Download (Citrus aurantium), Lavender (Lavandul officinalis)

The Antibacterial Effects of Essential Oils, Lemon (Citrus
lemon), Orange (Citrus aurantium), Lavender (Lavandul
officinalis) and Thyme (Thymus Vulgaris) Against GramNegative Bacteria, Escherichia coli, and Gram-Positive
Bacteria, Staphylococcus epidermidis
Cathy Billingsley
Biology 401
December 9, 2007
Final Draft
Table of Contents
Content
Page
Introduction
1
Current Research
1
Specific Goals
8
Material/Methods
9
Essential Oils
9
Bacteria
9
Procedure
9
Statistical Analysis
9
Flow Cart
10
Reference
11
2
Introduction
Plant products and their derivatives have been around for centuries; they have
been used for medicines, cleaning products and a range of other life necessities. Essential
oils made from plant products are just one of the by products that have been used as
medicines, and cleansing agents for thousands of years. The more commonly known oils
with biblical references are frankincense, myrrh and hyssop (Bible 1984), which can be
found, listed throughout the Bible.
In more recent history, and through current times, one of the more important
derivatives made from plants have been medicines; such as, vinblastine which is used in
chemotherapy treatment of breast cancer, lung cancer and various others ( Ajose, 2007 ).
Native Americans have used plants for more than just medicine; they have used them in
their religious ceremonies, for their homes and many other practices. In the ancient
beliefs of the Native Americans, plants came to the aid of man because man had upset the
animals by killing them and taking up their land. The animals got together and decided
to plague man with diseases, so plants decided to help man by providing a cure for all the
diseases that the animals put on man (Maxwell 1984).
Numerous essential oil, from various species of plants, have been tested in the
past for their antibacterial and antimicrobial properties. Cinnamon (Cinnamonum
zeylanicum), thyme (Thymus vulgaris and Thymus pectinatus), and oregano (Origanum
vulgare) are some of the more popular types of oils used, and the antibacterial activities
vary in each plant species (Lopez, 2007 and Vardar-Ünlü, 2002). The antibactetrial
activities of essential oils come from their chemical make-up, such as terpene alcohols
which is a major constituent of many oils (Inouye, 2001).
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Hammer et al. (1999) conducted a study using 52 plants oils and extracts, to test
the antimicrobial effect on yeast, gram-negative and gram-positive bacteria. There were
ten different microorganisms used and the organisms were received from the Department
of Microbiology at The University of Western Australia and the Western Australian
Centre for Pathology and Medical Research. Serial dilutions of bacteria were conducted
using saline solution to a concentration of approximately 108 (colony forming units) cfu/
ml-1 by both an agar dilution and broth microdilution method. The agar dilution method
is used to determine the minimum inhibitory concentration (MIC) used in antimicrobial
susceptibility testing such as the disk diffusion testing that I will be using (Eucast
Definitive Document, 2000). The broth dilution method use was conducted in the same
manner as the agar dilution method with a difference, the Mueller Hinton broth was used
instead of heart infusion broth. For most oils the highest concentration used was 4.0 %
(v/v); for some an 8.0 % (v/v) was used and the lowest concentration used was 0.008 %
(v/v). The essential oils were then autoclaved to help with the solubility of the oil prior to
inoculation. After inoculation, plates were incubated for 48 hours at 37° C and checked
for bacterial growth.
In the result of the study Hammer et al. (1999) found that many oils were
effective on inhibiting some or all bacterial growth. There where 6 oils that failed to
inhibit any bacterial growth at the highest concentration levels used, which was 2.0 %
volume per volume (v/v); although, the reason for the oils being inert was not explained.
In the resulting research, with the large number of plants used and the method of disc
diffusion being done this study follows a line of investigating the effects of essential oil
that I am interested in doing.
4
López et al. (2007) used three essential oils in a vapor-phase method to test for
the antibacterial activities of these oils. In a vapor-phase method, a single drop of the
organic material is suspended in an atmosphere from the tip of a microsyringe. The
sample drop (1-3 µl) is exposed to the atmosphere of a test tube or agar gel plate where
known samples of bacterium were cultured for an unknown amount of time in a known
temperature. The drop is then drawn back into the microsyringe and the sample is tested
through chromatographic analysis for the specific airborne bacteria. Although, this is not
a method I will be using, their use of thyme essential oil against both Escherichia coli
and Staphylococcus aureus, and the data collected they collected will be of great use in
comparing to the data that I collect in my experiment with the same essential oil and
gram-negative and gram-positive bacteria that will be in direct contact with the medium.
