Prelab
STUDENT GUIDE
Name
Date
Phenomenon
In 2016, plasmid-mediated resistance to the antibiotic colistin was first reported in bacteria causing an infection
in the United States. When treating common gram-negative bacterial infections that are resistant to multiple
antibiotics, colistin is the antibiotic used in the United States when the infection is resistant to all other antibiotics.
Gram-negative bacteria include E. coli, Salmonella, Neisseria gonorrheae, Vibrio cholerae (cholera), Yersinia pestis
(plague), Pseudomonas aeruginosa (commonly associated with pneumonia in cystic fibrosis patients) as well as
many others. Many of these bacteria cause significant disease in humans and are becoming increasingly difficult
to treat because of the spread of antibiotic resistance. Thus, the finding of colistin resistance in the U.S. especially
alarmed the medical community.
Prior to 2016, plasmid-mediated colistin resistance had been found in other countries in bacteria isolated from
humans. The first case of plasmid-mediated colistin resistance worldwide was found in animals raised for human
consumption.
Driving Question
What has led to the prevalence of bacterial antibiotic resistance in the environment? In this lab you will explore this
question and tie your observations into the larger process of evolution.
Background
Antibiotics’ Declining Effectiveness
The discovery of antibiotics is one of the most important breakthroughs in the history of medicine. Prior to the
discovery of antibiotics, many people died from bacterial infections, including pneumonia, tuberculosis, and
typhoid. With the introduction of antibiotics, tuberculosis and death from other bacterial infections have become
much less common. In many regions, tuberculosis and typhoid are practically nonexistent. In addition to causing
human disease, bacterial infections have also frequently killed or damaged valuable livestock, and these infections
have also been reduced by antibiotics. Some of the bacteria that infect livestock, such as anthrax and salmonella, can
also infect humans. The use of antibiotics and vaccines has lessened the negative impact of bacterial diseases both in
people and animals. However, the effectiveness
of many antibiotics, including those used
to treat tuberculosis and pneumonia, has
diminished over the years as bacteria have
Original gene
developed resistance to them.
Acquiring Antibiotic-resistance Genes
Genes conferring antibiotic resistance can
be acquired through multiple mechanisms.
Some of the genes that confer resistance are
variants of normal bacterial cell genes. These
gene variants may arise through random
mutation of the existing bacterial gene (figure
1). In addition, a bacterium may acquire
antibiotic resistance by obtaining an antibioticresistance gene from another microbe. During
a process called transformation, bacteria take
Evolution in Real Time: Bacteria and Antibiotic Resistance
Original
protein
Original gene
is mutated.
Original gene
with mutation
Original protein is now altered in a
way that confers antibiotic resistance.
Figure 1
S-1
©2018 Carolina Biological Supply Company
Prelab (continued)
STUDENT GUIDE
Gene conferring
antibiotic resistance
Protein
conferring
antibiotic
resistance
Bacteria dies and
disintegrates.
Fragments of its DNA
become present in
the environment.
up free DNA that became
part of the environment
when the bacterium that
previously contained the
DNA died and broke apart
(figure 2). A number of
factors limit the likelihood
that the DNA becomes a
permanent, functional part
of the bacterium’s genome,
but the process occurs often
enough that it accounts
for a significant amount of
bacterial genetic variation
and is known to be a way
of acquiring antibiotic
resistance.
Antibiotic-sensitive bacteria
DNA fragments with
gene conferring
antibiotic resistance
DNA fragments with gene conferring
antibiotic resistance is taken up by
antibiotic-sensitive bacteria.
Antibiotic resistance is integrated
into bacterial genome and antibioticresistance-conferring protein is made.
Figure 2
Plasmid with antibioticresistance gene
Another way to acquire
antibiotic-resistance genes
is through the uptake
of a plasmid (figure 3).
Plasmids are small loops of
extrachromosomal DNA
that exist in bacteria and
yeasts and can be transferred
between microbes. Bacteria
transfer them in a process
called conjugation, during
which there is physical
contact between the two
living microbes. Energy
is required in order for
replicating bacteria to
maintain the plasmids; if
having the plasmids provides
no advantage to the bacteria,
they often lose them.
Plasmid-containing bacterium conjugates
with antibiotic-sensitive bacterium.
The antibiotic-sensitive bacterium
is now antibiotic resistant.
