Respiration Lab

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					                                      Biological Processes

Outline of Lab
       Fermentation Experiment
       Photosynthesis Experiment
       Measuring the rate of photosynthesis
       Chromatography & pigments

Objectives of the Lab
       Understand fermentation
       Distinguish between aerobic and anaerobic respiration
       Describe the roles of light and pigment in photosynthesis.
       Name and describe pigments found in plant photosynthetic tissues.
       Determine how light affects photosynthetic rates
       Explain separation of pigments by the process of paper chromatography, based upon molecular
        structure and polarity of substances

For cells to function properly, energy must be supplied via the production of ATP. Cellular
respiration is the study of how cells convert macromolecules into useable energy packets of
ATP. Respiration has alternative pathways, depending upon the presence of a final electron
acceptor molecule, for example oxygen. If oxygen is present, a glucose molecule is broken down
through the processes glycolysis, Kreb’s cycle and electron transport chain to yield over 30
molecules of ATP. If oxygen is not present, however, the cell can still generate a few molecules
of ATP through glycolysis. After glucose is broken into pyruvate molecules, there are several
pathways that can occur, depending on the organism. Two common pathways are represented
below (fig. 1). Pyruvate is converted into lactate (lactic acid) in many animals, including
humans. Other organisms, such as yeast and some bacteria, convert pyruvate into alcohol
(ethanol) and carbon dioxide, a process called fermentation.

Figure 1. a) Anaerobic respiration resulting in formation of lactate. b) Anaerobic respiration
resulting in ethanol and carbon dioxide.

Bi101: Early History of Life             Biological Processes                                         1
For today’s respiration experiment, you will be assessing the rate of fermentation for glucose and
starch. Starch (amylose), a common storage carbohydrate, is a polymer of glucose units. The
polymer has the chemical formula of (C6H12O6)n where n represents a very large number. Starch
is broken down by the enzyme -glucosidase into glucose. For this experiment you will be
testing the fermentation rates of three scenarios: glucose, starch, and starch + -glucosidase.
Think about how the process of breaking down glucose works (see figure 1) and then determine
which molecule situation should be broken down into the products of fermentation at the fastest

Per student group (4):
     china marker                                           0.5% -glucosidase in bottle fitted
     3 50-ml beakers                                         with graduated pipet
     25-ml graduated cylinder                               3 glass stirring rods
     bottle of 10% glucose                                  ¼ teaspoon measure (optional)
     bottle of 1% starch                                    3 fermentation tubes
                                                             15-cm metric ruler
Per lab room:
     yeast
     scale and weighing paper (optional)
     37o incubator

Before you begin the procedure, write out a hypothesis and predictions based on fermentation
rates and the substance the yeast cells are digesting.

PROCEDURE: (working in groups)
1. Using a china marker, number three 50-ml beakers.
2. With a clean 25-ml graduated cylinder, measure out and pour 15 ml of the following solutions
   into each beaker: (Note: wash the graduated cylinder
   between solutions.)
         Beaker 1: 15 ml of 10% glucose
         Beaker 2: 15 ml of 1% starch
         Beaker 3: 15 ml of 1% starch; then, using the graduated
           pipet, add 5 ml enzyme, the 0.5% -glucosidase.
3. Wait 5 minutes and then to each beaker add ¼ teaspoon dry
   activated yeast. Stir with separate glass stirring rods.
4. When each is thoroughly mixed, pour the contents into three
   correspondingly numbered fermentation tubes, see the figure
   on next page. Cover the opening of the fermentation tube with
   your thumb and invert each fermentation tube so that the “tail”
   portion is filled with the solution.
5. Place the tubes in a 37o incubator or water bath.
6. At intervals of 5 minutes for 30 minutes after the start of the
   experiment, remove the tubes and, using a metric ruler,
   measure the distance (in mm) from the tip of the tail to the
   fluid level. Create a table and record your results.

What gas accumulates in the tail portion of the fermentation tube?                 (hint: check
  figure 1).

Bi101: Early History of Life           Biological Processes                                         2
Photosynthesis is the process by which photons of energy from the radiant energy of the sun is
channeled into organic molecules. As hydrogen gas on the sun is converted into helium, these
photons are liberated, pass to the Earth’s surface and absorbed by pigments in plant chloroplasts.

Plant photosynthesis combines carbon dioxide and water to produce glucose and oxygen. Cells
of living organisms use glucose as their primary source of energy, and plants may convert
glucose into sucrose and transport or store it as starch (a glucose polymer). Such organic
molecules are building blocks for plant growth and development. Animals consume plants,
converting plant molecules into animal organic molecules and energy sources. Oxygen, also a
by-product of photosynthesis, is essential for aerobic respiration in living cells of plants, animals
and other organisms.

Think about what the requirements and products are of photosynthesis?

Measuring the Rate of Photosynthesis in Spinach Leaf Discs
One way to measure the rate of photosynthetic reactions is by estimating O2 production in disks
cut from fresh spinach leaves. Leaf tissue is full of gas-filled intercellular spaces and sections of
leaf will float when placed in solution. If these leaf sections are subjected to a vacuum, the gases
in those spaces are removed (sucked out of the leaf) and the leaf disks sink. Then if
photosynthesis takes place the resulting oxygen gas diffuses into those intercellular spaces and
the liquid is replaced with gas. This allows the disks to float once more. You can use this
technique as a method of measuring the rate of photosynthesis. Sodium bicarbonate (NaHCO3),
which supplies CO2 for photosynthesis in this scenario, will be in the solution to infiltrate the
leaf disks once the gases have been removed.

