A study on the effects of changing different concentrations (0%, 2%, 4%, 6%, 8%,10%

and 12%) of Tulsi (Ocimum Sanctum) essential oil on Escherichia coli growth
inhibition zones (mm)

Research question: What is the effect of different concentrations of Tulsi (Ocimum

sanctum) (0%, 2%, 4%, 6%, 8%, 10% and 12%) on Escherichia coli grown on agar, by
measuring the maximum radii of inhibition zones (mm) from the disks formed over 24 hours.

Background information:
Ocimum sanctum commonly known as Tulsi or holy basil is a sacred herb in the Hindu
religion. It is used as a traditional medicinal herb to treat a variety of different diseases
including bronchitis, bronchial asthma, malaria, dysentery, skin diseases, arthritis, chronic
fever, insect bites, etc. It is believed to possess anticancer, antidiabetic, antispasmodic,
antifungal and antimicrobial properties (Prakash & Gupta, 2005).

Figure 1: The tulsi plant (source: Authors own)

Culturally, this herb has always been used in our

household as a natural alternative to prevent and treat
the common cold. I was therefore interested in
examining the antibacterial properties of this herb to see
whether it had an effect on bacterial growth.

Bacteria are single celled organisms that are

metabolically active and reproduce by binary fission
(Baron, 1998). E.coli belong to the family
Enterobacteriaceae and are gram-negative bacteria
meaning that they have a thin peptidoglycan layer. E.coli
also contain a hard protective outer capsule which
makes them harder to kill.

Gram-negative bacteria are a threat to global health, food security and development as they
are increasingly resistant to antibiotics (World Health Organization, 2020). Evolutionary
pressures caused by the overuse of antibiotics causes bacteria to mutate and develop
resistance mechanisms against antibiotics (Lupo et al., 2012). Globally, infections such as
pneumonia, tuberculosis, blood poisoning, gonorrhea and foodborne diseases are harder to
treat as antibiotics have become less effective (World Health Organization, 2020).

Antibiotic alternatives such as bacteriophage therapy, predatory bacteria, bacteriocins, and

competitive exclusion of pathogens do not consistently demonstrate efficacy compared to
antibiotic treatment (Allen, 2020). For this reason, research into alternative treatments such
as medicinal properties of plants is critical, as it may help reduce reliance on antibiotics. Tulsi
extract contains antimicrobial compounds including eucalyptol, phenols, quinones,
terpenoids and Flavonoids (Yamani et al., 2016). Phytochemicals such as ursolic acid,
rosmarinic acid and oleanolic acid also contribute to the antimicrobial properties of Tulsi
(Dixit, 2021).

Scientists at the Manipal College of Dental Sciences have found Tulsi to have efficacy on
periodontal bacteria. They found that as the concentration of Tulsi extract increased (from
0.5%,1%, 2%, 5%, and 10%), growth inhibition zones increased against A.
actinomycetemcomitans, P. gingivalis and P. intermedia. At 10%, the widest inhibition zone
of 22mm was created (Mallikarjun et al., 2016). Tantry et al (2015) found a 10%
concentration to create the widest inhibition zone of 13±0.32 mm whilst experimenting on
E.coli. However, Eshwar et al (2016) found the most significant concentration at 6% where
the inhibition zone was 22mm with Actinobacillus actinomycetemcomitans. Experiments on
the antibacterial activity of Tulsi using disk diffusion and E.coli are still in their infancy. As of
yet, research with this methodology has not been published. However, the Eswar study
1
 (2016) uses a similar methodology. Additionally, Actinobacillus actinomycetemcomitans is
gram-negative and will likely behave similarly to E.coli.

Hypothesis
Null hypothesis: Changing the extract concentration (0%, 2%, 4%, 6%, 8%, 10%, 12%) will
not affect the growth of E.coli.
Alternative hypothesis: Changing the concentration of tulsi extract concentration (0%, 2%,
4%, 6%, 8%, 10%,12%) will show a positive correlation with E.coli growth inhibition zones
with the widest zone of inhibition at a 6% concentration.

