Linda Peters
Belchertown HS, Belchertown, MA
Pat Ehrman
Davis HS, Yakima, WA
Belleville West HS, Belleville, IL
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Linda Peters Belchertown HS, Belchertown, MA Pat Ehrman Davis HS, Yakima, WA Belleville West HS, Belleville, IL |
Notes to the Teacher: to top
Preparation Time & Schedule
5 TO 2 WEEKS PRIOR TO CLASS USAGE:
1. Order the biologicals and
equipment needed for lab. Time lines for purchase orders getting
out of
the district varies. Allow plenty of time for the ordering task and adjust
your time line
accordingly.
2. Your first order to Sigma needs to be accompanied by a letter from
your principal supporting
your experiments.
2 WEEKS PRIOR TO CLASS USAGE:
1. All equipment and biologicals
are ordered and have arrived in your school
2. TSB broth preparation
3. Prepare 1X SSC
4. Prepare all antibiotic
stocks...store in freezer.
1 WEEK BEFORE:
1. Prepare all plates with antibiotics.
3 DAYS PRIOR TO EXPERIMENT:
1. Start overnight cultures
in broth for both bacteria before you leave
school
(this
is a system check).
All cultures are at 37 celsius with shaking if possible UNLESS specified.
2 DAYS PRIOR TO THE EXPERIMENT:
1. Check overnight growth of
last night's cultures. You should have heavy bacterial growth.
2. Start overnight cultures
in broth and antibiotics for both bacteria. Inoculate with bacteria
and the
appropriate antibiotic before you leave school.
THE DAY BEFORE THE EXPERIMENT:
1. If your have
a class doing the experiment during first period tomorrow
a. inoculate
2 new broth flasks in the morning before school starts each with a different
bacterial strain and appropriate
antibiotic.
b. Let this grow
during the teaching day. At the end of the teaching day before you
go
home inoculate 2 new broth flasks eachcorresponding to one of the bacterial
strains and
appropriate antibiotics.
Grow this at room temperature overnight. If you have classes
during the first two periods
of the day tomorrow, inoculate two cultures.
THE DAY OF THE EXPERIMENT
1. When you get to school calculate the starting times for two
hour cultures for classes later on
in the day.
2. Continue inoculating two hour cultures for each class you have approximately
2 hours
before the class starts.
3. Set out all your experimental materials in your classroom.
to top
Tryptic Soy Broth(TSB)
| AMOUNT | 500 ml per class of 30 students(400ml for overnight and 2 hour cultures, and 1ml/student for conjugation tubes. |
| PURPOSE | for the culturing of both bacterial cultures |
| VENDOR | SIGMA can be found at www.sigma.sial.com OR PO BOX 14508, St. Louis MO63178 |
| CATALOG # | T8261 |
| COST | $8.30 per 250g |
| STORAGE | room temperature after autoclaving |
| PREPARATION | prepare broth and pH following the directions on container, autoclave for 15min @ 15psi.add 250 ul of each antibiotic when you inoculate the broth with bacteria. |
TSB Plates(Spc and Km @ 25ug/ml)
| AMOUNT | 30 petrie dishes (600ml of TSB broth and 9 grams of Bacto Agar) |
| PURPOSE | negative control plates |
| VENDOR | SIGMA's Bacto Agar...see address above |
| CATALOG | A5036 |
| COST | $67.84 for 250g |
| STORAGE | store poured plates upside down at 4 Celsius after they harden @ room temperature |
| PREPARATION | Prepare 600ml of TSB broth. Add 9g of Bacto agar and autoclave for 15 minutes @ 15psi. Remove from autoclave and place in 45 Celsius water bath. Sterilize lab bench and label the bottom of each plate with "TSB/KmSpc" and the date. Add 300ul of each antibiotic stock and swirl. Pour 20ml of media into each plate. |
| AMOUNT | 30 petrie dishes (600ml of TSB broth and 9 grams of Bacto Agar) |
| PURPOSE | transformants plate |
| VENDOR | see TSB and Bacto Agar |
| CATALOG | see TSB and Bacto Agar |
| COST | see TSB and Bacto Agar |
| STORAGE | see TSB plates |
| PREPARATION | see TSB Plates(Spc and Km @ 25ug/ml) and change the antibiotics accordingly. |
| AMOUNT | Prepare 1 ml with a concentration of 50mg/ml which is enough for the entire lab. |
| PURPOSE | culturing or selective plating of transformants and controls |
| VENDOR | SIGMA can be found at www.sigma.sial.com OR PO BOX 14508, St. Louis MO63178 |
| CATALOG # | A2804 |
| COST | $8.90 for 50 mg |
| STORAGE | -20 Celsius |
