Most of the UV-C (the energy range that is most damaging to DNA) is filtered out of the sunlight by ozone. Normal cells--including those of humans and yeast--repair most of the damage produced by UV-B and UV-A wavelengths that penetrate the atmosphere. Consequently, they are resistant to the UV-B and UV-A in sunlight.
Most UV damage to DNA is in the form of pyrimidine dimers. Adjacent pyrimidine bases (thymine and/or cytosine) in become joined by covalent bonds. If a cell tries to replicate UV-damaged DNA with dimers present, it usually dies. A specific photoreactivating enzyme (photolyase) uses energy from visible light to split pyrimidine dimers. In other repair mechanisms, enzymes remove the dimers and then patch the affected DNA. Because these repair processes are so effective, only a source of high energy UV-C (such as a germicidal lamp) will produce substantial killing and mutation in normal yeast cells.
You can readily observe mutations in an adenine-requiring mutant strain which forms red colonies when grown on YED medium. Mutations in a number of genes can block the formation of the red pigment (Roman 1956, Jones & Fink 1982, Manney & Manney 1991, 1992) resulting in white colonies. Also, the original mutation occasionally reverts to wild-type by a back-mutation, also producing white colonies.
Experiment:
UV Lethality
To observe the lethal effects of UV,
you will spread red yeast cells on Petri plates
containing growth medium and expose them
to UV-C. After incubating the plates you
will represent the lethal effects of radiation
as a survival curve by plotting the fraction of
the irradiated cells that survive to form
colonies against the time of UV exposure.
UV-induced mutations
When a red mutant strain of yeast is
irradiated with UV-C, most of the surviving
colonies will be red, but a few will be white
mutants. The fraction of white mutants
among the surviving colonies is a measure
of the mutagenic effect.
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:
UV irradiation chamber with germicidal lamp Optional Materials:
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)
Dilute, Plate and Irradiate Cells
Time Line: Day 1: 50 min
Procedure:
1. Use the sample survival curve shown in Figure 2 to design your experiment:
Use the rule-of-thumb that you can get an
accurate count of a plate that contains between
30 and 300 colonies.
For a series of doses that seem reasonable,
calculate the expected number of survivors and
the appropriate number of cells to plate to
produce that many colonies.
Remember, it is easier to count a plate with too
few colonies than one with too many! Do several
of your lowest dose plates at the same cell count
as your unirradiated control plates.
Figure 1 illustrates how to adapt the dilution and plating procedure. Prepare a dilution series of strain HA2, or HB2 and spread the appropriate dilutions on your plates.
2. Expose all the plates except the unirradiated controls to the germicidal lamp. Take the lids off of the plates while you are irradiating them with UV-C. Plastic and glass lids will absorb UV-C wavelengths of light.
3. Incubate the plates until the colonies are large enough to count. At 30øC that will take about two days and at room temperature it will take three or four days.( Teacher Tips)
Figure 1 Serial Dilution and plating strategy for a survival curve.
Figure 2: UV-C survival curve for strain HB2
If your concentration
factors are 1 for the control tube (last
tube in the dilution series), 10 for the
next most concentrated tube, 100 for
the next, and so forth, then calculate the
surviving fraction for any irradiated
sample using the equation below.
Surviving Factor = Count Colonies and Analyze the
Results
Time Line: 4th Day: 50 min
1. Count the colonies and tabulate your
data:
Make a table of exposure time, concentration
factor, colonies/plate for each duplicate plate, and
mean colonies/plate on the form provided.
Calculate the surviving fraction for each exposure
time.
2. Use a piece of semi-log graph paper to
plot the surviving fractions against
their UV exposure times.
How does your survival curve compare to
Figure 2?
The curve in Figure 2 indicates that
approximately 21 seconds of exposure
produced a 0.1 surviving fraction. Does
your data indicate more or less than 21
seconds are needed to produce a 0.1
surviving fraction?
Compare your UV-C source to the one used
to produce the data in Figure 2.
Mean colonies / on irradiated plate
(Concentration Factor x Mean colonies) on unirradiated plates
Teacher tips
Figure 3: Plot of UV-induced mutations
against dose.
Mutation Results:
1. Count the total number of colonies
and the number of white mutant
colonies on your plates.
2. Pool the class data and calculate the
number of white mutants per 100
surviving colonies for each exposure
time.
3. Plot the pooled class data on linear
graph paper to make a graph like
Figure 3. It shows
mutants/100survivors plotted against
exposure time.
How does your graph compare to Figure 3?
Were your experimental UV-C conditions
more or less mutagenic than the ones that
produced the data in Figure 3?
Name of Investigators
Results:
Return to Contents
Technical Tip:
The concentration factor is based on
the dilution series. It is the ratio of the
number of cells plated on the irradiated
plate to the number of cells plated on
the control plate.
As you count each colony, mark its' position on
the bottom of the plate with a marking pen. Record and Analysis of Survival Data
Date
Time
Location
Ultraviolet Radiation Source
Yeast Strain
Growth medium
Incubation temperature
Diagram of Dilution and Plating Procedure:
Exposure
Time
Count
Plate 1
Count
Plate 2
Count
Plate 3
Mean
Count
Concentration
Factor
Surviving
Fraction
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
Last updated Friday July 11 1997