Extended Essay

Chemistry

Higher Level

Determination of Limonene Content from Different Citrus Fruits Skin and its
Antibacterial Potency: Comparative Investigation

“In lemon skin, lime skin, Mandarin Orange skin, Pacitan Orange skin, and Sunkist Valencia
Orange skin, how does dissolving Styrofoam time affect the area of bacterial inhibition, and how
effective is citrus essential oil in inhibiting bacterial activity based on its limonene content?”

Essay Word Count: 3869

 Table of Contents
Section Title Page
1 Introduction 1

2 Research Question 2

3 Background Information 2
3.1 Limonene 2
3.2 Essential Oil Extraction 5
3.3 Styrofoam test 7
3.4 Antimicrobial test 9

4 Variables 11
5 Materials and Apparatus 13

6 Methodology 14
6.1.1 Sample Preparation 14
6.1.2 Essential Oil Extraction 14
6.2 Styrofoam Test 15
6.3.1 Agar Medium Preparation 15
6.3.2 Sterilization of Apparatus 15
6.3.3 Antimicrobial Test (Streak Method) 16

7 Result and Analysis 17

7.1 Essential Oil extraction 17
7.2 Styrofoam Test 18
7.3 Antimicrobial Test 20

8 Conclusion 21

9 Evaluation and Improvement 22

10 Further Research 23

11 Bibliography 24

12 Appendix 26
13.1 Appendix 1 26
13.3 Appendix 2 32

ii
1. Introduction

In ancient times, severe diseases were incurable. Bacterial infection and cancer are the

example. Millions of people die as pathogens ate their body at a slow pace. Years later, the

Egyptians discovered antibiotic to compensate its hostile environment, in hopes of combating

bacteria and as time evolved Humans has since innovated the idea to keep up with the growing

mutations of bacteria. Nevertheless, antibiotics trigger development of multiple bacteria resistance,

which make the case worse. On the other hand, medication for cancer is somehow unknown.

Therefore, world needs new alternative to eradicate those problems. Despite low bacteria

inhibition activity performed by citrus essential oils, but it can prevent development of multidrug

resistance.1

By reason of its function for ruling pathogens away, citrus essential oils are also used in

demolishing Styrofoam due to presence of organic compound.2 Since its polymer causes terrible

effect for environment, degrading those will reduce pollution and amount of waste as one way of

recycling.3

High oil yield is great as it is proportional to the number of bacteria will be killed. Therefore,

choosing the highest yield is very significant. Nevertheless, limonene, a compound inside most

citrus essential oil, matters more than prominent. The first focus of this investigation is to compare

1
Kimball, Katie (n.d.). Why Essential Oils are Better Than Penicillin. Retrieved September 25, 2018, from
https://www.kitchenstewardship.com/essential-oils-antibiotic-resistance/
2
Cohen, Joe (2018, 21 June). 10 Surprising Health Benefits of Limonene – with Mechanisms and Side Effects.
Retrieved September 25, 2018, from https://www.selfhacked.com/blog/limonene-health-benefits/

3
Kinhal, V. (n.d.). How Styrofoam is Bad for the Environment. Retrieved November 30, 2018, from
https://greenliving.lovetoknow.com/How_Styrofoam_is_Bad_for_the_Environment

1
oil yield and content of the mentioned citrus fruits. Simple distillation can be applied to extract the

oil from the zest of fruits.

It is significant to know which sample has most limonene. Simple Styrofoam test will be

conducted, where it records time for essential oil to dissolve the Styrofoam, concluding the result

qualitatively. The last focus will be on antimicrobial activity in order to know the effect of E. coli

bacteria in each drop of essential oil, whether those are affected by abundance of limonene.

2. Research Question

“In lemon skin, lime skin, mandarin orange skin, Pacitan orange skin, and Arabian Valencia orange

skin, how does dissolving Styrofoam time affect the area of bacterial inhibition, and how effective

is citrus essential oil in inhibiting bacterial activity based on its limonene content?”

3. Background Information

3.1 Limonene

One of the most abundant monoterpenes in citrus essential oil is limonene. Due to its

benefit of curing diseases, maintaining health, reduce pests, and recycling polymers, citrus oil is

extracted by manufacturer and present in perfume, flavoring, or pesticides through distillation or

cold-pressing.

2
 Limonene molecules have one chiral carbon as asymmetric center, surrounded by 4 atoms

or group of atoms, which is H, CH2CH2, CH2CH, and C. Figure 3.1.1 shows the chiral carbon of

limonene molecule. 4

Figure 3.1.1 Chiral Carbon and its 4 functional groups in Limonene

Limonene forms 2 enantiomers, (+) limonene and (-) limonene, creating a 50:50 racemic

mixture. Those 2 types present in essential oil of different citrus fruit. Lime and lemon consist

majority of (-) limonene, where Sunkist Valencia Orange, Mandarin Orange, and Pacitan Orange

consist of (+). Both enantiomers have different structure, which is shown in its optical isomers.

Figure 3.1.2 shows the optical isomers of limonene

Figure 3.1.2 Optical isomers of limonene

4
Chapter 5 Stereochemistry The Arrangement of Atoms in Space; The Stereochemistry of Addition Reactions. - ppt
download. (n.d.). Retrieved December 1, 2018, from https://slideplayer.com/slide/6924027/

3
 Both limonene compounds have same physical properties, where they have same boiling

points. However, there are differences in smell of essential oil, which therefore performs different

task. Distinction in chemical properties also can be shown in reaction with other enantiomers

biologically.

As mentioned previously, limonene is present in citrus fruit skin in plenty amount. Its

biosynthesis involves HMG-CoA reductase pathway, since the reactants include isopentenyl

pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP). The reactions are shown in

figure 3.2.2 and figure 3.2.35

1. Both colored negative ions, isopentenyl

pyrophosphate (IPP) and dimethylallyl

pyrophosphate (DMAPP) produce Geranyl

pyrophosphate (GPP) with the help of enzyme

Geranyl pyrophosphate synthase by condensation

reaction6.

Figure 3.2.2. Condensation reaction of IPP and DMAPP

2. Electron from 𝜋 bond move to fill the

missing σ bond. Therefore, Geranyl

pyrophosphate (GPP) loses a proton of hydrogen

ion and limonene is formed in the end.7

Figure 3.2.3. Oxidation reaction of GPP to form limonene

5
Clinical Microbiology: Open Access. (n.d.). Retrieved November 2, 2018, from
https://www.omicsonline.org/scholarly/antimicrobial-activity-journals-articles-ppts-list.php

6
Isopentenyl pyrophosphate. (n.d.). Retrieved November 2, 2018, from
https://www.sciencedirect.com/topics/biochemistry-genetics-and-molecular-biology/isopentenyl-pyrophosphate

7
Harro J. Bouwmeester. (1998, July). Biosynthesis of the Monoterpenes Limonene and Carvone in the Fruit of
Caraway. Retrieved November 2, 2018, from https://www.ncbi.nlm.nih.gov/pmc/articles/PMC34944/

4
 Limonene product is then deposited in skins of the fruit after passing through the process

in leaves and seeds. It is the main component that made up citrus fruit peels, giving smooth and

soft texture.

