This document outlines the design, methods, and expected outcomes of an experiment to test the effects of three antibiotics (chloramphenicol, erythromycin, and tetracycline) on the growth of the bacteria E. coli. Sterile technique will be used to isolate E. coli and expose samples to antibiotic disks on agar plates. The zones of inhibited bacterial growth around each disk will be measured to determine the relative effectiveness of each antibiotic. The results will then be analyzed to identify which antibiotic most strongly inhibits E. coli growth based on its known classification, and evaluate weaknesses and ways to improve the experimental design.

1. Lab-growth inhibition of E. coli by various antibiotics
Design, Aspect 1: We wish to determine whether and why certain antibiotics will have an effect on the
growth of E. Coli, namely the antibiotics Chloramphenicol, Erythromycin, and Tetracycline. The
independent variable will be the type of antibiotic used (against a negative control of paper disk with no
antibiotic) and the dependent variable will be the measured inhibition of growth of bacterial lawn
(measured to the nearest mm) of E. coli around each paper disk.
Design, Aspect 2: We will use sterile technique to insure that only a single strain of E. Coli
(available from Carolina Biological Supply) is used, we will seal petri dishes with parafilm to inhibit
airborne microbial spores of any other species from entering our culture. There will be a negative
control disk containing no antibiotic to insure that the simple presence of a disk on the lawn of E. coli is
not a potential inhibitor of growth, thereby strengthening the case for the actual antibiotics themselves
and not what they are carried on. We will use uniform doses of E coli bacteria for each experiment by
sharing the same original culture in Nutrient Broth.
Design, Aspect 3: We will use four petri plates per experiment, all poured to a uniform depth of 4 mm
with sterilized nutrient Agar (Carolina Biological). An E. coli culture will be prepared by taking a sterile
loop and dipping it into our original Carolina E. coli culture, scraping a generous loopful, and distributing
it into an eppendorff tube (small conical plastic tube with connected snap on lid) then vortexing for 30
seconds. Light a Bunsen burner for sterile technique. The eppendorf is then poured into a sterile petri
dish with no agar and immediately covered to prevent airborne bacteria from infecting. A glass
spreader bar is then disinfected in alcohol, allowed to dry momentarily, sterilized by Bunsen burner
flame (rapid-don’t want too much heat), dipped into the bacteria in the Bacterial culture and then
spread onto a petri dish with agar and immediately re-covered. This sterization, dipping, and spreading
on an agar plate is done to three more plates. Flip the plates and label them with Your initials, E. coli,
the Antibiotic type for that plate or CONTROL. Now sterilize a pair of forceps (alcohol, allow to dry,
flame briefly) and get one Chloramphenicol disk with the forceps, put it in the middle of the plate you
labeled Chloramphenicol. Do the same, re-sterilizing the forceps each time, for each of the other two
antibiotics and for sterile paper disks. Seal your plate with some parafilm, flip it upside down, and put it
in the incubator.
Data Collection and Processing, Aspect 1: Make up a data table, using quantitative data including units
and uncertainties.
Data Collection and Processing, Aspect 2: Process your raw data. Attempt to apply some kind of math
to your findings or a combination of the class’s findings.

2. Data Collection and Processing, Aspect 3: Present PROCESSED data in a graph of some sort, including
errors (like perhaps error bars) and/or uncertainties in measurement.
Conclusion and Evaluation, Aspect 1: Make a conclusion about the antibiotics, and which had the most
effect on the growth of E. coli, based on a reasonable interpretation of the data (you look up what type
of bacteria E. coli is classified as, and which kind of antibiotics are specifically good for that type of
bacteria, and whether our findings (or just yours) agree with what research shows should happen.
Conclusion and Evaluation, Aspect 2: Evaluate the weaknesses and limitations of this experiment, as
done by us.
Conclusion and Evaluation, Aspect 3: Suggest realistic improvements to the experiment which would fix
the weaknesses you identify in Aspect 2.