ANTIBACTERIAL EFFECTIVENESS OF AROMATIC BOTANICAL ESSENTIAL OILS

ABSTRACT

Aromatic botanical essential oils are very concentrated extracts, naturally derived from surrounding plants. These plants are good sources of bioactive compounds with antiseptic and antimicrobial properties. In this experiment, the following essential oils are utilized: Tea tree oil, Penicillin, Eucalyptus oil, Cinnamon oil, Peppermint oil and Almond oil. This study explores the practical application of scientific methodology by testing the antibacterial effectiveness of these plant essential oils on Gram-Negative and Gram-Positive bacteria samples which were set to grow on lysogeny broth agar plates. Additionally, antimicrobial susceptibility testing, done by the disc diffusion method is thoroughly elaborated on, with emphasis on the results collected. The purpose of the disc diffusion method is to test the species of bacteria (Gram-negative and Gram-positive), over the nutrient-enriched agar plate. Generally, for results, predictions state that if the zone of inhibition exerts the following behaviours, then its effectiveness is stated as shown: 6mm= not antibacterial, >6mm-16mm =slightly antibacterial and >16mm= highly antibacterial.

INTRODUCTION

`This experiment is based on determining the antibacterial effectiveness of 6 different types of oils, by measuring the growth of bacteria (zone of inhibition). Therefore, the immediate purpose of this study is to investigate the antibacterial effectiveness of aromatic botanical essential oils, by measurement of its zone of inhibition, that is, the extent of its bacterial growth. These bacteria may be separated into Gram-Positive bacteria and Gram-Negative bacteria. Gram-Positive bacteria creates a purple colour appearance when observed under a light microscope, which is due to the retention of the purple crystal violet stain on the thick peptidoglycan layer of its cell wall, (Stewart, 2019). However, with regards to Gram-negative bacteria, this bacteria displays a pink colour appearance, when observed under a light microscope, which is due to the counterstain, safranin that is retained in the membrane, (Man, 2019).

In fact, gram-positive bacteria are more sensitive to herbal extracts than gram-negative bacteria. This may be because of Inherent tolerance of gram negatives and the nature and composition of herbs. According to the studies, the cell walls of gram-positive bacteria compared with gram-negative bacteria, are more sensitive to many of anti-boutiques, antimicrobial chemical compounds and even many herbal drugs. Lipopolysaccharides layer and periplasmic space of gram-negative bacteria are the reasons of relative resistance of gram-negative bacteria.

For results, predictions state that if the zone of inhibition exerts the following behaviours, then its effectiveness is stated as shown: 6mm= not antibacterial, >6mm-16mm =slightly antibacterial and >16mm= highly antibacterial. Therefore, the hypothesis of this experiment states that in this activity, Cinnamon Oil is the most effective aromatic botanical essential oil, and this is most successfully evidenced by use of Gram-positive bacteria.

