Name: winogradsky column microbial layers and gradients
Uploaded: 2023-04-30T07:09:27.000Z
Duration: 8 min 8 s
Channel: JoVE

1. 准备工作
<ol>
	<li>设置一个温诺格拉基柱（Winogradsky column）需要一些基本材料：
	<ul>
		<li>铁锹、桶和瓶子，用于在野外采集样品</li>
		<li>一个垂直的透明容器，例如约1L的量筒或塑料水瓶</li>
		<li>塑料保鲜膜和橡皮筋</li>
		<li>大号混合碗和大勺用于搅拌</li>
		<li>硫源（蛋黄或硫酸钙）</li>
		<li>有机碳源（以碎报纸形式提供的纤维素）</li>
		<li>光源（阳光充足的窗台或台灯）</li>
		<li>从沼泽、湿地、池塘或溪流中采集的土壤或泥样</li>
		<li>来自同一生境的水</li>
		<li>可选：以下材料适用于本实验方案中描述的某些可选实验：
		<ul>
			<li>食盐</li>
			<li>不同颜色的玻璃纸</li>
			<li>铁源（如铁钉或钢丝绒）</li>
			<li>带光源的冰箱</li>
			<li>靠近光源的暖气片</li>
		</ul>
		</li>
	</ul>
	</li>
	<li>如果使用塑料水瓶，需剪掉瓶口部分，使柱体呈圆柱形。移除所有标签，以确保光线可穿透塑料瓶壁。</li>
	<li>生鸡蛋可能含有沙门氏菌（Salmonella），应小心处理，并严格遵守正确的洗手方法。也可使用煮熟的鸡蛋替代。此外，无法完全确定泥样或沉积物是否被污水或其他有害物质污染，因此在混合泥样和组装柱体时应佩戴手套。</li>
</ol>
2. 组装一个温克尔曼柱
<ol>
	<li>使用铲子从水边挖掘并收集淤泥至桶中。所采集的沉积物应靠近水边，并完全被水饱和。所需淤泥量应足以填满每个 Winogradsky 柱。同时从同一水源采集部分水样至样品瓶中（每根柱子约需 3000 mL）。</li>
	<li>在实验室中，将足量淤泥转移至第一个混合碗中，以填满 1 升体积柱的约 75%。随后用勺子挑出大石块、小树枝或树叶，并打散结块。</li>
	<li>一边搅拌，一边向混合碗中加入部分采集的水，直至水和淤泥的混合物呈奶昔状。持续搅拌以确保无结块残留。</li>
	<li>将约三分之一的水-泥奶昔混合物转移至第二个碗中，加入蛋黄和一把撕碎的报纸，充分混合。</li>
	<li>将泥、蛋黄和报纸的混合物加入柱子中，直至柱子约填充至 1/4 处。</li>
	<li>将普通水-泥混合物加入柱子中，直至柱子约填充至 3/4 处。</li>
	<li>向柱子中补充额外的水，仅在顶部留出少量空间（约 ½ 英寸）的空气。</li>
	<li>用塑料薄膜覆盖柱子，并用橡皮筋固定密封。</li>
	<li>将柱子置于室温光照条件下培养。</li>
	<li>在接下来的 4 至 8 周内，观察 Winogradsky 柱中不同颜色层的形成以及气泡的产生情况（见表 1）。同时，应记录各色层出现所需的时间。</li>
</ol>
3. 对经典维诺格拉茨基柱的可选改进
<ol>
	<li>在加入水并搅拌之前（步骤 2.3），向收集的泥样中每 1L 加入 25–50g 盐。添加盐可筛选嗜盐（喜盐）细菌。</li>
	<li>可将其他底物（如铁，以铁钉或钢丝绒形式）与蛋黄和碎报纸一同加入柱中（步骤 2.4）。这将富集铁氧化细菌（如 Gallionella），并形成锈色层。</li>
	<li>柱体培养时（步骤 2.9），可不采用室温，而将其置于散热器附近以筛选嗜热（喜热）细菌，或置于带有光源的冰箱中以筛选嗜冷（喜冷）细菌。</li>
	<li>在培养过程中（步骤 2.9），可通过将不同柱体置于强光、弱光或黑暗环境中，调节其接受的光照强度。</li>
	<li>在培养过程中（步骤 2.9），可用不同颜色的玻璃纸覆盖柱体，以限制入射光的波长，从而确定哪些颜色可筛选出不同的细菌类群。</li>
</ol>
4. 数据分析
<ol>
	<li>经过1至3周后，经典温诺格拉斯基柱泥层表面应可见一些绿色着色（图2A）。这是蓝细菌层开始生长的最初迹象。</li>
	<li>随着时间推移，持续观察各层的外观及其演化过程，每一层均指示不同类型的细菌。提示：参考“原理”部分及表1，了解哪些细菌参与形成不同层次。</li>
</ol>
<img alt="沉积物岩心提取；用于地质分析的分层土壤层示意图。" class="xfigimg" src="/files/ftp_upload/10506/10506fig2a.jpg" data-altupdatedat="1761830439000" data-alt="Figure 2A"> 
图 2A： 一个经典的 Winogradsky 柱在室温下培养 21 天后的照片。注意柱体上部呈绿色的沉积物，表明存在蓝细菌。
<ol start="3">
	<li>如果还制备了对经典 Winogradsky 柱的修改版本，比较每个柱子的结果。
	<ol>
		<li>观察每个修改后的 Winogradsky 柱中的分层情况。注意以下几点：
		<ul>
			<li>各柱子的层数是否相同？</li>
			<li>各层的颜色和厚度是否一致？</li>
			<li>各层出现的深度是否相同？</li>
			<li>每个柱子发育所需的时间有多长？</li>
			<li>是否有某个柱子的发育速度比其他柱子更慢？</li>
		</ul>
		</li>
	</ol>
	</li>
</ol>

