<p class="jove_content">The funding source for the lab demonstration was NIGMS grant: GM077119. We thank Dr. Joseph R. Haywood and Dr. Peter Cobbett for their support of the Short Couse in Integrative and Organ Systems Pharmacology. We also thank Ms. Hannah Garver for her technical support of the laboratory demonstration.&#160;</p>

<p></p>
<p class="jove_content">Prior to any animal procedure, the institutional animal care and use committee (IACUC) must approve the protocol. This protocol was approved by the Michigan State University IACUC.</p>
<p class="jove_title">1. Pre-lab Preparation of FITC-inulin Solution</p>
<ol>
	<li>Warm 20 ml of saline to 70 &#176;C and slowly stir in 100 mg of FITC-inulin (5 mg/ml FITC-inulin) until all inulin is dissolved.</li>
	<li>Cool solution to RT&#160;and add 800 mg of bovine serum albumin (40 mg/ml BSA, lyophilized powder, essentially globulin free, low endotoxin, &#8805;98% purity by agarose gel electrophoresis).</li>
	<li>Filter the inulin-BSA solution with filter paper (grade 1). Place the filtered solution in a 20 ml syringe with a syringe-tip filter (0.2 &#181;m) and cover with foil to protect from light.</li>
</ol>
<p class="jove_title">2. Anesthesia and Surgery</p>
<ol>
	<li>Place the rat in an induction chamber filled with 5% isoflurane to induce anesthesia. Record body weight (250-350 g) and place the rat on a heated surgical platform designed to maintain 37 &#176;C body temperature throughout the experiment. Gently secure the rat to the platform with laboratory tape over the paws.&#160;Maintain anesthesia with 1-2% isoflurane with medical grade 100% O<sub>2</sub>&#160;at airflow rate of 0.8-1.0 L/min.</li>
	<li>Insert a tapered catheter (intravascular tip O.D., 2.7F)&#160;into the femoral artery for blood pressure and heart rate monitoring, and blood sampling.</li>
	<li>Insert a catheter (PE-50) into the femoral vein for inulin infusion. Secure the catheter to surrounding tissue with 5-O braided silk surgical suture<sup>6</sup>.</li>
	<li>Attach the arterial catheter to a strain gauge pressure transducer. Record blood pressure and heart rate using data acquisition software and display on a computer screen in real-time. This technique is demonstrated in detail on video<sup>6</sup>.</li>
	<li>Expose the bladder via a suprapubic incision. Cut a small hole in the tip of the bladder and insert a cannula (PE-190) with a heat flared tip inside the bladder for urine collection. Secure the cannula to the bladder with a purse-string suture.</li>
</ol>
<p class="jove_title">3. Urine and Blood Collection</p>
<ol>
	<li>Place the syringe of FITC-inulin in a syringe pump with flow rate set of 1 ml/hr per 100 g of body weight (3 ml/hr for a rat weighing 300 g). Attach the syringe to the femoral vein catheter. Start the inulin infusion and allow a 1-2 hr equilibration period. Keep syringe covered with foil to protect from light.</li>
	<li>Determine if urine flow rate is stable and adequate for sample analysis (20 &#181;l/min) by collecting a urine sample in a pre-weighed collection vial for a period of 10 min. Determine urine volume gravimetrically with a digital scale. An adequate urine volume for a 10 min collection period is 0.2 ml. Continue to collect urine samples until two consecutive collections indicate a urine flow rate of 20 &#181;l/min or more.</li>
	<li>Pre-Drug Samples
	<ol>
		<li>Collect a urine sample during a 20 min period. Collect a blood sample (0.5 ml) from the arterial catheter at the midpoint of the urine collection period. Be careful to completely clear the arterial catheter of saline before collecting a blood sample in a collection vial containing 1 U heparin. Use collection vials with volume markings to facilitate the collection of 0.5 ml&#160;of arterial blood.</li>
		<li>Flush the arterial catheter with heparin-saline (20 U/ml) to clear the catheter of blood (approx. 0.1 ml). The length of the arterial catheter should be as short as possible to limit the volume of heparin-saline required to flush.<br />
		Note: Diluted blood samples produce inaccurate calculations of GFR and fractional excretion of Na and K.</li>
		<li>Wait 10 min, and repeat the collection of a second Pre-drug urine and blood sample.</li>
	</ol>
	</li>
	<li>Following the collection of two Pre-drug samples, administer a diuretic drug, furosemide (10 mg/kg), via the arterial catheter. Flush the arterial catheter with heparinized saline to clear the catheter of drug. Take care to prevent the injection of air through the arterial catheter. Record the time of the furosemide injection.</li>
	<li>Post-drug Samples: At each of the 3 time points below, collect a urine sample during a 10 min collection period, and a blood sample (0.5 ml) at the midpoint of the urine collection period.
	<ol>
		<li>For Post-Drug Sample 1 &#8211; collect five min after furosemide.</li>
		<li>For Post-Drug Sample 2 &#8211; collect ten min after furosemide.</li>
		<li>For Post-Drug Sample 3 &#8211; collect fifteen min after furosemide.</li>
	</ol>
	</li>
	<li>After all samples have been collected, euthanize the rat in accordance with institutional procedures by thoracotomy and removal of the heart. Remove both kidneys. Decapsulate (remove the surrounding membrane) and blot the kidneys to remove excess blood. Weigh the kidneys.</li>
</ol>
<p class="jove_title">4. Sample Analysis</p>
<ol>
	<li>Measure all urine sample volumes gravimetrically with a digital scale, and record weights.</li>
	<li>Centrifuge whole blood samples with a table-top centrifuge (1,800 x g) to separate plasma. Transfer plasma samples to small labeled vials.</li>
	<li>Analyze Na and K concentrations in urine and plasma samples with a sodium/potassium analyzer.</li>
	<li>Measurement of FITC-inulin in plasma and urine
	<ol>
		<li>Dilute pre-drug urine (from 1:200 to 1:400), and post-drug urine (1:10) with HEPES buffer (500 mM, pH 7.4).</li>
		<li>Add 40 &#181;l of standard or sample and 60 &#181;l of HEPES buffer in a 96 well plate (one sample per well) and allow to mix for 10 min while covered with aluminum foil.</li>
		<li>Generate a standard curve for FITC-inulin for concentrations of 6.25, 12.5, 25, 50, 100, 200, 400 &#181;g/ml&#160;(<strong>Figure 1</strong>). Determine FITC-inulin fluorescence in samples and standards using a microplate reader with excitation and emission wavelengths of 485 and 538 nm, respectively.</li>
		<li>Fit the fluorescent values for the standards to a 4-paramter logistic function regression analysis. The regression function parameters are used to calculate FITC-inulin concentration in plasma and urine samples (<strong>Table 1</strong>).</li>
	</ol>
	</li>
</ol>
<p class="jove_title">5. Post-lab Analysis of Results: Calculations</p>
<ol>
	<li>Calculate Urine Flow Rate (UV; ml/min): [volume of urine collected (ml)] &#247; [time of collection (min)]</li>
	<li>Calculate Glomerular Filtration Rate (GFR; ml/min): [Urine inulin concentration (&#181;g/ml) x UV (ml/min)] &#247; [Plasma inulin conc. (&#181;g/ml)]</li>
	<li>Calculate Filtered Sodium Load (&#181;mol/min): Plasma sodium concentration (&#181;mol/ml) x GFR (ml/min)</li>
	<li>Calculate Sodium Excretion Rate (U<sub>Na</sub>V; &#181;mol/min): Urine sodium concentration (&#181;mol/ml) x UV (ml/min)</li>
	<li>Calculate Fractional Excretion of Sodium (FE Na; %): [U<sub>Na</sub>V (&#181;mol/min)] &#247; [Filtered Sodium Load (&#181;mol/min)] x 100</li>
	<li>Calculate Filtered Potassium Load (&#181;mol/min): Plasma potassium concentration (&#181;mol/ml) x GFR (ml/min)</li>
	<li>Calculate Potassium Excretion Rate (U<sub>K</sub>V; &#181;mol/min): Urine potassium concentration (&#181;mol/ml) x UV (ml/min)</li>
	<li>Calculate Fractional Excretion of Potassium (FE K; %): [U<sub>K</sub>V (&#181;mol/min)] &#247; [Filtered Potassium Load (&#181;mol/min)] x 100</li>
</ol>

<ol>
	<li>Silverthorn, D. U. <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'>Human Physiology: An integrated approach</em>. , (2012).</li><li>Hall, J. E. <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'>Guyton and Hall Textbook of Medical Physiology</em>. , 303-344 (2011).</li><li>Levey, A. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measurement+of+renal+function+in+chronic+renal+disease.">Measurement of renal function in chronic renal disease.</a> <em style='font-style: italic'>Kidney International</em>. <b>38</b> (1), 167-184 (1990).</li><li>Thurau, K., Valtin, H., Schnermann, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Kidney.">Kidney.</a> <em style='font-style: italic'>Annual Review of Physiology</em>. <b>30</b>, 441-524 (1968).</li><li>Shannon, J. A., Smith, H. 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+excretion+of+inulin,+xylose,+and+urea+by+normal+and+phoriziniaed+man.">The excretion of inulin, xylose, and urea by normal and phoriziniaed man.</a> <em style='font-style: italic'>Journal of Clinical Investigation</em>. <b>14</b>, 393-401 (1935).</li><li>Jespersen, B., Knupp, L., Northcott, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Femoral+arterial+and+venous+catheterization+for+blood+sampling,+drug+administration+and+conscious+blood+pressure+and+heart+rate+measurements.">Femoral arterial and venous catheterization for blood sampling, drug administration and conscious blood pressure and heart rate measurements.</a> <em style='font-style: italic'>Journal of Visualized Experiments</em>.  (59), (2012).</li><li>Sterner, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determining+'true'+glomerular+filtration+rate+in+healthy+adults+using+infusion+of+inulin+and+comparing+it+with+values+obtained+using+other+clearance+techniques+or+predictive+equations.">Determining 'true' glomerular filtration rate in healthy adults using infusion of inulin and comparing it with values obtained using other clearance techniques or predictive equations.</a> <em style='font-style: italic'>Scandinavian Journal of Urology and Nephrology</em>. <b>42</b>, 278-285 (2008).</li><li>Toto, R. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conventional+measurement+of+renal+function+utilizing+serum+creatinine,+creatinine+clearance,+inulin+and+para-aminohippuric+acid+clearance.">Conventional measurement of renal function utilizing serum creatinine, creatinine clearance, inulin and para-aminohippuric acid clearance.</a> <em style='font-style: italic'>Current Opinion in Nephrology and Hypertension</em>. <b>4</b> (6), 505-509 (1995).</li><li>Matavelli, L. C., Kadowitz, P. J., Navar, L. G., Majid, D. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Renal+hemodynamic+and+excretory+responses+to+intra-arterial+infusion+of+peroxynitrite+in+anesthetized+rats.">Renal hemodynamic and excretory responses to intra-arterial infusion of peroxynitrite in anesthetized rats.</a> <em style='font-style: italic'>Americam Journal of Physiology</em>. <b>296</b>, F170-F176 (2009).</li><li>Davidson, W. D., Sackner, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simplification+of+the+anthrone+method+for+the+determination+of+inulin+in+clearance+studies.">Simplification of the anthrone method for the determination of inulin in clearance studies.</a> <em style='font-style: italic'>Journal of Laboratory, &amp; Clinical Medicine</em>. <b>62</b>, 351-356 (1963).</li><li>Symes, A. L., Gault, M. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assay+of+inulin+in+tissues+using+anthrone.">Assay of inulin in tissues using anthrone.</a> <em style='font-style: italic'>Clinical Biochemistry</em>. <b>8</b> (1), 67-70 (1975).</li><li>Shalmi, M., Lunau, H. E., Petersen, J. S., Bak, M., Christensen, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Suitability+of+tritiated+inulin+for+determination+of+glomerular+filtration+rate.">Suitability of tritiated inulin for determination of glomerular filtration rate.</a> <em style='font-style: italic'>Americam Journal of Physiology</em>. <b>260</b> (2 Pt 2), F283-F289 (1991).</li><li>Denton, K. M., Anderson, W. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glomerular+untrafiltration+in+rabbits+with+superficial+glomeruli.">Glomerular untrafiltration in rabbits with superficial glomeruli.</a> <em style='font-style: italic'>EUropean Journal of Physiology</em>. <b>419</b> (3-4), 235-242 (1991).</li><li>Jobin, J., Bonjour, J. -. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measurement+of+glomerular+filtration+rate+in+conscious+unrestrained+rats+with+inulin+infused+by+implanted+osmotic+pumps.">Measurement of glomerular filtration rate in conscious unrestrained rats with inulin infused by implanted osmotic pumps.</a> <em style='font-style: italic'>Americam Journal of Physiology</em>. <b>248</b> (5 Pt 2), F734-F738 (1985).</li><li>Lorenz, J. N., Gruenstein, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple,+nonradioactive+method+for+evaluating+single-nephron+filtration+rate+using+FITC-inulin.">A simple, nonradioactive method for evaluating single-nephron filtration rate using FITC-inulin.</a> <em style='font-style: italic'>Americam Journal of Physiology</em>. <b>276</b> (1 Pt 2), F172-F177 (1999).</li><li>Qi, Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Serial+determination+of+glomerular+filtration+rate+in+conscious+mice+using+FITC-inulin+clearance.">Serial determination of glomerular filtration rate in conscious mice using FITC-inulin clearance.</a> <em style='font-style: italic'>Americam Journal of Physiology</em>. <b>286</b> (3), F590-F596 (2004).</li><li>Bivona, B. J., Park, S., Harrison-Bernard, L. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glomerular+filtration+rate+determinations+in+conscious+type+II+diabetic+mice.">Glomerular filtration rate determinations in conscious type II diabetic mice.</a> <em style='font-style: italic'>Americam Journal of Physiology</em>. <b>300</b> (3), F618-F625 (2011).</li><li>Rosen, S. M. <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+anaesthesia+and+surgery+on+renal+hemodynamics.">Effects of anaesthesia and surgery on renal hemodynamics.</a> <em style='font-style: italic'>British Journal of Anesthesiology</em>. <b>44</b>, 252-258 (1972).</li><li>Cousins, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anesthesia+and+the+kidney.">Anesthesia and the kidney.</a> <em style='font-style: italic'>Anaesthesia and intensive care</em>. <b>11</b> (4), 292-320 (1983).</li><li>Walter, S. J., Zewde, T., Shirley, D. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effect+of+anaesthesia+and+standard+clearance+procedures+on+renal+function+in+the+rat.">The effect of anaesthesia and standard clearance procedures on renal function in the rat.</a> <em style='font-style: italic'>Quarterly Journal of Experimental Physiology</em>. <b>74</b>, 805-812 (1989).</li><li>Rieg, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+high-throughput+method+for+measurement+of+glomerular+filtration+rate+in+conscious+mice.">A high-throughput method for measurement of glomerular filtration rate in conscious mice.</a> <em style='font-style: italic'>Journal of Visualized Experiments</em>.  (75), (2013).</li></ol>

