<p class="jove_content"> Die Autoren erkennen dankbar die geschickte Unterstützung von Frau Trine W. Mikkelsen, Frau Lise M. Fitting, und das Personal an Påskehøjgaard. Das dänische Medical Research Council, die Lundbeck-Stiftung, und die Novo Nordisk Foundation finanziell unterstützten die Studie. </p>

<p class="jove_content"> Tier Anästhesie und euthanesia wurden gemäß „Principles of Labortierhaltung“ (NIH Publikation Nr 86-23, überarbeitet 1985) und genehmigt vom dänischen Rat für Tierforschung Ethik durchgeführt. </p><p class="jove_title"> 1. Instrumente </p><ol><li> Sammeln Sie die Instrumente in dem Video dargestellt und in der <strong>Tabelle der Materialien</strong> aufgeführt. </li></ol><p class="jove_title"> 2. Enthauptung </p><p class="jove_content"> HINWEIS: Die Anästhesie wurde durch eine intramuskuläre Injektion von 5 ml Midazolam (5 mg / ml) und 5 ml Ketamin (25 mg / ml) induziert. 5-10 min später, als das Tier sediert tief war, wurde eine kanülierte Ohrvene und eine letale Überdosis (100 mg / kg Körpergewicht) von Natriumpentobarbital (200 mg / ml) wurde intravenös verabreicht. Um sicherzustellen , dass das Tier vollständig eingeschläfert wurde, wurde der Kammschmerzreflex , wie durch Ettrup <em>et al getestet.</em> (2011) <sup class="xref">20.</sup> Komplette Euthanasie wurde wie beschrieben gewährleistet inDie Ethik - Aussage über und durch eine transkardialer Perfusion mit 5 l isotonischer Kochsalzlösung folgte, wie von Ettrup <em>et al.</em> (2011) <sup class="xref">20.</sup> Alle zeigten Verfahren sind Obduktion durchgeführt, Ausschließen der Notwendigkeit, dass die Wohlfahrtsmaßnahmen notwendig für die Langzeit Anästhesie und postprocedural Überleben. </p><ol><li> Köpft das Schwein durch einen hohen Kreis cervikale Inzision, ein chirurgisches Skalpell, direkt unterhalb dem Unterkieferwinkel <strong>(1A).</strong> </li><li> Immer noch mit dem chirurgischen Skalpell, weiterhin die Inzision ventral durch das weiche Gewebe des Halses einschließlich dem Larynx und die Speiseröhre, bis die knöcherne Wirbelsäule erreicht ist, etwa auf der Höhe des kraniozervikalen Übergangs. </li><li> Voraus, um den Schnitt mit einem Chirurgisches Skalpell aus der vorderen Seite des kraniozervikalen Übergangs über dem anterioren Bogen von Atlas, und durch das vordere atlanto Membran, um dadurch den Spinalkanal Belichtungs- und das Rückenmark (<strong>1B).</strong> Gleichzeitig einen Assistenten bitten, den Schweinekörper vom Schwein Kopf zu ziehen, den Zugang zwischen der Schädelbasis und dem ersten Halswirbel zu erleichtern. </li><li> Weiterhin den chirurgischen Schnitt durch den Duralsack und dem Rückenmark <strong>(1B).</strong> Besondere Vorsicht, um sicherzustellen, dass ein vollständiger Querschnitt des Rückenmarks erreicht wird. <br /> HINWEIS: Bei Nichtbeachtung des vorhergehenden Schritt durchzuführen, in unerwünschtem Zuge auf das Rückenmark und Gehirn während der folgenden Schritte des Prozesses Dekapitation führen. </li><li> Zwangsweise erweitert den cranocervical Übergang an der Abschnittsebene <strong>(1C).</strong> Zur gleichen Zeit, verwenden, um das chirurgische Skalpell Abschnitt die restlichen Bänder atlanto das Gelenk zwischen dem Condylus occipitalis zu lösen und dem oberen artikulierten Prozess des Atlas. Trennt den Schweinekopf aus dem Körper. </li></ol><p><img alt="Abbildung 1" src="/files/ftp_upload/55511/55511fig1.jpg" style="font-size: 14px;" /><br style="font-size: 14px;" /> <strong style="font-size: 14px;">Abbildung 1: Minipig Enthaupten.</strong> <strong>(A)</strong> Halsschnitt (Pfeil, Kieferwinkel). <strong>(B)</strong> durch die Incision atlanto Bänder und die Dura-Rückenmark umgibt (SC) an dem kraniozervikalen Übergang (C1, anteriore Bogen von Atlas; OC, occipitalen Kondylen). <strong>(C)</strong> Der hintere Teil des Gelenkes atlanto wird durch eine kraftvolle Erweiterung (Pfeile) auf der Abschnittsebene freigegeben. <a href="https://ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/55511/55511fig1large.jpg" style="font-size: 14px;" target="_blank">Bitte klicken Sie hier</a> , um <a href="https://ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/55511/55511fig1large.jpg" style="font-size: 14px;" target="_blank">eine größere Version dieser Figur zu sehen.</a> </p><p class="jove_title"> 3. Schädelöffnung </p><ol><li> Positionieren Sie den Schweinekopf auf einem Tisch. </li><li> Führte einen dorsalen Längsinzision mit einem chirurgischen Skalpell durch die Haut und das darunterliegende Weichgewebe von der Rückseite des Mauls, über den Scheitel des Kopfes, und nach unten durch den hinteren Teildie Okzipitalregion. </li><li> Belichten des dorsalen und posterioren Teil des Schädels durch das Weichgewebe zu entfernen, um den anfänglichen Einschnitt mit einem chirurgischen Skalpell entfernt lateral. </li><li> Lassen Sie den Schläfenmuskel bilateral aus dem Schädel <strong>(2A)</strong> mit einem chirurgischen Skalpell. Stellen Sie sicher, dass der hintere Hinterhauptsbein ist aus weichem Gewebe gereinigt. </li><li> Verwenden Sie die posterior Eingang des Foramen Magnum okzipitalen Knochen mit einem Kerrison Knochenstanze und Knochen Rongeurs belichten und die Dura-bedeckten Cerebellum <strong>(2B)</strong> zu entfernen. </li><li> Kehrt zu der freiliegenden vorderen Seite des Schädels und wählen einen Eintrittspunkt in dem Stirnbein, direkt vor den Augen. An diesem Punkt wird eine Knochenmeißel mit einem Hammer den Schädel eindringt und die Stirnhöhle <strong>(2C)</strong> eingegeben werden . </li><li> Verwenden, um die Ausdehnung der Stirnhöhle das dorsoposterior Entfernen des äußeren Schädel Lamina mit einer Knochen-Rongeur oder Knochenstanze zu fördern und setzen die inneren, dünnenknöcherner Schädel Lamina das Cerebrum Belag <strong>(Figur 2D).</strong> </li><li> Öffnet sanft den inneren knöchernen Schädel lamina ventral mit einem Hammer und einem Meißel Knochen zur Dura bedeckte Cerebrum <strong>(Abbildung 2E)</strong> zu belichten. </li><li> Weiterhin die Knochenentfernung seitlich einen Knochen Meißel und einen Knochen - Rongeur durch die zeitliche und Scheitelbein , um das endgültige dorsoposterior Teil des Schädels freizugeben, der sich zwischen den bereits belichteten Teile der dura bedeckten Cerebrum und Cerebellum <strong>(Figur 2F).</strong> <br /> HINWEIS: Es ist oft möglich, im letzten Schritt dieses Verfahrens, den Meißel zu verwenden, öffnen Sie die restlichen hinteren Schädelknochen von einer Seite zu brechen, so wie man eine Tür öffnet. </li></ol><p><img alt="Figur 2" src="/files/ftp_upload/55511/55511fig2.jpg" style="opacity: 0.9; font-size: 14px;" /><br style="font-size: 14px;" /> <strong style="font-size: 14px;">Abbildung 2: Minipig Schädelöffnung.</strong> <strong>(A)</strong> Die Exposition der dorsoposterior Schädeloberfläche, einschließlich der Entfernung des okzipitalen und temporalen Muskeln. <strong>(B)</strong> Entfernen des Hinterhauptbeins (CB, dura bedeckten Cerebellum; OB, okzipitalen Knochen; OC, occipitalen Kondylen, und SC, Rückenmark). <strong>(C)</strong> ein Hammer und ein Meißel Knochen verwendet , um den Schädel ventral zu durchdringen und die Stirnhöhle auf der Höhe der Augen ein. <strong>(D)</strong> Das Ausmaß der Stirnhöhle (FS) werden verwendet , um die äußeren dicken Schädelknochen (1) zu entfernen, eine inneren dünnen Knochenplättchen (2) , die die Cerebrum aussetzt. <strong>(E)</strong> Entfernen der dünnen Knochen Lamina, Freilegung der Dura bedeckten Cerebrum (Pfeil). <strong>(F)</strong> Schließlich wird ein Hammer und ein Knochenmeißel verwendet , um die ventral seitlich anschließen und die hinteren Schädelöffnungen. <a href="https://ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/55511/55511fig2large.jpg" style="font-size: 14px;" target="_blank">Bitte klicken Sie hier</a> , um <a href="https://ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/55511/55511fig2large.jpg" style="font-size: 14px;" target="_blank">eine größere Version dieser Figur zu sehen.</a> </p><p class="jove_title"> 4. Gehirn Removal </p><ol><li> Verwenden chirurgische Zange der Dura heben und einen sanften Einschnitt nahe dem venösen Sinus sagittalis superior mit einem feinen Chirurgisches Skalpell <strong>(3A)</strong> zu erstellen. </li><li> Verwenden Mikro Schere oder ein Dura-Messer weiter die Dura öffnen Abdecken der dorsale Oberfläche des Gehirns. <br /> HINWEIS: Besondere Vorsicht ist geboten, wenn die Dura an das Klein tentorium (3B) entsprechende Entfernung, wie Erhaltung dieses dural Blattes wird nachfolgende Gehirnentfernung zu verhindern. </li><li> Platzieren Sie den Schweinekopf vertikal <strong>(3C).</strong> </li><li> Verwenden , um die Knochenmeißel oder Dissektor die ventroanterior Cerebrum zur Freigabe durch stumpfe Dissektion des Riechkolbens aus dem Dura-bedeckten Boden der Schädelhöhle <strong>(Abbildung 3D).</strong> </li><li> Verwenden , um ein feines Chirurgisches Skalpell Abschnitt des belichteten Chiasma <strong>(Figur 3E).</strong> Freizulegen, und den Abschnitt Hypophysenstiels und die oculomotor Nerven. </li><li> Lassen Sie die ventrale brainstem durch die lo sectioningWer Hirnnerven <strong>(Abbildung 3F)</strong> mit einem feinen chirurgischen Skalpell. Sicherstellen , dass die duralen cerebellar tentorium vollständig eingeschnitten wurde <strong>(3B),</strong> da diese sonst durch durale Blatthirnstamm während des Freisetzungsprozesses geschnitten wird. </li></ol><p><img alt="Abbildung 3" src="/files/ftp_upload/55511/55511fig3.jpg" style="font-size: 14px;" /><br style="font-size: 14px;" /> <strong style="font-size: 14px;">Abbildung 3: Minipig Hirnentfernung.</strong> <strong>(A)</strong> Dural Öffnung mit chirurgischer Pinzette und ein dura Messer. <strong>(B)</strong> Es muss vollständig genommen werden , um den Dural Blatt (Pfeil) einzuschneiden, zwischen Gehirn- und Cerebellum. <strong>(C)</strong> Der Schweinekopf positioniert ist , vertikal zur besseren Visualisierung der Schädelbasisstrukturen und damit die Schwerkraft in der beabsichtigten Verschiebung des Gehirns zu unterstützen. <strong>(D)</strong> Ein Dissektor oder ein Knochenmeißel wird verwendet , um den Riechkolben durch stumpfe Sektion von den Dura-covere zu entlastend Schädelbasis. <strong>(E)</strong> Die Präparation wird in einer posterioren Richtung entlang der Schädelbasis für die Belichtung und Sektionierung des Chiasma (Pfeil), infundibular Stiel und oculomotor Nerven fortgesetzt. <strong>(F)</strong> Das Gehirn Freisetzung wird mit dem Abschnitt der unteren Hirnnerven abgeschlossen , wie sie von der ventralen Oberfläche der brainstem abgehen (III, Oculomotorius; IV, Trochlearis; V, Trigeminus und VI, Abducens). <a href="https://ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/55511/55511fig3large.jpg" style="font-size: 14px;" target="_blank">Bitte klicken Sie hier</a> , um <a href="https://ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/55511/55511fig3large.jpg" style="font-size: 14px;" target="_blank">eine größere Version dieser Figur zu sehen.</a> </p><p class="jove_title"> 5. Hypophysen Removal </p><ol><li> Identifizieren Sie den Hypophysenstiel und seine Umgebung dural Blatt (die diapragma sellae) im Schädelboden <strong>(4A).</strong> </li><li> Einzuschneiden die durale Blatt lateral den Hypophysenstiels ein feines chirurgisches Skalpell <strong>(4B)</strong> verwendet wird . </li><li> Verwenden Sie einen Dissektor die Grube zu lösenuitary und hebt ihn aus der Hypophyse fossa <strong>(4C).</strong> </li></ol><p><img alt="Abbildung 4" src="/files/ftp_upload/55511/55511fig4.jpg" style="opacity: 0.9; font-size: 14px;" /><br style="font-size: 14px;" /> <strong style="font-size: 14px;">Abbildung 4:</strong> Minipig Hypophysen Entfernung. <strong>(A)</strong> Die Hypophyse fossa (*) in dem Schädelboden identifiziert (1, Bulbus olfactorius, 2, Chiasma; und PF, hintere Schädelgrube). <strong>(B)</strong> Die durale Abdeckung (sellar diagphragm, (Pfeil)) seitlich eingeschnitten. <strong>(C)</strong> Die Hypophyse (Pfeil) mit einem Dissektor freigegeben und aus der Hypophyse Fossa angehoben. Maßstabsbalken (AC) = 10 mm. <a href="https://ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/55511/55511fig4large.jpg" style="font-size: 14px;" target="_blank">Bitte klicken Sie hier</a> , um <a href="https://ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/55511/55511fig4large.jpg" style="font-size: 14px;" target="_blank">eine größere Version dieser Figur zu sehen.</a> </p>

