Antibacterial Activity of Mugwort Essential Oil’s Against E.

coli
1

Evaluation of the Antibacterial Activity of Mugwort (Artemisia vulgaris) Leaf Essential

Oil Against Escherichia coli

Jekka Roan O. Arciaga

Glaiza F. Cobcobo

Ruq Ezer B. Behis

Carl Jericho P. Garcia

Theo Joshua A. Herrera

Aishwarya Deneil B. Anama

Carl Wilson P. Naboye

Baguio City National Science High School

Science, Technology, Engineering and Mathematics Program

Authors’ Note

The researchers would like to express their gratitude to Ma'am Lisa Roxas for

the support given to the researchers while writing this research proposal.

Any questions, clarifications or suggestions should be addressed to the email

address of Theo Joshua A. Herrera at theoherrera76@gmail.com

Antibacterial Activity of Mugwort Essential Oil’s Against E.coli
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METHODOLOGY

Research Design

This study utilised a pure experimental research design to test the causal

relationship between the application of mugwort essential oil and inhibition of E. coli

in a controlled environment. The specified experimental design used was Completely

Randomised Design (CRD). It induced randomization best suited for an experiment

with a small number of treatments and it ensured the collection of unbiased data

through the absence of purposeful sorting.

Figure 1: Framework for the completely randomised research design to be used to evaluate the
antibacterial activity of A. vulgaris against E. coli

Data Gathering Materials

All apparatus and machinery used in this experiment was obtained from

Philippine Science High School Cordillera Administrative Region Campus (PSHS-

CARC) and from Baguio City National Science High School (BCNSHS) (Baguio City,

Cordillera Administrative Region, Philippines) apart from the plant material which was

acquired from a local vendor at the Baguio City Orchidarium and within the Upper

Quezon Hill Barangay.

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Distilled water was used in the washing of all apparatus in contact with the

extract, washing of plant samples, decoction of crude extract, and making of Mueller

Hinton Agar. A large metal pot and gas stove was used for the decoction of crude

extract. A hotplate, metal tray, and seven 50 ml beakers were used in the isolation of

the essential oil through a water bath. A 50 ml beaker, 5 g of anhydrous sodium

sulphate, and a strainer was used for drying the essential oil. Three 10 ml amber

vials and a refrigerator was used for the storage of the extract throughout the

experiment. 30% ethanol was used in the dilution of the extract to achieve different

test concentrations.

Alongside laboratory coats, latex gloves, laboratory goggles, and N95 masks

were worn by the researchers in preparation for handling the bacteria. Cooked

Mueller Hinton Agar was prepared by PSHS-CARC. An autoclave was used to

sterilise the glassware and agar, Thirteen 100 mm petri dishes were used for

bacterial isolation (1 petri dish) and well diffusion (12 petri dishes). A culture of E. coli

provided by PSHS-CARC was used for isolation onto one of the 100 mm petri dishes.

A 5 ml glass vial of saline solution, a vial of 0.5 McFarland turbidity standard solution

and a McFarland turbidity standard comparison card was used to standardise the

turbidity of the bacterial suspension.

Wooden cotton applicators were used to streak the bacteria onto twelve 100

mm petri dishes. A bunsen burner was used to sterilize the lip of the petri dishes.

Bond paper, masking tape, and a permanent marker was used in labelling the petri

dishes for storage.

During well diffusion, a 4 mm well cutter was used to bore holes into the agar.

A micropipette was used for the inoculation of the antibacterial agents. For recording

observations, an Ipad Air 4 was used to take pictures of the petri dishes from 1 ft

away, a distance that was measured using a 12-inch ruler. A fractional calliper was
Antibacterial Activity of Mugwort Essential Oil’s Against E.coli
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also used to record the diameter of bacterial growth inhibition around each well.

Data Gathering Procedure

Collection of plant materials. A. vulgaris was collected from a local vendor

at the Baguio City Orchidarium (Baguio City, Cordillera Administrative Region,

Philippines, 16.4110 N, 120.5924 E) and in the Upper Quezon Hill Barangay (Baguio

City, Cordillera Administrative Region, Philippines, 16.4167 N, 120.5760 E).

Artemisia vulgaris leaf essential oil preparation. Fresh mugwort leaves

were washed in distilled water repeatedly before being sun-dried for 5 days.

Extraction of crude extract was done through decoction. The leaves were put in a

large metal pot, which was filled with distilled water until fully submerged. The pot

was heated to 90°C and boiled at that temperature for 1 hour (Stéphane et al., 2022).

