作者感谢教授保罗COS和LMPH，安特卫普大学，比利时期间，在他的实验室拍摄过程中他的支持。这项工作是由芬兰工程学院（项目272266和282981）和瑞典技术科学院芬兰资助。硕士Janni Kujala的技术贡献得到承认。

1.成长的细菌<ol><li>预培养物在37℃下在胰蛋白酶大豆肉汤（TSB）中的细菌过夜以220rpm振荡（16-18小时）。 </li><li>稀释预培养100-1,000倍（取决于细菌的生长速率;在这里，1000倍，用于金黄色葡萄球菌 ）的新鲜TSB，让它生长在37℃和200rpm下达到指数生长（光密度在0.2和0.6）之间的595纳米（OD 595）。 注:此步骤需要特定应变优化。 </li></ol> 2.生物膜的形成:前和暴露后<ol><li>稀释成倍增长文化的100倍（这相当于每毫升约10 6个菌落形成单位（CFU /毫升））。 </li><li> 预曝光协议 <ol><li>对于未经处理的对照样品，加入200微升每孔稀释细菌培养无菌96微孔板的。 </li><li>添加测试化合物或对照的4微升抗生素（50X原液）和196微升每孔稀释细菌培养。 </li><li>在37℃下以200rpm搅拌18小时孵育它在板振荡器上。 注意:在这里我们使用2毫米的轨道振动筛。 </li></ol></li><li> 暴露后协议 <ol><li>对于所有的样品，加入200微升每孔稀释细菌培养无菌96微孔板的。 </li><li>在37℃下以200rpm搅拌18小时孵育它在板振荡器上。 注意:在这里我们使用2毫米的轨道振动筛。 </li><li>小心地取出整个浮游的解决方案，而不触及生物膜，使用多道移液器。 </li><li>添加4微升测试化合物或控制抗生素（50X原液），并每孔196微升TSB的。 </li><li>在37℃下孵育其上以200rpm板振荡器另外24小时。 注意:在这里我们使用2毫米的轨道振动筛。 </li></ol></li></ol> 3.刃天青染色Protocol对于生物膜的可行性分析<ol><li>制备的在无菌PBS 0.1毫克/毫升刃天青（0.4毫摩尔）的储备溶液。保持这个股票不育，从曝光的保护，并在4℃。 </li><li>稀释刃天青库存1:50在无菌磷酸盐缓冲盐水（PBS），以实现20微米的最终浓度。 </li><li>传送整个浮游液（离开生物膜在孔中）小心，不接触生物膜或产生气泡，要使用多道移液器一个单独的，干净的96孔板中。 </li><li>通过加入每孔200μl的用无菌PBS洗涤生物膜一次，并小心地用多道移液器将其删除。 </li><li>加入200μl每使用多道移液器生物膜板以及稀释刃天青。 </li><li>孵育它在黑暗中，在室温（RT），并以200rpm，约20分钟，振摇直到未处理的生物膜控制是均匀的粉红色。 </li><li>测量在荧光λEXC = 560纳米和λEM =用酶标仪（顶端读数）590纳米。 </li></ol> 4.结晶紫染色协议生物膜生物量定量使用同一板块如步骤3 <ol><li>使用多道移液器井小心取出刃天青染色。 </li><li>固定用200μl甲醇的生物膜15分钟。 </li><li>让板空气干燥10分钟。 </li><li>加入190微升0.02％（体积/体积，在去离子水稀释）龙胆紫液，小心使用多道移液器。避免与同时移液污渍接触孔的侧面和防止空气气泡的形成不按移液器完成吹出。 </li><li>孵育它在RT 5分钟。 </li><li>小心取出色斑，使用多道移液器。 </li><li>用去离子水（200每次微升）洗两次。 </li><li>让各孔在RT干燥5分钟，溶解剩余的污点在96％乙醇或33％乙酸。 </li><li>孵育它在RT 1小时，并在595nm处读取吸光度。 </li></ol> 5.浮游菌评价杀菌效果使用相同的样品板作为生物膜<ol><li>测量在含有从3.3浮游溶液板的595nm处的浊度。 </li><li>加入10微升刃天青股票，每孔用染色的刃天青浮游细菌。吹打拌匀。 </li><li>它孵育在黑暗中在室温约23分钟，直到被处理控件是均匀的粉红色。 </li><li>在测量EXCλ= 560nm的荧光，λ= EM 590纳米。 </li></ol> 6.麦胚凝集素染色矩阵量化和生物膜的荧光显微成像<ol><li> 量化矩阵 <ol><li>制备在无菌PBS WGA的探针的5微克/毫升的溶液。 </li><li>使用并行样品解放军忒这个染色。除去从孔浮游溶液并用无菌PBS（每孔200微升）洗一次，小心用多道移液器而不触及生物膜。 </li><li>加入200μl的WGA解决每个被染色良好。 </li><li>它孵育在黑暗中在4℃下2小时。 </li><li>通过用200μl的PBS清洗3次洗涤各孔除去未结合的污点。 </li><li>让板干燥在室温15分钟。 </li><li>溶解在33％乙酸结合的染色，使用每孔200μl的。 </li><li>密封该孔与带状帽和使用水浴超声波仪在室温30秒和40千赫超声处理它们。 </li><li>将培养板在RT 1小时。 </li><li>重复超声步骤。井可以在超声处理步骤之间保持密封。 </li><li>测量在λEX = 495nm处的荧光，λEM =用酶标仪（顶端读数）520纳米。 </li></ol></li><li> 可视化与基质荧光显微镜<ol><li>使用相同的协议，上面直到步骤6.1.6。 </li><li>在干燥步骤之后，可视化的样品用荧光显微镜，使用FITC标记的过滤器（或其它合适的绿色激发过滤器）。 </li></ol></li></ol>在生物膜内7.染色活细胞和死细胞与荧光显微镜和信号的定量绿色到红色比（G / R）成像<ol><li> 成像与荧光显微镜 <ol><li>制备各探针（一个染色的活细胞，另一种染色死细胞）的溶液中，根据制造商的指导方针。 </li><li>除去从孔浮游溶液，并用无菌PBS洗涤一次（每孔200微升）用多道移液器。 </li><li>加入6微升每孔的染色溶液中。 </li><li>孵育在黑暗中板15分钟。 </li><li>在显微镜之前，除去多余的液态间全线采用多道移液器。 </li><li>捕获使用荧光显微镜图像，使用例如FITC过滤器（绿色荧光，活细胞）或TRITC滤波器（红色荧光，死细胞）。 </li></ol></li><li> 信号作为绿色到红色荧光比的定量（G / R） <ol><li>按照相同的协议，在步骤7.1.1和7.1.2。 </li><li>加入200μl每孔的染色溶液的孵育它们在黑暗中15分钟。 </li><li>测量与激发/发射波长为绿色和红色荧光535分之485nm和635分之485，分别为（顶端读数）读板器的荧光。 </li></ol></li></ol>