López et al. (2007) used the vapor-phase technique to test for the reduction of
bacteria in the packaging of food and the preserving of packaged foods. The findings
showed that Thyme was the least inhibitory of the oils they used. In other studies done,
Thyme proved to be a strong inhibitory essential oil. It will be interesting to see of their
results correlate with my study and the use of Thyme as a cleaning product through the
home can decrease the bacteria. Possible future investigation may show that Thyme can
inhibit bacteria left by food preparation as well.
López et al. (2007) used bacterial strains from both gram-negative and grampositive bacteria, as well as yeast and molds. The results of the study showed that
oregano demonstrate to be highly inhibitory against bacteria, whereas, cinnamon showed
to be mostly effective against fungal strains. Thyme proved to be ineffective against
bacterial and fungal growth. In López et al. research, they tested essential oils that were
5
fortified, which is adding two oils together, and found that in doing so there is a greater
inhibitory factor against bacterial and fungal growth. This may suggest that to control all
forms of bacteria several essential oils would have to be used in correlation with each
other. This is due to no one essential oil controlling all forms of bacterial.
Vardar-Ünlü et al. (2002) looked at both gram-positive and gram-negative
antimicrobial organisms. In this research study, the essential oils were collected by
Clevenger distillation apparatus, instead of being purchased. Clevenger distillation is
done by heating and distilling a compound without any added solvents or water. The
plant that was used was Thymus pectinatus which has thirty-eight species within it
represented; pectinatus is a small shrub about 12 cm high and grows in the wild in Inner
Anatolia. This particular species is found in Turkey and the East Aegean Islands. In
Turkey it is used as an herbal tea and as a condiment. The plants were air dried and
ground up then distilled to obtain the needed essential oil.
Vardar-Ünlü et al. (2002) used two different methods of antimicrobial testing.
Three different microorganisms were used; gram-negative, gram-positive bacteria and a
yeast. The first test conducted utilized an agar well diffusion method. The wells were
divided into zones and after a “flood-inoculated” procedure the zones were examined for
bacterial growth. The second method incorporated is a disk diffusion method. Bacteria
were spread over an agar gel plate and a filter paper disk was soaked in the essential oil
solution and laid over the plate. The plates where then incubated for 24 hours for
bacteria and 48 hours for yeast. The area where no growth was observed was sectioned
and measurements of the bacterial growth retardations were taken.
6
Vardar-Ünlü et al. (2002) tested plant extractions with a polar subfraction (water
soluble portion) of the essential oil, which showed no antimicrobial activity; and a non
polar subfraction (water-insoluble portion) proved to be somewhat active. The essential
oil in a pure form was highly active against all samples of bacteria; this is due to the
chemical constituents of the essential oils such as their phenolic contents. The results
here would make a good comparative study to thyme essential oil purchased from a
commercial facility. This would be an excellent essential oil that would fit into my
hypothesis.
Dorman et al. (2000) studied the antibacterial effects of black pepper, thyme,
oregano, nutmeg, clove, and geranium essential oils on 9 gram-positive and 16 gramnegative bacteria. The pathogens that were used can be found in plants and animals, or in
organisms that cause food spoilage or food poisoning. Oils were collected by
hydrodistillation using essential oil distillation apparatus. Bacteria were transferred onto
agar gel plates and slant gel tubes. Bacteria were cultured every two weeks to create
subcultures. The bacteria was cultured and then pipetted onto an agar gel plate. The gel
plates were then incubated for 48 hours and the agar plates were cut into zones and each
zone was measured for growth rate using Vernier calipers; Vernier calipers are used to
get an additional digit of measurement from length.
Dorman et al. (2000) used both gram-negative and gram-positive bacteria in the
study. There findings showed that all essential oils showed some degree of inhibiting
bacterial growth. The findings show that the chemical structure configuration plays a big
part in the effect of the essential oils ability to control bacteria. Although I will not be
able to determine the chemical constituents of the compounds being used, the
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corresponding information as to the control of bacteria by the essential oils used is
conducive to the study that I am doing.
