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.

Figure 1: Four images of Saccharomyces cerevisiae cells.

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 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 10ml 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 ml 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.