3.2. Essential Oil Extraction

Essential oils are dissimilar with any other oil extracted from seeds and saturated oil from

animals. Terpene family are the major component of essential oil. It contains very high

unsaturation level due to eminent number of double bond. Thus, volatility of essential oil is

exceptional due to its ability to do comprehensive evaporation.

Several methods can be done to extract oil from citrus fruit

peels, which is cold pressing and distillation. Although cold pressing

is much simpler than distillation, the purity of the product is not

assured since it leaves residual oil and pulp. Also, cold pressed

essential oil is more phototoxic and volatile, which evaporate

Figure 3.2.1 Structure of citrus fruit peels
very easily under the sun and has higher chance of skin

irritation. Being more effective, simple distillation will be conducted for this investigation. 8

Essential oils are present in small pores surrounding the peels of the citrus fruit protected

by its compartments, shown in figure 3.2.1 Thus, it needs to be grated in order to open the pores

and release the oil.9 Due to its volatility, the oil can evaporate together with the water, despite the

8
Amy (2018, 10 April). Cold Pressed vs Steam Distilled Essential Oils: Which Is Best For You?. Retrieved September
25,2018, from http://essentialoilsguide.top5picks.net/cold-pressed-vs-steam-distilled-essential-oils-best/

9
Fruit Fireballs. (n.d.). Retrieved November 14, 2018, from https://www.thenakedscientists.com/get-
naked/experiments/fruit-fireballs

5
higher boiling point of oil. It is because of the very weak attractive force between the particle in

the substance caused by less hydrogen bonding compared to less volatile substances. The reason

is due to its absence of oxygen, nitrogen, or fluorine that creates double bond. Only London or

Van der Waals force is present, which is weak and temporary.

Figure 3.2.2 shows limonene structure.

3.2.2 Chemical structure of Limonene

Therefore, when a little amount of heat or energy is applied, these forces can break easily,

which cause particles to escape from liquid to gas phase.10

After vapor has been condensed by condenser, hydrosol and oil will be obtained as distillate.

Density of essential oil is slightly lower than water, which is 0.888 g/mL at 25 °C, so oil form on

top while hydrosol sinks at the bottom. Mass of essential oil is recorded for further calculation of

percentage with its total mass of citrus fruit skins.

10
(n.d.). Volatility of a Liquid. Retrieved November 2, 2018, from
https://sites.duke.edu/apep/module-4-alcohol-and-the-breathalyzer-test/biology-and-chemistry-
connections/volatility-of-a-liquid/

6
3.3 Styrofoam Test

Citrus essential oils consist mainly organic compound, which makes its reason to be called

as an excellent organic solvent. Organic compounds are non-polar molecules, since it doesn’t

contain any charges or partial charges. Therefore, dissolving other non-polar molecules is one of

its main properties, with the help of high energy and force to allow bombardment between

molecules.11

Styrofoam is a polymer of ethylbenzene that has a chemical

formula of C8H8, which belongs to aromatic hydrocarbons. It has

benzene ring in the middle since it is made by electrophilic

substitution of benzene and ethene, with hydrochloric acid as

catalyst. Its molecule only has carbon and hydrogen. Despite the
Figure 3.3.1 Structure of Ethylbenzene
positive charge present in hydrogen, its electron is shared equally

with carbon, causing the molecule to have very small (almost 0) electronegativity which

determined as non-polar.

Electronegativity of C-H can be calculated using electronegativity differences formula:

( 𝑥(𝐶) = 2.55, 𝑥(𝐻) = 2.2 )

∆𝑥𝑝 = 𝑥(𝐶) − 𝑥(𝐻)

= 2.55 − 2.2 = 0.35

11
Essential Oil University (n.d.). What are You Proving When you Dissolve Styrofoam with Lemon Oil?. Retrieved
November 13, 2018, from
https://www.facebook.com/notes/essential-oil-university/what-does-it-prove-to-dissolve-styrofoam-with-
essential-oils/10155627031143083/

7
 Ethylbenzene has resonance structure, where it allows delocalization of 𝜋 electrons to

happen. The presence of different resonance energy stabilizes benzene molecule. The stable

molecules indicate equal share of electrons in π and σ bond. Therefore, no charges or partial

charges present like in polar molecules.

This investigation also has its environmental advantage. Since ethylbenzene is hazardous

for human, production of Styrofoam is restricted. Despite its unavailability to biodegrade and

recycled, it causes diseases from chemical reaction of benzene with foods, which increases the

carcinogen concentration in organism blood that forms cancer. Therefore, Styrofoam needs to be

converted or mix with other substance to reduce its harmful effect and increase recycling efficiency.

Citrus essential oil can be one of the possible substance as it dissolves Styrofoam.12

Main organic compound in citrus essential oil is monoterpenes. Limonene occupies most

percentage of citrus essential oil composition. Thus, it is mostly in charge in non-polarity and

dissolving other non-polar molecules like ethylbenzene. The ability to dissolve is due to the

presence of London force and lacking of hydrogen bond and dipole-dipole forces. Since it is

temporary and weak, it can be easily broken down, causing intramolecular bond between polymer

of ethylbenzene in Styrofoam to break and dissolves. 13

12
Ethylbenzene. (2004, 16 April). Retrieved November 14, 2018, from
https://pubchem.ncbi.nlm.nih.gov/compound/ethylbenzene#section=Top

13
Noguchi, T., Miyashita, M., Inagaki, Y., & Watanabe, H. (1998, December 04). A new recycling system for
expanded polystyrene using a natural solvent. Part 1. A new recycling technique. Retrieved December 1, 2018,
from https://onlinelibrary.wiley.com/doi/pdf/10.1002/(SICI)1099-1522(199802)11:13.0.CO;2-5

8
 A metal mass is used as an indication of which essential oil can dissolve Styrofoam quickly.

Time needed for coin to fall from top of Styrofoam is recorded for further comparison with other

5 essential oils.14

3.4. Antimicrobial activity

Limonene is a chiral hydrocarbon molecule that is classified as cyclic monoterpene. It is

the main component for the detoxification, and immunity supports to fight against bacteria and

skin protection. High amount of limonene content increase the ability of killing bacteria.

