Name: rearing axenic delia antiqua with half-fermented sterile diets
Uploaded: 2023-12-22T14:55:00.000+00:00
Duration: 5 min 58 s
Description: Watch this Scientific Journal Video about Axenic Rearing of Delia antiqua to Detect the Impact of Intestinal Microbes on Its Growth and Development at JoVE.com

本研究得到了国家自然科学基金（32272530）、济南大学新二十政策项目（2021GXRC040）、山东省重大科技创新项目（2021TZXD002）和齐鲁工业大学科教融合项目（2022PYI009、2022PY016、2022PT105）的支持。

D. antiqua 产自泰安市樊镇田间。
1.无菌饮食的制备
<ol><li>剥掉葱的外层，丢弃绿叶。保留葱的白色部分（图1A）并用无菌水清洗，重复漂洗过程三次。用剪刀将葱的白色部分切成1-2厘米的丁块（见 材料表），用75%EtOH溶液（在步骤2.5中提到）灭菌，以备后用。</li>
	<li>称取 50 克葱丁并将它们放入食品加工机中（见 材料表）。加入 50 mL 灭菌水，研磨五次，每次 2 分钟。所有切成丁的葱都应磨成糊状稠度（图2A）。</li>
	<li>用镊子将10片3龄幼虫转移到含有10mL 1x PBS的10mL离心管中，并用一次性研磨杵研磨约5分钟，得到幼虫研磨液。</li>
	<li>将糊状葱液和幼虫研磨液倒入干净的 500 mL 锥形瓶中（参见 材料表）。用两层透明密封膜包裹锥形瓶口，然后将烧瓶置于振荡培养箱（参见 材料表）中，在25°C和180rpm下发酵12小时（图2B）。</li>
	<li>用剪刀剪下七层边长为10厘米的方形纱布（无需无菌），并将它们堆叠在一起，形成一个简单的过滤装置。将发酵的葱液慢慢倒在纱布上，然后将过滤后的液体挤出到新的500mL锥形瓶中。将剩余的葱渣用锡箔纸包裹在纱布上以备后用。</li>
	<li>使用真空泵（参见 材料表）和布氏漏斗过滤上一步中获得的滤液。将去离子水润湿的滤纸放在布氏漏斗上，然后倒入滤液，用不同的滤纸进行三次抽滤。将最终滤液收集在 500 mL 锥形瓶中。</li>
	<li>使用0.22μM滤瓶和真空泵进行另一次过滤。在超洁净工作台中拧紧抽吸过滤瓶的盖子（参见 材料表）以备进一步使用。此时，得到发酵的葱渣和滤液（图2C）。 注意:从此步骤获得的过滤液体将是无菌的。（0.22μM滤瓶是用于对滤液进行灭菌的无菌真空过滤系统）。</li>
	<li>重复上述步骤以获得未发酵的葱渣和滤液。这里的区别在于不需要发酵。</li>
	<li>在 10 mL 离心管中称取 0.24 g 氯化胆碱和 0.56 g L-抗坏血酸（参见 材料表）。加入 3 mL 去离子水，剧烈摇晃直至完全溶解。在超洁净工作台中，使用新的 5 mL 注射器并连接三个 0.22 μM 注射器过滤器（参见 材料表）对溶液进行灭菌，并将其放入无菌的 10 mL 离心管中以备后用。 注:氯化胆碱是昆虫生长的必需神经递质，而L-抗坏血酸则用作防腐剂。</li>
	<li>称取 2 g 琼脂粉末和 6 g TSB（参见 材料表），然后将它们倒入 500 mL 锥形瓶中。然后加入 100 mL 去离子水制备培养基。用去离子水冲洗玻璃棒后，用它来搅拌混合物，直到它充分混合。然后，用两层透明密封膜密封锥形瓶。</li>
	<li>将发酵的葱和未发酵的葱（在步骤1.5和步骤1.8中提到）包裹在铝箔中，并将它们与培养基一起在121°C的高压灭菌器中灭菌20分钟。</li>
	<li>在超净工作台中，将灭菌的氯化胆碱、L-抗坏血酸和无菌滤液倒入培养基中，同时轻轻旋转以充分混合。最后，加入发酵和未发酵的葱，用手剧烈摇晃以获得无菌饮食。将日粮以一定角度倒入50mL离心管（无菌;带有0.22μM PVDF疏水膜的排气盖）（参见 材料表）（图2D）。 注意:灭菌后，培养基的温度应保持在65-80°C之间，以防止在添加葱或其他成分时快速冷却和凝固。此外，在每个试管中，倒入大约 10-15 毫升的饮食。一旦饮食凝固，如果不立即使用，请将试管存放在4°C的冰箱中。</li>
</ol>2. 获取斧头卵
<ol><li>D. antiqua的实验室饲养系统
	<ol><li>将饲养笼置于25°C内部LED照明的孵化器中（24小时一个周期，明暗比例为16:8）以饲养雄性和雌性成虫，使它们交配并产卵。将饮水机（图1B）装满水，并用湿棉绒密封，为成年人提供水。</li>
		<li>将四个 35 mm x 12 mm 培养皿底部放入 94 mm x 16 mm 培养皿盖中。用离子水润湿棉絮，然后将湿棉皿放在两个培养皿底部，在棉絮上加入一汤匙蔗糖。</li>
		<li>用两汤匙蔗糖和酵母提取物填充另外两个培养皿底部（见 材料表），它们作为成虫的食物（图1C）。 注意:步骤2.1.2和步骤2.1.3中提到的蔗糖和酵母提取物不需要灭菌。</li>
		<li>将一个94毫米×16毫米的培养皿底部装有湿润的沙子和一块未生根的去皮蒜瓣（图1D）放入苍蝇笼内以收集卵。雌性会被大蒜的气味所吸引，并在其周围产卵。</li>
	</ol></li>
	<li>鸡蛋的收集<ol><li>从笼子中取出产卵装置，这是一个 94 mm x 16 mm 的培养皿，里面装有上面提到的沙子和大蒜（步骤 2.14）。将沙子和大蒜一起倒入 500 mL 烧杯中，然后用自来水将大蒜中剩余的鸡蛋冲洗到烧杯中。此时，鸡蛋漂浮在水中。</li>
		<li>取一个100目筛子（见 材料表），用自来水润湿。然后将含有漂浮鸡蛋的水从烧杯倒入筛子上。鸡蛋将留在筛子上。</li>
		<li>鸡蛋在筛子上自然干燥后，用刷子轻轻地将鸡蛋扫到培养皿上。</li>
