Name: a procedure to observe context-induced renewal of pavlovian-conditioned alcohol-seeking behavior in rats
Uploaded: 2014-09-19T14:00:00.000+00:00
Duration: 13 min 24 s
Description: Watch this Scientific Journal Video about A Procedure to Observe Context-induced Renewal of Pavlovian-conditioned Alcohol-seeking Behavior in Rats at JoVE.com

<p class='jove_content'>This work was supported by the National Institute on Alcohol Abuse and Alcoholism (RO1 AA014925 awarded to Dr. Patricia H. Janak at University of California, San Francisco, CA, USA), Fonds de recherche du Quebec &#8211; Sant&#233; (NC) and the Natural Sciences and Engineering Research Council (NC, 387224-2010). The open-access publication of this article was made possible by sponsorship from Med Associates Inc. </p>

<p class="jove_content"> Alle Verfahren werden von der Animal Research Ethics Committee an der Concordia Universität genehmigt und stimmen mit den Empfehlungen des Canadian Council on Animal Care. </p><p class="jove_title"> 1. Tiere </p><ol><li> Erhalten männlichen Long-Evans-Ratten (220 - 240 g bei Ankunft). </li><li> Pflegen Ratten in einem Temperatur (21 ° C) und Luftfeuchtigkeit (44%) kontrolliert Tierstation, auf einem 12 Stunden Licht-Dunkel-Zyklus mit Lichtern auf 7:00 Uhr. </li><li> Paar-Haus Ratten bei der Ankunft. Nach 3 Tagen, Hausratten einzeln in Plastikkäfigen Schuhkarton mit Beta-Chip-Bettwäsche, eine Nylon-Knochen und freien Zugang zu Standardrattenfutter und Wasser. Verwenden Käfig Deckel, die doppelte Ösen für die nachfolgende Platzierung einer zweiten Flasche ermöglichen, zusätzlich zu der Wasserflasche zu haben. HINWEIS: Die Ratten einzeln untergebracht werden, sobald sie in der Tierstation ankommen. </li><li> Beginnen, Ratten täglich behandeln. HINWEIS: Als Richtwert ist eine Woche der Behandlung in der Regel ausreichend, um sie als pi akklimatisierencked und vom Experimentator behandelt. </li></ol><p class="jove_title"> 2. Gelegentliche Ethanol-Zugang im Käfig </p><p class="jove_content"> HINWEIS: Verhaltens intermittierende Ethanol-Zugang in den eigenen Käfig, um sicherzustellen, dass Ratten wird physiologisch relevanten Mengen von Ethanol, bevor das Verhaltenstraining Phase des Experiments beginnt <sup>17-19</sup> trinken. Initiieren dieses Verfahren während der Lichtphase, obwohl sie jederzeit während der hellen oder dunklen Zyklus initiiert werden. </p><ol><li> Bereiten Sie zwei Flaschen für jede Ratte. <ol><li> Verwenden Sie ein 100-ml-Plastikmesszylinder als Ethanol-Flasche und einer 473 ml Standard-Kunststoff-Wasserflasche. Verwenden Sie eine relativ geringe Kapazität Ethanol-Flasche, fast auf maximale Lautstärke Kapazität gefüllt, um den Verlust von Ethanol durch Leckage und Verdunstung zu minimieren. </li><li> Füllen Sie eine Flasche mit 15% Ethanol, 95% durch Verdünnen Ethanol in Leitungswasser. HINWEIS: Andere Ethanolkonzentrationen, wie 10% oder 20%, können anstelle von 15% Ethanol verwendet werden. </li><li> Fiwerde die zweite Flasche mit Leitungswasser. </li><li> Legen Sie eine Gummistopfens mit einer Zuführungsschlauch, der ein Kugellager, um ein Auslaufen zu minimieren, in jede Flasche enthält. </li></ol></li><li> Bis zwei Steuer Käfige, identisch zu den Käfigen mit der Ausnahme, dass die Steuer Käfige haben Ratten oder Bettwäsche enthalten behandelt eingestellt. <ol><li> Bereiten Ethanol und Wasser-Flaschen für die Steuer Käfige wie in den Schritten 2.1.1 - 2.1.4. </li><li> Verwenden Sie die von den Steuer Käfige erhalten, um für Verschütten und / oder Verdampfung, die im Laufe des Ethanols Zugriffssitzung auftreten können (wie in den Schritten 2.8.1 beschrieben - 2.8.2) Schutzmaßnahmen. </li></ol></li><li> Wiegen alle Ethanol und Wasser-Flaschen. Notieren Sie alle Gewichte. </li><li> Wiegen alle Ratten. Notieren Sie alle Gewichte. </li><li> Legen Sie das Ethanol-Flasche und die Wasserflasche auf jedem Heimkäfig Käfig und Kontrolle zugleich. Lassen Sie beide Flaschen auf die Käfige für 24 Stunden. </li><li> Am Ende des 24-Stunden-Zeitraum, entfernen Sie die beiden Flaschen aus derKäfige gleichzeitig. </li><li> Wiegen alle Ethanol und Wasser-Flaschen. Notieren Sie alle Gewichte. </li><li> Um zu bestimmen, wie viel Ethanol und Wasser wurde während des 24 Stunden-Zeitraum verbraucht wird, subtrahiert das Gewicht jeder Flasche, wenn es aus dem Käfig aus dem Gewicht der Flasche entfernt werden, wenn es auf dem Käfig 24 Stunden früher für jede Ratte gegeben. <ol><li> Machen Sie dasselbe für die beiden Regel Käfige, und dann die Berechnung der Durchschnittsbetrag der Verschüttung für jede Flüssigkeit. </li><li> Subtrahieren Sie die durchschnittliche Menge von Ethanol und Wasser Verschütten bzw. aus den entsprechenden Ethanol-und Wasserverbrauch Maßnahmen für jede Ratte. </li><li> Verwenden Sie diese Verschütten gesteuert Unterschied Maßnahmen zur Ethanolkonsum in g / kg, Verbrauch von Wasser und Ethanol Vorlieben zu berechnen. </li></ol></li><li> Ethanol Verbrauch (g / kg), das erste Gewicht (g) Ethanol-Lösung in das Volumen (ml) verbraucht zu konvertieren, basierend auf der Dichte der gesamten Lösung (Ethanol + Wasser-Komponenten) zu berechnen, nachder Formel Volumen (ml) = Gewicht (g) / Dichte (g / ml). HINWEIS: Die Dichte der Lösung wird als Funktion der Ethanolkonzentration variieren. <ol><li> Als Nächstes multiplizieren das Volumen (ml) Ethanol-Lösung durch die Dichte (g / ml) Ethanol für die spezifische Anteil Lösung verwendet skaliert verbraucht, um das Gewicht (g) an Ethanol zu erhalten. </li><li> Teilen Sie das Gewicht (g) Ethanol durch das Rattengewicht (kg) verbraucht, um die g / kg Maß der Ethanolkonsum zu erhalten. </li></ol></li><li> Ethanol Vorlieben zu berechnen, ausdrücken Ethanolaufnahme (ml) als Prozentsatz des Gesamtflüssigkeit (Ethanol + Wasser, ml) Aufnahme. </li><li> Bringen Sie die Wasserflasche nur zurück nach Hause Käfig der Ratte. Für die nächsten 24 Stunden, geben die Ratten Zugang zu Wasser, aber nicht zu Ethanol. Statt einer Flasche Ethanol, legen Sie ein vorgewogen zweite Wasserflasche auf dem Käfig. </li><li> Am Ende des 24-Stunden-Zeitraum, entfernen Sie die beiden Wasserflaschen aus dem Käfig in der gleichen Zeit. </li><li> Wiegen beide Arten von Wasser-Flaschen. Notieren Sie alle weights. </li><li> Subtrahieren das Gewicht jeder Flasche, wenn es aus dem eigenen Käfig von dem Gewicht der Flasche entfernt werden, wenn sie auf der Heimkäfig 24 Stunden zuvor gelegt, Subtrahieren der durchschnittlichen Verschütten Maßnahme aus Steuer Käfige erhalten wie in den Schritten 2.8.1 beschrieben - 2.8.2. </li><li> Verwenden Sie diese Verschütten gesteuerte Unterschied Maßnahmen zur Wasserverbrauch zu berechnen. </li><li> Beginnen Sie den Zyklus erneut, indem sowohl Ethanol und Wasser-Flaschen an Ratten, wie in den Schritten 2.1 bis 2.14 beschrieben. <ol><li> Wiederholen Sie diesen Zyklus für mehrere (ca. 12) Sitzungen, um eine hohe und stabile Ebenen der Ethanolkonsum zu induzieren. Siehe <strong>Abbildung 1</strong> für repräsentative Ergebnisse der Ethanolkonsum (g / kg) im Käfig. </li><li> Abwechselnd die räumliche Lage der Ethanol und Wasserflasche auf dem Käfigdeckel über die Sitzungen, die Entwicklung von Seitenpräferenz zu verhindern. </li><li> Machen Sie eine frische Ethanollösung einmal pro Woche, um genaue und stabile Ethanolkonzentration über zu gewährleistenSitzungen. Minimierung der Auswirkungen der Verdampfung von Ethanol über eine längere Zeitdauer unter Verwendung dieses Ansatzes. </li></ol></li></ol><p class="jove_title"> 3. Geräte </p><ol><li> Haus jeder Klimakammer in einer belüfteten, schalldämpfenden Kabine. </li><li> Konstruieren jedes Konditionierungskammer aus einem Edelstahl-Metallstab Stock, eine klare Polycarbonat Decke, klarem Polycarbonat Rückwand und Fronttür und Aluminium Metallplatte Seitenwände. <ol><li> Installieren Sie eine Fluidöffnung zentral in der Nähe der Basis der rechten Seitenwand Metall. Rüsten den Eingang der Fluidanschluss mit einem Infrarot-Lichtschranke, um Einträge in die Öffnung zu erfassen. </li><li> Verbinden Polyethylenschlauch von der Rückseite des Fluidöffnung zu einer 20 ml Spritze mit Ethanol unter Verwendung eines stumpfen Nadel. Legen Sie die Spritze in eine Spritzenpumpe außerhalb der schalldämpfenden Schrank gelegen. Für Pawlow Diskriminierung Schulungen, laden Ethanol in die Spritze, bevor Sie es in die Pumpe. Prime der Schlauch by manuell Förderung der Pumpe, und wischen Sie das überschüssige Ethanol aus dem Fluidanschluss. </li><li> Auf der linken Seitenwand, installieren Sie einen weißen houselight zentral in der Nähe der Decke der Kammer. </li><li> Installieren Sie einen Clicker (2 Hz-Zyklus) und einen weißen Rauschgenerator mit Lautsprecher in der linken oberen Ecke des linken Seitenwand. </li></ol></li><li> Statten Sie jeden Klimakammer mit einer bestimmten Konfiguration von visuellen, olfaktorischen und taktilen Reizen zu einem unverwechselbaren Kontext zu schaffen. <ol><li> Verwenden Sie zwei unterschiedliche Kontexte, bezeichnet Kontext 1 und Kontext 2. Konfigurieren Sie die Hälfte der Klimakammern als Kontext 1 und die andere Hälfte als Kontext 2. </li><li> Um Kontext 1 zu erstellen, legen Sie schwarze Pappe Platten über die Decke und die vorderen und hinteren Wänden der Kammer, Spray Zitronenduft (10% v / v, durch Verdünnen Zitronenöl in Leitungswasser) in eine Petrischale und legen Sie sie in der Mitte eines Metallabfallfach unter dem Boden der Kammer, und legen Sie eine klare Polycarbonat Bodenplatte über dem Metall rod Boden. Säumen den Abfallbehälter mit einem saugfähigen Papiertuch oder Bank-Liner. </li><li> Um Kontext 2 zu erstellen, lassen Sie die Decke und Vorder-und Rückwände der Kammer aufgedeckt, so dass sie klar sind, sprühen Mandelgeruch (10% v / v, durch Verdünnen Benzaldehyd in Leitungswasser) in eine Petrischale und legen Sie sie zentral in einem Metallabfallfach unter dem Boden der Kammer, und legen Sie eine perforierte Metallbodenplatte über die Metallstange Boden. Säumen den Abfallbehälter mit einem saugfähigen Papiertuch oder Bank-Liner. </li></ol></li></ol><p class="jove_title"> 4. Habituation </p><p class="jove_content"> Gewöhnen Ratten auf die Verhaltenstraining Raum und jeder der beiden Kontexte innerhalb Anlage Kammern. In dieser Phase des Experiments nicht Ethanol in den 20 ml Spritzen geladen. </p><ol><li> Ratten zu gewöhnen, um den Prozess der von der Tierstation auf die Verhaltensschulungsraum im Labor in zwei kurzen Gewöhnungs Sitzungen transportiert. <ol><li> In der ersten Sitzung, laden die Ratten in ihre Käfige auf einem Rollwagen in der Tierstation, und transportieren sie zu den Verhaltens-Schulungsraum im Labor. Beleuchten die Raumbeleuchtung, schalten Sie auf die Fans in jedem schalldämpfenden Schrank, den Raum zu verlassen, schließen Sie die Tür und ließ die Ratten anpassen, um diese neue Umgebung für 20 Minuten, während sie in ihre Käfige auf dem Wagen zu bleiben. Am Ende dieser 20 Minuten Zeit, kehren die Ratten in die Tierstation. </li><li> In der zweiten Sitzung, bringen die Ratten auf die Verhaltensschulungsraum im Labor. Nach wie vor, das Licht und Schrank Fans in den Raum. Bleiben im Zimmer, bei geschlossener Tür, um Ablenkungen zu minimieren und zu behandeln und wiegen jede Ratte in der Verhaltensschulungsraum. Notieren Sie sich die Gewichte, und dann die Ratten zurück zum Tierstation. </li></ol></li><li> Gewöhnen Ratten zu den Klimakammern in zwei kurzen Gewöhnungs Sitzungen. <ol><li> Transportieren die Ratten auf die Verhaltensschulungsraum, notieren das Gewicht jeder Ratte,und laden Sie jede Ratte in die Klimakammer bezeichnet. </li><li> In der ersten Sitzung festgelegt Gewöhnung bis die Hälfte der Kammern als Kontext 1 und den Rest als Kontext 2. </li><li> In der zweiten Sitzung Gewöhnung jede Kammer einzurichten mit dem Kontext, die nicht in der vorherigen Sitzung verwendet wurde. </li><li> Gewöhnung führen Sitzungen in einer Art und Weise ausgeglichen, so dass die Hälfte der Ratten werden zunächst Kontext 1 ausgesetzt, gefolgt von Kontext 2, und die andere Hälfte sind erste Kontext 2 ausgesetzt, gefolgt von Kontext 1. Idealerweise führen beide Gewöhnung Sitzungen in der am gleichen Tag, auch wenn sie sich über zwei Tage verteilt werden könnte, wenn nötig. </li><li> Für Gewöhnung Sitzungen, verwenden Sie ein Computerprogramm, um die houselight 1 min zu beleuchten, nachdem das Programm ausgegeben wurde. Notieren Sie sich die Gesamtzahl der Fluidanschluss Einträge während eines 20 Minuten-Sitzung gemacht. Am Ende der Sitzung, programmieren die houselight zu deaktivieren. Ethanol oder jede Merkmale während Gewöhnung Sitzungen präsentieren nicht. </li></ol></li&gt; <li> Ratten zu entfernen aus den Kammern, kehren sie in ihre Heimatkäfige, und transportieren sie zurück in die Tierstation. </li><li> Reinigen Sie das Gerät <em>(zB</em> Bodenplatten, Abfallschalen, Petrischalen). Siehe Schritt 5.7 für weitere Details. </li></ol><p class="jove_title"> 5. Pawlowdiskriminierungstraining (PDT) </p><p class="jove_content"> Zug Ratten, die verhaltens unterscheiden zwischen einem Gehör Cue, die mit Ethanol und einem zweiten Gehör Cue, die nicht gepaart ist. In dieser Phase des Experiments, füllen die 20 ml Spritzen mit 15% Ethanol. </p><ol><li> Statten Sie jeden Klimakammer mit einer bestimmten Konfiguration von visuellen, olfaktorischen und taktilen Reizen zu einem unverwechselbaren Kontext zu schaffen, wie in den Schritten 3.3.2 und 3.3.3 beschrieben <ol><li> Weisen Sie die Hälfte der Ratten Kontext 1 und die andere Hälfte zum Kontext 2 in einem Gegengewichts Mode, basierend auf Ethanolkonsum und Vorzugs gemittelt über die letzten 2 Sitzungen des Heimkäfig Ethanol Zugangsverfahren. Beziehen sich auf die Fortsetzungext während PDT verwendet als Kontext A. HINWEIS: Jede Ratte sollte nur einen Kontext zu erleben in dieser Phase der Ausbildung. </li><li> Stellen Sie sicher, dass die Geruchsreizes ist die letzte Kontextreiz, jedem Behandlungsraum hinzugefügt werden. Schließen Sie die Türen der Kammer und dem schalldämpfenden Kabine, um den olfaktorischen Reiz enthalten und die Vermischungen künftig von verschiedenen Gerüche zu minimieren. </li></ol></li><li> Bringen die Ratten in der Verhaltensschulungsraum, notieren das Gewicht jeder Ratte, und laden Sie jede Ratte in die Klimakammer bezeichnet. </li><li> Starten Sie den Computer-Programm, das die folgende Sequenz von Ereignissen während jeder Sitzung PDT steuert. <ol><li> 5 min nach dem Programm ausgegeben wurde, beleuchten die houselight. </li><li> Präsentieren Sie das weiße Rauschen Reiz und der Clicker Reiz 16 Mal jeweils. Bestimmen Sie eine dieser auditive Reize, um die CS + und die andere, um die CS sein, in einer Art und Weise ausgeglichen über beide Kontexte. </li><li> Machen jedes CS + und CS-Studie last 10 sek. </li><li> Liefern 0.2 ml Ethanol in die Fluidöffnung in den letzten 6 sec des CS +. Keinerlei Ethanol während der CS nicht liefern. </li><li> Machen jedes PDT-Sitzung letzten 58 min im Durchschnitt. Präsentieren Sie die CS + und CS die jeweils auf einer variablen Zeit 67 sec Zeitplan. </li><li> Datensatz Fluidöffnung Eingaben während der 10 sec CS Intervalls (10 s bei CS Präsentation), die 10 Sekunden vor-CS Intervalls (10 s unmittelbar vor Beginn CS) und 10 sec nach der CS Intervalls (10 s sofort folgenden CS-Offset) sowohl für CS + und CS-Präsentationen, sowie der Gesamtfluidanschluss Einträge für die gesamte Sitzung. Auch aufzeichnen die Dauer der einzelnen Port-Eintrag in diesen Intervallen. </li><li> Am Ende der Sitzung, drehen Sie den houselight aus. </li></ol></li><li> Ratten zu entfernen aus den Kammern, kehren sie in ihre Heimatkäfige, und transportieren sie zurück in die Tierstation. </li><li> Überprüfen Sie die Fluidanschlüsse, um sicherzustellen, dass Ratten, die verbraucht 3,2 ml Ethanol in jeder PDT s geliefertession. </li><li> Spülen Sie das Ethanol aus dem Polyethylenschlauch mit einer 60 ml Spritze mit einer kleinen Menge Wasser. Dann passieren 60 ml Luft durch den Schlauch mit einem zweiten, leeren Spritze, um sicherzustellen, dass es trocken ist. </li><li> Reinigen Sie das Gerät. Für die tägliche Reinigung, waschen Sie die Bodenplatten, Abfallfächer und Petrischalen. Einmal in der Woche, reinigen Sie die gesamte Konditionierungskammer. Verwenden Sie keine Reinigungsmittel duftende Einführung zu vermeiden unerwünschte Gerüche in die Versuchsapparatur. </li><li> Weiter PDT-Sitzungen täglich bis Ratten zeigen stabile Diskriminierung in ihrem Port-Eintrag Antworten auf die CS + vs CS. HINWEIS: Port-Eintrag in Reaktion auf den CS + gemacht sollte über Sitzungen zu erhöhen, während Porteinträge in Reaktion auf die CS hergestellt tief bleiben. Die genaue Anzahl der PDT-Sitzungen notwendig, diskriminiert zu erreichen Anlage reagiert, kann variieren, aber, wie ein Führer, planen etwa 20 Sitzungen. </li></ol><p class="jove_title"> 6. Extinction </p><ol><li> Statten Sie jeden Anlage Chamber mit visuellen, olfaktorischen und taktilen Reizen, um einen Kontext, der sich von der einen die Ratten während der PDT-Sitzungen erlebt zu schaffen. Wenn zum Beispiel 1 wurde im Kontext PDT verwendet, konfigurieren die Kammer Kontext 2 zum Löschen Sitzungen. Erhalten Sie über die während der Extinktion als Kontext B verwendet </li><li> Führen Aussterben Sitzungen identisch PDT-Sitzungen, aber keine Ethanol während der CS + zu liefern. <ol><li> Verwenden Sie die gleiche Computerprogramm, das während der PDT verwendet wurde, um den CS + und CS zu präsentieren. </li><li> Um die Konsistenz über den Phasen des Experiments zu erhalten, legen Sie eine leere Spritze in die Spritzenpumpe, und lassen Sie die Pumpe eingeschaltet. Stellen Sie sicher, dass die Polyethylen-Schlauch nicht Ethanol enthalten vor Aussterben Sitzungen. </li></ol></li><li> Weiter Aussterben Sitzungen täglich bis Ratten nicht mehr diskriminiert Show reagiert zwischen dem CS + und CS. HINWEIS: Port-Eintrag in Reaktion auf den CS + gemacht schrittweise über Extinktion Sitzungen sinken, bis es keine differenz zwischen Anschlusseinträge in Reaktion auf die CS + gemacht und Anschlusseinträge in Reaktion auf die CS hergestellt. Die genaue Zahl der vom Aussterben Sitzungen notwendig, Anlage reagiert, kann variieren löschen, aber als Führer, planen etwa 8 Sitzungen. </li><li> Reinigen Sie das Gerät am Ende jeder Sitzung, wie in Schritt 5.7 beschrieben. </li></ol><p class="jove_title"> 7. Context-induzierte Renewal-Test </p><ol><li> Anlegen jeder Klimakammer mit den visuellen, olfaktorischen und taktilen Reizen, um den Kontext, die während der PDT-Sitzungen erlebt wurde erstellen. Zum Beispiel, wenn eine bestimmte Ratte erlebt Kontext 1 in der PDT-Sitzungen konfigurieren seiner Kammer als Kontext 1 in der Testsitzung. Erhalten Sie über die während der Erneuerung Test als Kontext verwendet A. </li><li> Führen Sie den Kontext-induzierte Erneuerung Test identisch mit PDT-Sitzungen, aber keine Ethanol während der Erneuerung Test liefern. Präsentieren die <br /> CS + und CS nach wie vor während der PDT-Sitzungen, aber zurückhalten, das Ethanol. <ol><li> Verwenden Sie den gleichen Computer prOgram, die während der PDT verwendet wurde, um den CS + und CS zu präsentieren. </li><li> Um die Konsistenz Phasen zu erhalten, laden Sie eine leere Spritze in die Spritzenpumpe, und lassen Sie die Pumpe eingeschaltet. Stellen Sie sicher, dass die Polyethylen-Schlauch nicht Ethanol enthalten, bevor der Testsitzung. </li></ol></li><li> Beachten Kontext-induzierte Erneuerung der Pawlowschen Anlage Alkoholsucht Verhalten. ANMERKUNG: Diese Erneuerung Effekt wird offensichtlich sein, durch eine selektive Erhöhung der Anzahl von Eingangsports in Abhängigkeit von der CS + gemacht, ohne Veränderung der Eingangsöffnung Verhalten in Reaktion auf die CS, bezogen auf CS + und CS hergestellt Reagieren auf die beobachtete Ende des Aussterbens Ausbildung. </li><li> Reinigen Sie das Gerät am Ende der Sitzung, wie in Schritt 5.7 beschrieben. </li></ol>

