Name: discovering middle ear anatomy by transcanal endoscopic ear surgery: a dissection manual
Uploaded: 2018-01-11T13:00:00.000+00:00
Duration: 10 min 40 s
Description: Watch this Scientific Journal Video about Discovering Middle Ear Anatomy by Transcanal Endoscopic Ear Surgery: A Dissection Manual at JoVE.com

<p class="jove_content">O presente estudo foi aprovado pelo Conselho de revisão institucional local (KEK-ser 00887-2016).</p>
<p class="jove_title">1. preparação da amostra</p>
<ol><li>Posicione um colchão a vácuo na mesa de dissecação e cubra-o com um cobertor estéril. Coloque o espécime anatômico na posição cirúrgica sobre o colchão com a cabeça ligeiramente rodada para o lado contralateral.</li>
	<li>Retrai o tragus com um gancho flexível para o lado contralateral.</li>
	<li>Cobri a amostra com um cobertor, poupando o ouvido externo, assim como na sala de cirurgia.<br>Nota: Consulte a <strong>Tabela de materiais</strong> , para obter uma lista completa de equipamentos necessários.</li>
</ol><p class="jove_title">2. como começar</p>
<ol><li>Adotar uma posição confortável ou posição sentada e ajustar a tela do endoscópio. Esta é uma questão importante para evitar o endurecimento dos músculos do pescoço e, portanto, mais rápido esgotamento.</li>
	<li>Executar o branco equilíbrio em seu menu de câmera e aplicar uma gota de solução de anti-fog na ponta do endoscópio.</li>
	<li>Segure os instrumentos cirúrgicos em sua mão dominante e o endoscópio na outra mão.</li>
	<li>Introduza o endoscópio de comprimento de 15 cm, 3 mm de diâmetro e 0° a EAC. Como uma primeira tarefa, limpe o EAC completamente de cera de ouvido eventualmente presente usando o tubo de sucção. Enxágue o EAC com água. Corte o cabelo dentro do EAC com um par de tesouras pequenas para evitar o endoscópio se tornar suja durante cada passagem.</li>
	<li>Uma vez que o EAC é limpo, inspecione a membrana timpânica (TM). Identifica:<br>-Malleus com processo curto, manúbrio e umbo<br>-Anel de membrana timpânica<br>-Pars flaccida (membrana Shrappnel)</li>
</ol><p class="jove_title">3. elevação do retalho Tympano-Meatal (TMF)</p>
<p class="jove_content">Nota: Em cirurgia endoscópica de ouvido, a TMF é executada ligeiramente menor e em uma forma triangular. A razão é o espaço limitado dentro do EAC.</p>
<ol><li>Faça uma incisão na pele EAC superiormente, ligeiramente anterior o processo curto do martelo usando uma foice ou uma faca em volta.</li>
	<li>Estender a incisão para a porção superior e posterior da EAC e prolongar a incisão para a fronteira anterior e inferior do TM para configurar um retalho em forma triangular.</li>
	<li>Com a faca redonda em ângulo de 45°, expor o osso e elevar a pele até alcançar o anel. É importante aderir ao osso.</li>
	<li>Eleve o anel com a faca de volta para acessar a cavidade timpânica (TC). A mucosa do TC é um pouco mais transparente do que a pele e é incisão ao nível da chorda tympani usando um gancho.</li>
	<li>Mobilize o ânulo inferiormente e anteriormente usando o Dissecador Thomassin.</li>
	<li>Identifica superiormente o ligamento lateral posterior do martelo (PLML). Este ligamento representa o limite inferior do espaço de Prussak. Agarre o ligamento com a pinça de Hartmann e puxe-a delicadamente anteriormente, no sentido do umbo até descobrir o processo curto do martelo.</li>
	<li>Delicadamente, disse o espaço de Prussak anterior até atingir a inserção superior anterior do annulus. Agora identifica:<br>-Anel<br>-Chorda tympani<br>-Maleolares posterior ligamento<br>-Lateral maleolares ligamentar dobra<br>-Maleolares anterior ligamentar dobra<br>-Espaço de Prussak</li>
</ol><p class="jove_title">4. desprendimento do TMF do identificador do Malleus</p>
<ol><li>Do processo curto do martelo, continuar a dissecação e entalha o periostium do punho em direção a umbo maleolares com um Dissecador de agulha. É importante destacar o TM no plano certo para evitar perfurações acidentais.</li>
	<li>Desanexe o TM completamente do manúbrio. Um cottonoid seco pode ser útil, uma vez que facilita a identificação da camada correta e sua dissecação.</li>
	<li>Uma vez atingido o umbo, corte a camada fibrosa restante da TM usando uma microtesoura.</li>
	<li>Agora Posicione a aba toda na parede anterior da EAC para permitir o acesso gratuito para o TC. Identifica:<br>-Fibrosa camada de membrana timpânica<br>-Anel superior anterior<br>-Umbo, manúbrio e processo curto malleus<br>-Chorda tympani anterior o martelo</li>
</ol><p class="jove_title">5. ouvido médio exploração</p>
<p class="jove_content">Nota: Esta parte das ajudas dissecação no reconhecimento e no ensaio da anatomia do ouvido médio. Cada subdivisão do TC deve ser explorado separadamente usando endoscópios de 0° e 45°. Eventualmente, apresentam dobras ou aderências cuidadosamente devem ser removidas.</p>
<ol><li>Epitympanum. Identifica:<br>-Martelo: Pescoço, processo curto, manúbrio e umbo<br>-Bigorna: Corpo, longo processo, processo lenticular, incudostapedial comum<br>-Epitympanic diafragma: ligamentos maleolares anterior e lateral, dobra ligamentar lateral incudomalleolar<br>-Anterior e posterior da coluna<br>-Chorda tympani<br>-Timpânicas padrões istmo e ventilação para o antro<br>-Processo Cochleariform<br>-Músculo de tímpanos tensor, tendão e canal ósseo<br>-Dobra tensor</li>
	<li>Mesotympanum. Identifica:<br>-Estribo: Cápsula, anteriores e posteriores crus, estribo<br>-Tendão do músculo estapédica<br>-Eminência piramidal<br>-Osso promontório<br>-Nervo de Jacobson com artéria inferior timpânicas</li>
	<li>Retrotympanum: Se existirem aderências eles devem ser completamente removidos. Mudando a posição do cirurgião para o lado contralateral facilita acesso a retrotympanum a bobina em forma ótica (45°). Identifica:<br>-Recesso facial<br>-Sinus posterior<br>-Ponticulus<br>-Tímpano sinusal<br>-Subiculum<br>-Estiloide Eminência<br>-Sinus subtympanic<br>-Fustis óssea<br>-Área concamerata<br>-Nicho da janela redonda com tegmen, pilar anterior e posterior<br>-Membrana de janela redonda<br>-Canalículo Subcochlear<br>-Finiculus</li>
	<li>Hypotympanum<br>-Células Hypotympanic<br>-Estimar a localização da abelha se não visível</li>
	<li>Protympanum. Identifica:<br>-Protiniculus<br>-Artéria carótida interna<br>-Trompa de Eustáquio (ET)</li>
	<li>Desarticular a articulação incudostapedial e remover a bigorna. Com uma cureta remova o escudo. Identifica:<br>-Timpânicas segmento do nervo facial<br>-Lateral canal semicircular<br>-Antrum<br>-COG ou crista transversal<br>-Tímpano tegmen</li>
	<li>Cortar o tendão do músculo tensor tímpanos e remover o martelo. Identifica:<br>-Epitympanum anterior<br>-Gânglio geniculado</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">Os autores declaram não haverá conflito de interesses. LA tem uma bolsa de pesquisa pela Fundação Bangerter-Naraa, Bern, Suíça e por Karl Storz GmbH, Tuttlingen, Alemanha. Os financiadores não tinham qualquer papel no projeto de estudo, coleta de dados e análise, a decisão de publicar ou preparação do manuscrito.</p>