| PREPARATION | Add 1 ml of distilled water to the ampicillin container. Mix and filter sterilize with .45um syringe and disc. |
| AMOUNT | Prepare 1 ml with a concentration of 50mg/ml |
| PURPOSE | culturing, selective plating of transformants and controls |
| VENDOR | SIGMA can be found at www.sigma.sial.com OR PO BOX 14508, St. Louis MO63178 |
| CATALOG # | K0879 |
| COST | $8.90 for 50 mg |
| STORAGE | -20 Celsius |
| PREPARATION | Add 1 ml of distilled water to the ampicillin container. Mix and filter sterilize with .45um syringe and disc. |
| AMOUNT | Prepare 1 ml with a concentration of 50mg/ml |
| PURPOSE | culturing, selective plating of transformants and controls |
| VENDOR | SIGMA can be found at www.sigma.sial.com OR PO BOX 14508, St. Louis MO63178 |
| CATALOG # | S2647 |
| COST | $6.60 for 100 mg |
| STORAGE | -20 Celsius |
| PREPARATION | Add 2 ml of distilled water to the ampicillin container. Mix and filter sterilize with .45um syringe and disc. |
| AMOUNT | Prepare 1 ml with a concentration of 50mg/ml |
| PURPOSE | culturing, selective plating of transformants and controls |
| VENDOR | SIGMA can be found at www.sigma.sial.com OR PO BOX 14508, St. Louis MO63178 |
| CATALOG # | S2522 |
| COST | $6.60 for 50 mg |
| STORAGE | -20 Celsius |
| PREPARATION | Add 1 ml of distilled water to the ampicillin container. Mix and filter sterilize with .45um syringe and disc. |
| AMOUNT | Cultured in an agar stab. |
| PURPOSE | to culture the recipient strain of bacteria |
| VENDOR | Dr. Laura Frost is found at LauraFrost@ualberta.ca |
| CATALOG | NA |
| COST | NA |
| STORAGE | Stab is stored in a closed vial at room temperature. |
| PREPARATION | Prepare your selected amount of autoclaved broth with antibiotics. Flame a wire loop or needle. Stab the loop or needle into the visible puncture of the stab. Using sterile technique place the loop into the TSB broth. Grow at 37 Celsius overnight. |
| AMOUNT | Cultured in an agar stab. |
| PURPOSE | to culture the recipient strain of bacteria |
| VENDOR | Dr. Laura Frost is found at LauraFrost@ualberta.ca |
| CATALOG | NA |
| COST | NA |
| STORAGE | Stab is stored in a closed vial at room temperature. |
| PREPARATION | Prepare your selected amount of autoclaved broth with antibiotics. Flame a wire loop or needle. Stab the loop or needle into the visible puncture of the stab. Using sterile technique place the loop into the TSB broth. Grow at 37 Celsius overnight. |
| AMOUNT | The powder for 1 liter of 20X solution |
| PURPOSE | to make serial dilutions of your bacterial culture |
| VENDOR | SIGMA can be found at www.sigma.sial.com OR PO BOX 14508, St. Louis MO63178 |
| CATALOG# | S8015 |
| COST | $6.30 for powder to make 1 liter |
| STORAGE | room temperature |
| PREPARATION | Add distilled water to the powder in an appropriate container. It is not necessary to make the entire liter if you weight the powder and proportionally prepare the solution. |
60x 150mm Sterile Petrie Dishes
| AMOUNT | 2 per student |
| PURPOSE | plating control and transformant cultures |
| VENDOR | SIGMA can be found at www.sigma.sial.com OR PO BOX 14508, St. Louis MO63178 |
| CATALOG# | C6421 |
| COST | $55.30 for 100 plates |
| STORAGE | NA |
| PREPARATION | NA-these plates are available in a number of sizes from many vendors at variable prices. Shop carefully! Note: SIGMA requires letter from principal for initial order |
| AMOUNT | 5 per student |
| PURPOSE | for serial dilutions of 2 hour cultures |
| VENDOR | Carolina Biological |
| CATALOG# | K3-21-5220 |
| COST | $28.25 for 1000 tubes |
| STORAGE | no special conditions |
| PREPARATION | Autoclave for 15 minutes at 15psi. in a foil covered glass container. |
| AMOUNT | 2 per student |
| PURPOSE | to streak and spot the the bacterial cultures on antibiotic selective plates |
| VENDOR | Carolina Biological |
| CATALOG# | K3-21-5832 |
| COST | $38.75 for 250 loops |
| STORAGE | no special conditions |
| PREPARATION | NA |
| AMOUNT | 1 |
| PURPOSE | sterilize broth and agar solutions, disposal of plates at the end of the experiment |
| VENDOR | NA |
| CATALOG | NA |
| COST | NA |
| STORAGE | NA |
| PREPARATION | A pressure cooker, or asking other organizations in your community that have access will work if you do not have an incubator. |