<p class="jove_content">The authors declare that they have no competing financial interests. The opinions or assertions contained herein are the private views of the author and are not to be construed as official or as reflecting the views of the Department of the Army or the Department of Defense.</p>

<p class="jove_content">An appropriate marker for GFR measurement must meet four criteria: be freely filtered at the glomerulus, be unbound to plasma proteins, and neither be absorbed nor secreted in the nephron. Inulin is a fructose polymer which satisfies these criteria. As a result, the renal clearance of inulin is considered the gold standard for measuring GFR<sup>7</sup>. The demonstrated technique represents the traditional approach of determining the renal clearance of inulin using timed urine collections during a constant infusion of inulin<sup>8,9</sup>. Traditional inulin measurements have been made using the anthrone method to produce a quantitative colorimetric determination of inulin measured by spectrophotometer<sup>10,11</sup>. However, in an attempt to facilitate the measurement of inulin in smaller volumes of urine and plasma, inulin has been tagged with radioactive<sup>12-14</sup>, and fluorescent labels<sup>15-17</sup>. The lab demonstration presented in this video used FITC labelled inulin for the measurement of renal function because of the lack of risk of human radiation exposure and the ease of measuring FITC fluorescence<sup>15</sup>.</p>
<p class="jove_content">This lab demonstration is intended to provide a conceptual understanding of how to measure renal function to students with minimal laboratory skills. Therefore, the pre-lab preparation of FITC-inulin solution, and surgical preparation of the animals are performed by experienced technicians prior to the start of the demonstration. The students arrive for the demonstration at the end of the 1-2 hr inulin equilibration period. At this time, the students are presented with a Pre-lab overview and informed of the procedures that have been conducted on the animals. Two students are assigned to one animal experiment, and instructed on how to collect blood and urine samples before and after the administration of the diuretic drug. The analysis of blood and urine samples is conducted by experienced technicians and results are delivered to the students for calculations of renal function. Results are presented during a Post-lab discussion which can be scheduled after the demonstration.</p>
<p class="jove_content">There are several critical steps within the protocol to insure valid responses. Firstly, FITC inulin must be completely dissolved and filtered prior to animal administration. Ideally, FITC inulin should be dialyzed in water for 48 hr at RT&#160;to remove residual unbound FITC. Secondly, plasma samples must be free of saline. Students are instructed to collect a blood sample only after all of the saline in the arterial catheter has been expelled and only blood is flowing out of the catheter. Blood samples that are diluted with saline will provide inaccurate values for plasma inulin, sodium and potassium. Thirdly, urine flow must be steady and adequate to produce enough sample for analysis. A steady urine flow rate at baseline is critical because it is an indication of a stable experimental preparation. If urine flow is too low, the infusion rate of inulin can be increased prior to sample collections. However, the infusion of inulin must be constant during the course of the experiment, <em>i.e.</em>, inulin infusion rate should not be adjusted during the experiment. Finally, the measurement of inulin fluorescence in plasma and urine samples by microplate reader is critical to a successful experiment. Since the specifications of the microplate reader will determine if samples require dilutions, it is recommended that a test run of the inulin assay be conducted prior to the lab demonstration in an effort to optimize the specifications of the microplate reader and ensure that sample fluorescence values are within the mid-range of the standard curve.</p>
<p class="jove_content">While assessing renal function based on the renal clearance of inulin is considered the gold standard, this technique has limitations because the animals must be anesthetized, and instrumented with vascular and bladder catheters. Anesthethetic agents have been shown to affect renal hemodynamics and GFR<sup>18,19</sup>; however isoflurane and inactin are typically used in renal function experiments due to their minimal effects on the kidney<sup>19,20</sup>. The inulin clearance technique also requires a constant infusion of inulin and multiple blood and plasma samples which can be prohibitive in smaller animals such as mice. Modifications of this technique have been developed to allow the measurement of plasma clearance from a single injection of inulin in conscious animals<sup>21</sup>. These modifications also require smaller volumes of blood samples for analysis, and provide an alternate method for assessing renal function in mice.</p>
<p class="jove_content">The measurement of renal function is applicable to studies of physiology, pathology, toxicology, pharmacology and disease states. Students who participate in the Renal Function demonstration will learn the gold standard technique of renal clearance of inulin to assess renal function. By mastering this technique, students will understand the principles of renal function and allow them to apply the technique to their own research and determine if modifications to the technique are appropriate for their studies.</p>

<p class='jove_content'>The most important function of the kidney is the homeostatic regulation of extracellular water and electrolyte content. The kidneys closely regulate extracellular water, sodium (Na) and potassium (K) to maintain normal physiological levels. Disturbances in renal function can result in serious metabolic disorders which can be fatal. The basic renal process occurs in the nephron and begins with the filtration of plasma at the glomerulus and ends with the excretion of urine. Other processes that determine the final concentration of water, Na and K in the urine are secretion and reabsorption within the nephron. Measurements of glomerular filtration rate (GFR) and the fractional excretion of Na and K are critical in assessing renal function in health and disease. The reader is referred to previously published review articles and textbooks for a more thorough discussion of kidney function<sup>1-4</sup>. </p>
<p class='jove_content'>GFR can be measured as the steady state renal clearance of inulin which is filtered at the glomerulus, but not secreted or reabsorbed along the nephron<sup>5</sup>. While this technique requires anesthesia, surgical preparation, and a terminal experiment, it is considered the gold standard of GFR measurement. Using inulin that is tagged with fluorescein-isothiocyanate (FITC), plasma and urine concentration of FITC-inulin can be easily measured in small volumes and used to calculate GFR during multiple time points of an experiment. The fractional excretion of Na and K can be determined from the concentration of Na and K in plasma and urine. </p>
<p class='jove_content'>The conceptual understanding of how to measure renal function can easily be demonstrated in a short lab designed to allow students to actively participate in some aspects of the experiment. This video depicts the pre-lab preparation, the renal function demonstration, and the post-lab evaluation of results. The surgical techniques necessary for making measurements of GFR are demonstrated in an anesthetized rat. In addition, example calculations for GFR, and the fractional excretion of Na and K are shown before and after administration of a diuretic drug.</p>

