Topic: To study the effect of varying concentrations of Tea Tree Oil on bacterial growth of E.

coli to
investigate the antibacterial properties of tea tree oil.

Personal engagement:
Growing up, I was heavily influenced by my mother’s constant affection towards essential oils. one thing
that stood out to me the most was the different applications of Tea Tree Oil. It intrigued me into
researching more about it. There are numerous functions of tea tree oil, it can be used as cleaning agents,
or diffusing in the air to kill mold. Moreover, tea tree oil can be used in many occasions aiming to achieve
different purposes, but most of the effects were to kill or decrease the growth of different microorganisms.
Upon my findings, I noticed that Tea Tree Oil contains antibacterial properties. Keeping the different
methods of applications in mind, I wanted to find out more about the effects of the antiseptic properties
when the concentrations were altered.

Research Question: What effect will varying tea tree oil concentrations have on the growth of E. coli by
measuring the diameter of the zone of inhibition surrounding the paper discs?

Tea Tree Oil:

Figure 1: Tea Tree Oil
Tea Tree Oil, which is derived from Melaleuca alternifolia, was
long used as a topical antiseptic1. Tea Tree Oil can be applied to
the skin for infections such as acne, and it is also used as a local
antiseptic for cuts and abrasions, for burns, insect bites. Some
people add it to bath water to treat cough, bronchial congestion,
and pulmonary inflammation.

In previous studies, Tea tree oil has been shown to have broad-
spectrum inhibitory activity2 against numerous bacteria and other
organisms. In gram-negative bacterium E. coli, tea tree oil was
found to disrupt the permeability of cell membrane structures,
stimulate leakage of cellular potassium ions and inhibit
respiration with lethal effects, which may explain tea tree oil’s
antimicrobial activity.

The major active antimicrobial components of Tea Tree Oil

consist of over 100 components, including α-pinene, terpinen-4-ol, linalool and α-terpineol3. It stimulates
the leakage of cellular potassium ions and inhibits respiration in E. coli cell suspensions, providing
evidence of a lethal action related to cytoplasmic membrane damage.

1 The mode of antimicrobial action of the essential oil of Melaleuca alternifolia (tea tree oil)
2 Effects of tea tree oil on Escherichia coli
3 Effects of tea tree oil on Escherichia coli
Escherichia coli:
E.coli4 is a type of bacteria that lives in the digestive systems of
humans and animals. It belongs to the family of Enterobacteriaceae
where most of them are harmless. Despite so, there are still some that
can cause bloody diarrhea, severe anaemia or kidney failure, all in
which can lead to death. In some other cases E. coli can also cause
urinary tract infections. Majority of them are restricted to the intestine
even though they are able to survive outside for a brief period of time.

Figure 2: E. coli

E.coli is a gram negative bacterium5. The Gram-

negative ‘has a peptidoglycan layer that is 2–3
nm thick, which is thinner than in the cell wall
of Gram-positive bacteria, and composes
approximately 20% of the dry weight of the
cell’6. The outer membrane contains porin
proteins that enables hydrophilic solutes to pass
through.

Figure 3: Cell membrane of Gram-Negative and Gram-Positive Bacteria7

The purpose of this investigation is to learn about the antibacterial properties of Tea Tree Oil and how it
may inhibit the growth of E Coli. And how potent tea tree oil solutions need to be in order for to be
effective, are lower concentrations enough? Or higher concentrations are needed to have effective results.

Hypothesis
1. Tea tree oil will have an effect on reducing bacterial growth.
2. Increasing concentration of tea tree oil will have increasing effectiveness in reducing bacterial
growth as measured by a ruler ±0.1𝑐𝑚.

4 "What is E. Coli?" WebMD. Accessed October 29, 2017

5 "Gram Staining." Microscopy. November 03, 2016. Accessed October 29, 2017.
6 Effect of Essential Oils on Pathogenic Bacteria
7 https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3873673/figure/pharmaceuticals-06-01451-f001/
Variables:
Table 1: To identify the need of controlling the experiment
Control Variable
The temperature the agar plates
were kept in
The concentration of nutrients in Nutrients are needed in bacterial
agar plates
The bacteria cultures
The type of paper for the test
disks
The number of streaks of E.coli The more the streaks of bacteria,
when culturing the agar plates
Total volume of oil mixture
Significance of the variable
To maximize bacterial growth,
the agar plates containing E.coli
should be kept in optimum
temperature for bacteria growth.
growth. The higher to
concentration of nutrients in the
agar plate, the more rapid the
bacteria reproduce and therefore
are more likely to spread
throughout the agar plates.
Different species of bacteria
may react differently to Tea
Tree Oil, thus affecting the
reliability of the experiment.
Different type of paper might
have different permeability and
it might resulting in different
rate of diffusion of Tea Tree Oil
to the agar
the more bacteria can be
cultured. It will consequently
lead to different numbers of
colonies present on the agar
plate. A variation in the
intensity? Of the colonies.
To keep the concentration ratio
of Tea Tree Oil equal, thus
increasing validity of the results.
How can it be controlled
All plates cultured with E.coli
were be kept in the incubator at
25℃ to grow for 24 hours.
The concentration of nutrients in
the agar plates were kept
constant throughout the
experiment to ensure higher
validity of the results.
Ensure only E.coli were cultured
by choosing the correct visual
characteristics.
Keep the type of the papers used
constant, sticking to only one
brand.
Keep the number of streaks
constant across all plates to
ensure validity.
This was controlled by mixing a
total of 10 cm3 mixture each
time. This was to ensure that the
concentration variation of tea
tree oil are consistent, increasing
validity.

