UAB-CORD “BioTeach” MODULE #2
ULTRAVIOLET MUTAGENESIS LABORATORY
aka. Sunlight can be bad for your health
Teacher’s Guide……………………………………… …………..page 2
Part I: UV Dose Response Experiment (Day 1) …… … … ……..page 3
Part II: UV Blocking Agents Experiment (Day 2)… ………..….. page 6
Part I: Data Sheet …………………………………………………page 7
Part II: Data Sheet ……………………………………………..…page 8
Appendix 1: Autoclave Instructions………………………………page 9
Appendix 2: Incubator Instructions … ………………….………page 10
Contents of Module 2 …………………………………….………page 11
If you have questions or comments, please don't hesitate to call or email Ryan Reardon!
Ryan Reardon
(205) 714-8479 (McWane Office)
(205) 714-8387 (McWane Lab)
(205) 243-8795 (Cell)
GENEius@mcwane.org (Email)
This project is supported in part by a Science Education Partnership Award (SEPA) from the National Center for
Research Resources, National Institutes of Health.
Ultraviolet Mutagenesis Laboratory
TEACHER’S GUIDE
ADVANCED PREPARATION OF MATERIALS
1
Objectives
Step 1 Prepare agar plates
Time required
At least 1 hour
When
1 day before Lab 1
Step 2 Set up workstations
Obtain sunblockers
10 minutes
Shortly before lab
This protocol is designed for use with 4-6 groups of 4-6 students. Each group will use 5
LB Agar plates for the first experiment and 4 LB Agar plates for the second experiment.
Thus, for a class or 30 divided into 6 groups of 5, at least 54 plates would be required.
One liter of LB Agar should be sufficient for one class.
Pour agar plates (LB)
1. Prepare LB Agar For 1 L LB Agar, weigh 40g LB Agar, add to 1L H2O and mix.
Pour into two 500 ml bottles. Top with lid, very loosely screwed on. Autoclave as per
directions for autoclave.
Stack the empty plates 4 to 8 high. Starting with the bottom plate, lift the lid and the
upper plates straight up and to the side with one hand and pour the LB/agar into the
bottom plate with the other. Fill the plate about one-third (~15 ml) with agar, replace the
lid and continue up the stack. Pour the entire 1 L of LB agar in this fashion. Let the
plates cool in this stacked configuration.
2. Plate storage
After the plates have cured overnight at room temperature they can be used or stacked up
twenty high and the plastic sleeve bag slipped back down over them. Invert the stack,
tape the bag closed, and store the plates upside-down in the refrigerator until needed.
Safety Reminders:
WEAR SAFETY GLASSES AT ALL TIMES!
WASH YOUR HANDS AFTER THE EXPERIMENT!
Ultraviolet Mutagenesis Laboratory
aka. Sunlight can be bad for your health
STUDENT GUIDE
Important Safety Reminders:
WEAR SAFETY GLASSES AT ALL TIMES!
WASH YOUR HANDS AFTER THE EXPERIMENT!
Part I: UV DOSE RESPONSE
Introduction:
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More than ever, people are becoming aware of the effects of UV radiation on humans. It
is known that UV radiation can cause human genes to mutate and cause skin cancer.
Today we are going to do an experiment that shows the effects of UV radiation on a
bacterium called E. coli.. E. coli can be used to show mutation since it can form
mutations similar to those found in human genes.
Dose Response Experiment (Day 1)
1. One member of each group should obtain the following from the front of the room:
5 plates
2 inoculating loops
1 Sharpie marking pen
4 index cards
1 box
2. Label the bottoms of the plates as indicated.
S
0
S
0
R
Control
2
S
4
5
R
S
10
15
R
A
S
30
R
B
C
30
60
R
D
3. Get the UV Sensitive (UV S) and UV Resistant (UV R) E. coli bacteria from the
instructor.
Part I: UV Dose Response
(cont’d)
4. Select a plate. Remove the inoculating from the package, taking care to not touch the
loop end to anything. Dip the loop into the UV Sensitive bacteria culture. Make a
broad streak in one direction on the surface of the agar, across the “S”, then in the
opposite direction, as shown below (left). Streak the bacteria ACROSS the agar, not
into it! Repeat the above steps across the “R” using the UV Resistant culture, as
shown below (right).
