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Advancing 2094-9243
ISSNPharmacy
2449-4844
Research Advancing Pharmacy Research
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ISSN 2244-047X
2094-9588

The Antimicrobial Activity of a Formulated Liquid

Spray Preparation from Allium sativum (Garlic)
ZOE MAE L. OLACO
ORCID No. 0000-0002-5868-9395
olacozoemae@gmail.com

VINT PAULINE A. TAGOD

ORCID No. 0000-0003-0109-5112
tagodvintpauline@gmail.com

KHAIRA S. TAURAC
ORCID No. 0000-0002-0541-5163
khairataurac@gmail.com

AMELIA T. VALENDEZ
ORCID No. 0000-0001-8155-7342
valendezamelia@gmail.com
Liceo de Cagayan University
Cagayan de Oro City, Philippines

ABSTRACT

The study focused on the antimicrobial activity of a formulated liquid

spray preparation from Allium sativum (Garlic) on a toilet bowl. TThe
researchers determined which of the following concentrations is effective
against microorganisms found in the toilet bowls: 50%, 75% and 100%. The
antimicrobial activity of the formulated Allium sativum toilet spray cleaner was
compared versus the positive control and the negative control through the zone
of inhibitions. The macerated garlic bulb extract exhibets an antimicrobial
property, which is capable of inhibiting the growth of microbes in culture media
from the toilet bowl.

Keywords: Allium sativum, antimicrobial activity

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INTRODUCTION

Many infectious diseases are taken from anywhere most especially in the toilet
bowl. Every house, school, office and other facilities have a toilet, but some are
actually not fond of cleaning them. Many are using water flush, as it only requires
a push of a button. In this regular way of cleaning may not be enough, toilet bowl
may require extra effort to make them clean.
Based on evidences that toilet bowl is one of the main source of diseases in
the community, such as shigellosis causes infections, severe diarrhea, abdominal
cramping and other gastrointestinal distress that may lose for week, also
staphylococcus bacteria that causes skin infection, pneumonia and lingers around
on the toilet surfaces for longer period which increasing the chances of disease
transmission. The clean bowl will just simply mean that people will not expose to
various germs that may cause sickness or diseases.
Synthetic preparation is commonly used toilet bowl cleaner nowadays which
is acidic and expensive. Many of these preparations are available in the market
that can kill bacteria and viruses that could be found in the toilet, but harmful to
the skin, not safe for a human used and the environment. Toilet bowl cleaner that
comes from organic materials can be effective as a synthetic product, less acidic
that is safe for human use, less irritating to the skin, safe to the environment and
effective against bacteria.
Garlic is widely known as a holy and miraculous plant, which is an effective
organic antimicrobial agent. Naturally, occurring plant has played important role
in the discovery of new therapeutic agent since ancient time. It has been known
for treating some infectious diseases, important dietary and medicinal role for
centuries.

OBJECTIVES OF THE STUDY

The study aimed to determine the antimicrobial activity of a formulated liquid

spray preparation from Allium sativum (Garlic) on a toilet bowl. Specifically, it
sought to: 1) culture bacteria from the toilet bowl; 2) determine the antimicrobial
activity of the garlic extract regarding zone of inhibition; 3) Determine the
antimicrobial activity from garlic extract using different concentrations: 3.1)
50%; 3.2) 75%; 3.3) 100%; and 4) determine the significant difference in the
antimicrobial activity between the experimental concentrations, positive control,
and negative control.

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MATERIALS AND METHODS

Research Setting
The experiment was conducted at the Pharmacy Laboratory of Liceo De
Cagayan University located at Rodolfo N. Pelaez Blvd, Kauswagan, Cagayan de
Oro City. The Laboratory provided the instruments needed. The microorganisms
were taken from the toilet bowl of Taurac’s residence in Macanhan, Cagayan de
Oro City

Research Design
The researchers utilized an experimental design; this was used in the
formulation of an organic Toilet spray preparation, and to determine the efficacy
of garlic as an antimicrobial agent.

Collection of Plant Material

The native Allium sativum (Garlic) bulbs were purchased in Bulua Market.
The bulbs were used as sample for the experiment.

Preparation of Extract
Two hundred sixty-five grams of garlic bulb was weighed, washed and peeled.
Clove of garlic was crushed using a mortar and pestle and placed in a glass
container. Enough amount of 95% ethanol was added to the crushed garlic to
make 100 ml.
It was allowed to stand for ten days. After ten days of maceration, macerated
garlic was filtered and placed in a beaker. Using a hot plate, the filtrate (extractive)
was concentrated on a water bath, until the alcohol was being totally evaporated
and extract remained. The extract obtained was then divided into three parts 50g,
75g, and 100g.