Burt et al. (2003) looked at the effects of essential oils against Escherichia coli
0157:H7. The bacteria was isolated from bovine faeces and maintained on tryptone soya
broth agar slants at 4° C. The inocula were prepared by culturing them in MuellerHinton Broth at 37° for 16 hours. There were five essential oils obtained from C.
Melchers Essential Oils Handels-GmbH, Bremen, Germany. The essential oils were bay
(Pimenta racemosa), clove bud (Eugenia caryophyllata), Oregano (Origanum vulgare)
and red and light thyme oils (Thymus vulgaris).
Burt et al. (2003) used a disc diffusion assay in their test. A 16 hour inocula was
diluted with sterile physiological saline solution [PS; 0.85 % (w/v) sodium chloride], this
is a reference to the McFarland standard which is used in adjusting the density of
bacterial suspension. A 5-ml portion of the inoculum was placed over the agar gel plates
which had been pre-dried and allowed to remain in contact for 1 minute; the excess
inoculum was removed by a sterile syringe. The plates were allowed to dry at room
temperature for 20 minutes. Sterile 6mm filter paper discs were placed on the plates and
a 15μl portion of the essential oil was added to the filter paper discs. The plates were
allowed to sit for 1 hour so the essential oil could diffuse across the gel plates; the plates
were then incubated at 37°C for 24 hours. Each test was carried out three times and the
inhibition zone was measured in millimeters. The control used was a sterile
physiological saline solution, which was 0.85% (w/v) of sodium chloride. This was to
achieve a solution of 106 cfu ml-1. A 5 ml portion of the sample was then placed onto a
dried agar plate and allowed to remain for 1 minute. The excess was then removed with a
8
sterile syringe and the plate was allowed to dry for 20 min at room temperature. The 6
mm filter discs were then placed on the plate and 15 µl of essential oils were added to the
disc. This is an excellent procedural method to use for testing my hypothesis of the
effects of essential oils. It is simple and could be repeated easily, I also believe it would
be cost effective.
I will be able to use the findings in all the previous research studies to help me
compare results in my own research. A comparable analysis will be done to see if the
amount of bacterial control in the essential oils I have chosen to use is equivalent to the
essential oils that have been used previously.
The essential oils in a liquid form will be purchased from Youngs Living Oils,
Utah. Bacteria, both gram-negative and gram-positive will be purchased from Carolina
Science, and will need to be cultured by a broth dilution method. The control groups and
test groups will be cultured by a spread plate method from the broth dilution (Madigan et
al. 2000, Bert et al., 2003). Both control and test population, after having the broth
dilution spread on the agar gel plates will have a sterile filter disk that has been saturated
in either the controls or essential oils placed on the quartered plate, incubated and the
inhibited growth area will be measured. The sample plates will also be compared to the
control group to make sure that it is the essential oils that are destroying the bacteria and
not over colonization of the bacteria or depletion of nutrients. Control plates may need to
be cultured onto new plates to keep the bacteria samples growing.
I hypothesize that essential oils will effectively inhibit the growth of both gramnegative and gram-positive bacteria giving a more natural way to clean and disinfect
surfaces in the home.
9
Specific goals:

To culture a gram-negative (Escherichia coli) and gram-positive
(Staphylococcus epidermidis) bacteria on nutrient agar gel plates.

Culture test population from control groups.

Use essential oils Lemon (Citrus lemon), Orange (Citrus aurantium),
Lavender (Lavandul officinalis) and Thyme (Thymus Vulgaris) to determine
inhibitory effects on test population of bacteria.

Collect data and do statistical analysis on results.
Materials/Methods:
Essential Oils
Essential oils to be used will be purchased from Youngs Living Oils (Utah). The
oils from Youngs have been some of the highest quality I have found. Essential oils that
will be used in this experiment will be Lemon (Citrus lemon), Orange (Citrus
aurantium), Lavender (Lavandul officinalis) and Thyme (Thymus Vulgaris). All essential
oils are already in liquid form and are some of the highest quality that I have found.
