6. Cell Energy - Plant Pigments

Chlorophyll extractionSummary
Chlorophyll is the pigment in plants that captures sunlight energy and uses it to drive photosynthesis. While chlorophyll does give plants their characteristic green color, chlorophyll actually comes in many colors and subtypes ranging from green to yellow to orange to red. In this experiment, students use paper chromatography to separate the many pigments from one another. First the pigments are extracted from the plants by simply crushing the plant cells open on the filter paper with the edge of a penny. When the filter paper is then immersed in rubbing alcohol, the pigments are carried upwards through capillary action. The smallest pigments travel more quickly and thus separate from the larger pigments that remain closer to the origin line.

Chlorophyll is an amazing chemical that is the essential ingredient in photosynthesis, the process through which plants capture light from the sun to create glucose. When photons of light hit chlorophyll, the electrons in the central magnesium atom donate an electron to a series of chemical reactions – the electron transport chain – that produce ATP, the cells primary unit of energy currency. The chlorophyll gets this electron back by taking one from water, resulting in the release of oxygen gas as a byproduct.

Chlorophyll comes in two main forms: chlorophyll a and chlorophyll b. Each has a slightly different chemical structure and therefore absorbs light of different wavelengths. In this paper chromatography experiment, chlorophyll a will appear as a bright yellow-green band while chlorophyll b will appear as a dull green band. In addition, there are many other accessory pigments in plants that absorb light and help transfer photons to the chlorophyll. These include carotene (an orange band) and xanthophyll (a yellow band). A final pigment that may be detected is anthocyanin (a red-brown band) that acts as a type of sunscreen for plants, protecting the plant from UV damage.

For more information on paper chromatography and its use in chlorophyll extraction, a fabulous scientific description may be found on the Science Buddies website. Also see this photosynthesis lesson plan by Sara Swisher, Barb Wilson, and Jean Wilson. Finally, Cheryl Massengale has a great write up for chlorophyll extraction including a table illustrating how to calculate the R_f value for each band and use that as a quantitative means of identifying the various pigments.

Materials

• Filter paper cut into 9 cm x 2 cm strips (coffee filters work but not as well)
• Pennies
• Beaker or cup no more than 9 cm tall
• Small stick - bamboo skewer, pencil, plastic stirrer, drinking straw, etc.
• Rubbing alcohol
• Tape
• Ruler
• Assorted fresh plant leaves – spinach, lettuce, or leaves collected from around the neighborhood (unusually colored leaves such as white, purple, red, and yellow are particularly interesting)

Procedure

1. Use a pencil to draw a line across the filter paper strip, 2 cm from one end.
2. Place the leaf you want to test on top of the filter paper strip, remembering where the line is.
3. Using the edge of a penny, trace the penny across the leaf right on top of where you drew the line. Push down hard enough to leave a green smear on the paper on top of the original pencil mark.
4. Repeat 2 more times with fresh sections of leaf to make the smear darker. Try to keep the smear as close to the original pencil line as possible.
5. Fill the cup or beaker to a depth of 1 cm with rubbing alcohol.
6. Carefully lower the filter paper strip, green smear end in first, into the beaker until the bottom just touches the alcohol. Make sure that the green smear does not actually touch the alcohol, only the tip of the paper should actually be in contact with the alcohol.
7. Lay the stick across the mouth of the beaker like a bridge from one edge to the other.
8. Tape the filter paper strip to the stick so that the paper is held in place just touching the alcohol but not touching the sides of the beaker.
9. Wait. The alcohol should gradually move up the paper, bringing many of the pigments along with it.
10. Remove the stick and paper when the alcohol has almost reached the stick. The actual length of time will depend on the type of paper you use.
11. Mark how far up the paper the alcohol traveled with a pencil.
12. Determine how many bands of pigment you have, what color they are, and measure how far each band traveled from the origin line. Some of the pigments will fade and disappear over time. It may help to trace around each band in pencil while they are still clear so that the strip can still be analyzed if and when the bands fade.
13. Calculate an R_f value for each band (R_f value = distance that band traveled divided by distance the alcohol front traveled).
14. Identify the pigments based on their colors or by their R_f value. The R_f values allow you to compare one pigment band to another from strip to strip.
15. Compare what was found in different types of leaves.