Variables
Independent variables: The percentage of Tusli extract concentration (0%, 2%, 4%, 6%,
8%, 10%, 12%) made by dilution with ethanol.
These concentrations were chosen as preliminary trials found a 6% concentration to be the
most significant concentration. This was similar to the findings by Eshwar et al (2016).
Inhibition zones continued to increase at 8%, 10% and 12% concentrations, however, there
was only a marginal increase in their size. 6% was therefore chosen as the median value.
Ocimum sanctum was chosen for this experiment as opposed to other varieties of
tulsi for its increasing popularity as a ‘natural’ medicine. Additionally, this variety is easily
grown and available to local communities in rural areas.

Dependent variables: The maximum radius from the edge of the filter paper disk to the
edge of the inhibition zone. Bacterial growth inhibition zones are measured using a stainless
steel caliper formed over 24 hours. This time period has been chosen as preliminary trials
showed that it was a sufficient amount of time for bacterial growth.
Non-pathogenic E. coli was used in this experiment as it reproduces quickly, was
easily available, inexpensive and safe to use in the school laboratory (Cooper, 2000).

Figure 2: Control variable, reason for controlling and method of controlling

Control variable Reason for controlling Method of controlling

The source of Tulsi extract
Due to genetic variation amongst
different Tulsi plants range in nutritional
composition. The amount of antibacterial
properties will therefore vary from
individual plants.
Tulsi extract was sourced from
Banyan Botanicals which organically
farms Tulsi in south, central India.
Leaves and stems of different Tulsi
plants are powdered through a milling
machine ensuring homogeneity.

Temperature of Incubation
Temperature affects metabolic activity of
bacteria. Growth and binary fission of
E.coli change at different temperatures.
Incubation at the same temperature will
ensure variation in E.coli growth is due to
Tulsi concentration rather than variations
in temperature.
E.coli are able to grow in
temperatures ranging from 21°C to
49°C. They have an optimum growing
temperature of 37°C. Due to potential
human contamination (see safety
section), bacteria will be grown in an
incubator set at 25°C.

Duration of experiment
As time increases, bacterial growth and
division will also increase. To ensure
comparison between Tulsi extract on
E.coli growth inhibition, time has to be
standardized.
All petri dishes containing E.coli are
incubated for 24 hours. Time is
measured using a clock.

Distribution of bacteria
Different bacterial concentrations of
E.coli will result in varying levels of
growth. This must be controlled to ensure
that inhibition is due to extracts rather
than a lack of bacteria.
The entire agar surface is inoculated
for 30 seconds by streaking E.coli
using an inoculum (see method
section). Due to different inoculation
techniques amongst individuals, the
same person is used in each trial.
2
The time and temperature
each disc was saturated in
the extract
The longer the disks are soaked, more
Tulsi extract will be absorbed by the
disks. Additionally, temperature affects
the rate of diffusion which would affect
saturation of the disks.
Filter paper disks were placed in
different concentrations and placed in
an incubator at 25°C for 24 hours.

Agar exposure to air Microorganisms in the air could All petri dishes are covered with glass
contaminate agar plates by inhibiting lids and stored upside down for the
E.coli growth or culturing other duration of the experiment. However,
microorganisms species. they were not further sealed.

Sterilization of materials To prevent contamination of Surfaces are cleaned with 100%

microorganisms affecting results and to ethanol. All equipment was placed in
prevent the growth of pathogenic a heat oven at 200°C for 20 minutes.
microorganisms that could raise safety The inoculator was sterilized using a
concerns. bunsen burner flame.

Size of filter paper disks
Surface area of filter paper disks affects
the amount of liquid being absorbed.
Hence, diffusion of the Tulsi extract
would vary depending on the size of the
filter paper.
Prior to experimentation, the same
hole puncher was used to make all
the filter paper disks. The filter paper
disks have a 7mm diameter.

Source of agar solution Nutritional composition in agar affects All agar plates used the same stock
bacterial growth. solution.

Control group
A control group is needed to ensure that variation of growth inhibition zones is due to the
manipulation of Tulsi extract concentrations rather than other variables. Pure ethanol without
Tulsi powder was used as a control to test the effects on E.coli growth.

Notes from preliminary trials:

The first preliminary trials showed no effects on bacterial growth. I thought that this could be
due to the lack of a prior extraction process to break down the Tulsi leaf, releasing its active
compounds. I included a process of maceration using ethanol to counter this in my second
trial. However, this did not seem to have an effect. Additionally, I was using a bought powder
which had sat on a shelf for a long time. I hypothesized that this could have reduced its
potency resulting in insignificance on bacterial growth. In my next trial, I collected my own
leaves from a Tulsi tree. However, there was still no effect on growth inhibition zones. Finally,
I changed the disc soaking time from 30 minutes to 24 hours using the Banyan Botanicals
Tulsi. Growth inhibition zones were then created. I experimented with using dimethyl
sulfoxide, ethanol and water as solvents. Ethanol was finally chosen as it showed the most
significant results.

