In the experiment Observing the Effects of Solar Ultraviolet Radiation on Cells you observed that when yeast cells are exposed to sunlight, all, some, or none of them may be killed. If you use the serial dilution method to refine the, procedure you can measure the fraction of the irradiated cells that survive. With the surviving fraction data and a standard dose-survival curve you can estimate the amount of UV-B in sunlight. This is a bioassay for solar UV-B and gives a better measure of DNA-damaging UV than most physical meters because it is based directly on UV damage to yeast DNA. Most meters don't measure just those energies of UV.
Experiment:
In this experiment the number of yeast
cells that are killed by being exposed to
sunlight depends on the intensity of UV-B in
the sunlight. Therefore, you can estimate
the amount of UV-B by measuring how
many cells are killed. You measure the
killing indirectly by determining the fraction
of cells in a sample that survive a particular
exposure time.
We assume you have some experience
with yeast, we have left some of the details
for you to design. You will make a yeast suspension with
a known number of cells per mL, and then
dilute that suspension by making a set of
serial dilution tubes so that the number of
cells per mL in each tube is 1/10th of the
number in the previous tube (see Figure 2).
You will use the diluted yeast suspension in
the tubes to plate between 300 and 3000
cells on each plate. That will yield between
30 and 300 survivors for an exposure time
that gives a 0.1 surviving fraction. These are
the techniques described in Serial Dilutions
and Viable Cell Counts.
This experiment will require some
practice. To select the best dilutions for
each dose, you must know the approximate
outcome of the experiment in advance.
Make your starting guesses using the
survival curve in Figure 1. In the first trial,
plate different dilutions and try several
exposure times. Use the results to make
better choices in subsequent trials. After
several trials, you'll be getting good
results and you will be able to monitor solar
UV-B. The goal is to get skilled enough so
that you only need two plates each time you
take a UV reading. (One control plate and
one timed exposure plate)
Time Line:
Day before:10 min Getting Ready
45 min Discussion of the
strategy and
objectives
Day 1: 50 min Dilution, Plating and
Irradiation of Cells
Day 3 or 4: 50 min Counting Colonies
and Analyzing
Results
Materials:
For each student or team:
Common Materials:
Getting Ready:
Time Line:Day before: 10 min
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 agar slant in the vial.
4. Replace the lid. Tighten. ( Store in a
refrigerator to keep the cells 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 Tips)
Technical Tip:
You can count how many cells
survive UV-exposure because when a
suspension of living yeast cells is
spread on agar medium, each cell will
grow into a visible colony after two to
three days. Cells that form colonies are
called viable cells. Some of the cells
exposed to UV will be killed and so will
not be viable. To determine the fraction
of viable cells on an irradiated plate
compare the number of surviving
colonies with the number of colonies on
an unirradiated plate (control).
Dilution, Plating and Irradiation of
Cells
1st Day: 50 min
1. Use the sample survival curve (Figure
1: Yeast strain G948-1C/U exposed to sunlight)
to design your experiment:
Choose several exposure times that look
reasonable and will produce a surviving fraction
close to 0.1 The times in Figure 1 are for the
middle of the summer. You will need to use
longer exposure times during the other seasons of
the year.
Use the rule-of-thumb that you can get an
accurate count of a plate that contains between 30
and 300 colonies. Calculate the appropriate
number of cells to plate to have that many
surviving colonies at each exposure time. For
example if you want to shoot for 100 surviving
colonies at 3 min exposure (0.1 surviving
fraction) you need to put 1000 cells on the plate.
Remember it is easier to count a plate with too
few colonies than one with too many! Be sure to
make several unirradiated control plates from the
dilution tube with the least cells per mL.(Teacher
Tips)
2. Prepare your dilutions and plates. Using the dilution and plating procedure illustrated in Figure 2, prepare a dilution series of strain G948-1C/U. Spread the appropriate dilutions on your plates. Label the bottom of each plate with the dilution and the exposure time.
3. Expose all the plates to the sun except for the unirradiated control plates. Point the plates directly at the sun for the desired exposure time and then place them in a dark container such as a cardboard box or paper bag.
4. Incubate the plates until the colonies are
large enough to count.
At 30xC that will take about two days and at
room temperature it will take three or four days.
Figure 2: Serial dilution and plating strategy for a survival curve
Teacher tip
Technical Tip:
The concentration factor is based
on the dilution series and is the ratio of
the number of cells plated on the
irradiated plate to the number of cells
plated on the control plate.
Time Line: 3rd or 4th Day: 50 min
1. Count the colonies and tabulate your
data:
As you count each colony, mark its position on the
bottom of the plate with a marking pen.
Surviving fraction:
Exposure time: 5 min
From standard survival curve (Figure 1), a surviving fraction of 0.12 is produced by an
exposure of 80 J/m2. The UVB irradiance is the amount of UVB energy (in units of
joules) falling on 1 square meter in one second. The irradiance that would produce and
exposure of 80 J/m2 in 5 minutes would be
( Teacher Tips
)
Teacher Tips
Technical Tip:
When you have your technique
calibrated, you can get good results
from a single exposed plate and a
control. It is practical to make frequent
measurements to monitor the solar UVB
in your area. As you accumulate more
measurements it will become possible
for you to look for trends. Your class
may want to make a large wall chart and
plot irradiance against the day of the
year or plot irradiance against the hours
of a single day.
Procedure:
Return to contents
Counting Colonies and Analyzing
Results
Make a table of exposure time, concentration
factor, colonies/plate for each duplicate plate, and
mean colonies/plate.
Calculate the surviving fraction for each exposure
time as described below.
If your concentration factor is 1 for the control
tube then it is 10 for the next higher tube, 100 for
the next, and so forth.
Sample calculations:
Concentration factor = 10
Mean colonies on irradiated plate = 124
Mean colonies on unirradiated plate = 103
Surviving fraction = (Mean colonies on irradiate plate)/ ( Concentration factor * Mean colonies on
irradiated plate)
Surviving fraction = 124 / (10 * 103) = 0.12
UVB Irradiance:)
Exposure for surviving fraction of 0.12 80 J/m2 1 min
Irradiance = --------------------------------------------------- = -------- * -------- = 0.34 J/m2/sec
Exposure Time 5 min 60 sec
UV Monitoring: Two Plate Method
From your initial trials you should have a
good idea of the dilution and exposure
conditions that will produce a surviving
fraction close to 0.1
1. Subculture G948-1C/U overnight on a
YED plate.
2. Make a 1 to 10 dilution series of the
cells in sterile water.
3. Spread an appropriate number of cells
on 1 control plate and 1 exposure
plate.
4. Place the control plate in a dark
container. Put the exposure plate in
the sunlight for a length of time that
should produce a surviving fraction
close to 0.1 Place the exposure plate
in a dark container and incubate the
plates for 2 to 3 days.
5. Count the colonies on both plates. Use
the surviving fraction data and the
standard curve (Figure 1) to determine
the irradiance.
The fraction of the cells surviving is plotted against the dose
of UV-B on a semi-logarithmic graph. The dose was measured with
a UV radiometer sensitive to wavelengths near 300 nm.
Last updated Tuesday July 07 1998