1

Running head: THE EFFECT OF ESSENTIAL OILS IN A GASEOUS STATE ON

ESCHERICHIA COLI

The Effect of Essential Oils in a Gaseous State on Escherichia coli

Brandon DeLoach & Kayla Pintro

Harrison High School

December 2018
 2
Running head: THE EFFECT OF ESSENTIAL OILS IN A GASEOUS STATE ON
ESCHERICHIA COLI

Abstract

Aromatherapy, the process of diffusing essential oils into the air, has become increasingly

popular in today’s societies and is believed by many to help cleanse the air of toxins and

pathogens. In our science fair project, The Effects of Essential Oils in a Gaseous State on

Escherichia Coli, we are exposing Escherichia coli bacteria to an assortment of essential oils

with antimicrobial potential to see how they affect the bacteria’s growth. The essential oils that

will be used in this experiment are cinnamon, citronella, eucalyptus, grapefruit, lemon, oregano,

and tea tree. Background information shows that these essential oils have antimicrobial

properties and are some of the strongest oils to use to topically fight bacteria. Since all of the past

experiments on this topic have been done topically, we decided to test them in a gaseous state

using an essential oil diffuser. Nonpathogenic E. coli was exposed to the individual essential oils

for a limited period of time and then incubated. After 24 hours of growth, we quantified the data

using a spectrophotometer and by counting colonies. Out of the plates that grew enough E. coli

to be quantified, the plates exposed to grapefruit oil most closely portrayed the predicted results.
 3
Running head: THE EFFECT OF ESSENTIAL OILS IN A GASEOUS STATE ON
ESCHERICHIA COLI

The Effect of Essential Oil in a Gaseous State on Escherichia coli

One common belief about aromatherapy, the atomization of water to suspend essential

oils in the air, is that it has antibacterial properties. Although ideally possible, this misconception

lacks the evidence needed to prove its significance. The growing popularity of aromatherapy

sparked our interest in the method’s actual effects. In our experiment, The Effects of Essential

Oils in a Gaseous on Escherichia coli, we test the overall antibacterial properties of seven

essential oils and their effect on E. coli growth using aromatherapy. The seven essential oils we

used, grapefruit, citronella, lemon, oregano, tea tree, cinnamon, and eucalyptus, all had evidence

showing some form of antibacterial resistance. According to Comparative in vitro efficacy of

eight essential oils as antibacterial agents against pathogenic bacteria isolated from pet-turtles

(De Silva, B., Et al., 2018), cinnamon, oregano, and lime all proved to have some form of

antimicrobial properties. When put against pathogenic bacteria, these oils negatively affected the

bacteria. Similar to citronella and eucalyptus oil, grapefruit and lemon essential oils were chosen

because of their prevalence in antibacterial products. The research, In Vitro Evaluation of

Antioxidant and Antimicrobial Activities of Melaleuca Alternifolja Essential Oils (Zhang, X.,

Guo, Y., Guo, L., Jiang, H., & Ji, Q., 2018), primarily tested tea tree oil. Tea tree oil has

antimicrobial properties making it a good choice for our research. The bacteria we chose to test

on was E. coli due to its availability and prevalence in America. Due to its popularity, it was easy

to find and obtain a non-pathogenic sample, making it a good choice for our research. Various

procedures were used throughout the experiment to conduct the research. The first procedure

used was the creation of agar plates. This created a base for the E. coli to be swabbed and grown

on. Another method used was serial dilution. The original E. coli sample was diluted 5 times to
 4
Running head: THE EFFECT OF ESSENTIAL OILS IN A GASEOUS STATE ON
ESCHERICHIA COLI

create a less concentrated sample of E. coli to swab. Before starting data collection all of the

plates were swabbed with the 1/32 E. coli dilution using 10L inoculating loops. To change the

state of the essential oils a diffuser was used. We used an ultrasonic water diffuser that works by

rapidly vibrating a small disc which then atomizes the water and suspends it in the air along with

the essential oils. One of the main procedures used in our experiment was spectrophotometry, a

method of transmitting light through a solution to determine the concentration. This technique

allowed us to measure the concentration of the 5 dilution samples compared to their light

absorbance. One downside of using a spectrophotometer is that if a plate has over 300 colonies

or less than 30 colonies they are not quantifiable because they are classified as too many to count

