<p class="jove_content"> Con el apoyo de subvenciones de la Fundación Rüdiger en 2008 y 2010 (BVS), BONFOR / Gerok Beca O-137.0015 (BVS), BONFOR / Gerok Beca O-137.0019 (FT), Deutsche Forschungsgemeinschaft / DFG (BVS) STA 1135 / 2-1, chino Consejo beca Nº 2008627116 (ZL) y una subvención sin restricciones por Geuder AG, Heidelberg (Fig. 2). Miembros del laboratorio de H. Skottman, Universidad de Tampere Finlandia se agradece para proporcionar HES RPE se muestra en la figura 2 se derivan. </p>

<p> La ética de la manipulación de los animales en la investigación oftálmica: Se obtuvo la aprobación del comité de ética de la Facultad de Medicina, Universidad de Bonn, y se adhieren a las directrices establecidas por la Asociación para la Investigación en Visión y Oftalmología (ARVO). Por otra parte, todos los procedimientos fueron aprobados por las autoridades reguladoras del estado de Renania del Norte-Westfalia. Los animales se mantuvieron bajo techo en un centro especializado en una habitación con aire acondicionado, con temperaturas entre 18 - 20 ° C, la exposición a la luz del día regular, en jaulas individuales estandarizados con acceso libre a comida y agua. </p><p></p><p> Nota: Para asegurarse de que los animales afinidad operativa, una hoja de resultados de salud de los animales se sigue que incluye los siguientes criterios de exclusión definitiva de animales: 20% de pérdida de peso en comparación con el peso sobre la admisión; cianosis aparente del animal; escalofríos animales, tiene calambres o no puede mover en coordinación; . ataxia / parestesia, <em>por ejemplo,</em> paraliza; apatía; la mutilación extrema automático (heridas en la piel, miembros amputados). </p><p> &lt;/ P&gt; <p class="jove_title"> 1. La esterilización de instrumentos </p><ol><li> Coloque los instrumentos reutilizables en el baño de ultrasonidos. </li><li> Añadir 500 ml de agua destilada y 2 ml de desinfectante instrumento. </li><li> Limpiar los instrumentos que utilizan la función de barrido durante 15 minutos. </li><li> Suprimir instrumentos de baño de ultrasonidos y enjuagar bien con agua destilada durante 5 minutos. </li><li> Insertar instrumentos en autoclave y utilizar el programa estándar (esterilización de instrumentos bajo 121 ºC durante 20 minutos). </li></ol><p class="jove_title"> 2. Preparación Instrumento </p><ol><li> Establecer y mantener un campo estéril, al trabajar en una habitación cerrada, el uso de batas quirúrgicas, máscara y cubierta de pelo. Desinfectar las manos antes de usar guantes quirúrgicos estériles. Para la aproximación detallada ver <sup>28.</sup> </li><li> Coloque los instrumentos esterilizados en un campo estéril. </li><li> Colocar 1 ml jeringa llena con 40 mg de triamcinolona unido a una aguja 27 G para la inyección, la jeringa 10 ml con solución salina equilibrada (BSS), y 5 ml jeringa of lubricante en cortina. </li><li> Coloque seda 3-0, 7-0 vicryl, palos oculares (para detener la hemorragia conjuntival / escleral), esponjas de gasa tornado, tiras de cierre de la herida (para fijar el tubo de punta vitrectomía), y el cable de lámpara de la fibra endoillumination en una sábana. </li><li> Desenvuelva 25 G araña endoiluminador y conectarse a la máquina de luz usando técnicas estériles (véase el paso 2.1). Conectar el set de vitrectomía incluyendo vitrector alta velocidad y casete de Venturi para equipo de vitrectomía utilizando técnicas estériles (véase el paso 2.1). </li><li> Abra la botella de 500 ml de BSS y conectarse a la solución Venturi casete según las instrucciones del fabricante. </li></ol><p class="jove_title"> 3. Preparación de la anestesia y el posicionamiento del Animal </p><ol><li> Pesar animales para asegurar la dosis del medicamento precisa. </li><li> Preparar la anestesia intramuscular (IM) usando 1 jeringa con aguja 27 G que contiene 0,35 mg / kg de ketamina y 0,25 mg / kg de medetomidina para el arranque. Voltear la jeringa para mezclar. </li><li> Preparar 2 jeringas con 1/3 tque las dosis para mantener la anestesia durante la operación. </li><li> Preparar jeringa que contiene 20 ml de solución de glucosa al 5% y 18 aguja G para la inyección subcutánea como una alternativa infusión intravenosa. </li><li> Dar 3 x 1 gota de ojo midriático falla antes de la vitrectomía para la dilatación de la pupila. </li><li> conejo de la cubierta con una manta a la calma antes de la inyección de anestesia, se inyectan en la extremidad posterior (músculo glúteo) y masaje alrededor del sitio de la inyección durante 30 segundos. <br /> Nota: El primer tiro de la anestesia IM dura aproximadamente 1 - 2 horas. dependiendo del tamaño del conejo, tolerancia a las drogas, la capa de grasa, el estrés y la temperatura corporal. El primer signo de la anestesia desvanecimiento es un nistagmo (deberá ser supervisado por el cirujano), las inyecciones posteriores duran alrededor de 30 - 45 min. </li><li> Confirmar la anestesia apropiada, mediante la verificación de la hipnosis, hiporreflexia, analgesia y relajación muscular del animal. </li><li> Dar una inyección subcutánea (Pt. 3.3) en el pliegue cutáneo del cuello, una vez que el conejo es inconsciente. </li><li> Añadir metilClubricante ellulose cada 5 - 10 minutos a en el ojo operado, añadir lubricante y cinta de tapas en el ojo no operado. </li><li> Coloque la cubierta de conejo en la mesa quirúrgica cubierta con una cubierta tal como una manta de algodón en una posición óptima (nariz ligeramente elevada a través de un molde de la manta, por lo que está a nivel con la superficie ocular) bajo el microscopio quirúrgico. Alinear el ojo perpendicular al microscopio objetivo. </li><li> Garantizar la adecuada temperatura corporal utilizando un termómetro rectal (normotermia 39 ± 1 ºC) <sup>29.</sup> </li><li> pestañas de corte con unas tijeras (un poco de ungüento en la lámina) para reducir las infecciones postoperatorias. </li><li> Desinfectar el ojo utilizando 2 - 3 gotas de 0,1 g / ml povidona yodada tópica durante 1 min y aclarar con BSS estéril. </li><li> Cubrir el ojo con paño estéril con la apertura de pre-corte en el medio para el ojo y luego cubrir con caída (pegajoso) incisión quirúrgica 12 x 17 cm. </li></ol><p class="jove_title"> 4. La vitrectomía </p><ol><li> Proptose y el ojo seguro con seda 3-0 usando invpinza y erted, lleve a cabo una peritomía conjuntival. <ol><li> Incisión en la conjuntiva con una tijera Vannas cerca del limbo, pero lo suficientemente lejos de los vasos sanguíneos (~ 1 mm de distancia). </li><li> Diseccionar la conjuntiva mediante la creación de una &quot;T-corte&quot;. En primer lugar ampliar la peritomía con la tijera paralelo al limbo y luego una incisión en la conjuntiva verticalmente en forma de una &quot;T&quot; durante aproximadamente 6 - 7 mm. separe con cuidado la conjuntiva sin rodeos. </li></ol></li><li> Realizar una sclerotomy utilizando una cuchilla de 23 G microvitreoretinal (MVR) a las 8 de la mañana del ojo derecho / OD (4 horas el ojo izquierdo / OS) insertando cuidadosamente la punta afilada de la cuchilla en dirección hacia el nervio óptico. retraer lentamente la cuchilla en la misma dirección y evitar la ampliación de la esclerotomía. </li><li> Insertar y lado de encargo de sutura puerto de infusión cánula <sup>27</sup> mediante una sutura de seda 7-0 y ajuste de la presión intraocular (PIO) a los 24 mmHg. </li><li> Realizar una sclerotomy con 25 g de trocar cabeza plana a las 2 en punto de OD (10 O &#39;; Reloj en OS) similar a la etapa 4.2. </li><li> Inserte 25 G luz de la lámpara en trocar de punta plana, fijar con cinta adhesiva y encienda la fuente de luz a <em>aproximadamente</em> 30%. </li><li> Si es necesario, retire epitelio de la córnea edematosa utilizando un bisturí # 20 para una mejor visualización intraocular. </li><li> Realizar una sclerotomy similar a la etapa 4.2 a las 10 en punto de OD (dos en OS), (pre) punta de la cuchilla de vitrectomía lugar 7-0 suturas alrededor sclerotomy sin atar el nudo en forma de U, y la inserción. </li><li> Iniciar la vitrectomía <sup>30</sup> alrededor del orificio de entrada, y luego continuar sobre el disco óptico y los medullares fibrae utilizando vitrector alta velocidad por cortar el humor vítreo en trozos pequeños en el máximo. 2.000 - 3.000 cortes / min, aspiración a la max. 200 mmHg mediante la configuración del parámetro indicado de la máquina de vitrectomía <strong>(Tabla 1)</strong> </li><li> Realizar un desprendimiento del vítreo posterior (DVP) separando el humor vítreo de la retina mediante la celebración de la alta velocidad a través de vitrector la parte posterior del poste unaND (si es posible con suavidad) superior del disco <sup>31</sup> mientras se aspira únicamente a la max. 200 mmHg sin cortar. </li><li> Inyectar <em>ca.</em> 50 l (20 mg) de triamcinolona o fluoresceína diluido <em>(ca.</em> 0,1 mg / ml) por vía intravítrea para visualizar y facilitar (cerca total) eliminación del vítreo que flota sobre el polo posterior y periferia media durante la vitrectomía. Evitar cruzar debajo de la lente. Se recomienda el sangrado para afeitarse vítreo periférico por un asistente (especializada), si se desea taponamiento de gas. </li><li> Añadir 20 unidades / ml de heparina y 0,5 mg de epinefrina a concentración final de 0,001 mg / ml en la solución de infusión BSS en paralelo o después de la etapa 4.10. <br /> Nota: Como la heparina / epinefrina no se inyectan intraocular sus efectos se retrasan dependiendo de la tasa de flujo de infusión. </li></ol><p class="jove_title"> 5. Carga tirador </p><p> Nota: El trabajo descrito en este documento no entra dentro de los principios de la Declaración de Helsinki; no involucró pacientes humanos. Aquí, staándar células RPE fueron aisladas de los ojos humanos fetales, cultivadas y diferenciadas en poliéster no revestida de 10 micras de espesor (PET) inserta de acuerdo con el protocolo publicado previamente <sup>14.</sup> Un permiso para trabajar con el material fetal humano se obtuvo del comité de ética de la Universidad de Bonn. Alternativamente, HES-RPE fueron enviados desde el laboratorio Skottman (manuscrito en preparación.), Donde se cultivan de acuerdo con la técnica descrita por Vaajasaari <em>et al</em> <sup>32.;</sup> para estas células un permiso se ha obtenido del Instituto Koch R., Berlín, Alemania. </p><ol><li> Enjuague de cultivo celular antes de la preparación de la 3x implante con BSS oftálmica grado. </li><li> Llene una placa de cultivo celular estándar (100 x 20 mm) con 10 ml de BSS oftálmica grado. </li><li> Añadir la inserción de cultivo celular en el BSS y el centro del plato con un microscopio óptico. </li><li> Perforar un implante de 2,4 x 1,1 mm con un óvalo, aguja roma, hecho a medida para obtener un sustrato plano, en forma de frijolcon dos bordes largos y dos bordes redondos. </li><li> Inundar suavemente la aguja a través del segundo puerto con BSS para hacer salir el implante en la estación de carga por encargo BSS llenado <strong>(Figura 1).</strong> </li><li> Opcionalmente cortar un extremo redondo del implante (&lt;0,5 mm), para obtener un tercer borde. </li><li> Asegúrese de que el implante está en la orientación correcta, asegurando que la monocapa está al revés en el soporte celular. Para cambiar el posicionamiento de utilizar cuidadosamente dos escalpelos. </li><li> Empuje el implante suave y completamente en el instrumento tirador utilizando el soporte de la aguja hasta que todo el implante se fija en el interior de la punta. El émbolo debe permanecer retraída. </li><li> Mantenga la punta de disparos &quot;cargado&quot; en la estación de carga bajo BSS hasta el momento de la implantación. </li></ol><p class="jove_title"> 6. Implantación </p><ol><li> Enfoque de retina neural con extensible 41 G aguja de inyección subretinal conectado a una jeringa estanca a los gases (asegúrese de que todas las burbujas de aire han sido evacuadosa partir de tubo!). </li><li> Inyectar BSS (con calcio y magnesio / CM) subretinally y de ese modo crear un desprendimiento de retina de la ampolla (BRD) de aproximadamente 2-3 diámetro de disco (DD). Dos BRD por ojo se puede levantar con seguridad. </li><li> Agrandar retinotomía a 1,5 mm con 23 g verticales VR-tijeras. El espacio subretinal es ahora accesible para la implantación o más maniobras. </li><li> Extender sclerotomy (precisamente) con un cuchillo de incisión de 1,4 mm a 20 G enfoque. </li><li> Intentar pasar a través del uso de un tirador sclerotomy maniquí 20 G, ampliar según sea necesario para garantizar una transición sin problemas, sin embargo, ceñido del tirador cargado. </li><li> Pasar con el tirador cargado <sup>27</sup> a través de sclerotomy idealmente a 24 mmHg. </li><li> Enfoque retinotomía borde y expulsar el implante subretinally desde una posición epirretiniana. </li><li> Ajustar el implante con semicerrados 23 g tijeras, pinzas o aguja 41 G para asegurarse de que se sitúe bajo la retina- razonablemente lejos de la retinotomía. </li></ol><p class="jove_title"> 7.Poner fin a la operación </p><ol><li> Retire 25 G de la lámpara y la cánula de infusión. </li><li> Suturar todas las esclerotomías. </li><li> Inyectar 25 l (10 mg) de triamcinolona por el sclerotomy 8:00 (antes de la sutura última sclerotomy). </li><li> Comprobar / ajustar la PIO mediante palpación e inyectar BSS a través de 30 G de la aguja / jeringa, si es necesario. </li><li> conjuntiva sutura con Vicryl 7-0. </li><li> Retire proptosing 3-0 cabestrillo de seda lentamente (Evitar profunda plexo venoso orbital!). </li><li> Añadir dexametasona ungüento / antibiótico bajo la tapa. </li><li> Posición durante 1 hora cubierto con una manta con el ojo operado hacia arriba (w / o gas), o hacia abajo (con aire / gas). </li><li> No deje desatendida animales hasta que recupere la conciencia suficiente para mantener la posición decúbito esternal. </li><li> No transporte de conejo antes de la anestesia se desvanece por completo, esto puede acelerarse mediante la inyección de medetomidina agente de igualdad de marcha atrás a la cantidad de medetomidina dado. </li></ol><p class="jove_title"> 8. Post-operatorio Cuidado de Animales </p><ol><li> Guardarconejos en condiciones adecuadas (temperatura, luz, alimento, agua, espacio, <em>etc.)</em> y una estrecha vigilancia en una instalación especializada. </li><li> Asegúrese de que los animales esté bien descansado, <em>es decir.,</em> No hay largos períodos de privación de alimentos o agua. </li><li> Busque cualquier heridas o lesiones, especialmente en los sitios de inyección. </li><li> Mantenga las heridas secas para prevenir infecciones. Dar antibióticos cuando se sospecha de infección: dexametasona 1 mg / g, neomicina sulfato de 3.500 UI / g, sulfato de polimixina B se aplicó 6.000 UI / g de ungüento dos veces al día durante 1 semana postoperatoria sobre la superficie ocular. </li><li> Añadir dexametasona ungüento / antibiótico para el próximo 7 días postoperatorios dos veces al día para una mejor regeneración de la superficie ocular y la reducción del dolor post-operatorio. </li><li> Dar analgésicos sistémicos (Carprofene 4 mg / kg dos veces al día) durante 48 h inicial. </li><li> No deje un animal sin vigilancia hasta que se haya recuperado el conocimiento suficiente para mantener la posición decúbito esternal. </li><li> No devuelva un animal quese ha sometido a una cirugía para la compañía de otros animales hasta que esté completamente recuperado. </li><li> No exponer a los animales a sufrimientos innecesarios. </li></ol><p class="jove_title"> 9. SLO / SD-OCT Orientación </p><ol><li> Preparar e inyectar la anestesia intramuscular (IM) usando 1 jeringa con aguja de 27 G que contiene 0,175 mg / kg de ketamina y 0,125 mg / kg de medetomidina para el arranque. Voltear la jeringa para mezclar. </li><li> Añadir un lubricante por lo menos cada 5 minutos para hidratar los ojos y mantener una clara imagen SD-OCT, opcionalmente una lente de contacto personalizado puede ser utilizado. </li><li> Adjuntar una plataforma de acero en el reposacabezas para estabilizar el animal en la posición requerida. </li><li> Colocar el conejo en la plataforma de acero, colocando su ojo perpendicular a la sonda. </li><li> Mantenga la cabeza del conejo del hueso de la mejilla inferior (mandíbula) evitando la tráquea. </li><li> la cabeza del animal de la inclinación de aproximadamente 45º hacia la OCT SD-sonda para optimizar el ángulo de visión sobre el implante. </li><li> Utilizar una lente de 30 grados y los siguientes parámetros para opttremo de imágenes de OCT [SA]: 30 grados ajustes para sola línea escanea con el modo de ART se ajusta al 100 (de promedio) y 20 x 20 ajustes de grado para las exploraciones de volumen con el modo de arte fijado a 15; No se requiere el modo de alta resolución. </li><li> Utilice SLO formación de imágenes de infrarrojos de reflectancia para encontrar el plano focal del implante <strong>(Fig 2A.);</strong> un enfoque óptimo se alcanza cuando (todos) los bordes del implante son agudos <sup>14.</sup> </li><li> Añadir dexametasona 1 mg / g, neomicina sulfato de 3.500 UI / g, polimixina B sulfato de 6.000 UI / g pomada debajo de la tapa cuando haya terminado. </li></ol>