<table><tbody><tr><td>5-0 Braided Silk Surgical Suture</td><td>Surgical Specialties Corp</td><td>SP1033</td><td></td></tr><tr><td>Assay Plate, 96-Well</td><td>Costar&amp;nbsp;</td><td>3922</td><td></td></tr><tr><td>Bovine Serum Albumin</td><td>Sigma Chemical Co</td><td>A2934-25G</td><td></td></tr><tr><td>Centrifuge</td><td>Beckman Coulter</td><td>MicroFuge 18, 357160</td><td></td></tr><tr><td>Conical Sample Tubes</td><td>Dot Scientific Inc.&amp;nbsp;</td><td>#711-FTG</td><td></td></tr><tr><td>Cotton Tipped Applicators</td><td>Solon Manufacturing Co</td><td>56200</td><td></td></tr><tr><td>Data Acquisition Software</td><td>ADInstruments</td><td>LabChart Pro 7.0</td><td></td></tr><tr><td>Digital Scale&amp;nbsp;</td><td>Denver Instrument</td><td>APX-4001</td><td></td></tr><tr><td>FITC-Inulin</td><td>Sigma Chemical Co</td><td>F3272-1G</td><td></td></tr><tr><td>Gauze Sponges</td><td>Covidien</td><td>2146</td><td></td></tr><tr><td>Heated Surgical Bed</td><td>EZ-Anesthesia</td><td>EZ-212</td><td></td></tr><tr><td>Heparin</td><td>Sagnet</td><td>NDC 25021-402-10</td><td></td></tr><tr><td>HEPES</td><td>Sigma Chemical Co</td><td>H3375</td><td></td></tr><tr><td>Isoflurane</td><td>Abbott Animal Health</td><td>IsoFlo, 5260-04-05</td><td></td></tr><tr><td>Isoflurane Vaporizer</td><td>EZ-Anesthesia</td><td>EZ-190F</td><td></td></tr><tr><td>Micro Dissecting Forceps</td><td>Biomedical Research Instruments Inc.</td><td>70-1020</td><td></td></tr><tr><td>Microplate Reader - Fluoroskan</td><td>ThermoScientific</td><td>Ascent FL, 5210460</td><td></td></tr><tr><td>NOVA 5+ Sodium/Potassium Analyzer</td><td>NOVA BioMedical</td><td>14156</td><td></td></tr><tr><td>Olsen-Hegar Needle Holders with Scissors</td><td>Fine Science Tools</td><td>12002-12</td><td></td></tr><tr><td>PE-190 (for bladder catheter)</td><td>BD Medical</td><td>427435</td><td></td></tr><tr><td>Pressure Transducer&amp;nbsp;</td><td>ADInstruments</td><td>MLT1199</td><td></td></tr><tr><td>Pyrex Culture Tubes</td><td>Corning Inc.</td><td>99445-12</td><td></td></tr><tr><td>Rat Femoral Tapered Artery Catheter</td><td>Strategic Applications Inc.</td><td>RFA-01</td><td></td></tr><tr><td>Salix Furosemide 5%</td><td>Intervet</td><td>#34-478</td><td></td></tr><tr><td>Strabismus Scissors</td><td>Fine Science Tools</td><td>14075-11</td><td></td></tr><tr><td>Student Surgical Scissors</td><td>Fine Science Tools</td><td>91402-12</td><td></td></tr><tr><td>Surgical Gloves</td><td>Kimberly-Clark</td><td>Sterling Nitrile Gloves</td><td></td></tr><tr><td>Syringe pump</td><td>Razel Scientific</td><td>R99-E</td><td></td></tr><tr><td>Tissue Forceps</td><td>Fine Science Tools</td><td>91121-12</td><td></td></tr><tr><td>Tissue Scissors</td><td>George Tiemann&amp;nbsp; Co</td><td>105-420</td><td></td></tr><tr><td></td><td></td><td></td><td></td></tr><tr><td colspan="4">&lt;p&gt;5-0 Braided Silk Surgical Suture Surgical Specialties Corp SP1033 Assay Plate, 96-Well Costar&amp;nbsp; 3922 Bovine Serum Albumin Sigma Chemical Co A2934-25G Centrifuge Beckman Coulter MicroFuge 18, 357160 Conical Sample Tubes Dot Scientific Inc.&amp;nbsp; #711-FTG Cotton Tipped Applicators Solon Manufacturing Co 56200 Data Acquisition Software ADInstruments LabChart Pro 7.0 Digital Scale&amp;nbsp; Denver Instrument APX-4001 FITC-Inulin Sigma Chemical Co F3272-1G Gauze Sponges Covidien 2146 Heated Surgical Bed EZ-Anesthesia EZ-212 Heparin Sagnet NDC 25021-402-10 HEPES Sigma Chemical Co H3375 Isoflurane Abbott Animal Health IsoFlo, 5260-04-05 Isoflurane Vaporizer EZ-Anesthesia EZ-190F Micro Dissecting Forceps Biomedical Research Instruments Inc. 70-1020 Microplate Reader - Fluoroskan ThermoScientific Ascent FL, 5210460 NOVA 5+ Sodium/Potassium Analyzer NOVA BioMedical 14156 Olsen-Hegar Needle Holders with Scissors Fine Science Tools 12002-12 PE-190 (for bladder catheter) BD Medical 427435 Pressure Transducer&amp;nbsp; ADInstruments MLT1199 Pyrex Culture Tubes Corning Inc. 99445-12 Rat Femoral Tapered Artery Catheter Strategic Applications Inc. RFA-01 Salix Furosemide 5% Intervet #34-478 Strabismus Scissors Fine Science Tools 14075-11 Student Surgical Scissors Fine Science Tools 91402-12 Surgical Gloves Kimberly-Clark Sterling Nitrile Gloves Syringe pump Razel Scientific R99-E Tissue Forceps Fine Science Tools 91121-12 Tissue Scissors George Tiemann&amp;nbsp; Co 105-420&lt;/p&gt;</td></tr></tbody></table>

physiology lab demonstration: glomerular filtration rate in a rat

<p class='jove_content'>Measurements of glomerular filtration rate (GFR), and the fractional excretion of sodium (Na) and potassium (K) are critical in assessing renal function in health and disease. GFR is measured as the steady state renal clearance of inulin which is filtered at the glomerulus, but not secreted or reabsorbed along the nephron. The fractional excretion of Na and K can be determined from the concentration of Na and K in plasma and urine. The renal clearance of inulin can be demonstrated in an anesthetized animal which has catheters in the femoral artery, femoral vein and bladder. The equipment and supplies used for this procedure are those commonly available in a research core facility, and thus makes this procedure a practical means for measuring renal function. The purpose of this video is to demonstrate the procedures required to perform a lab demonstration in which renal function is assessed before and after a diuretic drug. The presented technique can be utilized to assess renal function in rat models of renal disease. </p>

<p class="jove_content">The diuretic used in the lab demonstration was furosemide which very quickly inhibits the reabsorption of Na and K filtered by the kidney resulting in increased Na, K, and water excretion within minutes of drug administration. By its primary mechanism, furosemide should have minimal effects on GFR and the filtered load of Na and K, but will increase urine flow, and fractional excretion of Na and K.</p>
<p class="jove_content">The representative results in <strong>Table 3</strong> show that in an anesthetized rat, the average of the pre-drug values for GFR was 3.2 ml/min, Na excretion was 0.58 &#181;mol/min (0.1% of the filtered load), and K excretion was 4.4 &#181;mol/min (27% of the filtered load). Five minutes after furosemide (post-drug 1), GFR and the filtered load of Na and K were unaffected. However, the fractional excretion of Na increased to 11.5%, and the fractional excretion of K increased to 63% of the respective filtered loads. The measurements of MAP and HR indicate that furosemide had minimal effects on MAP and HR (<strong>Table 2</strong>).</p>
<p class="jove_content">The indices of renal function assessed in the laboratory demonstration were the GFR, defined as the rate by which plasma is filtered by the kidney; the filtered Na and K, defined as the rate by which Na and K are filtered by the kidney; the Na and K Excretion Rate, defined as the rate by which Na and K are excreted by the kidney; and the fractional Excretion of Na and K, defined as the percentage of filtered Na and K that is excreted by the kidney</p>
<p class="jove_content"><img alt="Figure 1" src="/files/ftp_upload/52425/52425fig1.jpg" /><br />
<strong>Figure 1: Inulin Standard Curve. </strong>FITC fluorescence values are shown for standards containing 6.25, 12.5, 25, 50, 100, 200 and 400 &#181;g/ml inulin. A 4-paramter logistic function regression analysis generates the best-fit curve. The regression function parameters from this curve were used to calculate FITC-inulin concentration in plasma and urine samples.</p>
<p class="jove_content"></p>
<p class="jove_content"></p>
<table border="0" cellpadding="0" cellspacing="0" height="515" width="592">
	<tbody>
		<tr>
			<td></td>
			<td colspan="3">FITC-Inulin fluorescence</td>
			<td nowrap="nowrap">Concentration</td>
			<td></td>
			<td>Result</td>
		</tr>
		<tr>
			<td>Standard</td>
			<td>replicate 1</td>
			<td>replicate 2</td>
			<td>Mean</td>
			<td>&#956;g/ml</td>
			<td>Dilution</td>
			<td>&#956;g/ml</td>
		</tr>
		<tr>
			<td>Blank</td>
			<td align="right">63.9</td>
			<td align="right">64.8</td>
			<td align="right">64.4</td>
			<td align="right">0.4</td>
			<td align="right">1</td>
			<td align="right">0.4</td>
		</tr>
		<tr>
			<td>6.25</td>
			<td align="right">253.2</td>
			<td align="right">264.1</td>
			<td align="right">258.7</td>
			<td align="right">5.9</td>
			<td align="right">1</td>
			<td align="right">5.9</td>
		</tr>
		<tr>
			<td>12.5</td>
			<td align="right">474.0</td>
			<td align="right">491.3</td>
			<td align="right">482.7</td>
			<td align="right">12.5</td>
			<td align="right">1</td>
			<td align="right">12.5</td>
		</tr>
		<tr>
			<td>25</td>
			<td align="right">854.8</td>
			<td align="right">881.3</td>
			<td align="right">868.1</td>
			<td align="right">24.4</td>
			<td align="right">1</td>
			<td align="right">24.4</td>
		</tr>
		<tr>
			<td>50</td>
			<td align="right">1617.1</td>
			<td align="right">1618.0</td>
			<td align="right">1617.6</td>
			<td align="right">50.3</td>
			<td align="right">1</td>
			<td align="right">50.3</td>
		</tr>
		<tr>
			<td>100</td>
			<td align="right">2813.1</td>
			<td align="right">2846.1</td>
			<td align="right">2829.6</td>
			<td align="right">101.3</td>
			<td align="right">1</td>
			<td align="right">101.3</td>
		</tr>
		<tr>
			<td>200</td>
			<td align="right">4367.3</td>
			<td align="right">4588.7</td>
			<td align="right">4478.0</td>
			<td align="right">198.2</td>
			<td align="right">1</td>
			<td align="right">198.2</td>
		</tr>
		<tr>
			<td>400</td>
			<td align="right">6258.0</td>
			<td align="right">6650.0</td>
			<td align="right">6454.0</td>
			<td align="right">401.6</td>
			<td align="right">1</td>
			<td align="right">401.6</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Urine Sample</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Pre-drug 1</td>
			<td align="right">2443.9</td>
			<td align="right">2062.3</td>
			<td align="right">2253.1</td>
			<td align="right">88.5</td>
			<td align="right">200</td>
			<td align="right">17700</td>
		</tr>
		<tr>
			<td>Pre-drug 2</td>
			<td align="right">2266.5</td>
			<td align="right">1707.0</td>
			<td align="right">1986.8</td>
			<td align="right">76.3</td>
			<td align="right">200</td>
			<td align="right">15250</td>
		</tr>
		<tr>
			<td>Post-drug 1</td>
			<td align="right">1208.9</td>
			<td align="right">1391.2</td>
			<td align="right">1300.1</td>
			<td align="right">44.7</td>
			<td align="right">10</td>
			<td align="right">447</td>
		</tr>
		<tr>
			<td>Post-drug 2</td>
			<td align="right">2753.4</td>
			<td align="right">2120.5</td>
			<td align="right">2437.0</td>
			<td align="right">97.0</td>
			<td align="right">10</td>
			<td align="right">970</td>
		</tr>
		<tr>
			<td>Post-drug 3</td>
			<td align="right">2888.3</td>
			<td align="right">3178.0</td>
			<td align="right">3033.2</td>
			<td align="right">124.4</td>
			<td align="right">10</td>
			<td align="right">1244</td>
		</tr>
	</tbody>
</table>
<p class="jove_content"><strong>Table 1: Sample Results of Inulin Assay.</strong> FITC-Inulin fluorescence values are shown for the reagent blank, 7 standards, and 5 urine samples. Standards and samples were assayed in duplicate and diluted as needed. The average fluorescence for each sample was used to calculate the concentration of inulin. The inulin concentrations in these urine samples are included in the table of measurements (<strong>Table 2</strong>).</p>
<p class="jove_content"></p>
<p class="jove_content"></p>
<p class="jove_content" fo:keep-together.within-page="always"><img alt="Table 2" src="/files/ftp_upload/52425/52425table2.jpg" /><br />
<strong>Table 2: Measurements Recorded during the Renal Function Lab Demonstration.</strong> The variables recorded during five time periods (two Pre-drug and three Post&#8211;drug) of the renal function lab demonstration are right and left kidney weight, mean arterial pressure (MAP), heart rate (HR), sample time, urine volume, plasma and urine sodium (Na), potassium (K), and inulin concentrations. The urine inulin concentrations were determined from the inulin assay shown in <strong>Table 1</strong>.</p>
<p class="jove_content"></p>
<p class="jove_content"></p>
<p class="jove_content" fo:keep-together.within-page="always"><img alt="Table 3" src="/files/ftp_upload/52425/52425table3.jpg" /><br />
<strong>Table 3: Renal Function Parameters Calculated from Recorded Measurements.</strong> Using the formulas shown in Protocol Section 5, the recorded variables (<strong>Table 2</strong>) are used to calculate urine flow rate, glomerular filtration rate (GFR), GFR/g kidney weight, excretion rate, filtered load, and fractional excretion of sodium (Na) and potassium (K) during the two Pre-drug and three Post&#8211;drug periods.</p>

Watch this Scientific Journal Video about Physiology Lab Demonstration: Glomerular Filtration Rate in a Rat at JoVE.com

Physiology Lab Demonstration: Glomerular Filtration Rate in a Rat | Biology | Video

Physiology Lab Demonstration: Glomerular Filtration Rate in a Rat

<p class='jove_content'>The purpose of this protocol is to demonstrate the principles and techniques for measuring and calculating glomerular filtration rate, urine flow rate, and excretion of sodium and potassium in a rat. This demonstration can be used to provide students with an overall conceptual understanding of how to measure renal function. </p>

Measurements of glomerular filtration rate (GFR), and the fractional excretion of sodium (Na) and potassium (K) are critical in assessing renal function ...

physiology-lab-demonstration-glomerular-filtration-rate-in-a-rat

Universidad San Sebastian

Research

Education

JoVE Journal

JoVE Core

Biology

Human Biology

Osmoregulation and Excretion

SCIENTISTS IN ACTION

17.2K Views.  U.S. Army Institute of Surgical Research. The purpose of this protocol is to demonstrate the principles and techniques for measuring and calculating glomerular filtration rate, urine flow rate, and excretion of sodium and potassium in a rat. This demonstration can be used to provide students with an overall conceptual understanding of how to measure renal function. 