(TMTC) and too few to count (TFTC). The plates exposed to tea tree, cinnamon, and eucalyptus

oil all were unquantifiable. Out of the plates that grew enough E. coli to be quantified, the plates

exposed to grapefruit oil most portrayed the predicted results.

Literature Review

In the experiments, Effectiveness of Ginger Essential Oil on Postoperative Nausea and

Vomiting in Abdominal Surgery Patients (Lee, Y. R. & Shin, H. S., 2017) and Effect of

Aromatherapy with Peppermint Oil on the Severity of Nausea and Vomiting in Pregnancy: A

Single-blind, Randomized, Placebo-controlled trial (Joulaeerad, N., Ozgoli, G., Hajimehdipoor,

H., Ghasemi, E., & Salehimoghaddam, F., 2018), aromatherapy was used to alleviate nausea and

vomiting in post-surgical patients and pregnant women. They used ginger and peppermint

essential oil because they claim to help with nausea and are the most well-known for aiding
 5
Running head: THE EFFECT OF ESSENTIAL OILS IN A GASEOUS STATE ON
ESCHERICHIA COLI

nausea. E. coli mainly targets the digestive system and causes extreme pain and stomach

problems in the people it affects. The scientists involved in Effectiveness of Ginger Essential Oil

on Postoperative Nausea and Vomiting in Abdominal Surgery Patients (Lee, Y. R. & Shin, H. S.,

2017) concluded that the ginger essential oil contained biological components that alleviate

nausea. While Joulaeerad and Et al. (2018) used a single blind clinical trial to test the

effectiveness of peppermint oil, Lee, Y. R. and Shin, H. S. (2017) used 2 control groups in their

experiment to test the effects that the ginger essential oil had. In both experiments, one group

was exposed to the essential oil through inhalation while the other one was exposed to saline to

keep the experiment controlled.

The study, Comparative in vitro efficacy of eight essential oils as antibacterial agents

against pathogenic bacteria isolated from pet-turtles (De Silva, B., Et al., 2018), exposed

different essential oils to pathogenic bacteria gathered from turtles. The scientist that were a part

of this experiment used bacterial assays to measure the bacterial activity of the pathogen after

being exposed to each essential oil. Unlike our experiment, the scientists directly add the

essential oils to the bacteria sample, instead of changing its state of matter. The essential oils

used in this experiment were cinnamon, clove, ginger, lemongrass, lime, lavender, and oregano.

They concluded that out of the 8 essential oils used oregano, clove, and lemongrass were the

most effective against the strain of bacteria they were exposed to.

The research, In Vitro Evaluation of Antioxidant and Antimicrobial Activities of

Melaleuca Alternifolja Essential Oils (Zhang, X., Guo, Y., Guo, L., Jiang, H., & Ji, Q., 2018),

experiments were done on the essential oil to test its overall antimicrobial and antioxidant

activity. Melaleuca Alternifolia, also known as tea tree oil, is claimed to have strong antibacterial
 6
Running head: THE EFFECT OF ESSENTIAL OILS IN A GASEOUS STATE ON
ESCHERICHIA COLI

properties. When exposed to E. Coli, Melaleuca Alternifolia lessened the growth rate

significantly; therefore, these claims are true. Melaleuca Alternifolia had many biological

components that showed antibacterial properties. The oils used in this experiment were extracted

directly from the plant rather than commercially bought.

