Evaluation of Antibacterial Activity of Neolamarckia macrophylla Leaf Extract

Izzarul Atirah binti Boestamam

(44943)

Bachelor of Science with Honours (Resource Biotechnology)

2016

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Evaluation of Antibacterial Activity of Neolamarckia macrophy/la Leaf Extract

Izzarul Atirah binti Boestamam

(44943)

This project is submitted

in partial fulfillment of the requirements for the Degree of Bachelor Science with

Honours

(Resource Biotechnology)

Faculty of Resource Science and Technology Universiti Malaysia Sarawak

2016

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ACKNOWLEDGEMENT

Above all, I would like to express my special and smcere gratitude to my

supervisor, Mdm. Fazia binti Mohd Sinang for her guidance and complete support

throughout my final year project. The insight and experience was certainly appreciated.

Next, I would like to express my gratitude to my co-supervisor, AP Dr. Ho Wei Seng for

his advices and supports in completing my research and giving me opportunity to do this

project. Additionally, I would like to express my appreciation to Dr. Hashimatul Fatma

binti Hashim and Dr. Elexson Nillian for their advices and concerns during completing my

research.

Special thanks to Mr. Iskandar and Ms. Kamaliawati for the guidance in the

laboratory and kindly preparing apparatus and materials that needed during my research. I

am also grateful to Mr. Kelvin Toh Seng Chiew (Msc Student), Ms. Habsah Hassan (Msc

Student), Ms. Suzella Demie (Msc Student) and Ms. Nur Adila binti Muradi (Msc Student)

for their suggestions to improve my skills and knowledge to complete my project. I am

indebted to you for the knowledge you have bestowed upon me as a reflection of your

experience.

Last but not least, I would like to thank myself for my efforts, my parents for their

support, my friends especially Nadiaizzati binti Mohamad and all the members of my

laboratory for their help and companionship while performing this research.

r

DECLARATION

I hereby declare that no portion of this dissertation has been submitted in the support of an

application for another degree of qualification of this or any other university or institution

of higher learning.

O~J ~:, ..............{.. ....... ................ .

(lZZARUL A TlRAH BINTI BOEST AMAM)

Resource Biotechnology Programme

Department of Molecular Biology

Faculty of Resource Science and Technology

Universiti Malaysia Sarawak

II

"usat Khidmat MakJumat Akademik UNlVERSm MALAYSIA SARAWAK

T ABLE OF CONTENT

Acknowledgement

IIDeclaration

IIITable of Content

VList of Abbreviations

VIList of Tables

VIIList of Figures

Abstract

21.0 Introduction

52.0 Literature Review

2.1 Potential antibacterial component from plant leaves 5

2.2 Taxonomy of Neolamarckia macrophylla 5

2.3 Medicinal use ofNeolamarckia macrophylla 7

2.4 Evaluation of antibacterial activity ofN. macrophylla leaf extract 8

93.0 Materials and Methods

93.1 Materials

3.2 Preparation of leaf powder 10

103.3 Crude Extraction

103.3.1 Maceration

3.3 .1.1 Ethanol Extraction 10

3.3.1.2 Aqueous Extraction 11

3.3.2 Hot Continuous Extraction (Soxhlet) 11

3.3.3 Direct Use of Aqueous Extraction 12

3.4 Preparation of culture media 12

3.5 Preparation of bacterial culture 12

3.6 Antibacterial Assay 13

3.6.1 Preparation of Disc for the Assay 14

3.7 Positive and Negative Control 15

III

II

4.0 Results and Discussion 16

4.1 Plant Materials 16

4.2 Comparison between aqueous and ethanol crude extract using maceration 17

technique

4.3 Soxhlet Technique for Ethanol Extraction 22

4.4 Direct Use of Aqueous Filtrate 26

4.5 Comparison between zone inhibition ofN. macrophylla leaves and current 28

antibiotics

4.6 Disc Diffusion Method 29

4.7 Recommendations 30

5.0 Conclusions 31

6.0 References 32

Appendix 37

IV

C

DMSO

MRSA

Mrc

g

ml

mm

rpm

LIST OF ABBREVIATIONS

Dimethyl sulfoxide

Methicillin Resistant Staphylococcus aureus

Minimal Inhibitory Concentration

Gram

Millilitres

Millimetres

Rotation per minute

Celsius

v

LIST OF TABLES

Table 4.1 Inhibition zone diameter (mm) of aqueous and ethanol crude 17

extract ofN. macrophylla leaves used at different concentrations.

