<p class='jove_content'>We thank Dr. Joseph R. Fetcho for his input throughout the development of the procedure.We would like to acknowledge funding from the US National Institutes of Health (R01 EB002019 to C.B.S and DP OD006411 to Joseph R. Fetcho) and the National Science and Research Council of Canada (to M.J.F.) for financial support.</p>

<p class="jove_content"> NOTA: La cura e la manipolazione sperimentale di tutti i topi in questo documento è stato approvato dal Institutional Animal Care and Use Committee Cornell University. </p><p class="jove_title"> 1. Impostazione di Chirurgia </p><ol><li> Sterilizzare tutti gli strumenti necessari tramite autoclave o procedure di sterilizzazione approvate. Come minimo, includere un bisturi, forbici, forbici Vanna, pinze, cacciavite del gioielliere, tamponi di cotone, un insieme di divaricatori, l&#39;impianto <strong>(Figura 1A)</strong> e il suo sistema di erogazione <strong>(Figura 1D, E).</strong> </li><li> Pulire tutte le superfici, comprese le manopole stereoscopio, con il 70% di etanolo e / o disinfettanti idonei. </li><li> Creare un campo sterile sovrapponendo il stereotax personalizzato <strong>(Figura 1D)</strong> e da banco circostante con teli sterili. Una piastra elettrica (preferibilmente retroazione controllata) deve essere posto sulla parte superiore della stereotax e sotto i teli sterili. </li></ol><p class="jove_title"> 2. Preparazioneil mouse di Chirurgia </p><ol><li> Anestetizzare un topo sotto 5% isoflurano / ossigeno in una camera di induzione fino tasso di respirazione rallenta a circa 1 respiro / sec. </li><li> Trasferimento del mouse in un&#39;area intermedia di distanza dal campo sterile, con un cono di continuare l&#39;esposizione isoflurano. Ridurre isoflurano al 2%. Applicare collirio per evitare l&#39;essiccazione della cornea. </li><li> Somministrare glicopirrolato (un farmaco anticolinergico) a 0.05 mg / 100 g del mouse intramuscolare nelle arti posteriori utilizzando 29 - 30 aghi G e 3/10 siringa cc, fornendo circa il 50% del volume di iniezione per ogni arto. <br /> NOTA: Glicopirrolato serve per evitare l&#39;accumulo di liquido nei polmoni, mentre il mouse è sotto anestesia. </li><li> Utilizzando tagliatori elettrici, radere una porzione della faccia dorsale del mouse, lunga circa 3 cm di 2 cm di larghezza e centrate sull&#39;arco toracica. Pulire tutti i capelli tagliati ben lontano dalla zona rasata. </li><li> Amministrare i seguenti iniezioni sottocute e distale al rasatapatch con 29 - 30 aghi G e 3/10 siringhe cc: 1 ml / 100 g di mouse 5% di glucosio in soluzione salina (per l&#39;idratazione), 5 mg / kg ketoprofene mouse (un FANS per analgesia), 0,2 mg / kg di mouse desametasone ( uno steroide antinfiammatori per ridurre l&#39;infiammazione). </li><li> Utilizzando tamponi di cotone, effettuare tre lavaggi alternati di iodio e il 70% di etanolo, in attesa 1 min tra lavaggi. </li><li> Somministrare 100 ml di 0,125% bupivacaina (un blocco nervoso periferico) per via sottocutanea con 29-30 aghi G e 3/10 cc siringa al centro della patch rasata per ridurre il dolore al sito di incisione. </li><li> Trasferimento del mouse al stereotax personalizzato <strong>(Figura 1D),</strong> inserire termometro rettale, e attendere circa 10 minuti per bupivicaina abbia effetto. </li></ol><p class="jove_title"> 3. Esporre e pulire la Dorsale lamine 3 Adiacente Vertebra toracica </p><ol><li> Usando il bisturi, creare un piccolo (5 mm o meno) incisione sopra le vertebre di interesse <strong>(Figura 2A).</strong> </li><li&gt; Utilizzando sia pinze o inserendo la punta di forbici chirurgiche e diffondere le maniglie, aumentare la dimensione della apertura nella pelle per dissezione. </li><li> Usando un bisturi o forbici chirurgiche, dissociare delicatamente fascia connettivo tra la pelle e il muscolo sottostante. </li><li> Utilizzare divaricatori per trattenere la pelle, consentendo la più ampia area di lavoro possibile <strong>(Figura 2B).</strong> Evitare di aumentare il carico sul petto e quindi ostacolare la respirazione. </li><li> Utilizzando un paio di pinze, afferrare il rostrale-più vertebra che sarà esposto attraverso il muscolo sovrastante. Usando un bisturi, tagliare tessuti distanza su entrambi i lati del processo dorsali da ogni 3 vertebre <strong>(figura 2c).</strong> </li><li> Usando una combinazione di un bisturi, tamponi di cotone, e / o raschietto osso, rimuovere tutto il tessuto sovrastante dal lamine dorsale <strong>(Figura 2D).</strong> </li><li> Utilizzando le forbici chirurgiche, accuratamente tagliato via i tendini collegati alle vertebre su entrambi Lateral bordi della colonna vertebrale <strong>(Figura 2E).</strong> </li><li> Usando il bisturi, rimuovere delicatamente più tessuto dai bordi laterali della colonna vertebrale possibile al fine di fornire una superficie di bloccaggio pulita. </li><li> Sbrigliare delicatamente le vertebre di un tessuto molle rimanente con un raschietto osseo e / o tampone di cotone. Tagliare via qualsiasi tessuto incongrua altrove con le forbici chirurgiche <strong>(Figura 2F).</strong> <br /> NOTA: E &#39;essenziale sia per il bloccaggio e l&#39;esecuzione della laminectomia dorsale che le vertebre sono esposti in modo pulito. Un cauterizer può essere utile nel controllo successiva sanguinamento. </li><li> Spostare divaricatori per trattenere il tessuto circostante la colonna vertebrale in aggiunta alla pelle <strong>(Figura 2F).</strong> </li><li> Fissare le barre laterali di impianto per le punte sui posti di consegna e caricare questi in post-titolari (come in <strong>Figura 1E).</strong> </li><li> Bloccare il 3 vertebre utilizzando le barre laterali, applicando una pressione sufficiente per tenere securely <strong>(Figura 2G, H).</strong> Utilizzare pinze per contribuire a garantire un livello di bloccaggio del 3 vertebre. Si noti che diversi tentativi possono essere necessarie. Assicurarsi che le barre laterali sono sia di livello e parallelamente prima di eseguire la laminectomia. </li><li> Pulire e asciugare la superficie dorsale delle vertebre bloccato utilizzando tamponi di cotone. </li></ol><p class="jove_title"> 4. Eseguire una dorsale laminectomia </p><ol><li> Inserire le punte delle forbici Vännäs nello spazio epidurale della vertebra bloccato mediale e stringere le impugnature leggermente; se l&#39;osso è asciutto, dovrebbe crepa lungo la lunghezza della lamina dorsale. Ripetere questa procedura sul lato controlaterale per rilasciare la lamina. </li><li> Presa delicatamente la lamina sciolto dal processo dorsale con una pinza, tagliare attentamente tutta tessuto connettivo alle estremità rostrale e caudale della lamina e toglierlo. </li><li> Usando 0,9% soluzione salina e / o artificiale cerebrale liquido spinale (ACSF), lavare via ogni overlyi sangueng il midollo spinale. Usare tamponi di cotone per assorbire il liquido in eccesso. </li><li> Utilizzando forbici Vännäs, ritagliare con attenzione i bordi laterali della spina dorsale alle barre laterali della protesi. Anche in questo caso, lavare via e controllare il sanguinamento con tamponi di cotone e saline / ACSF. </li><li> Nel caso in cui una porzione di periostio è staccato e sovrasta il midollo spinale, rimuovere delicatamente questo tessuto utilizzando tamponi di cotone e / o un 29 - ago G 30. Fare attenzione a non danneggiare il midollo spinale in questa parte della procedura. Non confondere il periostio sciolto con la dura madre strettamente avvolto. </li><li> Cautela, sigillare i bordi rimanenti esposte della osso con uno o entrambi acrilico e cianoacrilato dentale <strong>(Figura 2I).</strong> Fare attenzione a non consentire le colle di scorrere sul midollo spinale esposto. </li></ol><p class="jove_title"> 5. Seal la Camera </p><ol><li> Posizionare la piastra superiore con attenzione in modo che i 4 slot si allineano con i 4 fori sulle barre laterali e l&#39;apertura sovrappone lo spin espostoal cavo <strong>(Figura 1B</strong> e <strong>2J).</strong> </li><li> Utilizzando un cacciavite il gioielliere, avviare accuratamente la prima vite, stabilizzando l&#39;albero del cacciavite con un altro paio di pinze. Non serrare la vite in questa fase, ma inserirla in modo che i primi fili vengono impegnati. Ripetere questa procedura per gli altri 3 viti. <br /> NOTA: È utile usare titanio o altre pinze non magnetizzabili per posizionare le viti in questa fase. </li><li> Con tutti i 4 viti in atto, serrare ogni vite turno dalla stessa quantità di piccoli incrementi fino viti sono saldamente fissate <strong>(Figura 2K).</strong> Si noti che non è insolito per la piastra superiore di flettersi durante questo processo. Fare attenzione a non stringere le viti troppo, come la testa della vite può rompere e l&#39;eccessiva pressione al ribasso sloggiare il morsetto e / o provocare lesioni del midollo spinale. </li><li> Utilizzare un elastomero siliconico per riempire lo spazio tra il midollo spinale e la finestra di vetro. Perché does non producono acido acetico durante la polimerizzazione, utilizzare KwikSil marca elastomero e punte di miscelazione. Applicare una porzione abbondante di silicone al midollo spinale esposto. Fare attenzione a sbarazzarsi di qualsiasi silicone contenente bolle d&#39;aria dalla punta di miscelazione prima dell&#39;applicazione al midollo spinale. </li><li> Lavorando in fretta, premere delicatamente a 5 mm # 0 coprioggetto nel riquadro nella piastra superiore. Applicare una pressione sufficiente a comprimere gradualmente l&#39;elastomero liquido circondare il midollo spinale, ma non provocare compressione midollare <strong>(Figura 2L).</strong> Tempo di presa per l&#39;elastomero è di circa 90 sec, in modo da assicurarsi il vetrino viene applicato rapidamente. </li><li> In caso di tenuta fallito, attendere che l&#39;elastomero ha fissato, rimuovere delicatamente dall&#39;impianto con una pinza, e ripetere 5,4-5,5. </li><li> Coprire i bordi di elastomero agglomerato con dentale acrilico / cianoacrilato e rispettare l&#39;osso circostante il più possibile per evitare l&#39;elastomero sposti sulla superficie del midollo spinale. </li><li> Rimuovere divaricatori e tirare la pelle attorno al bordo dell&#39;impianto. Utilizzando cianoacrilato, aderire alla pelle ai bordi dell&#39;impianto <strong>(Figura 2M, N).</strong> </li><li> Inserisci # 0-80 - 1/4 &quot;set viti nelle ali della piastra superiore <strong>(Figura 1C</strong> e 2 <strong>O).</strong> </li><li> Utilizzando acrilico dentale, sigillare eventuali lacune nelle fessure delle viti della piastra superiore <strong>(Figura 2P).</strong> </li></ol><p class="jove_title"> 6. Cura postoperatoria </p><ol><li> Rimuovere animali da anestesia e posto in una grande gabbia pulita. Assicurarsi che la gabbia è sufficientemente grande che la camera non intoppo su nulla (come il dip a V per una bottiglia di acqua e / o cibo) nel corso del normale locomozione tutta la gabbia. <br /> NOTA: Una gabbia di ratto standard è l&#39;ideale. </li><li> Posizionare la gabbia su una superficie riscaldata mentre l&#39;animale recupera. Non restituire il mouse per la compagnia di altri animali fino a quando non ha pienamente recuperato. </li><li> Controllare Beha animaliVior entro 1 - 2 ore postoperation per i segni di dolore o angoscia e l&#39;integrità dell&#39;impianto. <br /> NOTA: In condizioni normali, l&#39;animale deve essere in grado di deambulare con modeste perturbazioni al movimento. </li><li> Continuare a controllare animale ogni 8-12 ore per il primo 72 ore, misurare il peso e il comportamento di monitoraggio. Verificare che gli animali sono attivi e mobili durante la notte. Cibo umido in forma di terra Chow mescolato con acqua o gel nutrizionali, pellets Chow, e l&#39;acqua potabile dovrebbe essere fornita a livello del suolo. Ulteriori idratazione con iniezioni sottocutanee o intraperiotoneal dovrebbe essere fornita, se necessario. </li><li> Somministrare 5 mg / kg del mouse ketoprofene e 0,2 mg / kg per via sottocutanea del mouse desametasone a 24 e 48 punti di tempo ora, dopo l&#39;intervento. Inoltre, ,005-,010 mg / 100 g mouse deve essere somministrato ogni 8 ore per iniezione sottocutanea per 72 hr. </li></ol><p class="jove_title"> 7. <em>In Vivo</em> Imaging </p><ol><li> Anestetizzare un topo sotto5% isoflurano / ossigeno in una camera di induzione frequenza respiratoria rallenta fino a circa 1 respiro / sec. </li><li> Trasferire il mouse per il stereotax personalizzato <strong>(Figura 1D)</strong> con feedback controllato piastra elettrica e inserire il termometro rettale. Ridurre isoflurano al 2%. </li><li> Somministrare glicopirrolato 0,05 mg / 100 g del mouse intramuscolare nelle arti posteriori utilizzando 29 - 30 aghi G e 3/10 siringa cc, fornendo circa il 50% del volume di iniezione per ogni arto. </li><li> Somministrare 1 ml / 100 g del mouse 5% di glucosio (per idratazione sottocutanea) in soluzione salina volta per ora. </li><li> Inserire i titolari camera filettati nei post-titolari utilizzati per la chirurgia <strong>(Figura 1F).</strong> Regolare l&#39;altezza e girare i posti sulle viti nella piastra superiore. </li><li> Con entrambi i supporti attaccati, elevare i titolari nel post-titolari fino al torace può espandersi liberamente su ispirazione. <br /> NOTA: Sia il fermo attaccamento dei titolari e la libera espansione della significan pettoTLY ridurre artefatti di movimento a causa di respirazione. </li><li> Eseguire l&#39;imaging come desiderato. </li><li> Quando l&#39;imaging è completo, posti più bassi, twist off titolari camera, rimuovere termometro rettale, e posto il mouse su una superficie riscaldata per recuperare. Non restituire il mouse per la compagnia di altri animali fino a quando non ha pienamente recuperato. </li></ol>

