<p class="jove_content">The authors are grateful for the artistic contributions of Hyewon Cooper.</p>

<p class="jove_title">1. Dissection and Recording Electrical Activity from the Propodite-dactylopodite (PD) Nerve</p>
<ol>
	<li>Hold the crab across the carapace from behind, and avoiding the claws, pinch the proximal part of the meropodite with forceps. The leg will autotomize to prevent the animal from bleeding to death. Use caution when handling blue crabs as they are rather aggressive and very fast.<br />
	<img alt="Figure 1" fo:content-width="5in" src="/files/ftp_upload/51050/51050fig1.jpg" width="500px" /><br />
	<strong>Figure 1.</strong> <strong>First walking leg of a crab</strong>. The anatomical location of chordotonal organs (hatched regions) are superimposed on this schematic. The double arrow head indicates where to transect the leg for the PD nerve experiments. <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/51050/51050fig1highres.jpg" target="_blank">Click here to view larger image</a>.</li>
	<li>Make a cut between the propodite and carpodite. Discard the carpopodite and the attached meropodite (<strong>Figure 1</strong>).</li>
	<li>Cut a large window in the cuticle on the pigmented (lateral) side of the propodite with a scalpel with a #11 blade (<strong>Figure 2</strong> and <strong>3</strong>). <strong>Note:</strong> Do not cut deeply.</li>
	<li>Remove the cuticle layer by sliding the scalpel blade beneath and parallel to the cuticle. This severs the muscle fibers attached to the cuticle.</li>
	<li>Using the same technique cut a smaller window on the pigmentless (medial) side of the propodite, but leave the condyle (the socket joint or hinge between segments) attachment intact.<br />
	<img alt="Figure 2" fo:content-width="5in" src="/files/ftp_upload/51050/51050fig2.jpg" width="500px" /><br />
	<strong>Figure 2.</strong> <strong>Cut along the dotted line on the propodite</strong>. <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/51050/51050fig2highres.jpg" target="_blank">Click here to view larger image</a>.<br />
	<img alt="Figure 3" fo:content-width="5in" src="/files/ftp_upload/51050/51050fig3.jpg" width="500px" /><br />
	<strong>Figure 3.</strong> <strong>Expose the nerve in the window (the arrows outline the nerve bundle)</strong>. <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/51050/51050fig3highres.jpg" target="_blank">Click here to view larger image</a>.</li>
	<li>Prepare a Sylgard-lined dish containing crab saline to pin the preparation down. <strong>Note:</strong> See <strong>Table 1</strong> for species specific saline recipes.</li>
	<li>Locate the PD organ by carefully probing with the fire-polished glass needles. The elastic strand spanning the joint has a silver appearance.</li>
	<li>Remove muscle fibers that obscure your view of the organ from both sides of the tendon. Be very careful not to injure the PD organ or its nerve.</li>
	<li>Once this has been accomplished, firmly reattach the preparation to the dish with the pigment side (lateral) facing up (<strong>Figure 4</strong>).<br />
	<img alt="Figure 4" fo:content-width="5in" src="/files/ftp_upload/51050/51050fig4.jpg" width="500px" /><br />
	<strong>Figure 4.</strong> <strong>Exposed PD organ and nerve</strong>. <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/51050/51050fig4highres.jpg" target="_blank">Click here to view larger image</a>.</li>
	<li>Follow the PD organ nerve in the propodite as far proximally as possible in order to free-up a long length of nerve (1.5 cm) for recording purposes. This is best done while the PD nerve is still attached to the main leg nerve.</li>
	<li>After separating the PD nerve from the main leg nerve with the aid of glass needles, sever the PD nerve proximally with the iris scissors. <strong>Note:</strong> Do not stretch or pull on the nerve during the dissection.</li>
	<li>Move the dactylopodite to an extended and fully flexed position. Take note about where the extreme flexion and extension positions are and a half-way point for later use.</li>
	<li>Place a ground wire inside the saline bath.</li>
	<li>Turn on the electrophysiology recording hardware/software. <strong>Note:&#160;</strong>Our setup has been described previously<sup>23</sup>.</li>
	<li>Position the microscope so that it is overlooking the microscope stage. Once the prep dish is placed on the stage, you will need to adjust the position of the high intensity illuminator beam to best visualize the preparation.</li>
	<li>Position the micromanipulator so the attached suction electrode assembly will have easy access to the saline bath and preparation. The suction electrode is constructed as shown in an online video<sup>24</sup>.</li>
	<li>To detect neural activity, draw the cut end of the PD nerve into the suction electrode.</li>
	<li>Move the dactyl throughout extended and flexed positions for several cycles with the aid of a glass probe or wooden dowel.</li>
	<li>Next observe activity when the dactyl is pinned in the extended, flexed, and mid positions.</li>
</ol>
<p class="jove_title">2. Recording Electrical Activity from the Tension Nerve while Monitoring Force Generation</p>
<ol>
	<li>Determine the lateral and medial side of the leg. The medial side has a soft texture that can be felt by pinching gently in the meropodite region with a fingernail.</li>
	<li>Place this soft cuticle side up in the dish. The excitor motor nerve that innervates the opener muscle also innervates the stretcher muscle in the carpopodite.</li>
	<li>In order to stimulate the opener muscle the stretcher motor nerve in the carpodite region is isolated and stimulated with a suction electrode. The proximal part of the leg is removed by transecting the meropodite with scissors.</li>
	<li>Remove a section of the cuticle in the carpus region on the inner side (medial side, <strong>Figure 5A</strong>).</li>
	<li>Cut the apodeme of the bender muscle and remove the muscle carefully as not to pull the main leg nerve out of the leg cavity (<strong>Figure 5B, C</strong> note the arrows where to bender apodeme is separated). The main leg nerve and a branch to the stretcher muscle can then be observed (<strong>Figure 5D</strong>).</li>
	<li>Find the nerve branching from the main nerve bundle to the stretcher muscle (<strong>Figure 5E, </strong>at arrow)<strong>. </strong>This can be cut close to the muscle and pulled into a suction electrode to stimulate (<strong>Figures 5F </strong>and<strong>&#160;G, </strong>arrow depicts the branch).</li>
	<li>Tease out a section of the nerve that projects towards the opener without pinching the nerve. Transect the stretcher/opener motor nerve.</li>
	<li>Pull the motor nerve into the suction electrode and then stimulate it.</li>
	<li>To expose the opener muscle and tension nerve remove the closer muscle and the ventral region of the propodite. Cut off the closer muscle with a scalpel with a #11 blade in the propodite (<strong>Figure 6</strong>). <strong>Note:</strong> Do not cut deeply in the proximal region because it might damage the opener motor nerve.<br />
	<img alt="Figure 5" fo:content-width="5in" src="/files/ftp_upload/51050/51050fig5.jpg" width="500px" /><br />
	<strong>Figure 5.</strong> <strong>Dissection steps for exposing the motor neuron for stimulating the opener muscle</strong>. <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/51050/51050fig5highres.jpg" target="_blank">Click here to view larger image</a>.<br />
	<img alt="Figure 6" fo:content-width="5in" src="/files/ftp_upload/51050/51050fig6.jpg" width="500px" /><br />
	<strong>Figure 6. Cut the cuticle on the ventral half of the propodite to expose the closer muscle so it can be removed</strong>. <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/51050/51050fig6highres.jpg" target="_blank">Click here to view larger image</a>.</li>
	<li>Exchange the saline with fresh cooled saline throughout the dissection process to keep the neurons alive. For further dissection, place the preparation dish under a dissecting microscope and use fiber-optic illumination.</li>
	<li>Carefully cut the closer tendon from its attachment to the dactyl using sharp-pointed medium-size scissors.</li>
	<li>Be very careful not to disturb the branches to the opener muscle from the main leg nerve which should be clearly visible.</li>
	<li>Remove and discard the closer muscle and tendon.</li>
	<li>Make a hole in the dactyl with a dissection pin. This hole will be used later to hook the metal pin on the tension (force) transducer, but it is necessary to make it at this stage of the procedure.</li>
	<li>Locate the nerve branch that projects to the opener muscle and apodeme in the distal region of the opener muscle. Carefully probe the nerve with the fire-polished glass tool.</li>
	<li>Observe the tension nerve that arises from the distal end of the apodeme and proceeds to the motor nerve bundle.</li>
	<li>To detect neural activity, fill a suction electrode with crab saline and draw the cut end of the opener tension nerve into the electrode. Ensure that the suction electrode fits tightly on the nerve.</li>
	<li>Examine for the neural correlate of passive tension on the opener apodeme. While recording electrical activity from the tension nerve, rotate the dactyl rapidly into an extended joint (<em>i.e.</em> stretching the opener muscle).</li>
	<li>Next, test active force development in relation to stimulation frequency.</li>
	<li>Firmly attach a metal hook so that the tip of the hook goes through a small hole in the dactyl.</li>
	<li>Attach the other end of the metal hook to the force transducer. <strong>Note:</strong> Make sure the transducer is at a 90&#176; angle each time so that the pin is perpendicular to the transducer for maximal detection of the force generated.</li>
	<li>Pull the hook and dactyl so that it is at about a 45&#176; angle of the opener joint.</li>
	<li>Now stimulate the motor nerve at 100 Hz for 250 msec and measure the force as well as the firing frequency of the tension nerve.</li>
	<li>Place the joint into a fully extended position so that the opener muscle fibers are flaccid.</li>
	<li>Stimulate the motor nerve at 100 Hz for 250 msec and measure the force as well as the firing frequency of the tension nerve.</li>
	<li>Bend/flex the joint so that it is fully flexed (~90&#176;). In this position the muscle fibers of the opener are fully stretched. There may be some force measured by the transducer due to this passive stretch of the muscle.</li>
	<li>Stimulate the motor nerve at 100 Hz for 250 msec and measure the force as well as the firing frequency of the tension nerve.</li>
	<li>After measuring a force, proceed in a series of various frequencies: 20, 40, 60, 80, and 100 Hz for 8-10 sec&#160;in each joint position.</li>
	<li>Measure the response with a quick tension release. Arrange the preparation so that the hook on the force transducer can easily be pushed out of the hole in the dactyl.</li>
	<li>Bend/flex the joint so that it is fully flexed (~90&#176;). Now stimulate the motor nerve at 100 Hz with a continuous stimulation of 5 sec.</li>
	<li>After the first second or less, as tension has built up on the opener muscle, push the pin out of the hole in the dactyl.</li>
	<li>Examine the tension nerve recording before and after release of the pin holding the dactyl.</li>
	<li>Modulatory effects of neuroactive substances such as octopamine, serotonin, and proctolin, which alter the output of the tension neurons, can be determined by adding these molecules to the bathing media over the exposed opener muscle and repeating the experiments.</li>
	<li>Use a pipette and drip 1-2 ml over the preparation.</li>
</ol>
<p class="jove_title">3. Staining Peripheral Nervous System Structures in Crustacean Walking Legs</p>
<ol>
	<li><strong>Methylene blue technique</strong>
	<ol>
		<li>Dilute one part methylene blue chloride stock solution (0.25%) with two parts of distilled water.</li>
		<li>Add this mixture to five parts of buffered saline. Thoroughly irrigate the area of interest.</li>
		<li>Dissect a leg according to the protocol in section 1. Incubate the preparation in the methylene blue solution at 12-13 &#176;C.</li>
		<li>Examine the preparation every 10-15 min&#160;with the dissecting microscope using low intensity illumination to follow the progress of the staining. In some cases staining will be completed in an hour, in others it may take overnight.</li>
	</ol>
	</li>
	<li><strong>4-di-2-ASP technique</strong>
	<ol>
		<li>Fluorescent dyes can be used to back-fill the PD neuron or directly stain the preparation from the bath. However a suitable microscope with abilities to view the fluorescent stain is required.</li>
		<li>Dissect a leg according to a protocol in section 1.</li>
		<li>Incubate the preparation in a 10 &#181;M concentration of 4-Di-2-ASP solution and leave the preparation in the refrigerator for 15 min. Use just enough solution to cover the preparation.</li>
		<li>Photograph the preparations quickly and avoid over exposure to the mercury light. This fluorescent dye fades away relatively quickly.</li>
	</ol>
	</li>
	<li><strong>Cobalt chloride technique</strong>
	<ol>
		<li>Expose the PD nerve according to the protocol in section 1 and keep it immersed in cold saline.</li>
		<li>Make a petroleum jelly well to hold the CoCl<sub>2</sub>. If any CoCl<sub>2</sub> spills into the saline bath the entire preparation will stain black, and the preparation should be discarded.</li>
		<li>Slip a small cut of a polystyrene sheet, to make a plastic platform, and pin it in such a manner that it will not float away or become immersed in the saline bath (<strong>Figure 7</strong>).<br />
		<img alt="Figure 7" fo:content-width="5in" src="/files/ftp_upload/51050/51050fig7.jpg" width="500px" /><br />
		<strong>Figure 7.</strong> <strong>Polystyrene sheet with pins to hold in place in dish</strong>. <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/51050/51050fig7highres.jpg" target="_blank">Click here to view larger image</a>.</li>
		<li>Eject petroleum jelly from a fine hypodermic needle fastened to a disposable syringe, then make a barrier (a circle might work well), which should be about 1-1.5 mm high except for a shallow &#34;V&#34; at midpoint where the nerve will be draped across, on top of the slip of polystyrene.</li>
		<li>Make a small puddle of saline on either side of the barrier near the &#34;V&#34;. Taking care not to stretch or pinch the nerve, lift the nerve carefully from the dish and place it in the saline puddle in the petroleum jelly well.</li>
		<li>Work quickly so that the section of nerve does not dry out, carefully eject petroleum jelly to cover the exposed nerve. Now test the barrier with saline and make sure it is not leaking.</li>
		<li>Blot away saline on the inside of the barrier and see if it fills up when the bath saline is high around the wall of petroleum jelly to be sure that the two sides are isolated from one another (<strong>Figure 8</strong>).<br />
		<img alt="Figure 8" fo:content-width="5in" src="/files/ftp_upload/51050/51050fig8.jpg" width="500px" /><br />
		<strong>Figure 8.</strong> <strong>Petroleum jelly well with saline and PD nerve spanning the well</strong>. The PD nerve has not been cut yet. <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/51050/51050fig8highres.jpg" target="_blank">Click here to view larger image</a>.</li>
		<li>Wick away the saline in the well using a piece of tissue paper. Avoid letting the paper wrap up the nerve.</li>
		<li>Make a new puddle using a few small drops of distilled water, and then cut the nerve end. Be very careful not to pull the nerve through the barrier when making the cut. The osmotic shock of the distilled water will &#34;balloon&#34; the axons.</li>
		<li>Within 30 sec&#160;add a small drop of CoCl<sub>2</sub> to the water, soak up this solution, and then add enough CoCl<sub>2</sub> to form a puddle over this shortened section of nerve (<strong>Figure 9</strong>). Preparations are best kept refrigerated at 13&#160;&#176;C for 12-24 hr.<br />
		<img alt="Figure 9" fo:content-width="5in" src="/files/ftp_upload/51050/51050fig9.jpg" width="500px" /><br />
		<strong>Figure 9.</strong> <strong>The cut PD nerve being exposed to CoCl<sub>2</sub></strong>. The cut nerve swells up in the presence of water that facilitates and accelerates the movement of dye molecules through the axons into the neuron cell bodies. <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/51050/51050fig9highres.jpg" target="_blank">Click here to view larger image</a>.</li>
		<li>Remove the humidifying puddles. Blot away the cobalt solution using a tissue and wash away the remnants of cobalt with several changes of saline.</li>
		<li>Transfer the isolated preparation to a small glass Petri dish containing about 10 ml of crab saline. The neurons are washed and the following steps are performed <em>in situ</em>. Good metal tools are not to be used to handle the preparation after this step (you should use specific tools that are not to be used at a later time for physiology).</li>
		<li>Add 1-2 drops of ammonium sulfide (NH<sub>4</sub>)<sub>2</sub>S to the saline. Cap the (NH<sub>4</sub>)<sub>2</sub>S bottle tightly and place back into the hood. Observe the reaction in the preparation under a dissecting microscope</li>
		<li>Within 1-2 min, cobalt-filled neurons and their processes should begin to appear because they will stain black.</li>
		<li>After 5-10 min, replace the development solution with fresh saline.</li>
		<li>Make certain that the development solution you have poured into the sink drain is followed by running tap water for a few minutes or in a waste bottle with a lid.</li>
		<li>Pour out the saline and fix the nerve preparation for about 15 min with two changes of Bouin&#8217;s solution fixative. For larger tissues like the opener muscle (to stain tension neurons), increase the duration of fixation to 30 min.</li>
		<li>Dehydrate in a series of ascending order of ethanol concentration beginning at 70% (<em>i.e.</em> 70%, 80%, 90%, 100%). About 10 min at each concentration is sufficient for small tissues.</li>
		<li>After about 10-15 min in two changes of 100% ethanol, clear the tissue by replacing ethanol with 100% methyl salicylate. The preparation will stay in this solution permanently for repeated viewing. With time the filled cells will become more apparent because the surrounding tissue will become clearer. Intensification methods can be used to help prevent fading over time<sup>25</sup>.</li>
		<li>Use the same process to fill the tension nerve with CoCl<sub>2</sub>. However, change ethanol solution multiple times in each ethanol dehydration step and incubate the preparation inside alcohol for about 20 min for each step to thoroughly dehydrate the muscles and allow them to clear well.</li>
	</ol>
	</li>
</ol>

