Name: a simple pit assay protocol to visualize and quantify osteoclastic resorption in vitro
Uploaded: 2022-06-16T13:40:00.000+00:00
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Description: Watch this Scientific Journal Video about A Simple Pit Assay Protocol to Visualize and Quantify Osteoclastic Resorption In Vitro at JoVE.com

<p class="jove_content">This work was partially funded by the China Scholarship Council [CSC No. 201808440394]. W.C. was financed by CSC.</p>

<p class="jove_content">The protocol was reviewed and approved by the local ethics committee (approval number 287/2020B02).</p>
<p class="jove_title"><strong>1. Preparation of calcium phosphate coated cell culture plates</strong></p>
<ol>
	<li>Preparation of calcium stock solution (25 mM CaCl<sub>2</sub>&#183;2H<sub>2</sub>O, 1.37 M NaCl, 15 mM MgCl<sub>2</sub>&#183;6H<sub>2</sub>O in Tris buffer)
	<ol>
		<li>Prepare 50 mM Tris buffer and adjust pH to 7.4 using 1 M HCl.</li>
		<li>Set up a glass beaker on a magnetic stirrer and add 100 mL of 50 mM Tris buffer.</li>
		<li>Weigh out 0.368 g of CaCl<sub>2</sub>&#183;2H<sub>2</sub>O, 8.0 g of NaCl, and 0.305 g of MgCl<sub>2</sub>&#183;6H<sub>2</sub>O and dissolve in Tris buffer one by one.</li>
		<li>Adjust pH to 7.4 using 1 M HCl. Store at room temperature.</li>
	</ol>
	</li>
	<li>Preparation of phosphate stock solution (11.1 mM Na<sub>2</sub>HPO<sub>4</sub>, 42 mM NaHCO<sub>3</sub> in Tris buffer)
	<ol>
		<li>Set up a glass beaker on a magnetic stirrer and add 100 mL of 50 mM Tris buffer.</li>
		<li>Weigh out 0.158 g of Na<sub>2</sub>HPO<sub>4</sub>&#160;and 0.353 g of NaHCO<sub>3</sub> and dissolve in Tris buffer one by one.</li>
		<li>Adjust pH to 7.4 using 1 M HCl. Store at room temperature.</li>
	</ol>
	</li>
	<li>Pre-calcification of 96-well cell culture plates
	<ol>
		<li>Prepare 30 mL of working solution by mixing 15 mL of 50 mM Tris buffer, 7.5 mL of calcium stock (step 1.1.4) solution, and 7.5 mL of phosphate stock solution (step 1.2.3). Filter the solution with a 0.2 &#181;m filter.</li>
		<li>Prepare a 96-well cell culture plate with flat bottom. Pipette 300 &#181;L of calcium phosphate solution (step 1.3.1.) to each well. Cover the plate with a lid and incubate the plate at 37 &#176;C for 3 days.</li>
	</ol>
	</li>
	<li>Preparation of calcium phosphate solution (2.25 mM Na<sub>2</sub>HPO<sub>4</sub>, 4 mM CaCl<sub>2</sub>&#183;2H<sub>2</sub>O, 0.14 M NaCl, 50 mM Tris base in ddH<sub>2</sub>O)
	<ol>
		<li>Add 2 mL of 1 M HCl to 40 mL of deionized water in a beaker with a magnetic stirring bead and dissolve 0.016 g of Na<sub>2</sub>HPO<sub>4</sub>, 0.0295 g of CaCl<sub>2</sub>&#183;2H<sub>2</sub>O, 0.409 g of NaCl, and 0.303 g of Tris base one by one.</li>
		<li>Adjust pH to 7.4 using 1 M HCl. Add deionized water to fill the volume to 50 mL. Filter the solution with a 0.2 &#181;m filter.</li>
	</ol>
	</li>
	<li>Calcification of 96-well cell culture plates
	<ol>
		<li>Aspirate pre-calcification solution from the pre-calcified 96-well cell culture plates and add 300 &#181;L of calcium phosphate solution (step 1.4.2) to each well. Cover the plates with a lid and incubate at 37 &#176;C for 1 day.</li>
		<li>Turn the plate over to pour out the solution and wash the plate three times with deionized water thoroughly. Dry the plate immediately using a hair dryer or compressed CO<sub>2</sub> or N<sub>2</sub> gas to maintain a uniform surface.</li>
		<li>Sterilize the coated plate by UV radiation in a clean bench for 1 h. Use the coated plate immediately or seal the plate with parafilm and store it at room temperature.</li>
	</ol>
	</li>
</ol>
<p class="jove_title"><strong>2. Isolation of PBMCs from human peripheral blood</strong></p>
<ol>
	<li>Draw 15 mL of blood from the vein after obtaining written informed consent from the blood donor (ethical vote: 287/2020B02).</li>
	<li>Dilute 15 mL of fresh blood with an equal volume of PBS and mix by inverting the tube several times or by drawing the mixture in and out of a pipette.</li>
	<li>Prepare a 50 mL conical tube containing 15 mL of density gradient solution (e.g., Ficoll, <strong>Table of Materials</strong>) per tube. Tilt the tube and carefully layer 30 mL of diluted blood sample onto the 15 mL of density gradient solution (diluted blood sample: density gradient solution, 1:0.5-1 ratio).<br />
	NOTE: When overlaying the sample, pay attention not to mix the density gradient solution with the diluted blood sample.</li>
	<li>Centrifuge at 810 x <em>g</em> for 20 min at 20 &#176;C in a swinging-bucket rotor without brake.</li>
	<li>Aspirate the upper layer leaving the mononuclear cell layer (lymphocytes, monocytes, and thrombocytes) undisturbed at the interphase.</li>
	<li>Carefully transfer the mononuclear cell layer to a new 50 mL conical tube.</li>
	<li>Fill the tube with PBS, mix, and centrifuge at 300 x <em>g</em> for 10 min at 20 &#176;C (brake can be used from this stage onwards).</li>
	<li>Carefully remove supernatant completely. Resuspend the cell pellet in 50 mL of PBS and centrifuge at 300 x <em>g</em> for 10 min at 20 &#176;C. Carefully remove the supernatant completely.</li>
	<li>For removal of platelets, resuspend the cell pellet in 50 mL of PBS and centrifuge at 200 x <em>g </em>for 10 min at 20 &#176;C. Carefully remove the supernatant completely.</li>
	<li>Transfer resuspended cells to cell culture flasks for expansion of OC progenitors.</li>
</ol>
<p class="jove_title"><strong>3. Expansion of OC progenitors</strong></p>
<ol>
	<li>Resuspend PBMCs in complete &#945;-MEM (10% FBS, 1% Pen/Strep, 1% amphotericin B) containing 20 ng/mL M-CSF. Seed PBMCs at a density of 2.5 x 10<sup>5</sup> cells/cm<sup>2</sup>. Usually, cells isolated from 15-20 mL fresh blood can be seeded in one 75 cm<sup>2</sup> flask (1.5-2 x 10<sup>7</sup> cells).</li>
	<li>Feed the cells with fresh complete &#945;-MEM containing 20 ng/mL M-CSF every third day until the attached cells reach a desired confluency. Typical yields are 1.5-2 x 10<sup>6</sup> cells/flask when cells are 95% confluent. Usually, the expansion period lasts 6 days.<br />
	&#8203;NOTE: The osteoclastogenic potential of the precursors can decrease with prolonged cultivation time.</li>
</ol>
<p class="jove_title"><strong>4. Induction of osteoclastogenesis in CaP coated plates</strong></p>
<ol>
	<li>To facilitate cell adhesion, incubate the CaP coated plates with 50 &#181;L of FBS for 1 h in a 37 &#176;C incubator.</li>
	<li>To detach the OC precursors, wash the flask with PBS twice to remove dead or non-adherent cells. Add 4 mL of trypsin (<strong>Table of Materials</strong>) per 75 cm<sup>2</sup> flask, for 30 min.
	<ol>
		<li>Add 4 mL of complete &#945;-MEM to stop the digestion, carefully detach the cells using a cell scraper and transfer the cells into a 50 mL tube.</li>
		<li>Determine the total cell number using a Neubauer chamber or similar.</li>
	</ol>
	</li>
	<li>Pellet the cells by centrifugation for 7 min at 350 x <em>g</em>. Resuspend the pellet in sufficient complete &#945;-MEM containing 20 ng/mL M-CSF and 20 ng/mL RANKL to get a concentration of 1 x 10<sup>6</sup> cells/mL.</li>
	<li>Aspirate FBS solution from the CaP coated 96-well plate and pipette 200 &#181;L of cell suspension per well (2 x 10<sup>5</sup> cells/well).</li>
	<li>Incubate the OC precursors for the desired time period or with the desired reagents relevant to the experimental design. Usually, high numbers of large and multinucleated OCs can be observed after 6 days and when resorption pits are forming. Feed cells with fresh complete &#945;-MEM medium containing 20 ng/mL M-CSF and 20 ng/mL RANKL every third day.</li>
	<li>At the end of incubation, wash cells with PBS twice, fix with 4% paraformaldehyde for 10 min, and wash with PBS again.</li>
	<li>Use the fixed OCs directly for fluorescence staining or store at 4 &#176;C.</li>
</ol>
<p class="jove_title"><strong>5. Fluorescence staining of OCs and CaP coating</strong></p>
<ol>
	<li>Incubate the fixed cells with permeabilization buffer (0.1% Triton in PBS) for 5 min.</li>
	<li>Stain actin filaments with 100 &#181;L of AlexaFluor 546 labeled phalloidin solution in PBS for 30 min and aspirate the staining solution. Add 100 &#181;L of Hoechst 33342 staining solution (10 &#181;g/mL in PBS) for 10 min to stain nuclei. DAPI can also be used.</li>
	<li>Stain CaP coating with 100 &#181;L of 10 &#181;M calcein in PBS for 10 min. Wash three times with PBS and take images.</li>
	<li>After fluorescence imaging the same plates can be used to quantify resorption pit area by Von Kossa staining. To do so, wash the plates with deionized water twice.</li>
</ol>
<p class="jove_title"><strong>6. Quantification of total resorption pit area</strong></p>
<ol>
	<li>To stain CaP coating with Von Kossa staining, incubate with 50 &#181;L of 5% AgNO<sub>3</sub> in deionized water per well under UV radiation for 1 h, until the coating on the bottom of the wells has turned brown.</li>
	<li>Wash the plate three times with deionized water and take brightfield images. An objective with little magnification, such as a 1.25x objective, can be used to capture the whole well in one image. This facilitates subsequent image analysis. Alternative methods to capture the whole area of the well can be applied.</li>
	<li>Open an image file with ImageJ: <strong>File | Open | Image | Type | 8-bit</strong>. Verify the scale unit in the lower right corner of the image using <strong>Straight Line | Analyze | Set Scale</strong>. Enter the length in the &#34;known distance&#34; blanket, enter new scale unit in <strong>Unit of length</strong>, and check the box <strong>Global</strong>.</li>
	<li>List measurement parameters to be analyzed: <strong>Analyze | Set Measurements</strong>. In the Set Measurements window, check the Area and Limit to threshold boxes.</li>
	<li>Measure the area of pits: <strong>Image | Adjust | Threshold</strong>. In the Threshold window, check box <strong>Dark background</strong> and click <strong>Auto</strong>. The area of pits turns red. Close the Threshold window and select <strong>Analyze | Measure</strong>. Save the result file: <strong>File | Save As</strong>.</li>
</ol>
<p class="jove_title"><strong>7. Quantification of OC number and size, and normalized resorption pit area</strong></p>
<ol>
	<li>Open a fluorescence image of osteoclasts with ImageJ and verify the scale unit (see step 6.3).</li>
	<li>List measurement parameters to be analyzed: <strong>Analyze | Set Measurements</strong>. In the Set Measurements window, check the <strong>Area </strong>box.</li>
	<li>Outline the OCs using ROI Manager and Polygon selections: <strong>Analyze | Tools | ROI Manager</strong>. Click Polygon selections in toolset, outline one OC (cells with actin ring and &#8805; three nuclei) and click <strong>Add [t]</strong> in ROI Manager window. Repeat outlining until all OCs are included.</li>
	<li>Measure number and size of OCs: Select all items in ROI Manager and click <strong>Measure</strong>. Save the result file: <strong>File | Save As</strong> (<strong class="xfig">Figure 3E</strong>).</li>
	<li>Measure the pit area of fluorescence images. Open the fluorescent image of coating correlated with the image in step 7.3 and verify the scale unit (see step 6.3).
	<ol>
		<li>List measurement parameters to be analyzed (see step 6.4). Select <strong>Image | Adjust | Threshold</strong>. In the Threshold window, uncheck all boxes and click <strong>Auto</strong>. The area of the pits turns red. Close the <strong>Threshold</strong> window and select <strong>Analyze | Measure</strong>. Save the result file.</li>
	</ol>
	</li>
	<li>Calculate normalized pit area by OC number.</li>
</ol>

