Antimicrobial Agents
Background Information
Bacteria have existed long before humans set foot on the earth, and will probably outlive the
human species. They are found on almost every surface available, on our desks, in the food we
eat, and even in and on our bodies! Some bacteria are very helpful, such as E. coli, which lives
in our intestines and helps with digestion and production of Vitamin K. Only a small number
of bacteria are considered harmful and actually cause disease. Attempting to eliminate bacteria
through the overuse of antibacterials could actually cause more harm than good. Removing the
natural bacterial population from our bodies provides an opportunity for those bacteria that
make us sick to take up residence and establish a foothold. We need bacteria to remain healthy,
and the overuse of antibacterials could have devastating effects on our health and the
environment.
Antiseptics, disinfectants, and antibiotics, can all be referred to as chemical antimicrobial
agents. Antiseptics are preparations of chemicals that are meant to be applied to skin or other
living tissues. Examples of antiseptics include: alcohol, iodine, hydrogen peroxide, heavy
metals such as silver nitrate and detergents. Disinfectants are preparations of chemicals
(usually liquids) that are intended for application to the surfaces of nonliving materials. Many
household cleaning agents such as ammonia and bleach are powerful disinfectants. Antibiotics
are chemical substances produced by microorganisms that stop the growth of other
microorganisms and are effective in very small doses. Antibiotic production by
microorganisms is common, and most medically valuable antibiotics are produced either by
fungi, especially those in the genus Penicillium, which produces antibiotics such as penicillin;
or bacteria in the genus Bacillus, which produce antibiotics such as bacitracin and polymyxin;
or bacteria in the genus Streptomyces, which produce a number of medically important
antibiotics.
Antimicrobial agents work in one of two ways: they are either bacteriostatic or bacteriocidal.
A bacteriostatic agent does not kill the bacteria, but inhibits growth by preventing cell wall
formation or interfering with biochemical cellular processes. A bacteriocidal agent kills
bacteria on contact by breaking the protective cell membrane and lysing the cell, or stopping
nucleic acid synthesis, or preventing the bacteria from carrying out normal chemical reactions.
Many products exhibit natural antimicrobial activity. Sugar, salt, spices and wood smoke have
traditionally been used to prohibit bacteria from inhabiting food. Plants develop antimicrobial
properties as a defense mechanism against invasions by bacteria.
In the following experiments you will be able to test some common antimicrobials to see how
effective they are at stopping bacterial growth using the zone-of-inhibition method. With this
method, a chemical is applied to a freshly inoculated plate and after incubation, bacterial
growth around the antimicrobial agent is observed. A clear area around the chemical agent
(zone-of-inhibition) indicates that bacteria were killed. The size of the zone is usually
proportional to the effectiveness of the antimicrobial.
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Materials
Cultures and Plates
Escherichia coli (broth)
Staphylococcus epidermidis (broth)
Nutrient Agar Plates
Supplies and Equipment
Sterile swabs
Forceps
Alcohol lamp
Antibiotic discs
Blank discs
Natural antimicrobials
Disinfectants
Clean weighing dishes
Beaker with 95% alcohol
Permanent marking pen
Disposal bags
10% bleach or disinfectant
The following chemicals can be used for testing. Check with your teacher to find out which
ones are available.
Goods and spices:
 Egg whites
 Cloves
 Cinnamon
 Allspice
 Garlic





Grapes
Onions
Coke
Horseradish
Tabasco sauce
Household chemicals:
 Bleach
 First aid ointment
 Iodine
 Hydrogen peroxide




Hand soap
Mouthwash
Hand sanitizing gel
Melaleuca oil
Antibiotics:
 Kanamycin
 Streptomycin
 Erythromycin
 Tetracylcine




