Heather Elia

Effect of Antibiotic Treatment on Microbial Growth

Abstract: In this experiment we tested broad range and narrow range spectrum
antibiotics with the microorganisms of B. cereus, E. aerogenes, and S. lutea.
Chloramphenicol and Tetracycline represents the broad spectrum and Penicillin
represents the narrow spectrum of the antibiotics. Each microbe was plated separately in
a petri dish using easy gel inoculated with the microbes. To each petri dish antibiotic
disks of; Chloramphenicol, Tetracycline, and Penicillin were placed on the gel in each
dish. Each plate was taped up after 45mins and incubation then occurred at 30oC for 24
hours to see how each type of microbe reacted with the antibiotic plate and if it was
resistant, intermediate or susceptible to it. Our results showed S. lutea was most
susceptible to all antibiotic disks and the broad-spectrum antibiotic Tetracycline had the
largest zone of inhibition for all microorganisms used.
Introduction: The purpose of this experiment is to develop an understanding of how
different antibiotics affect the growth of different microorganisms. Broad and narrow
spectrum antibiotics and gram positive and gram negative bacterium will be used in the
tests. The different bacterium was inoculated into easy gel and antibiotic disks were
placed on to the gel and zone of inhibition was used to find the results.
The zone of inhibition that is formed by the antibiotic is observed by how large or small
the zone is. The larger the zone of inhibition, the more susceptible the bacteria is the
antibiotic and the smaller the zone of inhibition, the more resistant the bacteria is the
antibiotic. If the bacterium is resistant it has no effect of antibiotic. If it is susceptible it
means that the antibiotic as the ability to inhibit the bacteria. If the zone of inhibition is
neither small nor large then the bacteria is intermediate, and this means that some
inhibition from the antibiotic occurred but not sufficiently enough to inhibit the growth of
the organism in the body. Antibiotics are drugs that kill or slow the growth of some
bacteria, they can fall under the categories of: Bactericide (kill) or Bacteriostatic (slow
growth). Antibiotics are produced by mold, other bacteria, or they could be produced in a
laboratory. Antibiotic resistance can occur easily so a way to prevent this can be to create
bad conditions for bacterial growth rather than attempting to kill them all with chemicals.
I hypothesize that the broad range spectrum antibiotics will have the greatest effect on
bacterial growth because that they act against a wide range of disease-causing bacteria
Materials:
3 petri dishes labeled 1-3
3 bottles of easy gel labeled
1-3
Bacteria: E. aerogenes, B.
cereus, S. lutea
Antibiotic disks: Tetracycline
(red), Penicillin (blue),
Chloramphenicol (black)
Methods:

Sterilized forceps
Alcohol
Clean sheet of paper
Burner
Ruler
Tape
Incubator

Heather Elia
Day 1
1. Obtain 3 petri dishes and 3 bottles of Easygel. Put petri dishes in an area that
will be comfortable for once initiate experiment they are not to be moved.
2. Swirl each bottle of Easygel and pour the liquid from bottle #1 into petri dish
#
1. Repeat with bottle and dish sets #2 and #3. Gently swirl and rock the dish
while if sits on the counter top until the bottom is covered by the liquid.
3. Once the bottom of the petri dish is covered, do not pick the dish off the
counter until instructed to do so.
4. Write your name or initials and the date on the top of the petri dish, as shown
below. Then write the name of the culture with which each dish is inoculated
(Dish #1 = E aerogens, dish #2 = B. cereus, dish #3 = S. lutea).
5. Using your forceps, take 3 tetracycline disks proviced by instructor and place
on a clean sheet of paper. Take them back to your counter and place one of
these disks on the gel in each dish. It is alright if they fall upside down on the
gel.
6. Repeat with the penicillin and chloramphenicol disks. Once they are in the
dish, do not try to pick them up or move them. At no time should the disks
or the gel be touched by hand.
7. After forty to forty-five minutes, seal the petri dishes with tape.
8. Incubate at 25 35oC for 24 hrs. Wash hands using soap and water.
Day 2
9. Measure the diameter of the zones of inhibition in millimeters by placing the
ruler against the bottom of the dishes. Record results in a table. Petri dishes
should not be opened under any circumstances.