Video: Physiology Lab Demonstration: Glomerular Filtration Rate in a Rat

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

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

JoVE Lab Manual

Chemistry

Scientific Measurement and Lab Skills

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

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

	Measuring Diameter and CircumferenceExpand
	Error can be introduced in any experiment, even ...

Lab Manual Sub Articles

Temperature Dependence

<p>Source: Smaa Koraym at Johns Hopkins University, MD, USA</p>
<ol class="note-items">
	<li data-note-item="1" data-parent-collapse="">Temperature Dependent Decomposition of Hydrogen Peroxide<button class="expand">Expand</button>
	<p><em>We suggest that students work in pairs for this experiment. Equipment controls may vary.</em></p>
	<p>In this lab, you&#39;ll perform a decomposition reaction where a single compound breaks down into two or more simpler products. You will observe the decomposition of hydrogen peroxide into water and oxygen. This decomposition happens very slowly, so you will use iron (III) nitrate as a catalyst to lower the activation energy.</p>
	<p>During this process, iron undergoes a redox reaction and then returns to its starting oxidation state. You will be able to see this as a color change in your solution during the reaction. You&#39;ll perform the same reaction at four different temperatures and track the rate of the reaction by recording the pressure inside the flask. This way you can measure how quickly oxygen was produced in each experiment. You&#39;ll use this to calculate the reaction&#39;s activation energy.</p>
	<p><strong>Table 1. Estimate the apparent activation energy for the decompostion of hydrogen peroxide</strong></p>
	<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
		<tbody>
			<tr>
				<td><strong>Trial&#160;</strong></td>
				<td><strong>Temperature (&#8451;)</strong></td>
				<td><strong>&#916;P (kPa/s)</strong></td>
				<td><strong>1/T&#160;</strong></td>
				<td><strong>ln (&#916;P)</strong></td>
			</tr>
			<tr>
				<td>1</td>
				<td></td>
				<td></td>
				<td></td>
				<td></td>
			</tr>
			<tr>
				<td>2</td>
				<td></td>
				<td></td>
				<td></td>
				<td></td>
			</tr>
			<tr>
				<td>3</td>
				<td></td>
				<td></td>
				<td></td>
				<td></td>
			</tr>
			<tr>
				<td>4</td>
				<td></td>
				<td></td>
				<td></td>
				<td></td>
			</tr>
		</tbody>
	</table>
	<p><a href="/CDNSource/labs/51/Table_1._Estimate_the_apparent_activation_energy_of.xlsx" target="_blank">Click Here to download Table 1</a> &#160;</p>
	<ul class="lab-items">
		<li data-sub-note-item="1-1">
		<div class="lab-step">Before starting this experiment, put on a lab coat, safety glasses, and nitrile gloves.</div>
		</li>
		<li data-sub-note-item="1-2">
		<div class="lab-step">Ensure that your hot plate is off and then place a 600-mL beaker on the hot plate.</div>
		</li>
		<li data-sub-note-item="1-3">
		<div class="lab-step">Connect vacuum tubing to the barbed sidearm of a 125-mL B&#252;chner filter flask, and carefully clamp the filter flask in the 600-mL beaker so that the sidearm is just above the top of the beaker.</div>
		</li>
		<li data-sub-note-item="1-4">
		<div class="lab-step">Fill a 400-mL beaker with deionized water., and pour the water into the 600-mL beaker until the water level is about 2&#8211;3 cm below the side arm.<br />
		<strong>NOTE</strong>: Make sure that the water is not able to get into the flask or vacuum line.</div>
		</li>
		<li data-sub-note-item="1-5">
		<div class="lab-step">Immerse the thermometer in the water, clamping it in place so that the end is in contact with the outer wall of the flask. Ensure that you can read the current temperature, as well as the 40, 60, and 80 &#176;C marks.</div>
		</li>
		<li data-sub-note-item="1-6">
		<div class="lab-step">Make sure that each hole of the 2-hole adapter is fitted with a tapered locking adapter. Check that all the adapters are firmly seated, since any air that escapes around them will affect your data.</div>
		</li>
		<li data-sub-note-item="1-7">
		<div class="lab-step">Lock a 2-way stopcock into one of the stopper adapters. Then, lock one end of the flexible tubing into the second adapter with the other end into the connector of the gas pressure sensor.</div>
		</li>
		<li data-sub-note-item="1-8">
		<div class="lab-step">Turn on the data acquisition device for the gas pressure sensor and ensure that the pressure is displayed in kPa.</div>
		</li>
		<li data-sub-note-item="1-9">
		<div class="lab-step">Set the acquisition rate to two samples per second and the duration to 300 s. Then, display the pressure in real-time.</div>
		</li>
		<li data-sub-note-item="1-10">
		<div class="lab-step">Label a 400-mL beaker as &#8216;waste&#8217;, a 50-mL beaker as &#8216;0.5 M Fe(NO<sub>3</sub>)<sub>3</sub>&#8217;, and a 100-mL beaker as &#8216;3% w/w hydrogen peroxide&#8217;.</div>
		</li>
		<li data-sub-note-item="1-12">
		<div class="lab-step">Place a few paper towels on the work surface as a clean area for glassware that you will reuse. Keep a supply of paper towels on hand for later.</div>
		</li>
		<li data-sub-note-item="1-13">
		<div class="lab-step">Then, bring the 50-mL and 100-mL beakers to the stock solution area. Pour about 30 mL of 0.5 M Fe(NO<sub>3</sub>)<sub>3</sub> in the 50-mL beaker and 100 mL of 3% w/w hydrogen peroxide in the 100-mL beaker.</div>
		</li>
		<li data-sub-note-item="1-14">
		<div class="lab-step">Back at your workspace, setup a 20-mL volumetric pipette and fill it to the mark with 3% w/w hydrogen peroxide. Dispense the hydrogen peroxide into the filter flask and set the volumetric pipette aside.</div>
		</li>
		<li data-sub-note-item="1-15">
		<div class="lab-step">Adjust the thermometer as needed so that it contacts the flask below the hydrogen peroxide solution level.</div>
		</li>
		<li data-sub-note-item="1-16">
		<div class="lab-step">Fit the rubber stopper into the mouth of the filter flask, being careful not to loosen the connections to the sensor and the 2-way stopcock.</div>
		</li>
		<li data-sub-note-item="1-17">
		<div class="lab-step">Check that the 2-way stop is closed. Open the vacuum line and monitor the pressure as it decreases. This will seal the stopper in the flask. Once the pressure reaches 10 kPa, close the vacuum.</div>
		</li>
		<li data-sub-note-item="1-18">
		<div class="lab-step">Monitor the pressure for at least 1 min to confirm there are no slow leaks.<br />
		<strong>NOTE</strong>: If the pressure immediately starts rapidly increasing, there is a leak in your setup, so tighten the connections and try again until the pressure holds at 10 kPa when the vacuum line is closed.</div>
		</li>
		<li data-sub-note-item="1-19">
		<div class="lab-step">Draw 5 mL of 0.5 M Fe(NO<sub>3</sub>)<sub>3</sub> into a 20-mL syringe. Expel all air from the syringe so that it only contains the solution.</div>
		</li>
		<li data-sub-note-item="1-20">
		<div class="lab-step">Lock the syringe into the top of the 2-way stopcock. You are now ready to start the room temperature trial, so record the water temperature in your lab notebook.</div>
		</li>
		<li data-sub-note-item="1-21">
		<div class="lab-step">Start acquiring gas pressure data. Allow the device to record data for about 15 s, then open the stopcock and quickly close it once all of the Fe(NO<sub>3</sub>)<sub>3</sub> has entered the flask. The observed increase in pressure is from the evolution of oxygen gas produced by the decomposition of hydrogen peroxide.</div>
		</li>
		<li data-sub-note-item="1-22">
		<div class="lab-step">Once the data collection finishes, save your data. Then, disconnect the syringe and open the stopcock to vent the flask.</div>
		</li>
		<li data-sub-note-item="1-23">
		<div class="lab-step">Take out the rubber stopper, carefully remove the thermometer from the clamp and the flask from the beaker, and disconnect the vacuum tubing from the filter flask.</div>
		</li>
		<li data-sub-note-item="1-24">
		<div class="lab-step">Empty the flask into the waste beaker. Try not to get liquid in the side arm. Dry the outside of the flask with paper towels.</div>
		</li>
		<li data-sub-note-item="1-25">
		<div class="lab-step">Rinse the interior of the flask with deionized water and pour the rinse into the waste beaker. If any liquid gets into the side arm, remove it with paper towels.</div>
		</li>
		<li data-sub-note-item="1-26">
		<div class="lab-step">Reconnect the flask to the vacuum line and clamp it in the 600-mL beaker in contact with the thermometer.</div>
		</li>
		<li data-sub-note-item="1-27">
		<div class="lab-step">Turn on the hotplate and heat the water around the flask until the thermometer reads 80 &#176;C. Then, turn off the heat.</div>
		</li>
		<li data-sub-note-item="1-28">
		<div class="lab-step">Then, add 20 mL of 3% w/w hydrogen peroxide to the flask.</div>
		</li>
		<li data-sub-note-item="1-29">
		<div class="lab-step">Dry the stopper with paper towels. Do this after each trial to ensure that the stopper has a snug fit in the neck of the beaker and fit the stopper in the flask.</div>
		</li>
		<li data-sub-note-item="1-30">
		<div class="lab-step">Check that the stopcock is closed and evacuate the flask to about 10 kPa. Then, close the vacuum and confirm that there are no leaks.</div>
		</li>
		<li data-sub-note-item="1-31">
		<div class="lab-step">Draw 5 mL Fe(NO<sub>3</sub>)<sub>3</sub> solution into the syringe, expel the air from the syringe, and connect it to the stopcock.</div>
		</li>
		<li data-sub-note-item="1-32">
		<div class="lab-step">Record the temperature shown on the thermometer in your lab notebook, then, start collecting data.</div>
		</li>
		<li data-sub-note-item="1-33">
		<div class="lab-step">Wait about 15 s and introduce the Fe(NO<sub>3</sub>)<sub>3</sub> solution in the same way as before.<br />
		<strong>NOTE:</strong> As the pressure approaches 150 kPa, the stopper may pop off!</div>
		</li>
		<li data-sub-note-item="1-34">
		<div class="lab-step">After data collection finishes, save the data, vent and clean the filter flask, and set up for the third trial.</div>
		</li>
		<li data-sub-note-item="1-35">
		<div class="lab-step">Fill the 400-mL beaker with ice and add some to cool the water to about 60 &#176;C.</div>
		</li>
		<li data-sub-note-item="1-36">
		<div class="lab-step">Perform the third trial in the same way as before. Remember to dry the stopper and the inside of the flask and to record the temperature prior to data collection.</div>
		</li>
		<li data-sub-note-item="1-37">
		<div class="lab-step">After the data is collected for that trial, cool the water to about 40 &#176;C and perform the fourth trial.</div>
		</li>
		<li data-sub-note-item="1-38">
		<div class="lab-step">Once you have finished all four trials, empty the filter flask and rinse it into the waste beaker one last time.</div>
		</li>
		<li data-sub-note-item="1-39">
		<div class="lab-step">Dispose of any excess 0.5 M Fe(NO<sub>3</sub>)<sub>3</sub> and the contents of the waste beaker in a container labeled for aqueous iron waste.</div>
		</li>
		<li data-sub-note-item="1-40">
		<div class="lab-step">Then, disassemble the apparatus and pour the leftover ice, water, and hydrogen peroxide in the sink. Wash your glassware following your lab&#39;s standard protocol.</div>
		</li>
	</ul>
	</li>
	<li data-note-item="1" data-parent-collapse="">Results<button class="expand">Expand</button>
	<p>The decomposition of hydrogen peroxide with iron is a complex, multi-step process that we can&#39;t easily describe in a single equation. However, we can estimate the apparent activation energy from the rate of oxygen production and compare it to the apparent activation energy of the un-catalyzed process.</p>
	<ul class="lab-items">
		<li data-sub-note-item="2-1">
		<div class="lab-step">Find the rate of pressure change, which is directly proportional to the rate of oxygen production. For each experiment, make a plot of pressure with respect to time and find the point where the reaction started.</div>
		</li>
		<li data-sub-note-item="2-2">
		<div class="lab-step">Identify the maximum pressure attained and determine the slope between the two points.</div>
		</li>
		<li data-sub-note-item="2-3">
		<div class="lab-step">Once you&#39;ve determined the slopes and the corresponding temperatures in Kelvin for all of the temperatures, use the Arrhenius equation to estimate the apparent activation energy of this reaction.</div>
		</li>
		<li data-sub-note-item="2-4">
		<div class="lab-step">Take the reciprocal of the temperatures in Kelvin and the natural log of the rate of change in pressure. Remember that the rate constant k is essentially equal to the change in pressure.</div>
		</li>
		<li data-sub-note-item="2-5">
		<div class="lab-step">Make an Arrhenius plot and find the slope of the line.</div>
		</li>
		<li data-sub-note-item="2-6">
		<div class="lab-step">The slope is equal to the negative activation energy over the universal gas constant, so multiply the slope by the negative universal gas constant to get the apparent activation energy of the iron catalyzed decomposition reaction. You&#39;ll see a value in the range of 35&#8211;60 kJ/mol because the iron catalyst made the decomposition take less energy. The apparent activation energy of the uncatalyzed decomposition of hydrogen peroxide is around 78 &#8211; 88 kJ/mol.</div>
		</li>
	</ul>
	</li>
</ol>