<ol>
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V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two-photon+laser+scanning+microscopy+imaging+of+intact+spinal+cord+and+cerebral+cortex+reveals+requirement+for+CXCR6+and+neuroinflammation+in+immune+cell+infiltration+of+cortical+injury+sites.">Two-photon laser scanning microscopy imaging of intact spinal cord and cerebral cortex reveals requirement for CXCR6 and neuroinflammation in immune cell infiltration of cortical injury sites.</a> <em style='font-style: italic'>Journal of Immunological Methods</em>. <b>352</b> (1-2), 89-100 (2010).</li><li>Dibaj, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+Vivo+imaging+reveals+distinct+inflammatory+activity+of+CNS+microglia+versus+PNS+macrophages+in+a+mouse+model+for+ALS.">In Vivo imaging reveals distinct inflammatory activity of CNS microglia versus PNS macrophages in a mouse model for ALS.</a> <em style='font-style: italic'>Plos One</em>. <b>6</b> (3), 17910 (2011).</li><li>Davalos, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fibrinogen-induced+perivascular+microglial+clustering+is+required+for+the+development+of+axonal+damage+in+neuroinflammation.">Fibrinogen-induced perivascular microglial clustering is required for the development of axonal damage in neuroinflammation.</a> <em style='font-style: italic'>Nature Communications</em>. <b>3</b>, 1227 (2012).</li><li>Dibaj, P., Steffens, H., Zsch&#252;ntzsch, J., Kirchhoff, F., Schomburg, E. D., Neusch, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+imaging+reveals+rapid+morphological+reactions+of+astrocytes+towards+focal+lesions+in+an+ALS+mouse+model.">In vivo imaging reveals rapid morphological reactions of astrocytes towards focal lesions in an ALS mouse model.</a> <em style='font-style: italic'>Neuroscience Letters</em>. <b>497</b> (2), 148-151 (2011).</li><li>Bareyre, F. M., Garzorz, N., Lang, C., Misgeld, T., Buning, H., Kerschensteiner, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+imaging+reveals+a+phase-specific+role+of+STAT3+during+central+and+peripheral+nervous+system+axon+regeneration.">In vivo imaging reveals a phase-specific role of STAT3 during central and peripheral nervous system axon regeneration.</a> <em style='font-style: italic'>Proceedings of the National Academy of Sciences</em>. <b>108</b> (15), 6282-6287 (2011).</li><li>Nikic, I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+reversible+form+of+axon+damage+in+experimental+autoimmune+encephalomyelitis+and+multiple+sclerosis.">A reversible form of axon damage in experimental autoimmune encephalomyelitis and multiple sclerosis.</a> <em style='font-style: italic'>Nature Medicine</em>. <b>17</b> (4), 495-499 (2011).</li><li>Misgeld, T., Nikic, I., Kerschensteiner, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+imaging+of+single+axons+in+the+mouse+spinal+cord.">In vivo imaging of single axons in the mouse spinal cord.</a> <em style='font-style: italic'>Nature Protocols</em>. <b>2</b> (2), 263-268 (2007).</li><li>Davalos, D., Akassoglou, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+Imaging+of+the+Mouse+Spinal+Cord+Using+Two-photon+Microscopy.">In vivo Imaging of the Mouse Spinal Cord Using Two-photon Microscopy.</a> <em style='font-style: italic'>J. Vis. Exp</em>. <b>59</b>, 2760 (2012).</li><li>Farrar, M. J., Bernstein, I. M., Schlafer, D. H., Cleland, T. A., Fetcho, J. R., Schaffer, C. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chronic+in+vivo+imaging+in+the+mouse+spinal+cord+using+an+implanted+chamber.">Chronic in vivo imaging in the mouse spinal cord using an implanted chamber.</a> <em style='font-style: italic'>Nature. Methods</em>. <b>9</b> (3), 297-302 (2012).</li><li>Fenrich, K. K., Weber, P., Hocine, M., Zalc, M., Rougon, G., Debarbieux, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long-term+in+vivo+imaging+of+normal+and+pathological+mouse+spinal+cord+with+subcellular+resolution+using+implanted+glass+windows.">Long-term in vivo imaging of normal and pathological mouse spinal cord with subcellular resolution using implanted glass windows.</a> <em style='font-style: italic'>The Journal of Physiology</em>. <b>590</b> (16), 3665-3675 (2012).</li><li>Figley, S. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Spinal+Cord+Window+Chamber+Model+for+In+Vivo+Longitudinal+Multimodal+Optical+and+Acoustic+Imaging+in+a+Murine+Model.">A Spinal Cord Window Chamber Model for In Vivo Longitudinal Multimodal Optical and Acoustic Imaging in a Murine Model.</a> <em style='font-style: italic'>Plos One</em>. <b>8</b> (3), 58081 (2013).</li><li>Fenrich, K. K., Weber, P., Rougon, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Implanting+Glass+Spinal+Cord+Windows+in+Adult+Mice+with+Experimental+Autoimmune+Encephalomyelitis.">Implanting Glass Spinal Cord Windows in Adult Mice with Experimental Autoimmune Encephalomyelitis.</a> <em style='font-style: italic'>Journal of Visualized Experiments</em>. <b>82</b>, 50826 (2013).</li><li>Romanelli, E., Sorbara, C. D., cacute, I. N., Dagkalis, A., Misgeld, T., Kerschensteiner, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular,+subcellular+and+functional+in+vivo+labeling+of+the+spinal+cord+using+vital+dyes.">Cellular, subcellular and functional in vivo labeling of the spinal cord using vital dyes.</a> <em style='font-style: italic'>Nature Protocols</em>. <b>8</b> (3), 481-490 (2013).</li></ol>

<p class='jove_content'>The authors having nothing to disclose, and no competing financial interests.</p>