<p class="jove_content">O manual de dissecação proposto é útil para a realização de uma dissecação completa do ouvido médio. Exibir um conhecimento profundo da anatomia do ouvido médio é um pré-requisito fundamental para qualquer intervenção cirúrgica do ouvido médio. Cadavérico dissecação permite treinar o manuseio da câmera e os instrumentos cirúrgicos na EAC. Para um iniciante no EEE, a coordenação entre o olho e o instrumento, bem como o manuseio correto do endoscópio (não dobra, manobrando entre estruturas anatômicas importantes como os ossículos em um espaço limitado e a imagem bidimensional) são críticos etapas para começar com esta técnica cirúrgica de emergentes.</p>
<p class="jove_content">A principal limitação deste modelo em comparação com a situação real cirúrgica é a falta de sangramento. Isto aplica-se para qualquer modelo de dissecação cadavérico. Recentemente, Dedmond <em>et al</em>. descreveu um modelo de osso temporal, onde o sangramento foi simulada durante a elevação do TMF. Este modelo na verdade pode ser uma boa opção para os cirurgiões avançadas já costumava EES. Em nossa experiência, a familiarização inicial com EES deve ser tão fácil quanto possível, uma vez que a manipulação dos instrumentos já é uma tarefa desafiadora. A dissecação cadavérico é considerada o padrão-ouro para a educação cirúrgica. No entanto, é sujeito a custos elevados e normas éticas. Em comparação, modelos animais ou sintéticos podem superar estas questões<sup class="xref">8</sup><sup>,</sup><sup class="xref">11</sup>. No entanto, nos modelos sintéticos a sensação tátil para os tecidos é difícil e a resolução de impressão 3D ainda não é capaz de fornecer todos os detalhes de que um espécime anatômico pode. Em contraste, o modelo animal fornece propriedades tecido excelente, mas tem uma anatomia diferente. Podemos considerar o modelo cadavérico o único modelo apropriado para ensino anatômico, Considerando que modelos sintéticos e animais fornecem alternativas baratas para treinamento cirúrgico.</p>
<p class="jove_content">Em comparação com a técnica microscópica para dissecação do ouvido médio, o endoscópio permite a observação e a preparação das estruturas delicadas ouvido médio através de um orifício natural, a EAC. Portanto, nenhum osso tem que ser removido para fins de acesso e do ouvido médio pode ser estudado em seu estado natural. Além disso, o endoscópio permite uma observação muito perto de qualquer estrutura anatômica e, portanto, também a ampliação sem perder a iluminação. Claro, a abordagem endoscópica não ensina mastoidectomy, que pode ser efectuada na mesma amostra após a dissecação endoscópica. A técnica endoscópica para cirurgia de ouvido internacionalmente está se espalhando e, portanto, a necessidade de formação cirúrgica adequada irá aumentar.</p>