	</ol></li>
	<li>第一天，冲洗产卵装置以收集卵子，而无需消毒或灭菌。第二天下午四点，再次冲洗鸡蛋，用清水过筛收集。 注意:在第二天收集鸡蛋是为了确保后续阶段的生长速度一致。下午四点是 D. antiqua 的产卵高峰期。因此，此时收集鸡蛋是理想的选择。</li>
	<li>将收集的卵放在超洁净工作台（图3A）中的无菌细胞筛（参见材料表）上。</li>
	<li>准备鸡蛋灭菌溶液<ol><li>取 5 mL 5.2% NaClO（参见 材料表）并将其转移到灭菌的 250 mL 锥形瓶中。加入 95 mL 灭菌水，使总体积为 100 mL，得到 0.26% NaClO 溶液。</li>
		<li>要制备 75% EtOH 溶液，取 75 mL 99.7% EtOH 并将其转移到灭菌的 250 mL 锥形瓶中。加入 25 mL 灭菌水，使总体积为 100 mL，得到 75% 的 EtOH 溶液。</li>
	</ol></li>
	<li>将 30 mL NaClO 倒入培养皿中，将 30 mL EtOH 倒入另一个培养皿中。将含有步骤2.4中提到的鸡蛋的细胞筛放入含有NaClO的培养皿中。将其浸泡在0.26%NaClO溶液中0.5分钟。然后将筛子转移到含有EtOH的培养皿中，使其在75%EtOH溶液中再浸泡0.5分钟。</li>
	<li>重复此过程三次，每0.5分钟在0.26%NaClO和75%EtOH之间交替。之后，将获得斧头蛋。此时，应将装有卵的筛子浸没在EtOH中。 注意:为确保彻底灭菌，请使用 1 mL 移液管吸出 NaClO 和 EtOH 并分散鸡蛋。多次重复冲洗过程。此步骤将进一步清洁鸡蛋表面的残留细菌和杂质，确保表面清洁无菌。</li>
</ol>3. 幼虫的饲养
<ol><li>要转移灭菌的卵子，请使用一把用酒精灯灭菌的剪刀切割1mL移液管的端尖（图3B）。确保切开的移液器的端部开口大于鸡蛋的直径（约2毫米）。然后，使用移液管将鸡蛋从EtOH溶液（鸡蛋与溶液一起）（图3C）转移到含有无菌饮食的离心管中，每个管应包含约20-50个鸡蛋（图3D）。重复此过程，直到所有卵子都转移完毕。 注意:移液器吸头的直径应尽可能大，以避免在转移过程中破坏卵子，这可能导致它们死亡。</li>
	<li>使用移液管从试管中除去多余的乙醇。将带盖的离心管放入自密封袋内，并在袋子的开口处放一块棉花，以允许空气流通。最后，将袋子放入25°C培养箱（相对湿度:50%-70%;光周期:16小时光照:8小时黑暗）中进行观察和培养。</li>
	<li>大约三天后，日粮中的斧头卵开始孵化（图4A）。孵化的幼虫将活跃，无菌饮食的表面将不再光滑。幼虫将开始以饮食为食。</li>
	<li>继续观察幼虫，直到它们达到2龄幼虫的阶段。在此期间，请继续关注他们的成长和发展。</li>
	<li>9-12天后，超过80%的卵发育成3龄幼虫（图4B）。这表明幼虫成功生长和发育。</li>
</ol>4. 用培养依赖性测定法验证无轴幼虫
<ol><li>从离心管中随机选择3只3龄 幼虫。</li>
	<li>将幼虫放入含有75%酒精的94mm x 16mm培养皿中进行清洁。之后，将它们转移到装有无菌水的容器中进行进一步冲洗。</li>
	<li>使用已用75%EtOH溶液消毒的消毒解剖钳抓住幼虫（头部和尾部如 图5A所示）。使用解剖剪刀去除头端和尾端（图5B）。用镊子握住幼虫的尾巴，用另一把镊子从尾巴轻轻挤压到头部，取出内脏（结果如 图5C所示）。将内脏置于去离子水中，用镊子按压脂肪组织轻轻拉出肠，并将提取的内脏（图5D）置于无菌水中以备进一步使用。</li>
	<li>将肠放入无菌的1.5mL离心管中，加入500μL灭菌的1x PBS缓冲液，并使用一次性研杵将肠组织研磨成匀浆。</li>
	<li>取 100 μL 匀浆并将其加入营养琼脂培养基中。使用L形吊具（见 材料表）对琼脂平板进行划线。</li>
	<li>将培养皿置于28°C的生化培养箱中，孵育72小时以观察菌落的生长。</li>
</ol>5. 使用培养非依赖性测定法验证轴幼虫
<ol><li>按照制造商的说明，使用DNA提取试剂盒（参见 材料表）从轴性幼虫的肠道组织中提取总DNA。</li>
	<li>使用紫外分光光度计测量DNA浓度。</li>
	<li>使用通用细菌16S rRNA引物（1492R:5'-GGTTACCTTGTTACGACTT -3'，27F:5'-AGAGTTTGATCATGGCTCAG -3'）进行PCR反应。总反应体积为 25 μL，由 1 μL DNA 模板、0.5 μL 正向引物、0.5 μL 反向引物、10.5 μL ddH2O 和 12.5 μL 2x Taq PCR 预混液组成（参见 材料表）。设置PCR反应条件如下:在94°C下初始变性3分钟;在94°C下变性30秒，在55°C下退火30秒，在72°C下延伸90秒;在72°C下最终延伸10分钟。将PCR产物储存在4°C进行进一步分析。</li>
	<li>使用通用真菌ITS引物（ITS1:5'-TCCGTAGGTGAACCTGCGG-3'，ITS4:5'-TCCTCCGCTTATTGATATGC -3'）进行PCR反应。总反应体积为 25 μL，由 1 μL DNA 模板、0.5 μL 正向引物、0.5 μL 反向引物、10.5 μL ddH2O 和 12.5 μL 2x Taq PCR 预混液组成。将PCR条件设定在95°C5分钟;95°C30秒，55°C1分钟，72°C90秒35次循环;然后在72°C下10分钟。将PCR产物储存在4°C进行进一步分析。</li>
	<li>使用Super Green核酸染料（见 材料表）与两种类型的PCR产物中的每一种均匀混合，并通过在1x TAE缓冲液中的1%琼脂糖凝胶上电泳进行分析（从50x TAE稀释，见材料表）。使用3μL DNA标记物（参见 材料表）作为参考。</li>
	<li>使用紫外透射仪观察凝胶并定位目标片段。</li>
</ol>