<p class="jove_content"> La tecnica dimostrata qui consente ripetute, temporizzata, imaging <em>in vivo</em> del mouse dorsali SC ad molte settimane post operativamente senza la necessità di ulteriori interventi chirurgici. Questa procedura rappresenta un notevole miglioramento rispetto studi di imaging repeat-chirurgia o su approcci istologici portmortem, dove le informazioni sulle dinamiche cellulari è perso. Abbiamo già <sup>30</sup> hanno dimostrato il valore di questa tecnica per lo studio SCI patologia <em>in vivo.</em> </p><p class="jove_content"> La portata massima longitudinale di imaging è stato determinato dalla crescita di un denso tessuto fibroso sulla superficie dorsale del SC. Nel tempo, questa crescita ha portato alla perdita di contrasto e risoluzione. Questa crescita è stata vista anche in preparazioni chirurgiche alternative <sup>31.</sup> Aneddoticamente, abbiamo osservato che questa crescita può essere minimizzato lavando accuratamente la superficie dorsale SC per rimuovere i prodotti del sangue, tenuta la superficie dil&#39;osso taglio con cianoacrilato, bordi della camera di tenuta bene con elastomero di silicone, e minimizzando lo spazio interstiziale tra la SC dorsale e il vetro di copertura. </p><p class="jove_content"> Recentemente, un altro approccio simile alla nostra con una camera di policarbonato è stata dimostrata <sup>32.</sup> L&#39;uso di policarbonato è vantaggioso poiché è compatibile con X-ray e modalità di imaging acustiche, che non è il caso per i nostri parti in acciaio inossidabile. Tuttavia, con la tecnologia ormai onnipresente di stampanti 3D, parti della camera possono essere fabbricati da un&#39;ampia varietà di materiali per soddisfare esigenze specifiche. Abbiamo recentemente stampato tutte le nostre parti in un chiaro fotopolimero. </p><p class="jove_content"> Uno svantaggio fondamentale di una camera chiusa è l&#39;incapacità di amministrare ripetuti dosaggi di farmaci o coloranti esogeni adatti per l&#39;imaging SC <sup>34.</sup> Tuttavia, capitalizzando sulla stabilità meccanica del nostro sistema attuale, abbiamo già usato la nostra camera presente per il successoly ancorare un catetere intratecale collegato ad una porta di iniezione sottocutanea impiantato, che ha permesso per la consegna della droga in diversi punti temporali anche in un sistema di camera chiusa (inedito). Inoltre, a causa della natura modulare della piastra superiore, versioni future di questa preparazione includeranno una camera richiudibile per consentire l&#39;applicazione ripetuta di entrambi gli agenti terapeutici e etichette fluorescenti. È anche possibile prevedere piastre superiori con supporti per la registrazione di elettrodi, inserti ottici e porte per drug delivery. Tali integrazioni sarebbe probabilmente più difficile attuare utilizzando il sistema più minimalista Fenrich <em>et al.</em> <sup>31.</sup> In conclusione, la nostra camera offre una piattaforma di gateway su cui diversi esperimenti possono essere basati. </p>

<p class="jove_content"> Time-lapse <em>in</em> microscopia <em>vivo</em> negli organismi intatti permette la visualizzazione diretta di processi biologici complessi che sono inaccessibili ad un&#39;analisi tradizionale unico punto-tempo, come immunoistochimica. In particolare, la microscopia multi-fotone (MPM) <sup>1</sup> permette l&#39;imaging in dispersione tessuti, come la neocorteccia roditore, dove è stato raggiunto immagini fino a <sup>2,3</sup> e in eccesso su <sup>4</sup> 1 mm. Quando combinato con preparazioni chirurgiche <sup>5-7</sup> in cui un singolo procedimento consente accesso ottico al cervello per settimane o mesi, questi approcci microscopia sono stati utilizzati per studiare processi dinamici nel cervello in stati sani e malati <sup>8-11.</sup> Inoltre, sono stati sviluppati protocolli <sup>12,13</sup> che prevedono l&#39;imaging <em>in vivo</em> in sveglio <em>(cioè,</em> non anestetizzati) animali, consentendo cellulari risoluzione imaging funzionale durante saggi comportamentali. Questi protocolli sono stati utilizzati per comparisons di correlata attività neuronale <sup>14,</sup> astrociti calcio segnalazione <sup>15</sup> in animali anestetizzati e sveglio, l&#39;identificazione di specifici task-clusters neuronali <sup>16,</sup> e la capacità dei neuroni di discriminare posizione dell&#39;oggetto su baffo stimolazione <sup>17.</sup> </p><p class="jove_content"> Dato il potenziale di questo approccio per chiarire i meccanismi sani e patologici, time-lapse imaging <em>in vivo</em> è stato applicato al midollo spinale del mouse (SC), consentendo l&#39;identificazione di degenerazione assonale acuta (AAD) come meccanismo malattia <sup>18.</sup> Studi successivi effetti di lesioni periferiche su gangli spinali (DRG) assone rigenerazione <sup>19,</sup> il ruolo dei vasi sanguigni in assonale rigenerazione <sup>20,</sup> chemiotassi gliali in risposta al danno <sup>21,</sup> la migrazione delle cellule T in encefalomielite autoimmune sperimentale (EAE) <sup>22,</sup> l&#39;attività indagati di microglia <sup>23,24</sup> e astrociti <sup>25</sup> in risposta a amyotrophsclerosi ic laterale (ALS), il ruolo di STAT-3 in sprouting assonale dopo la lesione SC (SCI) <sup>26,</sup> e un meccanismo di perdita di assoni e recupero in EAE <sup>27.</sup> Nonostante il successo di questi approcci, tutti questi studi sono stati limitati a una singola sessione di imaging, limitando così gli studi di dinamica a breve termine, o altrimenti richiesto ripetute aperture chirurgiche dell&#39;animale in ogni punto di tempo di imaging, limitando il numero di punti di tempo accessibili e aumentando la probabilità di artefatti sperimentali confondenti. Protocolli per questi interventi sono stati pubblicati in precedenza <sup>28,29.</sup> </p><p class="jove_content"> Recentemente, abbiamo pubblicato una tecnica <sup>30</sup> per l&#39;impianto di una camera spinale cronica che ha permesso time-lapse MPM immagini nel topo SC su più settimane senza la necessità di interventi di ripetizione. Brevemente, questa preparazione chirurgica inclusa eseguendo laminectomia nella colonna toracica inferiore e l&#39;impianto di una camera di quattro parti. La camera inclusotre parti in acciaio inox custom-lavorati che serrate le vertebre che circonda la laminectomia, ed un vetrino di vetro posto sopra la SC e fissato con elastomero siliconico. Questa tecnica ha permesso per l&#39;imaging di routine ad più di 5 settimane dall&#39;intervento in stati sani e feriti, senza la necessità di interventi di ripetizione. Il numero di punti di tempo di imaging è stato limitato soltanto dalla frequenza alla quale l&#39;animale può tollerare induzione anestetica. Imaging durata era limitata dalla crescita di una fitta, tessuto fibroso sulla superficie del SC. Inoltre, abbiamo verificato che l&#39;impianto chirurgico non ha avuto effetto a lungo termine sulla funzione motoria. </p><p class="jove_content"> Dato che la nostra prima pubblicazione, approcci alternativi che consentano anche di imaging a lungo termine nella SC sono stati descritti altrove <sup>31-33.</sup> Questo protocollo dimostra la nostra procedura per impiantare la camera di midollo abbiamo sviluppato. </p>

<table><tbody><tr><td>Vannas scissors</td><td>Fine Science Tools</td><td>15000-04</td><td></td></tr><tr><td>forceps, scissors, scalpel, etc.</td><td>Fine Science Tools</td><td>multiple&amp;nbsp;</td><td></td></tr><tr><td>retractor kit -magnetic fixator</td><td>Fine Science Tools</td><td>18200-02</td><td></td></tr><tr><td>retractor kit -retractor</td><td>Fine Science Tools</td><td>18200-09</td><td>other retractors may also be used</td></tr><tr><td>retractor kit -elastomer</td><td>Fine Science Tools</td><td>18200-07</td><td></td></tr><tr><td>feedback controlled heating blanket</td><td>CWE Inc.</td><td>Model: TC-1000 Mouse&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; Part No. 08-13000</td><td></td></tr><tr><td>stereotax</td><td></td><td>see Farrar et al, Nat. Meth., 9, 297, 2012, Supplement (online)</td><td></td></tr><tr><td>spinal chamber</td><td></td><td>see Farrar et al, Nat. Meth., 9, 297, 2012, Supplement (online)</td><td></td></tr><tr><td>spinal chamber holders</td><td></td><td>see Farrar et al, Nat. Meth., 9, 297, 2012, Supplement (online)</td><td></td></tr><tr><td>Cyanoacrylate glue</td><td>Loctite</td><td>Loctite 495</td><td>multiple suppliers</td></tr><tr><td>Teets Cold Cure Coral Powder (dental acrylic powder)</td><td>Teets</td><td>Mfg. Part: 8101</td><td>multiple suppliers</td></tr><tr><td>Teets Cold Cure Liquid (dental acrylic liquid)</td><td>Teets</td><td>Mfg. Part: 8501</td><td>multiple suppliers</td></tr><tr><td>Glycopyrrolate</td><td>MWI Veterinary Supply</td><td>MWI SKU: 29706 (Baxter 1001901602)</td><td></td></tr><tr><td>D-(+)-Glucose</td><td>Sigma-Aldrich</td><td>G5767</td><td></td></tr><tr><td>Bupivacaine</td><td>MWI Veterinary Supply</td><td>MWI SKU: 029841 (Hospira 116301)</td><td></td></tr><tr><td>Ketoprofen</td><td>MWI Veterinary Supply</td><td>MWI SKU: 002800 (Pfizer 2193)</td><td></td></tr><tr><td>Dexamethasone</td><td>MWI Veterinary Supply</td><td>MWI SKU: 501012 (VetOne 1DEX032)</td><td></td></tr><tr><td>KwikSil Elastomer</td><td>World Precision Inc.</td><td>KWIK-SIL</td><td></td></tr><tr><td>KwikSil Mixing Tips</td><td>World Precision Inc.</td><td>KWIKMIX</td><td></td></tr></tbody></table>

a procedure for implanting a spinal chamber for longitudinal <em>in vivo </em>imaging of the mouse spinal cord

<p class='jove_content'>Studies in the mammalian neocortex have enabled unprecedented resolution of cortical structure, activity, and response to neurodegenerative insults by repeated, time-lapse <em>in vivo</em> imaging in live rodents. These studies were made possible by straightforward surgical procedures, which enabled optical access for a prolonged period of time without repeat surgical procedures. In contrast, analogous studies of the spinal cord have been previously limited to only a few imaging sessions, each of which required an invasive surgery. As previously described, we have developed a spinal chamber that enables continuous optical access for upwards of 8 weeks, preserves mechanical stability of the spinal column, is easily stabilized externally during imaging, and requires only a single surgery. Here, the design of the spinal chamber with its associated surgical implements is reviewed and the surgical procedure is demonstrated in detail. Briefly, this video will demonstrate the preparation of the surgical area and mouse for surgery, exposure of the spinal vertebra and appropriate tissue debridement, the delivery of the implant and vertebral clamping, the completion of the chamber, the removal of the delivery system, sealing of the skin, and finally, post-operative care. The procedure for chronic <em>in vivo</em> imaging using nonlinear microscopy will also be demonstrated. Finally, outcomes, limitations, typical variability, and a guide for troubleshooting are discussed.</p>