<p class="jove_content"> RB, BVS, ZL, NE y Geuder AG han presentado una solicitud de patente europea sobre el tirador. NB es un empleado de Geuder AG. las tasas de publicación de este video-artículo fueron pagados por Geuder AG. </p>

<p class="jove_content"> El uso de un modelo de conejo, se presenta un método seguro y reproducible para la entrega transvítrea de RPE cultivadas en portadores de células en el espacio subretiniano con un instrumento de tirador de diseño personalizado. El método descrito ofrece una técnica quirúrgica corta / optimizado para facilitar el aprendizaje, ya que implica técnicas estándar en la vitrectomía con maniobras subretiniana. Resultado se ve facilitada en gran medida por una interfaz vitreorretiniana limpio, y la infusión intraocular que evita la turbulencia del fluido sobre el sitio de implantación, la inducción de la ampolla desprendimiento de retina (BRD) a baja presión intraocular, la prevención de retina y daños esclerótica a través de la sequedad, y el posicionamiento apropiado del conejo. </p><p class="jove_content"> Advertimos sin embargo, como varias complicaciones intraoperatorias pueden ocurrir en cualquier momento, lo que dificulta el éxito de la implantación, por ejemplo intra hemorragias oculares, la anestesia decoloración fuera durante las etapas vitales, tales como la implantación, el colapso de la BRD debido a la manipulación de instrumentos o hipotonía ocular, rabbit la muerte debido a las dosis excesivas de la anestesia, la presión arterial baja durante el funcionamiento a largo causando daño cerebral hipóxico, o hipertermia. Sin embargo, estas complicaciones disminuyen con el tiempo, ya que son rápidamente abordados y resueltos mediante el aumento de la experiencia del equipo quirúrgico. </p><p class="jove_content"> Algunas complicaciones podrían reducirse siguiendo unos simples, pero importantes pasos. El lubricante debe añadirse cada 5 - 10 minutos a para prevenir la córnea daños, escleral y conjuntival durante la operación, y mantener una clara medios intraoculares, como secado esclerótica / ennegrecido pueden ser una causa de dehiscencia de la herida, que a su vez conduce a la hipotonía ocular y / o fuga intraoperatoria de esclerotomías. La heparina se debe añadir para evitar la formación de una película de fibrina que hace que la implantación particularmente subretinal desafiante y adición simultánea de epinefrina para reducir el sangrado bajo heparina <sup>16.</sup> veces demasiado largo heparina / exposición epinefrina (&gt; 1 hora) se debe evitar para prevenir ede la córneama por descompensación endotelial <sup>33,</sup> crisis hipertensiva o la muerte intraoperatoria. eliminación meticulosa vítreo se debe realizar en el puerto de instrumento (entrada) para evitar la retina y / o desprendimientos coroideos. instrumentos intraoculares debe dirigirse hacia el polo posterior para evitar el contacto de la lente (provoca la formación de cataratas iatrogénica) o (sitio de entrada) daño en la retina. Una cánula de infusión de puerto lateral intraocular se debe utilizar, ya que atenúa la corriente en chorro alrededor de la zona de implantación, evitando así la rotura incontrolada de la retinotomía, y el colapso de la BRD. inducción BRD en la línea media (eje vertical del nervio óptico) o cerca de fibras ópticas medulares deben evitarse para prevenir extensos desprendimientos de retina iatrogénicas. Finalmente, el último pero no menos importante BRD debe ser inducida a baja presión intraocular, para evitar la inyección subretiniana BSS utilizando caudales excesivos que pueden dar lugar a un daño en la retina <em>(por ejemplo.,</em> Por el estiramiento). </p><p class="jove_content"> Muchas variables de estudio como Carrie celularr variantes, fetal, madre adultas o fuentes de células derivadas de células RPE, opciones para los inmunosupresores, <em>etc.,</em> pueden ser explorados <sup>14,26,27,34.</sup> Nuevas mejoras tales como los métodos de cultivo libre de suero RPE, caracterización de xenoRPE en el espacio subretiniano, la eliminación de la capa de RPE anfitrión <sup>14</sup> o estrategias para el anclaje del implante son trabajo actual en progreso. </p><p class="jove_content"> Hasta la fecha, las técnicas descritas se han utilizado en 5 cepas de conejo diferentes, incluyendo bastardo Chinchilla, Chinchilla bastardo / híbridos KBL, Nueva Zelanda Blanco / Cruz Roja, Nueva Zelanda Blanco (albino) y holandés con cinturón. Ambos conejos machos y hembras fueron operados, con los conejos al menos 1,5 kg o 2 meses de edad (dependiendo de la especie). La mayoría de las cirugías estaban en conejos pigmentados (bastardos de chinchilla o híbridos bastardos chinchilla) con pesos entre 2,5 - 3 kg. </p><p class="jove_content"> Todas las cepas de conejo que hemos tenido la oportunidad de trabajar con parecen tener algunas peculiaridades. Teniendo en cuenta los aive disponibilidad de conejos pigmentados de la cepa de chinchilla bastardo en Alemania en 2009-13, hemos recogido la mayor experiencia con estos animales. Por desgracia, ya no está disponible, ya que la reproducción se ha interrumpido, pero se compara muy bien a New Zealand White Cross / Red excepción de la esclerótica más grueso más ventajosa y volúmenes más grandes de los ojos en el segundo. Chinchilla híbridos bastardos tienen formación significativa de fibrina intraoperatoria y requieren el uso de heparina / epinefrina como se describe anteriormente para asegurar las maniobras subretiniana exitosas. Este protocolo también se ha realizado en conejos albinos no pigmentadas (blancos de Nueva Zelanda), la creación sin embargo particularmente BRD y la implantación subretinal es más difícil dado apreciación reducida de contraste. La viabilidad de inducir un desprendimiento vítreo posterior no parecía cepa dependiente de conejo en nuestras manos. </p><p class="jove_content"> subretiniana entrega transvítrea es probable que la futura estrategia quirúrgica de elección dado que es el más comm<html> en la ruta de hoy en día clínicamente para acceder a la retina. Como resultado, muchos otros grupos han presentado este tipo de técnicas para la RPE cultivadas en soporte apoya en <sup>11,15,23,35</sup> trabajo con animales. Aramant <em>et al.</em> <sup>36</sup> tienen un instrumento, que coloca en lugar de empuja su implante suave de hidrogel encapsulado a su sitio diana subretinal. El diseño de Thumann <em>y col.</em> Utiliza una espátula hueca, que libera el injerto portadora apoyada por flotando fuera a través de la inyección de fluido <sup>19.</sup> Ambos ex estrategias requieren la inserción subretiniano del instrumento, que en nuestra opinión es más propenso a las complicaciones, en comparación con un instrumento epiretinally appositioned. Montezuma <em>et al.</em> <sup>22</sup> describe un instrumento de inserción subretiniana para el suministro de implantes de chips subretiniana en los cerdos, pero ningún trabajo adicional se ha publicado desde a lo mejor de nuestro conocimiento. Hemos sido capaces de extender la técnica descrita con alguna modificación al cerdo. </p>jove_content &quot;&gt; Nuestros portadores celulares preferidas son 10 micras de espesor de tereftalato de poliéster (PET) membranas. Desde una perspectiva quirúrgica, este material tiene parámetros de rigidez y elasticidad favorables, además de su amplia versatilidad durante los experimentos de cultivo celular. Encontramos experiencias similares con tetrafluoroetileno expandido (ePTFE) <sup>37</sup> o nanofibras membranas electrospun de PET, poli-ácido láctico / capronolactic (PLCL) o poli -. láctico-co-glicólico (PLGA), así como de nanofibras compuesto (PLGA o PET) y PET ultrafino <sup>26</sup> Cuando membranas de PET se utilizan con nuestro instrumento tirador metálico, tienen una tendencia ocasional a exhibir carga electrostática, que pone a prueba su expulsión desde el tirador <sup>27.</sup> membranas de poliamida Ultrathin podrían en nuestras manos no ser implantados en el espacio subretiniano con el protocolo descrito anteriormente ( manuscrito en preparación). </p><p class="jove_content"> Marmor <em>et al.</em> han estudiado sistemáticamente reso espontánearption del líquido subretiniano en iatrogénicas desprendimientos de retina localizadas <sup>38-41.</sup> Incluso después de la manipulación en el espacio subretiniano se encontró que éstos absorban día postoperatorio 4 en cirugías sin incidentes. retinopexia con láser no se lleva a cabo para asegurar los bordes de la retinotomía. Aún en contra de si se compara con la cirugía humana, no se requiere taponamiento aire / gas. A menos que se puede lograr la eliminación meticulosa de vítreo periférico, en particular en el cuadrante superior, esto puede de hecho resultar en desgarros retinianos gigantes procedentes de la retinotomía. Sólo se recomienda para llevar a cabo el intercambio de aire fluido con la subsiguiente taponamiento de gas SF6 20% de salvar los desprendimientos de retina iatrogénicas intraoperatorias o en caso de una posición de implante en particular necesita ser asegurado. </p><p class="jove_content"> Aunque la ablación inducida mecánicamente de la retina neural puede causar daños en los fotorreceptores y RPE en conejos <sup>42,43,</sup> su extensión varía mucho (incluso con BSS regular) dependiente de factores tales como el tipo de IOP, jeringa usada, volumen de inyección con la retina de ese modo de extensión inducidos, <em>etc.</em> También hemos probado la frecuencia recomendada de Ca / Mg-libre BSS facilitaron desprendimiento <sup>42-44,</sup> pero se encontró que causa la opacificación del cristalino intraoperatoria (particularmente con temperatura elevada), y significativamente retrasa o incluso afecta la retina re-anexo <sup>27.</sup> Por lo tanto, se recomienda volumen de 30 l de BSS regular con una jeringa de 100 l - subretiniana inyección lenta de 20; movimientos de la aguja de inyección deben ser mínimos por lo que los sellos retinotomía alrededor de ella y evitar daños en la membrana de Bruch. Algunos de los daños iatrogénicos se pueden resolver por RPE cicatrización de la herida, y la preservación relativa observada de grosor ONL después de reinserción, sugiere que el complejo / fotorreceptor RPE puede tolerar este deterioro, como también se describe por otros <sup>45.</sup> </p><p class="jove_content"> productos terapéuticos basados ​​en células o prótesis de retina requieren ánima preclínical prueba antes de la aprobación regulatoria y el inicio de los estudios de seguridad humanos. El primero puede variar de país a país. El modelo de conejo descrito aquí puede servir como una plataforma rentable y menos exigente para el establecimiento o incluso llevar a cabo todos los requisitos de las autoridades reguladoras. Por otra parte, puede servir posteriormente para la formación de cirujanos en los ensayos clínicos multicéntricos o eventuales mejoras adicionales de la técnica a lo largo del camino. </p>

