我们感谢海角-巴西, CNPq 巴西, FAP/DF 为财政支持。我们感谢 Hugo Paes 博士修改了手稿。

1. 解决方案和媒体
<ol><li>准备2X 的罗斯威尔公园纪念研究所 (RPMI) 1640 中等, 磷酸盐缓冲盐水 (PBS), Sabouraud 葡萄糖汤和 Sabouraud 葡萄糖琼脂按表 1。</li>
</ol>2. 真菌接种生长条件
<ol><li>将所有真菌菌株储存在35% 甘油-80 摄氏度, 直到需要。</li>
	<li>在每个实验之前执行以下步骤。<ol>
<li>对于白色念珠菌菌株:<ol>
<li>解冻一个股票瓶和转移200µL 到10毫升 Sabouraud 葡萄糖汤在一个无菌50毫升聚丙烯管与帽和文化隔夜在30°c 与鼓动 (200 rpm)。记住要倾斜的管和离开帽稍微开放, 以更好地曝气的文化。</li>
		</ol>
</li>
		<li>对于隐球菌菌株:<ol>
<li>刮掉一瓶的冷冻表面。将细胞印在 Sabouraud 葡萄糖琼脂板上, 在30摄氏度孵育48小时。在被隔绝的殖民地的可见的成长以后, 保持板材在冰箱 (4 °c) 密封与石蜡膜至多15天。</li>
			<li>用无菌牙签或无菌接种环从盘子中收集一个中型的孤立菌落, 并在无菌50毫升圆锥管中接种10毫升的 Sabouraud 葡萄糖汤。孵育大约24小时在30°c 在鼓动之下 (200 转每分钟)。</li>
			<li>不要超过24小时的孵化。记住要倾斜的管和留下的帽子稍微开放, 以更好的曝气。在每次试验前, 标准化细胞的生长阶段总是好的, 因为这是影响抗菌素耐药性的一个重要因素。 注: 两种真菌生长在30摄氏度, 以快速繁殖在我们的实验, 但温度可以改变, 以反映研究的目的, 例如临床治疗 (37 °c)。最重要的是, 这最初的孵化时间应该事先建立在每个真菌分离和保持在所有的测试, 以确保重现性。</li>
		</ol>
</li>
	</ol>
</li>
	<li>真菌生长后, 在室温下用 1200 x g 离心锥形管以5分钟的速度收集细胞。丢弃上清, 加入10毫升的 PBS。</li>
	<li>并用重悬细胞和离心机在室温下再以 1200 x g 为5分钟。</li>
	<li>重复 PBS 洗涤和离心两次以上。第三次洗涤后, 并用重悬细胞在5毫升 (根据颗粒大小) 的 2X RPMI-1640 培养基。</li>
	<li>准备1毫升的1:100 或 1:1, 000 稀释在一个离心管 (取决于混浊的细胞悬浮)。</li>
	<li>整除10µL 的稀释, 把它放在一个 hemocytometer 室, 并计算在显微镜下四角象限的细胞总数。用公式计算浓度: (总细胞 number/4) x 稀释因子 x 104 (室稀释常数)。<ol>
<li>考虑到100% 的可行性, 如果生存能力计数和生长条件已有系统地相关的隔离被研究。 注: 可根据欧洲抗菌药敏试验委员会 (EUCAST) 对酵母的抗真菌性最低抑制浓度 (MIC) 方法, 对活性计数进行标准化和质量控制10.</li>
		<li>如果没有为研究中的隔离物建立生长/存活相关性, 则通过对 hemocytometer 中的活细胞进行计数来测量真菌的生存能力, 并借助染料选择性地着色死细胞, 如 phloxine B11、台盼蓝12或双面绿色13。仅当目前人口的生存能力为90% 或以上时, 才使用此协议的真菌细胞。 注: 死/活染料可能与选择的真菌没有很好的配合。请在使用前进行测试。例如, 台盼蓝染料与C. 隐球菌不太配合。</li>
	</ol>
</li>
	<li>然后, 在 2X RPMI-1640 培养基 (2X 调整后的接种 RPMI-1640 培养基) 中制备细胞悬浮液。对于96井板, 考虑每块板的体积为5毫升。<ol>
<li>对于所有的C. 白念珠菌菌株, 准备一个股票细胞悬浮 4 x 103细胞/毫升。这种浓度是每个井中最终细胞浓度的 2X (2 x 103细胞/毫升)。</li>
		<li>对于C. 隐球菌菌株, 准备 2 x 104细胞/毫升细胞的股票培养。这个浓度是两次的最终细胞浓度在每个井 (1 x 104细胞/毫升)。</li>
		<li>对于其他真菌, 测试与已知的抗真菌药物浓度将是理想的特定研究与孵化时间有关。考虑到真菌的新陈代谢和重复时间。</li>
	</ol>
</li>
</ol>3. 多肽 (未知剂)
