Name: quantification, viability assessment, and visualization strategies for acinetobacter biofilms
Uploaded: 2023-08-04T14:10:00.000+00:00
Duration: 7 min 41 s
Description: Watch this Scientific Journal Video about A Comprehensive Protocol for Acinetobacter Biofilm Quantification, Assessment, and Visualization at JoVE.com

这项研究得到了韩国食品研究所（KFRI）的主要研究计划（E0210702-03）的支持，该计划由科学和信息通信技术部资助。

1.细菌接种物的制备
<ol><li>取出储存在-80°C的甘油储备瓶。</li>
	<li>使用无菌移液器吸头从小瓶中取出细菌菌株（2-10μL）。 注： 不动杆菌 菌株， 布维 曲霉（13-1-1）， 朱氏 曲霉（13-1-2）， 皮蒂 曲霉（13-2-5）， 鲍曼 曲霉（13-2-9）， 放射性曲霉 （20-1）和 乌辛曲 霉（24-1）用于本协议。</li>
	<li>用细菌接种市售的血琼脂平板（补充有5%羊血的胰蛋白酶大豆琼脂，见 材料表）。 注意：也可以使用其他培养基，例如营养琼脂或MacConkey琼脂。</li>
	<li>使用间歇性燃烧的无菌环在琼脂表面划线以使其变薄。</li>
	<li>将琼脂平板倒置在培养箱中，并在30°C下孵育20-24小时。</li>
	<li>用无菌针头挑选细菌培养物的单个菌落，并用培养物接种5mL无菌脑心脏输注液（BHI，参见 材料表）。</li>
	<li>将接种的肉汤在30°C的振荡培养箱中以约150rpm孵育20-24小时。</li>
	<li>将 1 mL 过夜培养物转移到无菌微量离心管中，并在室温 （RT） 下以 6,000 × g 离心过夜培养物 10 分钟。</li>
	<li>用移液管除去上清液。</li>
	<li>使用涡旋，将沉淀重悬于 1 mL 无菌磷酸盐缓冲盐水 （PBS） 中。</li>
	<li>在室温下以6,000× g 离心10分钟，除去上清液。</li>
	<li>在600nm处使用涡旋至约0.1的光密度，将沉淀重悬于无菌的10倍稀释BHI中。 注：约 0.1 的光密度相当于约 107 CFU/mL。</li>
</ol>2. 使用结晶紫进行生物膜定量
<ol><li>向无菌聚苯乙烯 96 孔板的每个孔中加入 200 μL 接种物（参见 材料表），并向最外层的每个孔中加入 200 μL 去离子水以防止内孔变干15.</li>
	<li>向同一板的每个孔中加入 200 μL 10 倍稀释的 BHI 作为阴性对照。</li>
	<li>将板在25°C孵育24小时。</li>
	<li>用移液管除去上清液。 注意：多通道移液器通常更方便用于多个样品。</li>
	<li>小心地向每个孔中加入300μL PBS，并用移液管除去上清液。 注意：多通道移液器通常更方便用于多个样品。</li>
	<li>再重复步骤 2.5 两次。</li>
	<li>向每个孔中加入 200 μL 结晶紫溶液 （1%）（参见 材料表），并在室温下孵育 30 分钟。</li>
	<li>重复步骤 2.4-2.6。</li>
	<li>向每个孔中加入 200 μL 无水乙醇，并在室温下孵育 15 分钟，通过上下移液数次彻底混合。</li>
	<li>将 100 μL 洗脱液从每个孔转移到新的 96 孔板中。</li>
	<li>使用酶标仪测量595nm处的吸光度（参见 材料表）。</li>
	<li>当吸光度超过2.0时，将样品在无水乙醇中适当稀释至2.0以下的吸光度，并按步骤2.11进行测量。然后，通过将观察值乘以稀释因子来计算样品的吸光度（最终值）。</li>
	<li>通过从同一孔板上测试样品的每个孔的值中减去阴性对照的平均值来计算样品的真实吸光度。</li>
</ol>3. 生物膜活力计数
<ol><li>向无菌聚苯乙烯 12 孔板15 的每个孔中加入 1 mL 接种物（步骤 1）。将板在25°C孵育24小时。用移液管除去上清液。</li>
	<li>小心地向每个孔中加入 1.5 mL 无菌 PBS，并用移液管除去上清液。向每个孔中加入 1 mL 无菌 PBS。</li>
	<li>使用安装的细胞刮刀刮掉孔的底部和壁面。</li>
	<li>将收获的细胞悬液转移到含有 9 mL 盐水 （0.85% NaCl） 和 10-20 个玻璃珠的无菌塑料管 （10-1） 中（参见 材料表）。 注意：要收集残留在孔底面上的任何残留生物膜碎片，可以将其重新悬浮并使用10-1 管中的生理盐水通过移液管转移。</li>
	<li>以最大速度涡旋10-1 管60秒。</li>
	<li>通过将 1 mL 转移到下一个含有 9 mL 盐水 （0.85 % NaCl） 至 10-7 的无菌管 （10-2） 中，进行 10 倍的连续稀释。</li>
	<li>将每种稀释液中的100μL涂布在不动杆菌琼脂上（参见 材料表），并将琼脂平板在30°C下孵育24-42小时。</li>
	<li>手动计算每个板上典型红色菌落的数量，范围在 10 到 250 之间。</li>
	<li>使用以下公式计算每个孔的生物膜中的活细胞数： 活细胞 = 菌落数 × 稀释因子 × 10</li>
</ol>4. 使用共聚焦激光扫描显微镜（CLSM）进行生物膜可视化
<ol><li>向用于显微镜分析的无菌 96 孔板的每个孔中加入 200 μL 接种物。</li>
	<li>将板在25°C孵育24小时。</li>
	<li>用移液管除去上清液。</li>
	<li>小心地向每个孔中加入 300 μL 过滤灭菌的去离子水，并用移液管除去上清液。</li>
	<li>重复步骤 4.4。</li>
	<li>制备在过滤灭菌的去离子水中稀释至最终浓度分别为10μM和60μM的SYTO 9和碘化丙啶（见 材料表）的混合物。</li>
	<li>将混合物以 200 μL 加入每个孔中，并在室温下在黑暗中孵育板 20-30 分钟。</li>
	<li>重复步骤 4.3-4.4</li>
	<li>使用激发波长分别为 488 nm 和 561 nm，发射波长范围为 499-535 nm 和 568-712 nm 的 CLSM 可视化底表面附着的生物膜。</li>
</ol>

