytometry with antibodies specific to the cell surface markers expressed during hematopoiesis provides information about lineage and maturation stage. Morphological studies of individual cells under a microscope after Wright- Giemsa staining provide further information with regard to lineage and maturation. Comparison of cells transduced with control empty vector to those transduced with an oncogene reveals the effects of the oncogene on hematopoietic differentiation.

Colony Forming Cell CFC Assay for Human Hematopoietic Cells

The colony forming cell (CFC) assay is an in vitro assay in which hematopoietic progenitors form colonies in a semi-solid medium. A combination of colony morphology, cell morphology, and flow cytometry are used to assess the ability of the progenitors to proliferate and differentiate along the different hematopoietic lineages.

集落形成细胞（CFC）的含量对人体的造血细胞

Human hematopoietic stem/progenitor cells are usually obtained from bone marrow, cord blood, or peripheral blood and are used to study hematopoiesis and ...

科研

免疫与感染

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 ...

Growth of Mycobacterium tuberculosis Biofilms

Mycobacterium tuberculosis, the etiologic agent of human tuberculosis, has an extraordinary ability to survive against environmental stresses including antibiotics. Although stress tolerance of M. tuberculosis is one of the likely contributors to the 6-month long chemotherapy of tuberculosis 1, the molecular mechanisms underlying this characteristic phenotype of the pathogen remain unclear. Many microbial species have evolved to survive in stressful environments by self-assembling in highly organized, surface attached, and matrix encapsulated structures called biofilms 2-4. Growth in communities appears to be a preferred survival strategy of microbes, and is achieved through genetic components that regulate surface attachment, intercellular communications, and synthesis of extracellular polymeric substances (EPS) 5,6. The tolerance to environmental stress is likely facilitated by EPS, and perhaps by the physiological adaptation of individual bacilli to heterogeneous microenvironments within the complex architecture of biofilms 7.
In a series of recent papers we established that M. tuberculosis and Mycobacterium smegmatis have a strong propensity to grow in organized multicellular structures, called biofilms, which can tolerate more than 50 times the minimal inhibitory concentrations of the anti-tuberculosis drugs isoniazid and rifampicin 8-10. M. tuberculosis, however, intriguingly requires specific conditions to form mature biofilms, in particular 9:1 ratio of headspace: media as well as limited exchange of air with the atmosphere 9. Requirements of specialized environmental conditions could possibly be linked to the fact that M. tuberculosis is an obligate human pathogen and thus has adapted to tissue environments. In this publication we demonstrate methods for culturing M. tuberculosis biofilms in a bottle and a 12-well plate format, which is convenient for bacteriological as well as genetic studies. We have described the protocol for an attenuated strain of M. tuberculosis, mc27000, with deletion in the two loci, panCD and RD1, that are critical for in vivo growth of the pathogen 9. This strain can be safely used in a BSL-2 containment for understanding the basic biology of the tuberculosis pathogen thus avoiding the requirement of an expensive BSL-3 facility. The method can be extended, with appropriate modification in media, to grow biofilm of other culturable mycobacterial species.
Overall, a uniform protocol of culturing mycobacterial biofilms will help the investigators interested in studying the basic resilient characteristics of mycobacteria. In addition, a clear and concise method of growing mycobacterial biofilms will also help the clinical and pharmaceutical investigators to test the efficacy of a potential drug.

Growth of Mycobacterium tuberculosis Biofilms

Mycobacterium tuberculosis forms drug tolerant biofilms when cultured in certain conditions. Here we describe methods for culturing M. tuberculosis biofilms and determining the frequency of drug tolerant persisters. These protocols will be useful for further studies into the mechanisms of drug tolerance in M. tuberculosis.

增长结核分枝杆菌生物膜

Mycobacterium tuberculosis, the etiologic agent of human tuberculosis, has an extraordinary ability to survive against environmental stresses including ...

Isolation and Genome Analysis of Single Virions using 'Single Virus Genomics'

Whole genome amplification and sequencing of single microbial cells enables genomic characterization without the need of cultivation 1-3. Viruses, which are ubiquitous and the most numerous entities on our planet 4 and important in all environments 5, have yet to be revealed via similar approaches. Here we describe an approach for isolating and characterizing the genomes of single virions called 'Single Virus Genomics' (SVG). SVG utilizes flow cytometry to isolate individual viruses and whole genome amplification to obtain high molecular weight genomic DNA (gDNA) that can be used in subsequent sequencing reactions.

Single Virus Genomics (SVG) is a method to isolate and amplify the genomes of single virons. Viral suspensions of a mixed assemblage are sorted using flow cytometry onto a microscope slide with discrete wells containing agarose, thereby capturing the virion and reducing genome shearing during downstream processing. Whole genome amplification is achieved using multiple displacement amplification (MDA) resulting in genomic material that is suitable for sequencing.

