<p class="jove_content">本研究报告得到了当地机构审查委员会 (能源公司-2016-00887) 的批准。</p>
<p class="jove_title">1. 标本的准备</p>
<ol><li>将真空床垫放在解剖台上, 用无菌毯覆盖。将解剖标本放置在床垫上的外科位置, 头部微微旋转至对侧。</li>
	<li>收回耳屏与对侧的一个灵活的钩。</li>
	<li>把标本盖在外面的耳朵上, 就像在手术室里一样。<br>注: 请参阅<strong>材料表</strong>以了解所需设备的完整列表。</li>
</ol><p class="jove_title">2. 入门</p>
<ol><li>采用舒适的站立或坐姿, 调整内窥镜屏幕。这是一个重要的问题, 以防止颈部肌肉硬化, 因此更快的疲惫。</li>
	<li>在您的相机菜单中执行白平衡, 并在内窥镜提示上应用一滴雾溶液。</li>
	<li>在你的优势手和内窥镜的另一只手拿着手术器械。</li>
	<li>引入0°, 3 毫米直径, 15 厘米长度内窥镜进入选管会。作为第一项任务, 清洁选管会彻底从最终目前的耳蜡使用吸管。用水冲洗选管会。在选管会内用一把小剪刀剪下头发, 防止内窥镜在每段期间变得肮脏。</li>
	<li>一旦选管会清洁, 检查鼓膜 (TM)。确定:<br>-锤骨短流程, 柄和鳞<br>-鼓膜环<br>-松弛 (Shrappnel 膜)</li>
</ol><p class="jove_title">3. Tympano-道口皮瓣的标高 (TMF)</p>
<p class="jove_content">注: 在内窥镜耳手术中, TMF 的表现略小, 呈三角形。原因是选管会内的空间有限。</p>
<ol><li>用镰刀或圆刀切割锤骨的皮肤, 稍微在短的过程前。</li>
	<li>将切口延伸至选管会后上部, 并将切口延长至 TM 的前、下缘, 以配置三角形状皮瓣。</li>
	<li>用45°角度圆刀, 露出骨骼并将皮肤抬高直到到达环形环。严格遵守骨骼是很重要的。</li>
	<li>用圆刀抬高环隙以进入鼓室腔 (TC)。TC 的粘膜比皮肤略微透明, 在鼓索的水平上用钩切开。</li>
	<li>利用 Thomassin 解剖, 调动环下和前。</li>
	<li>鉴定锤骨 (PLML) 的后侧韧带。这韧带代表了 Prussak 空间的下极限。用哈特曼钳抓住韧带, 在鳞的方向轻轻前, 直到揭开锤骨的短过程。</li>
	<li>轻轻地解剖前 Prussak 的空间, 直到到达前高级插入环。现在确定:<br>环<br>-鼓索<br>-后踝韧带<br>-侧踝 ligamental 褶皱<br>-前踝 ligamental 折叠<br>-Prussak 空间</li>
</ol><p class="jove_title">4. TMF 从锤骨的手柄上脱离</p>
<ol><li>从锤骨的短过程, 继续解剖和切割的 periostium 踝手柄对鳞与针解剖。重要的是要分离 TM 在正确的平面, 以防止意外穿孔。</li>
	<li>从柄完全分离 TM。干燥的 cottonoid 也许是有用的, 因为它促进正确的层数和它的解剖的识别。</li>
	<li>一旦到达鳞, 用微剪刀切割 TM 的剩余纤维层。</li>
	<li>现在, 将整个皮瓣放在选管会的前壁上, 让他们可以自由进入 TC。确定:<br>-鼓膜纤维层<br>-前高级环<br>-锤骨短流程, 柄和鳞<br>-鼓索前锤骨</li>
</ol><p class="jove_title">5. 中耳探查</p>
<p class="jove_content">注意: 这部分解剖有助于识别和排练中耳解剖。TC 的每个细分应分别使用0°和45°内镜进行探讨。最终出现的褶皱或坚持应小心去除。</p>
<ol><li>Epitympanum确定:<br>-锤骨: 颈部、短过程、柄和鳞<br>-骨: 身体, 长过程, 透镜状过程, 砧关节<br>-上膈肌: 前外侧踝韧带, 侧 incudomalleolar ligamental 襞<br>-前后脊柱<br>-鼓索<br>-鼓膜和通气模式的窦<br>-Cochleariform 工艺<br>-张索肌, 肌腱和骨管<br>-张量褶皱</li>
	<li>Mesotympanum确定:<br>-镫: 小头, 前后小腿, 踏板<br>-镫肌肌腱<br>-金字塔隆起<br>-岬骨<br>-雅各布森的神经与下鼓室动脉</li>
	<li>Retrotympanum: 如果坚持存在, 他们应该彻底删除。将外科医生的位置改变为对侧, 便于角度光学 (45 °) retrotympanum。确定:<br>-面部休息<br>-后窦<br>-Ponticulus<br>窦索<br>-Subiculum<br>-茎隆起<br>-Subtympanic 窦<br>-Fustis 骨<br>-地区 concamerata<br>-tegmen、前后柱的圆形窗龛<br>-圆窗膜<br>-Subcochlear 小<br>-Finiculus</li>
	<li>下<br>-下细胞<br>-估计 JVB 的本地化 (如果不可见)</li>
	<li>Protympanum确定:<br>-Protiniculus<br>-颈内动脉<br>-咽鼓管 (ET)</li>
	<li>Disarticulate 砧接头, 取出骨。用刮去掉盾牌。确定:<br>-面神经鼓室段<br>-侧半规管<br>-窦<br>-齿轮或横向波峰<br>-Tegmen 索</li>
	<li>切开索肌的肌腱, 取出锤骨。确定:<br>-前 Epitympanum<br>-膝曲神经节</li>
</ol>

<ol>
	<li>Presutti, L., Marchioni, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> <em style='font-style: italic'>Endoscopic Ear Surgery</em>. , (2015).</li><li>Weiss, M. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Surgery+for+recurrent+and+residual+cholesteatoma.">Surgery for recurrent and residual cholesteatoma.</a> <em style='font-style: italic'>Laryngoscope</em>. <b>102</b>, 145-151 (1992).</li><li>Pickett, B. P., Cail, W. S., Lambert, P. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sinus+tympani:+anatomic+considerations,+computed+tomography,+and+a+discussion+of+the+retrofacial+approach+for+removal+of+disease.">Sinus tympani: anatomic considerations, computed tomography, and a discussion of the retrofacial approach for removal of disease.</a> <em style='font-style: italic'>Am J Otol</em>. <b>16</b>, 541-550 (1995).</li><li>James, A. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endoscope+or+microscope-guided+pediatric+tympanoplasty?+Comparison+of+grafting+technique+and+outcome.">Endoscope or microscope-guided pediatric tympanoplasty? Comparison of grafting technique and outcome.</a> <em style='font-style: italic'>Laryngoscope</em>. , (2017).</li><li>Tarabichi, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endoscopic+management+of+limited+attic+cholesteatoma.">Endoscopic management of limited attic cholesteatoma.</a> <em style='font-style: italic'>Laryngoscope</em>. <b>114</b> (7), 1157-1162 (2004).</li><li>Bonali, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+variants+of+the+retro-+and+hypotympanum:+an+endoscopic+anatomical+study.">The variants of the retro- and hypotympanum: an endoscopic anatomical study.</a> <em style='font-style: italic'>Eur Arch Otorhinolaryngol</em>. , (2017).</li><li>Bennett, M. L., Zhang, D., Labadie, R. F., Noble, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+Middle+Ear+Visualization+With+Endoscopy+and+Microscopy.">Comparison of Middle Ear Visualization With Endoscopy and Microscopy.</a> <em style='font-style: italic'>Otol Neurotol</em>. <b>37</b> (4), 362-366 (2016).</li><li>Anschuetz, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+Ovine+Model+for+Exclusive+Endoscopic+Ear+Surgery.">An Ovine Model for Exclusive Endoscopic Ear Surgery.</a> <em style='font-style: italic'>JAMA Otolaryngol Head Neck Surg</em>. <b>143</b> (3), 247-252 (2017).</li><li>Wang, M. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+costs+and+quality+of+operative+training+for+residents+in+tympanoplasty+type+I.">The costs and quality of operative training for residents in tympanoplasty type I.</a> <em style='font-style: italic'>Acta Otolaryngol</em>. <b>129</b>, 512-514 (2009).</li><li>Dedmon, M. M., Kozin, E. D., Lee, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+a+Temporal+Bone+Model+for+Transcanal+Endoscopic+Ear+Surgery.">Development of a Temporal Bone Model for Transcanal Endoscopic Ear Surgery.</a> <em style='font-style: italic'>Otolaryngol Head Neck Surg</em>. <b>153</b> (4), 613-615 (2015).</li><li>Barber, S. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=3D-printed+pediatric+endoscopic+ear+surgery+simulator+for+surgical+training.">3D-printed pediatric endoscopic ear surgery simulator for surgical training.</a> <em style='font-style: italic'>Int J Pediatr Otorhinolaryngol</em>. <b>90</b>, 113-118 (2016).</li></ol>

<p class="jove_content">作者声明没有利益冲突。LA 由 Bangerter-Rhyner 基金会, 伯尔尼, 瑞士和由卡尔 Storz GmbH, 图特林根, 德国。资助者在研究设计、数据收集和分析、决定出版或准备原稿方面没有作用。</p>

<p class="jove_content">所建议的解剖手册是有用的进行完整解剖的中耳。对中耳解剖的全面了解是任何中耳手术干预的基本前提。尸体解剖允许在选管会中训练照相机和手术器械的处理。对于一个新手在 EES, 眼睛和仪器之间的协调以及正确处理内窥镜 (没有弯曲, 在重要的解剖结构之间的听骨在一个有限的空间和 two-dimensional 图像) 是至关重要的这一新兴的外科技术开始的步骤。</p>
<p class="jove_content">与实际手术情况相比, 该模型的主要局限性是出血量不足。这适用于任何尸体解剖模型。最近, Dedmond <em>et al</em>。描述了一个颞骨模型, 其中出血是模拟在海拔的 TMF。这种模式实际上可能是一个很好的选择, 先进的外科医生已经习惯了 EES。根据我们的经验, 对 EES 的初步熟悉应尽可能简单, 因为处理这些文书已经是一项具有挑战性的任务。尸体解剖被认为是外科教育的黄金标准。然而, 它是受高费用和道德章程的。相比之下, 合成或动物模型可以克服这些问题<sup class="xref">8</sup><sup>,</sup><sup class="xref">11</sup>。然而, 在合成模型中, 组织的触觉感觉很困难, 3D 印刷的分辨率还不能提供解剖标本所能得到的所有细节。相比之下, 动物模型提供了优良的组织性能, 但有不同的解剖。我们可以考虑尸体模型作为解剖学教学的唯一合适的模型, 而合成和动物模型为外科训练提供便宜的选择。</p>
<p class="jove_content">与中耳解剖的显微技术相比, 内窥镜允许通过自然口、选煤厂观察和准备微妙的中耳结构。因此, 没有骨头必须被删除的目的和中耳可以研究它的自然状态。此外, 内窥镜允许非常密切的观察任何解剖结构, 从而也放大, 而不会失去照明。当然, 内镜检查方法不教授突, 这可能是在内窥镜解剖后同一个标本上进行的。鼻内窥镜技术是国际传播, 因此需要适当的外科训练将增加。</p>