Limonene can be transformed to other product in certain condition, such as perillyl alcohol

through hydroxylation process 15 . Perillyl alcohol is a monoterpene alcohol that is useful for

Figure 3.4.1 Hydroxylation of limonene

antimicrobial activity test, so a production of this compound is more favorable in this case. It

involves hydrogen peroxide catalyst that present in earth atmosphere. Transformation process is

shown below:

14
(n.d.). Retrieved November 14, 2018, from http://lemon-essential-oil.homestead.com/styrofoam-cup-tests---
Young-Living-Lemon-Essential-oil-vs-doTerra-vs-Ameo-Lemon-essential-oils-comparison.html

15
Alonso-Gutierrez, J., Chan, R., Batth, T., Adams, P., Keasling, J., Petzold, C. and Lee, T. (2019). Metabolic
engineering of Escherichia coli for limonene and perillyl alcohol production.
https://www.sciencedirect.com/science/article/pii/S109671761300058X

9
 E. coli bacteria is one of the most common gram negative bacteria which causes diarrhea.

It can be transmitted by ingestion to patient’s body, through saliva in our mouth or inhalation

through nose. It has double plasma membrane, making it more impermeable than gram positive

bacteria. Perillyl alcohol or limonene itself acts as protein denaturing or dehydrating agents, which

may involve denaturing the naked DNA and protein to decreases the cell size and kills the bacteria

eventually.16

Most potent antibiotics is ciprofloxacin, since it prevents DNA gyrase in bacteria to

separate double helix strand present is bacterial DNA. As a result, bacteria die due to lack of DNA

replication process.17 Nevertheless, Ciprofloxacin is quite expensive than other antibiotics because

of its potency towards bacteria. Other antibiotics for E. coli are not potent enough to kill those,

such as amoxicillin due to presence of beta-lactam ring. Hence, citrus essential oil may be the

alternative solution due to its abundance availability in nature.

Number of bacteria killed in the agar plate will be measured in unit radius and area, which

indicate effect of essential oil ability to inhibit bacteria. The radius of the circle will be recorded

to indicate the area of circles which indicates killed bacteria are measured using area of circle

formula. It is then compared to see which has the biggest area to determine its antimicrobial

ability.18

16
Dorman, H. and Deans, S. (2019). Antimicrobial agents from plants: antibacterial activity of plant volatile oils.
https://onlinelibrary.wiley.com/doi/full/10.1046/j.1365-2672.2000.00969.x
17
DNA Gyrase - Dna Replication Simulator. (n.d.). Retrieved December 1, 2018, from
https://sites.google.com/site/dnareplicationsimulator/dna

18
Obidi O. F., Adelowotan A. O., Ayoola G. A, Johnson O. O., Hassan M. O., Nwachukwu S. C. U. (2016).
ANTIMICROBIAL ACTIVITY OF ORANGE OIL ON SELECTED PATHOGENS. Retrieved on December 14, 2018, from
http://www.aessweb.com/pdf-files/ijb%202(6),%20113-122.pdf

10
4. Variables

Variables Factors Observation and Measurement

Independent Samples used There will be 5 different samples of different
kind of citrus fruits. Lemon, lime, Mandarin
orange, Pacitan orange, and Sunkist Valencia
Orange are the samples.
Dependent Mass of essential oil Mass of each essential oil is recorded using
digital balance after the extraction. Hence, oil
yield can be deduced
Time of dissolving Styrofoam Time required for essential oil to dissolve
Styrofoam will be shown by falling of metal
mass from the top of Styrofoam bowl,
resulting a hole its bowl. Time is recorded in
seconds
Area of inhibition Diameter of its area will be recorded using
metal ruler. By applying area of circle
formula, area of inhibition can be recorded as
part of investigation.
Control Essential oil Mass of grated 70.0 g, measured with digital balance, will
Extraction citrus fruit be the constant mass of citrus fruit peels used
peels to extract citrus essential oil.
Temperature of Temperature shown in heater will be kept
heater around 350-400˚C at the beginning. Then
decreased to 250˚C after few minutes to
prevent massive explosion
Volume ratio Ratio of distilled water and mass of peels
of distilled will be 2:3 or 1:1.5. This way, it will ensure
water and mass fairness of essential oil extraction, as too
of peels much water require much longer time to boil

11
 and essential oil will not be maximumly
extracted.
Styrofoam Volume of 0.2 ml will be measured with pipette and
test essential oils kept constant among all Styrofoam test.
Mass of metal 20 g of same metal mass will be used
throughout all samples.
Antimicrobial Type of ATCC 4000 E. Coli bacteria will be the only
test bacteria bacteria used, with the same manufacturing
date, on 10 October 2018. It will be used for
the whole test on each plate.

Temperature of Temperature of incubator will be maintained

Incubator at approximately 37˚C for all plates
Volume of 30 ml of agar solution will be poured into
agar medium each plate and will remain the same for all
each plate samples. Agar medium is the food of
bacteria, so growth and antimicrobial activity
of bacteria in each plate depends on its food.
Volume of Each plate consists of 4 filter paper
essential oil containing 5 different essential oils and
ciprofloxacin antibiotics. Every 4 filter
papers will be given a tiny drop of its
samples, since amount of sample is
extremely limited for this experiment.
Volume of In 300 ml of water, 8.3 g of agar powder will
water in agar be mixed, creating agar solution. Volume of
water will be measured with measuring
cylinder.
Time for In order to show its minimum inhibitory
bacteria to concentration, bacteria commonly require
18-24 hours to produce an effect. To make it

12
 react with its less complicated and more accurate, 18 hours
agent will be set as its time requirement.

5. Materials and Apparatus

Test type Materials Quantity (M) Apparatus Quantity (A)

Sample Citrus fruits 5-6 fruits, 40 Grater 1
preparation fruits for lime Brush 1
Spoon 1
Extraction of Citrus fruit peels 70 g Round bottom flask 1
Essential Oil Distilled water 105 ml Condenser 1
Separating funnel 1
Measuring cylinder 1
100 ml
Heater 1
Funnel 1
Beaker 2
Spoon 1
Cork 1
Retort stand 2
Clamp 2
Styrofoam Test Styrofoam bowl 15 bowls Pipette 1
Metal mass 20 g 1
Stopwatch 1
Antimicrobial Agar powder 8.3 g Incubator 1
test Distilled water 300 ml Petri disks 12
E. Coli Bacteria 1 tube Autoclave 1
Ciprofloxacin 1 tablet Beaker 500 ml 1
Alcohol 200 ml Inoculating loop 2
Spirit burner 2 Heater 1

13
 Alcohol 1
thermometer
Fire starter 1
Spoon 1
Beaker 100 ml 1

6. Methodology

6.1 Essential Oil Extraction

6.1.1 Sample Preparation

Lemon, lime, Pacitan Oranges, Baby Java Oranges, and Mandarin Oranges were grated to

obtain its skin using a cheese grater. The grated peels were then stored in container and refrigerated

overnight.