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<li>Ettrup, K. S., <em>et al.</em> <a target="_blank" href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3197034/">Basic Surgical Techniques in the Minipig: Intubation, Transurethral Bladder Catheterization, Femoral Vessel Catheterization, and Transcardial Perfusion.</a> <em>J Vis Exp</em>.  (52), e2652(2011).</li>
<li>Ettrup, K. S., Tornøe, J., Sørensen, J. C., Bjarkam, C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+surgical+device+for+minimally+invasive+implantation+of+experimental+deep+brain+stimulation+leads+in+large+research+animals%5BTitle%5D)+AND+%22J+Neurosci+Methods%22%5BJournal%5D)">A surgical device for minimally invasive implantation of experimental deep brain stimulation leads in large research animals.</a> <em>J Neurosci Methods</em>. <strong>200</strong> (1), 41-46 (2011).</li>
<li>Danielsen, E. H., <em>et al.</em> <a target="_blank" href="http://www.academia.edu/13111607/Positron_emission_tomography_of_living_brain_in_minipigs_and_domestic_pigs">Positron emission tomography of living brain in minipigs and domestic pigs.</a> <em>Scand J Lab Anim Sci Suppl</em>. <strong>25</strong> (1), 127-135 (1998).</li>
<li>Røhl, L., <em>et al.</em> <a target="_blank" href="http://onlinelibrary.wiley.com/doi/10.1002/jmri.10068/pdf">Time evolution of cerebral perfusion and ADC measured by MRI in a porcine stroke model.</a> <em>J Magn Reson Imaging</em>. <strong>15</strong> (2), 123-129 (2002).</li>
<li>Cumming, P., Gillings, N. M., Jensen, S. B., Bjarkam, C. R., Gjedde, A. <a target="_blank" href="http://europepmc.org/abstract/med/12831994">Kinetics of the uptake and distribution of the dopamine D2/3 agonist (R)-N-[1-11C]n-propylnorapomorphine in brain of healthy and MPTP-poisoned Gottingen miniature pigs.</a> <em>Nucl Med Biol</em>. <strong>30</strong> (5), 547-553 (2003).</li>
<li>Jensen, K. N., Deding, D., Sørensen, J. C., Bjarkam, C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Long-term+implantation+of+deep+brain+stimulation+electrodes+in+the+pontine+micturition+centre+of+the+minipig%5BTitle%5D)+AND+%22Acta+Neurochir%22%5BJournal%5D)">Long-term implantation of deep brain stimulation electrodes in the pontine micturition centre of the minipig.</a> <em>Acta Neurochir</em>. <strong>151</strong> (7), 785-794 (2009).</li>
<li>Rosendal, F., <em>et al.</em> <a target="_blank" href="https://www.ncbi.nlm.nih.gov/pubmed/16298581">Does chronic low dose treatment with ciclosporin influence the brain? A histopathological study in pigs.</a> <em>Transplantation Proc</em>. <strong>37</strong> (8), 3305-3308 (2005).</li>
<li>Nielsen, M. S., Sørensen, J. C., Bjarkam, C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+substantia+nigra+pars+compacta+of+the+minipig%3A+An+anatomical+and+stereological+study%5BTitle%5D)+AND+%22Brain+Struct+Funct%22%5BJournal%5D)">The substantia nigra pars compacta of the minipig: An anatomical and stereological study.</a> <em>Brain Struct Funct</em>.  (4-5), 481-488 (2009).</li>
<li>Sørensen, J. C., Bjarkam, C. R., Simonsen, C. Z., Danielsen, E., Geneser, F. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Oriented+sectioning+of+irregular+tissue+blocks+in+relation+to+computerized+scanning+modalities.+Results+from+the+domestic+pig+brain%5BTitle%5D)+AND+%22J+Neurosci+Methods%22%5BJournal%5D)">Oriented sectioning of irregular tissue blocks in relation to computerized scanning modalities. Results from the domestic pig brain.</a> <em>J Neurosci Methods</em>. <strong>104</strong>, 93-98 (2000).</li>
<li>Bjarkam, C. R., Pedersen, M., Sørensen, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((New+strategies+for+embedding%2C+orientation+and+sectioning+of+small+brain+specimens+enable+direct+correlation+to+MR-images%2C+brain+atlases%2C+or+use+of+unbiased+stereology%5BTitle%5D)+AND+%22J+Neurosci+Methods%22%5BJournal%5D)">New strategies for embedding, orientation and sectioning of small brain specimens enable direct correlation to MR-images, brain atlases, or use of unbiased stereology.</a> <em>J Neurosci Methods</em>. <strong>108</strong>, 153-159 (2001).</li>
<li>Bjarkam, C. R., Sørensen, J. C., Geneser, F. A. <a target="_blank" href="https://www.ncbi.nlm.nih.gov/pubmed/12625454">Distribution and morphology of serotonin-immunoreactive axons in the hippocampal region of the New Zealand white rabbit. I. Area dentata and hippocampus proper.</a> <em>Hippocampus</em>. <strong>13</strong> (1), 21-37 (2003).</li>
<li>Bjarkam, C. R., Glud, A. N., Orlowski, D., Sørensen, J. C., Palomero-Gallagher, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+telencephalon+of+the+minipig%2C+cytoarchitecture+and+cortical+surface+anatomy%5BTitle%5D)+AND+%22Brain+Struct+Funct%22%5BJournal%5D)">The telencephalon of the minipig, cytoarchitecture and cortical surface anatomy.</a> <em>Brain Struct Funct</em>. , e-pub ahead of print (2016).</li>
<li>Boltze, J., Nitzsche, B., Geiger, K. D., Schoon, H. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Histopathological+investigation+of+different+MCAO+modalities+and+impact+of+autologous+bone+marrow+mononuclear+cell+administration+in+an+ovine+stroke+model%5BTitle%5D)+AND+%22Transl+Stroke+Res%22%5BJournal%5D)">Histopathological investigation of different MCAO modalities and impact of autologous bone marrow mononuclear cell administration in an ovine stroke model.</a> <em>Transl Stroke Res</em>. <strong>2</strong>, 279-293 (2011).</li>
<li>Jortner, B. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+return+of+the+dark+neuron.+A+histological+artifact+complicating+contemporary+neurotoxicologic+evaluation%5BTitle%5D)+AND+%22Neurotoxicology%22%5BJournal%5D)">The return of the dark neuron. A histological artifact complicating contemporary neurotoxicologic evaluation.</a> <em>Neurotoxicology</em>. <strong>27</strong>, 628-634 (2006).</li>
</ol>