<p class="jove_content"> La degeneración macular asociada a la edad (DMAE) es la causa más común de discapacidad visual en los adultos de 50 años o más en los países industrialmente desarrollados, ya que causa la pérdida de la visión central. Alrededor del 15% de estos pacientes sufren de la forma &quot;húmeda&quot; de la enfermedad, en la que la neovascularización se origina en la coroides y altera la función de la retina <sup>1.</sup> Esta variante puede ser tratada mediante una terapia altamente eficaz con inyecciones repetidas intra-vítreo de los fármacos antiangiogénicos <sup>2.</sup> Sin embargo, la gran mayoría de los pacientes (~ 85%) sufren de la forma seca, que se caracteriza por depósitos extracelulares <em>(por ejemplo,</em> drusas) bajo el epitelio pigmentario de la retina (RPE). Estos depósitos causan la disfunción RPE que conduce a atrofia de la retina en la mácula. Dada la falta de opciones terapéuticas curativas, AMD se convirtió en un campo de investigación intensiva en desarrollo, donde se están ensayando diversos enfoques terapéuticos curativos. reemplazo RPE quirúrgica esuna atractiva posibilidad futura para derrotar esta enfermedad debilitante <sup>3.</sup> </p><p class="jove_content"> El trasplante autólogo de RPE RPE subretiniano reemplaza disfuncional o perdido en la mácula, y tiene el potencial para restaurar su función fisiológica <sup>4-9.</sup> Esta técnica quirúrgica tenía un gran avance con el desarrollo de protocolos de diferenciación de las células RPE madre embrionarias humanas (células madre) y células madre pluripotentes inducidas (IPSC), dando el científico una fuente de células de RPE ilimitada para el trasplante <sup>10.</sup> trasplante de RPE es ahora reconocida como una atractiva primera vez en seres humanos solicitud de productos terapéuticos derivados de células madre. El ojo ofrece un excelente acceso quirúrgico y sofisticadas herramientas de monitorización <em>in vivo en</em> <sup>11-13.</sup> </p><p class="jove_content"> Para trasplantar el RPE, una forma es con una entrega mínimamente invasiva utilizando una suspensión de células, de forma alternativa, para conservar mejor las características de RPE y la función del trasplante, arti fi substr portador cialAtes (andamios) para la sustitución del EPR están siendo considerados <sup>4,14,15.</sup> Se requieren grandes modelos animales para la validación preclínica, sin embargo, la información técnica detallada sobre el manejo de animales y la técnica quirúrgica que falta hasta la fecha <sup>16-23.</sup> </p><p class="jove_content"> Nosotros y otros <sup>11,24</sup> A pesar de algunas pruebas de lo contrario <sup>25,</sup> sugerimos el uso de un sustrato de soporte rígido pero elástico, ya que proporciona un manejo más seguro, preserva la integridad de la monocapa y funcionalidad. Con el tiempo hemos probado varios instrumentos diseñados a medida y técnicas auxiliares para la implantación de células portadoras trasplantes RPE apoyados en el espacio subretiniano (SRS). Hemos utilizado las grabaciones de vídeo intraoperatorias, <em>en</em> oftalmoscopio láser de barrido <em>vivo</em> combinado con dominio espectral tomografía de coherencia óptica (SLO / SD-OCT), y la histología para evaluar el éxito de la implantación <sup>14,26,27.</sup> Aquí proporcionamos nuestra recomendación actual para implantes RPE subretiniana en conejos,el cual se ensayaron en 5 cepas diferentes de conejo, 7 materiales de soporte de células y fuentes de células RPE 4 en más de 150 procedimientos. </p>

<table><tbody><tr><td>s30 ultrasonic cleaning unit</td><td>Elmasonic</td><td>100 4631</td><td>2.75 L</td></tr><tr><td>DE-23&amp;nbsp; autoclave</td><td>Systec</td><td>C 2209</td><td>23 L</td></tr><tr><td>Syringe&amp;nbsp;</td><td>BD&amp;nbsp;</td><td>300013&lt;br/&gt; 300995&amp;nbsp;&lt;br/&gt; 301285&lt;br/&gt; 300294&lt;br/&gt; 300330&amp;nbsp;</td><td>1 ml x3&lt;br/&gt; 2 ml x3&lt;br/&gt; 5 ml x1&lt;br/&gt; 10 ml x1&lt;br/&gt; 20 ml x1</td></tr><tr><td>Needle&amp;nbsp;</td><td>BD</td><td>305196&lt;br/&gt; 305136</td><td>18 G x1&amp;nbsp;&lt;br/&gt; 27 G x5</td></tr><tr><td>Scalpel</td><td>Feather</td><td>2975#20</td><td>blade#20 x3</td></tr><tr><td>Surgical drape</td><td>HARTMANN&lt;br/&gt; LOHMANN &amp;amp; RAUSCHER</td><td>277 502&lt;br/&gt; 25 440</td><td>60 x 40 cm x2&lt;br/&gt; 12 x 17 cm</td></tr><tr><td>Ocular sticks</td><td>LOHMANN &amp;amp; RAUSCHER</td><td>16 516</td><td>66 x 5 mm</td></tr><tr><td>Twister gauze sponges</td><td>HARTMANN</td><td>481 274</td><td>x2</td></tr><tr><td>Closure strips</td><td>HARTMANN</td><td>540 686</td><td>x4</td></tr><tr><td>Opmi Visu CS Microscope</td><td>Zeiss</td><td>N/a</td><td>incl. fundus imaging system BIOM II</td></tr><tr><td>Chandelier endoillumination</td><td>Geuder</td><td>G-S03503 +&lt;br/&gt; G-S03504</td><td>25 G incl. trocar</td></tr><tr><td>Light machine</td><td>Geuder</td><td>G-26033</td><td>Xenotron III</td></tr><tr><td>Vitrectomy machine</td><td>Geuder</td><td>G-60000</td><td>MegaTRON S4 S4/ HPS</td></tr><tr><td>Vitrector</td><td>Geuder</td><td>G-46301</td><td>MACH2 vitreous cutter 23 G</td></tr><tr><td>Venturi cassette&amp;nbsp;</td><td>Geuder</td><td>G-60700</td><td></td></tr><tr><td>Sideport-infusion cannula</td><td>Geuder</td><td>custom&amp;nbsp;</td><td>23 G x1</td></tr><tr><td>3-0 silk suture</td><td>ETHICON</td><td>V546G</td><td>x1</td></tr><tr><td>Caliper</td><td>Geuder</td><td>G-19135</td><td>x2</td></tr><tr><td>Vannas scissors</td><td>Geuder</td><td>G-19777</td><td>x1</td></tr><tr><td>Sclerotomie blade</td><td>Ziemer</td><td>21-2301</td><td>1x 23 G&lt;br/&gt; 1x 20 G</td></tr><tr><td>7-0 silk suture</td><td>ETHICON</td><td>EH6162H</td><td>x1</td></tr><tr><td>Needle holder</td><td>Geuder</td><td>G-32320</td><td>x2</td></tr><tr><td>Iris forceps</td><td>Geuder</td><td>G-18910</td><td>x1</td></tr><tr><td>Colibri forceps</td><td>Geuder</td><td>G-18950</td><td>x1</td></tr><tr><td>Extendible subretinal injection needle</td><td>DORC</td><td>1270.EXT</td><td>41 G</td></tr><tr><td>VR scissor</td><td>Geuder</td><td>G-36542</td><td>25 G</td></tr><tr><td>Grieshaber forceps holder</td><td>Alcon</td><td>712.00.41</td><td>23 G</td></tr><tr><td>Curved scissor forceps tips</td><td>Alcon</td><td>723.52</td><td>23 G</td></tr><tr><td>Implant loading station</td><td>Dow Corning</td><td>3097358-1004</td><td>SYLGARD 184 Silicone Elastomer Kit</td></tr><tr><td>Blunt oval implant trephine&amp;nbsp;</td><td>Geuder</td><td>custom-made&amp;nbsp;</td><td>2.4 x 1.1 mm&amp;nbsp;</td></tr><tr><td>Shooter dummy</td><td>Geuder</td><td>G-32227</td><td>x1</td></tr><tr><td>Shooter</td><td>Geuder</td><td>G-S03443</td><td>x1</td></tr><tr><td>Flute needle</td><td>DORC</td><td>1281.SD</td><td>20 G (Vacuum)</td></tr><tr><td>Manual microliter syringe</td><td>Hamilton</td><td>24535</td><td>100 &amp;micro;l</td></tr><tr><td>Tissue culture plates</td><td>Greiner bio-one</td><td>&amp;nbsp;664160</td><td>100 x 20 mm</td></tr><tr><td>Spectralis Multi-Modality&lt;br/&gt; Imaging System</td><td>Heidelberg&lt;br/&gt; Engineering</td><td>N/a</td><td>Spectralis HRA&lt;br/&gt; &amp;nbsp;+ OCT</td></tr><tr><td>&lt;strong&gt;Drugs and solutions&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Active agent&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>Mucadont-IS</td><td>Merz Hygiene</td><td>virucidal instrument disinfectant&amp;nbsp;</td><td>2 L</td></tr><tr><td>Mucocit&amp;nbsp;T</td><td>Merz Hygiene</td><td>Aldehyde-free instrument disinfectant</td><td>2 L</td></tr><tr><td>Ketamin 10%</td><td>WDT</td><td>Ketamine&amp;nbsp;</td><td>10 ml (100 mg/ml)</td></tr><tr><td>Domitor</td><td>Orion Pharma</td><td>Medetomidine hydrochloride</td><td>10 ml (1 mg/ml)</td></tr><tr><td>Antisedan</td><td>Orion Pharma</td><td>Atipamezole hydrochloride</td><td>10 ml (5 mg/ml)</td></tr><tr><td>Neosynephrin POS 10%</td><td>URSAPHARM</td><td>Phenylephrine HCl</td><td>10 ml</td></tr><tr><td>Mydriacyl</td><td>Alcon</td><td>Tropicamid</td><td>10 ml (5 mg/ml)</td></tr><tr><td>Methocel 2%</td><td>Omni Vision</td><td>hydroxypropyl methylcellulose</td><td>10 g</td></tr><tr><td>PURI CLEAR</td><td>ZEISS</td><td>Balance salt solution (BSS)&amp;nbsp;</td><td>500 ml</td></tr><tr><td>Glucose 5%&amp;nbsp;&amp;nbsp;</td><td>B.Braun</td><td>Glucose 5%&amp;nbsp; solution</td><td>100 ml</td></tr><tr><td>Heparin-Natrium-25,000</td><td>Ratiopharm</td><td>Heparin</td><td>5 ml (2,500 unit/ml)</td></tr><tr><td>Suprarenin</td><td>SANOFI</td><td>Epinephrine</td><td>1 ml (1 mg/ml)</td></tr><tr><td>Triamcinolone</td><td>University of Bonn pharmacy</td><td>preservative-free Triamcinolone&amp;nbsp;</td><td>1 ml (40 mg/ml)&amp;nbsp;</td></tr><tr><td>Isoptomax eye ointment</td><td>Alcon</td><td>dexamethasone 1 mg/g&lt;br/&gt; neomycin sulfate 3,500 IU/g&lt;br/&gt; polymyxin B sulfate 6,000 IU/g</td><td>10 ml</td></tr><tr><td>Betaisodona</td><td>Mundipharma</td><td>Povidon-Iod</td><td>30 ml (1 g/10 ml)</td></tr><tr><td>Optive</td><td>ALLERGAN</td><td>sodium carboxymethylcellulose glycerol</td><td>10 ml</td></tr></tbody></table>

a step by step protocol for subretinal surgery in rabbits

<p class="jove_content"> La degeneración macular relacionada (AMD), la retinitis pigmentosa y otras enfermedades relacionadas con el EPR son las causas más comunes de pérdida irreversible de la visión en adultos en los países industrialmente desarrollados. RPE trasplante parece ser una terapia prometedora, ya que puede reemplazar RPE disfuncional, restaurar su función, y por lo tanto la visión. </p><p class="jove_content"> Aquí se describe un método para el trasplante de una monocapa RPE cultivadas en un andamio en el espacio subretiniano (SRS) de los conejos. Después de xenotrasplantes de vitrectomía se dieron en el SRS utilizando un tirador por encargo que consta de una boquilla metálica de calibre 20 con un émbolo revestido de politetrafluoroetileno (PTFE). La técnica actual se desarrolló en más de 150 cirugías de conejo durante 6 años. Post-operatorio de seguimiento se puede obtener utilizando no invasiva y repetitiva <em>de</em> imágenes <em>in vivo</em> como dominio espectral tomografía de coherencia óptica (OCT-SD), seguido por la histología de perfusión-fijo. </p><p class="jove_content"> ThMétodo E tiene pasos bien definidos para facilitar el aprendizaje y la alta tasa de éxito. Los conejos se consideran un gran modelo animal del ojo útil en los estudios preclínicos para la traducción clínica. En este contexto, los conejos son una alternativa rentable y tal vez conveniente a otros modelos animales de ojos grandes. </p>