Temperature Dependence and Arrhenius Equation - Procedure

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

	Temperature Dependent Decomposition of Hydrogen PeroxideExpand
	We suggest that students work ...

Concentration Dependence

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

Concentration Dependence and Reaction Order - Procedure

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

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

Acid and Base Concentrations

<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 ~0.1 M NaOH<button class="expand">Expand</button>
	<p>In the first part of the lab, you will use a 50% w/w solution of NaOH to prepare 500 mL of ~0.1 M. The 50% w/w NaOH is indicative of its weight ratio. For example, if the instructor prepared 150 mL of the 50% w/w NaOH solution, then 150 g of NaOH was dissolved in 150 g of water, and the total weight of the solution is 300 g.</p>
	<ul class="lab-items">
		<li data-sub-note-item="1-2">
		<div class="lab-step">To begin, put on the appropriate personal protective equipment, including gloves, chemical splash goggles, and a lab coat.</div>
		</li>
		<li data-sub-note-item="1-3">
		<div class="lab-step">Calculate the molarity of 50% w/w NaOH, which is M<sub>1</sub> in the dilution formula.<strong> Note: </strong>The density of 50% w/w NaOH is 1.53 g/mL, and the molar mass of NaOH is 39.998 g/mole.
		<h4>Table 1. Preparation of 0.1 M NaOH from 50% w/w NaOH</h4>
		<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
			<tbody>
				<tr>
					<td>Density of 50% w/w stock solution</td>
					<td style="text-align: center;">1.53 g/mL</td>
				</tr>
				<tr>
					<td>Molar mass<sub>NaOH</sub></td>
					<td style="text-align: center;">39.998 g/mol</td>
				</tr>
				<tr>
					<td>Mass of NaOH in 50% w/w stock solution (mg)</td>
					<td style="text-align: center;"></td>
				</tr>
				<tr>
					<td>Total mass of 50% w/w stock solution</td>
					<td style="text-align: center;"></td>
				</tr>
				<tr>
					<td>Volume of 50% w/w stock solution (mL)</td>
					<td></td>
				</tr>
				<tr>
					<td>Moles of NaOH in 50% w/w solution (mol)</td>
					<td></td>
				</tr>
				<tr>
					<td>Molarity of 50% w/w stock solution (M<sub>1</sub>)</td>
					<td></td>
				</tr>
				<tr>
					<td>Volume of 50% w/w solution needed (V<sub>1</sub>)</td>
					<td></td>
				</tr>
			</tbody>
		</table>
		<a href="https://www-jove-com.bdigitaluss.remotexs.co/CDNSource/lm/labs/44/Table_1._Preparation_of_0.1_M_NaOH.xlsx" target="_blank">Click Here to download Table 1</a></div>
		</li>
		<li data-sub-note-item="1-4">
		<div class="lab-step">Use the dilution formula to determine the volume of 50% w/w NaOH needed to prepare 500 mL of ~0.1 M NaOH.</div>
		</li>
		<li data-sub-note-item="1-5">
		<div class="lab-step">Label the 500 mL polyethylene bottle as &#8216;~0.1 M NaOH&#8217;.</div>
		</li>
		<li data-sub-note-item="1-6">
		<div class="lab-step">Adjust the volume on a 1-mL pipette to the value calculated and use it to transfer the 50% w/w NaOH to the polyethylene bottle.</div>
		</li>
		<li data-sub-note-item="1-7">
		<div class="lab-step">Use a 100-mL graduated cylinder to measure the amount of water and pour it into the bottle containing the NaOH. Cap the bottle and invert it several times to mix the solution. <strong>Note:</strong> You will need 500 mL minus the calculated volume of NaOH.</div>
		</li>
	</ul>
	</li>
	<li data-note-item="2" data-parent-collapse="">Standardization of 0.1 M NaOH<button class="expand">Expand</button>
	<p>After you have prepared 0.1 M NaOH, determine its exact concentration, or standardize it, using the acid-base titration method. In this technique, a base like NaOH is slowly added to an acid like potassium hydrogen phthalate (KHP). The chemical reaction that takes place in the flask is a neutralization reaction. In this neutralization reaction, one mole of base neutralizes one mole of acid, resulting in salt and water. This reaction is performed in the presence of the indicator phenolphthalein, which is colorless at the start of the reaction when the pH is acidic. The indicator turns pink as soon as enough NaOH is added to the flask to make the pH basic.</p>
	<ul class="lab-items">
		<li data-sub-note-item="2-2">
		<div class="lab-step">Label the three Erlenmeyer flasks as &#8216;A&#8217;, &#8216;B&#8217;, and &#8216;C&#8217;.</div>
		</li>
		<li data-sub-note-item="2-3">
		<div class="lab-step">Weigh 0.5 &#8211; 0.7 g of KHP for each of the flasks and record the mass for each. <strong>Note: </strong>Try to measure the same mass of KHP for all three flasks.
		<h4><strong>Table 2. Standardization of NaOH</strong></h4>
		<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
			<tbody>
				<tr>
					<td><strong>Molar mass<sub>KHP</sub> = 204.23 g/mol </strong></td>
					<td style="text-align: center;"><strong>Flask A</strong></td>
					<td style="text-align: center;"><strong>Flask B</strong></td>
					<td style="text-align: center;"><strong>Flask C</strong></td>
				</tr>
				<tr>
					<td>Mass of KHP (g)</td>
					<td></td>
					<td></td>
					<td></td>
				</tr>
				<tr>
					<td>Volume<sub>initial NaOH</sub>(mL)</td>
					<td></td>
					<td></td>
					<td></td>
				</tr>
				<tr>
					<td>Volume<sub>final NaOH</sub> (mL)</td>
					<td></td>
					<td></td>
					<td></td>
				</tr>
				<tr>
					<td>Volume<sub>NaOH</sub> (mL)</td>
					<td></td>
					<td></td>
					<td></td>
				</tr>
				<tr>
					<td>Moles of KHP</td>
					<td></td>
					<td></td>
					<td></td>
				</tr>
				<tr>
					<td>Moles of NaOH</td>
					<td></td>
					<td></td>
					<td></td>
				</tr>
				<tr>
					<td>Molarity of NaOH</td>
					<td></td>
					<td></td>
					<td></td>
				</tr>
				<tr>
					<td>Average molarity</td>
					<td></td>
				</tr>
				<tr>
					<td>Standard deviation</td>
					<td></td>
				</tr>
			</tbody>
		</table>
		<a href="https://www-jove-com.bdigitaluss.remotexs.co/CDNSource/lm/labs/44/Table_2._Standardization_of_NaOH.xlsx" target="_blank">Click Here to download Table 2</a></div>
		</li>
		<li data-sub-note-item="2-4">
		<div class="lab-step">Add 50 mL of deionized water to each flask, and use a glass stirring rod to stir the solutions until they appear homogeneous.</div>
		</li>
		<li data-sub-note-item="2-5">
		<div class="lab-step">Add 2-3 drops of phenolphthalein to each of the three flasks.</div>
		</li>
		<li data-sub-note-item="2-6">
		<div class="lab-step">Set up the titration apparatus. Attach the burette clamp to the ring stand, and securely clamp the 50-mL glass burette to it. Make sure the valve of the burette is closed.</div>
		</li>
		<li data-sub-note-item="2-7">
		<div class="lab-step">Label a 400-mL beaker as &#8216;waste&#8217; and place it under the burette. Rinse the burette by pouring about 5 mL of 0.1 M NaOH into the burette. Open the burette valve to allow the NaOH to flow into the beaker.</div>
		</li>
		<li data-sub-note-item="2-8">
		<div class="lab-step">Close the valve and fill the burette with slightly more than 50 mL of NaOH. Release any air bubbles present in the tip of the burette by opening and closing the burette valve. Record the starting volume of NaOH.</div>
		</li>
		<li data-sub-note-item="2-9">
		<div class="lab-step">Place flask A below the tip of the burette, and titrate the solution using 1 mL volumes of NaOH. Swirl the solution after each addition. Continue adding 1 mL volumes to the flask until the pink color persists. This is considered the endpoint. Record the volume of 0.1 M NaOH added to achieve the endpoint.</div>
		</li>
		<li data-sub-note-item="2-10">
		<div class="lab-step">Repeat the titration for flasks B and C. This data will be used to calculate the actual concentration of NaOH.</div>
		</li>
	</ul>
	</li>
	<li data-note-item="3" data-parent-collapse="">Titration of phosphoric acid<button class="expand">Expand</button>
	<p>In this experiment, we will determine two of the three pKa values for the triprotic acid, phosphoric acid, using acid-base titration. In this neutralization reaction, phosphoric acid reacts with NaOH to form water and the salt, sodium phosphate.</p>
	<ul class="lab-items">
		<li data-sub-note-item="3-2">
		<div class="lab-step">Attach the drop counter to the ring stand, below the burette clamp. Secure the plastic burette so that its tip is just above the drop counter.</div>