©2018 Carolina Biological Supply Company
S-2
Figure 3
Plasmid is replicated and transferred
to the nonresistant bacteria.
Evolution in Real Time: Bacteria and Antibiotic Resistance
Prelab (continued)
Name
STUDENT GUIDE
Date
Ampicillin
In this lab you will use the antibiotic ampicillin. Ampicillin and other members of the penicillin family of antibiotics
interfere with the formation of bacterial cell walls and thus prevent bacteria from growing and replicating. Bacteria
resistant to ampicillin destroy ampicillin by using an enzyme called beta-lactamase to cleave the beta-lactam ring, a
chemical group in the antibiotic that is critical to its function. The ampicillin-resistant E. coli used in this lab carries
the gene for beta-lactamase on a plasmid.
Summary of the Lab
On day 1 of the lab you create a mixed population of E. coli by combining both ampicillin-sensitive and ampicillinresistant bacteria. You then divide this mixed population into two culture tubes and grow one culture in the presence
of ampicillin and the other in the absence of ampicillin. LB (Luria–Bertani) broth is used as the growth medium to
provide nutrients to the bacteria.
On days 2, 3, 4, 5, 6, and 7 you transfer a set amount of the bacteria from each of the two cultures to a new culture tube
containing fresh LB broth (and antibiotic, for the culture grown with ampicillin). This ensures that the bacteria have
nutrients to continue to reproduce, and that there is sufficient antibiotic present. At the end of these 7 days, you have
two cultures grown from the same mixed population of bacteria. One culture has been grown in the presence and one
in the absence of ampicillin.
On day 8, you assay the two cultures for antibiotic-resistant and sensitive bacteria by plating dilutions of the cultures
on LB and LB/amp plates.
On day 9, you count the number of colonies on the LB and the LB/amp plates and analyze your data to determine the
effect of the two different environments on the two cultures with respect to antibiotic resistance.
Evolution in Real Time: Bacteria and Antibiotic Resistance
S-3
©2018 Carolina Biological Supply Company
Prelab (continued)
STUDENT GUIDE
Name
Date
Prelab Questions
1. Read the Background and the Procedure. What question should you be able to answer using the data generated
in this lab?
2. On days 2–7 you transfer some of each culture to a new tube with fresh LB broth and, for the culture that was
grown in the presence of antibiotic, fresh ampicillin. Why in this experiment would it be critical to maintain
the concentration of ampicillin?
Question for deeper discussion: What might cause the level of ampicillin in the culture to decrease?
3. At the end of the experiment, what do you think the phenotype will be for the bacteria that are grown with
ampicillin? Explain your answer.
4. At the end of the experiment, what do you think the phenotype will be for the bacteria that are grown without
ampicillin? Explain your answer.
©2018 Carolina Biological Supply Company
S-4
Evolution in Real Time: Bacteria and Antibiotic Resistance
Laboratory Investigation
STUDENT GUIDE
Name
Date
Day 1: Set up the Culture
Materials
For each group:
sterile culture tubes, 2
culture tube rack
50-mL bottle sterile LB broth
22-μL aliquot of 100-mg/mL ampicillin
pipetting device and sterile pipets for sterilely measuring and dispensing 1 and 2 mL of LB broth
pipet and sterile tips for measuring 20 μL
disposable, sterile inoculating loops, 2
benchtop waste container
permanent marker
Bunsen burner (may be shared between two groups)
Shared:
LB plate containing ampicillin-sensitive colonies
LB/amp plate containing ampicillin-resistant colonies
37°C incubator
Safety
The bacterium used in this laboratory is Escherichia coli, the same one used in many molecular biology and
teaching labs. Many naturally occurring strains of E. coli inhabit the gut of many animals, including cattle,
swine, and humans. Some genetic variants of E. coli do cause disease; these variants contain disease-causing
genes (e.g., those that code for toxins causing intestinal upset). The laboratory strain used in this lab, is a
weakened version of the normal E. coli of the gut and does not contain these disease-causing genes. This
strain is harmless under normal conditions. If introduced into a cut or into the eye, laboratory strains might
conceivably cause infection, so standard safety precautions should be taken when handling the bacteria. With
this activity, it is especially important to destroy all the bacteria before disposal, to ensure that the antibioticresistance gene is not added to the environment outside of the lab.
Safety Tips for Handling E. coli
1. Do not place disposable plastic loops and pipets as well as any other materials that have come into contact
with E. coli on the bench. This will contaminate the workspace. Place them into a benchtop waste container
for later decontamination.
