Chapter 11:

Chemical Agents of Control:

Antibiotics and Disinfectants
Objectives
• Determine the antibiotic sensitivity profile of your unknowns
• Determine the disinfectant sensitivity profile of your unknowns
• Learn the Kirby-Bauer dis diffusion method

Chemotherapeutic Agents
Chemotherapeutic agents are chemical substances used in the treatment of infectious diseases.
Their mode of action is to interfere with microbial metabolism, thereby producing bacteriostatic
or bactericidal effect on the microorganisms, without producing a like effect in host cells.
Chemotherapeutic agents act on a number of cellular targets Their mechanisms of action include
inhibition of cell-wall synthesis, inhibition of protein synthesis, inhibition of nucleic acids
synthesis, disruption of the cell membrane, and inhibition of folic acid synthesis. These drugs can
be separated into two categories:

1. Antibiotics are synthesized and secreted by some true bacteria, actinomycetes, and fungi
that destroy or inhibit growth of other microorganisms. Today, some antibiotics are
laboratory synthesized or modified; however, their origins are living cells.

2. Synthetic drugs are synthesized in the laboratory.

To determine a therapeutic drug of choice, one must know its mode of action, possible adverse
side effects to the host, and the scope of its antimicrobial activity. The specific mechanism of
action varies among different drugs, and the short-term or long-term use of many drugs can
produce systemic side effects in the host. These vary in severity from mild to temporary upsets to
permanent tissue damage. See the table on the next page.
Synthetic Agents
Sulfadiazine (a sulfonamide) produces a static effect on a wide range of microorganisms by a
mechanism of action called competitive inhibition. The active component of the drug,
sulfanilamide, acts as an antimetabolite that competes with the essential metabolite, p-
aminobenzoic acid (PABA), during the synthesis of folic acid in the microbial cell. Folic acid is
an essential cellular coenzyme involved in the
synthesis of amino acids and purines. Many
microorganisms possess enzymatic pathways
for folic acids synthesis and can be adversely
affected by sulfonamides. Human cells lack
these enzymes, and the essential folic acid
enters the cell in a preformed state. Therefore,
these drugs have no competitive effect on
human cells. The similarity between the
chemical structure of the antimetabolite
sulfanilamide and the structure of the essential
metabolite PABA is shown to the right.
Kirby-Bauer Disc Diffusion Method
The available chemotherapeutic agents vary in their scope of antimicrobial activity. Some have a
limited spectrum of activity, being effective against only one group of microorganisms. Others
exhibit broad-spectrum activity against a range of microorganisms. The drug susceptibility of
many pathogenic microorganisms are known, but it is sometimes necessary to test several agents
to determine the drug of choice.

A standardized diffusion procedure with filter paper discs on agar, known as the Kirby-Bauer
method, is frequently used to determine the drug susceptibility of microorganisms isolated from
infectious processes. This method allows the rapid determination of the efficacy of a drug by
measuring the diameter of the zone of inhibition that results from the diffusion of the agent into
the medium surrounding the disc. In this procedure, filter-paper discs of uniform size are
impregnated with specified concentrations of different antibiotics and then placed on the surface
of an agar plate that has been seeded with the organism to be tested. The medium of choice is
Mueller-Hinton agar, with a pH of 7.2-7.4, which is poured into plates to a uniform depth of 5 mm
and refrigerated after solidification. Prior to use the plates transferred to an incubator set to 37°C
for 10 to 20 minutes to dry off the moisture that
develops on the agar surface. The plates are then
heavily inoculated with a standardized inoculum by
means of cotton swab to ensure the confluent
growth of the organism. The discs are aseptically
applied to the surface of the agar plate at well-
spaced intervals. Once applied, each disc is gently
touched with a sterile applicator stick to ensure its
firm contact with the agar surface.

Following incubation, the plates are examined for

the presence of growth inhibition, which is
indicated by a clear zone surrounding each disc. See
the image to the right.

