The experiment Observing the Effects of Solar Ultraviolet Radiation on Cells shows that when cells are exposed to sunlight all, some, or none of them may be killed. Many experimental questions can be answered with qualitative answers like "all, some, or none." Other questions may require quantitative answers. For example, in the next experiment you will use the sensitive yeast strain to measure the intensity of solar UV radiation by measuring the fraction of cells exposed that survive. To get quantitative answers about yeast survival you must put a known numbers of viable (living) cells onto the agar plates and then count the number that remain after being exposed. You can determine the number of viable cells by counting the colonies that grow up on the agar growth medium in a Petri plate by assuming that each colony grows from a single viable cell. This is usually a reasonable assumption.
Experiment:
In the experiment that follows you will learn
how to measure the number of viable cells
on a Petri plate. You will be able to use this
procedure whenever you need to measure
the number of cells that survive an exposure to radiation or some other treatment. First you will estimate the number of
cells in a liquid suspension in order to plate
a reasonable number of cells. For this you
will use one of the most sophisticated and
sensitive optical instruments in existence,
the human eye. With surprisingly little
practice you can learn to estimate the
number of cells in a suspension by just
looking at it.
You can estimate cell density because of your eyes' fairly sharp threshold for observing turbidity (cloudiness). When viewed in a standard 13 100 mm glass tube, yeast suspensions of less than about 1 million cells per mL are not visibly turbid. Above this threshold density, the suspension is cloudy. When you adjust the number of cells in a suspension until just barely visible, you obtain a suspension of known density (approximately 1 106 cells/ml).
When you have a suspension that contains approximately 1 106 cells/ml, you will dilute it to get the right concentration for plating. You will make the dilutions in known steps so you can calculate the number of cells in each dilution tube. This procedure helps you plate a countable number of colonies.
Time Line:
Day before:10 min Getting Ready
Day 1: 50 min Serial Dilution and
Plating Cells
Day 3: 30 min Counting Plates
Materials:
For each student or team:
12 to 15 sterile culture tubes,
13 100 mm with caps
5 to 10 polystyrene Petri dishes with
YED agar
1 Alcohol wipe
1-11 sterile pipets, either 1-mL
calibrated bulbed transfer pipets
or (1mL disposable serological
pipets calibrated in 0.1 mL steps
and pipet pump)
Common Materials:
Time Line:
Day before: 10 min
To have a freshly-grown culture of yeast
cells grow the yeast on YED growth
medium overnight at 30o C or 1 to 2 days at
room temperature.
1. Make a clean sterile work space by
wiping the table or bench with an
alcohol wipe. Because most
contamination is airborne select a
place free from drafts.
2. Open the yeast storage vial.
3. Using the broad end of a sterile
toothpick, pick up a small amount of
yeast from the slant.
4. Replace the lid. Tighten. ( Store in a
refrigerator to keep viable for up to
nine months.)
5. Open the YED Petri dish just enough
so that you can reach into it with the
toothpick full of cells.
6. Gently make several streaks of the
culture on the surface of the agar.
(Remember that you need not be able
to see the streaks to have enough to
grow into a visible culture overnight.)
7. Close the lid and incubate the culture
overnight at 30oC, or 2 days at room
temperature. (Most microbial cultures
should be incubated with the agar side
up to prevent condensation from
dropping on the colonies.)
Teacher Tip
Technical Tip:
By starting with an obviously turbid
cell suspension and diluting it in two-fold
steps, you eventually reach a dilution
that is not visibly turbid. We have found
that for most people, the last, or "just
turbid" tube will contain approximately
1 106 cells/mL.
1. Use a sterile pipet to place 2 mL of
sterile water into one tube and 1 ml
into each of six more tubes.
2. Make a turbid suspension in the first
tube.
Take a small amount of yeast--less than the size of
a pin head--on a toothpick and select the tube that
contains 2 mL of water.