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<p class="jove_content">The authors have nothing to disclose.</p>

<p class="jove_content">Here we describe a simple and reliable method for an osteoclastic resorption assay using OCs derived and expanded <em>in vitro</em> from PBMCs. The used CaP coated cell culture plates can be easily prepared and visualized using lab-available materials. In addition to unsorted PBMCs adopted in this protocol, OCs generated from murine monocytic cells<sup class="xref">21</sup> and bone marrow macrophage cells<sup class="xref">17</sup> have also been cultured on similar synthetic substrates for pit assay, thus these cell sources can be moved to this approach referring to the corresponding literature.</p>
<p class="jove_content">For this protocol, we used a simple in-lab fabricated CaP coating instead of expensive commercially available assay plates. Although bone and dentin slices are two common and affordable resorbable materials for resorption pit assay, the availability and complicated preparation are major drawbacks. As an easily prepared synthetic substrate, CaP coating is more convenient and readily available than the two original materials. Another advantage of this pit assay approach is that cells can be visualized under a bright field microscope during culture, whereas cells growing on opaque bone and dentin slices cannot be observed.</p>
<p class="jove_content">This CaP coating method can be flexibly adapted according to the required experimental needs to different cell culture plate sizes.</p>
<p class="jove_content">Unlike previously reported<sup class="xref">17</sup><sup>,</sup><sup class="xref">21</sup>, we incubate the plates with coating solution at 37 &#176;C instead of at room temperature, resulting in the spontaneous growth of apatite nuclei on cell culture plates at body temperature. CaP coated plates are available for experiments immediately after being dried and sterilized by UV radiation, which saves time compared to previously reported methods<sup class="xref">17</sup><sup>,</sup><sup class="xref">21</sup>.</p>
<p class="jove_content">Filtration of the coating solution (Step 1.3.1 and 1.4.2) is a crucial step, which helps to obtain a uniform particle size and reduce uncoated spots within the coating caused by air bubbles and impurities in the solution. Drying the plate immediately (step 1.5.2) is another critical step, which helps to make a uniform CaP coating. Otherwise, it is prone to form a thicker coating in the middle of the well.</p>
<p class="jove_content">It is worth pointing out that the thickness of the coating and the density of calcium phosphate deposits depend on the volume of coating solution within a certain range. Therefore, it is possible to control coating thickness by changing the volume of coating solution in the well. However, it is suggested to add as much volume of coating solution as possible when the 96-well cell culture plate is used because of the small total volume for this culture plate format. The inner wall of the well is also coated by CaP during both coating steps, therefore caution is required to not touch the bottom nor the wall with the pipette in order to protect the coating from damage.</p>
<p class="jove_content">According to our observation, most of OC precursors expanded from PBMCs are able to attach to the CaP coating without any problems. However, soaking the coated plate with FBS before cell seeding improves the cell adhesion efficiency, as previously reported<sup class="xref">21</sup>.</p>
<p class="jove_content">Detaching the OC precursors gently using a cell scraper (step 4.2) is also important because most of the OC precursors were still adherent, and minimizing the mechanical injury is favorable for cell viability.</p>
<p class="jove_content">A limitation of this method is that the bulk PBMCs that we used as a source of OC precursors is a highly mixed cellular source, of which only a small fraction of cells (CD14<sup>+</sup> cells) are actual OC precursors. Since other cells capable of affecting osteoclastogenesis (such as stromal cells and lymphocytes) are present amongst isolated PBMCs, this may impact assay results in ways that may be hard to predict, and might confound assay interpretation. To better investigate direct effects of growth factors, cytokines or glucocorticoids on resorptive activity of OCs, methods of OC precursor purification (e.g., fluorescence-activated cell sorting (FACS) and magnetic beads purification<sup class="xref">20</sup> based on specific cell-surface markers<sup class="xref">23</sup><sup>,</sup><sup class="xref">24</sup><sup>,</sup><sup class="xref">25</sup><sup>,</sup><sup class="xref">26</sup>) are recommended.</p>
<p class="jove_content">The limitations of the CaP substrate in obtaining brightfield images of both cells and underlying pits for the same area is caused by the incompatibility of TRAP and Von Kossa staining, so that normalized resorption area of the entire well is not available. But instead, fluorescence imaging can be used to visualize resorption area and cell number to obtain normalized resorption data. These data may provide important insight into whether the increase in total resorption area under experimental conditions is due to increased osteoclast number alone or increased resorption capacity of individual cells. In addition, it allows the study of the relative effects of osteoclast number, size, nucleus number, and resorption capacity.</p>
<p class="jove_content">In summary, we describe a useful and simple protocol for a resorption pit assay using a two-step calcium phosphate coating and OCs derived from primary human PBMCs. This protocol provides an easy method to establish an <em>in vitro</em> bone resorption model for studies related to osteoclastic resorption, and could be applied to study treatments for bone disorder diseases.</p>

