Experimental Procedure

Equipment:

•  Aquarium, gallon jars, or other “glass bells” that can be used to create a closed system.
•  Thermometers
•  Dissolved O₂  probes or kits for dissolved oxygen measurements. (Can be acquired from a science supply company).
•  pH indicators – pH paper or other indicators.
•  Light source
•  CO₂ measurement kits.
•  Slides and cover slips
•  Microscopes
•  Prepared microscope slides demonstrating leaf anatomy (Can be purchased from a supplier).
•  Environment to control temperature.

Materials:

• High CO₂ levels: soda water; vinegar and baking soda mixture; Alka-seltzer in water; or dry ice (walnut size piece should be sufficient  for a large aquarium.)
• Vaseline or silicone gel (tape and/or plastic wrap can be substituted) for sealing aquaria.
• C3 plants such as sunflowers, beans, barley, oats,  wheat, tomatoes etc. (plant size: 10 cm to 15 cm).
• C4 plants such as corn and sorghum. (plant size: 10 cm to 15 cm)
• When choosing plant types use plants with similar leaf structure
• Distilled water

Procedures:

• Obtain a number of C3 and C4 seedlings, grown in pots.
• Select an equal number of each type of plant, for each aquarium.
• Obtain the following data from each type of plant, and then set aside for experiment average leaf number. 1). average size of  leaves. 2). average weight (One method to measure leaf size is to trace leaf area on paper, cut out and weigh paper).

 

Measuring the length of leaves

• Using plants that will not be in the experiment, find the average mass as follows:
• Gently remove the soil from the roots, wash and pat dry.
• Mass plants and record data.
• Using the plants that were massed.
• Obtain a small sample of leaf from each plant and prepare them for microscopic observations employing the following method:

To view the epidermis of leaf apply a thin layer of nail polish 1 cm2,  allow to dry.

• Place scotch tape over polished area, pull leaf away from tape.  Place tape (sticky side up) on microscope slide, place cover slip on top.
• Examine and draw differences in morphology between the species.  This may be done once to see major differences or between the two plants or at the beginning and at  the end  to compare the changes in stomates during growth.

Example of Corn Stamates

Set-up

• Obtain 4 aquaria or bell jars for controlled environments.
• Two of the aquaria will be set-up in a warm environment and the other two in a cold environment.
• There should be at least 15^o C between each aquaria.
• Place a thermometers in each aquarium.

• In the first aquarium the CO₂ and O₂ in the atmosphere of the aquarium will equilibrate with the concentrations in the water, so that it will be possible to test for changes in the concentration of these gases due to photosynthesis and respiration.
• Put a small beaker with distilled water close to the plants (use the same amount of water in each aquarium).
• Place plants of each type in the aquarium.
• NOTE: Make sure to sufficiently water plants before sealing them in the aquarium and that water regime and light are the same for all treatments.
• Set-up second aquarium with a high concentration of CO₂.  Place a wide beaker with the CO₂ concentration in the aquarium. This will allow the large surface area of acidic water to exchange with the CO₂ in the atmosphere.
• Put a small beaker with distilled water close to the plants (use the same amount of water in each aquarium).

Example of an enhanced CO2 aquarium set-up.

• NOTE: The increased CO₂ environment should be created just before sealing aquarium in order to maintain high CO₂
• levels. (Refer to materials above for high CO₂ concentrations).
• Place a thermometers in each aquarium.
• Using the kits measure the dissolved O₂, CO₂, and pH from the water that is placed in each aquarium. (from this data you will be able to calculate both photosynthetic rates and the change in “atmospheric” concentrations of  CO₂  and O₂).
• Obtain temperatures higher than room temperature by  placing  light source close to system.  To prevent over heating, put a  flask full of water between the “system” and the light source.
• NOTE: It is important to emphasize that all parameters except those that are being tested for their effects on the experiment should remain as constant as possible for all treatments.
• Tightly seal each system so that gas exchange is limited to that in the closed system.
• Use the same methods for setting up cold temperature aquariums as used for warm temperature aquariums.
• After about five days open the aquaria.
• Immediately upon opening the aquaria measure dissolved O_2, CO₂, and pH before the water has time to change temperatures and  interact with external atmosphere.  The pH of the water will be used to calculate the CO₂ concentration in the atmosphere. (The pH in the beakers may be measured in the following ways: a.  pH meter – make sure water is stirred with a magnetic stirrer and that the beaker is sealed to prevent atmospheric exchange as much as possible (cover with parafilm).  (Optional: * pH indicators – these will give a measure of change in the pH by approximately 1 unit pH. * Use of  indicator dyes: Lime water – calcium hydroxide, forms precipitate when CO₂  is added to water, phenolphthalein  –  red at pH 10, colorless at pH 8.3, bromothymol blue: blue at pH 7.6, yellow at pH 6).
• The oxygen can be measured in two ways - dissolved oxygen meter or kit.
• Releaser leaf size and mass of plants.
• Record and Analyze Data

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