Table 2: identify the uncontrolled variables

Uncontrolled Variable
Temperature
The purity of the Tea Tree Oil
The significance of the variable
This was at room temperature which was at an approximate constant
of 29 ̊C so that the growth of E.coli is not affected by the
surrounding temperature.
The experiment is to test out the antibacterial properties of Tea Tree
Oil

Independent:
Concentrations of the tea tree oil solution achieved by mixing Tea Tree Oil and corn oil to create different
concentrations to test the effect of different concentrations on the inhibition of E. coli.
Corn oil was used as it has no antibacterial properties and can act as a neutral liquid for dilution.
Dependent:
The inhibitory effects of Tea Tree Oil by observing the clear zone formed surrounding the paper discs
(Zone of Inhibition) measured using a ruler ±0.1𝑐𝑚.

Materials and Apparatuses

The sign ‘-’ signifies not needed or does not have one
Table 3: the list of materials and apparatuses needed for the experiment

Name of apparatus Quantity Uncertainty

Test tube with bungs 2 -

Test tube rack 1 -

Bunsen burner 1 -

Filter paper discs 50 -

Saline solution 50ml 0.9M -

Forceps 2 -

Disposable rubber gloves 1 -

Inoculating loop 2 -

Ruler 1 ±0.1𝑐𝑚

Agar plates (prepared by the 5 -

school’s science technicians)

Tea tree oil 1 bottle (30ml) -

Corn oil 50ml -

Autoclave 1 -

Incubator 1 -

Refrigerator 1 -

Safety
Non-pathogenic E. coli were used in this experiment to avoid any infections to the human body.
Despite being found in the human body; E. coli should not have contact with skin as they are contagious.
Thus, lab goggles, lab coats and rubber gloves must be worn at all times when handling with E. coli to
avoid any direct contact with the bacteria.
According to aseptic methods, a Bunsen burner should be kept on safety flame throughout the duration of
the experiment. Hair should be tied up to avoid being burnt and also safety goggles should always be kept
on to avoid scorching the eyes.
Aseptic method.
1. Wipe working area with ethanol in 30 seconds interval to sanitize.
2. Put on gloves when working and remove when finish handling with E. coli both to prevent
bacteria from skin contaminating the culture and also prevent E. coli from spreading.
3. Keep Bunsen burner on with safety flame to kill airborne bacteria.
4. Keep lab coat on at all times when working in the lab.
5. Put on goggles to prevent flame from burning eye, and to prevent E.coli contact.
6. Autoclave to sterilize every possible equipment used.
7. Burn the inoculation loop until bright red and wait 30 seconds to cool down before scooping
E.coli. That is to kill any unwanted bacteria on the tool, and cooling it down before contact with
E.coli is to prevent killing E.coli.

Sub-culturing
Scrape a small amount of E. coli off the agar slope of a culture using a sterile inoculating loop. shake the
loop in a small volume of sterile nutrient broth

Method
1. E. coli was sub-cultured in test tubes of 0.9M saline solution which was pre-inoculated few days
prior to experiment.
2. Agar plates prepared by school science technicians, and inoculated at the day of use.
3. Tea Tree Oil is mixed with corn oil to achieve desired concentrations in a beaker previously
autoclaved. For 0%, 5 ml of corn oil is used, 25%, 1 ml of Tea Tree Oil mixed with 4 ml of corn
oil, 50% will have 2.5 ml of both, for 75% add 4 ml of Tea Tree Oil to 1 ml of corn oil, and for
100% use 5 ml of Tea Tree Oil.
Commercially prepared Tea Tree Oil with concentration of 100% is used in this process.