<p class="jove_content"> Die Autoren haben nichts zu offenbaren. </p>

<p class="jove_content"> Die meisten experimentellen Neurowissenschaften Studien werden in kleinen Tierarten, wie Mäuse und Ratten durchgeführt, in denen den Zugang zum ZNS durch einen dünnen Schädel- und Dural-Dicke erleichtert wird. In größeren Versuchstieren , wie Schweine <sup class="xref"><sup>1,</sup></sup> <sup class="xref"><sup>4,</sup></sup> <sup class="xref">8,</sup> Schafe <sup class="xref">32,</sup> aber, und nicht-menschliche Primaten, erfordern die beträchtlichen Dicke dieser Strukturen , die die Verwendung von robusten Instrumenten (Tabelle von Materialien) und die richtigen Einspeisepunkte für Schädelknochenentfernung <strong>(Abbildung 2).</strong> Die Kenntnis der Beschränkungs dural Blätter <strong>(3</strong> und <strong>4)</strong> erforderlich ist, bevor das CNS zugegriffen werden kann , und das Gehirn sicher entfernt. </p><p class="jove_content"> Es wird empfohlen, die Dura intakt während der Schädelknochenentfernung zu lassen, da dies das zugrunde liegende Gehirn vor Schäden zu schützen. Zurück transkardialer Fixierung <sup class="xref">20</sup> kann ebenfalls verhärten und slightly das Gehirn schrumpft, die Knochen und Dural Entfernungsprozess ermöglicht, mit mehr Leichtigkeit und Sicherheit durchgeführt werden. Eine Besonderheit des Schweins Schädels, im Gegensatz zu den Schafen und nicht-menschlichen Primaten, ist die fortschreitende Ausdehnung des Stirnhöhle mit dem Alter, das in den Schädelknochen - Entfernungsverfahren <strong>(2)</strong> , von Vorteil sein kann. Die vorgestellte Technik kann demnach auf allen großen Tierarten verwendet werden, aber nur bei Schweinen, vor allem denjenigen, die älter als 6 Monaten, wird die Stirnhöhle genug Hilfe zu leisten, in dem Schädelknochen Entfernungsprozess entwickelt werden. Schließlich vollständige Sektionierung des Rückenmarks während des Prozesses Dekapitation (1B) und die vollständigen Sektionierung des duralen cerebellar tentorium <strong>(3B)</strong> vor dem endgültigen Gehirn freisetzen , sind unbedingt erforderlich , um Folgeschäden an den Hirnstamm zu vermeiden. </p><p class="jove_content"> In einigen Untersuchungen kann es vorteilhaft sein, einen Teil der rostralen zervikalen Rückenmark an das Gehirn angebracht zu haben.Dies kann durch Platzieren des anfänglichen Einschnitts Dekapitation <strong>(1A)</strong> mehr caudal auf dem Hals erhalten werden, die den Zugang zu dem Rückenmark durch eine Bandscheibe zervikalen anstelle des kraniozervikalen Übergangs, wie in dem aktuellen Video demonstriert. Die hintere Knochenentfernung wird dann von dem freiliegenden Schwanz Lamina beginnen. Abgesehen davon, wird die Technik ähnlich sein, so ist es wichtig , sich daran zu erinnern , dass das Rückenmark muss vollständig geschnitten werden , bevor das Abtrennen des Kopfes mit kraftvollen Erweiterung auf der Abschnittsebene abgeschlossen ist <strong>(Abbildung 1).</strong> Das derzeitige Verfahren ist auf nicht fixierten Tieren gezeigt, wie die tatsächliche Hintergrundstudie HPLC-Analyse des abgeleiteten Hypophysen notwendig gemacht. Beachten Sie jedoch, dass genau die gleiche Technik , die auf Tiere transkardial mit Paraformaldehyd <sup class="xref"><sup>3,</sup></sup> <sup class="xref">5</sup> fixiert eingesetzt <sup>wird,</sup> <sup class="xref"><sup>14,</sup></sup> <sup class="xref"><sup>15,</sup></sup> <sup class = &quot;Xref&quot;&gt; <sup>16,</sup> <sup class="xref"><sup>17,</sup></sup> <sup class="xref"><sup>18,</sup></sup> <sup class="xref"><sup>19,</sup></sup> <sup class="xref"><sup>20,</sup></sup> <sup class="xref"><sup>25,</sup></sup> <sup class="xref"><sup>26,</sup></sup> <sup class="xref"><sup>27,</sup></sup> <sup class="xref"><sup>28,</sup></sup> <sup class="xref">31,</sup> obwohl alle Verfahren in einem solchen Fall muss von einem Abzug <sup class="xref">20</sup> versehen , unter guter Belüftung durchgeführt werden. Die Wahl des nativen Dissektion Vergleich <em>in toto</em> -Fixierung und anschließende Präparation sollte daher lediglich abhängig von den erforderlichen Nachbearbeitungsverfahren <em>(zB</em> herkömmliche Histologie, Immunhistochemie, HPLC und FISH) <sup class="xref">32.</sup> </p><p class="jove_content"> Wie in den folgenden Abschnitten besprochen haben wir chirurgische Instrumente zu verwenden nichtmotorisierten bevorzugt. Der Schädel von Schweinen im Alter von über 1-2 Jahren kann jedoch sein, so robust, dass Schädelentfernung mit dem demonstriert Instruments nicht möglich ist, was die Verwendung von motorisierten Instrumenten wie Craniotome, oszillierende Sägen und elektrische Bohrer <sup class="xref">32.</sup> In diesem Fall ist es immer noch empfohlen, die Protokollschritten oben angegeben zu folgen, damit die Vorteile des natürlich vorkommenden Schädel Einspeisepunkten und Stirnhöhle Entwicklung. Die vorgestellte vorderen Zugang zur Schädelbasis ist elegant und einfach, aber die beschriebene stumpfe Freigabe der Riechkolben muss ohne direkte visuelle Führung <strong>(3D),</strong> im Gegensatz zu den mehr posterior Freisetzung des Gehirns <strong>(Abbildung 3EF)</strong> durchgeführt werden. Die ventrale Teil der Riechkolben kann daher einen gewissen Grad an unkontrollierte Schäden erleiden, die nur vermieden werden können, wenn nötig, durch die vordere Schädelbasis Bohren ventral heraus zu den Leuchtmitteln, bevor der Lösevorgang eingeleitet wird. Beachten Sie, dass die Manipulation, insbesondere von nicht fixierten Hirngewebe während Schädelentfernung, wurde in der histologi führen angezeigt<html> cal dunklen neuron Artefakts, die <sup class="xref">33</sup> bis falschen Schlüssen in neurotoxikologischen Studien führen kann. </p><p class="jove_content"> Der Schädelknochen Entfernungsprozess kann auch mit Maschinen wie Craniotome, oszillierenden Sägen und elektrische Bohrer <sup class="xref">32</sup> durchgeführt werden. Diese können den Prozess beschleunigen, aber sie werden auch das Risiko von Schäden an den zugrunde liegenden neuralen Strukturen erhöhen. Solche Geräte können auch teuer sein und werden höchstwahrscheinlich zu den meisten Labors nicht zur Verfügung. Wir haben daher bevorzugt das derzeitige Verfahren zu demonstrieren , unter Verwendung von nicht-motorisierte chirurgische Geräte <strong>(Table of Materials)</strong> , die leicht zu erreichen und zu nutzen. </p><p class="jove_content"> Die beschriebene und dargestellte Technik wird, wenn sie richtig verwendet wird , ermöglichen , die Belichtung und der Entfernung des postmortalen Schweinehirn, Hypophyse, und / oder zervikalen Rückenmark <strong>(5),</strong> in Gewebestücke führt , die für die weitere makroskopische Analyse gut geeignet sind ,&quot;Xref&quot;&gt; <sup>5,</sup> <sup class="xref"><sup>6,</sup></sup> <sup class="xref"><sup>19,</sup></sup> <sup class="xref"><sup>26,</sup></sup> <sup class="xref">31,</sup> in dem Gehirn sectioning Brammen <sup class="xref">28</sup> und anschließende chemische Analyse und / oder Verarbeitung histologische <sup class="xref"><sup>6,</sup></sup> <sup class="xref"><sup>14,</sup></sup> <sup class="xref"><sup>15,</sup></sup> <sup class="xref"><sup>16,</sup></sup> <sup class="xref"><sup>17,</sup></sup> <sup class="xref"><sup>25,</sup></sup> <sup class="xref"><sup>26,</sup></sup> <sup class="xref"><sup>27,</sup></sup> <sup class="xref">31.</sup> </p>

<html><p class="jove_content"> Translationale Neurowissenschaften Studien bei Schweinen immer beliebter in den letzten zwei Jahrzehnten worden. Die große Größe des Schweinehirns ermöglicht die Verwendung von herkömmlich klinischen brain Imager und der unmittelbaren Nutzung und Prüfung von neurochirurgischen Verfahren und Geräten aus der menschlichen Klinik <sup class="xref"><sup>1,</sup></sup> <sup class="xref"><sup>2,</sup></sup> <sup class="xref"><sup>3,</sup></sup> <sup class="xref"><sup>4,</sup></sup> <sup class="xref"><sup>5,</sup></sup> <sup class="xref"><sup>6,</sup></sup> <sup class="xref"><sup>7,</sup></sup> <sup class="xref">8.</sup> In den letzten 20 Jahren, Schweine, insbesondere Minipigs <em>(zB</em> minipig Göttingen), wurden verwendet , neuromodulatorischen Behandlungsmodalitäten, wie beispielsweise Stammzelltransplantation zu untersuchen; viraler Vektor Transfektion; und tiefe Hirnstimulation gerichtet gegen Parkinson - Krankheit, Übergewicht, Depression und Alzheimer Krankheit <sup class="xref"><sup>2,</sup></sup> <sup class="xref"><sup>6,</sup></sup>= &quot;Xref&quot;&gt; <sup>9,</sup> <sup class="xref"><sup>10,</sup></sup> <sup class="xref"><sup>11,</sup></sup> <sup class="xref"><sup>12,</sup></sup> <sup class="xref"><sup>13,</sup></sup> <sup class="xref"><sup>14,</sup></sup> <sup class="xref"><sup>15,</sup></sup> <sup class="xref"><sup>16,</sup></sup> <sup class="xref">17.</sup> Dies wurde durch die Entwicklung von Stereotaxierahmen und chirurgische Ansätze folgte dem minipig CNS <sup class="xref"><sup>3,</sup></sup> <sup class="xref"><sup>18,</sup></sup> <sup class="xref"><sup>19,</sup></sup> <sup class="xref"><sup>20,</sup></sup> <sup class="xref">21</sup> zu manipulieren. Die eingeleiteten CNS Veränderungen in lebenden Tieren unter Verwendung von brain imaging (PET <sup class="xref"><sup>10,</sup></sup> <sup class="xref"><sup>13,</sup></sup> <sup class="xref"><sup>22,</sup></sup> <sup class="xref">24</sup> und MR <sup class="xref">23),</sup> Zystometrie <sup class="xref"><sup>11,</sup></sup> <sup class="xref"><sup>12,</sup></sup> <sup class="xref">25,</sup> Ganganalyse evaluiert<sup class="xref">17,</sup> neurologische Bewertung <sup class="xref"><sup>9,</sup></sup> <sup class="xref">17</sup> und Obduktion basierend auf der Histologie und stereologischen Analyse <sup class="xref"><sup>14,</sup></sup> <sup class="xref"><sup>15,</sup></sup> <sup class="xref"><sup>17,</sup></sup> <sup class="xref"><sup>26,</sup></sup> <sup class="xref"><sup>27,</sup></sup> <sup class="xref">31.</sup> Jedoch erfordert postmortale Analyse die Belichtung und die Entfernung des Schweinehirns, das keine leichte Aufgabe ist, als ein dicker, knöcherner Schädel und ein faseriger dural das Schweinehirn abdeckt umgeben. </p><p class="jove_content"> Das Ziel dieses Papiers und Lehr-Video ist zu beschreiben, wie die Obduktion Schweinehirn und Hypophyse können unter Verwendung von nicht-motorisierte chirurgische Instrumente in 15-20 min in einem intakten Zustand belichtet und entfernt werden. Das Lehr-Video und fotografische Abbildungen zeigen männlich minipigs (Alter: 6 Monate, Körpergewicht: 20-25 kg) für eine anatomische Studie über die minipig Hypophyse verwendet. </p>