不动杆菌生物膜形成的评估和生物膜的表征

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作者没有什么可透露的。

使用所描述的方案，测量、可视化不同程度的不动杆菌分离株的生物膜形成，并估计生物膜中的活细胞（图1、图2和图3）。
在该方案中，使用两种不同的温度，30°C用于生长，25°C用于不动杆菌的生物膜形成。使用30°C是因为许多研究使用超过30°C的不动杆菌的最佳生长，这对于获得足够的接种物是合适的12,13,14,17。然而，该温度通常高于会形成生物膜的环境温度，例如医院表面或食品加工环境。因此，使用25°C进行生物膜形成，这将更好地模拟这些环境条件。
该方案使用10倍稀释的BHI代替原始BHI形成生物膜（步骤1.12）。许多表面，如医院表面或食品加工环境，可能是低营养的18,19。此外，受污染表面上的微生物在清洁后仍留在表面上，并在低营养条件下持续存在20。因此，低营养条件（如10倍稀释的BHI）比高营养条件（如原始BHI）更可取。
结晶紫测定法是一种公认的、公认的测量生物膜质量的方法，本研究表明，通过使用这种方法， 不动杆菌 的生物膜形成性在不同水平上是可辨别的。
生物膜中的活细胞计数也很重要，因为生物膜中的活细胞是人类感染的原因，而人类感染更有可能由更多的细胞发生21。此外，活菌计数可能是区分不良生物膜产生者的良好工具，如 A. bouvetii 和 A. radioresistens 所示（图1 和 图2）。 A. radioresistens 的活菌数远高于 A. bouvetii，而生物膜质量差异不大，表明 A. radioresistens 感染的可能性更高（图 1 和 图 2）。此外，该方法可用于估计多物种生物膜中 不动杆菌 的比例，这在环境中更为常见。
不动杆菌不仅在底面形成生物膜，而且在侧壁上形成生物膜，特别是在管17,22的气液界面处。使用微量滴定板的CLSM分析仅限于底面附着的生物膜，这使得有时很难与结晶紫测定的结果进行比较，结晶紫测定法测量所有浸没表面，包括壁表面。在这些分离株中还发现了大量气液界面处的生物膜，尤其是强生物膜生产者 A. pittii。与其他分离株相比，它在底面形成的生物膜相对较差（图3）。因此，必须进一步开发一种显微分析技术，以可视化在侧壁形成的生物膜。
在这项研究中，使用了三种不同的测定法，即结晶紫、活菌计数和 CLSM，以表征 不动杆菌的生物膜。结晶紫测定法是定量生物膜的成熟方法，并且存在确定生物膜形成性的标准16。然而，它不仅测量活细胞和EPS，还测量死细胞，这在人类感染和毒力方面意义不大。生物膜中的活细胞可以通过活菌计数法进行测量。然而，生物膜的形成性不能通过活菌计数法确定，因为该方法不能测量EPS，而EPS是生物膜结构中的关键成分。因此，该方法不具备确定生物膜形成性的标准或临界值。此外，活菌计数方法仅测量细胞的可培养性，而不测量“活但不可培养状态”下的细胞11。生物膜的存在可以通过CLSM方法确认，该方法可以有效地可视化生物膜。然而，它只测量底面附着的生物膜，尽管生物膜也经常在侧壁上形成。因此，建议使用结晶紫测定法来确定生物膜的形成性。然后，可以通过活菌计数法对生物膜中的活细胞和可培养细胞进行定量，并通过CLSM方法确认或表征底表面附着的生物膜。

