GARLIC (ALLIUM SATIVUM L.

) AQUEOUS EXTRACT AS A SURFACE

DISINFECTANT

JOSHUA S. ESCALA
MEIEVA ANGELA MANAWAN
ANA MAE M. SANTIAS

Medical Laboratory Science Department

College of Health
Adventist University of the Philippines
Puting Kahoy, Silang, Cavite

2018
GARLIC (ALLIUM SATIVUM L.) AQUEOUS EXTRACT AS A
SURFACE DISINFECTANT

by

JOSHUA S. ESCALA
MEIEVA ANGELA MANAWAN
ANA MAE M. SANTIAS

A Thesis Submitted to the

Medical Laboratory Science Department
Adventist University of the Philippines

In Partial Fulfillment
of the Requirements for the Degree
Bachelor of Medical Laboratory Science

2018
 CERTIFICATE OF ACCEPTANCE

This is to certify that this undergraduate thesis, entitled: Garlic (Allium

Sativum L.) Aqueous Extract as a Surface Disinfectant and submitted by:
JOSHUA S. ESCALA, MEIEVA ANGELA MANAWAN, and ANA MAE M.
SANTIAS to fulfill part of the requirements for the degree of Bachelor in Medical
Laboratory Science, is hereby endorsed.

Princess Red Rose G. Rodriguez, RMT

Thesis Adviser

Ma. Kristine H. Mendoza, RMT, MSMT Gershon Mark G. Cortez, RMT

Panelist Panelist

Miriam R. Estrada, RND, DrPH

Chair

The Department of Medical Laboratory Science accepts this undergraduate

thesis as partial fulfillment of the requirement for the degree Bachelor in Medical
Laboratory Science.

Ma. Krisitine H. Mendoza, RMT, MPH

Chairperson, Medical Laboratory Science Department

Miriam R. Estrada, RND, DrPH

Dean, College of Health

ii
 ABSTRACT

Laboratories routinely use sodium hypochlorite that may be harmful to human

health as well as cause hazardous effect to the environment. An alternative is the use of

herbal products such as garlic (Allium sativum L.) extract. To investigate the

antimicrobial effect of garlic extract as an effective surface disinfectant, allicin was

obtained via aqueous extraction. The extract was wiped on groups of tiles containing five

common human pathogens, namely Eschericia coli, Staphylococcus aureus,

Pseudomonas aeruginosa, Klebsiella pneumoniae and Candida albicans, and cultured in

respective agar media. The effectiveness of garlic extract was compared with the decking

solution by evaluating the growth in the agar after 24 hours. Garlic aqueous extract

showed inhibitory action against all four bacteria as well as fungi. It has comparable

efficacy to that of the 10% sodium hypochlorite. Thus garlic extract can be used as an

alternative surface disinfectant for laboratory purposes.

iii
 ACKNOWLEDGEMENTS

We express our deep appreciation to Mary Mediatrix Medical Hospital

Laboratory for allowing us to use their facilities and materials for our research. Our great

thanks are also due Sir Keen Clark Bitong and Ma’am Ivy Rosita, who helped in

gathering the microbial strains needed and gave us ideas on how we can grow our

cultures. Lastly, we are grateful to Ma’am Red Rose Rodriguez for her patience, advice

and enthusiastic instructions.

iv
 TABLE OF CONTENTS

INTRODUCTION ........................................................................................................1

REVIEW OF LITERATURE .......................................................................................3

Allium sativum ....................................................................................................3

Method of Extraction ..........................................................................................3

Test Organisms ...................................................................................................4

Sodium Hypochlorite ..........................................................................................5

MATERIALS AND METHODS ..................................................................................6

Materials .............................................................................................................6

Preparation of Garlic Aqueous Extract ...............................................................6

Maintenance of Cultures .....................................................................................6

Preparation of Test Suspensions and Decking Solution .....................................7

Surface Sampling ................................................................................................7