<table><tbody><tr><td>Heavy Scalpel Handle #4</td><td>FST (Fine Science Tools)</td><td>10008-13</td><td>Good for skin incision and soft tissue removal</td></tr><tr><td>Non-Sterile Scalpel Blades #23</td><td>FST&amp;nbsp;</td><td>10023-00</td><td></td></tr><tr><td>Scalpel Handle #7</td><td>FST&amp;nbsp;</td><td>10007-12</td><td>Optimal for dural incision and precision work</td></tr><tr><td>Non-Sterile Scalpel Blades #11</td><td>FST&amp;nbsp;</td><td>10011-00</td><td></td></tr><tr><td>Surgical Forceps</td><td>FST&amp;nbsp;</td><td>11024-18</td><td>The tip of the surgical forceps ensure a firm grip&amp;nbsp;</td></tr><tr><td>Kerrison Bone Punch</td><td>Aesculap Neurosurgery</td><td>FF713R</td><td>Must be robust, bite size 3-5 mm</td></tr><tr><td>Bone Rongeur</td><td>Aesculap Neurosurgery</td><td>MD615</td><td>Must be robust, bite size 15 x 5 mm</td></tr><tr><td>Bone Rongeur</td><td>Aesculap Neurosurgery</td><td>FO551R</td><td>Must be robust, bite size 25 x 15 mm&amp;nbsp;</td></tr><tr><td>Bone Chisel</td><td>Lawton</td><td>67-0335</td><td>The size of the chisel head should not exceed 20 mm</td></tr><tr><td>Mallet (Hammer)</td><td>Millarco</td><td>5624108</td><td>Weigth 300 g, length 30 cm, head hit area size 2 x 2 cm</td></tr><tr><td>Micro-Scissor</td><td>FST&amp;nbsp;</td><td>14002-14&amp;nbsp;&amp;nbsp;</td><td></td></tr><tr><td>Dissector</td><td>Aesculap Neurosurgery</td><td>OL165R</td><td></td></tr><tr><td>G&amp;ouml;ttingen minipigs</td><td>Ellegaard G&amp;ouml;ttingen Minipigs A/S, Denmark</td><td></td><td></td></tr><tr><td>Euthanimal</td><td></td><td></td><td>pentobarbital</td></tr><tr><td>Ketamine</td><td>Pfizer</td><td></td><td></td></tr><tr><td>Midazolam&amp;nbsp;</td><td>Hameln Pharmaceuticals</td><td></td><td></td></tr></tbody></table>

exposure of the pig cns for histological analysis: a manual for decapitation, skull opening, and brain removal

<p class="jove_content"> Schweine haben in Großtier translationalen neurowissenschaftlichen Forschung als wirtschaftlich und ethisch möglich Ersatz für nichtmenschliche Primaten immer beliebter geworden. Die große Größe des Gehirn des Schweins ermöglicht die Verwendung von herkömmlich klinischem Gehirn Imager und der direkten Nutzung und Prüfung von neurochirurgischen Verfahren und Geräten aus der menschlichen Klinik. Weitere makroskopische und histologische Analyse erfordert jedoch postmortem Exposition des Schweins Zentralnervensystem (ZNS) und die anschließende Entfernung des Gehirns. Dies ist keine einfache Aufgabe, da das Schwein CNS durch einen dicken, knöchernen Schädels und der Wirbelsäule eingekapselt ist. Das Ziel dieses Papier und Lehr-Video ist zu beschreiben, wie die Obduktion Schweinehirn und die Hypophyse in einem intakten Zustand, geeignet für die anschließende makroskopische und histologische Analyse belichten und zu entfernen. </p>

<p class="jove_content" fo:keep-together.within-page="1"> Um das Gewebematerial vor dem Austrocknen zu verhindern, wird empfohlen, das Gehirn und Hypophyse entfernt in einem Glas mit Fixativ oder isotonischer Kochsalzlösung gefüllt zu speichern, unmittelbar nach makroskopischer Analyse durchgeführt wurde. Das Gewebematerial kann in der Fixiermittel für Jahre gespeichert werden, während der Lagerung in isotonischer Kochsalzlösung, auch in einem Kühlschrank wird auf Gewebezerfall mit der Zeit führen. </p><p class="jove_content" fo:keep-together.within-page="1"> Die Hypophyse entfernt auch durch Eintauchen wird in Trockeneis gekühlten Flüssigkeit 2-methylbutan unmittelbar eingefroren, während das intakte Schweinehirn zum direkten Gefrieren <sup class="xref">28</sup> zu groß ist. Stattdessen ist es empfehlenswert , das Schweinehirn zu schneiden, wie zuvor <sup class="xref">28</sup> gezeigt, in 9-15 mm dicke koronale parallel Gewebeplatten , die <em>in toto</em> eingefroren werden können und Kryostat Schnitt in 40 &amp; mgr; m dicke Abschnitte <sup class="xref"><sup>5,</sup></sup> <sup class="xref"><sup>18,</sup></sup> </sup&gt; <sup class="xref"><sup>26,</sup></sup> <sup class="xref">28.</sup> Alternativ können spezifische Hirnareale von dem Abnehm intakten Gehirn oder in Scheiben geschnitten brain Bramme und vorzulegenden weiterer histologischer Verarbeitung nach Vibratom sectioning <sup class="xref">30,</sup> Paraffin / Methacrylat - Einbettungs und Mikrotom sectioning <sup class="xref"><sup>6,</sup></sup> <sup class="xref"><sup>17,</sup></sup> <sup class="xref">27</sup> oder Einfrieren und Kryostat sectioning <sup class="xref">6</sup> frei seziert <sup>werden,</sup> <sup class="xref"><sup>14,</sup></sup> <sup class="xref"><sup>15,</sup></sup> <sup class="xref">25.</sup> In unserer Einstellung werden die Schweineschlachtkörper schließlich in bestimmten Kunststoffbehälter gegeben und in einem speziellen Kühlraum gelagert, bis sie zu einer biologischen abbau Anlage gesammelt und transportiert werden. </p><p class="jove_content" fo:keep-together.within-page="1"> Bei der Verwendung von nicht-motorisierten chirurgischen Werkzeugen (Tabelle von Materialien), die beschriebenen Technik <strong>(Figuren 1</strong> </stron<html> g&gt; - <strong>3)</strong> ermöglicht, in etwa 15-20 min, bleiben die Entfernung der intakten Schweinehirn <strong>(Abbildung 5AB),</strong> während die abgetrennten Hirnnerven und Hypophyse zum Schädel Boden <strong>(4A)</strong> verbunden ist . Ebenso kann die Hypophyse einfach entfernt werden, intakt, nachdem das darüberliegende Gehirn entfernt und das durale sellar Membran <strong>(Figur 4</strong> und <strong>5C)</strong> freigibt . </p><p class="jove_content" fo:keep-together.within-page="1"> Die sich ergebende Gehirn und / oder Hypophysen <strong>(Figur 5)</strong> kann anschließend auf makroskopische Analyse unterzogen werden , die, abgesehen von der direkten visuellen Inspektion, können Größe und Volumenmessungen <sup class="xref">31</sup> umfassen. Dies kann durch orientierte Schneiden in kleiner brain Brammen <sup class="xref"><sup>28,</sup></sup> <sup class="xref">29</sup> geeignet für die chemische Analyse und / oder weitere histologisches Präparat, Färbung und mikroskopische Analyse <sup class="xref">6</sup> <sup>folgen,</sup>s = &quot;Xref&quot;&gt; <sup>14,</sup> <sup class="xref"><sup>15,</sup></sup> <sup class="xref"><sup>17,</sup></sup> <sup class="xref"><sup>25,</sup></sup> <sup class="xref"><sup>26,</sup></sup> <sup class="xref">27.</sup> </p><p class="jove_content" fo:keep-together.within-page="1"><img alt="Abbildung 5" src="/files/ftp_upload/55511/55511fig5.jpg" /><br /> <strong>Abbildung 5:</strong> <strong> </strong> Das minipig Gehirn <strong>(A</strong> und <strong>B)</strong> und der Hypophyse <strong>(C).</strong> <strong>(A)</strong> , Gehirn, laterodorsal Ansicht (BS, brainstem; CB, Cerebellum, und CRB, Cerebrum). <strong>(B)</strong> Gehirn, ventral (BS, brainstem; CB, Cerebellum, und CRB, Cerebrum). <strong>(C)</strong> Pituitary, posterior Ansicht (AH, Adenohypophyse und NH, neurohypophyse). Maßstabsbalken (A und B) = 10 mm, (C) = 5 mm. <a href="https://ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/55511/55511fig5large.jpg" target="_blank">Bitte klicken Sie hier</a> , um <a href="https://ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/55511/55511fig5large.jpg" target="_blank">eine größere Version dieser Figur zu sehen.</a> </p>

Watch this Scientific Journal Video about Exposure of the Pig CNS for Histological Analysis: A Manual for Decapitation, Skull Opening, and Brain Removal at JoVE.com

Exposure of the Pig CNS for Histological Analysis: A Manual for Decapitation, Skull Opening, and Brain Removal

<p class="jove_content"> Das Ziel dieses Papier und Lehr-Video ist zu beschreiben, wie die Obduktion Schweinehirn und Hypophyse in einem intakten Zustand, geeignet für die anschließende makroskopische und histologische Analyse belichten und zu entfernen. </p>

Belichtung des Schweins CNS für die histologische Analyse: Ein Handbuch für Enthauptung, Schädel öffnen und Gehirn Removal

Pigs have become increasingly popular in large-animal translational neuroscience research as an economically and ethically feasible substitute to ...

exposure-pig-cns-for-histological-analysis-manual-for-decapitation

Universidad Científica del Sur

Research

Education

JoVE Core

JoVE Journal

Anatomy and Physiology

Neuroscience

Unit 1

The Axial Skeleton

SCIENTISTS IN ACTION

15.7K Aufrufe.  Aalborg University Hospital.  Das Ziel dieses Papier und Lehr-Video ist zu beschreiben, wie die Obduktion Schweinehirn und Hypophyse in einem intakten Zustand, geeignet für die anschließende makroskopische und histologische Analyse belichten und zu entfernen. 

