Lab 2, January 16: Osmosis & photosynthetic pigments

OSMOSIS & PHOTOSYNTHETIC PIGMENTS
INTRODUCTION
Cells, as the basic units of life, must perform all of the tasks of any living system. These tasks include the
intake of energy and nutrients, and the elimination of waste material. The outer boundary of the living cell
is the plasma membrane (also known as the plasmalemma, or as the cell membrane). Materials can pass
through the plasmalemma in several ways. Certain molecules pass through the membrane through
specialized protein carriers in a process called facilitated transport. Small, uncharged molecules may also
diffuse through the lipid bilayer of membranes. Water, oxygen and carbon dioxide can all diffuse through
membranes. The net diffusion of water across membranes is called osmosis. Under different
circumstances, more water may enter the cell than exit. In other circumstances, more water may exit the
cells than enter. This direction of water movement is extremely important to cells, due to the great
importance of water to all living things.
Water, like other molecules, diffuses from where it is abundant to where it is less common. When water is
mixed with other polar materials, it forms solutions that dissolve these other materials (called solutes”).
When more solutes are dissolved in water, the percent of water in the solution decreases because the
percent of other materials increases. This means that water will move FROM a low-concentrated solution
TO a highly concentrated solution. Cell cytoplasm has materials dissolved in it such as carbohydrates and
proteins. Cells are usually surrounded by aqueous (water) solutions that may be low or high in solute
concentration, so water might move into the cell or out of the cell. Hypotonic solutions contain less
solutes than cell cytoplasm; hypertonic solutions have more solutes that cell cytoplasm; isotonic
solutions have the same solute concentration as cell cytoplasm.
Your task in this lab is to make observations on plant and animal cells, determine which way water is
moving across the cell membrane by osmosis, and explain why water is moving in that direction. We will
use water plant and human blood cells.
We will also investigate a critical component of photosynthesis in this lab. Proteins, carbohydrates and
lipids are the most abundant classes of organic compounds in living things, but they are not the only
ones. Many other essential compounds exist within cells. For example, plant cells contain chlorophyll
molecules that absorb light to provide the energy for photosynthesis. Molecules such as chlorophyll have
unique chemical properties. For example, not all colors of light are equally absorbed by chlorophyll, so not
all colors are equally effective for plant photosynthesis.
Visible light waves range from 400 to 700 nanometers (nm) in length. (A nanometer is one-thousandth of
a micron.) Each color of light represents a different set of wavelengths. Violet and blue colors are in the
400 to 500nm range; yellow and green colors are in the 500 - 600nm range; orange and red colors are in
the 600 - 700nm range.
In this lab you will use a spectrophotometer to measure the light absorbance of chlorophyll by different
wavelengths. The spectrophotometer passes light of a certain wavelength through a solution. A detector
on the other side of the solution measures the light intensity. From comparing how much light shines on
the solution with how much light passes through the solution, the difference is how much light is absorbed
by the molecules in the solution.
You will shine lights of different wavelengths through a chlorophyll solution, construct a graph of
absorbance, and make several conclusions about the nature of leaves, chlorophyll and photosynthesis
with respect to different colors of light.
OBJECTIVES
1.
2.
3.
4.
5.
Define, characterize, and identify isotonic, hypertonic, and hypotonic solutions.
Graph, describe and explain processes such as plasmolysis and hemolysis resulting from
osmosis.
Use a spectrophotometer to determine chlorophyll’s absorbance of different wavelengths of light.
Construct a line graph of chlorophyll absorbance as a function of the wavelength of light.
Draw conclusions from experimental results to explain why leaves are green, and the most
effective colors of light for photosynthesis.
MATERIALS
Elodea in various concentrations of sucrose solutions test tubes (0.0M, 0.1M, 0.2M, 0.3M & 0.4M)
isotonic, hypertonic, and hypotonic solutions of NaCl solution
lancets
cotton swabs
alcohol
microscopes
slides and cover slips
spectrophotometers
chlorophyll solution in a spectrophotometer cuvette
“blank” solution without chlorophyll in a cuvette
PLASMOLYSIS OF ELODEA
1.
2.
3.
4.
5.
6.
7.
Obtain a leaf of Elodea in pure water, make a wet mount of the leave, and observe it under high
magnification. (Use your normal protocol by proceeding from scanning to low to high power).
Review the parts of the cells: cell wall; plasma membrane; vacuole & chloroplasts.
Obtain a leaf of Elodea from the 0.4M sucrose solution, and observe the leaf under high
magnification (using normal protocol by proceeding from scanning to low to high power).
Note how chloroplasts and other organelles are located in the center of the cell. Find the plasma
membrane, which is pulled away from the cell wall. This condition is called plasmolysis. You
may need to vary the light to see the plasma membrane accurately. See page 85 of your textbook
for explanation of how osmosis is affecting the cells.
Diagram and label one or two plasmolyzed Elodea cells.
View a section of Elodea from each solution. On each leaf, choose a section at random. Count the
total number of cells visible, and the number of cells which have plasmolyzed. Calculate the
percent of cells (if any) that have plasmolyzed
Construct a graph of Elodea plasmolysis, the horizontal axis being the concentration of the
solution, and the vertical axis being percent plasmolysis of Elodea cells.
Estimate the concentration at which 50% of the Elodea cells would plasmolyze.
HEMOLYSIS OF BLOOD CELLS
1.
2.
3.
Place a small amount of (about 5ml) of isotonic, hypertonic, and hypotonic NaCl solutions into
three numbered test tubes.
Sterilize a finger with alcohol. Lance the finger with a sterile lancet.
Place your finger over one of the tube and invert the tube to mix the solution with the drop of
blood.
4.
5.
6.
7.
Quickly dry your finger and press out a second drop of blood. Repeat step 3 with a second tube.
Repeat step 4 with the third tube.
Each solution should appear reddish from the drop of blood. Whole blood cells floating in a
solution make the cells appear cloudy. If the blood cells burst and fall out of solution, the solution
will appear clear, but will still appear reddish due to the hemoglobin released from the cells. See
page 85 of your text for additional explanation. Placing a page with letters behind the tube may
help you determine whether the solution is cloudy or clear.
Measure the time until hemolysis for each solution. You will recognize that hemolysis has
occurred when the solution is no longer cloudy, but is clear. Test for clarity by placing this paper
behind the test tube. If the letters are clearly visible, hemolysis has occurred.
DETERMINING THE CHLOROPHYLL ABSORBANCE SPECTRUM
1.
2.
3.
4.
5.
6.
7.
Set the wavelength of the spectrophotometer to 400nm.
Place a "blank" cuvette filled with only solvent into the chamber and close the chamber cover.
“Zero” the spectrophotometer by pressing the appropriate button on the control panel.
Remove the blank cuvette and place a cuvette filled with chlorophyll into the chamber.
Read the absorbance reading and record both the wavelength and absorbance.
Repeat the procedure for at 25nm increments from 400nm to 700nm.
Construct a line graph of chlorophyll absorbance at different wavelengths. Label the vertical axis
"absorbance" and the horizontal axis by both wavelength and color. Give the graph an
appropriate sentence title.
FOR THOUGHT AND DISCUSSION
Answer each in a sentence or two.
1.
Use the concept of osmosis to explain why Elodea cells in only certain sucrose solutions
plasmolyzed.
2.
Discuss why hemolysis occurs in some solutions and not in others.
3.
What colors of light are best absorbed by chlorophyll? Which colors are poorly absorbed?
4.
Explain why leaves have a green color.