<p><a href="https://app.jove.com/t/6601">This corrects</a> the article <a href="https://dx.doi.org/10.3791/64016">10.3791/64016</a></p>

<p>An erratum was issued for:&#160;<a href="https://www.jove.com/t/64016">A Simple Pit Assay Protocol to Visualize and Quantify Osteoclastic Resorption <em>In Vitro</em></a>. The Protocol section was&#160;updated.</p>

Erratum: A Simple Pit Assay Protocol to Visualize and Quantify Osteoclastic Resorption <em>In Vitro</em>

<p class="jove_content">Osteoclasts (OCs) are tissue-specific macrophages derived from hematopoietic stem cells (HSCs), playing a pivotal role in bone remodeling together with osteoblasts<sup class="xref">1</sup>. Sex hormone-induced, immunological, and malignant bone disorders that destroy bone systemically or locally are due to excess osteoclastic activity, including menopause-related osteoporosis<sup class="xref">2</sup>, rheumatoid arthritis<sup class="xref">3</sup>, periodontal disease<sup class="xref">4</sup>, myeloma bone disease<sup class="xref">5</sup>, and osteolytic bone metastasis<sup class="xref">6</sup>. In contrast, defects in OC formation and function can also cause osteopetrosis<sup class="xref">7</sup>. HSCs undergo differentiation into OC progenitors under macrophage colony-stimulating factor (M-CSF, gene symbol ACP5) stimulation. In the presence of both M-CSF and receptor activator of NF-&#954;B ligand (RANKL, gene symbol TNFSF11), OC progenitors differentiate further into mononuclear OCs and subsequently fuse to become multinucleated OCs<sup class="xref">8</sup><sup>,</sup><sup class="xref">9</sup><sup>,</sup><sup class="xref">10</sup>. Both cytokines M-CSF and RANKL are indispensable and sufficient for induction of osteoclastic markers such as calcitonin receptor (CT), receptor activator of nuclear factor &#954; B (RANK), proton pump V-ATPase, chloride channel 7 alpha subunit (CIC-7), integrin &#946;3, tartrate-resistant acid phosphatase (TRAP, gene symbol ACP5), lysosomal cysteine protease cathepsin K (CTSK), and matrix metallopeptidase 9 (MMP9). Activated OCs form a sealing zone on the bone surface through the formation of an actin ring with a ruffled border<sup class="xref">11</sup><sup>,</sup><sup class="xref">12</sup>. Within the sealing zone, OCs mediate resorption through secreting protons <em>via</em> the proton pump V-ATPase<sup class="xref">12</sup><sup>,</sup><sup class="xref">13</sup>, MMP9<sup class="xref">14</sup>, and CTSK<sup class="xref">15</sup>, leading to the formation of lacunae.</p>
<p class="jove_content">For <em>in vitro</em> experiments, OC progenitors can be obtained by expansion of bone marrow macrophages from mice&#39;s femur and tibia<sup class="xref">16</sup><sup>,</sup><sup class="xref">17</sup>, as well as by isolation of human peripheral blood mononuclear cells (PBMCs) from blood samples and buffy coats<sup class="xref">18</sup><sup>,</sup><sup class="xref">19</sup><sup>,</sup><sup class="xref">20</sup>, or by differentiation of the immortalized murine monocytic cells RAW 264.7<sup class="xref">21</sup><sup>,</sup><sup class="xref">22</sup>.</p>
<p class="jove_content">In the present protocol, we describe an osteoclastic resorption assay in CaP coated cell culture plates using OCs derived from primary PBMCs. The CaP coated cell culture plates method used here are adopted and refined from the method described previously by Patntirapong et al.<sup class="xref">17</sup> and Maria et al.<sup class="xref">21</sup>. To obtain OC precursors, PBMCs are isolated by density gradient centrifugation and expanded as described previously<sup class="xref">20</sup>.</p>

<table><tbody><tr><td>AgNO&lt;sub&gt;3&lt;/sub&gt;</td><td>SERVA Electrophoresis GmbH</td><td>35110</td><td>Silver nitrate</td></tr><tr><td>a-MEM</td><td>Gibco</td><td>32561-029</td><td>MEM alpha, GlutaMAX, no nucleosides</td></tr><tr><td>amphotericin B</td><td>Biochrom</td><td>03-028-1B</td><td>Amphotericin B Solution</td></tr><tr><td>CaCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Sigma-Aldrich</td><td>21097-50G</td><td>Calcium chloride Dihydrate</td></tr><tr><td>Calcein</td><td>Sigma-Aldrich</td><td>C0875</td><td>Calcein</td></tr><tr><td>FBS</td><td>Sigma-Aldrich</td><td>F7524</td><td>fetal bovine serum</td></tr><tr><td>Ficoll</td><td>Cytiva</td><td>17144002</td><td>Ficoll Paque Plus</td></tr><tr><td>Fixation buffer</td><td>Biolegend</td><td>420801</td><td>Paraformaldehyde</td></tr><tr><td>HCl</td><td>Merk</td><td>1.09057.1000</td><td>Hydrochloric acid</td></tr><tr><td>Hoechst 33342</td><td>Promokine</td><td>PK-CA707-40046</td><td>Hoechst 33342</td></tr><tr><td>M-CSF</td><td>PeproTech</td><td>300-25</td><td>Recombinant Human M-CSF</td></tr><tr><td>MgCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Sigma-Aldrich</td><td>7791-18-6</td><td>Magnesium chloride</td></tr><tr><td>Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;</td><td>AppliChem GmbH</td><td>A2943,0250</td><td>di- Sodium hydrogen phosphate anhydrous</td></tr><tr><td>NaCl</td><td>Merk</td><td>S7653-250G</td><td>Sodium chloride</td></tr><tr><td>NaHCO&lt;sub&gt;3&lt;/sub&gt;</td><td>Merk</td><td>K15322429</td><td>Bicarbonate of Soda</td></tr><tr><td>PBS</td><td>Lonza</td><td>17-512F</td><td>Dulbecco&#039;s Phosphate Buffered Saline (1X), DBPS without Calcium and Magnesium</td></tr><tr><td>Pen-Strep</td><td>Lonza</td><td>DE17-602E</td><td>Penicillin-Streptomycin Mixture</td></tr><tr><td>Phalloidin-Alexa Fluor 546</td><td>Invitrogen</td><td>A22283</td><td>Alexa Fluor 546 Phalloidin</td></tr><tr><td>RANKL</td><td>PeproTech</td><td>310-01</td><td>Recombinant Human sRANK Ligand (E.coli derived)</td></tr><tr><td>Tris</td><td>Sigma-Aldrich</td><td>93362</td><td>Tris(hydroxymethyl)aminomethan</td></tr><tr><td>Triton X-100</td><td>Sigma-Aldrich</td><td>T8787</td><td>Alkyl Phenyl Polyethylene Glycol</td></tr><tr><td>TrypLE Express</td><td>Gibco</td><td>12605010</td><td>Recombinant cell-dissociation enzymes</td></tr></tbody></table>

