Antibacterial Effects Of Different Essential Oils Biology Essay

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The project will involve an investigation into whether the following essential oils: lavender oil, tea tree oil, rosewood oil, chamomile oil and almond oil have any antibacterial properties against different strains of bacteria since these oils are frequently used in numerous skin products such as soaps and moisturising creams; I want to investigate whether they have any actual effect on common skin bacteria or whether they are just used for fragrance and aroma, they are also used in cosmetics, perfumes, food and drink. For decades these essential oils have been used as traditional remedies for various medical conditions as a natural plant extract, some individuals believe essential oils act as natural antibiotics. Previous research and published work on essential oils have found that "the majority of oils showed antibacterial activity against the tested strains.[1]" Another study found that essential oils were susceptible to both Gram-positive bacteria such as Staphylococcus aureus and Gram-negative bacteria such as Escherichia coli [2].

The essential oils mentioned above will be obtained in the purest form possible. The disc diffusion method will be used for screening antibacterial activity of each essential oil. Test organisms will be grown in a nutrient broth culture and these include: Staphylococcus epidermis, Staphylococcus aureus, E.coli and Corynebacterium sp. When a filter paper disc soaked with a chemical is placed on agar the chemical will diffuse from disc in to the agar [3]. If an organism placed on the agar does not grow in the area surrounding the disc it is said to be susceptible to the chemical. Many conditions can affect a disc diffusion susceptibility test. When performing these tests certain things are held constant so only the size of the zone of inhibition is variable. Conditions that must be constant from test to test include the agar used, the amount of organism used, the concentration of chemical used, and incubation conditions (time, temperature, and atmosphere).

All media and materials should be sterilised and prepared at least one day before procedures. The bacterial inoculums and disc soaking will all be standardised. As essential oils are generally lipophilic (oil-loving) compounds that are often unable to mix with water [4], in this experiment they will be diluted in solvents such as aqueous dimethyl sulfoxide and Tween 80. I will be using Müeller-Hinton agar in my methodological approach for this project as it is considered to be the best for routine susceptibility testing of non fastidious bacteria. The purpose of the disc diffusion susceptibility test is to determine the sensitivity or resistance of pathogenic aerobic and facultative anaerobic bacteria to various antimicrobial compounds. 

Disc Diffusion Test:

Preparation of Müeller-Hinton Agar:

Müeller-Hinton agar should be prepared.

Immediately after autoclaving (121˚C for 15 mins), allow it to cool in a 45 to 50°C water bath.

Pour the freshly prepared and cooled medium into glass or plastic, flat-bottomed petri dishes on a level, horizontal surface to give a uniform depth of approximately 4 mm. This corresponds to 60 to 70 ml of medium for plates with diameters of 150 mm and 25 to 30 ml for plates with a diameter of 100 mm.

The agar medium should be allowed to cool to room temperature and, unless the plate is used the same day, stored in a refrigerator (2 to 8°C), pH of the MH agar should fall between 7.2 and 7.4 at room temperature after solidification.

5. Plates should be used within seven days after preparation [5].

Preparation of inoculums:

Using a sterile inoculating loop or needle, touch four or five isolated colonies of the organism to be tested.

Suspend the organism in 2ml of sterile saline.

Mix thoroughly the saline tube to create a smooth suspension, using vortex making sure that no solid material from the colony is visible

Use this suspension within 15 minutes of preparation.


Obtain broth culture of one of the organism to be tested.

Using a sterile pipette transfer 0.1ml of broth culture containing the organism onto Mueller-Hinton agar plate, then spread evenly on to the surface of each agar plate using a sterile spreader for a lawn of growth.

Allow the surface of the agar plates to dry for about 5 minutes, no more than 15 minutes at room temperature.

Each essential oil will be dissolved in 10% aqueous dimethyl sulfoxide (DMSO) with Tween 80 (0.5% v/v for easy diffusion) and then 50µL of different concentrations (1:1, 1:5, 1:10) of the essential oils will be coated on a separate sterile filter paper disc and then dried. Sterile distilled water will be used as a negative control [3].

Place the impregnated discs on the surface of the agar by obtaining individual discs and placing them at least 24 mm from each other and 10 mm from the edge of the plates, using flame sterilised forceps. Sterilize forceps by dipping the tips in 95% ethanol and igniting the ethanol by rapid passage through a flame. Touch each disc on the plate to ensure complete contact with the agar surface. Replace the lid of the plate between discs to minimise exposure to air-borne contaminants. Do not move a disc once it has contacted the agar surface even if the disc is not in the proper location, because some of the drug begins to diffuse immediately upon contact with the agar. Avoid placing discs close to the edge of the plate as the zones will not be fully round and can be difficult to measure. No more than 8 discs on each agar plate. Label agar plates appropriately.