Evaluation of Effectiveness ................................................................................8

RESULTS .....................................................................................................................9

DISCUSSION .............................................................................................................14

CONCLUSION ...........................................................................................................15

RECOMMENDATIONS ............................................................................................15

REFERENCES ...........................................................................................................16

APPENDICES ............................................................................................................18

Documentation ..................................................................................................18

CURRICULUM VITAE .............................................................................................23

v
 INTRODUCTION

Sodium hypochlorite or commonly known as bleach is the most common surface

disinfectant used in households and laboratories. Prolonged exposure to bleach poses a

significant health risks not limited to headaches, burning sensations and neurological

disorders. In clinical laboratories, sodium hypochlorite is used for disinfecting surfaces

before and after work as part of the laboratory protocol to reduce the transmission of

microorganisms that cause healthcare-associated infections (HAIs). These

microorganisms spread from contaminated hands of health professionals and medical

equipment that are frequently in contact with environmental surfaces including machines,

table tops, floor and walls. While PPE is implemented, disinfection procedures is applied

to further protect laboratory workers from acquiring and transmitting infections.

To lessen the exposure of the laboratory workers to different chemicals, a safer

and more organic reagents and cleaning agents must be promoted in the laboratories,

hence this study. The aim of this study was to investigate the antimicrobial effect of

garlic aqueous extract as an effective surface disinfectant. Garlic (Allium sativum L.) has

a remarkable amount of organosulfur compounds called alliin. Alliin is then converted to

allicin which is responsible for the antimicrobial activity of garlic.

This study can help reduce chemical-based agents in the laboratory which may be

beneficial to the health of laboratory workers and to the environment.

Allicin was extracted through homogenization of fresh garlic bulbs using

domestic blender and straining using cheesecloth. Test suspensions of microorganism

were cultured for application on vinyl tiles.

1
 To test the antimicrobial ability of allicin extract against five strains of common

pathogens, vinyl tiles surface were treated with garlic extract and the growth was

compared with the vinyl treated with 10% sodium hypochlorite after 24 hours.

2
 REVIEW OF LITERATURE

Allium sativum

Garlic (Allium sativum L.) is a member of the Alliaceae family along with

mountain onion, leek, chive, and rakkyo.1,2 It is widely used as a natural spice and

medicine. Its distinct smell and taste is due to the presence of organosulfur compounds

such as allicin, alliin, ajoene, dially disulfide, dithiin and sallylcysteine.3 The extract has

50 - 90 mol% allicin, making it the most abundant among the thiosulfinates. Though

considered as the major antimicrobial component, it is not a found in the whole garlic

bulb. It is derived from a freshly crushed garlic when S(+)-allyl-L-cysteine sulfoxide

(alliin) escapes from the ruptured cell membranes and is converted into allicin by the

enzyme alliinase.4 This reaction forms pyruvic acid, ammonia and alkenyl sulfenic acid

as intermediates. Sulfenic acid is a sulfur containing compound that will undergo rapid

condensation to form thiosulfinates.5

Method of Extraction

The antimicrobial efficacy of garlic aqueous extract was estimated through in

vitro studies. A number of extraction methods were developed to isolate allicin including

steam distillation, organic solvent extraction, and supercritical extraction using garlic

homogenate.6 Aqueous extraction using distilled water was used to prevent interference

and decomposition of allicin.

3
Test Organisms

In this study the antimicrobial effect of garlic aqueous extract was tested against

five common human pathogens namely S. aureus, E. coli, P. aeruginosa, K. pneumoniae

and C. albicans.