温克尔曼柱是微生物相互依赖生态系统的范例。将泥浆、水以及额外的碳源和硫源在垂直柱中混合后，经过数周时间，该分层的生态系统将逐渐稳定，形成不同的稳定区域。这些区域由不同的微生物占据，它们在从底部富含硫化物的沉积物到顶部富含氧气的沉积物之间的梯度上特定位置生长旺盛。通过调控温克尔曼柱内的条件和底物，可以观察到嗜盐菌、嗜热菌、嗜冷菌、硫氧化菌、硫还原菌、铁氧化菌以及光合菌等不同微生物的存在与活性。

构建温诺格拉茨基柱：富集沉积物样品中微生物物种的一种方法

Creating a Winogradsky Column: A Method to Enrich the Microbial Species in a Sediment Sample

来源：Elizabeth Suter1，Christopher Corbo1，Jonathan Blaize1 
1 生物科学系，瓦格纳学院，1 Campus Road，纽约史泰登岛，10301
Winogradsky 柱是一种用于富集沉积物微生物群落的微型封闭生态系统，尤其适用于参与硫循环的微生物。该方法最早由 Sergei Winogradsky 于19世纪80年代使用，此后已广泛应用于研究多种参与生物地球化学过程的微生物，例如光合生物、硫氧化菌、硫酸盐还原菌、产甲烷菌、铁氧化菌、氮循环菌等（1,2）。
地球上大多数微生物被认为是不可培养的，这意味着它们无法在试管或培养皿中被分离培养（3）。造成这一现象的原因很多，其中包括微生物在某些代谢产物上依赖其他微生物。温诺格拉茨基柱中的条件 closely 模拟了微生物的自然生境，包括它们与其他生物之间的相互作用，从而使其能够在实验室中生长。因此，该技术使科学家能够研究这些微生物，并理解它们在地球生物地球化学循环中的重要作用，而无需将它们单独分离培养。
地球的环境中充满了微生物，它们在各种生境中繁衍生息，例如土壤、海水、云层以及深海沉积物。在所有生境中，微生物之间相互依赖。当微生物生长时，会消耗特定的底物，包括富含碳的燃料（如糖类）、营养物质、维生素以及氧气等呼吸气体。当这些重要资源耗尽时，具有不同代谢需求的其他微生物便开始增殖并繁荣发展。例如，在维诺格拉茨基柱（Winogradsky column）中，微生物首先消耗添加的有机物质，同时耗尽柱体底层的氧气。一旦氧气被耗尽，厌氧微生物便占据主导地位，并开始分解不同的有机物。这种随时间推移而依次出现的不同微生物群落的发展过程称为演替（4）。微生物演替在维诺格拉茨基柱中至关重要，因为微生物的活动会改变沉积物的化学环境，进而影响其他微生物的活动，如此循环往复。土壤和沉积物中的许多微生物也生活在梯度环境中，即基于底物浓度差异形成的两种不同生境之间的过渡区域（5）。在梯度的合适位置，微生物可以获得各种底物的最佳浓度。随着维诺格拉茨基柱的发育，它开始模拟这些自然梯度，尤其是氧气和硫化物的梯度（图1）。
<img alt="温克尔曼柱状图、微生物梯度、H2S 和 O2 浓度分析、教学用途" class="xfigimg" src="/files/ftp_upload/10506/10506fig1.jpg" data-altupdatedat="1761830439000" data-alt="图 1"> 
图 1： 温克尔曼柱中形成的氧气（O2）和硫化物（H2S）梯度示意图。
在Winogradsky柱中，将池塘或湿地的泥和水混合于一个透明柱形容器中，并通常在光照条件下进行培养。向该柱中添加额外的底物，为微生物群落提供碳源（通常以纤维素形式）和硫源。光合微生物通常开始在沉积物的上层生长。这些光合微生物主要由cyanobacteria（蓝细菌）组成，它们能够产生氧气，并呈现为绿色或红褐色的层状结构（图2，表1）。尽管光合作用会释放氧气，但氧气在水中的溶解度较低，因此在该层以下氧气浓度逐渐下降（图1）。这形成了从上层高浓度氧气到下层无氧的氧气梯度。含有氧气的区域称为aerobic（好氧）层，而无氧区域则称为anaerobic（厌氧）层。