6.1.2 Essential Oil Extraction

Distillation apparatus was set up with materials in Appendix 2. 70.00 g of prepared sample

was put in 200 ml beaker. 105 ml of distilled water (2:3 ratio) was poured into the beaker, and then

stirred. The mixture was then poured into round bottom flask and closed with cork stoppers. Heater

was set for approximately 350-400˚C for fast boiling process. After the mixture started boiling,

heater was lowered to 250˚C to prevent vigorous explosion. An immiscible liquid containing

hydrosol and essential oil was collected in separating funnel after 2-3 hours. The distillation

process was stopped until there is no oil that flows through the condenser (only hydrosol). Essential

oil could be collected using separating funnel which separated it from hydrosol. Mass of the oil

14
was recorded with digital balance in glass container. Percentage yield of oil extraction will also be

recorded each sample.19

6.2 Styrofoam test

A Styrofoam bowls with flat base were prepared facing upside down. 20.0 g of metal mass

was smeared with 1 drop of 0.2 ml of citrus essential oil. A drop of oil was applied to the Styrofoam

base as well. Smeared mass was placed on top of oil drop on the Styrofoam. Stopwatch was used

to record the time taken for certain area of Styrofoam to completely dissolve, showed by falling of

metal from Styrofoam. It was repeated 3 trials for each oil samples.20

6.3 Antimicrobial Test

6.3.1 Agar Medium preparation

8.3 g of agar powder was placed on 500 ml beaker. The beaker was then added 300 ml of

distilled water. The mixture was stirred using magnetic stirrer and heated until it boils. After 18

minutes, when all agar dissolved in water, solution was removed from heat.

6.3.2 Sterilization of Apparatus

Laboratory tables, utensils, and hand were sprayed with ethanol solution 70% before the

experiment start. Tap water were poured into bottom of autoclave container until the water level

sign. Perforated bottom plate was then placed and grid basket was placed on top. 12 petri disks

19
Agritech.tnau.ac.in. (2019). [online]. Retrieved on January 22, 2019, from
http://agritech.tnau.ac.in/horticulture/extraction_methods_natural_essential_oil.pdf
20
Lemon-essential-oil.homestead.com. (2019). styrofoam-cup-tests---Young-Living-Lemon-Essential-oil-vs-doTerra-
vs-Ameo-Lemon-essential-oils-comparison Young living Lemon essential oil vs doTerra Lemon Oil vs Mountain Rose
lemon vs Now Lemon vs Ameo Lemon vs essential lemon oils review. Retrieved from January 22, 2019, from
http://lemon-essential-oil.homestead.com/styrofoam-cup-tests---Young-Living-Lemon-Essential-oil-vs-doTerra-vs-
Ameo-Lemon-essential-oils-comparison.html

15
and a beaker of agar solution were put inside grid basket, arranged in order manner. The lid was

closed and autoclave was turned on, maintained with 120 kPa for 15 minutes. Afterward, petri

disks and agar solution were taken out from autoclave. Thermometer was placed in agar solution

until it showed 40˚C. Petri disks were sprayed with ethanol solution 70% then placed on top of

Bunsen burner for further sterilization.

6.3.3 Streaking Bacteria

Cooled agar solution was poured to warm petri disks. Agar was allowed to cool and thicken

for 5 minutes. Tube of ATCC 4000 E. coli bacteria were taken from -2˚C refrigerator and placed

on test tube stand. Close to Bunsen burner, inoculating loop was inserted to E. coli tube, streaking

the agar surface in bacteria tube, then streak it to solid agar in petri disks with arranged pattern.

Leave the petri disks containing bacteria and agar in 37˚C incubator for 10 minutes. Small circular

filter papers separated in porcelain, where each hole contained 4 papers. A drop of each oil extracts,

and ciprofloxacin 0.01% was added to a hole. Wet papers containing samples were then placed on

solid agar in petri disks, forming square pattern. Petri disks were put back in 37˚C incubator for

24 hours. 2 disks trial were done for each oil sample, creating 8 trials each sample.21

21
Obidi O. F., Adelowotan A. O., Ayoola G. A, Johnson O. O., Hassan M. O., Nwachukwu S. C. U. (2016).
ANTIMICROBIAL ACTIVITY OF ORANGE OIL ON SELECTED PATHOGENS. Retrieved on December 14, 2018, from
http://www.aessweb.com/pdf-files/ijb%202(6),%20113-122.pdf

16
7. Result and Analysis

Raw data and data processing are in Appendix 2

7.1 Essential Oil Extraction

3.50

3.00
2.57
2.50
Essential Oil Yield (%)

2.14
2.00
1.71

1.50 1.28

1.00 0.857

0.50

0.00
Lemon Lime Mandarin Orange Pacitan Orange Sunkist Valencia
Essential oil samples

Chart 5.1.1 Comparison of total oil yield by mass

From chart 5.1.1, it could be deduced that different citrus fruit skin produces different

amount of essential oil with different percentage of oil yield. Sample with oil yield in decreasing

order are Pacitan Oranges (2.57%) continued with Lemon (2.14%), Sunkist Valencia (1.71%),

Mandarin (1.28%), and Lime (0.857%) which has the lowest oil yield. From the colors, most

essential oils are clear, except for lime with yellowish-green color.

Pacitan orange skin has the most oil yield, which indicates the effectiveness for massive

production. On the contrary, lime has the least oil yield that makes it less effective for medical

purposes. Dissimilarities of oil yields could be caused by the number of pores present in citrus

17
skins and their growth environment and adaptation, as in hot temperature essential oil in skins may

evaporate.

As sample preparation, citrus fruit skins are grated with cheese grater. Hence, it was found

out some oil still left on the grater, as it becomes oily after grate. Since grated samples are placed

in container, some oil droplets also attach to the container, continued with the spoon and other

utensils. Moreover, small amount of oil also present on the hydrosol, which is not possible to be

extracted using separating funnel. These errors caused amount of oil extracted from distillation is

less than the actual value in fruit skin. However, grating increased the surface area, allowing more

collision between solvent and skin pores, which release essential oil from fruit skins and speed up

reaction.