4. About 10 filter paper disks (mm) were placed in beaker and was left soaking in the solutions for a
few hours. Seal the beakers to avoid any contaminations by air borne bacteria.
5. Using sterilized forceps, pick out the filter paper discs and place on a previously sterilized white
tile to remove excess and place onto the bacteria culture in petri dishes.
6. Petri dishes were kept in the incubator for 48 hours at 20°C.
7. After 48 hours, petri dishes were removed from the incubator and diameter of inhibition is
measured using a ruler(±0.01cm).

Data processing
Qualitative data
- First batch of subcultures got contaminated and there were random colonies of bacteria together
with E. coli.
- More bacteria colonies can be seen in concentrations 0, 25, and 50 compared to concentrations 75
and 100, indicating more bacteria growth suggesting the effect of low concentrations of tea tree
oil on bacteria
Table 4: Raw data, the diameter of the zone of inhibition (ZoI) around each paper discs, measured by a
ruler (±0.1cm)
Concentration Diameter of zone of inhibition
of TTO
(%)
1 2 3 4 5

0 0.00 0.00 0.00 0.00 0.00

25 0.00 0.00 0.00 0.00 0.00

50 0.00 0.13 0.00 0.00 0.00

75 13.17 8.0 6.33 8.17 (0.00)

100 14.32 10.33 (0.00) 8.50 15.00

*The data from the above table is the mean calculated through 6 measurements of the diameter at
different angle. The data(s) in brackets have suspicion of being anomaly as it does not follow the trend
observed and therefore will be excluded from data processing.

1. Calculating the mean of the data collected:

Example: For concentration of 75%, the result for trial 5 seems irregular, assumed to be an anomaly thus
left out from calculation. Number of trials: 4
∑x 𝑠𝑢𝑚 𝑜𝑓 𝑑𝑎𝑡𝑎 𝑓𝑟𝑜𝑚 𝑒𝑎𝑐ℎ 𝑡𝑟𝑖𝑎𝑙 13.2 + 8.0 + 6.3 + 8.2
=( )= = = 8.9𝑚𝑚(2𝑠. 𝑓. )
𝑛 𝑛𝑢𝑚𝑏𝑒𝑟 𝑖𝑠 𝑡𝑟𝑖𝑎𝑙 4

2. Calculation of Standard deviation:

Example: For concentration 75%. Excel spreadsheets are used as it allows an instant calculation of
standard deviation, therefore the excel spreadsheet would be used to calculate the standard deviation
through the formula =STDEV
13.172 +8.002 +6.332 +8.172
=√ 4
− (8.92)2 = 42.95

Table 5: Average diameter of Zone of Inhibition(ZoI) in each concentration.

Concentration of TTO (%) Mean diameter of ZoI (in mm) Standard Deviation

0 0.00 0.00

25 0.00 0.00

50 0.03 0.06

75 8.92 2.95

100 12.03 3.13

Figure 2:

Graph showing the average diameter of ZoI in different

concentrations of TTO
16
Average diameter measured by a
14
12
ruler(±0.001mm)

10
8
6
4
2
0
-2 Mean
Concentration of TTO (%)

𝑢𝑛𝑐𝑒𝑟𝑡𝑎𝑖𝑛𝑡𝑦 𝑜𝑓 𝑖𝑛𝑠𝑡𝑟𝑢𝑚𝑒𝑛𝑡
Percentage uncertainty (%) = 𝑚𝑒𝑎𝑠𝑢𝑟𝑒𝑑 𝑣𝑎𝑙𝑢𝑒
× 100

Example: Data collection for diameter of zone of inhibition uses a ruler that can measure up to 1 decimal
places, thus the uncertainty is 0.1cm. Average zone of inhibition for tea tree oil concentration 75% is
0.1
=(8.9×4) ×100 = 0.2808988764%

Conclusion
Tea tree oil can be used as an efficient antiseptic medium in our daily lives. The higher the concentration
of the tea tree oil will supposedly have a better antibacterial effect. The aim of this experiment, which is
visible from my research question, was to explore the antibacterial effects of tea tree oil on the growth of
E. coli by measuring the diameter of the zone of inhibition surrounding the paper discs and how will the
effect be different if the concentration of tea tree oil is varied. It was predicted that tea tree oil possesses
antibacterial effects and as the concentration of the tea tree oil increases the Zone of Inhibition
surrounding the filter paper disc will be larger.

My experiment supported the hypothesis where, tea tree oil will have an effect on reducing bacterial
growth, and that can be seen through the presence of the clear ring around the paper discs, as seen in
concentration 50%, 75% and also 100%. The increasing concentration of tea tree oil had an increased
effect in reducing bacterial growth. This can be observed from the increasing measurements of the Zone
of Inhibition in concentration 50%, 75%, and 100%. The absence of clear ring around the paper discs in
concentration 0% and 25% suggests that lower concentrations of tea tree oil was not affective in
inhibiting bacteria growth.

