<p class="jove_content">This work was supported by the Natural Sciences &#38; Engineering Council Discovery Program (US: RGPIN-2016-06694).</p>

<p class="jove_content">All rats are treated according to the guidelines of the Canadian Council on Animal Care. Approval for all the experimental procedures was granted by the Concordia University Animal Research Ethics Committee.</p>
<p class="jove_title"><strong>1. Animals</strong></p>
<ol>
	<li>Acquire Long Evans rats weighing 275-300 g (males) or 225-250 g (females). Double house rats in standard plastic cages with corncob bedding and shredded paper until surgery.</li>
	<li>Keep the rats on a reversed light-dark cycle (lights off at 9:30 AM, light on at 9:30 PM).</li>
	<li>Allow unlimited access to food and water, except during the food-deprivation test. Use regular rat chow (20.9% protein, 67.2% carbohydrates, and 11.8% fat).</li>
</ol>
<p class="jove_title"><strong>2. Intravenous surgeries</strong></p>
<ol>
	<li>Build catheters and perform intravenous surgery as described in Sedki et al. 2013<sup class="xref">17</sup> with the following differences: 1) Use Isofluorane 2-2.5% for anesthesia, 2) Change the dose of penicillin administered during surgery to 60 000 IU/rat, via subcutaneous injection, and 3) Increase the dose of gentamicin in the mixture of heparin and gentamicin used to flush the catheters to 7.5 IU + 800 &#181;g; 0.2- 0.3 mL.</li>
	<li>Inject ketoprofen or carprofen, an anti-inflammatory analgesic, intraperitoneally or subcutaneously (5 mg/kg) immediately after surgery (before recovery from the anesthesia) and on the following 3 days. During the 2 days recovery period, give mashed food and heat (if necessary) to help with the recovery. Weigh and supervise the animals daily to check for any abrupt loss of weight or any other signs of health issues.</li>
</ol>
<p class="jove_title"><strong>3. Behavioral procedure</strong></p>
<ol>
	<li>Operant conditioning chambers:
	<ol>
		<li>Set the operant conditioning chambers as described in Sedki et al. 2013<sup class="xref">17</sup>.</li>
	</ol>
	</li>
	<li>Habituation day:
	<ol>
		<li>House the rats in the operant training chambers after recovery from surgery and allow animals to habituate to the chamber for 24 h before initiating any protocol. Keep animals housed in the operant chambers throughout the whole experiment.</li>
		<li>Do not attach the rats to the metal spring and do not initiate any experimental protocol (the cue-light, the tone, and the levers are not available during habituation day).</li>
	</ol>
	</li>
	<li>Heroin self-administration:
	<ol>
		<li>Attach the 5-up connector to the Tygon tube and the metal spring.</li>
		<li>Set up daily training sessions of 6 h of heroin self-administration (0.1 mg/kg/infusion) according to the procedure described below.</li>
		<li>Start daily training sessions on the onset of the dark phase of the reversed light cycle (around 9:30 AM in the representative results) with the extension of the seek lever (or take lever on the first 2 days; see below), as well as turning off the houselight on.</li>
		<li>Self-administration with only the take lever available under fixed-ratio 1 (FR1; 2 days)
		<ol>
			<li>Program each trial to begin with the insertion of the take lever.</li>
			<li>Once the rat presses the take lever one time (FR1), retract the take lever, turn on the cue light above the lever and tone for a 20 s timeout period, turn off the houselight, and allow the delivery of a heroin infusion (0.1 mg/kg in 0.13 mL)</li>
			<li>Program a 30 s inter-trial interval in which no cues are presented, the houselight is turned off, and the lever is retracted. For the beginning of the next trial, insert the take lever, and turn on the houselight.</li>
		</ol>
		</li>
		<li>Self-administration with seek-take chain under FR1 (5 days)
		<ol>
			<li>Program the operant training system to insert only the seek lever and turn on the houselight to signal the beginning of the session. The take lever is in a retracted state.</li>
			<li>Once the rat presses one time (FR1) on the seek lever, the program retracts the seek lever and inserts the take lever.</li>
			<li>One press on the take lever (FR1) retracts the take lever and initiates the same procedure as in step 3.3.4.2 and a 30 s inter-trial interval.</li>
			<li>Program the operant training system to insert the seek lever at the conclusion of the inter-trial interval and turn on the houselight to signal the beginning of the next trial.</li>
			<li>Program the operant training system to initiate the inter-trial interval if a rat fails to accomplish the seek-take chain during 10 min (not pressing the seek or the take lever) to represent the end of a trial and the loss of the opportunity to administer the drug.</li>
		</ol>
		</li>
		<li>Self-administration with seek-take chain under Variable Intervals (VI5, VI30, and VI60)
		<ol>
			<li>Program all steps in section 3.3.5 but introduce a variable interval of 5 (VI5) as the new schedule of reinforcement on the seek lever, instead of FR1. Allow self-administration training under the VI5 schedule for 3 days.</li>
			<li>Set up the software to allow the first press on the seek lever to activate the VI5 schedule. For the VI5 schedule, let the software to randomly select an interval time from a list of 0.1 s, 5 s, and 10 s, which results in an average of 5 s interval. The first seek lever press after the selected interval has elapsed, retracts the seek lever and extends the take lever.</li>
			<li>Follow steps 3.3.4.2, 3.3.5.4., 3.3.5.5.&#160;for take lever and end of trial instructions.</li>
			<li>Next, train rats for 5 days under VI30 schedule for the seek link, followed by 3 days under VI60 schedule.</li>
			<li>For the VI30 schedule, allow the software to randomly select a time from a list of 15 s, 30 s, and 45 s, for an average of 30 s, whereas for the VI60 schedule, the software randomly selects a time from a list of 45 s, 60 s, and 75 s.</li>
			<li>Vary the inter-trial intervals along the different schedules of reinforcement. Start the inter-trial interval at 30 s during FR1 and VI5 but gradually increase to 7 min during VI30 and VI60.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Punishment-imposed abstinence
	<ol>
		<li>Start punishment sessions with insertion of seek lever and turning the houselight on. Allow punishment sessions for 8 days, with regular supervision to guarantee that animals do not show signs of abnormal pain or any health issues.</li>
		<li>Program the operant training system so that after completing the seek link under VI60, the seek lever retracts, the houselight is turned off, and a mild footshock is administered on 30% of the completed seek links instead of insertion of the take lever. The take lever extends on the other 70% of completed seek links, as in a normal self-administration trial.</li>
		<li>Program an inter-trial interval of 7 min with all cues and houselight off before inserting the seek lever and turning the houselight on to signal the beginning of the next trial.</li>
		<li>Set the footshock intensity at 0.2 mA and increase by 0.1 mA per punishment day until 1.0 mA.</li>
		<li>After rats press the take lever (on trials when it is available), repeat step 3.3.4.2 for retraction of the lever, delivery of the heroin infusion, and presentation of cues, followed by a 7 min inter-trial interval.</li>
		<li>Start the next trial with the insertion of the seek lever and turning the houselight on.</li>
		<li>If the rat does not press any lever for 10 min, follow step 3.3.5.5.</li>
	</ol>
	</li>
	<li>Acute food deprivation (24 h)
	<ol>
		<li>On the day following the last day of punishment-imposed abstinence, divide rats into two matched groups (according to body weight and the average number of seek lever and infusions over the last 3 days of self-administration training): Food Deprived (FD) and Sated.</li>
		<li>Remove food hoppers from the chambers (around 9:30 AM) for rats that undergo food deprivation-induced relapse test and keep food hoppers for the sated group that has unlimited access to food. Provide unlimited access to water in both groups and supervise animals to guarantee that they do not show any health problems during the food deprivation period.</li>
	</ol>
	</li>
	<li>Food deprivation-induced heroin seeking tests
	<ol>
		<li>The morning after the 24 h of food deprivation, perform a 3 h heroin-seeking test, under a VI60 schedule with an inter-trial interval of 7 min with no shock or heroin infusions being delivered. Return food hoppers to the food-deprived rats after the 3 h session.</li>
		<li>On the morning after the first heroin seeking test, remove food hoppers from the chambers of the rats that were sated on the first relapse test. Allow the rats that were previously food-deprived unlimited access to food (sated condition) before and during the test.</li>
		<li>Perform another 3 h heroin seeking test on the next day (after 24 h food deprivation), as in step 3.6.1.</li>
		<li>The day after the test euthanize the rats using a carbon dioxide chamber (100% CO<sub>2</sub> at fill rate of 30-70% of the chamber volume per minute).</li>
	</ol>
	</li>
</ol>

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<p class="jove_content">The authors declare that they have no competing financial interests. </p>

<p class="jove_content">There are two important demonstrations in this paper. First, the validation of the punishment-imposed abstinence using the seek and take chain with heroin. Second, it was demonstrated that stress-induced relapse could be observed in a punishment-imposed abstinence procedure. These are important demonstrations because (i) The punishment-induced abstinence procedure more closely mimics the human condition as it results in voluntary abstinence, i.e., not due to extinction of drug-seeking or forced removal from the drug-taki...