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<p class="jove_content"> Alle experimentellen Verfahren wurden von der Concordia University Animal Research Ethics Committee genehmigt und sind in Übereinstimmung mit den Richtlinien der Canadian Council on Animal Care. <span style="font-size: 14px; line-height: 28px;">Die Open-Access-Veröffentlichung dieses Artikels möglich durch das Sponsoring von Med Associates Inc. wurde</span> </p>

<p class="jove_content">Results from this procedure reveal that discrete environmental stimuli that routinely accompany alcohol delivery can acquire the capacity to drive alcohol-seeking behavior. They also demonstrate that contextual stimuli associated with the prior availability or absence of alcohol can guide conditioned behavioral responses to discrete alcohol-predictive cues.</p>
<p class="jove_step">Critical steps within the protocol</p>
<p class="jove_content">Contexts in the present task incorporate stimuli that are external to the rat. However, the interoceptive state of the rat at test can also constitute a &#8216;context&#8217; that could affect renewal<sup>20</sup>. It is therefore important to habituate rats to any procedures that may occur before the renewal test and change their interoceptive state, like intracranial microinfusions or systemic injections. Moreover, habituation to microinfusions or systemic injections should be conducted prior to both extinction and PDT sessions, to prevent such procedures from becoming associated with one specific phase of the experiment.</p>
<p class="jove_content">Temporal parameters, such as CS duration, CS-US interval, and intertrial interval (ITI), have long been known to influence conditioned responding in a variety of conditioning paradigms<sup>21-26</sup>. Similarly, temporal parameters may influence the observation of the renewal effect in our procedure. Any set of parameters other than those specified in the protocol should be experimentally validated in pilot studies before proceeding to employ them in larger investigations.</p>
<p class="jove_step">Modifications and troubleshooting</p>
<p class="jove_content">Previous research has found that the amount of ethanol that rats consume in the home cage can vary as a function of the supplier from which rats are purchased<sup>17,27</sup>. Supplier is therefore an important consideration for this phase of the study. Similarly, the choice of rat strain should be considered carefully, as there are numerous reports of strain differences in ethanol consumption<sup>28-31</sup>.</p>
<p class="jove_content">Prior work in an aversive conditioning paradigm has shown that the developmental age of rats influences the observation of renewal of a Pavlovian conditioned fear response<sup>32</sup>. The lack of a renewal effect in very young rats (younger than postnatal day 20) has been suggested to be due to impaired encoding of contextual information<sup>33</sup>. The developmental age of rats to be used in the present procedure should therefore be taken into consideration.</p>
<p class="jove_content">Renewal of Pavlovian conditioned alcohol-seeking behavior has been observed when 10%<sup>9,16</sup>, 15%<sup>14</sup>, or 20%<sup>15</sup> ethanol solutions were used during the home cage ethanol access and PDT phases of the experiment. Researchers could use any of these concentrations of ethanol for the purposes of establishing this procedure in their laboratories.</p>
<p class="jove_content">During the acquisition of Pavlovian discrimination training it is important to check fluid ports after each session to ensure that all the ethanol that was delivered has been consumed. Leaving rats in the conditioning chambers for an additional 10 - 15 min after each session may be done early in PDT to encourage consumption of any ethanol that was not consumed during the session. The number of rats leaving unconsumed ethanol in the ports should decrease as PDT progresses. If by the end of PDT rats are either (a) not consuming all the ethanol and/or (b) not responding to the CS+ then these subjects should be dropped from the study. Most rats reliably respond more to the CS+ than the CS-. However, some rats may respond at high levels to both cues, thereby not showing evidence of discrimination. Rats that show high responding to both the CS+ and CS- during PDT are not dropped from the study. In our experience, these subjects show remarkable discrimination during the first extinction session in the absence of ethanol, suggesting that their high levels of responding to the CS- during PDT are driven by the presence of ethanol in the fluid port. These subjects also tend to show robust renewal effects.</p>
<p class="jove_step">Limitations of the procedure</p>
<p class="jove_content">Because the renewal test is conducted in the absence of ethanol, conditioned port entries elicited by the CS+ gradually diminish across the test session. Therefore, the overall amount of behavior generated at test, against which experimentally induced changes can be assessed, is fairly low. Because in this task responding tends to be higher at the start versus the end of the test session, it is important to examine the effects of experimental manipulations on conditioned responding on a trial-by-trial basis.</p>
<p class="jove_content">Entries into the fluid receptacle provide the main dependent measure in this task. However, an erroneous port entry might be registered if an animal is close enough to the fluid receptacle to break the infrared beam across the opening of the fluid port with its whiskers. While infrequent, erroneous responses are obvious as a dramatic increase in total port entries relative to the subject&#8217;s behavior on prior sessions. Videotaping the animals would facilitate the detection of possible erroneous responses, and allow for a more refined analysis of the behaviors exhibited by the rats throughout training and testing.</p>
<p class="jove_step">Significance of technique with respect to existing/alternative methods</p>
<p class="jove_content">Context-induced renewal of drug-seeking behavior is largely studied using an instrumental conditioning task in which the primary dependent variable is an operant response, such as a lever press<sup>8,34</sup> or nose poke<sup>12</sup>. In the present procedure, the drug-seeking response is &#8216;conditioned approach&#8217; to the fluid port that is elicited by presentations of the CS+. This response is likely acquired through Pavlovian learning, which is the learning process that mediates the formation of associations between environmental stimuli and the pharmacological effects of drugs of abuse in humans.</p>
<p class="jove_step">Future applications</p>
<p class="jove_content">This task can be used in combination with neuropharmacology, optogenetics and neurochemistry to study the neural mechanisms that are involved in context-induced renewal of Pavlovian-conditioned alcohol-seeking behavior<sup>16</sup>. In addition, behavioral and neural mechanisms that regulate the acquisition and extinction of Pavlovian-conditioned alcohol-seeking behavior can be investigated. Lastly, this task can be used to explore manipulations of extinction that might prevent renewal, an important direction for translational research aimed at reducing the impact of drug associated environmental contexts on reactivity to drug predictive cues after cue exposure therapy in human addicts.</p>