7.2 Styrofoam Test

900
Mean time for essential oil to dissolve

800
724
5.3 Antimicrobial Test 675
700
styrofoam (second)

600

500
403
400
280
300
203
200

100

0
Lemon Lime Mandarin Orange Pacitan Orange Sunkist Valencia
Orange
Essential oil samples

Chart 5.2.1 Comparison of Styrofoam dissolving time

18
 Limonene is a non-polar compound that readily dissolves non-polar polymer like

Styrofoam. A hollow was created and glue-like texture was formed as the product of the

experiment. Solid Styrofoam was converted into semi-solid form, which allow 20 grams of mass

to fall on a certain time.

From chart 5.2.1, the time required for oil samples to dissolve Styrofoam for Lemon, lime,

Mandarin Orange, Pacitan Orange Sunkist Valencia Orange, are 403 seconds, 203 seconds, 280

seconds, 724 seconds, and 675 seconds. Only Mandarin Orange, lemon and lime required less than

500 seconds to dissolve a certain area of Styrofoam, others took more than 500 seconds. It can be

deduced that lime has shortest time to dissolve Styrofoam with 203 seconds, which might indicate

highest limonene content, followed with lemon, Mandarin Orange, Sunkist Valencia Orange, and

Pacitan Orange with the longest time of 724 seconds. This result will be compared with its

antimicrobial activity, to determine if there is a correlation.

ANOVA F-Test was also conducted to determine the significance of the result, shown in

appendix 1. It is proved that results were significant as F-stats is bigger than the critical value,

which is 58.4. Hence, results can be approved for further investigation.

Despite the same metal mass used, thickness of Styrofoam could differ on every trial,

which was included in manufacturing errors. Subsequently, a drop of essential oil may vary in

volume as it is only measured with pipette.

From 3 tests, mean was then recorded and deduced as its fixed result for comparison.

Fortunately, those 3 results were not far from each other and no anomalous result, which ensure

the accuracy of the mean value.

19
7.3 Antimicrobial Test

Antimicrobial test
1000
Average Area of inhibition (mm2)

800 717.5

600

400 294.5
202.03
200 154.7 123.1
81.4 103.7 74.3
0 0 0
0
Lemon Lime Mandarin Orange Pacitan Orange Sunkist Valencia Ciproflacaxin
-200 Orange 0.01%

Plate 1 Plate 2 Oil samples

Chart 5.3.1 Comparison of area of inhibition (mm2)

From chart 5.3.1, first plate bacterial activities were shown in decreasing order, lemon

(294.5 mm2), lime (123.1 mm2), Mandarin Orange (81.4 mm2), Pacitan Orange (0 mm2), and

Sunkist Orange (0 mm2). Second plate bacterial activities of lemon, lime, Mandarin Orange,

Pacitan Orange, and Sunkist Valencia Orange were 154.7 mm2, 203.03 mm2, 103.7 mm2, 0 mm2,

and 74.3 mm2. Compared to 5 samples, lemon caused the most area of Inhibition with an average

of 224.6 mm2, followed with lime (157.7 mm2), Pacitan Orange (52.9 mm2), and Sunkist Valencia

(39 mm2). 0.01% Ciprofloxacin has inhibition area for 717.5 mm2, which is more than 3 times of

lemon essential oil. Higher number of inhibition indicates its high effectivity to be used in

medicinal purposes, which can be said as good antimicrobial agent like antibiotics. It drove out

bacteria from its sample to bacterial zone with no agents. If agents effect is large, bacteria could

be killed as no bacterial zone available for survival

ANOVA F-Test was conducted, shown in appendix 1. It is shown that P-value of lemon is

bigger than 0.05, which tells insignificance of the result. In worse case, Mandarin Orange has F-

20
stat smaller than the P-value, with P-value more than 0.05. This insignificance can be caused by

uneven spread and number of bacteria in each plate and section, which affect each antimicrobial

activity

Based on the result, Pacitan Orange has the lowest average area due to absence of ability

to inhibit bacteria in both plate, that is diffused to all filter paper containing samples. It proves the

lack effectiveness being an antimicrobial agent. On the other hand, in appendix 3, it is also shown

that Sunkist Valencia’s petri disks have different forms of bacteria, where in the first plate bacteria

clump and form a colony to combat the sample, resulting no area of inhibition, while in second

plate bacteria grew individually. In this case, capability of essential oil to combat bacteria is more

on second plate, since attacking individual bacteria is easier than colonies. This case could be a

reason on the different result between plate 1 and plate 2 of other sample, where have no correlation.

Inhibition zone wasn’t always form circular area. It depends on the position of bacteria and

capability of sample to attack. Hence, it sometimes formed irregular shapes, where its rounded for

simpler measurement, as measuring area of irregular shape will be very complicated.

8. Conclusion

The research question “In lemon skin, lime skin, mandarin orange skin, Pacitan orange

skin, and Sunkist Valencia orange skin, how does dissolving Styrofoam time affect the area of

bacterial inhibition, and how effective is citrus essential oil in inhibiting bacterial activity based

on its limonene content?” can be answered.

Based on Styrofoam test, limonene content can be determined, where lime has the most

limonene among all. However, antimicrobial test proved lemon could best inhibit bacteria. It might

21
because of higher amount of other organic compound in lemon that also act as antimicrobial agent

than lime, such as linalool. This case also happens in Mandarin Orange, where dissolving time

does not affect area of inhibition. Despite that difference, there are correlation between Styrofoam

test and inhibition area of Pacitan Orange and Sunkist Valencia Orange, where both oil have the

lowest among 5. Hence, its fact in inaccuracy could not prove no correlation.

From the result above, lemon is the most potent antibacterial agent among all essential oil.

It also has high amount of oil yield. Despite the highest oil yield goes to Pacitan Orange, but has

no antimicrobial activity. However, compared to real antibiotics like ciprofloxacin, essential oils

are still far away from antibiotics potency, which is 3 times less, even if it’s only 0.01%

concentration. Therefore, it is extremely less effective to replace antibiotics with essential oil,

especially to cure diseases. Moreover, citrus essential oils are less edible due to volatile organic

compounds and have severe side effects, such as skin infection and poisoning. Hence, essential

oils are not recommended to substitute antibiotics.

9. Evaluation and Improvement

In this investigation, data obtained might not be precise because of several errors. Residual

oils in hydrosol might present which may require further extraction. Number of extraction could

be maximized. Also, using of other separating apparatus such as soxhlet is recommended to obtain

oil efficiently and force all oil that left on hydrosol. Unavailability of instruments and time

limitations are the reason these were not done.

Brand and thickness of Styrofoam used each test might be different, which caused

inaccurate result. Hence, using the same brand and thickness could reduce this error. Moreover,

22
measuring 0.2 ml using pipette was inaccurate. Using measuring cylinder would be better to

increase its accuracy. However, some oil droplets could stick to cylinder’s wall, and enhance the

inaccuracy further due to tiny amount of essential oil used. Therefore, using more volume of

essential oil would be more efficient and create more impact on the result. Lacking amount of

essential oils and availability of Styrofoam are the cause of these errors.

