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 border="0" cellpadding="0" cellspacing="0" width="543">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:197px;">50% Glycerol</td>
			<td style="width:161px;">Sigma-Aldrich</td>
			<td style="width:121px;">G5516</td>
			<td style="width:64px;"></td>
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			<td height="20" style="height:20px;">Immersion Oil (Type DF)</td>
			<td>Cragille&#160;</td>
			<td>16482</td>
			<td></td>
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			<td height="20" style="height:20px;">Lens Paper</td>
			<td>Fisherbrand</td>
			<td>11-995</td>
			<td></td>
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		<tr height="20">
			<td height="20" style="height:20px;">Sterile Water</td>
			<td>GeneMate</td>
			<td>G-3250-1L</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">100% Ethanol</td>
			<td>Sigma-Aldrich</td>
			<td>E7023</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Propidium Iodide</td>
			<td>Invitrogen</td>
			<td>P1304MP</td>
			<td></td>
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			<td height="20" style="height:20px;">Toothpicks (flat)</td>
			<td>Target</td>
			<td>081-22-1957</td>
			<td></td>
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			<td height="20" style="height:20px;">Pipette Tips (10 ml)</td>
			<td>GeneMate</td>
			<td>P-1240-10</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Pipette Tips (200 ml)</td>
			<td>GeneMate</td>
			<td>P-1240-200Y</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Pipette Tips (1250 ml)</td>
			<td>GeneMate</td>
			<td>P-1240-1250</td>
			<td></td>
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			<td height="20" style="height:20px;">Wooden Applicators 6&#39;&#39;</td>
			<td>Solon Care</td>
			<td>070809</td>
			<td></td>
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		<tr height="20">
			<td height="20" style="height:20px;">Cotton Swabs</td>
			<td>Fisherbrand</td>
			<td>23-400-124</td>
			<td></td>
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			<td height="20" style="height:20px;">Soy Flour&#160;</td>
			<td>Bob&#39;s Red Mill&#160;</td>
			<td>1516C164</td>
			<td></td>
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		<tr height="20">
			<td height="20" style="height:20px;">D-Mannitol</td>
			<td>Sigma-Aldrich</td>
			<td>M4125-1KG</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Agar</td>
			<td>Sigma-Aldrich</td>
			<td>A1296-1KG</td>
			<td></td>
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		<tr height="20">
			<td height="20" style="height:20px;">Glycerol 100%</td>
			<td>VWR amresco life science&#160;</td>
			<td>0854-1L</td>
			<td></td>
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		<tr height="20">
			<td height="20" style="height:20px;">0.8% NaCl (Saline)</td>
			<td>Sigma-Aldrich</td>
			<td>SLBB9000V</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">1.2 mL freezer tube</td>
			<td>NEST</td>
			<td>606101</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Ultra low (-80&#176;C) freezer</td>
			<td>SO-LOW</td>
			<td>U85-18</td>
			<td></td>
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			<td height="20" style="height:20px;">Cryo Safety Gloves</td>
			<td>Bel-Art</td>
			<td>H13201</td>
			<td></td>
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			<td height="20" style="height:20px;">Petri dishes&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>P5856-500EA</td>
			<td></td>
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			<td height="20" style="height:20px;">Cover slips #1.5</td>
			<td>Thomas Scientific</td>
			<td>64-0721</td>
			<td></td>
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			<td height="20" style="height:20px;">Slides</td>
			<td>Carolina</td>
			<td>63-2010</td>
			<td></td>
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		<tr height="20">
			<td height="20" style="height:20px;">Autoclave</td>
			<td>Tuttnauer</td>
			<td>2540E</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Phase-Contrast Microscope&#160;</td>
			<td>Olympus</td>
			<td>BX40</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Forceps</td>
			<td>Carolina Biological</td>
			<td>624504</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Bunsen Burner</td>
			<td>Carolina Biological</td>
			<td>706706</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Methanol</td>
			<td>Sigma-Aldrich</td>
			<td>34860-1L-R</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">PBS (phosphate buffer saline)</td>
			<td>Sigma-Aldrich</td>
			<td>P4417-50TAB</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">WGA-FITC</td>
			<td>Biotium</td>
			<td>29022-1</td>
			<td></td>
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			<td height="20" style="height:20px;">Clear nail polish</td>
			<td>OPI</td>
			<td>22001009000</td>
			<td></td>
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		<tr height="20">
			<td height="20" style="height:20px;">Image J</td>
			<td>NIH</td>
			<td>Free Software</td>
			<td></td>
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		<tr height="20">
			<td height="20" style="height:20px;">100 mL beaker</td>
			<td>Pyrex USA</td>
			<td>1000-T</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">1 L beaker</td>
			<td>Carolina Biological</td>
			<td>721253</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">1 L flask</td>
			<td>Fisherbrand</td>
			<td>S63274</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">250 mL flask</td>
			<td>Pyrex USA</td>
			<td>4980</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">100 mL graduated cylinder</td>
			<td>Carolina Biological</td>
			<td>721788</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">500 mL graduated cylinder&#160;</td>
			<td>Carolina Biological</td>
			<td>721792</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Stir Bar</td>
			<td>Fisher Scientific</td>
			<td>22-271825</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Centrifuge</td>
			<td>Eppendorf</td>
			<td>5810R</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Camera for Microscope</td>
			<td>Olympus</td>
			<td>DP72</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Nitrile Examination Gloves (Med)</td>
			<td>Bio Excell</td>
			<td>71011002</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Vortex Mixer</td>
			<td>Carolina Biological</td>
			<td>701077</td>
			<td></td>
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</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