a simple pit assay protocol to visualize and quantify osteoclastic resorption <em>in vitro</em>

<p class="jove_content">Mature osteoclasts are multinucleated cells that can degrade bone through the secretion of acids and enzymes. They play a crucial role in various diseases (e.g., osteoporosis and bone cancer) and are therefore important objects of research. <em>In vitro</em>, their activity can be analyzed by the formation of resorption pits. In this protocol, we describe a simple pit assay method using calcium phosphate (CaP) coated cell culture plates, which can be easily visualized and quantified. Osteoclast precursors derived from human peripheral blood mononuclear cells (PBMCs) were cultured on the coated plates in the presence of osteoclastogenic stimuli. After 9 days of incubation, osteoclasts were fixed and stained for fluorescence imaging while the CaP coating was counterstained by calcein. To quantify the resorbed area, the CaP coating on plates was stained with 5% AgNO<sub>3</sub> and visualized by brightfield imaging. The resorption pit area was quantified using ImageJ. </p>

<p class="jove_content">The calcium phosphate coating on the bottom of cell culture plates was performed in two coating steps comprising a 3-day pre-calcification and a 1-day calcification step. As shown in <strong class="xfig">Figure 1</strong>, uniformly distributed calcium phosphate was obtained on the bottom of the 96-well plates. The coating adhered very well to the bottom after the performed washing steps.</p>
<p class="jove_content biglegend" fo:keep-together.within-page="1"><img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/64016/64016fig01.jpg" /><br />
<strong class="xfig">Figure 1</strong><strong>: Representative brightfield image of the calcium phosphate coating on 96-well cell culture plates.</strong> The coating was carried out in two coating steps comprising a 3-day pre-calcification step and a 1-day calcification step. The scale bar represents 200 &#181;m. <a href="https://www.jove.com/files/ftp_upload/64016/64016fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a></p>
<p class="jove_content">OC precursors derived from human PBMCs were cultured on the CaP coated plates. Resorption pits and large OCs (blank area) were formed in the presence of M-CFS and RANKL after 9 days of culture (<strong class="xfig">Figure 2A</strong>). The multinucleated OCs located in the pits of the CaP coating expressed high levels of TRAP (<strong class="xfig">Figure 2B</strong>).</p>
<p class="jove_content biglegend" fo:keep-together.within-page="1"><img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/64016/64016fig02.jpg" /><br />
<strong class="xfig">Figure 2</strong><strong>: TRAP expression by OCs after maturation on the CaP coated cell culture plates.</strong> OC precursors were cultured on the CaP coated plates in the presence of 20 ng/mL M-CSF and 20 ng/mL RANKL for 9 days. (<strong>A</strong>) Blank area represented resorption pits. (<strong>B</strong>) Several multinucleated TRAP-positive (purple) OCs were observed within the resorption pits. The scale bar represents 200 &#181;m. <a href="https://www.jove.com/files/ftp_upload/64016/64016fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a></p>
<p class="jove_content">To further characterize the maturation of OCs on the CaP coated plates, cells were stained for actin (red fluorescence). Cell nuclei were stained with Hoechst (blue) and calcein was used for calcium visualization in green. Resorption pits are visible as black areas in the green CaP coating (<strong class="xfig">Figure 3B</strong>). Functional mature OCs showed three or more nuclei and the characteristic actin ring which is essential for the osteoclastogenic resorption (<strong class="xfig">Figure 3A</strong><strong>,C</strong>). The pit area of fluorescence images can be measured using the thresholding tool of ImageJ (<strong class="xfig">Figure 3D</strong>). The number and size of OCs can be calculated using the ROI Manager and Polygon selection tool of ImageJ (<strong class="xfig">Figure 3E</strong>).</p>
<p class="jove_content biglegend" fo:keep-together.within-page="1"><img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/64016/64016fig03.jpg" /><br />
<strong class="xfig">Figure 3</strong><strong>: Morphology of mature OCs on the CaP coating.</strong> OC precursors were cultured on the CaP coated plates for 9 days in the presence of 20 ng/mL M-CSF and 20 ng/mL RANKL. (<strong>A</strong>) OCs were stained for actin and nuclei by phalloidin-Alexa Fluor 546 and Hoechst 33342. (<strong>B</strong>) CaP coating was stained by calcein. Black areas represent resorption pits. (<strong>C</strong>) Merged image. (<strong>D</strong>) Quantification of pit area (red area) using the thresholding tool of ImageJ. (<strong>E</strong>) OC outlining and counting using the ROI Manager and Polygon selection tool of ImageJ. The scale bar represents 500 &#181;m. <a href="https://www.jove.com/files/ftp_upload/64016/64016fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a></p>
<p class="jove_content">For the quantification of the resorption pits on the CaP coated cell culture plates, calcium was stained by incubation with 5% AgNO<sub>3</sub> (Von Kossa staining). As shown in <strong class="xfig">Figure 4A</strong>, OC precursors did not reach full functionality in the absence of RANKL and were not able to form resorption pits. Osteoclastogenic pits were observed only in the presence of both factors M-CSF and RANKL (<strong class="xfig">Figure 4B</strong>). The resorption pit area was quantified with the thresholding tool of ImageJ (<strong class="xfig">Figure 4C</strong>).</p>
<p class="jove_content biglegend" fo:keep-together.within-page="1"><img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/64016/64016fig04.jpg" /><br />
<strong class="xfig">Figure 4</strong><strong>: Visualization and quantification of resorption pits.</strong> OC precursors were cultured on the CaP coated 96-well cell culture plates in the absence of RANKL (<strong>A</strong>) and in the presence of both factors M-CSF and RANKL (<strong>B</strong>). (<strong>C</strong>) Numbers of formed resorption pits were quantified using the thresholding tool of ImageJ. Scale bar represents 1,000 &#181;m. <a href="https://www.jove.com/files/ftp_upload/64016/64016fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a></p>

Watch this Scientific Journal Video about A Simple Pit Assay Protocol to Visualize and Quantify Osteoclastic Resorption In Vitro at JoVE.com

A Simple Pit Assay Protocol to Visualize and Quantify Osteoclastic Resorption In Vitro

<p class="jove_content">Here, we present a simple and effective assay procedure for resorption pit assays using calcium phosphate coated cell culture plates.</p>

A Simple Pit Assay Protocol to Visualize and Quantify Osteoclastic Resorption <em>In Vitro</em>

Mature osteoclasts are multinucleated cells that can degrade bone through the secretion of acids and enzymes. They play a crucial role in various diseases ...

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5.8K Views.  University Hospital Tübingen. Here, we present a simple and effective assay procedure for resorption pit assays using calcium phosphate coated cell culture plates.

Video: A Simple Pit Assay Protocol to Visualize and Quantify Osteoclastic Resorption In Vitro

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

Accuracy, Precision, Uncertainty, and Significant Figures - Procedure

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

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

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/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/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/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 ...