已知不动杆菌会引起医院感染，其人类感染，尤其是在医疗机构中，越来越多的报道1.它广泛存在于医院、医疗机构和食品相关环境中 2,3,4。它可以在包括医院表面（如床栏、床头柜、呼吸机表面和水槽）在内的环境中长期存活4.这种在环境表面的持久性可能是导致不动杆菌4 院内感染的重要因素之一。
生物膜是微生物生命的一种形式，是由活的微生物细胞和来自细胞的细胞外聚合物物质（EPS）组成的微生物基质5。微生物细胞嵌入基质中，通常对环境压力（如热、盐、干燥、抗生素、消毒剂和剪切力）具有很强的抵抗力6,7。
不动杆菌可以在表面上形成生物膜，这表明它可能有助于延长在环境表面（包括医院表面）的存活时间，并增强对抗生素治疗的耐药性8,9。此外，不动杆菌的生物膜形成可能与人类临床结果高度相关8。因此，不动杆菌菌株的生物膜形成性可能是预测环境生存和人类感染的指标之一8,10。
表面附着的生物膜可以被量化和可视化，以评估生物膜的形成性。为了量化表面附着的生物膜，通常用生物膜染色染料（如结晶紫）对生物膜进行染色，并将染料在溶液中洗脱并测量光密度11。生物膜的可视化是评估生物膜形成性的另一种好方法11。与其他技术（如 SEM12,13）相比，使用荧光染料的特异性共聚焦激光扫描显微镜 （CLSM） 可视化方法可能更有助于表征生物膜形态。
可以对生物膜中的活细胞进行计数，以估计生物膜中的活细胞数量11。将嵌入生物膜中的活细胞分离、稀释、铺在琼脂平板上、孵育和计数。由于较高的细胞数可能满足感染剂量，因此可以提供有关生物膜的更详细的信息，例如与细胞数相关的感染电位13,14。
本文介绍了 （1） 量化表面附着的生物膜，（2） 对生物膜中的活细胞进行计数，以及 （3） 使用 Acinetobacter 的 CLSM 可视化生物膜的分步方案。所提出的方案描述了评估 不动杆菌 分离株的生物膜形成性和表征其生物膜的方法。

<table><tbody><tr><td>96-well cell culture plate</td><td>SPL</td><td>30096</td><td>Polystyrene 96-well plate</td></tr><tr><td>BHI (Brain Heart Infusion) broth</td><td>Merck KGaA</td><td>1.10493.0500</td><td></td></tr><tr><td>Blood Agar Base Plate</td><td>KisanBio</td><td>MB-B1005-P50</td><td>Growth media for Acinetobacter</td></tr><tr><td>CHROMagar Acinetobacter</td><td>CHROMagar</td><td>AC092</td><td>Selective plate for Acinetobacter</td></tr><tr><td>Crystal violet solution</td><td>Sigma-Aldrich</td><td>V5265</td><td></td></tr><tr><td>Filmtracer LIVE/DEAD biofilm viability kit</td><td>Invitrogen</td><td>L10316</td><td>SYTO9 and propidium iodide</td></tr><tr><td>Microplate reader</td><td>Tecan</td><td>Infinite M200 PRO NanoQuant</td><td>Biofilm measurement</td></tr><tr><td>RBC Glass Plating Beads</td><td>RBC</td><td>RG001</td><td>Glass beads</td></tr><tr><td>&amp;mu;-Plate 96 Well Black</td><td>ibidi</td><td>89621</td><td>Microplate intended for CLSM</td></tr></tbody></table>

quantification, viability assessment, and visualization strategies for acinetobacter biofilms