<ol><li>将冻干肽贮存在-20 摄氏度, 在每次试验前将其溶解于去离子水中。一旦溶解在水中的最大贮存时间将取决于每种肽的性质。</li>
	<li>准备两次整除数的最高最终浓度测试 (2X)。理想情况下, 准备少量的整除数, 用足够多的肽一次使用, 以避免冻融循环。 注: 浓度的选择应以文献和肽的性质为基础。建议用大约100µM 的肽开始序列稀释, 然后根据得到的结果减少或增加这个浓度范围。</li>
</ol>4. 参考抗真菌药物 (积极控制)
<ol><li>为那些稀释在水: 准备一个2X 解决方案最高的集中分析 (参见部分 5: 抗真菌化验为稀释的步)。<ol>
<li>对于C. 白色念珠菌菌株, 准备128µg/毫升氟康唑或128µg/毫升的卡泊芬溶液。两个板块的浓度将是64µg/毫升。</li>
		<li>对于C. 隐球菌菌株, 准备32µg/毫升两性霉素 B (水溶性溶液) 的溶液。板块浓度将为16µg/毫升。 注意: 通常两性霉素 B 在二甲基亚砜 (亚砜) 中被稀释, 因为这种抗真菌在水中是不溶的。然而, 有水溶性两性霉素 B 制剂在商业上可用。</li>
	</ol>
</li>
	<li>对于在有机溶剂中稀释的抗真菌药物: 在亚砜中准备一个100X 的库存溶液, 然后将其稀释到10X 的水中使用。 注: 因此, 在井中, 亚砜的最终浓度不会超过1%。如果真菌在含有1% 亚砜的介质中生长, 就需要控制, 因为有些真菌不能很好地耐受这种浓度的溶剂。请记住, 亚砜是光敏的, 所以用箔盖住盘子, 或者把它放在一个黑暗的房间里, 在潜伏期期间。</li>
</ol>5. 抗真菌试验
注:体外抗真菌检测是根据临床和实验室标准研究所 (CLSI) M27-A3 指南中的稀释敏感性试验进行的, 并进行了一些修改。
<ol><li>制备每种肽的双倍序列稀释剂, 并控制96井聚苯乙烯微型中的抗真菌, 最终体积为50µL。<ol>
<li>使用吸管添加100µL 的抗真菌/肽的最高预期最终浓度的2X 浓度在1-3 列, 在行 a。</li>
		<li>使用多通道吸管, 将50µL 的无菌水添加到其他井中, 排在 B 至 H。</li>
		<li>去除50µL 的油井浓度最高 (A 排), 转移到下一个浓度 (行 B) 和融汇下井。</li>
		<li>重复上述步骤, 直到井与最低浓度和丢弃50µL 的这个井 (行 H)。将列11和 12 (行 A、B、C) 保留为空白控制或负/增长控制的唯一水。</li>
		<li>在 RPMI-1640 介质中, 将2X 调整后的接种添加50µL (1-3 列加11列 (负/生长控制));C. 白念珠菌的最终浓度为 2 x 103细胞/毫升, 最后浓度为C. 隐球菌菌株将为 104细胞/毫升。</li>
		<li>准备空白控制 (50 µL 水 + 50 µL 2X RPMI-1640 培养基无细胞) 和负/生长控制 (50 µL 水 + 50 µL 调整接种 2X RPMI-1640 培养基没有抗真菌), 并加载到井板如上所述。</li>
	</ol>
</li>
	<li>对每个要测试的化合物和所选择的抗真菌控制 (列 4-10) 重复该过程。 注意: 如果需要测试的给定化合物/提取物的稀释数量为八或更多, 则可以垂直于下面的实验或在板中水平 (即) 进行串行稀释。<ol>
<li>如果化合物、参考药物或其任何成分是光敏的, 则在还原光中进行检测, 用箔盖住盘子, 或在孵化期间放置在暗腔中。</li>
	</ol>
</li>
	<li>请考虑以下可选建议。<ol>
<li>用透明的盖板密封板, 允许气体交换, 因为这有助于减少蒸发。</li>
		<li>把盘子放在潮湿的房间里;这也将有助于减少蒸发。</li>
	</ol>
</li>
	<li>将37摄氏度的板材孵化为24小时或48小时, 用于所有c. 白念珠菌菌株, 48 h, 200 rpm 为c. 隐球菌而颤抖。较低的最终体积 (100 µL) 可确保不发生溢出。 注: C. 白念珠菌菌株的 MIC 读数在24小时和48小时之间没有显著差异。然而, 48 小时的读数更容易形象化。</li>
	<li>观察所有井, 在倒置光学显微镜的协助下, 在潜伏期之前和之后验证形态学的变化以及检查是否有污染的迹象。 注意: 读数可以在实验结束时进行视觉和拍照。在每次阅读之前, 融汇略有板块。如果没有观察到细胞团簇或成丝, 也可以通过测量其 OD 在 600 nm 进行读取。</li>
	<li>在不同的日期进行至少三次实验。</li>
</ol>

