Growth Inhibition of Escherichia coli. by Essential Oils of Rosemary (Rosmarinus officinalis) and Lavender (Lavandula augustifolia)

Scott Lilly and Jordan Meek

Department of Biological Sciences

Saddleback College

Mission Viejo, CA 92692

This study was undertaken to ascertain whether a statistical difference existed in growth inhibition of Escherichia coli, E coli. between Rosemary (Rosmarinus officinalis) and Lavender (Lavandula augustifolia) essential oils. We expected statistical significance to be observed regarding growth inhibition between both Rosemary and Lavender in addition to both groups showing significantly more inhibition then observed in control. Two sample groups and one control group containing 10 agar dishes each were plated with E coli. and three equidistantly plated chads immersed in Lavender oil, Rosemary oil, and DI water respectively. Plates were allowed to incubate for forty eight hours and growth inhibition was measured across the chads in millimeters with a ruler. Average inhibition (mm) in the control group was observed to be 0.14 ± 0.07 (± S.E.M.). Average inhibition (mm) in the Rosemary group found to be 2.9 ± 0.41 (± S.E.M.) and in the Lavender group to be 2.5 ± 0.55 (± S.E.M.). Results demonstrated no significant difference in growth inhibition of E. coli between the Lavender and Rosemary groups (p = 0.4205, ANOVA). As expected however, significant differences in inhibition were observed in comparison of both the lavender and control group (p = 0.000032, ANOVA), and the rosemary and control group (p= 0.000302, ANOVA). Results indicate that both Rosemary and Lavender essential oils significantly inhibit E coli. growth but do not vary significantly from each other in terms of inhibition.

Introduction

Aromatic plants, such as Rosemary, Lavender, Oregano, and Thyme have long been valued for the medicinal and aromatic uses of their essential oil extractions. In addition, a number of studies undertaken in the last twenty five years have looked at the antibacterial properties these plants possess. The bird species, blue tit (Cyanistes caerulas) has been found to include pieces of aromatic plants amongst its normal nest building material (Blondel et al. 2009). Upon examination of the effect of these plants on the bacteria present on the blue tits, it was found that the plants significantly altered the structure of the observed bacterial communities specifically in regards to reducing the density of colonies among hatchlings (Blondel et al. 2009). Variability in antibacterial activity has also been noted in these plants due to a variance in concentration of essential oils specifically in regards to ice nucleation active bacteria (Karamanoli et al., 2004). In regards to the effects of these plants on specific bacterial strains, the aromatic plant basil (Ocimum basillicum) has been shown to be effective in inhibiting the growth of E coli. (Lopez et al., 2005).

Relatively few studies have directly looked at the variance in effect that different essential oil extracts from these aromatic plants have on bacterial growth. The objective of this study was to observe the amount of growth inhibition of Escheridia coli. from the application of Lavender and Rosemary essential oils and whether the two differed significantly in their inhibition ability. It was hypothesized that both groups would significantly inhibit bacterial growth and that they would significantly vary between each other in the amount of inhibition.

Materials and Methods

1L of agar medium (Criterion Dehydrated Culture Media) was prepared and autoclaved for forty five minutes. The agar was then distributed evenly to 30 petri dishes and allowed to cool for a period of forty minutes. Three, single hole punch chads of filter paper were prepared for each Petri dish for a total of 90, these were subsequently autoclaved for forty five minutes. 20mL of Escherichia coli, E.coli. (precultured in the microbiology lab a few days prior) was distributed in 0.5mL increments to each dish in a lawn spread using a sterile spreader and standard procedure aseptic techniques, left over E. coli was disposed of properly. The 30 dishes were split into three groups: the control group, the rosemary group, and the lavender group. Three chads were placed on each dish using tweezers. Control group chads were dipped in water, whereas rosemary and lavender group chads were dipped in essential oil extracts of rosemary and lavender respectively. Oil extracts were sterilized via boiling and standard procedure Aseptic techniques were followed in transferring the chads to dish. All groups were incubated for 48 hours at a temperature of 37° Celsius. Upon removal from incubation, growth inhibition was measured as the diameter of E. coli absence across the center of the chads.

Results

Before evaluating the data, the diameter length of the Chad was subtracted from each measured zone of inhibition in order to get an accurate measurement. The average zone of inhibition on E.coli between the three groups was analyzed for comparison. The rosemary group had the greatest average growth inhibition at 2.91 mm ± .414 (± S.E.M) with the lavender group close behind at 2.45mm ± .546 (± S.E.M). As expected the control group had the smallest average, .14 mm ± .070 (± S.E.M)

(Figure 1).

Although the rosemary group had the largest average zone of inhibition, as seen in figure 1, it was found to have no statistical difference when compared to the lavender group (p= 4.2 x 10-1 , ANOVA). However, when the rosemary group was examined against the control group there was a significant statistical difference (p= 3.2 x 10-4, ANOVA). The results were also similar when the lavender group was compared to the control (p=3.0 x 10-4, ANOVA).

Figure 1 - Average growth inhibition (mm) of E.coli of the Lavender group was 2.45 ±

.546 (± S.E.M). The rosemary group was 2.91 ± .415 (± S.E.M) and the control group was 0.14 ± .070 (± S.E.M)

Discussion

The results presented provide evidence that refutes one aspect of the two part hypothesis and supports the other. The average growth inhibition between the Rosemary group and the control group was found to be significant as was the growth inhibition between the Lavender group and control group. This data supports the hypothesis that these particular aromatic plants significantly inhibit bacterial growth and reflect similar results to the study of E coli. inhibition by basil (Lopez et al., 2005).

No statistical difference was found in growth inhibition between the Lavender and Rosemary groups. This provides evidence for the validity of the null hypothesis, that there is not a significant difference in the E coli. growth inhibition ability of these two plants. These results also appear to run counter to the study conducted by Karamanoli et al., 2004 which found that variances in concentration of essential oils caused significant differences in antibacterial activity (Karamanoli et al., 2004). The differing results could potentially have been caused by human error in this study. Namely, chads may not have been uniformly coated in their respective oils due to a density difference amongst the two sample groups. (If one was more dense, when the immersed chad was shaken to remove excess oil prior to plating, less might have dripped off causing the two groups not to have equal representation). Also, while sterilizing the essential oils, the Rosemary oil was found to come to its boiling point faster then did the Lavender oil which could have skewed the results.

This experiment looked at the effects of two of these aromatic plants on a single strain of bacteria’s growth. Further experiments could employ a wider range of essential oils and a larger sample size to better ascertain if/how these aromatic plants differ in their effect on E coli. growth. In addition, a single essential oil could be used and tested against a variety of bacterial strains to see if the inhibitory effect is widespread to limited to E coli.

Literature Cited

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Karamanoli, K., Vokou, D., Menkissoglu, U., Constantinidou, H.-I. “Bacterial Colonization of Phyllosphere of Mediterranean Aromatic Plants”. Journal of Chemical Ecology (2004): 2035-2048

Lopez, P., Sanchez, C., Battle, R., Nerin, C. “Solid- and vapor-phase antimicrobial activities of six essential oils: suscepitibility of selected foodborne bacterial and fungal strains.” J Agric Food Chem (August 2005); 53(17):6939-46

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