这项工作由美国国立卫生研究院R21AG059117和密歇根大学Paul F. Glenn衰老生物学研究实验室资助。SB由T32AG000114资助。ESK由NSF DGE 1841052资助。

1.细菌接种
<ol><li>通过将 10 g 胰蛋白胨、5 g 酵母提取物和 10 g 氯化钠 （NaCl） 溶解在 950 mL 蒸馏水中来制备 Luria 肉汤 （LB）。</li>
	<li>通过加入5M氢氧化钠（NaOH）将LB的pH值调节至7.0。这应该只需要大约 0.2 mL 的 NaOH。</li>
	<li>在15psi下以液体循环高压灭菌pH调节的LB培养基45分钟。让溶液冷却并在室温下储存。</li>
	<li>在 500 mL 锥形瓶中接种 100 mL LB 中的单个菌落。将细菌在37°C振荡培养箱中培养过夜。</li>
	<li>根据细菌菌落的健康状况、烧瓶的大小和振荡器设置的速度，细菌生长所需的时间可能会有所不同。~14小时后，检查细菌在600nm（OD600）处的光密度（OD）。</li>
	<li>当OD600 为3.0（1×109 菌落形成单位（CFU）/ mL）时，从振荡器中除去细菌。如果 OD600 小于 3.0，则将烧瓶放回摇床培养箱，直到达到所需的 OD。</li>
	<li>将细菌分装在50mL锥形管中，并储存在4°C或进行下一步。</li>
</ol>2. 使用多聚甲醛
注意:使用的多聚甲醛 （PFA） 浓度和暴露时间可能因气候、位置和所治疗细菌的类型而略有不同。OP50 的良好起点是暴露于 0.5% PFA 1 小时，而 HT115 的 0.25% PFA 可能就足够了。
<ol><li>准备 32% PFA 原液或使用市售的 32% PFA 溶液。使用 PFA 时，请使用适当的个人防护设备 （PPE）。戴上手套和护目镜。</li>
	<li>在通风良好的化学通风橱内添加 PFA。将含有 PFA 的洗涤剂和吸头丢弃在通风橱中的适当化学危险容器中。</li>
</ol>3.用多聚甲醛进行细菌处理
<ol><li>一旦细菌达到 OD600 为 3.0，使用血清移液管将 50 mL 转移到新的 250 mL 锥形瓶中。将其余部分保存为活对照、模拟处理的对照（参见步骤 4），或根据需要用 PFA 处理。</li>
	<li>避免从一个烧瓶倒到另一个烧瓶，并注意不要用细菌溅到新烧瓶的侧面。烧瓶侧面的菌落可以接受较低剂量的PFA。</li>
	<li>在化学罩中，向 50 mL 细菌中加入 781 μL 32% PFA，使最终浓度达到 0.5%。处理固体废物化学危险容器中使用的吸头。</li>
	<li>用箔纸盖住烧瓶，然后返回37°C振荡器培养箱1小时。1小时后，从培养箱中取出烧瓶并继续进行步骤5。</li>
</ol>4.模拟处理对照
<ol><li>一旦细菌达到 OD600 为 3.0，使用血清移液管将 50 mL 细菌转移到 50 mL 锥形管中。</li>
	<li>继续执行步骤5.3以完成类似于PFA处理组的洗涤步骤。</li>
</ol>5.清洗细菌以去除残留的PFA
<ol><li>在化学罩中，使用血清移液管将处理过的细菌从锥形瓶转移到 50 mL 锥形管中。使用血清移液管代替倒入细菌将防止烧瓶边缘任何细菌菌落的污染，这些菌落可能避免了直接用PFA处理。</li>
	<li>将处理过的细菌以约3000× g 离心20分钟。将上清液丢弃在化学罩中的液体废物化学危害容器中，以去除上清液。</li>
	<li>加入 25 mL LB 并涡旋以重悬细菌沉淀（完全填充试管会使沉淀更难重悬）。重复离心和沉淀重悬 4 次。</li>
	<li>以适合不同测定的体积重悬沉淀。对于寿命测定，将装有 200 μL 细菌的 60 mm 培养板重新悬浮在 10 mL LB 中，将细菌重悬于 10 mL LB 中，可得到原始 50 mL 培养物的 5 倍浓度。</li>
	<li>将细菌储存在4°C。</li>
</ol>6. 细菌生长质量检查
<ol><li>在最终洗涤和重悬后，用准备好的细菌划出LB板（使用无菌移液器吸头）。最好将用于清洗和重悬的 LB 划在单独的板上，以确保使用的 LB 没有被污染。</li>
	<li>将板置于37°C培养箱中过夜。检查是否有任何增长。当菌落不在LB平板上生长时，细菌被认为是复制死亡的。</li>
</ol>7. 使用呼吸计进行细菌代谢质量检查
<ol><li>在步骤5.4的最终洗涤和重悬后，使用可用的工具（如呼吸计19,20）确认细菌代谢死亡并测量基础耗氧率（OCR）。</li>
	<li>制备M9溶液:将3g磷酸二氢钾（KH2PO 4），6g磷酸氢二钠（Na2HPO4）和5g氯化钠（NaCl）溶于950mL蒸馏水中。在15psi下在液体循环中高压灭菌45分钟，然后让溶液冷却至室温。加入 1 mL 的 1 M 硫酸镁 （MgSO4） 并在室温下储存。</li>
	<li>水合呼吸计墨盒:向 96 孔板的所有孔中加入 200 μL 校准品。将小柱置于96孔板中，并在37°C培养箱中孵育过夜。</li>
	<li>测定校准:第二天，将水合卡盒放入机器中并开始校准。</li>
	<li>测定测试板设置:使用新的 96 孔板，向测试孔中加入 160 μL M9 和 40 μL 制备的细菌 （1 x 109 CFU/mL）。向 4 个角孔中加入 200 μL M9 用作空白孔。加入 160 μL M9 和 40 μL LB 用于洗涤和重悬，用作阴性对照。将 200 μL M9 添加到其余不使用的孔中。</li>
	<li>运行检测:墨盒校准完成后，将步骤7.5中的检测板插入机器进行分析。这些设置包括混合、等待、测量和循环的步骤。结果将显示为耗氧率 （OCR）。当 OCR 为零时，细菌在代谢上死亡并准备使用。</li>
</ol><img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/65775/65775fig01.jpg"> 图 1.多聚甲醛处理的工作流程。大肠杆菌OP50细菌的单个菌落在一夜之间生长。将PFA加入至0.5%的终浓度，并将PFA处理的培养物在37°C下振荡1小时。 最后，通过用新鲜的LB 5x洗涤培养物来除去PFA。为了确认处理过的细菌是复制性无活性的，划出处理过的细菌的LB板并生长过夜。<a href="https://www.jove.com/files/ftp_upload/65775/65775fig01large.jpg" target="_blank">请点击这里查看此图的较大版本.</a>

