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

Utilization of Bright Field Microscopy and Electrical Conductivity for the Determination of Osmotic Potential Sherry Hebert BIOL-3221L-U2020F Lab Procedure Overview ● Preparation of solutions ● Determination of isosmotic thresholds using bright field microscopy ● Measurement of electrical conductivity Part A:

Solution Preparation Step 1: Make 750 ml of a 1M solution of NaCl (MW = 58.44 g/mol) Weight (g) = MW x Conc (M) x Vol (L) Step 1: Make 750 ml of a 1M solution of NaCl (MW = 58.44 g/mol) Weight (g) = MW x Conc (M) x Vol (L) = 58.44 g/mol x 1 M x 0.75 L = 43.83 gPart A:

Solution Preparation Step 1: Make 750 ml of a 1M solution of NaCl (MW = 58.44 g/mol) Weight (g) = MW x Conc (M) x Vol (L) = 58.44 g/mol x 1 M x 0.75 L = 43.83 g Always dissolve in smaller starting volume, then adjust!Part A:

Solution Preparation Step 2: Make required dilutions eg. Make 250 mL of a 0.05M NaCl solution from 1M starting solution Use C 1 V 1 = C 2 V 2Part A:

Solution Preparation Step 2: Make required dilutions eg. Make 250 mL of a 0.05M NaCl solution from 1M starting solution Use C 1 V 1 = C 2 V 2 This is the same as:

Vol needed (L) = Conc wanted (M) x Vol wanted (L) Conc have (M)Part A:

Solution Preparation Step 2: Make required dilutions eg. Make 250 mL of a 0.05M NaCl solution from 1M starting solution Vol needed (L) = Conc wanted (M) x Vol wanted (L) Conc have (M) Vol needed = 0.05 M x 0.25 L = 0.0125 L 1 MPart A:

Solution Preparation Step 2: Make required dilutions eg. Make 250 mL of a 0.05M NaCl solution from 1M starting solution Vol needed (L) = 12.5 mL 1M NaCl With 250 mL – 12.5 mL = 237.5 mL dH 2 OPart A:

Solution Preparation Part B: Determination of Isosmotic Thresholds Prepare slides of red onion epidermis and use bright field microscopy to determine the threshold.

Start with the most concentrated solution (0.5M) Plasmolysis will have occurred Use increasingly more dilute solutions until plasmolysis is no longer apparent This is the isosmotic threshold for that solute.

Conc inside = Conc outsidePart B: Determination of Isosmotic Thresholds Part C: Determination of Electrical Conductivity Calibrate the electrical conductivity meter Utilize the probe to determine the electrical conductivity of each solution Data Analysis for Part B ● Determine isosmotic threshold for each solution using the class data available on Nexus.

● Use the equation to determine the osmotic potential in each solution:

Ψ O = - C ϕ VRT Data Analysis for Part B ● Use the equation to determine the osmotic potential in each solution:

Ψ O = - C ϕ VRT where:

C = concentration (M) = isosmotic threshold ϕ = osmotic coefficient (dissociation) V = # of ions/molecule R = pressure constant = 0.0083 L MPa/mol K T = 273 + O C = O K Data Analysis for Part B Example: NaCl is used and an isosmotic threshold of 0.4 is determined C = 0.4M ϕ = 0.93 V = 2 R = 0.0083 L MPa/mol O K T = 273 + 22 O C = 295 O K Data Analysis for Part B Example: NaCl is used and an isosmotic threshold of 0.4 is determined Ψ O = - C ϕ VRT = - ( 0.4)( 0.93)(2)(0.0083)( 295) = -1.8239 MPa Data Analysis for Part B ● Average the osmotic potential calculated for each solution to determine the osmotic potential of the cell Data Analysis for Part C ● Convert the electrical conductivity data (in µ MHOS/cm 3 ) to osmotic potential (in MPa) by multiplying by the constant:

-3.6 x 10 -5 ● Prepare graphs of concentration (M) vs osmotic potential (MPa) for each of the solutes. Use a best-fit straight line, forced through zero.

● Use the line equation to determine the concentration of each solution required to reach the isosmotic threshold based on this data. Assignment ● Is available in the Lab 2 Nexus folder ● Is due by 11:59 PM on October 15 th and is worth 1.5% of your final grade ● Includes calculations, formal table & figure prep, and answering questions related to the theory and analysis of your data