Staphylococcus aureus causes a variety of diseases including skin infections, food

poisoning, toxic shock syndrome and nosocomial infections acquired during

hospitalization.7

Escherichia coli exists naturally as part of normal gut of healthy humans and

mammals. Relatively few strains of this organism are pathogenic to humans. It causes

enteric diseases and other food related illnesses.7

Pseudomonas aeruginosa is a rod-shaped, aerobic, Gram-negative bacterium

that thrives in soil and aquatic habitats and colonizes and invades plants, animals, and

human hosts. With highly adaptive physiology, Pseudomonas species are able to act as

opportunistic pathogens in humans with weakened immune systems, causing

life-threatening community-acquired pneumonia, nosocomial infections such as UTI and

bacteremia.8

Klebsiella pneumoniae is the second most common gram-negative bacterium

belonging to the Enterobacteriaceae family, which is comprised of Escherichia coli,

Salmonella species, Shigella species, and Yersinia species. It is associated with urinary

tract infections (UTI), bloodstream infections (BSI), pneumonia, meningitis, pyogenic

liver abscess (PLA).9,10

C. albicans is an opportunistic pathogen of humans and the primary cause of

candidiasis. It is dimorphic, growing either as unicellular budding yeast or in filamentous

form. Being the fourth most common cause of nosocomial infection, C. albicans are

known to grow on medical implants, such as blood and urinary catheters or heart valves,

4
by organizing into biofilms composed of a dense network of yeasts, hyphae,

pseudohyphae and extracellular polymeric materials. The cells in biofilms are resistant to

conventional antifungal agents which renders them ineffective in treating C. albicans

infections.11

Sodium hypochlorite

Despite the new products and new technologies for disinfection procedures,

sodium hypochlorite is still one of the most widely used and accessible products in terms

of cost and benefit. This study intends to seek evidence on their efficiency on

environmental surfaces in reducing contamination and prevention of

healthcare-associated infection.12 Sodium hypochlorite poses health effects both during

the preparation of decking solution and its actual use. It is corrosive and may irritate the

skin or cause burning pain, inflammation and blisters. Chronic exposure may lead to

allergic contact dermatitis.13 It was also found that regular bleach use increases the

chance of developing chronic obstructive pulmonary disease (COPD).14

A comparison to the efficacy of 10% sodium hypochlorite will help evaluate the

effectiveness of garlic aqueous extract as surface disinfectant.

5
 MATERIALS AND METHODS

Materials

Fresh garlic bulbs, distilled water, cloth for decking, cheesecloth, vinyl tiles, and

chemical disinfectant, Zonrox, were purchased from the local market.

One big vinyl tile was purchased from a local store in Manila, and it was divided

into 15 squares, each with an area of 3 x 3 inches. Five squares, one for each organism,

were assigned for each test groups—garlic and 10% sodium hypochlorite and another

five tiles for the control group which did not receive any treatment. Prior to its use the

vinyl tiles were autoclaved for 15 minutes at 121 C and 15 psi. All other materials used

were sterile: cotton swabs, spraying bottles, inoculating loops, cheesecloths, culture

plates and the cloth for decking.

Personal Protective Equipment such as laboratory gown, masks, and gloves were

used during the whole testing.

Preparation of Garlic Aqueous Extract

250 grams of fresh garlic bulbs were chopped in small pieces and homogenized in

1 L of distilled water using a domestic blender for 15 minutes. 25% aqueous solution of

garlic was obtained. The homogenized mixture was filtered through cheesecloth about

three times. The extract obtained was stored in the refrigerator at a temperature of 2-8°C

until use.

Maintenance of Cultures

Before proceeding to the experimentation proper, the identification of

microorganism colonies were assured via Vitek 2 Compact Workflow which is routinely

used in Mary Mediatrix Medical Hospital to isolate colonies and determine whether it is

6
Gram Positive or Gram Negative. Microorganisms were checked if they will grow on the

surface of the vinyl tiles exposed in an open area.

One Gram positive bacteria—Staphylococcus aureus), three Gram negative

bacteria—Escherichia coli, Pseudomonas aeruginosa, and Klebsiella pneumoniae) and

one pathogenic fungus—Candida albicans were obtained in cultures from Bacteriology

Section of Mary Mediatrix Medical Center, Lipa City, Philippines. Positive culture

results from the patients were subcultured into another agar media and incubated for 24

hours. The growth were maintained by replanting new colonies to new agar media every

48 hours.

