Im sorry sir but I'm afraid your cat has contracted curiosity.
Introduction
In today’s modern society, many of us wouldn’t think twice about downing a couple of pills of Panadol
and antibiotics the moment we feel slightly under the weather, or asking the doctor for extra tablets just so
we can stay at home for one extra day. Due to the availability and versatility of antibiotics, many people
often consume them even when they don’t actually need to. Over the years, this has led to the
development of antibiotic resistance of some bacteria. Therefore, the need arose for the investigation into
natural remedies has risen because of their direct and wide ranging effects on pathogens without bearing
some of the negative side effects. Plants produce a wide variety of compounds and metabolites used as a
defense mechanism against invasive pathogens; as such these compounds also exhibit medicinal
properties. Thus this investigation intends to explore the antimicrobial efficacy of cinnamon essential oil
Motivation for study
As a teen going through puberty, I often suffer from the odd pimple or acne. Trying several different
home remedies, I found that natural substances seem to be more soothing for the skin compared to creams
and other ointments. Thus this piqued my interest into research of natural essential oils which inhibit
bacterial growth and promote healthier skin. The bacteria which causes acne is P. acnes, however for this
investigation two species of bacteria, E. coli and B. subtillus will be used to model to effects of cinnamon
essential oil on P. acnes as both species of bacteria have individual similarities to P. acnes.
As such my research question is:
What is the effect of varying concentrations (0.1, 0.2, 0.4, 0.6, 0.8 and
0.9)% of Cinnamomum verum (cinnamon) essential oil on the growth of Escherichia coli (E. coli)
and Bacillus subtilis (B. subtillus) as measured by spectrophotometry at 600 nm, keeping the
temperature, incubation time, volume of tween 20 and nutrient broth solutions constant?
Background information
Cinnamon essential oil (Cinnamomum zeylanicum)
Cinnamon is a spice harvested from the inner bark wood of a variety of tree species belonging to the cinnamomum genus1 . The essential oil of cinnamon is produced through the steam distillation of cinnamon bark. Its unique aroma is derived from its essential oil which comes from the organic compound cinnamaldehyde which makes up more than 90% of cinnamon bark essential oil. Cinnamaldehyde mainly functions to inhibit microbial growth; hence high concentrations of cinnamaldehyde in cinnamon bark essential oil makes it an effective inhibitor of antimicrobial growth for a wide range of pathogens2 . The cinnamaldehyde in cinnamon bark essential oil acts by inhibiting cell division and cell wall synthesizing enzymes. It also inhibits the function of Beta-(1,3)- glucan synthase and chitin synthase by acting as a non-competitive inhibitor. At high concentrations, cinnamaldehyde causes the depolarization and increased permeability of cellular membranes, causing leakages of cytoplasmic material which leads to the bacteria cells dying.
E. coli bacterium
3 E. coli are gram negative, anaerobic, rod like bacteria that are most commonly found in the digestive tracts of mammals. E. coli have a cell wall containing a thin lipopolysaccharide layer and a peptidoglycan layer along amongst other structural features which differ from gram positive bacteria. These features allow E. coli to better cope and survive under bad or stressful condition which would be lethal towards gram positive bacteria. Furthermore, E. coli are capable of complex metabolism despite its simple structure to maintain higher rates of cellular growth and division. 4 Similarly, P. acnes bacteria are also gram negative; hence it will have a similar cell wall and membrane structure to E. coli. This makes it more likely that P acnes will have a similar response as E. coli to cinnamon essential oil.
B. subtillus bacterium
B. subtillus are gram-positive bacteria that are also rod shaped. Relative to E. coli, B. subtillus have a much thicker peptidoglycan layer constituting both carbohydrates and amino acids. In contrast to E. coli, B. subtillus do not have a lipopolysaccharide layer. Furthermore, they are strictly aerobic, hence cannot undergo cell division and growth in the absence of oxygen6 . Moreover since B. subtillus is a common cause of foot odor among people, hence the findings from this investigation can be carried over and applied for skin care remedies and act as a treatment for acne by inhibiting the growth of bacteria P. acnes.
