Antibiotic Resistance in E.coli
A laboratory exercise for undergraduate evolutionary biology courses
This laboratory outline includes
Instructor Notes
Evolution of Antibiotic Resistance
Suggestions for instructors are in
purple
This lab will use antibiotic resistance
as example on what evolutionary biologists do and how they do it.
Potential pre-lab readings
Brandon, R. N. 1996. Concepts
and Methods in Evolutionary Biology. Cambridge University
Press. Read: Chapter 9.
Theory and experiment in evolutionary
biology. Pp. 147-160.
Stearns, S. 1999. Evolution in Health and Disease
. Oxford University Press. Read: Chapter 1: Introducing evolutionary
thinking Pp. 3-15.
Garrett, L. 1994. The Coming Plague. Farrar, Straus,
and Giroux, N.Y. Read: Chapter 13: Revenge of the Germs. Pp.
422-428.
Khachatourians, G. G. 1998. Agricultural use of antibiotics
and the evolution and transfer of antibiotic resistant bacteria.
Canadian Medical Association Journal
159: 1129-1136.
Goal of Lab Exercise: To introduce experimental
methods for investigating evolution. To understand how evolutionary
biologists test hypotheses.
Background: Evolutionary biologists ask "why" rather
than "how". Causation: ultimate (“why”?) versus proximate (“how”?).
A “why” question is one that asks the historical sequence of causation.
A “how” question asks about the present day functional sequence.
Lead brief discussion with students
about the difference between ultimate and proximate mechanisms in
evolutionary biology. Pose a question that asks why a certain trait
exists in a modern population. Examples:
- Why do cheetahs run fast
- Proximate: Muscle development,
physiology, etc.
- Ultimate: Cheetah ancestors
who ran faster caught more gazelles, etc.
- Why do orchid flowers mimic the
females of many insect species?
- Proximate: developmental
cascade leading to production of various flower parts
- Ultimate: Ancestors of orchids
that resembled female insects had increased pollination rates and
hence increased reproduction
Ask how students would test the ultimate explanations generated
by the preceding questions.
Talk about difference between “hypothesis generation" and
"hypothesis testing".
- Hypothesis generation
- Observation of patterns and
phenomena
- Theoretical modeling (deductive)
- Failure to connect deduction
and observation
- Hypothesis testing
- Assign "levels of belief" based
on how well the data fit the observations, and based on our knowledge
of the reliability/accuracy of the data and of the observer.
- Big ideas tested by specific
examples. Have to assess generality of findings. The
bigger the idea the more doubtful that a specific example will test
that idea; need to assess the scope of the generalization.
- The more "controlled"
the experiments, the worse they may be. Trade off in precision
vs. realistic context.
- Talk about benefits vs. pitfalls
of models
The above discussion should stimulate ideas about the following
pre-lab question:
Questions to think about before lab:
- Why are evolutionary biologists more interested in
the historical sequence of causation than the present day functional
sequence?
- What can be gained by considering the historical
events that led to a certain trait?
- What can be gained by considering the current function
of a certain trait?
Now, consider the question: Why is there antibiotic resistance
in organism x?
Possible responses:
- Because there has been natural selection on organism
x. (Because antibiotic resistance conferred a reproductive/survival
advantage on organism x)
- Because organism x synthesizes enzymes that degrade
the antibiotic.
Which of the above responses best addresses the historical
events that led to the existence of antibiotic resistance as a trait?
(which is a “why” question and which is a “how” question?).
Introduce specific instructions of
antibiotic resistance lab (follows)
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Instructions for Antibiotic Resistance Lab
Isolation, culture, and transfer techniques for E. coli.
Isolation of Escherichia coli
Background: E. coli is a common bacterium in
the gut of many organisms. For this lab, we will use sterile
cotton swabs to isolate E. coli from ourselves. The swabs will be used
to inoculate petri dishes containing a selective and differential medium,
eosin-methylene blue (EMB). Using this medium, we can differentiate
E. coli from other bacteria based on two properties. First, EMB
supports the growth of gram-negative organisms and inhibits the growth
or gram positive organisms. Second, E. coli can be differentiated
from other coliform bacteria by its metallic green sheen when grown on
EMB (many other bacteria will have a pink mucousy appearance). Once
inoculated, the plates are incubated at 37°C (body temperature) to
support growth of the bacterial colony.
Week 1
Objectives:
Isolate up to 18 distinct individual colonies of E. coli
for use next week in antibiotic resistance assay.
Day 1
- Label the bottom of a petri dish with your name,
today’s date, and the contents of the plate (i.e. fecal swab on EMB).
- Moisten sterile cotton swab in sterile H2
- Swab desired surface and then swab the agar surface
once on one edge of the plate (see Figure 1). Students, if willing,
should head to the bathroom and acquire fecal swabs from themselves. Students
may also take swabs from the inside of the mouth, although mouth swabs typically
yield fewer colonies than fecal swabs.