3
 Materials:
● 1 Electronic weighing scale (±0.01) ● 1 Inoculator
● Powdered Banyan Botanicals Tulsi ● 1 non-pathogenic Escherichia coli
(Ocimum sanctum) (10.5g±0.01) growth culture on agar
● 1 Metal spatula ● 1 Forceps
● 1 Measuring cylinder (50ml ±0.5) ● 1 Stainless steel caliper (mm±0.01)
● Ethanol (175ml±0.5) ● 7 Empty petri dishes with lids
● 6 Glass rods ● 20 Filter paper disks with 7mm
● 1 Incubator set to 25°C diameter
● 1 Clock for timing ● 1 Lab coat
● 5 Glass beakers (100ml) ● 1 pair safety goggles
● 1 Bunsen Burner ● Kolibri Latex gloves
● 1 Match box/lighter ● 100% Ethanol for sterilizing
● 7 Premade Agar plates surface
Note: All materials were sterilized in a heat oven for 20 minutes at 200°C. Before
experimentation, 100% ethanol was used to sanitize the working area.

Method:
Preparation of concentrations
1.1 Place a 50 ml beaker on a weighing scale and set it to 0.
1.2 Weigh the appropriate amount of the Tulsi powder (see Figure 3) in the beaker using a
metal spatula. Avoid residue on the sides of the beaker.
1.3 Using a 25 ml measuring cylinder, add 25 ml of ethanol into each beaker. Thus resulting
in 6 different concentrations and a control group only containing ethanol.
1.4 Stir the solutions using a sterilized glass rod for 120 seconds each. Measure time using
a clock.
1.5 Immediately pour the solutions into the empty petri dishes avoiding sedimentation of the
extract
1.6 Using sterilized forceps, place 4 disks evenly spaced in each dish. Cover the dishes with
lids immediately to avoid contamination of the extracts.
1.7 Place the dishes in an incubator set to 25°C and leave them to soak for 24 hours, using
a clock to measure the time.

Figure 3: Tulsi extract and ethanol concentrations

Beaker concentration (% ±0.1) Tulsi extract (g ±0.01 Ethanol (ml ± 0.5)

1 (control) 0 0.00 25.0

2 2 0.5 25.0

3 4 1.0 25.0

4 6 1.5 25.0

5 8 2.0 25.0

6 10 2.5 25.0

7 12 3.0 25.0

Streaking of Agar plates with E.coli

2.1 After 24 hours have elapsed, divide agar plates into four equal segments using a ruler
and marker. Agar plates were prepared by the lab assistant prior to experimentation.
2.2 Flame the inoculation loop in the bunsen burner until red in color and hold for 5 seconds.
This sterilizes the inoculator from pre existing bacteria.
2.3 Cool the inoculation loop by placing the tip in the agar at the edge of the petri dish.
2.4 Swipe the inoculation loop across the non-pathogenic E.coli culture to obtain a pinhead
sample of the bacteria.
4
2.5 Continuously swipe the surface of the agar with the inoculation loop making sure to
create a uniform layer of bacteria. Measure a 30 seconds streaking time with a clock (see
Figure 4 for visual set up.)
2.6 Repeat steps 2.1 - 2.5 for 7 agar plates.
2.7 Cover the agar plates immediately with glass lids to prevent contamination.

Disk diffusion method

3.1 Place the disks in the middle of each divided quadrant of the agar plate using sterilized
forceps (see Figure 4 for the visual setup of the petri dish.)
3.2 Cover immediately, and incubate at 25ºC for 24 hours. Use a clock to measure time.

Determining the results

4.1 Using a stainless steel metal caliper, measure the distance (mm) from the edge of the
disk to the maximum radii of inhibition of E.coli growth.
4.2 Repeat the experiment twice on the same day to obtain 8 trials for each Tulsi
concentration.