在厌氧层中，多种不同的微生物群落能够增殖，其种类和数量取决于可利用底物的类型和数量、初始微生物的来源以及沉积物的孔隙度。在柱体底部，能够厌氧分解有机物的生物可以旺盛生长。微生物发酵作用通过纤维素降解产生有机酸。这些有机酸可被硫酸盐还原菌利用，后者以硫酸盐作为电子受体氧化这些有机物，并将硫化物作为代谢副产物。当沉积物变黑时，表明硫酸盐还原菌具有活性，这是因为铁与硫化物反应生成黑色的硫化铁矿物（图2，表1）。此外，硫化物还会向上扩散，在柱体中形成另一个浓度梯度，即柱体底部硫化物浓度高，顶部浓度低（图1）。
在柱体中部附近，硫氧化菌可利用来自上方的氧气和来自下方的硫化物。在适宜的光照条件下，光合硫氧化菌可在这些层次中生长。这类生物被称为绿色硫细菌和紫色硫细菌，常表现为绿色、紫色或紫红色的丝状体或斑块（图2，表1）。绿色硫细菌对硫化物的耐受性更高，通常生长在紫色硫细菌下方的层次中。在紫色硫细菌的上方，还可能生长有紫色非硫细菌。这类生物进行光合作用时以有机酸作为电子供体而非硫化物，常呈现为红色、紫色、橙色或棕色的层次。非光合硫氧化菌可在紫色非硫细菌的上方生长，通常表现为白色丝状体（图2，表1）。此外，Winogradsky 柱中也可能形成气泡。好氧层中的气泡表明蓝细菌正在产生氧气；而厌氧层中的气泡则可能源于产甲烷菌的活动，这类生物在厌氧条件下分解有机物，并以甲烷作为代谢副产物。 
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>柱中位置</td>
			<td>功能群</td>
			<td>生物示例</td>
			<td>视觉指示</td>
		</tr>
		<tr>
			<td>顶部</td>
			<td>光合生物</td>
			<td>Cyanobacteria</td>
			<td>绿色或红褐色层。有时可见氧气气泡。</td>
		</tr>
		<tr>
			<td rowspan="5"></td>
			<td>非光合作用的硫氧化菌</td>
			<td>Beggiatoa, Thiobacilus</td>
			<td>白色层。</td>
		</tr>
		<tr>
			<td>紫色非硫细菌</td>
			<td>Rhodomicrobium, Rhodospirilum, Rhodopseuodmonas</td>
			<td>红色、紫色、橙色或棕色层。</td>
		</tr>
		<tr>
			<td>紫色硫细菌</td>
			<td>Chromatium</td>
			<td>紫色或紫红色层。</td>
		</tr>
		<tr>
			<td>绿色硫细菌</td>
			<td>Chlorobium</td>
			<td>绿色层。</td>
		</tr>
		<tr>
			<td>硫酸盐还原菌</td>
			<td>Desulfovibrio, Desulfotomaculum, Desulfobacter, Desulfuromonas</td>
			<td>黑色层。</td>
		</tr>
		<tr>
			<td>底部</td>
			<td>产甲烷菌</td>
			<td>Methanococcus, Methanosarcina</td>
			<td>有时可见甲烷气泡。</td>
		</tr>
	</tbody>
</table>
表1： 从上到下典型的温诺格拉茨基柱中可能出现的主要细菌类群。列出了每个类群的生物示例以及各层生物的视觉特征。资料来源：Perry 等（2002）和 Rogan 等（2005）。