<p class="jove_content" fo:break-after="page">Relapse is the most challenging problem in the treatment of drug use<sup class="xref">1</sup><sup>,</sup><sup class="xref">2</sup>. However, only a handful of pharmacological treatments are approved to help avoid relapse in humans<sup class="xref">3</sup>. The opioid epidemic that North America is currently facing is a striking example of it, and it demands considering different approaches on animal models of relapse to opioids.</p>
<p class="jove_content">Acute stress has been shown to be a key trigger to relapse in humans<sup class="xref">4</sup>. One environmental stressor that is often associated with drug addiction is food deprivation. Drug users often choose to allocate resources towards obtaining drugs instead of food. Caloric deficit has been shown to be correlated with higher relapse to cigarettes<sup class="xref">5</sup> and alcohol<sup class="xref">6</sup> use. Due to ethical and practical issues, animal models have been developed over the last decades to facilitate research in the field. In animal models, acute food deprivation has been demonstrated to robustly reinstate extinguished heroin seeking<sup class="xref">7</sup>. Currently, most animal models of relapse are based on abstinence procedures that are either not representative of human abstinence (e.g., extinction-based models) or encompass only the small percentage of drug users that are forced to abstain due to incarceration or inpatient treatment (e.g., forced abstinence models). The main reason drug users choose to abstain is the negative consequences associated with drug-seeking and taking<sup class="xref">8</sup>. Punishment-imposed abstinence is an animal model that mimics the negative consequences associated with drug-seeking on self-imposed abstinence in humans. This model introduces an aversive stimulus, e.g., a mild footshock, with drug-seeking or taking, which leads the animal to stop taking the drug voluntarily. Another procedure that incorporates negative consequences for drug-seeking is the electrical barrier conflict model for drug abstinence and relapse<sup class="xref">9</sup>. The rat must cross an electrical barrier to perform the operant behavior linked with drug self-administration. The model was used successfully to demonstrate voluntary abstinence and relapse to psychostimulant and opioid drugs<sup class="xref">10</sup><sup>,</sup><sup class="xref">11</sup>. However, under the electrical barrier procedure, drug-seeking efforts are always associated with an aversive event, unlike the human condition. Moreover, drug-taking itself might overlap with the electrical footshock as the animal returns to the safe area following the infusion by crossing the barrier again.</p>
<p class="jove_content">Punishment-imposed abstinence has been used with other drugs of abuse such as cocaine<sup class="xref">12</sup>, alcohol<sup class="xref">13</sup>, methamphetamine<sup class="xref">14</sup>, remifentanil<sup class="xref">15</sup>, but it was never applied to heroin-trained animals. The model has been used to study relapse induced by priming<sup class="xref">14</sup> and drug-associated cues<sup class="xref">16</sup>, but it was not integrated into a stress-induced relapse procedure. The procedure described here is used to demonstrate acute food deprivation-induced relapse to heroin seeking after punishment-imposed abstinence in male rats.</p>

<table><tbody><tr><td>Anafen Injection 100 mg/mL Vial/50 mL</td><td>MERIAL Canada, Inc.</td><td>1938126</td><td>anti-inflammatory drug</td></tr><tr><td>Balance arm</td><td>Coulbourn Instruments</td><td>H29-01</td><td></td></tr><tr><td>Cannulae (22 G, 5-up)</td><td>Plastics One</td><td>C313G-5up</td><td></td></tr><tr><td>Environment connection board &amp;amp; Linc cable</td><td>Coulbourn Instruments</td><td>H03-04</td><td></td></tr><tr><td>Fixed speed infusion pump (3.3 RPM)</td><td>Coulbourn Instruments</td><td>A73-01-3.3</td><td></td></tr><tr><td>GE Marine Silicon</td><td>GE</td><td>SE-1134</td><td></td></tr><tr><td>Graphic State Notation 3</td><td>Coulbourn Instruments</td><td>GS3</td><td>Software</td></tr><tr><td>Habitest universal Linc</td><td>Coulbourn Instruments</td><td>H02-08</td><td></td></tr><tr><td>Heroin HCl</td><td>National Institute for Drug Abuse, Research Triangle Park, NC, USA</td><td></td><td></td></tr><tr><td>House light-Rat</td><td>Coulbourn Instruments</td><td>H11-01R</td><td></td></tr><tr><td>Isofluorane USP 99.9% Vial/250 mL</td><td>Fresenius Kabi Canada Ltd</td><td>2237518</td><td></td></tr><tr><td>Liquid Swivels, Plastic, 22 G</td><td>Lomir Biomedical, Inc.</td><td>RSP1</td><td></td></tr><tr><td>Rat test cage</td><td>Coulbourn Instruments</td><td>H10-11R-TC</td><td>Operant conditioning chambers</td></tr><tr><td>Retractable lever-Rat</td><td>Coulbourn Instruments</td><td>H23-17RA</td><td></td></tr><tr><td>Silastic tubing (ID 0.02, OD 0.037)</td><td>Fisher Scientific (Canada)</td><td>1118915A</td><td></td></tr><tr><td>Single high-bright cue-Rat</td><td>Coulbourn Instruments</td><td>H11-03R</td><td></td></tr><tr><td>Sound attenuation boxes</td><td>Concordia University</td><td>Home made</td><td></td></tr><tr><td>Stainless steal grid floor</td><td>Coulbourn Instruments</td><td>H10-11R-TC-SF</td><td></td></tr><tr><td>System controller 2</td><td>Coulbourn Instruments</td><td>SYS CTRL 2</td><td></td></tr><tr><td>System power base</td><td>Coulbourn Instruments</td><td>H01-01</td><td></td></tr><tr><td>Tone module 2.9 KHz</td><td>Coulbourn Instruments</td><td>H12-02R-2.9</td><td></td></tr><tr><td>Tygon tubing (ID 0.02, OD 0.060)</td><td>VWR</td><td>63018-044</td><td></td></tr></tbody></table>

a procedure to study stress-induced relapse of heroin seeking after punishment-imposed abstinence

<p class="jove_content">The punishment-imposed abstinence procedure models the self-imposed abstinence that humans initiate due to the adverse consequences associated with drug-taking. This model has been implemented in experiments using different types of substances of abuse such as methamphetamine, cocaine, and alcohol. However, punishment-induced abstinence in heroin-trained animals has not been demonstrated. Furthermore, acute stress is a key trigger for relapse in humans and animal models. It was previously demonstrated that acute food deprivation robustly induced reinstatement of extinguished cocaine and heroin seeking. The procedure described here can be used to assess the effects of acute stress exposure on heroin seeking after punishment-imposed abstinence. A total of 8 rats were implanted with chronic intravenous (i.v.) catheters and trained to self-administer heroin (0.1 mg/kg/infusion) for 18 days under a seek-take chained schedule. Completing the seek link gave access to the take lever, which was paired with a heroin infusion. The seek lever was programmed with a variable interval 60 schedule of reinforcement (VI60), and the take lever was programmed with a fixed-ratio 1 reinforcement schedule (FR1). Following self-administration training, a mild foot shock was delivered on 30% of the completed seek links instead of the extension of the take lever. Footshock intensity was increased by 0.1 mA per daily session from 0.2 mA to 1.0 mA. Heroin-seeking tests were performed after 24 h of food deprivation (FD) or sated conditions. Rats under acute food deprivation condition robustly increased heroin seeking after punishment-imposed abstinence.</p>

<p class="jove_content">Male rats demonstrated an increase in seek lever presses as the schedule of reinforcement increased over the training days, and a reliable, consistent number of heroin infusions over training days (<strong class="xfig">Figure 1</strong>). During punishment, rats decreased the number of seek lever presses and infusions with the increase of footshock intensity over 8 punishment days (<strong class="xfig">Figure 2</strong>). Food-deprivation condition significantly increased heroin seeking after punishment-imposed abstinenc...

Watch this Scientific Journal Video about A Procedure to Study Stress-Induced Relapse of Heroin Seeking after Punishment-Imposed Abstinence at JoVE.com

A Procedure to Study Stress-Induced Relapse of Heroin Seeking after Punishment-Imposed Abstinence

<p class="jove_content">A procedure that demonstrates a robust acute food deprivation-induced relapse to heroin seeking after punishment-imposed abstinence is described. A punishment-imposed abstinence model was successfully implemented using the seek and take chain schedule for heroin self-administration. Heroin-seeking tests are then performed following 24 h of food-deprivation stress.</p>

The punishment-imposed abstinence procedure models the self-imposed abstinence that humans initiate due to the adverse consequences associated with ...

a-procedure-to-study-stress-induced-relapse-heroin-seeking-after

Research

JoVE Journal

Behavior

2.5K Views.  Concordia University. A procedure that demonstrates a robust acute food deprivation-induced relapse to heroin seeking after punishment-imposed abstinence is described. A punishment-imposed abstinence model was successfully implemented using the seek and take chain schedule for heroin self-administration. Heroin-seeking tests are then performed following 24 h of food-deprivation stress.