Due to different ways of growth in bacteria, irregular shapes formed as zero zone, which

were rounded in order to calculate the area of inhibition. Those were not the actual area due to its

inaccuracy. Further calculation involving calculus could be required to calculate the exact area of

irregular shape. Moreover, for more significant ANOVA F-Test result, increasing number of trials

would increase probability of significant by decreasing the P-value to less than 0.05 and increase

the value of F-stats.

10. Further Research

Certain aspects of this investigation were not investigated. Ripeness of fruit samples used

were those normally accepted and edible by human. This might affect the actual oil yield of citrus

fruits. Therefore, it would be great to compare the result with different ripeness.

Investigating antimicrobial potency of essential oil would require to know the efficiency

of E. Coli bacteria’s growth and activity. Some bacteria might be damaged or injured due to

external effects, which affect the result comparison.

23
11. Bibliography

(n.d.). Clinical Microbiology: Open Access. Retrieved November 2, 2018, from

https://www.omicsonline.org/scholarly/antimicrobial-activity-journals-articles-ppts-
list.php

(n.d) Chapter 5 Stereochemistry The Arrangement of Atoms in Space; The Stereochemistry of

Addition Reactions. - ppt download. Retrieved December 1, 2018, from
https://slideplayer.com/slide/6924027/

(n.d) Ethylbenzene. (2004, 16 April). Retrieved November 14, 2018, from

https://pubchem.ncbi.nlm.nih.gov/compound/ethylbenzene#section=Top

(n.d.). Isopentenyl pyrophosphate. Retrieved November 2, 2018, from

https://www.sciencedirect.com/topics/biochemistry-genetics-and-molecular-
biology/isopentenyl-pyrophosphate

(n.d.). Fruit Fireballs. Retrieved November 14, 2018, from

https://www.thenakedscientists.com/get-naked/experiments/fruit-fireballs

(n.d.). Volatility of a Liquid. Retrieved November 2, 2018, from

https://sites.duke.edu/apep/module-4-alcohol-and-the-breathalyzer-test/biology-and-
chemistry-connections/volatility-of-a-liquid/

(n.d.). Retrieved November 14, 2018, from http://lemon-essential-oil.homestead.com/styrofoam-

cup-tests---Young-Living-Lemon-Essential-oil-vs-doTerra-vs-Ameo-Lemon-essential-
oils-comparison.html

(n.d.). DNA Gyrase - Dna Replication Simulator. Retrieved December 1, 2018, from
https://sites.google.com/site/dnareplicationsimulator/dna

Agritech.tnau.ac.in. (2019). Retrieved on January 22, 2019, from

http://agritech.tnau.ac.in/horticulture/extraction_methods_natural_essential_oil.pdf

Alonso-Gutierrez, J., Chan, R., Batth, T., Adams, P., Keasling, J., Petzold, C. and Lee, T. (2019).
Metabolic engineering of Escherichia coli for limonene and perillyl alcohol production.
https://www.sciencedirect.com/science/article/pii/S109671761300058X.

24
Amy (2018, 10 April). Cold Pressed vs Steam Distilled Essential Oils: Which Is Best For You?.
Retrieved September 25,2018, from http://essentialoilsguide.top5picks.net/cold-pressed-
vs-steam-distilled-essential-oils-best/

Cohen, Joe (2018, 21 June). 10 Surprising Health Benefits of Limonene – with Mechanisms and
Side Effects. Retrieved September 25, 2018, from
https://www.selfhacked.com/blog/limonene-health-benefits/

Dorman, H. and Deans, S. (2019). Antimicrobial agents from plants: antibacterial activity of
plant volatile oils. https://onlinelibrary.wiley.com/doi/full/10.1046/j.1365-
2672.2000.00969.x
Essential Oil University (n.d.). What are You Proving When you Dissolve Styrofoam with Lemon
Oil?. Retrieved November 13, 2018, from https://www.facebook.com/notes/essential-oil-
university/what-does-it-prove-to-dissolve-styrofoam-with-essential-
oils/10155627031143083/

Harro J. Bouwmeester. (1998, July). Biosynthesis of the Monoterpenes Limonene and Carvone in
the Fruit of Caraway. Retrieved November 2, 2018, from
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC34944/

Kimball, Katie (n.d.). Why Essential Oils are Better Than Penicillin. Retrieved September 25,
2018, from https://www.kitchenstewardship.com/essential-oils-antibiotic-resistance/

Kinhal, V. (n.d.). How Styrofoam is Bad for the Environment. Retrieved November 30, 2018, from
https://greenliving.lovetoknow.com/How_Styrofoam_is_Bad_for_the_Environment

Lemon-essential-oil.homestead.com. (2019). Lemon-Essential-Oils-Review.com. Retrieved from

January 22, 2019, from http://lemon-essential-oil.homestead.com/styrofoam-cup-tests---
Young-Living-Lemon-Essential-oil- vs-doTerra-vs-Ameo-Lemon-essential-oils-
comparison.html

Noguchi, T., Miyashita, M., Inagaki, Y., & Watanabe, H. (1998, December 04). A new recycling
system for expanded polystyrene using a natural solvent. Part 1. A new recycling
technique. Retrieved December 1, 2018, from
https://onlinelibrary.wiley.com/doi/pdf/10.1002/(SICI)1099-1522(199802)11:13.0.CO;2-
5

25
Obidi O. F., Adelowotan A. O., Ayoola G. A, Johnson O. O., Hassan M. O., Nwachukwu S. C.
U. (2016). ANTIMICROBIAL ACTIVITY OF ORANGE OIL ON SELECTED
PATHOGENS. Retrieved on December 14, 2018, from
https://mail.google.com/mail/u/0/#search/ee/FMfcgxvxBjRrDbFsWkSQjNXJXwlXklkC?
projector=1&messagePartId=0.1

12. Appendix

12.1. Appendix 1 – Raw Data and Data Processing

In order to obtain the mass of essential oil, mass of measuring cylinder and essential oil is subtracted with
mass of measuring cylinder.

