Download Attack of the Superbugs

Project 1.2.3: Attack of the Superbugs
Introduction
Streptococcus pneumoniae, commonly referred to as pneumococcus, is a bacterium
commonly found in the back of the nose in healthy people. Occasionally,
pneumococcal bacteria spread to other parts of the body and cause diseases such
as ear infections, sinus infections, pneumonia, and meningitis. Before the
introduction of penicillin in the early 1940s, a person might die from a pneumococcal
infection. After the introduction of penicillin, pneumococcal infections were effectively
treated. However, strains of pneumococcal bacteria resistant to penicillin were
discovered in the 1960s. Since then, new strains of pneumococcal bacteria resistant
to multiple antibiotics have been emerging.
Over the last decade, almost every type of bacteria known to cause disease has
become stronger and less responsive to antibiotic treatment. Antibiotic resistant
bacteria are threatening communities with strains of infectious diseases.
Tuberculosis, gonorrhea, malaria, skin infections, pneumonia, and ear infections are
just a few of the diseases that have become harder to treat. For this reason,
antibiotic resistance has been called one of the world’s most pressing public health
problems and is one of the Centers for Disease Control’s top concerns.
In this activity, you will investigate the mechanisms by which DNA from one bacterial
cell is transferred to another bacterial cell. When the DNA that is transferred carries
an antibiotic resistant gene, the intercellular transfer enables the new cell to become
antibiotic resistant. Over time, one bacterial cell containing an antibiotic resistant
gene could lead to an army of superbugs.
Equipment
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Computer with Internet access
Laboratory journal
Carolina Introductory Bacterial Conjugation Kit
o E. coli I broth culture
o E. coli II broth culture
o 2 LB agar plates
o 2 LB agar plates with streptomycin
o 2 LB agar plates with ampicillin
o 2 LB agar plates with streptomycin and ampicillin
o 10 sterile loops
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Alcohol in beaker
Alcohol lamp
Sterile loops
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MI Activity 1.2.3 Attack of the Superbugs – Page 1
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Matches
Permanent marker (Sharpie® fine point)
Disinfectant in squirt bottle
Incubator at 37º C
Safety goggles
Latex or Nitrile exam gloves
Roll of transparent tape (e.g. Scotch tape)
Chart paper
Markers
Modeling supplies, possible supplies include
o Styrofoam, balls or sheets
o Knife to cut the Styrofoam
o Pipe cleaners
o Colored construction paper
o Glue
o Tape
o Clay or play dough
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Design Process handout
Procedure
Part I: Exploring the Effects of Antibiotics
1. Read the first section of Daniel Zwerdling’s Antibiotics on the Farm article titled A
New Public Health Threat on the Farm found at:
http://americanradioworks.publicradio.org/features/farm/a1.html .
2. Work in groups of 3-4 and brainstorm why you believe CIPRO is not working on
Culbreath. Record your ideas on chart paper.
3. Share your group’s ideas with the class. Post your chart paper in front of the
class.
4. As a class, once each group has presented, discuss the validity of each idea and
eliminate any ideas that the class believes are inaccurate.
You learned in the previous activity that bacterial cells can carry genetic information in
two ways. The genes necessary for growth and reproduction are carried in the bacterial
chromosomes, but bacteria also contain genes carried on rings of DNA outside of the
nucleoid region, called plasmids. In the following experiment, you will work with two
strains of E. coli. The first strain, E. coli I, contains a gene found on the chromosomal
DNA coding for streptomycin resistance. The second strain, E. coli II, contains a gene
found on the plasmid DNA coding for ampicillin resistance.
5. Predict what will happen when the E. coli I strain is mixed with the E. coli II strain.
Record your prediction in your laboratory journal.
6. Put on safety gloves and goggles. Tie back any loose hair.
7. Wipe down your laboratory table using a 75% ethyl alcohol in water solution or
other effective disinfectant solution.