Antimicrobial Mechanism
The membrane structure of bacterial cells plays an integral role for their survival. Their cell membrane constitutes a phospholipid bilayer that regulates cell homeostasis through the use of proton pumps and protein channels. Cinnamaldehyde 7 contains aldehyde groups which contain reactive sites that covalently bond to amine groups in DNA molecules, hence disrupting their normal cell function. The depolarization of the membrane structure by high concentrations of cinnamaldehyde causes the leakage of important materials found in the cytoplasm as well as the loss of enzymes needed to catalyze metabolic reaction. 8 If the bacterial cells are exposed to cinnamaldehyde for prolonged period of time, this will eventually lead to death. Furthermore, lower concentrations of cinnamaldehyde in cinnamon bark essential oil have been found to inhibit the functions of ATPase9 . As such this investigation seeks to investigate the antibacterial efficacy of varying concentrations of cinnamon bark essential oil
Hypothesis
It is hypothesized that an increase in concentration of cinnamon essential oil will lead to a decrease in growth of E. coli and B. subtillus as higher concentrations are lethal to bacterial cells. As such higher concentrations of cinnamon essential oil should be exponentially more effective at inhibiting bacterial growth. Furthermore, Due to the inherent difference in cell structure, it is hypothesized that the cinnamon essential oil will be more effective at inhibiting bacteria growth of B. subtillus as compared to E. coli.
Table of chemicals and apparatus
Methodology
Preliminary trials
I initially used 48 hours for incubation allowing E. coli growth to take place. However after testing this out and collecting one set of replicates, I realized that 48 hours was too long as most of the % transmission readings were similar in value, indicating that the inhibitory effects of cinnamon essential oil on bacterial growth has reached its maximum. Furthermore the % transmission readings were also above 3 arbitrary units suggesting that there was too much bacteria present. Hence in light of this and upon consulting literature sources on ideal incubation periods by Sharmin Siddique Bhuiyan11, I settled on 18 hours of incubation which gave ample time for the bacteria to grow whilst not completely negating the inhibitory effects of cinnamon essential oil. During preliminary trials I also tried to find a suitable range for concentration of cinnamon essential oil to be used in order to best show its antimicrobial properties. To begin with, I used 100 to 200 micro liters of cinnamon essential oil. However upon collecting the first set of replicates I realized that this concentration was too high. Since high concentrations of cinnamon essential oil were lethal to bacteria, there was very little growth seen in the E. coli and their % transmission readings were also very closely related. By progressively decreasing the % concentration of essential oil by lowering the volume added and increasing the volume of water, I arrived at the suitable range from 10 to 90 micro liters which allowed the E. coli growth while also producing a suitable trend line. The following method below shows the final procedure used after reflecting on the preliminary trials.
Preparation of master plate
1. 15g Agar powder and 1 litre water were measured out and poured into a reagent bottle.
2. The bottle was autoclaved to kill all contaminants. Ethanol wipes and a Bunsen burner were used to ensure aseptic conditions during inoculation.
3. A sterile petri dish was half filled with agar solution and left to harden for 30 minutes.
4. E. coli samples from the lab technician were used to inoculate the agar plate.
5. This was left at room temperature for 24 hours to allow time for the E. coli master plate to grow.
6. After 24 hours, the E. coli master plate was placed in the lab refrigerator to prevent further growth.
7. The same procedures were also carried out for B. subtillus master plate.
Experimental procedures
1. E. coli liquid cultures were prepared by inoculating 10cm3 of sterile Nutrient Broth in a multipurpose bottle from the prepared master plate.
2. The culture was left to incubate for 24 hours before use in data collection.
3. Using a micro pipette, 10 micro liters of 0.1% tween solution, 10 micro liters of cinnamon essential oil, and 500 micro litres of E. coli liquid culture 1400 micro liters of nutrient broth and 90 micro liters of deionized water were added into an eppendorf tube.
4. The mixture was incubated for 18 hours in the lab at 25 oC.
5. After 18 hours had elapsed, 1 cm3 of the mixture is transferred into cuvettes and their % transmission was obtained.
6. Since the bacteria in the solution scatters light, more bacteria would thus produces a higher % transmission rating as more light is scattered.
7. This procedure was repeated with 10, 20, 40, 60, 80 and 90 micro liters of cinnamon oil and 5 replicates were obtained for each concentration.