| AMOUNT | 1 per student group |
| PURPOSE | used for sterile technique |
| VENDOR | several |
| CATALOG | NA |
| COST | NA |
| STORAGE | NA |
| PREPARATION | NA |
| AMOUNT | 4 or 5 per class |
| PURPOSE | precise measurement of liquids, for setting up serial dilutions |
| VENDOR | Rainin Instrument
Corp. www.rainin.com OR
Mack Rd. Box 4026, Woburn, MA 01888-4026 |
| CATALOG# | P200 |
| COST | $250.00 each |
| STORAGE | NA |
| PREPARATION | several other alternatives are available e.g.. Carolina Biologicals K3-21-4688 @ $10.90 . It will aliquot 200ul. Recent market products include inexpensive plastic barrel pipets that are calibrated. Shop carefully. |
| AMOUNT | 8 for the entire class |
| PURPOSE | sterilization of broth and cultures |
| VENDOR | |
| CATALOG | |
| COST | |
| STORAGE | |
| PREPARATION |
| AMOUNT | 2 per student |
| PURPOSE | dilution of 2 hour culture |
| VENDOR | |
| CATALOG | |
| COST | |
| STORAGE | |
| PREPARATION | Several options exist here. Standard lab tubes with foil covers that have been autoclaved with work well. Plastic disposable tubes with a plastic snap cap also work well(research labs throw them away, we might reuse them). |
Suggested vocabulary
transduction
transformation
conjugation
F-plasmid
sex pili
R-plasmid
antibiotic resistance
MRSA (methicilin-resistant Staphylococcus aureus)
vancomycin
serial dilution
Part 1: Conjugation
Bacterial infections are treated with a special group of drugs called
antibiotics. When doctors first started prescribing antibiotics to
their patients back in the 1950s, the drugs were a very powerful weapon
against the illnesses caused by bacterial infections. But sometimes
a mutation in the genes of a few bacteria would allow them to survive antibiotic
treatment. Over time, these antibiotic resistant bacteria
were able to continue to grow and multiply. Today there are many
types of bacteria that are resistant to antibiotics, so the drugs are less
effective in treating illness.
Scientists have learned that some bacteria have the ability to donate their drug-resistance genes to other non-resistant bacteria through a process called conjugation. Bacteria that have this ability often possess a special piece of DNA called an F-plasmid. This plasmid allows the bacterium to transfer certain genes to other bacteria without the F-plasmid. We refer to the bacterium with the F-plasmid as the "donor". The bacterium the receives the donor's genes is called the "recipient". Antibiotic resistance is just one characteristic that bacteria can share through the process of conjugation. In this laboratory experiment, you will observe the results of conjugation between an antibiotic resistant donor and and non-resistant recipient bacteria.
The bacteria known as pOX38-Km is resistant to the antibiotic called kanamycin (Km). If pOX38-Km is spread on an agar plate that has been treated with Km, pOX38-Km is not affected by the antibiotic and will grow. A different bacteria, ED24, is not resistant to Km. If ED24 is spread on an agar plate with Km, ED24 is affected by the antibiotic and will not grow. In this lab, you will add pOX38-Km to a sample of ED24 and allow them to carry out the exchange of antibiotic resistance through the process of conjugation.
If the exchange is successful, ED24 will now have the ability to grow in the presence of Km just like the pOX38. But how can you tell which bacteria is growing? Remember, you mixed the two types together to allow them to "mate", so your sample now contains both types.
To selectively grow the ED24 that has acquired the Km resistance, the
agar plate must contain an additional antibiotic that will kill the donor
(pOX38) without affecting the recipient (ED24). Spectinomycin (Spc)
is such an antibiotic. So, the agar plates used to test for successful
conjugation will contain two antibiotics: kanamycin and spectinomycin
(KmSpc).
Part 2: Mating Efficiency
After the bacteria have a chance to perform conjugation, the efficiency of the process can be calculated. Do all the pOX38-Km donate their antibiotic resistance to ED24, or will just a few? To find out, compare the number of possible pOX38-Km donors to the number of ED24 bacteria that actually display the resistance to Km determined in Part 1.
