Antibiotics Resistance Lab

Hi. I’m Sara, and I am going to explain
the procedure for the antibiotic resistance lab activity. Remember we learned that because
of natural selection, some bacteria can live in the presence of an antibiotic that previously
may have killed them. When the antibiotic does not kill the bacteria, the bacteria are
said to be resistant, as in the case of drug-resistant tuberculosis.
In lab, we will test the effectiveness of antibiotics on 3 bacterial species to see
which ones are resistant to any of the antibiotics. You will work with one partner to test the
effects of the antibiotics on one of three bacterial species. In another class, after
the bacteria have grown, you will observe your results and compare them to the results
of 2 other teams. Let’s get started. Here is a petri dish containing sterile agar,
commonly referred to as an agar plate. Agar is a gelatin-like substance made from seaweed.
It is moist and contains nutrients, so it is an ideal environment on which bacteria
can grow. Open the petri dish only as much as necessary, to avoid contaminating the agar
with organisms from the air. This package contains two swabs that are sterile. They
will remain sterile as long as they are in the package, so do not remove them until you
are ready to use them. The swabs have cotton on one end only; so open the package on the
opposite end to avoid contaminating the sterile cotton.
Each group of 2 students will receive an agar plate with one of these 3 bacterial species:
E.coli, B. mycoides, or S. marcescens, which are growing on the surface of the agar. Your
goal is to remove a small amount of the bacteria from the agar surface with the sterile swab,
and spread it evenly over the surface of the agar in your sterile plate, while minimizing
exposure of the agar surface to the air. As you spread the bacteria on the surface
of the agar, rotate the swab to be sure the sample is being transferred from the swab.
You have now made a “lawn” of bacteria on the surface of your agar plate.
Place the used swab in an orange biohazard bag to avoid contaminating any of the lab
surfaces, or yourself. Label the outside bottom of the petri dish (the half which contains
the agar) with a large X to divide the plate into 4 quadrants.
If you are allergic to any of the antibiotics we are using in this activity, your partner
should do the rest of the procedure. Each of these sterile petri dishes contains
a different antibiotic labeled with the initials of the antibiotic: Am for ampicillin, E for
erythromycin, and Te for tetracycline. A fourth petri dish contains blank disks. You will place one of each of these disks,
using forceps, on the surface of the agar. First, sterilize the forceps using an alcohol
pad. Then open the small petri dish, remove one of the disks, and place it on the surface
of the agar in the middle of one of the quadrants. In order to prevent the disks from falling
off the agar, gently press each one so that it sticks to the agar. Repeat the procedure
for the other 2 antibiotics, sterilizing the forceps before you remove a disk each time.
Finally, you will place one blank disk in the last quadrant, the same way you placed
the antibiotic disks. We will incubate your plate at room temperature
in an upside down position – that is to say, with the agar surface on the top. This
is to prevent condensation from the lid caused by the respiration of the bacteria from “raining
down” on the agar surface, and interfering with the bacterial growth.
Place your initials, and the initials of the bacteria on the lid of the petri dish, that
is, the part without the agar. The TA will collect your plate when you have finished.
You will observe the growth of the bacteria in the next class period.
So what does resistance look like? Here is an agar plate from a few days ago that was
swabbed with bacteria, and then antibiotic disks and a control disk were placed on the
surface. Here you see a clear zone around one of the antibiotic disks, called the “zone
of inhibition.” The antibiotic killed the bacterial cells that were swabbed in that
area. Where the bacteria have grown up to the edge of the disk, the antibiotic was ineffective,
that is to say, the bacteria were resistant to this antibiotic.
When you arrive at the lab, review these procedures before you prepare your agar plate.
See you next time. Don’t forget to wash your hands before you leave the lab.


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