𝑀𝑎𝑠𝑠 𝑜𝑓 𝑒𝑠𝑠𝑒𝑛𝑡𝑖𝑎𝑙 𝑜𝑖𝑙 = 𝑀𝑎𝑠𝑠 𝑜𝑓 𝑔. 𝑐𝑜𝑛𝑡𝑎𝑖𝑛𝑒𝑟 𝑎𝑛𝑑 𝑒𝑠𝑠𝑒𝑛𝑡𝑖𝑎𝑙 𝑜𝑖𝑙 (𝑔) − 𝑀𝑎𝑠𝑠 𝑜𝑓 𝑔. 𝑐𝑜𝑛𝑡𝑎𝑖𝑛𝑒𝑟 (𝑔)

Uncertainty of both data is obtained by adding the uncertainty of both data

±𝑀𝑎𝑠𝑠 𝑜𝑓 𝑒𝑠𝑠𝑒𝑛𝑡𝑖𝑎𝑙 𝑜𝑖𝑙 = ±𝑀𝑎𝑠𝑠 𝑜𝑓 𝑔. 𝑐𝑜𝑛𝑡𝑎𝑖𝑛𝑒𝑟 𝑎𝑛𝑑 𝑒𝑠𝑠𝑒𝑛𝑡𝑖𝑎𝑙 𝑜𝑖𝑙 + ±𝑀𝑎𝑠𝑠 𝑜𝑓 𝑔. 𝑐𝑜𝑛𝑡𝑎𝑖𝑛𝑒𝑟

Lemon Lime Mandarin Orange Pacitan Orange Sunkist

Valencia Orange
Mass of glass 10.8 9.9 10.2 11.1 10.5
container and
Essential oil (g)
±0.02
Mass of Essential 1.5 0.6 0.9 1.8 1.2
oil (g)
±0.01
Oil Yield (%) 2.14 0.857 1.28 2.57 1.71

Table 10.1.1. Volume of Essential Oil (ml) and its Oil Yield (%)
𝑀𝑎𝑠𝑠 𝑜𝑓 𝑒𝑠𝑠𝑒𝑛𝑡𝑖𝑎𝑙 𝑜𝑖𝑙 (𝑔)
𝑂𝑖𝑙 𝑌𝑖𝑒𝑙𝑑 (%) = × 100%
𝑀𝑎𝑠𝑠 𝑜𝑓 𝑐𝑖𝑡𝑟𝑢𝑠 𝑓𝑟𝑢𝑖𝑡 𝑝𝑒𝑒𝑙𝑠 (𝑔)

Mass of glass container = 9.3 g

Mass of citrus fruit peels = 70.0 g

26
 1.5 𝑔
For example, in lemon, Oil yield = × 100 = 2.14%
70 𝑔

There are other formula going to be used for the rest of data calculation. One of them is:

∑𝑛𝑖=1 𝑥𝑖
𝐴𝑣𝑒𝑟𝑎𝑔𝑒 (𝑥̅ ) =
𝑛

For example, Time for lime to dissolve Styrofoam is 220s, 119s, and 271s

220 + 119 + 271

𝐴𝑣𝑒𝑟𝑎𝑔𝑒 (𝑥̅ ) = = 203.3𝑠
3

Lemon Lime Mandarin Pacitan Sunkist Valencia

orange Orange Orange
1st Styrofoam test trial (s) 392 220 326 776 672
2nd Styrofoam test trial (s) 414 119 281 643 641
3rd Styrofoam test trial (s) 402 271 234 700 711
Average time 403 203 280 724 675

One other formula that is responsible for ANOVA calculation

𝐷𝐹(𝐵𝐺) = 𝑘 − 1 SS = Sum of squares

𝐷𝐹(𝑊𝐺) = 𝑁 − 𝑘 BG = Between Groups

𝑘
WG = Within Groups
2
𝑆𝑆𝐵𝐺 = ∑ 𝑛𝑖 (𝑥̅𝑖 − 𝑥̅ )
𝑖=1
k = number of groups
𝑘
𝑆𝑆𝑊𝐺 = ∑ (𝑛𝑖 − 1)𝑆𝑖 2 N = total number of subjects
𝑖=1
𝑛𝑖 = number of subjects in i-th group
𝑆𝑆𝐵𝐺 /(𝑁 − 𝑘)
𝐹 𝑠𝑡𝑎𝑡 = 𝑆𝑖 = standard deviation of i-th group
𝑆𝑆𝑊𝐺 /(𝑘 − 1)

27
ANOVA of Styrofoam test:

ANOVA summary
Source DF SS MS F-Stat P- Critical
Value Value
Between 4 623980.468 155995.117 58.361 0 3.48
Groups
(BG)
Within 10 26729.343 2672.943
groups
(WG)
Total 14 650709.811

Table 10.1.3. ANOVA summary determining F-stat for significance

Table 10.1.2. Time for essentiallevel
oils to dissolve Styrofoam

For example, in Styrofoam test:

𝐷𝐹(𝐵𝐺) = 5 − 1 = 4

𝐷𝐹(𝑊𝐺) = 15 − 5 = 10

623980.468/10
𝐹 𝑠𝑡𝑎𝑡 = = 58.361
26729.343/4

For the last investigation, there are uncertainties in the value of averages, which can be concluded
in formula:

𝑥𝑚𝑎𝑥 − 𝑥𝑚𝑖𝑛
𝑈𝑛𝑐𝑒𝑟𝑡𝑎𝑖𝑛𝑡𝑦 𝑜𝑓 𝐴𝑣𝑒𝑟𝑎𝑔𝑒 (±𝑥̅ ) =
2

For example, in first plate of lemon essential oil, area of inhibition has 113,1 mm 2 as minimum
294.5−113.1
and 294.5 mm2 as maximum, so 𝑈𝑛𝑐𝑒𝑟𝑡𝑎𝑖𝑛𝑡𝑦 𝑜𝑓 𝐴𝑣𝑒𝑟𝑎𝑔𝑒 (±𝑥̅ ) = = 90.7 𝑚𝑚2
2

Other formula involves Area of inhibition, where formula of area of circle is used:

28
 𝐴𝑟𝑒𝑎 𝑜𝑓 𝑐𝑖𝑟𝑐𝑙𝑒 = 𝜋𝑟 2

For example, zero zone surrounding 1st paper in the 1st plate of lemon essential oil has diameter
of 20 mm,

Radius = Diameter / 2 = 20/2 = 10 mm

Hence, 𝐴𝑟𝑒𝑎 𝑜𝑓 𝑖𝑛ℎ𝑖𝑏𝑖𝑡𝑖𝑜𝑛 𝑎𝑛𝑑 𝑝𝑎𝑝𝑒𝑟 = 𝜋102 = 314.2 𝑚𝑚2

This area includes the area of filter paper, with diameter 5 mm and radius 2.5 mm, so area obtained
above is subtracted with area of filter paper.