<p class="jove_content">O ouvido médio está localizado no centro do osso temporal e ostenta uma anatomia altamente complexa. As estruturas mais contíguas são o nervo facial (FN), a cóclea (CO), a cadeia ossiculary (OC), o bulbo da veia jugular (abelha) e a artéria carótida interna (ACI). A fissura do ouvido médio é anatomicamente dividida em cinco partes: o epitympanum está localizado superiormente e está interligado por antro para o sistema de célula de ar mastoide (MACS); o retrotympanum posterior é um sistema de pontes ósseas e seios mais ou menos superficiais; inferiormente, encontra-se a hypotympanum; anteriormente, é o protympanum com sua conexão ao tubo de Eustáquio; e no centro, encontra-se o mesotympanum<sup class="xref">1</sup>.</p>
<p class="jove_content">O acesso fisiológico através do canal auditivo externo (EAC) é estreito. Portanto, a abordagem cirúrgica padrão para o ouvido médio requer uma incisão retroauricular e perfuração dos MACS. Esses tipos de intervenções são executados usando um microscópio de operação. Sua introdução foi um marco na cirurgia otológica, pois permitiu o tratamento das estruturas microscópicas de orelha média. No entanto, o microscópio tem uma vista em linha reta forçada e algumas áreas, especialmente os tímpanos sinusal, são de difícil acesso. Isto traz o risco de doença residual especialmente durante colesteatoma cirurgia<sup class="xref">2</sup><sup>,</sup><sup class="xref">3</sup>. Além disso, os tecidos saudáveis da EAC, MACS, ouvido médio e têm de ser removidos simplesmente para fins de acesso. Isto não apenas estende o tempo de funcionamento, mas também responde por mais de morbidade cirúrgica e uma cura prolongada tempo<sup class="xref">4</sup>.</p>
<p class="jove_content">Avanços recentes levaram à introdução do endoscópio como uma poderosa ferramenta para a cirurgia de orelha. O endoscópio foi usado primeiramente para auxiliar o cirurgião convencionalmente para visualizar as áreas escondidas do ouvido médio. Refinamentos técnicos e cirúrgicos permitiu a introdução do endoscópio como a principal ferramenta<sup class="xref">5</sup>de funcionamento. As principais vantagens do endoscópio são as vistas panorâmicas e largo-angular do ouvido médio. Por exemplo, o caso contrário oculto retrô - e hypotympanum podem ser acessados usando uma abordagem exclusiva transcanal endoscópica sem um canaloplasty<sup class="xref">6</sup>. Um estudo recente mostrou uma visibilidade superior de todos os compartimentos do ouvido médio usando o endoscópio quando comparado com a técnica microscópica<sup class="xref">7</sup>. O uso de endoscópios angulados (30° e 45°), ainda mais, melhora a visibilidade e permite dissecação com vista para as áreas mais escondidas do ouvido médio.</p>
<p class="jove_content">No entanto, a cirurgia endoscópica de ouvido (EES) é uma técnica cirúrgica com uma mão, como o ponteiro dos segundos geralmente mantém o endoscópio. Esta questão e o espaço estreito dentro o EAC exigem formação para melhorar as habilidades do cirurgião para aplicar com segurança a técnica em pacientes. Em geral, o padrão ouro para treinamento cirúrgico é considerado dissecação cadavérico. Em caso de indisponibilidade de espécimes cadavéricos ou questões éticas locais, um modelo animal para a formação de cirurgia endoscópica de ouvido foi recentemente descrito<sup class="xref">8</sup>. Treinamento cirúrgico é considerado um fator importante na formação de estagiários em uma nova técnica de<sup class="xref">9</sup>.</p>
<p class="jove_content">Apesar da importância da formação cirúrgica, uma descrição concisa das considerações importantes sobre como realizar dissecação cadavérico ainda está faltando na literatura. O objetivo deste artigo é descrever a metodologia de exclusivamente endoscópica cadavérico médio ouvido dissecação. Além disso, nosso objetivo é fornecer um guia completo de anatomia endoscópica de ouvido médio.</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">O ouvido médio está localizado no centro do osso temporal e ostenta uma anatomia altamente complexa. A abordagem endoscópica exclusivamente transcanal recentemente introduzido para o ouvido médio é uma técnica minimamente invasiva, poupando o osso e a mucosa do osso mastoide, desde que o ouvido médio é acessado através do canal auditivo externo. Este método emergente tem várias vantagens sobre as abordagens tradicionais (microscópicas) para o ouvido médio como os grande angular vistas panorâmicas sobre a anatomia, a possibilidade de se aproximar e ampliar estruturas minúsculas e a possibilidade de olhar em torno do canto usando endoscópios angulados.</p>
<p class="jove_content">O método de dissecação cadavérico aqui apresentado consiste em uma visão geral sobre os requisitos técnicos e uma descrição exacta de um protocolo passo a passo para descobrir a anatomia do ouvido médio. Cada passo e a estrutura anatômica é descrita com cuidado a fim de fornecer um guia completo de anatomia endoscópica da orelha. Em nossa opinião, isto é particularmente importante para qualquer iniciante em cirurgia endoscópica de ouvido, pois fornece profundo conhecimento anatômico e pode melhorar habilidades cirúrgicas.</p>