8. From this we can plot a graph of % transmission against concentration of cinnamon used and obtain the relationship between bacteria growth and cinnamon. Thus obtaining the optimal concentration of Cinnamon needed to inhibit bacterial growth.
9. The same procedure was repeated, using B. subtillus in place of E. coli
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All the above procedures were carried out under aseptic conditions to prevent air borne microorganisms
from contaminating the broth.
Control experiment
The same procedure from the experiment was used for the control, replacing cinnamon essential oil with deionized water. The E.coli and B. subtillus were incubated for 18 hours at 25oC before their % transmission was taken. The purpose of this was to show that changes in the % transmission reading comes from the increasing concentration of cinnamon essential oil which inhibits bacterial growth and not limited by other factors such as amount of nutrient broth.
Qualitative observations
Quantitative data collection
Standard deviation is given to 4 decimal places for meaningful comparison In general the standard deviations remained fairly constant throughout experimentation. No general pattern could be observed in the standard deviation with increasing concentration of cinnamon essential oil which is to be expected as each replicate of bacteria would grow with slightly differing rates. Overall the Standard deviations were observed to be lesser for B. subtillus as compared to E. coli. This is also within expectation since by referring to table 1 and 2; B. subtillus had lesser growth then E. coli. The relatively low standard deviation suggests a low deviance in results between replicates to their mean. This serves to show that the replicates collected were similar. Since the data collected were reliably and replicable, this adds credibility to the methodology applied.
Table of formula
A bar graph was used over data points to better show the drop in % transmission between each data point. A bar graph was chosen over a line graph as it allowed for clearer comparison of difference in % transmission between E. coli and B. subtillus, particularly at higher concentrations of cinnamon essential oil where it would be difficult to distinguish between the data points for E. coli and B. subtillus.
The errors bar represent ± 1 Standard deviation for the 5 replicates collected Anova two factors and Tukey post-hoc using Microsoft Excel software were used to conduct statistical testing and determination of significant difference.
Statistical test
The data collected for E. coli and B. subtillus appeared to show a significant difference in their % transmission as seen in figure 6 where the difference is % transmission is greater than the error bars. Furthermore there also seems to be significant difference between each data point for E. coli and B. subtillus. Hence ANOVA two factor test was used to test the significance of differentiation between all the groups.
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Null hypothesis H0= there is no significant difference between the results for E. coli and B. subtillus
Alternative hypothesis Ha = there is significant difference between the results for E. coli and B. subtillus
The calculated p-value during comparison of essential oil concentrations was determined to be 6.1 X 10-38 . As the critical p-value tolerance is 0.05, the calculated p-value is smaller than the tolerance number. Thus the null hypothesis is rejected and the alternative is accepted, being there is significant variation in results between E. coli and B. subtillus.
Tukey post-hoc
A Tukey post-hoc test was conducted between each concentration of cinnamon essential oil for each type of bacteria respectively. This tests for significant difference between two specific concentrations of cinnamon essential oil for each type of bacteria. The Pcritical is taken to be 0.05. Null hypothesis H0= there is no significant difference between the results for E. coli and B. subtillus Alternative hypothesis Ha = there is significant difference between the results for E. coli and B. subtillus
Discussion
Graphical analysis
With reference to figure 10, initial analysis shows that there is an apparent inverse relationship between concentrations of cinnamon essential oil bacteria growth. This corroborates with the initial hypothesis that increasing concentrations of cinnamon essential oil reduces bacteria growth. Analyzing the graph further, there appears to be a general linear trend line between concentration and bacterial growth. However by analyzing both graphs closely, higher concentrations of cinnamon essential oil, 0.6 to 0.9% for B. subtillus and 0.8 to 0.9% for E. coli, showed negligible change in % transmission and began to plateau.
Whilst comparing the two graphs, a noticeable gap can be observed between each data point as the % transmission for B. subtillus is consistently lower than E. coli with a difference ranging from 0.388 to 0.012. Thus seems to show that cinnamon essential oil have varying effectiveness depending on the species of bacteria chosen. Thus the results collected seem to indicate that cinnamon essential oil is more effective at inhibiting B. subtillus bacterial growth compared to E. coli. This may be attributed to the structural differences between the two species of bacteria. A study by Siddiqua S on the antibacterial activity of cinnamaldehyde on food pathogens showed similar results and revealed that cinnamaldehyde was more effective at inhibiting gram positive bacteria species like B. subtillus 12 . Furthermore, the relatively insignificant difference in % transmission between the two bacteria at concentrations 0.8% and higher reflect that the maximum inhibitory effects of cinnamaldehyde have been reached, where the concentration is lethal towards bacteria.