The susceptibility of an organism to a drug is assessed by the size of this zone, which is affected
by other variables such as:

1. The ability and rate diffusion of the antibiotic into the medium and its interaction with the
test organism.

2. The number of organisms inoculated.

3. The growth rate of the organism.

A measurement of the diameter of the zone of inhibition in millimeters is made, and its size
compared to that contained in a standardized chart, which is shown on the next page. Based on this
comparison, the test organism is determined to be resistant, intermediate, or susceptible to the
antibiotic.
Disinfectants and Antiseptics
Antiseptics and disinfectants are chemical substances used to prevent contamination and infection.
Many are available commercially for disinfection and asepsis. The table below shows the major
groups of antimicrobial agents, their modes and ranges of action, and their practical use.
The efficacy of all disinfectants and antiseptics is influenced by a variety of factors, including the
following:

1. Concentration: The concentration of a chemical substance markedly influences its effect

of microorganisms, with higher concentrations producing a more rapid death.
Concentration cannot be arbitrarily determined; the toxicity of the chemical to the tissues
being treated and the damaging effect on nonliving materials must also be considered.

2. Length of exposure: All microbes are not destroyed within the same exposure time.
Sensitive forms are destroyed more rapidly than resistant ones. To longer the exposure to
the agent, the greater its antimicrobial activity. The toxicity of the chemical and
environmental conditions must be considered in assessing the length of time necessary for
disinfection or asepsis.

3. Type of microbial population to be destroyed: Microorganisms vary in their

susceptibility to destruction by chemicals. Bacterial spores are the most resistant forms.
Capsulated bacteria are more resistant than non-capsulated forms; acid-fast bacteria are
more resistant than non-acid-fast; and older, metabolically less-active cells are more
resistant than younger cells. Awareness of the types of microorganisms that may be present
will influence the choice of agent.

4. Environmental conditions: Conditions under which a disinfectant or antiseptic effects the

chemical agent are as follows:

a. Temperature: Cells are killed as the result of a chemical reaction the agent and the
cellular component. As increasing temperatures increase the rate of chemical
reactions, application of heat during disinfection markedly increases the rate at
which the microbial population is destroyed.

b. pH: The pH conditions during disinfection may affect not only the microorganisms
but also the compound. Extremes in pH are harmful to many microorganisms and
may enhance the antimicrobial action of a chemical. Deviation from a neutral pH
may cause ionization of the disinfectant; depending on the chemical agent, this may
serve to increase or decrease the chemical’s microbicidal action.

c. Type of material on which the microorganisms exist: The destructive power of

the compound on cells is due to its combination with organic cellular molecules. If
the material on which the microorganisms are found is primarily organic, such as
blood, pus, or tissue fluids, the agent will combine with these extracellular organic
molecules, and its antimicrobial activity will be reduced.

Numerous laboratory procedures are available for evaluating the antimicrobial efficiency of
disinfectants or antiseptics. They provide a general rather than an absolute measure of
effectiveness of any agent because test conditions frequently differ considerably from those seen
during practical use. Adapting the Kirby-Bauer disc diffusion method will be done in our
experiment.
Kirby-Bauer Disc Diffusion Method (for Antiseptics)
This procedure requires the heavily inoculum of an agar plate with the test organism. Sterile, paper-
discs are impregnated antiseptics by dipping them in the substance. The discs are then equally
spaced on the inoculated agar plate. Following incubation, the agar plate is examined for zones of
inhibition (areas of no microbial growth) surrounding the discs. A zoned of inhibition is indicative
of microbicidal activity against the organism. Absence of a zone of inhibition indicates that the
chemical was ineffective against the test organism. Note: The size of the zone of inhibition is not
indicative of the degree of effectiveness of the chemical agent. Antiseptic susceptibility is
represented in the figure below.
Antibiotic Discs Protocol
Materials: Amount:
Bunsen burner 1 set up / pair
Markers 1 box / class
One roll of tape 1 roll / pair
Sterile cotton swabs 1 box / class
Forceps in 95% ethanol 1 beaker / set up
Antibiotic discs 1 dispenser / set up
Gentamycin
Tetracycline
Penicillin
Erythromycin
Chloramphenicol
Ampicillin

