作者要向 GVK 和 SGK 承认 Otterbein 大学本科生研究奖学金和学生研究基金奖;和 Otterbein 教授吉尔伯特 e. 米尔斯纪念馆颁发的休假项目奖和生物和地球科学学院的研究和奖学金授予的奖励。伯特和简角赋予学生研究基金在科学被授予 GVK 和 SGK。作者还要感激地感谢 GVK 和 SGK 大学资助的本科研究项目奖学金。前 Juniata 大学本科生, 瑞安约翰逊和林赛德雷柏分别贡献了图2和3的显微图像。

1. 准备仪器、培养基、溶液和培养皿
<ol><li>在50毫升的烧杯中, 用铝箔覆盖的高压釜牙签保持不育。</li>
	<li>在高压釜中消毒所有将使用的干货 (小贴士、棍子、玻璃器皿等)。 注意: 小心!按照使用的高压釜模型的所有安全方向。高压釜对爆炸有危险, 从蒸汽中燃烧, 与热表面、材料和介质接触。对于高压釜的安全性, 请查看 "适当使用高压釜"34。</li>
	<li>
准备14的 MS 琼脂 (可持续的甘露醇) 培养。
	<ol>
<li>添加5克大豆粉和5克琼脂到1升瓶。</li>
		<li>在250毫升自来水中溶解5克甘露醇, 并将其放入含有大豆粉和琼脂的烧瓶中。</li>
		<li>添加一个泡沫插头和铝箔打开的烧瓶和高压釜在121摄氏度, 15 psi 至少30分钟。 注意: 这种介质容易沸腾。使用一瓶可以保持至少四倍的实际体积的 MS 琼脂是蒸压。一个标准的压力锅可以用来代替有类似安全考虑的高压釜。另外, 如果进入高压釜的可能性不是35、36, 微波炉可以用来对介质和材料进行消毒。高中教师也可能希望与当地的学院或大学合作, 以获得蒸压供应。</li>
		<li>很酷的媒体, 直到足够舒服处理。小心不要冷却太久, 因为琼脂固化在温度低于50摄氏度。</li>
		<li>将 MS 琼脂从烧瓶倒入无菌培养皿, 直到每个培养皿的底部大约半满 (每培养皿大约25毫升的培养基)。在使用琼脂板之前, 请先固化。将琼脂盘存放在室温下的塑料袋中, 直到需要。 注意: 琼脂盘子也可以储存在冰箱里, 尤其是在培养基中需要抗生素的时候。</li>
	</ol>
</li>
</ol>2. 条纹链霉菌到板材传播
<ol><li>条纹链霉菌菌株到 MS 琼脂板块的单一殖民地使用标准方法, 如象限条纹方法, 如桑德斯, 201237。 注: 可使用接种循环或无菌牙签。可选: 其他媒介通常用于链霉菌类, 例如 R2YE 和极小的媒介14。</li>
	<li>孵育板在30°c。</li>
	<li>Restreak 每第4-6 天采摘一个殖民地。随着殖民地时代的发展, 它们变得容易发生随机变异。</li>
</ol>3. 创建甘油菌丝储存细菌贮存
注: 可以为所有链霉菌菌株 (包括栋菌株和其他无法完成孢子的发育突变体) 创建菌丝种群。
<ol><li>浸渍15–20殖民地在200–500µL 20% 甘油使用无菌木钉涂抹器。</li>
	<li>使用微或无菌转移吸管将浸渍菌落的浆料转移到1.5 毫升冷冻培养管。 注意: 微使用的基本知识在其他地方覆盖38。</li>
	<li>添加足够的20% 甘油, 使最终体积1毫升和轻轻混合。将样品存放在摄氏-80 摄氏度。 注意: 小心!使用低温安全手套和实验室大衣保护皮肤免受严寒的接触。另外,-20 °c 冰箱可用于长期贮存时间的潜在减少。避免过度结冰和解冻, 以延长生存能力。</li>
</ol>4. 为能孢子的菌株创建甘油孢子储存
注: 孢子储存比长期存活更可取, 但仅适用于能够完成孢子的菌株。
<ol><li>
将100µL 的浸渍材料 (从步骤 3.1) 延伸到琼脂板上, 获得生长37的汇合草坪。 注: 在桑德斯37中显示电镀 (修改: 不需要转盘)。可以用一只手转动盘子, 同时用另一只手在琼脂介质上轻轻地来回移动吊具。<ol>
<li>吸管100µL 的浸渍菌丝的中心的 MS 琼脂板块。</li>
		<li>对玻璃或金属传播工具进行消毒, 将其部分浸入70% 乙醇中, 并通过本生燃烧器火焰缓慢传递, 以加速乙醇的蒸发。 注意: 乙醇是高度易燃的。不要将燃烧的吊具放入乙醇或让燃烧的乙醇落入乙醇盘中。或者, 使用一次性无菌棉签将细菌扩散到盘子上, 这不需要乙醇或火焰。</li>
		<li>用一只手转动 MS 琼脂盘, 同时使用来回运动来给盘子接种无菌的吊具。</li>
		<li>在30摄氏度孵育5–7天, 直到菌株形成孢子。 注意: 潜伏期会因种类和菌株而异。</li>
	</ol>
</li>
	<li>将2毫升的无菌生理盐水 (0.8% NaCl) 添加到已经历孢子的链霉菌细胞的汇合草坪中心。</li>
	<li>用不育的接种回路 (或棉花芽) 轻轻地摩擦菌丝表面, 慢慢地向草坪边缘移动, 收获孢子。</li>
	<li>将含有孢子的盐水吸管成15毫升锥形管。漩涡有力地将孢子链分解成单个细胞。</li>
	<li>离心机在室温下为5分钟左右, 约 2500 x克。</li>
	<li>吸管上清并丢弃。在少量剩余液体中, 通过剧烈的涡流搅动分散孢子颗粒。</li>
	<li>通过用吸管向上和向下绘制溶液, 并用重悬1毫升20% 甘油中的孢子。</li>