Mating Efficiency = Number of Km resistant ED24 / Number of Donors
In order to selectively grow and count only the donors in our sample, we will take advantage of another antibiotic resistance of pOX38-Km. This particular strain also carries streptomycin (Sm) resistance, but ED24 does not. When our mixture of pOX38-Km and ED24 bacteria are introduced to an agar plate with both Km and Sm antibiotics, only the pOX38-Km will grow.
In summary, the purpose of this experiment is to
1. Demonstrate transfer of kanamycin resistance present in pOX38-Km
to ED24, a bacterium not normally resistant to kanamycin
2. Calculate the "mating" efficiency between pOX38-Km and ED24
by selectively growing the donor bacteria and comparing them to the number
of transformed ED24 bacteria.
Materials:
2 ml pOX38-Km
2 ml ED24
2 TSB plates containing Km and Spc antibiotics (1 for control, 1 for
growing transformed ED24)
1 TSB plate containing Km Sm antibiotics (for growing pOX38 donors)
Eppendorf tubes
micropipettor and tips
transfer loop
discard container
Bunsen burner or alcohol lamp
1xSSC stock solution
water bath (37 deg C)
incubator
Procedure:
Caution! You will be working with bacteria. It is extremely important that you read all directions first and follow the safety rules your teacher has demonstrated. Wash the lab table approiate disinfectant and wash your hands with soap and water before and after experiment.
A. Conjugation
1. Obtain a test tube from your teacher containing 800 ml TSB
(broth for bacteria). Label this
tube with your group name and "ED24 + pOX38"
2. Using a micropipettor and fresh tip, add 100 ml ED24 to the
test tube. Discard tip.
Replace cap on ED24 sample.
3. Attach a fresh tip and add 100 ml pOX38 to the test tube.
Discard tip. Replace cap on
pOX38 sample.
4. Place the tube in the water bath for 30 minutes.
5. While the bacteria conjugate, set up the control condition according to directions below:
B. Setting up the Control
1. Label a KmSpc plate according to the following diagram:
2. Use a micropipettor and fresh tip to drop 20 ml ED24 on the
side of the plate labeled
"ED24". Be sure to lift and lower the
cover quickly to prevent contamination.
Discard the pipettor tip immediately.
3. Using a disposable or flame-sterilized metal transfer loop,
gently spread the sample evenly
to cover the that side.
4. Repeat steps 2 and 3 using 20 ml pOX38-Km on the appropriate side of the plate.
5. Set the plate aside and do not disturb until the agar surface has dried thoroughly.
6. Prepare for the next stage of the experiment according to the directions below:
C. Plating the conjugated bacteria
1. Label the second KmSpc plate according to the following diagram
with your Group Name (top), type of antibiotics (KmSpc), five points to
spot your dilution samples (-1 through -5) and the type of bacteria that
should grow ("Recipients--ED24")
2. Label the KmSm plate according to the following diagram with
your Group Name (top), type of antibiotics (KmSm), five points to spot
your dilution samples (-1 through -5) and the type of bacteria that should
grow ("Donors--pOX38-Km")
3. Using a micropipettor and fresh tip, fill each of five Eppendorf
tubes with 180 ml 1xSSC.
These will be used to successively dilute
your bacterial samples.
4. After 30 minutes, remove your test tube with the conjugating
bacteria from the water bath.
Gently tap the tube to interrupt the mating
process.
5. Using a micropipettor and fresh tip, fill the first Eppendorf
tube with 20 ml of your sample.
Close the cap, and tap to mix well.
Also tap on the table top.
6. Draw 20 ml from the first Eppendorf tube and add this to the
second Eppendorf tube.
Mix well.
7. Repeat the procedure for tubes 3-5.
8. Starting with Eppendorf tube #5, draw 10 ml of the sample and
spot the -5 spot on the
KmSpc "Recipient" plate. Repeat with
10 ml samples from tubes 4, 3, 2, 1. Discard tip.
9. Cover the plate and set aside. Do not disturb until samples have dried completely.
10. Using a fresh tip, repeat steps 8 and 9 with the KmSm "Donor" plate.
11. Place all your plates in the incubator (37 deg C) for 24 hours.
12. After 24 hours, remove your plates and draw the results of
each plate. Write a summary
for each picture explaining your
results and answer the following questions:
POST-LAB QUESTIONS
1. Why is the KmSpc plate allow only ED24 to grow?
2. Why is the KmSm plate allow only pOX38-Km to grow?
3. Why are the pOX38-Km bacteria called "Donors"?
4. Why are the ED24 bacteria called "Recipients"?
5. If the transfer of Km resistance was NOT successful, what
would the KmSpc plate look
like after 24 hours? Draw your predictions.