在本实验中，从淡水生境中采集了水样和沉积物。构建了两个温克尔曼柱并使其发育：一个经典的温克尔曼柱在室温光照条件下培养（图2A），另一个温克尔曼柱在室温避光条件下培养（图2B）。
<img alt="沉积物柱分析；生物地球化学梯度研究；显示分层结构的实验。" class="xfigimg" src="/files/ftp_upload/10506/10506fig2b.jpg" data-altupdatedat="1761830439000" data-alt="图 2B"> 
图 2B：经典维诺格拉茨基柱（左侧），在室温光照条件下培养 68 天，以及在室温黑暗条件下培养 68 天的维诺格拉茨基柱（右侧）的照片。
在培养7至9周后，可将经典型柱中的分层结构与避光培养的柱进行比较（图2B）。在经典的维诺格拉茨基柱中，可在管子顶部附近观察到绿色的蓝细菌层。在管子中部附近，可见一层红紫色，表明存在紫色非硫细菌。在此层下方，可观察到一层紫红色，表明存在紫色硫细菌。紧接该层之下，在柱的厌氧区域可观...

来源：Elizabeth Suter1，Christopher Corbo1，Jonathan Blaize1  
1 纽约州史泰登岛瓦格纳学院生物科学系，校园路1号，邮编10301