Video: A Procedure to Study Stress-Induced Relapse of Heroin Seeking after Punishment-Imposed Abstinence

Extraction

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

Wydobywanie

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

	Cellulose RecoveryExpand
	Extraction and filtration can ...

Education

JoVE Lab Manual

Chemistry

Lab Manual Sub Articles

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="">Esterification<button class="expand">Expand</button>
	<p>In this lab, you will synthesize an ester from a carboxylic acid and an alcohol in the presence of sulfuric acid. This reaction is called Fischer esterification. The sulfuric acid makes the carboxylic acid more reactive towards the alcohol. Without it, esterification would be slow and unfavorable. Esterification is highly reversible, so you&#39;ll use an excess of alcohol to drive the reaction towards the ester.</p>
	<p>You will be assigned one of seven carboxylic acid-alcohol pairs for your reaction. Each of the seven possible product testers has a distinct fruity scent. When you finish the reaction, you will waft your products vapor towards yourself and identify it based on the fruit that it smells like.</p>
	<ul class="lab-items">
		<li data-sub-note-item="1-3">
		<div class="lab-step">Before you get started, put on a lab coat, safety glasses, and nitrile gloves. <strong>Note: </strong>Your instructor will demonstrate wafting before handing out the reagents. Always smell compounds by wafting, rather than direct inhalation.</div>
		</li>
		<li data-sub-note-item="1-4">
		<div class="lab-step">Once you are ready to begin, get your assigned carboxylic acid and alcohol and bring the vials to your fume hood. The vial labeled &#8216;A&#8217;, &#8216;B&#8217;, or &#8216;C&#8217; is your carboxylic acid. The other vial is the alcohol. Record the letters of your carboxylic acid and alcohol in your lab notebook.
		<h4><strong>Table 1: Scent of Carboxylic Acid, Alcohol, and Ester</strong></h4>
		<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
			<tbody>
				<tr>
					<td></td>
					<td><strong>Letter </strong></td>
					<td><strong>Scent </strong></td>
				</tr>
				<tr>
					<td>Assigned carboxylic acid</td>
					<td></td>
					<td></td>
				</tr>
				<tr>
					<td>Assigned alcohol</td>
					<td></td>
					<td></td>
				</tr>
				<tr>
					<td>Synthesized ester</td>
					<td></td>
					<td></td>
				</tr>
			</tbody>
		</table>
		<a href="https://www.jove.com/CDNSource/lm/labs/68/Table_1._Scent_of_carboxylic_acid,_alcohol,_and_ester.xlsx" target="_blank">Click Here to download Table 1</a></div>
		</li>
		<li data-sub-note-item="1-5">
		<div class="lab-step">Now, get a piece of filter paper and a Pasteur pipette. In the fume hood, use the Pasteur pipette to apply a drop of your alcohol to the filter paper.</div>
		</li>
		<li data-sub-note-item="1-6">
		<div class="lab-step">Bring the filter paper to the front of the hood and waft the scent of the alcohol towards yourself. Be careful because it might smell unpleasant.</div>
		</li>
		<li data-sub-note-item="1-7">
		<div class="lab-step">Then, put the filter paper back in the hood and write a brief description of the scent of the alcohol in your lab notebook. Do the same for your carboxylic acid if it&#39;s a liquid using a new pipette and piece of filter paper. <strong>Note:</strong> If you were assigned a solid carboxylic acid, write &#39;solid&#39; in your lab notebook and skip this step.</div>
		</li>
		<li data-sub-note-item="1-8">
		<div class="lab-step">Now, attach an open 3-prong clamp to a lab stand in your fume hood. Place a lab jack at the base of the stand and set a stir plate and a heating mantle on top of it. Move the temperature controller next to the setup.</div>
		</li>
		<li data-sub-note-item="1-9">
		<div class="lab-step">Then, place a small stir bar in a 50-mL round-bottom flask and securely clamp the flask over the heating mantle.</div>
		</li>
		<li data-sub-note-item="1-10">
		<div class="lab-step">Add the carboxylic acid to the flask using a funnel. Rinse the carboxylic acid vial into the flask with 1 or 2 mL of your alcohol, and then add the rest of the alcohol to the flask.</div>
		</li>
		<li data-sub-note-item="1-11">
		<div class="lab-step">Next, bring a 10-mL graduated cylinder to the fume hood reserved for acids, and carefully measure 4 mL of 3 M sulfuric acid. <strong>Note:</strong> It&#39;s highly toxic and corrosive, so be careful with it.</div>
		</li>
		<li data-sub-note-item="1-12">
		<div class="lab-step">Cover your graduated cylinder with plastic paraffin film. Then, bring the acid back to your hood and add it to the flask. It&#39;s OK if the liquids form two layers.</div>
		</li>
		<li data-sub-note-item="1-13">
		<div class="lab-step">Now, use one piece of tubing to connect the lower arm of the standard condenser to the water tap in your fume hood. Attach the other piece of tubing to the upper arm and direct it to your drain. Make sure that the connections are secure.</div>
		</li>
		<li data-sub-note-item="1-14">
		<div class="lab-step">Apply vacuum grease to the lower ground glass joint of the condenser. Fit the condenser into the round-bottom flask and ensure that the grease forms a good seal.</div>
		</li>
		<li data-sub-note-item="1-15">
		<div class="lab-step">Secure the condenser in place with a second 3-prong clamp and make sure that the tubing is out of the way. Use a laboratory wipe to remove excess grease, and then secure the connection with a joint clip.</div>
		</li>
		<li data-sub-note-item="1-16">
		<div class="lab-step">Now, slowly open the water tap until water is flowing smoothly through the condenser. Make sure that the water is flowing into the drain and that there are no leaks.</div>
		</li>
		<li data-sub-note-item="1-17">
		<div class="lab-step">Then, raise the lab jack until the round-bottom flask is sitting in the heating mantle. You are now ready to start heating your reaction mixture to reflux.</div>
		</li>
		<li data-sub-note-item="1-18">
		<div class="lab-step">Set the stir motor to medium speed and start heating the mixture on medium heat or with a set point of about 110 &#176;C.</div>
		</li>
		<li data-sub-note-item="1-19">
		<div class="lab-step">The first boiling point of your mixture will be between 65 and 100 &#176;C. So, keep an eye on it as it heats and adjust the temperature setting as needed.</div>
		</li>
		<li data-sub-note-item="1-20">
		<div class="lab-step">Once the mixture reaches an even boil, let it reflux for 50 min.</div>
		</li>
		<li data-sub-note-item="1-21">
		<div class="lab-step">Then, turn off the heat, lower the lab jack, and replace the heating mantle with a round bottom flask holder.</div>
		</li>
		<li data-sub-note-item="1-22">
		<div class="lab-step">Let the mixture cool to room temperature with the condenser still in place, which usually takes about 10 min. During that time, prepare an ice bath in a 600-mL beaker and bring it to your fume hood.</div>
		</li>
		<li data-sub-note-item="1-23">
		<div class="lab-step">Once the mixture has cooled completely, turn off the flow of water to the condenser. Carefully separate the condenser from the flask and move it out of the way. Then, raise the ice bath up to the flask and let it cool for 10 min.</div>
		</li>
		<li data-sub-note-item="1-24">
		<div class="lab-step">After that, bring a clean 10-mL graduated cylinder to the base hood and obtain 5 mL of saturated sodium bicarbonate.</div>
		</li>
		<li data-sub-note-item="1-25">
		<div class="lab-step">Back at your hood, use a lab wipe to remove vacuum grease from the inside of the neck of the round bottom flask.</div>
		</li>
		<li data-sub-note-item="1-26">
		<div class="lab-step">Then, obtain several clean Pasteur pipettes and some pH paper. Use a pipette to slowly add 1 mL of saturated sodium bicarbonate to the flask and stir the mixture with a stir bar or a glass rod.</div>
		</li>
		<li data-sub-note-item="1-27">
		<div class="lab-step">Add the rest of the sodium bicarbonate solution a milliliter at a time, stirring after each addition.</div>
		</li>
		<li data-sub-note-item="1-28">
		<div class="lab-step">Then, use a new pipette to check the pH of the solution. If the pH is &#60; 7, get a few more milliliters of sodium bicarbonate and add it 1 mL at a time, stirring and checking the pH after each addition.</div>
		</li>
		<li data-sub-note-item="1-29">
		<div class="lab-step">Once the solution is at a neutral pH, remove the ice bath and dry the outside of the flask with a paper towel.</div>
		</li>
		<li data-sub-note-item="1-30">
		<div class="lab-step">Let the mixture sit for a few minutes to see whether it settles into two layers.</div>
		</li>
		<li data-sub-note-item="1-31">
		<div class="lab-step">Then, obtain a clean Pasteur pipette and a new piece of filter paper. Carefully draw up a small amount of the product solution and apply one or two drops to the filter paper. If your product mixture separated, test the top layer first.</div>
		</li>
		<li data-sub-note-item="1-32">
		<div class="lab-step">Bring the filter paper to the edge of the fume hood and waft the set towards yourself. The seven possible scents are apple, banana, grape, orange, pear, pineapple, and strawberry.</div>
		</li>
		<li data-sub-note-item="1-33">
		<div class="lab-step">Record the fruit that you think your ester smells like in your lab notebook. If you aren&#39;t sure which fruit it is, ask your instructor for the reference scents to help you decide.</div>
		</li>
		<li data-sub-note-item="1-34">
		<div class="lab-step">Once you&#39;ve identified the smell of your ester, retrieve the stir bar, and dispose of your reaction mixture in the non-halogenated organic waste container. Clean your glassware and stir bar using your usual methods.</div>
		</li>
		<li data-sub-note-item="1-35">
		<div class="lab-step">Next, unclamp the condenser and carefully tilt it to let the water drain through the outlet tubing. Disconnect the inlet tubing from the water tap, keeping the end raised to avoid spills, and let the remaining water flow through the condenser to the drain.</div>
		</li>
		<li data-sub-note-item="1-36">
		<div class="lab-step">Then, disconnect the condenser from the tubing and remove the vacuum grease on the bottom joint with lab wipes. Clean the inner walls of the condenser using your usual methods.</div>
		</li>
		<li data-sub-note-item="1-37">
		<div class="lab-step">Leave the filter papers in the hood until they stop smelling strongly of fruit. Dispose of used Pasteur pipettes in the glass waste and throw out any other trash in the lab trash container.</div>
		</li>
		<li data-sub-note-item="1-38">
		<div class="lab-step">Lastly, put away your equipment and clean your hood before you leave.</div>
		</li>
	</ul>
	</li>
	<li data-note-item="2" data-parent-collapse="">Results<button class="expand">Expand</button>
	<p>Here are the seven possible esters that you could have made and their scents: propyl acetate smells like pear, isoamyl acetate smells like banana, octyl acetate smells like orange, butyl butyrate smells like pineapple, ethyl butyrate smells like strawberry, methyl butyrate smells like apple, and methyl anthranilate smells like grape.</p>
	<ul class="lab-items">
		<li data-sub-note-item="2-2">
		<div class="lab-step">Compare the scent of your ester to find the corresponding structure. For example, a strong scent of pears means that you made propyl acetate.</div>
		</li>
		<li data-sub-note-item="2-3">
		<div class="lab-step">Determine your starting carboxylic acid and alcohol. <strong>Note: </strong>Remember that alcohol attacks the carboxylic acid in this reaction. The resulting intermediate rearranges and eliminates a water molecule to form an ester.</div>
		</li>
		<li data-sub-note-item="2-4">
		<div class="lab-step">Studies have shown that the alkoxy oxygen comes from the alcohol, not the carboxylic acid. So, we&#39;ll split the ester diagram at the carbon-oxygen single bond and fill in the missing OH and H to get the starting materials. For example, if we split propyl acetate at that bond, add an OH to the carbonyl carbon and add a hydrogen to the alkoxy oxygen, we get acetic acid and 1-propanol as the starting materials.</div>
		</li>
	</ul>
	</li>
</ol>