研究 Axenic Delia antiqua 的开发并确保无细菌生长

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作者没有需要披露的利益冲突。

昆虫具有高度复杂的肠道微生物群20,21，因此需要使用接种特定肠道微生物菌株的昆虫来研究昆虫与微生物的相互作用。昆虫的制备对于此类研究工作至关重要。抗生素治疗是一种用于消除肠道微生物群的方法。例如，Jung 和Kim 22 用青霉素喂养草地贪夜蛾，而Raymond 23 用含有利福平的人工饮食喂养木叶假单胞菌以清除肠道细菌。然而，抗生素治疗不能完全消除肠道细菌，并且对宿主昆虫有许多副作用。这些包括代谢酶活性的变化、肠道功能受损、生长、发育和繁殖的抑制，以及死亡率的增加 23,24,25。因此，在抗生素治疗后，昆虫肠道微生物群的功能可能会被掩盖。幸运的是，这个问题可以通过对卵的表面进行化学消毒来克服，从而相对容易地产生无虫16。相比之下，对鸡蛋进行表面消毒，然后用无菌饮食喂养的组合比抗生素治疗更有效和可行26,27。
获得 axenic D. antiqua 主要依赖于利用 axenic 卵和无菌饮食。与其他昆虫物种相比， D. antiqua 的卵相对较小。以前采用的消毒方法，例如将卵短暂浸泡在0.1%十二烷基苯磺酸钠中，然后用2%过氧化氢溶液处理5分钟，得到蟑螂卵28，或使用10%次氯酸钠溶液10分钟，对红棕榈象鼻虫29的卵进行表面消毒，都不适合对 赤蟑螂 进行消毒蛋。以前关于双翅目卵消毒的研究，例如利用过氧化氢溶液作为手部消毒剂和过氧化氢作为表面消毒剂对 Lucilia sericata （双翅目:Calliphoridae）卵30进行消毒。因此，优化消毒剂的选择和消毒时间变得必要。经实验发现，使用0.26%的NaClO和75%的EtOH，对卵进行消毒0.5分钟，重复该过程3次，即可达到 对杜松 卵的杀菌效果。
在获得斧头虫卵后，饲养斧头 虫 的另一个关键方面是为它们提供体外幼虫的无菌饮食。尽管人工饮食不能完全复制天然食物，但研究31、32、33 证明了使用人工饮食饲养昆虫的可行性。人工饮食的营养成分会影响昆虫的免疫系统和适应性34.因此，人工制备的饮食必须含有与天然食物相似的营养成分。这将有助于确保饲养的昆虫获得发育、免疫反应和整体健康所需的营养。在这项研究中，大葱是 D. antiqua 无菌饮食的主要成分。半发酵日粮为 D. antiqua提供更好的营养，有助于它们的生长和发育。此外，将幼虫匀浆与日粮混合旨在促进体外肠道微生物群对日粮的预消化。这是因为肠道细菌对昆虫的摄食和消化有影响35.使用这种方法制备 D. antiqua 的无菌日粮简化了过程，并显着提高了日粮制备的效率。
在细胞内母体传播的细菌共生体广泛存在于昆虫中 36. 沃尔巴克氏体是一种母体传播的细胞内细菌，存在于许多昆虫中 37。 沃尔巴克氏体 最广为人知的是它在生殖操纵中的作用，因为它可以使宿主后代的性别比例偏向于产生更多受感染的雌性38。在验证了 D. antiqua 幼虫的轴心状态后发现，除内共生细菌 Wolbachia 外，消毒后的幼虫基本上是 axonic 的，它存在于 D. antiqua 卵巢组织细胞的细胞质中，并通过卵细胞的细胞质垂直传播。至于消除 沃尔巴克氏体的方法，抗生素治疗是一种常见的方法。综上所述，本文提出了一种陈螨的饲养 方法。根据协议，可以成功培育出 axenic D. antiqua 。此外，该方法还具有简单、成本低廉的特点，为饲养其他昆虫提供了一种新的方法，提高了研究昆虫与肠道微生物群相互作用的可行性。

Axenic 动物，定义为无法检测到活微生物或寄生虫的动物，是研究宿主-微生物相互作用的有价值的实验模型 1,2。昆虫是最大的无脊椎动物类群，可以与微生物形成共生关系3.Axenic 昆虫可用于研究共生系统中的宿主-共生体相互作用4.例如，Nishide等人[5]为恶臭蠕虫Plautia stali建立了一种实用的无菌饲养程序，能够可靠而严格地分析模型共生系统中的宿主-共生体相互作用。Axenic昆虫可以通过对卵阶段进行消毒并向幼虫和成虫提供无菌食物来产生6,7。Axenic昆虫具有重要意义，在生物学研究中被广泛使用。例如，Somerville等人进行的一项研究[8]表明，接种阴沟肠杆菌的菱形蛾提高了转基因雄性的适应性。
Delia antiqua Meigen 是全球洋葱和其他百合科作物的一种具有重要经济意义的害虫，其幼虫会破坏洋葱和其他百合科作物的鳞茎9.D. antiqua 主要分布在温带气候中，广泛分布于美洲、欧洲和亚洲的洋葱种植区。如果控制不当，可导致洋葱（洋葱）、大蒜（Allium sativum L.）、青葱（Allium fistulosum L.）和韭菜（Alliumchoenoprasum L.）的作物损失，从50%到100%不等10,11。幼虫以植物的地下部分为食，这种进食导致幼苗枯萎并最终死亡。此外，受损的植物可以使病原体进入，导致鳞茎腐烂12。即使这些植物没有被幼虫完全吃掉，它们造成的损害也会使洋葱植物无法销售并导致经济损失。
昆虫与肠道微生物群密切相关，大多数昆虫肠道含有多种共生细菌，这些细菌在宿主提供的营养物质中茁壮成长13,14。Jing等[15]表明，肠道共生群落的主要功能是提供必需的营养物质，其次是消化和解毒相关的功能。在某些情况下，肠道细菌可以作为害虫管理目的的微生物资源。因此，研究单个肠道细菌在 D. antiqua 体内的表现和特定功能是可取的。因此，制备轴幼虫对于研究特定细菌菌株与昆虫之间的相互作用尤为重要16。目前，消除昆虫肠道细菌的常用方法是使用抗生素组合来根除相关微生物17,18,19。与单独使用抗生素只能减少微生物数量不同，昆虫的轴性饲养可以控制微生物的组成和数量，从而更准确地验证肠道微生物群的功能。
因此，本文介绍了一种制备和饲养 axenic D. antiqua 的方案。Axenic幼虫食品是通过利用天然饮食的高温杀菌与半发酵食品相结合而获得的。按照实验方案对卵进行灭菌以获得斧头虫卵，最后，从斧头卵中培养斧头幼虫。在实验中，轴性饲养系统仅进行了一代。这将为研究昆虫与肠道微生物群之间的相互作用提供便利。