Without touching the mouth of the tube, place the
yeast as far down the inside wall of the tube as
you can.
3. Mix the suspension thoroughly by
thumping it.
Holding the tube loosely near its top between
thumb and forefinger, and thump it near the
bottom with the palm side of one or more fingers
of the other hand, imparting a swirling motion.
The thumping motion approximates the gesture
one would make to beckon someone to come
hither.
4. Add yeast until the suspension is
noticeably turbid or cloudy.
5. Make a series of two-fold dilutions
until you have a tube that is clear.
Transfer 1 mL of the suspension from each tube to
the next, beginning with the one containing the
cells.
Mix each tube between steps by thumping it.
6. Compare each tube with a tube that
contains 1mL of clear water. The last
turbid tube will contain between 1 and
2 million cells/mL. Use this tube for
the rest of the experiment.
Teacher Tip
Technical Tip:
This procedure is called a serial
dilution. For making the "just turbid"
suspension the dilution steps are 1 mL
into 1 mL so that each tube is twice as
dilute as the previous one. Each
dilution step is two-fold. To dilute the
cell suspension further, for plating, dilute
0.1 mL into 0.9 mL to obtain more
convenient ten-fold dilution steps
Technical Tip:
Three drops from the bulbed transfer
pipet is equal to 0.1 mL.
Technical Tip:
Label the plates using a marking pen
(a Pilot SC-UF or a Sharpie). Use the
same kind of pen for marking the
colonies when you count them. Identify
each plate with the dilution plated and
your initials. Write in small letters on the
edge of the bottom leaving the middle
clear for marking the colonies when you
count them.
Prepare and Plate a series of ten-fold dilutions:
1. Start with the most dilute turbid tube
(just barely turbid tube from step 6)
Use the following procedure to make
six serial ten-fold dilutions from this
tube into sterile water. (see Figure 2)
2. Set up a series of dilution tubes.
Pipet 0.9 ml of sterile water into each of six tubes
and label them.
Make a diagram in your notebook showing the
procedure and explaining your labels.
3. Dilute the just turbid suspension into the first tube. Thump the tube containing the cells. Remove 0.1 ml, and transfer the 0.1 mL to one of the tubes of water. Then thump that tube to suspend the cells.
4. Repeat the procedure serially, using a
fresh pipet for each of the remaining
tubes. The sixth dilution (which will
be the seventh tube) should contain
approximately 1 100 cells/ml (1 cell!).
5. Spread samples of the cells from each
tube in your ten-fold dilution series on
agar plates by the following procedure:
Resuspend the cells in each tube by thumping the
tubes.
Pour the entire contents of each tube
onto an agar plate.
Replace the lid and distribute the suspension over
the surface by tilting and rotating the plate.
6. Incubate the plates for two days at
30 C or for three or four days at room
temperature until the colonies are large
enough to count.(Teacher
Tips)
Counting Colonies:
Time Line:
Day 3: 30 min
1. Count the colonies on each plate
Mark the position of each colony on the bottom of
the plate with a marking pen as you count it.
Plates containing more than a few hundred
colonies are considered "too numerous to count"
(recorded as TNTC in data records).
2. Calculate ratio for each dilution step
by dividing the number of colonies on
each plate by the number on the next
more dilute plate.
Questions:
1. How well do your colony counts agree
with the expected dilution steps of ten?
2. If the agreement isn't perfect (and we
don't expect it to be, even with a "perfect"
technician) do you think the errors could
be explained by "random chance"?
Explain.
3. Is there any pattern to the errors? Do the
ratios systematically get larger or smaller
as the number of colonies increases?
4. Write down any reasons you can think of
for such systematic errors to occur in
this procedure.
5. If you have a culture with 1x106 cells/mL
and you wanted to dilute it to 1x102
cells/mL, would it be better to just make a
single 10,000-fold dilution or four ten-fold dilutions? Explain the advantages
and disadvantages of each method.(answers)
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Last updated Friday July 11 1997