地球上的大多数微生物无法在实验室中培养，通常是因为它们依赖于其原始群落中的其他微生物。由发明者谢尔盖·维诺格拉茨基（Sergei Winogradsky）命名的维诺格拉茨基柱是一种微型封闭生态系统，可富集沉积物样品中的微生物群落，使科学家能够在无需单独分离和培养的情况下，研究许多在地球生物地球化学过程中起关键作用的微生物。通常，将来自某种生态系统（如池塘或沼泽）的泥浆和水进行混合。作为可选实验，可在该混合物中加入盐，以富集各种嗜盐微生物。接下来，取少量该混合物并添加碳源（通常以报纸中的纤维素形式提供）和硫源（通常来自蛋黄）。在另一项可选实验中，可在该混合物中加入一根铁钉，以富集特定的嘉利翁氏菌（Gallionella）物种。然后将此新混合物加入透明柱中，使柱体填充至约四分之一高度。最后，将剩余的泥浆混合物和更多的水加入柱中，直至柱体几乎充满。演替是指不同微生物群落在时间上的连续发展过程，这一过程可通过瓦诺根斯基柱（Winogradsky column）实时观察。随着微生物在柱内的生长，它们消耗特定的底物，并改变其周围环境的化学条件。当原有底物被耗尽后，最初的微生物逐渐死亡，而具有不同代谢需求的微生物则能在改变后的环境中繁盛起来。随着时间推移，柱内开始形成肉眼可见的不同层次，每一层都包含具有不同微环境需求的细菌群落组成部分。例如，光合微生物主要由蓝细菌组成，在柱体顶部附近形成绿色或红褐色的层。由于光合作用产生氧气，常在柱体上部观察到气泡，从而形成一个溶氧梯度，即柱体顶部氧气浓度最高，底部最低。根据可利用底物的不同，不同的微生物群落可在无氧的底层中生长。该层中的气泡可能表明存在产甲烷菌，它们通过发酵作用产生甲烷气体。在此过程中，微生物对纤维素的发酵产生有机酸。硫酸盐还原菌氧化这些有机酸生成硫化物，其活动表现为黑色沉积物。硫化物在柱体中向上扩散，形成另一个浓度梯度，即柱体底部硫化物浓度最高，顶部最低。在柱体中段，硫氧化菌利用上方的氧气和下方的硫化物进行代谢。在充足光照条件下，光合型硫氧化菌（如绿色和紫色硫细菌）得以生长。绿色硫细菌对较高浓度的硫化物具有耐受性，因此它们直接生长在紫色硫细菌的下方。紧邻该层上方，紫色非硫细菌形成一层红橙色的菌层。非光合型硫氧化菌则通过白色丝状体的存在得以识别。光照和温度等条件也可以进行调整，以富集其他微生物群落。在本视频中，您将学习如何构建温哥华斯基柱（Winogradsky column），并通过改变培养条件和底物来富集特定的微生物群落。首先，寻找一个合适的水生生态系统，例如池塘或沼泽。沉积物样品应取自靠近水边的区域，并且要完全被水饱和。然后，使用铲子和桶采集一到两升饱和的淤泥。接着，从同一水源获取约三升淡水，并将野外样品带回实验室。在实验室中，穿戴适当的个人防护装备，包括实验服和手套。然后，将约750毫升的泥浆转移至一个搅拌碗中。接着，筛检泥浆以去除较大的石块、小树枝或树叶，并用勺子将任何结块捣碎。随后，向搅拌碗中加入一些淡水，用大勺搅拌。继续加水，直至水与泥浆混合物的稠度类似于奶昔。在此过程中，持续确保无结块存在。作为可选实验，可通过向泥浆混合物中添加 25 至 50 毫克食盐来筛选嗜盐细菌。然后，将约三分之一的水-泥混合物转移至第二个搅拌碗中。向碗中加入一个蛋黄和一把碎报纸。接着，将此混合物加入柱中，直至柱体约四分之一满。随后，将不含蛋黄和报纸的水-泥混合物加入柱中，直至柱体约四分之三满。然后，向柱中加入更多水，顶部留出约1/2英寸的空间。用塑料薄膜盖住柱体，并用橡皮筋固定。将该培养柱置于室温下靠近窗户有光处孵育4至8周。在孵育期间，每周至少观察一次Winogradsky培养柱的变化，记录不同颜色层的形成以及气泡的产生情况。此外，记录各色层形成所需的时间。另一种可进行的改进是将柱体置于暖气附近孵育以筛选嗜热细菌，或置于冰箱中孵育以筛选嗜冷细菌。通过将不同的柱体置于强光、弱光或黑暗条件下孵育，来改变光照条件。或者，可用不同颜色的玻璃纸覆盖柱体，以限制入射光的波长，从而确定哪些颜色有利于不同细菌类群的生长。在另一个可选实验中，若要富集铁氧化细菌，可在加入报纸和蛋黄之前，向泥水混合物中加入一枚铁钉。经过一到两周后，经典温诺格拉斯基柱中泥层表面出现绿色或红褐色的膜，表明蓝细菌层开始生长。随着时间推移，可观察到不同层次的形成与演变，每一层均指示着特定类型细菌的存在。当将置于黑暗条件下培养的温诺格拉斯基柱与传统的温诺格拉斯基柱进行比较时，可见黑暗处理组在柱体底部形成黑色层，表明存在硫酸盐还原细菌。深色柱层还可能产生其他层，具体取决于其他孵育条件。此外，深色柱层不会产生绿色的蓝细菌层，也不会产生分别指示紫色非硫细菌、紫色硫细菌和绿色硫细菌的红色、紫色或绿色层。这些微生物类群的生长依赖于光照。

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创建维诺格拉茨基柱:在沉积物样本中丰富微生物物种的方法

winogradsky column microbial layers and gradients

A Winogradsky column shows how microbial layers form in mud and water over time. It is a clear, enclosed ecosystem that helps scientists grow sediment microbes that are hard to culture in a test tube or on a petri dish. By keeping conditions close to a microbe’s natural habitat, the column makes it possible to study organisms that help drive Earth’s biogeochemical cycles.

The column is built from pond or wetland sediment mixed with water in a transparent container. Extra sources of carbon, usually cellulose, and sulfur are added, and the column is incubated in light. As microbes use these resources, they go through succession, which means one community replaces another as conditions change. This activity also creates gradients, or zones with changing chemical levels, especially for oxygen and sulfide.

At the top of the column, photosynthesizers grow first. These are mostly cyanobacteria, which make oxygen and often appear as a green or reddish-brown layer. Because oxygen is not very soluble in water, its concentration drops quickly below this zone. That creates an aerobic layer with oxygen at the top and an anaerobic layer with no oxygen below.

Deeper in the column, microbes that do not need oxygen can become active. At the bottom, organisms break down organic matter without oxygen, and fermentation of cellulose produces organic acids. Sulfate reducers then use these organics with sulfate and release sulfide. When iron reacts with sulfide, the sediment turns black and forms iron-sulfide minerals.