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B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tension+receptors+on+the+closer+muscle+apodeme+in+the+walking+legs+of+the+blue+crab+Callinectes+sapidus.">Tension receptors on the closer muscle apodeme in the walking legs of the blue crab Callinectes sapidus.</a> <em style='font-style: italic'>J. Comp. Physiol. A</em>. <b>157</b> (3), 355-362 (1985).</li><li>Holsinger, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> <em style='font-style: italic'>The effect of regional phenotypic differences of Procambarus clarkii opener muscle on sarcomere length, fiber diameter, and force development MSc thesis [dissertation]</em>. , (2013).</li><li>Parsons, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+morphology+and+ultrastructure+of+tension+receptors+in+the+walking+legs+of+the+crab,+Carcinus+maenas.">The morphology and ultrastructure of tension receptors in the walking legs of the crab, Carcinus maenas.</a> <em style='font-style: italic'>Cell Tissue Res</em>. <b>211</b> (1), 139-149 (1980).</li><li>Parsons, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Responses+and+central+interactions+of+tension+receptors+in+the+leg+muscle+of+Carcinus.">Responses and central interactions of tension receptors in the leg muscle of Carcinus.</a> <em style='font-style: italic'>Comp. Biochem. Physiol. A Physiol</em>. <b>72</b> (2), 391-399 (1982).</li><li>Tryba, A. K., Hartman, H. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamic+responses+of+series+force+receptors+innervating+the+opener+muscle+apodeme+in+the+blue+crab,+Callinectes+sapidus.">Dynamic responses of series force receptors innervating the opener muscle apodeme in the blue crab, Callinectes sapidus.</a> <em style='font-style: italic'>J. Comp. Physiol. A</em>. <b>180</b> (3), 215-221 (1997).</li><li>Whitear, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+fine+structure+of+crustacean+proprioceptors.+I.+The+chordotonal+organs+in+the+legs+of+the+shore+crab,+Carcinus+meanas.">The fine structure of crustacean proprioceptors. I. The chordotonal organs in the legs of the shore crab, Carcinus meanas.</a> <em style='font-style: italic'>Philos. Trans. R. Soc. Lond. B Biol. Sci</em>. <b>245</b> (725), 291-325 (1962).</li><li>Whitear, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+fine+structure+of+crustacean+proprioceptors.+II.+The+thoracico-coxal+organs+in+Carcinus,+Pagurus+and+Astacus.">The fine structure of crustacean proprioceptors. II. The thoracico-coxal organs in Carcinus, Pagurus and Astacus.</a> <em style='font-style: italic'>Philos. Trans. R. Soc. Lond. B Biol. Sci</em>. <b>248</b> (752), 437-456 (1965).</li><li>Leksrisawat, B., Cooper, A. S., Gilberts, A. B., Cooper, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Muscle+Receptor+Organs+in+the+Crayfish+Abdomen:+A+Student+Laboratory+Exercise+in+Proprioception.">Muscle Receptor Organs in the Crayfish Abdomen: A Student Laboratory Exercise in Proprioception.</a> <em style='font-style: italic'>J. Vis. Exp</em>.  (45), e2323 (2010).</li><li>Baierlein, B., Thurow, A. L., Atwood, H. L., Cooper, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Membrane+Potentials,+Synaptic+Responses,+Neuronal+Circuitry,+Neuromodulation+and+Muscle+Histology+Using+the+Crayfish:+Student+Laboratory+Exercises.">Membrane Potentials, Synaptic Responses, Neuronal Circuitry, Neuromodulation and Muscle Histology Using the Crayfish: Student Laboratory Exercises.</a> <em style='font-style: italic'>J. Vis. Exp</em>.  (47), e2322 (2011).</li><li>Delaney, K., Gelperin, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cerebral+interneurons+controlling+fictive+feeding+in+Limaxmaximus;+I.+Anatomy+and+criteria+for+re-identification.">Cerebral interneurons controlling fictive feeding in Limaxmaximus; I. Anatomy and criteria for re-identification.</a> <em style='font-style: italic'>J. Comp. Physiol. A</em>. <b>166</b>, 297-310 (1990).</li><li>Hartman, H. B., Cooper, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regeneration+and+molting+effects+on+a+proprioceptor+organ+in+the+Dungeness+crab,+Cancer+magister.">Regeneration and molting effects on a proprioceptor organ in the Dungeness crab, Cancer magister.</a> <em style='font-style: italic'>J. Neurobiol</em>. <b>25</b> (5), 461-471 (1994).</li><li>Hartman, H. B., Austin, W. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proprioceptor+organs+in+the+antennae+of+Decapoda+Crustacea.">Proprioceptor organs in the antennae of Decapoda Crustacea.</a> <em style='font-style: italic'>J. Comp. Physiol</em>. <b>81</b> (2), 187-202 (1972).</li><li>Cooper, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+sensory+processes+during+limb+regeneration+in+adult+crayfish.">Development of sensory processes during limb regeneration in adult crayfish.</a> <em style='font-style: italic'>J. Exp. Biol</em>. <b>201</b> (Pt 11), 1745-1752 (1998).</li><li>Chung, Y. -. S., Cooper, R. M., Graff, J., Cooper, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+acute+and+chronic+effect+of+low+temperature+on+survival,+heart+rate+and+neural+function+in+crayfish+(Procambarus+clarkii)+and+prawn+(Macrobrachium+rosenbergii)+species.">The acute and chronic effect of low temperature on survival, heart rate and neural function in crayfish (Procambarus clarkii) and prawn (Macrobrachium rosenbergii) species.</a> <em style='font-style: italic'>Open J. Mol. Int. Physiol</em>. <b>2</b> (3), 75-86 (2012).</li><li>Blundon, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+temperature+and+thermal+history+on+neuromuscular+properties+of+two+crustacean+species.">Effects of temperature and thermal history on neuromuscular properties of two crustacean species.</a> <em style='font-style: italic'>J. Comp. Physiol. B</em>. <b>158</b> (6), 689-696 (1989).</li><li>Cooper, R. M., Schapker, H., Adami, H., Cooper, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heart+and+ventilatory+measures+in+crayfish+during+copulation.">Heart and ventilatory measures in crayfish during copulation.</a> <em style='font-style: italic'>Open J. Mol. Int. Physiol</em>. <b>1</b> (3), 36-42 (2011).</li><li>Bierbower, S. M., Cooper, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+mechanistic+action+of+carbon+dioxide+on+a+neural+circuit+and+NMJ+communication.">The mechanistic action of carbon dioxide on a neural circuit and NMJ communication.</a> <em style='font-style: italic'>Journal of Experimental Zoology</em>. , (2013).</li><li>Bierbower, S. M., Shuranova, Z. P., Viele, K., Cooper, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sensory+systems+and+environmental+change+on+behavior+during+social+interactions.">Sensory systems and environmental change on behavior during social interactions.</a> <em style='font-style: italic'>Brain Behav</em>. <b>3</b> (1), 4-13 (2013).</li></ol>