不动杆菌 会引起院内感染，其生物膜的形成有助于在医院环境等干燥表面上存活。因此，生物膜定量和可视化是评估 不动杆菌菌 株引起医院感染潜力的重要方法。在微孔板表面形成的生物膜可以根据体积和细胞数量进行量化。生物膜体积可以通过使用结晶紫染色、洗涤、使用乙醇脱色，然后使用酶标仪测量溶解染料来定量。为了量化嵌入生物膜中的细胞数量，使用细胞刮刀将生物膜刮掉，在盐水中收获，在玻璃珠存在下剧烈搅拌，并涂抹在不动杆菌琼脂上。然后，将板在30°C孵育24-42小时。孵育后，计数红色菌落以估计生物膜中的细胞数。这种可行的计数方法也可用于计数混合物种生物膜中的 不动杆菌 细胞。 不动杆菌 生物膜可以使用荧光染料进行可视化。使用设计用于显微分析的市售微孔板来形成生物膜。然后，用SYTO9和碘化丙啶染料染色底表面附着的生物膜，洗涤，然后用共聚焦激光扫描显微镜观察。

按照该方案，最初从厨房表面分离的 不动杆菌 分离株的生物膜在聚苯乙烯96孔板上形成，用结晶紫染色，并将染料溶解在乙醇中并测量生物膜质量（图1）。生物膜的数量因菌株而异，范围从OD 0.04到1.69（图1）。根据 Stepanović 等人 16 建立的标准，除 A. bouvetii 外，所有分离株都形成了生物膜。 A.放射性抗性剂 形成弱生物膜。 朱 氏曲霉和 鲍曼 曲霉形成中等生物膜，而 皮蒂 曲霉和 乌尔辛吉曲 霉形成强生物膜。
为了对生物膜中的活细胞进行计数，使用细胞刮刀刮掉生物膜，高速涡旋，稀释并在 不动杆菌 选择性板上铺展。孵育后，计算菌落数以估计生物膜细胞的数量（图2）。除 A. bouvetii 外，所有分离株的生物膜中都具有相当于 7-8 Log CFU 的细胞。与结晶紫测定显示的生物膜非形成特性一致， 布维曲 霉在 4.4 Log CFU 时的细胞数量水平要低得多。
使用CLSM可视化底面附着的生物膜（图3）。在 黄芪、 鲍曼苜蓿和 熊芥 中发现了大量的生物膜，具有不同的生物膜形态。虽然 A. pittii 在结晶紫测定中是一种强生物膜形成者，但它在底面没有形成太多生物膜。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/65517/65517fig01.jpg"> 图 1：使用结晶紫测定法测量在聚苯乙烯 96 孔板上形成的 不动杆菌 的生物膜质量。 误差线表示与一式三份的标准偏差。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/65517/65517fig01large.jpg" target="_blank">请点击这里查看此图的较大版本.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/65517/65517fig02.jpg"> 图 2：在聚苯乙烯 12 孔板上形成的 不动杆 菌生物膜的活菌计数。 误差线表示与重复项的标准偏差。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/65517/65517fig02large.jpg" target="_blank">请点击这里查看此图的较大版本.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/65517/65517fig03.jpg"> 图 3：使用 SYTO 9 和碘化丙啶染料通过 CLSM 可视化的 96 孔板上形成的底表面附着 不动杆菌 生物膜。 比例尺：50 μm。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/65517/65517fig03large.jpg" target="_blank">请点击这里查看此图的较大版本.</a>

Watch this Scientific Journal Video about A Comprehensive Protocol for Acinetobacter Biofilm Quantification, Assessment, and Visualization at JoVE.com