<p class="jove_content" fo:keep-together.within-page="1"> Los resultados del método descrito para el implante subretiniano se muestra en la <strong>Tabla 2.</strong> El injerto debajo de la retina tuvo una tasa de éxito de <em>aproximadamente</em> 61% cuando se realiza una vitrectomía del núcleo y se elevó hasta el 76%, cuando se indujo desprendimiento vítreo posterior. Estos números incluyen <em>ca.</em> 21% de los animales que murieron ya sea durante la operación o durante los 3 primeros días del postoperatorio. Esta técnica se puede utilizar para implantar dos andamios sobre diferentes áreas de la retina de un ojo de forma simultánea. </p><p class="jove_content" fo:keep-together.within-page="1"> Los conejos fueron sometidos postoperatorio <em>en vivo</em> seguimiento utilizando SD-OCT y el procesamiento histológico como se describe por Stanzel <em>et al.</em> <sup>27</sup> <strong>(Figura 2).</strong> <strong>Fig.</strong> Escaneo oftalmoscopio láser imagen reflectancia en el infrarrojo de RPE cultivadas implantado en una membrana de poliéster (PET) <strong>2A</strong> muestra después de un trasplante sin complicaciones. loshalo alrededor del implante corresponde a la atrofia de los fotorreceptores. <strong>fig. 2B</strong> muestra adelgazamiento correspondiente SD-OCT, la notificación de la retina, la capa nuclear principalmente externa (ONL), banda reflectante hiper en SD-OCT por encima del implante, mientras que la retina neural adyacente al implante muestra casi normales bandas de reflexión. Estos resultados sugieren una entrega atraumática. <strong>Fig. 2C</strong> muestra Hematoxilina / Eosina (H / E) del implante, que muestra la cicatrización subretiniana y atrofia ONL alrededor del sitio retinotomía probablemente como resultado de la manipulación iatrogénica, una capa pigmentada todavía irregular contiguos de más de PET. la membrana de Bruch por debajo del implante también parecía ser contigua, y el coriocapilar contiene algunos eritrocitos dispersos. Estos resultados morfológicos son comparables a la SD-OCT y reforzar la tesis de un parto traumático. </p><p class="jove_content" fo:keep-together.within-page="1"><img alt="Figura 1" src="/files/ftp_upload/53927/53927fig1.jpg" /><br /> <strong>Higo</strong><html> Ure 1: <strong>Cargando tirador con Human IPSC-RPE cultivadas en PET Soporte celular. A)</strong> muestra punzonado de un implante utilizando por encargo de la aguja. <strong>B)</strong> del implante en forma de grano de corte del cultivo de células. <strong>C)</strong> Posicionamiento del implante antes de la carga. <strong>D)</strong> Carga de un tirador con implante. <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/53927/53927fig1large.jpg" target="_blank">Haga clic aquí para ver una versión más grande de esta figura .</a> </p><p class="jove_content" fo:keep-together.within-page="1"><img alt="Figura 2" src="/files/ftp_upload/53927/53927fig2.jpg" /><br /> <strong>Figura 2:</strong> <strong>humana cultivadas HES-RPE en PET Cell Carrier 4 semanas de conejo Subretiniana espacio. A)</strong> Imagen <strong>de</strong> reflectancia en el infrarrojo SLO Muestra, la línea verde demarca la sección transversal mostrada en la <strong>Fig. 2B.</strong> <strong>B)</strong> Corresponde SD-OCT. <strong>C)</strong> H / E mancha, véase el texto o <sup>26,27</sup> para más detalles.ove.com/files/ftp_upload/53927/53927fig2large.jpg &quot;target =&quot; _ blank &quot;&gt; Haga clic aquí para ver una versión más grande de esta figura. </p><table fo:keep-together.within-page="1"><colgroup><col /><col /></colgroup><tbody><tr height="21"><td height="21" style="height:21px;width:97px;"> <strong>Parámetro</strong> </td><td style="width:131px;"> <strong>configuración utilizada</strong> </td></tr><tr height="21"><td height="21" style="height:21px;"> vitrectomía </td><td> 6.000 cortes / min </td></tr><tr height="21"><td height="21" style="height:21px;"> Vacío </td><td> 200 mmHg </td></tr><tr height="21"><td height="21" style="height:21px;"> Hora de levantarse </td><td> 1 segundo </td></tr><tr height="21"><td height="21" style="height:21px;"> Aire </td><td> 24 mmHg </td></tr><tr height="21"><td height="21" style="height:21px;"> Irrigación </td><td> 24 cm de <sub>H2O</sub> </td></tr><tr height="21"><td height="21" style="height:21px;"> DiathErmy </td><td> 30% </td></tr></tbody></table><p class="jove_content" fo:keep-together.within-page="1"> <strong>Tabla 1:</strong> <strong>Configuración de parámetros de la máquina de vitrectomía.</strong> </p><table fo:keep-together.within-page="1"><tbody><tr><td> <strong>vitrectomía</strong> </td><td> <strong>Implante</strong> </td><td> <strong>conejos operados</strong> </td><td> <strong>implantes con éxito</strong> </td><td> <strong>implante fallido</strong> </td><td> <strong>Muerte</strong> </td><td> <strong>Tasa de éxito%</strong> </td></tr><tr><td> vitrectomía central </td><td> MASCOTA </td><td> 30 </td><td> 19 </td><td> 4 </td><td> 7 </td><td> 63.33 </td></tr><tr><td></td><td> PET + RPE </td><td> 70 </td><td> 42 </td><td> 12 </td><td> dieciséis </td><td> 60 </td></tr><tr><td> PVD, ± plasmina w / PPV </td><td> MASCOTA </td><td> 28 </td><td> 21 </td><td> 2 </td><td> 5 </td><td> 75 </td></tr><tr><td></td><td> PET + RPmi </td><td> 22 </td><td> 17 </td><td> 2 </td><td> 3 </td><td> 77.27 </td></tr></tbody></table><p class="jove_content" fo:keep-together.within-page="1"> <strong>Tabla 2:</strong> <strong>Resumen de los últimos 150 operaciones incluyendo Método y Tipo de implantes.</strong> </p>

Watch this Scientific Journal Video about A Step by Step Protocol for Subretinal Surgery in Rabbits at JoVE.com

A Step by Step Protocol for Subretinal Surgery in Rabbits

<p class="jove_content"> Epitelio pigmentario de la retina (EPR) y las estrategias de sustitución basado en la terapia génica son considerados por varias condiciones degenerativas de la retina. Para la traducción clínica, se requieren grandes modelos animales para estudiar el ojo técnicas quirúrgicas aplicables en los pacientes. Aquí presentamos un modelo de conejo para la cirugía subretiniana orientado hacia el trasplante de EPR, que es versátil y rentable. </p>

Un Paso a Paso Protocolo para la cirugía subretiniana en conejos

Age related macular degeneration (AMD), retinitis pigmentosa, and other RPE related diseases are the most common causes for irreversible loss of vision in ...

a-step-by-step-protocol-for-subretinal-surgery-in-rabbits

Universidad de Cartagena UDEC

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JoVE Journal

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15.3K vistas.  University of Bonn.  Epitelio pigmentario de la retina (EPR) y las estrategias de sustitución basado en la terapia génica son considerados por varias condiciones degenerativas de la retina. Para la traducción clínica, se requieren grandes modelos animales para estudiar el ojo técnicas quirúrgicas aplicables en los pacientes. Aquí presentamos un modelo de conejo para la cirugía subretiniana orientado hacia el trasplante de EPR, que es versátil y rentable. 

