Testing Antiseptics and Disinfectants: Disk Diffusion
Method
Copyright ©2009 by Gary Fromert, M.S.
PETRI Project, Northampton Community College
After completing this exercise, you should be able to:
1.
2.
3.
4.
5.
6.
Explain what is meant by an antiseptic.
Explain what is meant by a disinfectant.
Explain what is meant by the concentration effect.
Explain what is meant by disk diffusion.
Explain what is meant by a sterilant.
Determine the effectiveness of antiseptics and disinfectants.
As we know, the control and decontamination of microorganisms is one of the most critical aspects
in a clean room environment. We also know that the use of heat is an effective means of microbial
control but the use of heat has its limitation, for instance autoclaves and ovens are limited by the
number and size of the material or objects that can be placed into them. We would typically not try
to autoclave a bench top, floor, or a large piece of processing equipment so we need another means
for controlling microorganisms.
An effective means for the control of microorganisms is the use of chemical antiseptics and
disinfectants. Antiseptics are chemical agents typically used in preventing or arresting the growth
of microorganisms especially on living tissue. Disinfectants are chemical agents that destroy
vegetative forms of microorganisms especially on inanimate objects. Neither of these chemical
agent groups tend to kill spores so they are not considered sterilizing agents. Chemical agents that
kill all forms of microorganisms including spores are called sterilants. The efficiency and
effectiveness of these chemical agents are influenced by several factors:
1. Concentration - Concentration cannot be arbitrarily determined as the toxicity and the
effectiveness of the chemical in regard to the microorganism, tissue,
surface or material must be considered.
2. Length of Exposure (called “Dwell Time” in the biotechnology industry) –
The longer the exposure to the agent, the greater the antimicrobial activity. The
toxicity of the chemical and the environmental conditions must be considered in
assessing the length of time necessary for asepsis, disinfection, or sterilization.
3. Type of Microorganisms - Microorganisms vary in their susceptibility to chemical
agents.
4.
Environmental Conditions - Environmental conditions that can affect the chemical
agent are: temperature, pH, and the material on which microorganism are present.
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Numerous laboratory procedures are available for evaluating the antimicrobial efficiency of
antiseptics, disinfectants, and sterilants. These procedures provide a general rather than an
absolute measure of the effectiveness of any chemical agent, as test conditions frequently differ
considerably from those encountered during practical use. This being said, it is important to
characterize the chemical agent as to the conditions of contact time, temperature, concentration,
and type of microorganism for its usage as an antiseptic, a disinfectant, or a sterilant.
In this laboratory exercise you will be performing the disk diffusion susceptibility test; disks
containing known amounts of an antimicrobial agent are placed on the surface of an agar plate
containing a nonselective medium that has been inoculated with a suspension of a strain of bacteria
to give a confluent lawn of growth. The antimicrobial agent diffuses into the medium, causing a
zone of inhibition of growth of the strain around the disk corresponding to the susceptibility of the
strain to the agent.
In this laboratory exercise you will also be preparing a series of dilutions of an antimicrobial agent
and determining the effectiveness that the concentration has on a bacterial culture.
Materials Needed (per group):
24 Blank Diffusion Disks
1 Pure Culture Broth of 24 hour Pseudomonas fluorescens
1 Pure Culture Broth of 24 hour Staphylococcus epidermidis
1 Pure Culture Broth of 24 hour Bacillus subtilis
4 TSA or Nutrient Agar Plates
3-5% Solution Bleach
1-2% Solution Household Disinfectant Cleaner (e.g. Lysol®)
0.5-1% Solution Silver Nitrate
100% Antiseptic Mouthwash (e.g. Listerine®)
100% Liquid Antibacterial Soap
1-2% Solution of a Pine Cleaner (e.g. Pine-sol ®)
1 bottle of antibacterial soap
Sterile Cotton Swabs
Forceps (tweezers)
Alcohol for Flaming
China Marker or Sharpie
Inoculating/Transfer Loop
Bunsen Burner
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Procedure 1: Disk Diffusion Susceptibility Test Method
Acquire a pure culture broth of 24 hour Pseudomonas fluorescens.
Note: The surface of the plates should be dry before culture suspensions are inoculated. If
moisture is visible on the surface of the plates, dry them (with lids ajar) in a 37°C incubator
just prior to inoculation. There should be no visible droplets of moisture on the surface of
the agar or on the lids of the plates when they are inoculated.
Table 1. Antimicrobial Agents.
Label
A
B
C
D
E
F
Antimicrobial Agent
3-5% Solution Bleach
1-2% Solution Household Disinfectant
0.5-1% Solution Silver Nitrate
100% Antiseptic Mouth Wash
100% Liquid Antibacterial Soap
1-2% Solution Pine Cleanser
Active Ingredient
Sodium hypochlorite
Alkyl dimethyl benzyl ammonium chlorides
AgNO3
Thymol
Triclosan or other antibacterial agent
Natural pine oil
1. Label one of the TSA plates: "A", "B", "C", "D", "E", and "F" on the bottom of the plate in the
pattern shown in Figure 2, and with the culture name, your group initials and the date.