<p class="jove_content" fo:keep-together.within-page="1">Dissequei um total de cinco espécimes de toda cabeça e 12 amostras de osso temporal (n = 22 lados) usando as instruções descritas. As regiões diferentes do TC foram documentadas fotograficamente e mais analisadas. Todas as regiões foram exploradas usando endoscópios de 0° e 45°. Nós realizou uma exploração completa de todas as regiões anatômicas e identificados todos os mencionados pontos anatômicos.</p>
<p class="jove_content" fo:keep-together.within-page="1">As estruturas anatômicas são ilustradas de acordo com o progresso da dissecação. <strong class="xfig">A Figura 1</strong> ilustra o espaço de Prussak e os ligamentos maleolares, Considerando que a <strong class="xfig">Figura 2</strong> demonstra a anatomia após elevação completa do TMF. A visão grande angular permite a visualização do meso - e hypotympanum, bem como partes do epi, retro-e protympanum. Na <strong class="xfig">Figura 3</strong>, o retrotympanum superior é mostrado como ele aparece em um endoscópio de 45 °, enquanto o cirurgião está de pé no lado oposto da mesa. <strong class="xfig">A Figura 4</strong> ilustra detalhadamente o protympanum com um ICA deiscente. Após a remoção do OC e transcanal atticotomy o epitympanum é inspecionado até o canal semicircular lateral e o antro, conforme mostrado na <strong class="xfig">Figura 5</strong>.</p>
<p class="jove_content" fo:keep-together.within-page="1">Nenhum espécime necessário canaloplasty para realizar a exploração completa da cavidade timpânica. A identificação das estruturas anatômicas foi possível em todas as amostras usando o escopo de 0° e 45°. No entanto, a exploração completa da retrotympanum, especialmente o recesso facial e os tímpanos de seio, não foi possível em 27% dos casos (n = 6), uma vez que estas secções ósseas foram muito profundas para ser completamente explorado.</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">Figura 1</strong>. Orelha esquerda, endoscópio de 0º: vista do espaço de Prussak e as dobras ligamentares circundantes do martelo. TM: membrana do tímpano, um: anel, u: umbo, sp: curto processo de martelo, amf: maleolares anterior dobra, lmf: dobra lateral maleolares, plm: ligamento posterior do martelo, ct: chorda tympani, *: espaço de Prussak <a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56390/56390fig1large.jpg" target="_blank">por favor clique aqui para ver uma versão maior deste Figura.</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">Figura 2</strong>. Orelha esquerda, endoscópio de 0º: vista da cavidade timpânica após a elevação do retalho tympano-meatal. O meso - e hypotympanum bem como partes do epi, retrô - e protympanum são visualizadas. PR: promontório, jn: nervo de Jacobson, hy: hypotympanum, fi: finiculus, sst: sinus subtympanicus, se: Eminência estiloide, pe: Eminência piramidal, st: tendão estapédica, isj: incudostapedial conjunta, fn: nervo facial, u: umbo, m: manúbrio, sp: processo curto, n: pescoço de o martelo, ttm: músculo de tímpano tensor, ttf: tensor tímpanos dobra, ica. Artéria carótida interna <a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56390/56390fig2large.jpg" target="_blank">clique aqui para ver uma versão maior desta figura.</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">Figura 3</strong>. Bem ouvido, endoscópio de 45°, contralateral posição do cirurgião: A retrotympanum superior é ilustrado usando um endoscópio angulado. FN: nervo facial, s: estribo, st: tendão estapédica, ps: sinus posterior, fp: estribo, po: ponticulus, pe: Eminência piramidal, sente-se: tímpanos sinus, sub, subiculum, se: Eminência estiloide, sst: sinus subtympanicus, ap: pilar anterior, teg: tegmen, pp: posterior Pilar, rw: janela redonda, fu: fustis, tu: túnel de subcochlear <a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56390/56390fig3large.jpg" target="_blank">por favor clique aqui para ver uma versão maior desta figura.</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">Figura 4</strong>. Orelha esquerda, endoscópio de 45°: detalhada vista sobre o protympanum. Observe a artéria carótida deiscente. et: Eustáquio, ica: artéria carótida interna, pro: protiniculus, co: cóclea, ttm: músculo de tímpano tensor <a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56390/56390fig4large.jpg" target="_blank">clique aqui para ver uma versão maior desta figura.</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">Figura 5</strong>. Orelha esquerda, endoscópio de 0º: após a remoção da bigorna e maléolo e curetagem do scutum, uma visão ampla do tímpano o tegmen e o antro é ilustrada aqui. Observe o canal semicircular lateral e o músculo de tímpano tensor sendo em uma linha com a passagem do nervo facial. TEG: tegmen, CG: COG ou crista transversal, gg: gânglio geniculado, cp: cochleariform processo, ttm: músculo de tímpano tensor, lsc: lateral canal semicircular, fn: timpânicas segmento do nervo facial, s: estribo, co: cóclea, ica: artéria carótida interna <a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56390/56390fig5large.jpg" style="font-size: 14px;" target="_blank">por favor Clique aqui para ver uma versão maior desta figura.</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">O objetivo deste artigo é descrever a metodologia de exclusivamente endoscópica cadavérico médio ouvido dissecação. Além disso, nosso objetivo é fornecer um guia completo de anatomia endoscópica de ouvido médio.</p>