<p class="jove_content">中耳位于颞骨的中心, 具有高度复杂的解剖学。最毗邻的结构是面神经 (FN), 耳蜗 (CO), ossiculary 链 (OC), 颈静脉球 (JVB) 和颈内动脉 (ICA)。中耳裂在解剖学上分为五部分: epitympanum 位于优, 并由窦连接到乳突空气细胞系统 (mac);retrotympanum 后是一系统的骨桥和或多或少浅的鼻窦;下在于下;前是与咽鼓管连接的 protympanum;在中心, 位于 mesotympanum<sup class="xref">1</sup>。</p>
<p class="jove_content">通过外耳道 (选管会) 的生理通路是狭窄的。因此, 标准的手术方法对中耳需要一个耳切口和钻井的 mac。这类干预是使用操作显微镜进行的。它的介绍是一个里程碑在耳手术, 因为它允许治疗显微中耳结构。然而, 显微镜有一个牵强的平直的看法, 并且一些区域, 特别是窦索, 是难接近的。尤其在胆脂瘤手术中, 有残留疾病的风险<sup class="xref">2</sup><sup>,</sup><sup class="xref">3</sup>。此外, 选管会、互委会及中耳的健康组织, 必须简单地移除, 以供取用。这不仅延长了手术时间, 而且还为更多的外科发病率和延长愈合时间<sup class="xref">4</sup>。</p>
<p class="jove_content">最近的进展导致引入内窥镜作为一个强有力的工具, 耳外科。内窥镜是第一次用于协助传统的手术外科医生, 以可视化的隐蔽区域的中耳。技术和手术的改进使内窥镜的介绍成为主要操作工具<sup class="xref">5</sup>。内窥镜的主要优点是全景和宽角度的中耳的看法。例如, 在不成形<sup class="xref">6</sup>的情况下, 可以使用独占内窥 transcanal 方法访问其他隐藏的复古和下。最近的一项研究显示, 与显微技术<sup class="xref">7</sup>相比, 所有中耳腔室的可见性均优于内窥镜。使用倾斜的内窥镜 (30 °和45°) 进一步提高了能见度, 并允许对中耳最隐蔽的区域进行解剖。</p>
<p class="jove_content">然而, 内窥镜耳手术 (EES) 是一种 one-handed 外科技术, 因为第二手一般持有内窥镜。这个问题和在选管会内狭窄的空间需要训练, 以提高外科医生的技能, 安全地应用在病人的技术。一般来说, 手术训练的金标准被认为是尸体解剖。在没有尸体标本或当地伦理问题的情况下, 一种用于内窥镜耳外科训练的动物模型最近被描述为<sup class="xref">8</sup>。外科训练被认为是一个重要因素在教育的受训人员在一个新的技术<sup class="xref">9</sup>。</p>
<p class="jove_content">尽管外科训练的重要性, 对如何进行尸体解剖的重要考虑的简要描述仍然缺乏文献。本文的目的是描述完全内镜下尸体中耳解剖的方法。此外, 我们的目的是为内窥镜中耳解剖提供一个全面的指南。</p>

<table><tbody><tr><td>Endoscopes: 3mm diameter, 15cm length, 0&amp;deg; and 45&amp;deg;</td><td>Karl Storz</td><td></td><td></td></tr><tr><td>Otologic dissectors, round knifes, hooks, curette, microscissors (Bellucci) and microforceps (Hartmann)</td><td>Karl Storz</td><td></td><td></td></tr><tr><td>Straight and curved suction tubes</td><td>Karl Storz</td><td></td><td></td></tr><tr><td>Flexible hook</td><td></td><td></td><td></td></tr><tr><td>Scissors</td><td></td><td></td><td></td></tr><tr><td>Video Equipment&lt;br/&gt; - HD-scree&lt;br/&gt; - 3-CCD camera&lt;br/&gt; - Xenon light source</td><td>Karl Storz</td><td></td><td></td></tr><tr><td>Cadaveric Head</td><td>-</td><td></td><td></td></tr><tr><td>Vacuum matress</td><td></td><td></td><td></td></tr><tr><td>Aspirator</td><td></td><td></td><td></td></tr><tr><td>Consumables&lt;br/&gt; - Water to rinse&lt;br/&gt; - Antifog solution&lt;br/&gt; - Cotton pads&lt;br/&gt; - Cottonoid pledges&lt;br/&gt; - Gown&lt;br/&gt; - Gloves&lt;br/&gt; - Mask</td><td></td><td></td><td></td></tr></tbody></table>

discovering middle ear anatomy by transcanal endoscopic ear surgery: a dissection manual

<p class="jove_content">中耳位于颞骨的中心, 具有高度复杂的解剖学。最近引进的专门内窥镜 transcanal 方法的中耳是一种微创技术, 不遗余力的骨和粘膜的乳突骨, 因为中耳是通过外部听觉渠道访问。这种新兴的方法有几个优势优于传统的 (微观) 的方法, 以中耳, 如全景 wide-angle 的观点解剖, 可能接近和放大微小的结构, 并有可能环顾四周角落使用角度内窥镜。</p>
<p class="jove_content">这里提出的尸体解剖方法包括对技术要求的概述和一个 step-by 步协议的精确描述, 以发现中耳的解剖。每一个步骤和解剖结构被仔细描述, 以提供一个全面的指导, 内窥镜耳解剖。在我们看来, 这是特别重要的任何新手在内窥镜耳外科, 因为它提供了全面的解剖知识, 并可能提高手术技能。</p>

<p class="jove_content" fo:keep-together.within-page="1">我们使用所描述的指令解剖了总共五个头部标本和12颞骨标本 (n=22 侧)。对 TC 的不同区域进行了摄影和进一步分析。使用0°和45°内窥镜对所有区域进行了探索。我们对所有解剖区域进行了全面的探索, 并确定了所有的解剖标志。</p>
<p class="jove_content" fo:keep-together.within-page="1">解剖结构根据解剖的进展被说明。<strong class="xfig">图 1</strong>说明了 Prussak 空间和踝韧带, 而<strong class="xfig">图 2</strong>显示了完整的 TMF 提升后的解剖。wide-angle 视图允许可视化的中间-和下以及部分的 epi, 复古, 和 protympanum。在<strong class="xfig">图 3</strong>中, 高级 retrotympanum 显示在45°内窥镜中, 而外科医生则站在表的另一侧。<strong class="xfig">图 4</strong>详细说明了带有开裂 ICA 的 protympanum。在去除 OC 和 transcanal atticotomy 后, epitympanum 被检查到侧半规管和腔, 如<strong class="xfig">图 5</strong>所示。</p>
<p class="jove_content" fo:keep-together.within-page="1">没有标本需要成形进行完整的探查鼓膜腔。在所有使用0°和45°范围的标本中都可以识别解剖结构。然而, retrotympanum, 特别是面部凹槽和窦索的完整的探索, 是不可能的在27% 的案件 (n=6), 因为这些骨的海湾是太深, 无法完全探索。</p>
<p class="jove_content" fo:keep-together.within-page="1"><img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56390/56390fig1.jpg"><br><strong class="xfig">图 1</strong>。左耳朵, 0 °内窥镜: Prussak 的空间和周围的 ligamental 褶皱的锤骨的看法。tm: 鼓膜, a: 环, u: 鳞, sp: 锤骨的短的过程, 踝, 调频: 侧踝折叠, plm: 锤骨的后韧带, ct: 鼓索, *: Prussak 空间<a href="//cloudflare.jove.com/files/ftp_upload/56390/56390fig1large.jpg" target="_blank">请单击此处查看更大版本的此图.</a></p>
<p class="jove_content" fo:keep-together.within-page="1"><img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56390/56390fig2.jpg"><br><strong class="xfig">图 2</strong>。左耳, 0 °内窥镜: tympano 道口瓣抬高后鼓室的视野。中、下以及部分的 epi-, 复古和 protympanum 是可视化的。公关: 海角, 约: 雅各布森: 下, fi: finiculus, 海温: 窦 subtympanicus, 硒: 茎隆起, pe: 金字塔隆起, st: 镫腱, isj: 砧关节, fn: 面神经, u: 鳞, m: 柄, sp: 短过程, n: 颈部锤骨, ttm: 张量索肌, 专题信托: 张量索褶皱, ica。颈内动脉<a href="//cloudflare.jove.com/files/ftp_upload/56390/56390fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a></p>
<p class="jove_content" fo:keep-together.within-page="1"><img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/56390/56390fig3.jpg"><br><strong class="xfig">图 3</strong>。右耳、45°内窥镜、外科医生对侧位置: 使用角度内镜说明 retrotympanum 的优越性。fn: 面部神经, s: 镫, st: 镫肌腱, ps: 后窦, fp: 踏板, po: ponticulus, pe: 金字塔隆起, 坐: 窦索, 分, subiculum, se: 茎隆起, sst: 窦 subtympanicus, ap: 前柱, 三: tegmen, 聚丙烯: 后支柱, rw: 圆窗, 傅: fustis, tu: subcochlear 隧道<a href="//cloudflare.jove.com/files/ftp_upload/56390/56390fig3large.jpg" target="_blank">请单击此处查看此图的较大版本.</a></p>
<p class="jove_content" fo:keep-together.within-page="1"><img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/56390/56390fig4.jpg"><br><strong class="xfig">图 4</strong>。左耳, 45 °内窥镜: protympanum 的详细视图。注意颈动脉的开裂。et: 咽鼓管, ica: 颈内动脉, 临: protiniculus, co: 耳蜗, ttm: 张量索肌<a href="//cloudflare.jove.com/files/ftp_upload/56390/56390fig4large.jpg" target="_blank">请单击此处查看此图的较大版本.</a></p>
<p class="jove_content" fo:keep-together.within-page="1"><img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/56390/56390fig5.jpg"><br><strong class="xfig">图 5</strong>。左耳朵, 0 °内窥镜: 在去除骨和踝部和刮除盾牌之后, tegmen 索和窦的广泛的概要这里被说明。注意侧半规管和张量索肌在一条线上与面神经交叉。三: tegmen, 齿轮: 齿状或横脊, gg: 膝曲神经节, cp: cochleariform 过程, ttm: 张量索肌, 侧半规管, fn: 面神经鼓室段, s: 镫, co: 耳蜗, ica: 颈内动脉<a href="//cloudflare.jove.com/files/ftp_upload/56390/56390fig5large.jpg" style="font-size: 14px;" target="_blank">请单击此处可查看此图的较大版本.</a></p>

Watch this Scientific Journal Video about Discovering Middle Ear Anatomy by Transcanal Endoscopic Ear Surgery: A Dissection Manual at JoVE.com

Discovering Middle Ear Anatomy by Transcanal Endoscopic Ear Surgery: A Dissection Manual

<p class="jove_content">本文的目的是描述完全内镜下尸体中耳解剖的方法。此外, 我们的目的是为内窥镜中耳解剖提供一个全面的指南。</p>

Transcanal 内窥镜下耳部手术发现中耳解剖: 解剖手册

The middle ear is located in the center of the temporal bone and bears a highly complex anatomy. The recently introduced exclusively endoscopic transcanal ...

discovering-middle-ear-anatomy-transcanal-endoscopic-ear-surgery

Research

JoVE Journal

Medicine

64.4K 次观看.  University of Bern. 本文的目的是描述完全内镜下尸体中耳解剖的方法。此外, 我们的目的是为内窥镜中耳解剖提供一个全面的指南。

视频: Discovering Middle Ear Anatomy by Transcanal Endoscopic Ear Surgery: A Dissection Manual