使用多聚甲醛对 秀丽隐杆 线虫进行细菌代谢灭活

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作者没有什么可透露的。

相对于其他细菌杀灭方法，PFA杀灭的益处 PFA处理是一种高通量方法，可防止细菌代谢，同时保持秀丽隐杆线虫的营养食物来源。与其他方法相比，通过PFA处理杀死细菌具有多种优势。与紫外线处理不同，紫外线处理必须测试每个板才能成功杀灭，而一批 PFA 处理细菌中的单个板可以测试以验证第16 批。PFA处理在消除细菌代谢方面也非常有效（图2B），并且PFA处理的细菌相对于抗生素处理的细菌表现出较低的代谢活性19。PFA治疗的另一个好处是细菌保持其细胞结构和营养成分。 因此，蠕虫可以在 PFA 处理的细菌上发育，而从鸡蛋中喂养热杀灭的细菌会导致发育停滞13,16。
关键步骤和疑难解答 虽然PFA处理细菌是一个相对简单的方案，但重要的是要验证每批细菌是否具有复制性和代谢性死亡（步骤6和7），并确保PFA处理后的洗涤步骤彻底完成（步骤5）。如果菌落在步骤6中确实生长，表明细菌没有完全死亡，则可以在步骤3中对PFA的浓度和PFA处理的持续时间进行故障排除。在优化PFA治疗方案时，0.5%浓度的PFA是成功杀死OP50的理想选择，副作用最小。0.25% PFA 不足以代谢杀死所有细菌菌株（图 2C-D），并且将浓度增加到 0.5% PFA 不会改变 OP50 上蠕虫的发育时间、寿命或食物偏好相对于 0.25% PFA。此外，接种PFA的1小时是足以防止所有细菌生长的最短时间。如果用0.5%PFA接种1小时未完全杀死细菌，则可以增加接种时间和/或PFA浓度以解决杀灭效率问题。方案中的第二个关键步骤是在PFA处理后清洗细菌（步骤5）。完成所有五次洗涤并在每次洗涤时完全去除上清液以确保细菌中没有甲醛残留非常重要。如果蠕虫生长不良或没有泵送到 PFA 处理的细菌上，可以进行额外的清洗。
该方法的局限性 虽然PFA杀死的细菌是消除秀丽隐杆线虫研究中细菌代谢的混杂效应（图2B）的理想选择，但这种方法存在局限性。具体来说，与活细菌相比，秀丽隐杆线虫在PFA处理的细菌上的发育稍慢（图4A）16。如果存在FUdR（图4D），PFA处理也会延长蠕虫的寿命，但在NGM（图4C）板16上则不然。然而，其他方法也存在这些挑战;紫外线杀灭的细菌也会改变秀丽隐杆线虫的寿命。大多数报告表明，经过紫外线处理的细菌在 NGM12,23 和 FUdR 24 上延长了蠕虫的寿命，其他研究表明紫外线处理不会影响寿命 24，并且紫外线处理和 FUdR 之间存在相互作用，对秀丽隐杆线虫的寿命 25,26。除了PFA处理的细菌的寿命影响外，相对于PFA处理的细菌，蠕虫更喜欢花时间在活细菌上（图3C）16，但在PFA处理的食物上具有更高的泵送率（图3D）。这表明他们可能消耗相似数量的食物，或者PFA处理的细菌更容易或更难吃。其他杀死细菌的方法也观察到许多这些影响12,13,14，这表明代谢活跃的细菌缩短了发育时间，缩短了寿命，并且对秀丽隐杆线虫更具吸引力。
PFA处理细菌的潜在应用 总体而言，在 秀丽隐杆线虫 研究中，快速可靠地杀死大批量细菌的能力对于消除细菌代谢的混杂效应具有巨大的潜力。这种能力对于药物筛选、补充实验、毒性测定、代谢组学研究以及检查微生物代谢对宿主行为的影响特别有用。有关PFA处理细菌在每种应用中的使用的更多信息如下。
药物、补充剂和毒性实验:由于每批PFA处理的细菌只需测试一个平板即可杀灭成功，因此可以将PFA杀死的细菌添加到液体培养物中进行高通量药物筛选。PFA处理的细菌也可以接种到固体培养皿上，并将小分子或营养物质掺入琼脂培养基中。接种有PFA处理细菌的固体板也可用于毒性或应激反应测定。未发表的数据表明，PFA 处理的细菌有助于区分细菌应激反应途径和蠕虫应激抗性途径在百草枯和衣霉素耐药中的作用。在这些药物补充和毒性研究中使用PFA杀死的细菌可以防止细菌代谢目标分子，并确保观察到的任何表型都来自直接作用于 秀丽隐杆线虫 的化合物。使用代谢死亡的细菌进行药物和补充实验还可以最大限度地减少剂量的可变性，因为活细菌可以在不同实验之间代谢不同量的目标化合物。
代谢组学研究:秀丽隐杆线 虫代谢组学研究也可能受到细菌代谢的混淆:观察蠕虫代谢的变化需要没有细菌代谢活性29.即使是细菌食物来源的微小变化也会改变蠕虫的代谢组。例如，喂食活洗细菌的蠕虫的代谢组不同于喂食活的未洗细菌的蠕虫的代谢组16。与食用活食相比，食用 PFA 处理的细菌也会改变蠕虫代谢组16 ，但允许将实验条件对 秀丽隐杆线虫 代谢的影响与细菌代谢分开进行比较。
宿主-微生物行为研究:最后，PFA处理的细菌可用于鉴定细菌代谢与宿主行为之间的相互作用。致病菌和非致病菌分泌的代谢物对蠕虫具有吸引力或排斥性，从而影响其觅食和摄食行为10,11,30。此外，细菌可以产生神经调节剂，驱动运动30的变化。此外，在不同的细菌菌株上生长秀丽隐杆线虫会导致育雏大小、发育时间和生理机能的改变31,32。用PFA处理这些细菌可以帮助确定给定细菌对秀丽隐杆线虫的影响是否需要活跃的细菌代谢。