Descobrindo a anatomia do ouvido médio por cirurgia endoscópica de ouvido de Transcanal: um Manual de dissecação

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

Nanyang Technological University

Research

JoVE Journal

Medicine

64.3K visualizações.  University of Bern. O objetivo deste artigo é descrever a metodologia de exclusivamente endoscópica cadavérico médio ouvido dissecação. Além disso, nosso objetivo é fornecer um guia completo de anatomia endoscópica de ouvido médio.

Vídeo: 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

Estrutura Celular

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

Education

JoVE Lab Manual

Biologia

Fundamentais

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

Transformação bacteriana

Background

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

Genética

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.remotexs.ntu.edu.sg/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.remotexs.ntu.edu.sg/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>

Eussocialidade e Divisão do Trabalho

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

Ecologia

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.remotexs.ntu.edu.sg/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.remotexs.ntu.edu.sg/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.remotexs.ntu.edu.sg/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

Dinâmica Energética

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

Genética de Organismos

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

Relações evolutivas

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

Evolução

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.remotexs.ntu.edu.sg/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.remotexs.ntu.edu.sg/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.remotexs.ntu.edu.sg/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

Extinção

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.remotexs.ntu.edu.sg/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.remotexs.ntu.edu.sg/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

Diversidade microbiana e fúngica

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.remotexs.ntu.edu.sg/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.remotexs.ntu.edu.sg/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.remotexs.ntu.edu.sg/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.remotexs.ntu.edu.sg/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>

Medindo a biodiversidade

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

Transpiração

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