Serie: Exposure of the Pig CNS for Histological Analysis: A Manual for Decapitation, Skull Opening, and Brain Removal

Cell Structure

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

Cell Structure: Visualizing Onion and Human Cells - Concept

Zellstruktur

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

Lehre

JoVE Lab Manual

Biologie

GRUNDLAGEN

Lab Techniques

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

Accuracy, Precision, Uncertainty, and Significant Figures - Procedure

Labortechniken

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

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

Chemie

Lab Manual Sub Articles

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://www-jove-com.bdigitaluss.remotexs.co/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://www-jove-com.bdigitaluss.remotexs.co/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://www-jove-com.bdigitaluss.remotexs.co/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

Tierverhalten

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 ...

Ökologie

Extinction

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

Aussterben

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

Cell Division

<ol class="note-items"><li data-note-item="1" data-parent-collapse="">Observing the Cell Cycle in a Root Tip
<button class="expand">Expand</button><ul class="lab-items"><li data-sub-note-item="1-1"><div class="lab-step">Hypotheses: The experimental hypothesis is that in root tips slices that have been treated with nocodazole, a chemical that interferes with microtubular polymerization, all of the cells will be arrested at the same stage of the cell cycle and that in untreated onion tip slices all of the different stages of the cell cycle will be visualized. The null hypothesis is that there will be no differences in mitosis between the two groups and that different stages of the cell cycle will be visualized. 
</div></li><li data-sub-note-item="1-2"><div class="lab-step">To prepare for this experiment you will use an onion that has been placed in water for several days until new white root tips have begun to sprout. Use a razor blade to cut a 1 cm piece from the end of one of the root tips. 
</div></li><li data-sub-note-item="1-3"><div class="lab-step">Place the slice of onion into a microcentrifuge tube labeled &#8220;N&#8221; for the nocodazole condition. 
</div></li><li data-sub-note-item="1-4"><div class="lab-step">Then, cut a second 1 cm piece of onion root tip and place in a microcentrifuge tube labeled &#8220;C&#8221; for the control condition.
</div></li><li data-sub-note-item="1-5"><div class="lab-step">Add 2 &#181;L of the 5 mg/mL nocodazole to 1 mL of water in the microcentrifuge tube labeled N and 1 mL of water only to the tube labeled C. 
</div></li><li data-sub-note-item="1-6"><div class="lab-step">Leave the tubes containing the root tips to incubate for 12 hours and wipe down the workspace with 70% ethanol.
</div></li><li data-sub-note-item="1-7"><div class="lab-step">When incubation is complete, set a heat block to 60 &#186;C.
</div></li><li data-sub-note-item="1-8"><div class="lab-step">Wash the nocodazole treated tips with water and then aspirate the liquid.
</div></li><li data-sub-note-item="1-9"><div class="lab-step">Repeat this wash two more times, for a total of three washes. 
</div></li><li data-sub-note-item="1-10"><div class="lab-step">Fill both tubes with 1 mL of one normal hydrochloric acid. Incubate the tubes in a 60 &#186;C heat block for 12 - 15 min, then discard the acid into a waste flask.
</div></li><li data-sub-note-item="1-11"><div class="lab-step">Now, add 1 mL of distilled water drop wise to the tubes at least three times to rinse the samples.
</div></li><li data-sub-note-item="1-12"><div class="lab-step">Now add one microliter of DAPI stain to 10 mL of phosphate buffered saline (PBS).
</div></li><li data-sub-note-item="1-13"><div class="lab-step">Then place 50 &#181;L of this dilute DAPI stain into each tube, and leave them at room temperature to incubate for 12 min. 
</div></li><li data-sub-note-item="1-14"><div class="lab-step">After this, preform three distilled water washes as in step 10. 
</div></li><li data-sub-note-item="1-15"><div class="lab-step">Label one microscope slide as 'Control' and another as 'Nocodazole'. 
</div></li><li data-sub-note-item="1-16"><div class="lab-step">Transfer the stained roots onto their respective microscope slides and add a drop of water to the control slide and a drop of nocodazole to the nocodazole slide.
</div></li><li data-sub-note-item="1-17"><div class="lab-step">Use the razor blade to remove the unstained portions of each root tip and place a glass cover slip over each sample.
</div></li><li data-sub-note-item="1-18"><div class="lab-step">Carefully press down on each cover slip with a gloved fingertip, and then let the slides sit for 10 min. 
</div></li><li data-sub-note-item="1-19"><div class="lab-step">Finally, view the slides under the 40X objective of a fluorescence microscope and capture images of your root tip cell preparations.
</div></li></ul></li><li data-note-item="2" data-parent-collapse="">Understanding the Loss of Cell Cycle Control
<button class="expand">Expand</button><ul class="lab-items"><li data-sub-note-item="2-1"><div class="lab-step">NOTE: Before cells can pass through all of the stages of the cell cycle there are several checkpoints they must go through. These checkpoints are regulated by cyclin and cyclin-dependent kinase proteins, or CDKs. If the cyclins and CDKs determine that previous cellular events have not been adequately completed they will arrest the cells at this step and prevent them from proliferating. In cancer cells one or several of these molecular restraints for regulating cell division are lost and this allows the damaged cells to escape from cell proliferation control. These abnormal cells develop into tumors which are masses of uncontrolled proliferating cells that interfere with the normal functions of the body. Notice how before it divides this cancer cell rounds up and refracts light very strongly under the light microscope. This is because not all cancer cells successfully divide into daughter cells after each round of mitosis. This will mean that they often contain an excess of organelles and DNA and this increase in cellular content means they will refract light more intensely than normal cells do. 
</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">Examine your collected images and record in Table 1 the different cell structures and stages that could be identified in the onion tip slice that was treated with nocodazole. <a href="https://www-jove-com.bdigitaluss.remotexs.co/CDNSource/lm/labs/10/Table_1_Cell_division.xlsx" style="display:block;margin:20px;" target="__blank">Click Here to download Table 1</a>
</div></li><li data-sub-note-item="3-2"><div class="lab-step">If there are any cells undergoing mitosis, note what stages of cell division you see.
</div></li><li data-sub-note-item="3-3"><div class="lab-step">Record the percentage of cells in each stage of mitosis in Table 1. 
</div></li><li data-sub-note-item="3-4"><div class="lab-step">Next, examine the images of the untreated onion tip slice, and record what cell structures can you identify. 
</div></li><li data-sub-note-item="3-5"><div class="lab-step">If there are any cells undergoing mitosis, consider what stages of cell division you see, and record the percentages of cells in each stage of mitosis in the Table 1. 
</div></li></ul></li></ol>

Cell Division: Observing Cell Cycle in a Root Tip - Procedure

Zellteilung

Observing the Cell Cycle in a Root Tip
ExpandHypotheses: The experimental hypothesis is that in root tips slices that have been treated with nocodazole, a ...

