Part B: Basic Genetics Experiments
A Simple Cross
You may have heard the saying "What you see is what you get." Wouldn't
it be great if that was always true? Often we can't see what is happening
within organisms or inside individual cells even when we use a microscope.
However, yeast is an exception. One of the most appealing features of Baker's
yeast, Saccharomyces cerevisiae, is that in its life cycle "What
you see is what you get." In yeast, the complete sexual life cycle can
be seen, which makes it ideal for learning about the basic concepts of
cell biology and genetics. Yeast cells have unique shapes at key points
in the life cycle so you can learn to recognize each step. An outstanding
feature of the yeast life cycle is the ease with which you can manipulate
it. You can have yeast cells do whatever you want to study at a convenient
time. Control of the growth and reproduction of yeast cell cultures by
temperature changes or by type of food source is a most useful tool for
The entire experiment can be completed in less than two weeks. First
you will mix two haploid yeast cultures that have grown overnight on a
rich growth medium. Day-by-day you will change their growth conditions
(environment) and observe the cell mixtures as they appear on the agar,
and through the microscope. As you go along, you will be able to build
a diagram of the yeast life cycle. You will see the main events of the
sexual reproduction cycle and also examine the inheritance pattern for
a color trait. After completing the life cycle experiment, you will be
able to use your new skills to set up your own experiments to study influence
of the environment patterns of inheritance.
If you incubate your cultures at 30øC, you will be able to follow
this schedule. At room temperature, you may have to double the incubation
times suggested in each procedure.
1st Day : 5 min Getting Ready
2nd Day : 50 min Mating Two Haploid Strains and Observing Zygotes
3rd Day: 15 min Selecting the Diploids
4th Day: 15 min Presporulation
5th Day: 15 min Sporulating the Diploids
6th Day: 50 min Observation of Asci and Germination of Spores
10th Day: 15 min Looking for the Missing Color
Haploid yeast strain HA2
Haploid yeast strain HBT
YED agar plate
(Your teacher may have already done this for you.)
Time Line: First Day: 5 min
1. Touch a sterile toothpick to a colony of HA2 yeast. Make
a short (1/2 inch ) streak at the top of a YED agar plate. Discard the
2. Touch another toothpick to a colony of HBT yeast.
Make a short (1/2 inch ) streak on the left side of the YED agar
plate. Discard the toothpick.
3. Your plate should look like the plate diagramed in Figure 1.
4. Turn the plate over and label the bottom of the plate with "Getting
Ready", HA2, HBT, YED, your name and the date.
Teacher Tips )
Technical Tip :
When you mix haploid cells of opposite mating types a and
together on a nutrient agar medium, they become pear-shaped (we call them
"shmoos"). They then mate (conjugate) to form diploid zygotes. The zygotes
can be distinguished by their characteristic shapes. Before they start
budding they are peanut-shaped and after they bud, they look more like
2 & 3
Mating Two Haploid Strains and Observing Zygotes:
Time Line: 2nd Day: 50 min
1. Touch a sterile toothpick to the HBT streak on the YED plate and
then touch it to the agar at the center of the plate as illustrated in
Figure 2. Discard the toothpick.
YED plate with overnight subculture of parent strains (from Getting
Microscope, microscope slide, coverslip
2. Touch another sterile toothpick to the HA2 streak and touch it
to a spot close to the spot you just made at the center of the plate. Discard
3. Then use a third sterile toothpick to mix the two spots of yeast
together as illustrated in Figure 3. Be gentle, don't tear up the surface
of the agar. 4. Draw a sketch of the yeast cultures in the first circle
on the Data Record Sheet or in your lab journal. Label the yeast strains,
the streak color, the kind of medium, and the date of the procedure.
Time Shifting: Yeast cells follow a clock that depends on temperature,
so you can easily control how fast they grow and develop. If you can't
look at the mating mixture after three or four hours, you or a friend (ask
your teacher) can put the plate in the refrigerator until you have time.
Another way of slowing the growth of the yeast, is to put the plate in
the refrigerator as soon as you mix the cells and then taking it out four
or five hours before looking at the cells. If you leave the plate in the
refrigerator for one day, then you need to add another day to your experiment.
5. Make a wet-mount slide from either one of the parent strains and
look at it through the microscope.
6. Draw a sketch of about 10 of these cells in the first square
of your Data Record Sheet or in your lab journal. Label the square "Haploids".
7. After 3 hours, make a wet-mount slide of the mating mixture and
look at it through the microscope. See if you can find some zygotes (peanut
or clover-leaf shaped.) These zygotes are diploid. They contain the chromosomes
from both of their haploid parents. You may also see some shmoos that look
much like pear-shaped cells. Shmoos are haploid cells getting ready to
mate. Some cell shapes are shown in Figure 4.
8. Draw a sketch of about 10 cells in the second square of your
Data Record Sheet. Label the square "Mixture". Label unbudded zygotes with
"Z," budded zygotes with "BZ," and shmoos with "S."
9. Put the plate back in the incubator with the agar side up for
another day or two to let the diploid cells grow.
Geneticists use the word trait to mean any feature of an organism
that can be inherited such as color or the ability to grow on a certain
kind of food. Traits can come in different forms which are called variations.
For example, in the two haploid yeast strains you are studying, the trait
is the colony color while red and cream are the variations. The color of
the yeast is part of the
phenotype of the strain.