		</li>
		<li data-sub-note-item="3-3">
		<div class="lab-step">Connect the drop counter to the data acquisition system, and make sure the two valves of the plastic burette are both in the closed position.</div>
		</li>
		<li data-sub-note-item="3-4">
		<div class="lab-step">Place the waste container below the burette and pour a few mL of 0.1 M NaOH into the burette. Open both valves to drain the NaOH into the waste beaker. Then close the valves.</div>
		</li>
		<li data-sub-note-item="3-5">
		<div class="lab-step">Fill the plastic burette with 25 mL of 0.1 M NaOH. Drain about 5 mL into the waste beaker&#8212;enough so that NaOH fills the burette tip. Make sure there are no air bubbles, then close the valves.</div>
		</li>
		<li data-sub-note-item="3-6">
		<div class="lab-step">Calibrate the drop counter. Replace the waste beaker beneath the burette with a 10-mL graduated cylinder. Then, open the bottom valve on the burette, while keeping the top valve closed. Turn on the data acquisition system and set it to &#8216;drop counting mode&#8217;.</div>
		</li>
		<li data-sub-note-item="3-7">
		<div class="lab-step">Slowly open the top valve to very slowly release drops, ideally at one drop every 2 s. Allow the drops to empty from the burette until there is 9-10 mL of 0.1 M NaOH in the graduated cylinder.</div>
		</li>
		<li data-sub-note-item="3-8">
		<div class="lab-step">Close the bottom valve and leave the top valve as is. Read the volume of NaOH in the graduated cylinder to the first decimal place and enter this value in the data acquisition system. Record the value for &#8216;drops/mL&#8217;. Then, discard the NaOH in the graduated cylinder into the aqueous waste beaker.</div>
		</li>
		<li data-sub-note-item="3-9">
		<div class="lab-step">Calibrate the pH sensor before starting the titration. Connect the pH sensor to the data acquisition system, then select &#39;Calibrate&#39;. Rinse the bulb of the pH sensor with deionized water before inserting it into the pH 7 buffer. Leave the sensor submerged until the voltage stabilizes, then accept the measurement.</div>
		</li>
		<li data-sub-note-item="3-10">
		<div class="lab-step">Rinse the bulb with deionized water and insert it into the pH 10 buffer. Allow the voltage to stabilize, then accept the measurement.</div>
		</li>
		<li data-sub-note-item="3-11">
		<div class="lab-step">Rinse the bulb of the pH sensor again and slide it through the designated slot in the drop counter.</div>
		</li>
		<li data-sub-note-item="3-12">
		<div class="lab-step">Measure 40 mL of deionized water into a clean 100-mL beaker, then, transfer 1 mL of phosphoric acid into the beaker of water.</div>
		</li>
		<li data-sub-note-item="3-13">
		<div class="lab-step">Place the beaker on the stir plate under the drop counter. Carefully slide the pH sensor into the beaker.</div>
		</li>
		<li data-sub-note-item="3-14">
		<div class="lab-step">Add a stir bar to the beaker, and turn the stir setting onto high. Start collecting data on the acquisition device. Then, open the bottom valve on the burette. <strong>Note: </strong>The drop rate should be about 1 drop every 2 s. After the first drop is released, check to see data are being recorded.</div>
		</li>
		<li data-sub-note-item="3-15">
		<div class="lab-step">Continue the titration until the pH meter reads pH 12. Then, stop acquiring data and close the valve on the burette. Save your data on a flash drive.</div>
		</li>
		<li data-sub-note-item="3-16">
		<div class="lab-step">To clean up your workspace, check the pH of all waste solutions using pH paper. Neutralize all acidic aqueous waste with baking soda and all basic waste with citric acid. Add enough of either baking soda or citric acid to the solution until it stops bubbling.</div>
		</li>
		<li data-sub-note-item="3-17">
		<div class="lab-step">Flush all neutralized solutions down the sink with copious amounts of water.</div>
		</li>
		<li data-sub-note-item="3-18">
		<div class="lab-step">Wash all glassware.</div>
		</li>
	</ul>
	</li>
	<li data-note-item="4" data-parent-collapse="">Results<button class="expand">Expand</button>
	<ul class="lab-items">
		<li data-sub-note-item="4-1">
		<div class="lab-step">In the first part of this lab, you standardized a solution of NaOH using KHP to determine its actual concentration. Now, let&#39;s see how close the standardized concentration is to the 0.1 M concentration that was prepared. Determine the number of moles of KHP that was added to each flask, and by extension, the moles of NaOH. Once the solution is neutralized, the molar quantities of KHP and NaOH are equal.</div>
		</li>
		<li data-sub-note-item="4-2">
		<div class="lab-step">Calculate the molarity of NaOH based on the total volume of NaOH that was added to each flask. The actual concentration is lower than the expected 0.1 molarity. This is because NaOH is hygroscopic, so it is difficult to weigh it accurately.</div>
		</li>
		<li data-sub-note-item="4-3">
		<div class="lab-step">Plot the results for the phosphoric acid titration (pH vs. Volume of NaOH). Phosphoric acid is a weak triprotic acid, meaning that it has the potential to provide three protons per molecule when it dissociates in aqueous solutions. Phosphoric acid has three pKa values, one for when each proton is dissociated.</div>
		</li>
		<li data-sub-note-item="4-4">
		<div class="lab-step">Look at the data. There are two sigmoidal curves, indicating two equivalence points. Each equivalence point corresponds to a dissociation constant, Ka, of phosphoric acid. <strong>Note: </strong>You stopped the experiment once the pH reached 12, so you only measured two of the three Ka values.</div>
		</li>
		<li data-sub-note-item="4-5">
		<div class="lab-step">Plot the first derivative of the titration curve. The equivalence points are represented by the curve maximums.
		<h4><strong>Table 3. Titration of phosphoric acid</strong></h4>
		<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
			<tbody>
				<tr>
					<td>Volume<sub>1<sup>st</sup> equivalence point</sub> (mL)</td>
					<td style="text-align: center;"></td>
				</tr>
				<tr>
					<td>Volume<sub>1<sup>st</sup> half-equivalence point</sub> (mL)</td>
					<td style="text-align: center;"></td>
				</tr>
				<tr>
					<td>Volume<sub>2<sup>nd</sup> equivalence point</sub> (mL)</td>
					<td style="text-align: center;"></td>
				</tr>
				<tr>
					<td>Volume<sub>2<sup>nd</sup> half-equivalence point</sub> (mL)</td>
					<td style="text-align: center;"></td>
				</tr>
				<tr>
					<td>1<sup>st</sup> pKa<sub>measured</sub></td>
					<td style="text-align: center;"></td>
				</tr>
				<tr>
					<td>1<sup>st</sup> pKa<sub>theoretical</sub></td>
					<td style="text-align: center;">2.16</td>
				</tr>
				<tr>
					<td>2<sup>nd</sup> pKa<sub>measured</sub></td>
					<td style="text-align: center;"></td>
				</tr>
				<tr>
					<td>2<sup>nd</sup> pKa<sub>theoretical</sub></td>
					<td style="text-align: center;">7.21</td>
				</tr>
				<tr>
					<td>Moles of NaOH</td>
					<td style="text-align: center;"></td>
				</tr>
				<tr>
					<td>Moles of H<sub>3</sub>PO<sub>4</sub></td>
					<td style="text-align: center;"></td>
				</tr>
				<tr>
					<td>Molarity of H<sub>3</sub>PO<sub>4</sub></td>
					<td style="text-align: center;"></td>
				</tr>
			</tbody>
		</table>
		<a href="https://www-jove-com.bdigitaluss.remotexs.co/CDNSource/lm/labs/44/Table_3._Titration_of_Phosphoric_Acid.xlsx" target="_blank">Click Here to download Table 3</a></div>
		</li>
		<li data-sub-note-item="4-6">
		<div class="lab-step">Find the first half-equivalence point by taking the volume of NaOH corresponding to the first equivalence point and dividing it by 2. At the half-equivalence point, the concentrations of undissociated acid and its conjugate base are the same, and the pH is equal to the pKa.</div>
		</li>
		<li data-sub-note-item="4-7">
		<div class="lab-step">Look up the pH at this volume from your table of data to get a more precise value. This corresponds to the first pKa, which is reported in the literature as 2.16.</div>
		</li>
		<li data-sub-note-item="4-8">
		<div class="lab-step">Repeat this to find the second pKa. The second half-equivalence point is located midway between the first and second equivalence points, which should give a pKa of about 7.2.</div>
		</li>
	</ul>
	</li>
</ol>