秀丽隐杆线 虫最初于 1965 年被提出为模式生物1 ，此后被广泛用于遗传学、发育、行为、衰老和代谢的研究2。由于其育雏尺寸大和角质层透明， 秀丽隐杆线 虫特别适合使用荧光报告基因进行高通量筛选3。它们的短生命周期、雌雄同体的繁殖以及与人类的遗传同源性也使 秀丽隐杆线虫 成为研究发育4 和衰老生物学5 的有价值的模型系统。此外， 秀丽隐杆线虫 相对容易维护。蠕虫可以在液体培养物或固体琼脂平板上生长，并食用活 的大肠杆菌 OP50 细菌4。
然而， 秀丽隐杆线虫 的活食来源可能会混淆新陈代谢、药物补充和行为的研究。由于活细菌有自己的新陈代谢，因此影响细菌的实验条件也会改变蠕虫可用的营养物质和代谢物。例如，细菌铁、氨基酸和叶酸浓度的差异对 秀丽隐杆线虫的 发育、生理和寿命有不同的影响6。许多常见的实验室实践可以引起OP50产生的营养成分和代谢物的这种变化。具体而言，暴露于5-氟-2'-脱氧尿苷（FUdR）（一种常用于防止 秀丽隐杆线虫繁殖的化合物）会引起OP50基因表达的广泛变化，包括氨基酸生物合成途径7。活细菌也可能混淆秀 丽隐杆线 虫补充小分子的研究，因为细菌可以部分或完全代谢活性化合物。此外，这些小分子对细菌的影响反过来可以改变 秀丽隐杆线虫 的生理机能，正如延长寿命的药物二甲双胍8所报道的那样。最后，活细菌可以通过改变行为的方式改变蠕虫的环境，例如分泌有吸引力的气味剂9，产生外源性神经调节剂10，以及在密集的细菌草坪中产生氧气梯度11。
为了减轻细菌代谢对 秀丽隐杆线虫 研究的混杂影响，已经开发了多种杀死细菌的方法（表1）。杀死OP50的三种常见策略是紫外线照射、热杀灭和抗生素治疗。虽然简单且成本相对较低，但这些方法中的每一种都可能对细菌和 秀丽隐杆线虫 产生不良影响。通过UV交联剂12 进行UV杀灭是低通量的，其速率受UV交联剂中可容纳的板数的限制。此外，在批次内，紫外线杀灭的功效可能因板而异，并且在大型实验中测试所有板的生长可能变得困难。通过将培养物暴露在 >60 °C 的温度下来热杀灭 OP50 会带来一系列单独的挑战。高温会破坏蠕虫所必需的营养物质并破坏细菌的细胞结构，从而产生更柔软的质地，从而减少蠕虫在食物上花费的时间13.这种方法也不能在 秀丽隐杆 线虫的整个生命周期中使用，因为喂食热杀灭细菌的蠕虫可以在发育早期停滞13。抗生素治疗是抑制细菌代谢的第三种常用方法14，但抗生素也可以改变蠕虫的生长和代谢15。
消除活细菌代谢效应同时保留细菌结构和必需营养素的一种解决方案是用多聚甲醛 （PFA） 杀死 OP5016。PFA 是一种甲醛聚合物，可以在细胞内交联蛋白质17，以防止细菌复制，而不会破坏内部细胞结构，如内质膜18。由于这种内部细胞结构的保留，PFA 处理的细菌没有表现出生长或代谢活动，但仍然是秀丽隐杆线虫16 的可食用和营养丰富的食物来源。 在这里，提供了一个详细的方案，显示了如何使用多聚甲醛代谢灭活细菌。
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td>方法</td>
			<td>所需材料</td>
			<td colspan="2">可 伸缩？</td>
			<td>营养？</td>
			<td colspan="4">对蠕虫的影响？</td>
		</tr><tr><td rowspan="4">紫外线</td>
			<td rowspan="4">UV交联剂</td>
			<td colspan="2">受限于:</td>
			<td rowspan="4">是的</td>
			<td colspan="4">对 NGM12、23、24 寿命的可变影响</td>
		</tr><tr><td colspan="2">适合UV交联剂的板数</td>
			<td colspan="4">对 FUdR24、26、27 寿命的可变影响</td>
		</tr><tr><td colspan="2">每块板的照射时间</td>
			<td colspan="4" rowspan="2">食物偏好降低16</td>
		</tr><tr><td colspan="2">能够检查每个板的生长情况8</td>
		</tr><tr><td rowspan="3">热</td>
			<td rowspan="3">>60°C培养箱</td>
			<td colspan="2" rowspan="3">是的</td>
			<td rowspan="3">否:破坏细胞壁，降低营养价值</td>
			<td colspan="4">发育停滞 13</td>
		</tr><tr><td colspan="4">食物偏好降低13</td>
		</tr><tr><td colspan="4">延长 NGM31 的使用寿命</td>
		</tr><tr><td rowspan="3">抗生素</td>
			<td rowspan="3">抗生素（卡那霉素、羧苄西林等）</td>
			<td colspan="2" rowspan="3">是的</td>
			<td rowspan="3">是的</td>
			<td colspan="4">延迟生长发育15</td>
		</tr><tr><td colspan="4">延长液体介质的使用寿命19</td>
		</tr><tr><td colspan="4">延长 NGM15 的使用寿命</td>
		</tr><tr><td rowspan="3">全氟辛烷磺酸</td>
			<td rowspan="3">0.5% 多聚甲醛</td>
			<td colspan="2" rowspan="3">是的</td>
			<td rowspan="3">是的</td>
			<td colspan="4">小育雏减少16</td>
		</tr><tr><td colspan="4">开发时间增加小16</td>
		</tr><tr><td colspan="4">食物偏好降低16</td>
		</tr></tbody></table>表 1.杀死OP50的方法比较。 紫外线杀灭、热杀灭、抗生素治疗和 PFA 治疗对细菌的营养状况和喂食处理细菌的蠕虫的健康有不同的影响。这些复制灭活 大肠杆菌 的方法在所需的材料和可扩展性方面也有所不同。

