University of Pittsburgh at Bradford
Science In Motion
Biology Lab 016
Inhibition of Bacteria
Antibiotics and Antiseptics
Introduction:
In 1928 Alexander Fleming, a Scottish scientist discovered the antibiotic penicillin almost by
accident. He observed a mold growing on a bacterial culture in his lab. More importantly, Fleming
noticed a clear zone around the mold in which no bacteria appeared to be growing. Further study
indicated that the mold apparently produced some protective chemical, which inhibited or killed bacteria.
The mold was identified as Penicillium notatum and the chemical was called penicillin. Following this
discovery, other organisms were found that produced similar chemicals, which destroy or inhibit bacterial
growth. These chemicals were given the general name antibiotic because of their lethal effect on
microorganisms. Since this initial discovery, scientists have discovered dozens of microorganisms that
produce chemicals that destroy other microorganisms (bacteria). These chemicals (antibiotics) are
harvested by man to help fight infectious diseases caused by bacteria. Unfortunately, over the years
humans have developed a feeling that infectious bacterial diseases were under control because of the
variety of antibiotics we have discovered and used. Recently, we are finding that bacteria are mutating
and developing new strains that are resistant to known antibiotics. So the search is on for new antibiotics
that will protect the human race from bacterial infection in the future. If they are not found, pathogenic
bacteria might be the victors!
Antibiotics inhibit or destroy bacterial cells in different ways. Some affect the bacterial cell wall.
A bacterial cell wall is unique in construction because it is composed of a macromolecular network called
pepticogylcan. Certain antibiotics, such as bacitracin and vancomycin, prevent growing cells from
producing this chemical. As a result, the bacterial cell wall is weak and the cell ruptures. Other
antibiotics, such as penicillin and cephalosporin interfere with peptidoglycan linkages thus inhibiting cell
wall production.
A second way in which antibiotics inhibit bacteria is by interfering with protein production at the
ribosomes within the bacterial cell. Antibiotics that use this mode of attack are chloramphenicol,
erythromycin, streptomycin, and tetracycline. These antibiotics generally have a broad spectrum of
activity except for erythromycin, which does not penetrate the gram-negative cell wall. A third way
antibiotics inhibit is to disrupt the way bacterial ribosomes read messenger RNA, which leads to protein
errors. Examples of such antibiotics are gentamicin and streptomycin.
Generally speaking, antibiotics, which affect the peptidoglycan structure in bacterial cells, have no
negative affect on human cells since we have no cell walls. Likewise the antibiotics, which affect protein
production in bacterial cells, have little negative effect on human cells because of differences in the
ribosomes of bacteria and humans. However, some antibiotics interfere with the function of human
mitochondria, which possess ribosomes similar to those found in bacteria. It is important that antibiotic
use be monitored closely and limited to only severe infections. Doctors and patients both must realize that
overuse of antibiotics will lead to increases in bacterial resistance and the development of new strains of
bacteria for which we have no effective antibiotic!
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Antibiotics which inhibit cell wall synthesis
penicillin
ampicillin
methicillin
oxacillin
gentamicin
cephalosporin
bacitracin
vancomycin
Antibiotics which inhibit protein synthesis
streptomycin
neomycin
chloramphenicol
erythromycin
Objectives:
1.
2.
3.
4.
To understand the necessity of controlling or inhibiting bacterial growth.
To determine if inhibition by an agent is slight or extensive.
To compare inhibition of a single species of bacteria by different antibiotics and antiseptics.
To compare inhibition of different bacterial species by similar antibiotics and antiseptics.
Safety Notes:
1. Review and follow all safety precautions as outlined in the Aseptic Techniques lab.
2. Make sure you keep ethanol away from the Bunsen burners- Avoid fires!
Teacher Notes: - Instructors should assign the types of antibiotics and antiseptics used by lab groups
so that all chemicals are tested on both types of bacteria used. You may also want to split your class so
that half of them are testing antibiotics on bacteria #1 and antiseptics on bacteria #2 and the other half are
performing opposite tests.
Materials:
2 sterile nutrient agar plates
sharpie
metric ruler
biohazard bag
distilled water
alcohol burner
sterile cotton swabs
forceps
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2 types of bacterial broth cultures- M. leuteus, E. coli,.
or B Megaterium
sterile paper disks
commercial antiseptics: mouthwashes, soaps,
creams, toothpaste, etc.
commercial antibiotic disks: Erythromycin,
Tetracycline, Chloramphenicol,
Streptomycin, Novobiocin, others possible
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Procedure:
1. Obtain 2 large petri plates filled with nutrient agar, invert each and use a sharpie to mark the bottom
side into quarters. Do not open until necessary!
2. Label the lid with your name, date, time, and the species of bacteria you will grow in that plate.
3. Using aseptic technique, inoculate your plates:
a. Obtain broth cultures of the bacteria you are using along with two sterile
cotton swabs.
b. Remove the cap from one of the broth culture tubes. DO NOT place the cap
on the lab counter. Hold the top part of the cap between your fingers.
c. Flame the mouth of the tube in a Bunsen burner. Avoid spilling any of the
broth.
d. Dip a cotton swab into the bacterial broth.
e. Reflame the mouth of the tube and replace the cap.
f. Raise the petri plate cover slightly and then rub the swab over the entire
surface of the petri plate.
g. Replace the cover of the plate and discard the swab in the biohazard bag.
h. Repeat this procedure for your second plate.
4. Label one plate antibiotic (AB) and the other antiseptic (AS)
5. Select three antibiotics and three antiseptics for testing (your instructor will probably assign these).
6. If the antibiotics are on presoaked disks, use forceps to firmly press 3 different types onto the agar in 3
of the quarters on the AB plate. Sterilize the forceps each time by dipping in ethanol and then flaming.
7. The fourth quadrant of the AB plate will receive a plain, sterile, paper disk and will be the control.
8. Invert the AB plate making sure the disks don’t fall off and set it aside.
9. Obtain the three selected antiseptics and four sterile paper disks.
10. Using sterile forceps, dip each paper disk in a selected antiseptic and press it onto one quarter on the
AS plate. Again, remember to sterilize the forceps with ethanol and flame between each transfer.
11. The fourth quarter of the AS plate will receive a plain, sterile paper disk and be the control.
12. Invert the plate making sure the disks stick; make sure you have labeled properly and incubate both
the AB and AS plates at 37 degrees C. for 24 - 48 hours.
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Name________________________
Inhibition of Bacteria - Antibiotics and Antiseptics
Student Evaluation
1. Examine your petri plates looking for clear zones (zones of inhibition) around the disks. Bacteria
should be growing in “lawn growth” all over the remainder of the plates.
2. Measure the diameters of these zones of inhibitions in millimeters and record in the table below. No
zone equals 0mm.
Group Data
AB petri dish section
1
2
3
4
antibiotic used
zone of inhibition for ___________ ________
AS petri dish section
1
2
3
4
antiseptic used
zone of inhibition for ___________ ________
3. Record your data on the board along with all other group results. Compute averages for the zones of
inhibition of all antibiotics/antiseptics on both species of bacteria. Construct a line or bar graph for
your class data (it may be computer generated).
Analysis/Conclusions:
1. Why are some antibiotics effective against E. coli but not against Bacillus cereus? Give examples
from your observations.
2. How do antibiotics kill bacteria?
3. Are antiseptics as effective in the inhibition of bacteria as antibiotics? Use your observations to back
up your answer.
4. Why must researchers continuously search for new antibiotics?
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