<p class="jove_content"> Rest nüchtern ist eine große Herausforderung durch Personen, die unter Alkoholmissbrauch Erkrankungen konfrontiert. Abstinenz ist eine Zeit der Anfälligkeit für den Verhaltens, psychologischen und physiologischen Auswirkungen von Umweltreizen, die routinemäßig Alkoholkonsum zu begleiten, die können, durch Pawlowschen Konditionierung, sich mit Rausch <sup>1,2</sup> verbunden. Exposition gegenüber Alkohol prädiktiven Cues bedingter Reaktionen wie Alkohol Begierde, die Alkohol-Rückfall der Suche nach Verhaltensweisen, die <sup>3,4</sup> erleichtern fördern kann zu entlocken. </p><p class="jove_content"> Die stereotype Verhaltenssequenzen, die den Alkoholkonsum führen kann bestimmte Arten von Stimuli, routinemäßig unmittelbar vor den pharmakologischen Wirkungen von Alkoholkonsum erlebt werden. Zum Beispiel der Anblick, den Geruch und Geschmack von Alkohol sind die sensorischen Eigenschaften von Alkohol, die zuverlässig voraus Rausch. Zusätzlich zu solchen Signale, die als &quot;diskrete&quot; oder &quot;proxima bezeichnet werdenl &#39;Cues, Umwelt Kontexte, in denen Medikamente werden regelmäßig verwendet wird, kann auch provozieren Verlangen <sup>5,6.</sup> Exposition gegenüber physikalischen Standorten, an denen Medikamente haben bisher verwendet wurden, kann daher eine kritische Auslöser für den Rückfall <sup>7</sup> sein. </p><p class="jove_content"> Tiermodelle wurden entwickelt, um die neuronalen Mechanismen, die die Auswirkungen der Droge verbunden Kontexte auf Drogen-sucht Verhalten <sup>8-13</sup> vermitteln studieren. Das hierin beschriebene Verfahren erlaubt die Untersuchung, wie Kontexte mit Alkoholkonsum kann Alkohol-sucht Verhalten, das durch eine diskrete, Alkohol prädiktiven Pavlovian Cue ausgelöst wird modulieren. </p><p class="jove_content"> Pawlow Diskriminierung Ausbildung in einem spezifischen ökologischen Kontext, wo Ratten sind verhaltens zwischen zwei Gehöranlage Reize, eine CS +, die mit Alkohol kombiniert wird, und ein CS, die nicht zu unterscheiden geschult durchgeführt. Extinktion Einheiten werden dann in einem anderen Zusammenhang, in dem die Reaktion auf den CS durchgeführt+ Abnimmt, wenn ein Ergebnis der Alkohol zurückgehalten. Anschließend Reexposition zum Alkohol assoziiert, Pavlovian Ausbildung Kontext löst eine selektive Zunahme des Alkoholsuchverhalten der CS + hervorgerufen, ohne Änderung in Reaktion auf die CS. Dieses Ergebnis, das haben wir konsequent repliziert <sup>9,14-16</sup> erstreckt sich Erkenntnisse aus der instrumentellen Konditionierungsverfahren, in dem Drogen Kontexten gefunden worden, um die Erneuerung der operanten Reaktionen mit Drug-Delivery <sup>10,13</sup> assoziiert zu stimulieren. </p>

<table><tbody><tr><td>Heavy-duty utility cart</td><td>Rubbermaid</td><td>#4520-88</td><td>Used to transport rats in their home cage from the colony room to the behavior room or other facilities</td></tr><tr><td>Rats, Long-Evans</td><td>Harlan</td><td></td><td>Rats weighed 220-240 g on arrival</td></tr><tr><td>Ohaus scale</td><td>Fisher</td><td>#S402421</td><td>For weighing rats; tare the bottom of a plastic container first, then put the rat in it to weigh it</td></tr><tr><td>Beta-chips, aspen</td><td>Harlan</td><td>#7090A</td><td>Bedding for home cage</td></tr><tr><td>Nylabone</td><td>Bio-Serv</td><td>#K3580</td><td>Enrichment provided in the home cage</td></tr><tr><td>Autoclavable rodent chow</td><td>Agribrands, Charles River</td><td>#5075</td><td>For feeding rats</td></tr><tr><td>Wire bar cage lid</td><td>Ancare</td><td>#R20SS2B</td><td>Custom made with two slots for&amp;nbsp; bottles</td></tr><tr><td>Cage bottom</td><td>Ancare</td><td>#R20PC</td><td>To house rats</td></tr><tr><td>Stoppers</td><td>Ancare</td><td>#8.5</td><td>Rubber stoppers to occlude opening in water bottles &amp;amp; graduated cylinders; goes with ball point sipper tubes&amp;nbsp;</td></tr><tr><td>Ball point tubes, 1.5&quot; length</td><td>Ancare</td><td>#TD-99</td><td>Inserted in stoppers, enables rats to drink from water bottles and graduated cylinders</td></tr><tr><td>Water bottles</td><td>Ancare</td><td>#FSPC8HT</td><td>473 ml (16 oz) capacity, needs to be occluded with stopper</td></tr><tr><td>Graduated cylinders, 100 ml</td><td>Fisher</td><td>#0300741</td><td>To contain ethanol during alcohol exposure in the home cage; spouted end needs to be cut off to enable occlusion with stoppers</td></tr><tr><td>Mettler scale</td><td>Mettler Toledo</td><td>#MS6001S</td><td>For weighing water bottles and ethanol cylinders</td></tr><tr><td>Polyethylene tubing, 1/32&quot; inner diameter,&amp;nbsp; 1/32&quot; wall thickness, 3/32&quot; outside diameter</td><td>Fisher</td><td>#14-169-1A</td><td>To connect 20 ml ethanol syringe to fluid port</td></tr><tr><td>Needle, 18 g, 1.5&quot; length</td><td>Fisher</td><td>#B305199</td><td>To connect polyethylene tubing to ethanol syringes; needs to be filed</td></tr><tr><td>Syringes, 20 ml, Luer-Lok non-sterile</td><td>Fisher</td><td>#1482316J</td><td>To contain ethanol on the fluid pumps; needs to be connected to the polyethylene tubing by a 18 G 1 1/2 needle</td></tr><tr><td>Syringes, 60 ml, Luer-Lok non-sterile</td><td>Fisher</td><td>#14-820-11</td><td>To flush polyethylene tubing after PDT sessions</td></tr><tr><td>Single speed syringe pump, 3.33 rpm</td><td>Med Associates Inc.</td><td>#ENV-018MD</td><td>To hold 20 ml ethanol syringes and deliver ethanol</td></tr><tr><td>Fluid port</td><td>Med Associates Inc.</td><td>#ENV-254-CB</td><td>To enable ethanol delivery during PDT sessions; connected to ethanol syringes with polyethylene tubing</td></tr><tr><td>Port entry infrared detector</td><td>Med Associates Inc.</td><td>#ENV-215M</td><td>Fixed on both sides of&amp;nbsp; fluid port; need 2 per fluid port</td></tr><tr><td>Houselight, 28 V, 100 mA</td><td>Med Associates Inc.</td><td>#ENV-135M</td><td>Provides general lighting in conditioning chamber</td></tr><tr><td>Clicker module</td><td>Med Associates Inc.</td><td>#ENV-225SM</td><td>Auditory stimulus for conditioning chamber</td></tr><tr><td>White noise generator with speaker</td><td>Med Associates Inc.</td><td>#PHM-100</td><td>Auditory stimulus for conditioning chamber</td></tr><tr><td>Bar floor</td><td>Med Associates Inc.</td><td>#ENV-009A-GF</td><td>To go with modular test chamber</td></tr><tr><td>Bench coat, absorbent</td><td>VWR</td><td>#89126-790</td><td>Absorbent paper to put in metal tray, needs to be cut to fit the metal tray</td></tr><tr><td>Polycarbonate sheet, 1/8&quot; thick, 12-5/8&quot; x 10&quot;</td><td>Johnston Plastics</td><td>#30102515</td><td>To make smooth transparent floor insert used for context 1. Needs to be cut in 11.5&quot; x 12.25&quot; pieces</td></tr><tr><td>Aluminium sheet perforated, 0.063&quot; thick, 48&quot; x 96&quot;, 0.250&quot; holes x 0.375&quot; centers</td><td>Anica Steel</td><td></td><td>To make perforated floor insert used for context 2. Needs to be cut in 11.5&quot; x 12.25&quot; pieces</td></tr><tr><td>Black cardboard, 0.053&quot; thick, 28&quot; x 44&quot;</td><td>Omer DeSerres</td><td>#P1909</td><td>To construct panels covering the ceiling, door and back wall of the conditioning chamber in context 1. Needs to be cut to size. Each chamber needs three panels measuring approximately 11.5&quot; x 13.25&quot;. The panels can be joined by the narrower side using duct tape.</td></tr><tr><td>All-purpose strength duct tape, 1.88&quot; x 45 yd.</td><td>Duck Tape</td><td></td><td>Adhesive material to connect the black cardboard panels used to cover the back wall, ceiling and door in context 1.</td></tr><tr><td>Modular test chamber</td><td>Med Associates Inc.</td><td>#ENV-009A</td><td>Conditioning chamber</td></tr><tr><td>Sound attenuating cubicles</td><td>Med Associates Inc.</td><td>#ENV-200R3AM</td><td>Comes with installed 28 V DC fan (#ENV-025F)</td></tr><tr><td>Petri dish</td><td>Fisher</td><td>#08748B</td><td>Bottom only, to contain the sprayed solution that provides the odor stimulus; needs to be centered in the metal tray in conditioning chambers</td></tr><tr><td>PCI interface package</td><td>Med Associates Inc.</td><td>#DIG-700P2-R2</td><td>Connects tabletop interface cabinet to computer; includes 1 interface card, 1 Decode card, 1 ribbon cable and 1 28 V DC power cable</td></tr><tr><td>Large tabletop cabinet and power supply</td><td>Med Associates Inc.</td><td>#SG-6510D</td><td>Used to contain the 12 cards included in the SmartCrtrl 8 Input, 16 Output package</td></tr><tr><td>SmartCtrl 8 Input, 16 Output package</td><td>Med Associates Inc.</td><td>#DIG-716P2</td><td>Used to connect and control the devices in the conditioning chamber. The package includes all the cables, the interface module and the connection panel.</td></tr><tr><td>Med PC IV software</td><td>Med Associates Inc.</td><td>#SOF-735</td><td>Software to run programs. Needs to be installed on the computer.</td></tr><tr><td>Monitor LG, 19&quot; LCD</td><td>Concordia computer store</td><td>#W1942TQ-BF</td><td>Computer monitor; to go with computer running Med PC IV</td></tr><tr><td>HP Z200 tower workstation</td><td>Concordia computer store</td><td>#BZ788US#ABA</td><td>Computer on which Med PC IV is run</td></tr><tr><td>Benzaldehyde, &amp;ge;99%</td><td>Les produits chimiques OMEGA Chemical Company Inc.</td><td>#B37-50</td><td>Dilute in tap water for almond context odor</td></tr><tr><td>Lemon oil, cold pressed, California</td><td>Sigma Aldrich</td><td>#W262528-1KG-K</td><td>Dilute in tap water for lemon context odor</td></tr><tr><td>Ethyl alcohol, 95%</td><td>Commercial Alcohols</td><td></td><td>Dilute in tap water to desired concentration</td></tr></tbody></table>

a procedure to observe context-induced renewal of pavlovian-conditioned alcohol-seeking behavior in rats