2. To avoid inhaling any aerosol that might be created, when pipetting suspension cultures keep your nose
and mouth away from the tip of the pipet.
3. Wipe down the lab bench with 10% bleach solution or 70% ethanol at the end of laboratory sessions.
4. Wash your hands before leaving the laboratory.
5. Your instructor will treat the bacterial waste in the benchtop containers to kill the bacteria before disposal.
Evolution in Real Time: Bacteria and Antibiotic Resistance
S-5
©2018 Carolina Biological Supply Company
Laboratory Investigation (continued)
STUDENT GUIDE
Name
Date
Procedure
1. Label one sterile culture tube “LB” and the other, “LB+AMP.”
2. Also, write the date and your lab group’s number on each tube.
3. Using sterile technique, complete the following steps in order to pipet 2 mL of LB broth into the tube labeled
“LB.”
a. Loosen the cap on the LB broth, and on the culture tubes.
b. Have your pipet ready in one hand. With the other hand, remove the cap from the LB broth and hold it
with the little finger of the hand holding the pipet.
c. Flame the opening of the LB broth bottle and withdraw 2 mL of LB broth.
d. Reflame the mouth of the bottle and replace the cap.
e. Remove the cap from the tube labeled “LB”. Hold the cap with the little finger of the hand holding the pipet
or with the thumb and a free finger of the hand holding the tube. Expel the 2 mL of LB broth into the tube.
4. Using sterile technique, complete the following steps to inoculate the 2 mL of LB broth with an ampicillinsensitive colony from the LB plate and an ampicillin-resistant colony from the LB/AMP plate. To avoid
contamination, perform the following steps quickly and do not allow the lower part of the loop to touch
anything other than the colony and the LB broth you are inoculating.
a. Partly open the wrapper of one disposable loop from the pointed end of the loop (the non-working end).
b. Withdraw the loop from the wrapper and inoculate the loop with bacteria from the plate containing the
ampicillin-sensitive bacteria by touching the circular end of the loop to a bacteria colony. Lift the lid off the
bacteria plate just long enough to inoculate the loop.
c. Remove the lid from the culture tube and hold it using the pinkie finger of the hand with the loop or with
the free fingers of the hand holding the tube.
d. Dip the loop into the 2 mL of LB broth and agitate it to dislodge the bacteria from the loop.
e. Remove the loop from the tube, replace the cap on the tube, and place the loop, circle side down, into the
benchtop waste container.
f. Repeat steps a–e with a new loop to inoculate the 2 mL of LB broth with an ampicillin-resistant colony
from the LB/amp plate. At the end of this step, the 2 mL of LB broth in the tube labeled “LB” should have
been inoculated with both ampicillin-sensitive and ampicillin-resistant bacteria.
5. Snap the cap of the tube tightly shut, and disperse the bacteria throughout the broth by gently and quickly
agitating the bottom of the tube back and forth. Do not tip or invert the tube.
6. Use sterile technique to transfer 1 mL of the culture you just created to the tube labeled “LB+AMP.”
7. Add 20 μL of ampicillin to the LB+AMP tube.
8. Incubate both culture tubes at 37°C overnight. The overfitting caps have two positions—tightly sealed and
loose-fitting. Make sure that the caps are secured in the loose position (i.e., they can be spun and jiggled up and
down without coming off).
9. Wipe down your area with 70% ethanol and wash your hands.
©2018 Carolina Biological Supply Company
S-6
Evolution in Real Time: Bacteria and Antibiotic Resistance
Laboratory Investigation (continued)
STUDENT GUIDE
Name
Date
Procedure for Day 1
1. Label both sterile
culture tubes.
4. Agitate the tube of LB
broth containing both
kinds of bacteria.
2. Using sterile technique,
pipet 2 mL of LB broth
into the tube labeled “LB.”
5. Transfer 1 mL of the
inoculated LB broth to the
tube labeled “LB + amp.”
Evolution in Real Time: Bacteria and Antibiotic Resistance
3. Inoculate the tube labeled LB
with ampicillin-resistant and
ampicillin-sensitive bacteria.
6. Add 20 µL of 100-mg/mL
ampicillin to the tube
labeled “LB + amp.”
S-7
7. Incubate both the “LB”
and “LB + amp” tubes
for 24 hours at 37°C.