<p class="jove_content"> Seguendo la procedura sopra, ogni fase della chirurgia dovrebbe imitare i risultati per ogni fase della chirurgia delineato nella <strong>Figura 2.</strong> Particolare cura deve essere presa quando si applica l&#39;elastomero siliconico per eliminare le bolle d&#39;aria sotto la lente, o almeno garantire che siano situato alla periferia dell&#39;area di interesse <strong>(Figura 2L).</strong> Per un chirurgo esperto, le complicanze chirurgiche che richiedono l&#39;eutanasia sia durante l&#39;intervento chirurgico o entro il primo 24 ore dopo l&#39;intervento chirurgico si verificano in circa il 20% delle operazioni. Queste complicazioni più frequentemente derivano dall&#39;eccessiva emorragie tessuti molli o mancata fissare saldamente la camera alla colonna vertebrale. </p><p class="jove_content"> In camere impiantati con successo, la finestra rimane tipicamente otticamente trasparente per più settimane <strong>(Figura 3).</strong> Nonostante gli sforzi, una opaca, fibrosa, la crescita neoplastica costituisce spesso entro pochi giorni, con conseguente parziale obscuring della finestra <strong>(Figura 3C</strong> - <strong>F,</strong> nero linea tratteggiata). Strutture fluorescenti sono difficili da vedere al microscopio 2PEF sotto questo tessuto fibroso. Troviamo una distribuzione bimodale dei risultati, con circa il 50% delle finestre impiantati con successo rimanendo chiaro per più di 5 settimane dopo l&#39;intervento, e circa il 50% diventando offuscato entro 1 - 3 settimane. Per le finestre che rimangono chiare, la maggior parte dei posti all&#39;interno della finestra di esperienza visiva solo un modesto grado di neovascolarizzazione in o al di sopra della dura <strong>(Figura 3D</strong> - <strong>F,</strong> asterischi verde), che non ostacola l&#39;imaging 2PEF. Il nostro studio precedente <sup>30</sup> ha mostrato un aumento di microglia, ma non la densità astrocyte sotto la finestra e vertebre adiacenti, indicando lieve infiammazione analogo a quello visto in finestra preparati cranici <sup>5.</sup> </p><p class="jove_content"> Nei topi transgenici che esprimono la proteina fluorescente gialla (YFP) in un sottogruppo di dorsale radice Ganglia (DRG) neuroni (linea YFPH; Jackson Labs), gli assoni erano chiaramente distinguibili per settimane dopo l&#39;intervento <strong>(Figura 4A</strong> e b), e fino a quattro mesi in un animale (dati non riportati). I vasi sanguigni <strong>(Figura 4A</strong> e <strong>B)</strong> sono stati resi visibili dal retro-orbitale iniezione fluorescente Texas Red etichettato destrano nel plasma sanguigno. Microglia sono stati visualizzati in topi che esprimono GFP in un CX3CR1 topo knock-in (CX3CR1-GFP; Jackson Labs) attraversato la linea YFPH <strong>(Figura 4C).</strong> Contrasto e risoluzione deteriorata modestamente nel tempo <strong>(Figura 4C)</strong> a causa della formazione di una proliferazione fibrosa <sup>30</sup> precedentemente descritto. Abbiamo regolarmente ripreso per 3 ore in una singola sessione, senza mortalità e come spesso come ogni 12 ore. La durata della sessione e frequenza è limitata dalla tolleranza dell&#39;animale per l&#39;induzione di anestesia, in concomitanza con le considerazioni etiche accompagnano dolore e disagio per animali da laboratorio. </p> &lt;<html> p class = &quot;jove_content&quot; fo: keep-together.within-page = &quot;always&quot;&gt; <img alt="Figura 1" src="/files/ftp_upload/52196/52196fig1highres.jpg" /><br /> <strong>Figura 1. Una suite chirurgica personalizzata facilita consegna camera durante l&#39;impianto e stabilizza la camera durante sessioni di imaging successive.</strong> Due barre laterali e una piastra superiore <strong>(A)</strong> sono montate <strong>(B)</strong> utilizzando piccole viti nella camera spinale <strong>(C).</strong> Un tavolo operatorio con posti estensibili <strong>(D)</strong> consente per la consegna ed il bloccaggio delle barre laterali per le vertebre con spine di tenuta <strong>(E).</strong> Sessioni di imaging successive invece utilizzano i messaggi filettati internamente per collegare alla camera tramite le viti di fermo sulle ali della protesi <strong>(F).</strong> Panel b di questa figura è stata modificata da Farrar <em>et al., Nature Methods,</em> 2012 <sup>30</sup> con il permesso di Nature Publishing Group.96 / 52196fig1large.jpg &quot;target =&quot; _ blank &quot;&gt; Clicca qui per vedere una versione più grande di questa figura. </p><p class="jove_content" fo:keep-together.within-page="always"><img alt="Figura 2" src="/files/ftp_upload/52196/52196fig2highres.jpg" /><br /> <strong>Figura 2. Un laminectomia dorsale è eseguito e una camera di imaging cronica è impiantato per fornire accesso ottico al midollo spinale.</strong> La procedura è descritta nel montaggio. Innanzitutto, la pelle (pannelli <strong>A, B;</strong> sezioni protocollo 3.1- 3.4 -) e muscoli <strong>(C;</strong> 3,5) vengono retratti per esporre tre vertebre sottostante. Il tessuto sovrastante questi tre vertebre viene rimossa <strong>(D, E, F;</strong> 3,6-3,10) ei processi laterali sono raschiati pulite prima di essere fissati su entrambi i lati <strong>(G, H,</strong> 3.12). Attenzione, la lamina dorsale della vertebra centrale è rimosso ei bordi dell&#39;osso sigillato <strong>(I</strong> </strong&gt;; 4. 6). Una piastra superiore viene applicata <strong>(J,</strong> 5.1) e le viti fissata in posizione <strong>(K;</strong> 5.3) per fissare la camera prima di sigillare la camera con elastomero di silicone e vetro di copertura <strong>(L;</strong> 5.5). Dopo che le spine di consegna sono rimossi <strong>(M;</strong> 5.8), la pelle è incollato ai lati della camera <strong>(M, N,</strong> 5,8) e le viti sono inserite nelle ali <strong>(O;</strong> 5.9). Infine, gli slot delle viti sono sigillati con materiale acrilico dentale <strong>(P;</strong> 5.10). E il mouse è posto in una gabbia pulita per recuperare <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/52196/52196fig2large.jpg" target="_blank">Cliccate qui per vedere una versione più grande di questa figura.</a> </p><p class="jove_content" fo:keep-together.within-page="always"><img alt="Figura 3" src="/files/ftp_upload/52196/52196fig3highres.jpg" /></p><p class="jove_content"> <strong>Figura 3. Una camera spinale cronica rimane ov otticamente trasparente</strong>er più settimane. Immagini luce bianca del midollo spinale, che vanno da alcuni minuti <strong>(A)</strong> a tre settimane dopo l&#39;intervento <strong>(F).</strong> Punti di riferimento vascolari sono identificabili in tutti i tempi (frecce gialle). Piccolo cambiamento è visto in un giorno dopo l&#39;intervento chirurgico <strong>(B).</strong> Dense proliferazione fibrosa <strong>(C</strong> - <strong>F,</strong> delimitata da linea tratteggiata in basso a destra) è presente fin da tre giorni e risultati di offuscamento parziale l&#39;area di esposizione. Neovascolarizzazione Mild <strong>(D, E, F,</strong> asterischi verdi) è presente, ma non oscura la vascolarizzazione originale in luce bianca o di imaging 2PEF. <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/52196/52196fig3large.jpg" target="_blank">Cliccate qui per vedere una versione più grande di questa figura.</a> </p><p class="jove_content" fo:keep-together.within-page="always"><img alt="Figura 4" src="/files/ftp_upload/52196/52196fig4highres.jpg" /></p><pclass = &quot;jove_content&quot;&gt; <strong>Figura 4. Una camera spinale cronica consente ripetuto l&#39;imaging multi-photon senza la necessità di interventi di ripetizione.</strong> immagini ripetuta di topi transgenici esprimenti YFP in un sottoinsieme di assoni DRG (verdi) nelle funiculi dorsali del midollo spinale e vascolare (rosso) etichettati da intravascolare iniettabile colorante <strong>(A, B).</strong> L&#39;attività di microglia (malva) può anche essere seguito nel tempo Thy1-YFP / CX3CR1-GFP doppio topi transgenici <strong>(C).</strong> Mentre assoni e microglia rimangono visibili, il contrasto e la risoluzione declino modestamente nel corso del tempo <strong>(C).</strong> <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/52196/52196fig4large.jpg" target="_blank">Cliccate qui per vedere una versione più grande di questa figura.</a> </p>

Watch this Scientific Journal Video about A Procedure for Implanting a Spinal Chamber for Longitudinal In Vivo Imaging of the Mouse Spinal Cord at JoVE.com

A Procedure for Implanting a Spinal Chamber for Longitudinal In Vivo Imaging of the Mouse Spinal Cord

<p class="jove_content"> In questo video, si descrive un procedimento per impiantare una camera di imaging ottico cronica nel midollo spinale dorsale di un topo vivo. La camera, procedura chirurgica, e di imaging cronica sono rivisti e dimostrati. </p>

Una procedura di impianto del Camera spinale per longitudinale<em&gt; In Vivo</em&gt; Imaging del cavo del mouse spinale

Studies in the mammalian neocortex have enabled unprecedented resolution of cortical structure, activity, and response to neurodegenerative insults by ...

a-procedure-for-implanting-spinal-chamber-for-longitudinal-vivo

Universidad de Cartagena UDEC

Research

JoVE Journal

Neuroscience

A Procedure for Implanting a Spinal Chamber for Longitudinal <em>In Vivo </em>Imaging of the Mouse Spinal Cord

14.1K Views.  Cornell University.  In questo video, si descrive un procedimento per impiantare una camera di imaging ottico cronica nel midollo spinale dorsale di un topo vivo. La camera, procedura chirurgica, e di imaging cronica sono rivisti e dimostrati. 

Video: A Procedure for Implanting a Spinal Chamber for Longitudinal In Vivo Imaging of the Mouse Spinal Cord

Evolutionary Relationships

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

Evolutionary Relationships: Using BLAST to Test Evolutionary Hypotheses - Procedure