Esterification: Synthesis of Ester by Fischer Esterification - Procedure

Estryfikacji

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

	EsterificationExpand
	In this lab, you will synthesize an ester from a ...

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

Zależność od koncentracji

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

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

Recrystallization

<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="">Recrystallization of Acetanilide<button class="expand">Expand</button>
	<p>In this part of the lab, you&#39;ll purify acetanilide by recrystallization from an aqueous solution. Acetanilide is moderately soluble in boiling water, but it&#39;s much less soluble in room temperature and cold water. Aniline, a potential impurity, is much more soluble in room temperature water. Thus, you&#39;ll first make a saturated solution of acetanilide in boiling water. Then, you&#39;ll slowly cool the solution to room temperature.</p>
	<p>As the acetanilide&#39;s solubility decreases, it will slowly form flat colorless or white crystals. Water-soluble impurities will remain in solution. You will then cool the solution to further decrease the solubility of acetanilide. Finally, you&#39;ll recover the crystals with vacuum filtration and measure the melting point of your crystals.</p>
	<ul class="lab-items">
		<li data-sub-note-item="1-3">
		<div class="lab-step">Before you start the lab, put on a lab coat, safety glasses, and nitrile gloves.</div>
		</li>
		<li data-sub-note-item="1-4">
		<div class="lab-step">To begin, bring a 125-mL Erlenmeyer flask and your lab notebook to the analytical balance to obtain your acetanilide.</div>
		</li>
		<li data-sub-note-item="1-5">
		<div class="lab-step">Measure 2 g of acetanilide in a tared weighing boat and record the precise mass in your lab notebook. Add the acetanilide to the flask and bring it back to your fume hood.
		<h4><strong>Table 1: Recrystallization of acetanilide</strong></h4>
		<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
			<tbody>
				<tr>
					<td><strong>Compound</strong></td>
					<td><strong>Mass (g) </strong></td>
					<td><strong>Melting point (&#176;C) </strong></td>
				</tr>
				<tr>
					<td>Acetanilide</td>
					<td></td>
					<td></td>
				</tr>
				<tr>
					<td>Seed crystal (if used)</td>
					<td></td>
					<td></td>
				</tr>
				<tr>
					<td>Recrystallized acetanilide<br />
					(<em>subtract seed crystal if used</em>)</td>
					<td></td>
					<td></td>
				</tr>
				<tr>
					<td></td>
					<td></td>
					<td></td>
				</tr>
				<tr>
					<td><strong>Percent recovery</strong></td>
					<td></td>
					<td></td>
				</tr>
			</tbody>
		</table>
		<a href="https://www.jove.com/CDNSource/lm/labs/58/Table_1._Recrystallization_of_acetanilide.xlsx" target="_blank">Click Here to download Table 1</a></div>
		</li>
		<li data-sub-note-item="1-6">
		<div class="lab-step">Then, measure 30 mL of deionized water and carefully pour it into the flask of acetanilide.</div>
		</li>
		<li data-sub-note-item="1-7">
		<div class="lab-step">Place the flask on a stirring hotplate, add a stir bar, and set the hotplate to medium heat and stirring. Let the mixture boil for 4 &#8211; 5 min to dissolve the acetanilide.</div>
		</li>
		<li data-sub-note-item="1-8">
		<div class="lab-step">If you see solid acetanilide after the solution has boiled for 5 min, measure another 10 mL of deionized water. Use a Pasteur pipette to add water dropwise until either the solid dissolves or you&#39;ve added all 10 mL.</div>
		</li>
		<li data-sub-note-item="1-9">
		<div class="lab-step">Let the solution boil for at least a minute after each milliliter before adding more water.<strong> Note:</strong> It&#39;s important to add water slowly so that you don&#39;t cool the solution too much or add more water than you need.</div>
		</li>
		<li data-sub-note-item="1-10">
		<div class="lab-step">If you still see some solid after that, those are impurities that you must remove by hot filtration. If you do not see solid, continue the lab at step 14.</div>
		</li>
		<li data-sub-note-item="1-11">
		<div class="lab-step">To perform hot filtration, place about 15 mL of deionized water and one or two boiling chips in a 50-mL beaker and start heating it on a hotplate.</div>
		</li>
		<li data-sub-note-item="1-12">
		<div class="lab-step">Clamp another 125-mL Erlenmeyer flask on a hotplate and place a wide stem or stemless glass funnel in the flask. Fold a large piece of circular filter paper in quarters, place it point down in the funnel, and open it into a cone.</div>
		</li>
		<li data-sub-note-item="1-13">
		<div class="lab-step">Once the water boils, use a pipette to wet the filter paper with hot water. Then, use tongs to pick up the flask of acetanilide solution and pour the hot liquid into the funnel, being careful not to let it overflow. <strong>Note:</strong> If the stir bar falls into the funnel, retrieve it with tweezers and rinse it with hot water.</div>
		</li>
		<li data-sub-note-item="1-14">
		<div class="lab-step">Then, rinse the inside of the first flask with a few milliliters of hot water, pour the contents into the funnel, and dissolve any crystals that form with hot water.</div>
		</li>
		<li data-sub-note-item="1-15">
		<div class="lab-step">Once the funnel is empty, use tongs to move it to the first flask. Carefully unclamp the second flask. You can now continue with the lab as usual.</div>
		</li>
		<li data-sub-note-item="1-16">
		<div class="lab-step">Once there is no visible solid in your hot acetanilide solution, turn off the heat and stir motor and use tongs to place the flask on the floor of the hood.</div>
		</li>
		<li data-sub-note-item="1-17">
		<div class="lab-step">Leave the solution undisturbed as it cools to room temperature, which usually takes at least 15 min. Moving or shaking the flask will make it harder for crystals to form.</div>
		</li>
		<li data-sub-note-item="1-18">
		<div class="lab-step">While you wait, place a small amount of the starting acetanilide material in a weighing boat and bring it to the melting point apparatus.</div>
		</li>
		<li data-sub-note-item="1-19">
		<div class="lab-step">Pack a capillary tube with the acetanilide and measure and record its melting point.</div>
		</li>
		<li data-sub-note-item="1-20">
		<div class="lab-step">Once the solution cools, look for crystals without moving the flask too much. If you don&#39;t see any, use a glass rod to gently scratch the inside of the flask under the solution. It&#39;s easier for crystals to start to form on scratched or rough surfaces.</div>
		</li>
		<li data-sub-note-item="1-21">
		<div class="lab-step">If crystals still haven&#39;t formed after 10 more min, ask your instructor for a seed crystal of high purity acetanilide. Measure and record the mass of the seed crystal, and then drop it into the room temperature acetanilide solution. Crystal growth will then occur on the seed crystal.</div>
		</li>
		<li data-sub-note-item="1-22">
		<div class="lab-step">Once you see several crystals in the flask, prepare two ice baths in 600-mL beakers. One ice bath should nearly fill the beaker, and the other should occupy about half of the beaker.</div>
		</li>
		<li data-sub-note-item="1-23">
		<div class="lab-step">Measure 5 mL of deionized water and place it in the full ice bath. Confirm that the flask is cool to the touch and that several crystals have formed before gently placing it in the half-full ice bath. Leave the flask undisturbed to improve crystal growth.</div>
		</li>
		<li data-sub-note-item="1-24">
		<div class="lab-step">Check the crystal growth progress every 15 min. Once it looks like most of your acetanilide has crystallized, or you don&#39;t see much crystal growth over a 15-min period, set up for a vacuum filtration using a 125- or 250-mL filter flask.</div>
		</li>
		<li data-sub-note-item="1-25">
		<div class="lab-step">Wet the filter paper with 1 mL of ice-cold water. Then, start the vacuum and pour the contents of the flask into the B&#252;chner funnel of the vacuum filtration setup.</div>
		</li>
		<li data-sub-note-item="1-26">
		<div class="lab-step">Scrape crystals from the flask into the funnel using a glass rod or flexible spatula and rinse the remaining crystals into the funnel with about 1 mL of cold deionized water.</div>