Zellprozesse

Photosynthesis

<ol class="note-items"><li data-note-item="1" data-parent-collapse="">Plant Pigment Chromatography
<button class="expand">Expand</button><ul class="lab-items"><li data-sub-note-item="1-1"><div class="lab-step">NOTE: In this experiment you will separate pigments from spinach leaves using chromatography paper. Individual pigments travel along the paper at different rates and may have different colors. By calculating the relative distance the pigments travel, their resolution factor, and comparing them with literature values, you can identify different pigments.  HYPOTHESES: In this exercise the experimental hypothesis is that there will be multiple pigments within the spinach leaves that absorb different wavelengths of sunlight. The null hypothesis is that there is only one type of pigment within the spinach leaf.
</div></li><li data-sub-note-item="1-2"><div class="lab-step">Use a pencil to make a line two centimeters from one end of the chromatography paper.
</div></li><li data-sub-note-item="1-3"><div class="lab-step">Then, lay a pipe cleaner horizontally across the top of a clean 400 mL beaker.
</div></li><li data-sub-note-item="1-4"><div class="lab-step">Place the pencil-marked side of the chromatography strip at the bottom of the beaker.
</div></li><li data-sub-note-item="1-5"><div class="lab-step">Next, wrap the paper around the pipe cleaner so that the bottom edge is barely touching the bottom of the beaker and then secure it with a paperclip.
</div></li><li data-sub-note-item="1-6"><div class="lab-step">When the paper is secured around the pipe cleaner, remove it from the beaker, and then place a patted-dry spinach leaf over the marked line on the chromatography paper.
</div></li><li data-sub-note-item="1-7"><div class="lab-step">Roll a coin over the spinach leaf along the pencil line going back and forth multiple times and applying steady pressure. When the leaf is removed, a green line should be clearly present.
</div></li><li data-sub-note-item="1-8"><div class="lab-step">Next, place 8 mL of chromatography solvent in the beaker.
</div></li><li data-sub-note-item="1-9"><div class="lab-step">Lower the chromatography strip into the beaker so that the edge of the paper touches the solvent but the green line does not. Adjust the pipe cleaner if needed.
</div></li><li data-sub-note-item="1-10"><div class="lab-step">Without disturbing the beaker, observe the solvent as it moves up the paper and the individual pigments separate.
</div></li><li data-sub-note-item="1-11"><div class="lab-step">When the solvent has traveled half way up the chromatography paper, which will take approximately 10 minutes, and the pigments have separated into well-defined bands, remove the paper from the beaker.
</div></li><li data-sub-note-item="1-12"><div class="lab-step">Mark how far the solvent traveled with a pencil and then allow the paper to dry. NOTE: The solvent evaporates quickly.
</div></li><li data-sub-note-item="1-13"><div class="lab-step">Next, record the number of visible bands and describe their color and relative size.
</div></li><li data-sub-note-item="1-14"><div class="lab-step">Measure how far the solvent and pigments traveled, and record this information for each pigment in Table 1.  <a href="https://www-jove-com.bdigitaluss.remotexs.co/CDNSource/lm/labs/7/Table_1_Photosynthesis.xlsx" style="display:block;margin:20px;" target="__blank">Click Here to download Table 1</a>
</div></li><li data-sub-note-item="1-15"><div class="lab-step">Dispose of the chromatography solvent in a waste container under a fume hood. Throw the chromatography strips into the regular trash, and then clean the beakers with soap and water.
</div></li></ul></li><li data-note-item="2" data-parent-collapse="">Floating Leaf Discs in a Vacuum
<button class="expand">Expand</button><ul class="lab-items"><li data-sub-note-item="2-1"><div class="lab-step">NOTE: In this experiment you will indirectly observe photosynthesis and cellular respiration using a floating leaf disc in a solution. During photosynthesis, air bubbles will cause the leaves to float, and during respiration, the discs will sink. HYPOTHESES: In this exercise, the experimental hypothesis is that the leaf discs will have a greater rate of photosynthesis in the bicarbonate solution, because bicarbonate provides added CO<sub>2</sub> to fuel photosynthesis, causing more leaf discs to float. Additionally, all of the discs will sink in dark conditions as they perform cellular respiration. The null hypothesis is that there will be no difference in the rate of photosynthesis, and therefore the number of floating discs, between the bicarbonate and water, or light and dark treatments.
</div></li><li data-sub-note-item="2-2"><div class="lab-step">To place leaf discs under vacuum, first remove the plungers from two 20 mL syringes, and then place 10 leaf discs inside each syringe tube. Label one syringe &#8220;bicarbonate&#8221;, and label the other syringe &#8220;water&#8221;.
</div></li><li data-sub-note-item="2-3"><div class="lab-step">Replace the plungers and push the plunger until only a small amount of air remains in the syringe. Take care not to damage the leaf discs.
</div></li><li data-sub-note-item="2-4"><div class="lab-step">Pull 5 mL of the bicarbonate solution into one of the syringes. Invert and swirl the syringe to suspend the leaf discs in solution.
</div></li><li data-sub-note-item="2-5"><div class="lab-step">Push as much air out as possible without expelling the solution or damaging the leaf discs.
</div></li><li data-sub-note-item="2-6"><div class="lab-step">Then pull 5 mL of the water solution into the other syringe and swirl it as previously described (step 3).
</div></li><li data-sub-note-item="2-7"><div class="lab-step">To create a vacuum, hold one finger over the tip of the syringe while pulling back on the plunger. Hold this for 10 seconds while swirling the syringe to keep the leaf discs in suspension.
</div></li><li data-sub-note-item="2-8"><div class="lab-step">Then, release the vacuum.  NOTE: The discs should have absorbed the solution into the air spaces in their tissues and you should see them sink. If the discs don&#39;t sink, you can repeat the vacuum creation up to three times.
</div></li><li data-sub-note-item="2-9"><div class="lab-step">Next, add 50 mL of bicarbonate solution to a plastic cup or a glass beaker, and then gently add the discs from the bicarbonate vacuum syringe.
</div></li><li data-sub-note-item="2-10"><div class="lab-step">For the control, add the same amount of water to an identical cup, and then add the leaf discs from the water vacuum syringe. Label the containers appropriately.
</div></li><li data-sub-note-item="2-11"><div class="lab-step">Place both cups under a light source.
</div></li><li data-sub-note-item="2-12"><div class="lab-step">Every five minutes record the number of discs floating on the surface of the cup in Table 3 until 20 minutes have passed.  <a href="https://www-jove-com.bdigitaluss.remotexs.co/CDNSource/lm/labs/7/Table_3_Photosynthesis.xlsx" style="display:block;margin:20px;" target="__blank">Click Here to download Table 3</a>
</div></li><li data-sub-note-item="2-13"><div class="lab-step">Next, remove the cups from the light source and then swirl them so that the discs at the surface intermix with any gases also at the surface.
</div></li><li data-sub-note-item="2-14"><div class="lab-step">Move the cups to a dark place. Every five minutes record the number of leaf discs floating at the surface until 20 minutes have passed. Swirl the cup each time before placing it back in the dark.
</div></li><li data-sub-note-item="2-15"><div class="lab-step">To clean up, dispose of the leaf discs in the trash, and pour the bicarbonate solution down the drain. Wash the syringes and cups thoroughly.
</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">NOTE: In the first experiment, you observed how far pigments from spinach leaves traveled on chromatography paper. Different pigments absorb light at different wavelengths.
</div></li><li data-sub-note-item="3-2"><div class="lab-step">Using colored pens or pencils, draw the positions of the pigment bands and the solvent on Figure 3.
</div></li><li data-sub-note-item="3-3"><div class="lab-step">Calculate the retention factor, or Rf values for the pigments, which is done by dividing the distance the pigment in question moved up the paper from the line by the distance the solvent moved up the paper from the line.
</div></li><li data-sub-note-item="3-4"><div class="lab-step">Compare your calculated Rf values to those in Table 2 to determine the identity of the pigment. <a href="https://www-jove-com.bdigitaluss.remotexs.co/CDNSource/lm/labs/7/Table_2_Photosynthesis.xlsx" style="display:block;margin:20px;" target="__blank">Click Here to download Table 2</a>
</div></li><li data-sub-note-item="3-5"><div class="lab-step">Record these data in Table 1. NOTE: In the second experiment, you observed floating and sinking leaf discs as an indirect measurement of photosynthesis and respiration.
</div></li><li data-sub-note-item="3-6"><div class="lab-step">Graph the results with time and minutes on the x-axis and number of floating discs on the y-axis. Use two different lines to represent the water control and the bicarbonate treatment.
</div></li><li data-sub-note-item="3-7"><div class="lab-step">Add a line to the graph to indicate the point where the discs were removed from the light condition and placed into the dark.
</div></li><li data-sub-note-item="3-8"><div class="lab-step">Next, starting with the bicarbonate condition, use the graph to determine the point at which 50% of the leaf discs were floating. This is referred to as the effective time, or ET50.  NOTE: You will notice that the discs likely hit the 50% floating mark once in the light condition and then again in the dark condition.
</div></li><li data-sub-note-item="3-9"><div class="lab-step">Your water samples may or may not have reached the ET50 mark. If they did, add the line for this sample also.
</div></li><li data-sub-note-item="3-10"><div class="lab-step">Finally, compare your ET50 values and graphs with the rest of the class.
</div></li></ul></li></ol>

Studying Photosynthesis by Leaf Disc Experiment-Procedure

Photosynthese

Plant Pigment Chromatography
ExpandNOTE: In this experiment you will separate pigments from spinach leaves using chromatography paper. Individual pigments ...

Microbial and Fungal Diversity

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

Microbial Colony and Fungal Diversity - Procedure

Mikrobielle und Pilzvielfalt

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

Evolution

Animal Diversity

<ol class="note-items"><li data-note-item="1" data-parent-collapse="">Form and Function of Crickets and Crayfish
<button class="expand">Expand</button><ul class="lab-items"><li data-sub-note-item="1-1"><div class="lab-step">NOTE: To observe the relationship between form and function in animals, you will be examining and dissecting a cricket and a crayfish. Each pair of students will receive one of each of these organisms. When dissecting, be sure to wear gloves, closed-toed shoes, and safety goggles. Take care when handling dissecting tools, as they may be sharp. HYPOTHESES: In this experiment, the experimental hypothesis could be that the cricket and crayfish have similar body structures because they are both arthropods, but that they will have some specific adaptations for their different habitats and lifestyles. The null hypothesis might be that the cricket and crayfish have identical adaptations and body plans.
</div></li><li data-sub-note-item="1-2"><div class="lab-step">To begin, place the cricket on its back and observe its appendages.
</div></li><li data-sub-note-item="1-3"><div class="lab-step">In Table 1, make detailed notes about your observations and try to predict the potential function of each of the forms you observe. <a href="https://www-jove-com.bdigitaluss.remotexs.co/CDNSource/lm/labs/27/Table_1_Animal_Diversity.xlsx" style="display:block;margin:20px;" target="__blank">Click Here to download Table 1</a>
</div></li><li data-sub-note-item="1-4"><div class="lab-step">Do the same with the wings of the cricket if they are present.
</div></li><li data-sub-note-item="1-5"><div class="lab-step">Consider how the structure of the wings benefits the cricket. 
</div></li><li data-sub-note-item="1-6"><div class="lab-step">If the cricket does not have wings, consider the reasons behind their absence. 
</div></li><li data-sub-note-item="1-7"><div class="lab-step">If the cricket is female, it will have an ovipositor. This can be observed as a long dark tube protruding from the posterior end and it is used to deposit eggs.
</div></li><li data-sub-note-item="1-8"><div class="lab-step">Consider why the ovipositor is useful. 
</div></li><li data-sub-note-item="1-9"><div class="lab-step">Next, flip the cricket over onto its back and observe the mouth parts under a microscope. NOTE: Use a probe to move structures when necessary.
</div></li><li data-sub-note-item="1-10"><div class="lab-step">Make a hypothesis about the size and type of prey the cricket consumes based upon your observations.
</div></li><li data-sub-note-item="1-11"><div class="lab-step">Continue to examine the external parts of the cricket, making hypotheses about their function until you have filled at least five rows in the table with your observations.
</div></li><li data-sub-note-item="1-12"><div class="lab-step">Using tweezers to hold the cricket down, carefully remove the wings and legs from the body with the scissors.
</div></li><li data-sub-note-item="1-13"><div class="lab-step">Next, position the body of the cricket so that its underside is facing down.
</div></li><li data-sub-note-item="1-14"><div class="lab-step">Again with the scissors, cut the thin outer exoskeleton from the anus to the head trying not to damage any of the organs underneath.
</div></li><li data-sub-note-item="1-15"><div class="lab-step">Use the tweezers to open the incision and dissecting pins to hold open the exoskeleton.
</div></li><li data-sub-note-item="1-16"><div class="lab-step">Next, use the water pitcher to carefully add water to the Petri dish until it reaches just below the top edge. This should make the exposed organs of the cricket easier to see.
</div></li><li data-sub-note-item="1-17"><div class="lab-step">Move the Petri dish to the stage of the dissecting microscope and turn on the light.
</div></li><li data-sub-note-item="1-18"><div class="lab-step">Use the cricket figure as a reference and observe the organs under the dissecting microscope. Where necessary, use the probe to move organs that may be blocking others.
</div></li><li data-sub-note-item="1-19"><div class="lab-step">Identify as many internal structures as possible and fill out the form and predicted function for at least three internal structures.
</div></li><li data-sub-note-item="1-20"><div class="lab-step">Next, you&#39;ll observe the outer and inner structures of the crayfish. Begin with the crayfish on its back and observe the appendages.
</div></li><li data-sub-note-item="1-21"><div class="lab-step">In Table 1, take notes about the structures you notice.
</div></li><li data-sub-note-item="1-22"><div class="lab-step">Observe the tail. Devise a hypothesis for its jointed structure. Consider how a crayfish moves in its environment.
</div></li><li data-sub-note-item="1-23"><div class="lab-step">Next, with the crayfish still on its back, look for the swimmerets under its tail.  NOTE: If the crayfish is male, the swimmerets nearest the legs are long extending between the legs. These are specialized reproductive structures that transfer sperm to the female. Females do not have these long structures, but instead have swimmerets that store developing embryos.
</div></li><li data-sub-note-item="1-24"><div class="lab-step">Consider the benefits of this strategy. Be sure to observe both a male and female crayfish, checking with neighboring students to identify these structures.
</div></li><li data-sub-note-item="1-25"><div class="lab-step">Next, observe the mouth parts of your crayfish using a probe.
</div></li><li data-sub-note-item="1-26"><div class="lab-step">Based on these structures, make a hypothesis about the size and type of prey you think crayfish consume.
</div></li><li data-sub-note-item="1-27"><div class="lab-step">Before beginning the dissection, be sure to fill in Table 1 with at least five external structures you observed in the crayfish along with their predicted function. With the organism lying legs down, use scissors to cut an incision in the exoskeleton from the tail to between the eyes. Take care to not disturb the organs underneath.
</div></li><li data-sub-note-item="1-28"><div class="lab-step">Next, remove the carapace as demonstrated in the video.
</div></li><li data-sub-note-item="1-29"><div class="lab-step">Open the tail and use the pins to secure the exoskeleton down to the dissecting tray exposing the organs.
</div></li><li data-sub-note-item="1-30"><div class="lab-step">Observe the organs first with the naked eye. Use the figure to identify the different body parts of the animal.
</div></li><li data-sub-note-item="1-31"><div class="lab-step">Then, if there is enough space on the stage, place the dissecting tray with the crayfish under the dissecting microscope to view these structures more closely.
</div></li><li data-sub-note-item="1-32"><div class="lab-step">Fill out at least three rows of Table 1 with these observations and predicted functions of internal structures in the crayfish.
</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 analyze the data, first separate into groups. Within your groups, compare observations from your tables.
</div></li><li data-sub-note-item="2-2"><div class="lab-step">Using these observations, create class predictions that relate the observed forms to the hypothesized functions for both the cricket and the crayfish. As a group, fill out Table 2 with at least five of these predictions and discuss how you would go about testing these hypotheses. <a href="https://www-jove-com.bdigitaluss.remotexs.co/CDNSource/lm/labs/27/Table_2_Animal_Diversity.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 the internal and external structures you observed in the cricket and crayfish. Fill out Table 3 with similarities and differences you noticed in the structures of these organisms. <a href="https://www-jove-com.bdigitaluss.remotexs.co/CDNSource/lm/labs/27/Table_3_Animal_Diversity.xlsx" style="display:block;margin:20px;" target="__blank">Click Here to download Table 3</a>
</div></li></ul></li></ol>