Table 4.2 Inhibition zone diameter (mm) of ethanol crude extract (Soxhlet) of 22

N. macrophylla leaves used at different concentrations.

Table 4.3 Inhibition zone diameter (mm) from direct use of aqueous filtrate of 26

N. macrophylla leaves used at different concentrations.

Table 4.4 Comparison between zone inhibition ofN. macrophylla leaves and 28

current antibiotics

Table 5 Raw data on diameter of size inhibition zone, extracted using 37

maceration technique and 80% ethanol as the solvent.

Table 6 Raw data on diameter of size inhibition zone, extracted using 37

maceration technique and sterile distilled water as the solvent.

Table 7 Raw data on diameter of inhibition zone; extracted using Soxhlet 38

technique and 80% ethanol as the solvent.

Table 8 Raw data on diameter of inhibition zone in aqueous extraction 38

using maceration technique (direct use of the sample filtrate).

Table 9 The diameter (mm) ofzone inhibition produced by antibiotics using 39

disc diffusion method on all bacteria tested.

VI

LIST OF FIGURES

Figure 2.1 Red kelampayan at the age of7 to 8 month. 6

Figure 3.1 Flow chart represents an overview process for evaluation of

antibacterial activity of N. macrophylla 9

Figure 3.2 Flow chart represents the methodology of antibacterial assay. 14

Figure 3.3 The position of disc that impregnated onto each bacterial lawn. 15

Figure 4.1 Zone of inhibition by N. macrophylla ethanol crude extract on

Salmonella typhimurium. 18

Figure 4.2 Zone of inhibition by N. macrophylla aqueous crude extract on

Pseudomonas aeruginosa.(The zone were clearly seen with 19

better source of light)

-igure 4.3 Zone of inhibition by N. macrophylla ethanol crude extract

(Soxhlet) on Salmonella typhimurium. 23

Figure 5 Zone of inhibition produced by the antibiotic (Gentamycin) on

bacteria used in this bioassay (a). Escherichia coli (b).

Salmonella typhimurium (c). Klebsiella sp. (d). Pseudomonas 39

aeruginosa (e) Listeria monocytogenes (t). Staphylococcus

aureus (g). Bacillus cereus

Figure 6 Images represents (a) and (b) Fresh leaves of N. macrophylla

before proceeds to drying (c) Drying process of N. macrophylla

and (d) the leaves were cut into smaller pieces, dried in the oven 40

at constant temperature, 40 °e.

VII

Evaluation of Antibacterial Activity of Neolamarckia macrophylla Leaf Extract

Izzarul Atirah binti Boestamam

Resource Biotechnology Molecular Biology Department

Facult)' of Resource Science and Technology Universiti Malaysia Sarawak

ABSTRACT

Tremendous research were done to extract bioactive compound from natural product e pecially plant which can be used as an alternative drugs to treat infection. The evaluation of antibacterial activity of Neolamarckia macrophylla leaves crude extract by using three method; maceration, Soxhlet and direct use was determined using disc diffusion method against medically important bacteria such as Escherichia coli, Klebsiella sp., Pseudomonas aeruginosa, Salmonella typhimurium, Listeria monocytogenes, Staphylococcus aureus and Bacillus cereus with different concentrations (12.5%, 50%, 75% and 100%). The results showed that the inhibition growth varies among tested bacteria with maceration method gives better result compared to other techniques used. Two solvents were used; ethanol and distilled water, and ethanol extraction has better antibacterial activity compared to aqueous extraction. This study provides preliminary results suggesting that N. macrophylla can be used as a source of drugs to fight infection.

Keywords: Neolamarckia macrophylla, antibacterial activity, disc diffusion method, Gram-negative bacteria, Gram-positive bacteria.

ABSTRAK

Banyak penyelidikan dijalankan untuk mengek<itrak bio-aktif kompaun dari alam semula jadi terutamanya tumbuhan digunakan sebagai ubat alternatif. Penilaian aktiviti antibakteria Neolamarckia macrophylla ekstrak mentah dengan menggunakan tiga kaedah; kehabisan tenaga, Soxhlet dan kegunaan langsung telah ditentukan dengan menggunakan kaedah cakera penyebaran terhadap bakteria perubatan penting seperti Escherichia coli, Klebsiella sp., Pseudomonas aeruginosa, Salmonella typhimurium, Listeria monocytogenes, Staphylococcus aureus dan Bacillus cereus dengan kepekatan yang berbeza (12.5 %, 50 %, 75 % dan 100 %). Hasil kajian menunjukkan bahawa perencatan pertumbuhan yang berbeza antara bakteria diuji dengan kaedah kehabisan tenaga memberikan hasil yang lebih baik antara tiga teknik yang digunakan. Dua pelarut yang digunakan; etanol dan air suling, dan pengeluaran etanol mempunyai aktiviti anti­bakteria yang lebih baik dibandingkan dengan pengek<itrakan akueus. Penyelidikan ini mencadangkan N. macrophylla boleh digunakan sebagai salah satu sumber ubat.

