The Sporulation Response in Yeast
Biology 102 Lab #1
Before Coming to Lab:
• Carefully read the entire laboratory, both ‘Week 1’ and ‘Week 2’. Ask questions about any
parts that you don’t understand.
• Read pages 69-74 of the Biology Student Handbook. Pay particular attention to the “Using the
Microscope” section as we’ll be using the compound microscope extensively today.
Background:
There are many different eukaryotic fungal species that are classified as yeasts. They are found
around the world, inhabiting a variety of different niches. Yeasts can be found living on plants, in soil,
even on or in animals. Some yeasts are symbiotic, some are parasitic and some are even pathogenic.
Yeasts generally grow as single-celled microorganisms, but some will form long, thread-like,
multicellular mycelia. The best-understood yeast is Saccharomyces cerevisiae, and that is the species
of yeast that we will use in the BIO102 laboratory over the next two weeks.
S. cerevisiae is a fungus belonging to the phylum Ascomycetes in the class Hemiascomycetes
that is often found growing on plants around the world. This yeast is surrounded by a cell wall and
reproduces very rapidly by budding. Humans
have been using Saccharomyces cerevisiae for
more than 6,000 years without realizing it. This
species of yeast lives and grows in bread dough
and fermenting beverages. The carbon dioxide
gas produced by the yeast in bread dough is what
causes the bread to rise. Ethanol is produced as a
metabolic waste product by growing yeast and is
the source of alcohol in beer and wine.
Saccharomyces cerevisiae is commonly
used as a model organism, meaning that it is an
organism that can be easily studied and much of
Figure 1: Four images of Saccharomyces cerevisiae cells.
the resulting knowledge can be applied to other organisms, like people. There are many reasons that
yeast is a favorite eukaryote to study: yeast are easy to grow, they are nonpathogenic, it’s relatively
easy to add or remove genes and their biology is remarkable similar to human biology. Additionally,
yeast has been highly studied yielding a lot of background (more than 53,000 scientific papers in the
last 40 years) and the DNA sequence of the entire genome has been available since 1996. From this
sequence, we know that yeast has just 6,145 genes (compared to about 30,000 genes in humans).
Because it’s such a small organism, we can easily analyze a billion organisms (which is impossible in
humans or mice!). Because of these many useful traits, scientists will often turn to the “awesome
power of yeast genetics” to carry out experiments that are impossible in other organisms.
A healthy, actively growing yeast culture can double the number of cells present every 90
minutes! When they are rapidly growing like this, all of the cells in the culture are observed to be nice,
round cells or round cells with a bud emerging (Figure 1). That bud will grow larger and larger rather
like a balloon inflating. Eventually, the bud gets large enough that it will separate from the ‘mother’
cell and is a new cell in the population. Like any organism, S. cerevisiae can respond to changes in its
environment by altering the proteins it makes, its metabolism and/or its growth rate. Under some
conditions, a striking change occurs: the yeast cells can be observed to produce spores. Over the next
two weeks, we will investigate why yeast cells form spores.
Procedure:
Work in pairs for these experiments. In the first week, we will get used to using the compound
microscopes and will measure the sporulation efficiency of yeast exposed to some different conditions.
In the second week, you and your partner will carry out your own independent experiment that
investigates factors that might influence the sporulation efficiency.
Week #1:
Introduction to Microscopy and Yeast
How would you go about determining the size of microscopic objects? One good way to estimate size
is to know the size of the field of view, as well as the apparent length of the pointer. These are different
at each magnification (the length of the pointer doesn’t change, obviously, but how big it appears
changes with magnification).
1. If you haven’t read the “Using the Microscope” section of the Biology Student Handbook, stop
and read it now. You are responsible for the proper use and care of your microscope. Please ask
questions as we go along if things aren’t clear
2. Instead of a slide, place a transparent ruler on the microscope stage. Using the 4× objective,
focus on the millimeter scale. Using this objective lens, what is the total magnification of the
instrument? Record it in your notebook.
3. In your notebook, record the diameter of the field of view and the apparent length of the pointer.
Record both in millimeters.
4. If you switch to the 10× objective lens, what is the new total magnification of the microscope?
How large would you predict the field of view will be? What you predict for the apparent
length of the pointer? Calculate these values and record them in your notebook.
5. Now switch to the 10× objective and measure to see if you were right. Record the actual values
in your notebook.
6. Calculate what the size of the field of view and the apparent length of the pointer would be if
you switched to the 40× objective. You don’t have to try to actually measure this (why not??).
Observing Yeast
1. Place 10μl of actively dividing yeast cells on to a clean microscope slide. Place a cover slip on
top.
