Utilization of Bright Field Microscopy & Electrical Conductivity for the Determination of Osmotic Potential

In order to further explore this topic, it is essential that you have an understanding of the tonicity of solutions. Tonicity refers to whether the solution contains more or less solute than the cell interior. A solution in which the relative amount of solute is equivalent is referred to as an isotonic solution . As the solute concentration is equivalent within and outside of the cell, there is no net movement of water across the cell membrane, which results in the cell volume remaining unchanged . If the amount of solute outside the cell is less than that in the cell, the solution is hypotonic , and there will be a net increase in the amount of water within . This is due to the process of osmosis resulting in the movement of water across the semi-permeable cell membrane from a region of high concentration (low solute concentration) to one of low concentration (high solute concentration). While this will result in animal cells and others which lack a cell wall to potentially burst, cells with both cell membranes and cell walls will become turgid due to the pressure within. This state is essential for numerous plants, which rely on turgor pressure to support their tissues (such as stems). Finally, a solution which contains more solutes is hypertonic and the net movement of water will be out of the cell . In plant cells, this will result in a state of plasmolysis , which is the movement of the membrane away from the cell wall as the cell volume decreases, and the plant wilts. These states are depicted in Figure 1 below.

Figure 1 : Diagram depicting the net movement of water for a plant cell in solutions of various tonicity. (Public Domain) Following plasmolysis, if the cell is then moved into a hypotonic solution, it may undergo deplasmolysis , as water will begin to re-enter the cell. This process will also occur over time, as solutes are transported into the cell through a variety of mechanisms, including diffusion (passive or facilitated) and active transport. The speed at which this occurs is dependent upon the solute’s chemical nature, size, and the availability of specific transport proteins within the cell membrane. The expected observations for our red onion cells are shown in Figure 2 .

Figure 2 : Red onion cells undergoing plasmolysis and deplasmolysis. In (a), red onion cells were placed in an isotonic solution (0.85% NaCl). In a hypertonic solution (10% NaCl), plasmolysis will occur (b). Deplasmolysis occurs when moved to a hypotonic solution (distilled water) (c).

In the first portion of the lab (part A), we will review how to make solutions of various concentrations. We will then use these solutions to examine osmosis using bright field microscopy of red onion ( Allium sp. ) epidermis (part B) . This species is particularly useful for studies of water movement since it has large, translucent cell membranes, each surrounded by a cell wall . Most of the volume of an individual cell is occupied by a large central vacuole . The vacuoles of the cells comprising the first few outer layers are filled with anthocyanins, water-soluble red and purple pigments that impart the red colour to the onion. These pigments make it very easy to see the volume change of the vacuole under different osmotic conditions using a common bright field light microscope. In this lab, you would have observed the process of plasmolysis in red onion cell vacuoles by placing cells in solutions of different sucrose, NaCl, and CaCl 2 concentrations. In this manner, you would have determined the isosmotic threshold for each solution, which is the concentration of a given solution at which the cell no longer undergoes plasmolysis (ie. the solution is isotonic to the cell’s interior). This data will be used to determine the osmotic potential for these solutes at an isotonic concentration, from which the osmotic potential of a red onion cell can then be determined. The final portion of the lab (part C) will allow us to observe how the chemical nature of a solute will also alter its electrical conductivity . Molecules formed through ionic bonding may dissociate into positively and negatively charged ions, which will allow the conductance of an electrical current. The degree to which free ions are formed by a solute allow their classification into strong or weak electrolytes , while those which do not dissociate are classified as nonelectrolytes . This variance in dissociation can be represented by the specific osmotic coefficient for that solute and will be discussed further when we cover the subsequent calculations for the lab. The number of ions/molecule will also alter both the electrical conductivity and the isosmotic threshold, and must be taken into account as well. In this portion, we will examine the electrical conductivity of the same solutions which were 2a b c a b c a b c applied to the red onion cells, which can then be converted to osmotic potentials through the application of a constant. This will allow us to note any differences observed between these two alternate methods of determination.