	<li>将甘油孢子料转移到1.5 毫升冷冻管。存储在-80 摄氏度。 注意: 使用低温安全手套和实验室大衣保护皮肤免受严寒的接触。另外,-20 °c 冷冻机可用于长期贮存时间的潜在降低。避免过度结冰和解冻, 以延长生存能力。</li>
</ol>5. 进行诱变
<ol><li>根据导言中所提到的预期结果进行诱变, 并于最近的 Baltz 201639审查。 注意: 一些分型实验将不需要突变, 例如从环境中识别新物种的意图。随机诱变的方法包括使用座子8,9,10,11,12,13, 紫外线辐射, 或化学品14。站点定向突变15、16、17、18、19、20技术可用于创建特定点突变、删除或其他类型的突变。</li>
</ol>6. 比较和记录外观
<ol><li>注意每个突变体的菌落形态, 与在步骤2中裸奔所有菌株后的野型母菌株相比。将新的隔离物与一个研究良好的物种 (如天蓝色或s. venezuelae)进行比较, 以表征新的链霉菌物种。注意特征 (图 1), 如基本形状, 菌落表面 (模糊, 秃头, 皱纹等), 不透明度, 海拔和色素沉着 (区分植物菌丝, 空气菌丝和/或周围培养基)。</li>
	<li>在楔形图案上标明 MS 琼脂板和条纹野型和突变株 (图 1)。小心, 没有应变接触到另一个菌株, 以避免交叉污染。记下应变在盘子上的日期和时间。将盘子孵化在30摄氏度。</li>
	<li>将生长的培养皿放在彩色或白色的纸上, 以融汇背景。写在纸上的应变名称, 日期, 孵化温度和时间从第一板裸奔。将印版的数字图片与纸张背景上的后一种信息结合起来, 使混淆最小化。 注: 如果要用于图的准备工作, 可以在以后的时间从照片中裁剪出文字。</li>
</ol>7. 执行相位对比显微镜
<ol><li>用70% 乙醇洗净镊子, 然后通过本生燃烧器火焰蒸发乙醇。允许冷却。</li>
	<li>用70% 乙醇洗涤盖玻片, 然后让它们在无菌培养皿中干燥。</li>
	<li>
准备一个盖玻片的细菌生长的升降机将被检查。
	<ol>
<li>用无菌镊子拿起无菌盖玻片, 将盖玻片放在细菌生长致密的 MS 琼脂板上。用镊子轻轻按压盖玻片的背部, 以确保细菌孢子和空中菌丝有足够的转移。</li>
	</ol>
</li>
	<li>从盘子里拿起盖玻片, 使它的侧面与细胞材料朝向表面的显微镜幻灯片, 与15µL 下降50% 甘油准备在水中安装。将盖玻片放在45°角上, 减少气泡, 让它落到50% 甘油上。</li>
	<li>选用透明的指甲油密封以保存滑轨。</li>
	<li>
在生命周期的不同时间间隔执行 Frohlich40所描述的相衬显微术。 注意: 重复成像允许进行更完整的分析, 并能够检测开发中的延迟。独立重复观察, 确保准确性和重现性。<ol>
<li>将准备好的幻灯片放在显微镜阶段, 并在盖子滑动的中心添加一滴浸入油。旋转100X 相目标到位, 并设置冷凝器炮塔正确的 "匹配" 的阶段设置。一旦物镜与油接触, 只用微调旋钮将图像聚焦。</li>
		<li>检查几个领域, 以辨别突变菌株和野生类型之间的明显和一致的差异, 如形成孢子的能力, 孢子大小和形状, 以及孢子的总数量 (见图 2)。 注: 相对比显微术的替代品包括差分干涉造影 (DIC) 显微镜40和简单染色技术, 用于明亮的场显微镜41, 如水晶紫染色。</li>
	</ol>
</li>
</ol>8. 执行荧光显微术
<ol><li>用70% 乙醇洗净镊子, 然后在本生燃烧器火焰中运行。</li>
	<li>用不育的镊子拿起不育的盖玻片, 并将其放置在细菌生长明显的 MS 琼脂板上。用镊子轻轻地触摸盖玻片的背部, 以确保细菌孢子和空中花丝有足够的转移。</li>
	<li>从盘子里拿起盖玻片, 使它的侧面与细胞材料正朝上, 在卡片, 以方便地操纵盖玻片。</li>
	<li>洪水盖玻片与冰冷甲醇 (~ 300 µL 为 20 x 20 毫米2盖玻片) 并且让它完全地空气干燥。 注意: 甲醇是诱变。避免与皮肤直接接触。</li>
	<li>在磷酸盐缓冲盐水 (PBS)42中, 通过轻轻的配药和去除溶液, 洗涤盖玻片三次。</li>
	<li>添加15µL 100 微克/毫升碘碘化物和/或10微克/毫升小麦胚芽凝集素 (WGA-FITC), 用50% 甘油制成一张标签的显微镜幻灯片。 注: 碘碘化物染色 dna 红检测染色体 dna 位置, 而 WGA-FITC 污渍细胞壁绿色。</li>
	<li>将盖玻片面倒置到玻璃滑梯上的荧光着色滴上。(可选) 用透明指甲油密封以保持滑动。 由于染料对光敏感, 在昏暗或昏暗的房间里孵育15分钟。</li>
	<li>观察100X 物镜下的幻灯片, 使用相对比或 DIC 显微镜, 配备荧光励磁立方体碘碘化 (' 德州红 ' 过滤器集或 535/617 nm 激发/发射极大值) 和 FITC (FITC 过滤器集或 490/525 nm激发/发射极大值)。</li>
	<li>将幻灯片放在舞台上, 并使用粗、细调整旋钮。</li>
	<li>观察荧光显示在别处43。</li>
	<li>使用免费的图像分析软件包, 可以帮助识别低荧光强度的孢子44。</li>
</ol>