Cell Structure

<h4>Background</h4>
<p>Cells represent the most basic biological units of all organisms, whether it be simple, single-celled organisms like bacteria, or large, multicellular organisms like elephants and giant redwood trees. In the mid 19<sup>th</sup> century, the Cell Theory was proposed to define a cell, which states:</p>
<ul>
	<li>Every living organism is made up of one or more cells.</li>
	<li>The cells are the functional units of all organisms.</li>
	<li>All cells arise from preexisting cells.</li>
</ul>
<p>All cells share common features such as having a plasma membrane, a cytoplasm, DNA, and ribosomes. A plasma membrane is a phospholipid bilayer that surrounds the cell. This thin and fluid layer around the cells serves to isolate the cell&#8217;s contents from its environment and regulates the material exchange with its environment, while also facilitating interactions with other cells. Inside the plasma membrane, the cell is filled with a gel-like fluid called cytoplasm that contains organic molecules, salts, and other materials that are vital for the cell&#8217;s functions. Therefore, biochemical reactions that support life, which are known as metabolic processes, take place inside the cytoplasm. The types of metabolic processes that a cell can execute depend on its genetic information. All cells use DNA as the genetic material, which is the hereditary blueprint to construct cellular structures and products. Finally, all cells use ribosomes to synthesize their protein products.</p>
<p>There are two types of cells based on their genetic material location: prokaryotic, which means &#8220;before nucleus&#8221;, and eukaryotic, which means &#8220;true nucleus&#8221;. Therefore, while both types of organisms have DNA, prokaryotes like bacteria have nucleoids, or &#8220;nucleus-like&#8221; components instead of a nucleus, whereas eukaryotes possess true, membrane-bound nuclei to contain their DNA. Moreover, prokaryotes are relatively small, around 0.1&#8211;5.0 micrometers (&#181;m), in comparison to eukaryotes that can typically range from 10-100 &#181;m in size. The small size of prokaryotes allows quick and effortless distribution of materials within the cell and execution of metabolic processes, as well as swift removal of waste or other products from the cell. Hence, eukaryotic cells possess specialized structures known as organelles, such as mitochondria or Golgi apparatus, to enable the execution of vital functions.</p>
<h4>The Eukaryotic Cell</h4>
<p>The eukaryotic cell is a shared derived trait of all eukaryotes, meaning that it had a single origin that has since been inherited by all eukaryotes. The earliest eukaryotic cells are seen in fossils about 2.4 billion years ago, and are recognizable because they are larger than prokaryotic cells<sup>1</sup>. The origin of this cell type resulted from an endosymbiotic event in which one amoeba-like cell engulfed a micrococcal bacteria and formed a stable coexistence<sup>2</sup>. The engulfed bacteria evolved into the first energy-producing organelles, mitochondria, which are the aerobic metabolism organelles in the cell. Mitochondria have their own separate genome and are similar in size to prokaryotes. They contain two layers of membranes that enclose two distinct compartments. Some of the reactions that break down high-energy biomolecules occur in the inner compartment, whereas the outer compartment houses the reactions that capture the energy released from these compounds into adenosine triphosphate (ATP) molecules to be used as the energy currency of the cell.</p>
<p>Nuclei and mitochondria are not the only shared structures of eukaryotic cells. Other ubiquitous eukaryotic organelles are the smooth and rough endoplasmic reticulum (ER), Golgi apparatus, lysosomes and vacuoles. Endoplasmic reticulum simply means &#8220;network inside the plasma&#8221; and as its name indicates it is a large network of membranes inside the cell, especially around the nucleus. Parts of the rough ER extend from the nuclear membrane and are distinguished from the smooth ER by their rough appearance due to numerous ribosomes on their surface. Rough ER is the site for protein synthesis, such as the proteins that are embedded in the plasma membrane or the proteins that are secreted from the cell. In contrast, smooth ER produces lipid-based products but also contains enzymes to detoxify harmful chemicals. Hence liver cells contain abundant smooth ER. Also, muscle cells contain significant amounts of smooth ER because of the calcium-storing function of this organelle, which is essential for muscle contraction. The Golgi apparatus sorts, modifies, and packages cellular products inside vesicles, which fuse with the plasma membrane to release the products. Some of the proteins that are produced in the rough ER are intracellular digestive enzymes. These enzymes are packed in the Golgi apparatus into special vesicles called lysosomes. The major function of the lysosomes is to digest the food particles engulfed by the cell as well as old cell parts. Vacuoles are sacs of cell membrane that serve as storage units within cells. They may serve to store water to regulate the cell&#8217;s water content as well as store metabolic products or even poisonous molecules, depending on the cell type and the organism.</p>
<h4>Kingdom-specific Organelles</h4>
<p>Eukaryotic cells also developed distinct organelles, specific for each kingdom. For instance, kingdom Plantae and Animalia are both eukaryotic, however the organelles of plant and animal cells differ in key ways that allow them to carry out their lives as producers and consumers, respectively. Terrestrial plants need to grow tall and have rigid stems to hold leaves, which they use for photosynthesis. They also need to be able to retain water taken up by roots. Their cells reflect these specific needs. Unlike animal cells, plant cells have chloroplasts, which are used for photosynthesis and often contain the green pigment chlorophyll. Additionally, they are surrounded by cell walls, which are rigid outer layers made of cellulose to support growth and water retention. Because they need to store large amounts of water to maintain the water pressure in the cell, they have larger vacuoles than animal cells. In addition, plant cells also have another type of specialized storage organelles called plastids, which contain pigments as well as photosynthetic products such as starch. These differences are noticeable and distinguish plant cells from animal cells: plant cells generally have a regular, rectangular shape due to their rigid cell walls, while animal cells are rounded and more irregular.</p>
<h4>Microscopy</h4>
<p>Some cells, such as frog oocytes, are large enough to be seen with the naked eye, yet most cells cannot be seen without any visual aid. Therefore, scientists use microscopy techniques to study cell structures and distinguish cell types from one another. While microscopes are able to magnify objects that are difficult or impossible to be see with the human eye, most tissues naturally lack pigmentation. Therefore, solutions have been created that can selectively stain cells based on their molecular composition. This allows researchers to distinguish between organelles in a cell, tissue types in a plant stem, and fat layers in animals, just to name a few examples. Methylene blue dye stains nucleic acids of dead cells, binding to negatively charged DNA. Safranin solution is another biological dye that stains cell nuclei red. Cells only need to be in the staining solutions for a short period of time, and can be mounted immediately following the staining step. The commonly used mounting techniques are wet mount and oil immersion. A wet mount is created by collecting a sample and putting it on a glass slide with liquid between the slide and the coverslip. Cell samples are suspended in liquids like water or glycerol. Glycerol is better to use with live cultures, because it prevents the proliferation of bacteria<sup>3</sup>. Immersion oil can be added on top of the coverslip to improve viewing of the sample at high magnification. This is accomplished because the oil has the same refractive index as glass, which means that it allows light to pass through it as well as glass would. The glass-air interface scatters light more than oil or glass, so the clarity of the image is affected when samples are mounted &#8220;dry&#8221;, or without oil. Once the cells are stained and mounted, they are ready to be studied under the microscope.</p>
<p>There are various microscopy techniques from electron scanning technology that has allowed researchers to view objects on an atomic level to fluorescent live cell imaging that allows real-time monitoring of the movement of molecules within individual cells<sup>4</sup>. Bright-field microscopy is the simplest microscopy technique, requiring only a halogen source light, a condenser lens to focus the light, an ocular lens to view the image, and an objective lens to magnify the image. With any microscopy technique, it is important to understand the parts of a microscope before using one. In general, compound microscopes used for bright-field imaging have an eyepiece at the top of the scope, which is attached to the head and objectives. The eyepiece has a magnification of 10X, and objective lenses are set to a particular magnification in a range of 4X-100X. There are between three and five objectives on a standard microscope. The objectives point down to the stage, which is where a specimen is placed for viewing. The stage often has mechanical parts and stage clips to hold a slide and move it around while viewing. An aperture is a hole in the stage for light to pass through. This light is controlled by an adjustable condenser lens above an illuminator, or light source. To control the zoom of the stage for the object being viewed, microscopes feature coarse and fine focus adjustment knobs. The coarse focus knob moves on a larger scale than the fine focus, but they are on the same axis. The fine focus is useful when the object on the stage has been brought close to the objectives. It is important not to let the objective lens touch the object on the stage, as it can scratch the lens. Objects should always be first viewed on the lowest magnification objective and clearly focused before switching to higher magnification objectives.</p>
<p>Microscopy is an important tool for many aspects of the medical field including research, diagnosis, and treatment. This has the application of using nanotechnology in medicine, as a new treatment method in place of a more invasive surgery<sup>5</sup>. Surgeons also make use of microscopes, some of which have been modified to be mounted on a surgeon&#8217;s head and are operated with foot pedals. These are of much lower magnification than even the light microscopes used today, but they facilitate the safe execution of delicate procedures, such as optical and neurosurgery.</p>
<h4>References</h4>
<ol class="references">
	<li><strong>Bengtson S, Rasmussen B, Ivarsson M, Muhling J, Broman C, Marone F, Stampanoni M, Bekker A.</strong> Fungus-like mycelial fossils in 2.4-billion-year-old vesicular basalt. <em>Nature Ecology &#38; Evolution. </em>2017, Vol. 1, Article number: 0141.</li>
	<li><strong>Vellai T, Vida G.</strong> The origin of eukaryotes: the difference between prokaryotic and eukaryotic cells. <em>Proc. R. Soc. Lond. B. </em>1999, Vol. 266, 1571-1577.</li>
	<li><strong>Gouet V, Roger G, Fonty C, Andre P.</strong> Effects of glycerol on the growth, adhesion, and cellulolytic activity of rumen cellulolytic bacteria and anaerobic fungi. <em>Current Microbiology. </em>24, 1992, Vol. 4, 197-201.</li>
	<li><strong>Cognet L, Leduc C, Lounis B.</strong> Advances in live-cell single-particle tracking and dynamic super-resolution imaging. <em>Curr Opin Chem Biol. </em>2014, Jun; 20:78-85.</li>
	<li><strong>Asiyanbola B, Soboyejo W.</strong> For the surgeon: an introduction to nanotechnology. <em>J Surg Educ. </em>2008, Vol. 65, 2 (155-61).</li>
</ol>

Cell Structure: Visualizing Onion and Human Cells - Concept

单元结构

Background
Cells represent the most basic biological units of all organisms, whether it be simple, single-celled organisms like bacteria, or large, ...

教育

JoVE Lab Manual

生物学

科学探究

Bacterial Transformation

<h4>Background</h4>

<p>In early 20<sup>th</sup> century, pneumonia was accountable for a large portion of infectious disease deaths<sup>1</sup>. In order to develop an effective vaccine against pneumonia, Frederick Griffith set out to study two different strains of the <em>Streptococcus pneumoniae: </em>a non-virulent strain with a rough appearance (R-strain) and a virulent strain with a smooth appearance (S-strain) due to an outer polysaccharide capsule<sup>2</sup>. This outer layer of the S-strain bacteria enabled them to withstand the host immune system, eventually leading to a life-threatening illness. When Griffith separately injected mice with heat-killed bacteria of either strain, the mice lived. However, when he injected mice with a combination of the heat-killed S-strain with live R-strain, the mice died<sup>2</sup>. When he analyzed the samples obtained from dead mice injected with the combination, he observed live S-strain bacteria to be present. In 1928, Griffith noted that a &#8220;transformation&#8221; process must have occurred to change the nonvirulent bacteria into the virulent strain. As the first known discovery of bacterial transformation, his discovery paved the way for the development of an essential tool in genetic engineering - transformation<sup>3</sup>.</p>

<p>Transformation is the genetic change in a cell due to the intake of DNA from the environment. In Griffith&#8217;s experiment, the DNA that encodes the protective polysaccharide coating of the S-strain bacteria did not break down from the heat shock and was introduced into the R-strain, allowing the latter to bypass the immune system of the mouse. While this process constantly occurs in the wild between organisms and even different species, scientists transform bacteria in laboratory settings for research purposes<sup>4</sup>.</p>

<h4>Bacterial Transformation using Plasmids</h4>

<p>Bacteria are the ideal organisms for transformation as they can easily take in exogenous genetic material into their genome and quickly amplify it<sup>3,5</sup>. They have one circular chromosome and multiple small circular pieces of double-stranded DNA called plasmids within the cytoplasm. These plasmids can replicate independently from the chromosomal DNA  and generally provide certain functional benefits, such as resistance to antibiotics<sup>6,7</sup>. In their natural environment, bacteria undergo &#8220;bacterial transformation&#8221; by taking in plasmids from other bacteria in a process called conjugation<sup>8</sup>. Moreover, when they multiply, each of their progeny receives a copy of the new plasmid.</p>