The last article, Chemical Composition of Herbal Macerates and Corresponding

Commercial Essential Oils and Their Effect on Bacteria Escherichia coli (Białoń, M., Krzyśko-

Łupicka, T., Pik, A., & Wieczorek, P., 2017), addressed the biological composition of essential

oils on E. coli. Similar to our experiment, the researchers used commercial essential oils rather

than extracting the oils directly from the plants. In this experiment the scientists used the

essential oils in a liquid state when exposing them to the bacteria. They chose to test clove,

oregano, juniper, and marjoram oils. By doing a gas chromatography mass spectrometry analysis

for each essential oil, they were able to figure out all of the components that make up the

essential oils. They concluded that the effectiveness of the essential oils against the bacteria

depended on the ratio of terpenes, terpenoids, and sesquiterpenes along with its antibacterial

activity.

Method

Materials list

 2-liter beaker
 Nutrient agar powder
 Distilled water
 Petri dishes
 7
Running head: THE EFFECT OF ESSENTIAL OILS IN A GASEOUS STATE ON
ESCHERICHIA COLI

Agar plates

1. Weigh out 23 grams of nutrient agar powder

2. Add to 1 liter of distilled water in a 2-liter flask
3. Sterilize at 121° C for 20-25 minutes
4. Cool to 50° C
5. Swirl thoroughly to mix agar and nutrients
6. Pour 25-35 ml per petri dish
7. Allow to cool until firm

Materials list

 Non-iodized salt
 500 ml beaker
 Distilled water
 Eppendorf tubes
 Eppendorf tube stand
 Mechanical pipet with pipet tips
 Escherichia coli, K-12 Strain, Living, Tube (liquid) from Carolina.com

Serial dilution

1. Put .85g of salt into a 500mL beaker, then fill the beaker up to 100 mL with distilled

water to create the serial dilution solution

2. Place 5 Eppendorf tubes in an Eppendorf tube stand

3. Put 500 microliters of the serial dilution solution into each of the 5 Eppendorf tubes

4. Collect 500 microliters of E. coli with a pipet

5. Extrude the E. coli into the first Eppendorf tube

6. Push the pipet up and down 20 times to fully mix the dilution solution and the E. coli

together

7. Collect 500 microliters from the first Eppendorf tube and repeat step 6 in the second

Eppendorf tube
 8
Running head: THE EFFECT OF ESSENTIAL OILS IN A GASEOUS STATE ON
ESCHERICHIA COLI

8. Collect 500 microliters from the second Eppendorf tube and repeat step 6 in the third

Eppendorf tube

9. Collect 500 microliters from the third Eppendorf tube and repeat step 6 in the fourth

Eppendorf tube

10. Collect 500 microliters from the fourth Eppendorf tube and repeat step 6 in the fifth

Eppendorf tube creating the final dilution of 1/32

Materials list

 Sterile inoculating loops

 1/32 E. coli serial dilution
 Sterile nutrient agar plates

Swabbing plates

1. Dip a sterile inoculating loop into the 1/32 E. coli dilution

2. Take the lid of one petri dish off and using the inoculating loop from step one, swab the

agar starting on one side and zig-zagging to the opposite side

3. Use a new inoculating loop for each plate swabbed
4. After swabbing the plate, place the lids back on
5. Move onto the next procedure immediately

Materials

 E. coli swabbed nutrient agar plates

 Diffuser
 Distilled water
 Essential oil
o Lemon (Citrus Limonum)
o Tea tree (Melaleuca Alternifolia)
o Eucalyptus (Eucalyptus Globulus)
o Grapefruit (Citrus Racemose)
o Oregano (Origanum Vulgare L)
o Cinnamon (Cinnamomum Cassia)
o Citronella (Cymbopogon Winterianus)
 9
Running head: THE EFFECT OF ESSENTIAL OILS IN A GASEOUS STATE ON
ESCHERICHIA COLI

 Clear plastic box

 Sanitizing wipes/spray
 Incubator
 Timer

Essential oil testing

1. Label three plates as variables for the essential oil being tested
2. Set up the diffuser and put 200 mL of distilled water and 10 drops of the chosen oil into it
3. Place the diffuser and the three swabbed petri dishes onto a sterilized surface and place a

clear box on top of them, refer to figure 1.