		</li>
		<li data-sub-note-item="1-27">
		<div class="lab-step">Then, stop the vacuum, break the seal, and pour the rest of the cold deionized water into the funnel. Gently stir the crystals with the glass rod for a few seconds to wash them, being careful not to tear the filter paper.</div>
		</li>
		<li data-sub-note-item="1-28">
		<div class="lab-step">Turn on the vacuum to drain the funnel. Then, stop the vacuum, break the seal, and empty the filtrate into a waste beaker. Turn the vacuum back on, and leave the washed crystals in the funnel while the vacuum pump pulls air through them until they are dry, which usually takes about half an hour. You&#39;ll start working on your next recrystallization while you wait.</div>
		</li>
	</ul>
	</li>
	<li data-note-item="2" data-parent-collapse="">Recrystallization of <em>trans</em>-Cinnamic Acid<button class="expand">Expand</button>
	<p>In the second half of the lab, you&#39;ll recrystallize <em>trans</em>-cinnamic acid, which we&#39;ll just call cinnamic acid. Cinnamic acid is soluble in ethanol and nearly insoluble in water. So, you&#39;ll use a mixture of 95% ethanol and 5% water so that cinnamic acid is still soluble at high temperatures but less soluble at low temperatures.</p>
	<ul class="lab-items">
		<li data-sub-note-item="2-2">
		<div class="lab-step">To get started, fill a 5- or 10-mL graduated cylinder with deionized water and bring it to your hood.</div>
		</li>
		<li data-sub-note-item="2-3">
		<div class="lab-step">Then, weigh 0.5 g of cinnamic acid, record its mass, and place it in a 25-mL Erlenmeyer flask.
		<h4><strong>Table 2: Recrystallization of<em> trans</em>-Cinnamic acid</strong></h4>
		<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
			<tbody>
				<tr>
					<td><strong>Compound</strong></td>
					<td><strong>Mass (g) </strong></td>
					<td><strong>Melting point (&#176;C) </strong></td>
				</tr>
				<tr>
					<td><em>trans</em>-Cinnamic acid</td>
					<td></td>
					<td></td>
				</tr>
				<tr>
					<td>Seed crystal (if used)</td>
					<td></td>
					<td></td>
				</tr>
				<tr>
					<td>Recrystallized<em> trans</em>-cinnamic acid<br />
					<em>(subtract seed crystal if used)</em></td>
					<td></td>
					<td></td>
				</tr>
				<tr>
					<td></td>
					<td></td>
					<td></td>
				</tr>
				<tr>
					<td><strong>Percent recovery</strong></td>
					<td></td>
					<td></td>
				</tr>
			</tbody>
		</table>
		<a href="https://www.jove.com/CDNSource/lm/labs/58/Table_2._Recrystallization_of_trans-Cinnamic_acid.xlsx" target="_blank">Click Here to download Table 2</a></div>
		</li>
		<li data-sub-note-item="2-4">
		<div class="lab-step">Next, obtain 4 mL of 95% ethanol, and pour it into the flask containing cinnamic acid. Place the flask on a hotplate set to medium heat and stir the solution with a glass rod until the cinnamic acid dissolves, which usually takes 7 &#8211; 10 min.</div>
		</li>
		<li data-sub-note-item="2-5">
		<div class="lab-step">Then, turn off the heat and use a Pasteur pipette to add 5 drops of deionized water to the solution. This reduces cinnamic acid solubility. <strong>Note:</strong> If you don&#39;t see precipitation, add up to 5 more drops of water one by one. Stop adding water if you see solids starting to form.</div>
		</li>
		<li data-sub-note-item="2-6">
		<div class="lab-step">Then, remove the flask from the hotplate and let it cool to room temperature undisturbed.</div>
		</li>
		<li data-sub-note-item="2-7">
		<div class="lab-step">While you wait, get a small amount of the starting cinnamic acid and measure its melting point.</div>
		</li>
		<li data-sub-note-item="2-8">
		<div class="lab-step">Once the flask has cooled to room temperature, inspect it for crystals. If you don&#39;t see any, gently scratch the inside of the flask under the solution with the glass rod and wait for 10 min. <strong>Note:</strong> If that still doesn&#39;t work, get a seed crystal of cinnamic acid from your instructor. Remember to weigh the seed crystal and record its mass before you add it to your solution.</div>
		</li>
		<li data-sub-note-item="2-9">
		<div class="lab-step">Once the flask is cool to the touch and contains several crystals, remake the ice baths with one ice bath shallow enough that the 25-mL flask will not be completely submerged.</div>
		</li>
		<li data-sub-note-item="2-10">
		<div class="lab-step">Refill the graduated cylinder with deionized water. Place the flask of cinnamic acid crystals in the shallow ice bath and the water-filled graduated cylinder in the other ice bath. Let the crystals grow for at least 15 min.</div>
		</li>
		<li data-sub-note-item="2-11">
		<div class="lab-step">Your acetanilide crystals should be dry by now, so measure their mass and melting point while you wait for your cinnamic acid to finish crystallizing. Turn off the vacuum and break the vacuum seal.</div>
		</li>
		<li data-sub-note-item="2-12">
		<div class="lab-step">Gently place the B&#252;chner funnel in a clean beaker and bring it to the balance. Measure and record the mass of your pure acetanilide crystals. Remember to subtract the mass of the seed crystal if you used one.</div>
		</li>
		<li data-sub-note-item="2-13">
		<div class="lab-step">Then, bring your crystals to the melting point apparatus and measure and record the melting point.</div>
		</li>
		<li data-sub-note-item="2-14">
		<div class="lab-step">Now, place a clean, dry B&#252;chner funnel with a new piece of filter paper in the filter flask and check on your cinnamic acid. Once it looks like most of the cinnamic acid has crystallized, wet the filter paper with cold deionized water.</div>
		</li>
		<li data-sub-note-item="2-15">
		<div class="lab-step">Start the vacuum and pour the contents of the cinnamic acid flask into the funnel. Scrape the remaining crystals out of the flask with the glass rod and rinse the flask with about 1 mL of cold water.</div>
		</li>
		<li data-sub-note-item="2-16">
		<div class="lab-step">Now, turn off the vacuum, break the seal, and pour the rest of the cold water into the funnel. Gently stir the crystals with the glass rod to wash them, being careful not to damage the filter paper.</div>
		</li>
		<li data-sub-note-item="2-17">
		<div class="lab-step">Turn on the vacuum to drain the funnel. Leave the vacuum running until the cinnamic crystals are dry.</div>
		</li>
		<li data-sub-note-item="2-18">
		<div class="lab-step">Place the B&#252;chner funnel in a beaker and bring it to the balance. Measure and record the mass of your pure cinnamic acid crystals. Remember to subtract the mass of the seed crystal if you used one. Then, measure the melting point of your recrystallized cinnamic acid.</div>
		</li>
		<li data-sub-note-item="2-19">
		<div class="lab-step">Now, dispose of your acetanilide and cinnamic acid in the designated solid waste container. Flush the filtrate down the drain with running water and pour leftover ice and water into the sink.</div>
		</li>
		<li data-sub-note-item="2-20">
		<div class="lab-step">Clean your glassware using your usual methods and put away your other lab equipment.</div>
		</li>
		<li data-sub-note-item="2-21">
		<div class="lab-step">Lastly, discard your used pipettes in the glass waste and throw out used filter paper and other trash in the lab trash container.</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">First, calculate the percent recovery for each compound. We expect that some of each compound stayed in solution, so these percentages will be below the reported purity of your starting material.</div>
		</li>
		<li data-sub-note-item="3-2">
		<div class="lab-step">Now, look at the differences in melting points between the starting material and the crystals. For each compound, the less pure starting material has a lower melting point and a broader range than the recrystallized compound.</div>
		</li>
		<li data-sub-note-item="3-3">
		<div class="lab-step">Lastly, compare your results to the literature values. The melting point of acetanilide is 114 &#176;C, and the melting point of <em>trans</em>-cinnamic acid is 133 &#176;C. The melting points of your crystals should span 1 &#8211; 2 &#176;C and should be at or just below those values.</div>
		</li>
	</ul>
	</li>
</ol>

