Part B : Yeast Experiments

Environmental Effects on Colony Color

Genes, which are made of DNA, carry the information for making a cell or a complete organism. But the information by itself is not enough. It takes raw materials and energy, too. Making a cell is somewhat like baking a cake but more complicated. The genes are the recipe, calling for the proper ingredients at the right time. If you leave something out or substitute one ingredient for another, you get a different kind of cake or no cake at all. Leave out the chocolate and the cake is a different color and flavor; leave out the baking powder and you have a flat cake; leave out the flour and you have no cake.

We can apply this kind of logic to genetic mutation as seen in our red mutant yeast. Mutations are changes in genes; something gets left out so the cells can't grow. Whatever this ingredient is, it is in the YED medium but not in the MV medium. The red mutant cells cannot make this ingredient but the normal cream-colored ones can.

Besides genetic information, the environment also plays a key role in transmitting genetic traits. The traits of all organisms--from yeast to people--are the result of both the information carried in their genes and the environment in which the genes function. In the previous experiments you may have explored the effect of changing some of the genetic information by using cells that have mutant genes. Now you will see what happens to these mutants when you change their environment.

The biologists who first discovered these red mutants did many tests to see exactly what it was in YED that allowed the mutant cells to grow. They discovered that it is a compound called adenine, that is commonly found in nearly all food. It is used by cells to make DNA, RNA and also ATP, a molecule that moves energy around. The red mutants cannot grow unless adenine is supplied in their food. The normal cream-colored strains can make adenine from simpler chemicals.

Experiments:

You can easily study two environmental variables to see their effects on the growth and color of these mutants: A) The amount of adenine in the medium affects not only the growth, but also the color. You can study this by making a gradient of adenine in a plate. B) The presence of oxygen (O2) is not required for growth, so you can explore its possible effect on the red color trait.

A. Effect of Adenine Concentration on Pigment Formation
Time Line:
If you incubate your cultures at 30 C, you will be able to follow this schedule. At room temperature, you may have to double the time between steps.

1st Day: 30 min. Make an adenine gradient on a lawn of red mutant cells.

3rd Day: 15 min. Observe the development of growth and color rings. Retest the white yeast.

4th-6th Day:15 min. Observe and analyze the results.

Materials:

Common Materials:

Sterile water, sterile container, sterile toothpicks, sterile 1mL dropping pipet, Haploid strain HA2 or HB2

Individual Materials:

1 MV agar Petri plate
Adenine-impregnated filter paper disks

Making a Lawn of Cells and an Adenine Gradient Time Line: 1st day: 30 min.
1. Make a visibly turbid suspension of the cells:
Scrape some yeast cells from an overnight culture with a toothpick, Wipe them onto a spot on the inside of a sterile jar, Add about 35 mL of sterile water,
Replace the lid and swirl the water to suspend the yeast cells. If the suspension is not visibly cloudy, add more yeast cells.
2. Swirl the jar to resuspend the cells before removing any samples.
Remove the plate lid and pipet 1 mL of the suspension from the jar directly onto the surface of an MV agar plate.
Replace the lid of the plate.
3. Tilt and rotate the plate to spread the cells over the surface of the agar. If there are places the liquid does not cover, use the blunt end of a toothpick to guide the suspension to cover those areas.
4. Allow the agar to absorb the liquid for about 10 minutes, or until the liquid disappears. Teacher Tips
Making and Adenine Gradient:
1.Open the plate and put an adenine-impregnated filter paper disk in the middle on the surface of the agar. Replace the lid.
2.Incubate the plate right-side-up with the agar on the bottom, for two days.

Observe and Respond:

Carefully observe and record for several days a description of the MV plate to which the adenine-impregnated filter paper disk was added. When the adenine in the filter paper dissolved and entered the agar there was a large excess of adenine near the center. This excess diffused outward, forming a concentration gradient.

1.How can you explain the white growth near the center where the adenine concentration is excessive? Do these white cells require adenine? How can you test your answer experimentally?

2.Describe the nutritional conditions in the red zone.

3.Why is there no growth at the edges of the MV plate?