作者聚焦：不动杆菌生物膜定量、评估和可视化的综合方案 

该方案描述了接种物的制备，使用结晶紫染料在微量滴定板上的生物膜定量，生物膜中的活菌计数以及 不动杆菌生物膜的可视化。

不动杆菌生物膜的定量、活力评估和可视化策略

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

生物膜由活的微生物细胞组成，很难从表面去除。不动杆菌菌株常见于我们的环境中，引起医院感染，也已知会在表面形成生物膜。因此，我们的目标是开发量化和可视化不动杆菌生物膜的方法。我们的方案提供了一种简单易行的方法来量化和可视化不动杆菌的生物膜。通过使用活菌计数方法量化生物膜内细胞的活菌数。我们的研究结果表明，天然存在的不动杆菌载体菌株具有相当不同的生物膜形成潜力。此外，各种生物膜内将存在明显的形态差异。这为研究不动杆菌菌株之间如此大差异的原因铺平了道路。

quantification-viability-assessment-visualization-strategies-for

Universidad San Sebastian

Research

JoVE Journal

Biology

Quantification, Viability Assessment, and Visualization Strategies for Acinetobacter Biofilms

3.0K 次观看.  Korea Food Research Institute. 生物膜由活的微生物细胞组成，很难从表面去除。不动杆菌菌株常见于我们的环境中，引起医院感染，也已知会在表面形成生物膜。因此，我们的目标是开发量化和可视化不动杆菌生物膜的方法。我们的方案提供了一种简单易行的方法来量化和可视化不动杆菌的生物膜。通过使用活菌计数方法量化生物膜内细胞的活菌数。我们的研究结果表明，天然存在的不动杆菌载体?...

视频: 作者聚焦：不动杆菌生物膜定量、评估和可视化的综合方案 

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 ...

科研

免疫与感染

Use of a High-throughput In Vitro Microfluidic System to Develop Oral Multi-species Biofilms

There are few high-throughput in vitro systems which facilitate the development of multi-species biofilms that contain numerous species commonly detected within in vivo oral biofilms. Furthermore, a system that uses natural human saliva as the nutrient source, instead of artificial media, is particularly desirable in order to support the expression of cellular and biofilm-specific properties that mimic the in vivo communities. We describe a method for the development of multi-species oral biofilms that are comparable, with respect to species composition, to supragingival dental plaque, under conditions similar to the human oral cavity. Specifically, this methods article will describe how a commercially available microfluidic system can be adapted to facilitate the development of multi-species oral biofilms derived from and grown within pooled saliva. Furthermore, a description of how the system can be used in conjunction with a confocal laser scanning microscope to generate 3-D biofilm reconstructions for architectural and viability analyses will be presented. Given the broad diversity of microorganisms that grow within biofilms in the microfluidic system (including Streptococcus, Neisseria, Veillonella, Gemella, and Porphyromonas), a protocol will also be presented describing how to harvest the biofilm cells for further subculture&#160;or DNA extraction and analysis. The limits of both the microfluidic biofilm system and the current state-of-the-art data analyses will be addressed. Ultimately, it is envisioned that this article will provide a baseline technique that will improve the study of oral biofilms and aid in the development of additional technologies that can be integrated with the microfluidic platform.

Use of a High-throughput In Vitro Microfluidic System to Develop Oral Multi-species Biofilms

The goal of this methods paper is to describe the use of a microfluidic system for the development of multi-species biofilms that contain species typically identified in human supragingival dental plaque.&#160;Methods to describe biofilm architecture, biofilm viability, and an approach to harvest biofilm for culture-dependent or culture-independent analyses are highlighted.

使用高通量的<em&gt;在体外</em&gt;微流控系统开发口服多物种生物膜

There are few high-throughput in vitro systems which facilitate the development of multi-species biofilms that contain numerous species commonly detected ...