Recrystallization of Acetanilide and trans-Cinnamic Acid - Procedure

Rekrystalizacja

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

	Recrystallization of AcetanilideExpand
	In this part of ...

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

Zachowanie grupy

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 ...

Biology

Ecology

Sexual Selection and Mate Choice

<ol class="note-items"><li data-note-item="1" data-parent-collapse="">Sexual Selection Class Simulation
<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 and your classmates will portray a sexually reproducing population consisting of an equal number of male and female animals. You will simulate several different mating scenarios to determine how relative quality, attractiveness, sex, and parental commitment affect mate choice and reproductive success. The goal of this activity is to maximize your progeny. 
</div></li><li data-sub-note-item="1-2"><div class="lab-step">Hypotheses: There are several overall hypotheses for female and male behavior for this activity. The main experimental hypothesis for female behavior is that females will mate with males of high quality more frequently than those of low quality, yielding a higher reproductive success. The null hypothesis is that females will not demonstrate preference towards high- or low-quality males in any of the four rounds of mating. For males, one experimental hypothesis is that males with a higher visible score will have greater reproductive success. Another could be that males will be more selective for females in scenarios in which they join females to care for offspring as opposed to scenarios in which they exhibit no parental commitment. The null hypothesis for male behavior might be that males will not demonstrate preference towards high- or low-quality females in any of the four rounds of mating. 
</div></li><li data-sub-note-item="1-3"><div class="lab-step">Activity Overview: To begin, each student will be assigned a sex as well as a relative quality and attractiveness score. Remember - the goal is to maximize your reproductive success in each round. You do this by mating with partners of the highest possible quality which is rated on a scale of one to three with three being the highest possible score. Attractiveness scores, which follow the previously described scoring paradigm, correlate with quality but do not exactly predict the score. Though useful, they are not a perfect way to choose the best mate.  If you are assigned a male ID holder, you will have 16 gametes. However, if you are assigned a female ID holder, you will only have four. A successful mating occurs when one sperm and one egg are paper-clipped together and deposited at the fertilization area. 
</div></li><li data-sub-note-item="1-4"><div class="lab-step">Note - Rules of the activity: 
<ul>
	<li>Assigned male and female identities for these experiments are not representative of students&#39; personal gender identities. </li>
	<li>Running is not permitted. </li>
	<li>Talking is not permitted.</li>
	<li>Hidden values cannot be shared but individuals can attempt to hide their visible scores. </li>
	<li>Respect the space around each individual and remain an arm&#39;s length away. </li>
	<li>Offspring will only be counted towards an individual score if both gametes are paper-clipped together and dropped off in the designated fertilization area before the end of the two-minute round. </li>
</ul>
</div></li></ul></li><li data-note-item="2" data-parent-collapse="">Round 1 &#8211; Hidden Quality Score
<button class="expand">Expand</button><ul class="lab-items"><li data-sub-note-item="2-1"><div class="lab-step">To begin the first simulation, take the clip-on ID holder you were randomly assigned and make sure it contains four gametes if you are assigned female or sixteen gametes if male. Males should also have a strip of white paper and two paperclips. 
</div></li><li data-sub-note-item="2-2"><div class="lab-step">Next, males should paperclip the white strip of paper over their quality score to hide it, but keep the attractiveness score visible. 
</div></li><li data-sub-note-item="2-3"><div class="lab-step">Record your ID number for round one in table two.  <a href="https://www.jove.com/CDNSource/lm/labs/35/Table_2_sexual_selection.xlsx" style="display:block;margin:20px;" target="_blank">Click Here to download Table 2</a>
</div></li><li data-sub-note-item="2-4"><div class="lab-step">Next, clip on the ID holder and move to the designated mating area. 
</div></li><li data-sub-note-item="2-5"><div class="lab-step">When the instructor says begin, you may commence the mating simulation. You will have exactly two minutes to produce as many offspring as possible. Males may freely give their sperm to females, and females may choose to use these sperm immediately after which they would walk to the fertilization area and paperclip it to one of their eggs, or they may choose to wait until they receive other sperm to do so. 
</div></li><li data-sub-note-item="2-6"><div class="lab-step">Once the gametes have been deposited in the fertilization area, females may resume mating. 
</div></li><li data-sub-note-item="2-7"><div class="lab-step">When the two minutes have elapsed, all matings must stop and any gametes not in the fertilization area at this time will not be counted. 
</div></li><li data-sub-note-item="2-8"><div class="lab-step">The instructor should now tally all of the matings and record the numbers of the individuals who produced each offspring.
</div></li><li data-sub-note-item="2-9"><div class="lab-step">Now, find all the gametes that came with your ID holder and repackage them for the next round. 
</div></li></ul></li><li data-note-item="3" data-parent-collapse="">Round 2 &#8211; Hidden Attractiveness Score
<button class="expand">Expand</button><ul class="lab-items"><li data-sub-note-item="3-1"><div class="lab-step">For the second round, obtain a new randomly assigned ID holder and again ensure it contains the proper number of gametes. 
</div></li><li data-sub-note-item="3-2"><div class="lab-step">Next, males should use the paperclip and strip of white paper to hide their attractiveness score (instead of the quality score, which was covered in round one).
</div></li><li data-sub-note-item="3-3"><div class="lab-step">Record the ID number of your clip in table two under round two.  Note: The mating simulation process for round two will be the same as round one.
</div></li><li data-sub-note-item="3-4"><div class="lab-step">Move to the designated mating area and with for the instructor to signal the start of the round. As before, males may freely give away their sperm while females will choose to use it now or wait for different sperm. When a female decides to fertilize an egg, she will walk to the fertilization area and paperclip the gametes together. 
</div></li><li data-sub-note-item="3-5"><div class="lab-step">Proceed with the simulation for two minutes, and at the signal from the instructor all mating activity should stop. 
</div></li><li data-sub-note-item="3-6"><div class="lab-step">The instructor should now tally all of the matings from this second round.
</div></li><li data-sub-note-item="3-7"><div class="lab-step">Now, find and return the gametes to your ID holder, and then return the holder to the instructor.
</div></li></ul></li><li data-note-item="4" data-parent-collapse="">Round 3 &#8211; Visible Quality and Attractiveness Scores
<button class="expand">Expand</button><ul class="lab-items"><li data-sub-note-item="4-1"><div class="lab-step">For the third simulation, you should again obtain a new clip-on ID holder and check that it contains the appropriate number of gametes. Record the ID number of your clip in table two.
</div></li><li data-sub-note-item="4-2"><div class="lab-step">For this round, leave both attractiveness and quality scores visible on male IDs. Note: The mating simulation process for round three will be the same as the previous two rounds.
</div></li><li data-sub-note-item="4-3"><div class="lab-step">Move to the mating area and wait for the signal to begin the simulation.
</div></li><li data-sub-note-item="4-4"><div class="lab-step">Once the instructor starts the timer and gives the signal, commence mating. As before, males are allowed to freely give away their sperm to females and females may use the sperm immediately or wait for different sperm. Again, fertilized eggs only count if placed in the fertilization area. 
</div></li><li data-sub-note-item="4-5"><div class="lab-step">After the two-minute time limit, the instructor should tally all matings. 
</div></li><li data-sub-note-item="4-6"><div class="lab-step">Now, find and return the gametes to your ID holder, and then return the holder to the instructor.
</div></li></ul></li><li data-note-item="5" data-parent-collapse="">Round 4 &#8211; Increased Male Parental Care
<button class="expand">Expand</button><ul class="lab-items"><li data-sub-note-item="5-1"><div class="lab-step">In the final round, males will again leave their attractiveness and quality scores visible. 
</div></li><li data-sub-note-item="5-2"><div class="lab-step">Obtain an ID holder and ensure it contains the appropriate number of gametes. 
</div></li><li data-sub-note-item="5-3"><div class="lab-step">Clip on the ID holder and move to the mating area. Note: As before, males may freely give away their sperm to females. However, in this round, once a female chooses to mate with a male, both the male and female will walk to the fertilization area to paperclip their gametes together. After placing the paper-clipped gametes on the table, both male and female actors may return to the mating area. 
</div></li><li data-sub-note-item="5-4"><div class="lab-step">The instructor should signal the start of the round and begin the timer &#8211; at this point participants can begin the simulation.
</div></li><li data-sub-note-item="5-5"><div class="lab-step">Once the two-minute time limit is up, all matings must stop. 
</div></li><li data-sub-note-item="5-6"><div class="lab-step">The instructor should tally all mating events.
</div></li><li data-sub-note-item="5-7"><div class="lab-step">Find and return the gametes to your ID holder. Give your ID holder to the instructor and notify them if any gametes or supplies are missing. 
</div></li></ul></li><li data-note-item="6" data-parent-collapse="">Results
<button class="expand">Expand</button><ul class="lab-items"><li data-sub-note-item="6-1"><div class="lab-step">To analyze the data generated during the mating activities, first split up into groups of four. 
</div></li><li data-sub-note-item="6-2"><div class="lab-step">Collect the class data from the instructor for ONE of the four rounds of mating. Note: In this activity, there were several types of females and males distinguished by different letters. These letter types correspond to specific combinations of quality and attractiveness scores. In the compiled data, the average number of matings and reproductive success has already been sorted by letter type for you by the instructor. 
</div></li><li data-sub-note-item="6-3"><div class="lab-step">Use these data to generate four graphs for the round you were assigned. First plot two graphs for the average number of matings against female and male type. 
</div></li><li data-sub-note-item="6-4"><div class="lab-step">Then plot the reproductive success for males and females against male or female type, giving a total of four graphs.
</div></li><li data-sub-note-item="6-5"><div class="lab-step">Once these graphs are complete, each group should present their data to the class, making sure to state the original hypothesis for your assigned round and whether or not the hypothesis was upheld.
</div></li><li data-sub-note-item="6-6"><div class="lab-step">After each group presents, discuss the overall results of the four rounds together. 
</div></li><li data-sub-note-item="6-7"><div class="lab-step">Note the affect that hiding the quality score had on the overall quality of mates chosen.
</div></li><li data-sub-note-item="6-8"><div class="lab-step">Note how hiding or revealing the attractiveness score affected the results.
</div></li><li data-sub-note-item="6-9"><div class="lab-step">Discuss the effect of male parental investment on the number and overall quality of mating pairs.
</div></li></ul></li></ol>