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Microbiol. 68, 2950-2958 (2002).</li><li>Li, X., Yan, Z., Xu, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+variation+of+biofilms+among+strains+in+natural+populations+of+Candida+albicans.">Quantitative variation of biofilms among strains in natural populations of Candida albicans.</a> Microbiolog. 149, 353-362 (2003).</li><li>Barbosa, I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improved+and+simple+micro+assay+for+sulfated+glycosaminoglycans+quantification+in+biological+extracts+and+its+use+in+skin+and+muscle+tissue+studies.">Improved and simple micro assay for sulfated glycosaminoglycans quantification in biological extracts and its use in skin and muscle tissue studies.</a> Glycobiology. 13, 647-653 (2003).</li><li>Tot&#233;, K., Vanden Berghe, D., Maes, L., Cos, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+colorimetric+microtitre+model+for+the+detection+of+Staphylococcus+aureus+biofilms.">A new colorimetric microtitre model for the detection of Staphylococcus aureus biofilms.</a> Lett. Appl. Microbiol. 46, 249-254 (2008).</li><li>Arciola, C. R., Campoccia, D., Ravaioli, S., Montanaro, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polysaccharide+intercellular+adhesin+in+biofilm:+structural+and+regulatory+aspects.">Polysaccharide intercellular adhesin in biofilm: structural and regulatory aspects.</a> Front Cell Infect Microbiol. 5 (7), (2015).</li><li>Ausbacher, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Staphylococcus+aureus+biofilm+susceptibility+to+small+and+potent+beta(2,2)-amino+acid+derivatives.">Staphylococcus aureus biofilm susceptibility to small and potent beta(2,2)-amino acid derivatives.</a> Biofouling. 30, 81-93 (2014).</li></ol>