生物工程

Methodologies for Studying B. subtilis Biofilms as a Model for Characterizing Small Molecule Biofilm Inhibitors

This work assesses different methodologies to study the impact of small molecule biofilm inhibitors, such as D-amino acids, on the development and resilience of Bacillus subtilis biofilms. First, methods are presented that select for small molecule inhibitors with biofilm-specific targets in order to separate the effect of the small molecule inhibitors on planktonic growth from their effect on biofilm formation. Next, we focus on how inoculation conditions affect the sensitivity of multicellular, floating B. subtilis cultures to small molecule inhibitors. The results suggest that discrepancies in the reported effects of such inhibitors such as D-amino acids are due to inconsistent pre-culture conditions. Furthermore, a recently developed protocol is described for evaluating the contribution of small molecule treatments towards biofilm resistance to antibacterial substances. Lastly, scanning electron microscopy (SEM) techniques are presented to analyze the three-dimensional spatial arrangement of cells and their surrounding extracellular matrix in a B. subtilis biofilm. SEM facilitates insight into the three-dimensional biofilm architecture and the matrix texture. A combination of the methods described here can greatly assist the study of biofilm development in the presence and absence of biofilm inhibitors, and shed light on the mechanism of action of these inhibitors.

Methodologies for Studying B. subtilis Biofilms as a Model for Characterizing Small Molecule Biofilm Inhibitors

This study presents the development of reproducible methodologies to study biofilm inhibitors and their effects on Bacillus subtilis multicellularity.

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

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

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

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

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

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

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

Visualizing the Effects of Sputum on Biofilm Development Using a Chambered Coverglass Model

Biofilms consist of groups of bacteria encased in a self-secreted matrix. They play an important role in industrial contamination as well as in the development and persistence of many health related infections. One of the most well described and studied biofilms in human disease occurs in chronic pulmonary infection of cystic fibrosis patients. When studying biofilms in the context of the host, many factors can impact biofilm formation and development. In order to identify how host factors may affect biofilm formation and development, we used a static chambered coverglass method to grow biofilms in the presence of host-derived factors in the form of sputum supernatants. Bacteria are seeded into chambers and exposed to sputum filtrates. Following 48 hr of growth, biofilms are stained with a commercial biofilm viability kit prior to confocal microscopy and analysis. Following image acquisition, biofilm properties can be assessed using different software platforms. This method allows us to visualize key properties of biofilm growth in presence of different substances including antibiotics.

This protocol describes the visualization of biofilm development following exposure to host-factors using a slide chamber model. This model allows for direct visualization of biofilm development as well as analysis of biofilm parameters using computer software programs.

可视化痰生物膜发展影响使用腔室盖玻片型号

Biofilms consist of groups of bacteria encased in a self-secreted matrix. They play an important role in industrial contamination as well as in the ...

A Platform of Anti-biofilm Assays Suited to the Exploration of Natural Compound Libraries

Biofilms are regarded as one of the most challenging topics of modern biomedicine, and they are potentially responsible for over 80% of antibiotic-tolerant infections. Biofilms have displayed an exceptionally high tolerance for chemotherapy, which is thought to be multifactorial. For instance, the matrix provides a physical barrier that decreases the penetration of antibiotics into the biofilm. Also, cells within the biofilms are phenotypically diverse. Likely, biofilm resilience arises from a combination of these and other, yet unknown, mechanisms. All of the currently existing antibiotics have been developed against single-cells (planktonic) bacteria. Therefore, so far, a very limited repertoire of molecules exists that can selectively act on mature biofilms. This situation has driven a progressive paradigm shift in drug discovery, in which searching for anti-biofilms has been urged to occupy a more prominent place. An additional challenge is that there are a very limited number of standardized methods for biofilm research, especially those that can be used for large-throughput screening of chemical libraries. Here, an experimental anti-biofilm platform for chemical screening is presented. It uses three assays to measure biofilm viability (with resazurin staining), total biomass (with crystal violet staining), and biofilm matrix (using a wheat germ agglutinin, WGA-fluorescence-based staining of the poly-N-acetyl-glucosamine, PNAG, fraction). All the assays were developed using Staphylococcus aureus as the model bacteria. Examples of how the platform can be used for primary screening as well as for functional characterization of identified anti-biofilm hits are presented. This experimental sequence further allows for the classification of the hits based upon the measured end-points. It also provides information on their mode of action, especially on long-term versus short-term chemotherapeutic effects. Thus, it is very advantageous for the quick identification of high-quality hit compounds that can serve as starting points for various biomedical applications.

Biofilm infections show high tolerance towards chemotherapy. No single assay captures the complexity of biofilms. Instead, complementary assays are needed. We present a screening platform (developed for S. aureus) that combines assays for viability, biomass, and biofilm matrix. It allows anti-biofilm drug discovery, including the assessment of long-term chemotherapeutic effects.

适合与天然化合物库的探索抗生物试验的平台

Biofilms are regarded as one of the most challenging topics of modern biomedicine, and they are potentially responsible for over 80% of ...