<p class="jove_content"> Umwelt Kontexte, in denen Drogen konsumiert werden kann Verlangen, eine subjektive Pawlowschen-konditionierte Reaktion, die Drogen-sucht Verhalten und prompte Rückfall in abstinent Drogenkonsumenten erleichtern auslösen können. Wir haben ein Verfahren, um die Verhaltens-und neuronale Prozesse, die die Auswirkungen der Kontext auf Alkohol-seeking Verhalten bei Ratten zu vermitteln studieren entwickelt. Nach Akklimatisierung an den Geschmack und die pharmakologischen Wirkungen von 15% Ethanol im Käfig, erhalten männlichen Long-Evans-Ratten Pawlow Diskriminierung Ausbildung (PDT) in Klimakammern. In jeder täglich (Mo-Fr) PDT-Sitzung, die jeweils von zwei verschiedenen 10 sec Gehöranlage Reize 16 Studien auftreten. Während eines Stimulus, der CS + 0,2 ml 15% Ethanol wird in einen Fluidanschluss für den oralen Verzehr geliefert. Der zweite Reiz, der CS, wird nicht mit Ethanol gepaart. Über die Sitzungen, Einträge in die Fluidöffnung während der CS + Zunahme, während Einträge in der CS-Stabilisierung auf einem niedrigeren Niveau, das anzeigt, dass ein Predictive Assoziation zwischen der CS + und Ethanol gewonnen. Während PDT jede Kammer mit einer bestimmten Konfiguration von visuellen, taktilen und olfaktorischen Stimuli kontextuellen ausgestattet. Nach PDT ist vom Aussterben Ausbildung in der gleichen Kammer, die jetzt mit einer anderen Konfiguration von Kontextreizen ausgestattet ist durchgeführt. Die CS + und CS sind nach wie vor präsentiert, aber Ethanol wird zurückgehalten, die einen allmählichen Rückgang in Port-Einträge während der CS + verursacht. Bei Test werden Ratten wieder in den Kontext gestellt und PDT mit dem CS + und CS nach wie vor präsentiert, aber ohne Ethanol. Diese Manipulation löst eine stabile und selektive Erhöhung der Anzahl von Eingangsports während des Alkohols prädiktiven CS +, ohne Änderung in der CS antwortet. Dieser Effekt, der als Kontext-induzierte Erneuerung genannt, veranschaulicht die leistungsstarke Kapazität von Zusammenhängen mit dem Alkoholkonsum assoziiert zu Alkohol-sucht Verhalten in Reaktion auf Alkohol Pawlow Hinweise zu stimulieren. </p>

<p class="jove_content"> Intermittierende Ethanol Zugang im Käfig: Ethanol Verbrauch (g / kg) und Ethanol bevorzugt Anstieg über Sitzungen der intermittierenden Ethanol Zugang im Käfig, in der Regel erreicht ein stabiles Niveau in 8 - 12 Sitzungen <strong>(Abbildung 1).</strong> In der in <strong>1</strong> gezeigten Datensatz gemittelt stabile Ethanolkonsum in den letzten beiden Sitzungen Käfig Ethanol Zugang reichten von 1,33 bis 6,44 g / kg für einzelne Ratten. Der Käfig Ethanolkonsum Phase des Experiments kann gestoppt werden, sobald die g / kg Verbrauch ein Plateau erreicht hat, und Ratten sind in diesem stabilen Niveau von Trinkwasser für eine Anzahl von Sessions gehalten. </p><p class="jove_content"> Um Daten aus dieser Phase zu analysieren, führen getrennte wiederholter Maßnahmen Varianzanalyse (ANOVA) über die intraindividuelle Faktor Sitzung für Ethanolkonsum (g / kg) und Ethanol Präferenz. </p><p class="jove_content"> Schematische Darstellung der Kontext-induzierte Verlängerungsverfahren: Jede Phase desVersuchsdurchführung (PDT, Aussterben, Erneuerung Test) ist in <strong>Abbildung 2</strong> veranschaulicht. Siehe die Bildunterschrift für weitere Details. </p><p class="jove_content"> Reaktionsmaßnahmen und Datenanalyse: In allen nachfolgend beschriebenen Verhaltensphasen, sind Port-Eintrag Antworten während der CS + und CS gemacht normiert mit Bezug auf Eintrag Antworten Baseline-Port-CS vor. Um die normierten CS Maßnahmen erstellen, ziehen Sie die Anzahl der Anschlusseinträge während der Pre-Baseline-Intervall CS gemacht (die 10 Sekunden unmittelbar vor der CS Beginn) aus der Anzahl der Port-Einträge während der Präsentationen des CS + und CS gemacht. Die normalisierte CS + und CS-normierten Maßnahmen dienen als Indikatoren für Verhalten hervorgerufen insbesondere durch CS-Präsentationen, über den Ausgangswerten erhöht. </p><p class="jove_content"> Führen getrennte wiederholte Messungen ANOVA mit wiederholten intraindividuelle Faktoren der Sitzung und CS-Typ (CS + vs CS) für die PDT und Aussterben Phasen. Für Vergleiche zwischen Phase (PDT, Aussterben, erneuernal-Test), gemittelt Verwendung von Daten über den letzten beiden Sitzungen der PDT und Extinktion und einer Sitzung der Erneuerung Testdaten in wiederholten ANOVA Messungen. Im Nachgang zu signifikanten Haupteffekten und Wechselwirkungen, die Verwendung <em>post hoc</em> t-Tests für unabhängige oder gepaarte Stichproben, die für multiple Vergleiche korrigiert. Kriterium für die Bedeutung ist bei α = 0,05 festgelegt. </p><p class="jove_content"> PDT: Port Eintrag Antworten auf die CS + Anstieg über PDT-Sitzungen, während Port-Eintrag Antworten auf die CS nach wie vor niedrig und stabil, insgesamt. Beachten Sie, dass Port-Eintrag Antworten auf die CS in den ersten Sitzungen der PDT Training leicht erhöhen; Jedoch Anschlusseinträge in Reaktion auf die CS hergestellt Stabilisierung bei einem viel niedrigeren Niveau als die in Reaktion auf die CS + hergestellt. Erfolgreicher Diskriminierung wird durch eine statistisch signifikante Erhöhung in der Anschlusseinträge bei der CS + belegt verglichen mit dem CS <strong>(Abbildung 3).</strong> Dieses Ergebnis zeigt, dass Pawlowanlage Alkoholsucht Verhalten haten auf den CS +, nicht jedoch für die CS. </p><p class="jove_content"> Extinction: Port Eintrag Antworten auf die CS + allmählich über Aussterben Sitzungen sinken. Port-Eintrag Antworten auf die CS bleiben niedrig und stabil über Aussterben Sitzungen. Idealerweise unterschieden Reaktion wird durch das Ende der Löschung Ausbildung beseitigt, wie durch das Fehlen einer statistisch signifikanten Unterschied zwischen den Eingangsports zu den CS + und CS belegt. Es ist jedoch nicht ungewöhnlich gewissen diskriminiert reagiert bleiben, auch am Ende der Extinktion Ausbildung. In diesem Fall ist es wichtig, am Ende des Aussterbens Ausbildung sind statistisch niedriger als Port-Eintrag Antworten auf die CS + zu Beginn des Aussterbens, als auch am Ende der PDT zu überprüfen, dass die Port-Eintrag Antworten auf die CS + <strong>(Abbildung 3)</strong> . Dieses Muster der Ergebnisse zeigt, dass Pawlowanlage Alkoholsucht Verhalten wurde deutlich ausgelöscht. </p><p class="jove_content"> Kontext-induzierte Erneuerung Test: PortEintrag Antworten auf die CS + Zunahme Rahmen induzierte Verlängerung Test, bezogen auf CS + am Ende der Ausbildung Extinktion beobachtet Anschlusseinträge. Dieser Anstieg ist auf die selektive CS + als Hafen Einträge in die CS unverändert bleiben. Zusätzlich sind Anschlusseinträge zum CS + statistisch erhöhte im Vergleich zu Portangaben zu der CS im Rahmen induzierte Verlängerung Test <strong>(Abbildung 4).</strong> Statistische Vergleiche der Bestätigung der CS + Porteinträge zwischen Aussterben und Test, sowie Vergleiche von CS + und CS-Anschluss Einträge innerhalb Test, ein Indikator für eine Erneuerung der Pawlowschen Anlage Alkohol-sucht Verhalten durch den Alkohol zugeordneten Kontext bei Test induziert. </p><p class="jove_content" fo:keep-together.within-page="always"><img alt="Figur 1" fo:content-width="4in" src="/files/ftp_upload/51898/51898fig1highres.jpg" width="400" /><br /> <strong>Abbildung 1. Eskalation der Ethanolkonsum und Vorzugs durch intermittierende Ethanol-Zugang in den Heimkäfig induziert.</strong> Bedeuten und# 177; SEM <strong>(A)</strong> Ethanol Verbrauch (g / kg) und <strong>(B)</strong> Ethanol bevorzugt über 12 Sitzungen der intermittierenden Ethanol Zugang im Käfig. Beide Maßnahmen in der Regel über mehrere Sitzungen zu erhöhen und dann zu stabilisieren. Stabilen Ethanolkonsum gemittelt über die letzten beiden Sitzungen des Heimkäfig Ethanol Zugang reichten von 1,33 bis 6,44 g / kg für einzelne Ratten. Diese Daten wurden unter Verwendung von männlichen Long-Evans-Ratten Trink 15% (v / v) Ethanol. </p><p class="jove_content" fo:keep-together.within-page="always"><img alt="Figur 2" fo:content-width="4in" src="/files/ftp_upload/51898/51898fig2highres.jpg" width="400" /><br /> <strong>. Abbildung 2. Ein Schema des Versuchsverfahren</strong> Das <strong>Verfahren</strong> besteht aus drei Phasen: Pawlow Diskriminierung Ausbildung (PDT), Aussterben, und Kontext-induzierte Erneuerung Test. Während jeder Sitzung PDT erhalten Ratten 16 Studien, die ein Gehör bedingten Reiz (CS +), die die Lieferung von Ethanol und 16 Studien von Signaleneine zweite auditorischen Reizes (CS), die nicht mit Ethanol Lieferung verbunden ist. PDT-Sitzungen finden in Kontext A, der eine bestimmte Konfiguration von visuellen, olfaktorischen und taktilen Reizen zusammen. Während Extinktion Sitzungen, die im Kontext B durchgeführt werden, von einem anderen Satz von visuellen, Geruchs und taktilen Reizen besteht, die CS + und CS dargestellt, aber Ethanol zurückgehalten. Für den Kontext-induzierte Erneuerung Test werden Ratten Kontext Ein zurückgegeben und die CS + und CS sind ohne Ethanol vorgestellt. <a href="https://www.jove.com/files/ftp_upload/51898/51898fig2highres.jpg" target="_blank">Bitte klicken Sie hier, um eine größere Version dieser Figur zu sehen.</a> </p><p class="jove_content" fo:keep-together.within-page="always"><img alt="Figur 3" fo:content-width="4in" src="/files/ftp_upload/51898/51898fig3highres.jpg" width="400" /><br /> <strong>Abbildung 3. Erwerb und Erlöschen der Pawlowschen diskriminiert Anlage reagiert für Ethanol.</strong> Mittelwert ± SEM normiert Porteinträge made während der CS + und CS für PDT (Sitzungen 1-18) im Kontext A und Extinktion (Sessions 19-26) in Zusammenhang B. Diese Daten wurden erhalten unter Verwendung von männlichen Long-Evans-Ratten mit 10% ausgebildet (v / v) Ethanol als unbedingter Reiz (US), die in Verbindung mit der CS + während der PDT präsentiert wurde. * P &lt;0,05, im Vergleich zu CS + CS. Abbildung ursprünglich in Chaudhri, Sahuque &amp; Janak (2008) veröffentlicht. Verwendung mit Genehmigung von Elsevier. </p><p class="jove_content" fo:keep-together.within-page="always"><img alt="Figur 4" fo:content-width="5in" src="/files/ftp_upload/51898/51898fig4highres.jpg" width="500" /><br /> <strong>Abbildung 4. Kontext-induzierte Erneuerung der Pawlowschen Anlage reagiert für Ethanol.</strong> Mittelwert ± SEM normiert Porteinträge während der CS + und CS in der PDT-Sitzungen im Kontext A, Aussterben Sitzungen im Kontext B und Erneuerung Test in Context A. Daten sind gemittelt über die letzten beiden Sitzungen für die PDT und Aussterben; die Erneuerung Test ist eine einzige Sitzung. DieseDaten wurden unter Verwendung von männlichen Long-Evans-Ratten mit 10% (v / v) Ethanol als unbedingter Reiz (US), die in Verbindung mit der CS + bei PDT dargestellt ausgebildet wurde, erhalten. * P &lt;0,05; ** P &lt;0,001, im Vergleich zu CS + CS. #p &lt;0,05, CS + PDT gegenüber CS + Aussterben bedroht. ^ P &lt;0,05, CS + Aussterben gegen CS + Test. Abbildung ursprünglich in Chaudhri, Sahuque &amp; Janak (2008) veröffentlicht. Verwendung mit Genehmigung von Elsevier. </p>

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A Procedure to Observe Context-induced Renewal of Pavlovian-conditioned Alcohol-seeking Behavior in Rats

<p class="jove_content"> Ein Verfahren, um die Kapazität von einem Alkohol verbundenen Umweltkontextstudie zur Auslösung der Erneuerung der Alkoholsucht bei der Ratte beschrieben. </p>

Eine Vorgehensweise, um Context-induzierte Erneuerung der Pawlowschen Anlage Alkoholsucht bei Ratten Verhalten beobachten

Environmental contexts in which drugs of abuse are consumed can trigger craving, a subjective Pavlovian-conditioned response that can facilitate ...

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12.8K Aufrufe.  Concordia University.  Ein Verfahren, um die Kapazität von einem Alkohol verbundenen Umweltkontextstudie zur Auslösung der Erneuerung der Alkoholsucht bei der Ratte beschrieben. 