<table><tbody><tr><td>Aluminum Foil</td><td>Staples</td><td>2549291</td><td></td></tr><tr><td>Bunsen burner</td><td>VWR</td><td>470121-700&amp;nbsp;</td><td></td></tr><tr><td>Cell Density Meter</td><td>Denville</td><td>80-3000-45&amp;nbsp;</td><td></td></tr><tr><td>Centrifuge</td><td>Eppendorg</td><td>5430</td><td></td></tr><tr><td>Chemical fume hood</td><td>Labcono</td><td>975050411384RG</td><td></td></tr><tr><td>Conincal tubes (50 mL)</td><td>Fisher</td><td>339652</td><td></td></tr><tr><td>Cuvettes&amp;nbsp;</td><td>Fisher</td><td>14-955-127</td><td></td></tr><tr><td>E. coli OP50</td><td>CGC</td><td>OP50</td><td></td></tr><tr><td>Erlenmyer flasks</td><td>Fisher</td><td>250 mL: FB501250&lt;br/&gt; 500 mL: FB501500&lt;br/&gt; 1000 mL: FB5011000</td><td></td></tr><tr><td>Inoculation loop</td><td>Fisher</td><td>22-363-605</td><td></td></tr><tr><td>LB Agar</td><td>Fisher</td><td>BP1425500</td><td></td></tr><tr><td>Liquid waste collection bottle</td><td>Thomas Scientific</td><td>1230G50</td><td></td></tr><tr><td>Magnesium Sulfate (MgSO&lt;sub&gt;4&lt;/sub&gt;)</td><td>Sigma</td><td>M7506</td><td></td></tr><tr><td>Paraformaldehyde (32%)</td><td>Electron Microscopy Sciences</td><td>15714-S</td><td>Paraformaldehyde &amp;ndash; methanol free solution</td></tr><tr><td>Pipettor</td><td>Eppendorf</td><td>Eppendorf Easypet 3</td><td></td></tr><tr><td>Plastic dishes (100 mm)</td><td>Fisher</td><td>FB0875712</td><td></td></tr><tr><td>Potassium Phosphate Monobasic (KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;)</td><td>Fisher</td><td>P2853</td><td></td></tr><tr><td>Seahorse XF Calibrant</td><td>Agilent</td><td>100840-000</td><td></td></tr><tr><td>Seahorse XFe96 Extracellular Flux Assay Kit and Cell Culture Microplate</td><td>Agilent</td><td>101085-004</td><td></td></tr><tr><td>Serological pipettes (50 mL)</td><td>Genesee Scientific</td><td>12-107</td><td></td></tr><tr><td>Shaker incubator</td><td>Thermo</td><td>11 676 083</td><td></td></tr><tr><td>Sodium Chloride (NaCl)</td><td>Fisher</td><td>S640-3</td><td></td></tr><tr><td>Sodium Hydroxide (NaOH)</td><td>Fisher</td><td>S318500</td><td></td></tr><tr><td>Sodium Phosphate Dibasic Anhydrous (Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;)</td><td>Sigma</td><td>S374-500</td><td></td></tr><tr><td>Solid waste collection bucket</td><td>M&amp;amp;M Industries</td><td>&amp;nbsp;5.0 Gallon M1 Traditional Pail</td><td></td></tr><tr><td>Tryptone</td><td>Genesee Scientific</td><td>20-251</td><td></td></tr><tr><td>Vortex</td><td>Thermo</td><td>11676331</td><td></td></tr><tr><td>Weighing balance</td><td>C Goldenwall</td><td>HZ10K6B</td><td></td></tr><tr><td>Yeast Extract</td><td>Genesee Scientific</td><td>20-255</td><td></td></tr></tbody></table>

methodology to metabolically inactivate bacteria for caenorhabditis elegans research

秀丽隐杆线虫是一种常见的模式生物，用于遗传学、发育、衰老、代谢和行为研究。由于秀丽隐杆线虫以活细菌为食，因此其食物来源的代谢活动可能会混淆寻找各种干预措施对蠕虫的直接影响的实验。为了避免细菌代谢的混杂效应，秀丽隐杆线虫研究人员使用了多种方法来代谢灭活细菌，包括紫外线 （UV） 照射、热杀灭和抗生素。紫外线处理的通量相对较低，不能用于液体培养，因为必须检查每个培养板是否成功杀灭细菌。第二种治疗方法是热杀灭，会对细菌的质地和营养质量产生负面影响，导致秀丽隐杆线虫的发育停滞。最后，抗生素治疗除了防止细菌生长外，还可以直接改变秀丽隐杆线虫的生理机能。本手稿描述了一种使用多聚甲醛 （PFA） 代谢灭活细菌的替代方法。PFA处理使细菌细胞内的蛋白质交联，以防止代谢活动，同时保持细胞结构和营养成分。该方法通量高，可用于液体培养或固体培养板，因为测试一板 PFA 处理过的细菌的生长可以验证整个批次。通过PFA处理的代谢灭活可用于消除细菌代谢对药物或代谢物补充、应激抗性、代谢组学和秀丽隐杆线虫行为研究的混杂影响。