Live Cell Analysis of Shear Stress on Pseudomonas aeruginosa Using an Automated Higher-Throughput Microfluidic System

A higher-throughput microfluidic in vitro bioreactor coupled with fluorescence microscopy has been used to study bacterial biofilm growth and morphology, including Pseudomonas aeruginosa (P. aeruginosa). Here, we will describe how the system can be used to study the growth kinetics and the morphological properties such as the surface roughness and textural entropy of P. aeruginosa strain PA01 that expresses an enhanced green fluorescent protein (PA01-EGFP). A detailed protocol will describe how to grow and seed PA01-EGFP cultures, how to set up the microscope and autorun, and conduct the image analysis to determine growth rate and morphological properties using a variety of shear forces that are controlled by the microfluidic device. This article will provide a detailed description of a technique to improve the study of PA01-EGFP biofilms which eventually can be applied towards other strains of bacteria, fungi, or algae biofilms using the microfluidic platform.

Live Cell Analysis of Shear Stress on Pseudomonas aeruginosa Using an Automated Higher-Throughput Microfluidic System

Here, we describe the use of a higher-throughput microfluidic bioreactor coupled with a fluorescent microscope for the analysis of shear stress effects on Pseudomonas aeruginosa biofilms expressing green fluorescent proteins, including instrument set up, the determination of biofilm coverage, growth rate, and morphological properties.

利用自动高通量微流控系统对铜绿假单胞菌剪切应力的活细胞分析

A higher-throughput microfluidic in vitro bioreactor coupled with fluorescence microscopy has been used to study bacterial biofilm growth and morphology, ...

Quantitative Examination of Antibiotic Susceptibility of Neisseria gonorrhoeae Aggregates Using ATP-utilization Commercial Assays and Live/Dead Staining

The emergence of antibiotic resistant Neisseria gonorrhoeae (GC) is a worldwide health threat and highlights the need to identify individuals who fail treatment. This Gram-negative bacterium causes gonorrhea exclusively in humans. During infection, it is able to form aggregates and/or biofilms. The minimum inhibitory concentration (MIC) test is used for to determine susceptibility to antibiotics and to define appropriate treatment. However, the mechanism of the eradication in vivo and its relationship to laboratory results are not known. A method that examines how GC aggregation affects antibiotic susceptibility and shows the relationship between aggregate size and antibiotic susceptibility was developed. When GC aggregate, they are more resistant to antibiotic killing, with bacteria in the center surviving ceftriaxone treatment better than those in the periphery. The data indicate that N. gonorrhoeae aggregation can reduce its susceptibility to ceftriaxone, which is not reflected using the standard agar plate-based MIC methods. The method used in this study will allow researchers to test bacterial susceptibility under clinically relevant conditions.

Quantitative Examination of Antibiotic Susceptibility of Neisseria gonorrhoeae Aggregates Using ATP-utilization Commercial Assays and Live/Dead Staining

A simple ATP-measuring assay and live/dead staining method were used to quantify and visualize Neisseria gonorrhoeae survival after treatment with ceftriaxone. This protocol can be extended to examine the antimicrobial effects of any antibiotic and can be used to define the minimal inhibitory concentration of antibiotics in bacterial biofilms.

atp 利用商业检测和染色法定量检测淋病奈瑟菌骨料的抗生素敏感性

The emergence of antibiotic resistant Neisseria gonorrhoeae (GC) is a worldwide health threat and highlights the need to identify individuals who fail ...

Quantifying the Effects of Antimicrobials on In vitro Biofilm Architecture using COMSTAT Software

Biofilms are aggregates of microorganisms that rely on a self-produced matrix of extracellular polymeric substance for protection and structural integrity. The nosocomial pathogen, Pseudomonas aeruginosa, is known to adopt a biofilm mode of growth, causing chronic pulmonary infection in patients with cystic fibrosis (CF). The computer program, COMSTAT, is a useful tool for quantifying antimicrobial-induced changes in P. aeruginosa biofilm architecture by extracting data from three-dimensional confocal images. However, standardized operation of the software is less commonly addressed, which is important for optimal reporting of biofilm behavior and cross-center comparison. Thus, the aim of this protocol is to provide a simple and reproducible framework for quantifying in vitro biofilm structures under varying antimicrobial conditions via COMSTAT. The technique is modeled using a CF P. aeruginosa isolate, grown in the form of biofilm replicates, and exposed to tobramycin and the anti-Psl monoclonal antibody, Psl0096. The step-by-step approach aims to reduce user ambiguity and minimize the chance of overlooking crucial image-processing steps. Specifically, the protocol emphasizes the elimination of subjective variations associated with the manual operation of COMSTAT, including image segmentation and the selection of appropriate quantitative analysis functions. Although this method requires users to spend additional time processing confocal images prior to running COMSTAT, it helps minimize misrepresented biofilm heterogenicity in automated outputs.