Figure 4: Visual of petri dish setup

Safety, Ethics and Environmental impact

Safety regulations were carried out to ensure the proper measures were taken while
handling E.coli bacteria. Firstly, the E.coli obtained was non-pathogenic and cultured for
experimentation. Hair was tied up, and latex gloves and a lab coat were worn. Additionally,
hands were washed and disinfected. Prior to experimentation, all equipment used was
placed in a heat oven for one heat cycle at 200 degrees for 20 minutes and surfaces were
disinfected with 100% ethanol. During experimentation, the bunsen burner was handled with
care to prevent burning and an experienced lab technician lit the bunsen burner. All E.coli
cultures were incubated at 25 degrees to prevent ethical concerns relating to incubating
bacteria at body temperatures. Agar plates were then disposed of by the lab technician in
accordance with safety protocols. There may be ethical concerns associated with
experimenting on a sacred and worshiped herb. However, the possible insight provided by
experimenting on Tulsi could increase understanding of its antimicrobial properties. Thus,
the benefits of such experimentation outweigh the ethical concerns.

5
 Qualitative Observation
● The growth inhibition zones created were not uniformly circular in any of the
concentrations
● There were circular cultures of E.coli around streaked line colonies
● No mold growth was observed
● There were areas of streaked E.coli that grew more densely than other areas.

Results:
Figure 5: The size of growth inhibition zones (mm ±0.5) with altering different
concentration of Tulsi extract (%±0.1)
Substance Percentage E.coli growth inhibition zones (mm ±0.5)
concentration
(%±0.1) Trial 1 Trial 2 Trial 3 Trial 4 Trial 5 Trial 6 Trial 7 Trial 8
0 1.84 1.10 1.15 0.68 0.00 0.24 0.00 0.00
2 2.04 4.00 1.03 0.80 0.78 2.48 1.22 2.23
4 11.31 9.42 8.38 10.63 7.98 6.36 7.87 4.92
Tulsi ethanolic
6 11.34 6.15 9.49 7.26 9.49 11.46 7.45 14.10
extract
8 9.48 12.59 10.13 14.10 5.90 8.76 7.92 10.00
10 10.35 13.46 6.47 7.66 8.81 8.54 14.30 11.75
12 14.67 12.82 11.63 8.52 8.79 9.31 10.05 11.58

Figure 6: Mean size of growth inhibition zones mm ±0.5) with altering different
concentration of Tulsi extract (%±0.1) and its standard deviations
Percentage
Substance Mean (mm ±0.5) Standard deviations (mm)
concentration (%0.01)
0 0.63 0.67
2 1.82 1.10
4 8.36 2.11
Tulsi ethanolic extract 6 9.59 2.64
8 9.86 2.57
10 10.17 2.80
12 10.92 2.15

Sample Calculation: (using 2% concentration as an example)

Mean: 1. Add up the growth inhibition zones
∑𝑥
of all the trials in one particular
𝑥= concentration
𝑛
2. 04 + 4. 00 + 1. 03 + 0. 80 + 0. 78 + 2. 48
+ 1. 22 + 2. 23 = 14. 58
Where 𝑥 = is the growth inhibition zone 2. Divide by the sum by the total
(mm) amount of trials
14.58
𝑛 = is the number of trials growth inhibition 8
= 1. 8225
zones were measured 3. Round to two decimal places
= 1. 82

6
 Standard deviations: 1. Square the distance from the mean
to each data point
2
(
∑ 𝑥 𝑖−µ )
σ = 𝑁 (2. 04 − 2)
2
= 0. 0016
2
(4. 00 − 2) = 4
Where 𝑁 = the number of trials growth 2
inhibition zones were measured (1. 03 − 2) = 0. 9409
2
(0. 80 − 2) = 1. 44
𝑥 𝑖
= each value of the inhibition zones (0. 78 − 2)
2
= 1. 4884
2
(2. 48 − 2) = 0. 2304
µ = The mean value of E.coli growth 2
inhibition zones (1. 22 − 2) = 1. 6084
2
(2. 23 − 2) = 0. 529

2. Find the average of the results from

step 1 (figures rounded to 2 d.p.)

0.00+ 4.00+0.94+1.44+1.49+0.23+1.61+0.53
8
= 1. 10

3. Find the square root

1= 1

Figure 7: Graph showing the mean radii of E.coli growth inhibition zones (±0.5%) with
varying concentrations of Tulsi extract (±0.1) and its standard deviations

Statistical Analysis
A statistical test is needed as there is an overlap in the standard deviations of growth
inhibition zones as seen in Figure 7. This will determine whether there is enough evidence to
accept the alternative hypothesis that differing Tulsi contractions affect E.coli growth
inhibition zones. An ANOVA variance test (see appendix A for formula) was performed on all
concentrations (0-12%) as opposed to a t-test, as there are more than two sets of data
(there are seven concentrations being tested.)