Bacteria
Two different bacteria, a gram-negative and gram-positive, will be tested for their
susceptibility to the essential oils (Dorman et al. 2000). A gram-negative bacteria,
Escherichia coli, and gram-positive bacteria, Staphylococcus epidermidis will be
purchased from Carolina Science (Burlington, NC). The bacteria that were selected for
use were chosen do to the ability that they can be handle safely. A control group of each
type of bacteria will be cultured at 37° C for 24 to 48 hours on nutrient agar gel plates per
Caroline Science lab manual.
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Procedures
Plate Preparation:
All plates will be prepared with a nutrient agar gel. The gel will be prepared per
the lab description from Carolina Science. Plates will be allowed to set and solidify prior
to being cultured with bacteria. Plates will be marked by permanent marker and divided
into quadrants on the bottom of the plate to prevent any spillage of the inoculum.
Culturing Bacteria:
Bacteria will be cultured using a broth dilution method. Four test tubes per
bacteria will have a nutrient broth added to them; Escherichia coli will be added to the
first test tube with the nutrient broth in it, using a pipette the mixed contents will be then
subsequently added to each of the remaining test tubes with the nutrient broth in them.
This process will be repeated using Staphylococcus epidermidis. Each of the test tubes
will then be cultured at 35° for 24 hours per the lab manual. Additional test tubes will be
cultured to help maintain the integrity of the bacterial colonies.
Preparing Control Plates:
Eighty agar plates will be cultured for the control group forty each Escherichia
coli (E. coli) and Staphylococcus epidermidis (S. epidermis). For the control groups the
spread plate method will be used; each plate will be divided into quarters with a
permanent marker on the back side of the plate. There will be four different controls
used; one control will have a 6 mm filter disk placed into each quadrant with no oils for
twenty plate, the second control will have a 6 mm filter disk soaked with distilled water
placed in each quadrant for twenty plates, the third control will have a 6 mm filter disk
11
placed in each quadrant soaked with olive oil for twenty plates, and the forth control will
have a 6 mm filter disk placed in each quadrant soaked with corn oil for twenty plates.
Inoculating Test Plates:
Eighty plates will be used for the test population. Forty plates each for E. coli
and S. epidermidis, the spread plate method will be used and the plates will be divided
into quarters with permanent marker on the back of the plate. The test population will
have 50-100 ml of cultured bacteria pipette onto the nutrient agar plate and spread over
the entire plate; the plate will be allowed to sit undisturbed for 1 min to allow the
inoculum to fuse across the plate, the excess inoculum will be syringed off (Burt et al.,
2003).
Preparing Test Plates:
Each test population will contain twenty agar gel plates one set of twenty for each
of the 4 essential oil being used. After the plate has been inoculated and allowed to sit, a
6 mm filter disk will be placed in each of the four quadrants and saturated with 15-20 µl
of the non-diluted lavender essential oil (Burt et al.). Only one type of essential oil will
be used for each plate in each of the four quadrants. After the lavender oil has been
added to each of the four quadrants of all twenty plates, the plates will be allowed to set
for 1 hour to give the lavender oil time to fuse across the plate (Burt et al.). The test
population will then be incubated at 37° C, per laboratory instructions, for 24 to 48 hours
to obtain a proper growth of the bacteria for sampling. This process will be repeated for
the Orange, Lemon, and Thyme essential oils that are being used. This will give twenty
plates for each of the four oils being tested, for a total of eighty plates per bacterium
being tested.
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Culturing Plates:
After incubation the test populations will be observed for growth inhabitation and
measurements in millimeters, using a millimeter ruler, will be taken (Burt et al. 2003).
The test population will be compared to the control groups to ensure proper growth
patterns; plates with no growth will be recultered and the process repeated as mentioned
above. This is to ensure that there will be ample data collected for analysis.
Statistical Analysis
Statistical analysis will be drawn by a comparison of the test population against
the control group for inhibitory growth. A Q-Test will be run for any outlining data that
is collected from each set of test population agar plates; outlining data will be deleted
from the samples collected. Outlining data is any data that is inconsistent with the rest of
the data collected. A Q-Test can be done by calculating the difference between the data
range and the gap in the data. A Q table can be used to check for a 90 % confidence
level. The calculation for the Q-Test is: Qcalculation = gap/range, to determine if the data is
outside the data range Qcalculation > Qtable (Harris, 2007). Then I will be using an ANOVA
test to determine any statistical significance in the data, if there is a difference the Tukey
test will be used to determine where the differences are. Significant differences will have
addition ANOVA test and Tukey test run to determine what essential oils have the largest
inhibitory factors. The significant data will determined by the growth inhibition
measured in millimeters around the growth of inhibition.