Going Further

1. Try this on purple plants. Do they have chlorophyll too?
2. Try this on white-leafed plants. Do they have chlorophyll too?
3. Try this on leaves from trees before and after they change color in the fall. What happens to the chlorophyll as the leaves change color? Do new pigments emerge or were they there all along?
4. Cover half of a wide green leaf with tin foil. Leave the plant in a sunny window for at least 4-5 days. Remove the tin foil and see what happened to the leaf. Compare chlorophyll extracted from the uncovered half of the leaf with chlorophyll extracted from the covered half. Is there any difference? On a separate leaf that underwent the same treatment, uncover the leaf and leave it in a sunny window. Does the chlorophyll come back?

6. Cell Energy - Bubbling Plants

Elodea nuttallii: Image courtesy of Christopher Fischer.Summary
Students often believe that only animals “breathe”, but all things exchange gases with their environment. It’s just that the process is not so obvious in plants. Elodea is a very common water plant that can be found in aquarium stores. As photosynthesis occurs, oxygen is produced as a by-product. Elodea releases bubbles of oxygen as it photosynthesizes. In fact, the number or volume of bubbles in a certain amount of time can be used as a rough measure of photosynthetic rate.

The measure is rough because oxygen dissolves in water so may not always appear as a gas. Moreover, most light sources produce heat, which causes the water temperature to increase, which in turn causes lower gas solubility, and thus may produce bubbles just by turning the light on. Finally, the size of a bubble is not constant thus counting the number of bubbles per unit time is only a very rough measure. The total volume of gas produced is a better measure but again falls victim to the other caveats. The best measure is to use a dissolved oxygen meter which unfortunately costs a fair bit of money (between $400 to over $1000). You can also try disposable dissolved oxygen tests (see the Sources section of the Water Analysis activity).

An important note is that freshly cut Elodea stems produce more bubbles than the leaves. That is because Elodea stems contain large intracellular air passageways. As oxygen is produced, the plant transports the oxygen away from the leaves towards the roots. Thus, a freshly cut stem will produce oxygen bubbles at an observable rate. The cells in the leaves are much more tightly packed together and provide greater resistance to the emerging oxygen gas than the stems. Thus, to maximize oxygen gas production in this experiment, cut the stem of the Elodea then place the plant upside-down in the test tube. For more information about why the bubbles emerge from the stems, not the leaves, see this article by David Hershey of the Mad Scientist Network.

An excellent resource with more information about the use of Elodea in this experiment can be found at the Clifton College Science School website. There you will find detailed information about Elodea, how oxygen is produced, experiments by Frost Blackman, practical advice and more.

Materials

• Elodea plants
• Scissors
• Large beaker
• Clear test tube, preferably with fine graduations (< 1 ml) in order to measure the volume of the gas produced
• Water
• Sunlight or a lamp (best to find a light source that produces as little heat as possible)
• Optional: heavy black cloth

Procedure

1. Fill the large beaker almost to the top with water.
2. Cut the stem of the Elodea plant at an angle (for greater surface area) so that it is just a little shorter than the test tube.
3. Insert the plant, cut stem end in first, into the test tube.
4. Fill the test tube to overflowing with water. Try to avoid introducing any bubbles to the test tube as you do this. Gently tap the test tube on a tabletop to dislodge any bubbles trapped between the leaves.
5. Carefully cover the top of the test tube with your thumb, squeezing out some water as you do. Invert the test tube over the beaker and put the test tube with your thumb still covering the opening into the beaker.
6. Release your thumb. And gently settle the test tube on the bottom of the container. Check to make sure there are no bubbles in the test tube.
7. Place the beaker and test tube in sunlight or under a bright lamp. Observe what happens.
8. At 5 minute intervals for a total of 15 minutes, record either the number of bubbles produced or the volume of bubbles produced.

Going Further

1. Investigate the effect of light intensity. Compare bubble production between a plant in sunlight versus a plant in darkness, covered in a black cloth.
2. Investigate the effect of temperature. Compare bubble production between a plant in the fridge versus a plant at room temperature.
3. Investigate the effect of carbon dioxide levels. Compare bubble production between a plant in water that has been boiled and left to cool back to room temperature (little to no carbon dioxide) versus a plant in water that a person has blown bubbles through a straw for 3 minutes (lots of dissolved carbon dioxide). See the Colorful Respiration activity for more ideas along these lines.

6. Cell Energy - Photosynthesis in a Jar

Summary
These experiments use a bell jar (or any other very large, clear, glass jar) to determine the identity of the gas produced by plants. It mirrors the famous experiments of Joseph Priestley and Jan Ingenhousz from the 1700’s that first demonstrated the existence of oxygen and its importance to plants and animals.