•
S
S
R
R
Streak the UV Sensitive across the “S” and the UV Resistant across the “R”.
Place the plate in the box, upside down, with the lid on.
3
5. Repeat step 4 with each of the other plates.
6. Lift the cover of the light box and cover the surface of the transilluminator with a
piece of plastic wrap.
4
7. Remove plates A-D from the box. Remove the covers of the plates. Place the plates,
upside down, on the light box. Slide an index card under the left half of each plate, as
shown below. Close the cover to the transilluminator.
32
S
4
35
S
10
3 15
S
30
3 30
S
60
R
R
R
R
A
B
C
D
Always have the light box cover closed and wear your safety glasses when you
switch on the UV lamp box!!!!
(continued on page 8)
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Part I: UV Dose Response
(cont’d)
8. As you begin counting up with the timer, turn on the switch of the UV light box to
analytical.
Remember to restart the timer each time you turn the lamps back on.
After 2 seconds, stop the timer, turn off the UV light, and remove the card under plate A.
After 4 sec., stop the timer, turn off the UV light, remove plate A, and put it in the box.
After 5 sec., stop the timer, turn off the UV light, and remove the card under plate B.
After 10 sec., stop the timer, turn off the UV light, remove plate B, and put it in the box.
After 15 sec., stop the timer, turn off the UV light, and remove the card under plate C.
After 30 sec., stop the timer, turn off the UV light, remove the card under plate D,
remove plate C, and put plate C in the box.
After 60 sec., stop the timer, turn off the UV light, remove plate D, and put it in the box.
NOTE: The above procedure is provided as a guideline for establishing a dose response
curve for both UVR and UVS bacteria. If you have a better way to produce a dose
response curve, or if you set this up differently during BioTeach, use your own method.
Remember you want the dose response curve so you can perform experiment 2. There is
no “best way” to get it, just do what’s comfortable and effective.
Loosely wrap the stack of plates in aluminum foil and incubate upside down at 37oC
overnight.
REMEMBER TO STORE YOUR BACTERIAL STOCKS IN THE REFRIGERATOR
UNTIL THE NEXT PART OF THE EXPERIMENT:
UV MUTAGENESIS EXP (DAY 2)
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Part II: UV BLOCKING AGENTS
UV Mutagenesis Experiment (Day 2)
1. Examine the plates from yesterday’s dose response lab. Look for growth of bacterial
colonies on the plates. Where there is reduced growth in a streaked area, the UV
exposure was significant enough to slow or stop the growth of the E. coli.
Draw the growth patterns on the data sheet plate diagrams (pg. 7)
2. Observe your plates and drawings. What was the shortest amount of time needed to
inhibit the growth of both the UV Sensitive “S” and the UV Resistant “R” bacteria?
3. Obtain 4 CLEAN plates from the instructor, label with “S” and “R” and streak with
UV Sensitive and UV Resistant bacteria as before.
S
S
R
R
4. Remove the original lids and make covers with plastic wrap.
5. Completely cover the UV light box with a sheet of plastic wrap.
Apply a rectangular area about the size of an index card with UV blockers (lotions,
sunscreens, cloth, etc…be creative!).
6. Invert the plates and place them on the light box so that 1/2 of each plate (both UV
Sensitive and UV Resistant streaks) is covered by one of the UV blocking agents.
Close the light box cover and wear your safety glasses!
7. Expose the plates for the minimum time needed to slow or stop bacterial growth, as
determined in step #2 (the dose response curve).
8. Remove the plastic wrap and put the lids back on the plates.
9. Loosely wrap the stack of plates in aluminum foil and incubate at 37oC overnight.
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ULTRAVIOLET MUTAGENESIS
LABORATORY DATA SHEET
PART 1: UV DOSE RESPONSE
Results and Conclusions
1. Examine the plates from the UV Dose Response lab.
In the space provided below, draw the growth patterns found on each plate.