METHOD

Two agar plates, two metal bacteria spreaders, two beakers with ethanol, an ethanol lamp, micropipettes (set to 130 microliters), 2 cotton swabs, a ruler and a permanent marker were obtained. Each agar plate contained a different sample of bacteria (either Gram-positive or Gram-negative), in order to avoid cross contamination of the bacteria. Each petri dish was then labelled as either Gram-negative or Gram-positive. The agar plate was also kept closed when not in use. Next, the Petri dish loaded with lysogeny broth (LB) agar was turned side up. Then, with a permanent marker and ruler, the dish was marked with a straight line from the centre to the outer edge. By rotating 60 degrees, 6 equal-sized wedge sections were drawn and numbered sections 1-6. The LB agar was inoculated by using a micropipette (130 microliters of the assigned liquid bacterial culture) and the Petri dish lid was replaced. After, a metal bacteria spreader was sterilized by dipping it in a beaker with ethanol and quickly passing it over the flame of the ethanol lamp. Thirty seconds passed until the spreader was cooled. Additionally, the spreader was placed lightly on the LB agar, which allowed it to be parallel to the lines drawn on the plate. The plate was moved in order to spread the bacteria evenly throughout the agar. The spreader was then placed back into the beaker with ethanol and the dish was left undisturbed, agar side down for 5 minutes. At the table, there was a set-up of 1 forceps, 1 beaker with ethanol, sterile filter paper discs, 1 ethanol lamp, as well as a vial of essential oil. Afterwards, the forceps was dipped into the ethanol beaker and passed over the flame of the ethanol lamp, in order to sterilize it. Thirty seconds passed for the forceps to cool. Then, the forceps holding the filter paper disc, was dipped into the vial of the essential oil and pressed against its walls as means of removing any excess liquid. By using a cotton swab, the disc was pressed on the center of the corresponding wedge of agar. The agar plate was then closed. The above-mentioned procedure was then done for the remaining 5 other essential oils. Ultimately, after the lid was placed on the agar plate, the agar side down was left undisturbed for 5 minutes. After the five minutes, the Petri dish was labelled and placed into an incubator, set at 37 degrees Celsius for 48 hours. After Incubation, the Petri dishes were stored at 4 degrees Celsius for one week, (Pitzer, 2019).

RESULTS

Figure 1

As shown above, “Figure 1” is a visual representation of the results obtained for Cinnamon Oil. Here, the red colored bars indicate the results of 3 plates for Gram-Positive bacteria, while the yellow plates indicate the results for 3 plates with Gram-Negative bacteria. In both cases, the pink bars display the averages of the results from the 3 plates, respectively.

Figure 2

As shown above, “Figure 2” is a visual representation of the results obtained for Eucalyptus Oil. Here, the red colored bars indicate the results of 3 plates for Gram-Positive bacteria, while the yellow plates indicate the results for 3 plates with Gram-Negative bacteria. In both cases, the pink bars display the averages of the results from the 3 plates, respectively.

Figure 3

As shown above, “Figure 3” is a visual representation of the results obtained for Tea tree Oil. Here, the red colored bars indicate the results of 3 plates for Gram-Positive bacteria, while the yellow plates indicate the results for 3 plates with Gram-Negative bacteria. In both cases, the pink bars display the averages of the results from the 3 plates, respectively.

Figure 4

As shown above, “Figure 4” is a visual representation of the results obtained for Almond Oil. Here, the red colored bars indicate the results of 3 plates for Gram-Positive bacteria, while the yellow plates indicate the results for 3 plates with Gram-Negative bacteria. In both cases, the pink bars display the averages of the results from the 3 plates, respectively.

Figure 5

As shown above, “Figure 5” is a visual representation of the results obtained for Peppermint Oil. Here, the red colored bars indicate the results of 3 plates for Gram-Positive bacteria, while the yellow plates indicate the results for 3 plates with Gram-Negative bacteria. In both cases, the pink bars display the averages of the results from the 3 plates, respectively.

Figure 6

As shown above, “Figure 6” is a visual representation of the results obtained for Penicillin. Here, the red colored bars indicate the results of 3 plates for Gram-Positive bacteria, while the yellow plates indicate the results for 3 plates with Gram-Negative bacteria. In both cases, the pink bars display the averages of the results from the 3 plates, respectively.

DISCUSSION

The purpose of this study is to investigate the antibacterial effectiveness of aromatic botanical essential oils, by measurement of its zone of inhibition, that is, the extent of its bacterial growth. My predictions state that if the zone of inhibition exerts the following behaviours, then its effectiveness is stated as shown: 6mm= not antibacterial, >6mm-16mm =slightly antibacterial and >16mm= highly antibacterial. Cinnamon Oil is evidently seen to be the most effective essential oil, with regards to its activity on Gram-positive bacteria.