Novobiocin
Penicillin G
Chloramyphenicol
Neomyacin
The two bacterial available for testing are Escheridia coli, a gram negative bacterium that is a
common inhabitant of the gut, and Staphylococcus epidermidis, a gram positive bacterium
commonly found on the skin.
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Procedure:
Day 1
1. Select three sterile agar plates and a bacterial culture tube. Using a permanent marker,
label the bottom of each plate with your name, date, and bacteria. Designate one plate
for food, the second for household chemicals, and the third for antibiotics.
2. On the bottom of the plate, divide each plate into six equal sections. Use the permanent
pen to draw three lines across the bottom of the plate so they go through the center, as
you might cut a pie.
3. Choose five chemicals from each of the groups to be tested (food, household chemicals,
antibiotics). Record their names or use a number code to label each of the sections on the
corresponding plate. The remaining section on each plate will be left as a positive
control.
4. Remove a sterile swab from the protective paper, without touching the cotton end.
5. Using sterile technique, remove the top on the broth culture and dip the swab into the
broth. Rotate it several times, with firm pressure on the inside wall of the tube above the
fluid level, to remove excess liquid.
6. Replace the top on the liquid culture.
7. Just lifting the cover of the agar plate, streak the swab over the entire plate, rotating the
plate to cover the entire surface and ensure even distribution of inoculum. Repeat for the
other two plates using a clean sterile swab.
8. Place swabs and bacterial culture in a disposable bag.
9. Apply the antimicrobials in the following way:
For liquid agents:
i.
Place a small amount of each liquid in separate, clean weighing dishes.
ii.
Light the alcohol lamp.
iii.
Soak the tips of a forceps in 95% ethanol for 30 seconds, and then remove
them from the ethanol and pass the forceps tips through the flame. Hold
the points down so the burning ethanol does not run onto your hand.
When the flame goes out, the forceps are sterile.
iv.
Use the forceps to remove one of the blank disks from the glass vial and
touch the disk to the liquid in the weighing dish. Allow the disk to soak in
the liquid for 10 seconds.
v.
Remove the disk from the liquid and allow any excess liquid to drain off.
vi.
Place the disk in the middle of the section of the agar plate corresponding
to the antimicrobial. Once the disk touches the agar, do not remove it.
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Use the forceps to press on the disk so that the entire bottom surface of the
disk is in contact with the agar.
For solid agents:
i.
Light the alcohol lamp.
ii.
Soak the tips of a forceps in 95% ethanol for 30 seconds, and then remove
them from the ethanol and pass the forceps tips through the flame. Hold
the points down so the burning ethanol does not run onto your hand.
When the flame goes out, the forceps are sterile.
iii.
Pinch off a small piece of the sample with the forceps.
iv.
Use the forceps to transfer the sample to the proper section on the agar
plate.
For antibiotic disks:
i.
Light the alcohol lamp.
ii.
Soak the tips of a forceps in 95% ethanol for 30 seconds, and then remove
them from the ethanol and pass the forceps tips through the flame. Hold
the points down so the burning ethanol does not run onto your hand.
When the flame goes out, the forceps are sterile.
iii.
Use the forceps to pick up one of the antibiotic disks from the container..
iv.
Place the disk in the middle of the section of the agar plate corresponding
to the antibiotic. Once the disk touches the agar, do not remove it since
the medication will disperse into the agar immediately. Use the
forceps to gently press on the disk so that the entire bottom surface of the
disk is in contact with the agar.
10. Incubate the plates for 18-24 hours at 37 C inverted (agar side UP) for those plates with
disks and agar side DOWN for the plates with solid agents.
Day 2
11. Examine the plates for zones of inhibition.
12. Use your permanent marker to trace the zones of inhibition on the bottoms of the plates.
13. Compare each zone of inhibition to the diagram below and record the corresponding
scores on the chart that follows.
14. Repeat on Day 3 if directed to do so by your teacher.
15. Dispose of your plates properly and wipe down the lab station with 10% bleach.
16. Always wash your hands before leaving the laboratory when working with bacteria.
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Results & Discussion
Use the following diagram for comparing each zone of inhibition. Then place the
corresponding scores for each in the chart below. Note: Only fill in the blanks of the
agents and bacteria that you used.
Size of Zone of
Inhibition
none
Score
(–)
(+)
(++)
(+++)
Diagram 1
Foods & spices (solid agents)
E. coli
S. epidermidis
E. coli
S. epidermidis
Egg Whites
Cloves
Cinnamon
Allspice
Grapes
Garlic
Onion
Coke
Horseradish
Tabasco
Household chemicals (liquid agents)
Bleach
First aid ointment
Iodine
Hydrogen peroxide
Hand soap
Mouthwash
Hand sanitizing gel
Melaleuca oil
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Antibiotic disks
E. coli
S. epidermidis
Kanamycin
Streptomycin
Erythromycin
Tetracylcine
Novobiocin
Penicillin G
Chloramyphenicol
Neomyacin
Questions
1. What do the terms antiseptic, disinfectant, and antibiotic mean?
2. Why do some colonies grow inside the zone of inhibition?
3. Would you expect antibiotics to be effective against viral infections? Why of why not?
4. What would be the most effective agent to disinfect your countertop?
5. Which antimicrobial agent would you use to clean a wound?
6. In your opinion, which antimicrobial was the most effective on the bacteria that you
chose?
7. What other agents seemed to work well for others in your class?
8. Any surprises?
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