该协议的详细工作流程如图1所示。开发并优化了一种高通量方法，以一致地灭活细菌复制（图2A）和代谢（图2B），用于使用多聚甲醛16进行秀丽隐杆线虫研究的代谢和药物研究。目标是确定所需的最低PFA浓度和持续杀死细菌所需的最短时间，而不会影响蠕虫的各种健康措施。这些值可能因实验室环境而异，也因细菌菌株而异。例如，将HB101细菌暴露于0.25%1小时足以使其代谢失活（图2C），而粪肠球菌（EF）需要1.0%PFA浓度（图2D）才能保持一致的效果。先前已经建立了一种用于单个实验室优化的方法16，并在当前工作中进行了详细介绍。
食物吸引力和消费 随着细菌的生长和复制，它们会释放出各种对蠕虫有吸引力的代谢物和信息素。为了确定蠕虫是否仍然被 PFA 处理的 OP50 所吸引，用 PFA 处理或模拟处理的 OP50 播种平板，并将食物草坪上存在的蠕虫与草坪外存在的蠕虫百分比制成表格。数据显示，蠕虫被PFA处理的OP50吸引，因为大多数蠕虫在1小时后仍留在细菌草坪上（图3A），类似于模拟处理的对照（图3B）观察到的情况。然而，正如预期的那样，灵敏的成对测定21 表明，当在同一平板上接种 PFA 处理的活对照时， 秀丽隐杆线虫 对模拟处理的活对照（图 3C）有更强的偏好。在确定蠕虫被PFA杀死的细菌所吸引后，尽管比活细菌少，但必须确定它们吃PFA杀死的食物。为了确定蠕虫是否消耗PFA处理的OP50，测量了蠕虫在不同食物类型上的泵送速率。 如图 3D 所示，PFA 处理的 OP50 上的蠕虫比模拟处理的对照上的蠕虫具有更高的泵送率 （+25%）。这表明蠕虫并没有故意减慢对PFA杀死的食物的进食速度，并且可能表明进食速度较高，或者是对泵送处理食物的难易程度变化的补偿性反应。
繁殖力、发育和寿命 食物条件的变化会对蠕虫产生生理影响13,22。为了测试广泛的影响，测量了发育速度、繁殖力和寿命的变化。PFA 处理的 OP50 略微延迟野生型 N2 蠕虫的发育时间（~4 小时），如图 4A 所示。监测在不同细菌条件下生长的蠕虫的产卵能力显示，喂食PFA处理的OP50的蠕虫的繁殖力（-21%）略有但显着下降（图4B）。对于对秀丽隐杆线虫衰老感兴趣的实验室，然后确定PFA处理的OP50对寿命的影响。有趣的是，在标准线虫生长培养基（NGM）平板上喂食PFA处理的OP50的野生型蠕虫的寿命与喂食模拟处理的OP50的蠕虫没有显着差异（图4C），但是，当FUdR存在时，PFA处理的OP50增加了野生型寿命（+23%）（图4D）。与野生型蠕虫相比，将PFA处理的OP50喂食到长寿命的fmo-2过表达（OE）菌株20中不会改变其寿命（图4E）。紫外线、热和抗生素杀死的 OP50 也被证明可以改变秀丽隐杆线虫的寿命。具体来说，据报道，经过紫外线处理的细菌可以延长 NGM12,23 和 FUdR 24 的寿命。然而，关于 FUdR 和紫外线杀伤细菌25,26 的相互作用存在相互矛盾的数据，这可能是由于不同的紫外线杀伤方法和细菌中代谢不活跃的验证。此外，热灭菌可延长 NGM27 的寿命，抗生素可延长 NGM15 和液体培养物的寿命28。
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/65775/65775fig02.jpg"> 图2.用PFA治疗细菌可抑制增殖和代谢。 （A） 模拟处理和 0.5% PFA 处理的 OP50 的菌落形成单位 （CFU） 中的细菌生长。（B） 模拟处理和 0.5% PFA 处理的 OP50 的细胞外酸化率 （ECAR），单位为 mpH/min。（C） 模拟处理、0.25% PFA 处理和 0.5% PFA 处理 HB101 的基础耗氧率 （OCR），单位为 pmol/min。（D） 模拟处理、0.25% PFA 处理、0.5% PFA 处理和 1.0% PFA 处理粪肠球菌 （EF） 的基础耗氧率 （OCR），单位为 pmol/min。图中显示的所有误差线均表示平均值的标准误差（SEM）;采用双尾t检验推导p值。表示 p 值< 0.0001。这个数字是从 16修改过来的。 <a href="https://www.jove.com/files/ftp_upload/65775/65775fig02large.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/65775/65775fig03.jpg"> 图3.蠕虫被 PFA 处理的 OP50 吸引。 被吸引到 （A） PFA 处理或 （B） 模拟处理的 OP50 细菌草坪的蠕虫百分比。（C） 成对敏化测定测试蠕虫对模拟处理和 PFA 处理的 OP50 的偏好。（D） PFA 处理或模拟处理的 OP50 上蠕虫的泵送速率（每 30 秒泵）。使用双尾 t 检验分析来推导所有比较的 p 值。图中显示的所有误差线均表示平均值的标准误差（SEM）;表示 p 值< 0.0001。面板 A-C 已从 16 修改。 <a href="https://www.jove.com/files/ftp_upload/65775/65775fig03large.jpg" target="_blank">请点击这里查看此图的较大版本.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/65775/65775fig04.jpg"> 图4.蠕虫在 PFA 处理的 OP50 上生长和发育。 （A）在模拟处理和PFA处理的OP50上从卵到产卵成虫的蠕虫的发育时间（h）。（B） 喂食模拟处理或PFA处理的OP50的蠕虫的平均后代（蠕虫数量）。（C，D）在 （C） NGM 和 （D） FUdR 寿命板上喂食模拟处理或 PFA 处理的 OP50 的蠕虫的存活百分比。（E） 喂食经 PFA 处理的 OP50 的野生型和 fmo-2 OE 蠕虫的存活百分比。使用双尾 t 检验分析推导发育和繁殖力比较的 p 值。log-rank 检验用于推导寿命比较的 p 值。图中显示的所有误差线均表示平均值的标准误差（SEM）;* 表示 p 值 < 0.05，*** 表示 p 值 <0.001，**** 表示 p 值< 0.0001。图A、B和D从图16修改而来。 <a href="https://www.jove.com/files/ftp_upload/65775/65775fig04large.jpg" target="_blank">请点击这里查看此图的较大版本.</a>