©2018 Carolina Biological Supply Company
SG Page Title
RIGHT
Laboratory Investigation
(continued)
STUDENT GUIDE
Name
Date
Days 2, 3, 4, 5, 6, and 7:
Subculturing the Cultures Grown in the Presence and Absence of Ampicillin
Materials
For each group:
cultures set up the day
before, 2
culture tube rack
sterile culture tubes, 2
50-mL bottle sterile LB
broth
22-μL aliquot of 100-mg/
mL ampicillin
pipetting devices and
sterile pipets for
sterilely measuring and
dispensing 1 mL of LB
broth
pipet and sterile tips for
measuring 10 μL and
20 μL
benchtop waste container
permanent marker
Bunsen burner
(may be shared between
two groups)
Procedure
1. Obtain your two E. coli cultures from the previous day.
2. Label one new sterile culture tube “LB” and the other, “LB+AMP.” Also,
write the date and the number identifying your lab group on each tube.
3. Using the same sterile technique that you used on Day 1, add 1 mL of LB
broth to each of the tubes that you just labeled.
4. Use sterile technique to add 20 μL 100-mg/mL ampicillin to the tube
labeled “LB+AMP.”
5. Using the same sterile technique you used on day 1, transfer 10 μL of
culture from the “LB” tube from the previous day to the fresh tube
labeled “LB.”
6. Also transfer 10 μL of culture from the “LB+AMP” tube from the
previous day to the fresh tube labeled “LB+AMP.”
7. Return the tubes with the current date to the 37°C incubator.
8. Wipe down your area with 70% ethanol and wash your hands.
Shared:
37°C incubator
©2018 Carolina Biological Supply Company
S-8
Evolution in Real Time: Bacteria and Antibiotic Resistance
Laboratory Investigation (continued)
STUDENT GUIDE
Procedure for Days 2–7
1. Obtain the two cultures
from the previous day.
3. Add 1 mL of LB broth
to each labeled tube.
5. Transfer 10 µL of culture from
the “LB” tube from the previous
day to the new “LB” tube.
2. Label two new sterile
culture tubes.
4. Add 20 µL of ampicillin to new
tube labeled “LB + amp.”
6. Transfer 10 µL of culture from the
“LB + amp” tube from the previous
day to the new “LB + amp” tube.
Evolution in Real Time: Bacteria and Antibiotic Resistance
S-9
7. Incubate the new “LB” and
“LB + amp” tubes for 24 hours
at 37°C.
©2018 Carolina Biological Supply Company
Laboratory Investigation (continued)
STUDENT GUIDE
Name
Date
Day 8: Assaying the Culture for Antibiotic Resistance
Materials
For each group:
2 cultures from lab day 7
sterile culture tubes, 6
LB plates, 2
LB/amp plates, 2
50-mL bottle sterile LB
broth
pipetting devices and
sterile pipets for
sterilely measuring and
dispensing 0.9 mL and
1 mL of LB broth
pipet and sterile tips for
measuring 10 μL, 20 μL,
and 100 μL
culture tube rack
benchtop waste container
permanent marker
Bunsen burner (may be
shared between 2 groups)
Shared:
37°C incubator
spreading beads
Reflection Questions
•• Why do the cultures need to be diluted before they are plated?
•• If you did not resuspend the culture before plating, how would that affect the
results?
Procedure
Use sterile technique for all of the manipulations with bacteria and any plates
or LB broth.
When making serial dilutions refer to the diagram below to clarify the steps.
1. Label the six sterile culture tubes as follows:
L, 1:10
L, 1:1000
L, 1:100,000
LA, 1:10
LA, 1:1000
LA, 1:100,000
Note: “L” designates cultures grown in LB and “LA” designates cultures
grown with LB+amp.
2. Add 0.9 mL of LB broth to the tubes labeled “L, 1:10”, and “LA, 1:10.”
3. Add 1.0 mL of LB broth to the remaining four tubes.
4. Dilute the culture labeled “LB” by 1:10 as follows: agitate the tube
labeled “LB” and immediately transfer 100 μL of the culture from this
tube to the 0.9 mL of LB broth in the tube labeled “L, 1:10.” Mix well.
Resuspending the bacteria just prior to pipetting is critical for the
accuracy of the assay.