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作者没有什么可透露的。

稀释试验可以用少量的化合物分析靶化合物的潜在抗真菌活性, 同时在浓度范围内对其进行测试。因此, 建议将本议定书作为筛选潜在新的抗真菌化合物的第一步。这里提出的协议是基于 M27-A3 协议, 最初旨在帮助选择在诊所的抗真菌疗法, 并可适应各种新的抗真菌化合物。总的来说, 该协议可以集中于提取物或化合物的物理化学特征。例如, 由于介质中的化合物可能阻止其与真菌细胞的相互作用, 如...

真菌感染已成为近几十年来一个重要的医疗问题, 主要是由于免疫机能丧失者的数量增加, 如接受癌症治疗的人和患有艾滋病毒/艾滋病者或移植的器官1,2。然而, 一组非常有限的可用抗真菌药物和越来越多的真菌耐药性报告导致了关于系统性部真菌病3 治疗的主要问题。
新的抗真菌化合物的潜在来源是抗菌肽 (安培), 许多生物体产生的小阳离子肽, 作为其对感染的先天免疫反应的一部分4。然而, 检测这些化合物对真菌病原体的筛选方法并不标准化。不同的程序已经被用来评估安培的抗真菌活性, 有时对于相同的模型微生物5,6,7。在某些协议中, 这些差异和细节的缺乏使化合物的比较复杂化, 并阻碍了重现性。
对新药候选者进行标准化测试的一种方法是遵循在临床环境中确定抗真菌敏感性的指导方针, 如临床和实验室标准研究所 (CLSI) M27-A3 指南。然而, 这些抗真菌敏感性测试是太限制性的, 并没有考虑到不同物种的新陈代谢变化, 因为它们只是建立了一些选择剂。例如, 它们不考虑非发酵酵母的代谢需要。
该协议允许评估潜在的抗真菌化合物的活性, 并在这里实施, 以寻找抗真菌肽。它的基础上的汤稀释敏感性测试从 CLSI M27-A3 指南与修改, 优化筛选新化合物8,9。这些变化允许使用少量的化合物, 温度或初始接种的变化, 和不同的介质, 以获得最佳的预试生长, 同时使用参照抗真菌药物作为控制来标准化结果。这种方法, 利用多井培养板, 使其能够快速和可靠地筛选出大量的化合物。
由于其固有的灵活性, 本议定书可用于不同的化学类化合物和其他微生物, 几乎没有适应。