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

<p class="jove_content">The purpose of this set of experiments is 1) to teach and exhibit the fundamental principles of extracellular recordings from an identifiable proprioceptive organ and tension nerve and 2) to stress the importance of anatomical mapping in relation to physiological function of particular sensory systems. This experimental approach and the animal models utilized are inexpensive and relatively easy to conduct in neurophysiology teaching laboratories.</p>
<p class="jove_content">The neurons of chordotonal organs are of two specific functional types, those that respond to movement and those responding to static positions. Single cell recordings from a variety of chordotonal organs, no matter which joint is examined, have shown this to be the case<sup>3,5</sup>. Indeed, chordotonal organs associated with the antennal joints of lobsters reveal the same two sensory types and basic anatomy<sup>27</sup>.&#160;In addition to there being two neuron types (movement and position), the neurons share the same anatomical arrangement on their respective elastic strands. The large somata located proximally on the strand tend to belong to the dynamic movement sensitive neurons. Neurons that signal static positions have small somata and are located distally. These cells are tonically active. The PD joint only contains a single chordotonal organ while there are two chordotonal organs in the carpus-propodus (CP) and merus-carpus (MC) joints.</p>
<p class="jove_content">The dissection to expose proprioceptive structures in blue crabs (<em>C. sapidus</em>) for electrophysiological recording requires a strategy that allows joint movements to take place in the natural positions while recording from sensory neurons. The tension nerve for the opener muscle in the walking leg is a very fine nerve made up of several neurons. Unless care is taken, the tension nerve as well as the motor nerve innervating the muscle to be stimulated, can be damaged during this dissection. For optimal recordings the suction electrodes need to be tailored to the size of the nerve. Recordings are readily accessible in a student laboratory using a 30-40X magnification dissecting microscope and low-end micromanipulators.</p>
<p class="jove_content">Future experiments that would be interesting to pursue with the joint chordotonal organs would be to examine the structural and physiological profiles during leg regeneration in various species at different stages in the life cycle as a follow up to an initial study that used <em>Cancer magister</em><sup>19,26</sup>. Questions remaining to be addressed are 1) does the distribution and organization of regenerated neurons depend on the age of the animal when regenerating a limb, 2) are the axonal projections to the CNS (ventral nerve cord) in a regenerating limb functional or does it take time and joint use to establish functional connections, and 3) what happens to the severed axons proximal to the autotomy plane when the limb is autotomized?<sup>28</sup></p>
<p class="jove_content">Crustaceans conform to environmental conditions and their surrounding temperature, but it is unclear how they maintain coordination within a neural circuit as neurons alter their activity in response to temperature changes. A slow rate of change might allow the animal some time for acclimatization whereas a rapid change may not29,30.&#160;Physiological changes in pH or osmolarity due to metabolism, behavior<sup>31</sup>, or environmental impact may present similar challenges to neural circuits involved in proprioception. These crustacean preparations are ideal for addressing these types of problems because their function is well characterized at a single cell level.</p>
<p class="jove_content">In this protocol we have demonstrated the physiological importance of tension neurons in monitoring force generated by the opener muscle. These tension receptors can be traced to their location within the apodeme by using staining procedures. These neurons, as in mammals, detect force at various levels and recruit additional neurons as the force increases. The frequency in activity is related to the stimulation frequency of the motor neuron until saturation in reception is reached. Using a quick release protocol with the flexed dactylus joint, tension activity quickly disappears but then returns upon regaining tension in a fully extended joint. This is a classic experimental procedure to illustrate the force measured by tension receptors. Various neuromodulators can be applied to the preparation to see how it effects the development of force and neuronal response. One of the important aspects is how the neural responses are processed and integrated in the central nervous system and their impact on activity of motor neurons. The techniques we have shown allow one to start to address more information about the tension (sensory) nerve-motor neuron circuit function, <em>i.e.</em>&#160;signal in an intact leg to the ganglion and back to the muscle.</p>
<p class="jove_content">The staining procedures demonstrated are key to understanding the physiology of sensory neurons that innervate proprioceptive organs. Anatomical arrangement of the neurons based on function and the size of the soma are similar in the various chordotonal organs within the crab legs. It is not known if similar neuronal arrangements are also found in other crustacean species or insects. Combining physiological recordings from single cells and mapping the location allows direct structure function relationships. The long term preservation of the anatomical arrangement with CoCl<sub>2</sub> staining and fixation allows one to repetitively make measures and assess the structural arrangement.</p>
<p class="jove_content">Proprioception and tension reception of skeletal muscles are sensory modalities that enable coordinated behaviors and responses to external and internal environment for articulated animals in a variety of skeletal muscle configurations. The muscle receptor organ in the abdomen of the crayfish is another well-documented preparation (see the Crawdad Project; <a href="http://www.crawdad.cornell.edu/">http://www.crawdad.cornell.edu/</a>) for teaching purposes of proprioception with only two neurons per abdominal hemi-segment<sup>23</sup>.&#160;Being able to record from single neurons to sensory nerve bundles provides further details that aid in understanding the basic principles of sensory reception. These relatively simple crustacean preparations allow one to address fundamental aspects of proprioception and tension monitoring, with the potential to determine the neural circuits that enable central integration of proprioceptive and other sensory inputs<sup>9-12, 32, 33</sup>.</p>