The authors have no conflicts of interest to disclose.

没有可以同时测量上的可行性，生物质，和生物膜基质的化合物的效果单一方法。因此，有必要在为了检测在三个端点的效果，优选在初次筛选阶段结合测定。 刃天青是仅由加入氧化还原探针的一个非常简单的染色方案。然而，建立与刃天青生物膜的最佳温育时间是该测定的成功是至关重要的。在某些细菌菌株，刃天青探针的粉红色，荧光试卤灵的减少发生得非常快，而在?...

细菌可以两种非常不同的生活方式，浮游和固着，其中生物膜是最常见的例子之间进行切换。在生物膜，细菌形成嵌入在自产矩阵1结构化的社区。此自产矩阵是细菌，它们的外部环境之间的屏障，它保护了的微生物细胞，使它们在接近。生物膜基质的组合物之间，甚至在物种而异，但它主要是由脂多糖，胞外DNA和蛋白质的紧密网。基质用作物理屏障抑制有害剂的入口，但它也可以防止脱水生物膜并防止营养物质逸出的小区2。 生物膜被视为现代生物医学的最具挑战性的课题之一，它们是对抗生素耐受感染3超过80％的据称负责。他们disp已铺设针对外部威胁的固有的高耐受性:湿度，渗透压，机械应力4，热，紫外线辐射5，消毒剂，抗菌剂，和宿主免疫系统1。例如，为了杀死生物膜所需的所需的抗微生物剂的浓度已显示相比较，以杀死浮游细菌所需的要高的1000倍。此更高公差的解释似乎是多因素的。基质提供了降低抗生素渗透到生物膜的物理屏障。另外，生物膜内的细胞表型多样;他们由于氧，营养物，和生物膜6的内和外部分之间的代谢物的现有梯度不同的代谢状态之间转变。因此，在一些生物膜地区，如核心，细菌被剥夺氧气和营养，生活在一个代谢活性较低或完全休眠状态<suP&gt; 7。完全休眠细胞被称为持留细胞，它们是不易于常规抗微生物治疗8。因此，它很可能是生物膜的韧性源自的当前建议等，未知，机制的组合。 葡萄球菌属。仍然是最有问题的革兰氏阳性菌中，引起严重的，通常生物膜相关的，感染4。有人建议，所有的细菌高达99％的生物膜内的关联，使得它的主要细菌生活方式3。但是，所有的现有的抗生素已经开发针对单小区（浮游）细菌。迄今，分子的一个非常有限的剧目存在可在成熟生物膜选择性作用。这种情况已经在推动药物发现一个渐进的转变，其中的抗生物膜搜索已敦促占据了更加突出的位置。 从methodologiCal中的角度来看，额外的挑战存在，因为只有生物膜的方法有限数目已经由标准制定组织，特别是那些适用于化学文库的高通量筛选开发的。所有的标准化测定（只有一个例外）都基于生物膜反应器中，并且这些方法需要大量的工作容积和大量的化合物进行测试，这通常是在早期研究性阶段9-12不可用。现有的唯一标准筛选适用的测定法是所谓的卡尔加里生物膜的装置，从该商购的最小生物膜消除浓度（MBEC）系统的开发13-15。然而，该测定的限制是该生物膜生长在钉，并且不是所有的细菌物种或同一物种内甚至菌株能够形成这个装置上生物膜。而且，可以特别适用于天然化合物的探索方法必要的。天然产品已经在抗菌药物发现在过去的16世纪创新的主要来源。它们可提供具有独特的作用机制也可以是针对持留细胞有效新颖抗生物化合物。因此，自然和自然的启发库的探索有前途的生产和独特的抗生物引线的高机会。 这里，我们提出测定的一个平台，是为使用三个测定法来测量上的可行性，总生物量，和金黄色葡萄球菌生物膜基质的影响抗生物化合物的化学筛选开发的实验细节。第一测定测量生物膜的可行性，并且它是基于刃天青染色。刃天青是氧化还原染色是蓝色和非荧光在其氧化态和由细菌的代谢活性降低时变为粉红色，高荧光试卤灵。这是一个非常简单和快速米ethod适合初级筛查17-20。第二个实验中，根据结晶紫染色，测量总质量的生物膜。结晶紫为在生物膜19,21-23研究细菌和细菌广泛使用的污点。该测定法是基于廉价的试剂和具有简单的吸光度端点读数。最后，第三个测定目标通过小麦胚凝集素生物膜（WGA），其特异性结合聚-N-乙酰葡糖胺残基（PNAG）存在于葡萄球菌生物膜24的基体的胞外聚合物物质（EPS） -矩阵。 WGA的结合有可使用荧光强度读者25被检测的荧光团。我们在座的基本原理和我们开发，包括示例应用程序的平台的详细信息。

<table><tbody><tr><td>Resazurin</td><td>Sigma Aldrich</td><td>R7017</td><td></td></tr><tr><td>Crystal violet</td><td>Sigma Aldrich</td><td>HT90132&amp;nbsp;</td><td></td></tr><tr><td>Wheat Germ Agglutinin, Alexa Fluor 488 Conjugate</td><td>Thermo Fisher Scientific</td><td>W11261</td><td></td></tr><tr><td>LIVE/DEAD &lt;em&gt;BacLight&lt;/em&gt;&amp;nbsp;</td><td>Molecular Probes</td><td>L7012</td><td>SYTO 9 for staining viable cells green and propidium iodide for staining dead cells red</td></tr><tr><td>Phosphate Buffered Saline</td><td></td><td></td><td></td></tr><tr><td>Tryptone soy agar</td><td>Lab M, Neogen</td><td>LAB011</td><td></td></tr><tr><td>Tryptine soy broth</td><td>Lab M, Neogen</td><td>LAB004</td><td></td></tr><tr><td>F96 Well Plate Polystyrene Sterile Clear Flat bottom</td><td>Thermo Fisher Scientific</td><td>161093</td><td></td></tr><tr><td>BRAND caps, strips of 8</td><td>Sigma Aldrich</td><td>BR781413-300EA</td><td></td></tr><tr><td>Branson CPX series ultrasonic bath</td><td>Sigma Aldrich</td><td>Z769428-1EA</td><td></td></tr><tr><td>Multipipette</td><td>Thermo Fisher Scientific</td><td></td><td></td></tr><tr><td>Multidrop dispenser</td><td>Thermo Fisher Scientific</td><td></td><td></td></tr><tr><td>Biomek 3000</td><td>Beckman Coulter</td><td></td><td></td></tr><tr><td>Varioskan Flash Multiplate reader</td><td>Thermo Fisher Scientific</td><td></td><td></td></tr><tr><td>&lt;em&gt;Staphylococcus aureus&lt;/em&gt;</td><td>ATCC&amp;nbsp;</td><td>25923</td><td></td></tr></tbody></table>

a platform of anti-biofilm assays suited to the exploration of natural compound libraries

生物膜被视为现代生物医学的最具挑战性的课题之一，它们是潜在的负责抗生素耐受性感染的80％以上。生物膜已显示化疗极高的耐受性，这被认为是多因素的。例如，基体提供了降低抗生素渗透到生物膜的物理屏障。另外，生物膜内的细胞的表型多样性。可能的是，生物膜韧性源于这些和其他，未知，机制的组合。所有的现有的抗生素已经开发针对单细胞（浮游）细菌。因此，到目前为止，分子的一个非常有限的剧目存在可在成熟生物膜选择性作用。这种情况已经在推动药物发现一个渐进的转变，其中的抗生物膜搜索已敦促占据了更加突出的位置。一个额外的挑战是，有一非常有限的生物膜研究标准化方法，尤其是那些可用于化学文库的大通量筛选号码。在这里，药剂筛选试验的抗生物平台提出。在聚-N-乙酰氨基葡萄糖，PNAG的它采用三个实验使用小麦胚芽凝集素测量生物膜活力（带刃天青染色），总生物量（用结晶紫染色），和生物膜矩阵（，WGA-荧光染色基础， 分数）。所有的测定用金黄色葡萄球菌作为模型细菌开发的。的平台如何可以用于初步筛选以及用于鉴定抗生物命中功能表征实例。这个实验序列还允许根据测量的终点命中的分类。它还提供了对他们的行动模式的信息，特别是在长期和短期化疗的效果。因此，它是非常ADVAntageous用于快速识别，可以作为起点关于各种生物医学应用的高质量的命中化合物。

在所提出的平台上的可行性，生物质，和生物膜基质的影响量化。在工作序列（ 图1），一个样品板沾有刃天青，随后用结晶紫同时评价对细菌生物膜存活和总生物膜的生物量的影响。这两个实验可以连续在同一个板块，因为它被证明早些时候与刃天青第一染色对结晶紫染色结果（P = 0.4149无统计学显著影响水晶紫激光印版之间最大的信号吸光度单位的?...

Watch this Scientific Journal Video about A Platform of Anti-biofilm Assays Suited to the Exploration of Natural Compound Libraries at JoVE.com

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

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 ...

a-platform-anti-biofilm-assays-suited-to-exploration-natural-compound

Research

JoVE Journal

Immunology and Infection

17.6K 次观看.  University of Helsinki. 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. 

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

A 96 Well Microtiter Plate-based Method for Monitoring Formation and Antifungal Susceptibility Testing of Candida albicans Biofilms

Candida albicans remains the most frequent cause of fungal infections in an expanding population of compromised patients and candidiasis is now the third most common infection in US hospitals. Different manifestations of candidiasis are associated with biofilm formation, both on host tissues and/or medical devices (i.e. catheters). Biofilm formation carries negative clinical implications, as cells within the biofilms are protected from host immune responses and from the action of antifungals. We have developed a simple, fast and robust in vitro model for the formation of C. albicans biofilms using 96 well microtiter-plates, which can also be used for biofilm antifungal susceptibility testing. The readout of this assay is colorimetric, based on the reduction of XTT (a tetrazolium salt) by metabolically active fungal biofilm cells. A typical experiment takes approximately 24 h for biofilm formation, with an additional 24 h for antifungal susceptibility testing. Because of its simplicity and the use of commonly available laboratory materials and equipment, this technique democratizes biofilm research and represents an important step towards the standardization of antifungal susceptibility testing of fungal biofilms.

A 96 Well Microtiter Plate-based Method for Monitoring Formation and Antifungal Susceptibility Testing of Candida albicans Biofilms

We describe a simple, rapid and robust method for the formation of Candida albicans biofilms using 96 well microtiter plates and its utility in antifungal susceptibility testing of cells within biofilms.

一个96孔微孔板为基础的方法监测的形成和抗真菌的药敏试验白色念珠菌生物膜

Candida albicans remains the most frequent cause of fungal infections in an expanding population of compromised patients and candidiasis is now the third ...

科研

免疫与感染

Microtiter Dish Biofilm Formation Assay

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

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

酶标菜生物膜形成实验

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

An Analytical Tool-box for Comprehensive Biochemical, Structural and Transcriptome Evaluation of Oral Biofilms Mediated by Mutans Streptococci

Biofilms are highly dynamic, organized and structured communities of microbial cells enmeshed in an extracellular matrix of variable density and composition 1, 2. In general, biofilms develop from initial microbial attachment on a surface followed by formation of cell clusters (or microcolonies) and further development and stabilization of the microcolonies, which occur in a complex extracellular matrix. The majority of biofilm matrices harbor exopolysaccharides (EPS), and dental biofilms are no exception; especially those associated with caries disease, which are mostly mediated by mutans streptococci 3. The EPS are synthesized by microorganisms (S. mutans, a key contributor) by means of extracellular enzymes, such as glucosyltransferases using sucrose primarily as substrate 3.
Studies of biofilms formed on tooth surfaces are particularly challenging owing to their constant exposure to environmental challenges associated with complex diet-host-microbial interactions occurring in the oral cavity. Better understanding of the dynamic changes of the structural organization and composition of the matrix, physiology and transcriptome/proteome profile of biofilm-cells in response to these complex interactions would further advance the current knowledge of how oral biofilms modulate pathogenicity. Therefore, we have developed an analytical tool-box to facilitate biofilm analysis at structural, biochemical and molecular levels by combining commonly available and novel techniques with custom-made software for data analysis. Standard analytical (colorimetric assays, RT-qPCR and microarrays) and novel fluorescence techniques (for simultaneous labeling of bacteria and EPS) were integrated with specific software for data analysis to address the complex nature of oral biofilm research.
The tool-box is comprised of 4 distinct but interconnected steps (Figure 1): 1) Bioassays, 2) Raw Data Input, 3) Data Processing, and 4) Data Analysis. We used our in vitro biofilm model and specific experimental conditions to demonstrate the usefulness and flexibility of the tool-box. The biofilm model is simple, reproducible and multiple replicates of a single experiment can be done simultaneously 4, 5. Moreover, it allows temporal evaluation, inclusion of various microbial species 5 and assessment of the effects of distinct experimental conditions (e.g. treatments 6; comparison of knockout mutants vs. parental strain 5; carbohydrates availability 7). Here, we describe two specific components of the tool-box, including (i) new software for microarray data mining/organization (MDV) and fluorescence imaging analysis (DUOSTAT), and (ii) in situ EPS-labeling. We also provide an experimental case showing how the tool-box can assist with biofilms analysis, data organization, integration and interpretation.

Biofilms formed on tooth surfaces are highly complex and exposed to constant innate and exogenous environmental challenges, which modulate their architecture, physiology and transcriptome. We developed a toolbox to examine the composition, structural organization and gene expression of oral biofilms, which can be adapted to other areas of biofilm research.

口腔生物膜的生物化学，结构和转录综合评价分析工具盒介导变形链球菌

Biofilms are highly dynamic, organized and structured communities of microbial cells enmeshed in an extracellular matrix of variable density and ...

Candida albicans Biofilm Chip (CaBChip) for High-throughput Antifungal Drug Screening

Candida albicans remains the main etiological agent of candidiasis, which currently represents the fourth most common nosocomial bloodstream infection in US hospitals1. These opportunistic infections pose a growing threat for an increasing number of compromised individuals, and carry unacceptably high mortality rates. This is in part due to the limited arsenal of antifungal drugs, but also to the emergence of resistance against the most commonly used antifungal agents. Further complicating treatment is the fact that a majority of manifestations of candidiasis are associated with the formation of biofilms, and cells within these biofilms show increased levels of resistance to most clinically-used antifungal agents2. Here we describe the development of a high-density microarray that consists of C. albicans nano-biofilms, which we have named CaBChip3. Briefly, a robotic microarrayer is used to print yeast cells of C. albicans onto a solid substrate. During printing, the yeast cells are enclosed in a three dimensional matrix using a volume as low as 50 nL and immobilized on a glass substrate with a suitable coating. After initial printing, the slides are incubated at 37 &deg;C for 24 hours to allow for biofilm development. During this period the spots grow into fully developed "nano-biofilms" that display typical structural and phenotypic characteristics associated with mature C. albicans biofilms (i.e. morphological complexity, three dimensional architecture and drug resistance)4. Overall, the CaBChip is composed of ~750 equivalent and spatially distinct biofilms; with the additional advantage that multiple chips can be printed and processed simultaneously. Cell viability is estimated by measuring the fluorescent intensity of FUN1 metabolic stain using a microarray scanner. This fungal chip is ideally suited for use in true high-throughput screening for antifungal drug discovery. Compared to current standards (i.e. the 96-well microtiter plate model of biofilm formation5), the main advantages of the fungal biofilm chip are automation, miniaturization, savings in amount and cost of reagents and analyses time, as well as the elimination of labor intensive steps. We believe that such chip will significantly speed up the antifungal drug discovery process.