5. Transfer 10 μL of the dilute culture you just made (in the tube labeled
L, 1:10) to the tube labeled “L, 1:1000.” Mix well. Again, remember to
resuspend the bacteria just before pipetting.
6. Transfer 10 μL of the dilute culture in the “L, 1:1000” tube to the tube
labeled “L, 1:100,000.” Mix well. As in the steps above, agitate the tube the
transfer is made from just prior to making the transfer.
©2018 Carolina Biological Supply Company
S-10
Evolution in Real Time: Bacteria and Antibiotic Resistance
Laboratory Investigation (continued)
Transfer 10 µL
and mix.
STUDENT GUIDE
Transfer 10 µL
and mix.
Add 100 µL from
tube labeled
“LB” and mix.
20 µL
0.9 mL
medium
1 mL
medium
1 mL
medium
LB/amp plate
labeled “L”
20 µL
LB plate
labeled “L”
7. Repeat steps 4–6, using the culture labeled “LB+amp” and the tubes labeled “LA, 1:10”, “LA, 1:1000”, and “LA,
1:100,000.”
Plating the 1:100,000 Dilutions of the Cultures
1. Write your group number and the date on the side of the two LB plates and the two LB/amp plates.
2. Write “L” (for culture grown in LB) on the edge of the bottom of one LB plate and on one LB/amp plate. These
two plates will be used to determine how many ampicillin-resistant and ampicillin-sensitive colonies are
present in the culture grown in LB. Make labeling small to avoid interfering with your view of the colonies on
the plate.
3. In small letters, write “LA” (for culture grown in LB+amp) on the edge of the bottom of the other LB plate and
the other LB/amp plate. These two plates will be used to test how many ampicillin-resistant and ampicillinsensitive colonies are present in the culture grown in LB+amp.
4. Using sterile technique, add 4–5 spreading beads to each of the four plates. Add the beads while the plates are
lid-side down, since the beads have a tendency to bounce on the agar. Once the beads have been added, flip the
plate back over.
5. Remove 20 μL of the culture from the “L, 1:100,000” tube and pipet it onto the LB/amp plate that you labeled
“L.” Be sure to agitate the culture just prior to removing the 20 μL from the tube. Using a fresh tip and the same
procedure, plate 20 μL of the same culture onto the LB plate labeled “L.”
6. Spread the bacteria evenly across the plate by shaking the spreading beads across the plate. Use a back-andforth motion (not round and round). Shake for 1–2 minutes.
7. Remove the beads by slightly opening the plates over the waste container.
Evolution in Real Time: Bacteria and Antibiotic Resistance
S-11
©2018 Carolina Biological Supply Company
Laboratory Investigation (continued)
STUDENT GUIDE
8. Allow the liquid to soak into the plates for a few minutes, and then flip the plates over to incubate them, lidside down, overnight at the temperature indicated by your instructor.
9. Using the same procedure as in steps 5–8, plate 20 μL of culture from the “LA, 1:100,000” tube onto the LB and
LB/amp plates that you labeled “LA.”
10. Wipe down your area with 70% ethanol and wash your hands.
Predict the Result
Predict what type of colonies you expect to see on each plate and explain the reasoning behind each prediction.
©2018 Carolina Biological Supply Company
S-12
Evolution in Real Time: Bacteria and Antibiotic Resistance
Laboratory Investigation (continued)
Name
STUDENT GUIDE
Date
Day 9: Tabulating the Results
Materials
For each group:
LB and LB/amp plates from
day 8
permanent marker,
1 or more
Procedure
1. Create a data table to record your colony counts.
2. Carefully count the number of colonies on each plate and fill in the data
table. To count accurately, use a permanent marker to put a dot by each
colony as you count it. If the numbers are high, try to come up with a
simple strategy for keeping track of colonies as you count.
Analysis
1. Questions 1a–d pertain to the plates that were plated with the culture
grown in LB/amp. These plates are the plates labeled “LA.”
a. What is the phenotype of the colonies on the LB/amp plate with
respect to ampicillin resistance?
b. What is the phenotype of the colonies on the LB plates? Explain your
answer.
c. How does the number of colonies on the LB/amp plate compare with
the number on the LB plate? What do these relative numbers suggest
to you? Explain.
d. Can you think of a way to verify the phenotype of the colonies on the
LB plate? Explain your method.