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8. Working with your partners, obtain the following materials from your teacher:
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1 LB agar plate
1 LB agar plate with streptomycin
1 LB agar plate with ampicillin
1 LB agar plate with streptomycin and ampicillin
8 sterile loops
Alcohol lamp
Matches
Permanent marker (Sharpie® fine point)
9. Write your initials or first names on the outside edge of each Petri dish. Do not
write too large or your writing will interfere with your observation of the results.
10. On the bottom surface of each agar plate, draw a line down the middle as shown
in the figure below. Write “I” on one side of the line, and “II” on the other side.
11. Obtain the E. coli I and E. coli II broth cultures from your teacher. You will have to
share these cultures with another group.
12. Remove a loop from its wrapper. Do this by opening the package at the end
farthest from the inoculating loop, being careful not to touch anything.
13. Watch as your teacher demonstrates the sterile technique.
14. Use the sterile technique to remove and hold the cap from the E. coli I broth
culture bottle.
15. Dip the sterile loop into the culture while holding the cap so that it does not touch
anything.
16. Withdraw the loop and replace the cap immediately. Be careful not to touch the
loop to anything before placing it into the bottle or after withdrawing it. Note that
you will not need the culture tube again.
17. Touch the loop lightly to the agar at the top edge of the plate in the section
marked “I” on one of the four plates. Note that in the following steps you will be
instructed on how to streak the plate. The purpose of streaking a plate in this
manner is to dilute a concentrated sample and grow isolated bacterial colonies.
18. Gently move the loop back and forth across the agar surface, creating a series of
parallel lines at the top of the plate.
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MI Activity 1.2.3 Attack of the Superbugs – Page 3
19. Turn the plate 90 degrees.
20. Repeat the streaking procedure beginning at the top edge of the plate (but do not
dip the loop into the culture tube). Gently move the loop back and forth across
the agar surface, making sure to cross the previous material two to three times to
pick up cells from the first section you streaked.
21. Turn the plate another 90 degrees in the same direction.
22. Repeat the streaking procedure beginning at the top edge of the plate. Gently
move the loop back and forth across the agar surface, making sure to cross the
previous material one to two times to pick up cells from the second section you
streaked.
23. Follow the same procedure to inoculate and streak the E. coli I culture onto all of
the sections marked “I” on the remaining three plates. Make sure to use a new
loop for each plate.
© 2010 Project Lead The Way, Inc.
MI Activity 1.2.3 Attack of the Superbugs – Page 4
24. Using a new loop, follow the same procedure to inoculate and streak the E. coli II
culture onto the sections marked “II” on each agar plate.
25. Follow your teacher’s directions to properly dispose of the contaminated loops.
26. Allow the liquid to soak into the agar.
27. Invert the plates and tape the four plates together for incubation. It is important to
incubate the plates upside down to prevent condensation from dripping onto the
culture surface.
28. Incubate the plates at 37˚C for 24 hours.
29. Spray disinfectant on the work area and wipe it clean.
30. Remove your gloves and dispose of them as directed by your teacher.
31. Wash your hands and forearms thoroughly with soap and warm water.
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32. Predict what growth you will see on both sections for each of the four plates.
Record your predictions in your laboratory journal. Give your reasons for your
predictions.
33. Create the table below in your laboratory journal.
Agar Plates
E. coli I Strain Observed
Results
E. coli II Strain Observed
Results
LB agar
LB agar + streptomycin
LB agar + ampicillin
LB agar + streptomycin +
ampicillin
34. Retrieve your plates from the incubator and observe the bacterial growth. Record
the observed results in the table in your laboratory journal. Use (+) for bacterial
growth and (-) for lack of growth.
35. Working with your partners, obtain the following materials from your teacher:
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Agar plates from previous class
1 LB agar plate
3 sterile loops
Permanent marker (Sharpie® fine point)
36. Write your initials or first names on the outside edge of the Petri dish. Label this
dish Mix Plate.
37. Note that the streaking procedures completed in the previous steps should have
resulted in the growth of individual bacterial colonies. Bacterial colonies can be
seen as separated clusters on the agar plates.