使用"单个病毒基因组的单病毒颗粒的分离和基因组分析&#39;

Whole genome amplification and sequencing of single microbial cells enables genomic characterization without the need of cultivation 1-3. Viruses, which ...

Ratiometric Imaging of Extracellular pH in Dental Biofilms

The pH in bacterial biofilms on teeth is of central importance for dental caries, a disease with a high worldwide prevalence. Nutrients and metabolites are not distributed evenly in dental biofilms. A complex interplay of sorption to and reaction with organic matter in the biofilm reduces the diffusion paths of solutes and creates steep gradients of reactive molecules, including organic acids, across the biofilm. Quantitative fluorescent microscopic methods, such as fluorescence life time imaging or pH ratiometry, can be employed to visualize pH in different microenvironments of dental biofilms. pH ratiometry exploits a pH-dependent shift in the fluorescent emission of pH-sensitive dyes. Calculation of the emission ratio at two different wavelengths allows determining local pH in microscopic images, irrespective of the concentration of the dye. Contrary to microelectrodes the technique allows monitoring both vertical and horizontal pH gradients in real-time without mechanically disturbing the biofilm. However, care must be taken to differentiate accurately between extra- and intracellular compartments of the biofilm. Here, the ratiometric dye, seminaphthorhodafluor-4F 5-(and-6) carboxylic acid (C-SNARF-4) is employed to monitor extracellular pH in in vivo grown dental biofilms of unknown species composition. Upon exposure to glucose the dye is up-concentrated inside all bacterial cells in the biofilms; it is thus used both as a universal bacterial stain and as a marker of extracellular pH. After confocal microscopic image acquisition, the bacterial biomass is removed from all pictures using digital image analysis software, which permits to exclusively calculate extracellular pH. pH ratiometry with the ratiometric dye is well-suited to study extracellular pH in thin biofilms of up to 75 &#181;m thickness, but is limited to the pH range between 4.5 and 7.0.

A pH-sensitive ratiometric dye is used in combination with confocal laser scanning microscopy and digital image analysis to monitor extracellular pH in dental biofilms in real-time.

在口腔生物膜细胞外pH值成比例成像

The pH in bacterial biofilms on teeth is of central importance for dental caries, a disease with a high worldwide prevalence. Nutrients and metabolites ...

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 ...

Extraction of Hemocytes from Drosophila melanogaster Larvae for Microbial Infection and Analysis

During the pathogenic infection of Drosophila melanogaster, hemocytes play an important role in the immune response throughout the infection. Thus, the goal of this protocol is to develop a method to visualize the pathogen invasion in a specific immune compartment of flies, namely hemocytes. Using the method presented here, up to 3 &#215; 106 live hemocytes can be obtained from 200 Drosophila 3rd instar larvae in 30 min for ex vivo infection. Alternatively, hemocytes can be infected in vivo through injection of 3rd instar larvae followed by hemocyte extraction up to 24 h post-infection. These infected primary cells were fixed, stained, and imaged using confocal microscopy. Then, 3D representations were generated from the images to definitively show pathogen invasion. Additionally, high-quality RNA for qRT-PCR can be obtained for the detection of pathogen mRNA following infection, and sufficient protein can be extracted from these cells for Western blot analysis. Taken together, we present a method for definite reconciliation of pathogen invasion and confirmation of infection using bacterial and viral pathogen types and an efficient method for hemocyte extraction to obtain enough live hemocytes from Drosophila larvae for ex vivo and in vivo infection experiments.

Extraction of Hemocytes from Drosophila melanogaster Larvae for Microbial Infection and Analysis

This method demonstrates how to visualize pathogen invasion into insect cells with three-dimensional (3D) models. Hemocytes from Drosophila larvae were infected with viral or bacterial pathogens, either ex vivo or in vivo. Infected hemocytes were then fixed and stained for imaging with a confocal microscope and subsequent 3D cellular reconstruction.

从果蝇幼虫中提取包囊微生物感染及分析

During the pathogenic infection of Drosophila melanogaster, hemocytes play an important role in the immune response throughout the infection. Thus, the ...

Complementary Use of Microscopic Techniques and Fluorescence Reading in Studying Cryptococcus-Amoeba Interactions

To simulate Cryptococcus infection, amoeba, which is the natural predator of cryptococcal cells in the environment, can be used as a model for macrophages. This predatory organism, similar to macrophages, employs phagocytosis to kill internalized cells. With the aid of a confocal laser-scanning microscope, images depicting interactive moments between cryptococcal cells and amoeba are captured. The resolution power of the electron microscope also helps to reveal the ultrastructural detail of cryptococcal cells when trapped inside the amoeba food vacuole. Since phagocytosis is a continuous process, quantitative data is then integrated in the analysis to explain what happens at the timepoint when an image is captured. To be specific, relative fluorescence units are read in order to quantify the efficiency of amoeba in internalizing cryptococcal cells. For this purpose, cryptococcal cells are stained with a dye that makes them fluoresce once trapped inside the acidic environment of the food vacuole. When used together, information gathered through such techniques can provide critical information to help draw conclusions on the behavior and fate of cells when internalized by amoeba and, possibly, by other phagocytic cells.