Video: A Step by Step Protocol for Subretinal Surgery in Rabbits

Buffers

<p>Source: Smaa Koraym at Johns Hopkins University, MD, USA</p>
<ol class="note-items">
	<li data-note-item="1" data-parent-collapse=""><strong>Preparing 50 mM NaH<sub>2</sub>PO<sub>4</sub> Buffer, pH 7</strong><button class="expand">Expand</button>
	<p>In the first part of this experiment, you will prepare a sodium phosphate solution buffered at pH 7.0. Monosodium phosphate is a weak acid with the conjugate base, disodium phosphate. Unadjusted monosodium phosphate solutions usually have a pH of about 4 - 6.</p>
	<p>Buffers are most effective close to their pKa, which is 6.8 to 7.2 for monosodium phosphate. So, you will use NaOH to push the equilibrium towards the conjugate base without altering the overall composition of the buffer equilibrium.</p>
	<ul class="lab-items">
		<li data-sub-note-item="1-3">
		<div class="lab-step">Before starting the experiment, calculate the mass of monosodium phosphate that you will need to make 200 mL of a 50 mM solution.
		<h4><strong>Table 1: Preparation of 50 mM NaH<sub>2</sub>PO<sub>4</sub> Buffer, pH 7.0</strong></h4>
		<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always" height="247" width="358">
			<tbody>
				<tr>
					<td colspan="2"><strong>Molar mass of NaH<sub>2</sub>PO<sub>4</sub> = 119.98 g/mol</strong></td>
				</tr>
				<tr>
					<td>Solution volume (mL)</td>
					<td style="text-align: center;">200</td>
				</tr>
				<tr>
					<td>Molarity (mM)</td>
					<td style="text-align: center;">50</td>
				</tr>
				<tr>
					<td>Moles of NaH<sub>2</sub>PO<sub>4</sub> (mol)</td>
					<td style="text-align: center;"></td>
				</tr>
				<tr>
					<td>Mass needed (mg)</td>
					<td style="text-align: center;"></td>
				</tr>
				<tr>
					<td>Initial volume of 1 M NaOH (mL)</td>
					<td style="text-align: center;"></td>
				</tr>
				<tr>
					<td>Final volume of 1 M NaOH (mL)</td>
					<td style="text-align: center;"></td>
				</tr>
				<tr>
					<td>Volume of NaOH used (mL)</td>
					<td style="text-align: center;"></td>
				</tr>
				<tr>
					<td>Volume of DI water needed (mL)</td>
					<td style="text-align: center;"></td>
				</tr>
			</tbody>
		</table>
		<a href="https://jove.unicartagenaproxy.elogim.com/CDNSource/lm/labs/45/Table_1._Preparation_of_50_mM_NaH2PO4_Buffer,_pH_7.0.xlsx" target="_blank">Click Here to download Table 1</a></div>
		</li>
		<li data-sub-note-item="1-4">
		<div class="lab-step">To begin, put on the necessary personal protective equipment, including a lab coat, splash-resistant safety glasses, and gloves. <strong>Note: </strong>This section of the lab uses NaOH, which is corrosive and toxic. Use caution when pouring and transporting NaOH.</div>
		</li>
		<li data-sub-note-item="1-5">
		<div class="lab-step">Fill a 250-mL plastic wash bottle with deionized water.</div>
		</li>
		<li data-sub-note-item="1-6">
		<div class="lab-step">Label two 100-mL beakers, one for neutral aqueous waste and one for basic aqueous waste.</div>
		</li>
		<li data-sub-note-item="1-7">
		<div class="lab-step">Calibrate your pH meter using the provided buffers. Store the probe in its storage solution when you are done.</div>
		</li>
		<li data-sub-note-item="1-8">
		<div class="lab-step">Tare weighing paper, and use a clean spatula to measure out the required amount of monosodium phosphate according to your calculations. Record the exact amount of monosodium phosphate in your lab notebook. <strong>Note: </strong>Monosodium phosphate absorbs moisture from the air, so work quickly to obtain an accurate measurement and close the container tightly when you are done with it.</div>
		</li>
		<li data-sub-note-item="1-9">
		<div class="lab-step">Place the monosodium phosphate in a 400-mL beaker and clean the spatula with a laboratory wipe. Throw out the weighing paper and the laboratory wipe before returning to your fume hood.</div>
		</li>
		<li data-sub-note-item="1-10">
		<div class="lab-step">Use a graduated cylinder to measure out 175 mL of deionized water. Pour the water into the beaker of monosodium phosphate and add a magnetic stir bar.</div>
		</li>
		<li data-sub-note-item="1-11">
		<div class="lab-step">Stir the solution until the salt has dissolved completely and the solution appears homogeneous. This usually takes 2 - 3 min.</div>
		</li>
		<li data-sub-note-item="1-12">
		<div class="lab-step">Measure 15 mL of deionized water and pour it into the beaker. Continue stirring the solution until it appears homogeneous again (about 1 &#8211; 2 min). Turn off the stir motor.</div>
		</li>
		<li data-sub-note-item="1-13">
		<div class="lab-step">Rinse the pH probe with deionized water, and then clamp it in the solution with the sensor above the stir bar.</div>
		</li>
		<li data-sub-note-item="1-14">
		<div class="lab-step">Bring a 10-mL graduated cylinder and watch glass to the dispensing hood and measure out 10 mL of 1 M NaOH. Note the exact volume in the graduated cylinder.</div>
		</li>
		<li data-sub-note-item="1-15">
		<div class="lab-step">Cover your NaOH with the watch glass, and carefully bring it to your fume hood.</div>
		</li>
		<li data-sub-note-item="1-16">
		<div class="lab-step">Resume stirring the monosodium phosphate solution. While monitoring the pH reading, use a disposable pipette to slowly add NaOH to the stirring solution in a drop-wise manner. Once the buffer pH reaches 7.0, return any NaOH still in the pipette to the graduated cylinder.</div>
		</li>
		<li data-sub-note-item="1-17">
		<div class="lab-step">Calculate the volume of NaOH that you added to the buffer. Subtract that volume from 10 mL to determine how much deionized water you must add to your buffer to reach a total solution volume of 200 mL.</div>
		</li>
		<li data-sub-note-item="1-18">
		<div class="lab-step">Measure the deionized water with another 10-mL graduated cylinder and add it to the stirring solution to finish making the buffer.</div>
		</li>
		<li data-sub-note-item="1-19">
		<div class="lab-step">Rinse the pH sensor with deionized water, cover it with the storage solution filled cap, and unplug or turn off the probe.</div>
		</li>
		<li data-sub-note-item="1-20">
		<div class="lab-step">Label a 250-mL polyethylene bottle as &#39;50 mM monosodium phosphate buffer, pH 7.0&#39;. Retrieve the magnetic stir bar from the solution.</div>
		</li>
		<li data-sub-note-item="1-21">
		<div class="lab-step">Use a funnel to pour the buffer solution into the labeled bottle and cap it tightly.</div>
		</li>
	</ul>
	</li>
	<li data-note-item="2" data-parent-collapse=""><strong>Absorption Spectroscopy of Free and Bound Neutral Red</strong><button class="expand">Expand</button>
	<p>Buffers can be used to evaluate compounds at specific pH values. In this section, you will record the absorbance spectrum of the indicator neutral red in various buffers. The protonated form of neutral red is red, and the deprotonated form is yellow-orange, meaning that they absorb green and blue-violet light, respectively. Thus, the acidic and basic forms have distinct absorption wavelengths. Protein binding alters the properties of neutral red, changing both its absorbance and its pKa.</p>
	<p>After measuring the absorbance spectrum of free neutral red at several pH values, you will add riboflavin-binding protein, or RP, to the solutions and measure the absorbances again. Absorbance intensity is related to concentration, so you will use the spectra to determine the pKa of free and bound neutral red after the lab.</p>
	<ul class="lab-items">
		<li data-sub-note-item="2-3">
		<div class="lab-step">Draw the following table in your lab notebook. Number the cuvettes 1 through 10 and list the nine buffer pH values that you will use. The 10th cuvette will be a deionized water blank. <strong> Note: </strong>Always hold cuvettes by the textured sides and wipe the transparent sides just before putting the cuvette in the spectrophotometer. Remember to align the transparent sides with the beam of light in the spectrophotometer.
		<h4><strong>Table 2: Absorbance of Free and Bound Neutral Red</strong></h4>
		<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
			<tbody>
				<tr>
					<td style="text-align: center;"><strong>Cuvette #</strong></td>
					<td style="text-align: center;"><strong>Buffer pH </strong></td>
					<td style="text-align: center;"><strong>Abs. at &#955;<sub>max </sub>(Free NRH<sup>+</sup>)</strong></td>
					<td style="text-align: center;"><strong>Abs. at &#955;<sub>max</sub> (Bound NRH)</strong></td>
				</tr>
				<tr>
					<td style="text-align: center;">1</td>
					<td style="text-align: center;">5.0</td>
					<td style="text-align: center;"></td>
					<td style="text-align: center;"></td>
				</tr>
				<tr>
					<td style="text-align: center;">2</td>
					<td style="text-align: center;">5.5</td>
					<td></td>
					<td></td>
				</tr>
				<tr>
					<td style="text-align: center;">3</td>
					<td style="text-align: center;">6.0</td>
					<td></td>
					<td></td>
				</tr>
				<tr>
					<td style="text-align: center;">4</td>
					<td style="text-align: center;">6.5</td>
					<td></td>
					<td></td>
				</tr>
				<tr>
					<td style="text-align: center;">5</td>
					<td style="text-align: center;">7.0</td>
					<td></td>
					<td></td>
				</tr>
				<tr>
					<td style="text-align: center;">6</td>
					<td style="text-align: center;">7.5</td>
					<td></td>
					<td></td>
				</tr>
				<tr>
					<td style="text-align: center;">7</td>
					<td style="text-align: center;">8.0</td>
					<td></td>
					<td></td>
				</tr>
				<tr>
					<td style="text-align: center;">8</td>
					<td style="text-align: center;">8.5</td>
					<td></td>
					<td></td>
				</tr>
				<tr>
					<td style="text-align: center;">9</td>
					<td style="text-align: center;">11</td>
					<td></td>
					<td></td>
				</tr>
				<tr>
					<td style="text-align: center;">10</td>
					<td style="text-align: center;">blank</td>
					<td></td>
					<td></td>
				</tr>
			</tbody>
		</table>
		<a href="https://jove.unicartagenaproxy.elogim.com/CDNSource/lm/labs/45/Table_2._Absorbance_of_Free_and_Bound_Neutral_Red.xlsx" target="_blank">Click Here to download Table 2</a></div>
		</li>
		<li data-sub-note-item="2-4">
		<div class="lab-step">Obtain ten 1.5-mL cuvettes and caps and label the caps 1 through 10 to match the table in your lab notebook.</div>
		</li>
		<li data-sub-note-item="2-5">
		<div class="lab-step">Label a 25-mL beaker as &#8216;DIH2O&#8217; and fill it with deionized water.</div>
		</li>
		<li data-sub-note-item="2-6">
		<div class="lab-step">Flush your neutral aqueous waste down the drain and relabel the beaker as &#39;aqueous buffer waste&#39;.</div>
		</li>
		<li data-sub-note-item="2-7">
		<div class="lab-step">Label a 400-mL beaker for used micropipette tips.</div>
		</li>
		<li data-sub-note-item="2-8">
		<div class="lab-step">Attach a tip to a 1-mL micropipette and use it to dispense 1000 &#181;L of deionized water into cuvette 10. This is your solvent blank.</div>
		</li>
		<li data-sub-note-item="2-9">
		<div class="lab-step">Place 925 &#181;L of your pH 7.0 monosodium phosphate buffer in cuvette 5.</div>
		</li>
		<li data-sub-note-item="2-10">
		<div class="lab-step">Bring the remaining empty cuvettes to the buffer table. Guided by the table in your lab notebook, dispense 925 &#181;L of each buffer into the appropriate cuvette using the labeled micropipettes. Be careful not to use the same pipette tip for different buffers.</div>
		</li>
		<li data-sub-note-item="2-11">
		<div class="lab-step">Once you have finished, bring the buffers back to your workbench. There, set a 200-&#181;L micropipette to dispense 75 &#181;L, and attach a tip to the micropipette.</div>
		</li>
		<li data-sub-note-item="2-12">
		<div class="lab-step">Dispense 75 &#181;L of neutral red into each of the nine buffer cuvettes. Invert each cuvette several times to thoroughly mix the solutions. <strong>Note: </strong>Replace the pipette tip if it touches a buffer.</div>
		</li>
		<li data-sub-note-item="2-13">
		<div class="lab-step">Once you have mixed neutral red with every buffer, take a picture, or write down the colors of the solutions.</div>
		</li>
		<li data-sub-note-item="2-14">
		<div class="lab-step">Turn on a hand-held spectrophotometer and allow the light source to warm up. Once it is ready, create a new experiment to measure absorbance versus wavelength for the free neutral red.</div>
		</li>
		<li data-sub-note-item="2-15">
		<div class="lab-step">Insert the cuvette of deionized water and acquire a spectrum of the deionized water. Set it as a solvent background or blank.</div>
		</li>
		<li data-sub-note-item="2-16">
		<div class="lab-step">Then, remove the blank and insert the cuvette of neutral red in the lowest pH buffer, which will have the highest concentration of protonated neutral red.</div>
		</li>
		<li data-sub-note-item="2-17">
		<div class="lab-step">Acquire a spectrum, which should show one strong peak. Identify the wavelength corresponding to the highest point of this peak, or the maximum. Record this wavelength in your lab notebook as lambda max for protonated free neutral red.</div>
		</li>
		<li data-sub-note-item="2-18">
		<div class="lab-step">Save the data and remove the cuvette. Record the absorbance in your notebook, then collect spectra for cuvettes 2 - 9 using the same procedure.</div>
		</li>
		<li data-sub-note-item="2-19">
		<div class="lab-step">Record the wavelength of the isosbestic point in your lab notebook. That is where all the spectra cross at a single wavelength. If a spectrum does not cross at that point, empty and clean the cuvette, prepare a fresh sample, and try again.</div>
		</li>
		<li data-sub-note-item="2-20">
		<div class="lab-step">Once you finish collecting spectra for all nine cuvettes, save and export the data.</div>
		</li>
		<li data-sub-note-item="2-21">
		<div class="lab-step">Create a new experiment to measure absorbance versus wavelength for the bound neutral red.</div>
		</li>
		<li data-sub-note-item="2-22">
		<div class="lab-step">Fit a new tip to the 200-&#181;L micropipette and add 75 &#181;L of riboflavin-binding protein solution to each cuvette, including the blank. Eject the tip, secure the cuvette caps, and invert the cuvette several times to mix the solutions.</div>
		</li>
		<li data-sub-note-item="2-23">
		<div class="lab-step">Insert cuvette 10 into the spectrophotometer and set it as the solvent blank.</div>
		</li>
		<li data-sub-note-item="2-24">
		<div class="lab-step">Acquire a spectrum of the lowest pH sample, and identify the wavelength corresponding to the maximum of the most intense peak. Record it in your lab notebook as the lambda max of protonated-bound neutral red.</div>
		</li>
		<li data-sub-note-item="2-25">
		<div class="lab-step">Acquire spectra for cuvettes 2 - 9 in the same way as you did for the free neutral red samples. Record the intensities at lambda max for protonated-bound neutral red in your table, along with the wavelength at the isosbestic point.</div>
		</li>
		<li data-sub-note-item="2-26">
		<div class="lab-step">When you are done, save your data, export it for later analysis, and shut down the spectrophotometer.</div>
		</li>
		<li data-sub-note-item="2-27">
		<div class="lab-step">Put away all equipment and dispose of your used pipette tips in an approved waste container or trash bin.</div>
		</li>
		<li data-sub-note-item="2-28">
		<div class="lab-step">Empty the cuvettes into the aqueous buffer waste beaker and rinse the cuvettes into the beaker with deionized water.</div>
		</li>
		<li data-sub-note-item="2-29">
		<div class="lab-step">Empty the excess NaOH into the basic waste and rinse the graduated cylinder with water.</div>
		</li>
		<li data-sub-note-item="2-30">
		<div class="lab-step">Flush the basic aqueous waste down the drain with copious tap water, along with the other aqueous waste.</div>
		</li>
		<li data-sub-note-item="2-31">
		<div class="lab-step">Wash your glassware, cuvette caps, and stir bar per your lab standard procedures. Clean your work surfaces with a damp paper towel and throw out used paper towels and lab wipes in the lab trash.</div>
		</li>
	</ul>
	</li>
	<li data-note-item="3" data-parent-collapse=""><strong>Results</strong><button class="expand">Expand</button>
	<p>Now, let&#39;s analyze our absorbance data to determine the pKa&#39;s of neutral red.</p>
	<h4><strong>Table 3: Determination of Neutral Red pKa&#39;s</strong></h4>
	<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
		<tbody>
			<tr>
				<td></td>
				<td>Free NRH<sup>+</sup></td>
				<td>Bound NRH<sup>+</sup></td>
			</tr>
			<tr>
				<td>&#160;&#955;<sub>max</sub> (nm)</td>
				<td></td>
				<td></td>
			</tr>
			<tr>
				<td>&#160;&#916;A (nm)</td>
				<td></td>
				<td></td>
			</tr>
			<tr>
				<td>&#160;Midpoint (nm)</td>
				<td></td>
				<td></td>
			</tr>
			<tr>
				<td>&#160;pKa</td>
				<td></td>
				<td></td>
			</tr>
		</tbody>
	</table>
	<p><a href="https://jove.unicartagenaproxy.elogim.com/CDNSource/lm/labs/45/Table_3._Determination_of_Neutral_Red_pKa.xlsx" target="_blank">Click Here to download Table 3</a></p>
	<ul class="lab-items">
		<li data-sub-note-item="3-2">
		<div class="lab-step">Plot both sets of intensity data with absorbance intensity at lambda max on the y-axis and pH on the x-axis, with the points joined by smooth lines.</div>
		</li>
		<li data-sub-note-item="3-3">
		<div class="lab-step">Calculate the difference between the starting and ending absorbance intensities for each data series.</div>
		</li>
		<li data-sub-note-item="3-4">
		<div class="lab-step">Determine the absorbance midway between the starting and ending absorbances for each series, or the absorbance midpoints.</div>
		</li>
		<li data-sub-note-item="3-5">
		<div class="lab-step">Find where the midpoint occurs on each line and identify the pH value at that point. These pH values are the pKa&#39;s of free and bound neutral red.</div>
		</li>
		<li data-sub-note-item="3-6">
		<div class="lab-step">Here, we see an increase in pKa of about one between free and bound neutral red, indicating that bound-protonated neutral red is a weaker acid than free protonated neutral red by an order of magnitude.</div>
		</li>
	</ul>
	</li>
</ol>

Buffers: Common Ion Effect, Henderson-Hasselbalch Equation, and Buffer Capacity - Procedure

Búferes

Source: Smaa Koraym at Johns Hopkins University, MD, USA

	Preparing 50 mM NaH2PO4 Buffer, pH 7Expand
	In the first part of this experiment, you will ...