Serie: A Procedure to Observe Context-induced Renewal of Pavlovian-conditioned Alcohol-seeking Behavior in Rats

Esterification

<p>Source: Lara Al Hariri at the University of Massachusetts Amherst, MA, USA</p>
<ol class="note-items">
	<li data-note-item="1" data-parent-collapse="">Preparation of the Laboratory<button class="expand">Expand</button>
	<p><em>Here, we show the laboratory preparation for 10 students working in pairs, with some excess. Please adjust quantities as needed.</em></p>
	<ul class="lab-items">
		<li data-sub-note-item="1-2">
		<div class="lab-step">First, put on a lab coat, safety glasses, and nitrile gloves.<strong> Note:</strong> Some of the reagents are volatile or have unpleasant odors, so you will handle them in a fume hood.</div>
		</li>
		<li data-sub-note-item="1-3">
		<div class="lab-step">Ensure that you have glass and organic waste containers and that each sink has paper towels.</div>
		</li>
		<li data-sub-note-item="1-4">
		<div class="lab-step">Follow the table to designate codes for the carboxylic acids and alcohols used in this lab. The carboxylic acids are coded A through C, and the alcohols are coded D through I. The students will use a 3:1 molar ratio of alcohol to carboxylic acid in the reaction.
		<table align="center" height="224" style="border-collapse: collapse; font-size: 17px; line-height: 24px;">
			<tbody>
				<tr>
					<td style="border: 1px solid; padding: 0px; text-align: center;"><strong>Code</strong></td>
					<td style="border: 1px solid; padding: 0px; text-align: center;"><strong>Compound</strong></td>
					<td style="border: 1px solid; padding: 0px; text-align: center;"><strong>MW<br />
					(g/mol) </strong></td>
					<td style="border: 1px solid; padding: 0px; text-align: center;"><strong>Density at RT (g/cm<sup>3</sup>)</strong></td>
					<td style="border: 1px solid; padding: 0px; text-align: center;"><strong>Moles per vial</strong></td>
					<td style="border: 1px solid; padding: 0px; text-align: center;"><strong>Grams per vial</strong></td>
					<td style="border: 1px solid; padding: 0px; text-align: center;"><strong>cm<sup>3</sup> per vial </strong></td>
					<td style="border: 1px solid; padding: 0px; text-align: center;"><strong>Number of vials</strong></td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px; text-align: center;">A</td>
					<td style="border: 1px solid; padding: 0px; text-align: center;">Acetic acid</td>
					<td style="border: 1px solid; padding: 0px; text-align: center;">60.05</td>
					<td style="border: 1px solid; padding: 0px; text-align: center;">1.049</td>
					<td style="border: 1px solid; padding: 0px; text-align: center;">0.07</td>
					<td style="border: 1px solid; padding: 0px; text-align: center;">4.2035</td>
					<td style="border: 1px solid; padding: 0px; text-align: center;">4.0</td>
					<td style="border: 1px solid; padding: 0px; text-align: center;">3</td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px; text-align: center;">B</td>
					<td style="border: 1px solid; padding: 0px; text-align: center;">Butyric acid</td>
					<td style="border: 1px solid; padding: 0px; text-align: center;">88.11</td>
					<td style="border: 1px solid; padding: 0px; text-align: center;">0.953</td>
					<td style="border: 1px solid; padding: 0px; text-align: center;">0.07</td>
					<td style="border: 1px solid; padding: 0px; text-align: center;">6.1677</td>
					<td style="border: 1px solid; padding: 0px; text-align: center;">6.5</td>
					<td style="border: 1px solid; padding: 0px; text-align: center;">3</td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px; text-align: center;">C</td>
					<td style="border: 1px solid; padding: 0px; text-align: center;">Anthranilic acid</td>
					<td style="border: 1px solid; padding: 0px; text-align: center;">137.14</td>
					<td style="border: 1px solid; padding: 0px; text-align: center;">-</td>
					<td style="border: 1px solid; padding: 0px; text-align: center;">0.07</td>
					<td style="border: 1px solid; padding: 0px; text-align: center;">9.5998</td>
					<td style="border: 1px solid; padding: 0px; text-align: center;">-</td>
					<td style="border: 1px solid; padding: 0px; text-align: center;">1</td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px; text-align: center;">D</td>
					<td style="border: 1px solid; padding: 0px; text-align: center;">1-Propanol</td>
					<td style="border: 1px solid; padding: 0px; text-align: center;">60.10</td>
					<td style="border: 1px solid; padding: 0px; text-align: center;">0.803</td>
					<td style="border: 1px solid; padding: 0px; text-align: center;">0.21</td>
					<td style="border: 1px solid; padding: 0px; text-align: center;">12.6210</td>
					<td style="border: 1px solid; padding: 0px; text-align: center;">15.7</td>
					<td style="border: 1px solid; padding: 0px; text-align: center;">1</td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px; text-align: center;">E</td>
					<td style="border: 1px solid; padding: 0px; text-align: center;">Isoamyl alcohol</td>
					<td style="border: 1px solid; padding: 0px; text-align: center;">88.15</td>
					<td style="border: 1px solid; padding: 0px; text-align: center;">0.810</td>
					<td style="border: 1px solid; padding: 0px; text-align: center;">0.21</td>
					<td style="border: 1px solid; padding: 0px; text-align: center;">18.512</td>
					<td style="border: 1px solid; padding: 0px; text-align: center;">22.8</td>
					<td style="border: 1px solid; padding: 0px; text-align: center;">1</td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px; text-align: center;">F</td>
					<td style="border: 1px solid; padding: 0px; text-align: center;">1-Butanol</td>
					<td style="border: 1px solid; padding: 0px; text-align: center;">74.12</td>
					<td style="border: 1px solid; padding: 0px; text-align: center;">0.810</td>
					<td style="border: 1px solid; padding: 0px; text-align: center;">0.21</td>
					<td style="border: 1px solid; padding: 0px; text-align: center;">15.565</td>
					<td style="border: 1px solid; padding: 0px; text-align: center;">19.2</td>
					<td style="border: 1px solid; padding: 0px; text-align: center;">1</td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px; text-align: center;">G</td>
					<td style="border: 1px solid; padding: 0px; text-align: center;">Ethanol</td>
					<td style="border: 1px solid; padding: 0px; text-align: center;">46.07</td>
					<td style="border: 1px solid; padding: 0px; text-align: center;">0.789</td>
					<td style="border: 1px solid; padding: 0px; text-align: center;">0.21</td>
					<td style="border: 1px solid; padding: 0px; text-align: center;">9.6747</td>
					<td style="border: 1px solid; padding: 0px; text-align: center;">12.3</td>
					<td style="border: 1px solid; padding: 0px; text-align: center;">1</td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px; text-align: center;">H</td>
					<td style="border: 1px solid; padding: 0px; text-align: center;">Methanol</td>
					<td style="border: 1px solid; padding: 0px; text-align: center;">32.04</td>
					<td style="border: 1px solid; padding: 0px; text-align: center;">0.791</td>
					<td style="border: 1px solid; padding: 0px; text-align: center;">0.21</td>
					<td style="border: 1px solid; padding: 0px; text-align: center;">6.7284</td>
					<td style="border: 1px solid; padding: 0px; text-align: center;">8.5</td>
					<td style="border: 1px solid; padding: 0px; text-align: center;">2</td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px; text-align: center;">I</td>
					<td style="border: 1px solid; padding: 0px; text-align: center;">1-Octanol</td>
					<td style="border: 1px solid; padding: 0px; text-align: center;">130.23</td>
					<td style="border: 1px solid; padding: 0px; text-align: center;">0.830</td>
					<td style="border: 1px solid; padding: 0px; text-align: center;">0.21</td>
					<td style="border: 1px solid; padding: 0px; text-align: center;">27.348</td>
					<td style="border: 1px solid; padding: 0px; text-align: center;">32.9</td>
					<td style="border: 1px solid; padding: 0px; text-align: center;">1</td>
				</tr>
			</tbody>
		</table>
		</div>
		</li>
		<li data-sub-note-item="1-5">
		<div class="lab-step">Prepare the three carboxylic acids: acetic acid, butyric acid, and anthranilic acid. Each group will need 0.07 moles of their assigned carboxylic acid in a vial.</div>
		</li>
		<li data-sub-note-item="1-6">
		<div class="lab-step">Tare a 10-mL glass vial on an analytical balance in a fume hood. Use a Pasteur pipette to measure 4.2035 g of acetic acid, which will be about 4 mL. Cap the vial and label it &#8216;A&#8217;. Prepare two more labeled vials of acetic acid in the same way.</div>
		</li>
		<li data-sub-note-item="1-7">
		<div class="lab-step">Weigh 6.1677 g of butyric acid, which will be about 6.5 mL, in a tared 10-mL vial. Cap the vial and label it &#8216;B&#8217;. Prepare two more vials of butyric acid in the same way.</div>
		</li>
		<li data-sub-note-item="1-8">
		<div class="lab-step">Use a small spatula to weigh 9.5998 g of solid anthranilic acid in a tared 10-mL vial. Cap the vial and label it &#8216;C&#8217;. You should now have a total of seven vials of carboxylic acid.</div>
		</li>
		<li data-sub-note-item="1-9">
		<div class="lab-step">Prepare the 7 alcohols. Each group will need 0.21 moles of their assigned alcohol in a vial. Use the table as a guide to prepare and label one vial of each alcohol. Prepare and label two vials of methanol.</div>
		</li>
		<li data-sub-note-item="1-10">
		<div class="lab-step">You should now have a total of seven vials of alcohol. Keep the reagent vials in the instructor&#39;s hood for now and put away the stock bottles of the carboxylic acids and alcohols.</div>
		</li>
		<li data-sub-note-item="1-11">
		<div class="lab-step">Assign each student group one of these seven combinations of reagents.
		<table align="center" height="224" style="border-collapse: collapse; font-size: 17px; line-height: 24px;">
			<tbody>
				<tr>
					<td style="border: 1px solid; padding: 0px; text-align: center;"><strong>Ester code</strong></td>
					<td style="border: 1px solid; padding: 0px; text-align: center;"><strong>Carboxylic acid </strong></td>
					<td style="border: 1px solid; padding: 0px; text-align: center;"><strong>Alcohol</strong></td>
					<td style="border: 1px solid; padding: 0px; text-align: center;"><strong>Ester product</strong></td>
					<td style="border: 1px solid; padding: 0px; text-align: center;"><strong>Scent</strong></td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px; text-align: center;">AD</td>
					<td style="border: 1px solid; padding: 0px; text-align: center;">Acetic acid</td>
					<td style="border: 1px solid; padding: 0px; text-align: center;">1-Propanol</td>
					<td style="border: 1px solid; padding: 0px; text-align: center;">Propyl acetate</td>
					<td style="border: 1px solid; padding: 0px; text-align: center;">Pear</td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px; text-align: center;">AE</td>
					<td style="border: 1px solid; padding: 0px; text-align: center;">Acetic acid</td>
					<td style="border: 1px solid; padding: 0px; text-align: center;">Isoamyl alcohol</td>
					<td style="border: 1px solid; padding: 0px; text-align: center;">Isoamyl acetate</td>
					<td style="border: 1px solid; padding: 0px; text-align: center;">Banana</td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px; text-align: center;">AI</td>
					<td style="border: 1px solid; padding: 0px; text-align: center;">Acetic acid</td>
					<td style="border: 1px solid; padding: 0px; text-align: center;">1-Octanol</td>
					<td style="border: 1px solid; padding: 0px; text-align: center;">Octyl acetate</td>
					<td style="border: 1px solid; padding: 0px; text-align: center;">Orange</td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px; text-align: center;">BF</td>
					<td style="border: 1px solid; padding: 0px; text-align: center;">Butyric acid</td>
					<td style="border: 1px solid; padding: 0px; text-align: center;">1-Butanol</td>
					<td style="border: 1px solid; padding: 0px; text-align: center;">Butyl butyrate</td>
					<td style="border: 1px solid; padding: 0px; text-align: center;">Pineapple</td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px; text-align: center;">BG</td>
					<td style="border: 1px solid; padding: 0px; text-align: center;">Butyric acid</td>
					<td style="border: 1px solid; padding: 0px; text-align: center;">Ethanol</td>
					<td style="border: 1px solid; padding: 0px; text-align: center;">Ethyl butyrate</td>
					<td style="border: 1px solid; padding: 0px; text-align: center;">Strawberry</td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px; text-align: center;">BH</td>
					<td style="border: 1px solid; padding: 0px; text-align: center;">Butyric acid</td>
					<td style="border: 1px solid; padding: 0px; text-align: center;">Methanol</td>
					<td style="border: 1px solid; padding: 0px; text-align: center;">Methyl butyrate</td>
					<td style="border: 1px solid; padding: 0px; text-align: center;">Apple</td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px; text-align: center;">CH</td>
					<td style="border: 1px solid; padding: 0px; text-align: center;">Anthranilic acid</td>
					<td style="border: 1px solid; padding: 0px; text-align: center;">Methanol</td>
					<td style="border: 1px solid; padding: 0px; text-align: center;">Methyl anthranilate</td>
					<td style="border: 1px solid; padding: 0px; text-align: center;">Grape</td>
				</tr>
			</tbody>
		</table>
		</div>
		</li>
		<li data-sub-note-item="1-12">
		<div class="lab-step">Prepare or obtain seven vials of the fruit scents and label them with the fruit names as references.</div>
		</li>
		<li data-sub-note-item="1-13">
		<div class="lab-step">Set out the following glassware and equipment at each student lab station (we suggest that students work in pairs):</div>
		</li>
		<li data-sub-note-item="1-14">
		<div class="lab-step">Put bottles of saturated sodium bicarbonate and 3 M sulfuric acid in a dedicated fume hood, along with a roll of plastic paraffin film and scissors.</div>
		</li>
		<li data-sub-note-item="1-15">
		<div class="lab-step">Set out filter paper and Pasteur pipettes in a central area so that every student group can easily access them.
		<table align="center" height="224" style="border-collapse: collapse; font-size: 17px; line-height: 24px;">
			<tbody>
				<tr>
					<td style="border: 1px solid; padding: 0px;">&#160;1&#160;&#160;&#160;&#160;Lab stand</td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px;">&#160;2&#160;&#160;&#160;&#160;3-prong clamps</td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px;">&#160;1&#160;&#160;&#160;&#160;Stir plate</td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px;">&#160;1&#160;&#160;&#160;&#160;Lab jack</td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px;">&#160;1&#160;&#160;&#160;&#160;Heating mantle</td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px;">&#160;1&#160;&#160;&#160;&#160;Temperature controller</td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px;">&#160;2&#160;&#160;&#160;&#160;Pieces of tubing (for condenser)</td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px;">&#160;1&#160;&#160;&#160;&#160;Joint clip</td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px;">&#160;1&#160;&#160;&#160;&#160;Tube of vacuum grease</td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px;">&#160;1&#160;&#160;&#160;&#160;Box of laboratory wipes</td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px;">&#160;1&#160;&#160;&#160;&#160;10-mL graduated cylinder</td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px;">&#160;1&#160;&#160;&#160;&#160;600-mL beaker</td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px;">&#160;1&#160;&#160;&#160;&#160;50-mL round-bottom flask and flask stand</td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px;">&#160;1&#160;&#160;&#160;&#160;Standard condenser</td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px;">&#160;1&#160;&#160;&#160;&#160;Small stir bar</td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px;">&#160;1&#160;&#160;&#160;&#160;Glass rod</td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px;">&#160;1&#160;&#160;&#160;&#160;Package of pH paper</td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px;">&#160;2&#160;&#160;&#160;&#160;Pasteur pipette bulbs</td>
				</tr>
			</tbody>
		</table>
		</div>
		</li>
		<li data-sub-note-item="1-16">
		<div class="lab-step">Just before the lab, fill a large insulated cooler with crushed ice. Place the cooler and an ice scoop in a central area in the lab.</div>
		</li>
		<li data-sub-note-item="1-17">
		<div class="lab-step">When the students arrive, show them how to safely waft vapors before giving them their assigned reagents.</div>
		</li>
	</ul>
	</li>
</ol>

Esterification: Synthesis of Ester by Fischer Esterification - Prep

Veresterung

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

	Preparation of the LaboratoryExpand
	Here, we show the laboratory preparation ...