The extracted essential oil was isolated by water bath using seven 50 ml beakers

over the course of 3 weeks in the BCNSHS laboratory. The remaining water in the

essential oil was absorbed through spooning in 5 g of anhydrous sodium sulphate,

letting it absorb the water, and straining out the final extract. The essential oil was

kept in three amber vials and stored in a refrigerator until use (Judžentienė &

Būdienė, 2020).

Before experimentation, the essential oils were diluted in 30% ethanol using a

micropipette to achieve the following concentrations: (1) 50% mugwort essential oil,

50% thirty percent ethanol; (2) 75% mugwort essential oil, 25% thirty percent ethanol;

(3) 100% mugwort essential oil. The filtered substances were labelled as mugwort

leaf extract (MLE) alongside their specific concentrations. (Xi et al., 2022).

Culture media and inoculum preparation. PPE was worn in the laboratory

in preparation for handling the bacteria, these included proper latex gloves,

laboratory goggles, laboratory coats, and N95 masks (Personal Protective

Equipment (PPE) in Laboratories | EHS, n.d.). All glassware to be used in the

Antibacterial Activity of Mugwort Essential Oil’s Against E.coli
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experiment was sterilized in an autoclave at its highest temperature (°C) for 1 hour.

Cooked Mueller Hinton Agar (MHA) was pre-prepared in the PSHS-CARC lab. The

mixture was poured inside a 500 ml erlenmeyer flask, covered with a piece of cotton,

this was sterilized by autoclaving at the highest temperature (°C) for 1 hour to

eliminate potential contaminants. The mixture was then cooled to room temperature.

The cooled Mueller Hinton Agar was poured into a graduated cylinder to measure 20

ml, depending on how much the MHA can fill the 12 petri dishes. Then, it was poured

into sterile petri dishes until they were filled with 20 ml of MHA. Afterward, the dishes

were left on a flat surface to cool while completely lidded.

A sample of E. Coli was retrieved from PSHS-CARC. The bacteria was

isolated onto a fresh agar dish using wooden cotton applicators. Before closing the

dishes, all petri dishes were held close to the bunsen burner for sterilization. The

isolation dish was then incubated at room temperature for 48 hours (Bacterial

Isolation - Microbiology Resource Center - Truckee Meadows Community College,

n.d.). The isolated bacteria was dissolved in a 5 ml glass vial of saline solution using

an inoculation loop until the turbidity matched the 0.5 McFarland Standard after

comparison with a vial of 0.5 McFarland turbidity standard solution and a McFarland

Turbidity Standard Comparison Card.

The resulting solution was streaked onto twelve 100 mm petri dishes with

wooden cotton applicators. The petri dishes were lidded, wrapped in bond paper, and

taped closed. The plates were then randomly labelled 1 to 12 near the edge of each

petri dish using a permanent marker, the plates were also labelled with the name of

the researchers’ representative, institution, grade, and section. The experimental

petri dishes were kept in an incubator at room temperature for another 48 hours to

propagate the lawn culture of E. coli.

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Testing antibacterial effectivity through well diffusion. Each agar plate

was divided into 4 quadrants of equal area. In the middle of each quadrant, wells

were made using a 7 mm sterile borer. Then the bottom of the agar was sealed with

sterile molten agar to avoid leakages. After the agar had set, the wells were filled with

0.1ml of each antibacterial agent. The plates were then incubated at 37ºC for 24

hours (Naz et al., 2010).

Observation and collection of data. The diameter of the zones of inhibition

around each of the wells was taken as a measure of the antibacterial activity. The

mean diameter of the inhibition zone was recorded for each antibacterial agent.

Photos were taken exactly 1 foot above the petri dish lids using an Ipad Air 4

for later visual reference. To accurately record data, the zone of inhibition around

each well was measured using a fractional calliper. Results were recorded in the

table below.

Table 1: Mean zones of inhibition on E. coli when exposed to 50% MLE, 75% MLE, 100% MLE, and
Amoxicillin

Petri Dishes Antibacterial Agents

50% MLE 75% MLE 100% MLE Amoxicillin

8
Antibacterial Activity of Mugwort Essential Oil’s Against E.coli
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10

11

12

Mean ZOI:

Data Analysis

This study utilised the inferential statistical analysis tool, one-way ANOVA

using the software application Jamovi with a significance level of p=0.05. This was

used to compute the distribution or variance of the mean zones of inhibition of each

antibacterial agent used in order to ascertain their consistent efficacy in comparison

to each other.
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Bacterial Isolation - Microbiology Resource Center - Truckee Meadows

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