Educación

JoVE Lab Manual

Química

Lab Manual Sub Articles

Lab Techniques

<p>Source: Smaa Koraym at Johns Hopkins University, MD, USA</p>
<ol class="note-items">
	<li data-note-item="1" data-parent-collapse=""><strong>Determining the Density of an Egg</strong><button class="expand">Expand</button>
	<p>Density is a measure of how compact a substance or object is, and it is calculated as mass divided by volume. When a dense object is placed in a liquid with a lower density, the object sinks; when the object is less dense than the liquid, it floats. When the densities are the same, the object will be suspended in the liquid. In this experiment, we&#39;ll determine the density of an egg by creating a solution with the same density. We&#39;ll measure the density of the salt solution using different volume measurement glassware and determine which is the most accurate.</p>
	<ul class="lab-items">
		<li data-sub-note-item="1-1">
		<div class="lab-step">To begin, wear the appropriate personal protective equipment, including close-toed shoes, long pants, long-sleeved shirt, lab coat, chemical splash goggles, and gloves.</div>
		</li>
		<li data-sub-note-item="1-2">
		<div class="lab-step">Measure the mass of the egg. Set a weigh boat on the balance and tare it. Carefully set the egg in the weigh boat while you record the mass in your notebook.</div>
		</li>
		<li data-sub-note-item="1-3">
		<div class="lab-step">Prepare the salt solution that will be used to suspend your egg. Use the 600-mL beaker and fill it 3/4 of the way full with deionized water.</div>
		</li>
		<li data-sub-note-item="1-4">
		<div class="lab-step">Gently place your egg in the beaker, and then add about one teaspoon of NaCl into the water. Stir the solution gently until the NaCl dissolves. Continue to add NaCl until the egg floats just below the surface.</div>
		</li>
		<li data-sub-note-item="1-5">
		<div class="lab-step">Use different glassware to measure the volume of the salt solution. Weigh the 50-mL graduated cylinder on the top loading balance and record the mass. Then, measure 20 mL of your salt solution and record the volume in your notebook. <strong>Note: </strong>Place a blank sheet of paper behind the graduated cylinder to read the volume more clearly. Always view the measurement at eye level and measure the volume from the bottom of the meniscus. The lines on the graduated cylinder are 0.2 mL apart, meaning that you can read the volume to the hundredths place.
		<h4><b>Table 1: Measuring Mass</b></h4>
		<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
			<tbody>
				<tr>
					<td style="text-align: center;"><strong>Container</strong></td>
					<td style="text-align: center;"><strong>Mass of empty container (g)</strong></td>
					<td style="text-align: center;"><strong>Mass of full containter (g)</strong></td>
					<td style="text-align: center;"><strong>Mass of solution (g)</strong></td>
					<td style="text-align: center;"><strong>Percent error</strong></td>
				</tr>
				<tr>
					<td>50-mL graduated cylinder</td>
					<td style="text-align: center;"></td>
					<td style="text-align: center;"></td>
					<td style="text-align: center;"></td>
					<td style="text-align: center;"></td>
				</tr>
				<tr>
					<td>10-mL volumetric pipette</td>
					<td style="text-align: center;"></td>
					<td style="text-align: center;"></td>
					<td style="text-align: center;"></td>
					<td style="text-align: center;"></td>
				</tr>
				<tr>
					<td>50-mL beaker</td>
					<td style="text-align: center;"></td>
					<td style="text-align: center;"></td>
					<td style="text-align: center;"></td>
					<td style="text-align: center;"></td>
				</tr>
				<tr>
					<td>20-mL volumetric flask</td>
					<td style="text-align: center;"></td>
					<td style="text-align: center;"></td>
					<td style="text-align: center;"></td>
					<td style="text-align: center;"></td>
				</tr>
			</tbody>
		</table>
		<a href="https://jove.unicartagenaproxy.elogim.com/CDNSource/lm/labs/41/Table_1._Measuring_Mass.xlsx" target="_blank">Click Here to download Table 1</a></div>
		</li>
		<li data-sub-note-item="1-6">
		<div class="lab-step">Weigh the filled graduated cylinder and record the mass in your notebook. Then, pour the salt solution back into the 600-mL beaker.</div>
		</li>
		<li data-sub-note-item="1-7">
		<div class="lab-step">Use the volumetric pipette to measure volume. First, weigh the empty 50-mL beaker and record the mass in your notebook. Then, carefully measure 10 mL of the salt solution using the volumetric pipette and dispense the salt solution into the beaker.</div>
		</li>
		<li data-sub-note-item="1-8">
		<div class="lab-step">Measure another 10 mL and add it to the beaker. Weigh the filled beaker and record the mass in your notebook. Then, empty, rinse, and dry the beaker.</div>
		</li>
		<li data-sub-note-item="1-9">
		<div class="lab-step">Use the volumetric flask to measure volume. Weigh the empty 20-mL volumetric flask and record the mass. Then, carefully fill the flask with the salt solution to just below the 20-mL fill mark. Do not fill it all the way. Use an eyedropper to finish filling the flask so that the bottom of the meniscus just touches the fill line on the neck of the flask. Then, weigh the full flask and record the weight.</div>
		</li>
		<li data-sub-note-item="1-10">
		<div class="lab-step">Lastly, use the 50-mL beaker to measure volume. Weigh the empty beaker, then fill the beaker with salt solution to the 20-mL line and weigh the full beaker. Record the masses in your notebook.</div>
		</li>
		<li data-sub-note-item="1-11">
		<div class="lab-step">Measure the displacement of the egg when it is submerged in the salt solution. First, remove the egg and look at the level of the salt solution in the 600-mL beaker. <strong>Note:</strong> If it is not at a marked line, add deionized water to the solution so that the meniscus is at a readable volume and record the volume.&#160;
		<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
			<tbody>
				<tr>
					<td><strong>Measurements</strong></td>
					<td><strong>Salt solution using 20-mL volumetric flask&#160;</strong></td>
					<td><strong>Egg</strong></td>
				</tr>
				<tr>
					<td>Mass (g)</td>
					<td></td>
					<td></td>
				</tr>
				<tr>
					<td>Displacement of water (cm)</td>
					<td style="text-align: center;">-</td>
					<td></td>
				</tr>
				<tr>
					<td>Inner diameter of 600-mL beaker (cm)</td>
					<td style="text-align: center;">-</td>
					<td></td>
				</tr>
				<tr>
					<td>Volume (cm&#179;)</td>
					<td style="text-align: center;">-</td>
					<td></td>
				</tr>
				<tr>
					<td>Volume (mL)</td>
					<td style="text-align: center;">20</td>
					<td></td>
				</tr>
				<tr>
					<td>Density (g/mL)</td>
					<td></td>
					<td></td>
				</tr>
			</tbody>
		</table>
		<strong><a href="https://jove.unicartagenaproxy.elogim.com/CDNSource/lm/labs/41/Table_2._Measuring_Density.xlsx" target="_blank">Click Here to download Table 2</a> </strong></div>
		</li>
		<li data-sub-note-item="1-12">
		<div class="lab-step">Gently place the egg in the beaker.</div>
		</li>
		<li data-sub-note-item="1-13">
		<div class="lab-step">Use a ruler to measure the new height of the solution level and record the height. Then, place the ruler across the top of the beaker to measure the inner diameter.</div>
		</li>
		<li data-sub-note-item="1-14">
		<div class="lab-step">Calculate the new volume from these measurements. Subtract the initial volume to obtain the displacement of the egg (volume).</div>
		</li>
		<li data-sub-note-item="1-15">
		<div class="lab-step">To clean up from the experiment, return the egg to your instructor and pour the salt solution down the sink. Rinse all used glassware with tap water and set them out to dry. Finally, return the sodium chloride to your instructor.</div>
		</li>
	</ul>
	</li>
	<li data-note-item="2" data-parent-collapse=""><strong>Results</strong><button class="expand">Expand</button>
	<ul class="lab-items">
		<li data-sub-note-item="2-1">
		<div class="lab-step">Calculate the mass of the solution in each of the different volumetric glassware by subtracting the empty glassware mass from the full glassware mass.</div>
		</li>
		<li data-sub-note-item="2-2">
		<div class="lab-step">Keep track of uncertainty in your measurement by using significant figures. For example, when we look at the graduated cylinder measurement, the weight of the empty container is 90.92, and the weight of the full container is 111.59. The weight measurements have significant figures to the hundredths place, so it is important that significant figures are maintained throughout our calculations. Thus, the mass of the 20-mL salt solution is 20.67 g.</div>
		</li>
		<li data-sub-note-item="2-3">
		<div class="lab-step">The 50-mL beaker weighed 28.65 g when it was empty and 47.96 g when it contained 20 mL of salt solution. Therefore, the mass of 20 mL of the salt solution is 19.31 g.</div>
		</li>
		<li data-sub-note-item="2-4">
		<div class="lab-step">Repeat the calculation to obtain the mass of 20 mL of salt solution that was measured using the 10-mL pipette and the 200-mL volumetric flask.<b> Note:</b> Generally speaking, volumetric measurements are more accurate when using a volumetric flask.</div>
		</li>
		<li data-sub-note-item="2-5">
		<div class="lab-step">Look at the accuracy of the other methods compared to the volumetric flask in terms of percent error. Percent error is calculated by subtracting the theoretical value from the experimental value, then dividing by the theoretical value times 100.
		<ul>
			<li><img src="https://jove.unicartagenaproxy.elogim.com/CDNSource/lm/labs/41/41_Procedure_1b.jpg" /></li>
		</ul>
		</div>
		</li>
		<li data-sub-note-item="2-6">
		<div class="lab-step">Use the volumetric flask measurement as the theoretical value to determine the percent error for the other volume measurement methods. As expected, the volume measurement that uses the markings on the beaker is the least accurate.<strong> Note: </strong>We also know this because the known density of pure water is 1 g/mL. Thus, 20 mL of pure water weighs 20 g. The density of the salt solution must be higher than 20 g since salt was added to the water.</div>
		</li>
		<li data-sub-note-item="2-7">
		<div class="lab-step">Calculate the density of the salt solution using the volumetric flask measurement. Divide the mass of the solution by the volume of the solution. Thus, the density of the salt solution is 1.053 g/mL.</div>
		</li>
		<li data-sub-note-item="2-8">
		<div class="lab-step">Compare the calculated density of the salt solution to the density of the egg. The volume calculated using the displacement of water in a beaker was found to be 61.14 cm<sup>3</sup> or 61.14 mL. The weight of the egg was measured to be 60.15 g, so its density is 0.9838 g/mL. The disparity between the density of the egg and the density of the salt solution arises from error introduced in both calculations, primarily due to volume measurement readings.</div>
		</li>
	</ul>
	</li>
</ol>

Accuracy, Precision, Uncertainty, and Significant Figures - Procedure

Técnicas de laboratorio

Source: Smaa Koraym at Johns Hopkins University, MD, USA

	Determining the Density of an EggExpand
	Density is a measure of how compact a substance or ...

Extraction

Extraction: Liquid-Liquid and Acid-Base Extraction - Procedure

Extracción

Source: Lara Al Hariri and Ahmed Basabrain at the University of Massachusetts Amherst, MA, USA

	Cellulose RecoveryExpand
	Extraction and filtration can ...

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

Extinction: Diversity and Extinction during the Paleogene Epoch - Procedure

Extinción

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

Biología

Ecología

Optimal Foraging

<ol class="note-items"><li data-note-item="1" data-parent-collapse="">Simulating Foraging with Artificial Habitats and Prey
<button class="expand">Expand</button><ul class="lab-items"><li data-sub-note-item="1-1"><div class="lab-step">NOTE: The foragers will hunt for prey represented by pinto beans in four buckets of rice with varying prey densities. Without knowing what these densities are, foragers must obtain as many prey items as possible in as little time as possible. 
</div></li><li data-sub-note-item="1-2"><div class="lab-step">HYPOTHESES: In this experiment, the experimental hypothesis could be that foragers will catch the most prey in the higher prey density bucket and also spend the most time foraging there.  The null hypothesis would then be that foragers will catch an equal number of prey in each bucket and that foragers will spend an equal amount of time foraging in each patch. 
</div></li><li data-sub-note-item="1-3"><div class="lab-step">To begin the foraging activity, record the names and role of each participant at the top of the student data collection sheet.  NOTE: Before starting the experiment, the forager is allowed to become familiar with the foraging process &#8211; the following steps are a practice round. 
</div></li><li data-sub-note-item="1-4"><div class="lab-step">The forager should kneel or sit in front of the average density bucket. 
</div></li><li data-sub-note-item="1-5"><div class="lab-step">Once the timer says start, the forager may begin looking for the pinto beans representing the prey. The forager can spend as much time as they like foraging in this bucket. 
</div></li><li data-sub-note-item="1-6"><div class="lab-step">Once a prey item is located, the forager must signal that one has been found and place the prey in the cup next to the bucket.
</div></li><li data-sub-note-item="1-7"><div class="lab-step">Once they believe they have acquired most of the prey in the patch, they should tell the timer to stop the time and the practice round is over. 
</div></li><li data-sub-note-item="1-8"><div class="lab-step">To begin the simulation proper, the forager should sit or kneel in front of bucket A. 
</div></li><li data-sub-note-item="1-9"><div class="lab-step">The timer must be ready to start the stopwatch. 
</div></li><li data-sub-note-item="1-10"><div class="lab-step">The recorder should be ready to record the time each prey item is located, in addition to the time that the forager arrived at and left each bucket. 
</div></li><li data-sub-note-item="1-11"><div class="lab-step">When all parties are ready, the timer should start the stopwatch and say &#8220;start&#8221;,  and the forager should begin foraging. NOTE: The stopwatch will run through the rest of the round for buckets B and C.
</div></li><li data-sub-note-item="1-12"><div class="lab-step">As the forager signals finding pinto beans and places them in the cup next to the bucket, the timer should tell the recorder the time.
</div></li><li data-sub-note-item="1-13"><div class="lab-step">The recorder should then write the time in the student data collection sheet.  <a href="https://jove.unicartagenaproxy.elogim.com/CDNSource/lm/labs/34/Table_1_Optimal_foraging.xlsx" style="display:block;margin:20px;" target="_blank">Click Here to download Table 1</a>
</div></li><li data-sub-note-item="1-14"><div class="lab-step">Once the forager has decided to move on to the next bucket, they should tell the timer &#8220;done&#8221;, and begin moving to the next patch. 
</div></li><li data-sub-note-item="1-15"><div class="lab-step">The recorder should record the time the forager left and the time they arrived at bucket B in the student data collection sheet.
</div></li><li data-sub-note-item="1-16"><div class="lab-step">Repeat the same procedure for buckets B and C. Once the forager has completed the activity, it is time for the next forager to begin using the same procedure. 
</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">Before performing any data analysis, review the data collection sheet and compile your individual data for captured prey, foraging time, and travel time for each bucket in your student calculations table. <a href="https://jove.unicartagenaproxy.elogim.com/CDNSource/lm/labs/34/Table_2_Optimal_foraging.xlsx" style="display:block;margin:20px;" target="_blank">Click Here to download Table 2</a>
</div></li><li data-sub-note-item="2-2"><div class="lab-step">After this, calculate your individual capture rate for each bucket by dividing the number of prey captured by the sum of the foraging time and travel time and record these values in your table. Individual capture rate =  Number of prey captured / (foraging time + travel time)
</div></li><li data-sub-note-item="2-3"><div class="lab-step">To calculate your Giving-Up-Time or a GUT for each bucket, subtract the time that the last prey item was acquired from the time you left the bucket.  GUT = time forager left bucket  -  time last prey item acquired
</div></li><li data-sub-note-item="2-4"><div class="lab-step">Next, calculate the mean GUT for your foraging group and record the values in your table.
</div></li><li data-sub-note-item="2-5"><div class="lab-step">Finally, calculate your scaled GUT for each bucket by dividing the bucket GUT by your group&#39;s mean GUT. Scaled GUT =  Bucket GUT / Group mean GUT NOTE: The scaled GUT will allow class data to be pooled later on without differences in individual foragers skewing the data. 
</div></li><li data-sub-note-item="2-6"><div class="lab-step">The instructor should now compile the data into the classroom spreadsheet.  <a href="https://jove.unicartagenaproxy.elogim.com/CDNSource/lm/labs/34/Table_3_Optimal_foraging.xlsx" style="display:block;margin:20px;" target="_blank">Click Here to download Table 3</a>
</div></li><li data-sub-note-item="2-7"><div class="lab-step">Plot a bar graph of the average number of prey collected in each bucket. On the x-axis, A should represent the low prey density bucket, B should represent the medium prey density bucket, and C the high prey density bucket. Plot the y-axis as the number of prey items caught. 
</div></li><li data-sub-note-item="2-8"><div class="lab-step">Next, plot the graph of the class average scaled GUT, where the x-axis represents the buckets with varying levels of prey density and the y-axis shows the values for the average scaled GUT. 
</div></li><li data-sub-note-item="2-9"><div class="lab-step">Plot a third graph of the average time spent foraging in each bucket. 
</div></li><li data-sub-note-item="2-10"><div class="lab-step">Now review the Marginal Value Theorem (MVT) from the concepts video and examine the three graphs that you generated from your class data. Take note of whether the graphs conform to the predictions of MVT. 
</div></li></ul></li></ol>

