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The Effect of essential oil of Oregano (Origanum vulgare) on the in vitro growth of
the bacteria Escherichia coli, Salmonella typhimurium, and Staphylococcus aureus
Eric T. Rueda
Department of Biological Sciences
Saddleback College
Mission Viejo, CA 92692
Abstract
Oregano oils a very potent source of the antimicrobial substance Carvacrol. Three
bacteria were exposed to this natural active chemical; Escherichia coli, Salmonella
typhimurium, and Staphylococcus aureus.They were incubated at 37.0°C and data
was collected by subtracting the total area of inhibition (including the area occupied
by the chad) and subtracting the area of the oregano (Origanum vulgare) oil dipped
chad. Results were calculated as the average area of inhibition and graphed
however they did not stand up to par with previous experiments. The growth of the
bacteria was mildly effected by the presence of the oregano oil.
Introduction
Our bodies are in a constant state of attentive and destructive microscopic
responses, fighting to achieve homeostasis. These functions will continue regardless of
the macroscopic environment that we consciously expose ourselves to. The body thrives
under control of the brain and its collective use of complex systems, such as the nervous,
lymphatic, and digestive systems. Electrical and chemical messages relay from exterior
or interior stimuli and the body will react accordingly.
In the case of an unwanted microbe or bacterium within our body, the immune
system works to destroy this parasite by surface markers, cytokines, phagocytosis, or
antibodies (Campbell et al., 2008). Multiple cells and tissues are affected by these
changes; such as if the body has no memory of the parasite, then an acquired immunity
develops. This allows for cells to retain information of the parasite so that it can be
destroyed faster and more efficiently. Our body is good at managing a broad spectrum of
infection, however, when a parasite reproduces too rapidly within our body or it is
extremely unfamiliar, we may be working to catch up to it to destroy it. If this is the case,
then we seek the aid of medicine.
Medical practice has developed and grown exponentially over an incredible
amount of time. Dating back to before 1400 B.C. (McCall et al., 2003), society made use
of simple, yet misunderstood blood letting techniques to remove excess bile. Currently,
the procedure of a standard infection follows that a prescription medication is
administered to the patient in hope, and through multiple trials and experience, that the
parasite will be vanquished. Prescription medication is used regularly and has become a
multibillion dollar industry (Wechsler, 2010). Through private investors and government
funding, this field targets FDA approval of microbiological based medicines. Scientists
draw inspiration from nature to fuel cognition and fabricate new medications and medical
techniques. Over time, plants and fungus have adapted to extreme environments,
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developing abilities to keep them free of certain microbes. In particular, the oregano plant
(Origanum vulgare) (Morris 2005) makes use of two phenol compounds: Carvacrol
(C10H14O) and Thymol (C10H14O) (Preuss et al. 2005). The main body constructed in a
benzene ring with different appendage hydroxyl groups. Carvacrol, specifically, has been
shown through many in vitro experiments to be a strong antibacterial compound
(Nutraceuticals, 2001; Preuss et al., 2005; Ott et al., 2008; Sarac et al., 2008; Erickson,
2009; Wong et al., 2008; Patrone, 2010). Carvacrol shows repeated antimicrobial activity
against related bacteria found in animals and plants, such as strains of Aspergillus,
Streptococcus, Bacillus, and Candida. These concentrations are as low as 0.625 mg/mL
(Can Baser, 2008). Carvacrol inhibits bacteria by creating an ionic imbalance on the cell
membrane by allowing K+ ions to leak from the cell resulting in unstable cytoplasmic pH
levels and osmotic pressure. This membrane now leaks ATP due to the breakdown of the
cellular membrane leaving the cell unable to function. Carvacrol will also inhibit ATPase,
the enzymatic active-transport pump located on the cellular membrane which functions to
regulate proton distribution between the cytoplasm and extracellular fluid. (Campbell et
al., 2008) With such a long standing and well documented history of antimicrobial
qualities, the objective of the study was to examine if oregano oil would inhibit the
growth of three bacteria: Escherichia coli, Salmonella typhimurium, and Staphylococcus
aureus. It is hypothesized that the growth of the bacteria would be greatly affected the
presence of the oil.
Materials and Methods
Forty nutrient agar plates were prepared using Difco nutrient agar. Three bacteria:
Escherichia coli, Salmonella typhimurium, and Staphylococcus aureus, were cultured and
provided by the Saddleback Biology Department. Sterile water was used as the control.
Supplies and methods were completed using aseptic technique; gloves and eye
shield were dawned and all materials used were from sterile packaging or have been
autoclaved. Serological pipettes were used to transfer 0.3 mL of E. coli to the nutrient
agar plates. A glass rod spreader was used to distribute the bacteria evenly around the
petri dish. After each use, the glass spreader was dipped in 95% ethanol, passed through a
Bunsen burner flame before being used on the next agar plate. The procedure was
repeated for S. typhimurium, S. aureus, and water control plates.
Three chad disks were dipped in the oregano oil and placed onto each agar plate
using forceps. The brand Nature’sAnswer alcohol-free oregano oil, purchased from The
Vitamin Shoppe in Mission Viejo, California, was combined with olive oil and contained
active ingredient Carvacrol at 7.0 mg per 0.2 mL of oil. This dropper bottle was
purchased from The Vitamin Shoppe in Mission Viejo, California. Forceps were dipped
in 95% ethanol and passed through a Bunsen burner flame before handling each disk. All
agar plates were incubated at 37.0°C for 48 hours at Saddleback College.
Data was collected by measuring the entire diameter of the chad and area of
inhibition. Measurements were taken using a metric ruler (millimeters) and values were
recorded to the tenths. Area of inhibition of one chad disk was calculated by subtracting
the chad disk diameter, 7.0 mm, from the inhibition diameter. The summation of these
values was then divided by the total number (n=30) to obtain the mean diameter of
inhibition. Statistical test ANOVA was run comparing the bacterial growth to each other.
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Results
The oregano oil had a similar effect on the mean area of inhibition on each
bacteria. ANOVA statistical test was run comparing the area of inhibition of all bacteria
to each other. A significant P-Value of 1.04E-09 was found between the groups. The
Bonferroni Correction Test with MS Residual of 14.63211111 and DS Residual of 3.0,
and a confidence interval of 95% was run to compare each bacteria to each other.
Compared to one another the bacteria showed no statistical significance, with a P-Value >
0.05 (Figure 1).
Figure 1. The comparison of inhibition of growth of bacteria (n=30)
Staphylococcus aureus, Escherichia Coli, and Salmonella
typhimurium against oregano (Origanum vulgare) oil (Carvacrol at
7.0 mg per 0.2 mL). Control was water and showed zero inhibition.
P-Value was statistically insignificant (> 0.05) (Bonferroni
Correction Test) and bacteria are graphed with a 95% confidence
interval. S.aureus standard error of 0.158832, E.coli standard error of
0.172239, and S.typhimurium standard error of 0.229767.
Discussion
The expected results are not supported by the outcome of the collected data.
Through previous studies such as Can Baser (2008) and Wong (2008), oregano oil
inhibited the growth of these three bacteria significantly. Factors that may have caused
such a great deviation from the normal results are largely thought to be due to the low
concentration of Carvacrol in the sample tested. A small range of inhibition was due to
the presence of Carvacrol, however this does not stand to say that it had a significant
effect on the growth of the three bacteria Staphylococcus aureus, Escherichia Coli, and
Salmonella typhimurium.
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