Tetracycline (red)

Name
Date
Penicillin (blue)
B. cereus

Results:

Chloramphenicol
(black)

Heather Elia
Zones of Inhibition (Diameter in mm)
Organism
Chloramphenicol (black) Penicillin (blue)
Tetracycline (Orange)
E. aerogenes (-)
25mm
9mm
22mm
B. cereus (+)
12mm
13mm
34mm
S. lutea (+)
53mm
48mm
70mm
S. Lutea showed the largest zone of inhibition to all of the antibiotics indicating that it is
suseceptible to all of them.
Discussion:
There are many divisions and classifications of bacteria that assist in identifying
them. The bacteria that we used were E. aerogenes, B. cereus, and S. lutea. E. aerogenes
is a gram-negative bacterium and B. cereus and S. lutea are both gram positive bacterium.
Antibiotics also have different divisions. The antibiotics that we used are
Chloramphenicol and Tetracycline which are both broad-range spectrum antibiotics and
we also used Penicillin which is a narrow range antibiotic.
The first plate containing the gram negative bacterium E. aerogenes, shows that
Chloramphenicol gave the best zone of inhibition, which inhibited the bacteria the most.
The antibiotic, which showed the second most inhibited amount of bacteria, was the
Tetracycline. This indicates that E. aerogenes is most susceptible to Chloramphenicol
and Tetracycline, but there was a very small zone of inhibition with penicillin, which
shows that the bacterium is resistant to it.
Since this is a gram negative bacterium it has a unique outer membrane, a thinner layer of
peptidoglycan, and a periplasmic space between the cell wall and the membrane and in
the outer membrane has lipopolysaccharides. The lipopolysaccharides are endotoxins
which blocks antibiotics, dyes, and detergents protecting the sensitive inner membrane
and cell wall which is why it is resistant to penicillin since penicillin cannot penetrate the
Gram-negative outer membrane.
In the second plate with the gram positive bacterium B. cereus, Tetracycline had
the best zone of inhibition and Chloramphenicol and Penicillin having a very small zone
of inhibition. This would indicate that B. cereus is susceptible to the antibiotic
Tetracycline and probably resistant to Chloramphenicol and Penicillin. Bacillus Cereus
are a spore forming type of bacteria. They are gram-positive and contain rods. Due to the
fact that this bacteria is known to survive because of the spores, which could be a
possibility of why it may be resistant to both Chloramphenicol and Penicillin.
In the last plate with the gram positive bacteria S. lutea there was a large zone of
inhibition for all antibiotics, the largest with Tetracycline. The smallest zone of inhibition
was shown with Penicillin, meaning that the bacteria can be resistant to it. The second
largest zone of inhibition was with the Chloramphenicol, which can indicate that it is
intermediate to this antibiotic.
Chloramphenicol and Tetracycline are both broad spectrum antibiotics, a clear advantage
to the use of broad-spectrum antibiotics is that there is less of a need to identify the
infecting pathogen with real certainty before commencing treatment. Chloramphenicol
and Tetracycline are also both bacteriostatic antibiotics, which means that they inhibit the
ability of the bacteria to grow which makes these antibiotics bacteriostatic. Bacteriostatic
antibiotics inhibit growth and reproduction of bacteria without killing them; killing is

Heather Elia
done by bactericidal agents. Penicillin is bactericidal which refers to a substance or a
condition capable of killing bacteria. We have learned that antibiotics are chemical
substances that can be natural or synthetic and are able to destroy or kill bacterial cells, or
even inhibit their growth. Most antibiotics act by inhibiting the formation of the bacteria;
in this case the broad-spectrum antibiotics. Over the years, antibiotic distribution has
resulted in resistance, as shown from the results. Antibiotics have the ability to
kill/destroy and resistance is becoming a world problem for curing diseases. Unless
antibiotic resistance problems are detected and actions are taken immediately to contain
them, society could be faced with a large problem of becoming unable to treat diseases
that were once treatable. This is why it is a wiser option to kill a bacterium not by using
chemicals, but to create an environment with bad conditions for the bacteria to grow in it
to try to prevent resistance.
***This discussion was based off our class results. Based off of the hypothesis made: I
hypothesize that the broad range spectrum antibiotics will have the greatest effect on
bacterial growth because that they act against a wide range of disease-causing bacteria,
It was shown that Tetracycline, a broad spectrum antibiotic had the best results, which
can show that it was true. I am unable to tell whether a bacteria is actually resistant,
intermediate, or susceptible to these antibiotics. Results can vary by numerous types of
human error, like the preparation of the experiment, or by the conditions of the
experiment. Different conditions may affect the results of this experiment. It will take
extensive testing with multiple trials to find more accurate results then the ones made in
class.
Acknowledgements:
Dr. Lisa Lucente
Bibliography:
Dr. Lisa Lucentes class slides Antibiotic Assay
Antibiotic Effects Kit Student Worksheet
http://www.colby.edu/biology/BI163/163Lab/163Ecoli09.pdf
http://www.textbookofbacteriology.net/resantimicrobial.html
http://www.abxlist.com/chloramphenicol.htm
http://www.hhmi.org/biointeractive/Antibiotics_Attack/bb_5.html