<p>Plasmids that are used for experimental purposes are called plasmid vectors. In a laboratory setting, scientists can artificially create &#8220;recombinant plasmids&#8221; that are approximately 5,000-10,000 base pairs in length by inserting DNA fragments into a plasmid vector. These recombinant plasmids usually have certain components: an origin of replication (ORI), an antibiotic resistance gene, a multiple cloning site, a promoter, a selection marker, and the gene of interest. The origin of replication is where replication begins. The antibiotic resistance gene allows the bacteria that take in the plasmid to survive on plates in the presence of a certain antibiotic drug. Although plasmids are relatively small pieces of DNA, scientists need to treat the host cells to enable the plasmid penetration through the cell membrane. Hence, the efficiency of the transformation is directly related to porosity of the host membrane.  One common approach is to heat-shock the bacteria that have been treated with a calcium chloride solution<sup>9</sup>. The bacteria that do not incorporate the plasmid will not transform, and therefore have no resistance to survive on the plate and be visible. The multiple cloning sites aid in DNA insertion by containing sites for restriction enzymes to cut the plasmid where the gene of interest can be inserted and ligated. The promoter drives transcription of the gene of interest. It is tagged by a marker, usually a fluorescent protein such as a green fluorescent protein (GFP), or can be an additional antibiotic resistance gene. The antibiotic resistance gene and the other selection markers help us determine whether the collected bacteria contain the plasmid of interest.</p>

<h4>Applications</h4>

<p>Effective transformation methods enabled scientists to isolate and profile genes and gene products and led to many advancements in life sciences and medicine, such as development of effective drugs, generation of genetically-modified crops, and advanced diagnostic tools<sup>10</sup>.  In addition, with technological advancements, new methods of transformation have emerged. For instance, Gateway Cloning allows insertion of multiple DNA fragments into different vectors as well as transfer of DNA sequences between plasmids<sup>11</sup>. Furthermore, clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 is a gene-editing technique that directly modifies nucleotides in the genome and does not require the use of plasmids<sup>12</sup>. After transformation, researchers often isolate and profile the gene of interest and its products. Subsequently, the whole process of genetic cloning has opened up a new field of genetic manipulation. Thanks to genetic cloning, researchers can manipulate bacteria to produce large amounts of specific human proteins, such as insulin to treat diabetes patients<sup>10</sup>. Cloning also has served a major importance in modern day agriculture. Genetically modified organisms (GMO) are a direct result of genetic cloning and bacterial transformation<sup>10</sup>. For instance, scientists are working to generate genetically modified crops with nitrogen-fixing genes incorporated into their genome to boost food production and reduce fertilizer use, thus, diminishing the economic and environmental impact of fertilizers<sup>13</sup>. In sum, bacterial transformation is the first step of modern-day biotechnology and the foundation of future research discoveries.</p>

<h4>References</h4>

<ol class="references">
	<li><strong>GL, Armstrong, LA, Conn and RW, Pinner.</strong> Trends in Infectious Disease Mortality in the United States During the 20th Century. <em>JAMA. </em>1999, Vol. 281, 1 (61-66).</li>
	<li><strong>F, Griffith.</strong> The Significance of Pneumococcal Types. <em>J Hyg (Lond). . </em>1928 , 2 (113-59).</li>
	<li><strong>Lorenz, MG and Wackernagel, F.</strong> Bacterial gene transfer by natural genetic transformation in the environment. <em>Microbiol Rev. . </em>1994 Sep;, Vol. 58, 3: (563-602).</li>
	<li><strong>S, Domingues, et al.</strong> Natural Transformation Facilitates Transfer of Transposons, Integrons and Gene Cassettes between Bacterial Species. <em>PLOS Pathogens. </em>2012, 8(8): e1002837.</li>
	<li><strong>Dubnau, D.</strong> DNA uptake in bacteria. <em>Annu Rev Microbiol. . </em>1999, 53: (217-44).</li>
	<li><strong>Solar, G del, et al.</strong> Replication and control of circular bacterial plasmids. <em>Microbiol Mol Biol Rev. . </em>1998 , Vol. 62, 2: (434-64).</li>
	<li><strong>Bennett, PM.</strong> Plasmid encoded antibiotic resistance: acquisition and transfer of antibiotic resistance genes in bacteria. <em>Br J Pharmacol. . </em>2008 , Vol. 153, S1: (S347-S357).</li>
	<li><strong>MLlosa, et al.</strong> Bacterial conjugation: a two-step mechanism for DNA transport. <em>Mol Microbiol. </em>2002 Jul;, Vol. 45, 1: (1-8).</li>
	<li><strong>Roychoudhury, A, Basu, S and Sengupta, DN.</strong> Analysis of comparative efficiencies of different transformation methods of E. coli using two common plasmid vectors. <em>Indian J Biochem Biophys. . </em>2009 , Vol. 46, 5: (395-400).</li>
	<li><strong>Khan, S, et al.</strong> Role of Recombinant DNA Technology to Improve Life. I. <em>nt J Genomics. . </em>2016, 2016:2405954.</li>
	<li><strong>Marsischky, G and LaBaer, J.</strong> Many Paths to Many Clones: A Comparative Look at High-Throughput Cloning Methods. <em>Genome Res. </em>2004, Vol. 14, 2020-28.</li>
	<li><strong>Doudna, JA and Charpentie, E.</strong> The new frontier of genome engineering with CRISPR-Cas9. <em>Science. </em>2014, Vol. 346, 6213-1258096.</li>
	<li><strong>Good, A.</strong> Toward nitrogen-fixing plants. <em>Science. </em>2018, Vol. 359, 6378: 869-70.</li>
</ol>


Bacterial Transformation using Plasmids - Concept

细菌转化

Background

In early 20th century, pneumonia was accountable for a large portion of infectious disease deaths1. In order to develop an effective vaccine ...

减数分裂

Eusociality and Division of Labor

<ol class="note-items"><li data-note-item="1" data-parent-collapse="">Simulating Eusocial and Solitary Behavior
<button class="expand">Expand</button><ul class="lab-items"><li data-sub-note-item="1-1"><div class="lab-step">Note: In this simulation, you will portray either a eusocial or solitary animal. The goal of this simulation is to maximize your fitness by reproducing as many offspring as possible during the allotted five minutes. To do this, you must forage for resources and reproduce within the constraints of your assigned role.
</div></li><li data-sub-note-item="1-2"><div class="lab-step">Assuming a class size of 10, 4 individuals should form the solitary, or the carpenter bee group, and the remaining 6 students should form the eusocial, or the honeybee group. Note: The eusocial honeybee group will include one queen, one drone, and four workers. The queen and the drone will reproduce and protect the resources, while the workers will forage and steal resources. The solitary carpenter bee students must perform all of the tasks in this activity, with the exception of reproduction, by themselves.
</div></li><li data-sub-note-item="1-3"><div class="lab-step">Store your resources for reproduction in the nest bowl provided by your instructor. These resources are represented by assorted beans that can be collected by foraging in the environment buckets or by stealing from other nests.
</div></li><li data-sub-note-item="1-4"><div class="lab-step">The blue cups are male and the red cups are female, and they will be used to produce offspring. This is done by combining all four bean types in one red or blue cup to symbolize the resources needed to reproduce, and then stacking one completed cup inside a cup of the opposite color that also contains one of each bean type, to simulate a mating that produces two-cup offspring, one from each parent.
</div></li><li data-sub-note-item="1-5"><div class="lab-step">To reproduce, the solitary students will need to seek out another solitary student with an opposite-colored cup. Conversely, the eusocial queen and drone always reproduce with each other. HYPOTHESES: The hypotheses for this lab activity are based on the possible benefits of eusocial groups over solitary individuals. The alternate hypothesis could be that one group, either eusocial or solitary, will have an advantage over the other, given the ecological constraints, and this will be reflected in the number of offspring per capita. The null hypothesis might be that there will be advantage conferred by either of the strategies.
</div></li><li data-sub-note-item="1-6"><div class="lab-step">If you are a worker in the honeybee group or a solitary student in the carpenter bee group, walk to the other side of the room to search through the rice on the foraging side of the room for one bean and bring the bean back to your bowl on the nest side of the room. Note: Even though this activity has a 5-minute time limit, running is not permitted. 
</div></li><li data-sub-note-item="1-7"><div class="lab-step">If a solitary student is not present at a neighboring bowl, you can try to steal one bean at a time, using a cup to scoop up the bean without touching the bean or the bowl with anything but the cup. You must immediately stop trying to steal if the solitary student makes it back to their bowl before you finish stealing. Note: Neighbors cannot steal from eusocial nests because the queen and drone do not leave their nest.
</div></li><li data-sub-note-item="1-8"><div class="lab-step">You can create offspring at any time during the activity. Remember to label each cup that is contributed to an offspring with your name to keep track of your reproduction.  Note: Eusocial groups produce two offspring for their group every time they reproduce, while solitary students produce only one offspring per reproductive coupling.
</div></li></ul></li><li data-note-item="2" data-parent-collapse="">Results
<button class="expand">Expand</button><ul class="lab-items"><li data-sub-note-item="2-1"><div class="lab-step">If you were a solitary carpenter bee, record the number of offspring you produced in the appropriate row of the table. <a href="https://www.jove.com/CDNSource/lm/labs/36/Table_1_Eusociality_and_division_of_labor.xlsx" style="display:block;margin:20px;" target="_blank">Click Here to download Table 1</a>
</div></li><li data-sub-note-item="2-2"><div class="lab-step">If you were a part of the eusocial honeybee group, record the number of offspring produced by your queen and drone.
</div></li><li data-sub-note-item="2-3"><div class="lab-step">To calculate the fitness for a solitary carpenter bee student, simply count the number of offspring that the student produced.
</div></li><li data-sub-note-item="2-4"><div class="lab-step">To calculate the fitness for a eusocial honeybee student, divide the number of offspring produced by the queen and drone by the number of members in the group, in this case 6, to get a per-student fitness value.
</div></li><li data-sub-note-item="2-5"><div class="lab-step">When each student has calculated their respective fitness value, calculate the average number of offspring produced for each social category and plot these as a bar graph. <a href="https://www.jove.com/CDNSource/lm/labs/36/Table_2_Eusociality_and_division_of_labor.xlsx" style="display:block;margin:20px;" target="_blank">Click Here to download Table 2</a>
</div></li><li data-sub-note-item="2-6"><div class="lab-step">Next, use Hamilton&#39;s equation to analyze the data. To do this, set r equal to the degree of relatedness, B to the class average eusocial fitness, and C to the class average solitary fitness.  r * B > C
</div></li><li data-sub-note-item="2-7"><div class="lab-step">Use these results to determine if altruistic behavior should persist in your eusocial honeybee group. Discuss the variables in Hamilton&#39;s equation that may contribute to this observation. Consider circumstances under which you would expect to see different results. 
</div></li></ul></li></ol>

社会性与劳动分工

Simulating Eusocial and Solitary Behavior
ExpandNote: In this simulation, you will portray either a eusocial or solitary animal. The goal of this ...