Buffers

<p>Source: Smaa Koraym at Johns Hopkins University, MD, USA</p>
<ol class="note-items">
	<li data-note-item="1" data-parent-collapse=""><strong>Preparing 50 mM NaH<sub>2</sub>PO<sub>4</sub> Buffer, pH 7</strong><button class="expand">Expand</button>
	<p>In the first part of this experiment, you will prepare a sodium phosphate solution buffered at pH 7.0. Monosodium phosphate is a weak acid with the conjugate base, disodium phosphate. Unadjusted monosodium phosphate solutions usually have a pH of about 4 - 6.</p>
	<p>Buffers are most effective close to their pKa, which is 6.8 to 7.2 for monosodium phosphate. So, you will use NaOH to push the equilibrium towards the conjugate base without altering the overall composition of the buffer equilibrium.</p>
	<ul class="lab-items">
		<li data-sub-note-item="1-3">
		<div class="lab-step">Before starting the experiment, calculate the mass of monosodium phosphate that you will need to make 200 mL of a 50 mM solution.
		<h4><strong>Table 1: Preparation of 50 mM NaH<sub>2</sub>PO<sub>4</sub> Buffer, pH 7.0</strong></h4>
		<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always" height="247" width="358">
			<tbody>
				<tr>
					<td colspan="2"><strong>Molar mass of NaH<sub>2</sub>PO<sub>4</sub> = 119.98 g/mol</strong></td>
				</tr>
				<tr>
					<td>Solution volume (mL)</td>
					<td style="text-align: center;">200</td>
				</tr>
				<tr>
					<td>Molarity (mM)</td>
					<td style="text-align: center;">50</td>
				</tr>
				<tr>
					<td>Moles of NaH<sub>2</sub>PO<sub>4</sub> (mol)</td>
					<td style="text-align: center;"></td>
				</tr>
				<tr>
					<td>Mass needed (mg)</td>
					<td style="text-align: center;"></td>
				</tr>
				<tr>
					<td>Initial volume of 1 M NaOH (mL)</td>
					<td style="text-align: center;"></td>
				</tr>
				<tr>
					<td>Final volume of 1 M NaOH (mL)</td>
					<td style="text-align: center;"></td>
				</tr>
				<tr>
					<td>Volume of NaOH used (mL)</td>
					<td style="text-align: center;"></td>
				</tr>
				<tr>
					<td>Volume of DI water needed (mL)</td>
					<td style="text-align: center;"></td>
				</tr>
			</tbody>
		</table>
		<a href="https://www.jove.com/CDNSource/lm/labs/45/Table_1._Preparation_of_50_mM_NaH2PO4_Buffer,_pH_7.0.xlsx" target="_blank">Click Here to download Table 1</a></div>
		</li>
		<li data-sub-note-item="1-4">
		<div class="lab-step">To begin, put on the necessary personal protective equipment, including a lab coat, splash-resistant safety glasses, and gloves. <strong>Note: </strong>This section of the lab uses NaOH, which is corrosive and toxic. Use caution when pouring and transporting NaOH.</div>
		</li>
		<li data-sub-note-item="1-5">
		<div class="lab-step">Fill a 250-mL plastic wash bottle with deionized water.</div>
		</li>
		<li data-sub-note-item="1-6">
		<div class="lab-step">Label two 100-mL beakers, one for neutral aqueous waste and one for basic aqueous waste.</div>
		</li>
		<li data-sub-note-item="1-7">
		<div class="lab-step">Calibrate your pH meter using the provided buffers. Store the probe in its storage solution when you are done.</div>
		</li>
		<li data-sub-note-item="1-8">
		<div class="lab-step">Tare weighing paper, and use a clean spatula to measure out the required amount of monosodium phosphate according to your calculations. Record the exact amount of monosodium phosphate in your lab notebook. <strong>Note: </strong>Monosodium phosphate absorbs moisture from the air, so work quickly to obtain an accurate measurement and close the container tightly when you are done with it.</div>
		</li>
		<li data-sub-note-item="1-9">
		<div class="lab-step">Place the monosodium phosphate in a 400-mL beaker and clean the spatula with a laboratory wipe. Throw out the weighing paper and the laboratory wipe before returning to your fume hood.</div>
		</li>
		<li data-sub-note-item="1-10">
		<div class="lab-step">Use a graduated cylinder to measure out 175 mL of deionized water. Pour the water into the beaker of monosodium phosphate and add a magnetic stir bar.</div>
		</li>
		<li data-sub-note-item="1-11">
		<div class="lab-step">Stir the solution until the salt has dissolved completely and the solution appears homogeneous. This usually takes 2 - 3 min.</div>
		</li>
		<li data-sub-note-item="1-12">
		<div class="lab-step">Measure 15 mL of deionized water and pour it into the beaker. Continue stirring the solution until it appears homogeneous again (about 1 &#8211; 2 min). Turn off the stir motor.</div>
		</li>
		<li data-sub-note-item="1-13">
		<div class="lab-step">Rinse the pH probe with deionized water, and then clamp it in the solution with the sensor above the stir bar.</div>
		</li>
		<li data-sub-note-item="1-14">
		<div class="lab-step">Bring a 10-mL graduated cylinder and watch glass to the dispensing hood and measure out 10 mL of 1 M NaOH. Note the exact volume in the graduated cylinder.</div>
		</li>
		<li data-sub-note-item="1-15">
		<div class="lab-step">Cover your NaOH with the watch glass, and carefully bring it to your fume hood.</div>
		</li>
		<li data-sub-note-item="1-16">
		<div class="lab-step">Resume stirring the monosodium phosphate solution. While monitoring the pH reading, use a disposable pipette to slowly add NaOH to the stirring solution in a drop-wise manner. Once the buffer pH reaches 7.0, return any NaOH still in the pipette to the graduated cylinder.</div>
		</li>
		<li data-sub-note-item="1-17">
		<div class="lab-step">Calculate the volume of NaOH that you added to the buffer. Subtract that volume from 10 mL to determine how much deionized water you must add to your buffer to reach a total solution volume of 200 mL.</div>
		</li>
		<li data-sub-note-item="1-18">
		<div class="lab-step">Measure the deionized water with another 10-mL graduated cylinder and add it to the stirring solution to finish making the buffer.</div>
		</li>
		<li data-sub-note-item="1-19">
		<div class="lab-step">Rinse the pH sensor with deionized water, cover it with the storage solution filled cap, and unplug or turn off the probe.</div>
		</li>
		<li data-sub-note-item="1-20">
		<div class="lab-step">Label a 250-mL polyethylene bottle as &#39;50 mM monosodium phosphate buffer, pH 7.0&#39;. Retrieve the magnetic stir bar from the solution.</div>
		</li>
		<li data-sub-note-item="1-21">
		<div class="lab-step">Use a funnel to pour the buffer solution into the labeled bottle and cap it tightly.</div>
		</li>
	</ul>
	</li>
	<li data-note-item="2" data-parent-collapse=""><strong>Absorption Spectroscopy of Free and Bound Neutral Red</strong><button class="expand">Expand</button>
	<p>Buffers can be used to evaluate compounds at specific pH values. In this section, you will record the absorbance spectrum of the indicator neutral red in various buffers. The protonated form of neutral red is red, and the deprotonated form is yellow-orange, meaning that they absorb green and blue-violet light, respectively. Thus, the acidic and basic forms have distinct absorption wavelengths. Protein binding alters the properties of neutral red, changing both its absorbance and its pKa.</p>
	<p>After measuring the absorbance spectrum of free neutral red at several pH values, you will add riboflavin-binding protein, or RP, to the solutions and measure the absorbances again. Absorbance intensity is related to concentration, so you will use the spectra to determine the pKa of free and bound neutral red after the lab.</p>
	<ul class="lab-items">
		<li data-sub-note-item="2-3">
		<div class="lab-step">Draw the following table in your lab notebook. Number the cuvettes 1 through 10 and list the nine buffer pH values that you will use. The 10th cuvette will be a deionized water blank. <strong> Note: </strong>Always hold cuvettes by the textured sides and wipe the transparent sides just before putting the cuvette in the spectrophotometer. Remember to align the transparent sides with the beam of light in the spectrophotometer.
		<h4><strong>Table 2: Absorbance of Free and Bound Neutral Red</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>Cuvette #</strong></td>
					<td style="text-align: center;"><strong>Buffer pH </strong></td>
					<td style="text-align: center;"><strong>Abs. at &#955;<sub>max </sub>(Free NRH<sup>+</sup>)</strong></td>
					<td style="text-align: center;"><strong>Abs. at &#955;<sub>max</sub> (Bound NRH)</strong></td>
				</tr>
				<tr>
					<td style="text-align: center;">1</td>
					<td style="text-align: center;">5.0</td>
					<td style="text-align: center;"></td>
					<td style="text-align: center;"></td>
				</tr>
				<tr>
					<td style="text-align: center;">2</td>
					<td style="text-align: center;">5.5</td>
					<td></td>
					<td></td>
				</tr>
				<tr>
					<td style="text-align: center;">3</td>
					<td style="text-align: center;">6.0</td>
					<td></td>
					<td></td>
				</tr>
				<tr>
					<td style="text-align: center;">4</td>
					<td style="text-align: center;">6.5</td>
					<td></td>
					<td></td>
				</tr>
				<tr>
					<td style="text-align: center;">5</td>