As concentration of Tea Tree Oil increases, the larger the clear zone surrounding the paper discs,
suggesting higher inhibition of bacteria. This is might be because tea tree oil acts as an inhibitor of E. coli
and disturbs cell structure. Hence, the by increasing the concentration of Melaleuca alternifolia the rate of
respiration of will decrease. This can be seen from the data in Table 5 and Figure 2. When in
concentrations 0% and 25%, no inhibition can be seen, while in 50%, the average diameter of the clear
zone was 0.03. In 75% there is a clear zone with the mean of 8.92mm and 100% with a Zone of Inhibition
of 12.04mm.

The results show that Tea Tree Oil is effective for inhibiting bacterial growth but only when in high
concentrations. However, the data is considered not reliable due to the high standard deviation values that
I obtain while excluding the anomalies. Moreover, the error bars seen in Figure 2 suggest the results were
not statistically significant. Although the experiment was carried with full aseptic procedures,
contamination still occurred to the pilot experiment.

While I was successful in identifying the positive correlations between the increase in concentration of tea
tree oil and the antibacterial properties, observed through the increase in the average diameter of the zone
of inhibition surrounding the paper discs.

Table 7: Evaluation Table

Systematic Errors

Weakness Significance of error Way(s) of improvement

Uncertainties when measuring
the zone of inhibition of tea tree
oil on bacteria with a ruler
As the uncertainties determine Measure the diameter along the
the percentage uncertainty of the circumference of the clear zone
whole investigation, the and take the average diameter of
reliability of the investigation the
depends on the percentage
uncertainty of the instruments
used.

Number of trials Less trials often have more
errors and by increasing number
of trials, a more obvious trend
can be observed and anomalies
can be excluded.
Carry out more trials so that
errors can be minimised and
easier identification of
anomalies.
Table 7 continued
Weakness
The delay between opening the
bung of the test tube and
submerging cotton swab in the
culture.
Excess Tea Tree Oil on the
paper discs
Weakness
Inaccurate reading of the ruler’s
value when measuring the
diameter, in cm, of the zone of
inhibition around the filter paper
discs due to parallax error.
Contamination in the incubator
Possible Expansions to the Experiment
Due to the small range of bacteria species used, it would be rather difficult to generalize the antibacterial
properties of tea tree oil. It weakens the support towards H1 as the relationship between the Zone of
Inhibition and Tea Tree Oil is not studied broadly. Therefore, by increasing the variety of bacteria, a
Random Errors
Significance of error
The delay might increase
chances of contamination as
period of direct contact to the air
is increased, leading to possible
air borne contaminations.
Excess oil will diffuse out from
the paper onto the agar plates
causing inequality in the starting
point of the Zone of Inhibition,
consequently leading to errors
when measuring the diameter of
the clear ring surrounding the
paper discs.
Human Errors
Significance of error
The angle caused misreading of
values when measuring the
diameter, in cm. Human
judgement is not accurate
enough; it includes factors such
as the angle of reading and the
estimation in decimal places etc.
This will make the data collected
inaccurate.
As the experiment was held in
the school and equipment are to
be shared with others, the
incubator might be contaminated
with other’s experiment which
leads to the bacterial
contamination.
Way(s) of improvement
During the process of culturing
the bacteria, everything must be
done quickly with sterilized
equipment. Or run the opening
of the test tube over fire to force
out any unwanted bacteria
before sealing with a bung.
Leave soaked paper discs on
filter papers to absorb excess tea
tree oil before adding to the agar
plate. Ensure the work place is
constantly disinfected to prevent
contamination.
Way(s) of improvement
Taking multiple measurements
along the circumference of the
paper disc and deduce an
average value.
Arranging a timetable to have
set times when using the
incubator
wider range of data can be generated. Since E.coli is a gram negative bacteria8, it would be interesting to
test out the effects of Tea Tree Oil on gram positive bacteria and find correlations between gram positive
and negative bacteria.

Additionally, by increasing the bacterial population, the data generated would be more reliable as more
trials were used to determine the presence of a trend and the identification of anomalies would be easier.
Another possible expansion I think would be to add different types of essential oil that might or might not
possess antiseptic properties to have a comparison between different types of oils. There is a huge variety
of essential oils and some might have better antiseptic properties than others. Doing that can allow more
data to be generated and find out other essential oil that potentially have better antibacterial properties.

Further research is needed to have a better understanding of the exact tea tree oil induced killing event in
bacteria and also to determine whether tea tree oil will have a similar effect on other microorganisms.

8
"Gram Staining." Microscopy. November 03, 2016. Accessed October 29, 2017.
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