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

Facilitating the Analysis of Immunological Data with Visual Analytic Techniques

Visual analytics (VA) has emerged as a new way to analyze large dataset through interactive visual display. We demonstrated the utility and the flexibility of a VA approach in the analysis of biological datasets. Examples of these datasets in immunology include flow cytometry, Luminex data, and genotyping (e.g., single nucleotide polymorphism) data. Contrary to the traditional information visualization approach, VA restores the analysis power in the hands of analyst by allowing the analyst to engage in real-time data exploration process. We selected the VA software called Tableau after evaluating several VA tools. Two types of analysis tasks analysis within and between datasets were demonstrated in the video presentation using an approach called paired analysis. Paired analysis, as defined in VA, is an analysis approach in which a VA tool expert works side-by-side with a domain expert during the analysis. The domain expert is the one who understands the significance of the data, and asks the questions that the collected data might address. The tool expert then creates visualizations to help find patterns in the data that might answer these questions. The short lag-time between the hypothesis generation and the rapid visual display of the data is the main advantage of a VA approach.

Visual analytics (VA) is a new approach of analyzing data interactively. In this video, we discuss the data overload problem brought on by high-throughput biological experiments, and propose VA as a solution to such problem. The video demonstrates analysis within and between immunological datasets using a VA tool called Tableau.

促进免疫学数据分析与视觉分析技术

Visual analytics (VA) has emerged as a new way to analyze large dataset through interactive visual display. We demonstrated the utility and the ...