4. Turn the diffuser on and let it run for 20 minutes
5. After 20 minutes turn the diffuser off but leave the

plates under the box for 10 minutes

6. After 10 minutes take the plates out and put them in

the incubator at 37° C for 24 hours

7. While completing steps 1-7, label three new plates
Figure 1: 2:
Figure Step 3 setup
Step 3 setup
as controls for the essential oil being tested and place them under a box in a separate

room and leave for 30 minutes

8. After 30 minutes place the controls into the incubator at 37° C
9. Clean the box, table, and diffuser
10. Repeat steps 1-9 for each essential oil being tested
11. After 24 hours take the plates out of the incubator
12. Count the colonies on each of the plates and record

Materials

 Venier Spectrophotometer with cuvettes

 Smartphone with Bluetooth
 Serial dilutions (1, 1/2, 1/4, 1/8, 1/16, 1/32)

Figure 3: Venier Spectrophotometer with a

cuvette in place.
 10
Running head: THE EFFECT OF ESSENTIAL OILS IN A GASEOUS STATE ON
ESCHERICHIA COLI

Spectrophotometer

1. Download the “Spectral Analysis” app on your

smartphone and connect it via Bluetooth to the

Spectrophotometer

2. Fill a cuvette (about 3⁄4 full) with the .85 salt solution

3. Insert the cuvette in the sample chamber, making sure that the clear side of the cuvette is

in the path of the light

4. On the top menu, click on Experiment. Scroll down to calibrate and select Spectrometer 1

5. Wait 60 seconds for the lamp to warm up

6. Click on Finish Calibration and then OK. The spectrophotometer is now calibrated

7. On the top bar, find the Configure Spectrophotometer icon

8. Select Absorbance vs. Concentration as the Collection Mode

9. Below the concentration menu, select Individual Wavelengths

10. Click the box and type 600 nm

11. A graph will appear with concentration on the X-axis and absorbance at 600 on the Y-axis

12. REMOVE the blank cuvette

 11
Running head: THE EFFECT OF ESSENTIAL OILS IN A GASEOUS STATE ON
ESCHERICHIA COLI

st
13. With the cuvette containing the original E. coli solution (1 dilution) in the chamber,

click on Collect on the top menu

14. Wait until a red dot appears on the screen and click on keep on the top bar

15. A menu appears, asking for the concentration of the sample. Use 1 for this dilution

16. Click on KEEP with the1/2 dilution of bacteria, enter .5 for the concentration

17. Click on KEEP with the 1/4 dilution of bacteria, entering .25 for the concentration

18. Click on KEEP with the 1/8 dilution of bacteria, entering .125 for the concentration

19. Click on KEEP with the 1/16 dilution of bacteria, entering .0625 for the concentration

20. Click on KEEP with the 1/32 dilution of bacteria, entering .03125 for the concentration

21. Click Stop on the top bar menu

Data Analysis Number of colonies Number of

bacteria/µL

Table 1: Tea tree

Tea Tree 1 Greater than 300 N/A
The three tea tree variable
Tea Tree 2 Greater than 300 N/A
plates all had over 300 colonies

and the three constants had Tea Tree 3 Greater than 300 N/A

181, 198, and 269 colonies. Tea Tree 4 181 579.2

Tea Tree 5 269 860.8

Tea Tree 6 198 633.6

 12
Running head: THE EFFECT OF ESSENTIAL OILS IN A GASEOUS STATE ON
ESCHERICHIA COLI

The bacteria on the variable plates are not isolated and cover the entire plate. The constants had

an bacteria/µL count of 579.2, 860.8, and 633.6.