Complementary Use of Microscopic Techniques and Fluorescence Reading in Studying Cryptococcus-Amoeba Interactions

This paper details a protocol for preparing a co-culture of cryptococcal cells and amoebae that is studied using still, fluorescent images and high-resolution transmission electron microscope images. Illustrated here is how quantitative data can complement such qualitative information.

在研究隐球菌-阿米巴相互作用中,显微技术和荧光读数的补充应用

To simulate Cryptococcus infection, amoeba, which is the natural predator of cryptococcal cells in the environment, can be used as a model for ...

Applying Live Cell Imaging and Cryo-Electron Tomography to Resolve Spatiotemporal Features of the Legionella pneumophila Dot/Icm Secretion System

The Dot/Icm secretion system of Legionella pneumophila is a complex type IV secretion system (T4SS) nanomachine that localizes at the bacterial pole and mediates the delivery of protein and DNA substrates to target cells, a process generally requiring direct cell-to-cell contact. We have recently solved the structure of the Dot/Icm apparatus by cryo-electron tomography (cryo-ET) and showed that it forms a cell envelope-spanning channel that connects to a cytoplasmic complex. Applying two complementary approaches that preserve the native structure of the specimen, fluorescent microscopy in living cells and cryo-ET, allows in situ visualization of proteins and assimilation of the stoichiometry and timing of production of each machine component relative to other Dot/Icm subunits. To investigate the requirements for polar positioning and to characterize dynamic features associated with T4SS machine biogenesis, we have fused a gene encoding superfolder green fluorescent protein to Dot/Icm ATPase genes at their native positions on the chromosome. The following method integrates quantitative fluorescence microscopy of living cells and cryo-ET to quantify polar localization, dynamics, and structure of these proteins in intact bacterial cells. Applying these approaches for studying the Legionella pneumophila T4SS is useful for characterizing the function of the Dot/Icm system and can be adapted to study a wide variety of bacterial pathogens that utilize the T4SS or other types of bacterial secretion complexes.

Applying Live Cell Imaging and Cryo-Electron Tomography to Resolve Spatiotemporal Features of the Legionella pneumophila Dot/Icm Secretion System

Imaging of bacterial cells is an emerging systems biology approach focused on defining static and dynamic processes that dictate the function of large macromolecular machines. Here, integration of quantitative live cell imaging and cryo-electron tomography is used to study Legionella pneumophila type IV secretion system architecture and functions.

应用活细胞成像和低温电子断层扫描解决军团菌肺瘤点/Icm分泌系统的时空特征

The Dot/Icm secretion system of Legionella pneumophila is a complex type IV secretion system (T4SS) nanomachine that localizes at the bacterial pole and ...

Clarifying and Imaging Candida albicans Biofilms

The microbial fungus Candida albicans can undergo a change from commensal colonization to virulence that is strongly correlated with its ability to switch from yeast-form growth to hyphal growth. Cells initiating this process become adherent to surfaces as well as to each other, with the resulting development of a biofilm colony. This commonly occurs not only on mucosal tissue surfaces in yeast infections, but also on medical implants such as catheters. It is well known that biofilm cells are resistant to antifungal drugs, and that cells that shed from the biofilm can lead to dangerous systemic infections. Biofilms range from heavily translucent to opaque due to refractive heterogeneity. Therefore, fungal biofilms are difficult to study by optical microscopy. To visualize internal structural, cellular, and subcellular features, we clarify fixed intact biofilms by stepwise solvent exchange to a point of optimal refractive index matching. For C. albicans biofilms, sufficient clarification is attained with methyl salicylate (n = 1.537) to enable confocal microscopy from apex to base in 600 &#181;m biofilms with little attenuation. In this visualization protocol we outline phase contrast refractometry, the growth of laboratory biofilms, fixation, staining, solvent exchange, the setup for confocal fluorescence microscopy, and representative results.

Clarifying and Imaging Candida albicans Biofilms

To view and quantify the internal features of Candida albicans biofilms, we prepare fixed intact specimens that are clarified by refractive index matching. Then, optical sectioning microscopy can be used to obtain three-dimensional image data though the full thickness of the biofilm.

澄清和成像念珠菌生物膜

The microbial fungus Candida albicans can undergo a change from commensal colonization to virulence that is strongly correlated with its ability to switch ...