Evolution in Real Time: Bacteria and Antibiotic Resistance
S-13
©2018 Carolina Biological Supply Company
Laboratory Investigation (continued)
STUDENT GUIDE
Name
Date
2. Read the following paragraph and then answer the question.
Mechanisms of Antibiotic Resistance
Bacteria develop resistance to antibiotics through a variety of mechanisms:
•• The target of the antibiotic is modified so that the antibiotic can no longer work. For example, some
antibiotics kill bacteria by binding to their ribosomes and inhibiting translation. Some bacteria are resistant
to these antibiotics because their ribosomes have been altered such that the antibiotic no longer binds.
•• The bacteria evolve in a way that prevents the antibiotic from entering or from staying in the cell, for
example, by eliminating the surface molecules through which the antibiotics enter or by developing surface
proteins that pump the antibiotic out of the cell before it reaches its target.
•• Some develop ways to alter the antibiotic molecule itself so that it no longer binds to its target efficiently. For
example, if an antibiotic binds to the bacterial ribosome to stop bacterial growth, the bacteria will alter the
antibiotic, so it can no longer bind.
•• Some produce enzymes that are able to destroy the antibiotic. For example the beta-lactamase made by the
ampicillin-resistance bacteria used in this kit destroys ampicillin. The bacteria also secrete the beta-lactamase
into the surrounding medium.
How did the observed phenotypic ratios for the bacterial culture grown in the presence of ampicillin come
about? Give a step-by-step explanation. Include any of the information in the paragraph above that is relevant
to your argument.
3. Questions 3a–e pertain to the plates plated with the culture grown in LB. The plates are labeled “L.”
a. With respect to ampicillin resistance, what is the phenotype of the colonies on the LB/amp plate?
Explain the results that you got.
b. What is the likely phenotype of the colonies on the LB plates? Explain your answer.
©2018 Carolina Biological Supply Company
S-14
Evolution in Real Time: Bacteria and Antibiotic Resistance
Laboratory Investigation (continued)
Name
STUDENT GUIDE
Date
c. Compare the number of colonies on the LB/amp plate and the LB plate.
d. What does that comparison suggest about how the bacterial population in the culture evolved when grown
in the environment without ampicillin?
e. Why do you think the ampicillin-sensitive bacteria came to dominate the culture? Put another way, in this
environment what advantage might ampicillin-sensitive bacteria have over ampicillin-resistant ones?
Read back through the introductory material for a clue.
4. A student performs the experiment in this lab and finds that there are still ampicillin-sensitive colonies in the
culture after the culture has been grown in the presence of ampicillin for a week. Explain how this would be
possible.
5. A single E. coli bacterium like the ones used in this kit can divide into two new cells in 20 minutes when grown
at 37°C.
a. How many generations does the bacterial population go through in 24 hours?
Evolution in Real Time: Bacteria and Antibiotic Resistance
S-15
©2018 Carolina Biological Supply Company
Laboratory Investigation (continued)
STUDENT GUIDE
Name
Date
b. How many progeny can a single bacterium produce in 24 hours? Assume that growth conditions are not
limiting (i.e., the bacteria do not run out of food and space).
6. During the 7 days you grew your cultures, the bacteria go though many generations. Do you think the relative
numbers of ampicillin-sensitive and ampicillin-resistant bacteria in the culture grown in the absence of
antibiotic for 7 days would be different from what you observed if each bacterium required 6 days to divide?
Why? For this question, assume that the bacterium cannot lose the plasmid.
7. A correlation is a relationship between two variables, but that relationship is not necessarily causative; in
other words, a change in one of the variables does not necessarily cause a change in the other. For example, a
rise in the number of car crashes over a period of five years might correlate almost exactly with a decrease in
expenditure on highway maintenance. The correlation does not necessarily mean that the neglect caused the
crashes. Further analysis might indicate that the cause is something entirely different, such as a change in speed
limit or a general increase in the number of cars and drivers on the roadways.
Make an argument for why the experiment you performed in this lab demonstrates that the loss of antibiotic
resistance by the bacteria is truly caused by the absence of the ampicillin in the medium.
©2018 Carolina Biological Supply Company
S-16
Evolution in Real Time: Bacteria and Antibiotic Resistance
Assessment
Name
STUDENT GUIDE
Date
1. Given what you observed in this lab, would you argue for or against treating livestock with an antibiotic even
when they show no sign of disease? Explain your reasoning.