Relazioni evolutive

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

Didattica

JoVE Lab Manual

Biologia

Evoluzione

Lab Manual Sub Articles

Species Distribution and Biogeography

<ol class="note-items"><li data-note-item="1" data-parent-collapse="">Simulating Colonization and Extinction on Islands
<button class="expand">Expand</button><ul class="lab-items"><li data-sub-note-item="1-1"><div class="lab-step">Form groups of three. One member of the group will simulate colonization by throwing balls into the groups of cups representing the islands. Another will simulate extinction by rolling dice to randomly remove balls from the cups. The third group member will record the data.
</div></li><li data-sub-note-item="1-2"><div class="lab-step">Take time to familiarize yourself with the four island stations. NOTE: There are two large islands with 12 cups and two small islands with 6 cups. Two of the stations will have tape lines that are 2 m away from the cups. These are the far islands. The other two stations will have tape lines that are 1 m away from the cups. These are the near islands. Thus, the stations are far large and near large and far small and near small. HYPOTHESES: In this experiment, one alternate hypothesis might be that the larger islands with more cups will support a greater number of species than the smaller islands. The null hypothesis might be that all islands will support this same number of species regardless of size. Another alternate hypothesis might be that islands closer to the mainland will have a higher colonization rate than islands farther from the mainland. The null hypothesis might be that all islands will have the same colonization rate regardless of their distance from the mainland. Additionally, in all trials we may hypothesize that the colonization rate will be expected to decrease as more species inhabit an island whereas the extinction rate will increase as more species are found on the island. The null hypothesis might be that the colonization rate and extinction rates are expected to stay the same across all numbers of species on the island.
</div></li><li data-sub-note-item="1-3"><div class="lab-step">First, choose one of the four stations as a starting place.
</div></li><li data-sub-note-item="1-4"><div class="lab-step">From the line of tape at this location, the colonization simulator must throw the ball to the island and try to get the ball into a cup. NOTE: One round consists of five throws, each throw representing a colonization attempt. If the ball lands in a cup that does not already have a ball in it, this is a successful colonization attempt and the ball should be left in the cup.
</div></li><li data-sub-note-item="1-5"><div class="lab-step">The data collector should place a tally on the colonizers box for the round in the recording sheet found at the station. <a href="https://jove.unicartagenaproxy.elogim.com/CDNSource/lm/labs/29/Tables_1-4_Island_biogeography.xlsx" style="display:block;margin:20px;" target="_blank">Click Here to download Tables 1-4</a>
</div></li><li data-sub-note-item="1-6"><div class="lab-step">If the ball does not land in a cup, retrieve it. This is an unsuccessful colonization attempt.
</div></li><li data-sub-note-item="1-7"><div class="lab-step">If a ball lands in a cup that already has a ball in it, retrieve it. This is also an unsuccessful colonization attempt.
</div></li><li data-sub-note-item="1-8"><div class="lab-step">Add the number of species at the beginning of the round with the tally of colonizers.
</div></li><li data-sub-note-item="1-9"><div class="lab-step">The data collector should record this number in the column entitled &#8220;Number of species after colonization&#8221; in the recording sheet found at the station.
</div></li><li data-sub-note-item="1-10"><div class="lab-step">The extinction simulator should now roll a die. If the trial is occurring at a large island site, choose one of the two colored dice at random with eyes closed.
</div></li><li data-sub-note-item="1-11"><div class="lab-step">Remove the ball if there is one present from the cup that is of the same color as the die and labeled with the number rolled by that die.
</div></li><li data-sub-note-item="1-12"><div class="lab-step">The data collector should count the removal as an extinction in the station recording table. NOTE: In a trial at the small island station, follow the same procedure except that it is only necessary to match the number rolled by the die to the cup. If there is no ball in the cup, no extinction is recorded. For both the large and small islands, repeat the extinction roll for a total of two rolls if there are 3 - 8 species present on the island and three rolls if there are 9 - 12 species on the island. If there are fewer than three species on the island, do not roll the die again.
</div></li><li data-sub-note-item="1-13"><div class="lab-step">This is the end of one round. After this, count the total number of species currently in the cups at this station and then tally this number into the &#8220;Number of species at beginning of round&#8221; column on the station recording sheet for the next round.
</div></li><li data-sub-note-item="1-14"><div class="lab-step">Groups should switch stations after five rounds at one table to control for the throwing ability of the colonization simulators. Repeat the trials and rotation through the stations until 20 rounds have been recorded in total for all groups combined at all four stations.
</div></li><li data-sub-note-item="1-15"><div class="lab-step">Count the tally marks for the number of species at the beginning of the round, colonizers, total number of species after colonization, and extinctions for each round at each island station.
</div></li><li data-sub-note-item="1-16"><div class="lab-step">Look at your data recording sheet. How many times were there zero species at the beginning of a round? How many times was there only one species at the beginning of a round? Repeat this calculation for 0 through 12 species present at the beginning of a round.
</div></li><li data-sub-note-item="1-17"><div class="lab-step">Then, tally these values in the corresponding rows of the &#8220;Rounds with this many species&#8221; column in the appropriate colonization rate calculation table. <a href="https://jove.unicartagenaproxy.elogim.com/CDNSource/lm/labs/29/Tables_5-8_Island_biogeography.xlsx" style="display:block;margin:20px;" target="_blank">Click Here to download Tables 5-8</a>
</div></li><li data-sub-note-item="1-18"><div class="lab-step">In the colonization attempts column, multiply these values by five to represent the total number of Ping-Pong balls thrown.
</div></li><li data-sub-note-item="1-19"><div class="lab-step">Look at your data recording sheet once more and calculate the total number of colonizers for each value in the number of species at beginning of round column.
</div></li><li data-sub-note-item="1-20"><div class="lab-step">Record these values in the &#8220;Successful colonization attempts&#8221; column of the appropriate colonization rate calculation table.
</div></li><li data-sub-note-item="1-21"><div class="lab-step">Divide the successful colonization attempts by the colonization attempts for each initial species value and record this in the &#8220;Colonization rate&#8221; column of the appropriate colonization rate calculation table.
</div></li><li data-sub-note-item="1-22"><div class="lab-step">Repeat the process for determining the colonization rate for all of the island trials. 
</div></li><li data-sub-note-item="1-23"><div class="lab-step">To calculate the extinction rate for each of the trials, return to the data recording sheet for the near large island trial. Count the number of times that the number of species after colonization matched each number from 1 to 12, as previously described.
</div></li><li data-sub-note-item="1-24"><div class="lab-step">Record these values in the &#8220;Rounds with this many species&#8221; column in the appropriate extinction rate calculation table. <a href="https://jove.unicartagenaproxy.elogim.com/CDNSource/lm/labs/29/Tables_9-12_Island_biogeography.xlsx" style="display:block;margin:20px;" target="_blank">Click Here to download Tables 9-12</a>
</div></li><li data-sub-note-item="1-25"><div class="lab-step">To calculate extinction attempts, multiply the value in the &#8220;Rounds with this many species&#8221; cells by 1 if the species after colonization value is 0 &#8211; 2, by 2 if the value is 3 &#8211; 8, and by 3 if the value is 9 or above. NOTE: These values correspond with the number of times the die was rolled during the extinction simulation.
</div></li><li data-sub-note-item="1-26"><div class="lab-step">Then, using the data contained in the data recording sheet, count the number of extinctions that occurred for each value in the &#8220;Number of species after colonization&#8221; column.
</div></li><li data-sub-note-item="1-27"><div class="lab-step">Record these values in the &#8220;Extinctions&#8221; column of the appropriate extinction rate calculation table.
</div></li><li data-sub-note-item="1-28"><div class="lab-step">Record these values in the &#8220;Extinctions&#8221; column of the appropriate extinction rate calculation table.
</div></li><li data-sub-note-item="1-29"><div class="lab-step">Repeat the process for calculating the extinction rate for all of the island trials. 
</div></li><li data-sub-note-item="1-30"><div class="lab-step">After this, plot the colonization and extinction rates versus the number of species for each of the island stations.
</div></li><li data-sub-note-item="1-31"><div class="lab-step">Draw the best-fit lines for the colonization and extinction rates in each of the four graphs.
</div></li><li data-sub-note-item="1-32"><div class="lab-step">Compare the colonization rate between the near and far islands. Determine the distance that had a greater dynamic equilibrium of species and develop a hypothesis to explain your observation. 
</div></li><li data-sub-note-item="1-33"><div class="lab-step">Next, compare the extinction rates between the large and small islands. Determine the size that had a greater dynamic equilibrium number of species.
</div></li><li data-sub-note-item="1-34"><div class="lab-step">Observe the slopes of the colonization rates in the plots. Determine if they are positive or negative. 
</div></li><li data-sub-note-item="1-35"><div class="lab-step">Observe the slopes of the extinction rates in the plots. Determine if they are positive or negative. 
</div></li></ul></li></ol>

Distribuzione delle specie e biogeografia

Simulating Colonization and Extinction on Islands
ExpandForm groups of three. One member of the group will simulate colonization by throwing balls into ...