<table><tbody><tr><td>&lt;strong&gt;Media and Reagents&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>RPMI 1640 medium with l-glutamine, without sodium bicarbonate</td><td>Thermo Fisher</td><td>31800-022</td><td></td></tr><tr><td>3-(&lt;em&gt;N&lt;/em&gt;-morpholino) propane sulfonic acid&lt;em&gt; (MOPS) (o que a gente usa tem um s&amp;oacute;dio, completa o nome dele please)&lt;/em&gt;</td><td>Sigma-Aldrich</td><td></td><td>Use to buffer 2X RPMI medium</td></tr><tr><td>Sodium chloride (NaCl)</td><td>Din&amp;acirc;mica</td><td>1528-1</td><td>137 mM for Phosphate buffered saline (PBS)</td></tr><tr><td>Potassium chloride (KCl)</td><td>J.T.Baker</td><td>3040-01</td><td>2.7 mM for Phosphate buffered saline (PBS)</td></tr><tr><td>Sodium phosphate dibasic (Na2HPO4)</td><td>Sigma-Aldrich</td><td>V000129</td><td>10 mM for Phosphate buffered saline (PBS)</td></tr><tr><td>Potassium dihydrogen phosphate (KH2PO4)</td><td>Sigma-Aldrich</td><td>60230</td><td>2 mM for Phosphate buffered saline (PBS)</td></tr><tr><td>BD Difco Sabouraud dextrose broth</td><td>BD</td><td>238230</td><td></td></tr><tr><td>BD Difco Sabouraud Dextrose Agar</td><td>BD</td><td>210950</td><td></td></tr><tr><td>Glycerol</td><td>Sigma-Aldrich</td><td>V000123</td><td>35% for (solu&amp;ccedil;&amp;atilde;o de estoque? Criopreserva&amp;ccedil;&amp;atilde;o?)</td></tr><tr><td>Sterile water</td><td></td><td></td><td>Para dilui&amp;ccedil;&amp;atilde;o das drogas na dilui&amp;ccedil;&amp;atilde;o seriada</td></tr><tr><td>&lt;strong&gt;Antifungal drugs&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Amphotericin B</td><td>Sigma-Aldrich</td><td>A2942</td><td></td></tr><tr><td>Fluconazole</td><td>Sigma-Aldrich</td><td>F8929</td><td></td></tr><tr><td>Caspofungin</td><td>Sigma-Aldrich</td><td>PHR1160</td><td></td></tr><tr><td>&lt;strong&gt;Plastics&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>50 mL conical tube</td><td>Sarstedt</td><td>62.547.254</td><td></td></tr><tr><td>Dish petri</td><td>J.Prolab</td><td>0304-5</td><td></td></tr><tr><td>96 well plate</td><td>Corning</td><td>3595</td><td></td></tr><tr><td>Sterile Solution Reservoir</td><td>KASVI</td><td>K30-208</td><td>Use to pippet the solutions using the multichannel pippet</td></tr><tr><td>&lt;strong&gt;Equipment and other materials&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Optical microscope</td><td>Nikon</td><td>E200MV</td><td></td></tr><tr><td>Centrifugue</td><td>Thermo Fisher</td><td>MegaFuge 16R</td><td></td></tr><tr><td>Incubator</td><td>Ethik Technology</td><td>403-3D</td><td>Set to 37&amp;deg; C</td></tr><tr><td>Shaker</td><td>New Brunswick Scientific</td><td>Excella E25</td><td>Set to 37&amp;deg; C, 200 RPM</td></tr><tr><td>Cell counting chamber, Neubauer</td><td>BOECO Germany</td><td>BOE 13</td><td></td></tr><tr><td>Multichannel pipette</td><td>HTL</td><td>5123</td><td></td></tr></tbody></table>

broth microdilution in vitro screening: an easy and fast method to detect new antifungal compounds