7
 Null hypothesis: Changing the extract concentration (0%, 2%, 4%, 6%, 8%, 10%,
12%) will not affect the growth of E.coli.

Alternative hypothesis: Changing the concentration of tulsi extract concentration

(0%, 2%, 4%, 6%, 8%, 10%,12%) will show a positive linear correlation with E.coli
growth inhibition zones with the widest zone of inhibition at a 6% concentration.

The F-value of 31.556 is greater than the decision value of 0.000 at p<0.05. Although the
graph shows overlap in the standard deviations of inhibition zones, the null hypothesis is
rejected as the F-value is not in the 95% region of acceptance. Moreover, the small P-value
indicates that the alternative hypothesis can be strongly supported.

Data Analysis
The graph shows a positive correlation between the radii of growth inhibition zones and
increasing Tulsi extract concentrations. The trend further is supported by the ANOVA test
which showed the F-value at 31.556 which is significantly larger than the decision value of
p=0.000. Additionally, the presence of smaller to no inhibition zones with pure ethanol,
suggest that Tulsi extract did have an effect on growth of bacteria as inhibition zones
significantly increased with rising concentrations. The results obtained displayed the
maximum antimicrobial potential at a 6% concentration, after which inhibition zones
increased marginally with high standard deviations. These findings support the results of the
Eshwar study (2016) which also found a 6% concentration to have the most significance on
bacterial growth. However, these results differ from the findings of the Tantry et al (2015) and
Mallikarjun (2015) studies that found a 10% to have the maximum antimicrobial potential.
The highest average of growth inhibition zones were much larger in the Mallikarjun (2015)
and Eswar study (2016). These studies both found the highest average of inhibition zone
radii at 22mm compared to 10.92mm in this experiment. However, the highest radii in this
experiment was more closely aligned with the results of Tantry et al (2015), which found an
inhibition zone of 13mm whilst also experimenting on E.coli. This could indicate that E.coli
has lower susceptibility to Tulsi compared to other bacterial strains. The differences in these
studies could be explained by differences in methodology between the three studies as well
as in individual variation between Ocimum sanctum plants. It is likely that the Tulsi had lost
some potency due to age as the other studies used fresh leaves. The inhibition zones
generated from the use of ethanol were somewhat unexpected as they were small or not
present. This could have been because the ethanol may have evaporated from the disks as
agar plates were not completely sealed.

Factors that contribute to the antibacterial properties of Tulsi

Quantitative analysis has found the presence of several phytochemicals that contribute to
the antibacterial properties of the Tulsi plant. These include phenols, quinone, terpenoids,
flavonoids, eucalyptol, and various acids. Phenols such as eugenol disrupt cellular
membranes and inhibit microbial enzymes. This causes cell leakage and leads to an
increased cellular permeability (Heipieper et al., 1991). Quinone are biological pigments that
have antimicrobial potency. Their structure contains heteroatoms and hydrogen making them
more reactive and combative against microorganisms (Dahlem et al., 2022). Terpenoids are
a set of secondary metabolites that are found in Tulsi (Dixit et al., 2021). They contain
monoterpene, diterpene and carvone which have also shown antimicrobial potency (Yang et
al., 2020). Flavonoids are found in photosynthesising cells of Tulsi and have been found to
inhibit the synthesis of nucleic acids, proteins and lipids within bacterial cells. It is proposed
to affect hydrogen bonding with the stacking of nucleic acid bases during the synthesis of
DNA and RNA. Flavonoids also inhibit cytoplasmic membrane function by increasing
membrane fluidity and therefore killing bacteria (Cushnie et al., 2005). Small quantities of
eucalyptol have been found in Tulsi which induce oxidative stress and disrupt the bacterial
membrane (Moo, 2021;Yamani et al., 2016). Tulsi also contains rosmarinic acid and
oleanolic acids which have shown efficacy against bacterial growth, however the reason is
unclear (Adamczak et al,. 2019). Ursolic acid, also found in Tulsi, has shown a synergistic
effect on other antibacterial agents (Nascimento et al., 2014).