Acid-Base Titration: Calculating pH, Strength, and Concentration - Procedure

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

	Preparation of ~0.1 M NaOHExpand
	In the first part of the lab, you will use a 50% w/w solution ...

Population Growth

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

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

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

Ecology

Photosynthesis

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

Studying Photosynthesis by Leaf Disc Experiment-Procedure

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

Cellular Processes

Diffusion and Osmosis

<ol class="note-items"><li data-note-item="1" data-parent-collapse="">Diffusion in Agar
<button class="expand">Expand</button><ul class="lab-items"><li data-sub-note-item="1-1"><div class="lab-step">NOTE: In this exercise, you will be given agar containing an indicator chemical called phenolphthalein. When phenolphthalein is exposed to the normal alkaline conditions in the agar, it will look pink. But when it is exposed to neutral or acidic conditions, it changes from pink to clear. You will make different size and shaped agar cubes as a model for cells to study the impact of cell size and shape on diffusion rate.
</div></li><li data-sub-note-item="1-2"><div class="lab-step">To make the first set of cells, measure out and cut a small cube of agar where each side measures one centimeter. 
</div></li><li data-sub-note-item="1-3"><div class="lab-step">Next, measure and cut out a medium cell cube with sides of 2 cm and a large cell of 3 cm on each side.
</div></li><li data-sub-note-item="1-4"><div class="lab-step">Knowing the length of the sides of your cube cells, calculate their surface area using this equation, where lower case a represents the length of the sides: Surface Area = 6a^2
</div></li><li data-sub-note-item="1-5"><div class="lab-step">Record these values in the appropriate column in Table 1. <a href="https://www-jove-com.bdigitaluss.remotexs.co/CDNSource/lm/labs/6/Table_1_Diffusion_and_Osmosis.xlsx" style="display:block;margin:20px;" target="__blank">Click Here to download Table 1</a>
</div></li><li data-sub-note-item="1-6"><div class="lab-step">Then, use the same length value and the equation below to calculate the volume of each cube and add these to the table: Volume = a^3 HYPOTHESES: The experimental hypothesis might be that the acid will diffuse completely to the center of the small cell faster than the medium and large cells. The null hypothesis could be that the acid will diffuse to the center of the small and two larger cubes at around the same time.
</div></li><li data-sub-note-item="1-7"><div class="lab-step">Add 100 mL of 0.1 M HCl to each of the three 400 mL beakers to make the diffusion baths.
</div></li><li data-sub-note-item="1-8"><div class="lab-step">Working in a team, have one experimenter ready with the timer and the second and third experimenters ready to drop each cube into one of the beakers.
</div></li><li data-sub-note-item="1-9"><div class="lab-step">When the first experimenter says go, simultaneously drop all three cubes into their respective beakers and start the timer.
</div></li><li data-sub-note-item="1-10"><div class="lab-step">Observe carefully until one of the cubes becomes completely clear or 10 min have passed.
</div></li><li data-sub-note-item="1-11"><div class="lab-step">Stop the timer, remove the agar cubes from the beakers and place the cubes into a Petri dish.
</div></li><li data-sub-note-item="1-12"><div class="lab-step">Make a note of which of the three cells became clear or had the smallest remaining pink area. Then, also note which cell had the most remaining pink agar.
</div></li><li data-sub-note-item="1-13"><div class="lab-step">Next, in Table 1, calculate the surface area to volume ratio for each cell. Surface Area: Volume Ratio = (surface area)/volume
</div></li><li data-sub-note-item="1-14"><div class="lab-step">As the cell size increases, note whether the surface area to volume ratio increases or decreases. Also consider whether this correlates with your observation of the depth of diffusion into the agar cells. If cells rely on diffusion to deliver essential nutrients and molecules to the whole cell, discuss with your group if it would be better to have a smaller or larger surface area to volume.
</div></li><li data-sub-note-item="1-15"><div class="lab-step">Now, with the remaining agar, cut three rectangular shaped blocks of different sizes and record their length, width, and height. This will test what happens when the shapes of cells are different. 
</div></li><li data-sub-note-item="1-16"><div class="lab-step">Calculate the surface area of your rectangular cells using the formula below, where length is l, width is w, and height is h. Surface Area = 2lw + 2lh + 2wh
</div></li><li data-sub-note-item="1-17"><div class="lab-step">Then, calculate the volume of your rectangles using this formula: Volume = l * w * h
</div></li><li data-sub-note-item="1-18"><div class="lab-step">Repeat the experiment by dropping the new shapes into the hydrochloric acid solution for 10 min or until one cube becomes completely clear.
</div></li><li data-sub-note-item="1-19"><div class="lab-step">Remove the cell shapes from the solutions and observe the depth that the hydrochloric acid diffused into each of these cells, and which shapes have the smallest and largest remaining pink areas not reached by the solute.
</div></li><li data-sub-note-item="1-20"><div class="lab-step">Using the surface area and volume data you recorded for your rectangular shapes, calculate the surface area to volume ratio of these cells. Surface Area: Volume Ratio = (surface area)/volume
</div></li><li data-sub-note-item="1-21"><div class="lab-step">Consider whether these values correlate to which cells had the most and least complete diffusion. Additionally, discuss with the group whether these rectangular cells displayed a similar or different pattern of diffusion to that observed with the cube shaped cells, and what this might mean.
</div></li></ul></li><li data-note-item="2" data-parent-collapse="">Movement of Molecules Across a Semi-Permeable Membrane
<button class="expand">Expand</button><ul class="lab-items"><li data-sub-note-item="2-1"><div class="lab-step">Before beginning the experiment, add 250 mL of distilled water to each of four 1 L beakers.
</div></li><li data-sub-note-item="2-2"><div class="lab-step">Then, label the beakers from 1-4, and add 0.5 mL of iodine to the first beaker. HYPOTHESES: In this experiment, the experimental hypothesis is that some of the solutes will be able to pass through the dialysis tubing membrane and others will not. The null hypothesis is that there will be no difference in the ability to diffuse through the dialysis tubing membrane between the solutes.
</div></li><li data-sub-note-item="2-3"><div class="lab-step">To prepare the dialysis tubing, remove the pieces one at a time from the distilled water bath and tie a tight knot at one end of each tube. These tubes, when filled, will act as model cells with the dialysis tubing acting like the semipermeable membrane.
</div></li><li data-sub-note-item="2-4"><div class="lab-step">Add 10 mL of starch solution to the first tube and tie off the open end, making sure to leave space in case the tubing expands during the experiment.
</div></li><li data-sub-note-item="2-5"><div class="lab-step">Then add 10 mL of the NaCl and dextrose solutions to the second and third pieces of tubing, respectively, and tie off both tubes, again, leaving space in case of expansion.
</div></li><li data-sub-note-item="2-6"><div class="lab-step">After adding 10 mL of distilled water and tying off the fourth tube, weigh each of your model cells.
</div></li><li data-sub-note-item="2-7"><div class="lab-step">Record the initial weight values in grams and the colors of the starting solution in each tube in the appropriate columns of Table 2. <a href="https://www-jove-com.bdigitaluss.remotexs.co/CDNSource/lm/labs/6/Table_2_Diffusion_and_Osmosis.xlsx" target="__blank" style="display:block;margin:20px;">Click Here to download Table 2</a>
</div></li><li data-sub-note-item="2-8"><div class="lab-step">After quickly rinsing the outside with tap water, place each piece of tubing in its corresponding beaker for 1 h at room temperature. NOTE: For example, the starch solution tube should be placed into the beaker containing the iodine.
</div></li><li data-sub-note-item="2-9"><div class="lab-step">At the end of the diffusion period, weigh the tubes again.
</div></li><li data-sub-note-item="2-10"><div class="lab-step">Then, observe the tubes carefully, noting any color changes. 
</div></li><li data-sub-note-item="2-11"><div class="lab-step">Record all of these data in Table 2. 
</div></li><li data-sub-note-item="2-12"><div class="lab-step">Next, to perform a Benedict&#39;s Reagent test for simple sugars, make a water bath by adding 250 mL of water to a 600 mL beaker and placing it onto a hot plate.
</div></li><li data-sub-note-item="2-13"><div class="lab-step">Set the plate to high, to boil the water. 
</div></li><li data-sub-note-item="2-14"><div class="lab-step">Label two new glass test tubes as H<sub>2</sub>O and dextrose, respectively.
</div></li><li data-sub-note-item="2-15"><div class="lab-step">Use a graduated cylinder to transfer 1 mL of solution from the water and dextrose beakers into the corresponding test tubes.
</div></li><li data-sub-note-item="2-16"><div class="lab-step">Then, add 2 mL of Benedict&#39;s Reagent to each tube. 
</div></li><li data-sub-note-item="2-17"><div class="lab-step">Once the water is boiling, place each test tube into the water bath for 3-5 minutes.
</div></li><li data-sub-note-item="2-18"><div class="lab-step">After this time, note the color of the solution in each tube. 
</div></li><li data-sub-note-item="2-19"><div class="lab-step">Then use this key to assess whether the test is positive or negative and record these data in the appropriate column in Table 2. <a href="https://www-jove-com.bdigitaluss.remotexs.co/CDNSource/lm/labs/6/Lab_6_Diffusion_Figure_1.png" style="display:block;margin:20px;" target="__blank">Click Here to download Figure 1</a>
</div></li></ul></li><li data-note-item="3" data-parent-collapse="">Results
<button class="expand">Expand</button><ul class="lab-items"><li data-sub-note-item="3-1"><div class="lab-step">First, look at the mass of your four dialysis tube cells at the beginning versus the end of the experiment. Calculate the change in mass for each of the four cells and plot it onto a bar chart.
</div></li><li data-sub-note-item="3-2"><div class="lab-step">Note which cells demonstrated the most change, and whether any of the cells appeared visibly different in size.
</div></li><li data-sub-note-item="3-3"><div class="lab-step">For the experiment with the starch and iodine indicator, note whether there was a color change in the fluid in the artificial cell. Also consider whether there was a color change in the water in the beaker, and what both of these observations say about the properties of the dialysis tubing membrane.
</div></li><li data-sub-note-item="3-4"><div class="lab-step">Finally, in the Benedict&#39;s Reagent test for dextrose, note whether this simple sugar was able to pass through the semipermeable membrane of the &#8220;cell&#8221; into the water in the beaker. Discuss with the class which of the molecules you think could and could not pass through the semipermeable membrane.
</div></li></ul></li></ol>

Diffusion in Agar
ExpandNOTE: In this exercise, you will be given agar containing an indicator chemical called phenolphthalein. When phenolphthalein is ...