Watch this Scientific Journal Video about Advancing Caenorhabditis elegans Research Using Paraformaldehyde-Treated Bacteria at JoVE.com

作者聚焦:使用多聚甲醛处理的细菌推进秀丽隐杆线虫研究 

实验室中 秀丽隐杆线虫 的食物来源是活 的大肠杆菌。由于细菌具有代谢活性，因此它们在 秀丽隐杆线虫 的代谢和药物研究中存在混杂变量。本文描述了使用多聚甲醛代谢灭活细菌的详细方案。

秀丽隐杆线虫研究的代谢灭活细菌的方法

Caenorhabditis elegans is a common model organism for research in genetics, development, aging, metabolism, and behavior. Because C. elegans consume a ...

海洋优雅研究的一个挑战是将活细菌饮食的影响与实验操作的影响隔离开来。很难确定操作是直接影响宿主还是通过宿主微生物组相互作用间接影响蠕虫。多聚甲醛处理的细菌解决了将细菌代谢的影响与宿主生理学隔离开来的挑战。多聚甲醛处理是高通量的，并始终如一地使细菌复制和代谢不活跃。除了一致性之外，PFA处理的细菌仍然是一种可口且营养丰富的食物来源。

methodology-to-metabolically-inactivate-bacteria-for-caenorhabditis

Nanyang Technological University

Research

JoVE Journal

Biology

Methodology to Metabolically Inactivate Bacteria for Caenorhabditis elegans Research

2.1K 次观看.  University of Michigan, Ann Arbor. 海洋优雅研究的一个挑战是将活细菌饮食的影响与实验操作的影响隔离开来。很难确定操作是直接影响宿主还是通过宿主微生物组相互作用间接影响蠕虫。多聚甲醛处理的细菌解决了将细菌代谢的影响与宿主生理学隔离开来的挑战。多聚甲醛处理是高通量的，并始终如一地使细菌复制和代谢不活跃。除了一致性之外，PFA处理的细菌仍然是一种可口且营养丰富的食物来源?...

视频: 作者聚焦:使用多聚甲醛处理的细菌推进秀丽隐杆线虫研究 

A Simple Method for High Throughput Chemical Screening in Caenorhabditis Elegans

Caenorhabditis elegans is a useful organism for testing chemical effects on physiology. Whole organism small molecule screens offer significant advantages for identifying biologically active chemical structures that can modify complex phenotypes such as lifespan. Described here is a simple protocol for producing hundreds of 96-well culture plates with fairly consistent numbers of C. elegans in each well. Next, we specified how to use these cultures to screen thousands of chemicals for effects on the lifespan of the nematode C. elegans. This protocol makes use of temperature sensitive sterile strains, agar plate conditions, and simple animal handling to facilitate the rapid and high throughput production of synchronized animal cultures for screening.

A Simple Method for High Throughput Chemical Screening in Caenorhabditis Elegans

Here we describe a simple protocol for rapidly producing hundreds of nematode growth media agar, 96-well culture plates with consistent numbers of Caenorhhabditis elegans per well. These cultures are useful for the phenotypic screening of whole organisms. We focus here on using these cultures to screen chemicals for pro-longevity effects.

一种简单的高通量化学筛选方法在秀丽线虫中的应用

Caenorhabditis elegans is a useful organism for testing chemical effects on physiology. Whole organism small molecule screens offer significant advantages ...

科研

生物化学

Plate-based Large-scale Cultivation of Caenorhabditis elegans: Sample Preparation for the Study of Metabolic Alterations in Diabetes

Culturing Caenorhabditis elegans (C. elegans) in a large-scale manner on agar plates can be time-consuming and difficult. This protocol describes a simple and inexpensive method to obtain a large number of animals for the isolation of proteins to proceed with a western blot, mass spectrometry, or further proteomics analyses. Furthermore, an increase of nematode numbers for immunostainings and the integration of multiple analyses under the same culturing conditions can easily be achieved. Additionally, a transfer between plates with different experimental conditions is facilitated. Common techniques in plate culture involve the transfer of a single C. elegans using a platinum wire and the transfer of populated agar chunks using a scalpel. However, with increasing nematode numbers, these techniques become overly time-consuming. This protocol describes the large-scale culture of C. elegans including numerous steps to minimize the impact of the sample preparation on the physiology of the worm. Fluid and shear stress can alter the lifespan of and metabolic processes in C. elegans, thus requiring a detailed description of the critical steps in order to retrieve reliable and reproducible results. C. elegans is a model organism, consisting of neuronal cells for up to one-third, but lacking blood vessels, thus providing the possibility to investigate solely neuronal alterations independent of vascular control. Recently, early neurodegeneration in diabetic retinopathy was found prior to vascular alterations. Thus, C. elegans is of special interest for studying general mechanisms of diabetic complications. For example, an increased formation of advanced glycation end products (AGEs) and reactive oxygen species (ROS) is observed, which are reproducibly found in C. elegans. Protocols to handle samples of adequate size for a broader spectrum of investigations are presented here, exemplified by the study of diabetes-induced biochemical alterations. In general, this protocol can be useful for studies requiring large C. elegans numbers and in which liquid culture is not suitable.

Plate-based Large-scale Cultivation of Caenorhabditis elegans: Sample Preparation for the Study of Metabolic Alterations in Diabetes

This protocol describes a method for the large-scale cultivation of Caenorhabditis elegans on solid media. As an alternative to liquid culture, this protocol allows obtaining parameters of different scales under plate-based cultivation. This increases the comparability of results by omitting the morphological and metabolic differences between liquid and solid media culture.

基于板块的秀丽线虫规模化栽培: 糖尿病代谢变化研究的样品制备

Culturing Caenorhabditis elegans (C. elegans) in a large-scale manner on agar plates can be time-consuming and difficult. This protocol describes a simple ...