Preparation of Test Suspensions and Decking Solution

Test suspensions of fresh cultures of E. coli, S. aureus, P. aeruginosa, K.

pneumoniae and C. albicans were prepared for sampling by suspending one medium

colony of the organism in 5 mL of sterile saline solution. The suspension was compared

to 0.5 Mcfarland. The suspensions were labeled accordingly and were mixed until it

became homogenous.

To prepare a 10% sodium hypochlorite solution, 50 mL of Zonrox bleach was

transferred to a sterile beaker containing 500 mL of distilled water. The fresh decking

solution was used within the day of the testing.

Surface Sampling

Cotton swabs were dipped on each of the prepared test suspension and were

streaked on the entire surface of the three groups of vinyl tiles. The tiles were swabbed

onto three different directions by rotating the vinyl tile at different angles. Afterwards,

each tile was air dried and was left for five minutes. Meanwhile, the prepared

7
disinfectant—garlic and 10% sodium hypochlorite, were soaked with a clean cloth for 1

minute and the cloth was squeezed and compressed to remove the excess liquid.

After five minutes of drying the tiles, the first group of vinyl tiles was wiped with

a clean cloth soaked in garlic aqueous extract; the second group was wiped with a clean

cloth soaked in 10% sodium hypochlorite, while the third group of tiles was left untreated.

After the application of the disinfectants to the two groups of tiles, the tiles were left for

air drying.

Evaluation of Effectiveness

After air drying, the surfaces of the experimental tiles were swabbed again with

sterile cotton swabs to assess the effectiveness of the garlic aqueous disinfectant. Using

the swab, the specimen was transferred onto the culture plates by half of the plate because

the agar plates consisted of two culture media (Figure1). The culture media were labeled

properly and were incubated at 37OC for 24 hours. After 24 hours of incubation, the

growth in all the culture media were observed and were graded semi-quantitatively using

the grading in table 1.

Table 1
Guide to Grading the Growth Rate
Growth Rating Colony Count
No Growth ( - ) 0
Light (+) >1
Moderate (++) >5
Heavy (+++) > 10

8
 RESULTS

After 24 hours of incubation, the growth of the organisms were observed and

graded semi-quantitatively. The following are the results of the observation.

Table 2 shows the semi-quantitative grading results of the growth of the organism

on the three groups of vinyl tiles.

Table 2
Semi-Quantitative Grading of the Growth Rate
Growth
Test Organisms Control Garlic Extract 10% Sodium
Hypochlorite
S. aureus Moderate NG NG
P. aeruginosa Light NG NG
E. coli Heavy NG NG
K. pneumoniae Heavy NG NG
C. albicans Heavy NG Heavy

The culture media of control group were covered with colonies while there were

no colonies were observed in the experimental plates exposed to garlic extract, whereas

very heavy growth of C. albicans were seen in the 10% sodium hypochlorite. There were

medium-sized colonies seen in some plates. These colonies were proven to be an NPI

(No Pathogen Isolated) through the help of our Bacteriology Section Head.

9
Positive Control Group

Plate 1. C. albicans Plate 2. S. aureus

Plate 3. K. pneumoniae Plate 4. E. coli

10
Plate 5. P. aeruginosa

Garlic Extract Treated Group

Plate 1. P aeruginosa Plate 2. S. aureus and C. albicans

11
Plate 3. E. coli and K. pneumoniae

10% Sodium Hypochlorite Treated Group

12
Plate 1. C. albicans Plate 2. S. aureus

13
Plate 3. P. aeruginosa Plate 4. E. coli and K.pneumoniae

14
 DISCUSSION

As the result has shown, there was no growth in all culture media treated with the

garlic aqueous extract. It is known that Allium sativum is used widely because of its

antimicrobial activity, and according to the WHO Allium sativum is still being used in

folk medicine. The presence of organosulfur compounds such as allicin, alliin, ajoene,

dially disulfide, dithiin and sallylcysteine contributed to the antimicrobial property of the

garlic. (discuss about the smell of the garlic after application to the tiles)

Allium sativum has comparable efficacy to that of the 10% sodium hypochlorite.

