The Antibacterial Effects of Essential Oils, Lemon (Citrus lemon), Orange (Citrus aurantium), Lavender (Lavandul officinalis) and Thyme (Thymus Vulgaris) Against Gram-Negative 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

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 agentsfor 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 (Maxwell1984).

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).

Hammer et al. (1999) conducted a study using52 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 byboth 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 48hours 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.

Lópezet al. (2007) used three essential oils in a vapor-phase method to test for the antibacterial activities of these oils. Ina 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 comparingto 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 thebacteria. 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 gram-positive 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 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 theEastAegeanIslands. 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 conductedutilized 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 incorporatedis 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.

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)provedto 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 9gram-positive and 16 gram-negative bacteria. The pathogens that were used can be found in plants and animals,or inorganisms that cause food spoilage or food poisoning. Oils were collected by hydrodistillation using essential oil distillation apparatus. Bacteria weretransferred 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 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 Mueller-Hinton 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 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 formwill 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 havea 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 inhibitthe growth of both gram-negative and gram-positive bacteria giving a more natural way to clean and disinfect surfaces in the home.

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 epidermidiswill 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.

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 usingStaphylococcus 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 methodwill 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 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).