作者没有什么可透露的。

在这里, 我们提出了启动链霉菌研究人员的协议, 通过包括传播菌株和准备储存长期储存所需的步骤来开始研究。然后我们描述了链霉菌菌株的视觉和显微特征的协议。分型发育突变体的一些典型初始步骤为: 1) 与琼脂培养基上野生型菌落相比的突变体的目视检查;2) 相衬显微镜;3) sporogenic 空气菌丝的荧光显微学。根据这三个步骤显示的表型, 可以使用多种技术进一步辨别特定菌株的表型。
最初的分型实验通常被用来表征新物种, 识别感兴趣的突变体, 部分地表征变种人, 并开始辨别某一特定基因的典型作用, 其基础是确定的突变体的表型。这里描述的方法已经被用于大学教学实验室, 以识别和表征各种各样的链霉菌突变体, 包括那些有缺陷的细胞分裂48,49,50,51,52,53,54, 孢子55,56, 空中菌丝形成57,58, 抗生素生产59, 第二信使信号47, 和染色体分离60. 这些技术是确定突变体的一般表型的重要第一步, 它揭示了有关基因的作用的大量重要信息。这些方法很容易推广到其他种类的链霉菌, 已经被用来描述灰色、 venezuelae、疥疮和许多其他 streptomycetes 的菌株。这里描述的视频协议有望成为新研究人员进入链霉菌研究领域的重要资源, 例如在药物发现领域。这包括新的导师, 他们正在努力抗击抗菌素耐药性危机, 并教育了无数的新本科生研究员加入了世界范围内的小全球倡议的努力。
这里描述的技术可以很容易地适应大学和高中课堂使用, 除了研究实验室, 使用的修改在视频和文本。在一所小型文科学院的第一年显微镜模块的学生能够条纹菌株, 拍摄在琼脂培养基上生长的菌株的数码照片, 并执行相对比和荧光显微镜, 这最终在提交一个在3周模块结束时, 多镶板数字的组合, 代表实验室工作的15小时。大约160个第一年学生负责最初的分型320个新的座子变种人。在三个机构的本科生研究学生参加了分型的新变种人的初始化, 以及随后的许多菌株的鉴定。在相对较短的时间内获得的综合数据, 说明了此处描述的协议的价值。数以百计的变种人被储存作为甘油菌丝的股票, 以供未来的表征。
在这里描述的初步实验之后, 可以使用多种方法来扩展与感兴趣菌株相关的信息的质量。如果突变是未知的, 应使用基因分型方法来确定突变的类型和/或位置。例如, 随机座子突变的野生型染色体6,7,8,9,10,11,12,13结果在殖民地, 应该经历最初的表型屏幕, 如上文所述。然后, 座子的位置应该使用一种技术, 如逆聚合酶链反应 (iPCR)61。确定新发现突变体的基因型是继初步鉴定后的重要步骤。
一些常用的先进方法, 随后的分型分析, 可能会在课堂上提到或通过进一步的研究, 包括绿色荧光蛋白 (GFP) 标记, 以确定的蛋白质的本地化模式的兴趣,基因表达分析, 如实时定量 PCR (qPCR) 和全球基因表达模式的野生型与突变通过 rna 排序 (rna 序列)。基因互补分析也需要分型技能。在互补实验中, 将基因的野生型拷贝引入变异菌株中, 以确定新添加的等位基因是否能补偿突变等位基因的功能损失。将补充菌株的表型与原突变体和亲本的表现形式进行比较, 需要野生型菌株。

Streptomycetes 是革兰阳性, 丝状的土壤细菌, 他们的能力生产各种次生代谢产物, 包括超过2/3 的商用抗生素, 以及抗肿瘤, 抗 HIV, 抗寄生虫药1.天蓝色是2、3属最具基因特征的成员, 是此处描述的方法中使用的物种。天蓝色有一个复杂的生命周期, 始于单一孢子的萌发, 进展为一个广泛的, 分支植物菌丝生长成琼脂培养基。随着生命周期的推移, 形成了空气花丝, 打破基体菌丝的表面张力, 最终被分为长链的细胞, 最终转化为成熟的, 灰色色素孢子。这些新形成的孢子的分散构成了下一个生命周期4的开始。
由于其复杂的分化模式,天蓝色是研究细菌发育的优良模型。历史上, 突变导致两个主要发展阶段的区块, 并产生不同的视觉表型。栋(秃头) 突变体被阻塞为空中菌丝形成, 并导致缺乏一个模糊的空气菌丝传授一个 "秃" 殖民地的外观。突变体在孢子形成和成熟过程中受到抑制 , 被称为白度突变体, 因为它们通常不能产生野生类型的灰孢子色素, 而空中菌丝保持白色。其他有趣的突变体在抗生素生产, 细胞分裂, 染色体分离, 或其他重要过程中被抑制4,5。
尽管在链霉菌物种中发现了许多发育基因, 但根据突变筛的缺乏饱和度, 还有许多人认为存在。我们的实验室继续使用我们构建的微型座子系统来识别新的发育基因。在随机诱变实验中分离出的突变菌株与我们的座子进行表型筛选, 以确定每个新基因的可能作用发现6,7。这里描述的细菌分型方法与座子突变8、9、10、11、12分离的链霉菌突变体有关, 13和其他随机方法, 如化学和紫外线 (UV) 诱变14, 以及定向构造突变, 如基因删除使用重组工程15,16或CRISPR-Cas9 (聚集定期 Interspaced 短回文重复) 基因组编辑技术17,18,19和点突变20。
随着病原体耐药性的日益普遍, 对新抗生素的需求变得越来越紧迫,21,22。"小世界倡议" (或) 是一项有效的科学、技术、工程和数学 (茎) 学习23和研究策略24 , 通过大学生的众包来对抗抗生素抗药性, 以及更多最近高中的学生。支持的课程工作包括确定新的土壤微生物, 产生新型抗生素 (http://www.smallworldinitiative.org)。据信, 一个主要来源的未发现的抗生素将继续是链霉菌物种在广泛的土壤和水生境25,26,27,28, 29,31。最近, 我们的实验室和其他人的工作已经发现和特征的信号基因的链霉菌物种, 调节形态学和发展, 包括抗生素生产7,32, 33。这些基因表达的变化导致了抗生素数量和时间的变化。新物种的熟练分型和新的抗生素产生突变体将继续是重要的。随着新教师和学生成为药物发现领域的主要贡献者, 细菌分型的训练对于这些新手的成功是必要的。此外, 这些实验是适合于高中茎或大学微生物学教育实验室。它们代表了教学实验室环境中基本微生物遗传原理的演示。