The results showed that both can inhibit the growth of the selected pathogens and as

observed after the 24 hour incubation.

Surprisingly, the strain of C. albicans grew on the medium for the 10% sodium

hypochlorite. There could be a technical error in the decontamination. Insufficient

amount of the decking solution was used.

15
 CONCLUSIONS

In the current investigation Allium sativum extract with the ratio of 1:4 was

compared to 10% sodium hypochlorite. The extract was found to be effective on all the

clinically isolated microorganisms. This present study justified the claimed uses of garlic

in the traditional system and proved that garlic can be used as an alternative surface

disinfectant for laboratory and household purposes.

RECOMMENDATIONS

Further studies are needed to better evaluate the potential effectiveness of the

Allium sativum as an antimicrobial agent. Active components may be isolated using

distillation and compared the effectiveness to that of the aqueous extract. Inclusion of

viruses such as Hepaviridae and other fungi in the testing of the inhibitory or virucidal

property of garlic must be explored. The aroma of garlic may be improved by using

commercial or natural scents.

16
 REFERENCES

1. Gafar MK, Itodo AU, Warra AA and Abdullahi L. Extraction and Physicochemical
Determination of Garlic (Allium sativum L) Oil. International journal of food and
nutrition science. 2012; 1(2):4-7.

2. Nejad ASM, Shabani S, et al. Antibacterial Effect of Garlic Aqueous Extract

on Staphylococcus aureus in Hamburger. Jundishapur J Microbiol. 2014; 7(11):
e13134.

3. Gafar MK, Itodo AU, Warra AA and Abdullahi L. Extraction and Physicochemical
Determination of Garlic (Allium sativum L) Oil. International journal of food and
nutrition science. 2012; 1(2):4-7.

4. Chong K, Zamora MP, Tilakawardane DA, et al. Investigation of Allicin Stability in

Aqueous Garlic Extract by High Performance Liquid Chromatography Method.
Journal of Scientific Research & Reports.2015; 4(7): 590-598.

5. Mansor M, Herng HJ, et al. Quantification and Characterization of Allicin in Garlic

Extract. Journal of Medical and Bioengineering. 2016; 5(1): 24-17.

6. Yanga L, Hanb L and Zhuo Y. Microwave-Assisted Extraction of Garlic Essential Oil

from Garlic. Applied Mechanics and Materials. 2012; 117-119: 1022-1026.

7. Abiy1 E and Berhe A. Anti-Bacterial Effect of Garlic (Allium sativum) against

Clinical Isolates of Staphylococcus aureus and Escherichia coli from Patients
Attending Hawassa Referral Hospital, Ethiopia. Journal of Infectious Diseases and
Treatment. 2016; 2(2): 2472-1093.

8. Yayan J, Ghebremedhin B, and Rasche K. Antibiotic Resistance of Pseudomonas

aeruginosa in Pneumonia at a Single University Hospital Center in Germany over a
10-YearPeriod. 2015; PLoSONE10(10):e0139836. doi:10.1371/journal.
pone.0139836.

9. Vading M, Naucle P, Kalin M, and Giske CG. Invasive infection caused by Klebsiella
pneumoniae is a disease affecting patients with high comorbidity and associated with
high long-term mortality. 2017; PLoS ONE 13(4):e0195258.

10. Martin R and Bachman M, Colonization, Infection, and the Accessory Genome of
Klebsiella pneumoniae. Front. Cell. Infect. 2018; Microbiol. 8:4. doi:
10.3389/fcimb.2018.00004.