Expected results:
The following pictures are representative data from our experiments. Check students' plates, their drawings, their calculations for mating efficiency, and answers to postlab questions.
1. Recipient (ED24) plate with KmSpc (both streak and serial dilutions)
2. Donors (pOX38-Km) on KmSm plate
3. Negative Control (both ED24 and pOX38) on KmSpc plate--no growth
to top
Objective:
The students will simulate a serial dilution using
water and food coloring to represent the 1xSSC and the bacterial culture
to be diluted in order to reinforce serial dilution technique and measurement
skills.
Materials:
10 clear Eppendorf tubes (1.5 ml), 2 samples of
colored water solutions that will represent the bacterial culture and the
1xSSC, a measurement device (micropipetors & tips, 1 ml in 1/100 glass
or plastic pipettes with pi-pump, 1 ml disposable plastic pipettes), test
tube rack, and marking pen.
Procedure:
1. Assemble all the above materials before beginning lab!
2. Label the test tubes 10-1, 10-2, 10-3,
10-4, 10-5 (Extremely important in case of
accidental mix-up).
3. Dispense .9 ml (180 ul) of 1xSSC solution (yellow liquid) into each
of the 5
Eppendorf tubes.
4. Dispense .1 ml (20 ul) of the bacterial culture (blue liquid) into
the Eppendorf tube
labeled 10-1.
5. Close lid of tube and gently tap the tube on the work bench to ensure
through mixing
of the bacterial culture (blue) and the 1xSSC solution
(yellow).
6. Now extract .l ml (20 ul) from the 10-1 tube and dispense
into the 10-2 tube, close
tube and gently tap on the work bench.
7. Repeat #6, but Now extract .1 ml (20 ul) from the 10-2
tube and dispense into the
10-3 tube, close tube and gently tap tube on
work bench.
8. Next extract, .1 ml (20 ul) from the 10-3 tube and dispense
into the 10-4 tube, close
and gently tap tube on work bench.
9. Finally, extract, .1 ml (20 ul) from the 10-4 tube and
dispense into the 10-5 tube,
close and gently tap tube on work bench.
10. Congratulations! Now have your lab partner complete the same procedure
using
the remaining Eppendorf tubes.
11. Compare the tubes with each other ....... Hint: Look for color changes
and
discrepancies in the quantity of liquid
in the tubes.
12. If at this point you fell comfortable with this procedure and your
ability to accurately
measure and perform a serial dilution,
compare your results with the teacher's
standard for evaluation or TRY AGAIN!
Teacher Background:
We included this simulation to be used prior to the
lab. Hopefully, this will reduce problems and errors that could occur
on the actual lab day. For those classes expert in these skills feel
free to move directly to the lab. This activity could also be used
as a stand alone. Many states are requiring performance assessment
components in their state tests. This is not only simple but cheap!
The time required for the actual teacher set up
is minimal. Mixing food coloring and water (6 drops of food coloring
per 1 ml) to represent the bacterial cultures and the 1xSSC solutions.
Dispense the materials in any manner you feel comfortable, make at
least 1 classroom set of directions so that they can be reused (Recycle.....Recycle).
Teacher Background:
Obtain a copy of the article
above. One can either read article (the first three paragraphs and skip
the six recommendations then complete the article) to the class as a whole
or make copies for each group.
Objective:
The students will discuss the article and come to
consensus on a set of recommendations to the WHO to tackle the Drug Resistant
Disease Problem.
Student Page:
Directions:
1. After reading the article make a list of recommendations to WHO
about possible
strategies that could be incorporated in the fight
against Drug Resistant Diseases.
2. Each group must come to consensus on the recommendations for your list.
3. Write the recommendations at the bottom of this page.
4. Select a representative to present the recommendations to the class.
Objective:
The students will practice inferring and hypothesizing.
Student Page:
Pre Lab Directions:
1. In your groups, discuss the implications of the headline -.
2. Make a list of the implications in the space provided below.
3. Does combining the antibiotics boost their killing power? Explain.
Post Lab Directions:
1. How can you relate the information gathered from the lab activity
to the headline?
Explain:
2. Will using more antibiotics reduce or increase the antibiotic resistant
strains of
bacteria? Explain:
3. If you were a doctor what procedures would you follow in prescribing
antibiotics to
your patience's?
Further Research: