Note A: If large quantities of
plates are out of budgetary considerations for you, you
might consider running this lesson as a whole class demonstration.
B: Although E. coli K12 is used in this lesson because
of its safety and accessibility, other forms of bacteria
can be used. If you decide to use something other than
E.coli K12 be sure to check safety information and follow
appropriate safety precautions.
C: Contamination can be a problem in this exercise.
To reduce contamination do the following:
- Have students use cotton swabs once and only to touch
the bacteria cultures and agar plates.
not to touch the agar plates or culture broth with
anything other than the swabs.
- Have students keep the Petri dishes closed at all times
except during transfer of bacteria. Even
should keep the lids opened briefly and narrowly.
When the lid is open, it should help to be as
close to the agar
surface as possible.
- When swirling the cotton swab over the agar to make
a bacterial lawn, have students rub
the agar and then dispose of the swabs in a
designated waste container. All liquid cultures
should be placed in the same container and
disposal and labeled as biological waste.
- Have students wash their hands thoroughly with soap
(preferably triclosan-free) and
after handling bacterial
D: The use of paper disks in this lab helps students
get more accurate results. Paper disks allow the treatment
to diffuse through the agar in a predictable manner and
give students a clear visual about the treatment gradient
caused by diffusion.
Using cotton swabs gives students the ability to transfer
bacteria without faculty supervision and make spreading
bacteria onto the agar plates very easy.
1. As students enter the room, have the following question
on the board, “How do new diseases come into being?” Allow
students a few minutes to record and share answers. Hopefully
students will being up the idea of bacteria and diseases
evolving into stronger, better adapted strains. If not,
you will want to ask students higher-level thinking questions
to get them to that idea
2. Tell students that today they will read a brief article
about bacteria and diseases. [If you want to provide more
background, you may want to copy several articles so that
each student in each lab group has a different piece of
information to bring to the discussion.]
3. Distribute the article to the students and have them
read over the report. Then ask them “What have scientists
found? What do students think abut the research? How might
they conduct an experiment to test the scientists’ work?”
4. Divide students into groups and tell them they will
be following a protocol to test for antibacterial resistance
in E. Coli K12. Be sure to tell students that while this
is a relatively harmless strain of bacteria, it is still
important that they follow standard safety protocol and
wear lab coats, gloves, and protective eyewear.
5. Distribute protocol
sheets to students and have them
read over the material. What questions (if any) do they
6. Have students follow steps 1-3 and then return to the
front of the class for a group discussion.
7. Discuss steps 4-7, which will be conducted tomorrow.
What is the zone of inhibition? [This is the area where
growth of the bacteria is inhibited by the treatment on
the disk.] What does it mean if the zone is “large” or “small?” If
the zone decreased what does that tell them? What if the
zone has increased? What if it has stayed the same? [Change
in the zone is related to resistance. As the zone decreases
in size, bacterial resistance is increasing.] Where on
the plate will students be able to find bacteria likely
to be resistant to the treatment they are applying? [The
bacteria nearest the zone of inhibition are more likely
to be resistant to the treatment. They have been put under
selective pressure by the treatment and have survived.
If the reason for survival is a genetic mutation, they
will pass that on to their offspring as they are reproducing.]
What is the purpose of taking bacteria and transferring
it to a liquid culture? [This mixes the bacteria and allows
for a more even growth of bacteria on the next plate.]
students continue to collect bacteria and re-plate
them following the protocol sheet. Over the weekends,
students leave bacteria sealed on a counter. Bacteria
will grow slower at room temperature. If possible,
solution would be to place the plates in a refrigerator
either before or after a 24-hour incubation period.
- When students are not collecting data or preparing
the plates for the next culture, this is an appropriate
for students to begin conducting research for the
did students discover? How did bacteria respond to the
What does that tell us about
resistance? How do the different treatments affect the
natural evolution of bacteria? How does that affect humans,
who ultimately act as “hosts” to bacteria as
they reproduce? What are the benefits and drawbacks of
the use of antibacterial, antimicrobials, and antibiotics?
their science notebooks, have students write a reflective
What did they learn? What new questions do
they have? How does the lab connect to “real life?”
students explain how antibiotics affect the evolution
Can they explain how misuse of antibiotics can lead to
the evolution of antibiotic resistant bacteria?