Ecologia

Population Growth

<ol class="note-items"><li data-note-item="1" data-parent-collapse="">Exponential Population Growth Model
<button class="expand">Expand</button><ul class="lab-items"><li data-sub-note-item="1-1"><div class="lab-step">NOTE: In these experiments you will use computer software to simulate different types of growth models. HYPOTHESES: The experimental hypothesis might be that if we increase either R, the reproductive rate or the initial population size, NT, that this will increase the population size after 10 generations in an exponential growth model compared to unmanipulated control populations. The null hypothesis may be that populations of different R and initial population size will have the same population size as control groups after 10 generations.
</div></li><li data-sub-note-item="1-2"><div class="lab-step">To build an exponential population growth model, first open a new workbook in Excel.
</div></li><li data-sub-note-item="1-3"><div class="lab-step">Save the file to a secure location and name it &#8220;exponential growth&#8221;.
</div></li><li data-sub-note-item="1-4"><div class="lab-step">Label cell A1 &#8220;generation&#8221;, and cell B1 &#8220;population size&#8221;.
</div></li><li data-sub-note-item="1-5"><div class="lab-step">Type the number zero into cell A2. This row will represent the initial generation.
</div></li><li data-sub-note-item="1-6"><div class="lab-step">In cell A3 type this formula: = 1 + A2
</div></li><li data-sub-note-item="1-7"><div class="lab-step">Now hit enter.
</div></li><li data-sub-note-item="1-8"><div class="lab-step">Click on cell A3 and drag the small box in the bottom right corner to cell A12 for 10 generations.
</div></li><li data-sub-note-item="1-9"><div class="lab-step">Type the number two in cell B2. This will represent the initial population size in the model.
</div></li><li data-sub-note-item="1-10"><div class="lab-step">Label cell E1 &#8220;growth rate&#8221;, and in cell E2 type the number 0.05. This value represents the proportion of new offspring produced by each individual for each generation, or R in the exponential growth model.
</div></li><li data-sub-note-item="1-11"><div class="lab-step">NOTE: In the classic exponential growth equation, T is the generation, and NT is the number of organisms in the current generation. NT+1 will be is the number of individuals in the next generation.
</div></li><li data-sub-note-item="1-12"><div class="lab-step">Enter the exponential growth equation in cell B3 as the formula shown here: = B2 + (1 + E2) * B2
</div></li><li data-sub-note-item="1-13"><div class="lab-step">Now, hit enter.
</div></li><li data-sub-note-item="1-14"><div class="lab-step">Click cell B3 and drag the small square in the bottom right corner down to cell B12 to simulate 10 generations of exponential growth.
</div></li><li data-sub-note-item="1-15"><div class="lab-step">To observe the results, select all data, along with the headers, in columns A and B.
</div></li><li data-sub-note-item="1-16"><div class="lab-step">Click &#8220;insert&#8221; and select &#8220;scatter plot with smooth lines and markers&#8221; from the toolbar. A scatter plot should appear in the sheet. TIP: If it does not, check to see if a new sheet was created in the workbook.
</div></li><li data-sub-note-item="1-17"><div class="lab-step">Label the Y axis &#8220;population size&#8221; and the X axis &#8220;generation&#8221;. Title the chart &#8220;exponential growth of a population&#8221;.
</div></li><li data-sub-note-item="1-18"><div class="lab-step">Observe how the population increases in a classic exponential growth pattern.
</div></li><li data-sub-note-item="1-19"><div class="lab-step">Next, create Table 1 labeling the &#8220;growth rate R&#8221; as the header of column A, and &#8220;population size after 10 generations&#8221; as the second column.
</div></li><li data-sub-note-item="1-20"><div class="lab-step">Record the population size at generation 10 for the growth rate of 0.05, the rate we just simulated.
</div></li><li data-sub-note-item="1-21"><div class="lab-step">Now, set the growth rate in cell E2 to 0.1, and repeat the steps to generate a new scatter plot.
</div></li><li data-sub-note-item="1-22"><div class="lab-step">Record the population size at generation 10.
</div></li><li data-sub-note-item="1-23"><div class="lab-step">Continue to alter the growth rate using the values in table one, noticing how the Y axis changes to accommodate the increasing values.
</div></li><li data-sub-note-item="1-24"><div class="lab-step">After completing Table 1, set the growth rate back to 0.05 and change the initial population size in cell B2 to 100 individuals.
</div></li><li data-sub-note-item="1-25"><div class="lab-step">Record the population size at generation 10 in a new table, Table 2.
</div></li><li data-sub-note-item="1-26"><div class="lab-step">Proceed to change the initial population size using the values in Table 2. Once the table is complete, save the Excel sheet and close it.
</div></li></ul></li><li data-note-item="2" data-parent-collapse="">Logistic Population Growth Model
<button class="expand">Expand</button><ul class="lab-items"><li data-sub-note-item="2-1"><div class="lab-step">HYPOTHESES: In a logistic growth model, the experimental hypothesis could be that populations will approach and meet the carrying capacity, and any populations with sizes greater than the carrying capacity will decrease in the subsequent generation. The null hypothesis might be that population growth will not follow a logistic S-shaped curve in the logistic models, and that generations with population sizes greater than the carrying capacity will continue to increase or stay the same in the next generation.
</div></li><li data-sub-note-item="2-2"><div class="lab-step">To build a logistic population growth model, open a new spreadsheet in Excel.
</div></li><li data-sub-note-item="2-3"><div class="lab-step">Save the file as &#8220;logistic growth&#8221; in the same folder as before.
</div></li><li data-sub-note-item="2-4"><div class="lab-step">Then label cell A1 &#8220;generation&#8221; and B1 &#8220;population size&#8221;.
</div></li><li data-sub-note-item="2-5"><div class="lab-step">Type the number zero in cell A2. This row will represent the initial generation.
</div></li><li data-sub-note-item="2-6"><div class="lab-step">In cell A3 type the formula: = 1 + A2
</div></li><li data-sub-note-item="2-7"><div class="lab-step">Now, hit enter.
</div></li><li data-sub-note-item="2-8"><div class="lab-step">Now, click on cell A3 and drag the small box in the bottom right corner to cell A12 to number up to the 10th generation.
</div></li><li data-sub-note-item="2-9"><div class="lab-step">Type the number two in cell B2 to represent the initial population size.
</div></li><li data-sub-note-item="2-10"><div class="lab-step">Label cell E1 as &#8220;max growth rate&#8221; and cell F1 as &#8220;carrying capacity&#8221;.
</div></li><li data-sub-note-item="2-11"><div class="lab-step">Finally, set the max growth rate in cell E2 to 0.05. This value will represent the maximum growth rate the population may achieve &#8211; &#8220;R max&#8221; in the discrete logistic equation. NOTE: In the classic logistic growth equation the term K represents carrying capacity. As the population size of the current generation or NT, approaches the carrying capacity, the growth of the population begins to slow.
</div></li><li data-sub-note-item="2-12"><div class="lab-step">Now set the carrying capacity in cell F2 to 50.
</div></li><li data-sub-note-item="2-13"><div class="lab-step">Enter the logistic growth equation in cell B3 as the formula below: = B2 + ((1 + E$2) * B2) * ((F$2 - B2) /F$2)
</div></li><li data-sub-note-item="2-14"><div class="lab-step">Now hit enter.
</div></li><li data-sub-note-item="2-15"><div class="lab-step">Drag the formula down to cell B12 to add it to all 10 generations.
</div></li><li data-sub-note-item="2-16"><div class="lab-step">To observe the results of this exercise, highlight all data and headers in columns A and B, then click &#8220;insert&#8221; and select &#8220;scatter plot with smooth lines and markers&#8221;.
</div></li><li data-sub-note-item="2-17"><div class="lab-step">Label the Y axis population size and the X axis generation.
</div></li><li data-sub-note-item="2-18"><div class="lab-step">Title the chart as logistic growth of a population.
</div></li><li data-sub-note-item="2-19"><div class="lab-step">Observe the growth of the population as it nears carrying capacity and record the generation that the population meets or exceeds carrying capacity in a new table.
</div></li><li data-sub-note-item="2-20"><div class="lab-step">Now set the max growth rate in cell E2 to 1.
</div></li><li data-sub-note-item="2-21"><div class="lab-step">Notice how the population responds when it rises over carrying capacity.
</div></li><li data-sub-note-item="2-22"><div class="lab-step">Continue to set the max growth rate to the values in Table 3 and record the generation at which the population rises two or above carrying capacity.
</div></li><li data-sub-note-item="2-23"><div class="lab-step">Now set the max growth rate back to 0.05 and set the carrying capacity to 1,000.
</div></li><li data-sub-note-item="2-24"><div class="lab-step">Notice how the graph changes as the growth approaches the carrying capacity.
</div></li><li data-sub-note-item="2-25"><div class="lab-step">Drag the formulas in A12 and B12 down to A14 and B14.
</div></li><li data-sub-note-item="2-26"><div class="lab-step">Adjust the plot to include these new data points to show when the population reaches carrying capacity.
</div></li><li data-sub-note-item="2-27"><div class="lab-step">Save the Excel sheet and close it.
</div></li></ul></li><li data-note-item="3" data-parent-collapse="">Predator-Prey Model
<button class="expand">Expand</button><ul class="lab-items"><li data-sub-note-item="3-1"><div class="lab-step">Hypotheses: In our third simulation of predator-prey logistics, the experimental hypothesis could be that predator populations will increase as prey populations increase and that the two populations will experience growth and decline dependent on the population of the other species. The null hypothesis may be that the two populations will show patterns of growth unrelated to the other species&#39; population size.
</div></li><li data-sub-note-item="3-2"><div class="lab-step">To build a predator-prey model, open a new spreadsheet in Excel.
</div></li><li data-sub-note-item="3-3"><div class="lab-step">Save the file in the same folder as the other two models and name it &#8220;predator-prey model&#8221;.
</div></li><li data-sub-note-item="3-4"><div class="lab-step">Label cell A1 &#8220;time&#8221;, cell B1 &#8220;prey&#8221; and cell C1 &#8220;predator&#8221;.
</div></li><li data-sub-note-item="3-5"><div class="lab-step">In cell A2 type zero and hit enter.
</div></li><li data-sub-note-item="3-6"><div class="lab-step">In cell A3 type the formula below: = 1 + A2
</div></li><li data-sub-note-item="3-7"><div class="lab-step">Now hit enter.
</div></li><li data-sub-note-item="3-8"><div class="lab-step">Drag the formula down to cell A802 as the model will require many time points to visualize changes of both populations.
</div></li><li data-sub-note-item="3-9"><div class="lab-step">Now type 100 into cell B2 to represent the initial prey population and type 25 into cell C2 as the initial predator population.
</div></li><li data-sub-note-item="3-10"><div class="lab-step">Label cell E1 &#8220;prey growth rate&#8221;, cell F1 &#8220;consumption of prey by predator&#8221;, cell G1 &#8220;conversion efficiency&#8221; and cell H1 &#8220;starvation of predator&#8221;.
</div></li><li data-sub-note-item="3-11"><div class="lab-step">Type the number 0.02 in cell E2. This value represents the growth rate of the prey species or R in the prey population model. NOTE: Here the prey population is represented as VT and the predator population as CT. The value A is the per capita attack rate of the predator species on the prey. In the predator population model, variables are introduced for predators produced per prey consumed, F or conversion efficiency as well as for the starvation rate of the predators noted as Q.
</div></li><li data-sub-note-item="3-12"><div class="lab-step">Type 0.0005 in cell F2 for the attack rate in both the prey population and the predator population model.
</div></li><li data-sub-note-item="3-13"><div class="lab-step">Then type 0.8 in cell G2 to represent the conversion efficiency, and 0.05 in cell H2 for the starvation rate in the predator population equation.
</div></li><li data-sub-note-item="3-14"><div class="lab-step">Now type the prey growth equation in cell B3 as the formula below: =B2+(E$2*B2)-(F$2*C2*B2)
</div></li><li data-sub-note-item="3-15"><div class="lab-step">Hit enter.
</div></li><li data-sub-note-item="3-16"><div class="lab-step">Then type the prey growth equation in cell C3 as the displayed formula: =C2 + (F$2*G$2*B2*C2)-(H$2*C2)
</div></li><li data-sub-note-item="3-17"><div class="lab-step">Hit enter.
</div></li><li data-sub-note-item="3-18"><div class="lab-step">Hold down the shift keys and click to select both cells B3 and C3.
</div></li><li data-sub-note-item="3-19"><div class="lab-step">Click the small box at the bottom right corner of cell B3 and drag the two formulas down to row 802 to simulate the predator and prey populations over 800 generations.
</div></li><li data-sub-note-item="3-20"><div class="lab-step">To visualize the data, select all of the data and headers in columns A, B and C.
</div></li><li data-sub-note-item="3-21"><div class="lab-step">Click &#8220;insert&#8221; and select &#8220;scatter plot with smoothed lines&#8221;.
</div></li><li data-sub-note-item="3-22"><div class="lab-step">A chart showing the population change of the predator and the prey populations should appear.
</div></li><li data-sub-note-item="3-23"><div class="lab-step">Label the Y axis of the plot population size and the X axis time.
</div></li><li data-sub-note-item="3-24"><div class="lab-step">Title the plot &#8220;predator-prey interactions&#8221;.
</div></li><li data-sub-note-item="3-25"><div class="lab-step">Observe the interactions between the predator and prey populations.
</div></li><li data-sub-note-item="3-26"><div class="lab-step">Set the initial predator population to zero and observe what happens in the plot.
</div></li><li data-sub-note-item="3-27"><div class="lab-step">Now set the initial predator population to 120 and observe what happens to the predator population when there are more predators than prey.
</div></li><li data-sub-note-item="3-28"><div class="lab-step">Finally set the initial prey population to zero and observe what happens to the population of the predators.
</div></li><li data-sub-note-item="3-29"><div class="lab-step">Experiment with other values to see what other interactions can be created.
</div></li><li data-sub-note-item="3-30"><div class="lab-step">When finished, save the workbook and close it.
</div></li></ul></li><li data-note-item="4" data-parent-collapse="">Results
<button class="expand">Expand</button><ul class="lab-items"><li data-sub-note-item="4-1"><div class="lab-step">In the exponential growth model built in the first activity, observe the shape of the resulting population growth curve, and consider whether you think this type of growth could be sustained in nature.
</div></li><li data-sub-note-item="4-2"><div class="lab-step">Note how the growth rate R affected the population size after 10 generations in the exponential growth model.
</div></li><li data-sub-note-item="4-3"><div class="lab-step">Also consider how the initial population size affected the population size after 10 generations in the exponential growth model.
</div></li><li data-sub-note-item="4-4"><div class="lab-step">Now, consider the logistic growth model, and observe the shape of this graph, noting how it differs from the exponential growth model.
</div></li><li data-sub-note-item="4-5"><div class="lab-step">Then, note how the populations behave as they approach the carrying capacity in your logistic growth model.
</div></li><li data-sub-note-item="4-6"><div class="lab-step">Decide if you think that the maximum per capita growth rate, R max, influenced how quickly populations reached carrying capacity in the logistic growth model.
</div></li><li data-sub-note-item="4-7"><div class="lab-step">Also, consider what happens to population size when populations are over carrying capacity in the logistic growth model, or what might happen if the carrying capacity is lowered.
</div></li><li data-sub-note-item="4-8"><div class="lab-step">In the predator-prey model, note whether the prey population responded to increasing predator population. Conversely, consider how the predators then respond to decreasing prey populations.
</div></li></ul></li></ol>

Population Growth: Exponential and Logistic Growth and Predator-Prey Model - Procedure

Crescita della popolazione

Exponential Population Growth Model
ExpandNOTE: In these experiments you will use computer software to simulate different types of growth models. ...