Lehre

JoVE Lab Manual

Chemie

Lab Manual Sub Articles

Identifying Alcohols

<p>Source: Lara Al Hariri and Ahmed Basabrain at the University of Massachusetts Amherst, MA, USA</p>
<ol class="note-items">
	<li data-note-item="1" data-parent-collapse="">Ferric Chloride Test for Phenols<button class="expand">Expand</button>
	<p>In this lab, you will identify an unknown alcohol using the ferric chloride test, the Jones test, and the Lucas test. You&#39;ll test known alcohols alongside the unknown alcohol as examples of positive and negative results for each test.</p>
	<p>The four known alcohols are 1-butanol, a primary alcohol, 2-butanol, a secondary alcohol, 2-methyl-2-propanol, a tertiary alcohol, and phenol. The four possible unknown alcohols are 1-propanol, 2-propanol, 2-methyl-2-butanol, and para-chlorophenol.</p>
	<p>You&#39;ll start with the ferric chloride test for the presence of phenols. Iron(III) chloride, or ferric chloride, forms a brown complex when it&#39;s dissolved in water. If you add a phenol to this solution, a purple iron(III)-phenol complex will form instead. This effect is not observed with non-aromatic or aliphatic alcohols.</p>
	<ul class="lab-items">
		<li data-sub-note-item="1-4">
		<div class="lab-step">Put on a lab coat, safety glasses, and gloves before you begin. <strong>Note:</strong> The alcohols that you will test are toxic and flammable, and the reagents that you will use are toxic and corrosive, so you will perform this lab in a fume hood.</div>
		</li>
		<li data-sub-note-item="1-5">
		<div class="lab-step">Now, get your assigned unknown alcohol from your instructor and bring it to your fume hood. Label a 250-mL beaker &#8216;aqueous metal waste&#8217;.</div>
		</li>
		<li data-sub-note-item="1-6">
		<div class="lab-step">Next, put five small test tubes in a rack and label them &#8216;1-butanol&#8217;, &#8216;2-butanol&#8217;, &#8216;2-methyl-2-propanol&#8217;, &#8216;phenol&#8217;, and &#8216;unknown&#8217;. Bring the rack to the analytical balance to obtain phenol.</div>
		</li>
		<li data-sub-note-item="1-7">
		<div class="lab-step">If your unknown is a solid, bring it with you. Weigh about 0.2 g of phenol and pour it into the corresponding tube. Do the same for your unknown if it is a solid and then return to your hood.</div>
		</li>
		<li data-sub-note-item="1-8">
		<div class="lab-step">Now label three small vials &#8216;1-butanol&#8217;, &#8216;2-butanol&#8217;, and &#8216;2-methyl-2-propanol&#8217;.</div>
		</li>
		<li data-sub-note-item="1-9">
		<div class="lab-step">Bring the vials to the solvent hood and use clean Pasteur pipettes to fill each vial with about 1 mL of the matching alcohol.<strong> Note: </strong>Do not reuse pipettes.</div>
		</li>
		<li data-sub-note-item="1-10">
		<div class="lab-step">Next, obtain at least four Pasteur pipettes and a 250-mL beaker. Get more pipettes whenever you need them, and don&#39;t put used pipettes back in the beaker.</div>
		</li>
		<li data-sub-note-item="1-11">
		<div class="lab-step">In your fume hood, pipette 3 &#8211; 4 drops of 1-butanol, 2-butanol, and 2-methyl-2-propanol into the corresponding tubes.</div>
		</li>
		<li data-sub-note-item="1-12">
		<div class="lab-step">To the phenol tube, add several drops of deionized water and shake to mix. This will provide better visual results for the ferric test.</div>
		</li>
		<li data-sub-note-item="1-13">
		<div class="lab-step">If your unknown alcohol is a liquid, use a clean pipette to place 3 &#8211; 4 drops of it in the unknown test tube.</div>
		</li>
		<li data-sub-note-item="1-14">
		<div class="lab-step">Now, obtain the bottle of iron(III) chloride solution and add 2 &#8211; 3 drops to each test tube.</div>
		</li>
		<li data-sub-note-item="1-15">
		<div class="lab-step">Stir each mixture well with a glass rod. Use deionized water to rinse off residual alcohol before mixing the contents of the next tube. Make sure that the phenol dissolves completely along with the unknown if it is a solid.</div>
		</li>
		<li data-sub-note-item="1-16">
		<div class="lab-step">Wait 5 min before comparing your unknown to the known solutions. <strong>Note:</strong> If your unknown solution is purple, like the phenol solution, then your unknown is a phenol derivative. If it looks like the other three tubes, it is not a phenol derivative. Record the results of the test in your lab notebook.
		<h4><strong>Table 1: Identifying Unknown Alcohol</strong></h4>
		<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
			<tbody>
				<tr>
					<td style="text-align: center;"><strong>Alcohol</strong></td>
					<td style="text-align: center;"><strong>Ferric Chloride Test</strong></td>
					<td style="text-align: center;"><strong>Jones Test</strong></td>
					<td style="text-align: center;"><strong>Lucas Test</strong></td>
				</tr>
				<tr>
					<td>1-butanol</td>
					<td style="text-align: center;"></td>
					<td style="text-align: center;"></td>
					<td style="text-align: center;"></td>
				</tr>
				<tr>
					<td>2-butanol</td>
					<td style="text-align: center;"></td>
					<td style="text-align: center;"></td>
					<td style="text-align: center;"></td>
				</tr>
				<tr>
					<td>2-methyl-2-propanol</td>
					<td style="text-align: center;"></td>
					<td style="text-align: center;"></td>
					<td style="text-align: center;"></td>
				</tr>
				<tr>
					<td>Phenol</td>
					<td style="text-align: center;"></td>
					<td style="text-align: center;"></td>
					<td style="text-align: center;"></td>
				</tr>
				<tr>
					<td>Unknown</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/CDNSource/lm/labs/71/Table_1._Identifying_unknown_alcohol.xlsx" target="_blank">Click Here to download Table 1</a></div>
		</li>
		<li data-sub-note-item="1-17">
		<div class="lab-step">The next tests don&#39;t work with phenols, so you&#39;ll only test aliphatic alcohols for the rest of the lab. If your unknown is a phenol derivative, skip the instructions for the unknown and test only the three known aliphatic alcohols.</div>
		</li>
	</ul>
	</li>
	<li data-note-item="2" data-parent-collapse="">The Jones Test for Aliphatic Primary and Secondary Alcohols<button class="expand">Expand</button>
	<p>In this section, you&#39;ll perform the Jones test for primary and secondary alcohols. Jones&#39; reagent is made with chromium trioxide and sulfuric acid in water, which forms chromic acid (H<sub>2</sub>CrO<sub>4</sub>)<em> in situ</em>. This powerful reagent oxidizes secondary alcohols to ketones, primary alcohols to aldehydes, which after forming an aldehyde hydrate, are further reduced to carboxylic acids.</p>
	<p>The oxidation state of chromium is the key to this test. Chromium is in the +6 oxidation state in the Jones&#39; reagent. The Cr(VI) complexes in the reagent give it its bright reddish, orange color.</p>
	<p>When Cr(VI) oxidizes an alcohol, chromium is reduced to the +3 oxidation state. First, the alcohol and chromic acid form a chromate ester. Then, a base (H<sub>2</sub>O) cleaves the C-H bond of the alcohol, forming the carbonyl group while reducing Cr(VI) to Cr(IV). Because the carbon of the alcohol undergoes a 2-electron oxidation, and Cr(VI) a 2-electron reduction, this step is a reduction-oxidation step. Cr(IV) participates in further oxidation steps and is eventually reduced to Cr(III). Cr(III) is often present as hexaaquachromium(III) ions&#160;&#8212; [Cr(H<sub>2</sub>O)<sub>6</sub>]<sup>3+</sup> &#8212; and Cr(III) complexes, where H<sub>2</sub>O molecules are replaced by one or more sulphate ions, [Cr(H<sub>2</sub>O)<sub>5</sub>(SO<sub>4</sub>)]<sup>+</sup>. These complexes give Cr(III) the characteristic green color. So, when you mix Jones&#39; reagent with a primary or secondary alcohol in acetone, the orange solution will turn green.</p>
	<p>Jones oxidation of alcohols doesn&#39;t work with tertiary alcohols because the -OH group is already bonded to three carbon atoms and cannot form an extra C-O bond. Thus, you won&#39;t see a color change if you combine Jones&#39; reagent with a tertiary alcohol because chromium isn&#39;t reduced.</p>
	<ul class="lab-items">
		<li data-sub-note-item="2-5">
		<div class="lab-step">Label four clean test tubes &#8216;1-butanol&#8217;, &#8216;2-butanol&#8217;, &#8216;2-methyl-2-propanol&#8217;, and &#8216;unknown&#8217; and place 10 drops of acetone in each. Acetone is the preferred solvent for Jones oxidation.</div>
		</li>
		<li data-sub-note-item="2-6">
		<div class="lab-step">Then, use clean pipettes to add 3 &#8211; 4 drops of the known aliphatic alcohols and your unknown alcohol to the corresponding tubes.</div>
		</li>
		<li data-sub-note-item="2-7">
		<div class="lab-step">Stir each solution well with the glass rod and rinse the rod with water after mixing each one.</div>
		</li>
		<li data-sub-note-item="2-8">
		<div class="lab-step">Now, obtain the bottle of Jones&#39; reagent and carefully add 2 &#8211; 3 drops to each tube. The reagent is strongly acidic and corrosive, so be careful with it and tightly cap the bottle when you&#39;re done. Then, mix each solution well, cleaning the glass rod after each one.</div>
		</li>
		<li data-sub-note-item="2-9">
		<div class="lab-step">Let the solutions react for 10 min, and then compare your unknown to the knowns. <strong>Note:</strong> If the unknown solution turned green, like 1-butanol and 2-butanol, the test is positive for a primary or secondary alcohol. If it stayed orange, like 2-methyl-2-propanol, the test is negative.</div>
		</li>
		<li data-sub-note-item="2-10">
		<div class="lab-step">Record the results in your lab notebook.</div>
		</li>
	</ul>
	</li>
	<li data-note-item="3" data-parent-collapse="">The Lucas Test for Classifying Small Saturated Alcohols<button class="expand">Expand</button>
	<p>The Jones test doesn&#39;t distinguish between primary and secondary alcohols, so you&#39;ll use the Lucas test to identify which is which.</p>
	<p>Lucas&#39; reagent, which is a mixture of zinc chloride and hydrochloric acid, converts secondary and tertiary alcohols to chloroalkanes at room temperature. Chloroalkanes are nearly insoluble in water, so a positive result appears as the mixture separates into a cloudy chloroalkane-containing layer over a clear layer.</p>
	<p>The rate-determining step of the reaction involves converting the alcohol to a carbocation, so the speed of the reaction depends on how stable the carbocation is.</p>
	<p>Tertiary carbocations are very stable, so tertiary alcohols give a positive reaction almost immediately. Secondary carbocations are less stable, so secondary alcohols give a positive result after a few seconds to a few minutes. Primary carbocations are too unstable for this reaction, so primary alcohols give a negative result.</p>
	<ul class="lab-items">
		<li data-sub-note-item="3-5">
		<div class="lab-step">Now, label for clean test tubes as &#8216;1-butanol&#8217;, &#8216;2-butanol&#8217;, &#8216;2-methyl-2-propanol&#8217;, and &#8216;unknown&#8217;.</div>
		</li>
		<li data-sub-note-item="3-6">
		<div class="lab-step">Obtain the bottle of Lucas&#39; reagent and use a graduated cylinder to measure 2 mL of the reagent for each tube.</div>
		</li>
		<li data-sub-note-item="3-7">
		<div class="lab-step">Next, add 2 drops of 2-methyl-2-propanol to the appropriate tube. Immediately, stir the mixture for a few seconds and place it in the rack. The mixture will promptly separate into a clear lower layer and a cloudy upper layer.</div>
		</li>
		<li data-sub-note-item="3-8">
		<div class="lab-step">Thoroughly rinse the glass rod and record the immediate positive result in your lab notebook.</div>
		</li>
		<li data-sub-note-item="3-9">
		<div class="lab-step">Mix the remaining alcohols with Lucas&#39; reagent in the same way, using a fresh pipette for each alcohol and rinsing the glass rod each time. <strong>Note:</strong> Don&#39;t add too much alcohol or jostle the tubes because that can slow down the reaction.</div>
		</li>
		<li data-sub-note-item="3-10">
		<div class="lab-step">Watch carefully after mixing the unknown alcohol with Lucas&#39; reagent to ensure that you can distinguish an immediate positive result from a positive result after a brief delay.</div>
		</li>
		<li data-sub-note-item="3-11">
		<div class="lab-step">Check on the tubes every few minutes and record positive results as you see them. Note approximately how long it took for delayed positive results to appear.</div>
		</li>
		<li data-sub-note-item="3-12">
		<div class="lab-step">Once the tubes have sat for at least 15 min, you can assume that the results are final. Record the results for 1-butanol, 2-butanol, and for your unknown if applicable.</div>
		</li>
		<li data-sub-note-item="3-13">
		<div class="lab-step">Now, let&#39;s clean up. Your instructor will collect the Cr(VI) solutions, so leave any tubes of orange solution from the Jones test in your hood.</div>
		</li>
		<li data-sub-note-item="3-14">
		<div class="lab-step">Empty the other test tubes into the waste beaker and rinse them with deionized water. Pour the waste into the aqueous metal waste container using a funnel and rinse the beaker with deionized water.</div>
		</li>
		<li data-sub-note-item="3-15">
		<div class="lab-step">Now, wash your glassware as usual and dispose of used pipettes in the glass waste. Lastly, clean your hood and throw out any trash in the lab trash container.</div>
		</li>
	</ul>
	</li>
	<li data-note-item="4" data-parent-collapse="">Results<button class="expand">Expand</button>
	<p>Identify your unknown alcohol. The four possibilities are 1-propanol, 2-propanol, 2-methyl-2-butanol, and para-chlorophenol.</p>
	<ul class="lab-items">
		<li data-sub-note-item="4-2">
		<div class="lab-step">Consider the results of the ferric chloride test first. If the mixture with your unknown alcohol turned purple, like phenol did, your alcohol must be para-chlorophenol. If you saw a negative result, your unknown is one of the three aliphatic alcohols.</div>
		</li>
		<li data-sub-note-item="4-3">
		<div class="lab-step">Look at the results of the Jones test. If the mixture with the unknown alcohol turned green, the unknown could be primary or secondary. If the mixture with your unknown alcohol stayed orange, your unknown alcohol is tertiary. Therefore, a negative Jones result means that your alcohol must be 2-methyl-2-butanol.</div>
		</li>
		<li data-sub-note-item="4-4">
		<div class="lab-step">Lastly, look at the results of the Lucas test. If you had a negative result in the Jones test, you should also have seen an immediate positive result in the Lucas test. This confirms that your alcohol was tertiary. If your unknown had a positive result after a short delay, then your unknown alcohol is secondary, and therefore, must be 2-propanol. Finally, if your unknown had a negative result in the Lucas test, your alcohol is primary, and therefore, must be 1-propanol.</div>
		</li>
	</ul>
	</li>
</ol>