Table 2: Grapefruit
The variable plates for
Number of colonies Number of
grapefruit oil had one plate bacteria/µL
with less than 30 colonies, and
Grapefruit 1 Less than 30 N/A
the other two plates grew 34
Grapefruit 2 34 108.8
and 120 colonies. The
Grapefruit 3 120 384.0
constants for grapefruit all

grew over 300 colonies except Grapefruit 4 183 585.6

for one which grew 183. The Grapefruit 5 Greater than 300 N/A

grapefruit variable’s colonies

Grapefruit 6 Greater than 300 N/A
are very small compared to the

constants. Two of the variables had a bacteria per microliter count of 108.8 and 384 and the

constant had a count of 565.6.

Table 3: Lemon
The plates exposed to the
Number of colonies Number of
bacteria/µL
lemon essential oils had one

plate that grew 289 colonies Lemon 1 Greater than 300 N/A

and two that grew over 300 Lemon 2 289 924.8

colonies. Two of the constants

Lemon 3 Greater than 300 N/A
grew over 300 colonies and
Lemon 4 Greater than 300 N/A

Lemon 5 Greater than 300 N/A

Lemon 6 82 262.4
 13
Running head: THE EFFECT OF ESSENTIAL OILS IN A GASEOUS STATE ON
ESCHERICHIA COLI

one of them grew 82 colonies. The lemon variable’s colonies are less dense and a lot smaller than

the constants. The variable had a bacteria/µL count of 924.8 and the constant had 262.4.

One of the oregano constants Table 4: Oregano Number of colonies Number of

bacteria/µL
grew over 300 colonies. The

other two grew 274 and 192 Oregano 1 Greater than 300 N/A

colonies. The plates exposed Oregano 2 205 656.0

to the oregano essential oils Oregano 3 Greater than 300 N/A

grew over 300 colonies except
Oregano 4 274 876.8
for one that grew 205
Oregano 5 292 934.4
colonies. In one of the
Oregano 6 Greater than 300 N/A
oregano’s variable plates the

bacteria are not isolated and covers almost the entire plate. In the other variable plates there are a

few big colonies with a lot of smaller colonies. The variable plate had 656 bacteria/µL while the

constants had 876.8 bacteria/µL and 934.4 bacteria/µL.

Table 5: Cinnamon
All of the cinnamon variable Number of colonies Number of
bacteria/µL
plates were un-quantifiable
Cinnamon 1 Less than 30 N/A
because they grew less than

30 colonies, whereas, one of Cinnamon 2 Less than 30 N/A

the constants grew less than Cinnamon 3 Less than 30 N/A

30 colonies and the other two Cinnamon 4 Greater than 300 N/A
grew 141 colonies and over
Cinnamon 5 141 451.2
300 colonies. The variable
Cinnamon 6 34 108.8
 14
Running head: THE EFFECT OF ESSENTIAL OILS IN A GASEOUS STATE ON
ESCHERICHIA COLI

plates have a few small colonies and they are very small while the constant plates had a lot of big

colonies. The two constants had 451.2 bacteria/µL and 108.8 bacteria/µL.

Two of the eucalyptus

Number of colonies Number of
bacteria/µL
variables had less than 30

colonies while one had over Eucalyptus 1 Less than 30 N/A

300. The eucalyptus constants Eucalyptus 2 Greater than 300 N/A

had 104, 260, and over 300

Eucalyptus 3 Less than 30 N/A
colonies. Two of the variable
Eucalyptus 4 Greater than 300 N/A
plates have small colonies
Eucalyptus 5 260 832.0
randomly along the plate while

one variable plate has many Eucalyptus 6 104 332.8

very small colonies with a few big ones. The constants have a lot of big colonies. The two

constants had 332.8 bacteria/µL and 832 bacteria/µL.

Number of colonies Number of

One of the citronella variables bacteria/µL

had 103 colonies and the other

Citronella 1 Less than 30 N/A
two had less than 30 colonies.
Citronella 2 103 329.6
The citronella constants had
Citronella 3 Less than 30 N/A
48, 87, and over 300 colonies.