					<td style="text-align: center;">7.0</td>
					<td></td>
					<td></td>
				</tr>
				<tr>
					<td style="text-align: center;">6</td>
					<td style="text-align: center;">7.5</td>
					<td></td>
					<td></td>
				</tr>
				<tr>
					<td style="text-align: center;">7</td>
					<td style="text-align: center;">8.0</td>
					<td></td>
					<td></td>
				</tr>
				<tr>
					<td style="text-align: center;">8</td>
					<td style="text-align: center;">8.5</td>
					<td></td>
					<td></td>
				</tr>
				<tr>
					<td style="text-align: center;">9</td>
					<td style="text-align: center;">11</td>
					<td></td>
					<td></td>
				</tr>
				<tr>
					<td style="text-align: center;">10</td>
					<td style="text-align: center;">blank</td>
					<td></td>
					<td></td>
				</tr>
			</tbody>
		</table>
		<a href="https://www.jove.com/CDNSource/lm/labs/45/Table_2._Absorbance_of_Free_and_Bound_Neutral_Red.xlsx" target="_blank">Click Here to download Table 2</a></div>
		</li>
		<li data-sub-note-item="2-4">
		<div class="lab-step">Obtain ten 1.5-mL cuvettes and caps and label the caps 1 through 10 to match the table in your lab notebook.</div>
		</li>
		<li data-sub-note-item="2-5">
		<div class="lab-step">Label a 25-mL beaker as &#8216;DIH2O&#8217; and fill it with deionized water.</div>
		</li>
		<li data-sub-note-item="2-6">
		<div class="lab-step">Flush your neutral aqueous waste down the drain and relabel the beaker as &#39;aqueous buffer waste&#39;.</div>
		</li>
		<li data-sub-note-item="2-7">
		<div class="lab-step">Label a 400-mL beaker for used micropipette tips.</div>
		</li>
		<li data-sub-note-item="2-8">
		<div class="lab-step">Attach a tip to a 1-mL micropipette and use it to dispense 1000 &#181;L of deionized water into cuvette 10. This is your solvent blank.</div>
		</li>
		<li data-sub-note-item="2-9">
		<div class="lab-step">Place 925 &#181;L of your pH 7.0 monosodium phosphate buffer in cuvette 5.</div>
		</li>
		<li data-sub-note-item="2-10">
		<div class="lab-step">Bring the remaining empty cuvettes to the buffer table. Guided by the table in your lab notebook, dispense 925 &#181;L of each buffer into the appropriate cuvette using the labeled micropipettes. Be careful not to use the same pipette tip for different buffers.</div>
		</li>
		<li data-sub-note-item="2-11">
		<div class="lab-step">Once you have finished, bring the buffers back to your workbench. There, set a 200-&#181;L micropipette to dispense 75 &#181;L, and attach a tip to the micropipette.</div>
		</li>
		<li data-sub-note-item="2-12">
		<div class="lab-step">Dispense 75 &#181;L of neutral red into each of the nine buffer cuvettes. Invert each cuvette several times to thoroughly mix the solutions. <strong>Note: </strong>Replace the pipette tip if it touches a buffer.</div>
		</li>
		<li data-sub-note-item="2-13">
		<div class="lab-step">Once you have mixed neutral red with every buffer, take a picture, or write down the colors of the solutions.</div>
		</li>
		<li data-sub-note-item="2-14">
		<div class="lab-step">Turn on a hand-held spectrophotometer and allow the light source to warm up. Once it is ready, create a new experiment to measure absorbance versus wavelength for the free neutral red.</div>
		</li>
		<li data-sub-note-item="2-15">
		<div class="lab-step">Insert the cuvette of deionized water and acquire a spectrum of the deionized water. Set it as a solvent background or blank.</div>
		</li>
		<li data-sub-note-item="2-16">
		<div class="lab-step">Then, remove the blank and insert the cuvette of neutral red in the lowest pH buffer, which will have the highest concentration of protonated neutral red.</div>
		</li>
		<li data-sub-note-item="2-17">
		<div class="lab-step">Acquire a spectrum, which should show one strong peak. Identify the wavelength corresponding to the highest point of this peak, or the maximum. Record this wavelength in your lab notebook as lambda max for protonated free neutral red.</div>
		</li>
		<li data-sub-note-item="2-18">
		<div class="lab-step">Save the data and remove the cuvette. Record the absorbance in your notebook, then collect spectra for cuvettes 2 - 9 using the same procedure.</div>
		</li>
		<li data-sub-note-item="2-19">
		<div class="lab-step">Record the wavelength of the isosbestic point in your lab notebook. That is where all the spectra cross at a single wavelength. If a spectrum does not cross at that point, empty and clean the cuvette, prepare a fresh sample, and try again.</div>
		</li>
		<li data-sub-note-item="2-20">
		<div class="lab-step">Once you finish collecting spectra for all nine cuvettes, save and export the data.</div>
		</li>
		<li data-sub-note-item="2-21">
		<div class="lab-step">Create a new experiment to measure absorbance versus wavelength for the bound neutral red.</div>
		</li>
		<li data-sub-note-item="2-22">
		<div class="lab-step">Fit a new tip to the 200-&#181;L micropipette and add 75 &#181;L of riboflavin-binding protein solution to each cuvette, including the blank. Eject the tip, secure the cuvette caps, and invert the cuvette several times to mix the solutions.</div>
		</li>
		<li data-sub-note-item="2-23">
		<div class="lab-step">Insert cuvette 10 into the spectrophotometer and set it as the solvent blank.</div>
		</li>
		<li data-sub-note-item="2-24">
		<div class="lab-step">Acquire a spectrum of the lowest pH sample, and identify the wavelength corresponding to the maximum of the most intense peak. Record it in your lab notebook as the lambda max of protonated-bound neutral red.</div>
		</li>
		<li data-sub-note-item="2-25">
		<div class="lab-step">Acquire spectra for cuvettes 2 - 9 in the same way as you did for the free neutral red samples. Record the intensities at lambda max for protonated-bound neutral red in your table, along with the wavelength at the isosbestic point.</div>
		</li>
		<li data-sub-note-item="2-26">
		<div class="lab-step">When you are done, save your data, export it for later analysis, and shut down the spectrophotometer.</div>
		</li>
		<li data-sub-note-item="2-27">
		<div class="lab-step">Put away all equipment and dispose of your used pipette tips in an approved waste container or trash bin.</div>
		</li>
		<li data-sub-note-item="2-28">
		<div class="lab-step">Empty the cuvettes into the aqueous buffer waste beaker and rinse the cuvettes into the beaker with deionized water.</div>
		</li>
		<li data-sub-note-item="2-29">
		<div class="lab-step">Empty the excess NaOH into the basic waste and rinse the graduated cylinder with water.</div>
		</li>
		<li data-sub-note-item="2-30">
		<div class="lab-step">Flush the basic aqueous waste down the drain with copious tap water, along with the other aqueous waste.</div>
		</li>
		<li data-sub-note-item="2-31">
		<div class="lab-step">Wash your glassware, cuvette caps, and stir bar per your lab standard procedures. Clean your work surfaces with a damp paper towel and throw out used paper towels and lab wipes in the lab trash.</div>
		</li>
	</ul>
	</li>
	<li data-note-item="3" data-parent-collapse=""><strong>Results</strong><button class="expand">Expand</button>
	<p>Now, let&#39;s analyze our absorbance data to determine the pKa&#39;s of neutral red.</p>
	<h4><strong>Table 3: Determination of Neutral Red pKa&#39;s</strong></h4>
	<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
		<tbody>
			<tr>
				<td></td>
				<td>Free NRH<sup>+</sup></td>
				<td>Bound NRH<sup>+</sup></td>
			</tr>
			<tr>
				<td>&#160;&#955;<sub>max</sub> (nm)</td>
				<td></td>
				<td></td>
			</tr>
			<tr>
				<td>&#160;&#916;A (nm)</td>
				<td></td>
				<td></td>
			</tr>
			<tr>
				<td>&#160;Midpoint (nm)</td>
				<td></td>
				<td></td>
			</tr>
			<tr>
				<td>&#160;pKa</td>
				<td></td>
				<td></td>
			</tr>
		</tbody>
	</table>
	<p><a href="https://www.jove.com/CDNSource/lm/labs/45/Table_3._Determination_of_Neutral_Red_pKa.xlsx" target="_blank">Click Here to download Table 3</a></p>
	<ul class="lab-items">
		<li data-sub-note-item="3-2">
		<div class="lab-step">Plot both sets of intensity data with absorbance intensity at lambda max on the y-axis and pH on the x-axis, with the points joined by smooth lines.</div>
		</li>
		<li data-sub-note-item="3-3">
		<div class="lab-step">Calculate the difference between the starting and ending absorbance intensities for each data series.</div>
		</li>
		<li data-sub-note-item="3-4">
		<div class="lab-step">Determine the absorbance midway between the starting and ending absorbances for each series, or the absorbance midpoints.</div>
		</li>
		<li data-sub-note-item="3-5">
		<div class="lab-step">Find where the midpoint occurs on each line and identify the pH value at that point. These pH values are the pKa&#39;s of free and bound neutral red.</div>
		</li>
		<li data-sub-note-item="3-6">
		<div class="lab-step">Here, we see an increase in pKa of about one between free and bound neutral red, indicating that bound-protonated neutral red is a weaker acid than free protonated neutral red by an order of magnitude.</div>
		</li>
	</ul>
	</li>
</ol>