Cinnamon oil is produced by the Cinnamoumum Cassia tree, primarily extracted from the leaves and bark and later refined into an essential oil. According to (Healthline, 2019), this botanical essential oil is primarily used as an anti-inflammatory, antioxidant as well as mood enhancer. Evidently, Cinnamon oil possessed the most constant results, with only one plate showing an abnormal figure. Generally, the oil displayed a high average of 34.67mm towards being antibacterial. Against Gram-negative bacteria, it showed an average zone of 33.125mm and against gram positive bacteria the average was 34.67mm, hence it shows that this oil is a great option as an antiseptic. Cinnamon bark oil’s antibacterial properties allows it to be termed as a safe and effective alternative that can be used for the preservation products on grocery shelves in order to increase their shelf life, (Zahiri, 2015). However, there are also limitations to this essential oil. According to the American Cancer Society, Cinnamon oil has a lot of strong effects on the body, in that, if it is utilized too frequently, the individual has a high risk of skin cancer, commencing with symptoms of skin rashes and burning.

Furthermore, Eucalyptus oil is investigated. It is an oil that is extracted from the eucalyptus plant. This oil is used mainly as a natural deodorant, perfume, cough suppressant, soothes cold sores and repeals bugs but not too widely used for its antibacterial properties. The results show that the eucalyptus oil showed a zone to gram negative bacteria of 19.625mm and towards gram positive bacteria it showed a zone of 8.67mm. As we can see eucalyptus oil is a good antibacterial towards gram negative bacteria but not too well against gram positive.

Tea tree oil is used as a natural sanitizer, insect repellent, natural deodorant, and as an antiseptic for minor cuts and wounds. As we test tea tree oil against bacteria, we can see that tea tree oil is very good as an antiseptic. The results show that the average zone displayed by the tea tree oil towards gram positive 19.333mm and against gram negative, there was an average zone of 22.125mm. As we can see the tea tree oil makes a substance to fight bacteria wherever it is applied.

Almond oil is extracted from the almond nut and it is used most commonly for skin care and to help stabilize blood sugar in the body. Within our experiments we can see that almond oil is not a very antibacterial oil. The results showed that the average zone for almond oil against gram negative bacteria was 4.25mm and against gram positive bacteria there was an average zone of 4.67mm. After analysing the results, we can see that almond oil is not very antibacterial.

This oil can be extracted from the leaf of the peppermint plant, this plant can be found all across

North America and Europe.

Peppermint oil is usually used to treat irritable bowel syndrome, nausea, common colds, itch relief, muscle ache, and a verity of soaps and cosmetics, (Healthline, 2019). As we experimented with the oil we can infer that the peppermint oil was very effective on gram negative bacteria but not as effective on gram positive, even though it was very antibacterial all around, there was a sure difference between the two types of bacteria. As shown in our results the peppermint oil showed an average zone of 15.5mm towards the Gram- positive bacteria and a zone of 30.5mm towards gram negative. After viewing the averages, we can see that peppermint oil is highly antibacterial towards gram negative bacteria.

Finally, Pencillin was investigated in this experiment. This essential oil was one of the first medications to fight bacterial infections and is still widely used to this day, as shown on www.AmericanChemicalsociety.org. As we experimented with penicillin, we witnessed a trend throughout our results. As a collective group we experienced that this oil was very effective to gram positive bacteria but not very effective against gram negative bacteria. The average size of the zone of inhibition with penicillin towards the Gram-positive bacteria 39.5mm and the average towards the gram negative was22mm. Even though by the standards of what is highly antibacterial or not, over 16mm is highly antibacterial, we can see the drastic difference in averages of the zone from the gram negative and Gram-positive bacteria. Gram-negative (penicillin can’t get to the cell wall. Hence, it’s not effective.) The gram-positive would be more effective.

Even though several studies were performed in vitro to assess antibacterial and antifungal properties of EOs, only few studies reported on their bioactivity in vivo; food components (fats, carbohydrates, proteins, salts) and pH could reduce the antimicrobial effects of EOs in food systems. In fact, the same effect observed in vitro is achieved in food matrix only with higher concentrations (Tyagi et al., 2014).

Most effective>

-cinnamon oil, peppermint oil

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