2. Look at the cells through the 4× objective lens. Then change to the 10× lens and observe them
again. Increase the magnification again by switching to the 40× lens to look at fewer cells in
more detail. Also try viewing your cells using phase-contrast illumination, as described in the
Biology Student Handbook.
3. Using the information that you gathered above about the apparent size of the field of view and of
the pointer, estimate the size of the cells.
4. Draw several yeast cells as viewed under 400× magnification. Include some budding cells and
some unbudded cells. Take some time to carefully observe the cells and make a drawing that
shows what you really see—you do not need to be a Great Artist, but we’re looking for more
than just a circle and some dots. Make your drawing larger than life and use a pencil so that you
can use shading to show details. Label any cell structures that are visible and be sure to
indicate the magnification used to make the drawing.
Observing Yeast Spores
1. Place 10 μl of sporulating yeast cells on to a clean microscope slide. Place a cover slip on top.
2. Just like with the culture of actively dividing yeast, locate the cells under low power before
shifting to higher power. Do you see some cells that look different? What do they look like?
Draw several spores and indicate the magnification at which they were observed. In addition to
the pictures, explain what you see in words as descriptively as possible. Estimate the sizes of
these spores.
3. How does this culture look different from the actively dividing culture? Do you see any cells
budding? Count the fraction of the cells in this population have sporulated.
4. With your partner discuss some possible environmental conditions that may have led to spore
formation in yeast. In essence, these possibilities that you come up with are hypotheses. How
can we test the various hypotheses? Record at least one hypothesis and how it might be tested
in your notebook.
When all of the groups have reached this point, the entire class will discuss the various hypotheses and
how they might be tested. As a class, we will choose one hypothesis to test during the rest of today’s
lab. Before we get too much farther, let’s make sure that our hypothesis makes some biological sense.
In other words, what’s the biological justification or rationale for our hypothesis? What sorts of
observations that we made, biological facts that we know or good logical deductions led us to this
hypothesis?
The hypothesis is only really useful if it is a testable hypothesis. Thus, our next step is to come up
with a protocol or the specific means with which to test our hypothesis. As a group, we will discuss
what it is that we will do including important details like amounts, concentrations and times. We need
to try and anticipate all the technical details that will be important for completing our experiment.
Before we actually run the experiment, let’s look at the possible outcomes or the predictions made by
our hypothesis. What kind of data would support our hypothesis? What kind of data would refute our
hypothesis? Is there any outcome that might be ambiguous, neither supporting nor rejecting our
hypothesis?
If so, is there some way to tweak our protocol to minimize the possibility of an
ambiguous outcome?
Each group will now run the experiment(s) that we decided on to actually test the hypothesis. By the
end of the lab today, we should have generated a substantial dataset for your analysis. So what do you
know? Was our hypothesis supported or refuted? Record your conclusions in your lab notebook and
start planning ahead to design your own experiment for next week.
Before Leaving Lab:
• Clean up! Plastic microfuge tubes (closed) can be simply thrown in the trash with their contents.
Microscope slides need to be washed, rinsed in distilled water from a squirt bottle and dried
well and returned to the box. Coverslips can be disposed of in the glass trash box by the door.
• Check your lab bench drawer. Did everything get returned to it? Get in the habit of doing this
at the end of every lab period for every course.
• Collect the data from all of the groups in your lab section.
• Be sure that your notebook is complete! Review the notebook guidelines in the Biology Student
Handbook for the requirements. We take these guidelines seriously; even stuff that seems small
(like numbered pages, writing in ink and listing dates) is absolutely essential.
• Look ahead to what you need to do after lab and to prepare for next week’s lab. Be sure that you
have all the necessary information to complete your assignments. Ask questions as needed!
• You’ve got quite a lot of information about what causes sporulation in yeast. What more do you
want to know? In science, most answers usually lead to more questions. What new questions
do you now have about sporulation in yeast? How can we experimentally address these
questions?
After Lab:
• Be sure that your notebook has your complete analysis. This will vary a bit depending on what
hypothesis and protocols the class chose.
Be sure that your notebook contains enough
information on how the experiment was done so that you could walk into lab with only your
notebook (and without this lab guide) and repeat the experiment successfully.
• You will almost certainly need to calculate some averages and standard deviations of the data.
You may use Microsoft Excel (or some other computer program) to do the calculations and
simply tape the printout into your notebok.
• You will almost certainly want to plot the class data as a graph. Be sure to follow the guidelines
for good graphs listed in the Biology Student Handbook. (What kind of graph would be best for
these data? bar graph? line graph? pie graph? why?) Use the standard deviation data for error
bars: the error bars should show one standard deviation from top to bottom. The graph may not
be hand-drawn. Tape this graph into your notebook.
• Comment on the reliability of your data. Does everything seem dependable? Why or why not?
What do the standard deviations tell you about your data?