Once all discs are in place, replace the lid, leave to dry for 20 minutes at room temperature then invert the plates, and incubate at 37ËšC for 18 hours.

Dilution susceptibility testing will involve determining the minimum amount of essential oil that inhibits the visible growth of an isolate or minimum inhibitory concentration (MIC). The varying bacteria will be subjected to a range of dilutions of essential oils. The highest dilution of antibiotic (essential oil) that has inhibited the growth of bacteria is considered as MIC. These tests can be done on either agar or broth media. The Minimum Bactericidal Concentration (MBC) is the lowest concentration of antibiotic required to kill a particular bacterium [6].

Agar dilution method:

A serial two-fold dilution of each oil should be prepared in Mueller-Hinton agar at 48ËšC and also add 0.5% Tween 20, before inoculating allow plates to dry for 30 minutes at room temperature. Using a wire loop inoculate a volume of 0.001-0.002 ml and spread over a small area on the surface of agar, the culture should be diluted to contain 105 to 106 organisms per ml, then incubate at 37ËšC overnight. Experiments will be carried out in triplicate. The lowest concentration of essential oil that inhibits visible growth on surface of agar is taken as MIC and compared to blank control plates [7].

Statistical Analysis

The diameters of zones of inhibition will be measured to the nearest mm using a ruler or calliper (if accessible at university) and the mean value will be calculated as the experiment will be done in triplicate, results will be interpreted using standard deviation.

T-test will also be calculated to understand the significant differences between the antimicrobial effectiveness of each oil. MIC (minimum inhibitory concentration), MBC values (minimum bactericidal concentration) will be compared.

Ingredients needed for nutrient broth [8] :

Peptone - 5g

Beef extract/yeast extract - 3g

NaCl (sodium chloride) - 5g

distilled water

pH 7 at 25 °C

Equipment and Laboratory Requirements:

x1 Bunsen Burner

x1 Sterile Inoculating loop

x1 Incubator

x5 different essential oils (see names above)

x80 Petri dishes

x2 Sterile pipettes

x4 Sterile tubes

Saline solution

Ingredients needed for Müller-Hinton agar [9] :

Beef Extract - 2g

Acid hydrolysate of casein - 17.5g

Starch - 1.5g

Agar - 17g

pH 7.3 at 25 °C

x1 Sterile forceps

x120 Sterile blank discs 6mm

Sterile distilled water

x20 Sterile plastic spreaders

x40 Mueller Hinton agar plates

x1 Test tube rack

x1 Vortex

x1 Ruler or calliper

95% ethanol in beaker

10% aqueous dimethyl sulfoxide (DMSO)

0.5% Tween 80



Study Location

Practical to be undertaken

Pre-session or after session


On-site at university campus

Obtain all 5 essential oils, get any final forms filled out with the help of supervisor and discuss any potential problems with lab technicians.

Get final method prepared and ready for lab practicals


On-site at university campus in laboratory

Prepare nutrient broth, Mueller-Hinton agar and grow bacteria.

Incubate overnight


On-site at university campus in laboratory

Standardise the bacterial inoculums and then carry out disc diffusion test.

Incubate discs in agar plates overnight


On-site at university campus in laboratory

Measure zone sizes and record data.

Take note of any problems occurred


On-site at university campus in laboratory

Carry out dilution susceptibility test- Agar dilution. Record data (MIC and MBC)

Start write up of results, note any problems


At home

Carry out necessary statistical analysis and calculations

Finish write up of results


On-site at university campus in laboratory

Repeat same procedures with any adjustments and improvements made as a result of any problems faced in previous weeks. Prepare nutrient broth, Mueller-Hinton agar and grow bacteria.

Incubate overnight


On-site at university campus in laboratory

Carry out disc diffusion test.

Incubate discs in agar plates overnight


On-site at university campus in laboratory

Measure zone sizes and record data.


On-site at university campus in laboratory

Carry out dilution susceptibility test- Agar dilution. Record data (MIC and MBC)

Write up of results, compare with original results obtained


Probably on-site at university

Leave week free to catch up with any work or just incase of absence in a previous week


At home

Start write up of Discussion

Start completing final project work

In second Semester

On-site at university

If lab practicals need to be repeated with any amendments then do so in the same order and format as previous lab sessions outlined

Important Ethical and Health and Safety Considerations:

For this project category 2 bacterial strains will be used in the laboratory.

All chemicals and organisms will be used in minimum amounts and at minimum concentrations as possible; this experimental design is the best possible one. This project work will not be harmful to me, the environment or those around me; we will be protected from any harm. There will be no participants required for this project experiment. All materials should be disposed of correctly once used.