One variable plate has a lot of Citronella 4 Greater than 300 N/A

very small colonies and the Citronella 5 87 278.4

other two have very few tiny

Citronella 6 48 153.6
colonies. The constant’s
 15
Running head: THE EFFECT OF ESSENTIAL OILS IN A GASEOUS STATE ON
ESCHERICHIA COLI

colonies are bunched up and are relatively big compared to the variables. The variable hd 329.6

bacteria/µL while the two constants had 278.4 bacteria/µL and 153.6 bacteria/µL.

The absorbance of the .5 dilution is .304. The Dilution Absorbance @ 600

nm
absorbance of the .25 dilution is .161. The
1/2 .304
absorbance of the .125 dilution is .080. The
1/4 .161
absorbance of the .0625 dilution is .049. The

absorbance of the .03125 dilution is .018. 1/8 .080

1/16 .049

1/32 .018

Discussion

In order for bacteria colonies to be quantifiable they have to have between 30-300

colonies. Any plates that have less than 30 colonies do not have enough colonies to provide a

valid sample and any plates that have more than 300 colonies have too many to be accurately

counted. The plates exposed to tea tree oil were not quantifiable because they all grew over 300

colonies. The growth of the variable plates was unlike the growth of the constant plates, though.

Instead of being isolated colonies it was one big grouping of colonies that was impossible to

count. The plates exposed to tea tree oil either over grew because the oil augmented the rate of

growth or because of one or more of our sources of error. The group of plates that had results

most similar the data hypothesized were the plates exposed to grapefruit oil. When compared to

the other oils, the grapefruit sample showed the least amount of growth compared to their

constants that were quantifiable. The lemon essential oil data was very unexpected and was most
 16
Running head: THE EFFECT OF ESSENTIAL OILS IN A GASEOUS STATE ON
ESCHERICHIA COLI

likely caused by our sources of error. The growth was very unusual and we theorize that it was

caused by excess water on the plate that spread the colonies (see figure ). We expected the lemon

essential oil to decrease the growth of the bacteria but it actually caused it to increase compared

to the constants. The cinnamon variable plates worked as expected by decreasing the growth

compared to the constants. Although colony counts less than 30 aren’t quantifiable, we can tell

that the essential oil caused less growth on the plates. Compared to the constant’s growth, the

oregano’s variables had almost no effect. The growth on the variable plates wasn’t that much

different than the constants so we can’t say that it increased growth rate, but it didn’t decrease it.

The data found in Chemical Composition of Herbal Macerates and Corresponding Commercial

Essential Oils and Their Effect on Bacteria Escherichia coli (Białoń, M., Krzyśko-Łupicka, T.,

Pik, A., & Wieczorek, P., 2017) showed that oregano oil had various antibacterial properties, but

in our data the Oregano oil didn’t have the expected effect on the E. coli. This means that

antibacterial properties in the oregano may have lessened when changed from a liquid state to a

gaseous state. Two of the eucalyptus variable plates worked as expected, however, one plate was

an outlier and grew over 300 colonies which was probably caused by a source of error. Since

only two of the plates portray the predicted results we can’t prove that the oil did indeed work

but we can say that there is a strong chance that they were corelated. When looking at the

quantitative data, the citronella didn’t reflect the wanted results because one of the plates grew

103 colonies, but when looking at the qualitative data, the colonies on all of the variable plates

are very small compared to the constant plates. The constant plates didn’t grow as rampant as the

constants for the other oils which means that there was most likely a swabbing error that

occurred. The data for most of the plates swabbed is un-quantifiable and this could be a result
 17
Running head: THE EFFECT OF ESSENTIAL OILS IN A GASEOUS STATE ON
ESCHERICHIA COLI

from the experiment itself or sources of error. The spectrophotometer gave us the absorbance

values from the dilution solutions and prove that the serial dilution truly worked. The absorbance

indirectly shows the concentration by showing how much light was absorbed into the solution, so

the lower the absorbance the lower the concentration.