In 1771 and 1772, Priestly conducted a series of experiments using a bell jar. It was known that a candle placed in a sealed bell jar would eventually burn out and could not be relighted while still in the jar. Priestly discovered that a plant can survive indefinitely within a jar. Thus, he tried placing a plant into the jar with the burning candle. The candle went out as before and could not be relit right away. Priestly waited several days and tried again. The candle could be relit! The plant had restored the air inside the jar! (Do not try the next series of experiments since it harms animals!) Next priestly investigated what would happen to animals. He found that a mouse placed inside a sealed jar will eventually collapse. However, a mouse can survive in a sealed jar with a plant since the plant restores the air. Priestly was the first to demonstrate that oxygen is necessary for fire and animals but that given time, plants can create oxygen, allowing fires to burn and animals to breathe.

A few years later, Jan Ingenhousz investigated the effect of light on a plant’s ability to restore air. He found that a plant left in darkness cannot restore the air for a candle. To demonstrate this, he burned up all the oxygen in a jar with a plant then left the plant in sunlight for a few days to restore the air. Then without relighting the candle, he put the plant into darkness for several more days. At the end of the dark period, he was unable to relight the candle. He concluded that a plant in darkness acts like animals, using up the oxygen that it had created. Ingenhousz had discovered that plants can photosynthesize and create oxygen but only in the light. If left in the dark, plants do not photosynthesize and thus no oxygen is produced. Moreover plants are always performing respiration, just like animals. In sunlight, the rate of photosynthesis outstrips respiration so there is an excess of oxygen being produced. But in darkness, no photosynthesis takes place but respiration continues to occur. Thus, by keeping a plant in darkness you can demonstrate that plants need oxygen and use it up, just like animals.

For more information, see the wonderful website of Julian Rubin which describes many of these experiments and offers links and resources on how to recreate them in the classroom. Also, NSTA has produced a great set of photosynthesis related inquiry activities including the bell jar experiments.

Materials

• Small plant in a pot, well watered
• Candle or row of matches (I found the matches easier to light with the converging lens although it produces significantly more smoke.)
• Flood lamp with a very high wattage bulb or bright sunlight
• Magnifying glass
• Bell jar or other large glass jar (Don’t use a plastic container like I did initially. Not only will it collapse with the vacuum produced following the burning of the candle but the smoke from the candle flame often deposits itself on the inside of the plastic, obscuring everything inside from view.)
• Vacuum plate or large tray full of water (If you use a tray of water, beware that as the candle heats the air, the expanding gas will escape out from under the rim of the jar. When the air cools again, the level of the water inside the jar will rise so be sure to prop your plant up on a pedestal of some sort to prevent the whole thing from getting swamped. Also use a lot of water in the tray initially or air from outside will be pulled into the jar as the air cools.)
• Heavy black cloth or dark closet

Procedure
A candle uses up the oxygen in the jar:

1. Place a burning candle inside the bell jar and seal it. The candle will eventually go out.
2. Focus a beam of light on the candle wick with the converging lens to show that the candle cannot be relit.

A plant restores the oxygen in the jar:

1. Place a burning candle and a plant inside the bell jar and seal it. The candle will eventually go out.
2. Try relighting the candle to show that the candle cannot be relit.
3. Place the setup in a sunny window for 2 days.
4. Try relighting the candle. The candle should relight.

A plant in darkness does not restore oxygen:

1. Place a burning candle and a plant inside the bell jar and seal it. The candle will eventually go out.
2. Try relighting the candle to show that the candle cannot be relit.
3. Place the setup in the dark for 2 days.
4. Try relighting the candle. The candle should not relight.

A plant in darkness uses oxygen:

1. Place a burning candle and a plant inside the bell jar and seal it. The candle will eventually go out.
2. Try relighting the candle to show that the candle cannot be relit.
3. Place the setup in a sunny window for 2 days.
4. Remove the setup from the sunny window and place it in darkness for 2 more days.
5. Try relighting the candle. The candle should not relight.

6. Cell Energy - Colorful Respiration

Summary
Blow through a straw into bluish liquid and watch it turn green then yellow before your eyes. Put some plants into the yellow liquid, leave it in a sunny window, come back the next day and the liquid is green. What if you leave the plants in the dark? What if you put some pond snails in? What if you put both pond snails and plants? What’s going on?