Sulfide also moves upward, so the middle of the column becomes a place where oxygen from above and sulfide from below meet. With enough light, sulfur-using photosynthesizers grow here. Green sulfur bacteria and purple sulfur bacteria often form green, purple, or purple-red layers. Green sulfur bacteria usually sit below purple sulfur bacteria because they tolerate more sulfide.

Above the purple sulfur bacteria, purple nonsulfur bacteria may form red, purple, orange, or brown layers. These microbes use organic acids as electron donors instead of sulfide. Higher up, nonphotosynthetic sulfur oxidizers can appear as white filaments. Bubbles may also form in the column. Oxygen bubbles in the aerobic layers come from cyanobacteria, while methane bubbles in the anaerobic layers are likely made by methanogens.

A mature Winogradsky column can show several microbial groups from top to bottom. Cyanobacteria are at the top, followed by nonphotosynthetic sulfur oxidizers, purple nonsulfur bacteria, purple sulfur bacteria, green sulfur bacteria, sulfate-reducing bacteria, and methanogens near the bottom. The visible colors, filaments, black sediment, and bubbles make the column a useful model for studying how microbes depend on one another in layered environments.

Winogradsky Column Microbial Layers and Gradients

winogradsky-column-microbial-layers-and-gradients

creating a winogradsky column: a method to enrich the microbial species in a sediment sample

A Winogradsky column shows how microbial layers form in mud and water over time. It is a clear, enclosed ecosystem that helps scientists grow sediment microbes that are hard to culture in a test tube or on a petri dish. By keeping conditions close to a microbe’s natural habitat, the column makes it possible to study organisms that help drive Earth’s biogeochemical cycles. The column is built from pond or wetland sediment mixed with water in a transparent container. Extra sources of carbon,...

serial dilutions and plating: microbial enumeration

serial-dilutions-and-plating-microbial-enumeration

Counting Bacteria with Dilution Plates

Counting bacteria with dilution plates helps microbiologists estimate how many cells are in a sample. Because bacteria can grow quickly and may be hard to count directly, serial dilution and plating make the concentration easier to measure. These methods have been used for more than a century to study bacterial and viral loads in clinical, industrial, pharmaceutical, and academic labs. Serial dilution reduces a sample in steps. A small volume of the original solution is moved into a fixed...

enrichment cultures: culturing aerobic and anaerobic microbes on selective and differential medias

enrichment-cultures-culturing-aerobic-anaerobic-microbes-on-selective

Selective Media for Bacterial Isolation

Selective media help scientists isolate bacteria by supporting some microbes and blocking others. This lab also uses differential media, which reveal a visible chemical trait in the organism being grown. Together, these methods help identify bacteria from mixed samples. Prokaryotic cells live in almost every environment on Earth. They also show wide metabolic diversity, so the growth medium must supply the molecules they need for energy. Some microbes are extremophiles, meaning they live in...

pure cultures and streak plating: isolation of single bacterial colonies from a mixed sample

pure-cultures-streak-plating-isolation-single-bacterial-colonies-from

Streak Plate Method for Isolating Bacteria

The streak plate method is used to isolate single bacterial colonies from a mixed sample. It helps separate one bacterial type from another so a pure culture can be grown. This is important in both clinical and research settings, where bacteria often grow together in mixed cultures. Pure cultures are needed for later genetic and protein studies. They are also used to check whether a sample is pure and to identify infectious agents from clinical samples. Because different strains can affect...

16s rrna sequencing: a pcr-based technique to identify bacterial species

16s-rrna-sequencing-pcr-based-technique-to-identify-bacterial

Bacterial ID with 16S rRNA Gene Sequencing

Bacterial identification with 16S rRNA gene sequencing helps scientists tell bacterial species apart. It is used in microbial ecology to study biodiversity in environmental samples and in medical microbiology to help diagnose infected patients. Traditional methods include microscopy, growth on specific media, biochemical and serological tests, and antibiotic sensitivity assays. Molecular methods have changed how bacteria are identified. 16S ribosomal RNA (rRNA) gene sequencing is often faster...

growth curves: generating growth curves using colony forming units and optical density measurements

growth-curves-generating-growth-curves-using-colony-forming-units

Bacterial Growth Stages and Cell Counts

Bacterial growth stages and cell counts show how a culture changes over time. This page follows a batch culture as it moves through lag, exponential, stationary, and death phases. It also shows how colony forming units, or CFU, and optical density can be used to measure that growth. Lag phase is the time bacteria need to adjust to a new environment before fast division begins. During exponential phase, cells grow and divide at the fastest rate, and DNA replication, RNA transcription, and...