8
 Conclusion
All in all, the results support the alternative hypothesis that changing the concentration of
Tulsi extract concentration (0%, 2%, 4%, 6%, 8%, 10%, 12%) would show a positive
correlation with radii of E.coli growth inhibition zones. There is enough evidence found from
the ANOVA variance test and processed data to accept the alternative hypothesis.

Evaluation
One strength of this investigation was that all materials, aside from E.coli and Tulsi powder,
were easily obtained and could be found in a standard laboratory. Additionally, having a
control in the experiment allowed for comparison between an already known bactericide and
the Tulsi extract. Moreover, the experiment had eight trials for each concentration. This
reduced the effects of anomalies, decreased the standard deviations and also increased the
precision of results. Furthermore, the experiment was conducted in a school laboratory
where variables were highly controlled. A strength of this investigation is its efficacy in
relation to global health. Tulsi is commonly found in rural areas of India where modern
medical intervention may not be available. This experiment highlighted that Tulsi extract is
an effective bactericide providing medical alternatives in rural communities where the plant is
present. In regards to public health, Tulsi may reduce reliance on antibiotics thus reducing
bacterial resistance. However, there are limitations to this experiment that are discussed in
figure 8.

Figure 8: Limitations, impact of limitations and suggested improvements

Limitations Impact of limitations Suggested improvement

Lack of a negative control
(systematic error)
The effects of normal bacterial growth
without bactericides was not observed
as ethanol has created inhibition
zones. Thus, what would have
happened without intervention is not
clear.
Having a negative control such as
disks soaked in water would enable
further comparison and ensure that the
results were solely based on the Tulsi
extract.

Control of agar thickness Studies (Flanagan, 2017) have shown Standardizing the agar thickness by
(random error) that agar thickness can affect the using the Kirby-bauer disk diffusion
reliability of microbial assay. method with 4mm agar plates.

Limited concentration range There were only five concentrations Increasing the range of concentrations
(systematic error) which were all within 12%. The data would allow for greater analysis on the
was thus restricted. effects of Tulsi at different
concentrations.

Sedimentation of the solution The sedimentation of the powder in Using a heavier solvent such as
while the disks were soaking the ethanol means that concentrations dimethyl sulfoxide would reduce
(systematic error) would not be accurate. sedimentation.

Inconsistency in the streaking
of agar plates (random error)
Depending on the streaking technique
of the individual, E.coli growth would
vary in density. Growth inhibition
zones will vary depending on the
amount of bacteria present.
Having an E.coli stock solution that is
evenly spread would ensure that there
is equal distribution of E.coli.

Solution droplets of disks Droplets of the solution balanced on Patting the disks to remove excess
(random error) the disk which led to drops spilling on solution would have prevented spillage
agar during application which mildly distorting inhibition zones.
distorted growth inhibition zones.

9
Possible Extensions
This experiment was successful in showing how different concentrations affect the inhibition
of bacterial growth. However, it would be interesting to experiment on the effects of
temperature on bacterial growth inhibition. Tulsi is commonly drunk as a herbal tea in hot
water. Hot water often limits the potency of plant extracts, as Tulsi is commonly drunk as a
herbal tea in hot water, it may be interesting to see whether rising temperatures reduces
potency. Additionally, this experiment was limited in the bacterial stains used due to ethical
concerns when using pathogenic bacteria. However, the epidemic of antibacterial resistance
encompasses more bacterial strains than non-pathogenic E.coli. Experimenting on gram
positive bacteria would provide further insight and determine whether the effects of Tulsi
were E.coli specific.

Works Cited

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APPENDIX
Appendix A: Calculations of ANOVA variance test

*Note: An online ANOVA calculator was used to find the P and F values. However, the F
value was checked using the ANOVA formula below

𝑀𝑆𝑇
𝐹 = 𝑀𝑆𝐸
𝐾
2 2
∑ (𝑇 𝐼 /𝑛 1)−𝐺 /𝑛
𝐼=1
𝑀𝑆𝑇 = 𝑘−1

𝑘 𝑛 1 𝑘
2 2
∑ ∑𝑦 𝑖𝑗
− ∑ (𝑇 1/𝑛 1)
𝑖−1𝑗−1 𝑖−1
𝑀𝑆𝐸 = 𝑛−𝑘

11