Dobór płciowy i wybór partnera

Sexual Selection Class Simulation
ExpandNote: In this activity, you and your classmates will portray a sexually reproducing population consisting of an ...

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

Optymalne żerowanie

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

Hardy-Weinberg and Genetic Drift

<ol class="note-items"><li data-note-item="1" data-parent-collapse="">Class Simulation
<button class="expand">Expand</button><ul class="lab-items"><li data-sub-note-item="1-1"><div class="lab-step">Begin by opening a new spreadsheet file. Following the Hardy-Weinberg equation where p is the frequency of a dominant allele A in a population, and q is defined as the frequency of a recessive allele B, input frequency p of allele A into cell B2, and frequency q of allele B into cell B3.
</div></li><li data-sub-note-item="1-2"><div class="lab-step">Assign the value 0.5 to cell C2.
</div></li><li data-sub-note-item="1-3"><div class="lab-step">Following the 1 &#8211; p = q equation, enter the formula &#8220;= 1 &#8211; C2&#8221; into cell C3 to calculate the frequency q of allele B. NOTE: Cells C2 and C3 will represent the gene pool used in the next few steps.
</div></li><li data-sub-note-item="1-4"><div class="lab-step">Label cells E2 and F2 &#8220;Allele 1&#8221; and &#8220;Allele 2&#8221; respectively, then enter the RAND code for random formula into cell E3. =IF (RAND()</div></li><li data-sub-note-item="1-5"><div class="lab-step">Select cell E3 and drag the bottom right corner of the cell down to E27 to duplicate the formula into 25 cells, creating an allele for the other offspring, and enter the same formula that was used in cell E3 into cell F3.
</div></li><li data-sub-note-item="1-6"><div class="lab-step">Add a third column of data starting with the description &#8220;Genotype&#8221; in cell G2, and add the CONCATENATE function to cell G3 to combine the two randomly generated alleles and to create a genotype.
</div></li><li data-sub-note-item="1-7"><div class="lab-step">Drag this formula down for 25 cells and label cells H2, I2, and J2 AA, AB, and BB respectively.
</div></li><li data-sub-note-item="1-8"><div class="lab-step">Then, input one IF function as shown here into cells H3, I3, and J3. Drag the formulas down for 25 rows per column. The formulas will return a 1 if the genotype in this row matches the genotype in the header row, and a 0 if the genotype in this row is one of the other two genotypes. H3: = IF (G3=&#8221;AA&#8221;,1,0) I3: =IF(OR(G3=&#8221;AB,G3=&#8221;BA&#8221;),1,0) J3: =IF(G3=&#8221;BB&#8221;,1,0)
</div></li><li data-sub-note-item="1-9"><div class="lab-step">Then, label cell D28 as &#8220;SUM&#8221;, add the formula to cell H28, and drag the formula to cells I28 and J28 to get the total number of each genotype.
</div></li><li data-sub-note-item="1-10"><div class="lab-step">Next, label cells H30 and J30 &#8220;A&#8221; and &#8220;B&#8221; respectively and label cell D31 &#8220;Number of alleles&#8221;.
</div></li><li data-sub-note-item="1-11"><div class="lab-step">Add code to cells H31 and J31 to obtain the total number of alleles in the simulated generation, which is two times the frequency of the respective homozygous genotype, plus the frequency of the heterozygous genotype.
</div></li><li data-sub-note-item="1-12"><div class="lab-step">Then, label cell E32 &#8220;Next gen allele frequency&#8221; and add code to cells H32 and J32 to obtain the ratio of alleles in the next generation.
</div></li><li data-sub-note-item="1-13"><div class="lab-step">Using the gene frequencies generated in the mathematical model you just built, adjust the gene pool values in cells C2 and C3 to those from H32 and J32, and run 10 more generations, updating the gene pool with the resulting allele frequency each time.
</div></li><li data-sub-note-item="1-14"><div class="lab-step">Record the ratio of the two variables at each time point in the spreadsheet on the table, and create a line graph to see how a small population changes over time. Hypotheses: The experimental hypothesis in this study is that smaller populations will deviate more from the Hardy-Weinberg expected frequencies of the alleles than will larger populations, because smaller populations are more susceptible to genetic drift. The null hypothesis is that the allele frequencies within the populations will not differ from the Hardy-Weinberg equilibrium equation expected frequencies, meaning that the allele frequencies will be the same from generation to generation.
</div></li><li data-sub-note-item="1-15"><div class="lab-step">Set the frequency of gene A to 0.3 and gene B to 0.7, and run the model 10 more times, recording the ratio of the genes in the next generation in the table after each run.
</div></li><li data-sub-note-item="1-16"><div class="lab-step">Then, drag the formulas for the cells further down to alter the model to include 100 zygotes and run the model another 10 times.
</div></li><li data-sub-note-item="1-17"><div class="lab-step">After the last run, save all of the working data to a personal storage device and exit the spreadsheet program.
</div></li></ul></li><li data-note-item="2" data-parent-collapse="">Simulation for Hardy-Weinberg and Genetic Drift
<button class="expand">Expand</button><ul class="lab-items"><li data-sub-note-item="2-1"><div class="lab-step">To begin, find a partner and then collect one bag from the instructor.
</div></li><li data-sub-note-item="2-2"><div class="lab-step">Verify that the bag contains beads of two different colors and an even number of each color. 
</div></li><li data-sub-note-item="2-3"><div class="lab-step">Designate one color as allele capital A and the other as small a. Hypotheses: The experimental hypothesis for this simulation is that allele frequencies will not change over the 10 generations of the experiment, and Hardy-Weinberg equilibrium will be observed. The null hypothesis is that equilibrium will not be seen in the population.
</div></li><li data-sub-note-item="2-4"><div class="lab-step">Note the initial frequency in Table 1 next to generation zero. Using the Hardy-Weinberg equation, calculate the initial genotype frequencies of the population and note this in Table 1. <a href="https://www.jove.com/CDNSource/lm/labs/4/Lab_4_Table_A.xlsx" target="__blank" style="display:block;margin:20px;">Click Here to download Table A</a>
</div></li><li data-sub-note-item="2-5"><div class="lab-step">Then, discuss with your partner about the expected final allele and genotype frequencies after 10 generations of Hardy-Weinberg equilibrium. Note this next to prediction in Table 1.
</div></li><li data-sub-note-item="2-6"><div class="lab-step">Draw a pair of beads from the bag. This represents the genotype of one individual in the next generation.
</div></li><li data-sub-note-item="2-7"><div class="lab-step">Place a tally mark in the appropriate column next to Generation 1.
</div></li><li data-sub-note-item="2-8"><div class="lab-step">Now, keeping each pair of beads out of the bag every time, repeat the drawing 20 times to fill out the generation one row of Table 1.
</div></li><li data-sub-note-item="2-9"><div class="lab-step">Using the tallies, calculate the allele frequency for that generation, remembering to count both alleles in each genotype. Make a note of the new allele frequency, and then adjust the 100 beads to reflect the population after Generation 1 by adding and removing beads as necessary.
</div></li><li data-sub-note-item="2-10"><div class="lab-step">Repeat the drawing of pairs from this adjusted population for another generation of 20 picks, recording all of the observations in Table 1.
</div></li><li data-sub-note-item="2-11"><div class="lab-step">After calculating the allele frequency for this generation, adjust the population again to set up a third generation draw.
</div></li><li data-sub-note-item="2-12"><div class="lab-step">Keep drawing, calculating allele frequencies, and adjusting the starting population until 10 generations have been recorded in total.
</div></li></ul></li><li data-note-item="3" data-parent-collapse="">Violations of Hardy-Weinberg 
<button class="expand">Expand</button><ul class="lab-items"><li data-sub-note-item="3-1"><div class="lab-step">To test the effect of different physical and ecological scenarios on the Hardy-Weinberg equilibrium, first draw a slip of paper to determine which Hardy-Weinberg assumption you will be testing.