行为

Lab Manual Sub Articles

Energy Dynamics

<ol class="note-items"><li data-note-item="1" data-parent-collapse="">Caterpillar Energy Dynamics
<button class="expand">Expand</button><ul class="lab-items"><li data-sub-note-item="1-1"><div class="lab-step">NOTE: In this lab you will grow caterpillars in conditions with and without food to examine the transfer of biomass in a typical trophic system.
</div></li><li data-sub-note-item="1-2"><div class="lab-step">Weigh and record the wet mass of enough leaves of cabbage to line the bottom of a plastic tub.
</div></li><li data-sub-note-item="1-3"><div class="lab-step">Then, collect two batches of five caterpillars from their cabbage habitat, and weigh and record the wet mass of each batch separately in individual weigh boats. Make sure to tare the balance before each measurement. HYPOTHESES: The experimental hypothesis is that only a fraction of energy will be transferred between the trophic levels of the cabbage and the caterpillars and a significant amount will be lost as waste and heat energy. The null hypothesis is that there will be no significant difference between the energy input from the cabbage, and the obtained energy stored as biomass in the caterpillars.
</div></li><li data-sub-note-item="1-4"><div class="lab-step">Place the weighed cabbage and five caterpillars in a plastic tub lined with damp paper towels, leaving the lid slightly cracked open for ventilation, but not wide enough for the animals to escape.
</div></li><li data-sub-note-item="1-5"><div class="lab-step">Place the other five caterpillars in a second clean container prepared the same way, but with no cabbage.
</div></li><li data-sub-note-item="1-6"><div class="lab-step">After three days, transfer the caterpillars to new weigh boats, and weigh and record the final wet mass of both groups of caterpillars and the cabbage.
</div></li><li data-sub-note-item="1-7"><div class="lab-step">Then place the caterpillar waste, or frass, and cabbage into two separate dishes, and put both dishes into a 37 &#176;C incubator to dry for 48 hours.
</div></li><li data-sub-note-item="1-8"><div class="lab-step">Euthanize the weighed caterpillars by putting them into a minus 20 &#176;C freezer for 24 hours.
</div></li><li data-sub-note-item="1-9"><div class="lab-step">Then, transfer the dishes to a 37 &#176;C incubator for 48 hours.
</div></li><li data-sub-note-item="1-10"><div class="lab-step">After drying, weigh and record the dry mass of the uneaten cabbage, the two groups of caterpillars, and the frass, and use these data to calculate the percent biomass of the caterpillars.
</div></li><li data-sub-note-item="1-11"><div class="lab-step">Finally, discard all of the dried organisms and plates into the hazardous waste bin, and wipe down your workspace with 70% ethanol.
</div></li></ul></li><li data-note-item="2" data-parent-collapse="">Results
<button class="expand">Expand</button><ul class="lab-items"><li data-sub-note-item="2-1"><div class="lab-step">Take a look at the data you collected. Ensure the initial wet mass, the final wet mass, and the final dry mass columns are accurately filled out in your table. <a href="https://www.jove.com/CDNSource/lm/labs/13/Table_1_Energy_Dynamics.xlsx" style="display:block;margin:20px;" target="__blank">Click Here to download Table 1</a>
</div></li><li data-sub-note-item="2-2"><div class="lab-step">NOTE: Remember an object&#39;s biomass is equal to its dry mass. Therefore, percent biomass for each group is the final dry mass divided by the final wet mass times 100. For example, obtain the percent biomass of the cabbage by dividing the final dry mass by the final wet mass and multiplying by 100. This means that approximately 15% of the cabbage&#39;s total weight is biomass.
</div></li><li data-sub-note-item="2-3"><div class="lab-step">After this, estimate the initial biomass of the cabbage and caterpillars by multiplying the percent biomass by the initial wet mass, and put the values in this new table. NOTE: Remember, biomass equals dry mass. Therefore, the final dry mass is equal to the final biomass of each group.
</div></li><li data-sub-note-item="2-4"><div class="lab-step">To calculate the approximate initial and final energy in the form of kilocalories within the cabbage, caterpillars, and frass, multiply the biomass values recorded in your table by the conversions, and record the calculated values in the table. <a href="https://www.jove.com/CDNSource/lm/labs/13/Table_2_Energy_Dynamics.xlsx" style="display:block;margin:20px;" target="__blank">Click Here to download Table 2</a>
</div></li><li data-sub-note-item="2-5"><div class="lab-step">After this, subtract the initial energy from the final energy to obtain the change in energy for each group.
</div></li><li data-sub-note-item="2-6"><div class="lab-step">NOTE: The negative sign denotes loss in energy, and positive indicates gain.
</div></li><li data-sub-note-item="2-7"><div class="lab-step">Using your data table, calculate the energy in kilocalories that the fed caterpillars lost to respiration. The formula is the energy gained from cabbage, which is the positive of the energy lost by cabbage, minus the biomass gained from cabbage, minus the energy lost via frass. <a href="https://www.jove.com/CDNSource/lm/labs/13/Table_3_Energy_Dynamics.xlsx" style="display:block;margin:20px;" target="__blank">Click Here to download Table 3</a>
</div></li><li data-sub-note-item="2-8"><div class="lab-step">Then, repeat the same calculation for the unfed group of caterpillars. Make another column in the table and note this down.
</div></li><li data-sub-note-item="2-9"><div class="lab-step">Finally, add up all of the energy gained via biomass and cabbage consumption, and the energy lost via frass and respiration for each group of caterpillars. NOTE: For both groups of caterpillars the net energy is zero, meaning that this experiment obeyed the first law of thermodynamics.
</div></li></ul></li></ol>

Studying Energy Dynamics and Productivity - Procedure

能源动力学

Caterpillar Energy Dynamics
ExpandNOTE: In this lab you will grow caterpillars in conditions with and without food to examine the transfer of biomass in a ...

Genetics of Organisms

<ol class="note-items"><li data-note-item="1" data-parent-collapse="">REF and WEF Fly Genetics
<button class="expand">Expand</button><ul class="lab-items"><li data-sub-note-item="1-1"><div class="lab-step">NOTE: At your station, you should have four vials of flies. They will be labeled as male or female. They will also be labeled with the notation REF for red-eyed flies or WEF for white-eyed flies. The female vial should also be labeled virgin as these flies should be unmated for the purpose of this experiment.
</div></li><li data-sub-note-item="1-2"><div class="lab-step">Make sure you have around five flies in each vial. NOTE: You will perform crosses using these flies to determine if red or white eyes is dominant and if the trait is sex-linked. You should also have two fly anesthesia wands, a paint brush, magnifying glass, fly anesthetic, some empty fly vials, and a sheet of white paper.
</div></li><li data-sub-note-item="1-3"><div class="lab-step">Draw a line in the middle of the paper to make a male side and a female side.
</div></li><li data-sub-note-item="1-4"><div class="lab-step">Dip the fly anesthesia wand into the solution and insert it into the red-eyed female vial until the flies stop moving.
</div></li><li data-sub-note-item="1-5"><div class="lab-step">Then, dip the same wand into the white-eyed male vial until these flies are also anesthetized.
</div></li><li data-sub-note-item="1-6"><div class="lab-step">Tap the flies from the two vials onto the sheet - Females on one side and males on the other.
</div></li><li data-sub-note-item="1-7"><div class="lab-step">Check to make sure that all the flies from the female vial are female and likewise, confirm that the male flies are male.  NOTE: Males are slightly smaller and have a darker solid pigment on the end of their abdomen. Females are larger and have stripes rather than a solid spot.
</div></li><li data-sub-note-item="1-8"><div class="lab-step">If there are any male flies in the female vial, obtain a new vial as these females likely have sperm from males from their own strain and are unusable.
</div></li><li data-sub-note-item="1-9"><div class="lab-step">Now, move the five red-eyed females and five white-eyed males into a new vial using the paintbrush.
</div></li><li data-sub-note-item="1-10"><div class="lab-step">Label this vial to indicate that this is a cross between red-eyed females and white-eyed males. HYPOTHESES: Use Punnett squares to formulate hypotheses for this experiment. Consider what you will see in a few days if the white allele is dominant and there is no effect of parental sex. What if red is dominant? Under the potential offspring genotype in each box, write the expected phenotype.
</div></li><li data-sub-note-item="1-11"><div class="lab-step">To set up the reverse cross, again dip the fly anesthesia wand into the solution and insert it this time into the white-eyed female vial and then the red-eyed male vial until all the flies have stopped moving.
</div></li><li data-sub-note-item="1-12"><div class="lab-step">Tap the flies onto a sheet of white paper and check to make sure that all the flies from the female vial are female and likewise with the male flies.
</div></li><li data-sub-note-item="1-13"><div class="lab-step">Now, move the five white-eyed females and five red-eyed males into a new vial using the paintbrush.
</div></li><li data-sub-note-item="1-14"><div class="lab-step">Label this vial to indicate that this is a cross between white-eyed females and red-eyed males.
</div></li><li data-sub-note-item="1-15"><div class="lab-step">Finally, place the two tubes containing the new crosses into a rack for incubation at room temperature. NOTE: After about three days, larvae should be moving around in the vial.
</div></li><li data-sub-note-item="1-16"><div class="lab-step">Remove the 10 adult flies and dispose of them into the morgue tube containing 70% ethanol.
</div></li><li data-sub-note-item="1-17"><div class="lab-step">Now, put the tubes back in the rack and let the larvae mature until they reach the adult stage in up to two weeks.
</div></li><li data-sub-note-item="1-18"><div class="lab-step">Once all of the larvae from these crosses have matured to adulthood, anesthetize the flies in one of the tubes using the wand.
</div></li><li data-sub-note-item="1-19"><div class="lab-step">Then, on a piece of white paper, sort the flies by sex and phenotype and then record these data.
</div></li><li data-sub-note-item="1-20"><div class="lab-step">Now, repeat this process and tally the sexes and phenotypes for the second cross (steps 1 &#8211; 18).
</div></li><li data-sub-note-item="1-21"><div class="lab-step">Finally, once you have counted all of your flies, dispose of them in the morgue vial.
</div></li></ul></li><li data-note-item="2" data-parent-collapse="">Results
<button class="expand">Expand</button><ul class="lab-items"><li data-sub-note-item="2-1"><div class="lab-step">To begin investigating your results, first compare the data you collected with the hypothesis you made using the Punnett squares.
</div></li><li data-sub-note-item="2-2"><div class="lab-step">Did your data match the simple Punnett square hypothesis where red or white is dominant and there is no effect of sex? Compare your data with other lab groups.
</div></li><li data-sub-note-item="2-3"><div class="lab-step">If your data does not match what your Punnett squares predicted, does this indicate that the gene may be sex-linked?
</div></li><li data-sub-note-item="2-4"><div class="lab-step">If the red gene were dominant and sex-linked, when we cross red-eyed females with white-eyed males, all of the offspring would have red eyes. Assuming all of the females were homozygous for the color allele, does this match your data for the first cross?
</div></li><li data-sub-note-item="2-5"><div class="lab-step">If white were dominant and the gene were sex-linked, we would see the exact opposite. So all of the flies would have white eyes. From your data, can you conclude from your first cross whether red or white is dominant?
</div></li><li data-sub-note-item="2-6"><div class="lab-step">When the reciprocal cross of white-eyed females and red-eyed males is performed, if the red phenotype is dominant, all of the females would be red and all of the males would be white. This would also confirm that the gene is specifically x-linked because white-eyed females could then only be produced in crosses with white-eyed males. Did your second cross produce any white-eyed females?
</div></li><li data-sub-note-item="2-7"><div class="lab-step">Finally, if the gene were x-linked and white was dominant, crossing white-eyed females with red-eyed males would result in all of the offspring having white eyes. Does this hypothesis fit with your second cross?
</div></li><li data-sub-note-item="2-8"><div class="lab-step">Look over your data and conclude which of the hypotheses best fits the patterns you found in your crosses. If your data does not fit with any of these, hypothesize why this might be the case.
</div></li></ul></li></ol>

Genetics of Organisms: REF and WEF Fly Genetics - Procedure

生物体的遗传学

REF and WEF Fly Genetics
ExpandNOTE: At your station, you should have four vials of flies. They will be labeled as male or female. They will also be ...

