The Effects of Varying Tea Tree Oil Concentrations on Staphylococcus aureus Growth
Yasmin Ghaemmaghami, Nicole Peters, Stevie White
Department of Biological Sciences
Saddleback College
Mission Viejo, CA 92692
Staphylococcus aureus has become increasingly resistant to antibiotics. As a
result, new methods are being explored to find approaches to fight against antibiotic
resistant S. aureus. One approach to fighting against S. aureus is by using essential oils like
tea tree oil. The objection of this study is to determine the minimum concentration of TTO
needed to inhibit S. aureus growth. There could be a statistical difference in the amount of
Staphylococcus aureus in the pure nutrient broth versus the Staphylococcus aureus in the
nutrient broth combined with the Tea Tree Oil at various concentrations. Fifty test tubes
were placed into five different groups based on the TTO concentration used in the
experiment: control, 3, 1, 0.5, 0.25 (% v/v). A spectrophotometer was used to measure
absorbance and percent transmittance of bacteria growth in different tea tree oil
concentrations. Absorbance and percent transmittance were used to calculate optical
density, which correlated to the amount of bacteria present The mean optical density for
the control was 0.0105 ± 0.003582 (±SEM), 0.25 M of TTO was 0.0303 ± 0.0058 (±SEM),
0.50 M of TTO was 0.0243 ± 0.004973 (±SEM), 1.0 M of TTO was 0.0247 ± 0.004028
(±SEM), and 3.0 M of TTO was 0.025 ± 0.004624 (±SEM). There was no significant
difference (p=0.055, single factor ANOVA) between any of the five groups. If the variation
within the four experimental groups had been closer to each other, then there would have
been a higher likelihood of there being a statistical difference.
Introduction
Staphylococcus aureus, known to cause cutaneous infections, has become increasingly
resistant to antibiotics forming dangerous strands like methicillin-resistant S. aureus (MRSA)
(Ansari et al. 2012; Leszczyńska et al. 2010). S. aureus is a bacteria commonly found on the skin
of the human body (Harris et al. 2002). However, S. aureus can cause health issues like
abscesses and superficial lesions if the bacteria enters the underlying skin (Ansari et al. 2012;
Harris et al. 2002). This occurs when the skin is breached due to cuts, surgery, or other causes
(Ansari et al. 2012; Harris et al. 2002). The threat of antibiotic-resistant S. aureus has led to the
exploration of new antibacterial agents (Ansari et al. 2012; Leszczyńska et al. 2010).
Currently, antibiotics are being tested to determine how effective they are against
antibiotic resistant S. aureus. Katarzyna Leszczyńska from Medical University of Biaystok,
Biaystok, Poland assessed “…the antibacterial activity of amoxicillin with clavulanic acid
(AMC), tetracycline (T), erythromycin (E) and amikacin (AN)… against different clinical
isolates of Staphylococcus aureus in combination with synthetic LL-37” (Leszczyńska et al.
2010). Synthetic LL-37 is the antimicrobial protein cathelicidin LL-37, a natural antimicrobial
molecule made by the body that can kill bacteria cells by negatively effecting bacterial cell walls
(Leszczyńska et al. 2010). Results suggested that “AMC was strongly potentiated when added in
combination with LL-37” (Leszczyńska et al. 2010). Though these antibiotics could provide
optimal treatment, the researchers “…did not observe any decrease in the MIC values of T and E,
particularly against methicillin-resistant S. aureus…in the presence of LL-37” (Leszczyńska et
al. 2010). MIC is Minimal Inhibitory Concentration used to determine the minimal concentration
of antibiotics needed to inhibit bacterial growth (Leszczyńska et al. 2010). But, effectiveness of
antibiotic drugs are questionable due to antibiotic resistance (Edwards et al. 2004). This is only
an example of the difficulty of finding antibiotics able to properly inhibit antibiotic-resistant
bacteria.