One source of error that was most likely the cause of overgrowth on the plates was

the forming on condensation on the agar plates. When we swabbed the E. coli, it mixed with the

water droplets already on the plates causing the water and E. coli to mix spreading out the

sample. The spreading of the sample caused the E. coli to not grow isolated colonies resulting in

overgrowth. Another possible source of error was the inoculation loops. When swabbing, the

bacteria wouldn’t always be spread evenly on the agar plates by the inoculating tubes, causing

large amounts of the sample on one part of the plate and smaller portions of the sample on

another part of the plate. This unevenness in plating many have caused large colonies to grow

and to bind together rather than creating even isolated colonies. Another source of error was the

amount of dilutions conducted. Doing 5 dilutions did not dilute the original E. coli sample

enough, causing a lot of the plates to grow more than 300 colonies in a 24-hour time span. The

researches limitations also could have led to the sources of error. One example of a limitation

that was experienced in the research was that not every essential oil could be tested. This limited

our results to the seven essential oils tested, meaning that it is possible that essential oils decrease

or stop the grow of E. coli, but since our experiment was limited to the seven oils tested, we

cannot for sure say that the use of essential oils doesn’t work.

In future research, different essential oils will be used to test their effect. Along

with testing new oils, we might also test previous oils used against different bacteria to get a
 18
Running head: THE EFFECT OF ESSENTIAL OILS IN A GASEOUS STATE ON
ESCHERICHIA COLI

wider range of data. One of the main next steps is to try new methods and procedure when

experimenting, but also to fix the sources of error made in this research. For example. if done

again more dilutions will occur when conducting the serial dilution portion of the experiment.

One of the main errors experienced in the current research was over growth of colonies which

may have been caused by swabbing a sample that was too concentrated. Doing more serial

dilutions will allow for more even and controlled growth of E. coli on the plates. More

background research will also be needed to start any further experimentation, which will allow

us to get a broader and more valid expectation of the results. Overall next steps for this research

project include widening our range of essential oils and bacteria, along with fixing sources of

error, and doing more research.

References

Białoń, M., Krzyśko-Łupicka, T., Pik, A., & Wieczorek, P. (2017). Chemical Composition of Herbal

Macerates and Corresponding Commercial Essential Oils and Their Effect on Bacteria

Escherichia coli. Molecules, 22(11), 1887. doi:10.3390/molecules22111887

De Silva, B., Hossain, S., Wimalasena, S., Pathirana, H., Dahanayake, P., & Heo, G. (2018).

Comparative in vitro efficacy of eight essential oils as antibacterial agents against pathogenic

bacteria isolated from pet-turtles. Veterinární Medicína, 63(No. 7), 335-343.

doi:10.17221/142/2017-vetmed
 19
Running head: THE EFFECT OF ESSENTIAL OILS IN A GASEOUS STATE ON
ESCHERICHIA COLI

Joulaeerad, N., Ozgoli, G., Hajimehdipoor, H., Ghasemi, E., & SAlehimoghaddam, F. (2018). Effect of

Aromatherapy with Peppermint Oil on the Severity of Nausea and Vomiting in Pregnancy: A

Single-blind, Randomized, Placebo-controlled trial. J Reprod Infertil, 19(1).

Lee, Y. R., & Shin, H. S. (2017). Effectiveness of Ginger Essential Oil on Postoperative Nausea and

Vomiting in Abdominal Surgery Patients. The Journal of Alternative and Complementary

Medicine, 23(3), 196-200. doi:10.1089/acm.2015.0328

Reynolds, J. (2011). Counting bacteria. Retrieved from

http://wiki.hackuarium.ch/images/0/09/Bacterial_counts.pdf

Thiel, T. (1999). Nutrient broth, agar plates and slants. Retrieved from

https://www.sciencebuddies.org/science-fair-projects/project_ideas/microbio_nutrientagar.pdf

Zhang, X., Guo, Y., Guo, L., Jiang, H., & Ji, Q. (2018). In Vitro Evaluation of Antioxidant and

Antimicrobial Activities of Melaleuca alternifolia Essential Oil. BioMed Research International,

2018, 1-8. doi:10.1155/2018/2396109