Formulation of liquid spray preparation for Toilet Bowl

On a 150mL beaker, Garlic residue was dissolved in distilled water and stirred
using a stirring rod (Essential Oil was added, optional) and placed in a suitable
liquid spray container. The solubility of the extract in water was sparingly soluble.

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Formulations of different concentration

On three separated 150 ml beakers, 50g of garlic residue was added sufficiently
with distilled water to make 100mL, 75g of garlic residue was added sufficiently
with distilled water to make 100mL, and 100g of garlic residue was added
sufficiently with distilled water to make 100mL. Mixed using a stirring rod.

Preparation of Culture Media

A. Fluid Thioglycollate Medium

On a 1000ml-distilled water, 29.75 grams of FTM was suspended. It was
Heated to dissolve the medium completely and sterilized by autoclaving at 15 lbs
pressure (121°C) for 15 minutes and allowed to cool at 25°C.

B. Nutrient Agar Medium

Fifteen grams (15g) of nutrient agar was suspended in 1000ml-distilled water.
It was heated to dissolve the medium completely and sterilized by autoclaving at
15 lbs pressure (121°C) for 15 minutes and allowed to cool at 25°C.

C. Mueller Hinton Agar (MHA)

Thirty-eight gram (38g) of Mueller Hinton Medium being suspended to a
1000ml of distilled water. Mixed and boiled for 1 minute with constant stirring.
The solution was being sterilized in an autoclave set at 15 psi at 121°C for 15
mins. The solution was poured into the selected Petri dish to a depth of 4mm,
cooled and solidified.

D. Filter Paper Disc

Whatman no. 1 filter paper was used for the preparation of the filter paper disc
using a puncher with 6mm diameter and sterilized using an autoclave set at 15
psi at 121°C for 15 minutes.

Source of microorganism
Together with the medical laboratory technician, the researchers collected
sample of the microorganisms from the toilet bowl of Taurac’s residence,
Swabbing was done and this was inoculated in Fluid Thioglycollate Medium and

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incubated for 24 hours at 37°C.

With the incubated FTM, all the Nutrient Agar plates were inoculated by
streaking the entire surface of the plate, turning the plate 90 degrees, swabbing a
second time, turning plates 45 degrees and incubated and cultured for 24 hours
at 37°C.

Disc Diffusion Method

The cultured bacteria in the Nutrient agar were inoculated in a MacFarland
until turbidity was obtained and swabbing in a Mueller Hinton agar was done.
The Discs (Whatman filter paper), was soaked in different concentrations of a
formulated garlic extract. Sterilized forceps used to place the discs into the plate,
gently pressing the disc towards the surface of the agar to ensure complete contact
between the disc and the agar surface then was covered. It was being incubated
for 24 hours at 37°C.

MacFarland Solution
The solution for incubation was prepared by inoculating the diluents with
one to five isolated colonies from Nutrient Agar until turbidity was obtained.

Zone of Inhibition
The cultured plate was filled with the discs and incubated at 37°C for about
24 hours. After incubating, it was observed and measured using a ruler.

Statistical Techniques
For validation of results, the data obtained from the experiment was subjected
to statistical analysis employing Analysis of Variance (ANOVA).

RESULTS AND DISCUSSIONS

Objective 1. Culture bacteria from the toilet bowl.

Gram (-) bacteria

The cultured bacteria were obtained from the toilet bowl using the culture
media; swabbing was done and the sample was placed in a Fluid Thioglycollate
Medium incubated for 24hrs at 37°C, after 24hrs the inoculums were inoculated
in a Nutrient Agar for culturing bacteria incubated for 24hrs at 37°C, using
MacFarland solution the cultured bacteria from the Nutrient agar were sub-

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cultured and inoculated to a Mueller Hinton Agar for the tests. Gram (-) bacteria
were identified in the toilet bowl.

Objective 2. Determine the antimicrobial activity of the garlic extract in

terms of zone of inhibition.

Table 1. Zone of Inhibition of the Garlic Extract

The garlic extract underwent three different trials to show and identify the
zone of inhibition that showed the effect against the cultured bacteria that was
present in the toilet bowl.
In the 1st trial, it showed a zone of inhibition of 13mm, 2nd trial showed a
zone of inhibition of 12mm and 3rd trial showed 18mm. This implies that the
garlic extract’s a zone of inhibition in the three different trials indicate a positive
result of its antimicrobial activity and its efficacy against the cultured bacteria
that were present in the toilet bowl.

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The three trials show three different results because each plate of Petri dish
produced a different amount of bacteria present. The plate of the second trial had
numerous amounts of bacteria that were thicker than the plates of the first trial
and the third trial. The results reveal that the pure garlic extract demonstrated on
antimicrobial activity.

Objective 3. Determine the antimicrobial activity from garlic extract using

different concentrations.