𝐴𝑟𝑒𝑎 𝑜𝑓 𝑓𝑖𝑙𝑡𝑒𝑟 𝑝𝑎𝑝𝑒𝑟 = 𝜋2.52 = 19.64 𝑚𝑚2

𝐴𝑟𝑒𝑎 𝑜𝑓 𝑖𝑛ℎ𝑖𝑏𝑖𝑡𝑖𝑜𝑛 = 𝐴𝑟𝑒𝑎 𝑜𝑓 𝑖𝑛ℎ𝑖𝑏𝑖𝑡𝑖𝑜𝑛 𝑎𝑛𝑑 𝑓. 𝑝𝑎𝑝𝑒𝑟 − 𝐴𝑟𝑒𝑎 𝑜𝑓 𝑓. 𝑝𝑎𝑝𝑒𝑟

So, Area of inhibition = 314.2 – 19.64 = 294.5 mm2

No. No. Diameter of inhibition and filter paper (mm) ± 0.1 mm

plates Trials
Lemon Lime Mandarin Pacitan Sunkist
Orange Orange Valencia
orange
1st 1 20 10 11 5 5
plate 2 18 14 11.5 5 5
3 14 11 15 5 5
4 13 12 6 5 5

2nd 1 16 15 13 5 12
plate 2 14 18 10 5 11
3 14 18 17 5 10.5
4 12.5 16 16 5 10

Table 10.1.3. Diameter of inhibition and filter paper

29
 Since diameter is measured with metal ruler, with uncertainty of ±0.1 mm, hence uncertainty of
area of inhibition = (±0.1) + ±0.1 mm = ±0.2 mm
No. No. Area of Inhibition and Filter paper (mm2) Area of Inhibition (mm2) ± 0.2 mm
plates trials ± 0.1 mm

Lemon Lime Mandarin Pacitan Sunkist Lemon Lime Mandarin Pacitan Sunkist
Orange Orange Valencia Orange Orange Valencia
Orange Orange
1st 1 314.2 66.5 95 19.6 19.6 294.5 96.9 75.4 0 0
plate 2 254.5 232.7 103.9 19.6 19.6 234.8 113.1 84.3 0 0
3 153.93 95 176.7 19.6 19.6 134.3 102.0 157.1 0 0
4 132.7 98.1 28.3 19.6 19.6 113.1 88.5 8.7 0 0

2nd 1 203.6 183.8 132.7 19.6 113.1 184 164.2 113.1 0 93.5
plate 2 156.7 255.5 78.5 19.6 95 137.1 235.9 58.9 0 75.4
3 156.7 249.1 80.9 19.6 86.6 137.1 229.5 61.3 0 67
4 123.1 198.1 201.1 19.6 80.9 160.6 178.5 181.5 0 61.3

Table 10.1.4. Area of inhibition and filter paper and Area of inhibition

No. plates Average area of inhibition (mm2)

Lemon Lime Mandarin Pacitan Sunkist
Orange Orange Valencia
Orange
1st plate 294.5 123.1 81.4 0 0
(± 90.7) (±12.3) (±148.4)
2nd plate 154.7 202.03 103.7 0 74.3
(±23.45) (± 35.85) (±138.3) (± 16.1)

Table 10.1.5. Average area of Inhibition and its uncertainties

30
Sample SS (BG) DFx SS (WG) DFy F-Stat P-Value Critical
Value
Lemon 3038.101 23506.7109 0.776 0.406 5.99
Lime 20767.22 4203.9583 29.6395 0.0016
Mandarin 996.8113 1 21006.1615 6 0.2847 0.613
Orange
Pacitan 0 0 - -
Orange
Sunkist 11040.98 592.1401 111.8754 0
Valencia
Orange

Table 10.1.6 ANOVA summary of

antimicrobial data

Control (0.01% Ciprofloxacin)

Diameter of filter paper and Area of filter paper and Area of inhibition (mm2) ±
inhibition (mm) ± 0.1 mm inhibition (mm2) ± 0.1 mm 0.2 mm

35 962.1 942.5
28 615.8 596.2
31 754.8 735.2
31 615.8 596.2
Average 717.5 (±346.3)

Table 10.1.7 Antimicrobial test raw and processed data of

Ciprofloxacin 0.01% (control)

Diameter of filter paper

= 5 mm
Area of filter paper
= 19.6 mm2

31
 Appendix 2 – Documentation of the Investigation

10.3.1 Essential oil extraction

Image 10.3.1.3 Sunkist Valencia

Image 10.3.1.1 Distillation set Image 10.3.1.2 Lemon essential essential oil in separating funnel
up oil in separating funnel

Image 10.3.1.4 Pacitan essential Image 10.3.1.5 Lime essential

oil in separating funnel oil in separating funnel Image 10.3.1.6 Mandarin
essential oil in separating funnel

32
 Image 10.3.1.6 Essential oil
placed in small bottle

10.3.2 Styrofoam Test

Image 10.3.2.1 20 g mass

containing essential oil at the
bottom of mass placed on
styrofoam

33
Image 10.3.2.2 Lemon essential oil
dissolves Styrofoam (3 trial)

Image 10.3.2.3 Lime essential oil

dissolves Styrofoam (3 trial)

Image 10.3.2.4 Mandarin essential oil

dissolves Styrofoam (3 trial)

Image 10.3.2.5 Pacitan essential oil

dissolves Styrofoam (3 trial)

34
 Image 10.3.2.6 Valencia essential oil
dissolves Styrofoam (3 trial)

10.3.3. Antimicrobial test

Image 10.3.3.2 Autoclave is maintained at

Image 10.3.3.1 Making of Image 10.3.3.3 Arrangement
120 kpa
agar solution of apparatus inside autoclave

Image 10.3.3.4 Agar solution in

petri disks Image 10.3.3.6 Sterilize inoculating loop
Image 10.3.3.5 E. Coli Bacteria with spirit burner

35
 Image 10.3.3.7
Streaking Bacteria Image 10.3.3.8
into agar medium Filter paper
containing
essential oils in
porcelain

Image 10.3.3.9 Minimum inhibitory

concentration of lemon essential oil (1)

Image 10.3.3.10 Minimum inhibitory

concentration of lemon essential oil (2)

Image 10.3.3.11 Minimum inhibitory

concentration of lime essential oil (2) Image 10.3.3.12 Minimum inhibitory concentration of
lime essential oil (1)

36
 Image 10.3.3.13 Minimum inhibitory Image 10.3.3.14 Minimum inhibitory
concentration of Sunkist Valencia essential oil (1) concentration of Sunkist Valencia essential oil (2)

Image 10.3.3.15 Minimum inhibitory Image 10.3.3.16 Minimum inhibitory

concentration of Pacitan essential oil (2) concentration of Pacitan essential oil (1)

Image 10.3.3.17 Minimum inhibitory

Image 10.3.3.18 Minimum inhibitory
concentration of Mandarin essential oil (1)
concentration of Mandarin essential oil (2)

37