( Teacher Tips )

Retesting the white yeast:

You have found that under certain conditions the so-called "red mutants" can turn white . Is this a genetic change or the result of a different environment? Time Line: 3rd Day: 15 min.
1. Touch a sterile toothpick to some of the white yeast near the center of the plate and streak it onto a YED plate.
Drag a new sterile toothpick through the first streak and make a zig zag streak.
Continue to use new sterile toothpicks to make more zig zag streaks on the plate (refer to the diagram on the left).
2. Incubate the plate for two or three days to see if the yeast will turn red on YED.

Analyze your observations:

1. Explain whether the change from red to white near the center of the plate was a genetic or an environmental effect? 2. Give an explanation for this change or suggest another experiment you might do to learn more about it. ( Teacher Tips)



B. Effect of Anaerobic Growth on Pigment Formation

Time Line:

1st Day: 15 min. Plate cells and grow the red strain anaerobically.

3rd-4th Day: 30 min (work time): Expose the cells to air and observe during the next 24 hours.

Materials:

Common Materials:
Sterile water, sterile container, sterile toothpicks, sterile 1mL dropping pipet, haploid strain HA2 or HB2

Individual materials:
1 Petri plate of YED Technical Tip: Yeast cells can grow either in the presence of oxygen (aerobically) or in the absence of oxygen (anaerobically). When cells grow in the absence of oxygen, they are unable to make some compounds that are made by the chemical process of oxidation. You might ask whether the red pigment can form in yeast cells that are growing in the absence of oxygen since it is common for colored compounds to be formed by oxidation. A simple way to avoid oxidation is to grow the yeast between two layers of agar.

Growing the red strain anaerobically:

Time Line: 1st Day: 15 min.
1. Make a visibly turbid suspension of the cells:
Scrape some yeast cells from an overnight culture with a toothpick, Wipe them onto a spot on the inside of a sterile jar, Add about 35 mL of sterile water,
Replace the lid and swirl the water to suspend the yeast cells. If the suspension is not visibly cloudy, add more yeast cells.

( Teacher Tips)

Figure 3: STEPS IN SETTING UP AN ANAEROBIC ENVIRONMENT

Step 1

Step 2

Step 3

2. Swirl the jar to resuspend the cells before removing any samples.
Remove the plate lid and pipet 1 mL of the suspension from the jar directly onto the surface of a YED agar plate.
Replace the lid of the plate to maintain sterility.
3. Tilt and rotate the plate to spread the cells over the surface of the agar. If there are places the liquid does not cover, use the blunt end of a toothpick to guide the suspension to cover those areas.
4. Allow the agar to absorb the liquid (usually about 10 minutes).
5. Cut the agar across the plate with a sterile toothpick. Divide it so that one part is slightly smaller than the other.
6. Fold the smallest portion over the large portion using a sterile toothpick.
Use another sterile toothpick to press the agar to seal the "sandwich" of cells.
Replace the lid.
7. Incubate the plate agar side down for two or three days. ( Teacher Tips )

STEPS IN RESTORING THE AEROBIC ENVIRONMENT

Effect of Aerobic growth on color
Exposing the cells to air (O2):

1. Make a sketch of the plate and show where the yeast is pink and where cream colored. Also, note any other features such as gas bubbles between the layers of agar.
2. Cut the top layer of the folded agar in half with a sterile toothpick.
Figure
Use another toothpick to lift one of the new quarters.
Lay the new quarter back in its original position with the cells facing upward.
3. Watch for change in the color of the cells over the next few hours, if possible, or on the following day.
4. Make a sketch of the plate and jot down any changes you see in the yeast color.

Analyze your observations:

1. Is there any part in the sandwich where the yeast has not turned pink or red?

2. Is there a reason to think that the yeast growing there might be anaerobic?

3. Do you see any gas bubbles between the layers of agar? If so, can you think of an explanation for them?

4. What conclusion, if any, can you draw from this experiment about the role of oxygen in the formation of the red pigment?

5. Explain whether the change from white to red was a genetic or an environmental effect?

6. Give an explanation for this change or suggest another experiment you might do to learn more about it.(Teacher Tips )
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Last updated Wednesday, 04-Dec-2002 20:58:28 UTC