However, upon cross referencing to literature sources, the trend line acquired appears to differ slightly from literature sources. In particular, a paper on Antibacterial activity of cinnamon essential oil on S.aureus bacteria by the University of Zhenjiang13 showed an exponential trend between % transmission and concentration of cinnamon oil using similar preparation set ups. One reason for this deviation could be the different species of bacteria used which has lower resistance to the antibacterial action of cinnamon essential oil. Alternatively, in the research paper, a much lower concentration of cinnamon essential oil was used between 0.01% and 0.09% as compared to this investigation which used approximately 10 times the concentration, from 0.1% to 0.9%. Hence it is possible that lower concentrations of cinnamon essential oil below 0.1% will bring about a much more rapid change in concentration of bacteria.
Structural comparison of E. coli and B. subtillus
The primary structural difference between E. coli and B. subtillus is that E. coli cells are gram negative while B. subtillus are gram positive, showing that they have differing cell wall compositions. B. subtillus cell walls contain a thick layer of peptidoglycan. Short peptides link the peptidoglycan layer together, providing structural stability. Conversely, E. coli cells have a thin layer of peptidoglycan relative to B. subtillus cells and have an outer lipopolysaccharide membrane. This additional outer membrane serves to protect the E. coli against harmful substances which include the action of cinnamaldehyde on the plasma membrane. Thus this increases the resistance of E. coli and other gram negative bacteria against essential oils, requiring a higher concentration of such oils to inhibit cell division and growth. Hence this would explain the gap in % transmission between E. coli liquid cultures and B. subtillus liquid cultures
Antibacterial efficacy
Referring again to figure 10, a noticeable drop in % transmission is observed between the concentrations 0.6% and 0.8% for E. coli with a difference of 0.378. This sudden drop in % transmission value indicates that there is a sharp decrease in the number of bacteria, suggesting that 0.8% concentration is vastly more effective at inhibiting bacteria growth compared to 0.6% concentration. Cross referencing to qualitative observations, this sudden drop in % transmission is observed by a distinct difference in colour between the two cuvettes. The cuvette containing 0.8% cinnamon essential oil is noticeably lighter in colour compared to the cuvette containing 0.6% cinnamon essential oil. Thus these findings show that 0.8% concentration of cinnamon essential oil is the ideal concentration for inhibiting E. coli growth. The results found were within expectations and by Cross referencing to the P-values collected during statistical testing, a similar noticeable drop in P value from 5.29 X 10-6 to 3.90 X 10-13 for E. coli suggest that 0.8% and 0.9% concentration are more closely related relative to previous comparison of 0.6% and 0.9%.
A possible explanation for the large gap between 0.6 and 0.8% is that the threshold between a low and high concentration of cinnamaldehyde in cinnamon essential oil lies within that region. As explained previously, a higher concentration of cinnamaldehyde is lethal towards bacterial cells while lower concentrations slow down the rate of cell division of bacterial cells by inhibiting the function of ATPase. Thus the threshold for the concentration of cinnamaldehyde transitioning from having inhibitory to lethal effects can be hypothesized to be at 0.7% concentration. This theory is substantiated by the negligible difference in % transmission from 0.8% to 0.9% concentration with only a 0.042 change in % transmission values which is significantly lower relative to other concentrations. A study by D.K manu also supports these findings as it was found that a concentration from 13.6 microgram/ml to 1362 microgram/ml were effective at inhibiting E. coli growth15 .