Optimal Foraging: Foraging with Artificial Habitats and Prey - Procedure

Forrajeo óptimo

Simulating Foraging with Artificial Habitats and Prey
ExpandNOTE: The foragers will hunt for prey represented by pinto beans in four buckets of rice with ...

Group Behavior

<ol class="note-items"><li data-note-item="1" data-parent-collapse="">The Hawk-Dove Game
<button class="expand">Expand</button><ul class="lab-items"><li data-sub-note-item="1-1"><div class="lab-step">NOTE: In this activity, you will play the hawk and dove game with a single partner and then with multiple partners to observe hypothetical costs and benefits of the antagonist or cooperative behavior respectively. 
</div></li><li data-sub-note-item="1-2"><div class="lab-step">Before the activity starts, review the cost-benefit table so that you understand how to score the game. <a href="https://jove.unicartagenaproxy.elogim.com/CDNSource/lm/labs/33/Table_1_Group_behavior.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">HYPOTHESES: The experimental hypothesis for this exercise could be that the dove strategy will become more frequent between repeated partners, and the hawk strategy will become more frequent when interactions are with a variety of partners. The null hypothesis could be that there will be no change in strategy over time with repeat or different partner combinations. NOTE: Benefit can be determined by looking at the left column and choosing the row that corresponds to the card you played. The box that corresponds to the choice of your partner can be found in the top row. For the purposes of this game, B equals four and C equals three. So depending on the cards played, player one here, which is you, would gain a score of two, four, zero or one half. 
</div></li><li data-sub-note-item="1-4"><div class="lab-step">Collect a set of two game cards. 
</div></li><li data-sub-note-item="1-5"><div class="lab-step">Take out the cards labeled hawk and dove and place them face down on the table or conceal them in your hands. 
</div></li><li data-sub-note-item="1-6"><div class="lab-step">When the instructor gives the signal show either of the two cards to the student sitting across from you. Try to strategize how to receive the highest benefit from every interaction on your own but do not communicate with your partner. 
</div></li><li data-sub-note-item="1-7"><div class="lab-step">In the Single Partner Recording Table (Table 2) provided by your instructor, note the cards played as well as the benefit you received for that round.
</div></li><li data-sub-note-item="1-8"><div class="lab-step">Repeat showing cards to the same partner and determining benefit on your instructor&#39;s signal for a total of 15 rounds.
</div></li><li data-sub-note-item="1-9"><div class="lab-step">At the end of those rounds, the student with the highest total benefit value was determined to be the &#8220;winner&#8221;, or the most fit.
</div></li><li data-sub-note-item="1-10"><div class="lab-step">As a class, determine the percentages of hawks and doves played as well as the average benefit received for each round, using this formula: Class average benefit=(sum total benefit)/(number of players)
</div></li><li data-sub-note-item="1-11"><div class="lab-step">For the second game, and then for each round of play, one row of students should rotate to their right, leaving all players with a new partner.
</div></li><li data-sub-note-item="1-12"><div class="lab-step">Continue playing for 15 rounds or until students end up back in their starting positions, recording the data in the Multiple Partner Recording Table (Table 3) given to you by your instructor.
</div></li><li data-sub-note-item="1-13"><div class="lab-step">For each round, determine the percentages of hawks and doves played that round, as well as the average benefit received.
</div></li></ul></li><li data-note-item="2" data-parent-collapse="">Cheating and Cooperation Exercise
<button class="expand">Expand</button><ul class="lab-items"><li data-sub-note-item="2-1"><div class="lab-step">NOTE: In this activity, you will exchange benefits, represented by colored or similar items, with your classmates. In this simulation, a cheater is defined as a person who gives fewer benefits than they receive. During this activity, you will observe the frequency of cheating behavior over time. 
</div></li><li data-sub-note-item="2-2"><div class="lab-step">HYPOTHESES: The experimental hypothesis is that cheaters will receive fewer benefits over time and cooperators will continue to provide equal benefits to one another. The null hypothesis is that the frequency of cheaters and cooperators does not change over time. 
</div></li><li data-sub-note-item="2-3"><div class="lab-step">Each person should collect beads of one color to represent a tangible benefit, as well as one paper bag and a three-ring binder.
</div></li><li data-sub-note-item="2-4"><div class="lab-step">Get into a group of three so that all members have objects of the same color.
</div></li><li data-sub-note-item="2-5"><div class="lab-step">Now find another group with a different color to pair with. In each round, partners will exchange benefits without knowing what they are receiving from their partner and without discussion within their group.  NOTE: Benefits of a different color from your starting color are worth twice as much.
</div></li><li data-sub-note-item="2-6"><div class="lab-step">Turn face to face with a member from the other group while still being able to conceal the objects in your possession.  TIP:<strong> </strong>Setting up a screen using a notebook or sitting behind a desk can be helpful here.
</div></li><li data-sub-note-item="2-7"><div class="lab-step">When given a signal from the instructor, place zero to two benefits into the paper bag and pass it to your partner.
</div></li><li data-sub-note-item="2-8"><div class="lab-step">Simultaneously, receive the bag from your partner and then remove the benefits from the bag.
</div></li><li data-sub-note-item="2-9"><div class="lab-step">Make a note of how many benefits you gave away and how many you received in the Cheating and Cooperation Table (Table 4) provided by your instructor.
</div></li><li data-sub-note-item="2-10"><div class="lab-step">Turn to a new member from the other group and repeat the exchange.
</div></li><li data-sub-note-item="2-11"><div class="lab-step">Rotate through all of the members of the other group for as many rounds as the instructor determines - which will not be revealed until the game ends so as not to alter player behavior.
</div></li><li data-sub-note-item="2-12"><div class="lab-step">Once the game has ended, note for each round in the cheating and cooperation table whether you gave more than your partner, received more from them or if you gave the same amount.
</div></li><li data-sub-note-item="2-13"><div class="lab-step">Now total up all of the benefits in your possession, with benefits you&#39;ve received from the other group being worth twice as much.
</div></li><li data-sub-note-item="2-14"><div class="lab-step">For each round, the whole class should total up the number of cheaters - with cheaters defined as those who gave less than they received.
</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">For both the single partner hawk-dove game and the multiple partners hawk-dove game, use the whole class data to plot the percentages as two separate lines - one for the percentage of hawks, and one for the percentage of doves, for each of the 15 rounds.
</div></li><li data-sub-note-item="3-2"><div class="lab-step">Take note of whether the cards played changed as more rounds were played.
</div></li><li data-sub-note-item="3-3"><div class="lab-step">Manipulate the values for B and C to see if you can find the point at which values of B and C would render the hawk strategy no longer effective (this would be the point when there is no benefit to choosing the hawk strategy over always choosing the dove strategy).
</div></li><li data-sub-note-item="3-4"><div class="lab-step">As a class, use the data from the cheating and cooperation activity to make a graph showing the percentage of rounds each individual player cheated versus the amount of benefits they ended with.
</div></li><li data-sub-note-item="3-5"><div class="lab-step">Consider whether it is better to be a cheater or a cooperator in this scenario and discuss as a class what makes this scenario different from the hawk-dove game.
</div></li><li data-sub-note-item="3-6"><div class="lab-step">Now, use the class data to graph the percentage of cheaters against the number of rounds, and look for any trends over time.
</div></li><li data-sub-note-item="3-7"><div class="lab-step">Consider which type of strategy would most likely evolve in a setting where partners interact repeatedly.
</div></li><li data-sub-note-item="3-8"><div class="lab-step">Then, note which tactic might be beneficial for a species that relies on cooperation. 
</div></li><li data-sub-note-item="3-9"><div class="lab-step">Discuss as a class what would likely happen if you increased or decreased the relative value of the benefits you receive.
</div></li></ul></li></ol>

Group Behavior: Hawk-Dove Game and Trading Exercise - Procedure

Comportamiento grupal

The Hawk-Dove Game
ExpandNOTE: In this activity, you will play the hawk and dove game with a single partner and then with multiple partners to observe ...

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 los 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 ...