38. Use the sterile technique to transfer one E. coli I colony from the LB agar +
streptomycin plate to the Mix Plate. Make sure to use a new sterile loop. Open
the lid of the plate just enough to slip the loop under, being careful not to touch
anything. Do not take the lid completely off the plate. Gently touch the end of the
inoculating loop to one of the isolated colonies. Withdraw the inoculating loop
from the plate, being careful not to touch the loop to anything. The bacterial
colony is now on the end of the inoculating loop. Open the lid of the Mix Plate just
© 2010 Project Lead The Way, Inc.
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enough to slip the loop under and touch the loop lightly to the agar in the middle
of the plate. Follow your teacher’s directions on how to dispose of the
contaminated loop. Use a permanent marker to mark the spot where the colony
is on the plate.
39. Follow the procedure in the previous step and use a new sterile loop to transfer
one E. coli II colony from the LB agar + ampicillin plate to the Mix Plate
approximately ½ inch apart for the strain I colony. Use a permanent marker to
mark the spot where the colony is on the plate.
40. Use a new sterile loop to mix strains I and II. Streak the plate by gently sweeping
the loop across the top of the agar to mix the two bacterial cultures. Use the
diagram below as your guide.
41. Properly dispose of the contaminated loop.
© 2010 Project Lead The Way, Inc.
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42. Seal the Petri dish using transparent tape.
43. Invert the plate and incubate at 37˚C for 24 hours.
44. Spray disinfectant on the work area and wipe it clean.
45. Remove your gloves and dispose of them as directed by your teacher.
46. Wash your hands and forearms thoroughly with soap and warm water.
47. Answer Conclusion questions 1 – 4.
48. Working with your partners, obtain the following materials from your teacher:
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1 LB agar plate with streptomycin
1 LB agar plate with ampicillin
1 LB agar plate with streptomycin and ampicillin
Plate with both E. coli strains from previous class
3 sterile loops
Permanent marker (Sharpie® fine point)
49. Write your initials or first names on the outside edge of each Petri dish.
50. Remove one sterile loop from the package using the handle end. Do not touch
the loop to anything.
51. Slightly lift the lid of the agar plate labeled Mix Plate and use the sterile loop to
pick up some bacteria. Close the lid of the plate.
52. Streak some of these bacteria onto the LB agar plate with streptomycin. Note
that for this step, you are not streaking for isolation but instead streaking in a
random pattern to test for growth. Streak the plate using the pattern shown in the
diagram below.
53. Repeat this procedure to streak the LB agar plate with ampicillin and the LB agar
plate with streptomycin and ampicillin. Make sure to use a new sterile loop for
each plate. Properly dispose of each loop.
54. Invert the plates and tape the four plates together for incubation.
55. Incubate the plates at 37˚C for 24 hours.
56. Spray disinfectant on the work area and wipe it clean.
57. Remove your gloves and dispose of them as directed by your teacher.
58. Wash your hands and forearms thoroughly with soap and warm water.
© 2010 Project Lead The Way, Inc.
MI Activity 1.2.3 Attack of the Superbugs – Page 8
59. Predict what growth you will see on each of the three plates. Record your
predictions in your laboratory journal. Give your reasons for your predictions.
60. Create the table below in your laboratory journal:
RESULTS FOR MIXED CULTURE
Agar Plates
Bacterial Growth
LB agar + streptomycin
LB agar + ampicillin
LB agar + streptomycin + ampicillin
61. Retrieve your plates from the incubator and observe the bacterial growth. Record
the observed results in the table in your laboratory journal. Use (+) for bacterial
growth and (-) for lack of growth.
62. Follow your teacher’s directions on how to properly dispose of Petri dishes.
63. Spray disinfectant on the work area and wipe it clean.
64. Wash your hands and forearms thoroughly with soap and warm water.
65. Answer Conclusion question 5.
Part II: Mechanisms for Antibiotic Resistance
66. Given what you know about bacterial cells and antibiotics, brainstorm answers to
the following questions with your partner:
o How would a genetic mutation in the bacteria’s DNA cause the cell
to become resistant to a particular antibiotic?
o How would a bacterial cell “share” this mutation with other bacterial
cells?