真菌感染已成为近几十年来的重要医学条件, 但现有抗真菌药物的数量有限。在这种情况下, 寻找新的抗真菌药物是必要的。此处报告的协议详细介绍了一种筛选肽的抗真菌性质的方法。它是基于从临床和实验室标准研究所 (CLSI) M27-A3 指导方针的汤稀释敏感性测试, 以适应研究抗菌肽作为潜在的新抗真菌药物。该协议描述了一种功能性的检测方法来评估抗真菌化合物的活性, 并且可以很容易地修改, 以适应任何特定的分子类别的调查。由于检测是用小体积的96井板进行的, 所以大规模的筛分可以在短时间内完成, 特别是在自动化环境下进行。这一过程说明了标准化和可调节的临床协议如何帮助新分子的板凳工作追求, 以改善真菌疾病的治疗。

MIC 被定义为在潜伏期结束时完全抑制可见真菌生长的最低抗菌化合物浓度。由于本议定书的目的是要有一个快速的方法来筛选潜在的抗真菌药物, 任何与透明介质类似的空白井被认为是一个积极的结果, 而任何与浊度类似的负/生长控制井是被认为是负面的。然而, 如果有兴趣知道一个给定的安培是否是抑菌作用或杀菌剂, 从积极的水井的媒体也可以镀到 Sabouraud 葡萄糖琼脂?...

Watch this Scientific Journal Video about Broth Microdilution In Vitro Screening: An Easy and Fast Method to Detect New Antifungal Compounds at JoVE.com

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

一种简便、适应性强的肉汤稀释法筛选抗真菌化合物和提取物。

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 ...

broth-microdilution-vitro-screening-an-easy-fast-method-to-detect-new

Research

JoVE Journal

Biology

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

18.7K 次观看.  University of Brasília. 一种简便、适应性强的肉汤稀释法筛选抗真菌化合物和提取物。

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

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

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

Measuring Volatile and Non-volatile Antifungal Activity of Biocontrol Products

The protocol described is based on a plug-transfer technique that allows accurate determination of microorganism quantities and their developmental stages. A specified number of spores are spread on an agar plate. This agar plate is incubated for a defined period to allow the fungi to reach the expected developmental stage, except for spores where incubation is not required. Agar plugs covered by spores, hyphae, or mycelium are next withdrawn and transferred onto agar media containing the antifungal compound to be tested either placed at a distance from the fungi or in contact. This method is applicable to test both liquid extracts and solid samples (powders). It is particularly well suited for quantifying the relative contributions of volatile and non-volatile agents in bioactive mixtures and for determining their effects, specifically on spores, early hyphae, and mycelium.
The method is highly relevant for the characterization of the antifungal activity of biocontrol products, notably plant-derived products. Indeed, for plant treatment, the results can be used to guide the choice of mode of application and to establish the trigger thresholds.

We describe a modified agar-based method designed to quantify the antifungal effects of plant-derived products. Both volatile and non-volatile contributions to the antifungal activity can be assessed through this protocol. In addition, efficacy against fungi can be measured at key developmental stages in a single experimental setup.

测量生物控制产品的挥发性和非挥发性抗真菌活性

The protocol described is based on a plug-transfer technique that allows accurate determination of microorganism quantities and their developmental ...