Buffers: Common Ion Effect, Henderson-Hasselbalch Equation, and Buffer Capacity - Procedure

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

	Preparing 50 mM NaH2PO4 Buffer, pH 7Expand
	In the first part of this experiment, you will ...

Solubility

Solubility: Solubility Equilibrium and Thermodynamics - Procedure

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

	Creating a Saturated SolutionExpand
	In this experiment, you will create a saturated solution ...

Recrystallization

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

Recrystallization of Acetanilide and trans-Cinnamic Acid - Procedure

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

	Recrystallization of AcetanilideExpand
	In this part of ...

Cell Structure

<ol class="note-items"><li data-note-item="1" data-parent-collapse="">Visualizing Onion and Human Cells
<button class="expand">Expand</button><ul class="lab-items"><li data-sub-note-item="1-1"><div class="lab-step">In this experiment you will prepare different types of cells from a plant and animal, onion and human respectively, and then visualize them under a microscope. HYPOTHESES: The experimental hypothesis is that the cells will appear different in overall shape and membrane structure, but there will be some shared similar structures, like nuclei, in both cells. The null hypothesis of the experiment is that there will be no difference in structure between the two cell types.
</div></li><li data-sub-note-item="1-2"><div class="lab-step">First, to prepare an animal cell slide, start by pouring 30 mL of distilled water into a beaker.
</div></li><li data-sub-note-item="1-3"><div class="lab-step">Then, use a glass dropper to place 2 - 3 drops of the water onto the center of a clean microscope slide.
</div></li><li data-sub-note-item="1-4"><div class="lab-step">Next, take a clean toothpick and scrape the inside of your cheek to gather cells.  CAUTION: Do not scrape your cheeks too aggressively. The scraping should not cause discomfort or bleeding. 
</div></li><li data-sub-note-item="1-5"><div class="lab-step">Put the end of the toothpick with the cheek cells onto the wetted area of the slide and mix the water and cheek cells together.
</div></li><li data-sub-note-item="1-6"><div class="lab-step">Next, add two drops of methylene blue staining solution to the wetted area of the slide and mix it in with a clean toothpick.
</div></li><li data-sub-note-item="1-7"><div class="lab-step">Let the mixture sit at room temperature for 3 minutes.
</div></li><li data-sub-note-item="1-8"><div class="lab-step">After 3 minutes have passed, use a glass dropper to add a drop of glycerol to the mixture.
</div></li><li data-sub-note-item="1-9"><div class="lab-step">Carefully, place a cover slip over the mix on the slide by first standing one end of the cover slip vertically and aligning it along to one side of the mixture. Then, carefully lower the other end of the cover slip down until the mixture is completely covered.
</div></li><li data-sub-note-item="1-10"><div class="lab-step">Finally, remove any excess liquid with blotting paper.
</div></li><li data-sub-note-item="1-11"><div class="lab-step">Next, prepare a slide of plant cells by placing five drops of distilled water into a clean watch glass.
</div></li><li data-sub-note-item="1-12"><div class="lab-step">Then, with forceps, take a thin strip of onion and place it into the water.
</div></li><li data-sub-note-item="1-13"><div class="lab-step">Apply 5 drops of safranin solution to another watch glass.
</div></li><li data-sub-note-item="1-14"><div class="lab-step">Now using a pair of forceps transfer the piece of onion from the distilled water into the safranin.
</div></li><li data-sub-note-item="1-15"><div class="lab-step">Allow the onion to soak for 30 seconds.
</div></li><li data-sub-note-item="1-16"><div class="lab-step">Then, again with forceps, move the onion back into the distilled water.
</div></li><li data-sub-note-item="1-17"><div class="lab-step">Now, place 3 drops of glycerol onto the center of a glass microscope slide.
</div></li><li data-sub-note-item="1-18"><div class="lab-step">Using the forceps again, transfer the onion into the glycerol on the slide.
</div></li><li data-sub-note-item="1-19"><div class="lab-step">Gently place a cover slip over the onion on the slide and blot away any excess liquid with a piece of blotting paper.
</div></li><li data-sub-note-item="1-20"><div class="lab-step">Next, you will visualize the slides you have prepared using a compound microscope. First, turn on the scope and set the objective lens to the lowest level of magnification.  NOTE: A compound microscope typically has two levels of magnification.
</div></li><li data-sub-note-item="1-21"><div class="lab-step">Place the cheek tissue slide onto the stage of the microscope and bring the cells into focus by adjusting the stage and using the coarse adjustment knob.
</div></li><li data-sub-note-item="1-22"><div class="lab-step">When the cells are in focus, observe the individual cells and draw what you see. Repeat these observations at medium and high magnifications adjusting the focus with the fine adjustment knob. IMPORTANT: Do NOT use the coarse adjustment knob at high magnifications. 
</div></li><li data-sub-note-item="1-23"><div class="lab-step">Next, rotate the nose piece so that the oil lens is almost in place.
</div></li><li data-sub-note-item="1-24"><div class="lab-step">Place a drop of immersion oil on top of the cover slip of the cheek cell slide.
</div></li><li data-sub-note-item="1-25"><div class="lab-step">Use the oil lens to view the cheek cells and record your observations.
</div></li><li data-sub-note-item="1-26"><div class="lab-step">Repeat these steps to view the onion plant cell, moving again from low to medium, high, and oil lenses (steps 20 &#8211; 24).
</div></li><li data-sub-note-item="1-27"><div class="lab-step">Record all of your observations and note the differences you see between animal and plant cells.
</div></li><li data-sub-note-item="1-28"><div class="lab-step">When you are finished, place the cheek cell slide and toothpicks into a diluted bleach solution. Dispose of the cover slips into a glass waste container.
</div></li><li data-sub-note-item="1-29"><div class="lab-step">Finally, turn off the microscope and clean the lenses with lens cleaning paper.
</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">Calculate the total magnification for each of the viewing levels. Remember to account for both the eyepiece magnification and the objective lens magnification. NOTE: Eye piece: 10X magnification, Objective lens: 4X, 10X, and 40X magnification
</div></li><li data-sub-note-item="2-2"><div class="lab-step">Then, identify the structural differences between plant and animal cells using the observations made under the microscope.
</div></li><li data-sub-note-item="2-3"><div class="lab-step">Label the nuclei in both cell types and then highlight or note any differences that you see between them.
</div></li></ul></li></ol>