Table 2. Zone of Inhibition of Formulated Garlic Extract

Table 2 shows the efficacy of the formulated concentrations with the negative
and positive control. In the first trial, only the positive control showed a zone

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of inhibition that is 18mm. This reveals that the three experimental group and
negative control showed no zone of inhibition. The findings denote that the
50%, 75%, 100% concentrations and negative control have no effect.
In the second trial, 75% concentration, 100% concentration, and positive
control showed a zone of inhibition. The 75% concentration showed an effect of
8mm zone of inhibition, 100% concentration showed 10mm zone of inhibition
and the positive control showed 16mm zone of inhibition. The following
concentration denotes that the positive control has a wider zone of inhibition
compared to the experimental group, this shows that the positive control is more
effective than the experimental group and negative control.
In the third trial, 50%, 100%, and positive control showed a zone of
inhibition. The 50% showed an effect of 8mm zone of inhibition, 100% showed
12mm zone of inhibition and positive control showed 21mm zone of inhibition.
This implies that the positive control, which is the Lysol, known for its 99.9%
antibacterial activity, showed a wider zone of inhibition than the experimental
group.

Objective 4. Determine the significant difference in the antimicrobial activity

between the experimental concentrations, positive control, and negative
control.

Table 3. Test of Significant Difference between Positive Control,

Negative Control and Experimental Group

Table 3 presents the test of significant difference between positive control,

negative control and experimental group.
As shown on the table, the null hypothesis is rejected at the level of significance
equal to 0.05, since the P-value, which is 0.0026, is lesser than the maximum
risk level of significance. This implies that there was a significant difference
between positive control, negative control and Experimental group. The
effectivity of the formulated liquid spray from garlic extract showed a significant

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difference in terms of zone of inhibition. This reveals that in three trials the three
concentrations showed different effects. Not all concentration showed a zone of
inhibition. There is a significant difference between the negative control and the
50% concentration of the extract, and 75% concentration of the extract, since it
showed a mean difference of 15.67, this means that the two concentrations are
effective as antimicrobial compared to the negative control. However, the positive
control did not show any significant difference among the three concentrations,
this implies that the three concentrations are not effective as with its positive
control in the experiment.

Table 4. Scheffe One-way Analysis of Variance

The result underwent scheffe analysis. The mean difference between the
concentration of 50% and 75% showed zero and the P-value is greater than the
alpha value which this indicates that there is no significant difference in the zone
of inhibition that resulted between the two concentrations.
In the comparison, the concentration between the 100% and the 50% and
75% the mean difference showed 4.67, and the P-value is 0.769, which indicates

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that there is no significant difference among the three concentrations in terms of

the zone of inhibitions.
The negative control when compared to the 50% and 75% concentration has
the mean difference of 0.67 and the P-value is 0.017 which indicates significant
difference. However, there is no significant difference between the negative
control compared with 100% concentration with the mean difference of 11 and
the P-value of 0.108. There is no significant difference in the zone of inhibition
of the negative control, the 50% and 75% concentration as indicated of the
-2.67 mean difference and the P-value of 0.960. The 100% concentration has a
mean difference of -7.33 and a P-value of 0.402, which means that there is no
difference between the three concentrations. The mean difference between the
positive control and the negative control is -18.33 and the P-value is 0.006 which
is significantly different. This means that the three formulated concentrations
showed no significant difference in the zone of inhibition with the positive
control, while there is a significant difference between the results showed by the
zone of inhibition between the positive control and the negative control. The
significant analysis connotes that the results of the experimental study of the
formulated antimicrobial liquid spray preparation using garlic extract which
gives a positive result, showed a zone of inhibition against the cultured microbes
but not as effective compared to the positive control which showed a wider zone
of inhibition compared to the three different concentrations formulated.

CONCLUSION

The garlic extract was formulated into liquid spray preparation. Based on the
results, the researchers conclude that garlic extract can be used as an alternative
ingredient that can be used as toilet bowl cleaner that is safer to human and
environmentally friendly because it is organic than the synthetic products.

RECOMMENDATIONS

Based on the findings of the study, the following are recommended:

1. A similar study about antimicrobial activity of Allium sativum (Garlic) can be

done with the different microorganism.

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2. To utilize Allium sativum (Garlic) for future study to probe deeper into its
antimicrobial effect.

3. To determine the exact temperature in obtaining the garlic extract on its

antimicrobial property.