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Results:
Tests were conducted on both E. coli and B. cereus with four different essential
oils. Twenty plates were prepared for each of the four oils that were tested per bacteria;
each plate was sectioned into four quadrants and a filter disk was placed in each quadrant
with oil placed on the disk. Measurements were taken in centimeters after 24 hours of
incubation. An ANOVA and Tukey test were run to determine the statistical
significance of the measurements taken. The ANOVA test showed that there was no real
statistical difference between the orange and lemon oils on either the E. coli or B. cereus
bacteria. But there was a significant difference between the lavender and Thyme oils.
Flow Cart
Effects of essential oils on gram-negative and gram-positive bacteria
↓
Prepare nutrient agar gel plates
↓
Culture bacteria in nutrient broth at 37° C for 24 to 48 hours
↓
Transfer bacterial colonies to nutrient agar gel plates for growth test population
↓
Use 15-20 µl essential oils on sterile filter disc for inhibitory control
↓
↓
↓
↓
Lemon oil
Orange Oil
Lavender oil
Thyme oil
↓
↓
↓
↓
Use sterile filter
Use sterile filter
Use sterile filter
Use sterile filter
disk soaked in oil disk soaked in oil
disk soaked in oil
disk soaked in oil
and placed on
and placed on
and placed on
and placed on
quartered agar
quartered agar
quartered agar
quartered agar
plates
plates
plates
plates
↓
Incubate for 24 to 48 hours at 37° C.
↓
Measure test population in millimeters.
14
↓
Run statistical analysis on data collected.
Reference:
Ajose, F. M.D. 2007. Some Nigerian Plants of Dermatologic Importance. International
Journal of Dermatology. 46: 48-55.
Burt, S.A.; Reinders, R.D. 2003. Antibacterial activity of selected plant essential oils
against Escherichia coli 0157:H7. Letters in Applied Microbiology. 36, 162-167.
Dorman, H.J.D., Deans, S.G. 2000. Antimicrobial agents from plants: antibacterial
activity of plant volatile oils. Journal of Applied Microbiology. 88, 308-316.
Eucast Definitive Document. Determination of Minimum Inhibitory Concentrations
(MICs) of antibacterial agents by agar dilutions. Eurpion Socity of Clinical
Microbiology and Infectious Deseases. 6, 509-515
Hammer, K.A., Carson, C.F., Riley, T.V. 1999. Antimicrobial activity of essential
oils and other plant extracts. Journal of Applied Microbiology. 86, 985-990.
Harris, D. 2007. Qunatitative Chemical Analysis. Seventh ed. W.H. Freeman and
Company. New York, NY. pp. 65
HOLY BIBLE, NEW INTERNATIONAL VERSION® NIV® Copyright © 1984
International Bible Society.
Inouye, S.; Takizawa, T.; Yamaguchi, H. 2001. Antibacterial Activities of Essential Oils
and Their Major Constituents Against Respiratory Track Pathogens by Gasesous
Contact. J. Antimicrob. Chemother. 47: 565-573.
López, P., Sánchez, C., Batlle, R., Nerín, C. 2007. Vapor-Phase Activities of
Cinnamon, Thyme, and Oregano Essential Oils and Key Constituents against
Foodborne Microorganisms. J. Agric. Food Chem. 55, 4348-4356.
15
Madigan, M.T., Martinko, J. M., Parker, J. 2000. Brock Biology of Microorganisms.
ninth ed. Prentice Hall, Inc. New Jersey, pp. 141-144
Maxwell, J.A. Reader’s Digest. 1984. America’s Fascinating Indian Heritage. second
ed. Reader’s Digest Association, N. Y. pp. 89
Vardar-Ünlü, G., Candan, F., Sökmen, A., Daferera, D., Polissiou, M., Sökmen, M.,
Dönmez, E., Tepe, B. 2002. Antimicrobial and Antioxidant Activity of the
Essential Oil and Methanol Extracts of Thymus pectinatus Fisch. Et Mey. Var.
pectinatus (Lamiaceae). J. Agric. Food Chem. 51: 63-67.
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