- Using a sterile toothpick, drag the loop across
the initial swab, making three streaks. To make the second
streak, turn the plate ~ 60 °, take a new sterile toothpick, and
drag it across the first streak several times.
- Repeat for the third streak, being sure not to touch
the initial swab with your third set or streaks (see Figure
1).
- Wrap plates in parafilm and place in incubator.
Figure 1
Day 2
Hopefully today you will have E. coli and possibly other
coliform bacterial
colonies on your plate. E. coli will be metallic
green (see below). If you do not have any bacteria, grab another
swab and try harder!
E. coli colonies - GREEN !!
Not E.coli - PINK !!
Instructors should have a picture
or sample of a “good” plate of colonies (see above).
We will now use the following protocol to isolate pure
cultures of E. coli. We need to go through these steps to ensure that:
a) there are no contamination by other
bacterial species
b) our cultures are not mixtures of lines
of E.coli
If you have E. coli on your plate, you will need 4 fresh
EMB plates.
You should label these plates A1, A2, B1, and B2.
(Also label the plates with your name and the date and “E. coli”)
To keep track of sequential cultures, make a mark on the
bottom of each plate denoting which direction is ‘UP’ on the plate
(see Figure 2). This mark will always be placed pointing away
from you.
Figure 2
Instructors should reproduce a copy of
Figure 2 that is the same size as petri dishes for students to use
as pattern.
- You have two transfer patterns at the end of this
handout.
- Place plate A1 over transfer pattern 1, being sure
to align the ‘UP’ marks on the pattern and the plate. You should
be able to see the pattern through the agar.
- Place plate A2 over pattern 2, and again, align the
‘UP’ position. Streak position 1 on the patterns is in the upper
left corner.
- Using a sterile toothpick, lightly touch a single
colony of E. coli on your plate. Transfer this to the first streak
position on plate A1.
- Take a second sterile toothpick and drag it perpendicularly
across this streak. Next, transfer this bacteria to plate A2
by streaking position 1 on plate A2 with this toothpick (see Figure 3).
- Repeat this step with 8 more colonies on this plate
and 9 more colonies on plates B1 and B2. If you don’t have at
least 10 nice colonies total, swab again!
- By keeping the ‘UP’ positions aligned, you can return
to these plates and identify streaks isolated from the same initial
colony.
- If you have enough E. coli from yesterday, you now
have 18 isolates spread across four plates. The number 2 plates
are replicates of the number one plates, but with lower concentrations
of bacteria in each streak. Hopefully, as a result, you will have
pure colonies to transfer tomorrow!
- Wrap the plates with parafilm, invert, and return
to incubator (you now have 5 plates).
- Dispose of toothpicks in autoclave bag and clean your
bench.
Figure 3
Day 3
- Today you should be able to isolate single, pure colonies
from the four plates you made yesterday.
- If you have no colonies on the plates from yesterday,
repeat day 2 procedures with 4 new plates.
- If you have good colonies:
- Take 2 new plates from the shelf. Label these
C and D. Also label with your name, date, and E. coli, as well
as marking the ‘UP’ position. If you are very lucky, you will
have bacteria growing on all 36 streak marks (don’t forget, these are
only from 18 different colonies).
- You want to transfer single colonies from as many
of these 18 colonies as possible. If you don’t have colonies
growing from all 18 initial streaks, don’t worry about it.
Get as many as you can. If you only have a few colonies, repeat
yesterday’s protocol to get more.
- Place plates C and D over the transfer patterns.
Touch the tip of the toothpick to a colony from streak one on plate
A. The best colony may be on plate A1 or A2. These are
replicates, so just use a colony on plate 1 or 2, not both. Now,
touch the toothpick to plate C at position 1 and make a small streak.
Repeat with as many of the remaining 17 colonies as possible.
- Invert plates, seal with parafilm, and return to incubator.
Dispose of trash and clean the bench.
Day 4
If you have >10 colonies in pure culture
on plates C and D, place plates in refrigerator until the next scheduled
lab. We will use these colonies to assay for antibiotic resistance.
Student plating colonies
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Week 2
Objectives
- Test strains cultured from last week for antibiotic
resistance. We will plate today, come in tomorrow, construct data
sheets, record resistances, and place plates in the fridge.
- Discuss possible follow-up independent experiments.
Testing strains for antibiotic resistance
- Transfer single strains to array of antibiotic containing
EMB plates by "replica-plating".
- Use a new toothpick for each transfer (6 x 9 x 2
= 108 toothpicks)
- Antibiotics are:
- Mark each plate as before, with an up or down,
etc.
- Wrap with parafilm and put in incubator.
- Rewrap your original plates and put them back
in fridge.
- Come in tomorrow, make data sheets, record resistances,
and put plates in fridge.