antibiotic susceptibility testing: epsilometer tests to determine mic values of two antibiotics and evaluate antibiotic synergy

antibiotic-susceptibility-testing-epsilometer-tests-to-determine-mic

Measuring Bacterial Resistance with E-Tests

Measuring bacterial resistance with E-tests helps show how strongly an antibiotic can stop growth. The method is used to find the minimal inhibitory concentration, or MIC, which is the lowest antibiotic level that prevents visible bacterial growth. It is a practical way to study resistance in bacteria and compare how different antibiotics perform. The E-test uses a plastic strip that contains a built-in antibiotic gradient. The strip is placed on an agar plate that has been inoculated with...

microscopy and staining: gram, capsule, and endospore staining

microscopy-and-staining-gram-capsule-and-endospore-staining

Bacterial Stains for Cell Walls and Spores

Bacterial stains help scientists see cell walls, capsules, and endospores with a compound light microscope. Many bacteria are hard to tell apart with light alone because they are small, colorless, and can look similar under the lens. Staining makes these hidden features visible and helps identify different bacteria. A compound microscope uses more than one lens to magnify a sample. Its objective lens collects light near the specimen, and the eyepiece enlarges the image. Many compound...

plaque assay: a method to determine viral titer as plaque forming units (pfu)

plaque-assay-method-to-determine-viral-titer-as-plaque-forming-units

Phage Plaque Test for Counting Viruses

Bacteriophages, or phages, are viruses that infect prokaryotic cells. The plaque assay is a classic way to count these viruses by measuring viral titer, which is the amount of virus in a sample. It was first described in 1952 and is still one of the most reliable methods for determining viral titer. Phages were identified in the early 20th century by Twort and d'Hérelle. They have since been recognized for their therapeutic value and for their role in human and global ecosystems. Interest in...

transformation of e. coli cells using an adapted calcium chloride procedure

transformation-e-coli-cells-using-an-adapted-calcium-chloride

Calcium Chloride DNA Uptake in E. coli

Calcium chloride treatment can help E. coli take up foreign DNA. This chemical transformation method is used in molecular biology to move a plasmid into bacterial cells and then identify the cells that received it. The protocol described here uses chemically competent E. coli DH5α cells and the plasmid pUC19 to measure transformation efficiency. Bacteria can exchange genetic material through horizontal gene transfer. The three main routes are transformation, transduction, and conjugation.

conjugation: a method to transfer ampicillin resistance from donor to recipient e. coli

conjugation-method-to-transfer-ampicillin-resistance-from-donor-to

Bacterial Gene Transfer by Conjugation in E. coli

Bacterial conjugation is a form of horizontal gene transfer in E. coli that moves DNA from one cell to another through direct contact. It was first discovered by Lederberg and Tatum in 1946. Unlike transformation or transduction, conjugation is a natural, one-way transfer of DNA from a donor cell to a recipient cell. This process can increase bacterial genetic diversity. It is often described as a type of bacterial mating. It has also helped drive the spread of antibiotic-resistant bacteria.

phage transduction: a method to transfer ampicillin resistance from donor to recipient e. coli

phage-transduction-method-to-transfer-ampicillin-resistance-from

Using P1 Phage to Move Antibiotic Resistance in E. coli

Bacteriophage transduction moves DNA between bacteria by using a phage, or bacterial virus, as the carrier. In this process, a phage can deliver genetic material from one bacterium to another. The method is a form of horizontal gene transfer and is important in both bacterial evolution and laboratory genetics. Phages were first described in the early 1900s, and transduction was named in 1951 by Norton Zinder and Joshua Lederberg. The transcript describes two main phage classes. Lytic phages...

microbiology

该集合展示了微生物调查的关键工具，包括适当的无菌技术和电镀，如何使用选择性培养基和丰富样品，以及混合或纯样品的培养方法。此外，它着眼于识别微生物分离物的常见方法，以及细菌基因操作的程序。

Explore how a Winogradsky column forms microbial layers, oxygen and sulfide gradients, and visible signs of cyanobacteria, sulfur bacteria, and methanogens.

Winogradsky Column Microbial Layers and Gradients

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