PROCEDURES A. Solution Preparation It is necessary to be able to perform basic calculations for the purpose of preparing solutions of a given concentration. In the lab, you would have prepared 750 ml volumes of 1M NaCl and CaCl 2 solutions. This is easily done if you know their molecular weight (MW), which are 58.44 g/mol and 110.98 g/mol, respectively, and apply the formula:

weight needed (g) = molecular weight (g/mol) x volume (L) x concentration (mol/L or M) Add this amount of salt to your flask and dissolve in slightly less than the final volume of water required. Once fully dissolved, adjust the volume to 750 ml.

You then use the 1 M solution to make solutions ranging from 0.05M to 0.5M, increasing in 0.05M increments. A 250 mL volume of each solution should be prepared. Determine the appropriate volumes for dilution using C 1 V 1 = C 2 V 2 , or V 1 = C 2 V 2 /C 1 , where:

Volume needed (L) = Concentration wanted (M) x Final volume wanted (L) Concentration you have (M) This provides you with the volume of concentrated solution needed. To determine the amount of water to use for the dilution, simply subtract the volume needed from the final volume wanted.

*You will need to perform these calculations for your Lab 2 Assignment and may also need to know how to apply both formulas for your lab exam.

B. Determination of Isosmotic Thresholds Using Bright Field Microscopy 1. Prepare a small section of red onion epidermis and place it on a slide. Add several drops of 0.5M sucrose to cover the epidermis and allow the slide to sit for 5 minutes . Add a coverslip and remove the excess solution. Place the slide on the microscope and determine if the cells have undergone plasmolysis. Repeat this process for the remaining sucrose solutions (0.45M to 0.05M) until reaching the solution at which plasmolysis is no longer detected. This is the isosmotic threshold for sucrose. You do not need to test solutions past the determined threshold.

2. Repeat the procedure above for both NaCl and CaCl 2 .

3 C. Measuring Electrical Conductivity for Determining the Osmotic Potential of Solutes Use the solutions from part A to determine the electrical conductivity. Instructions for the use of the electrical conductivity meter can also be found in Appendix D .

1. Calibrate the meter by turning the switch to redline and adjust the meter needle to the red line on the scale.

2. Plug in the probe and place in the provided KCl calibration solution. You should obtain a reading of approximately 1325 μ MHOS/cm 3 . As temperature will alter the electrical conductivity, the temperature of your solutions will need to be determined as well. We are expecting all of our solutions to be at room temperature (22 o C).

3. Due to the high concentration of the solutions we are measuring, turn the multiplier knob to adjust it to x100. If at any point one of your solutions is out of range adjust the multiplier. If that does not bring the readings on scale, perform a 1 in 2 dilution and take another reading, being sure to note the dilution on your data sheet.

4. Rinse the probe and submerge it into your first solution. Once the meter stabilizes, record your reading and repeat for the remaining solutions.

C. Data Analysis 1. Determine the average isosmotic threshold for each of the solutions from the class data for Part B on Nexus.

2. Determine the osmotic potential of the cell in each solution by applying the formula:

Ψ O = -C ϕ VRT where Ψ O = osmotic potential (MPa) C = concentration (M) ϕ = osmotic coefficient (based on dissociation) V = # of ions/molecule R = pressure constant = 0.0083 L MPa/mol o K T = temperature ( o K) = 273 + o C An example of the use of this equation will be provided in the Zoom tutorial 3. Average the osmotic potentials determined for the three solutions to determine the cellular osmotic potential.

4. Convert the electrical conductivity data on Nexus into osmotic potentials by multiplying the recorded meter values (μ MHOS/cm 3 ) by the conversion constant of -3.6 x 10 -5 (-0.000036).

5. Prepare a graph of concentration (M) versus osmotic potential (bars). Include all three solutions on the same graph. Use a best-fit straight line forced through zero for each line. Similar as to how you utilized your standard curves in Lab 1, the line equation can be used to determine the concentration of that solution which correlates to the previously determined cellular osmotic potential.

4 Prior to the Lab 2 Tutorial , please be sure to view the Bright Field Microscopy video so that you have observed the procedure for Part B. You should also read Appendix C: Bright Field Microscopy and review Appendix D: Use of the Electrical Conductivity Meter .

Lab 2 Assignment Your lab 2 Assignment will require answering a number of questions and performing the analyses described above using the data posted on Nexus. The analysis of data will be reviewed during the Lab 2 Tutorial. The data and assignment can be found in the Lab 2 folder . This assignment is worth 1.5% of your final grade and is due by 11:59 PM on October 15 th .

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