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

Comportamento di gruppo

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 ...

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

Comportamento animale

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 ...

Cell Structure

<ol class="note-items"><li data-note-item="1" data-parent-collapse="">Visualizing Onion and Human Cells
<button class="expand">Expand</button><ul class="lab-items"><li data-sub-note-item="1-1"><div class="lab-step">In this experiment you will prepare different types of cells from a plant and animal, onion and human respectively, and then visualize them under a microscope. HYPOTHESES: The experimental hypothesis is that the cells will appear different in overall shape and membrane structure, but there will be some shared similar structures, like nuclei, in both cells. The null hypothesis of the experiment is that there will be no difference in structure between the two cell types.
</div></li><li data-sub-note-item="1-2"><div class="lab-step">First, to prepare an animal cell slide, start by pouring 30 mL of distilled water into a beaker.
</div></li><li data-sub-note-item="1-3"><div class="lab-step">Then, use a glass dropper to place 2 - 3 drops of the water onto the center of a clean microscope slide.
</div></li><li data-sub-note-item="1-4"><div class="lab-step">Next, take a clean toothpick and scrape the inside of your cheek to gather cells.  CAUTION: Do not scrape your cheeks too aggressively. The scraping should not cause discomfort or bleeding. 
</div></li><li data-sub-note-item="1-5"><div class="lab-step">Put the end of the toothpick with the cheek cells onto the wetted area of the slide and mix the water and cheek cells together.
</div></li><li data-sub-note-item="1-6"><div class="lab-step">Next, add two drops of methylene blue staining solution to the wetted area of the slide and mix it in with a clean toothpick.
</div></li><li data-sub-note-item="1-7"><div class="lab-step">Let the mixture sit at room temperature for 3 minutes.
</div></li><li data-sub-note-item="1-8"><div class="lab-step">After 3 minutes have passed, use a glass dropper to add a drop of glycerol to the mixture.
</div></li><li data-sub-note-item="1-9"><div class="lab-step">Carefully, place a cover slip over the mix on the slide by first standing one end of the cover slip vertically and aligning it along to one side of the mixture. Then, carefully lower the other end of the cover slip down until the mixture is completely covered.
</div></li><li data-sub-note-item="1-10"><div class="lab-step">Finally, remove any excess liquid with blotting paper.
</div></li><li data-sub-note-item="1-11"><div class="lab-step">Next, prepare a slide of plant cells by placing five drops of distilled water into a clean watch glass.
</div></li><li data-sub-note-item="1-12"><div class="lab-step">Then, with forceps, take a thin strip of onion and place it into the water.
</div></li><li data-sub-note-item="1-13"><div class="lab-step">Apply 5 drops of safranin solution to another watch glass.
</div></li><li data-sub-note-item="1-14"><div class="lab-step">Now using a pair of forceps transfer the piece of onion from the distilled water into the safranin.
</div></li><li data-sub-note-item="1-15"><div class="lab-step">Allow the onion to soak for 30 seconds.
</div></li><li data-sub-note-item="1-16"><div class="lab-step">Then, again with forceps, move the onion back into the distilled water.
</div></li><li data-sub-note-item="1-17"><div class="lab-step">Now, place 3 drops of glycerol onto the center of a glass microscope slide.
</div></li><li data-sub-note-item="1-18"><div class="lab-step">Using the forceps again, transfer the onion into the glycerol on the slide.
</div></li><li data-sub-note-item="1-19"><div class="lab-step">Gently place a cover slip over the onion on the slide and blot away any excess liquid with a piece of blotting paper.
</div></li><li data-sub-note-item="1-20"><div class="lab-step">Next, you will visualize the slides you have prepared using a compound microscope. First, turn on the scope and set the objective lens to the lowest level of magnification.  NOTE: A compound microscope typically has two levels of magnification.
</div></li><li data-sub-note-item="1-21"><div class="lab-step">Place the cheek tissue slide onto the stage of the microscope and bring the cells into focus by adjusting the stage and using the coarse adjustment knob.
</div></li><li data-sub-note-item="1-22"><div class="lab-step">When the cells are in focus, observe the individual cells and draw what you see. Repeat these observations at medium and high magnifications adjusting the focus with the fine adjustment knob. IMPORTANT: Do NOT use the coarse adjustment knob at high magnifications. 
</div></li><li data-sub-note-item="1-23"><div class="lab-step">Next, rotate the nose piece so that the oil lens is almost in place.
</div></li><li data-sub-note-item="1-24"><div class="lab-step">Place a drop of immersion oil on top of the cover slip of the cheek cell slide.
</div></li><li data-sub-note-item="1-25"><div class="lab-step">Use the oil lens to view the cheek cells and record your observations.
</div></li><li data-sub-note-item="1-26"><div class="lab-step">Repeat these steps to view the onion plant cell, moving again from low to medium, high, and oil lenses (steps 20 &#8211; 24).
</div></li><li data-sub-note-item="1-27"><div class="lab-step">Record all of your observations and note the differences you see between animal and plant cells.
</div></li><li data-sub-note-item="1-28"><div class="lab-step">When you are finished, place the cheek cell slide and toothpicks into a diluted bleach solution. Dispose of the cover slips into a glass waste container.
</div></li><li data-sub-note-item="1-29"><div class="lab-step">Finally, turn off the microscope and clean the lenses with lens cleaning paper.
</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">Calculate the total magnification for each of the viewing levels. Remember to account for both the eyepiece magnification and the objective lens magnification. NOTE: Eye piece: 10X magnification, Objective lens: 4X, 10X, and 40X magnification
</div></li><li data-sub-note-item="2-2"><div class="lab-step">Then, identify the structural differences between plant and animal cells using the observations made under the microscope.
</div></li><li data-sub-note-item="2-3"><div class="lab-step">Label the nuclei in both cell types and then highlight or note any differences that you see between them.
</div></li></ul></li></ol>

Cell Structure: Visualizing Onion and Human Cells - Procedure

Struttura cellulare

Visualizing Onion and Human Cells
ExpandIn this experiment you will prepare different types of cells from a plant and animal, onion and human ...

Fondamenti

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

Genetica degli organismi

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 ...