科研

免疫与感染

A Genetic Screen to Isolate Toxoplasma gondii Host-cell Egress Mutants

The widespread, obligate intracellular, protozoan parasite Toxoplasma gondii causes opportunistic disease in immuno-compromised patients and causes birth defects upon congenital infection. The lytic replication cycle is characterized by three stages: 1. active invasion of a nucleated host cell; 2. replication inside the host cell; 3. active egress from the host cell. The mechanism of egress is increasingly being appreciated as a unique, highly regulated process, which is still poorly understood at the molecular level. The signaling pathways underlying egress have been characterized through the use of pharmacological agents acting on different aspects of the pathways1-5. As such, several independent triggers of egress have been identified which all converge on the release of intracellular Ca2+, a signal that is also critical for host cell invasion6-8. This insight informed a candidate gene approach which led to the identification of plant like calcium dependent protein kinase (CDPK) involved in egress9. In addition, several recent breakthroughs in understanding egress have been made using (chemical) genetic approaches10-12. To combine the wealth of pharmacological information with the increasing genetic accessibility of Toxoplasma we recently established a screen permitting the enrichment for parasite mutants with a defect in host cell egress13. Although chemical mutagenesis using N-ethyl-N-nitrosourea (ENU) or ethyl methanesulfonate (EMS) has been used for decades in the study of Toxoplasma biology11,14,15, only recently has genetic mapping of mutations underlying the phenotypes become routine16-18. Furthermore, by generating temperature-sensitive mutants, essential processes can be dissected and the underlying genes directly identified. These mutants behave as wild-type under the permissive temperature (35 &deg;C), but fail to proliferate at the restrictive temperature (40 &deg;C) as a result of the mutation in question. Here we illustrate a new phenotypic screening method to isolate mutants with a temperature-sensitive egress phenotype13. The challenge for egress screens is to separate egressed from non-egressed parasites, which is complicated by fast re-invasion and general stickiness of the parasites to host cells. A previously established egress screen was based on a cumbersome series of biotinylation steps to separate intracellular from extracellular parasites11. This method also did not generate conditional mutants resulting in weak phenotypes. The method described here overcomes the strong attachment of egressing parasites by including a glycan competitor, dextran sulfate (DS), that prevents parasites from sticking to the host cell19. Moreover, extracellular parasites are specifically killed off by pyrrolidine dithiocarbamate (PDTC), which leaves intracellular parasites unharmed20. Therefore, with a new phenotypic screen to specifically isolate parasite mutants with defects in induced egress, the power of genetics can now be fully deployed to unravel the molecular mechanisms underlying host cell egress.

A Genetic Screen to Isolate Toxoplasma gondii Host-cell Egress Mutants

Forward genetics is a powerful method to unravel the molecular level of how Toxoplasma egresses from its host cell. Protocols are provided to chemically mutagenize parasites, enrich for mutants with defects in induced egress, and validate the phenotype of cloned mutants.

遗传筛选隔离弓形虫宿主细胞的出口突变

The widespread, obligate intracellular, protozoan parasite Toxoplasma gondii causes opportunistic disease in immuno-compromised patients and causes birth ...

A Visual Assay to Monitor T6SS-mediated Bacterial Competition

Type VI secretion systems (T6SSs) are molecular nanomachines allowing Gram-negative bacteria to transport and inject proteins into a wide variety of target cells1,2. The T6SS is composed of 13 core components and displays structural similarities with the tail-tube of bacteriophages3. The phage uses a tube and a puncturing device to penetrate the cell envelope of target bacteria and inject DNA. It is proposed that the T6SS is an inverted bacteriophage device creating a specific path in the bacterial cell envelope to drive effectors and toxins to the surface. The process could be taken further and the T6SS device could perforate other cells with which the bacterium is in contact, thus injecting the effectors into these targets. The tail tube and puncturing device parts of the T6SS are made with Hcp and VgrG proteins, respectively4,5.
The versatility of the T6SS has been demonstrated through studies using various bacterial pathogens. The Vibrio cholerae T6SS can remodel the cytoskeleton of eukaryotic host cells by injecting an "evolved" VgrG carrying a C-terminal actin cross-linking domain6,7. Another striking example was recently documented using Pseudomonas aeruginosa which is able to target and kill bacteria in a T6SS-dependent manner, therefore promoting the establishment of bacteria in specific microbial niches and competitive environment8,9,10.
In the latter case, three T6SS-secreted proteins, namely Tse1, Tse2 and Tse3 have been identified as the toxins injected in the target bacteria (Figure 1). The donor cell is protected from the deleterious effect of these effectors via an anti-toxin mechanism, mediated by the Tsi1, Tsi2 and Tsi3 immunity proteins8,9,10. This antimicrobial activity can be monitored when T6SS-proficient bacteria are co-cultivated on solid surfaces in competition with other bacterial species or with T6SS-inactive bacteria of the same species8,11,12,13.
The data available emphasized a numerical approach to the bacterial competition assay, including time-consuming CFU counting that depends greatly on antibiotic makers. In the case of antibiotic resistant strains like P. aeruginosa, these methods can be inappropriate. Moreover, with the identification of about 200 different T6SS loci in more than 100 bacterial genomes14, a convenient screening tool is highly desirable. We developed an assay that is easy to use and requires standard laboratory material and reagents. The method offers a rapid and qualitative technique to monitor the T6SS-dependent bactericidal/bacteriostasis activity by using a reporter strain as a prey (in this case Escherichia coli DH5&alpha;) allowing a-complementation of the lacZ gene. Overall, this method is graphic and allows rapid identification of T6SS-related phenotypes on agar plates. This experimental protocol may be adapted to other strains or bacterial species taking into account specific conditions such as growth media, temperature or time of contact.