The liquid is bromthymol blue (BTB) a non-toxic acid-base indicator that can be used to indirectly measure levels of dissolved carbon dioxide (CO₂). The amount of CO₂ in a solution changes the pH. An increase in CO₂ makes a solution more acidic (the pH gets lower). A decrease in CO₂ makes a solution more basic (the pH gets higher). The reason for this is that carbon dioxide that is dissolved in water is in equilibrium with carbonic acid (H₂CO₃).

CO₂ + H₂O ↔ H₂CO₃

In any solution, while the majority of CO₂ stays as CO₂, some of it is converted to H₂CO₃, turning the solution slightly acidic. If CO₂ is added to the water, the level of H₂CO₃ will rise and the solution will become more acidic. If CO₂ is removed from the water, the amount of H₂CO₃ falls and the solution becomes more basic. Thus, acid-base indicators such as BTB can indirectly measure the amount of CO₂ in a solution.

For more than you ever wanted to know about carbonic acid, see the Wikipedia article on carbonic acid. For the example lesson plans developed by Bob Culler through Access Excellence at the National Health Museum. For a great time lapse video showing BTB color changes using elodea and snails, see Activity C13 from Addison-Wesley’s Science 10 curriculum.

Materials

• Bromthymol blue (BTB can be ordered from any science supply company such as Flinn Scientific $9 for 1 liter 0.04% BTB solution).
• Several 2 liter soda bottles
• Test tubes
• 500 ml beakers or disposable plastic or paper cups
• Water (since the pH of tap water varies, you may wish to use distilled water for your master BTB solution)
• Drinking straws
• Plastic wrap
• Elodea
• Pond snails

Procedure

1. Before the lesson, the teacher should mix a master BTB solution in one or more 2 liter soda bottles. For each 2 liter bottle, mix 120 ml 0.04% BTB with 1800 ml water. The end result should be a medium blue master BTB solution, dilute enough to be safe for plants and snails but dark enough to see the color changes.
2. Pour 200 ml diluted BTB in a beaker or cup.
3. Take a deep breath then blow bubbles in the BTB solution through a drinking straw. What happened? Why?
4. Set up a test tube rack with 3 tubes. In tube #1 put unbubbled BTB solution (blue). In tube #2 put bubbled BTB solution (yellow). Tubes #1 and #2 will be your comparison tubes. In tube #3 you have a choice of what to do. Choose one option from each of the following columns:
   

   BTB solution	Living things	Light conditions
   bubbled BTB (yellow)	spring of Elodea	Sunny window/bright light
   unbubbled BTB (blue)	5 pond snails	Dark closet/drak heavy cloth
   	both Elodea and 5 pond snails

5. Make a hypothesis about what will happen to your tube.
6. After 24 hours, check the color of your tube. What happened? Why?

Going Further

1. Investigate the effect of exercise. Compare blowing bubbles in BTB for 3 seconds before and after vigorous exercise (such as doing 2 minutes of jumping jacks).
2. Investigate the effect of holding your breath. Compare blowing bubbles in BTB for 3 seconds before and after holding your breath for as long as possible (without passing out).
3. Try this lesson developed by NASA. It describes how to capture various gases (room air, human exhalation, car exhaust, or carbon dioxide from a chemical reaction) in a balloon and use BTB to measure the carbon dioxide content.

6. Cell Energy - Bubbling Yeast

Summary
Bubbling Yeast: Thanks to Ellen Loehman for creating this image.Yeast are a single celled fungi that are a great model organism for studying respiration in the classroom. The species Saccharomyces cerevisiae is commonly used for leavening bread and fermenting beer but other species such as Candida albicans are known to cause infections in humans (vaginal yeast infections and diaper rash being the most common). In this investigation, students fill the bulb of a disposable pipet (eyedropper) with yeast, then submerge the pipet in a test tube of water. They can then measure the rate of respiration by counting the number of bubbles of carbon dioxide gas that emerge from the tip of the pipet in a certain length of time. By varying the temperature and the nutrient source, students can discover what variables affect the rate of respiration in yeast. By submerging the pipet in bromthymol blue (see Colorful Respiration activity), students can identify the gas being produced as carbon dioxide.

For more information about yeast in classroom experiments, see this experiment from the Exploratorium or this one from PBS Kids that has snippets of what different kids saw with different manipulations. The idea of using inverted disposable pipets to contain the yeast and measure their respiration rate came from a workshop led by Steve Ribisi of the University of Massachusetts – thanks Steve!