环境科学

A Reference Broth Microdilution Method for Dalbavancin In Vitro Susceptibility Testing of Bacteria that Grow Aerobically

Antimicrobial susceptibility testing (AST) is performed to assess the in vitro activity of antimicrobial agents against various bacteria. The AST results, which are expressed as minimum inhibitory concentrations (MICs) are used in research for antimicrobial development and monitoring of resistance development and in the clinical setting for antimicrobial therapy guidance. Dalbavancin is a semi-synthetic lipoglycopeptide antimicrobial agent that was approved in May 2014 by the Food and Drug Administration (FDA) for the treatment of acute bacterial skin and skin structure infections caused by Gram-positive organisms. The advantage of dalbavancin over current anti-staphylococcal therapies is its long half-life, which allows for once-weekly dosing. Dalbavancin has activity against Staphylococcus aureus (including both methicillin-susceptible S. aureus [MSSA] and methicillin-resistant S. aureus [MRSA]), coagulase-negative staphylococci, Streptococcus pneumoniae, Streptococcus anginosus group, &#946;-hemolytic streptococci and vancomycin susceptible enterococci. Similar to other recent lipoglycopeptide agents, optimization of CLSI and ISO broth susceptibility test methods includes the use of dimethyl sulfoxide (DMSO) as a solvent when preparing stock solutions and polysorbate 80 (P80) to alleviate adherence of the agent to plastic. Prior to the clinical studies and during the initial development of dalbavancin, susceptibility studies were not performed with the use of P-80 and MIC results tended to be 2-4 fold higher and similarly higher MIC results were obtained with the agar dilution susceptibility method. Dalbavancin was first included in CLSI broth microdilution methodology tables in 2005 and amended in 2006 to clarify use of DMSO and P-80. The broth microdilution (BMD) procedure shown here is specific to dalbavancin and is in accordance with the CLSI and ISO methods, with step-by-step detail and focus on the critical steps added for clarity.

A Reference Broth Microdilution Method for Dalbavancin In Vitro Susceptibility Testing of Bacteria that Grow Aerobically

Here, we present a protocol for determination of dalbavancin susceptibility of clinically relevant Gram-positive bacteria using a broth microdilution reference methodology.

参考微量稀释法达巴万星<em&gt;体外</em&gt;细菌敏感性测试生长有氧

Antimicrobial susceptibility testing (AST) is performed to assess the in vitro activity of antimicrobial agents against various bacteria. The AST results, ...

System for Efficacy and Cytotoxicity Screening of Inhibitors Targeting Intracellular Mycobacterium tuberculosis

Mycobacterium tuberculosis, the causative agent of tuberculosis (TB), is a leading cause of morbidity and mortality worldwide. With the increased spread of multi drug-resistant TB (MDR-TB), there is a real urgency to develop new therapeutic strategies against M. tuberculosis infections. Traditionally, compounds are evaluated based on their antibacterial activity under in vitro growth conditions in broth; however, results are often misleading for intracellular pathogens like M. tuberculosis since in-broth phenotypic screening conditions are significantly different from the actual disease conditions within the human body. Screening for inhibitors that work inside macrophages has been traditionally difficult due to the complexity, variability in infection, and slow replication rate of M. tuberculosis. In this study, we report a new approach to rapidly assess the effectiveness of compounds on the viability of M. tuberculosis in a macrophage infection model. Using a combination of a cytotoxicity assay and an in-broth M. tuberculosis viability assay, we were able to create a screening system that generates a comprehensive analysis of compounds of interest. This system is capable of producing quantitative data at a low cost that is within reach of most labs and yet is highly scalable to fit large industrial settings.

System for Efficacy and Cytotoxicity Screening of Inhibitors Targeting Intracellular Mycobacterium tuberculosis

We have developed a modular high-throughput screening system for discovering novel compounds against Mycobacterium tuberculosis, targeting intracellular and in-broth growing bacteria as well as cytotoxicity against the mammalian host cell.