<p class="jove_content">Proprioception is the sensation of limb position and movement that enables coordinated motor behavior. Proprioceptors consist of position (static) and movement (kinesthetic) receptors. In insects and crustaceans, chordotonal organs are the structures that provide that information to the CNS<sup>1</sup>. Not all chordotonal organs span a joint but they can still monitor joint movements due to their attachment on the apodemes (tendon like structures) which span the joint and move in association with the skeletal muscle and joint articulation. Crab legs have six joints, each having one or two chordotonal organs<sup>2</sup>. Typically a chordotonal organ has 60-100 or more sensory neurons embedded within an elastic strand, neurons that signal static joint position, direction and speed of movement<sup>3-6</sup>. The input from chordotonal organs at each joint and leg is then centrally processed allowing coordinated movements by the animal.</p>
<p class="jove_content">The forces that leg muscles produce during isometric and isotonic contractions are detected by tension receptors associated with muscle fibers and their attachments to apodemes7-9.&#160;In the crustacean walking leg protocols that follow we present methodology for recordings from primary sensory neurons that monitor proprioception and the neurons that respond to forces generated by muscle fibers. A technique for activating leg movements and quantifying force generation is also presented, as well as anatomical techniques that can be used to characterize the arrangement of these peripheral nervous system structures.</p>
<p class="jove_content">The procedures demonstrated below enable structural and functional analysis of the neurons that innervate both types of receptors relative to their location on a chordotonal elastic strand and apodeme. To illustrate, we use the propodite-dactylopodite (PD) chordotonal organ, the organ that spans the distal most segment of the crab leg<sup>3</sup>. Though detailed electrophysiological studies began in the 1930s and are still being carried out today, some aspects have become known about the segmental connections of proprioceptors in the various joints and their roles in coordinated control of muscles10-16.&#160;Establishing the structure-function relationship between the proprioceptive organs, muscles and the nervous system will further help define these roles. For instance, labeling the somata and distal endings of tension neurons inserted into the apodeme will reveal their location relative to muscle fibers<sup>8,17-21</sup>.</p>
<p class="jove_content">We present three staining techniques for crustacean legs that can be used in research or academic laboratories. Methylene blue staining provides suitable contrast for muscles and nerves and is recommended as a simple technique for students to learn anatomy. Labs that have fluorescence microscopy setups can accomplish more selective neuronal staining by briefly exposing the nerves to the vital dye 4-di-2-ASP. The third alternative is CoCl<sub>2</sub> backfill, which stains and fixes the neurons, and does not require fluorescence imaging. Though it is labor and time intensive, this staining process gives high contrast and specificity for the nerves that are filled. Together these techniques can be used for comparing various chordotonal organs, not only within a limb or between limbs, but also among other crustacean and insect species<sup>20-22</sup>. Blue crabs (<em>C. sapidus</em>) used in physiological recordings and for anatomical staining are readily available all around the Southern and Southeastern border of the United States. This species serves as a representative of the chordotonal and tension nerve arrangements found in most crabs. Laboratories on the west coast will prefer to use the much larger Dungeness crab (<em>Cancer magister</em>) for these experiments.</p>

<table><tbody><tr><td>Sylgard</td><td>Dow Corning</td><td>182 silicone kit</td><td></td></tr><tr><td>NaCl</td><td>Sigma-Aldrich</td><td>S7653</td><td></td></tr><tr><td>KCl</td><td>Sigma-Aldrich</td><td>P9333</td><td></td></tr><tr><td>CaCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Sigma-Aldrich</td><td>C5670</td><td></td></tr><tr><td>HEPES acid</td><td>Sigma</td><td>3375</td><td>Free acid, crystalline</td></tr><tr><td>HEPES base</td><td>Sigma</td><td></td><td></td></tr><tr><td>D-Glucose</td><td>Sigma</td><td>G7021</td><td></td></tr><tr><td>MgSO&lt;sub&gt;4&lt;/sub&gt;&amp;bull;7H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Sigma</td><td>M2643</td><td></td></tr><tr><td>Na&lt;sub&gt;2&lt;/sub&gt;SO&lt;sub&gt;4&lt;/sub&gt;&amp;bull;10H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Sigma</td><td>246980</td><td></td></tr><tr><td>Bouin&amp;rsquo;s solution fixative</td><td>Sigma</td><td>HT10-1-32</td><td>Caution: Hazardous material (Special shipping cost required)</td></tr><tr><td>CoCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Sigma</td><td></td><td>Caution: Hazardous material. Please follow proper disposal according to local and federal regulations.</td></tr><tr><td>Methylene blue chloride</td><td>Matheson Co., Inc</td><td>Basic Blue 9, C.I. 52015</td><td></td></tr><tr><td>4-Di-2-ASP</td><td>Molecular Probes</td><td></td><td>4-(4-diethylaminostyryl)-N-methylpyridinium iodide</td></tr><tr><td>Bleach</td><td>Sigma-Aldrich</td><td></td><td>To chloride silver wire</td></tr><tr><td>NaOH</td><td>Sigma-Aldrich</td><td>221465</td><td>To adjust pH</td></tr><tr><td>HCl</td><td>Sigma-Aldrich</td><td>H1758</td><td>To adjust pH</td></tr><tr><td></td><td></td><td></td><td></td></tr><tr><td>Materials</td><td></td><td></td><td></td></tr><tr><td>Dissecting tools</td><td>World Precision Instruments</td><td>assortment</td><td></td></tr><tr><td>Intracellular electrode probe</td><td></td><td></td><td></td></tr><tr><td>Faraday cage</td><td></td><td></td><td></td></tr><tr><td>Insect Pins</td><td>Fine Science Tools, Inc</td><td>26001-60</td><td></td></tr><tr><td>Dissecting microscope (100X)</td><td></td><td></td><td></td></tr><tr><td>Fiber optic lamp</td><td></td><td></td><td></td></tr><tr><td>Small adjustable mirror</td><td></td><td></td><td>To direct light beam toward the preparation.</td></tr><tr><td>Glass electrodes</td><td>Sigma-Aldrich</td><td>CLS7095B5X</td><td>Box of 200, suction electrodes</td></tr><tr><td>Micromanipulator</td><td>World Precision Instruments</td><td>MD4-M3-R</td><td>Can fix for base or on a metal rod</td></tr><tr><td>Raised preparation stand</td><td></td><td></td><td></td></tr><tr><td>Silver wire (10/1,000 inch)</td><td>A-M Systems</td><td>782500</td><td></td></tr><tr><td>Computer</td><td>Any company</td><td></td><td></td></tr><tr><td>AC/DC differential amplifier</td><td>A-M Systems</td><td>Model 3000</td><td></td></tr><tr><td>PowerLab 26T</td><td>AD Instruments</td><td>27T</td><td></td></tr><tr><td>Force transducer</td><td>AD Instruments</td><td>0-50g</td><td>MLTF050/ST</td></tr><tr><td>Head stage</td><td>AD Instruments</td><td></td><td>Comes with AC/DC amplifier</td></tr><tr><td>LabChart7</td><td>AD Instruments</td><td></td><td></td></tr><tr><td>Electrical leads</td><td>Any company</td><td></td><td></td></tr><tr><td>Glass tools</td><td>Make yourself</td><td></td><td>For manipulating nerves</td></tr><tr><td>Cable and connectors</td><td>Any company</td><td></td><td></td></tr><tr><td>Pipettes with bulbs</td><td>Fisher Scientific</td><td>13-711-7</td><td>Box of 500</td></tr><tr><td>Beakers</td><td>Any company</td><td></td><td></td></tr><tr><td>Wax or modeling clay</td><td>Any company or local stores</td><td></td><td></td></tr><tr><td>Stimulator</td><td>Grass Instruments</td><td>SD9 or S88</td><td></td></tr><tr><td>Plastic tip for suction electrode</td><td>Local hardware store (Watt&amp;rsquo;s brand)</td><td>&amp;frac14; inch OD x 0.170 inch ID</td><td>Cut in small pieces. Pull out over a flame and cut back the tip to the correct size</td></tr></tbody></table>

proprioception and tension receptors in crab limbs: student laboratory exercises

<p class="jove_content">The primary purpose of these procedures is to demonstrate for teaching and research purposes how to record the activity of living primary sensory neurons responsible for proprioception as they are detecting joint position and movement, and muscle tension. Electrical activity from crustacean proprioceptors and tension receptors is recorded by basic neurophysiological instrumentation, and a transducer is used to simultaneously measure force that is generated by stimulating a motor nerve. In addition, we demonstrate how to stain the neurons for a quick assessment of their anatomical arrangement or for permanent fixation. Staining reveals anatomical organization that is representative of chordotonal organs in most crustaceans. Comparing the tension nerve responses to the proprioceptive responses is an effective teaching tool in determining how these sensory neurons are defined functionally and how the anatomy is correlated to the function. Three staining techniques are presented allowing researchers and instructors to choose a method that is ideal for their laboratory.</p>