Using Caenorhabditis elegans for Studying Trans- and Multi-Generational Effects of Toxicants

Information about toxicities of chemicals are essential in their application and waste management. For chemicals at low concentrations, the long-term effects are very important in judging their consequences in the environment and on human health. In demonstrating long-term influences, effects of chemicals over generations in recent studies provide new insight. Here, we describe protocols for studying effects of chemicals over multiple generations using free-living nematode Caenorhabditis elegans. Two aspects are presented: (1) trans-generational (TG) and (2) multi-generational effect studies, the latter of which is separated to multi-generational exposure (MGE) and multi-generational residual (MGR) effect studies. The TG effect study is robust with a simple purpose to determine whether chemical exposure to parents can result in any residual consequences on offspring. After the effects are measured on parents, sodium hypochlorite solutions are used to kill the parents and keep the offspring so as to facilitate effect measurement on the offspring. The TG effect study is used to determine whether the offspring are affected when their parent is exposed to the pollutants. The MGE and MGR effect study is systematical used to determine whether continuous generational exposure can result in adaptive responses in offspring over generations. Careful pick-up and transfer are used to distinguish generations to facilitate effect measurement on each generation. We also combined protocols to measure locomotion behavior, reproduction, lifespan, biochemical and gene expression changes. Some example experiments are also presented to illustrate the trans- and multi-generational effect studies.

Using Caenorhabditis elegans for Studying Trans- and Multi-Generational Effects of Toxicants

Trans- and multi-generational effects of persistent chemicals are essential in judging their long-term consequences in the environment and on the human health. We provide novel detailed methods for studying trans- and multi-generational effects using free-living nematode Caenorhabditis elegans.

使用卡内哈布迪炎酶研究有毒物质的跨代和多代效应

Information about toxicities of chemicals are essential in their application and waste management. For chemicals at low concentrations, the long-term ...

环境科学

Studying Oxidative Stress Caused by the Mitis Group Streptococci in Caenorhabditis elegans

Caenorhabditis elegans (C. elegans), a free-living nematode, has emerged as an attractive model to study host-pathogen interactions. The presented protocol uses this model to determine the pathogenicity caused by the mitis group streptococci via the production of H2O2. The mitis group streptococci are an emerging threat that cause many human diseases such as bacteremia, endocarditis, and orbital cellulitis. Described here is a protocol to determine the survival of these worms in response to H2O2 produced by this group of pathogens. Using the gene skn-1 encoding for an oxidative stress response transcription factor, it is shown that this model is important for identifying host genes that are essential against streptococcal infection. Furthermore, it is shown that activation of the oxidative stress response can be monitored in the presence of these pathogens using a transgenic reporter worm strain, in which SKN-1 is fused to green fluorescent protein (GFP). These assays provide the opportunity to study the oxidative stress response to H2O2 derived by a biological source as opposed to exogenously added reactive oxygen species (ROS) sources.

Studying Oxidative Stress Caused by the Mitis Group Streptococci in Caenorhabditis elegans

The nematode Caenorhabditis elegans is an excellent model to dissect host-pathogen interactions. Described here is a protocol to infect the worm with members of the mitis group streptococci and determine activation of the oxidative stress response against H2O2 produced by this group of organisms.

线虫中耳炎群链球菌引起氧化应激的研究

Caenorhabditis elegans (C. elegans), a free-living nematode, has emerged as an attractive model to study host-pathogen interactions. The presented ...

发育生物学

测量 秀丽隐杆 线虫食物摄入量和寿命的高通量测定

Quantifying Food Intake in Caenorhabditis elegans by Measuring Bacterial Clearance

Feeding is an essential biological process for an organism&#39;s growth, reproduction, and survival. This assay aims to measure the food intake of Caenorhabditis elegans&#160;(C. elegans), an important parameter when studying the genetics of aging or metabolism. In most species, feeding is determined by measuring the difference between the amount of food provided and the amount left after a given time interval. The method presented here uses the same strategy to determine the feeding of C. elegans. It measures the amount of bacteria, the food source of C. elegans, cleared within 72 h. This method uses 96-well microtiter plates and has allowed the screening of hundreds of drugs for their ability to modulate food intake at a speed and depth not possible in other animal models. The strength of this assay is that it allows to measure feeding and lifespan simultaneously and directly measures the disappearance of food and, thus, is based on the same principles used for other organisms, facilitating species-to-species comparison.

作者聚焦：探索脂毒性与 HFpEF 之间的关系 

This protocol describes an assay to quantify Caenorhabditis elegans feeding rate based on measuring the clearance of bacteria in liquid culture.

通过测量细菌清除率来量化 秀丽隐杆线虫 的食物摄入量

Feeding is an essential biological process for an organism&#39;s growth, reproduction, and survival. This assay aims to measure the food intake of ...

行为学

Selective Cleaning of Wild Caenorhabditis Nematodes to Enrich for Intestinal Microbiome Bacteria

Caenorhabditis elegans (C. elegans)&#160;has proven to be an excellent model for studying host-microbe interactions and the microbiome, especially in the context of the intestines. Recently, ecological sampling of wild Caenorhabditis nematodes has discovered a diverse array of associated microbes, including bacteria, viruses, fungi, and microsporidia. Many of these microbes have interesting colonization or infection phenotypes that warrant further study, but they are often unculturable. This protocol presents a method to enrich the desired intestinal microbes in C. elegans and related nematodes and reduce the presence of the many contaminating microbes adhering to the cuticle. This protocol involves forcing animals into the dauer stage of development and using a series of antibiotic and detergent washes to remove external contamination. As dauer animals have physiological changes that protect nematodes from harsh environmental conditions, any intestinal microbes will be protected from these conditions. But, for enrichment to work, the microbe of interest must be maintained when animals develop into dauers. When the animals leave the dauer stage, they are singly propagated into individual lines. F1 populations are then selected for desired microbes or infection phenotypes and against visible contamination. These methods will allow researchers to enrich unculturable microbes in the intestinal lumen, which make up part of the natural microbiome of C. elegans and intracellular intestinal pathogens. These microbes can then be studied for colonization or infection phenotypes and their effects on the host fitness.