系统抑制剂靶向细胞内的疗效和毒性筛选<em&gt;结核分枝杆菌</em

Mycobacterium tuberculosis, the causative agent of tuberculosis (TB), is a leading cause of morbidity and mortality worldwide. With the increased spread ...

A Soluble Tetrazolium-Based Reduction Assay to Evaluate the Effect of Antibodies on Candida tropicalis Biofilms

Candida species are the fourth-most common cause of systemic nosocomial infections. Systemic or invasive candidiasis frequently involves biofilm formation on implanted devices or catheters, which is associated with increased virulence and mortality. Biofilms produced by different Candida species exhibit enhanced resistance against various antifungal drugs. Therefore, there is a need to develop effective immunotherapies or adjunctive treatments against Candida biofilms. While the role of cellular immunity is well established in anti-Candida protection, the role of humoral immunity has been studied less.
It has been hypothesized that inhibition of biofilm formation and maturation is one of the major functions of protective antibodies, and Candida albicans germ tube antibodies (CAGTA) have been shown to suppress in vitro growth and biofilm formation of C. albicans earlier. This paper outlines a detailed protocol for evaluating the role of antibodies on biofilms formed by C. tropicalis. The methodology for this protocol involves C. tropicalis biofilm formation in 96-well microtiter plates, which were then incubated in the presence or absence of antigen-specific antibodies, followed by a 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-carboxanilide-2H-tetrazolium (XTT) assay for measuring the metabolic activity of fungal cells in the biofilm.
The specificity was confirmed by using appropriate serum controls, including Sap2-specific antibody-depleted serum. The results demonstrate that antibodies present in the serum of immunized animals can inhibit Candida biofilm maturation in vitro. In summary, this paper provides important insights regarding the potential of antibodies in developing novel immunotherapies and synergistic or adjunctive treatments against biofilms during invasive candidiasis. This in vitro protocol can be used to check the effect of potential new antifungal compounds on the metabolic activity of Candida species cells in biofilms.

A Soluble Tetrazolium-Based Reduction Assay to Evaluate the Effect of Antibodies on Candida tropicalis Biofilms

A 96-well microtiter plate-based protocol using a 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-carboxanilide-2H-tetrazolium (XTT) reduction assay is described herein, to study antibodies' effects on biofilms formed by C. tropicalis. This in vitro protocol can be used to check the effect of potential new antifungal compounds on the metabolic activity of Candida species cells in biofilms.

一种基于四唑的可溶性还原测定，用于评估抗体对 热带念珠菌 生物膜的影响

Candida species are the fourth-most common cause of systemic nosocomial infections. Systemic or invasive candidiasis frequently involves biofilm formation ...

An In Vitro Assay to Evaluate the Effect of Antibodies on Candida tropicalis Biofilm

Source: Chandley, P., et al. <a href="https://app.jove.com/v/64425">A Soluble Tetrazolium-Based Reduction Assay to Evaluate the Effect of Antibodies on Candida tropicalis Biofilms.</a> J. Vis. Exp. (2022).
This video demonstrates an in vitro technique to study the effect of antibodies on fungal biofilms. The pathogenic fungus Candida tropicalis secretes a virulence factor secreted aspartyl proteinase 2 (Sap2), facilitating biofilm formation. Upon introducing a heat-inactivated serum containing Sap2-specific antibodies that inhibit biofilm maturation, the viability of the fungal cells is assessed using a chromogenic assay.

评估抗体对热带念珠菌生物膜影响的体外测定

1. C. tropicalis&#160;biofilm formation

	Prepare&#160;Candida&#160;biofilms in a 96-well flat-bottomed polystyrene microtiter plate as described earlier ...