Materials

• Fast-acting bread yeast (1 packet or 1/4 teaspoon per group)
• 1 cup water
• 2 tablespoons table sugar
• Disposable plastic pipets (These can be ordered from most science supply companies such as Science Kit and Boreal Labs, around $6 for 100 pipets or $18 for 500 pipets. Note: don’t get the “microtip” style since the yeast solution is too viscous to be sucked into the tip.)
• Metal washers from the hardware store (These will weigh the pipets down so that they don’t float up to the top of the tube. Make sure that the hole in the washers is large enough to sit around the neck of the pipet and rest on top of the bulb.)
• Small test tubes for mixing yeast solution with sugar solution
• Large test tubes (These must be wide enough to accommodate the pipets and washers comfortably but also tall enough to submerge the whole pipet. If you don’t have large enough test tubes, try using graduated 15 ml centrifuge tubes, a 100 ml graduated cylinder, or a small beaker.)
• Optional: other nutrient sources for the yeast such as milk, apple juice, soda, Kool-aid, salt water, potato starch solution, flour in water, chicken broth, etc. Most of these work better if diluted in water 1:1.
• Optional: bromthymol blue solution

Procedure

1. an hour before the activity, mix 1 packet of bread yeast with 1/4 cup of luke warm water. Stir around 2 minutes until all the yeast is dissolved. Stir again just before use.
2. Dissolve 1 tablespoon sugar in 1/2 cup of luke warm water. Stir around 1 minute until all the sugar is dissolved.
3. In a small test tube, mix equal quantities of the yeast solution and sugar solution. Stir gently to combine. Use separate droppers for each solution to avoid contaminating the original stock solutions.
4. Suck up some of this solution into a pipet. Invert the pipet and let the solution run down into the bulb. Carefully squeeze out the air and suck up some more yeast-sugar solution. Try to fill exactly half of the pipet bulb.
5. Thread 2 washers over the neck of the pipet so that they come to rest on top of the bulb.
6. Gently drop the pipet with washers into the large test tube.
7. Fill the large test tube with luke warm water until the pipet is completely submerged.
8. Wait 5 minutes to allow the yeast time to equilibrate and begin respiration.
9. Count how many bubbles emerge from the top of the pipet each minute for 10 minutes.

Going Further

1. Investigate the effect of temperature. Compare the respiration rate in yeast in cold water, luke warm water, and scalding hot water.
2. Investigate the effect of different nutrient sources. Compare the respiration rate in yeast dissolved in different nutrient sources.
3. Investigate the identity of the gas produced. Fill the test tube with BTB rather than water and see what happens to the color of the indicator over time. (See the Colorful Respiration activity.)

6. Cell energy - Standards

Standards
Grade 7
Cell Biology
1. All living organisms are composed of cells, from just one to many trillions, whose details usually are visible only through a microscope. As a basis for understanding this concept:
a. Students know cells function similarly in all living organisms.
b. Students know the characteristics that distinguish plant cells from animal cells, including chloroplasts and cell walls.
d. Students know that mitochondria liberate energy for the work that cells do and that chloroplasts capture sunlight energy for photosynthesis.

Investigation and Experimentation
7. Scientific progress is made by asking meaningful questions and conducting careful investigations. As a basis for understanding this concept and addressing the content in the other three strands, students should develop their own questions and perform investigations. Students will:
a. Select and use appropriate tools and technology (including calculators, computers, balances, spring scales, microscopes, and binoculars) to perform tests, collect data, and display data.
c. Communicate the logical connection among hypotheses, science concepts, tests conducted, data collected, and conclusions drawn from the scientific evidence.
e. Communicate the steps and results from an investigation in written reports and oral presentations.

Grade 8 Life Science
5. Chemical reactions are processes in which atoms are rearranged into different combinations of molecules. As a basis for understanding this concept:
a. Students know reactant atoms and molecules interact to form products with different chemical properties.
b. Students know the idea of atoms explains the conservation of matter: In chemical reactions the number of atoms stays the same no matter how they are arranged, so their total mass stays the same.
c. Students know chemical reactions usually liberate heat or absorb heat.

Grades 9-12 Biology
Cell Biology
1. The fundamental life processes of plants and animals depend on a variety of chemical reactions that occur in specialized areas of the organism's cells. As a basis for understanding this concept:
f. Students know usable energy is captured from sunlight by chloroplasts and is stored through the synthesis of sugar from carbon dioxide.
g. Students know the role of the mitochondria in making stored chemical-bond energy available to cells by completing the breakdown of glucose to carbon dioxide.