<table><tbody><tr><td>0.22 &amp;mu;M filter bottle</td><td>Thermo Scientific</td><td>450-0045</td><td></td></tr><tr><td>0.22 &amp;mu;M Syringe Filter</td><td>Biosharp</td><td>BS-QT-011</td><td></td></tr><tr><td>100-mesh sieve</td><td>Zhejiang Shangyu Jinding Standard Sieve Factory</td><td>No Catalog numbers</td><td></td></tr><tr><td>1x PBS solution</td><td>Solarbio</td><td>P1020</td><td></td></tr><tr><td>2x Taq PCR Master Mix</td><td>GENVIEW</td><td>GR1113-1ML</td><td></td></tr><tr><td>5.2% NaClO solution</td><td>Sinopharm Chemical Reagent Co., Ltd.</td><td>80010428</td><td></td></tr><tr><td>500 mL Conical flask</td><td>Thermo Scientific</td><td>4103-0500</td><td></td></tr><tr><td>50 mL vented centrifuge tube</td><td>JET BIOFIL</td><td>BRT-011-050</td><td></td></tr><tr><td>50x TAE buffer</td><td>GENVIEW</td><td>GT1307</td><td></td></tr><tr><td>Agar powder</td><td>Ding Guo</td><td>DH010-1.1</td><td></td></tr><tr><td>Biochemical incubator</td><td>STIK</td><td>21040121500010</td><td></td></tr><tr><td>Cell sieve</td><td>SAINING</td><td>5022200</td><td></td></tr><tr><td>Choline chloride</td><td>Sangon Biotech</td><td>A600299-0100</td><td></td></tr><tr><td>ddH&lt;sub&gt;2&lt;/sub&gt;O</td><td>Ding Guo</td><td>PER018-2</td><td></td></tr><tr><td>Disposable grinding pestle</td><td>JET BIOFIL</td><td>CSP-003-002</td><td></td></tr><tr><td>DNA extraction kit</td><td>Sangon Biotech</td><td>B518221-0050</td><td></td></tr><tr><td>DNA Marker</td><td>Sangon Biotech</td><td>B600335-0250</td><td></td></tr><tr><td>Ethanol absolute</td><td>Sinopharm Chemical Reagent Co., Ltd.</td><td>10009218</td><td></td></tr><tr><td>Filter paper</td><td>NEWSTAR</td><td>1087309025</td><td></td></tr><tr><td>Food processor</td><td>Guangdong Midea Life Electric Appliance Manufacturing Co., Ltd.</td><td>WBL25B26</td><td></td></tr><tr><td>Illuminated&amp;nbsp; incubator</td><td>Shanghai ESTABLISH Instrumentation Co., Ltd.</td><td>A16110768</td><td></td></tr><tr><td>L-Ascorbic acid</td><td>Sangon Biotech</td><td>A610021-0100</td><td></td></tr><tr><td>L-shaped spreader</td><td>SAINING</td><td>6040000</td><td></td></tr><tr><td>Nutrient agar medium</td><td>Hope Bio</td><td>HB0109</td><td></td></tr><tr><td>Scissors</td><td>Bing Yu</td><td>&amp;nbsp;BY-103</td><td>Purchase on Jingdong</td></tr><tr><td>Shock incubator</td><td>Shanghai Zhichu Instrument Co., Ltd.</td><td>2020000014</td><td></td></tr><tr><td>Sucrose</td><td>GENVIEW</td><td>CS326-500G</td><td></td></tr><tr><td>Super Green nucleic acid dye</td><td>Biosharp</td><td>BS355A</td><td></td></tr><tr><td>Super-clean table</td><td>Heal Force</td><td>AC130052</td><td></td></tr><tr><td>TSB</td><td>Hope Bio</td><td>HB4114</td><td></td></tr><tr><td>Vacuum pump</td><td>Zhejiang Taizhou Seeking Precision Vacuum Pump Co., Ltd.</td><td>22051031</td><td></td></tr><tr><td>Yeast extract</td><td>Thermo Scientific</td><td>LP0021B</td><td></td></tr></tbody></table>

rearing axenic delia antiqua with half-fermented sterile diets

Axenic昆虫是从使用无菌培养基的无菌人工饲养系统中获得的。这些昆虫的特点是体型小、生长周期短、饲料要求低，是研究微生物与宿主之间关系的理想选择。肠道菌群显著影响昆虫宿主的生理特性，将特定菌株引入昆虫中提供了一种验证肠道微生物功能的方法。 Delia antiqua 是双翅目、Anthomyiidae 科和 Delia 属的一种威胁性害虫，主要以洋葱、大蒜、韭菜和百合科的其他蔬菜为食。它的幼虫以鳞茎为食，导致整株植物腐烂、枯萎甚至死亡。通过饲养蛤蜊幼虫，可以进行后续研究，观察肠道菌群对 古蛾生长发育的影响。与涉及抗生素消除相关微生物的方法不同，本文提出了一种低成本和高效的方法来培养 axenen D. antiqua。对 古蛾 卵进行表面灭菌后，采用半发酵无菌日粮饲养幼虫，通过培养依赖性和培养非依赖性试验验证 了古蛾 的轴质状态。总之，昆虫卵灭菌和幼虫培养无菌日粮的制备相结合，能够开发出一种有效和简单的方法来获得 Axenic D. antiqua。该方法为研究昆虫-微生物群落的相互作用提供了一种强有力的方法。