JoVE Encyclopedia of Experiments

Immunology

Antibody-Based Technologies

Characterization and Functional Analysis

An In Vitro Broth Microdilution Assay for the Screening of Antifungal Compounds

Source: de-Souza-Silva, C. M., et al. <a href="https://app.jove.com/v/57127">Broth Microdilution In Vitro Screening: An Easy and Fast Method to Detect New Antifungal Compounds.</a> J. Vis. Exp. (2018).
This video demonstrates an in vitro assay to screen antifungal compounds using a broth microdilution assay. Test compounds are serially diluted in a microtiter plate and incubated with a fungal inoculum. Post-incubation, the plate is examined for wells without visible fungal growth to assess the antifungal potential of the test compounds and their minimum inhibitory concentration (MIC).

用于筛选抗真菌化合物的体外肉汤微量稀释测定法

1. Solutions and Media

	Prepare 2X Roswell Park Memorial Institute (RPMI) 1640 medium, phosphate-buffered saline (PBS), Sabouraud dextrose broth, and ...

Immunodiagnostics

In Vitro and In Vivo Models

Real-Time Assessment of the Antifungal Activity of Primary Human Immune Cells

Source: Brunel, S. F. et al., <a href="https://app.jove.com/v/55444">Live Imaging of Antifungal Activity by Human Primary Neutrophils and Monocytes in Response to A. fumigatus.</a>J. Vis. Exp. (2017)
This video demonstrates live cell imaging for the analysis of antifungal activity in human neutrophils using fluorescent Aspergillus reporter conidia. Initially, both red and magenta fluorescence are detected within neutrophils, while red fluorescence decreases over time, indicating the degradation of conidia and confirming the antifungal activity of neutrophils.

实时评估原代人免疫细胞的抗真菌活性

All procedures involving human participants have been performed in compliance with the institutional, national, and international guidelines for human ...

Cellular Immunodiagnostics

Evaluating the Effectiveness of a Test Compound against Mycobacteria in Broth

Source: Zheng, X., et al. <a href="https://app.jove.com/v/55273">System for Efficacy and Cytotoxicity Screening of Inhibitors Targeting Intracellular Mycobacterium tuberculosis.</a> J. Vis. Exp. (2017)
This video demonstrates an in-broth assay to determine antibiotic efficacy against Mycobacterium tuberculosis. The bacterial culture is incubated with different concentrations of a bactericidal antibiotic. The bactericidal efficacy is measured by assessing the presence of viable bacteria using a resazurin assay.

评估测试化合物对肉汤中分枝杆菌的有效性

1. Bacterial Strain and Growth Medium

	Make albumin dextrose and salt stock solution (ADS) by solubilizing 25 g of bovine serum albumin, 10.0 g of ...

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

摘要

探索更多视频

此视频中的章节

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

白色念珠菌生物膜芯片（CA BChip）为高通量的抗真菌药物筛选

测量生物控制产品的挥发性和非挥发性抗真菌活性

参考微量稀释法达巴万星

系统抑制剂靶向细胞内的疗效和毒性筛选

一种基于四唑的可溶性还原测定，用于评估抗体对热带念珠菌生物膜的影响

评估抗体对热带念珠菌生物膜影响的体外测定

用于筛选抗真菌化合物的体外肉汤微量稀释测定法

实时评估原代人免疫细胞的抗真菌活性

评估测试化合物对肉汤中分枝杆菌的有效性

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

摘要

探索更多视频

此视频中的章节

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

白色念珠菌生物膜芯片（CA BChip）为高通量的抗真菌药物筛选

测量生物控制产品的挥发性和非挥发性抗真菌活性

参考微量稀释法达巴万星

系统抑制剂靶向细胞内的疗效和毒性筛选

一种基于四唑的可溶性还原测定，用于评估抗体对 热带念珠菌 生物膜的影响

评估抗体对热带念珠菌生物膜影响的体外测定

用于筛选抗真菌化合物的体外肉汤微量稀释测定法

实时评估原代人免疫细胞的抗真菌活性

评估测试化合物对肉汤中分枝杆菌的有效性

一种基于四唑的可溶性还原测定，用于评估抗体对热带念珠菌生物膜的影响