Quantifying the Effects of Antimicrobials on In vitro Biofilm Architecture using COMSTAT Software

Antimicrobial-induced changes to Pseudomonas aeruginosa biofilm architecture differ among clinical isolates cultured from patients with cystic fibrosis and chronic pulmonary infection. Following confocal microscopy, COMSTAT software can be utilized to quantify variations in biofilm architecture (e.g., surface area, thickness, biomass) for individual isolates to assess the efficacy of anti-infective agents.

使用 COMSTAT 软件量化抗菌剂对 体外 生物膜结构的影响

Biofilms are aggregates of microorganisms that rely on a self-produced matrix of extracellular polymeric substance for protection and structural ...

Tools for the Real-Time Assessment of a Pseudomonas aeruginosa Infection Model

Pseudomonas aeruginosa (Pa) is one of the most common opportunistic pathogens associated with cystic fibrosis (CF). Once Pa colonization is established, a large proportion of the infecting bacteria form biofilms within airway sputum. Pa biofilms isolated from CF sputum have been shown to grow in small, dense aggregates of ~10-1,000 cells that are spatially organized and exhibit clinically relevant phenotypes such as antimicrobial tolerance. One of the biggest challenges to studying how Pa aggregates respond to the changing sputum environment is the lack of nutritionally relevant and robust systems that promote aggregate formation. Using a synthetic CF sputum medium (SCFM2), the life history of Pa aggregates can be observed using confocal laser scanning microscopy (CLSM) and image analysis at the resolution of a single cell. This in vitro system allows the observation of thousands of aggregates of varying size in real time, three dimensions, and at the micron scale. At the individual and population levels, having the ability to group aggregates by phenotype and position facilitates the observation of aggregates at different developmental stages and their response to changes in the microenvironment, such as antibiotic treatment, to be differentiated with precision.

Tools for the Real-Time Assessment of a Pseudomonas aeruginosa Infection Model

Synthetic cystic fibrosis sputum medium (SCFM2) can be utilized in combination with both confocal laser scanning microscopy and fluorescence-activated cell sorting to observe bacterial aggregates at high resolution. This paper details methods to assess aggregate populations during antimicrobial treatment as a platform for future studies.

伪多莫纳斯航空感染模型实时评估工具

Pseudomonas aeruginosa (Pa) is one of the most common opportunistic pathogens associated with cystic fibrosis (CF). Once Pa colonization is established, a ...

不动杆菌生物膜的定量、活力评估和可视化策略

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细胞和生物膜细胞外成分的定量并发

使用高通量的

方法学研究

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

可视化痰生物膜发展影响使用腔室盖玻片型号

适合与天然化合物库的探索抗生物试验的平台

利用自动高通量微流控系统对铜绿假单胞菌剪切应力的活细胞分析

atp 利用商业检测和染色法定量检测淋病奈瑟菌骨料的抗生素敏感性

使用 COMSTAT 软件量化抗菌剂对体外生物膜结构的影响

伪多莫纳斯航空感染模型实时评估工具

不动杆菌生物膜的定量、活力评估和可视化策略

副本

摘要

探索更多视频

此视频中的章节

细胞和生物膜细胞外成分的定量并发

使用高通量的

方法学研究

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

可视化痰生物膜发展影响使用腔室盖玻片型号

适合与天然化合物库的探索抗生物试验的平台

利用自动高通量微流控系统对铜绿假单胞菌剪切应力的活细胞分析

atp 利用商业检测和染色法定量检测淋病奈瑟菌骨料的抗生素敏感性

使用 COMSTAT 软件量化抗菌剂对 体外 生物膜结构的影响

伪多莫纳斯航空感染模型实时评估工具

使用 COMSTAT 软件量化抗菌剂对体外生物膜结构的影响