We describe a qualitative assay to monitor bacterial competition mediated by the Pseudomonas aeruginosa type VI secretion system (T6SS). The assay relies on the survival/killing of Escherichia coli target cells carrying a lacZ-reporter. This technique is adjustable to assess the bactericidal/bacteriostasis activity of T6SS-proficient microorganisms.

的视觉检测，以监测T6SS介导的细菌竞争

Type VI secretion systems (T6SSs) are molecular  nanomachines allowing Gram-negative bacteria to transport and inject proteins  into a wide variety of ...

High-throughput Quantitative Real-time RT-PCR Assay for Determining Expression Profiles of Types I and III Interferon Subtypes

Described in this report is a qRT-PCR assay for the analysis of seventeen human IFN subtypes in a 384-well plate format that incorporates highly specific locked nucleic acid (LNA) and molecular beacon (MB) probes, transcript standards, automated multichannel pipetting, and plate drying. Determining expression among the type I interferons (IFN), especially the twelve IFN-&#945; subtypes, is limited by their shared sequence identity; likewise, the sequences of the type III IFN, especially IFN-&#955;2 and -&#955;3, are highly similar. This assay provides a reliable, reproducible, and relatively inexpensive means to analyze the expression of the seventeen interferon subtype transcripts.

This protocol describes a high-throughput qRT-PCR assay for the analysis of type I and III IFN expression signatures. The assay discriminates single base pair differences between the highly similar transcripts of these genes. Through batch assembly and robotic pipetting, the assays are consistent and reproducible.

高通量实时定量RT-PCR检测方法的确定I型和III型干扰素亚型的表达谱

Described in this report is a qRT-PCR assay for the analysis of seventeen human IFN subtypes in a 384-well plate format that incorporates highly specific ...

Generation and Multi-phenotypic High-content Screening of Coxiella burnetii Transposon Mutants

Invasion and colonization of host cells by bacterial pathogens depend on the activity of a large number of prokaryotic proteins, defined as virulence factors, which can subvert and manipulate key host functions. The study of host/pathogen interactions is therefore extremely important to understand bacterial infections and develop alternative strategies to counter infectious diseases. This approach however, requires the development of new high-throughput assays for the unbiased, automated identification and characterization of bacterial virulence determinants. Here, we describe a method for the generation of a GFP-tagged mutant library by transposon mutagenesis and the development of high-content screening approaches for the simultaneous identification of multiple transposon-associated phenotypes. Our working model is the intracellular bacterial pathogen Coxiellaburnetii, the etiological agent of the zoonosis Q fever, which is associated with severe outbreaks with a consequent health and economic burden. The obligate intracellular nature of this pathogen has, until recently, severely hampered the identification of bacterial factors involved in host pathogen interactions, making of Coxiella the ideal model for the implementation of high-throughput/high-content approaches.

Generation and Multi-phenotypic High-content Screening of Coxiella burnetii Transposon Mutants

Coxiella burnetii is an obligate intracellular Gram-negative bacterium responsible for the zoonotic disease Q fever. Here we describe methods for the generation of Coxiella fluorescent transposon mutants as well as the automated identification and analysis of the resulting internalization, replication and cytotoxic phenotypes.