11. Li WR, Shi QS, Dai HQ, et al. Antifungal activity, kinetics and molecular mechanism
of action of garlic oil against Candida albicans. Scientific Reports. 2016; 6:22805. doi:
10.1038/srep22805.

12. Samantha Storer Pesani Pereira1, Hadelândia Milon de Oliveira, et al. Disinfection
with sodium hypochlorite in hospital environmental surfaces in the reduction of
contamination and infection prevention: a systematic review. Rev Esc Enferm USP.
2015; 49(4):675-681.

17
13. Compendium of Chemical Hazards: Sodium Hypochlorite. Public Health England.
May 2015; 4-10

14. Regular bleach use linked to increased risk of fatal lung disease, says study.
Independent. Web site
http://www.independent.co.ul/life-style/health-and-families/health-news/bleach-use-i
ncreased-risk-fatal-lung-disease-health-a7939896.html%3famp Accessed September
11, 2017

18
APPENDICES

19
20
21
22
23
 CURRICULUM VITAE

Personal Information

Name: Joshua Serrano Escala

Date of Birth: November 27, 1997

Place of Birth: Calamba City, Laguna

Civil Status: Single

Citizenship: Filipino

Religion: Roman Catholic

Permanent address: #997 Kamagong St. Miramonte Village Pansol Calamba City
Laguna

Email address: jescala04@yahoo.com

Contact Number: 09983946345

Educational Background

Inclusive Date School Attended Degree Completed Honors/Awards Received

2004-2010 Canossa Academy Elementary Diploma With Honors
Calamba
2010-2014 Canossa Academy High school Diploma 10th Special Honor
Calamba Award
2014-Present Adventist University
of the Philippines
Work Experience

Inclusive Date Company/Organization Position Status

2015-2017 Special Program for Tourism Office Retired
the Employment of Assistant
Student (Department
of Labor and
Employment)
May 2018 COMELEC Team Leader for Retired
Watchers

Others
Church Activities/ Position: Mater Dei Youth Choir 2013-2014
Extracurricular Activities/Role: Canossian Volunteer of the Philippines 2014-2016
Samahan ng Kabataan ng Canossa (SKC) President of SPAC

24
Personal Information

Name: Meieva Angela Manawan

Date of Birth: May 01, 1995

Place of Birth: Kotamobagu, Indonesia

Civil Status: Single

Citizenship: Indonesian

Religion: Seventh-Day Adventist

Permanent address: AUP, Puting Kahoy, Silang, Cavite

Email address: evhamanawan@gmail.com

Contact Number: 09951719609

Educational Background

Inclusive Date School Attended Degree Completed Honors/Awards Received

2011-2013 Adventist high school High School Diploma
of north sulawesi
2007-2010 Unklab junior hgh
scool
2002-2007 Unklab elemantary Elementary Diploma
school
2014-Present Adventist University
of the Philippines

Others

Church Activities/ Position: 1000 Missionary Movement Indonesian Church

2012-2018

25
Personal Information

Name: Ana Mae Malapad Santias

Date of Birth: October 8, 1996

Place of Birth: Lumangbayan, Pinamalayan, Or. Mindoro

Civil Status: Single

Citizenship: Filipino

Religion: Seventh-Day Adventist

Permanent address: Panicuason, Naga City, Camarines Sur

Email address: amsantias@yahoo.com.ph

Contact Number: 09212357060

Educational Background

Inclusive Date School Attended Degree Completed Honors/Awards Received

2002-2003 Pinamalayan
Adventist Elementary
School
2003-2009 Naga View Adventist Elementary 3rd Honorable Mention
College-Elementary Completion
Certificate
2009-2013 Naga View Adventist High School Diploma 10th Honorable Mention
College-Academy
2013-2014 Mapua Institute of
Technology
2014-Present Adventist University
of the Philippines

26