Selective Cleaning of Wild Caenorhabditis Nematodes to Enrich for Intestinal Microbiome Bacteria

Wild Caenorhabditis nematodes are associated with many microbes, often in the gut lumen or infecting the intestine. This protocol details a method to enrich unculturable microbes colonizing the intestine, taking advantage of the resistance of the dauer cuticle.

选择性清洁野生 卡诺菌线 虫以富集肠道微生物组细菌

Caenorhabditis elegans (C. elegans)&#160;has proven to be an excellent model for studying host-microbe interactions and the microbiome, especially in the ...

免疫与感染

Studying Inherited Immunity in a Caenorhabditis elegans Model of Microsporidia Infection

Inherited immunity describes how some animals can pass on the "memory" of a previous infection to their offspring. This can boost pathogen resistance in their progeny and promote survival. While inherited immunity has been reported in many invertebrates, the mechanisms underlying this epigenetic phenomenon are largely unknown. The infection of Caenorhabditis elegans by the natural microsporidian pathogen Nematocida parisii results in the worms producing offspring that are robustly resistant to microsporidia. The present protocol describes the study of intergenerational immunity in the simple and genetically tractable N. parisii -C. elegans infection model. The current article describes methods for infecting C. elegans and generating immune-primed offspring. Methods are also given for assaying resistance to microsporidia infection by staining for microsporidia and visualizing infection by microscopy. In particular, inherited immunity prevents host cell invasion by microsporidia, and fluorescence in situ hybridization (FISH) can be used to quantify invasion events. The relative amount of microsporidia spores produced in the immune-primed offspring can be quantified by staining the spores with a chitin-binding dye. To date, these methods have shed light on the kinetics and pathogen specificity of inherited immunity, as well as the molecular mechanisms underlying it. These techniques, alongside the extensive tools available for C. elegans research, will enable important discoveries in the field of inherited immunity.

Studying Inherited Immunity in a Caenorhabditis elegans Model of Microsporidia Infection

The infection of Caenorhabditis elegans by the microsporidian parasite Nematocida parisii enables the worms to produce offspring that are highly resistant to the same pathogen. This is an example of inherited immunity, a poorly understood epigenetic phenomenon. The present protocol describes the study of inherited immunity in a genetically tractable worm&#160;model.

秀 丽隐杆线虫 感染模型中的遗传免疫研究

Inherited immunity describes how some animals can pass on the "memory" of a previous infection to their offspring. This can boost pathogen resistance in ...

Culturing Caenorhabditis elegans in Axenic Liquid Media and Creation of Transgenic Worms by Microparticle Bombardment

In this protocol, we present the required materials, and the procedure for making modified&#160;C. elegans&#160;Habituation and Reproduction media (mCeHR). Additionally, the steps for exposing and acclimatizing C. elegans grown on E. coli to axenic liquid media are described. Finally, downstream experiments that utilize axenic C. elegans illustrate the benefits of this procedure. The ability to analyze and determine C. elegans&#160;nutrient requirement was illustrated by growing N2 wild type worms in axenic liquid media with varying heme concentrations. This procedure can be replicated with other nutrients to determine the optimal concentration for worm growth and development or, to determine the toxicological effects of drug treatments. The effects of varied heme concentrations on the growth of wild type worms were determined through qualitative microscopic observation and by quantitating the number of worms that grew in each heme concentration. In addition, the effect of varied nutrient concentrations can be assayed by utilizing worms that express fluorescent sensors that respond to changes in the nutrient of interest. Furthermore, a large number of worms were easily produced for the generation of transgenic C. elegans using microparticle bombardment.

Culturing Caenorhabditis elegans in Axenic Liquid Media and Creation of Transgenic Worms by Microparticle Bombardment

C. elegans is usually grown on solid agar plates or in liquid cultures seeded with E. coli. To prevent bacterial byproducts from confounding toxicological and nutritional studies, we utilized an axenic liquid medium, CeHR, to grow and synchronize a large number of worms for a range of downstream applications.

秀丽隐杆线虫研究的代谢灭活细菌的方法

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一种简单的高通量化学筛选方法在秀丽线虫中的应用

基于板块的秀丽线虫规模化栽培: 糖尿病代谢变化研究的样品制备

使用卡内哈布迪炎酶研究有毒物质的跨代和多代效应

线虫中耳炎群链球菌引起氧化应激的研究

通过测量细菌清除率来量化秀丽隐杆线虫的食物摄入量

通过测量细菌清除率来量化秀丽隐杆线虫的食物摄入量

通过测量细菌清除率来量化秀丽隐杆线虫的食物摄入量

选择性清洁野生卡诺菌线虫以富集肠道微生物组细菌

秀丽隐杆线虫感染模型中的遗传免疫研究

培养

秀丽隐杆线虫研究的代谢灭活细菌的方法

副本

摘要

探索更多视频

此视频中的章节

一种简单的高通量化学筛选方法在秀丽线虫中的应用

基于板块的秀丽线虫规模化栽培: 糖尿病代谢变化研究的样品制备

使用卡内哈布迪炎酶研究有毒物质的跨代和多代效应

线虫中耳炎群链球菌引起氧化应激的研究

通过测量细菌清除率来量化 秀丽隐杆线虫 的食物摄入量

通过测量细菌清除率来量化 秀丽隐杆线虫 的食物摄入量

通过测量细菌清除率来量化 秀丽隐杆线虫 的食物摄入量

选择性清洁野生 卡诺菌线 虫以富集肠道微生物组细菌

秀 丽隐杆线虫 感染模型中的遗传免疫研究

培养

通过测量细菌清除率来量化秀丽隐杆线虫的食物摄入量

通过测量细菌清除率来量化秀丽隐杆线虫的食物摄入量

通过测量细菌清除率来量化秀丽隐杆线虫的食物摄入量

选择性清洁野生卡诺菌线虫以富集肠道微生物组细菌

秀丽隐杆线虫感染模型中的遗传免疫研究