<p class="jove_content">When the PD organ is stretched by fully extending the joint, activity in the PD nerve is robust during the movement as shown for the first second in <strong>Figure 10.</strong> Some activity remains while it is held still in the open position. This activity is from the static position sensitive neurons (second half of the recording shown in <strong>Figure 10</strong>). Movement evokes a response during displacement, and the firing is mostly present during the stretching of the chordotonal strand (<strong>Figure 11</strong>).</p>
<p class="jove_content">Further analysis of the spikes can be readily approached by sorting the relative amplitudes. This is an approach to demonstrate different populations of sensory neurons being recruited for positions or types of movements<sup>5</sup>.&#160;Typical amplitudes range from 0.25-1.5 mV, but these values are dependent on the resistance (<em>i.e.</em>&#160;tightness) of the suction electrode seal. Frequency of spikes in the various size ranges can also be graphically represented for analysis.</p>
<p class="jove_content">Forces generated by the opener muscle with respect to the stimulation frequency can be compared by superimposing the respective voltage-time traces on top of each other (<strong>Figures 13A </strong>and<strong>&#160;B</strong>). This can also be performed for each joint position at each given stimulation frequency. Activity of the tension nerve can then be correlated with the amount of relative force generated at each stimulation frequency, and for each joint position. As in the PD nerve, a variety of spike amplitudes are seen in response to contraction of the opener muscle (<strong>Figure 13C</strong>).</p>
<p class="jove_content">Anatomical arrangement of neurons in the walking leg is clearly observed with methylene blue staining (<strong>Figure 14</strong>). Note the elastic chordotonal strand and tension neuron that is close to the apodeme. Several somata with different diameters and specific locations are also visible in this figure. The entire course of the tension nerve and stretcher motor nerve are shown in <strong>Figure 15</strong>. Individual neurons of the PD nerve are shown with higher contrast using 4-Di-2-ASP (<strong>Figure 16</strong>) and CoCl<sub>2</sub> (<strong>Figure 17</strong>) backfill techniques. At high magnification the sensory endings can be seen inside of the supportive scolopales (<strong>Figures 16B</strong>)<sup>21,22,26</sup>.</p>
<p class="jove_content" fo:keep-together.within-page="always"><img alt="Figure 10" fo:content-width="5in" src="/files/ftp_upload/51050/51050fig10.jpg" width="500px" /><br />
<strong>Figure 10.</strong> <strong>Move and hold at 0&#176;</strong>. The dynamic neurons are robust in firing during the movement and spikes from static sensitive neurons are present while the joint is held open. <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/51050/51050fig10highres.jpg" target="_blank">Click here to view larger image</a>.</p>
<p class="jove_content" fo:keep-together.within-page="always"><img alt="Figure 11" fo:content-width="5in" src="/files/ftp_upload/51050/51050fig11.jpg" width="500px" /><br />
<strong>Figure 11.</strong> <strong>Rapid open and closing from fully flexed to extended (90-0&#176;) position</strong>. <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/51050/51050fig11highres.jpg" target="_blank">Click here to view larger image</a>.</p>
<p class="jove_content" fo:keep-together.within-page="always"><img alt="Figure 12" fo:content-width="5in" src="/files/ftp_upload/51050/51050fig12.jpg" width="500px" /><br />
<strong>Figure 12.</strong> <strong>Extracellular spikes recorded from the PD nerve</strong>. The joint is fully extended and then quickly moved to a &#189; flexed position and then held still. Notice the activity during the movement and decreased activity when static. <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/51050/51050fig12highres.jpg" target="_blank">Click here to view larger image</a>.</p>
<p class="jove_content" fo:keep-together.within-page="always"><img alt="Figure 13" fo:content-width="5in" src="/files/ftp_upload/51050/51050fig13.jpg" width="500px" /><br />
<strong>Figure 13. The relative forces that are developed with the joint fully flexed and stimulated at the various frequencies.</strong> (<strong>A</strong>) Voltage-time traces from the force transducer are shown with each stimulation frequency. (<strong>B</strong>) The traces in panel A are superimposed in different colors for ease in comparison. (<strong>C</strong>) Voltage-time traces of electrical activity recorded from the tension nerve when the motor nerve was stimulated at 80 Hz. Note the regular pattern of the stimulus artifacts as compared to the neural activity. Also, note the various amplitudes of the neural responses. <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/51050/51050fig13highres.jpg" target="_blank">Click here to view larger image</a>.</p>
<p class="jove_content" fo:keep-together.within-page="always"><img alt="Figure 14" fo:content-width="5in" src="/files/ftp_upload/51050/51050fig14.jpg" width="500px" /><br />
<strong>Figure 14.</strong> <strong>Methylene blue stain of the walking leg preparation</strong>. Individual somas are shown with their sensory endings projecting into the elastic strand. Close to the apodeme a tension neuron is shown. <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/51050/51050fig14highres.jpg" target="_blank">Click here to view larger image</a>.</p>
<p class="jove_content" fo:keep-together.within-page="always"><img alt="Figure 15" fo:content-width="5in" src="/files/ftp_upload/51050/51050fig15.jpg" width="500px" /><br />
<strong>Figure 15.</strong> <strong>The tension nerve arising from distal end (red arrows) and joining the motor nerve (green arrow).</strong> <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/51050/51050fig15highres.jpg" target="_blank">Click here to view larger image</a>.</p>
<p class="jove_content" fo:keep-together.within-page="always"><img alt="Figure 16" fo:content-width="5in" src="/files/ftp_upload/51050/51050fig16.jpg" width="500px" /><br />
<strong>Figure 16.</strong> (<strong>A</strong>)<strong> A back-fill of the PD nerve in&#160;<em>Cancer magister</em> with 4-Di-2-ASP. </strong>(<strong>B</strong>) Higher magnification of sensory endings.&#160;<a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/51050/51050fig16highres.jpg" target="_blank">Click here to view larger image</a>.</p>
<p class="jove_content" fo:keep-together.within-page="always"><img alt="Figure 17" fo:content-width="5in" src="/files/ftp_upload/51050/51050fig17.jpg" width="500px" /><br />
<strong>Figure 17.&#160;Neurons that were filled with CoCl<sub>2</sub> and processed </strong>(<strong>A</strong>). Traced outline of the stained preparation shown (<strong>B</strong>). <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/51050/51050fig17highres.jpg" target="_blank">Click here to view larger image</a>.</p>
<table border="0" cellpadding="0" cellspacing="0" width="392">
	<colgroup>
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:235px;"><strong>Saline</strong></td>
			<td style="width:157px;"><strong>g/L</strong></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:235px;">NaCl</td>
			<td style="width:157px;">27.29</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:235px;">KCl</td>
			<td style="width:157px;">0.81</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;width:235px;">MgSO<sub>4</sub>&#8226;7H<sub>2</sub>O</td>
			<td style="width:157px;">4.81</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;width:235px;">CaCl<sub>2</sub>&#8226;2H<sub>2</sub>O</td>
			<td style="width:157px;">1.85</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;width:235px;">Na<sub>2</sub>SO<sub>4</sub>&#8226;10H<sub>2</sub>O</td>
			<td style="width:157px;">0.97</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:235px;">Dextrose (D-Glucose)</td>
			<td style="width:157px;">1.982</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:235px;">HEPES acid</td>
			<td style="width:157px;">0.476</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:235px;">HEPES salt</td>
			<td style="width:157px;">2.08</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:235px;">Adjust to pH 8.1 with NaOH or HCl</td>
			<td style="width:157px;"></td>
		</tr>
	</tbody>
</table>
<p><strong>Table 1.</strong> <strong>Recipes for crab saline.</strong></p>

Watch this Scientific Journal Video about Proprioception and Tension Receptors in Crab Limbs: Student Laboratory Exercises at JoVE.com

Receptory propriocepcji i napięcia w kończynach kraba | Neuronauka | Wideo

Proprioception and Tension Receptors in Crab Limbs: Student Laboratory Exercises

<p class="jove_content">Physiological and anatomical techniques are demonstrated to address function and structure for joint proprioceptors and muscle tension receptors in crustacean walking limbs.</p>

The primary purpose of these procedures is to demonstrate for teaching and research purposes how to record the activity of living primary sensory neurons ...

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25.1K Views.  University of Kentucky. Physiological and anatomical techniques are demonstrated to address function and structure for joint proprioceptors and muscle tension receptors in crustacean walking limbs.