S
0
S
0
R
Control
2
S
4
5
S
10
15
S
30
30
60
R
R
R
R
A
B
C
D
2. What was the smallest amount of time needed to inhibit both the UV Sensitive and
UV Resistance bacteria? ________________________________________________
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ULTRAVIOLET MUTAGENESIS
LABORATORY DATA SHEET
PART 2: UV BLOCKING AGENTS
Results and Conclusions
1. Examine the test plates and draw the pattern of bacteria colony growth on each plate.
S
0
S
0
R
Blocker
-----------------------
2
S
4
R
Blocker
-----------------------
5
S
10
R
Blocker
-----------------------
15
S
30
R
Blocker
-----------------------
30
60
R
Blocker
-----------------------
2. Why was it necessary to remove the lids before exposing your plates to UV light?
_________________________________________________________________
_________________________________________________________________
_________________________________________________________________
3. According to your results, what was the BEST sun (UV) blocker?
_________________________________________________________________
4. What other factors could affect bacterial growth?
_________________________________________________________________
_________________________________________________________________
5. Will the results of this experiment affect your sun -tanning behavior? If so, describe
any NEW behaviors you consider “safe”?
_________________________________________________________________
_________________________________________________________________
6. Looking at your results as a consumer, can you say that the more money spent on a
UV blocker provides better protection? Why or why not?
_________________________________________________________________
_________________________________________________________________
_________________________________________________________________
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APPENDIX 1: USING THE AUTOCLAVE
(Short Version)
1. Remove cover by turning the wing nuts. Always undo two opposite wing nuts at a
time (this also applies when tightening the cover).
2. Make sure support stand is in the bottom of the autoclave. Add water to about 2
inches.
3. Place articles to be sterilized into the container. Be sure to loosen caps on bottles!
Do not tighten caps until media or water has cooled.
4. Make certain that the Air exhaust tube channel is in a position on the right side of
the container when placed in the autoclave.
5. Place autoclave cover on unit, making sure that the index arrow on the cover
aligns with the index line-mark on the side of the bottom. Make certain that the
flexible tube is inserted into the guide channel on the inside wall of the container.
Tighten wing nuts – remember do this two opposites at a time. Never use a
wrench when tightening – finger tight is good.
6. Plug in unit. Turn on the toggle switch. (Red light on) Place the Control Valve
(on top of the lid) in the open position – that will be with the Petcock upright! It
will take about 30 minutes before steam begins to vent out the top. At this time
close control valve – petcock horizontal.
7. Pressure will increase in chamber – the Heat control knob on the front of the unit
should be positioned to have the pointer pointing straight up. This setting should
result in pressures increasing to 17-19 PSI. Adjust the heating control knob to
give desired pressure.
8. Sterilization begins once pressure reaches 17-19 PSI. Continue for 35 minutes.
9. After sterilization period turn off autoclave with toggle switch and wait for
pressure to drop to zero. Do not vent by opening the control valve if you have
liquids in the autoclave. Once pressure is at zero open control valve and open lid.
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APPENDIX 2: ASSEMBLY OF THE INCUBATOR/SHAKER
There are 6 components: (1) The shaker, (2) the heater blower, (3) the top and (4) the
bottom of the plastic cover, (5) a plastic air flow diverter, (6) platform with clips for
flasks (which can be removed to make this simply an incubator).
For this experiment, will only use the incubator/shaker as an incubator. Simply remove
the platform with the flask clips and set temperature on the heater/blower. Be sure not to
turn on shaker while plates are being incubated!
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CONTENTS OF MODULE #2
BOX #1
UV Light Box
Petri Dishes
500 ml bottles
Aluminum Foil
Index Cards (UV shields)
BioHazzard Bag
Protocol
Support Literature
1
3 sleeves
4
1 roll
6
1
BOX #2
Balance
Spatulas
UV Glasses
Plastic Wrap
Sharpies
Inoculating Loops
LB Agar
Gloves,medium
1
2
24
2 rolls
30
30
2 tubes
1 box
BOX #3 (GRAY CRATE)
Shaker/Incubator
1
ADDITIONAL EQUIPMENT
Autoclave (Pressure Cooker Style)
1
SHIPPED ON ICE
Bacterial cultures:
UV “Resistant” TE824-9A03
UV “Sensitive Strain” TE1749-9A04
For GENEius Use Only
Packed:_____________
Shipped:_____________
Received:_____________
Re- Checked In:_____________
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