Evolutionary Relationships

<ol class="note-items"><li data-note-item="1" data-parent-collapse="">Forming an Evolutionary Hypothesis
<button class="expand">Expand</button><ul class="lab-items"><li data-sub-note-item="1-1"><div class="lab-step">NOTE: A cladogram is an important tool for forming an evolutionary hypothesis. A cladogram is a tree-shaped chart used to depict the hypothetical genealogical relationships between species. The tips or leaves of the chart represent specific species and the branches of the tree are different lengths. The different lengths represent the degree of change between each of the species. The common ancestor of all of the species that a specific line branches from is located at a node, where the branches intersect. Species that share a node are called a sister group.
</div></li><li data-sub-note-item="1-2"><div class="lab-step">To begin, look at the provided blank cladogram and make a hypothesis about where the assigned animals should be placed on the tree. If a blank cladogram has not been provided please <a target="_blank" href="/files/ftp_upload/10562/Blank_Cladogram.png">download one here</a>.
</div></li><li data-sub-note-item="1-3"><div class="lab-step">Next, observe the image of the fossilized animal.
</div></li><li data-sub-note-item="1-4"><div class="lab-step">Using the morphology, or physical characteristics, of the fossil, make a hypothesis about where on your cladogram it should be placed. Add a new line or mark to indicate your proposed position for the fossil.
</div></li></ul></li><li data-note-item="2" data-parent-collapse="">Comparing Gene Sequences Using BLAST
<button class="expand">Expand</button><ul class="lab-items"><li data-sub-note-item="2-1"><div class="lab-step">NOTE: Historically, cladograms have been constructed through the comparison of morphology. Now, genetic sequences can also be compared between species of interest to construct cladograms. DNA sequences are not usually preserved in fossils, so for this lab you will use the BLAST database to compare the DNA sequences of living species that are closely related to the fossil to thousands of other living species.
</div></li><li data-sub-note-item="2-2"><div class="lab-step">Before beginning this lab, your instructor will have downloaded the three preserved gene sequences to a folder on your desktop.
</div></li><li data-sub-note-item="2-3"><div class="lab-step">Navigate to the BLAST homepage, which the instructor should have left open in your browser, and click on Saved Strategies from the menu at the top of the page.
</div></li><li data-sub-note-item="2-4"><div class="lab-step">Click Open on the Evolutionary_Relationships_Gene1.txt file.
</div></li><li data-sub-note-item="2-5"><div class="lab-step">Then, click View to go to the Standard Nucleotide BLAST search page. You should be able to see the DNA sequence in the Query Sequence box at the top-right of the screen.
</div></li><li data-sub-note-item="2-6"><div class="lab-step">Scroll to the bottom of the screen and click the BLAST button. Note: It will take a few moments to process and analyze the data. 
</div></li><li data-sub-note-item="2-7"><div class="lab-step">When the data has been processed, a graphic summary will appear.  NOTE: Your query sequence is represented by the blue line at the top of the box. Each of the lines below the blue line represents another sequence in the BLAST database that matches your query. The length and location of these bars represent where and how much of the sequence match up to the query. The color of the bar represents its score, or how identical the sequence is to the query. Below the graphic summary is the Sequences producing significant alignments list, which contains descriptions of the DNA sequences retrieved from the database that most closely align with the query sentence. These are listed in descending order, from the most similar at the top to the least similar at the bottom. On the right, there are several statistics on how closely related each database sequence is to the experimental sequence. The higher the Max, Total, Query Coverage, and Identity scores are, the more similar the query and retrieved sequences are to each other. Similarly, the lower the E value number, the less likely the sequences match was found by random chance and more significantly the alignment is accurate. An E value of 0 indicates a highly significant match.
</div></li><li data-sub-note-item="2-8"><div class="lab-step">Click on the name of the description of the most similar alignment listed. This will take you further down the page to the exact DNA sequence alignment between the retrieved sequence and the query sequence.
</div></li><li data-sub-note-item="2-9"><div class="lab-step">Click on the Sequence ID number. This will open a new tab with more specific information on the retrieved gene sequence.
</div></li><li data-sub-note-item="2-10"><div class="lab-step">Identify and record the scientific and common names of the organism, which should be listed next to Source.
</div></li><li data-sub-note-item="2-11"><div class="lab-step">Identify and record the protein that the gene codes for, which should be listed next to Definition.
</div></li><li data-sub-note-item="2-12"><div class="lab-step">Close the tab and hit Back to return to the Sequences producing significant alignments list.
</div></li><li data-sub-note-item="2-13"><div class="lab-step">Then, repeat steps 7 &#8211; 9 for the next most similar sequences.
</div></li><li data-sub-note-item="2-14"><div class="lab-step">After collecting several species names, return to the Sequences producing significant alignments list and click Select: All to check all of the boxes next to the names of each listed alignment sequence.
</div></li><li data-sub-note-item="2-15"><div class="lab-step">Click on Distance of tree results to create a phylogram based on the similarity of all of the BLAST result gene sequences to the query gene sequence.  Note: Your query fossil relative sequence will be highlighted in yellow.
</div></li><li data-sub-note-item="2-16"><div class="lab-step">Record your sequence&#8217;s location in relation to the other taxon groups shown in the tree.
</div></li><li data-sub-note-item="2-17"><div class="lab-step">Finally, return to the Saved Strategies tab and upload the next saved gene sequence.
</div></li><li data-sub-note-item="2-18"><div class="lab-step">Query the remaining two fossil relative DNA sequences as previously demonstrated (steps 1 &#8211; 16).
</div></li></ul></li><li data-note-item="3" data-parent-collapse="">Data Analysis
<button class="expand">Expand</button><ul class="lab-items"><li data-sub-note-item="3-1"><div class="lab-step">Based on your gene sequence results, come up with a second hypothesis about where your fossil specimen fit in the original cladogram.
</div></li><li data-sub-note-item="3-2"><div class="lab-step">Compare this placement with your first hypothesis.
</div></li><li data-sub-note-item="3-3"><div class="lab-step">Compare your tree with your classmates&#8217;. If any of your classmates put the fossil on a different branch, ask them how they came to that decision.
</div></li></ul></li></ol>

Evolutionary Relationships: Using BLAST to Test Evolutionary Hypotheses - Procedure

进化关系

Forming an Evolutionary Hypothesis
ExpandNOTE: A cladogram is an important tool for forming an evolutionary hypothesis. A cladogram is a tree-shaped chart ...

进化

Extinction

<ol class="note-items"><li data-note-item="1" data-parent-collapse="">Diversity and Extinction during the Paleogene Epoch
<button class="expand">Expand</button><ul class="lab-items"><li data-sub-note-item="1-1"><div class="lab-step">Begin this procedure by reading the extinction reading supplement that has been printed out for you. <br /><strong>NOTE:</strong> Alternatively, you may download the reading supplement at the link below.&nbsp; <a href="https://www.jove.com/CDNSource/lm/labs/24/Lab_20_Reading Supplement.docx" style="display:block;margin:20px;" target="_blank">Click here to download the extinction reading supplement</a>
</div></li><li data-sub-note-item="1-2"><div class="lab-step">Using the information contained in the extinction reading supplements pertaining to the taxa of interest and the Chicxulub impact event, make predictions for each taxon listed in Table 1. Consider whether diversity in each taxon increased, decreased, or stayed the same immediately following the Cretaceous-Paleogene boundary and later on in the Paleogene epoch. <a href="https://www.jove.com/CDNSource/lm/labs/24/Table_1_Extinction.xlsx" style="display:block;margin:20px;" target="_blank">Click Here to download Table 1</a>
</div></li><li data-sub-note-item="1-3"><div class="lab-step">Then, fill out the reasons for each of your predictions in the Contributing Factors column. HYPOTHESES: The experimental hypothesis is that many of the different taxa will show changes in diversity after the Cretaceous-Paleogene event, and again at the end of the Paleogene, with some increasing in diversity, while others decrease. The null hypothesis is that there will be no changes in diversity between taxa following the Cretaceous-Paleogene event, or at the end of the Paleogene.
</div></li><li data-sub-note-item="1-4"><div class="lab-step">Gather diversity data by going to the Paleobiology Database: <a href="http://www.paleobiodb.org" target="_blank">www.paleobiodb.org</a>.
</div></li><li data-sub-note-item="1-5"><div class="lab-step">In the center of the screen, click the yellow box labeled Download Data. Under the italicized heading, &#8220;What do you want to download?&#8221;, select Diversity over time on the left side and Comma-separated values on the right side.
</div></li><li data-sub-note-item="1-6"><div class="lab-step">Scroll down to the heading &#8220;Select by taxonomy&#8221;.
</div></li><li data-sub-note-item="1-7"><div class="lab-step">In the first box labeled 'Taxon or taxa to include', type in the name of the taxon that data is being gathered on.  NOTE: Ensure the spelling is correct.
</div></li><li data-sub-note-item="1-8"><div class="lab-step">Now, go to the dropdown menu right below that box labeled &#8220;Taxonomic resolution&#8221;, and select genera.
</div></li><li data-sub-note-item="1-9"><div class="lab-step">Under the next heading, Select by time, type Maastrichtian into the first box and Chattian in the box directly below. Make sure the dropdown menu labeled Time rule is set to major and the Temporal resolution is set to stage.
</div></li><li data-sub-note-item="1-10"><div class="lab-step">Select &#8220;Assume extant taxa range through to present&#8221;. Scroll back up to the middle of the page and click download. A save window will open.
</div></li><li data-sub-note-item="1-11"><div class="lab-step">Save the .csv file to a safe and accessible place and rename it to make it easy to identify.
</div></li><li data-sub-note-item="1-12"><div class="lab-step">Open the file and scroll down to the main dataset under Records.  NOTE: The data are arranged in reverse time order by geological stage. The second column, interval_name, gives the accepted name of the stage in question. The next two columns give the appropriate dates of the stage in millions of years before the present. The final column, number of occurrences, gives a total count of the genera extant during that geological stage as determined from occurrence of fossils.
</div></li><li data-sub-note-item="1-13"><div class="lab-step">To more easily work with this data, copy and paste each column into a new sheet. Be sure to label this sheet with the taxon in question, the data and time the data were obtained, and the data source.
</div></li><li data-sub-note-item="1-14"><div class="lab-step">Repeat this process for each of the taxa in Table 1 (steps 4 &#8211; 12). NOTE: Data from multiple taxa can be kept on the same sheet, so long as they are kept distinct and labeled appropriately.
</div></li><li data-sub-note-item="1-15"><div class="lab-step">To create line plots for each of the taxa showing the change in diversity over time, select the max_ma column and the number of occurrences column simultaneously.
</div></li><li data-sub-note-item="1-16"><div class="lab-step">In Excel, select Insert from the top toolbar and find the section labeled Charts. Select the bottom center icon, Insert Scatter Chart.
</div></li><li data-sub-note-item="1-17"><div class="lab-step">Then, select the Scatter with Straight Lines and Markers and a graph should appear. NOTE: Since the axis of this graph is in units of millions of years ago, the axis is in the reverse direction of time when put in numerical order.
</div></li><li data-sub-note-item="1-18"><div class="lab-step">To fix this, click on the green plus sign in the top-right corner of the graph and hover over Axes. This should reveal a small black arrow. Click the arrow to open up a small menu and select More Options.
</div></li><li data-sub-note-item="1-19"><div class="lab-step">This will open the Axis Options menu. Under Axis Options, click the box next to Values in reverse order. NOTE: It is also possible to change the axis scales from this menu, which can be used to eliminate the excess time that is not being analyzed in this lab. Keep any changes consistent between different graphs.
</div></li><li data-sub-note-item="1-20"><div class="lab-step">Double-click on the title at the top of the graph and change it to the taxon being represented.
</div></li><li data-sub-note-item="1-21"><div class="lab-step">Make a separate graph for each of the taxa in Table 1. Keep them organized and in one location. A separate sheet for graphs is recommended.
</div></li></ul></li><li data-note-item="2" data-parent-collapse="">Results
<button class="expand">Expand</button><ul class="lab-items"><li data-sub-note-item="2-1"><div class="lab-step">For each graph, identify the location of the Cretaceous-Paleogene boundary and draw a vertical line through the graph at this point.
</div></li><li data-sub-note-item="2-2"><div class="lab-step">Using the information from the graphs, fill out Table 2. In some cases, it may be helpful to go back to the original dataset and compare actual values, particularly when they are small. <a href="https://www.jove.com/CDNSource/lm/labs/24/Table_2_Extinction.xlsx" style="display:block;margin:20px;" target="_blank">Click Here to download Table 2</a>
</div></li><li data-sub-note-item="2-3"><div class="lab-step">Compare these values to the predictions made in Table 1 and discuss if the original contributing factors are still likely candidates.
</div></li><li data-sub-note-item="2-4"><div class="lab-step">Using the dataset, calculate the percentage of total taxa that went extinct over the Cretaceous-Paleogene boundary using this equation.
</div></li><li data-sub-note-item="2-5"><div class="lab-step">There is an event in the middle of the Paleogene known as the Paleocene-Eocene Thermal Maximum about 55.5 million years ago. This represents a high point in temperature during the Paleogene epoch at approximately 8 &#176;C higher than today&#39;s average temperature. Does this point seem to have an effect on diversity for any of the taxa? If yes, then which ones?
</div></li></ul></li></ol>

Extinction: Diversity and Extinction during the Paleogene Epoch - Procedure

灭绝

Diversity and Extinction during the Paleogene Epoch
ExpandBegin this procedure by reading the extinction reading supplement that has been printed out for ...