D. antiqua 的生命阶段如图 4 所示。完整的生命周期包括卵、幼虫、蛹（图4C）和成虫（图4D）。它们在无菌离心管中培养，其外观和存活率与在非轴性条件下饲养的 D. antiqua 没有区别。D. antiqua每个阶段的生长和发育时间如图6所示。非轴培的古蛾的卵期为2.83±0.29 d，轴向饲养的 D. antiqua 的卵期为 2.83 ± 0.29 d，两者之间无显著差异（独立 t 检验，t = 0.00，p = 1.00）。正常幼虫期持续13.83±0.76天，而无源幼虫期持续14.50天±0.50天，两者之间无显著差异（独立t检验，t = 1.27，p = 0.27）。正常饲养的蛹期为17.33天±0.76天，轴心饲养的蛹期为18.00天±0.50天，两者之间无显著差异（独立t检验，t=1.27，p=0.27）。正常饲养成虫存活时间为81.50±1.00 d，无脊椎饲养成虫存活时间为80.33±0.76 d，两者差异无统计学意义（独立t检验，t = 1.61，p = 0.18）。可以看出，在正常条件下饲养的斧头D. antiqua在发育时间上没有显著差异。
培养依赖性测定表明，在幼虫肠道中未检测到菌落（图7）。此外，基于细菌16S rRNA的PCR分析显示，在1500 bp左右存在一条条带（图8A），被鉴定为 沃尔巴克氏体 （该核苷酸序列的GenBank登录号为:OR564190），一种内共生细菌。然而，在靶向真菌ITS区域的PCR分析中未观察到条带（图8B）。这表明幼虫已经达到了无轴状态。Axenic幼虫对研究 古蛾 与微生物的相互作用机制，以及防控 古蛾造成的损害具有重要意义。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/66259/66259fig01.jpg"> 图1: 古蛾的饲养装置和食物。 （A）幼虫的葱白色部分。（B） 成人饮水机。（C） 成人食品。（D） 鸡蛋收集装置。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/66259/66259fig01large.jpg" target="_blank">请点击这里查看此图的较大版本.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/66259/66259fig02.jpg"> 图2:饮食制备的组成部分。 （A） 磨碎的葱。（B） 发酵葱。（三）发酵葱渣和滤液。（D） 准备好的饮食。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/66259/66259fig02large.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/66259/66259fig03.jpg"> 图3:卵子转移的过程。 （A）细胞筛中的卵。（B） 1 mL 移液器，不带端头。（C） 1 mL 移液管和鸡蛋。（D） 将鸡蛋转移到日粮中。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/66259/66259fig03large.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/66259/66259fig04.jpg"> 图4:无脊椎D. antiqua的生命阶段。 （A） 鸡蛋。（B） 幼虫。（C） 蛹。（D） 成年女性和男性。<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/66259/66259fig04large.jpg" target="_blank">请点击这里查看此图的较大版本.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/66259/66259fig05.jpg"> 图5:解剖幼虫肠道的过程。 （A）幼虫的头部和尾部。（B）去掉头和尾的幼虫。（C） 分离的肠道和身体组织。（D） 解剖后完整的肠道。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/66259/66259fig05large.jpg" target="_blank">请点击这里查看此图的较大版本.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/66259/66259fig06.jpg"> 图6:正常饲养和轴向饲养的 D. antiqua每个生长阶段的发育时间。 轴向饲养和正常饲养的 D. antiqua 在发育时间上没有显着差异。独立 t 检验， p < 0.05。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/66259/66259fig06large.jpg" target="_blank">请点击这里查看此图的较大版本.</a>
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/66259/66259fig07.jpg"> 图 7:通过随机选择 3 只幼虫并在 TSA 和 PDA 琼脂平板上孵育肠道匀浆来确认 D. antiqua 的不育性。 在喂食无菌饮食的幼虫中没有观察到细菌。（A） TSA上的Axenic幼虫。（B） TSA上的正常幼虫。（C） PDA上的Axenic幼虫。（D） PDA上的正常幼虫。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/66259/66259fig07large.jpg" target="_blank">请点击这里查看此图的较大版本.</a>
<img alt="Figure 8" class="xfigimg" src="/files/ftp_upload/66259/66259fig08.jpg"> 图 8:使用通用 16S rRNA 引物和真菌 ITS 引物通过 PCR 分析确认 D. antiqua 的不育性。 16S rRNA 基因的靶条带为 ~1,500 bp。观察到一个大约 1500 bp 的条带，并被鉴定为内共生细菌 沃尔巴克氏体。ITS基因的靶带为500-750 bp。在 AF 组中未观察到目标条带。（A）16S rRNA电泳图。（B） ITS电泳图。缩写:M = 标记;NC = 阴性对照;NF = 正常喂养;AF = 轴性进料。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/66259/66259fig08large.jpg" target="_blank">请点击这里查看此图的较大版本.</a>

Watch this Scientific Journal Video about Axenic Rearing of Delia antiqua to Detect the Impact of Intestinal Microbes on Its Growth and Development at JoVE.com

作者聚焦:Antiqua 的轴系饲养以检测肠道微生物对其生长发育的影响 

描述了用半发酵无菌日粮饲养 axenic Delia antiqua 的简单程序。使用PCR方法在每龄的Axenic D. antiqua 中仅检测到一种沃尔巴克氏菌株。

用半发酵无菌日粮饲养 Axenic Delia antiqua

Axenic insects are obtained from sterile artificial rearing systems using sterile media. These insects, characterized by their small size, short growth ...

我们的研究领域是微生物生态学，我们希望通过饲养 axenic Delia antiqua 来探索微生物内部的相互作用。将回答关于Delia antiqua肠道真菌的多样性及其对Delia antiqua生长发育的影响的问题。已经建立了一种用半发酵无菌日粮饲养斧头Delia antiqua的简单方法，并且通过PCR在Delia antiqua的每个龄期中仅检测到一种已鉴定的沃尔巴克氏菌株。与抗生素治疗相比，该协议完全消除了肠道细菌，并且不会对宿主昆虫造成许多副作用。昆虫肠道微生物群的功能也没有标记。它比抗生素治疗更有效、更实惠。

rearing-axenic-delia-antiqua-with-half-fermented-sterile-diets

Nanyang Technological University

Research

JoVE Journal

Biology

Rearing Axenic Delia antiqua with Half-Fermented Sterile Diets

592 次观看.  Qilu University of Technology (Shandong Academy of Sciences). 我们的研究领域是微生物生态学，我们希望通过饲养 axenic Delia antiqua 来探索微生物内部的相互作用。将回答关于Delia antiqua肠道真菌的多样性及其对Delia antiqua生长发育的影响的问题。已经建立了一种用半发酵无菌日粮饲养斧头Delia antiqua的简单方法，并且通过PCR在Delia antiqua的每个龄期中仅检测到一种已鉴定的沃尔巴克氏菌株。与抗生素治疗相比，该协议完全消除了肠道?...