A similar trend line is also observed for B. subtillus bacteria. However one significant difference between the two trend lines is that the drop in % transmission is observed earlier for B. subtillus, between 0.4 and 0.6% concentration of cinnamon essential oil, thus showing that the ideal concentration for inhibiting B. subtillus bacterial growth is at 0.6% concentration. This is within expectations since cinnamon essential oil is more effective against B. subtillus, it would be expected that it would have a lower ideal concentration to inhibit B. subtillus. A similar drop in P-value is also observed for B. subtillus, occurring between 0.6% and 0.4%. These finding are substantiated by a paper by Lertsatitthanakorn P. & Satayavongthip B. which found that B. subtillus was more susceptible to the action of cinnamaldehyde. 1
Conclusion
In conclusion, answering the research question, increasing the concentration of cinnamon essential oil reduces the growth of E. coli and B. subtillus bacteria, hence proving my initial hypothesis to be true. However past a certain threshold; the benefits appear to deteriorate as there are little changes in bacterial growth. Cinnamon essential oil is evidently more effective towards B. subtillus which could possibly be attributed to the difference in cell structure between gram positive and gram negative bacteria, this also Page 12 of 13 supports my initial hypotheses. Thus one clear finding would be that essential oils have differing antibacterial efficacies depending on the species of bacteria due to differences in their cell structure. Another clear finding from this investigation is that 0.8 and 0.6% concentration of cinnamon essential oil are the most effective at inhibiting E. coli and B. subtillus growth respectively. Reason being that the concentrations of cinnamaldehyde was high enough to be lethal towards bacteria cells
Area for further research and safety precautions
One possible extension to this investigation would be to use different mixtures of essential oils rather than using pure cinnamon essential oil to test their efficacy against bacteria. This is known as synergy which occurs when the combined effects of two substances surpasses the sum of the effects of the two substances would have on their own. Hence a mixture of essential oils such as cinnamon and clover could have a greater antibacterial efficacy rather if the two essential oils were used independently.
Since bacteria cultures were used in this investigation, safety precautions were taken to prevent infection by bacteria as well as contamination. As highlighted in the methodology aseptic conditions were followed and the desk was cleaned before and after experimentation to prevent contamination of lab materials. Gloves and safety goggles were also worn throughout experimentation to prevent infection and injury during handling of bacteria near the open flame of the Bunsen burner. Furthermore after data collection each day, excess solution of liquid culture was properly disposed of in the biochemical waste bin to prevent contamination of water in the sink, furthermore the apparatus was rinsed with water and cleaned with ethanol to prevent contamination of bacteria.
Strengths
A strength for this investigation would be the use of the spectrophotometer to measure the % transmission for replicates. Hence this directly measures the amount of bacteria present in each replicate and indirectly measures the rate of bacteria growth. Furthermore, the use of a spectrophotometer minimizes random errors in data collection, thus making the results more reliable. Furthermore, the relatively low uncertainty of the spectrophotometer allows for accurate measurements of the % transmission. Furthermore, 100% pure cinnamon bark essential oil was used to ensure reliable results collected since lower purities of cinnamon essential oil would have weak antibacterial properties due to a lower concentration of cinnamaldehyde and hence cause the antibacterial bacterial efficacy of cinnamon essential oil to be lower than expected. Thus using 100% pure extract ensures both accurate and reliable data. Adding on to this point, tween20 solution was used to ensure the essential oil was able to dissolve properly into the nutrient broth mixture. Thus this maximizes surface area in contact with bacteria allowing maximal inhibition by cinnamaldehyde.
Limitations
One limitation that arises during data collection is that spectrophotometry is unable to distinguish between live and dead bacterial cells; hence it is unsure whether the change is % transmission is due to an increasing number of dead bacteria cells or an increase in the number of living bacteria. This makes it difficult to quantify the antibacterial efficacy of cinnamon essential oil. Thus this may lead to an underestimation of the effectiveness of cinnamon essential oil as the since the difference in % transmission was assumed to be due to higher levels of bacterial growth. One possible way around this would be to use colony counting to count the number of colonies that emerge before and after a 24 hour incubation period with varying concentrations of cinnamon essential oil. Hence a lower colony count would mean a better inhibition. Living and dead bacteria are able to be differentiated using propidium iodide, staining living bacteria green and dead bacteria red17 . Another limitation would be the pH. Cinnamon essential oil has a pH of approximately 4.5 at 25oC 18; hence this inhibits bacteria growth as the ideal pH for bacteria would be 6.5 to 719 . This leads to an overestimation of the antibacterial efficacy of cinnamon essential oil as the low pH partially inhibits Page 13 of 13 bacterial growth. A solution to this would be adding an alkaline compound into the liquid culture that is not harmful towards the bacteria to increase the pH of the liquid culture.
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