Microbial and Fungal Diversity

<ol class="note-items"><li data-note-item="1" data-parent-collapse="">Examining Microbial Colony Structures
<button class="expand">Expand</button><ul class="lab-items"><li data-sub-note-item="1-1"><div class="lab-step">To begin, collect your allocated culture plates and then remove the Parafilm and lid from one of them.
</div></li><li data-sub-note-item="1-2"><div class="lab-step">Place the uncovered plate under a dissecting microscope and then observe the individual communities within each quadrant of the plate.
</div></li><li data-sub-note-item="1-3"><div class="lab-step">As the colonies are identified, fill out the observed colony properties in the appropriate column in the table.  <a href="https://www.jove.com/CDNSource/lm/labs/28/Table_1_Microbial_diversity.xlsx" style="display:block;margin:20px;" target="__blank">Click Here to download Table 1</a>
</div></li><li data-sub-note-item="1-4"><div class="lab-step">Then, repeat the observations for each of the other two bacterial species.
</div></li><li data-sub-note-item="1-5"><div class="lab-step">To perform Gram staining of your three bacterial species, first label the ends of three microscope slides with the initials of each bacteria species, your initials, and the date. HYPOTHESES: In this experiment, the experimental hypothesis might be that it is possible to elucidate different bacterial species and shapes using Gram staining, and that Gram-positive bacteria will stain purple with crystal violet whereas Gram-negative bacteria will appear pink due to the safranin counterstain. The null hypothesis could be that all of the bacterial types will retain both stains equally, and staining will not help elucidate their species or shape.
</div></li><li data-sub-note-item="1-6"><div class="lab-step">To begin the first stain, attach a clip to the slide for ease of handling. For each bacterial smear, remember to be sure to use a cleaned and flame-sterilized inoculating loop.
</div></li><li data-sub-note-item="1-7"><div class="lab-step">Place two loopfuls of sterile distilled water onto the center of the slide.
</div></li><li data-sub-note-item="1-8"><div class="lab-step">After sterilizing the loop again, cool the loop by touching an uninoculated portion of the agar several times.
</div></li><li data-sub-note-item="1-9"><div class="lab-step">Then, remove a small amount of culture from an isolated colony and mix it with the water on the slide.
</div></li><li data-sub-note-item="1-10"><div class="lab-step">Allow the slide to dry at room temperature and heat-fix the smear by quickly passing it through a flame.
</div></li><li data-sub-note-item="1-11"><div class="lab-step">Once dry, pipette several drops of crystal violet onto the slide and let it sit for two to three minutes.
</div></li><li data-sub-note-item="1-12"><div class="lab-step">Carefully rinse the slide with distilled water by aiming the direct flow towards the top of the slide, allowing the water to gently flow down. Do not aim the water flow directly at the smear.
</div></li><li data-sub-note-item="1-13"><div class="lab-step">Next, cover the slide with Gram&#39;s iodine, leave it to sit again for two minutes, and then gently rinse with distilled water.
</div></li><li data-sub-note-item="1-14"><div class="lab-step">Decolorize the slide with 95% ethanol until stain no longer washes away.
</div></li><li data-sub-note-item="1-15"><div class="lab-step">Immediately, rinse the slide with distilled water. This will limit over-decolorizing.
</div></li><li data-sub-note-item="1-16"><div class="lab-step">Then, add several drops of safranin counterstain and leave for 30 s. This will stain any Gram-negative cells present.
</div></li><li data-sub-note-item="1-17"><div class="lab-step">Gently rinse the slide with distilled water and blot dry with absorbent paper.
</div></li><li data-sub-note-item="1-18"><div class="lab-step">After staining the other two slides in the same manner (steps 6 &#8211; 17), observe each with a microscope.
</div></li><li data-sub-note-item="1-19"><div class="lab-step">Use a low power objective first to make coarse adjustments. When you find an ideal section, move on to the higher magnifications.
</div></li><li data-sub-note-item="1-20"><div class="lab-step">After further imaging and adjusting the smear with a medium power objective, add immersion oil directly to the smear. NOTE: The oil is needed for high power objectives that will provide the best micrographs.
</div></li><li data-sub-note-item="1-21"><div class="lab-step">Now, use colored pencils to draw the observed bacteria in the appropriate magnification circles and fill in the observed characteristics in the appropriate column, noting the bacteria&#39;s shape and whether it is Gram-positive, identified by darker purple staining, or Gram-negative, identified with pink staining. <a href="https://www.jove.com/CDNSource/lm/labs/28/Table_2_Microbial_diversity.xlsx" style="display:block;margin:20px;" target="__blank">Click Here to download Table 2</a>
</div></li><li data-sub-note-item="1-22"><div class="lab-step">Finally, repeat the observation, drawing, and characterization for the other two stained bacterial slides.
</div></li></ul></li><li data-note-item="2" data-parent-collapse="">Examining Fungal Structures
<button class="expand">Expand</button><ul class="lab-items"><li data-sub-note-item="2-1"><div class="lab-step">Remove the <em>S. cerevisiae</em> plate from the incubator and place a drop of water onto a microscope slide.
</div></li><li data-sub-note-item="2-2"><div class="lab-step">Using a sterilized inoculating loop, smear a film of yeast into the drop of water and place a cover slip over the slide.
</div></li><li data-sub-note-item="2-3"><div class="lab-step">Place the slide under the microscope at the lowest magnification, increasing the magnification to the oil immersion setting to see the individual yeast cells.
</div></li><li data-sub-note-item="2-4"><div class="lab-step">Observe the general shape and structure of the yeast and identify any cells that are undergoing asexual reproduction by budding.
</div></li><li data-sub-note-item="2-5"><div class="lab-step">Now, draw the cells in the appropriate magnification circles.
</div></li><li data-sub-note-item="2-6"><div class="lab-step">When you are done observing the yeast, replace the <em>S. cerevisiae</em> slide with a prepared Ascomycetes apothecium slide and set the microscope to the lowest magnification.
</div></li><li data-sub-note-item="2-7"><div class="lab-step">Using colored pencils, draw the structures at low and high magnifications, including the asci and ascospores.
</div></li><li data-sub-note-item="2-8"><div class="lab-step">Finally, place a button mushroom, <em>Agaricus bisporus</em>, under the dissecting microscope and draw the features, including the pileus, or cap, the gills, and the stipe, or stalk.
</div></li></ul></li><li data-note-item="3" data-parent-collapse="">Results
<button class="expand">Expand</button><ul class="lab-items"><li data-sub-note-item="3-1"><div class="lab-step">Using the coloring of the bacteria recorded in the table, determine which species are Gram-positive and which are Gram-negative.
</div></li><li data-sub-note-item="3-2"><div class="lab-step">Then, look at the shapes recorded in the table to determine which bacterial species are bacillus, coccus, or spirillum.
</div></li><li data-sub-note-item="3-3"><div class="lab-step">Next compare your illustrations of the yeast cells to your illustrations of the bacterial cells. NOTE: Yeast from the Ascomycetes phylum, such as <em>S. cerevisiae</em>, can reproduce asexually, which can be observed through the budding of the cells under a microscope.
</div></li><li data-sub-note-item="3-4"><div class="lab-step">Record any observations of yeast cells budding.  NOTE: Ascomycetes can also reproduce sexually via mitosis and haploid spore formation.
</div></li><li data-sub-note-item="3-5"><div class="lab-step">Record any observations of spore or asci formation you observed in the table.
</div></li><li data-sub-note-item="3-6"><div class="lab-step">NOTE: Fungus from the Basidiomycota phylum exhibit clearly observable gills under the pileus.
</div></li><li data-sub-note-item="3-7"><div class="lab-step">Record any fungal species that demonstrated gills under the pileus.
</div></li></ul></li></ol>

Microbial Colony and Fungal Diversity - Procedure

微生物和真菌多样性

Examining Microbial Colony Structures
ExpandTo begin, collect your allocated culture plates and then remove the Parafilm and lid from one of them.
Place ...