Commercial antiseptics have also been explored as methods to inhibit bacterial growth.
Elaine L. Larson and Barbara E. Laughon from the School of Nursing and Division of Infectious
Disease of John’s Hopkins University compared four antiseptic products containing gluconate on
bacteria growth on the skin (Larson and Laughon 2010). Various participants were given
different CHG antimicrobial soaps to test their efficacy on killing bacteria on the skin (Larson
and Laughon 2010). The study suggests there was no statistical difference between the CHG
products in their inhibition of bacterial growth on the skin, meaning that all the four CHG
products were effective in inhibiting general bacteria growth (Larson and Laughon 2010). While
certain commercial products are still widely used, the fear is that there could be a day when S.
aureus could become resistant to all antibacterial drugs and products (Edwards et al. 2004).
Essential oils have been acknowledged for their antimicrobial properties (Carson et al.
2002; Bassole & Juliani 2012). Generally, essential oils, like geranium and lavender, attack the
cell membrane of bacteria and cause the cell to lyse (Carson et al. 2002). These essential oils
have various components responsible for the oils’ medicinal effects (Edwards et al. 2004). One
of the most widely researched oils that have the most potential of inhibiting bacterial growth is
tea tree oil.
Tea tree oil, extracted from the leaves of Melaleuca alternifolia through steam
distillation, has been suggested to have antimicrobial effects (Carson et al. 2002; and Raman and
Bloomfield 2012). Tea tree oil and its components work by compromising the cytoplasmic
membrane of bacterial cells through various processes; certain methods include inducing the cell
to lose essential ions and causing a negative effect on cellular respiration (Carson et al. 2002).
Tea tree oil also had the largest zone of inhibition when compared to other oils such as patchouli,
lavender, geranium, and Citricidal (Edwards et al. 2004). A zone of inhibition shows the
potency of how well a substance’s antimicrobial properties have affected the organism’s growth.
Since S. aureus has been known to cause many different types of infections, using tea tree oil as
a natural way to treat and prevent these infections, as well as being a natural preservative, is
certainly desirable (Herman et al. 2013).
Studies have reported that S. aureus is susceptible at 0.5% TTO (Hammer et al 1999) or
concentrations greater than 2% TTO (Carson et al. 2006) in MIC testing (Minimum Inhibitory
Concentration). Therefore in this study concentrations of 3, 1, 0.5, 0.25 (% v/v) are utilized,
since these values are above, below, and in between the range of 0.5% and 2%. The objection of
this study is to determine the minimum concentration of TTO needed to inhibit S. aureus growth.
The implication of this project will hopefully provide better clarification in the amount of TTO
needed to inhibit S. aureus growth. There could be a statistical difference in the amount of
Staphylococcus aureus in the pure nutrient broth versus the Staphylococcus aureus in the
nutrient broth combined with the Tea Tree Oil at various concentrations.
TM
Materials and Methods
Fifty 0.5 mL samples of Staphylococcus. aureus and one 59 mL bottle of De La Cruz Tea
Tree Oil were used in the study. The S. aureus was provided to the researchers by Saddleback
College Biology Department and the tea tree oil was purchased at Walgreens in Mission Viejo,
California. Fifty test tubes were filled with 10 mL of Criterion nutrient broth (lot number 12185).
On October 14, 2015, tea tree oil dilution factors were prepared. Four test tubes filled
with 10 mL of sterile deionized water were provided to researchers by the Saddleback College
Biology Department. Using aseptic technique, each test tube was pipetted with one of the
following amounts of De La Cruz TTO: 300, 100, 50, and 25 microliters. The ratio of the TTO to
the 10 mL of DI water correlates to the TTO concentration used in the experiment: 3, 1, 0.5, 0.25
(% v/v). Each test tube was then gently shaken to uniformly mix the DI water and the TTO.
