Objectives

In this lab, your task will be to develop hypotheses and test them with experiments that you design and carry out. This will be an example of the scientific process which underlies all of the information presented in Bio 111.

Introduction

The lab begins with a phenomenon: a patch of yeast growing on a plate. This patch has a red center and a white edge.

Your task is to figure out as much as you can about why the middle is red and the edge is white. In this case, the objective is not to find the complete right answer - there isn't enough time, your tools are not sophisticated enough, and, in science, you never know the compete answer - you're always just adding more pieces to the picture. Instead, your objective is to find some piece of the answer that the community of scientists in your lab section can agree on.

Click here for more pictures of the yeast experiment.

Yeast is a single-celled organism, a type of fungus. The particular species of yeast we will be using is Saccharomyces cerevisiae, the species of yeast used to bake bread. When you buy yeast for baking at the supermarket, it is dried, but still living, cells of Saccharomyces cerevisiae.

Yeast cells are tiny, roughly 1/100 of a millimeter in diameter and approximately round. They "grow" by budding off new cells - that is, they grow by making more yeast, not by each yeast growing larger (like humans do). This is shown below:

Each time the cells bud, the number of cells doubles: 1, 2, 4, 8, 16, 32, 64, 128, etc. This is called exponential growth. Budding occurs roughly every 2 hours. That means in 24 hours, one cell would give rise to roughly 4000 decendants. Between experiments, we will let the cells grow for 7 days, which would mean that one cell would give rise to a block of yeast roughly 1 mile on a side! However, the cells eventually use up the nutrients we've fed them and stop growing long before that.

We will grow the yeast on petri dishes full of gelled nutrient medium; "plates" for short. In the picture on the previous page, you can see the edge of the dish and the gelled medium. The medium consists of simple nutrients which the yeast can use. The medium is gelled with agar which gives is a firm consistency.

The patch you study (a fully-grown one is shown on the previous page) starts as a puddle of yeast cells. The photo below shows how it develops:

When the puddle dries, it is a barely-visible film of yeast cells spaced far apart. This is the part of the plate marked "DAY 0" above; you can't see the film in the picture. After 3 days, the yeast budded many times and the progeny yeast piled up, producing the more dense patch marked "DAY 3". Finally, after 7 days, you see the dense patch marked "DAY 7" where the yeast have crowded and piled up on one another. You will use patches that are 7 days old.

The only tools you will use are toothpicks that have been sterilized - all microorganisms on them have been killed. The experiments you will do will be to take samples of yeast and put them on fresh plates to grow for 7 days. By carefully choosing what you pick, where you put it, and seeing what grows, you can learn a lot about why the center is red and the edge is white. The toothpicks have been sterilized so that you can be sure that the only microorganisms you put on your plates are the ones you intend.

This lab will span three meetings. Today will be the first; you will look at the plates and do the first round of experiments. Next week, you will look at the results, interpret them, and do a second round of experiments to firm up your conclusions. In the last week, you will look at the results of the second round of experiments and have a final discussion of what you've concluded. Your lab report will be a 2 page paper reflecting on what you found out (more on this later).

Procedure

(1) Today, you will be given 8 plates. Two will look like the one shown above and will be the source of samples for your experiments; you may want to use one to "mess around" and get a feel for the material and save the other as a source of samples for your experiments. Six will be blank, for you to do your experiments on.

(2) The first phase of the lab is to come up with some hypotheses to explain why the center is red and the edge is white. Remember that the patch started off as a uniform thin film; that is, the starting cells were distributed randomly and the film was too thin to determine its color. Begin by working in groups of three to come up with your own hypotheses. The class will then discuss each group's models and the TA will write some of them on the board.

(3) Then, in groups, come up with a few experiments you will do to address these hypotheses. The groups will then share their experiments with each other in a discussion. You should consider doing as many experiments as you can and are encouraged to do experiments invented by other groups; no experiment is any one group's "property".

(4) Do the experiments you have chosen to do. A few tips:

Don't count on your memory.

You will find that you will be unable to remember details which seemed obvious a week ago. So be very careful to keep records of what you did and thought. Two useful hints:

Label your plates with the markers we provide. Write something to identify your group, the date, what you put on the plate, and where you put it. Be sure to write on the bottom of the plate (the part with the medium on it) since the covers can get mixed up.

Take careful notes of what you did as well as the hypotheses you were testing (this second part will be essential for writing your lab report).

Contamination by other microbes is a big problem.

The plates are very rich medium, so almost any other microorganism that lands there will grow. Since we grow these plates for a long time (one week), a single spore of bread mold will have grown to cover the entire plate in a week. This kind of contamination will make your results uninterpretable. To help prevent this, or at least minimize the tragedy it causes:

Do more than one replicate of each experiment and have the replicates on different plates. Put the different plates in separate bags - one set of experiments in each bag - so that if one set gets contaminated and the contamination spreads throughout the bag, all will not be lost.

Use sterile technique to reduce the chance of contamination:

- before starting to work, remove all extra stuff from the lab tables and wipe the tabletop with alcohol using a squirt bottle and paper towels. This will kill most microorganisms on the tabletop.

- tie back your long hair & roll up your sleeves. This will prevent dust (with the microbes in it) from falling on your plates.

- avoid too much bustling in the lab to keep the dust down.

- when you must open your plates, open them for only the minimum time necessary. You may want to have one person hold the lid over the plate while another takes samples, etc.

- do not touch the nutrient medium in the plates with your fingers or anything except a toothpick.

- take toothpicks out of the tubes carefully. Keep the cap on until you need a toothpick. Then, take the cap off and tap the tube to shake out a toothpick (like tapping a cigarette out of a pack). Then, be sure to hold the toothpick only by the end that will not be touching the yeast or the plates.

- when you're done with a toothpick, put it in the paper cup or can. We re-sterilize them.

- if you even think that you've contaminated the toothpick (left it out too long, brushed something, etc.), put it in the paper cup & get a new one.

Use your imagination

Any experiment you can do with the toothpicks and plates may yield interesting results. Here are a few that have been tried (you should definitely think of your own as well):

put just red and just white separated on the plate

put samples on the lid of the plate

put samples next to each other

 put samples under blocks of medium that you have cut out

When you are done

Put the experiment plates in bags. Remember to bag duplicates separately. Wrap the extra bag around the plates and tape loosely to allow some air exchange. Also, be sure to bag the plates so that they are upside down when the bag sits on the table. This is shown below:

Give your bags to your TA.

Take all the other plates (the original patch ones, and the unused ones) and put them in the special trash bags. These must be sterilized before we can throw them away.

Clean up.