• What can you conclude about starvation and control of the sporulation response in yeast?
• After finishing your notebook, review it. Specifically, look and make sure that you have included
everything asked for in the Biology Student Handbook and everything asked for in this section
of the Lab Guide (especially this page!).
• Read next week’s lab carefully! Notice that you will need to turn in an experimental procedure
in class on Wednesday, March 26th. Thus, you and your partner need to start thinking and
making specific plans today.
• Sign-up for a time to meet with your lab instructor (either Dr. Visick or Dr. Johnston) for a few
minutes on either Wednesday afternoon or Thursday. We will discuss your experimental plan
together, ask a few questions and perhaps make some suggestions to be certain that your plan is
a good one.
• Your lab notebook is due Friday, March 28th in class.
Week #2:
In this week’s lab, we will again be measuring the sporulation efficiency of yeast, but we will
be asking different questions. The variable that you investigate will be chosen by you and your partner!
For most of these variables, you will probably want to test more than one level or amount so you can
find if any trends exist. You should generally plan on testing each experimental condition three times
so you can calculate standard deviations and get a feeling for what kinds of differences are
reproducible. That means you’ll probably be doing a lot of assays in week #2. However, if you come
to lab well-prepared, you should be able to do a large number of assays in a short amount of time
because you already know how the assay works and what you’re looking for. Note that you should set
up your experiment so that you are testing (changing) only one variable and keeping everything else
constant (controlled).
Turn in a specific, detailed protocol (including volumes, concentrations, temperatures, etc.) for
your experiment in class on Wednesday, March 26th for approval. Only one proposal per pair of
students is required, and it is expected that the proposal will be a group effort. The template for the
proposal can be found on the course Web site. Be sure that you have enough detail in your protocol so
you are actually ready to carry out your experiment. For example, if you want to test the effect of
increased salt concentrations, how much salt do you want? We are requiring you to turn in your plan
in advance for a couple of reasons. First, this forces you to think through your plan before coming to
class. Secondly, we can make sure that we have the appropriately stressed cells ready for you in lab.
Finally (and most importantly), your instructor may make some suggestions that will improve the
quality of your data. You need to schedule a brief meeting with your lab supervisor on Wednesday
afternoon or Thursday to discuss your experimental plan and make any alterations that might be
necessary.
When you come to lab during Week 2, go ahead and carry out your plan. I strongly encourage
you to do some quick calculations in lab and see if your data are making sense. If needed, go ahead and
repeat some experiments or add some additional treatments to help make a complete story.
Before Leaving Lab:
• Be sure your lab notebook is complete!
• Since you designed this experiment, only you know all the materials and methods. Be sure you
wrote everything down: solutions, volumes, etc. If anything isn’t clear, ask now.
After Lab:
You will need to write and submit a lab report for these experiments. You should write as if
you are part of a research team doing actual research on yeast, and use the style of a manuscript to
be submitted for publication in a professional scientific journal. Guidelines are given in your
Biology Student Handbook; please follow those guidelines carefully. Pay special attention to the
specified formats for tables, figures, citations and references. Although you can certainly discuss
your results with your lab partner (and your instructor), each individual must write his or her own
report, including making his or her own figures. In addition, specific suggestions to help you with
this lab report are given in the “Lab Reports” section of this manual.
Stay focused on the efficiency of sporulation. Before you start writing, think about what
your data are telling you and what you want to convince your reader of. Then decide how best to
present your results to make your case. You will probably need to plot and include at least one
graph, similar to the graph that you made during the experiment from the first week. Your report
may include the experiments from the first week if it is helpful to your argument. You are
responsible for thinking about how to present your data clearly. Do not rely on data from the first
week unless you show those data.
In a scientific paper, any background material that doesn’t come from your experiment
must be properly credited with a citation in the text and a reference giving all the source
information at the end of the article. Many of the things you will want to talk about in your
introduction may be background material for which you will need proper sources. Often, your
textbook (or another text from the library or the lab) will work very well. You do not need to use
outside sources for this report (though you may), but you must cite your lab manual, textbook or
any other sources of information that you use. Don’t wait until the last minute to try to find this
background material. And remember that websites are never acceptable sources.
You will be required to turn in a rough draft of your paper and then revise it based on the
comments you receive from your instructor (who will play the role of the independent reviewers
who would read your manuscript if you were actually submitting it for publication). If you’re smart,
you won’t turn in your first draft: you’ll write two or three drafts and work them over carefully
before you turn anything in. Then you’ll be prepared to take the suggestions and produce a truly
polished, professional final product. The draft is due in class on Monday, April 7th. I will make
many comments on your draft and return it to you during the week. The final lab report is due one
week later, on Monday, April 14th at the start of class. Please hand back in your draft with
comments at this time too.