A P-1000 micropipette was used to pipette 500 µL of S. aureus into each test tube that
had the previously prepared sterile nutrient broth using aseptic technique. After the transfer of S.
aureus each tube was swirled to distribute S. aureus. The tubes were labeled into five groups:
control and four experimental groups based on different TTO concentrations.
A Beckman Coulter DU 730 Spectrophotometer was used to measure the absorbance and
percent of transmittance at 532 nm. A P-1000 micropipette was used to pipette 1 mL out of each
tube into 1.5 mL disposable polystyrene PMMA cuvettes. A cuvette filled with 1 mL of nutrient
broth only was used as a blank. The initial absorbance and the percent of transmittance values
were recorded.
A P-200 micropipette was used afterwards to aseptically pipette four sets of tubes (n=10)
with 0.05 mLs of TTO with the following concentrations: 3, 1, 0.5, 0.25 (% v/v). All test tubes
were incubated at 37°C for 48 hours. At 9:20 AM on October 16, 2015 the fifty test tubes were
removed from the incubator to measure bacterial growth of each final group.
A P-1000 micropipette was used to pipette 1 mL out of each test tube into 1.5 mL
disposable polystyrene PMMA cuvettes. Each set of test tubes (n=10) had a different blank, a 1:1
ratio of TTO concentration used for the particular set and Criterion nutrient broth were placed
into the cuvette to be used as the blank.
The final absorbance and the percent of transmittance values were recorded in the lab
notebook. Optical density was determined with the following formula OD = (2-LOG
(%Trans))/abs; optical density was used to quantify the amount of bacteria in solutions. Once
calculated, the final optical density of each tube within all five groups was subtracted by its
initial optical density. A single factor ANOVA was run to determine if there was a significant
statistical difference between the concentrations of each group.
Results
The mean optical density for the control was 0.0105 ± 0.003582 (±SEM), 0.25 M of TTO
was 0.0303 ± 0.0058 (±SEM), 0.50 M of TTO was 0.0243 ± 0.004973 (±SEM), 1.0 M of TTO
was 0.0247 ± 0.004028 (±SEM), and 3.0 M of TTO was 0.025 ± 0.004624 (±SEM). A single
factor ANOVA test was used to determine if there was significant statistical difference between
all five groups containing different concentrations of tea tree oil. There was no significant
difference (p=0.055, single factor ANOVA) between any of the five groups (figure 1).
Figure 1. Comparison of the mean optical densities between five groups. Error bars are mean ±
SEM. There was not a significant statistical difference among any of the five groups (p=0.055,
single factor ANOVA).
Discussion
The premise was that there is a there could be a statistical difference in the amount of
Staphylococcus aureus in the pure nutrient broth versus the Staphylococcus aureus in the
nutrient broth combined with the Tea Tree Oil at various concentrations. Since studies have
reported that S. aureus is susceptible at 0.5% TTO (Hammer et al. 1999) or concentrations
greater than 2% TTO (Carson 2006) in MIC testing (Minimum Inhibitory Concentration),
concentrations of 3, 1, 0.5, 0.25 (% v/v) were utilized. The reason is 3, 1, 0.5, 0.25 (% v/v)
concentrations are above, below, and in between the range of 0.5% (Hammer et al 1999) and 2%
(Carson 2006). The objection of this study was to determine the minimum concentration of TTO
needed to inhibit S. aureus growth. TTO is a great inhibitor of S. aureus due to the high numbers
of the optical densities. However, Tea Tree Oil did not inhibit S. aureus significantly enough to
demonstrate a difference between the control and the various concentrations.
The greatest inhibition was 0.25% TTO concentration. The three other test groups (0.5,
1.0,3.0%) were relatively close to each other according to Figure 1. However, there was no
statistical difference between the control with S. aureus and the four experimental groups.
A possible reason may have been due to data variation. There was not a lot of variation in
the data. If there was even less variation within the data, there may have been a statistical
difference. Sample size may have been an issue. Although data suggests that there wasn’t a large
variation, more samples in the group may have decreased the variation further.
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