生成和多表型高内涵筛选<em&gt;贝氏柯克斯体</em&gt;转座子突变体

Invasion and colonization of host cells by bacterial pathogens depend on the activity of a large number of prokaryotic proteins, defined as virulence ...

Evaluation of Extracellular Vesicle Function During Malaria Infection

Malaria is a life-threatening disease caused by Plasmodium parasites, with P. falciparum being the most prevalent on the African continent and responsible for most malaria-related deaths globally. Several factors including parasite sequestration in tissues, vascular dysfunction, and inflammatory responses influence the evolution of the disease in malaria-infected people. P. falciparum-infected red blood cells (iRBCs) release small extracellular vesicles (EVs) containing different kinds of cargo molecules that mediate pathogenesis and cellular communication between parasites and host. EVs are efficiently taken up by cells in which they modulate their function. Here we discuss strategies to address the role of EVs in parasite-host interactions. First, we describe a straightforward method for labeling and tracking EV internalization by endothelial cells, using a green cell linker dye. Second, we report a simple way to measure permeability across an endothelial cell monolayer by using a fluorescently labeled dextran. Finally, we show how to investigate the role of small non-coding RNA molecules in endothelial cell function.

In this work, we describe protocols to investigate the role of extracellular vesicles (EVs) released by Plasmodium falciparum infected erythrocytes. In particular, we focus on the interactions of EVs with endothelial cells.

疟疾感染中胞外囊泡功能的评价

Malaria is a life-threatening disease caused by Plasmodium parasites, with P. falciparum being the most prevalent on the African continent and responsible ...

Paraffin Embedding and Thin Sectioning of Microbial Colony Biofilms for Microscopic Analysis

Sectioning via paraffin embedding is a broadly established technique in eukaryotic systems. Here we provide a method for the fixation, embedding, and sectioning of intact microbial colony biofilms using perfused paraffin wax. To adapt this method for use on colony biofilms, we developed techniques for maintaining each sample on its growth substrate and laminating it with an agar overlayer, and added lysine to the fixative solution. These optimizations improve sample retention and preservation of micromorphological features. Samples prepared in this manner are amenable to thin sectioning and imaging by light, fluorescence, and transmission electron microscopy. We have applied this technique to colony biofilms of Pseudomonas aeruginosa, Pseudomonas synxantha, Bacillus subtilis, and Vibrio cholerae. The high level of detail visible in samples generated by this method, combined with reporter strain engineering or the use of specific dyes, can provide exciting insights into the physiology and development of microbial communities.

We describe fixation, paraffin embedding, and thin sectioning techniques for microbial colony biofilms. In prepared samples, biofilm substructure and reporter expression patterns can be visualized by microscopy.

微生物菌落生物膜的石蜡嵌入和薄切片显微分析

Sectioning via paraffin embedding is a broadly established technique in eukaryotic systems. Here we provide a method for the fixation, embedding, and ...

Replication of the Ordered, Nonredundant Library of Pseudomonas aeruginosa strain PA14 Transposon Insertion Mutants

Pseudomonas aeruginosa is a phenotypically and genotypically diverse and adaptable Gram-negative bacterium ubiquitous in human environments. P. aeruginosa is able to form biofilms, develop antibiotic resistance, produce virulence factors, and rapidly evolve in the course of a chronic infection. Thus P. aeruginosa can cause both acute and chronic, difficult to treat infections, resulting in significant morbidity in certain patient populations. P. aeruginosa strain PA14 is a human clinical isolate with a conserved genome structure that infects a variety of mammalian and nonvertebrate hosts making PA14 an attractive strain for studying this pathogen. In 2006, a nonredundant transposon insertion mutant library containing 5,459 mutants corresponding to 4,596 predicted PA14 genes was generated. Since then, distribution of the PA14 library has allowed the research community to better understand the function of individual genes and complex pathways of P. aeruginosa. Maintenance of library integrity through the replication process requires proper handling and precise techniques. To that end, this manuscript presents protocols that describe in detail the steps involved in library replication, library quality control and proper storage of individual mutants.