Candida albicans Biofilm Chip CaBChip for High-throughput Antifungal Drug Screening

We have developed a high-density microarray platform consisting of 3D nano-biofilms of C. albicans called CaBChip. The susceptibility profile of drugs tested on a CaBChip is comparable to the conventional 96-well plate model, suggesting that the fungal chip is ideally suited for true high-throughput screening of antifungal drugs.

白色念珠菌生物膜芯片（CA BChip）为高通量的抗真菌药物筛选

Candida  albicans remains the main etiological agent of candidiasis, which  currently represents the fourth most common nosocomial bloodstream infection ...

生物工程

Targeting Biofilm Associated Staphylococcus aureus Using Resazurin Based Drug-susceptibility Assay

Most pathogenic bacteria are able to form biofilms during infection, but due to the difficulty of manipulating and assessing biofilms, the vast majority of laboratory work is conducted with planktonic cells. Here, we describe a peg plate biofilm assay as performed with Staphylococcus aureus. Bacterial biofilms are grown on pegs attached to a 96-well microtiter plate lid, washed through gentle submersion in buffer, and placed in a drug challenge plate. After subsequent incubation they are again washed and moved to a final recovery plate, in which the fluorescent dye resazurin serves as a viability indicator. This assay offers greatly increased ease-of-use, reliability, and reproducibility, as well as a wealth of data when conducted as a kinetic read. Moreover, this assay can be adapted to a medium-throughput drug screening approach by which an endpoint fluorescent readout is taken instead, offering a path for drug discovery efforts.

Targeting Biofilm Associated Staphylococcus aureus Using Resazurin Based Drug-susceptibility Assay

Most bacterial infections produce a biofilm. By virtue of their environment, biofilm associated bacteria are often phenotypically drug resistant. Novel antibacterial molecules that kill bacteria in biofilms are thus a high priority. We establish an assay to quickly screen for antimicrobial compounds that are effective at eradicating biofilms.

针对相关生物膜<em&gt;金黄色葡萄球菌</em&gt;使用基于刃天青药物敏感性测定

Most pathogenic bacteria are able to form biofilms during infection, but due to the difficulty of manipulating and assessing biofilms, the vast majority ...

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 ...

Broth Microdilution In Vitro Screening: An Easy and Fast Method to Detect New Antifungal Compounds

Fungal infections have become an important medical condition in the last decades, but the number of available antifungal drugs is limited. In this scenario, the search for new antifungal drugs is necessary. The protocol reported here details a method to screen peptides for their antifungal properties. It is based on the broth microdilution susceptibility test from the Clinical and Laboratory Standards Institute (CLSI) M27-A3 guidelines with modifications to suit the research of antimicrobial peptides as potential new antifungals. This protocol describes a functional assay to evaluate the activity of antifungal compounds and may be easily modified to suit any particular class of molecules under investigation. Since the assays are performed in 96-well plates using small volumes, a large-scale screening can be completed in a short amount of time, especially if carried out in an automation setting. This procedure illustrates how a standardized and adjustable clinical protocol can help the bench-work pursuit of new molecules to improve the therapy of fungal diseases.

Broth Microdilution In Vitro Screening: An Easy and Fast Method to Detect New Antifungal Compounds

An easy and adaptable broth microdilution method for screening antifungal compounds and extracts.