Fundamentals

Enzyme Activity

<ol class="note-items"><li data-note-item="1" data-parent-collapse="">Baseline Peroxidase Enzyme Experiment
<button class="expand">Expand</button><ul class="lab-items"><li data-sub-note-item="1-1"><div class="lab-step">To begin, the baseline for the peroxidase enzyme reaction must be determined. Make the substrate by adding 7 mL of distilled water to a clean test tube and then add 0.2 mL of guaiacol. Note: Guaiacol is a color-changing indicator that becomes more yellow-orange as the enzyme reaction progresses.
</div></li><li data-sub-note-item="1-2"><div class="lab-step">Next, add 0.3 mL of 0.1% H2O2.
</div></li><li data-sub-note-item="1-3"><div class="lab-step">Using a marker, label the test tube substrate and then cover the tube with a piece of sealing film.
</div></li><li data-sub-note-item="1-4"><div class="lab-step">Holding the cover in place, invert the tube four times to mix the tube contents.
</div></li><li data-sub-note-item="1-5"><div class="lab-step">In a second test tube, add 6.0 mL of distilled water and 1.5 mL of turnip peroxidase and label this tube &#8220;Enzyme&#8221;.
</div></li><li data-sub-note-item="1-6"><div class="lab-step">Cover this tube with a new piece of sealing film and mix these solutions by four inversions as previously described (step 4).
</div></li><li data-sub-note-item="1-7"><div class="lab-step">When both tubes have been mixed, place a timer on the bench, next to the test tube rack, then pour the contents from the substrate tube into the enzyme tube, being careful not to spill any liquid.
</div></li><li data-sub-note-item="1-8"><div class="lab-step">Next, pour the entire volume from the enzyme tube back into the substrate tube to thoroughly mix the solutions and cover the substrate tube with another piece of sealing film.
</div></li><li data-sub-note-item="1-9"><div class="lab-step">Put the test tube in the rack and start the timer.
</div></li><li data-sub-note-item="1-10"><div class="lab-step">Observe the color in the substrate tube at 0, 1, 2, 3, 4 and 5 minutes, post-mixing.
</div></li><li data-sub-note-item="1-11"><div class="lab-step">To quantify the relative change in color and the rate of the reaction, match the observed color to the closest color in the standard. NOTE: Taking a photo of the observed color intensity at each sample point may make it easier to record the results.
</div></li><li data-sub-note-item="1-12"><div class="lab-step">Record the colors you see in the table. <a href="https://www-jove-com.bdigitaluss.remotexs.co/CDNSource/lm/labs/16/Table_1_Enzyme_Dynamics.xlsx" style="display:block;margin:20px;" target="__blank">Click Here to download Table 1</a>
</div></li></ul></li><li data-note-item="2" data-parent-collapse="">Investigating the Effect of pH on Peroxidase Activity
<button class="expand">Expand</button><ul class="lab-items"><li data-sub-note-item="2-1"><div class="lab-step">HYPOTHESES: In this exercise, the experimental hypothesis is that the reaction rate will reach a maximum or optimum at a mid-range neutral pH and that these rates will be higher than or roughly equal to the baseline rate. The null hypothesis is that there will be no differences in the reaction rates between the baseline and the pH treatments.
</div></li><li data-sub-note-item="2-2"><div class="lab-step">First, label six clean test tubes for each of the six pH buffer levels 3 - 8 and the word &#8220;Enzyme&#8221;.
</div></li><li data-sub-note-item="2-3"><div class="lab-step">Next, add 6 mL of the corresponding pH buffer solutions provided by your instructor to each of the test tubes.
</div></li><li data-sub-note-item="2-4"><div class="lab-step">Then, add 1.5 mL of the turnip peroxidase to each tube, being careful not to contaminate the dropper with the buffer solution.
</div></li><li data-sub-note-item="2-5"><div class="lab-step">Cover the enzyme tubes with sealing film and gently invert each tube to mix the tube contents.
</div></li><li data-sub-note-item="2-6"><div class="lab-step">As described in the baseline reaction experiment, make the substrates for these reactions by adding 7 mL of distilled water, 0.2 mL of guaiacol and 0.3 mL of 0.1% H<sub>2</sub>O<sub>2 </sub>to six new clean test tubes.
</div></li><li data-sub-note-item="2-7"><div class="lab-step">Next, label six additional clean test tubes with the pH levels 3 - 8 and the word &#8220;Solution&#8221; and get a timer ready.
</div></li><li data-sub-note-item="2-8"><div class="lab-step">Working quickly, pour a substrate tube and a pH tube into the corresponding solution tube. Repeat this step for each of the different pH levels.
</div></li><li data-sub-note-item="2-9"><div class="lab-step">Then, cover the tubes with sealing film and mix each tube with four inversions. Place each tube in a tube rack, as they are mixed.
</div></li><li data-sub-note-item="2-10"><div class="lab-step">Observe the color in each tube at time 0 and immediately start the timer. Continue to observe the color in each tube at every minute interval, up to 5 minutes.
</div></li><li data-sub-note-item="2-11"><div class="lab-step">At each interval, use the color code in the standard to record the observed colors in your table.
</div></li></ul></li><li data-note-item="3" data-parent-collapse="">Investigating the Effect of Temperature on Peroxidase Activity
<button class="expand">Expand</button><ul class="lab-items"><li data-sub-note-item="3-1"><div class="lab-step">HYPOTHESES: In this exercise, the experimental hypothesis is that the reaction rate will reach an optimum above room temperature, or 23 &#176;C, but below enzyme denaturation rate and that this maximum will be higher than the baseline rate. The null hypothesis is that there will be no differences in the reaction rates between the baseline and the temperature treatments.
</div></li><li data-sub-note-item="3-2"><div class="lab-step">Fill an 800 mL beaker about halfway to the top with ice and pour cold water into the beaker to create an ice bath. Label this beaker as &#8220;Ice&#8221;.
</div></li><li data-sub-note-item="3-3"><div class="lab-step">Then, label four new 600 mL beakers with the same temperatures as your substrate tubes and add 150 mL of tap water to each.
</div></li><li data-sub-note-item="3-4"><div class="lab-step">Using a hot plate and a thermometer, bring the beakers to 25, 35, 45 and 60 &#176;C, as per their labels.
</div></li><li data-sub-note-item="3-5"><div class="lab-step">Prepare five more pairs of substrate and enzyme tubes, as described for the baseline reaction, using distilled water in the enzyme tube, instead of a pH buffer.
</div></li><li data-sub-note-item="3-6"><div class="lab-step">Label the substrate tubes as &#8220;Ice&#8221;, &#8220;25 &#176;C&#8221;, &#8220;35 &#176;C&#8221;, &#8220;45 &#176;C&#8221;, and &#8220;60 &#176;C&#8221;.
</div></li><li data-sub-note-item="3-7"><div class="lab-step">When all of the water baths have reached their appropriate temperatures, mix the substrate and enzyme solutions together by first pouring each substrate tube contents into an enzyme tube and then pouring the enzyme tube contents back into the substrate tube.
</div></li><li data-sub-note-item="3-8"><div class="lab-step">Cover the substrate tubes with sealing film and observe the colors at time 0.
</div></li><li data-sub-note-item="3-9"><div class="lab-step">Then, stand the tubes in their corresponding beaker baths, based on the temperature label each tube was given and start a timer.
</div></li><li data-sub-note-item="3-10"><div class="lab-step">For each 1-minute interval, remove the tubes from the water and record their color, using the stabdard as a reference.
</div></li><li data-sub-note-item="3-11"><div class="lab-step">Then, place them into their baths until each time point, up to 5 minutes, has been recorded.
</div></li></ul></li><li data-note-item="4" data-parent-collapse="">Results
<button class="expand">Expand</button><ul class="lab-items"><li data-sub-note-item="4-1"><div class="lab-step">To determine the baseline for the reaction, graph the change in color intensity against time. NOTE: The number of units that the color increased by over the 5-minute time period will serve as the baseline rate of reaction for relative comparison.
</div></li><li data-sub-note-item="4-2"><div class="lab-step">Now graph the difference in color intensity against the pH levels used in the first experiment.
</div></li><li data-sub-note-item="4-3"><div class="lab-step">Add in the baseline for comparison and then determine whether the optimal reaction rate was higher than, lower than or equal to the baseline.
</div></li><li data-sub-note-item="4-4"><div class="lab-step">Using this graph, determine the optimum pH for the reaction. Note: The reaction may not occur at every pH. The optimum pH for the peroxidase enzyme is typically in the range of 6 - 7. By exposing the enzyme to a substrate with different levels of pH, you will notice a drop-off in the reaction rate as the pH of the enzyme becomes more basic, making the enzyme more unstable.
</div></li><li data-sub-note-item="4-5"><div class="lab-step">Finally, graph the difference in color intensity over time for the different temperatures used in the second experiment, using the same baseline for comparison.
</div></li><li data-sub-note-item="4-6"><div class="lab-step">Using this graph, determine the optimum temperature for the reaction and compare it to the baseline. NOTE: The reaction may not occur at every temperature. In this experiment, the reaction appears most efficient around 45 &#176;C, and above this, the peroxidase enzyme begins to denature. For this reason, it should not produce a color change in the 60 &#176;C beaker. The reaction rate will also slow or stop in the ice bath, as there is not enough energy for the enzyme to catalyze the reaction.
</div></li></ul></li></ol>

Enzyme Activity: Effect of pH and Temperature on Peroxidase Activity - Procedure

Baseline Peroxidase Enzyme Experiment
ExpandTo begin, the baseline for the peroxidase enzyme reaction must be determined. Make the substrate by adding 7 ...

Transpiration

<ol class="note-items"><li data-note-item="1" data-parent-collapse="">Comparing Transpiration Rates of Leaves
<button class="expand">Expand</button><ul class="lab-items"><li data-sub-note-item="1-1"><div class="lab-step">To begin the experiment to measure the transpiration rates of leaves from different plant species, you will first create a tool called a potometer, using a piece of plastic tubing and 5 mL calibrated pipette.
</div></li><li data-sub-note-item="1-2"><div class="lab-step">To assemble the device, simply attach the rubber tubing to the tip of the pipette.
</div></li><li data-sub-note-item="1-3"><div class="lab-step">Then, submerge the device into a bucket of water and move the pipette around under the water to fully fill the tube, taking care not to create bubbles.
</div></li><li data-sub-note-item="1-4"><div class="lab-step">Next, cap the open end of the tube with a pipette bulb to keep the water in.
</div></li><li data-sub-note-item="1-5"><div class="lab-step">Now, taking care not to spill water from the potometer, use a clamp and stand to hold the capped end of the pipette around 1 - 3 inches below the pipette bulb.
</div></li><li data-sub-note-item="1-6"><div class="lab-step">Then, use a second clamp attached lower on the same stand to secure the open end of the rubber tubing.
</div></li><li data-sub-note-item="1-7"><div class="lab-step">Next, use a transfer pipette or dropper to remove water from the open end of the tubing until the water level in the pipette portion of the potometer rests at the zero line. HYPOTHESES: In this experiment, the experimental hypothesis might be that leaves from plant species more adapted to hotter or arid environments will have lower transpiration rates than those from humid or wet regions. The null hypothesis would be that leaves of the different plant species will not differ in their transpiration rates.
</div></li><li data-sub-note-item="1-8"><div class="lab-step">To begin the experiment, carefully use scissors to cut the end of the petiole, or stalk, of the first leaf and then insert the leaf into the tubing.
</div></li><li data-sub-note-item="1-9"><div class="lab-step">Then, place a layer of lubricant around the area where the plant stem and rubber tubing meet to make a watertight seal.
</div></li><li data-sub-note-item="1-10"><div class="lab-step">Next, remove the pipette bulb and taking care not to disturb the setup, let the experiment run for the appropriate experimental period, typically 30 - 60 minutes.
</div></li><li data-sub-note-item="1-11"><div class="lab-step">At the end of the allocated time, record the level of the water in the pipette, noting how much the level dropped in milliliters.
</div></li><li data-sub-note-item="1-12"><div class="lab-step">Then, refill and reset the potometer water level to zero, and then repeat the procedure for each of the remaining test leaves until the transpiration rates of all four leaves have been measured.
</div></li><li data-sub-note-item="1-13"><div class="lab-step">Next, to determine the surface area of the leaf samples, lay each leaf on a blank piece of paper and carefully trace the outlines.
</div></li><li data-sub-note-item="1-14"><div class="lab-step">Then, cut the outlines out and record the mass of each tracing.
</div></li><li data-sub-note-item="1-15"><div class="lab-step">Finally, cut out and weigh one four by four centimeter square of the same type of paper used to make the leaf tracings. This will act as a reference with known area and weight.
</div></li><li data-sub-note-item="1-16"><div class="lab-step">Next, you will need to quantify the number of stomata per unit area on each leaf. To do this, first prepare a slide mount by painting at least one square centimeter of the underside of each leaf with clear nail polish. IMPORTANT: Ensure you paint the bottom of the leaf, since the majority of the stomata are located there. 
</div></li><li data-sub-note-item="1-17"><div class="lab-step">When the nail polish is completely dry, carefully press a small piece of cellophane tape directly onto each patch and then gently peel off the tape to remove the painted on polish. NOTE: Use a piece of tape that is smaller than the microscope slide. 
</div></li><li data-sub-note-item="1-18"><div class="lab-step">Then, place each impression and tape onto a separate, clean microscope slide. HYPOTHESES: In this exercise, the alternative hypothesis could be that the leaves of the plants adapted to drier environments will have fewer stomata per unit area than the leaves of plants adapted to wetter environments. The null hypothesis might be that all plants species will have an equal number of stomata per unit area.
</div></li><li data-sub-note-item="1-19"><div class="lab-step">To observe each impression under the microscope, first use a low magnification to find areas of the leaf impression that contain stomata. These will appear as darker dots or impressions.
</div></li><li data-sub-note-item="1-20"><div class="lab-step">Then, switch to a higher magnification, keeping the sample viewing window centered on the stomata-containing area.
</div></li><li data-sub-note-item="1-21"><div class="lab-step">Draw and label your observations, making sure to label each sketch with the plant species.
</div></li><li data-sub-note-item="1-22"><div class="lab-step">Then, count and record the number of stomata in the field of view. You should perform this count a total of four times in four different areas of the leaf impression, and then determine the average number of stomata per counted area on the leaf.
</div></li><li data-sub-note-item="1-23"><div class="lab-step">To calculate the number of stomata per mm<sup>2</sup>, place a transparent plastic ruler on the microscope stage under the objective and measure the diameter of the field of view.
</div></li><li data-sub-note-item="1-24"><div class="lab-step">You can then use this number, the diameter or D, to calculate the area of the field of view in mm<sup>2</sup>.
</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">First, calculate the surface area of the 4x4 cm paper square to obtain the number of square centimeters.
</div></li><li data-sub-note-item="2-2"><div class="lab-step">Then, divide the weight of the paper square by the number of centimeters squared it covers to obtain the weight of 1 cm<sup>2</sup> of paper.
</div></li><li data-sub-note-item="2-3"><div class="lab-step">Finally, to calculate the surface area of each leaf, divide the weight of the leaf tracing by the weight of 1 cm<sup>2</sup> of paper. This will give you leaf area in centimeters squared.
</div></li><li data-sub-note-item="2-4"><div class="lab-step">You should now convert this number to meters squared, the standard unit used for calculating transpiration rate.
</div></li><li data-sub-note-item="2-5"><div class="lab-step">To calculate the transpiration rate for each leaf, divide the total water loss you measured in the potometer in mL by the time the experiment was run for in minutes.
</div></li><li data-sub-note-item="2-6"><div class="lab-step">Then, divide this by the surface area of the leaf in m<sup>2</sup>.
</div></li><li data-sub-note-item="2-7"><div class="lab-step">Make a bar graph with the four different leaf species on the X axis, and their corresponding transpiration rates on the Y axis. Do these rates appear different?
</div></li><li data-sub-note-item="2-8"><div class="lab-step">Next, add a second Y axis to your chart and plot the average number of stomata per millimeter squared for each leaf that was observed under the microscope. Do you see a correlation between transpiration rate and number of stomata? If so, does this seem to have any relationship to the native environment the tree would typically inhabit?
</div></li></ul></li></ol>

Assessing Transpiration Rates using Potometer - Procedure

Comparing Transpiration Rates of Leaves
ExpandTo begin the experiment to measure the transpiration rates of leaves from different plant species, you will ...