For today’s experiment, you will expose spinach leaves to three different light conditions:
intense, ambient (room lighting) and no light. Think about how light intensity could alter
photosynthetic rates.

Devise a testable hypothesis concerning light intensity and photosynthesis and record it on the
assignment sheet.

Fresh spinach leaves                                   Sodium bicarbonate
250 ml side-arm Erlenmeyer flask (for                  Balance
vacuum)                                                Light source
cork-borer (4mm diameter)                              Beakers
glass Petri dishes                                     Small brush or forceps

1. Prepare the lamp so that it is approximately 25 cm from the top of the Petri dish.
2. Pour 0.2% NaHCO3 solution into three Petri dishes so each is about 2/3 full. Pour
   approximately 150 ml of the same solution into the 250 ml flask. This amount does not have
   to be exact.
3. With a few healthy spinach leaves, place them on the plastic cutting board and cut at least 45
   disks with the cork-borer. Do not include large veins in the disks. Place the disks into the
   flask with the bicarbonate solution as quickly as possible.
4. Use a water aspirator set-up to sink the floating disks of spinach (see instructor for directions).
   a) attach the vacuum tubing to the sidearm of the flask. Put the rubber stopper firmly in to the
   mouth of the flask and press the tape securely over the stopper holes.

General Biology Laboratory               Biological Processes                                        3
   b) Turn on the water faucets to full on. After several seconds, you should be able to observe
   bubbles coming from the disk edges. Continue to vacuum for approximately 15-20 seconds
   once bubbling takes place. Then release the vacuum by peeling back the tape.
   c) swirl the flask and wait to see whether or not all disks sink. Leaf disks will continue to
   float as long as they are under a vacuum.
   d) You may have to re-vacuum two or three times to sink the majority of the spinach disks.
   When most of the disks are sunk, discontinue vacuuming. Over aspiration can cause damage
   to the leaf disks. You should have at least 10 disks per Petri dish.
5. Pour the contents of the flask into a large beaker. Discard any disks that remain floating
   and pour off the solution. With a brush or forceps (tweezers), gently transfer 10 or more
   disks to each Petri dish. Put the lids on and place each dish in the appropriate light condition.
   Fill the large beaker with water and set on top of the Petri dish under the lamp light. The heat
   from the glowing lights could affect the rate of photosynthesis.
6. Check each dish after 5, 10, 15, 20, 25 and 30 minutes to determine the number of disks
   floating and record your data in a table.

Separation and Identification of Plant Pigments by Paper Chromatography
Plant pigments on chromatographic paper can be separated by this method. As organic solvents
petroleum ether and acetone climb the paper, they will carry the pigments along. The organic
molecules in plant pigments will move at different rates, depending on their different
solubility in the solvents used and the degree of attraction to the paper. The leading edge of
the solvent is called the front. Discrete pigment bands will be formed from the leading edge
back to the point where pigments were added to the paper.

The following will be helpful information to help you make predictions and interpret results:
        a) Polar molecules or substances dissolve (or are attracted to) polar molecules.
        b) Nonpolar molecules are attracted to nonpolar molecules to varying degrees.
        c) Chromatography paper (cellulose) is a polar (charged) substance.
        d) Acetone, the solvent, is nonpolar.
        e) The most nonpolar substance will stay dissolved in the nonpolar acetone the longest
           and travels farthest up the paper.
        f) The most polar substance will be attracted to the polar chromatography paper,
           therefore, it will come out of solution (you will be able to see the pigment) the
           quickest and travels least up the paper.

Use this information and the molecular structure of major leaf pigments to predict the relative
solubility and separation patterns for the pigments and to identify the pigment bands. Study the
molecular structure of the four common plant pigments. Polar groups include O=CH (aldehyde),
C=O (ketone), OH (hydroxyl), and O-CH3 (esters). Hint: what do all these chemical groups
have in common?

General Biology Laboratory              Biological Processes                                       4
Rank the pigments according to polarity from most polar to least polar

        Most polar                                                                 Least polar

Construct a hypothesis relating concerning pigments, polarity, and attraction to polar or
nonpolar substances. Write this hypothesis on your assignment sheet.

fresh green leaves (parsley, spinach, fig)                Chromatography solvent (9 pts.
forceps                                                   petroleum ether, 1 pt. acetone)
Whatman #3 chromatography paper strips
Large test tube with hook in cork

1. Being careful not to handle the paper (handle with a paper towel, touch only at top edge) cut
   approximately 15 x 150 mm strip of chromatography paper.
2. Trim one end to a point with scissors.
3. Lay the strip on paper towel on your work table.
4. Using either a scissors handle or cork borer handle, crush a narrow band of leaf tissue into the
   chromatography paper near the pointed end (see figure below). Move intact leaf tissue over

General Biology Laboratory              Biological Processes                                          5
   this band or spot and repeat the procedure a few times to build up a conspicuous, narrow dark
5. After adding the pigment band to the paper, hook the paper to the cork stopper may add
   enough chromatography solvent to the large test tube to just reach the lower part of the filter
   paper strip you are preparing. Cork the tube to prepare a saturated environment. Do not let
   the pigment band dry. Hang the strip from a wire hook in a cork, and insert this into
   the large test tube.
6. Position the test tube vertically, and watch the pigment bands emerge as the solvent “climbs”
   the filter paper strip.
7. Remove the paper from the tube (or dish), allow it to air dry.

Analyze your results and draw conclusions about the pigments and their location on the paper.

Chromatography paper and pigment setup

General Biology Laboratory             Biological Processes                                      6

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