Cell Structure: Visualizing Onion and Human Cells - Procedure

Visualizing Onion and Human Cells
ExpandIn this experiment you will prepare different types of cells from a plant and animal, onion and human ...

Biology

Fundamentals

Cellular Respiration

<ol class="note-items"><li data-note-item="1" data-parent-collapse="">Quantifying Respiration using Microrespirometers
<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 measure the rate of cellular respiration for germinating seeds by measuring the rate of exchange for oxygen. As oxygen is consumed to provide energy, germinating seeds release carbon dioxide. This carbon dioxide is absorbed by potassium carbonate and thus the overall gaseous pressure of the respirometer will be reduced. HYPOTHESES: The experimental hypothesis is that germinating seeds will show a greater rate of respiration than control glass beads. Additionally, that at higher temperatures, the rate of cellular respiration in the seeds will increase. The null hypotheses are that both glass beads and germinating seeds will display a similar rate of cellular respiration and that temperature has no effect on respiration.
</div></li><li data-sub-note-item="1-2"><div class="lab-step">To set up the control, first remove the plunger from a prepared microrespirometer and label it with a &#8220;CR&#8221; or &#8220;CH&#8221; respectively, depending on whether it is the control room temperature or heated treatment.
</div></li><li data-sub-note-item="1-3"><div class="lab-step">Add the glass microbeads to each of the microrespirometers up to the 0.5 mL mark, and push the plunger in the syringe to the 1 mL mark.
</div></li><li data-sub-note-item="1-4"><div class="lab-step">Place 3 - 4 washers onto the flared end of each microrespirometer to weigh them down while in the water bath.  NOTE: The microrespirometer will sit capillary tube end up.
</div></li><li data-sub-note-item="1-5"><div class="lab-step">Set the control devices to the side.
</div></li><li data-sub-note-item="1-6"><div class="lab-step">To set up the experimental seated respirometers, remove the plunger from a prepared microrespirometer and label it with &#8220;ER&#8221; or &#8220;EH&#8221; respectively, depending on whether it is the experimental room temperature or heated treatment.
</div></li><li data-sub-note-item="1-7"><div class="lab-step">Measure 0.5 mL of germinating seeds into the tuberculin syringe of the microrespirometer.
</div></li><li data-sub-note-item="1-8"><div class="lab-step">Then, push the syringe to the 1 mL mark.
</div></li><li data-sub-note-item="1-9"><div class="lab-step">Place 3 - 4 washers onto the flared end of each microrespirometer to weigh them down while in the water bath.
</div></li><li data-sub-note-item="1-10"><div class="lab-step">To use the microrespirometers, carefully press the &#8220;CR&#8221; and &#8220;ER&#8221; microrespirometers into the room temperature water bath. The entire syringe should be submerged, but the capillary tubes should be entirely exposed. Remove water from the bath until this position is achieved. Be sure the top of the capillary tube is open.
</div></li><li data-sub-note-item="1-11"><div class="lab-step">Perform the same procedure (step 9) for the &#8220;CH&#8221; and &#8220;EH&#8221; microrespirometers to set up the higher temperature experimental group.
</div></li><li data-sub-note-item="1-12"><div class="lab-step">Wait 5 minutes before moving on to allow the temperature in the microrespirometers to equalize with the water bath.
</div></li><li data-sub-note-item="1-13"><div class="lab-step">Add a single drop of manometer fluid to the top of each of the four capillary tubes which seals the microrespirometer chambers.  NOTE: If the chambers are working properly, the manometer fluid will be sucked into the capillary chamber.
</div></li><li data-sub-note-item="1-14"><div class="lab-step">Use the plunger set in the bath to suck the manometer fluid into the capillary. Stop when the fluid is about halfway down the capillary tube. IMPORTANT: The microrespirometers should not be disturbed once they are added to the bath. Do not bump the table or insert or remove anything to or from the water bath once the chambers have been sealed and equalized.
</div></li><li data-sub-note-item="1-15"><div class="lab-step">Mark the bottom edge of the manometer fluid that is closer to the chamber. This represents time point zero.
</div></li><li data-sub-note-item="1-16"><div class="lab-step">Set a timer to sound in 5 minutes.
</div></li><li data-sub-note-item="1-17"><div class="lab-step">After 5 minutes, create a new mark where the manometer fluid is on each microrespirometer. Repeat every 5 minutes until 25 minutes has passed or until the manometer fluid has traveled the entire length of the capillary tube.
</div></li><li data-sub-note-item="1-18"><div class="lab-step">Then, remove the respirometers from the water baths.
</div></li><li data-sub-note-item="1-19"><div class="lab-step">Measure the distance between each of the marks and record them in Table 1 next to the correct time point. <a href="https://www.jove.com/CDNSource/lm/labs/8/Table_1_Cellular_respiration.xlsx" style="display:block;margin:20px;" target="__blank">Click Here to download Table 1</a>  
</div></li><li data-sub-note-item="1-20"><div class="lab-step">During clean up, manometers should either be deconstructed to be reused or disposed of in the trash.
</div></li></ul></li><li data-note-item="2" data-parent-collapse="">Results
<button class="expand">Expand</button><ul class="lab-items"><li data-sub-note-item="2-1"><div class="lab-step">To analyze the data you collected, first plot the data points with the x-axis as time and the y-axis as the distance the manometer moved, representing oxygen consumption.
</div></li><li data-sub-note-item="2-2"><div class="lab-step">Calculate the slope of each line using this equation.
</div></li><li data-sub-note-item="2-3"><div class="lab-step">Record these values for each microrespirometer in Table 2. NOTE: This value represents the amount of cellular respiration that took place over the course of the experiment.  <a href="https://www.jove.com/CDNSource/lm/labs/8/Table_2_Cellular_respiration.xlsx" style="display:block;margin:20px;" target="__blank">Click Here to download Table 2</a>
</div></li><li data-sub-note-item="2-4"><div class="lab-step">Plot the respiration rates as a bar graph.
</div></li><li data-sub-note-item="2-5"><div class="lab-step">Examine both of the graphs. Consider how your results fit with the hypotheses. Note any observed differences in respiration rate between the control beads and the germinating seeds. Determine if increasing the temperature had any effect on the respiration rate.
</div></li></ul></li></ol>

Cellular Respiration: Quantification using Microrespirometers - Procedure

Quantifying Respiration using Microrespirometers
ExpandNOTE: In this experiment, you will measure the rate of cellular respiration for germinating seeds ...

Cellular Processes

Cell Division

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

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

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

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/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/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/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 ...