Media: Amount:
Mueller-Hinton II 2 plates / pair

Cultures: Amount:
Unknown #1 2 broths / pair
Unknown #2 2 broths / pair

Creating a lawn of bacteria:

1. Obtain two plates of Mueller-Hinton II agar.

2. Label each plate with:

a. Media type
b. Unknown #1 or 2
c. Incubation temperature
d. Today’s date
e. Group #

3. When the plates are labeled flip them over, so the agar side is on the bench top.

4. Take your broth with Unknown 1 growing in it and vortex to mix the culture up.

5. When the culture is in suspension, open a sterile cotton swab and place it in the broth.

6. Now, like a painter’s brush, apply the organism to every part of the agar. Make sure to
twist and rotate the brush over the entire agar surface.

7. Let this plate sit, agar side down and covered, for 5-10 minutes.
Application of antibiotic discs:

1. Flame sterilize a forceps by dipping it in ethanol (or picking it up from ethanol) and passing
the forceps through the flame.

2. You should see the alcohol burn off. This is indicative that the forceps are now sterile.

3. Pick up an antibiotic disc dispenser and use one tong of the sterilized forceps to push the
paper disc out one side.

4. When enough of the disc is hanging off to the side use the forceps to grab the disc and then
place it, gently, on the surface of the Mueller-Hinton II agar.

5. Next, lightly tap the disc into place to get it situated in the agar.

6. Repeat steps 1-5 with a new antibiotic disc. Make sure to leave enough space between discs
so if a zone of inhibition is present you are able to accurately measure it.

7. When finished, flame sterilize your forceps and repeat the process with Unknown 2.

8. Tape up the inoculated Unknown 1 and 2 plates together but do not invert them. Dr. Siegert
will invert them to ensure the paper discs do not slide around.
Disinfectant Discs Protocol
Materials: Amount:
Bunsen burner 1 set up / pair
Markers 1 box / class
One roll of tape 1 roll / pair
Sterile cotton swabs 1 box / class
Forceps in 95% ethanol 1 beaker / set up
Sterile filter-paper-discs 1 container / set up
Disinfectant solutions in beakers 1 beaker / set up
Hibiclens
Lysol
10% bleach
2% iodine
10% acetic acid
5% bacdown

Media: Amount:
Mueller-Hinton II 2 plates / pair

Cultures: Amount:
Unknown #1 2 broths / pair
Unknown #2 2 broths / pair

Creating a lawn of bacteria:

1. Obtain two plates of Mueller-Hinton II agar.

2. Label each plate with:

a. Media type
b. Unknown #1 or 2
c. Incubation temperature
d. Today’s date
e. Group #

3. When the plates are labeled flip them over, so the agar side is on the bench top.

4. Take your broth with Unknown 1 growing in it and vortex to mix the culture up.

5. When the culture is in suspension, open a sterile cotton swab and place it in the broth.

6. Now, like a painter’s brush, apply the organism to every part of the agar. Make sure to
twist and rotate the brush over the entire agar surface.

7. Let this plate sit, agar side down and covered, for 5-10 minutes.
Application of disinfectant-impregnated discs:

1. Flame sterilize a forceps by dipping it in ethanol (or picking it up from ethanol) and passing
the forceps through the flame.

2. You should see the alcohol burn off. This is indicative that the forceps are now sterile.

3. Pick up a paper disc with the sterilized forceps.

4. Dip the disc into a disinfectant beaker to soak in the liquid.

5. Before removing the disc from the beaker, squeeze out excess disinfectant by tamping the
disc against the side of the beaker.

6. Use the forceps to place the impregnated disc, gently, on the surface of the Mueller-Hinton
II agar.

7. Next, lightly tap the disc into place to get it situated in the agar.

8. Repeat steps 1-5 with a new disinfectant. Make sure to leave enough space between discs
so if a zone of inhibition is present you are able to accurately measure it.

9. When finished, flame sterilize your forceps and repeat the process with Unknown 2.

10. Tape up the inoculated Unknown 1 and 2 plates together but do not invert them. Dr. Siegert
will invert them to ensure the paper discs do not slide around.