视频: 作者聚焦:Antiqua 的轴系饲养以检测肠道微生物对其生长发育的影响 

Rearing the Fruit Fly Drosophila melanogaster Under Axenic and Gnotobiotic Conditions

The influence of microbes on myriad animal traits and behaviors has been increasingly recognized in recent years. The fruit fly Drosophila melanogaster is a model for understanding microbial interactions with animal hosts, facilitated by approaches to rear large sample sizes of Drosophila under microorganism-free (axenic) conditions, or with defined microbial communities (gnotobiotic). This work outlines a method for collection of Drosophila embryos, hypochlorite dechorionation and sterilization, and transfer to sterile diet. Sterilized embryos are transferred to sterile diet in 50 ml centrifuge tubes, and developing larvae and adults remain free of any exogenous microbes until the vials are opened. Alternatively, flies with a defined microbiota can be reared by inoculating sterile diet and embryos with microbial species of interest. We describe the introduction of 4 bacterial species to establish a representative gnotobiotic microbiota in Drosophila. Finally, we describe approaches for confirming bacterial community composition, including testing if axenic Drosophila remain bacteria-free into adulthood.

Rearing the Fruit Fly Drosophila melanogaster Under Axenic and Gnotobiotic Conditions

A method for rearing Drosophila melanogaster under axenic and gnotobiotic conditions is presented. Fly embryos are dechorionated in sodium hypochlorite, transferred aseptically to sterile diet, and reared in closed containers. Inoculating diet and embryos with bacteria leads to gnotobiotic associations, and bacterial presence is confirmed by plating whole-body Drosophila homogenates.

饲养果蝇<em&gt;果蝇</em&gt;在无菌和悉生条件

The influence of microbes on myriad animal traits and behaviors has been increasingly recognized in recent years. The fruit fly Drosophila melanogaster is ...

科研

发育生物学

Solid Plate-based Dietary Restriction in Caenorhabditis elegans

Reduction of food intake without malnutrition or starvation is known to increase lifespan and delay the onset of various age-related diseases in a wide range of species, including mammals. It also causes a decrease in body weight and fertility, as well as lower levels of plasma glucose, insulin, and IGF-1 in these animals. This treatment is often referred to as dietary restriction (DR) or caloric restriction (CR). The nematode Caenorhabditis elegans has emerged as an important model organism for studying the biology of aging. Both environmental and genetic manipulations have been used to model DR and have shown to extend lifespan in C. elegans. However, many of the reported DR studies in C. elegans were done by propagating animals in liquid media, while most of the genetic studies in the aging field were done on the standard solid agar in petri plates. Here we present a DR protocol using standard solid NGM agar-based plate with killed bacteria.

Solid Plate-based Dietary Restriction in Caenorhabditis elegans

Here we present a protocol for performing solid plate-based dietary restriction in C. elegans with killed bacteria.

实心板为基础的饮食限制在线虫</em

Reduction of food intake without malnutrition or starvation is known to increase lifespan and delay the onset of various age-related diseases in a wide ...

生物学

Production of Germ-Free Fast-Growing Broilers from a Commercial Line for Microbiota Studies

Studies of the gut microbiota contribution to the host physiology and immunocompetence are facilitated by the availability of germ-free animal models, which are considered the gold standard. Nesting birds are ideal models for the production of germ-free animals since there is no need to raise their relatives under sterile conditions. Germ-free chickens are mainly generated from specific-pathogen-free (SPF) experimental lines, which are poorly representative of commercial chicken lines.&#160;The method proposed here allowed the production of germ-free chickens from the fast growing broiler line Ross PM3, commonly used by the poultry industry.&#160;Eggs were quickly collected after laying at a broiler breeder farm.&#160;They underwent a strict decontamination process from the collection to the introduction in a sterile egg hatching isolator.&#160;The chicks have been hatched and kept in these sterile isolators during the period necessary to control their sterility. Originally developed for an experimental SPF white leghorn line, the present protocol has been adapted not only to the Ross PM3 broiler line but also to quails. It therefore represents a robust and readily adaptable procedure to other poultry species and nesting birds of economic, biological or ecological relevance.

Here we describe a method for the generation of germ-free chicks from eggs of a commercial broiler line, the Ross PM3. This method can be adapted for the generation of germ-free animals from other poultry species.

从用于微生物群研究的商业生产线上生产无菌快速生长的肉鸡

Studies of the gut microbiota contribution to the host physiology and immunocompetence are facilitated by the availability of germ-free animal models, ...

免疫与感染

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.

培养<em&gt;秀丽隐杆线虫</em &gt;在无菌液体介质与创造转基因蠕虫通过微粒轰击

In this protocol, we present the required materials, and the procedure for making modified&#160;C. elegans&#160;Habituation and Reproduction media ...

Establishment and Maintenance of Gnotobiotic American Cockroaches (Periplaneta americana)

Gnotobiotic animals are a powerful tool for the study of controls on microbiome structure and function. Presented here is a protocol for the establishment and maintenance of gnotobiotic American cockroaches (Periplaneta americana). This approach includes built-in sterility checks for ongoing quality control. Gnotobiotic insects are defined here as cockroaches that still contain their vertically transmitted endosymbiont (Blattabacterium) but lack other microbes that normally reside on their surface and in their digestive tract. For this protocol, egg cases (oothecae) are removed from a (nonsterile) stock colony and surface sterilized. Once collected and sterilized, the oothecae are incubated at 30 &#176;C for approximately 4&#8722;6 weeks on brain-heart infusion (BHI) agar until they hatch or are removed due to contamination. Hatched nymphs are transferred to an Erlenmeyer flask containing a BHI floor, sterile water, and sterile rat food. To ensure that the nymphs are not housing microbes that are unable to grow on BHI in the given conditions, an additional quality control measure uses restriction fragment-length polymorphism (RFLP) to test for nonendosymbiotic microbes. Gnotobiotic nymphs generated using this approach can be inoculated with simple or complex microbial communities and used as a tool in gut microbiome studies.

Establishment and Maintenance of Gnotobiotic American Cockroaches Periplaneta americana

This protocol is used in establishing and maintaining gnotobiotic American cockroaches (Periplaneta americana) by surface sterilizing the egg cases (oothecae) prior to hatching. These gnotobiotic insects contain their vertically transmitted Blattabacterium endosymbionts but have axenic guts.