</div></li><li data-sub-note-item="3-2"><div class="lab-step">Make a prediction of what you think might happen in your selected scenario before beginning the experiment, and record this on the board.
</div></li><li data-sub-note-item="3-3"><div class="lab-step">If you selected the mutation scenario, select 5 alleles at random from the set and replace them with 5 of a third color, allele capital B.
</div></li><li data-sub-note-item="3-4"><div class="lab-step">As before, perform the simulation by pulling pairs of beads from the bag for a generation of 20 picks, recording the results.  <a href="https://www.jove.com/CDNSource/lm/labs/4/Lab_4_Table_B.xlsx" style="display:block;margin:20px;" target="__blank">Click Here to download Table B</a>
</div></li><li data-sub-note-item="3-5"><div class="lab-step">After each generation, make adjustments to have the starting population match the new allele frequency as before.
</div></li><li data-sub-note-item="3-6"><div class="lab-step">Then, replace 5 random alleles again with the new color, and simulate the new generation for 20 picks.
</div></li><li data-sub-note-item="3-7"><div class="lab-step">If you picked the non-random mating test, draw 1 allele at a time instead of 2 and then pair it with another of the same color pulled from the bag.
</div></li><li data-sub-note-item="3-8"><div class="lab-step">As with the previous simulation, adjust the population to match the new allele frequencies at the end of the previous generation, and carry out the simulation for 10 generations in total.
</div></li><li data-sub-note-item="3-9"><div class="lab-step">For the gene flow condition, begin by removing 10 alleles from the population and replacing them with 10 randomly selected alleles from another set of 100 beads.
</div></li><li data-sub-note-item="3-10"><div class="lab-step">Adjust the allele frequency and repeat the gene flow of removing 10 beads and adding 10 beads from another bag at the beginning of each generation.
</div></li><li data-sub-note-item="3-11"><div class="lab-step">If you drew small population size, simply start each generation with 60 alleles instead of 100.
</div></li><li data-sub-note-item="3-12"><div class="lab-step">As usual, adjust allele frequencies each generation and record 10 generations. In the selection condition, draw from the original bag of beads but do not count any homozygous recessive pairs that are drawn when calculating the allele frequency at the end of each generation.
</div></li></ul></li><li data-note-item="4" data-parent-collapse="">Simulation of Genetic Drift
<button class="expand">Expand</button><ul class="lab-items"><li data-sub-note-item="4-1"><div class="lab-step">Make your predictions before running the simulations and record them on the board.
</div></li><li data-sub-note-item="4-2"><div class="lab-step">If you have the small population size test, run the standard Hardy-Weinberg simulation as before, but instead start each generation with 30 alleles and not 100.
</div></li><li data-sub-note-item="4-3"><div class="lab-step">If you drew the founder effect with two alleles test, start generation 1 by drawing only 5 pairs of beads from the bag.
</div></li><li data-sub-note-item="4-4"><div class="lab-step">Then, creating the gene pool for generation 2, include only 50 beads at the allele frequency determined in generation 1. When you create the gene pool for generation 3 and all subsequent generations, include 100 beads.
</div></li><li data-sub-note-item="4-5"><div class="lab-step">For a founder effect with 10 alleles, you will need to collect a bag containing random amounts of 10 different colored beads.
</div></li><li data-sub-note-item="4-6"><div class="lab-step">Designate an allele name for each of the new alleles, and then calculate their initial allele frequency.
</div></li><li data-sub-note-item="4-7"><div class="lab-step">Now, run the experiment in the same way as the founder effect with two alleles, by drawing just 5 pairs randomly for generation 1 and then creating the second generation with 50 alleles, and third and subsequent generations with 100. <a href="https://www.jove.com/CDNSource/lm/labs/4/Lab_4_Table_C.xlsx" style="display:block;margin:20px;" target="__blank">Click Here to download Table C</a>
</div></li><li data-sub-note-item="4-8"><div class="lab-step">If you have the natural disaster with 2 alleles, you will need to collect a paper plate from your instructor.
</div></li><li data-sub-note-item="4-9"><div class="lab-step">Draw a line down the middle of the plate. Before generation 1, pour out all of the beads from a standard 100 bead setup onto the plate, and mix them randomly, spreading them evenly.
</div></li><li data-sub-note-item="4-10"><div class="lab-step">Choose one side of the plate and take all of the beads from that side. You should draw from these to create generation 1.
</div></li><li data-sub-note-item="4-11"><div class="lab-step">For generation 2 and beyond, adjust the population and use a total of 100 beads.
</div></li><li data-sub-note-item="4-12"><div class="lab-step">Finally, if you selected the natural disaster with 10 alleles test, you will need to collect a bag containing 10 different colored beads.
</div></li><li data-sub-note-item="4-13"><div class="lab-step">Assign a name to each new allele, and then place them on the plate and select half. Draw from these 50 beads for generation 1, and then calculate the allele frequencies to make a population of 100 in each subsequent round.
</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">Using the entered functions, calculate the number of each allele by multiplying the number of the homozygous genotypes by 2 and adding the number of the heterozygous genotypes.
</div></li><li data-sub-note-item="5-2"><div class="lab-step">The frequency can then be calculated by dividing the number of the allele in question by the total number of alleles. Enter the results into the table.
</div></li><li data-sub-note-item="5-3"><div class="lab-step">Next, use the Hardy-Weinberg equation to calculate the expected genotype frequency of the next generation, and compare the results of the simulation with the expected frequency, using the table as a guide.
</div></li><li data-sub-note-item="5-4"><div class="lab-step">Plot the changes of the population gene frequencies over time that you recorded in the table. Note any changes in allele frequencies over time and if any alleles decrease in frequency to the point that they disappear from the population. 
</div></li><li data-sub-note-item="5-5"><div class="lab-step">Plot the changes in the populations of the 25 and 100 zygotes with initial gene frequencies of 0.5 for each allele. Compare the results of the smaller population and the larger populations.  NOTE: The observed allele frequencies are expected to differ slightly from the Hardy-Weinberg expected values as a result of chance from the RAND function.
</div></li><li data-sub-note-item="5-6"><div class="lab-step">Using separate lines for each allele, graph the allele frequencies found for the original Hardy-Weinberg experiment over 10 generations.
</div></li><li data-sub-note-item="5-7"><div class="lab-step">Compare the results across the class, and see if your results agree.
</div></li><li data-sub-note-item="5-8"><div class="lab-step">Next, again using one line per allele, graph the results of your second experiment testing violations of the Hardy-Weinberg equilibrium, and share your finding with the class. Compare your actual results with your prediction. 
</div></li><li data-sub-note-item="5-9"><div class="lab-step">Finally, graph the results of your genetic drift simulation scenario in the same manner.
</div></li><li data-sub-note-item="5-10"><div class="lab-step">Again, present your results to the group and hypothesize why your results did or did not match your prediction. Compare your results to those of your classmates&#8217;. 
</div></li></ul></li></ol>

Hardy-Weinberg Equilibrium Principle and Genetic Drift - Procedure

Hardy-Weinberg i dryf genetyczny

Class Simulation
ExpandBegin by opening a new spreadsheet file. Following the Hardy-Weinberg equation where p is the frequency of a dominant allele A in a ...

Evolution

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

Zachowanie zwierząt

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

Wymieranie

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