Genetica

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

Estinzione

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

Natural Selection

<ol class="note-items"><li data-note-item="1" data-parent-collapse="">Simulating Natural Selection
<button class="expand">Expand</button><ul class="lab-items"><li data-sub-note-item="1-1"><div class="lab-step">To begin, cut the white, black, and gray pipe cleaners into quarters, which should produce a total of 100 pieces that are 3 inches long.
</div></li><li data-sub-note-item="1-2"><div class="lab-step">Then, cut the green pipe cleaners in the quantities and lengths specified in the video, taking care to minimize waste. This approximates a normal distribution of lengths and should result in an additional 100 pieces total. NOTE: One 12-inch pipe cleaner should yield multiple lengths, for example, a 5-inch piece and a 7-inch piece. Use this advantage to reduce waste.
</div></li><li data-sub-note-item="1-3"><div class="lab-step">Now, cut one sheet of black and one sheet of white construction paper into approximately 3-inch strips measured along the long edge of the paper.
</div></li><li data-sub-note-item="1-4"><div class="lab-step">Tape these strips down to cover the base of a dissecting tray to create an alternating pattern of black and white strips. NOTE: In the first simulation, you will use the white, gray, and black pipe cleaner segments against a black and white background to simulate different selection pressures due to predation on a population. You will select your prey using two different strategies, eyes-open and eyes-closed. For each of these strategies, you will perform four rounds consisting of 25 selections, alternating between two partners. After each round you will restore the population back to its original size using the proportions of phenotypes present after selection.
</div></li><li data-sub-note-item="1-5"><div class="lab-step">To begin the first scenario, count out 12 white, 12 black, and 26 gray pipe cleaner segments with your partner. Hypotheses: Here the experimental hypothesis might be that different predation strategies, such as having the eyes open or closed, will result in distinct selection pressures on the populations. Further, different phenotypes may be positively selected in the alternate scenarios. The null hypothesis might be that different predation strategies will have no effect on the populations or phenotypes.
</div></li><li data-sub-note-item="1-6"><div class="lab-step">Mix the pipe cleaner segments together as best as possible. Then, sprinkle them into the papered tray. NOTE: Ideally, the pipe cleaners will be spread out. However, this may not occur due to their self-adhering nature.
</div></li><li data-sub-note-item="1-7"><div class="lab-step">Once the segments are in the tray, separate them as best as possible without moving them too far from where they landed. As few segments as possible should be touching.
</div></li><li data-sub-note-item="1-8"><div class="lab-step">Have the first partner close their eyes. When given an audible signal by the second partner, the first should open their eyes and grab the first pipe cleaner they see from the tray as quickly as possible.
</div></li><li data-sub-note-item="1-9"><div class="lab-step">Set the removed pipe cleaner to the side.
</div></li><li data-sub-note-item="1-10"><div class="lab-step">Alternating partners, repeat this process a total of 25 times (steps 8 &#8211; 9).
</div></li><li data-sub-note-item="1-11"><div class="lab-step">Count the remaining pipe cleaners by color and record this in the data collection table for Scenario 1. <a href="https://jove.unicartagenaproxy.elogim.com/CDNSource/lm/labs/23/Lab_23_Tables_1_and_2.xlsx" style="display:block;margin:20px;" target="__blank">Click Here to download Tables 1 and 2</a>
</div></li><li data-sub-note-item="1-12"><div class="lab-step">Then, restore the population to 50 by adding one pipe cleaner of the same color for each of the remaining pipe cleaners. Hold these to the side until they are all gathered.
</div></li><li data-sub-note-item="1-13"><div class="lab-step">Then, sprinkle the pipe cleaners into the tray as previously described (steps 6-7). Repeat this process for three more rounds consisting of 25 selections each and record the counts for each of the rounds in the data collection table for Scenario 1.
</div></li><li data-sub-note-item="1-14"><div class="lab-step">Remove all pipe cleaners from the tray after the fourth round.
</div></li><li data-sub-note-item="1-15"><div class="lab-step">For Scenario 2, again count out 12 white, 12 black, and 26 gray pipe cleaner segments and sprinkle them randomly into the tray.
</div></li><li data-sub-note-item="1-16"><div class="lab-step">Follow the same protocol as in Scenario 1. However, when given the signal, the grabbing partner should keep their eyes closed. To aid in accuracy, it may help to position the hand near to the tray before grabbing.
</div></li><li data-sub-note-item="1-17"><div class="lab-step">Repeat this process 25 times, switching partner roles, and record the remaining pipe cleaner amounts in the data collection table for Scenario 2.
</div></li><li data-sub-note-item="1-18"><div class="lab-step">Then, restore the population to 50 based on that count. Do this for all four rounds. NOTE: In the second simulation, you will be using the eyes-open and eyes-closed predation strategies as previously described to select pipe cleaners of various lengths.
</div></li><li data-sub-note-item="1-19"><div class="lab-step">For Scenario 3, gather all 100 of the pipe cleaners that have been cut to varying lengths to represent different phenotypes.
</div></li><li data-sub-note-item="1-20"><div class="lab-step">One partner should hold all the pipe cleaners upright and loosely in one hand resting on the tabletop, such that the bottom end of each pipe cleaner rests against the tabletop.
</div></li><li data-sub-note-item="1-21"><div class="lab-step">Now, the partner that is not holding the pipe cleaners should close their eyes. When given an audible signal, the partner should open their eyes and grab the first pipe cleaner they see from their partner&#39;s hand.
</div></li><li data-sub-note-item="1-22"><div class="lab-step">Repeat this process 50 times (step 21). However, the partners should not switch roles during the round this time. The partner holding the pipe cleaners should always be the one giving the signal and never grabbing from the bunch.
</div></li><li data-sub-note-item="1-23"><div class="lab-step">After the first round consisting of 50 selections, count the remaining pipe cleaners by length. Then, record this information in the data collection table for scenario three. <a href="https://jove.unicartagenaproxy.elogim.com/CDNSource/lm/labs/23/Lab_23_Tables_3_and_4.xlsx" style="display:block;margin:20px;" target="__blank">Click Here to download Tables 3 and 4</a>
</div></li><li data-sub-note-item="1-24"><div class="lab-step">Add one pipe cleaner of the same length as each of the remaining ones to restore the population to 100. This will likely require more pipe cleaners than were originally cut. Try to use scraps from the class to reduce waste.
</div></li><li data-sub-note-item="1-25"><div class="lab-step">Repeat this for three more rounds, alternating which partner holds or grabs each round.
</div></li><li data-sub-note-item="1-26"><div class="lab-step">To perform Scenario 4, gather the varied-length pipe cleaners in their original proportions.
</div></li><li data-sub-note-item="1-27"><div class="lab-step">Now repeat the grabbing process, as described in Scenario 3. However, only remove 30 pipe cleaners instead of 50.
</div></li><li data-sub-note-item="1-28"><div class="lab-step">Divide the count at the end of each round by two for each phenotype 4 inches or below, rounding fractions down.
</div></li><li data-sub-note-item="1-29"><div class="lab-step">Then, record these values in the data recording table for Scenario 4.
</div></li><li data-sub-note-item="1-30"><div class="lab-step">Use the relative proportions of the phenotypes in the final population to restore the population to 100. When restoring the population to 100, it may be necessary to calculate relative proportions of phenotypes.
</div></li><li data-sub-note-item="1-31"><div class="lab-step">Repeat this for three more rounds, again alternating holding and grabbing the pipe cleaners.
</div></li><li data-sub-note-item="1-32"><div class="lab-step">For each scenario calculate the survival for each phenotype, that is the proportion of the phenotype surviving.
</div></li><li data-sub-note-item="1-33"><div class="lab-step">Then, calculate the relative fitness, or w, between each round. As reproductive rate does not vary in this simulation, this should always be considered equal, or 1.
</div></li><li data-sub-note-item="1-34"><div class="lab-step">Also, calculate the selection coefficients for each phenotype.
</div></li><li data-sub-note-item="1-35"><div class="lab-step">Now, use the relative frequencies of each phenotype to create histograms with appropriately labeled axes for each scenario showing the population before any selection took place and then alongside these in a different color after four rounds of selection.
</div></li><li data-sub-note-item="1-36"><div class="lab-step">Identify the type of selection taking place in each scenario and label the graphs. 
</div></li><li data-sub-note-item="1-37"><div class="lab-step">Develop a hypothesis to explain your observations. 
</div></li></ul></li></ol>

Natural Selection: Calculating Fitness and Selection - Procedure

Selezione naturale

Simulating Natural Selection
ExpandTo begin, cut the white, black, and gray pipe cleaners into quarters, which should produce a total of 100 pieces that ...

Artificial Selection

<ol class="note-items"><li data-note-item="1" data-parent-collapse="">Observing Artificial Selection in Plants 
<button class="expand">Expand</button><ul class="lab-items"><li data-sub-note-item="1-1"><div class="lab-step">To begin the experiment, collect five two-week old grown plants from the generation one population that appear healthy. Note: Plants should appear green, not wilted, and not have damaged leaves. Experimental Hypothesis: The experimental hypothesis for this lab might be that the number of lead trichomes varies naturally, but the average number will increase over generations if only plants with the highest number of trichomes are interbred or decrease if those with the least are interbred. Null Hypothesis: The null hypothesis might be that the average number of trichomes will not change over subsequent generations after artificial selection.
</div></li><li data-sub-note-item="1-2"><div class="lab-step">Label each plant with a letter from A to E and with both partners&#8217; names.
</div></li><li data-sub-note-item="1-3"><div class="lab-step">Record any natural differences you see between your plant samples. Note: All of the plants are the same age, so any observed variation is due to expressed phenotype, not development.
</div></li><li data-sub-note-item="1-4"><div class="lab-step">Use a hand lens to count the number of trichomes found on the petiole of the lowest leaf, not including the round, first-grown cotyledons for each plant. Note: It can be helpful to shine a light onto the plant and to hold it against a dark background, such as a lab bench, during the trichome analysis. 
</div></li><li data-sub-note-item="1-5"><div class="lab-step">Both partners will conduct counts on each plant independently and compare answers. If the counts are different, enlist a third counter or take the average of the two results.
</div></li><li data-sub-note-item="1-6"><div class="lab-step">After this, determine the median number of trichomes for the entire class.  <a target="__blank" style="display:block;padding:20px;" href="https://jove.unicartagenaproxy.elogim.com/CDNSource/lm/labs/2/Lab_2_Table_A.xlsx">Click Here to download Table A</a>
</div></li><li data-sub-note-item="1-7"><div class="lab-step">Determine the top and bottom 10% of the plants from the class trichome count. These groups will make up the two artificially selected lines: high hairiness and low hairiness, respectively.
</div></li><li data-sub-note-item="1-8"><div class="lab-step">If more than 10% of the plants have no trichomes, select 10% of those plants randomly for the low hairiness plant line.
</div></li><li data-sub-note-item="1-9"><div class="lab-step">Separate the two groups of selected plants and label them clearly.
</div></li><li data-sub-note-item="1-10"><div class="lab-step">Move the plants to a well-lit area and keep them well watered until they develop flowers. Note: Flowers typically develop around 14 days.
</div></li><li data-sub-note-item="1-11"><div class="lab-step">When the flowers are fully developed, use a pollination wand or paintbrush to rub the stigma and anther of one plant&#8217;s flowers for a few seconds.
</div></li><li data-sub-note-item="1-12"><div class="lab-step">Using the same wand or brush, rub the inside of each flower of every other plant in the same selection line.
</div></li><li data-sub-note-item="1-13"><div class="lab-step">Using a different wand or brush, repeat steps 11 through 12 on the other line. Note: Take care not to cross the lines. Spread the pollen only among the appropriately selected plants.
</div></li><li data-sub-note-item="1-14"><div class="lab-step">Return the generation one plants to the growing areas. When the plants reach approximately 1 month old, they should produce mature seed pods.
</div></li><li data-sub-note-item="1-15"><div class="lab-step">Allow the plants to dry out once the seed pods turn yellow.
</div></li><li data-sub-note-item="1-16"><div class="lab-step">Break the seed pods open and collect the seeds.
</div></li><li data-sub-note-item="1-17"><div class="lab-step">Obtain new pots for the generation two seeds.
</div></li><li data-sub-note-item="1-18"><div class="lab-step">Fill the pots with moist potting soil about halfway and add a fertilizer pellet to each pot.
</div></li><li data-sub-note-item="1-19"><div class="lab-step">Fill the pots with more soil to fill just below the lip.
</div></li><li data-sub-note-item="1-20"><div class="lab-step">Make a shallow depression in the soil of each pot with a probe.
</div></li><li data-sub-note-item="1-21"><div class="lab-step">Touch a clean toothpick to a gluestick and use the toothpick to transplant the seeds into the soil one by one.
</div></li><li data-sub-note-item="1-22"><div class="lab-step">Cover the seeds with a thin layer of soil.
</div></li><li data-sub-note-item="1-23"><div class="lab-step">Label the pots with their hairiness line and place them in the designated growing area.
</div></li><li data-sub-note-item="1-24"><div class="lab-step">Use a plastic dropper to water the plants as needed to ensure they do not dry out.
</div></li><li data-sub-note-item="1-25"><div class="lab-step">When the plants are two weeks old, collect five plants from each line and label them A to E.
</div></li><li data-sub-note-item="1-26"><div class="lab-step">Then, count the trichomes on these plants as previously described (steps 4- 5).
<a target="__blank" style="display:block;padding:20px;" href="https://jove.unicartagenaproxy.elogim.com/CDNSource/lm/labs/2/Lab_2_Table_B.xlsx">Click Here to download Table B</a>
</div></li></ul></li><li data-note-item="2" data-parent-collapse="">Data Analysis
<button class="expand">Expand</button><ul class="lab-items"><li data-sub-note-item="2-1"><div class="lab-step">Create a frequency graph based on the generation one class data with the trichome count on the x axis and the number of plants on the y axis. Label the mean. 
</div></li><li data-sub-note-item="2-2"><div class="lab-step">Then, create frequency graphs for both of the selected hairiness lines.
</div></li><li data-sub-note-item="2-3"><div class="lab-step">Compare the frequency graphs for generation one and both hairiness lines.
</div></li><li data-sub-note-item="2-4"><div class="lab-step">Take note of phenotypic shifts observed in the selected hairiness lines.
</div></li></ul></li></ol>

Artificial Selection: Visualization in Plants and Data Analysis - Procedure

Selezione artificiale

Observing Artificial Selection in Plants 
ExpandTo begin the experiment, collect five two-week old grown plants from the generation one population that ...

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