67. Record your ideas in your laboratory journal.
68. Access the Animation of Antimicrobial Resistance produced by The Food and
Drug Administration’s Center for Veterinary Medicine found at:
http://www.fda.gov/AnimalVeterinary/SafetyHealth/AntimicrobialResistance
/ucm134359.htm.
69. View the following sections that provide information about the mechanisms of
antibiotic resistance. Note: antibiotics are referred to as antimicrobials throughout
the animation.
o Mutation
o Destruction or Inactivation
o Efflux
70. View the following sections that provide information about gene transfer between
bacterial cells:
o Conjugation
o Transformation
o Transduction
© 2010 Project Lead The Way, Inc.
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71. Work in a group to design and construct a 3-D model that demonstrates one of
the pathways, assigned by your teacher, that bacteria use to transfer beneficial
genes throughout a population.
72. Obtain a Design Process handout from your teacher.
73. Use the information provided in the animation as well as other reliable sources to
help you plan your model. You can also use the Design Process handout as a
reference.
74. Design and construct your 3-D model with your partners.
75. Present your model to the rest of the class. Take notes as the other groups
present their models in your laboratory journal.
76. In a well-crafted paragraph in your laboratory journal, summarize the information
about all of the mechanisms of antibiotic resistance. Your summary should
include the terms listed below. Underline each term in your paragraph.
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Mutation
Target protein
Genes
Antibiotic
Conjugation
Resistant gene
Plasmid
Pilus
Transformation
“Naked” DNA
Transduction
Bacteriophage
77. Answer the remaining Conclusion questions.
© 2010 Project Lead The Way, Inc.
MI Activity 1.2.3 Attack of the Superbugs – Page 10
Conclusion
1. What was the purpose of culturing bacteria on these four plates? (1)
LB was the control, the strep, amp, and strep/amp was to confirm resistance
of the two types of Ecoli to the antibiotics.
2. How did your predicted results compare to your observed results? (1)
They were correct. Both grew on the LB, only 1 grew on step, only 2 grew on
amp, and neither grew on strep/amp.
3. Why did the E. coli I strain grow on both the LB agar plate and the LB agar plate
with streptomycin but not grow on the LB agar plate with ampicillin? (2)
The LB had no antibiotics so it was able to grow. The strep plate had the
strep antibiotic in it, but this E. coli has the resistant gene for it so it grew.
The amp plate had the amp antibiotic in it and E coli was not resistant so it
did not grow.
4. Why did neither strain grow on the plate containing both streptomycin and
ampicillin? (1)
This because both antibiotics were present and neither E. coli was
resistant to both.
5. Explain what the results indicate about the new strain of bacteria produced when
both strains of E. coli were mixed together. (2)
Since the bacteria was now able to grow on the amp and strep plate this
means that one of the bacteria strains is resistant to both antibiotics. This
happened due to conjugation when E. coli II transferred its plasmid to E.
coli 1. E. coli I probably did not transfer its gene because it was on the
chromosome and this is not as viable.
6. Based on the results of the experiment as well as what you learned about the
mechanism of gene transfer between bacterial cells, was the streptomycin
resistant gene transferred from Strain I to Strain II, or was the ampicillin resistant
gene transferred from Strain II to Strain I? How do you know? (2)
This happened due to conjugation when E. coli II transferred its plasmid to
E. coli 1. E. coli I probably did not transfer its gene because it was on the
chromosome and this is not as viable.
7. What mechanism of gene transfer do you think was responsible for transferring
the antibiotic resistant gene between these two strains of bacteria? Explain your
answer. (3)
The mechanism was conjucation, during which the plasmid from E. coli II
was transferred to E. coli I. We did not put any extra DNA on the plate so it
was not transformation and we did not expose the DNA to a virus so it was
not transduction.
© 2010 Project Lead The Way, Inc.
MI Activity 1.2.3 Attack of the Superbugs – Page 11