<table><tbody><tr><td>50% Glycerol</td><td>Sigma-Aldrich</td><td>G5516</td><td></td></tr><tr><td>Immersion Oil (Type DF)</td><td>Cragille&amp;nbsp;</td><td>16482</td><td></td></tr><tr><td>Lens Paper</td><td>Fisherbrand</td><td>11-995</td><td></td></tr><tr><td>Sterile Water</td><td>GeneMate</td><td>G-3250-1L</td><td></td></tr><tr><td>100% Ethanol</td><td>Sigma-Aldrich</td><td>E7023</td><td></td></tr><tr><td>Propidium Iodide</td><td>Invitrogen</td><td>P1304MP</td><td></td></tr><tr><td>Toothpicks (flat)</td><td>Target</td><td>081-22-1957</td><td></td></tr><tr><td>Pipette Tips (10 mL)</td><td>GeneMate</td><td>P-1240-10</td><td></td></tr><tr><td>Pipette Tips (200 mL)</td><td>GeneMate</td><td>P-1240-200Y</td><td></td></tr><tr><td>Pipette Tips (1,250 mL)</td><td>GeneMate</td><td>P-1240-1250</td><td></td></tr><tr><td>Wooden Applicators 6&#039;&#039;</td><td>Solon Care</td><td>070809</td><td></td></tr><tr><td>Cotton Swabs</td><td>Fisherbrand</td><td>23-400-124</td><td></td></tr><tr><td>Soy Flour&amp;nbsp;</td><td>Bob&#039;s Red Mill&amp;nbsp;</td><td>1516C164</td><td></td></tr><tr><td>D-Mannitol</td><td>Sigma-Aldrich</td><td>M4125-1KG</td><td></td></tr><tr><td>Agar</td><td>Sigma-Aldrich</td><td>A1296-1KG</td><td></td></tr><tr><td>Glycerol 100%</td><td>VWR amresco life science&amp;nbsp;</td><td>0854-1L</td><td></td></tr><tr><td>0.8% NaCl (Saline)</td><td>Sigma-Aldrich</td><td>SLBB9000V</td><td></td></tr><tr><td>1.2 mL freezer tube</td><td>NEST</td><td>606101</td><td></td></tr><tr><td>Ultra low (-80 &amp;deg;C) freezer</td><td>SO-LOW</td><td>U85-18</td><td></td></tr><tr><td>Cryo Safety Gloves</td><td>Bel-Art</td><td>H13201</td><td></td></tr><tr><td>Petri dishes&amp;nbsp;</td><td>Sigma-Aldrich</td><td>P5856-500EA</td><td></td></tr><tr><td>Cover slips #1.5</td><td>Thomas Scientific</td><td>64-0721</td><td></td></tr><tr><td>Slides</td><td>Carolina</td><td>63-2010</td><td></td></tr><tr><td>Autoclave</td><td>Tuttnauer</td><td>2540E</td><td></td></tr><tr><td>Phase-Contrast Microscope&amp;nbsp;</td><td>Olympus</td><td>BX40</td><td></td></tr><tr><td>Forceps</td><td>Carolina Biological</td><td>624504</td><td></td></tr><tr><td>Bunsen Burner</td><td>Carolina Biological</td><td>706706</td><td></td></tr><tr><td>Methanol</td><td>Sigma-Aldrich</td><td>34860-1L-R</td><td></td></tr><tr><td>PBS (phosphate buffer saline)</td><td>Sigma-Aldrich</td><td>P4417-50TAB</td><td></td></tr><tr><td>WGA-FITC</td><td>Biotium</td><td>29022-1</td><td></td></tr><tr><td>Clear nail polish</td><td>OPI</td><td>22001009000</td><td></td></tr><tr><td>ImageJ</td><td>NIH</td><td>Free Software</td><td></td></tr><tr><td>100 mL beaker</td><td>Pyrex USA</td><td>1000-T</td><td></td></tr><tr><td>1 L beaker</td><td>Carolina Biological</td><td>721253</td><td></td></tr><tr><td>1 L flask</td><td>Fisherbrand</td><td>S63274</td><td></td></tr><tr><td>250 mL flask</td><td>Pyrex USA</td><td>4980</td><td></td></tr><tr><td>100 mL graduated cylinder</td><td>Carolina Biological</td><td>721788</td><td></td></tr><tr><td>500 mL graduated cylinder&amp;nbsp;</td><td>Carolina Biological</td><td>721792</td><td></td></tr><tr><td>Stir Bar</td><td>Fisher Scientific</td><td>22-271825</td><td></td></tr><tr><td>Centrifuge</td><td>Eppendorf</td><td>5810R</td><td></td></tr><tr><td>Camera for Microscope</td><td>Olympus</td><td>DP72</td><td></td></tr><tr><td>Nitrile Examination Gloves (Med)</td><td>Bio Excell</td><td>71011002</td><td></td></tr><tr><td>Vortex Mixer</td><td>Carolina Biological</td><td>701077</td><td></td></tr></tbody></table>

visual and microscopic evaluation of streptomyces developmental mutants

Streptomycetes 是一种丝状的土壤细菌, 属于在世界各地发现的放线菌, 并产生广泛的抗生素和其他次生代谢物。天蓝色链霉菌是一种特性良好的非致病性物种, 可以在实验室进行各种分析。这里描述的分型方法使用天蓝色作为模型链霉菌;然而, 这些方法适用于这一大属的所有成员以及一些紧密相关的放线菌。分型是有必要的特征的新的物种链霉菌确定在环境中, 它也是一个重要的第一步, 以表征新的分离突变株的链霉菌。熟练掌握分型对于进入链霉菌研究领域的许多新研究者来说是很重要的, 其中包括细菌发育、细胞分裂、染色体分离和第二信使信号的研究。最近, 通过隔离新的土壤微生物来进行抗生素发现的众包, 使得在分型的培训更需要为新的链霉菌研究领域的讲师和他们的学院或高中生。本手稿通过目视和显微检查来描述细菌菌种的传播、贮存和表征方法。阅读这篇文章后, 新的研究人员 (微生物学教育实验室和公民科学家) 应该能够操纵链霉菌菌株和开始视觉表征实验。

最初的分型实验是必要的, 以表征新的物种和菌株, 可以作为一个免费的方法, 系统学和 dna-dna 杂交实验, 用于表征新物种。由随机诱变方法产生的链霉菌突变体, 如化学, 紫外线, 或座子诱变, 通常是通过直接的, 视觉屏幕上的琼脂板块识别。与野生型、亲本菌株相比, 对链霉菌的菌落进行了检查, 以观察表型的变化。例如, 较浅的彩色空中菌丝可能表明孢子缺陷引起的灰色素含量较低, 或者缺乏模糊外观预示着空中菌丝形成块 (图 1)。许多 streptomycetes 在营养菌丝或周围琼脂中产生色素抗生素。天蓝色生产两种有色抗生素以及两种非色素的抗菌药物。Actinorhodin 是一种蓝色色素的抗生素, undecylprodigiosin 是一种红色色素的抗生素45。经历了最初的视觉蜂群屏幕的菌株, 然后通过裸奔为单一的殖民地传播。
在视觉上识别出可能有趣的突变体后, 菌株受到显微检查。相对比显微镜特别适合于检测链霉菌突变体的发育缺陷, 使用野生型菌株作为控制 (图 2)。野生型S. 天蓝色菌落通常在 MS 琼脂上生长大约两天, 在30摄氏度时产生一个空中菌丝, 长链孢子生长三天。在其他类型的媒体上, 生命周期会略有减慢或更快。在得出有关发育缺陷和延迟的结论时, 必须对突变体与野外类型相同的生长条件进行分析。在琼脂培养基上长时间生长后, 秃发突变体可能产生孢子。一类变种称为白色突变体可能会延迟孢子形成, 显示出孢子丰度的减少, 产生形状和/或大小缺陷的孢子, 或简单地产生较低水平的成熟, 灰色孢子颜料46.到目前为止调查的其他变种人可能在生命周期进展中被延迟或加速, 例如影响信号分子积聚的突变体 (例如, 循环-二级 GMP47)。
上述光镜技术的一个简单的后续行动是荧光显微镜, 利用碘碘化物染色染色体 DNA nucleoids 和荧光标记小麦胚芽凝集素染色孢子隔膜细胞壁。缺乏染色体的孢子将没有红色碘碘化物染色, 而含有比往常少的 DNA 的孢子可能被识别, 如果他们似乎有减少染色 (图 3)。小麦胚芽凝集素可用于染色细胞壁, 并可观察细胞壁染色模式 (即细胞分裂缺陷) 的差异。在图 4中, venezuelae 的野生型菌株,一种最近被用作另一种模式链霉菌的物种, 因为它的生命周期更快, sporulate 在液体45中的能力, 已经被 WGA FITC 染色, 以阐明通常在孢子期间早期看到的分裂间隔的阶梯状阵列。
这里描述的最初的分型策略应导致以下结果: 1) 鉴定突变株的兴趣, 进一步研究;2) 获得有关新发现的突变体的知识以及基因突变的潜在作用;3) 制定后续的一系列实验步骤, 可用于进一步澄清有关基因的作用。在新发现的环境链霉菌的情况下, 研究人员将获得与已有特征的链霉菌物种相比, 潜在新物种的知识。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57373/57373fig1.jpg"> 图 1: 有代表性的琼脂板块的照片, 展示了天蓝色的宏观菌落表型。琼脂板块显示野生型S. 天蓝色菌株 MT1110 的空气菌丝的模糊、灰色视觉形貌, 与各种随机座子插入突变体与发育型的比较。变种人要么缺少一个空中菌丝 [秃头 (栋)], 要么是一个孢子色素减少的空中菌丝 [白色]。座子突变体是使用 mini-Tn5 的插入突变6,7产生的。菌株在琼脂糖上生长了5天, 在30摄氏度。培养皿直径, 100 毫米。
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/57373/57373fig2.jpg"> 图 2: 相对比显微照片, 显示有代表性的天蓝色白色突变株, 含有随机座子插入突变的空气灯丝.(A) 显示的是一种具有代表性的野生型架空灯丝, 经过同步、定时间隔的细胞分裂, 产生均匀形状和大小的孢子 (MT1110)。(BF)其余的面板显示了不同的白色突变体的微观表型, 通过随机座子突变分离。面板B显示了一个没有经过孢子的架空灯丝, 而是在灯丝内产生了膨胀的区域。在C、 D和F中的长箭表明异常大孢子是突变体 MIC42、MIC43 和 TH49 的特征。面板D中的短箭头表示裂解孢子室的一个例子。面板E显示了部分收缩较小的舱室是典型的孢子链产生的突变 TH10。菌株在琼脂糖上生长了5天, 在30摄氏度。突变体与图 1所示的不相关。野生型孢子在长轴上约为1.1 µm。刻度条 = 2 µm.<a href="/files/ftp_upload/57373/57373fig2large.jpg" target="_blank">请点击这里查看这个数字的大版本.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/57373/57373fig3v2.jpg"> 图 3: 具有代表性的显微照片显示天蓝色突变体染色体分离表型。(a) 图表示法显示了一个孢子链 (每个孢子含有染色体 DNA) 的典型野生型、定期间隔染色的外观, 与显示染色体的突变体的间歇染色模式相比较。离析缺陷。(B) 每对面板都显示在左侧的差分干涉对比 (DIC) 图像观察到的同一孢子链, 右侧显示了碘碘化着色的荧光图像。野生型表型 (a, b) 与两个随机座子插入突变体 (c、 d和e、 f) 进行比较。箭表明没有 DNA 的孢子。菌株在琼脂糖上生长了5天, 在30摄氏度。所显示的突变体与图 1和图 2中的变种人无关。刻度条 = 1 µm.<a href="/files/ftp_upload/57373/57373fig3v2large.jpg" target="_blank">请点击这里查看这个数字的大版本.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/57373/57373fig4v3.jpg"> 图 4: WGA-FITC 染色的venezuelae野生型菌株的荧光显微图像.(a) 一张架空灯丝的示意图, 没有凹痕, 也没有明显的孢子迹象, 使用相对比显微镜比较与阶梯状的细胞壁染色阵列, 表明早期的孢子有在相同的灯丝开始使用 WGA-FITC 在荧光显微镜下。(B) 野生型venezuelae的显微照片。(a) 孢子链中存在平滑的架空花丝。(b) 在小组 a 所显示的菌丝内, 在发展的早期阶段, 一条架空灯丝具有阶梯状的细胞壁沉积阵列。箭头表示有规律地间隔形成的交叉墙染色的 WGA-FITC, 在一个单一的空中灯丝同步发展, 因为它经历发育相关的孢子。菌株生长在 MYM (maltose, Y东提取物, malt 萃取物) MYM 琼脂38小时在30摄氏度。刻度条 = 2 µm.<a href="/files/ftp_upload/57373/57373fig4v3large.jpg" target="_blank">请点击这里查看这个数字的大版本.</a>

Watch this Scientific Journal Video about Visual and Microscopic Evaluation of Streptomyces Developmental Mutants at JoVE.com

Visual and Microscopic Evaluation of Streptomyces Developmental Mutants

链霉菌发育突变体的视觉和显微评价

在这里, 我们提出了新手研究员的协议, 以启动分型药理重要的细菌属链霉菌。

Streptomycetes are filamentous soil bacteria belonging to the phylum Actinobacteria that are found throughout the world and produce a wide array of ...

visual-microscopic-evaluation-streptomyces-developmental

Universidad de Cartagena UDEC

Research

JoVE Journal

Immunology and Infection

Visual and Microscopic Evaluation of Streptomyces Developmental Mutants

17.6K 次观看.  Otterbein University. 在这里, 我们提出了新手研究员的协议, 以启动分型药理重要的细菌属链霉菌。

视频: Visual and Microscopic Evaluation of Streptomyces Developmental Mutants

Genetic Manipulation of the Plant Pathogen Ustilago maydis to Study Fungal Biology and Plant Microbe Interactions

Gene deletion plays an important role in the analysis of gene function. One of the most efficient methods to disrupt genes in a targeted manner is the replacement of the entire gene with a selectable marker via homologous recombination. During homologous recombination, exchange of DNA takes place between sequences with high similarity. Therefore, linear genomic sequences flanking a target gene can be used to specifically direct a selectable marker to the desired integration site. Blunt ends of the deletion construct activate the cell&#39;s DNA repair systems and thereby promote integration of the construct either via homologous recombination or by non-homologous-end-joining. In organisms with efficient homologous recombination, the rate of successful gene deletion can reach more than 50% making this strategy a valuable gene disruption system. The smut fungus Ustilago maydis is a eukaryotic model microorganism showing such efficient homologous recombination. Out of its about 6,900 genes, many have been functionally characterized with the help of deletion mutants, and repeated failure of gene replacement attempts points at essential function of the gene. Subsequent characterization of the gene function by tagging with fluorescent markers or mutations of predicted domains also relies on DNA exchange via homologous recombination. Here, we present the U. maydis strain generation strategy in detail using the simplest example, the gene deletion.

Genetic Manipulation of the Plant Pathogen Ustilago maydis to Study Fungal Biology and Plant Microbe Interactions

We describe a robust gene replacement strategy to genetically manipulate the smut fungus Ustilago maydis. This protocol explains how to generate deletion mutants to investigate infection phenotypes. It can be extended to modify genes in any desired way, e.g., by adding a sequence encoding a fluorescent protein tag.

植物病原体的遗传操作<em&gt;玉米黑粉菌</em&gt;研究真菌生物学和植物微生物相互作用

Gene deletion plays an important role in the analysis of gene function. One of the most efficient methods to disrupt genes in a targeted manner is the ...

科研

遗传学

Live Cell Imaging of Bacillus subtilis and Streptococcus pneumoniae using Automated Time-lapse Microscopy

During the last few years scientists became increasingly aware that average data obtained from microbial population based experiments are not representative of the behavior, status or phenotype of single cells. Due to this new insight the number of single cell studies rises continuously (for recent reviews see 1,2,3). However, many of the single cell techniques applied do not allow monitoring the development and behavior of one specific single cell in time (e.g. flow cytometry or standard microscopy).
Here, we provide a detailed description of a microscopy method used in several recent studies 4, 5, 6, 7, which allows following and recording (fluorescence of) individual bacterial cells of Bacillus subtilis and Streptococcus pneumoniae through growth and division for many generations. The resulting movies can be used to construct phylogenetic lineage trees by tracing back the history of a single cell within a population that originated from one common ancestor. This time-lapse fluorescence microscopy method cannot only be used to investigate growth, division and differentiation of individual cells, but also to analyze the effect of cell history and ancestry on specific cellular behavior. Furthermore, time-lapse microscopy is ideally suited to examine gene expression dynamics and protein localization during the bacterial cell cycle. The method explains how to prepare the bacterial cells and construct the microscope slide to enable the outgrowth of single cells into a microcolony. In short, single cells are spotted on a semi-solid surface consisting of growth medium supplemented with agarose on which they grow and divide under a fluorescence microscope within a temperature controlled environmental chamber. Images are captured at specific intervals and are later analyzed using the open source software ImageJ.

Live Cell Imaging of Bacillus subtilis and Streptococcus pneumoniae using Automated Time-lapse Microscopy

This protocol provides a step-by-step procedure to monitor single cell behavior of different bacteria in time using automated fluorescence time-lapse microscopy. Furthermore, we provide guidelines how to analyze the microscopy images.

活细胞成像枯草芽孢杆菌肺炎链球菌使用自动定时显微镜

During the last few years scientists became increasingly aware that average data obtained from microbial population based experiments are not ...

免疫与感染

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

Analysis of the Development of a Morphological Phenotype as a Function of Protein Concentration in Budding Yeast

Gene deletion and protein overexpression are common methods for studying functions of proteins. In this article, we describe a protocol for analysis of phenotype development as a function of protein concentration at population and single-cell levels in Saccharomyces cerevisiae. Although this protocol is based on the overexpression of a protein, it can easily be adapted for morphological phenotypes dependent on suppression of protein expression. Our lab is interested in studying the signaling properties of the endocytic adaptor protein epsin. To that purpose we used a dominant negative approach in which we over-expressed the conserved Epsin N-Terminal Homology (ENTH) domain in order to interfere with the functions of endogenous epsin-2 (Ent2 or YLR206W). We observed that overexpression of the ENTH domain of Ent2 (ENTH2) in wild type cells led to a cell division defect that is dependent on the mislocalization of a family of scaffolding proteins, septins.

Gene deletion and protein overexpression are common methods for studying functions of proteins. In this article, we describe a protocol for analysis of phenotype development as a function of protein concentration at population and single-cell levels in Saccharomyces cerevisiae.

在芽殖酵母中的蛋白浓度的功能形态学表型的发展分析

Gene deletion and protein overexpression are common methods for studying functions of proteins. In this article, we describe a protocol for analysis of ...

生物学

Fluorescence Time-lapse Imaging of the Complete S. venezuelae Life Cycle Using a Microfluidic Device

Live-cell imaging of biological processes at the single cell level has been instrumental to our current understanding of the subcellular organization of bacterial cells. However, the application of time-lapse microscopy to study the cell biological processes underpinning development in the sporulating filamentous bacteria Streptomyces has been hampered by technical difficulties. 
Here we present a protocol to overcome these limitations by growing the new model species, Streptomyces venezuelae, in a commercially available microfluidic device which is connected to an inverted fluorescence widefield microscope. Unlike the classical model species, Streptomyces coelicolor, S. venezuelae sporulates in liquid, allowing the application of microfluidic growth chambers to cultivate and microscopically monitor the cellular development and differentiation of S. venezuelae over long time periods. In addition to monitoring morphological changes, the spatio-temporal distribution of fluorescently labeled target proteins can also be visualized by time-lapse microscopy. Moreover, the microfluidic platform offers the experimental flexibility to exchange the culture medium, which is used in the detailed protocol to stimulate sporulation of S. venezuelae in the microfluidic chamber. Images of the entire S. venezuelae life cycle are acquired at specific intervals and processed in the open-source software Fiji to produce movies of the recorded time-series.

Fluorescence Time-lapse Imaging of the Complete S. venezuelae Life Cycle Using a Microfluidic Device

Streptomyces are characterized by a complex life cycle that has been experimentally challenging to study by cell biological means. Here we present a protocol to perform fluorescence time-lapse microscopy of the complete life cycle by growing Streptomyces venezuelae in a microfluidic device.

完整的荧光成像定时<em&gt;秒。 venezuelae</em&gt;生命周期使用微流体设备

Live-cell imaging of biological processes at the single cell level has been instrumental to our current understanding of the subcellular organization of ...

Preparation of Adult Drosophila Eyes for Thin Sectioning and Microscopic Analysis

Drosophila has long been used as model system to study development, mainly due to the ease with which it is genetically tractable. Over the years, a plethora of mutant strains and technical tricks have been developed to allow sophisticated questions to be asked and answered in a reasonable amount of time. Fundamental insight into the interplay of components of all known major signaling pathways has been obtained in forward and reverse genetic Drosophila studies. The fly eye has proven to be exceptionally well suited for mutational analysis, since, under laboratory conditions, flies can survive without functional eyes. Furthermore, the surface of the insect eye is composed of some 800 individual unit eyes (facets or ommatidia) that form a regular, smooth surface when looked at under a dissecting microscope. Thus, it is easy to see whether a mutation might affect eye development or growth by externally looking for the loss of the smooth surface ('rough eye' phenotype; Fig. 1) or overall eye size, respectively (for examples of screens based on external eye morphology see e.g.1). Subsequent detailed analyses of eye phenotypes require fixation, plastic embedding and thin-sectioning of adult eyes.
The Drosophila eye develops from the so-called eye imaginal disc, a bag of epithelial cells that proliferate and differentiate during larval and pupal stages (for review see e.g. 2). Each ommatidium consists of 20 cells, including eight photoreceptors (PR or R-cells; Fig. 2), four lens-secreting cone cells, pigment cells ('hexagon' around R-cell cluster) and a bristle. The photoreceptors of each ommatidium, most easily identified by their light sensitive organelles, the rhabdomeres, are organized in a trapezoid made up of the six "outer" (R1-6) and two "inner" photoreceptors (R7/8; R8 [Fig. 2] is underneath R7 and thus only seen in sections from deeper areas of the eye). The trapezoid of each facet is precisely aligned with those of its neighbors and the overall anteroposterior and dorsoventral axes of the eye (Fig. 3A). In particular, the ommatidia of the dorsal and ventral (black and red arrows, respectively) halves of the eye are mirror images of each other and correspond to two chiral forms established during planar cell polarity signaling (for review see e.g. 3).
The method to generate semi-thin eye sections (such as those presented in Fig. 3) described here is slightly modified from the one originally described by Tomlinson and Ready4. It allows the morphological analysis of all cells except for the transparent cone cells. In addition, the pigment of R-cells (blue arrowheads in Fig. 2 and 3) can be used as a cell-autonomous marker for the genotype of a R-cell, thus genetic requirements of genes in a subset of R-cells can readily be determined5,6.

Preparation of Adult Drosophila Eyes for Thin Sectioning and Microscopic Analysis

A standard approach to prepare adult Drosophila eyes for semi-thin sectioning and light microscopic analysis is presented here. The protocol can be used for gross morphological analysis of eye defects, or with the indicated adjustments can be used to determine genetic requirements of genes in specific cell types of the eye (e.g. clonal analysis of photoreceptors) or for electron microscopic analysis.

成人的制备果蝇</em薄切片和显微镜分析>的眼睛

Drosophila has long been used as model system to study development, mainly due to the ease with which it is genetically tractable. Over the years, a ...

Visualization of Germinosomes and the Inner Membrane in Bacillus subtilis Spores

The small size of spores and the relatively low abundance of germination proteins, cause difficulties in their microscopic analyses using epifluorescence microscopy. Super-resolution three-dimensional Structured Illumination Microscopy (3D-SIM) is a promising tool to overcome this hurdle and reveal the molecular details of the process of germination of Bacillus subtilis (B. subtilis) spores. Here, we describe the use of a modified SIMcheck (ImageJ)-assistant 3D imaging process and fluorescent reporter proteins for SIM microscopy of B. subtilis spores&#8217; germinosomes, cluster(s) of germination proteins. We also present a (standard)3D-SIM imaging procedure for FM4-64 staining of B. subtilis spore membranes. By using these procedures, we obtained unsurpassed resolution for germinosome localization and show that &#62;80% of B. subtilis KGB80 dormant spores obtained after sporulation on defined minimal MOPS medium have one or two GerD-GFP and GerKB-mCherry foci. Bright foci were also observed in FM4-64 stained spores&#8217; 3D-SIM images suggesting that inner membrane lipid domains of different fluidity likely exist. Further studies that use double labeling procedures with membrane dyes and germinosome reporter proteins to assess co-localization and thus get an optimal overview of the organization of Bacillus germination proteins in the inner spore membrane are possible.

Visualization of Germinosomes and the Inner Membrane in Bacillus subtilis Spores

Germinant receptor proteins cluster in &#8216;germinosomes&#8217; in the inner membrane of Bacillus subtilis spores. We describe a protocol using super resolution microscopy and fluorescent reporter proteins to visualize germinosomes. The protocol also identifies spore inner membrane domains that are preferentially stained with the membrane dye FM4-64.

枯草芽孢杆菌中细菌体和内膜的可视化

The small size of spores and the relatively low abundance of germination proteins, cause difficulties in their microscopic analyses using epifluorescence ...

生物工程

Live-Cell Forward Genetic Approach to Identify and Isolate Developmental Mutants in Chlamydia trachomatis

The intracellular bacterial pathogen Chlamydia trachomatis undergoes a developmental cycle consisting of two morphologically discrete developmental forms. The non-replicative elementary body (EB) initiates infection of the host. Once inside, the EB differentiates into the reticulate body (RB). The RB then undergoes multiple rounds of replication, before differentiating back to the infectious EB form. This cycle is essential for chlamydial survival as failure to switch between cell types prevents either host invasion or replication.
Limitations in genetic techniques due to the obligate intracellular nature of Chlamydia have hampered identification of the molecular mechanisms involved in the cell-type development. We designed a novel dual promoter-reporter plasmid system that, in conjunction with live-cell microscopy, allows for the visualization of cell type switching in real time. To identify genes involved in the regulation of cell-type development, the live-cell promoter-reporter system was leveraged for the development of a forward genetic approach by combining chemical mutagenesis of the dual reporter strain, imaging and tracking of Chlamydia with altered developmental kinetics, followed by clonal isolation of mutants. This forward genetic workflow is a flexible tool that can be modified for directed interrogation into a wide range of genetic pathways.

Live-Cell Forward Genetic Approach to Identify and Isolate Developmental Mutants in Chlamydia trachomatis

This protocol utilizes fluorescent promoter-reporters, live-cell microscopy, and individual inclusion extraction in a directed forward genetic approach to identify and isolate developmental mutants of Chlamydia trachomatis.

活细胞向前遗传方法，识别和分离衣 原体沙眼中的发育突变体

The intracellular bacterial pathogen Chlamydia trachomatis undergoes a developmental cycle consisting of two morphologically discrete developmental forms. ...

Use of In Vivo Imaging to Screen for Morphogenesis Phenotypes in Candida albicans Mutant Strains During Active Infection in a Mammalian Host

Candida albicans is an important human pathogen. Its ability to switch between morphologic forms is central to&#160;its pathogenesis; these morphologic changes are regulated by a complex signaling network controlled in response to environmental stimuli. These regulatory components have been highly studied, but almost all studies use a variety of in vitro stimuli to trigger filamentation. To determine how morphogenesis is regulated during the pathogenesis process, we developed an in vivo microscopy system to obtain high spatial resolution images of organisms undergoing hyphal formation within the mammalian host. The protocol presented here describes the use of this system to screen small collections of C. albicans mutant strains, allowing us to identify key regulators of morphogenesis as it occurs at the site of infection. Representative results are presented, demonstrating that some regulators of morphogenesis, such as the transcriptional regulator Efg1, have consistent phenotypes in vitro and in vivo, whereas other regulators, such as adenyl cyclase (Cyr1), have significantly different phenotypes in vivo compared to in vitro.

Use of In Vivo Imaging to Screen for Morphogenesis Phenotypes in Candida albicans Mutant Strains During Active Infection in a Mammalian Host

This manuscript describes a method for screening moderately sized Candida albicans mutant libraries for morphogenesis phenotypes during active infection in a mammalian host using non-invasive confocal microscopy.

使用 体内 成像筛选哺乳动物宿主活动性感染期间 白色念珠 菌突变菌株的形态发生表型

Candida albicans is an important human pathogen. Its ability to switch between morphologic forms is central to&#160;its pathogenesis; these morphologic ...

Microscopy Techniques for Interpreting Fungal Colonization in Mycoheterotrophic Plants Tissues and Symbiotic Germination of Seeds

Structural botany is an indispensable perspective to fully understand the ecology, physiology, development, and evolution of plants. When researching mycoheterotrophic plants (i.e., plants that obtain carbon from fungi), remarkable aspects of their structural adaptations, the patterns of tissue colonization by fungi, and the morphoanatomy of subterranean organs can enlighten their developmental strategies and their relationships with hyphae, the source of nutrients. Another important role of symbiotic fungi is related to the germination of orchid seeds; all Orchidaceae species are mycoheterotrophic during germination and seedling stage (initial mycoheterotrophy), even the ones that photosynthesize in adult stages. Due to the lack of nutritional reserves in orchid seeds, fungal symbionts are essential to provide substrates and enable germination. Analyzing germination stages by structural perspectives can also answer important questions regarding the fungi interaction with the seeds. Different imaging techniques can be applied to unveil fungi endophytes in plant tissues, as are proposed in this article. Freehand and thin sections of plant organs can be stained and then observed using light microscopy. A fluorochrome conjugated to wheat germ agglutinin can be applied to the fungi and co-incubated with Calcofluor White to highlight plant cell walls in confocal microscopy. In addition, the methodologies of scanning and transmission electron microscopy are detailed for mycoheterotrophic orchids, and the possibilities of applying such protocols in related plants is explored. Symbiotic germination of orchid seeds (i.e., in the presence of mycorrhizal fungi) is described in the protocol in detail, along with possibilities of preparing the structures obtained from different stages of germination for analyses with light, confocal, and electron microscopy.

This protocol aims to provide detailed procedures for collecting, fixing, and maintaining mycoheterotrophic plant samples, applying different microscopy techniques such as scanning and transmission electron microscopy, light, confocal, and fluorescence microscopy to study fungal colonization in plants tissues and seeds germinated with mycorrhizal fungi.

用于解释霉菌异养植物组织中真菌定植和种子共生萌发的显微镜技术

Structural botany is an indispensable perspective to fully understand the ecology, physiology, development, and evolution of plants. When researching ...

链霉菌发育突变体的视觉和显微评价

摘要

探索更多视频

此视频中的章节

植物病原体的遗传操作

活细胞成像枯草芽孢杆菌肺炎链球菌使用自动定时显微镜

线虫中耳炎群链球菌引起氧化应激的研究

在芽殖酵母中的蛋白浓度的功能形态学表型的发展分析

完整的荧光成像定时

成人的制备果蝇的眼睛

枯草芽孢杆菌中细菌体和内膜的可视化

活细胞向前遗传方法，识别和分离衣原体沙眼中的发育突变体

使用体内成像筛选哺乳动物宿主活动性感染期间白色念珠菌突变菌株的形态发生表型

用于解释霉菌异养植物组织中真菌定植和种子共生萌发的显微镜技术

链霉菌发育突变体的视觉和显微评价

摘要

探索更多视频

此视频中的章节

植物病原体的遗传操作

活细胞成像枯草芽孢杆菌肺炎链球菌使用自动定时显微镜

线虫中耳炎群链球菌引起氧化应激的研究

在芽殖酵母中的蛋白浓度的功能形态学表型的发展分析

完整的荧光成像定时

成人的制备果蝇的眼睛

枯草芽孢杆菌中细菌体和内膜的可视化

活细胞向前遗传方法，识别和分离衣 原体沙眼中的发育突变体

使用 体内 成像筛选哺乳动物宿主活动性感染期间 白色念珠 菌突变菌株的形态发生表型

用于解释霉菌异养植物组织中真菌定植和种子共生萌发的显微镜技术

活细胞向前遗传方法，识别和分离衣原体沙眼中的发育突变体

使用体内成像筛选哺乳动物宿主活动性感染期间白色念珠菌突变菌株的形态发生表型