Animal Diversity: Form and Function of Crickets and Crayfish - Procedure

Tierische Vielfalt

Form and Function of Crickets and Crayfish
ExpandNOTE: To observe the relationship between form and function in animals, you will be examining and ...

DNA Isolation and Restriction Enzyme Analysis

<ol class="note-items"><li data-note-item="1" data-parent-collapse="">DNA Isolation
<button class="expand">Expand</button><ul class="lab-items"><li data-sub-note-item="1-1"><div class="lab-step">NOTE: In this experiment you will perform DNA isolation under two experimental conditions: one using a buffer containing the detergent SDS and one without detergent. Hypotheses: The alternate hypothesis for this experiment might be that the sample prepared without SDS, a strong anionic detergent that breaks apart cell membranes, will yield less DNA than the sample prepared with SDS. The null hypothesis for this experiment might be that both samples will yield an equal amount of DNA.
</div></li><li data-sub-note-item="1-2"><div class="lab-step">To begin collect a tissue sample for DNA isolation. Here you will be using your own cheek cells. Use a Popsicle stick to scrape the insides of your cheeks for approximately 30 s. IMPORTANT: Do not scrape hard enough to cause pain or draw blood.
</div></li><li data-sub-note-item="1-3"><div class="lab-step">Position a funnel in a 15-mL conical tube. 
</div></li><li data-sub-note-item="1-4"><div class="lab-step">Then take 10 mL of the 1% saline solution provided to you in a labeled cup and swish it around in your mouth without swallowing. 
</div></li><li data-sub-note-item="1-5"><div class="lab-step">Continue swishing for 90 s and then spit the saline solution containing your cheek cells into the funnel to collect in the conical tube.
</div></li><li data-sub-note-item="1-6"><div class="lab-step">Next, label two empty microcentrifuge tubes with your initials. 
</div></li><li data-sub-note-item="1-7"><div class="lab-step">Label one of the tubes as +SDS and the other as -SDS to differentiate the two treatments. 
</div></li><li data-sub-note-item="1-8"><div class="lab-step">Then add 1.5 to 2 mL of the saline solution containing the cheek cells to each tube.
</div></li><li data-sub-note-item="1-9"><div class="lab-step">Spin the tubes in a microcentrifuge for 30 s. 
</div></li><li data-sub-note-item="1-10"><div class="lab-step">Remove the tubes from the microcentrifuge and carefully pour off the supernatant without disturbing the cells collected at the bottom of each tube. 
</div></li><li data-sub-note-item="1-11"><div class="lab-step">Fill the tubes with more of the saline solution containing the cheek cells and repeat the centrifugation. 
</div></li><li data-sub-note-item="1-12"><div class="lab-step">Repeat this process taking care to evenly distribute cells between the +SDS and -SDS tubes until all of the cheek cells have been collected.
</div></li><li data-sub-note-item="1-13"><div class="lab-step">Next, add 1 mL of lysis buffer containing SDS to the tube marked as +SDS, and then resuspend the cell pellet by pipetting up and down. 
</div></li><li data-sub-note-item="1-14"><div class="lab-step">Then add 1 mL of lysis buffer without SDS to the tube marked with -SDS and resuspend the cell pellet by pipetting up and down.
</div></li><li data-sub-note-item="1-15"><div class="lab-step">After this add 10 &#181;L of proteinase K to each tube and then carefully place the tubes in the 56 &#186;C water bath for 10 min. 
</div></li><li data-sub-note-item="1-16"><div class="lab-step">After 10 minutes carefully remove the tubes from the water bath and add 100 &#181;L of 2.5 M NaCl to each tube. 
</div></li><li data-sub-note-item="1-17"><div class="lab-step">Close the tubes firmly and invert several times to mix.
</div></li><li data-sub-note-item="1-18"><div class="lab-step">Next, label one 15 mL conical tube as +SDS and another with -SDS. 
</div></li><li data-sub-note-item="1-19"><div class="lab-step">Pour the contents of each microcentrifuge tube into the corresponding 15 mL conical tube. 
</div></li><li data-sub-note-item="1-20"><div class="lab-step">Obtain one 10 mL aliquot of 100% ethanol from the freezer. 
</div></li><li data-sub-note-item="1-21"><div class="lab-step">Hold one of the conical tubes at an angle and slowly dispense 1 mL of 100% ethanol into the tube to form a layer on top of the liquid already in the tube.
</div></li><li data-sub-note-item="1-22"><div class="lab-step">Repeat this process for the second conical tube. 
</div></li><li data-sub-note-item="1-23"><div class="lab-step">Now look at both of your tubes. There should be a stringy precipitate present at the interface of the ethanol and the aqueous layer below it. This stringy material contains the isolated DNA. Take pictures of the DNA in the tube and make clear descriptive notes and drawings of your observations in your notebook. 
</div></li><li data-sub-note-item="1-24"><div class="lab-step">Using a glass stir rod, pick up the DNA from the +SDS tube and examine it. Repeat this for the -SDS tube, again recording your observations. Take note of whether one of the tubes appears to have more precipitate than the other, and why this might be.
</div></li><li data-sub-note-item="1-25"><div class="lab-step">Finally, discard all liquid waste into the appropriate waste container and return all tools to their proper locations in the lab.
</div></li></ul></li><li data-note-item="2" data-parent-collapse="">Restriction Enzyme Virtual Digest
<button class="expand">Expand</button><ul class="lab-items"><li data-sub-note-item="2-1"><div class="lab-step">To study how restriction enzymes work, first navigate to the New England Biolabs website: <a href="https://www.neb.com/" target="__blank">https://www.neb.com/</a>
</div></li><li data-sub-note-item="2-2"><div class="lab-step">The resources available at this site allow researchers to run virtual experiments before they are performed in the lab. Click on &#8220;Tools and Resources&#8221; to find the NEBcutter tool and open the program.
</div></li><li data-sub-note-item="2-3"><div class="lab-step">To the right of the search box find the list that says &#8220;Viral + phage&#8221;. From that list select Lambda. This is the name of the DNA you will be digesting. Select the Linear option where the tool says &#8220;the sequence is&#8221; and the &#8220;NEB enzymes&#8221; option where the tool says, &#8220;Enzymes to use&#8221;. Then click Submit.
</div></li><li data-sub-note-item="2-4"><div class="lab-step">The next page will display a full restriction map of the lambda DNA. This restriction map can be used to predict what will happen if certain enzymes are used. Next, click on &#8220;Custom digest&#8221; under the &#8220;Main options&#8221; heading.
</div></li><li data-sub-note-item="2-5"><div class="lab-step">Search for the enzyme StuI and note that it works optimally in buffer 2.1 or CS buffer. Select the StuI enzyme and click digest.
</div></li><li data-sub-note-item="2-6"><div class="lab-step">In the next window click on &#8220;View gel&#8221; under the &#8220;Main options&#8221; header. Select the option to run a virtual 1% gel using a pBR322-BstN1 digest.
</div></li><li data-sub-note-item="2-7"><div class="lab-step">After the results appear, take a screenshot and paste it into a blank document.
</div></li><li data-sub-note-item="2-8"><div class="lab-step">After this, navigate back to the main page of the NEBcutter tool and select &#8220;New custom digest&#8221;.
</div></li><li data-sub-note-item="2-9"><div class="lab-step">This time search for the BsrGI enzyme and note that it performs optimally in buffer 2.1 or buffer 3.1. Select the enzyme and click digest.
</div></li><li data-sub-note-item="2-10"><div class="lab-step">Under the Main options header click on &#8220;View gel&#8221; and select the options to run a virtual 1% gel using a pBR322-BstN1 digest.
</div></li><li data-sub-note-item="2-11"><div class="lab-step">Take a screenshot of the results and save this in a text document.
</div></li><li data-sub-note-item="2-12"><div class="lab-step">Finally, repeat these steps using both StuI and BsrGI together in a single digest.
</div></li><li data-sub-note-item="2-13"><div class="lab-step">Again, take a screenshot and save the resulting gel.
</div></li></ul></li><li data-note-item="3" data-parent-collapse="">DNA Profiling
<button class="expand">Expand</button><ul class="lab-items"><li data-sub-note-item="3-1"><div class="lab-step">Before starting the restriction enzyme digests first put your aliquots of lambda DNA, NEB buffer 2.1, StuI enzyme, and BsrGI enzyme into an ice bucket, and take this to your work area.
</div></li><li data-sub-note-item="3-2"><div class="lab-step">Label a tube as StuI &#8211; this will contain the StuI digest. 
</div></li><li data-sub-note-item="3-3"><div class="lab-step">Label a tube as BsrGI to contain the BsrGI digest. 
</div></li><li data-sub-note-item="3-4"><div class="lab-step">Then, label a tube S+B, to contain the digest with both of the enzymes.
</div></li><li data-sub-note-item="3-5"><div class="lab-step">Use the appropriate micropipetters to set up the reactions listed in the Table 1:<a href="https://www-jove-com.bdigitaluss.remotexs.co/CDNSource/lm/labs/12/Lab_12_Table_1.xlsx" style="display:block;margin:20px;" target="__blank">Click Here to download Table 1</a>
</div></li><li data-sub-note-item="3-6"><div class="lab-step">First, pipette the 40 &#181;L of water into each tube.
</div></li><li data-sub-note-item="3-7"><div class="lab-step">Then, using a fresh pipette tip each time, dispense 5 &#181;L of buffer 2.1 into each tube and pipette up and down to mix.
</div></li><li data-sub-note-item="3-8"><div class="lab-step">Next, add 4 &#181;L of lambda DNA to each tube, and then pipette 0.5 &#181;L of each enzyme into the appropriate tubes.
</div></li><li data-sub-note-item="3-9"><div class="lab-step">Finally, add a sufficient amount of water to each tube to bring the total volume up to 50 &#181;L.
</div></li><li data-sub-note-item="3-10"><div class="lab-step">Next, incubate the tubes at 37 &#186;C for 15 - 60 min in a water bath, and return the buffers and enzymes to the freezer.
</div></li><li data-sub-note-item="3-11"><div class="lab-step">To prepare an agarose gel to run the digest, in a 250 mL flask mix 1 g of agarose powder with 100 mL of TAE buffer.
</div></li><li data-sub-note-item="3-12"><div class="lab-step">Microwave the solution for approximately 90 s at full power, taking care to prevent the solution from boiling over.
</div></li><li data-sub-note-item="3-13"><div class="lab-step">Next, carefully remove the solution from the microwave using a glove or heat pad and place it to cool for about 15 min. 
</div></li><li data-sub-note-item="3-14"><div class="lab-step">While the agarose solution cools set up a casting tray. 
</div></li><li data-sub-note-item="3-15"><div class="lab-step">When the agarose solution is cool add 5 &#181;L of 10,000X SYBR Safe gel stain to the solution.
</div></li><li data-sub-note-item="3-16"><div class="lab-step">Add the comb which is used to make the wells of the gel to the casting tray of the gel electrophoresis apparatus.
</div></li><li data-sub-note-item="3-17"><div class="lab-step">Pour the agarose gel solution into the casting tray and allow it to solidify. 
</div></li><li data-sub-note-item="3-18"><div class="lab-step">Once the gel is solid, pull the comb straight up to remove it from the gel, and then remove the dams from the casting tray if appropriate.
</div></li><li data-sub-note-item="3-19"><div class="lab-step">Place the gel in the electrophoresis chamber with the wells positioned closest to the negative or black electrode and fill the chamber with 1x TAE buffer to completely cover the gel.
</div></li><li data-sub-note-item="3-20"><div class="lab-step">Remove your samples from the water bath.
</div></li><li data-sub-note-item="3-21"><div class="lab-step">Then add 8.3 &#181;L of 6X loading dye to each sample.
</div></li><li data-sub-note-item="3-22"><div class="lab-step">Dispense 10 &#181;L of the pBR322-BstN1 digest which contains DNA fragments of known size into the first well.
</div></li><li data-sub-note-item="3-23"><div class="lab-step">Then load 25 &#181;L of sample StuI into the second well.
</div></li><li data-sub-note-item="3-24"><div class="lab-step">Place 25 &#181;L of sample BsRGI into the third well.
</div></li><li data-sub-note-item="3-25"><div class="lab-step">Load 25 &#181;L of sample S+B into the fourth well.
</div></li><li data-sub-note-item="3-26"><div class="lab-step">To run the gel plug the leads into the electrophoresis chamber and then into the power supply. The red positive lead should be at the end farthest away from the wells and the black negative lead should be nearest to the wells. This is because the samples will run from negative to positive.
</div></li><li data-sub-note-item="3-27"><div class="lab-step">Turn on the power supply and set the gel box to run at 170 volts for approximately 60 minutes.
</div></li><li data-sub-note-item="3-28"><div class="lab-step">After the run is complete turn off the power to the gel box and disconnect the electrodes from the tank.
</div></li><li data-sub-note-item="3-29"><div class="lab-step">Then remove the gel from the electrophoresis chamber and observe it with a UV transilluminator.  CAUTION: Protect eyes from the UV by wearing goggles or using the provided shield.
</div></li><li data-sub-note-item="3-30"><div class="lab-step">Finally, use a cell phone camera to take a picture of the gel from directly above and parallel to the gel surface.
</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">Compare the gel you ran in the lab to your virtual enzymatic digest with StuI.
</div></li><li data-sub-note-item="4-2"><div class="lab-step">The virtual digest with StuI resulted in seven bands. Count the bands in the StuI lane of your gel and note whether this matched the virtual digest or appeared to show a similar pattern. NOTE: Small bands on agarose electrophoresis gels are often invisible.
</div></li><li data-sub-note-item="4-3"><div class="lab-step">Now look at the results for your BsrGI digest. The virtual digest with BsrGI resulted in six bands. Count the bands in the BsrGI lane. Again, note whether the virtual digest and lab digest showed the same or a similar pattern.
</div></li><li data-sub-note-item="4-4"><div class="lab-step">Finally, look at the virtual digest with StuI and BsrGI. The virtual digest with both enzymes resulted in 12 bands. Count the bands in the corresponding lane of your gel, and consider whether the DNA banding pattern looks similar to the virtual digest.
</div></li></ul></li></ol>