2. Moisten a sterile applicator swab in the pure culture suspension, and express excess moisture by
rotating the swab against the glass above the liquid in the tube. Inoculate the entire surface of the
agar, inoculating the surface completely in three different directions to ensure uniform, confluent
growth, see Figure 1.
Figure 1. Inoculation Pattern. (http:// www.cdc.gov)
3. Allow the inoculated plates to sit at room temperature for 3 to 5 min to allow the moisture from
the inoculum to absorb into the medium. Inspect the inoculated plates to ensure that there is no
visible liquid on the surface of the medium; the surface of the medium must be dry before the disks
are applied.
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4. Using an alcohol flamed forceps, acquire a blank disk and immerse it half-way into
antimicrobial agent "A" (see Table 1) and remove immediately, do not over saturate the disk.
Allow the liquid to absorb throughout the entire disk.
5. Apply the disk to the surface of the medium directly over the corresponding letter on the bottom
of the plate and tamp it gently with the forceps to ensure that it is in complete contact with the agar
surface. Note: All disks should be applied approximately the same distance from the edge of the
plate and from each other, see Figure 2.
Figure 2. Disk Application Pattern.
6. Repeat steps 4 and 5 for the antimicrobials B, C, D, E, and F.
7. Repeat steps 1 through 6 for the Staphylococcus epidermidis and Bacillus subtilis.
8. Invert the inoculated plates (lid side down), and incubate the plates at 37°C for 24 hours.
9. Examine the disk diffusion plates from the back, viewed against a black background and
illuminated with reflected light. With a caliper or scale, measure and record the diameter of each
zone of inhibition to the nearest whole millimeter. The zones of inhibition are measured as shown
in Figure 3. Record your results.
Figure 3. Measuring the Zone of Inhibition. (http:// www.cdc.gov)
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Procedure 2: Concentration Test
Acquire a pure culture broth tube of 24 hour Staphylococcus epidermidis.
Table 2. Dilution Ratios.
Label
A
B
C
D
E
F
Dilution Ratio
No Dilution
1:10
1:25
1:50
1:100
1:500
1. Label one of the TSA plates: "A", "B", "C", "D", "E", and "F" on the bottom of the plate in the
pattern shown in Figure 2, and with the antimicrobial used, culture name, your group initials and
the date.
2. Moisten a sterile applicator swab in the pure culture suspension, and express excess moisture by
rotating the swab against the glass above the liquid in the tube. Inoculate the entire surface of the
agar, inoculating the surface completely in three different directions to ensure uniform, confluent
growth, see Figure 1.3
4. Using either Listerine or a liquid antibacterial soap, make dilutions as per Table 2 using DI H2O
(sterile DI H2O if available).
5. Using an alcohol flamed forceps, acquire a blank disk and immerse it half-way into dilution "A"
(see Table 2) and remove immediately, do not over saturate the disk. Allow the liquid to absorb
throughout the entire disk.
6. Apply the disk to the surface of the medium directly over the corresponding letter on the bottom
of the plate and tamp it gently with the forceps to ensure that it is in complete contact with the agar
surface. Note: All disks should be applied approximately the same distance from the edge of the
plate and from each other, see Figure 2.
7. Repeat steps 4 and 5 for dilution B, C, D, E, and F.
8. Invert the inoculated plates (lid side down), and incubate the plates at 37°C for 24 hours.
9. Examine the disk diffusion plates from the back, viewed against a black background and
illuminated with reflected light. With a caliper or scale, measure and record the diameter of each
zone of inhibition to the nearest whole millimeter. The zones of inhibition are measured as shown
in Figure 3. Record your results.
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Results and Observations
For the Disk Diffusion Susceptibility Test record your results below:
Label Antimicrobial Agent
A
3-5% Solution Bleach
B
1-2% Solution Household Disinfectant
C
0.5-1% Solution Silver Nitrate
D
100% Antiseptic Mouth Wash
E
100% Liquid Antibacterial Soap
F
1-2% Solution Pine Cleanser
Zone of Inhibition (mm)
For the Concentration Test record your results below:
Label
A
B
C
D
E
F
Dilution Ratio
No Dilution
1:10
1:25
1:50
1:100
1:500
Zone of Inhibition (mm)
Laboratory Review
1. Explain the differences between an antiseptic, a disinfectant, and a sterilant:
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2. Explain what effect concentration of an antimicrobial agent has on inhibiting
microorganisms:
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3. Speculate as to how the zone of inhibition correlates to the diffusion of antimicrobial agent and
the tolerance of the microorganism for that particular antimicrobial agent:
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