Identifying Alcohols: Ferric Chloride Test, Jones Test, and Lucas Test - Procedure

Identifizierung von Alkoholen

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

	Ferric Chloride Test for PhenolsExpand
	In this lab, you ...

Beer's Law

Beer-Lambert Law and Equilibrium Constant Determination - Procedure

Das Gesetz des Bieres

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

	Lab Setup for Beer&#8217;s LawExpand
	In this lab, you will combine aqueous solutions of FeCl3 ...

Concentration Dependence

<p>Source: Smaa Koraym at Johns Hopkins University, MD, USA</p>
<ol class="note-items">
	<li data-note-item="1" data-parent-collapse="">Concentration Dependence<button class="expand">Expand</button>
	<p>In this experiment, you will combine varying concentrations of aqueous HCl and sodium thiosulfate to make solid sulfur, which rapidly gathers into opaque yellow particles at a certain sulfur concentration. Since the reaction solution starts clear and colorless, you can easily tell when it reaches that concentration.</p>
	<p>You&#39;ll use the same test tube and total volume each time, so it will take the same amount of sulfur to make each solution completely opaque. Thus, you&#39;ll measure the reaction progress by timing how long that takes. You&#39;ll then use that data to estimate the reaction orders of the individual reactants and of the overall reaction.</p>
	<p>Before starting the lab, make a table listing the reactant concentrations, the time to solution opacity, and the solution temperature for the trials.</p>
	<h4><strong>Table 1: Reaction progress</strong></h4>
	<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
		<tbody>
			<tr>
				<td><strong>Trial</strong></td>
				<td><strong>[Na<sub>2</sub>S<sub>2</sub>O<sub>3</sub>] (M)</strong></td>
				<td><strong>[HCl] (M)</strong></td>
				<td><strong>Time (s)</strong></td>
				<td><strong>Temp (&#176;C)</strong></td>
			</tr>
			<tr>
				<td style="text-align: center;">1</td>
				<td style="text-align: center;">0.1 M</td>
				<td style="text-align: center;">3.0 M</td>
				<td></td>
				<td></td>
			</tr>
			<tr>
				<td style="text-align: center;">2</td>
				<td style="text-align: center;">0.1 M</td>
				<td style="text-align: center;">3.0 M</td>
				<td></td>
				<td></td>
			</tr>
			<tr>
				<td style="text-align: center;">3</td>
				<td style="text-align: center;">0.2 M</td>
				<td style="text-align: center;">3.0 M</td>
				<td></td>
				<td></td>
			</tr>
			<tr>
				<td style="text-align: center;">4</td>
				<td style="text-align: center;">0.15 M</td>
				<td style="text-align: center;">3.0 M</td>
				<td></td>
				<td></td>
			</tr>
			<tr>
				<td style="text-align: center;">5</td>
				<td style="text-align: center;">0.05 M</td>
				<td style="text-align: center;">3.0 M</td>
				<td></td>
				<td></td>
			</tr>
			<tr>
				<td style="text-align: center;">6</td>
				<td style="text-align: center;">0.1 M</td>
				<td style="text-align: center;">6.0 M</td>
				<td></td>
				<td></td>
			</tr>
			<tr>
				<td style="text-align: center;">7</td>
				<td style="text-align: center;">0.1 M</td>
				<td style="text-align: center;">4.5 M</td>
				<td></td>
				<td></td>
			</tr>
			<tr>
				<td style="text-align: center;">8</td>
				<td style="text-align: center;">0.1 M</td>
				<td style="text-align: center;">1.5 M</td>
				<td></td>
				<td></td>
			</tr>
		</tbody>
	</table>
	<p><a href="https://www.jove.com/CDNSource/lm/labs/50/Table_1._Reaction_progress.xlsx" target="_blank">Click Here to download Table 1</a></p>
	<p>We want to make sure that the reactions are all taking place at room temperature. You&#39;ll perform two benchmark trials, three trials with different sodium thiosulfate concentrations, and three trials with different HCl concentrations. You will vary the concentrations by diluting stock solutions of sodium thiosulfate and HCl, as shown in the following tables.</p>
	<h4><strong>Table 2: Sodium thiosulfate dilutions</strong></h4>
	<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
		<tbody>
			<tr>
				<td style="text-align: center;"><strong>Target Concentration</strong></td>
				<td style="text-align: center;"><strong>Volume of 0.2 M sodium thiosulfate</strong></td>
				<td style="text-align: center;"><strong>Volume of DI water</strong></td>
			</tr>
			<tr>
				<td style="text-align: center;">0.05 M</td>
				<td style="text-align: center;">5 mL</td>
				<td style="text-align: center;">15 mL</td>
			</tr>
			<tr>
				<td style="text-align: center;">0.10 M</td>
				<td style="text-align: center;">10 mL</td>
				<td style="text-align: center;">10 mL</td>
			</tr>
			<tr>
				<td style="text-align: center;">0.15 M</td>
				<td style="text-align: center;">15 mL</td>
				<td style="text-align: center;">5 mL</td>
			</tr>
			<tr>
				<td style="text-align: center;">0.20 M</td>
				<td style="text-align: center;">20 mL</td>
				<td style="text-align: center;">0 mL</td>
			</tr>
		</tbody>
	</table>
	<p><a href="https://www.jove.com/CDNSource/lm/labs/50/Table_2._Sodium_thiosulfate_dilutions.xlsx" target="_blank">Click Here to download Table 2</a></p>
	<h4><strong>Table 3: HCl Dilutions</strong></h4>
	<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
		<tbody>
			<tr>
				<td style="text-align: center;"><strong>Target Concentration</strong></td>
				<td style="text-align: center;"><strong>Volume of 6.0 M HCl</strong></td>
				<td style="text-align: center;"><strong>Volume of DI water</strong></td>
			</tr>
			<tr>
				<td style="text-align: center;">1.5 M</td>
				<td style="text-align: center;">2.5 mL</td>
				<td style="text-align: center;">7.5 mL</td>
			</tr>
			<tr>
				<td style="text-align: center;">3.0 M</td>
				<td style="text-align: center;">5.0 mL</td>
				<td style="text-align: center;">5.0 mL</td>
			</tr>
			<tr>
				<td style="text-align: center;">4.5 M</td>
				<td style="text-align: center;">7.5 mL</td>
				<td style="text-align: center;">2.5 mL</td>
			</tr>
			<tr>
				<td style="text-align: center;">6.0 M</td>
				<td style="text-align: center;">10 mL</td>
				<td style="text-align: center;">0 mL</td>
			</tr>
		</tbody>
	</table>
	<p><a href="https://www.jove.com/CDNSource/lm/labs/50/Table_3._HCl_Dilutions.xlsx" target="_blank">Click Here to download Table 3</a></p>
	<p>Remember to use caution when handling HCl, which is toxic and highly corrosive. Sulfur dioxide, which is a gaseous product of this reaction, is also toxic. You will leave the reaction waste in the fume hood overnight to let the sulfur dioxide escape harmlessly.</p>
	<ul class="lab-items">
		<li data-sub-note-item="1-6">
		<div class="lab-step">First, put on a lab coat, splash-proof safety glasses, and nitrile gloves.</div>
		</li>
		<li data-sub-note-item="1-7">
		<div class="lab-step">Fill a plastic wash bottle with deionized water and get a test tube marked with an X.</div>
		</li>
		<li data-sub-note-item="1-8">
		<div class="lab-step">Clamp the test tube over the center of a small stir plate. The label must be on the opposite side from you. Make sure that you can clearly see the X. Place a small magnetic stir bar in the test tube.</div>
		</li>
		<li data-sub-note-item="1-9">
		<div class="lab-step">Use a thermometer clamp to secure a glass thermometer in the lower third of the tube, keeping it clear of the X and the stir bar.</div>
		</li>
		<li data-sub-note-item="1-10">
		<div class="lab-step">Cut a sheet of about 20 squares of plastic paraffin film from a roll and bring the sheet back to your hood.</div>
		</li>
		<li data-sub-note-item="1-11">
		<div class="lab-step">Lay paper towels in the fume hood as a clean surface for labware to be reused.</div>
		</li>
		<li data-sub-note-item="1-12">
		<div class="lab-step">Label a 600-mL beaker as &#8216;waste&#8217; for the reaction, a 100-mL beaker as &#8216;0.2 M sodium thiosulfate&#8217;, and a 50-mL beaker as &#8216;6 M HCl&#8217;.</div>
		</li>
		<li data-sub-note-item="1-13">
		<div class="lab-step">Fill the 100-mL beaker with 0.2 M sodium thiosulfate from the stock bottle in the dispensing hood.</div>
		</li>
		<li data-sub-note-item="1-14">
		<div class="lab-step">After that, bring the 50-mL beaker and a watch glass to the acid-dispensing hood to obtain 50 mL of 6 M HCl. Cover the filled beaker with the watch glass and bring your portion of HCl back to your hood.</div>
		</li>
		<li data-sub-note-item="1-15">
		<div class="lab-step">Prepare 20 mL of 0.1 M sodium thiosulfate for the first benchmark trial. Use a 10-mL volumetric pipette to measure 10 mL of 0.2 M sodium thiosulfate and dispense it into a 20-mL volumetric flask. Fill the 20-mL volumetric flask with deionized water to the mark.</div>
		</li>
		<li data-sub-note-item="1-16">
		<div class="lab-step">Then, cut a square of plastic paraffin film, seal the flask, and invert it several times to thoroughly mix the solution.</div>
		</li>
		<li data-sub-note-item="1-17">
		<div class="lab-step">Remove the film and pour the solution into the test tube.</div>
		</li>
		<li data-sub-note-item="1-18">
		<div class="lab-step">Prepare 10 mL of 3 M HCl. Pipette 5 mL of 6 M HCl into a 10-mL graduated cylinder. Add deionized water to fill the graduated cylinder to the 10-mL line.</div>
		</li>
		<li data-sub-note-item="1-19">
		<div class="lab-step">Seal the top of the graduated cylinder with plastic paraffin film and thoroughly mix the HCl solution by inverting it several times.</div>
		</li>
		<li data-sub-note-item="1-20">
		<div class="lab-step">Now, start the stir motor and get a reset stopwatch. Confirm that you can see the X through the solution in the test tube.</div>
		</li>
		<li data-sub-note-item="1-21">
		<div class="lab-step">Simultaneously start the stopwatch and pour the 3 M HCl into the test tube.</div>
		</li>
		<li data-sub-note-item="1-22">
		<div class="lab-step">Watch the X through the solution, which will start becoming cloudy when enough sulfur has been produced to saturate it. Stop the stopwatch exactly when the solution becomes so opaque that the X disappears.</div>
		</li>
		<li data-sub-note-item="1-23">
		<div class="lab-step">Record the time in your lab notebook for benchmark trial 1 and then reset the stopwatch. Also, record the temperature of the solution.</div>
		</li>
		<li data-sub-note-item="1-24">
		<div class="lab-step">Now, turn off the stir motor, remove the thermometer, and rinse it with deionized water into the waste beaker.</div>
		</li>
		<li data-sub-note-item="1-25">
		<div class="lab-step">Then, empty the test tube into the waste beaker, retrieve the stir bar with long forceps, and rinse the test tube and stir bar with deionized water.</div>
		</li>
		<li data-sub-note-item="1-26">
		<div class="lab-step">Thoroughly clean the test tube with a test tube brush. Briefly rinse the volumetric flask and graduated cylinder with deionized water as well. Dry everything with paper towels.</div>
		</li>
		<li data-sub-note-item="1-27">
		<div class="lab-step">Repeat the trial in the exact same way for the second benchmark time. If the time is not within 3 - 5 s of the first trial, repeat the benchmark trial until you have two consistent trials.</div>
		</li>
		<li data-sub-note-item="1-28">
		<div class="lab-step">Follow the same overall procedure for each subsequent trial, changing the dilutions as indicated in your table and cleaning your glassware between each trial. Use a 5-mL volumetric pipette as needed when measuring the sodium thiosulfate stock solution in trials four and five.</div>
		</li>
		<li data-sub-note-item="1-29">
		<div class="lab-step">Once you have performed all eight trials, rinse your glassware and tools one last time and neutralize the reaction waste with baking soda. Use caution because sulfur dioxide is one of the reaction products.</div>
		</li>
		<li data-sub-note-item="1-30">
		<div class="lab-step">Transfer the neutralized reaction waste to the designated waste container. Then, pour any remaining sodium thiosulfate solution into its labeled waste container and rinse the beaker with deionized water.</div>
		</li>
		<li data-sub-note-item="1-31">
		<div class="lab-step">Neutralize any remaining HCl with baking soda before flushing it down the drain with tap water.</div>
		</li>
		<li data-sub-note-item="1-32">
		<div class="lab-step">Finally, clean and put away your labware according to your standard practices and throw out any trash remaining in your hood. Your instructor will dispose of the reaction waste later.</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">Now, estimate the reaction order of sodium thiosulfate and HCl. Calculate the starting concentration of sodium thiosulfate in the reaction mixture for trials 1 through 5. Looking at the concentration of sodium thiosulfate and the reaction times, we see that as the concentration doubles, the time to opacity is halved.
		<h4><strong>Table 4: Estimating reaction order sodium thiosulfate</strong></h4>
		<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
			<tbody>
				<tr>
					<td style="text-align: center;"><strong>Trial</strong></td>
					<td style="text-align: center;"><strong>[Na<sub>2</sub>S<sub>2</sub>O<sub>3</sub>] added</strong></td>
					<td style="text-align: center;"><strong>Na<sub>2</sub>S<sub>2</sub>O<sub>3</sub> volume (mL)</strong></td>
					<td style="text-align: center;"><strong>Total volume (mL)</strong></td>
					<td style="text-align: center;"><strong>[Na<sub>2</sub>S<sub>2</sub>O<sub>3</sub>] in mixture </strong></td>
					<td style="text-align: center;"><strong>Time (s)</strong></td>
					<td style="text-align: center;"><strong>Inverse time (s<sup>-1</sup>)</strong></td>
				</tr>
				<tr>
					<td style="text-align: center;">1, 2</td>
					<td style="text-align: center;">0.1</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>
					<td style="text-align: center;"></td>
				</tr>
				<tr>
					<td style="text-align: center;">3</td>
					<td style="text-align: center;">0.2</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>
					<td style="text-align: center;"></td>
				</tr>
				<tr>
					<td style="text-align: center;">4</td>
					<td style="text-align: center;">0.15</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>
					<td style="text-align: center;"></td>
				</tr>
				<tr>
					<td style="text-align: center;">5</td>
					<td style="text-align: center;">0.05</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>
					<td style="text-align: center;"></td>
				</tr>
			</tbody>
		</table>
		<a href="https://www.jove.com/CDNSource/lm/labs/50/Table_4._Estimating_reaction_order_sodium_thiosulfate.xlsx" target="_blank">Click Here to download Table 4</a></div>
		</li>
		<li data-sub-note-item="2-2">
		<div class="lab-step">Remember that each trial used the same volume, so it took the same amount of sulfur to make each solution opaque. Thus, the reaction is twice as fast when the sodium thiosulfate concentration is doubled.</div>
		</li>
		<li data-sub-note-item="2-3">
		<div class="lab-step">When the concentration increases by a factor of 4, the reaction rate also increases by a factor of 4. This one-to-one relationship suggests that the reaction was first order for sodium thiosulfate. If so, we would expect a plot of rate versus sodium thiosulfate concentration to be linear.</div>
		</li>
		<li data-sub-note-item="2-4">
		<div class="lab-step">We didn&#39;t measure how much sulfur it took to turn the solution opaque, but we assume that it was the same for each trial. This lets us use the inverse of time to opacity as a stand-in for the rate. The slope won&#39;t be equal to k, but that&#39;s OK for this lab.</div>
		</li>
		<li data-sub-note-item="2-5">
		<div class="lab-step">Plot inverse reaction time as a function of sodium thiosulfate concentration. This confirms the linear relationship between sulfur production and thiosulfate concentration. Thus, the reaction seems to be first order with respect to thiosulfate.</div>
		</li>
		<li data-sub-note-item="2-6">
		<div class="lab-step">Use the same method to estimate the reaction order of HCl. First, calculate the starting concentration of HCl in the benchmark trials and in trials 6 through 8.
		<h4><strong>Table 5: Estimating reaction order HCl</strong></h4>
		<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
			<tbody>
				<tr>
					<td style="text-align: center;"><strong>Trial</strong></td>
					<td style="text-align: center;"><strong>[HCl] added</strong></td>
					<td style="text-align: center;"><strong>HCl volume (mL) </strong></td>
					<td style="text-align: center;"><strong>Total volume (mL) </strong></td>
					<td style="text-align: center;"><strong>[HCl] in mixture </strong></td>
					<td style="text-align: center;"><strong>Time (s)</strong></td>
				</tr>
				<tr>
					<td style="text-align: center;">1, 2</td>
					<td style="text-align: center;">0.1</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 style="text-align: center;">3</td>
					<td style="text-align: center;">0.2</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 style="text-align: center;">4</td>
					<td style="text-align: center;">0.15</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 style="text-align: center;">5</td>
					<td style="text-align: center;">0.05</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/CDNSource/lm/labs/50/Table_5._Estimating_reaction_order_HCl.xlsx" target="_blank">Click Here to download Table 5</a></div>
		</li>
		<li data-sub-note-item="2-7">
		<div class="lab-step">Then, look at the concentration of HCl and the reaction times for these trials. The reaction times are nearly identical, so the HCl concentration has no effect on the reaction rate. This means that HCl seems to be zeroth order for this reaction.</div>
		</li>
		<li data-sub-note-item="2-8">
		<div class="lab-step">Lastly, add up the reaction orders of the reactants to get the overall reaction order.</div>
		</li>
	</ul>
	</li>
</ol>