DNA-Isolierung und Restriktionsenzymanalyse

DNA Isolation
ExpandNOTE: In this experiment you will perform DNA isolation under two experimental conditions: one using a buffer containing the detergent ...

Genetik

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">To prepare for the experiment, be sure to obtain cultures of <em>Drosophila melanogaster</em> at least two weeks ahead of the planned experiment day. NOTE: Because adult females store sperm after mating, you will need this time to generate virgin females by breeding the flies.
</div></li><li data-sub-note-item="1-2"><div class="lab-step">You should get two stock cultures: wildtype, that is homozygous dominant red eyed flies, and white chromosome one mutant, which are homozygous recessive white eyed flies.
</div></li><li data-sub-note-item="1-3"><div class="lab-step">Purchase or create <em>Drosophila </em>habitats consisting of a clean vial, 3 cm of prepared food media, and a stopper to close the open end of the vial.
</div></li><li data-sub-note-item="1-4"><div class="lab-step">When not being moved or counted, keep the flies in an incubator or room kept at 23 - 25 &#176;C, where the lifecycle will take around 12 days. NOTE: From the egg, <em>Drosophila </em>progress through four main stages of development: embryo, larva, pupa, and adult. The complete lifecycle takes about 10 days at 25 &#176;C, but it can be affected by temperature. For example, at 18 &#176;C, the lifecycle is about 19 days and at 29 &#176;C, the lifecycle is only seven days.
</div></li><li data-sub-note-item="1-5"><div class="lab-step">As an overview, to set up for the student experiment, you will generate vials for each student group containing white eyed virgin female flies only, white eyed males only, red eyed virgin females only, and red eyed males only. These will be referred to as the experimental vials.
</div></li><li data-sub-note-item="1-6"><div class="lab-step">When your vials from the supplier arrive, referred to as the stock vials, these will contain a mix of male and female flies, as well as larvae, pupae, and eggs.
</div></li><li data-sub-note-item="1-7"><div class="lab-step">Beginning with your stock vials, you will take around 3 - 5 males and the same number of females, keeping white eyed and red eyed flies separate at all times, and place these together into a new nursery vial containing food. NOTE: You can sex the flies using a magnifying glass. Look at the posterior of the fly. Males are slightly smaller and have 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">You will leave the flies in these nursery vials for around 3 - 5 days to mate and lay eggs.
</div></li><li data-sub-note-item="1-9"><div class="lab-step">After this, you will move them back to their stock vials.
</div></li><li data-sub-note-item="1-10"><div class="lab-step">Next, you will observe as the eggs and larvae in the nursery vials mature, checking the vials every 6 - 8 hours. As it takes females around 8 - 10 hours to become sexually mature, this should ensure that any females found in the nursery vials are virgin.
</div></li><li data-sub-note-item="1-11"><div class="lab-step">As the new adult flies appear, you should transfer them from the nursery to the appropriate experimental tube. For example, if it is a male, transfer it to the experimental male tube for the appropriate eye color. If it is a female, it should be placed in an experimental vial labeled with eye color and virgin female.
</div></li><li data-sub-note-item="1-12"><div class="lab-step">Keep adding flies to the appropriate vials until each experimental vial has at least five flies. NOTE: You will need to make enough experimental vials so that each student group has one of each eye color type and sex, with a few class spares. You should create enough nursery vials to account for this. A good rule of thumb is that it generally takes about two weeks for 25 healthy adults to produce about 50 new adults.
</div></li><li data-sub-note-item="1-13"><div class="lab-step">To begin the process, first label empty habitats to make the nursery vials to raise larvae and pupae for each strain of flies: red eyed and white eyed.
</div></li><li data-sub-note-item="1-14"><div class="lab-step">To move the adult flies into the nursery vial to lay eggs, use an anesthesia wand according to the manufacturer&#39;s instructions, placing it into the stock vial of mature flies. Wait until the animals become unconscious. Now they can be gently tapped onto a piece of white paper and sorted into piles of the same sex using a brush.
</div></li><li data-sub-note-item="1-15"><div class="lab-step">Transfer a few male and female flies to the appropriate vial labeled with their eye color phenotype and sex.
</div></li><li data-sub-note-item="1-16"><div class="lab-step">Place the nursery vials to incubate at 23 to 25 &#176;C and the flies will lay eggs.
</div></li><li data-sub-note-item="1-17"><div class="lab-step">Remove the adults after 3 - 4 days using anesthesia and put them back into the stock vial.
</div></li><li data-sub-note-item="1-18"><div class="lab-step">Remove any adult flies that have eclosed in the 6 - 8 hour period using an anesthesia wand.
</div></li><li data-sub-note-item="1-19"><div class="lab-step">Then, tip out the adults onto paper and sort them into groups based on their sex before placing each fly into an appropriate experimental vial marked male or virgin female and red or white for the phenotype. NOTE: Each experimental vial should contain five flies. Once this number is reached, then a new vial should be started. If you miss the 6 - 8 hour window, females may not be virgins, so these should be discarded into a morgue tube containing 70% ethanol or returned to the stock vial. Males do not need to be virgins, but do need to be kept in male only containers.
</div></li><li data-sub-note-item="1-20"><div class="lab-step">Each lab group will need one tube of five virgin red eyed females, one of five virgin white eyed females, one tube of red eyed males, and one of five white eyed males. In addition, they will need a morgue vial containing ethanol, two anesthesia wands, fly anesthetic, a paintbrush, a magnifying glass, some plain paper, and two empty fly vials for crosses.
</div></li></ul></li></ol>

Genetics of Organisms: REF and WEF Fly Genetics - Prep

Genetik der Organismen

REF and WEF Fly Genetics
ExpandTo prepare for the experiment, be sure to obtain cultures of Drosophila melanogaster at least two weeks ahead of the ...