Video: Proprioception and Tension Receptors in Crab Limbs: Student Laboratory Exercises

Lab Techniques

<h4>Measurement Accuracy</h4>
<p>The ability to repeat an experiment and get the same results, or <em>reproducibility</em>, is essential in scientific research. However, it&#8217;s impossible to repeat an experiment if you don&#8217;t know how you did it. Thus, scientists keep detailed records of their experiments in lab notebooks. These records include important information like the amount of each material used and descriptions of each step of the procedure.</p>
<p>Equipment, like balances and volumetric or graduated glassware, is used to measure solid and liquid compounds for experiments. The precision of each measurement is limited by the equipment used. Balances and other equipment with digital readouts automatically display values at the maximum precision that they can support. The precision of graduated glassware and rulers depends on the increment, which is the amount represented by the smallest distance between two marks.</p>
<p>The surface of most liquids curves upwards at the edges in glass containers. This type of curved liquid surface is called a concave meniscus. When the edges of the liquid curve down, it is called a convex meniscus. When measuring liquid volumes, the reference point for the top of the liquid is the bottom of a concave meniscus or the top of a convex meniscus. To accurately identify the position of the meniscus, liquid volumes are read by looking at the liquid&#8217;s surface from the side at eye-level. Looking at the liquid from above or below will cause the liquid surface level to appear higher or lower than it actually is.</p>
<p>When the reference point of the meniscus touches a mark on graduated or volumetric glassware, the glassware is holding the volume defined for that mark. Volumetric glassware is designed to measure a specific volume, so it only has one mark. Graduated glassware is designed to measure a range of volumes, so it has many marks, or <em>graduations</em>, which are labeled at regular intervals. Both graduated and volumetric glassware may have additional manufacturer-provided information about their precision.</p>
<h4>Calibration and Uncertainty</h4>
<p>Manufacturers calibrate equipment to ensure that the measurements are accurate to within a specified range of uncertainty. For example, a high-precision analytical balance may read out to four decimal places (0.0000 g) with an uncertainty of &#177; 0.0001 g. A reading of 0.0345 g indicates that the true value is between 0.0344 g and 0.0346 g.</p>
<p>Glassware typically has its measurement uncertainty printed on it. To accurately record volume measurements, you must consider both the increment and the uncertainty. For example, a precise volume measurement in a 100 mL graduated cylinder with 1 mL increments and an uncertainty of &#177; 0.5 mL would be recorded to the tenths place (000.0 mL &#177; 0.5 mL). If the reference point of the meniscus falls between two marks, you should estimate a value for the tenths place; otherwise, leave it as 0. Since temperature affects volume, the temperature for which the glassware is calibrated will be printed on it.</p>
<p>Glassware is calibrated either <em>to contain</em> (TC) or <em>to deliver</em> (TD). TC glassware holds the specified volume of liquid when it is filled to the mark, but a small amount of liquid will be left behind when it is poured into another container. Solutions are often prepared in TC glassware because the accuracy of the solution volume affects the accuracy of the concentration. Volumetric flasks are typically calibrated to contain.</p>
<p>TD glassware holds a little more than the amount of liquid specified when it is filled to the mark, but it dispenses only the specified volume. Thus, TD glassware should not be emptied completely when dispensing liquids. This type of glassware is useful for transferring a precise volume of liquid to another container. Both graduated and volumetric pipettes are typically calibrated to deliver.</p>
<p>Unless otherwise specified, the uncertainty of a number is assumed to be on the scale of the last digit. Thus, it&#8217;s particularly important to report the uncertainty of a measurement if the uncertainty is on a larger scale. For rulers and graduated glassware without a known uncertainty, the uncertainty is estimated as half of the smallest increment. The uncertain digit is based on where the meniscus falls between two tick marks.</p>
<h4>Significant Figures</h4>
<p>Significant figures are the numbers in a value that are <em>meaningful</em>, or essential for expressing that value with the appropriate precision. All numbers other than leading zeroes (<strong>0.00</strong>1), trailing zeroes (1,<strong>000</strong>), or scientific notation multipliers (<strong>10</strong><sup>x</sup>) are always significant.</p>
<p>Leading zeroes are never significant because they can be removed by rewriting the number either in scientific notation or, if it is an SI unit, in smaller units. For example, there are only three significant figures in the value 0.00123 m because it can be rewritten as either 1.23 &#215; 10<sup>-3</sup> m or 1.23 mm without losing any information. Note that the scaling factor between different multiples of the same SI unit does <em>not</em> affect the number of significant figures.</p>
<p>Trailing zeroes are significant when they are before or after a decimal point. For example, the measurement 100.110 mL has six significant figures and 100.0 mL has four. Trailing zeroes in a number written without a decimal point are assumed to be insignificant unless they are marked otherwise. There are several different conventions for marking significant zeroes in those cases, but to avoid ambiguity it is advisable to rewrite those values in scientific notation. If the measurement is in SI units, use larger units.</p>
<p>The precision of a measurement limits the precision of any values calculated from it. A calculated value has the same number of significant figures as the <em>least</em> precise measurement or value used in its calculation. If the result has too many significant figures, it is rounded to the appropriate precision. If the result has too few significant figures, it is extended with significant trailing zeroes to preserve the precision. The significant figures of experimentally derived values, like molar mass, density, or some unit conversion factors, must also be considered when they are used in calculations.</p>
<p>The number of significant figures in a calculated value is <em>not</em> limited by physical or mathematical constants in formulas or exact numbers, like the number of data points acquired in an experiment. For example, the formula for calculating the volume of a sphere is V = 4/3&#960;r<sup>3</sup>. The number of significant figures in the calculated volume is <em>only</em> affected by the number of significant figures in the radius measurement <strong>r</strong>. 4/3&#8201; and &#960; are constants, and <sup>3</sup> is simply notation for a mathematical operation.</p>
<p>Defined conversion factors are also treated as constants. For instance, the inch is defined as exactly 25.4 millimeters; therefore, the number of significant figures of the converted value depends only on the original measurement. Always check whether the conversion factor is exactly defined or an experimental value when converting between units.</p>
<p>For simple addition and subtraction of measurements, we use significance arithmetic to determine the number of significant figures. In this case, the answer has as many decimal places as the measurement with the fewest decimal places <em>regardless of the number of significant figures</em>. Thus, 15,643.7 mL + 0.613 mL = 15,644.3 mL.</p>
<p>For multi-step calculations or complex equations that you break into smaller parts to solve, you should keep at least one or two insignificant figures on the intermediate values. For example, if you calculate the values of the numerator and denominator of a fraction separately before dividing them, you should retain the insignificant figures of the numerator and denominator when you divide them. This minimizes <em>rounding error</em>, which is the difference from the number you would get if you entered the entire formula into your calculator at once. If you write down these intermediate values, you should note which digits are insignificant.</p>
<h4>References</h4>
<p>Harris, D.C. (2015). <em>Quantitative Chemical Analysis</em>. New York, NY: W.H. Freeman and Company.</p>

Accuracy, Precision, Uncertainty, and Significant Figures - Concept

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The ability to repeat an experiment and get the same results, or reproducibility, is essential in scientific research. However, ...

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Chemistry

Extraction

<p><strong>Extraction</strong></p>
<p>Extraction is a common technique used in organic chemistry to isolate a target compound. In the extraction process, a solute is transferred from one phase to another to separate it from unreacted starting materials or impurities. Extraction is also used to facilitate the isolation of a solute from a reaction solvent that is difficult to remove by evaporation, such as a solvent with a high boiling point.<span style="color: rgb(0, 0, 0); font-family: Roboto, RobotoDraft, Helvetica, Arial, sans-serif; font-size: 13px; font-style: normal; font-variant-ligatures: normal; font-variant-caps: normal; font-weight: 400; letter-spacing: normal; orphans: 2; text-align: start; text-indent: 0px; text-transform: none; white-space: pre-wrap; widows: 2; word-spacing: 0px; -webkit-text-stroke-width: 0px; background-color: rgb(255, 255, 255); text-decoration-style: initial; text-decoration-color: initial; display: inline !important; float: none;"> </span>Generally, there are three types of extractions. First, in solid-liquid extraction, the solute is transferred from a solid phase to a liquid phase. In liquid-liquid extraction, a solute is transferred from one liquid to another. In acid-base extraction, a solute is transformed into an ionic compound and transferred from an organic phase to an aqueous phase. A common example of extraction is the brewing coffee or tea. The solid phase, which contains the caffeine, plant flavors, and odors, is extracted by the hot water into the liquid phase.</p>
<p><strong>Liquid-Liquid Extraction</strong></p>
<p>A liquid-liquid extraction either transfers an organic compound that is dissolved in an aqueous phase to an organic solvent, or it is used to transfer unreacted reactants, salts, and other water-soluble impurities to the aqueous phase while leaving the organic compound of interest in the organic phase. Immiscible liquids are liquids that never form a homogenous solution, even when thoroughly mixed. Instead, immiscible liquids separate into different phases, like oil and water.</p>
<p>A liquid-liquid extraction transfers an organic compound that is dissolved in an aqueous phase to an organic solvent. To perform a liquid-liquid extraction, first, the aqueous solution containing the solute is added to a separatory funnel. Then, a non-water-soluble organic solvent is added to the separatory funnel. When the contents of the funnel are mixed well, the organic compound partitions into the organic phase based on its higher solubility in the organic phase than the aqueous phase.</p>
<p>Since the two solvents are immiscible, the two liquids form discrete layers, with the dense liquid on the bottom and the less dense liquid on the top. Once the two phases settle back into two layers, they are separated by opening the stopcock at the bottom of the separatory funnel and allowing one layer to flow out. The liquid that had the solute removed is called the raffinate, while the liquid that gained the solute is called the extract.</p>
<p>The organic compound is partitioned between the two layers based on its solubility in each phase. Equilibrium is reached when the chemical potential of the solute is the same in the two phases. The partition coefficient for a solute, K, is the ratio of the concentration of the sample in the organic layer divided by the concentration in the aqueous phase. The partition coefficient is a constant dependent on both the solute and the pair of solvents used in the extraction. The partition coefficient is an expression of preference of the solute for each of the two solvents. Solutes with a large partition coefficient have a higher tendency to be extracted into the organic solvent layer. Solutes with small partition coefficients prefer to transition into the aqueous phase.</p>
<p>Determining which solvent pair to use for a liquid-liquid extraction is a vital step. Consider the following when choosing which solvents to use: First, the solute must be more soluble in the solvent than in water. Therefore, knowing the partition coefficient of the solute in a potential solvent pair is necessary. Second, the solvent-pair must be immiscible in water and not form a homogeneous solution when mixed. Third, the solvents must be inert and not react with the solute. The solvent should also be volatile so that it can be removed from the solute easily. The water-immiscible organic solvent generally possesses a non-polar or low polarity.</p>
<p>It is also critical to know the densities of the solvents to determine the identities of the top and bottom layers. Most organic liquids have a lower density than water, with the exception of chlorinated organic solvents, and will settle to the bottom of the separatory funnel.</p>
<p><strong>Acid-Base Extraction</strong></p>
<p>Acid-base extraction is a type of liquid-liquid extraction that separates organic compounds based on their acid-base properties. If a solute is an acid or base, its charge changes as the pH is changed. Generally, most organic compounds are neutral, and therefore more soluble in organic solvents than they are in water. However, if the organic compound becomes ionic, then it becomes more soluble in water. This is useful in extracting an organic acid or base compound from an organic phase to an aqueous phase.</p>
<p>Acid-base extraction harnesses this property by transforming the solute into its water-soluble salt form, thereby changing its solubility. The solubility of the organic compound and its salt must be dramatically different in order for the technique to be effective.</p>
<p>For example, consider a mixture containing an organic carboxylic acid, an amine, and a neutral compound. Carboxylic acids consisting of six carbons or more are insoluble in water and entirely soluble in organic solvents. However, their conjugate bases (an ionic compound) are water-soluble and insoluble in organic solvents. An amine consisting of at least seven carbons is insoluble in water but soluble in organic solvents. The conjugate acid of that amine (an ionic compound) is water-soluble and insoluble in organic solvents.</p>
<p>When reacted with a base, the carboxylic acid is neutralized to its salt form. The other compounds in the mixture remain neutral. Once the carboxylic acid is transformed into a salt, it will partition to the aqueous phase, while the neutral compounds remain in the organic phase.</p>
<p>Acid-base extraction is also used to separate two weak acids or two weak bases with a significant difference in their pK<sub>a</sub>. In the case of the acids, the relatively stronger acid, having a small pKa value, is neutralized to a salt using a weak base. The weak base does not efficiently react with the weaker acid, and only the stronger acid is transformed to a salt. Then, the salt is extracted into the aqueous phase during extraction. The process follows similarly for weak bases, where the relatively stronger base is neutralized to a salt using a weak acid.</p>
<p><strong>References</strong></p>
<ol>
	<li style="font-size:17px">Harris, D.C. (2015). <em>Quantitative Chemical Analysis.</em> New York, NY: W.H. Freeman and Company.</li>
</ol>