Replication of the Ordered, Nonredundant Library of Pseudomonas aeruginosa strain PA14 Transposon Insertion Mutants

Pseudomonas aeruginosa infection causes significant morbidity in vulnerable hosts. The nonredundant transposon insertion mutant library of P. aeruginosa strain PA14, designated as PA14NR Set, facilitates analysis of gene functionality in numerous processes. Presented here is a protocol to generate high-quality copies of the PA14NR Set mutant library.

复制的有序, 非冗余图书馆的铜绿假单胞菌应变 PA14 座子插入突变体

Pseudomonas aeruginosa is a phenotypically and genotypically diverse and adaptable Gram-negative bacterium ubiquitous in human environments. P. aeruginosa ...

Complementary Use of Microscopic Techniques and Fluorescence Reading in Studying Cryptococcus-Amoeba Interactions

To simulate Cryptococcus infection, amoeba, which is the natural predator of cryptococcal cells in the environment, can be used as a model for macrophages. This predatory organism, similar to macrophages, employs phagocytosis to kill internalized cells. With the aid of a confocal laser-scanning microscope, images depicting interactive moments between cryptococcal cells and amoeba are captured. The resolution power of the electron microscope also helps to reveal the ultrastructural detail of cryptococcal cells when trapped inside the amoeba food vacuole. Since phagocytosis is a continuous process, quantitative data is then integrated in the analysis to explain what happens at the timepoint when an image is captured. To be specific, relative fluorescence units are read in order to quantify the efficiency of amoeba in internalizing cryptococcal cells. For this purpose, cryptococcal cells are stained with a dye that makes them fluoresce once trapped inside the acidic environment of the food vacuole. When used together, information gathered through such techniques can provide critical information to help draw conclusions on the behavior and fate of cells when internalized by amoeba and, possibly, by other phagocytic cells.

Complementary Use of Microscopic Techniques and Fluorescence Reading in Studying Cryptococcus-Amoeba Interactions

This paper details a protocol for preparing a co-culture of cryptococcal cells and amoebae that is studied using still, fluorescent images and high-resolution transmission electron microscope images. Illustrated here is how quantitative data can complement such qualitative information.

在研究隐球菌-阿米巴相互作用中,显微技术和荧光读数的补充应用

To simulate Cryptococcus infection, amoeba, which is the natural predator of cryptococcal cells in the environment, can be used as a model for ...

Rapid, Seamless Generation of Recombinant Poxviruses using Host Range and Visual Selection

Vaccinia virus (VACV) was instrumental in eradicating variola virus (VARV), the causative agent of smallpox, from nature. Since its first use as a vaccine, VACV has been developed as a vector for therapeutic vaccines and as an oncolytic virus. These applications take advantage of VACV&#8217;s easily manipulated genome and broad host range as an outstanding platform to generate recombinant viruses with a variety of therapeutic applications. Several methods have been developed to generate recombinant VACV, including marker selection methods and transient dominant selection. Here, we present a refinement of a host range selection method coupled with visual identification of recombinant viruses. Our method takes advantage of selective pressure generated by the host antiviral protein kinase R (PKR) coupled with a fluorescent fusion gene expressing mCherry-tagged E3L, one of two VACV PKR antagonists. The cassette, including the gene of interest and the mCherry-E3L fusion is flanked by sequences derived from the VACV genome. Between the gene of interest and mCherry-E3L is a smaller region that is identical to the first ~150 nucleotides of the 3&#8217; arm, to promote homologous recombination and loss of the mCherry-E3L gene after selection. We demonstrate that this method permits efficient, seamless generation of rVACV in a variety of cell types without requiring drug selection or extensive screening for mutant viruses.

This is a method to generate &#8220;scarless&#8221; recombinant vaccinia viruses using host-range selection and visual identification of recombinant viruses.