After your mating mixture has grown up, you can see its color but you
need some way to tell the diploids from the cream-colored haploid parent.
To make this easy, we have used yeast that need different food (growth
medium). Both of the haploid strains can grow on the rich YED medium, but
neither can grow on the nutritionally poor MV medium. But, the diploid
cells formed from these two haploid strains can grow on MV. By putting
the cells on MV you can be confident that you have a purified diploid colony.
Selecting the Diploids:
Time Line: 3rd Day: 15 min
Mating mixture plate from previous procedure
MV agar plate
1. First record the appearance of your plate by making a drawing
of it in the 2nd blank circle on your Data Record Sheet. Label the parents
and the mixture and describe their colors. Be sure to write down what kind
of medium (YED or MV) cells are growing on and today's date.
2. Make a copy (replica) of the YED plate by transfering the mating
mixture onto an MV plate. Make the streaks the same size and shape as those
on the original plate. With a sterile toothpick pick up some HBT cells
streak from your YED plate. Then make a streak on the left side of the
MV plate (See Figure 5). Discard the toothpick. With another sterile toothpick
pick up some HA2 cells from YED plate and make a streak of HA2 cells at
the top of the MV plate. Discard the toothpick. With a third sterile toothpick
pick up some of the mixture in the middle of the YED and then make a dot
of cells in the center of the MV plate. Discard the toothpick. Label this
MV plate with your name, the date and "Selecting the Diploids". Incubate
the plate overnight.
Before inducing the yeast to sporulate, it is better to transfer the
diploid cells back to a YED plate. Cells on YED medium will grow faster
than on MV medium and rapidly growing cells will sporulate better.
Time Line: 4th Day: 15 min
Diploid cells from previous procedure
YED agar plate
1. Make a sketch of the MV plate you made in the last procedure
in the 2nd empty circle on your Data Record Sheet. Record the color of
the diploid cells growing on the plate. Does one color phenotype (pink
or cream-colored) seem to hide the other phenotype?
2. Make a wet-mount slide from the diploid cells growing on MV and
look at them through the microscope.
3. Draw a sketch of about 10 of these cells in the third square
of your Data Record Sheet. Label the square "Diploids".
4. Use a sterile toothpick to transfer some of the mating mixture
(from the middle of the MV plate) to a plate of YED medium (see Figure
6). Discard the toothpick.
5. Incubate the plate overnight.
By this point you have seen half of the yeast life cycle: mating
between two haploids to form a stable diploid cell. The diploid cells will
divide and can be cultured just as you have done with the haploid parents.
You can now see the other half of the life cycle by sporulating the diploid,
to obtain four haploid spores. To do this you need to transfer the diploid
cells to sporulation medium (YEKAC). YEKAC contains no nitrogen source
and only a nonfermentable carbon source (acetate). When diploid cells try
to grow on YEKAC, they sporulate and go through meiosis. Meiosis produces
two important results:
1) The chromosome number is reduced from diploid to haploid, and
2) the resulting haploid cells have all possible combinations of
the adenine, tryptophan and mating-type genes.
Through the microscope, you can see the products of sporulation
as four spores enclosed in a sack called an ascus.
Sporulating the Diploids:
Time Line: 5th Day: 15 min
1. Draw a sketch of the YED plate you prepared in the previous procedure
on your Data Record Sheet and record the color of the diploid colony growing
Diploid cells grown on YED in the previous procedure
2. Use a sterile toothpick to pick up some of the freshly grown
diploid cells from the YED plate and make three streaks on the YEKAC plate
3. Incubate the plate for at least 3 days.
When you examine the sporulation culture through the microscope, the
asci will be easy to find. They look like lumpy cells. These are actually
sacks containing four spores. After you observe the asci you will grow
the spores into colonies to see if any of them have the original color
phenotypes of the parent strains.
Observation of Asci and Germination of Spores:
Time Line: 8th Day: 50 min
Sporulated cells from YEKAC plate
Draw a sketch of the YEKAC sporulation plate on your Data Record
Make a wet-mount slide of a sample from the YEKAC plate and
examine it with a microscope. Look for lumpy- cells that appear to have
two, three, or four round spores inside a membrane. These are the asci
containing ascospores. They should all have four spores, but sometimes
some of the spores don't develop. If most of the cells do not have spores,
incubate the plate for another day or two.
Draw a sketch of about 10 of the asci in the third square of
your Data Record Sheet. Label the square "Sporulation".
Touch a sterile toothpick to one of the streaks of the YEKAC
plate. Make a streak on a new YED plate (See Figure 8). Then use a new
sterile toothpick to make another zigzag streak across the first one on
your YED plate. Continue using fresh sterile toothpicks to make 4 or 5
more zigzag streaks in this manner. The last streaks should give you some
single colonies. Each colony will grow from a single ascus or from parts
of broken asci that may contain single spores.
When you put spores back onto YED growth medium, they germinate, begin
budding, and grow into colonies. Since some will be mating type a and some
mating type , they may
also mate. Therefore, the colonies that grow may be either haploid or diploid
cells and either pink or cream-colored.
Looking for the Missing Color:
Time Line: 10thDay: 15 min
1. Look for different colors among the colonies. Can you find both
of the colony color phenotypes expressed by the original haploid parent
2. Draw and label a sketch of this plate on your Data Record Sheet.
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Last updated Wednesday, 04-Dec-02 14:51:34