Measuring Biodiversity

<ol class="note-items"><li data-note-item="1" data-parent-collapse="">Quadrats and Transects
<button class="expand">Expand</button><ul class="lab-items"><li data-sub-note-item="1-1"><div class="lab-step">In this activity, you will be using quadrats along transect lines to take biodiversity measurements of the plant species in three distinct habitats near your classroom. Begin by splitting into groups. Four works best.
</div></li><li data-sub-note-item="1-2"><div class="lab-step">Select one person in your group to be the data recorder. The other three will be the data collectors.
</div></li><li data-sub-note-item="1-3"><div class="lab-step">Next, take one quadrat, or hula hoop, per group and place it on one side of one transect, or rope, next to a knot to allow identification of the plant species encircled by your quadrat. HYPOTHESES: For this experiment, the alternative hypothesis is that the alpha diversity will be smaller than gamma, meaning that there will be differences in the species that are present in each different habitat. The null hypothesis for this activity is that the alpha and gamma will be equal, meaning that there will be no species diversity difference between the communities sampled.
</div></li><li data-sub-note-item="1-4"><div class="lab-step">Each data collector must survey and identify the different plant species or any morphologically distinct varieties within your group&#39;s quadrat, giving the name of each plant type that you identify to the data recorder. It may seem like there are only a couple of species present at first, but look closer. It may take several minutes to find them all. 
</div></li><li data-sub-note-item="1-5"><div class="lab-step">In addition to recording the species name, the data recorder must also write a short description in the table, so that each species can be identified again in subsequent quadrats. <a href="https://www.jove.com/CDNSource/lm/labs/25/Table_1_Measuring_biodiversity.xlsx" style="display:block;margin:20px;" target="__blank">Click Here to download Table 1</a>
</div></li><li data-sub-note-item="1-6"><div class="lab-step">Once all of the species within a quadrat have been identified, pick up your hoop and move on to the next knot along the same transect.
</div></li><li data-sub-note-item="1-7"><div class="lab-step">Continue surveying and moving to the next knot until you have completed the entire rope transect.
</div></li><li data-sub-note-item="1-8"><div class="lab-step">Once you have completed the rope, move to a transect in a different habitat, and begin identifying the different species within each quadrat along the new transect, until each group has identified plants along three lengths of rope, one in each habitat, for a total of 15 quadrats surveyed.
</div></li><li data-sub-note-item="1-9"><div class="lab-step">When all of the species data have been collected, carry the transects and quadrats back to the classroom.
</div></li></ul></li><li data-note-item="2" data-parent-collapse="">Species-Accumulation Curves and Richness/Evenness 
<button class="expand">Expand</button><ul class="lab-items"><li data-sub-note-item="2-1"><div class="lab-step">In the next activity, you will be calculating and comparing the species richness and evenness in two distinct populations, represented here by two opaque bags containing beads of various colors. For this exercise, start by taking one paper bag of beads per group.
</div></li><li data-sub-note-item="2-2"><div class="lab-step">Have one person reach into the bag and randomly pull out three beads without looking.
</div></li><li data-sub-note-item="2-3"><div class="lab-step">Write down the bead colors, as well as the total number of colors pulled from the bag in the table. <a href="https://www.jove.com/CDNSource/lm/labs/25/Table_2_Measuring_biodiversity.xlsx" style="display:block;margin:20px;" target="__blank">Click Here to download Table 2</a>HYPOTHESES: In this activity, the alternative hypothesis states that species evenness in the two communities will be different, while the richness will be the same. The null hypothesis might be that species richness and evenness will be the same in both communities.
</div></li><li data-sub-note-item="2-4"><div class="lab-step">Place the beads back in the bag after their colors are recorded.
</div></li><li data-sub-note-item="2-5"><div class="lab-step">Then, shake the bag and have the next person in the group draw another set of three beads.
</div></li><li data-sub-note-item="2-6"><div class="lab-step">Record the colors and numbers of beads that they pulled in the table.
</div></li><li data-sub-note-item="2-7"><div class="lab-step">Record the colors in the second row and the cumulative number of colors drawn in the last column.
</div></li><li data-sub-note-item="2-8"><div class="lab-step">Continue to take turns drawing three beads from the bag until 20 samples have been drawn.
</div></li><li data-sub-note-item="2-9"><div class="lab-step">Next, switch bags with a group with a bag from the other community, and repeat the exercise of drawing 20 samples, recording the color data in the appropriate community column of the table.
</div></li><li data-sub-note-item="2-10"><div class="lab-step">When all of the data have been recorded, return the beads to the bag and return the bags to your instructor.
</div></li></ul></li><li data-note-item="3" data-parent-collapse="">Results
<button class="expand">Expand</button><ul class="lab-items"><li data-sub-note-item="3-1"><div class="lab-step">To calculate the alpha diversity of the transect and quadrat data collected by your group, use the data your data recorder entered for each species and enter it into your diversity table. <a href="https://www.jove.com/CDNSource/lm/labs/25/Table_3_Measuring_biodiversity.xlsx" style="display:block;margin:20px;" target="__blank">Click Here to download Table 3</a>
</div></li><li data-sub-note-item="3-2"><div class="lab-step">Then, fill out the alpha diversity for each habitat into the appropriate column in the table.
</div></li><li data-sub-note-item="3-3"><div class="lab-step">Use the species data from your quadrat and transect table and enter the beta diversity data into the appropriate data cells within the diversity table. NOTE: The beta diversity is the number of species that are unique between two habitats. In this video, there are three species in common between the two sites, but a total of six unique species, giving a beta diversity of six between site A and B.
</div></li><li data-sub-note-item="3-4"><div class="lab-step">To calculate the gamma diversity for your activity, use the data from your quadrat and transect table and enter the gamma diversity data into the appropriate data cell. NOTE: The gamma diversity is the number of species in all of the habitats within a single study. It is not the sum of all of the alpha diversities, because species that are common between habitats should be counted only once.
</div></li><li data-sub-note-item="3-5"><div class="lab-step">To construct species accumulation curves for both of the bead sampling communities, use your bead color selection data to plot the species by sampling effort. That is the number of total colors by sample in the graph for each bead bag community.
</div></li><li data-sub-note-item="3-6"><div class="lab-step">Then, estimate the asymptote of the plotted data by appearance. Predict the number of species you would expect to find in each community. Note any differences in appearance between the two curves. If it was easier to estimate the number of species in one of the communities, make a hypothesis to explain this observation.
</div></li><li data-sub-note-item="3-7"><div class="lab-step">To construct rank abundance curves for both of the bead sampling communities, use the color bead data to fill in the table for both community A and community B, listing the colors that were identified during the sampling in the color column, and adding the total number of beads of each color that was sampled in the abundance column. <a href="https://www.jove.com/CDNSource/lm/labs/25/Table_4_Measuring_biodiversity.xlsx" style="display:block;margin:20px;" target="__blank">Click Here to download Table 4</a>
</div></li><li data-sub-note-item="3-8"><div class="lab-step">Now, assign a rank to each color in the rank column, designating the color of the highest abundance the highest rank, rank one, and the color with the lowest abundance the lowest rank, rank 10. If there are multiple colors with the same number, just assign them consecutive ranks.
</div></li><li data-sub-note-item="3-9"><div class="lab-step">Plot individual rank abundance curves for the number of individuals by species ranked for the most to least common in both community A and community B. Compare the steepness of the two rank curves. Compare the richness and evenness between the two communities. Identify the community that you consider more biodiverse. 
</div></li></ul></li></ol>

衡量生物多样性

Quadrats and Transects
ExpandIn this activity, you will be using quadrats along transect lines to take biodiversity measurements of the plant species in ...

Transpiration

<ol class="note-items"><li data-note-item="1" data-parent-collapse="">Comparing Transpiration Rates of Leaves
<button class="expand">Expand</button><ul class="lab-items"><li data-sub-note-item="1-1"><div class="lab-step">To begin the experiment to measure the transpiration rates of leaves from different plant species, you will first create a tool called a potometer, using a piece of plastic tubing and 5 mL calibrated pipette.
</div></li><li data-sub-note-item="1-2"><div class="lab-step">To assemble the device, simply attach the rubber tubing to the tip of the pipette.
</div></li><li data-sub-note-item="1-3"><div class="lab-step">Then, submerge the device into a bucket of water and move the pipette around under the water to fully fill the tube, taking care not to create bubbles.
</div></li><li data-sub-note-item="1-4"><div class="lab-step">Next, cap the open end of the tube with a pipette bulb to keep the water in.
</div></li><li data-sub-note-item="1-5"><div class="lab-step">Now, taking care not to spill water from the potometer, use a clamp and stand to hold the capped end of the pipette around 1 - 3 inches below the pipette bulb.
</div></li><li data-sub-note-item="1-6"><div class="lab-step">Then, use a second clamp attached lower on the same stand to secure the open end of the rubber tubing.
</div></li><li data-sub-note-item="1-7"><div class="lab-step">Next, use a transfer pipette or dropper to remove water from the open end of the tubing until the water level in the pipette portion of the potometer rests at the zero line. HYPOTHESES: In this experiment, the experimental hypothesis might be that leaves from plant species more adapted to hotter or arid environments will have lower transpiration rates than those from humid or wet regions. The null hypothesis would be that leaves of the different plant species will not differ in their transpiration rates.
</div></li><li data-sub-note-item="1-8"><div class="lab-step">To begin the experiment, carefully use scissors to cut the end of the petiole, or stalk, of the first leaf and then insert the leaf into the tubing.
</div></li><li data-sub-note-item="1-9"><div class="lab-step">Then, place a layer of lubricant around the area where the plant stem and rubber tubing meet to make a watertight seal.
</div></li><li data-sub-note-item="1-10"><div class="lab-step">Next, remove the pipette bulb and taking care not to disturb the setup, let the experiment run for the appropriate experimental period, typically 30 - 60 minutes.
</div></li><li data-sub-note-item="1-11"><div class="lab-step">At the end of the allocated time, record the level of the water in the pipette, noting how much the level dropped in milliliters.
</div></li><li data-sub-note-item="1-12"><div class="lab-step">Then, refill and reset the potometer water level to zero, and then repeat the procedure for each of the remaining test leaves until the transpiration rates of all four leaves have been measured.
</div></li><li data-sub-note-item="1-13"><div class="lab-step">Next, to determine the surface area of the leaf samples, lay each leaf on a blank piece of paper and carefully trace the outlines.
</div></li><li data-sub-note-item="1-14"><div class="lab-step">Then, cut the outlines out and record the mass of each tracing.
</div></li><li data-sub-note-item="1-15"><div class="lab-step">Finally, cut out and weigh one four by four centimeter square of the same type of paper used to make the leaf tracings. This will act as a reference with known area and weight.
</div></li><li data-sub-note-item="1-16"><div class="lab-step">Next, you will need to quantify the number of stomata per unit area on each leaf. To do this, first prepare a slide mount by painting at least one square centimeter of the underside of each leaf with clear nail polish. IMPORTANT: Ensure you paint the bottom of the leaf, since the majority of the stomata are located there. 
</div></li><li data-sub-note-item="1-17"><div class="lab-step">When the nail polish is completely dry, carefully press a small piece of cellophane tape directly onto each patch and then gently peel off the tape to remove the painted on polish. NOTE: Use a piece of tape that is smaller than the microscope slide. 
</div></li><li data-sub-note-item="1-18"><div class="lab-step">Then, place each impression and tape onto a separate, clean microscope slide. HYPOTHESES: In this exercise, the alternative hypothesis could be that the leaves of the plants adapted to drier environments will have fewer stomata per unit area than the leaves of plants adapted to wetter environments. The null hypothesis might be that all plants species will have an equal number of stomata per unit area.
</div></li><li data-sub-note-item="1-19"><div class="lab-step">To observe each impression under the microscope, first use a low magnification to find areas of the leaf impression that contain stomata. These will appear as darker dots or impressions.
</div></li><li data-sub-note-item="1-20"><div class="lab-step">Then, switch to a higher magnification, keeping the sample viewing window centered on the stomata-containing area.
</div></li><li data-sub-note-item="1-21"><div class="lab-step">Draw and label your observations, making sure to label each sketch with the plant species.
</div></li><li data-sub-note-item="1-22"><div class="lab-step">Then, count and record the number of stomata in the field of view. You should perform this count a total of four times in four different areas of the leaf impression, and then determine the average number of stomata per counted area on the leaf.
</div></li><li data-sub-note-item="1-23"><div class="lab-step">To calculate the number of stomata per mm<sup>2</sup>, place a transparent plastic ruler on the microscope stage under the objective and measure the diameter of the field of view.
</div></li><li data-sub-note-item="1-24"><div class="lab-step">You can then use this number, the diameter or D, to calculate the area of the field of view in mm<sup>2</sup>.
</div></li></ul></li><li data-note-item="2" data-parent-collapse="">Results
<button class="expand">Expand</button><ul class="lab-items"><li data-sub-note-item="2-1"><div class="lab-step">First, calculate the surface area of the 4x4 cm paper square to obtain the number of square centimeters.
</div></li><li data-sub-note-item="2-2"><div class="lab-step">Then, divide the weight of the paper square by the number of centimeters squared it covers to obtain the weight of 1 cm<sup>2</sup> of paper.
</div></li><li data-sub-note-item="2-3"><div class="lab-step">Finally, to calculate the surface area of each leaf, divide the weight of the leaf tracing by the weight of 1 cm<sup>2</sup> of paper. This will give you leaf area in centimeters squared.
</div></li><li data-sub-note-item="2-4"><div class="lab-step">You should now convert this number to meters squared, the standard unit used for calculating transpiration rate.
</div></li><li data-sub-note-item="2-5"><div class="lab-step">To calculate the transpiration rate for each leaf, divide the total water loss you measured in the potometer in mL by the time the experiment was run for in minutes.
</div></li><li data-sub-note-item="2-6"><div class="lab-step">Then, divide this by the surface area of the leaf in m<sup>2</sup>.
</div></li><li data-sub-note-item="2-7"><div class="lab-step">Make a bar graph with the four different leaf species on the X axis, and their corresponding transpiration rates on the Y axis. Do these rates appear different?
</div></li><li data-sub-note-item="2-8"><div class="lab-step">Next, add a second Y axis to your chart and plot the average number of stomata per millimeter squared for each leaf that was observed under the microscope. Do you see a correlation between transpiration rate and number of stomata? If so, does this seem to have any relationship to the native environment the tree would typically inhabit?
</div></li></ul></li></ol>

Assessing Transpiration Rates using Potometer - Procedure

蒸腾作用

Comparing Transpiration Rates of Leaves
ExpandTo begin the experiment to measure the transpiration rates of leaves from different plant species, you will ...