Concentration Dependence and Reaction Order - Procedure

Konzentrationsabhängigkeit

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

	Concentration DependenceExpand
	In this experiment, you will combine varying concentrations of ...

Optimal Foraging

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

Optimal Foraging: Foraging with Artificial Habitats and Prey - Procedure

Optimale Nahrungssuche

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

Biologie

Ökologie

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/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/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/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 ...

Extinction

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

Extinction: Diversity and Extinction during the Paleogene Epoch - Procedure

Aussterben

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

Group Behavior

<ol class="note-items"><li data-note-item="1" data-parent-collapse="">The Hawk-Dove Game
<button class="expand">Expand</button><ul class="lab-items"><li data-sub-note-item="1-1"><div class="lab-step">NOTE: In this activity, you will play the hawk and dove game with a single partner and then with multiple partners to observe hypothetical costs and benefits of the antagonist or cooperative behavior respectively. 
</div></li><li data-sub-note-item="1-2"><div class="lab-step">Before the activity starts, review the cost-benefit table so that you understand how to score the game. <a href="https://www.jove.com/CDNSource/lm/labs/33/Table_1_Group_behavior.xlsx" style="display:block;margin:20px;" target="_blank">Click Here to download Table 1</a>
</div></li><li data-sub-note-item="1-3"><div class="lab-step">HYPOTHESES: The experimental hypothesis for this exercise could be that the dove strategy will become more frequent between repeated partners, and the hawk strategy will become more frequent when interactions are with a variety of partners. The null hypothesis could be that there will be no change in strategy over time with repeat or different partner combinations. NOTE: Benefit can be determined by looking at the left column and choosing the row that corresponds to the card you played. The box that corresponds to the choice of your partner can be found in the top row. For the purposes of this game, B equals four and C equals three. So depending on the cards played, player one here, which is you, would gain a score of two, four, zero or one half. 
</div></li><li data-sub-note-item="1-4"><div class="lab-step">Collect a set of two game cards. 
</div></li><li data-sub-note-item="1-5"><div class="lab-step">Take out the cards labeled hawk and dove and place them face down on the table or conceal them in your hands. 
</div></li><li data-sub-note-item="1-6"><div class="lab-step">When the instructor gives the signal show either of the two cards to the student sitting across from you. Try to strategize how to receive the highest benefit from every interaction on your own but do not communicate with your partner. 
</div></li><li data-sub-note-item="1-7"><div class="lab-step">In the Single Partner Recording Table (Table 2) provided by your instructor, note the cards played as well as the benefit you received for that round.
</div></li><li data-sub-note-item="1-8"><div class="lab-step">Repeat showing cards to the same partner and determining benefit on your instructor&#39;s signal for a total of 15 rounds.
</div></li><li data-sub-note-item="1-9"><div class="lab-step">At the end of those rounds, the student with the highest total benefit value was determined to be the &#8220;winner&#8221;, or the most fit.
</div></li><li data-sub-note-item="1-10"><div class="lab-step">As a class, determine the percentages of hawks and doves played as well as the average benefit received for each round, using this formula: Class average benefit=(sum total benefit)/(number of players)
</div></li><li data-sub-note-item="1-11"><div class="lab-step">For the second game, and then for each round of play, one row of students should rotate to their right, leaving all players with a new partner.
</div></li><li data-sub-note-item="1-12"><div class="lab-step">Continue playing for 15 rounds or until students end up back in their starting positions, recording the data in the Multiple Partner Recording Table (Table 3) given to you by your instructor.
</div></li><li data-sub-note-item="1-13"><div class="lab-step">For each round, determine the percentages of hawks and doves played that round, as well as the average benefit received.
</div></li></ul></li><li data-note-item="2" data-parent-collapse="">Cheating and Cooperation Exercise
<button class="expand">Expand</button><ul class="lab-items"><li data-sub-note-item="2-1"><div class="lab-step">NOTE: In this activity, you will exchange benefits, represented by colored or similar items, with your classmates. In this simulation, a cheater is defined as a person who gives fewer benefits than they receive. During this activity, you will observe the frequency of cheating behavior over time. 
</div></li><li data-sub-note-item="2-2"><div class="lab-step">HYPOTHESES: The experimental hypothesis is that cheaters will receive fewer benefits over time and cooperators will continue to provide equal benefits to one another. The null hypothesis is that the frequency of cheaters and cooperators does not change over time. 
</div></li><li data-sub-note-item="2-3"><div class="lab-step">Each person should collect beads of one color to represent a tangible benefit, as well as one paper bag and a three-ring binder.
</div></li><li data-sub-note-item="2-4"><div class="lab-step">Get into a group of three so that all members have objects of the same color.
</div></li><li data-sub-note-item="2-5"><div class="lab-step">Now find another group with a different color to pair with. In each round, partners will exchange benefits without knowing what they are receiving from their partner and without discussion within their group.  NOTE: Benefits of a different color from your starting color are worth twice as much.
</div></li><li data-sub-note-item="2-6"><div class="lab-step">Turn face to face with a member from the other group while still being able to conceal the objects in your possession.  TIP:<strong> </strong>Setting up a screen using a notebook or sitting behind a desk can be helpful here.
</div></li><li data-sub-note-item="2-7"><div class="lab-step">When given a signal from the instructor, place zero to two benefits into the paper bag and pass it to your partner.
</div></li><li data-sub-note-item="2-8"><div class="lab-step">Simultaneously, receive the bag from your partner and then remove the benefits from the bag.
</div></li><li data-sub-note-item="2-9"><div class="lab-step">Make a note of how many benefits you gave away and how many you received in the Cheating and Cooperation Table (Table 4) provided by your instructor.
</div></li><li data-sub-note-item="2-10"><div class="lab-step">Turn to a new member from the other group and repeat the exchange.
</div></li><li data-sub-note-item="2-11"><div class="lab-step">Rotate through all of the members of the other group for as many rounds as the instructor determines - which will not be revealed until the game ends so as not to alter player behavior.
</div></li><li data-sub-note-item="2-12"><div class="lab-step">Once the game has ended, note for each round in the cheating and cooperation table whether you gave more than your partner, received more from them or if you gave the same amount.
</div></li><li data-sub-note-item="2-13"><div class="lab-step">Now total up all of the benefits in your possession, with benefits you&#39;ve received from the other group being worth twice as much.
</div></li><li data-sub-note-item="2-14"><div class="lab-step">For each round, the whole class should total up the number of cheaters - with cheaters defined as those who gave less than they received.
</div></li></ul></li><li data-note-item="3" data-parent-collapse="">Results
<button class="expand">Expand</button><ul class="lab-items"><li data-sub-note-item="3-1"><div class="lab-step">For both the single partner hawk-dove game and the multiple partners hawk-dove game, use the whole class data to plot the percentages as two separate lines - one for the percentage of hawks, and one for the percentage of doves, for each of the 15 rounds.
</div></li><li data-sub-note-item="3-2"><div class="lab-step">Take note of whether the cards played changed as more rounds were played.
</div></li><li data-sub-note-item="3-3"><div class="lab-step">Manipulate the values for B and C to see if you can find the point at which values of B and C would render the hawk strategy no longer effective (this would be the point when there is no benefit to choosing the hawk strategy over always choosing the dove strategy).
</div></li><li data-sub-note-item="3-4"><div class="lab-step">As a class, use the data from the cheating and cooperation activity to make a graph showing the percentage of rounds each individual player cheated versus the amount of benefits they ended with.
</div></li><li data-sub-note-item="3-5"><div class="lab-step">Consider whether it is better to be a cheater or a cooperator in this scenario and discuss as a class what makes this scenario different from the hawk-dove game.
</div></li><li data-sub-note-item="3-6"><div class="lab-step">Now, use the class data to graph the percentage of cheaters against the number of rounds, and look for any trends over time.
</div></li><li data-sub-note-item="3-7"><div class="lab-step">Consider which type of strategy would most likely evolve in a setting where partners interact repeatedly.
</div></li><li data-sub-note-item="3-8"><div class="lab-step">Then, note which tactic might be beneficial for a species that relies on cooperation. 
</div></li><li data-sub-note-item="3-9"><div class="lab-step">Discuss as a class what would likely happen if you increased or decreased the relative value of the benefits you receive.
</div></li></ul></li></ol>

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

Gruppenverhalten

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

Artificial Selection

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

Artificial Selection: Visualization in Plants and Data Analysis - Procedure

Künstliche Selektion

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

Evolution

Natural Selection

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

Natural Selection: Calculating Fitness and Selection - Procedure

Natürliche Auslese

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