Extraction: Liquid-Liquid and Acid-Base Extraction - Concept

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Extraction
Extraction is a common technique used in organic chemistry to isolate a target compound. In the extraction process, a solute is transferred ...

Scientific Measurement and Lab Skills

<p>Source: Smaa Koraym at Johns Hopkins University, MD, USA</p>
<ol class="note-items">
	<li data-note-item="1" data-parent-collapse="">Preparation of the Laboratory<button class="expand">Expand</button>
	<p><em>Here, we show the laboratory preparation for 10 students working individually, with some excess. Please adjust quantities as needed.</em></p>
	<ul class="lab-items">
		<li data-sub-note-item="1-2">
		<div class="lab-step">To set up for this lab experiment, wear the appropriate personal protective equipment, including a lab coat, chemical splash goggles, and gloves.</div>
		</li>
		<li data-sub-note-item="1-3">
		<div class="lab-step">Prepare a 1.5 x 10<sup>-5</sup> M erythrosin B red dye solution. To prepare 500 mL of this solution, weigh 0.0066 g of erythrosin B on an analytical balance.</div>
		</li>
		<li data-sub-note-item="1-4">
		<div class="lab-step">Transfer the erythrosin B to a 250-mL Erlenmeyer flask. Then, measure 200 mL of deionized water and add it to the flask. Place a magnetic stir bar in the flask, set the flask on a stir plate, and mix the solution until it appears homogeneous.</div>
		</li>
		<li data-sub-note-item="1-5">
		<div class="lab-step">Use a funnel to pour the solution into a 500-mL volumetric flask, and carefully fill the flask to the line using deionized water. Seal the top of the flask with paraffin film and invert it several times until the solution is well-mixed.</div>
		</li>
		<li data-sub-note-item="1-6">
		<div class="lab-step">Label a 500-mL squeeze bottle with the concentration and transfer the solution into the bottle. Store the bottle on a mat in the hood.<strong> Note: </strong>Take significant figures into account.</div>
		</li>
		<li data-sub-note-item="1-7">
		<div class="lab-step">Place acetone in an area so that students have access to it during the lab. <strong>Note: </strong>Each student will need about 40 mL.</div>
		</li>
		<li data-sub-note-item="1-8">
		<div class="lab-step">Set weigh boats, scoops, and a container of sand next to a balance. <strong>Note: </strong>Each student will need about 3 g.</div>
		</li>
		<li data-sub-note-item="1-9">
		<div class="lab-step">Place an organic waste container in a central hood.</div>
		</li>
		<li data-sub-note-item="1-10">
		<div class="lab-step">Set out the following glassware and equipment at each student lab station (we suggest students work independently during this lab):
		<table align="center" style="border-collapse: collapse; font-size: 17px; line-height: 24px;">
			<tbody>
				<tr>
					<td style="border: 1px solid; padding: 0px; ">&#160;1&#160;&#160;&#160;&#160;50-mL beaker</td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px; ">&#160;1&#160;&#160;&#160;&#160;100-mL beaker</td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px; ">&#160;1&#160;&#160;&#160;&#160;250-mL beaker</td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px; ">&#160;1&#160;&#160;&#160;&#160;400-mL beaker</td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px; ">&#160;1&#160;&#160;&#160;&#160;600-mL beaker</td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px; ">&#160;1&#160;&#160;&#160;&#160;500-mL filter flask</td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px; ">&#160;1&#160;&#160;&#160;&#160;B&#252;chner funnel</td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px; ">&#160;1&#160;&#160;&#160;&#160;10-mL graduated cylinder</td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px; ">&#160;1&#160;&#160;&#160;&#160;Watch glass</td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px; ">&#160;1&#160;&#160;&#160;&#160;5-mL volumetric pipette</td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px; ">&#160;1&#160;&#160;&#160;&#160;10-mL volumetric pipette</td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px; ">&#160;1&#160;&#160;&#160;&#160;10-mL capacity pipetter</td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px; ">&#160;1&#160;&#160;&#160;&#160;10-mL volumetric flask</td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px; ">&#160;1&#160;&#160;&#160;&#160;String (18 in)</td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px; ">&#160;1&#160;&#160;&#160;&#160;Silicone vacuum tubing (18 in)</td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px; ">&#160;1&#160;&#160;&#160;&#160;12-in ruler</td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px; ">&#160;1&#160;&#160;&#160;&#160;Rubber adapter</td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px; ">&#160;1&#160;&#160;&#160;&#160;Rubber stopper</td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px; ">&#160;1&#160;&#160;&#160;&#160;Glass stirring rod</td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px; ">&#160;1&#160;&#160;&#160;&#160;Rubber policeman</td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px; ">&#160;1&#160;&#160;&#160;&#160;Disposable pipette</td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px; ">&#160;1&#160;&#160;&#160;&#160;Bottle of deionized water</td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px; ">&#160;1&#160;&#160;&#160;&#160;Lab mat</td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px; ">&#160;1&#160;&#160;&#160;&#160;Box of filter papers (1 filter paper per student)</td>
				</tr>
			</tbody>
		</table>
		</div>
		</li>
	</ul>
	</li>
</ol>

Two-Dimensional Graphs, Curve Fitting, and Standard Deviation - Prep

Pomiary naukowe i umiejętności laboratoryjne

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

	Preparation of the LaboratoryExpand
	Here, we show the laboratory preparation for 10 students ...