Genética

Animal Behavior

<ol class="note-items"><li data-note-item="1" data-parent-collapse="">Making Choice Chambers
<button class="expand">Expand</button><ul class="lab-items"><li data-sub-note-item="1-1"><div class="lab-step">Before beginning the experiments, use one of the Sharpies from the lab table to trace a line around the circumference of the bottom of both bottles. One line should be 1 cm from the base of the bottle and the other should be 1.5 cm from the base of the bottle.
</div></li><li data-sub-note-item="1-2"><div class="lab-step">Use a razor blade to make a small cut in each bottle at the mark. 
</div></li><li data-sub-note-item="1-3"><div class="lab-step">Then, insert the scissors and carefully cut along the line until the bottoms of the bottles are removed completely.
</div></li><li data-sub-note-item="1-4"><div class="lab-step">Fit one bottle bottom into the other.
</div></li><li data-sub-note-item="1-5"><div class="lab-step">Tape the bottles together to make a secure chamber.
</div></li><li data-sub-note-item="1-6"><div class="lab-step">Use the razor blade and scissors to poke a hole into the center of the chamber. 
</div></li><li data-sub-note-item="1-7"><div class="lab-step">Take a piece of tape and fold the edge onto itself to make a tab and place the tape over the hole.  Note: The tab helps with easy removal and reattachment of the tape to the chamber.
</div></li><li data-sub-note-item="1-8"><div class="lab-step">Use the Sharpie to label one side of the chamber A and the other side B.
</div></li><li data-sub-note-item="1-9"><div class="lab-step">Upon receiving the colony of <em>Drosophila</em>, perform a count to make sure there are 40 living individuals in the vial.
</div></li></ul></li><li data-note-item="2" data-parent-collapse="">Geotaxis
<button class="expand">Expand</button><ul class="lab-items"><li data-sub-note-item="2-1"><div class="lab-step">To perform a geotaxis assay, first attach a clean, empty bottle cap to each end of the choice chamber and turn the fly vial upside down onto its lid.
</div></li><li data-sub-note-item="2-2"><div class="lab-step">Open the tape flap in the center of the choice chamber.
</div></li><li data-sub-note-item="2-3"><div class="lab-step">Insert the funnel into the hole and tap all of the flies into the chamber. HYPOTHESES: The alternate hypothesis is that more flies will be moving against gravity, or up, regardless of the orientation of the choice chamber. Alternatively, if the flies are positively geotactic, then more flies will move down, or with gravity. The null hypothesis is that the flies will have no preference with regard to gravity and will move randomly within the chamber. 
</div></li><li data-sub-note-item="2-4"><div class="lab-step">Once the <em>Drosophila</em> are in the chamber, close the tape flap, rotate the chamber so that it stands vertically with side A on the bottom, and start the timer.
</div></li><li data-sub-note-item="2-5"><div class="lab-step">Immediately, count the number of <em>Drosophila</em> that are moving up and the number that are moving down. Record these data for time point 0 in the table. 
</div></li><li data-sub-note-item="2-6"><div class="lab-step">After 30 seconds, rotate the chamber so that side B is on the bottom and count the number of <em>Drosophila</em> that are moving up or down. Record these data for time point 30 in the table.
</div></li><li data-sub-note-item="2-7"><div class="lab-step">After 60 seconds, rotate the chamber so that side A is on the bottom again and record the number of moving flies for time point 60.  <a href="https://jove.unicartagenaproxy.elogim.com/CDNSource/lm/labs/15/Table_1_Animal_behavior.xlsx" style="display:block;margin:20px;" target="__blank">Click Here to download Table 1</a>
</div></li><li data-sub-note-item="2-8"><div class="lab-step">Hold the chamber over the fly vial and carefully open the cap to return all of the <em>Drosophila</em> back to their empty vial. 
</div></li></ul></li><li data-note-item="3" data-parent-collapse="">Phototaxis
<button class="expand">Expand</button><ul class="lab-items"><li data-sub-note-item="3-1"><div class="lab-step">To perform a phototaxis analysis, first cover side A of the choice chamber with tin foil, taking care that the foil reaches to the exact center between the two sides. 
</div></li><li data-sub-note-item="3-2"><div class="lab-step">Ensure the caps are secured on both ends of the chamber.  HYPOTHESES: In this exercise, the alternative hypothesis is that more flies will be found in the light side of the choice chamber, if the flues are attracted to light, or on the dark side if they are repelled by light. The null hypothesis is that <em>Drosophila</em> will have no preference with regard to either stimulus. Therefore, equal numbers of flies will be found on either side of the choice chamber.
</div></li><li data-sub-note-item="3-3"><div class="lab-step">Open the tape flap in the center of the choice chamber, insert the funnel into the hole, and tap all 40 flies into the chamber as previously described.
</div></li><li data-sub-note-item="3-4"><div class="lab-step">Set the timer to sound an alarm at 10 minutes.
</div></li><li data-sub-note-item="3-5"><div class="lab-step">When the alarm sounds, stop the timer, and count the number of flies in the open side B of the chamber.
</div></li><li data-sub-note-item="3-6"><div class="lab-step">Subtract that number from the total number of flies added to the choice chamber to calculate the number of flies in the covered side A.
</div></li><li data-sub-note-item="3-7"><div class="lab-step">Record these data in the table for time point 10.  <a href="https://jove.unicartagenaproxy.elogim.com/CDNSource/lm/labs/15/Table_2_Animal_behavior.xlsx" style="display:block;margin:20px;" target="__blank">Click Here to download Table 2</a>
</div></li><li data-sub-note-item="3-8"><div class="lab-step">Carefully, return all of the flies back to their empty vial.
</div></li></ul></li><li data-note-item="4" data-parent-collapse="">Chemotaxis
<button class="expand">Expand</button><ul class="lab-items"><li data-sub-note-item="4-1"><div class="lab-step">HYPOTHESES: In this exercise, the alternative hypothesis is that more flies will be found on the side of the chamber with the apple cider vinegar if the flies are attracted to the odor. Or, if the flies are repelled by the vinegar, then more of them will be found on the side of the chamber presenting the odorless water. The null hypothesis is that <em>Drosophila</em> will have no preference with regard to either stimulus and will move randomly in the chamber.  Use an individual disposable plastic pipette to dispense two drops of the apple cider vinegar onto one cotton ball for side A of the chamber. 
</div></li><li data-sub-note-item="4-2"><div class="lab-step">Use an individual disposable plastic pipette to dispense two drops of the distilled water onto a cotton ball for side B of the chamber. 
</div></li><li data-sub-note-item="4-3"><div class="lab-step">Fold two pieces of tape over themselves to create individual pieces of double-sided tape and use the tape to attach each cotton ball to the inside of the single bottle cap.
</div></li><li data-sub-note-item="4-4"><div class="lab-step">Screw the bottle caps with the cotton balls onto their corresponding sides of the chamber.
</div></li><li data-sub-note-item="4-5"><div class="lab-step">Transfer all of the flies into the chamber and set the timer to sound an alarm after 10 minutes. 
</div></li><li data-sub-note-item="4-6"><div class="lab-step">When the alarm sounds, stop the timer and record the number of flies in each side of the chamber. <a href="https://jove.unicartagenaproxy.elogim.com/CDNSource/lm/labs/15/Table_3_Animal_behavior.xlsx" style="display:block;margin:20px;" target="__blank">Click Here to download Table 3</a>
</div></li><li data-sub-note-item="4-7"><div class="lab-step">At the end of the final experiment, funnel the flies into a morgue tube containing 70% ethanol. 
</div></li><li data-sub-note-item="4-8"><div class="lab-step">Dismantle the choice chambers for recycling.  NOTE: Since all of the chemicals used in these experiments are lab safe, the liquids can be disposed of down the sink, the chambers rinsed, and the solids can be thrown away in the trash. 
</div></li></ul></li><li data-note-item="5" data-parent-collapse="">Results
<button class="expand">Expand</button><ul class="lab-items"><li data-sub-note-item="5-1"><div class="lab-step">Gather all of the data collected for the geotaxis, phototaxis, and chemotaxis experiments.
</div></li><li data-sub-note-item="5-2"><div class="lab-step">Plot these values as bar graphs for different conditions and different time points.
</div></li><li data-sub-note-item="5-3"><div class="lab-step">Use these data to draw inferences about the behavior of <em>drosophila melanogaster</em>. 
</div></li></ul></li></ol>

Animal Behavior: Studying Taxis Behavior of Drosophila - Procedure

Comportamiento Animal

Making Choice Chambers
ExpandBefore beginning the experiments, use one of the Sharpies from the lab table to trace a line around the circumference of the ...

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://jove.unicartagenaproxy.elogim.com/CDNSource/lm/labs/25/Table_1_Measuring_biodiversity.xlsx" style="display:block;margin:20px;" target="__blank">Click Here to download Table 1</a>
</div></li><li data-sub-note-item="1-6"><div class="lab-step">Once all of the species within a quadrat have been identified, pick up your hoop and move on to the next knot along the same transect.
</div></li><li data-sub-note-item="1-7"><div class="lab-step">Continue surveying and moving to the next knot until you have completed the entire rope transect.
</div></li><li data-sub-note-item="1-8"><div class="lab-step">Once you have completed the rope, move to a transect in a different habitat, and begin identifying the different species within each quadrat along the new transect, until each group has identified plants along three lengths of rope, one in each habitat, for a total of 15 quadrats surveyed.
</div></li><li data-sub-note-item="1-9"><div class="lab-step">When all of the species data have been collected, carry the transects and quadrats back to the classroom.
</div></li></ul></li><li data-note-item="2" data-parent-collapse="">Species-Accumulation Curves and Richness/Evenness 
<button class="expand">Expand</button><ul class="lab-items"><li data-sub-note-item="2-1"><div class="lab-step">In the next activity, you will be calculating and comparing the species richness and evenness in two distinct populations, represented here by two opaque bags containing beads of various colors. For this exercise, start by taking one paper bag of beads per group.
</div></li><li data-sub-note-item="2-2"><div class="lab-step">Have one person reach into the bag and randomly pull out three beads without looking.
</div></li><li data-sub-note-item="2-3"><div class="lab-step">Write down the bead colors, as well as the total number of colors pulled from the bag in the table. <a href="https://jove.unicartagenaproxy.elogim.com/CDNSource/lm/labs/25/Table_2_Measuring_biodiversity.xlsx" style="display:block;margin:20px;" target="__blank">Click Here to download Table 2</a>HYPOTHESES: In this activity, the alternative hypothesis states that species evenness in the two communities will be different, while the richness will be the same. The null hypothesis might be that species richness and evenness will be the same in both communities.
</div></li><li data-sub-note-item="2-4"><div class="lab-step">Place the beads back in the bag after their colors are recorded.
</div></li><li data-sub-note-item="2-5"><div class="lab-step">Then, shake the bag and have the next person in the group draw another set of three beads.
</div></li><li data-sub-note-item="2-6"><div class="lab-step">Record the colors and numbers of beads that they pulled in the table.
</div></li><li data-sub-note-item="2-7"><div class="lab-step">Record the colors in the second row and the cumulative number of colors drawn in the last column.
</div></li><li data-sub-note-item="2-8"><div class="lab-step">Continue to take turns drawing three beads from the bag until 20 samples have been drawn.
</div></li><li data-sub-note-item="2-9"><div class="lab-step">Next, switch bags with a group with a bag from the other community, and repeat the exercise of drawing 20 samples, recording the color data in the appropriate community column of the table.
</div></li><li data-sub-note-item="2-10"><div class="lab-step">When all of the data have been recorded, return the beads to the bag and return the bags to your instructor.
</div></li></ul></li><li data-note-item="3" data-parent-collapse="">Results
<button class="expand">Expand</button><ul class="lab-items"><li data-sub-note-item="3-1"><div class="lab-step">To calculate the alpha diversity of the transect and quadrat data collected by your group, use the data your data recorder entered for each species and enter it into your diversity table. <a href="https://jove.unicartagenaproxy.elogim.com/CDNSource/lm/labs/25/Table_3_Measuring_biodiversity.xlsx" style="display:block;margin:20px;" target="__blank">Click Here to download Table 3</a>
</div></li><li data-sub-note-item="3-2"><div class="lab-step">Then, fill out the alpha diversity for each habitat into the appropriate column in the table.
</div></li><li data-sub-note-item="3-3"><div class="lab-step">Use the species data from your quadrat and transect table and enter the beta diversity data into the appropriate data cells within the diversity table. NOTE: The beta diversity is the number of species that are unique between two habitats. In this video, there are three species in common between the two sites, but a total of six unique species, giving a beta diversity of six between site A and B.
</div></li><li data-sub-note-item="3-4"><div class="lab-step">To calculate the gamma diversity for your activity, use the data from your quadrat and transect table and enter the gamma diversity data into the appropriate data cell. NOTE: The gamma diversity is the number of species in all of the habitats within a single study. It is not the sum of all of the alpha diversities, because species that are common between habitats should be counted only once.
</div></li><li data-sub-note-item="3-5"><div class="lab-step">To construct species accumulation curves for both of the bead sampling communities, use your bead color selection data to plot the species by sampling effort. That is the number of total colors by sample in the graph for each bead bag community.
</div></li><li data-sub-note-item="3-6"><div class="lab-step">Then, estimate the asymptote of the plotted data by appearance. Predict the number of species you would expect to find in each community. Note any differences in appearance between the two curves. If it was easier to estimate the number of species in one of the communities, make a hypothesis to explain this observation.
</div></li><li data-sub-note-item="3-7"><div class="lab-step">To construct rank abundance curves for both of the bead sampling communities, use the color bead data to fill in the table for both community A and community B, listing the colors that were identified during the sampling in the color column, and adding the total number of beads of each color that was sampled in the abundance column. <a href="https://jove.unicartagenaproxy.elogim.com/CDNSource/lm/labs/25/Table_4_Measuring_biodiversity.xlsx" style="display:block;margin:20px;" target="__blank">Click Here to download Table 4</a>
</div></li><li data-sub-note-item="3-8"><div class="lab-step">Now, assign a rank to each color in the rank column, designating the color of the highest abundance the highest rank, rank one, and the color with the lowest abundance the lowest rank, rank 10. If there are multiple colors with the same number, just assign them consecutive ranks.
</div></li><li data-sub-note-item="3-9"><div class="lab-step">Plot individual rank abundance curves for the number of individuals by species ranked for the most to least common in both community A and community B. Compare the steepness of the two rank curves. Compare the richness and evenness between the two communities. Identify the community that you consider more biodiverse. 
</div></li></ul></li></ol>

Medición de la biodiversidad

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

Scientific Method

<ol class="note-items"><li data-note-item="1" data-parent-collapse="">Observation and Background Research
<button class="expand">Expand</button><ul class="lab-items"><li data-sub-note-item="1-1"><div class="lab-step">NOTE: The first two steps in the scientific process involve studying and observing natural phenomena of interest.
</div></li><li data-sub-note-item="1-2"><div class="lab-step">Observe the leaves and branches of the individual plant species. Each species comes from a different environment.
</div></li><li data-sub-note-item="1-3"><div class="lab-step">Compare each branch to one another and record your observations about any qualitative similarities or differences. <a target="__blank" style="display:block;padding:20px" href="https://jove.unicartagenaproxy.elogim.com/CDNSource/lm/labs/1/Table_A_Lab_1.xlsx">Click Here to download Table A</a>
</div></li><li data-sub-note-item="1-4"><div class="lab-step">Use leaf clippers to cut the leaves from the branches.
</div></li><li data-sub-note-item="1-5"><div class="lab-step">Make additional qualitative observations about the leaves from each species.
</div></li></ul></li><li data-note-item="2" data-parent-collapse="">Hypothesis Formulation
<button class="expand">Expand</button><ul class="lab-items"><li data-sub-note-item="2-1"><div class="lab-step">Think of some general questions that you might have after observing the different plant species.
</div></li><li data-sub-note-item="2-2"><div class="lab-step">Using these general questions, create a null and an experimental hypothesis.
</div></li></ul></li><li data-note-item="3" data-parent-collapse="">Experimentation and Data Collection
<button class="expand">Expand</button><ul class="lab-items"><li data-sub-note-item="3-1"><div class="lab-step">Weigh and record the mass of each leaf. <a target="__blank" style="padding:20px;display:block;" href="https://jove.unicartagenaproxy.elogim.com/CDNSource/lm/labs/1/Table_B_Lab_1.xlsx">Click Here to download Table B</a>
</div></li><li data-sub-note-item="3-2"><div class="lab-step">Measure and record the length of each leaf from the petiole to the tip in the table.
</div></li><li data-sub-note-item="3-3"><div class="lab-step">Next, carefully trace each leaf outline onto a blank piece of computer paper.
</div></li><li data-sub-note-item="3-4"><div class="lab-step">Then, cut out and weigh and 4 x 4 cm square of the same paper, weigh the cutout tracing, and record these data in the table.
</div></li></ul></li><li data-note-item="4" data-parent-collapse="">Data Analysis and Conclusion
<button class="expand">Expand</button><ul class="lab-items"><li data-sub-note-item="4-1"><div class="lab-step">To calculate the leaf surface area, divide the surface area of the 4 x 4 cm paper square by the mass of the paper square, and multiply the resulting number by the mass of the leaf tracing.
</div></li><li data-sub-note-item="4-2"><div class="lab-step">Next, create a bar graph with your five plant species listed on the x-axis, and the surface area in square centimeters indicated on the y-axis.
</div></li><li data-sub-note-item="4-3"><div class="lab-step">Draw a trend line through the graph if a relationship is evident.
</div></li><li data-sub-note-item="4-4"><div class="lab-step">Using your qualitative observations and quantitative measurements, draw conclusions about the data you collected.
</div></li></ul></li></ol>