肉汤稀释体外筛选: 一种简便快速检测新抗真菌化合物的方法

Fungal infections have become an important medical condition in the last decades, but the number of available antifungal drugs is limited. In this ...

生物学

Systematic Approach to Identify Novel Antimicrobial and Antibiofilm Molecules from Plants' Extracts and Fractions to Prevent Dental Caries

Natural products provide structurally different substances, with a myriad of biological activities. However, the identification and isolation of active compounds from plants are challenging because of the complex plant matrix and time-consuming isolation and identification procedures. Therefore, a stepwise approach for screening natural compounds from plants, including the isolation and identification of potentially active molecules, is presented. It includes the collection of the plant material; preparation and fractionation of crude extracts; chromatography and spectrometry (UHPLC-DAD-HRMS and NMR) approaches for analysis and compounds identification; bioassays (antimicrobial and antibiofilm activities; bacterial &#34;adhesion strength&#34; to the salivary pellicle and initial glucan matrix treated with selected treatments); and data analysis. The model is simple, reproducible, and allows high-throughput screening of multiple compounds, concentrations, and treatment steps can be consistently controlled. The data obtained provide the foundation for future studies, including formulations with the most active extracts and/or fractions, isolation of molecules, modeling molecules to specific targets in microbial cells and biofilms. For example, one target to control cariogenic biofilm is to inhibit the activity of Streptococcus mutans glucosyltransferases that synthesize the extracellular matrix&#8217; glucans. The inhibition of those enzymes prevents the biofilm build-up, decreasing its virulence.

Natural products represent promising starting points for the development of new drugs and therapeutic agents. However, due to the high chemical diversity, finding new therapeutic compounds from plants is a challenging and time-consuming task. We describe a simplified approach to identify antimicrobial and antibiofilm molecules from plant extracts and fractions.

从植物提取物和分数中识别新型抗菌和抗生物膜分子的系统方法，以防止牙科蛀牙

Natural products provide structurally different substances, with a myriad of biological activities. However, the identification and isolation of active ...

A Solid Phase Adherence Assay to Assess Biofilm Formation

Source: Nickerson, K. P. et al., <a href="https://app.jove.com/v/57322">Bile Salt-induced Biofilm Formation in Enteric Pathogens: Techniques for Identification and Quantification.</a>&#160;J. Vis. Exp.&#160;(2018)
The video demonstrates a solid-phase adherence assay for assessing biofilm formation. In a multi-well plate, bacteria in bile salt-supplemented media adhere to solid surfaces, forming biofilms, which is confirmed by colorimetry through crystal violet staining and ethanol treatment.

评估生物膜形成的固相粘附测定

1. Preparation of Reagents

	Bile salts medium: To prepare tryptic soy broth (TSB) containing 0.4% bile salts (weight/volume), resuspend 200 mg of bile ...

JoVE Encyclopedia of Experiments

Immunology

Immunodiagnostics

Specialized Assays

Detection of Biofilm Disassembly Through a Dispersion Assay

Record the OD600 values from the overnight biofilm plate on the plate reader by setting the control well as blank. Next, aspirate the culture medium using a vacuum line without disturbing the adherent population on the plastic surface. Gently wash the wells twice with 200 microliters of sterile PBS.
After washing with PBS at 130 microliters of the prewarmed PBS, PBS with glucose, PBS with bile salts, and PBS with bile salts and glucose to each of the three wells. Then incubate the plate at 37 degrees Celsius for 30 minutes. After 30 minutes, remove the plate from the incubator. Pipette the supernatant out to a fresh sterile 96-well plate.
Finally, prepare 1 to 10 serial dilutions of the supernatant with PBS in the 96-well plate or in a dilution block. Then use a multichannel pipette to transfer 5 microliters of each dilution on LB agar plates. Incubate the plates at 37 degrees Celsius overnight. The next day, count the number of colonies.

适合与天然化合物库的探索抗生物试验的平台

摘要

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