Lab Manual Sub Articles

Energy Dynamics

<ol class="note-items"><li data-note-item="1" data-parent-collapse="">Caterpillar Energy Dynamics
<button class="expand">Expand</button><ul class="lab-items"><li data-sub-note-item="1-1"><div class="lab-step">NOTE: In this lab you will grow caterpillars in conditions with and without food to examine the transfer of biomass in a typical trophic system.
</div></li><li data-sub-note-item="1-2"><div class="lab-step">Weigh and record the wet mass of enough leaves of cabbage to line the bottom of a plastic tub.
</div></li><li data-sub-note-item="1-3"><div class="lab-step">Then, collect two batches of five caterpillars from their cabbage habitat, and weigh and record the wet mass of each batch separately in individual weigh boats. Make sure to tare the balance before each measurement. HYPOTHESES: The experimental hypothesis is that only a fraction of energy will be transferred between the trophic levels of the cabbage and the caterpillars and a significant amount will be lost as waste and heat energy. The null hypothesis is that there will be no significant difference between the energy input from the cabbage, and the obtained energy stored as biomass in the caterpillars.
</div></li><li data-sub-note-item="1-4"><div class="lab-step">Place the weighed cabbage and five caterpillars in a plastic tub lined with damp paper towels, leaving the lid slightly cracked open for ventilation, but not wide enough for the animals to escape.
</div></li><li data-sub-note-item="1-5"><div class="lab-step">Place the other five caterpillars in a second clean container prepared the same way, but with no cabbage.
</div></li><li data-sub-note-item="1-6"><div class="lab-step">After three days, transfer the caterpillars to new weigh boats, and weigh and record the final wet mass of both groups of caterpillars and the cabbage.
</div></li><li data-sub-note-item="1-7"><div class="lab-step">Then place the caterpillar waste, or frass, and cabbage into two separate dishes, and put both dishes into a 37 &#176;C incubator to dry for 48 hours.
</div></li><li data-sub-note-item="1-8"><div class="lab-step">Euthanize the weighed caterpillars by putting them into a minus 20 &#176;C freezer for 24 hours.
</div></li><li data-sub-note-item="1-9"><div class="lab-step">Then, transfer the dishes to a 37 &#176;C incubator for 48 hours.
</div></li><li data-sub-note-item="1-10"><div class="lab-step">After drying, weigh and record the dry mass of the uneaten cabbage, the two groups of caterpillars, and the frass, and use these data to calculate the percent biomass of the caterpillars.
</div></li><li data-sub-note-item="1-11"><div class="lab-step">Finally, discard all of the dried organisms and plates into the hazardous waste bin, and wipe down your workspace with 70% ethanol.
</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">Take a look at the data you collected. Ensure the initial wet mass, the final wet mass, and the final dry mass columns are accurately filled out in your table. <a href="https://jove.unicartagenaproxy.elogim.com/CDNSource/lm/labs/13/Table_1_Energy_Dynamics.xlsx" style="display:block;margin:20px;" target="__blank">Click Here to download Table 1</a>
</div></li><li data-sub-note-item="2-2"><div class="lab-step">NOTE: Remember an object&#39;s biomass is equal to its dry mass. Therefore, percent biomass for each group is the final dry mass divided by the final wet mass times 100. For example, obtain the percent biomass of the cabbage by dividing the final dry mass by the final wet mass and multiplying by 100. This means that approximately 15% of the cabbage&#39;s total weight is biomass.
</div></li><li data-sub-note-item="2-3"><div class="lab-step">After this, estimate the initial biomass of the cabbage and caterpillars by multiplying the percent biomass by the initial wet mass, and put the values in this new table. NOTE: Remember, biomass equals dry mass. Therefore, the final dry mass is equal to the final biomass of each group.
</div></li><li data-sub-note-item="2-4"><div class="lab-step">To calculate the approximate initial and final energy in the form of kilocalories within the cabbage, caterpillars, and frass, multiply the biomass values recorded in your table by the conversions, and record the calculated values in the table. <a href="https://jove.unicartagenaproxy.elogim.com/CDNSource/lm/labs/13/Table_2_Energy_Dynamics.xlsx" style="display:block;margin:20px;" target="__blank">Click Here to download Table 2</a>
</div></li><li data-sub-note-item="2-5"><div class="lab-step">After this, subtract the initial energy from the final energy to obtain the change in energy for each group.
</div></li><li data-sub-note-item="2-6"><div class="lab-step">NOTE: The negative sign denotes loss in energy, and positive indicates gain.
</div></li><li data-sub-note-item="2-7"><div class="lab-step">Using your data table, calculate the energy in kilocalories that the fed caterpillars lost to respiration. The formula is the energy gained from cabbage, which is the positive of the energy lost by cabbage, minus the biomass gained from cabbage, minus the energy lost via frass. <a href="https://jove.unicartagenaproxy.elogim.com/CDNSource/lm/labs/13/Table_3_Energy_Dynamics.xlsx" style="display:block;margin:20px;" target="__blank">Click Here to download Table 3</a>
</div></li><li data-sub-note-item="2-8"><div class="lab-step">Then, repeat the same calculation for the unfed group of caterpillars. Make another column in the table and note this down.
</div></li><li data-sub-note-item="2-9"><div class="lab-step">Finally, add up all of the energy gained via biomass and cabbage consumption, and the energy lost via frass and respiration for each group of caterpillars. NOTE: For both groups of caterpillars the net energy is zero, meaning that this experiment obeyed the first law of thermodynamics.
</div></li></ul></li></ol>

Studying Energy Dynamics and Productivity - Procedure

Dynamika energii

Caterpillar Energy Dynamics
ExpandNOTE: In this lab you will grow caterpillars in conditions with and without food to examine the transfer of biomass in a ...

Ecology

Animal Behavior

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

Animal Behavior: Studying Taxis Behavior of Drosophila - Procedure

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 ...

Natural Selection

<ol class="note-items"><li data-note-item="1" data-parent-collapse="">Simulating Natural Selection
<button class="expand">Expand</button><ul class="lab-items"><li data-sub-note-item="1-1"><div class="lab-step">To prepare for the lab, set out workstations for each student pair containing 6 white, 6 black, and 13 gray 12 inch pipe cleaners.
</div></li><li data-sub-note-item="1-2"><div class="lab-step">Then, add 62 pipe cleaners in any color but black, white or gray, a roll of tape, and a pair of scissors.
</div></li><li data-sub-note-item="1-3"><div class="lab-step">Also, place one piece of black construction paper and one piece of white construction paper at each station.
</div></li><li data-sub-note-item="1-4"><div class="lab-step">Finally, print out tables so that there are enough for each set of partners in the class.
</div></li><li data-sub-note-item="1-5"><div class="lab-step">To save materials for subsequent labs, inform students to save the materials and to avoid damaging them as much as possible.
</div></li></ul></li></ol>

Natural Selection: Calculating Fitness and Selection - Prep

Selekcja naturalna

Simulating Natural Selection
ExpandTo prepare for the lab, set out workstations for each student pair containing 6 white, 6 black, and 13 gray 12 inch ...

Evolution

Scientific Method

<ol class="note-items"><li data-note-item="1" data-parent-collapse="">Demonstrating the Scientific Method with Tree Branches
<button class="expand">Expand</button><ul class="lab-items"><li data-sub-note-item="1-1"><div class="lab-step">Obtain branches containing a few dozen typical leaves from five different local tree species. The branches should represent a range of leaf and tree types. 
</div></li><li data-sub-note-item="1-2"><div class="lab-step">Label each branch and leaf with their species name. 
</div></li><li data-sub-note-item="1-3"><div class="lab-step">Distribute the branches to the students, so that each student has access to all five species. 
</div></li><li data-sub-note-item="1-4"><div class="lab-step">Provide some background information about the environment from which each branch was collected. 
</div></li></ul></li></ol>

Scientific Method: Steps and Applications - Prep

Metoda naukowa

Demonstrating the Scientific Method with Tree Branches
ExpandObtain branches containing a few dozen typical leaves from five different local tree species. ...

Measuring Biodiversity

<ol class="note-items"><li data-note-item="1" data-parent-collapse="">Quadrats and Transects Preparation
<button class="expand">Expand</button><ul class="lab-items"><li data-sub-note-item="1-1"><div class="lab-step">Start by tying a knot at the beginning, end and every 5 feet of six 20-foot pieces of rope.
</div></li><li data-sub-note-item="1-2"><div class="lab-step">Then, identify three outdoor areas near the classroom, each containing a variety of at least four plant species.  NOTE: Surprisingly, grassy areas often contain at least four plant species. Although most plants in these areas will be of a single grass species, there are usually many weeds, such as clovers or dandelions mixed in. Therefore, a well-kept grassy area without shade, a well-kept grassy area with shade, for example under a tree, and a weedy field could be used for the three different habitats that they students will be moving between.
</div></li><li data-sub-note-item="1-3"><div class="lab-step">Place two ropes parallel to each other within each of the habitats. It does not matter which direction the ropes face as long as the entire length of rope is within each area and a hula hoop can be placed on either side of the ropes within the habitat.
</div></li><li data-sub-note-item="1-4"><div class="lab-step">Finally, back in the lab set out enough plant identification guides for each student group to have at least one.
</div></li><li data-sub-note-item="1-5"><div class="lab-step">Also set out enough hula hoops so that each group has one to use as a sampling quadrat.
</div></li></ul></li><li data-note-item="2" data-parent-collapse="">Diversity Lab Simulation Preparation
<button class="expand">Expand</button><ul class="lab-items"><li data-sub-note-item="2-1"><div class="lab-step">Before beginning the species accumulation curves and richness evenness activity, place 50 colored beads into one paper bag. The bag should contain 15 black beads, 10 purple beads, 8 red beads, 5 green beads, 3 blue beads, 3 yellow beads, 2 pink beads, 2 brown beads, 1 orange bead and 1 white bead.
</div></li><li data-sub-note-item="2-2"><div class="lab-step">Label this bag Community A.
</div></li><li data-sub-note-item="2-3"><div class="lab-step">Then, add five beads of each color to the second bag and mark this bag Community B.
</div></li></ul></li></ol>

Pomiar różnorodności biologicznej

Quadrats and Transects Preparation
ExpandStart by tying a knot at the beginning, end and every 5 feet of six 20-foot pieces of rope.
Then, identify three ...

Animal Diversity

<ol class="note-items"><li data-note-item="1" data-parent-collapse="">Form and Function of Crickets and Crayfish
<button class="expand">Expand</button><ul class="lab-items"><li data-sub-note-item="1-1"><div class="lab-step">This lab involves dissection of both crickets and crayfish. You will need one cricket and one crayfish for each pair of students. IMPORTANT: Purchase the largest crickets available and place them in the freezer the night before.
</div></li><li data-sub-note-item="1-2"><div class="lab-step">On the day of the lab if they are frozen, allow the crickets to thaw for at least three hours before the dissections begin.
</div></li><li data-sub-note-item="1-3"><div class="lab-step">While waiting for the crickets to thaw, melt dissecting wax in a microwave or on a hotplate until it is completely liquefied.
</div></li><li data-sub-note-item="1-4"><div class="lab-step">Fill enough Petri dishes to have one per student pair to around halfway. Allow these plates to harden at room temperature.
</div></li><li data-sub-note-item="1-5"><div class="lab-step">Next set out a dissecting tray, 12 pins, scissors and a needle probe for each pair of students.
</div></li><li data-sub-note-item="1-6"><div class="lab-step">Also set up a few dissecting microscopes around the room and turn them on. NOTE: Generally, one microscope for four students is sufficient.
</div></li><li data-sub-note-item="1-7"><div class="lab-step">Then, set out a pitcher or beaker of water for students to use during the cricket dissection.
</div></li></ul></li></ol>

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

Różnorodność zwierząt

Form and Function of Crickets and Crayfish
ExpandThis lab involves dissection of both crickets and crayfish. You will need one cricket and one crayfish ...

Evolutionary Relationships

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

Evolutionary Relationships: Using BLAST to Test Evolutionary Hypotheses - Procedure

Związki ewolucyjne

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