建立和维护侏罗育美国蟑螂 （佩里普拉内塔美洲）

Gnotobiotic animals are a powerful tool for the study of controls on microbiome structure and function. Presented here is a protocol for the establishment ...

Preparing and Rearing Axenic Insects with Tissue Cultured Seedlings for Host-Gut Microbiota Interaction Studies of the Leaf Beetle

Insect guts are colonized by diverse bacteria that can profoundly impact the host&#39;s physiological traits. Introducing a particular bacterial strain into an axenic insect is a powerful method to verify gut microbial function and elucidate the mechanisms underlying gut microbe-host interactions. Administering antibiotics or sterilizing egg surfaces are two commonly used methods to remove gut bacteria from insects. However, in addition to the potential adverse effects of antibiotics on insects, previous studies indicated that feeding antibiotics could not eliminate gut bacteria. Thus, germ-free artificial diets are generally employed to maintain axenic insects, which is a tedious and labor-intensive process that cannot fully resemble nutritional components in natural food. Described here is an efficient and simple protocol to prepare and maintain axenic larvae of a leaf beetle&#160;(Plagiodera versicolora). Specifically, surfaces of the beetle eggs were sterilized, following which germ-free poplar leaves were used to rear axenic larvae. The axenic status of the insects was further confirmed via culture-dependent and culture-independent assays. Collectively, by combining egg disinfection and germ-free cultivation, an efficient and convenient method was developed to obtain axenic P. versicolora, providing a readily transferable tool for other leaf-eating insects.

To obtain an axenic insect, its egg surface is sterilized, and the hatched larva is subsequently reared using axenic leaves. This method provides an efficient way for axenic insect preparation without administering antibiotics or developing an artificial diet, which can also be applied to other leaf-eating insects.

用组织培养幼苗制备和饲养轴性昆虫用于叶甲虫的宿主 - 肠道微生物群相互作用研究

Insect guts are colonized by diverse bacteria that can profoundly impact the host&#39;s physiological traits. Introducing a particular bacterial strain ...

Long-Term Culture of Individual Caenorhabditis elegans on Solid Media for Longitudinal Fluorescence Monitoring and Aversive Interventions

Caenorhabditis elegans are widely used to study aging biology. The standard practice in C. elegans aging studies is to culture groups of worms on solid nematode growth media (NGM), allowing the efficient collection of population-level data for survival and other physiological phenotypes, and periodic sampling of subpopulations for fluorescent biomarker quantification. Limitations to this approach are the inability to (1) follow individual worms over time to develop age trajectories for phenotypes of interest and (2) monitor fluorescent biomarkers directly in the context of the culture environment. Alternative culture approaches use liquid culture or microfluidics to monitor individual animals over time, in some cases including fluorescence quantification, with the tradeoff that the culture environment is contextually distinct from solid NGM. The WorMotel is a previously described microfabricated multi-well device for culturing isolated worms on solid NGM. Each worm is maintained in a well containing solid NGM surrounded by a moat filled with copper sulfate, a contact repellent for C. elegans, allowing longitudinal monitoring of individual animals. We find copper sulfate insufficient to prevent worms from fleeing when subjected to aversive interventions common in aging research, including dietary restriction, pathogenic bacteria, and chemical agents that induce cellular stress. The multi-well devices are also molded from polydimethylsiloxane, which produces high background artifacts in fluorescence imaging. This protocol describes a new approach for culturing isolated roundworms on solid NGM using commercially available polystyrene microtrays, originally designed for human leukocyte antigen (HLA) typing, allowing the measurement of survival, physiological phenotypes, and fluorescence across the lifespan. A palmitic acid barrier prevents worms from fleeing, even in the presence of aversive conditions. Each plate can culture up to 96 animals and easily adapts to a variety of conditions, including dietary restriction, RNAi, and chemical additives, and is compatible with automated systems for collecting lifespan and activity data.

Long-Term Culture of Individual Caenorhabditis elegans on Solid Media for Longitudinal Fluorescence Monitoring and Aversive Interventions

Here, we present a protocol to culture isolated individual nematodes on solid media for lifelong physiological parameter tracking and fluorescence quantification. This culture system includes a palmitic acid barrier around single-worm wells to prevent animals from fleeing, allowing the use of aversive interventions, including pathogenic bacteria and chemical stressors.

在固体培养基上长期培养单个 秀丽隐杆线虫 ，用于纵向荧光监测和厌恶干预

Caenorhabditis elegans are widely used to study aging biology. The standard practice in C. elegans aging studies is to culture groups of worms on solid ...

Long-Term Culture and Monitoring of Isolated Caenorhabditis elegans on Solid Media in Multi-Well Devices

The nematode Caenorhabditis elegans is among the most common model systems used in aging research owing to its simple and inexpensive culture techniques, rapid reproduction cycle (~3 days), short lifespan (~3 weeks), and numerous available tools for genetic manipulation and molecular analysis. The most common approach for conducting aging studies in C. elegans, including survival analysis, involves culturing populations of tens to hundreds of animals together on solid nematode growth media (NGM) in Petri plates. While this approach gathers data on a population of animals, most protocols do not track individual animals over time. Presented here is an optimized protocol for the long-term culturing of individual animals on microfabricated polydimethylsiloxane (PDMS) devices called WorMotels. Each device allows up to 240 animals to be cultured in small wells containing NGM, with each well isolated by a copper sulfate-containing moat that prevents the animals from fleeing. Building on the original WorMotel description, this paper provides a detailed protocol for molding, preparing, and populating each device, with descriptions of common technical complications and advice for troubleshooting. Within this protocol are techniques for the consistent loading of small-volume NGM, the consistent drying of both the NGM and bacterial food, options for delivering pharmacological interventions, instructions for and practical limitations to reusing PDMS devices, and tips for minimizing desiccation, even in low-humidity environments. This technique allows the longitudinal monitoring of various physiological parameters, including stimulated activity, unstimulated activity, body size, movement geometry, healthspan, and survival, in an environment similar to the standard technique for group culture on solid media in Petri plates. This method is compatible with high-throughput data collection when used in conjunction with automated microscopy and analysis software. Finally, the limitations of this technique are discussed, as well as a comparison of this approach to a recently developed method that uses microtrays to culture isolated nematodes on solid media.

Long-Term Culture and Monitoring of Isolated Caenorhabditis elegans on Solid Media in Multi-Well Devices

Presented here is an optimized protocol for culturing isolated individual nematodes on solid media in microfabricated multi-well devices. This approach allows individual animals to be monitored throughout their lives for a variety of phenotypes related to aging and health, including activity, body size and shape, movement geometry, and survival.