Assignment for independent projects
Make sure students know what supplies
will be available for projects. Advise them to keep projects short
and with the possibility of a definitive result by the next scheduled
lab meeting. These projects could be turned into semester-long independent
research projects if time and facilities permit.
1. Write a paragraph or two describing what you plan to
do for your independent projects and discuss project with the laboratory
instructor.
2. Do background research and reading on your topic.
Suggested General Reading (before next lab)
Bonten, M., Stobberingh, J. P., Houben,
A. 1992. Antibiotic resistance of Escherichia coli in fecal samples
of healthy people in two different areas of an industrialized
country. Infection 20: 258-262
Graves, S. R., Kennelly-Merrit, S. A., Tidemann, C. R.,
Rawlinson, P. A., Harvey, K. J. and Thornton, I. W. B1988.
Anti-biotic resistance patterns of enteric
bacteria of wild mammals on the Krakatau Islands and West Java, Indonesia.
Philosophical Transactions of the Royal
Society, London, Series B 322: 339-353.
Levin, B. R., Lipsitch, M., Perrot, V., Schrag, S., Antia,
R., Simonsen, L., Moore Walker, N.,
Stewart, F. M. 1997. The population genetics
of antibiotic resistance. Clinical Infectious
Diseases 24 (Supplement): S9-S16.
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Week 3
Objectives
- Discuss results from antibiotic resistance tests.
- Independent project idea presentation to the class.
- Questions on projects for presenters, discussion of
readings.
- Introduce dilution procedures.
Encourage students to question
presenters about methods and potential interpretations and pitfalls
of proposed projects.
Instructors should provide directions about length and
scope of presentations that will be given at next lab period.
Dilution series
Today
- Each dilution tube contains 9.9 ml of 2% NaCl
- Adding 100 μl makes each tube up to 10 ml.
- Swirl a loop of your culture (take any for fun),
swirl into a dilution tube to make the original batch.
- Vortex
- Transfer 100ml to next tube, etc.
- Do this for 4 transfers (5 tubes total).
- Spread 100ml of each dilution onto petri plate
- Plates contain LB medium - good for E.coli, but not
selective
- Spread using loop and turntable
- Place in incubator
Tomorrow
- Count cells on plates, estimate concentration in original
tube, work out and describe what dilution would be needed to plate
50 colonies on one petri dish from your original batch.
- Set-up independent projects. Check on projects as needed.
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Week 4
Objectives
- Each lab group should present the results of their
project to the entire class.
- Discuss procedures for write-up of lab exercise.
Depending upon scope of course or
writing assignment, these lab reports could include significant literature
review as well as the inclusion of both the initial test of antibiotic
resistance that the class did as a whole along with the independent projects.
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Appendices for Instructors - Antibiotic Resistance Lab
Student Supplies
Week 1: E. coli isolation and pure culture
supplies per student:
7 EMB plates (for
18 isolates)
1 sterile swab
~75 sterile toothpicks
supplies at side station:
Bunsen burner
loop
parafilm
Week 2: antibiotic resistance
supplies per student:
2 of each antibiotic
EMB plates (we used 6 different antibiotics)
2 control EMB plates
~20 sterile toothpicks
supplies at station:
parafilm
introduction to liquid culture and counting
colonies:
Lab instructor or
TA makes 5 bacterial cultures in LB, transfer to MD25
for counting, students
need to dilute and plate:
supplies per student:
3 LB plates
3 dilution tubes
with salt solution
supplies at station:
P200 pipetteman
sterile pipette tips
spreader
Bunsen burner
ethanol bath
Week 3: independent projects
Varies based on independent projects
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Growth Media Recipes
Luria-Bertant (LB) Medium
10g bacto-tryptone
5g bacto-yeast extract
10g NaCl
15g agar
1 L deionized water
adjust pH to 7.0 with ~250 ml
1N NaOH
autoclave 20 minutes @ 15lb/sq inch
on liquid cycle
Eosin-methylene blue (EMB) agar, Levine (pH 7.2)
10g peptone
5g lactose
2g potassium phosphate dibasic
15g agar
0.4g eosin-Y
0.065g methylene blue
1 L deionozed water
adjust pH to 7.2
autoclave 20 minutes @ 15lb/sq
inch on liquid cycle
Dilution tubes
9.9 mL 2% NaCl solution
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Sample Results
Class resistance totals – UVA 2000
Antibiotics tested
1. ampicillin (Amp)
2. chloramphenicol (Chl)
3. kanamycin (Kan)
4. rifampicin (Rif)
5. streptomycin (Strep)
6. tetracycline (Tet)
Class size of 15
12 people with no resistance to any antibiotics
(total 216 lines with no resistance)
3 people with resistance
person A
17 lines resistant
to Amp, Strep, and Kan
1 line resistant
to Kan only
person B
1 line resistant
to Amp, Chl, Kan, and Strep
16 lines resistant
to Amp and Strep
person C
11 lines resistant
to Tet
Total number of lines grown = 262
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