2. Reread the description of the phenomenon at the beginning of the lab. Given the information in that
description and what you have observed in the lab, come up with a hypothesis for how plasmid-mediated
colistin resistance came to be found in bacteria infecting humans. In writing your hypothesis, try to use terms
that are used when discussing evolution.
3. A patient comes to the doctor’s office with symptoms identical to those of a routine respiratory infection that
is in the community. The respiratory infection is caused by a virus, not a bacterium. Viruses are not killed by
antibiotics. The patient wants to be given antibiotics. You are the doctor. Give two good reasons why the patient
should not have antibiotics.
4. There are two different hog farms, farm “A” and farm “B.” The hogs are raised the same way on each farm, with
one exception. On farm A, a small amount of antibiotic “D” is always added to their feed. This increases the
hogs’ growth rate, possibly by reducing infections. On farm B, no antibiotics are included in the animals’ feed.
a. What trait is likely to become more prevalent among the bacteria found in the hogs’ immediate
environment on farm A? Explain why.
b. On which farm is it more likely that antibiotic D will effectively treat any bacterial infection that may arise
in the hogs? Why?
Evolution in Real Time: Bacteria and Antibiotic Resistance
S-17
©2018 Carolina Biological Supply Company
Assessment (continued)
STUDENT GUIDE
Name
Date
5. To combat the problem of antibiotic resistance, researchers try to develop new antibiotics that will kill or stop
the growth of bacteria in novel ways. Aside from a knowledge of chemistry (necessary if the antibiotics are
synthesized or are modified from existing molecules), what kind of knowledge would be useful in developing
new antibiotics?
6. You are studying two different patches of forest with almost identical environments. Each patch contains a
population of a lizard species with both striped and solid-colored individuals. There are approximately equal
numbers of striped and solid lizards in each forest patch. A population of birds that show a preference for
eating the solid-colored lizards moves into forest patch A. The birds do not reach patch B. With respect to the
striped trait, how do you expect the lizard population in the two patches of forest to evolve? Explain.
©2018 Carolina Biological Supply Company
S-18
Evolution in Real Time: Bacteria and Antibiotic Resistance
Appendix
STUDENT GUIDE
Name
Date
Sterile Technique
Because many microorganisms grow under the same conditions as E. coli, it is important to maintain sterile conditions
that minimize the possibility of contamination with foreign bacteria or molds. In this experiment, the LB broth, LB agar,
ampicillin, prepoured plates, disposable inoculating loops, culture tubes, and petri plates, are sterile. There is no need
to flame the plastic ware. Flaming is necessary for sterilizing the wire inoculating loop when streaking the starter plates
before the first day of lab. Students should also flame the openings of the media bottles and slants just prior to inserting
any pipets or loops into them and before replacing the cap.
Good sterile technique involves the following and should be used throughout the experiment.
1. Always open the wrappers of the sterile pipets and loops so that you do not touch the working end. Open a pipet
wrapper from the end opposite the tip of the pipet; open a loop wrapper from the pointed end of the loop.
2. Never allow the unwrapped circular end of a loop, or the lower half of a pipet to contact a nonsterile object.
3. Do not reuse pipets or loops. To avoid contaminating your work surface, once materials have come into contact with
E. coli, do not place them on your work surface but in a benchtop waste container. Materials in the container and the
container should then later be autoclaved or soaked in bleach.
4. Wash your hands thoroughly before and after working with bacteria cultures.
5. Wipe your work area with 10% bleach or 70% ethanol before and after working.
6. When transferring things to or from a tube or vial, have the loop or bulb pipet ready in one hand. Pick up the tube
or vial you are transferring to or from and quickly remove the cap. Some people remove and hold the cap using the
pinkie finger of the hand holding the loop or bulb. Others are more comfortable removing and holding the cap using
the thumb and forefinger of the hand holding the vial or tube. Whichever technique you use, do not drop the cap or
put it down, and make sure that you hold it with the open side facing down. Also, make sure you keep your fingers
away from the rim of the cap. Replace the cap quickly once you have made your addition to the tube.
7. When putting loops or pipets into tubes or vials while transferring material do not touch the sides of the tube or vial.
8. When adding things to or collecting them from agar plates, hold the lid over the plate to prevent contaminants from
falling onto the surface of the plate. Open the dish the minimum amount needed to perform the manipulation.
Evolution in Real Time: Bacteria and Antibiotic Resistance
S-19
©2018 Carolina Biological Supply Company