多孔设备固体培养基上分离秀 丽隐杆线虫 的长期培养和监测

The nematode Caenorhabditis elegans is among the most common model systems used in aging research owing to its simple and inexpensive culture techniques, ...

Bradysia coprophila Lab Protocols and Chromosomal Research

Laboratory Maintenance of the Lower Dipteran Fly Bradysia (Sciara) coprophila: A New/Old Emerging Model Organism

Laboratory stocks of the lower dipteran fly, Bradysia (Sciara) coprophila, have been maintained for over a century. Protocols for laboratory upkeep of B. coprophila are presented here. These protocols will be useful for the rapidly increasing number of laboratories studying B. coprophila to take advantage of its unique biological features, which include (1) a monopolar spindle in male meiosis I; (2) non-disjunction of the X dyad in male meiosis II; (3) chromosome imprinting to distinguish maternal from paternal homologs; (4) germ line-limited (L) chromosomes; (5) chromosome elimination (paternal chromosomes in male meiosis I; one to two X chromosomes in early embryos; L chromosomes from the soma in early embryos); (6) sex determination by the mother (there is no Y chromosome); and (7) developmentally regulated DNA amplification at the DNA puff loci in larval salivary gland polytene chromosomes. 
It is now possible to explore these many unique features of chromosome mechanics by using the recent advances in sequencing and assembly of the B. coprophila genome and the development of transformation methodology for genomic engineering. The growing scientific community that uses B. coprophila for research will benefit from the protocols described here for mating the flies (phenotypic markers for mothers that will have only sons or only daughters; details of mass mating for biochemical experiments), checking embryo hatch, feeding larvae, and other comments on its rearing.

Author Spotlight: Exploring Bradysia coprophila's Unique Biology &#8211; A Guide to Laboratory Maintenance

This paper outlines the laboratory maintenance (including mating and feeding) of the lower dipteran fly Bradysia (Sciara) coprophila.

下双翅目蝇 Bradysia （Sciara） coprophila 的实验室维护:一种新的/旧的新兴模式生物

Laboratory stocks of the lower dipteran fly, Bradysia (Sciara) coprophila, have been maintained for over a century. Protocols for laboratory upkeep of B. ...

<meta charset="utf8"></head><body>鸡胃肠道中未培养微生物多样性的培养策略

Strategies to Enhance Cultivation of Anaerobic Bacteria from Gastrointestinal Tract of Chicken

The gastrointestinal tract (GIT) of chicken is a complex ecosystem harboring trillions of microbes that play a pivotal role in the host&#39;s physiology, digestion, nutrient absorption, immune system maturation, and prevention of pathogen intrusion. For optimal animal health and productivity, it is imperative to characterize these microorganisms and comprehend their role. While the GIT of poultry holds a reservoir of microorganisms with potential probiotic applications, most of the diversity remains unexplored. To enhance our understanding of uncultured microbial diversity, concerted efforts are required to bring these microorganisms into culture. Isolation and cultivation of GIT-colonizing microorganisms yield reproducible material, including cells, DNA, and metabolites, offering new insights into metabolic processes in the environment. Without cultivation, the role of these organisms in their natural settings remains unclear and limited to a descriptive level. Our objective is to implement cultivation strategies aimed at improving the isolation of a diverse range of anaerobic microbes from the chicken&#39;s GIT, leveraging multidisciplinary knowledge from animal physiology, animal nutrition, metagenomics, feed biochemistry, and modern cultivation strategies. Additionally, we aim to implement the use of proper practices for sampling, transportation, and media preparation, which are known to influence isolation success. Appropriate methodologies should ensure a consistent oxygen-free environment, optimal atmospheric conditions, appropriate host incubation temperature, and provision for specific nutritional requirements in alignment with their distinctive needs. By following these methodologies, cultivation will not only yield reproducible results for isolation but will also facilitate isolation procedures, thus fostering a comprehensive understanding of the intricate microbial ecosystem within the chicken&#39;s GIT.

<meta charset="utf8"></head><body>作者聚焦：推进家禽肠道细菌培养

The protocol presents two methodologies to improve the isolation of anaerobic intestinal bacteria. The first focuses on the isolation of a diverse range of bacteria using different culture media. The second focuses on the cultivation steps of a specific microbial group, possibly assimilating myo-inositol, to fully comprehend its ecological significance.

加强鸡胃肠道厌氧菌培养的策略

The gastrointestinal tract (GIT) of chicken is a complex ecosystem harboring trillions of microbes that play a pivotal role in the host&#39;s physiology, ...

用半发酵无菌日粮饲养 Axenic Delia antiqua

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饲养果蝇

实心板为基础的饮食限制在线虫

从用于微生物群研究的商业生产线上生产无菌快速生长的肉鸡

培养

建立和维护侏罗育美国蟑螂（佩里普拉内塔美洲）

用组织培养幼苗制备和饲养轴性昆虫用于叶甲虫的宿主 - 肠道微生物群相互作用研究

在固体培养基上长期培养单个秀丽隐杆线虫，用于纵向荧光监测和厌恶干预

多孔设备固体培养基上分离秀丽隐杆线虫的长期培养和监测

下双翅目蝇 Bradysia （Sciara） coprophila 的实验室维护:一种新的/旧的新兴模式生物

加强鸡胃肠道厌氧菌培养的策略

用半发酵无菌日粮饲养 Axenic Delia antiqua

副本

摘要

探索更多视频

此视频中的章节

饲养果蝇

实心板为基础的饮食限制在线虫

从用于微生物群研究的商业生产线上生产无菌快速生长的肉鸡

培养

建立和维护侏罗育美国蟑螂 （佩里普拉内塔美洲）

用组织培养幼苗制备和饲养轴性昆虫用于叶甲虫的宿主 - 肠道微生物群相互作用研究

在固体培养基上长期培养单个 秀丽隐杆线虫 ，用于纵向荧光监测和厌恶干预

多孔设备固体培养基上分离秀 丽隐杆线虫 的长期培养和监测

下双翅目蝇 Bradysia （Sciara） coprophila 的实验室维护:一种新的/旧的新兴模式生物

加强鸡胃肠道厌氧菌培养的策略

建立和维护侏罗育美国蟑螂（佩里普拉内塔美洲）

在固体培养基上长期培养单个秀丽隐杆线虫，用于纵向荧光监测和厌恶干预

多孔设备固体培养基上分离秀丽隐杆线虫的长期培养和监测