4. To use another solvent for extraction.

5. To use another vehicle such as sterile water in formulating the product instead
of distilled water.

6. To identify the bacteria presence in the toilet bowl.

7. To conduct phytochemical screening test for the presence of allicin with the
formulated 50%, 75%, 100% concentration.

LITERATURE CITED

Akintobi OA, Nwanze JC, Ogele JO, Idowu AA, Onianwa O, Okonko IO.
2012. Antimicrobial Activity of Allium sativum (Garlic) Extract
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www.sciencepub.net/nature

Allan Parachini. 2001. A Deadly Mix of Household Cleansers : Poisonings

Result When Products Are Combined Available at: http://articles.
latimes.com/1986-02-04/news/vw-4307_1_household-cleaner

Barbara Penner. 2012. Small Rooms and Big Systems Available at:
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things/?gclid=CPabjJj41M4CFU-VvQodcOYILQ

Charles P Gerba. 1975. Microbiological Hazards of Household Toilets:

Droplet Production and the Fate of Residual Organism.
Available from: hhtp://www.ncbi.nlm.nih.gov/pmc/articles/
PMC187159/pdf/applmicro00026-0083.pdf

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Daynea Wallock-Richards. 2014. Garlic Revisited: Antimicrobial Activity

of Allicin-Containing Garlic Extracts against Burkholderia cepacia
Complex http://journals.plos.org/plosone/article?id=10.1371/journal.
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D. Jonkers. 1999. Antibacterial effect of garlic and omeprazole on Helicobacter

pylori http://jac.oxfordjournals.org/content/43/6/837.long

Daynea Wallock-Richards. 2014. Garlic Revisited: Antimicrobial Activity of

Allicin-Containing Garlic Extracts against Burkholderia cepacia
Complex. Available from: http://journals.plos.org/plosone/article?id=
10.1371/journal.pone.0112726

Godofredo Stuart. 2015. Philippine List of Medicinal Plants http://www.

stuartxchange.com/Bawang.html

Garlic extract Literature review. n.d. A short review of recent scientific literature
highlighting the beneficial effects of garlic extract in improving plant
health and performance. Available from: http://solufeed.com/
media/46101/garlic-extract-literature-review.pdf

Goncagul, G. & Ayaz, E. 2010. Antimicrobial Effect of Garlic

(Allium sativum) and Traditional Medicine. Available from: http://
www.medwelljournals.com/fulltext/?doi=javaa.2010.1.4

Gilberto E. Flores. 2011. Microbial Biogeography of Public Restroom Surfaces;

Available from: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3223
236/

Iram Gull. 2012. Inhibitory effect of Allium sativum and Zingiber

officinale extracts on clinically important drug-resistant pathogenic;
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articles/PMC3418209/

Leyla Bayan 2014. Garlic: a review of potential therapeutic effects; Available

from : http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4103721/

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L. Barth Reller. 2009. Antimicrobial Susceptibility Testing: A Review of

General Principles and Contemporary Practices.

Melda Genç. 2009. The Evolution of Toilets and Its Current State
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Palaksha, M.N. 2010. Antibacterial activity of garlic extract on streptomycin-

resistant Staphylococcus aureus and Escherichia coli solely and in synergism
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articles/PMC3874089/

Philippine herbal Medicine. 2010. Bawang, Bauang | Garlic (Allium sativum)

http://www.philippineherbalmedicine.org/garlic.htm

Paul Bergner. 1995. Allium sativum: Antibiotic and Immune

Properties; Available from: http://www.medherb.com/Materia_
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tr/tezler/master/endustriurunleritasarimi/T000465.pdf

Sana Mukhtar and Ifra Ghori. 2012. International Journal of Applied

Biology and Pharmaceutical Technology; Antibacterial activity
of aqueous and ethanolic extract of garlic, cinnamon and tourmeric
against E.coli ATCC 25922 and Vacillus sabtilis DSM 3256

Sarah van Schagen. 2009. Natural toilet-bowl cleaner. Available from:

http://grist.org/article/2009-05-19-natural-toilet-bowl-cleaner/

Solufeed Ltd. 2015. Garlic extract Literature review a short review of

recent scientific literature highlighting the beneficial effects of
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literature-review.pdf

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Tyler Street. 2014. Antimicrobial range Susceptibility; Available from: http://

emedicine.medscape.com/article/2103786-overview#a4

Z. M. Ross. 2011. Antimicrobial Properties of Garlic Oil against Human

Enteric Bacteria: Evaluation of Methodologies and Comparisons
with Garlic Oil Sulfides and Garlic Powder http://aem.asm.org/
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the Kiwanis Club of Saugerties. Available from; http://hvgf.org/
types%20of%20garlic.html

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APPENDICES

Plate 1. Collection of Plant

Plate 2. Plant Extraction

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Plate 3. Formulation of Product

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Plate 4. Sterilization of Materials

Plate 5. Preparation of Culture Media

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Plate 6. Source of Microorganism

Plate 7. Cultured Bacteria

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Plate 8. Test for the Controls and the Different Concentration

of a Formulated Liquid Spray Preparation

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Plate 9. Zone of Inhibitions of Pure Garlic Extract in Three Trials

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Plate 10. Zone of Inhibitions of Different Concentrations of Garlic Extract,

Positive Control and Negative Control in Three Trials

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