Chemical Methods of Control Essential Oils as
Antimicrobials against Escherichia coli JM101: Measurement of the
Zone of Inhibition
Adapted from T. Johnson and C. Case. Laboratory Experiments in Microbiology, Brief Edition,
4th ed., Redwood City, CA: Benjamin/Cummings Publishing Co., 1995.
Objectives:
1. Evaluate the relative effectiveness of an essential oil as antimicrobial agent.
Background:
A wide variety of chemicals called antimicrobial agents are available for controlling the growth
of microbes. For example:
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Chemotherapeutic agents, including antibiotics, are used internally.
Disinfectants are chemical agents used on inanimate objects to lower the level of
microbes present on the object.
Antiseptics are chemicals used on living tissue to decrease the number of microbes
present in that tissue.
No single antimicrobial agent is most effective for use in all situations - different situations may
call for different agents. A number of factors affect selection of the best agent for any given
situation - Antimicrobial agents must be selected with specific organisms and environmental
conditions in mind. Additional variables to consider in the selection of an antimicrobial agent
include pH, solubility, toxicity, organic material present, and cost.
Once an agent has been selected, it is important to evaluate it's effectiveness. In evaluating the
effectiveness of antimicrobial agents, the concentration, length of contact, and whether it is lethal
(-cidal) or inhibiting (-static) at that concentration and exposure are the important criteria.
One method of measuring the effectiveness of a chemical agent is to determine its zone of
inhibition. In the agar diffusion method or disc diffusion method, one species of microbe is
uniformly swabbed onto a nutrient agar plate. Chemicals are impregnated on paper disks. These
discs are added to the surface of the agar. During incubation, the chemical diffuses from the disk
containing the agent into the surrounding agar. An effective agent will inhibit microbial growth,
and measurements can be made to quantify the size of the zones of inhibition around the disks.
The relative effectiveness of a compound is determined by comparing the diameter of the zone of
inhibition with values in a standard table.
The agar diffusion test is not used to determined whether a chemical is “-cidal” (kills bacteria) or
“-static” (inhibits bacteria) - instead this characteristic is determined by the dilution method. In
this method the microbe of interest is placed in a tube containing the chemical (at various
concentrations) which is being tested. The microbe is then added (subcultured) onto an agar
plate. If the microbe grows on the agar the chemical is “-static”; if not, it was killed by the
chemical which is then termed "-icidal."
Materials:
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6 Petri dishes containing LB agar
Sterile cotton swab
Forceps
Disinfectant filled beakers for used tips and contaminated tips and diluent
Disks (use a hole punch on filter paper) and antibiotic discs
Essential Oils
E. coli K12 JM101 which has been grown in LB broth
Pure dilution diluent (10% aqueous DMSO, 0.5% Tween 80) and rinse diluent
Microcentrifuge tubes
Micropipettes and tips
Parafilm
Ruler and sharpie
Procedure:
1. Obtain gloves and clean your work area with the disinfectant provided. Gather supplies.
2. With a marker, divide only the bottom of the petri dish into three sections and label the
outside as seen below. Also, label both the top and bottom as seen below. Follow
example for each dish - A, B, C, D, paper control dish (P), diluent solution control (S)
dish. Make writing small so the growth area is not obscured!
A2
A1
A3
Label on Top & Bottom:
A, Ms Rios, 10-5-09
3. Prepare the dilutions of the essential oil you choose using the diluents provided to you.
You will be carrying out a 1:10, three step serial dilution. Label your microcentrifuge
tubes as follows: A, B, C, D, the concentrations, your initials and the oil you are testing.
100 µL oil
100 µL oil
A
5-6 drops oil
100 µL oil
C
B
900 µL
diluent
900 µL
diluent
D
900 µL
diluent
4. You will be using approximately 50 microliters of each concentration to impregnate your
discs and these dilutions can be made in microcentifuge tubes.
a. Drop 5-6 drops of essential oil from the essential oil bottle into a microcentrifuge
tube labeled “A” which is 100% essential oil.
b. Next, make a 1:10 dilution of the oil using the diluents provided. Using a
micropipette and tip, take 100 µL of the concentrated essential oil from “A” and
place it into another microcentrifuge tube labeled “B”. With a new pipette tip,
add 900 µL of diluent. Mix by flicking with your finger.
c. Next, make a 1:100 dilution of the oil using the diluents provided. With a new
pipette tip, take 100µL of “B” and place it into a microcentrifuge tube labeled “C”
and with a new pipette tip add 900 µL of diluent. Mix by flicking with your
finger.
d. Next, make a 1:1000 dilution of the oil using the diluents provided. With a new
pipette tip, take 100µL of “C” and place it into a microcentrifuge tube labeled “D”
and with a new pipette tip add 900 µL of diluent. Mix by flicking with your
finger.
5. Obtain a sample of E.coli in LB broth.
6. Using a cotton swab, dip the swab into the E.coli suspension and spread it onto the first
plate making sure to evenly distribute it. Repeat for the other 5 plates. Make sure to keep
the lid of the agar plate closed as much as possible to avoid further contamination with
other microorganisms. Place the used swabs in a beaker of disinfectant.
7. Placing the filter paper discs.
a. Using forcepts (rinsing in pure diluent between) dip three discs into the four
concentrations of oil and place them onto the corresponding agar plate in the
center of each division.
Label on Top & Bottom:
A, Ms Rios, 10-5-09
b. Prepare the “paper control” (P) dish with three uncoated paper discs as a control
(properly labeled) and a “diluent solution control” (S) dish with three paper discs
dipped in new pure diluent .
c. Other controls will include cinnamon oil discs prepared identically and antibiotic
discs.
8. After 15 minutes, place the dishes, right side up, in the incubator at 37 degrees Celsius
for 24 hours. Wrap them in parafilm before incubation.
9. Clean up and place potential contaminated materials in autoclave. Discard disinfectants.
10. After 24 hours, the inhibition zone can be measured in millimeters from one side of the
zone across the disc to the other side of the zone (diameter). The effectiveness of the
essential oil may be determined as in Table 1. Your values are presented as means ± S D
of three parallel measurements in chart provided (Table 2).
Table 1: Interpretation of Inhibition Zones of Test Cultures
Effectiveness
Diameter of Zones of
Inhibition (mm)
Resistant
_____________or less
Intermediate Between __________
Susceptible
___________ or more
Table 2: Essential Oils as Antimicrobials against Escherichia
coli JM101: Measurement of the Zone of Inhibition
Zone of Inhibition (mm)
Disc
Essential Oil
Concentration 1
1X
.1X
.01X
.001X
Paper disc control
Diluent paper disc
control
1X cinnamon
.1X cinnamon
.01X cinnamon
.001X cinnamon
antibiotic
Disc
2
Disc
3
Average
Zone
Standard
Deviation
Effectiveness
Resistant,
Intermediate, or
Suseptible