Kala Kunci: Neolamarckia macrophylla, aktiviti anti-bakteria, kaedah cakera penyebaran.

1.0 Introduction

The increasing occurrence of multidrug resistant bacteria and emergence bacteria strain

with reduced susceptibility to antibacterial agent raises the threat of untreated bacterial

infections. Besides, many existing synthetic drugs accumulate in the body causing liver

damage and other tissue problems. However such problems does not exist when natural

antibacterial agent are used (Aparadh et aI., 2012). Therefore researchers are aiming to

explore potential plant as alternative antibacterial agents. Hence, drug development from

natural or organic material could be useful to meet the demand of newer drug that is safe to

use.

Tremendous research about antimicrobial properties has been done involving

various species of plants around the world such as, Merremia emarginata, Cyathula

prostrata, Baeckeafrutescens, Azadirachta indica (neem) and many more (Elumalai et aI.,

2011; Ogu et aI., 2012; Razmavar et aI., 2014; Mohammed and Orner, 2015). Those plant

tested were containing many components (bioactive and can be medicinal) that functions

as defensive mechanism which helping to protect them from microbial infection and other

diseases.

Neolamarckia macrophylla belong to Rubiaceae family and preliminary evidence

of anti-mycobacterial activity in several species of this family shown motivates further

studies (Aro et aI., 2015). A study done by Martins and Nunez, (2015) stated that

phytochemical properties that responsible for disease protective agents are also present in

Rubiaceae family. Some of them showed anti-inflammatory, analgesic, antibacterial,

2

,....

antivira l, antioxidant, and effect on vascular diseases and activity on the central nervous

system (Heitzman et af., 2005).

Neolamarckia cadamba is a species that commonly related with N. macrophylla

which also belongs to same family. Previous research done by Patel et af. (2011) shows

that N. cadamba has the antifungal properties from the leaves and its bark extract. A

research involving the antibacterial activity of N. cadamba fruits and its various part of the

plant also showed good antibacterial activity (Mishra, 20 \\ ; Mishra, 20 \3). Thus, it is

speculated that N. macrophylla may possess antibacterial properties with similar potential

as N. cadamba. However, there are no studies yet to report on antimicrobial properties

from N. macrophylla leaf.

The scarcity of infective diseases in wild plants indicates the successful defence

mechanism developed by them (Hemaiswarya et af., 2008). This show the various parts of

plant can be used to treat disease and leaves are the main part that commonly used. Based

on Patel et af. (20 II), the research proves that the leaf extract showed more activity of

antifungal activity than the bark extract of N. cadamba. Similarly, a study done by

Magassouba et af. (2007) showed 6 leaves extract exhibit antibacterial activity towards

various strains of bacteria. Advantageously, uses of N. macrophylla in timber industry

yield a large amount of leaf. Thus, it provides an easy access to get a large volume of the

leaf for extraction of antimicrobial compound.

3

The methods of extraction and solvent used to obtaining the crude extract are very

important to maximise yield product during extraction process. Therefore, numerous

research regarding antibacterial activity derived from leaf extract that been extracted by

ethanol showed bigger inhibition activity in comparison to aqueous extraction (Ekwenye

and Edeha, 2010; 1I0dibia et aI., 2015 and Valle et aI., 2015). The same study done by

Dubey et aI., (2012) and Razmavar et al., (2014) proved that ethanol extract exhibit

effective against isolated multidrug resistance bacteria such as Methicillin-resistant

Staphylococcus aureus (MRSA). This is due to the fact that ethanol has a better dissolving

capacity than water (Cheremisinoff, 2003).

Therefore, the objectives of this study are ;

1. To extract the antibacterial properties of N. macrophylla leaf using three technique;

maceration, Soxhlet and direct use by using two different solvent; sterile distilled

water and ethano I.

2. To evaluate antibacterial activity towards three Gram positive bacteria and four

Gram negative bacteria by using disc diffusion method.

4

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2.0 Literature Review

2.1 Potential antibacterial component from plant leaves

Natural products provide as an alternative to discover new substances especially for

medical purpose. Antibacterial agents that derived from plants have enormous therapeutic

potentia l. This is proved by many researches and their studies have been published

whereby mostly they were investigating on the bioactive compound on the leaf. Many of

extract tested have demonstrated good potential applications against diverse range of

mioroorganism (Shinde and Mulay, 2015~ Momtaz and Abdollahi, 2010~ Manikandan el

aI., 2011). The continuous emergence of multidrug-resistant bacteria towards antibacterial

agents used leads the effort to explore the new drug obtained from the natural resources. In

addition, rising awareness of using natural and safe product in new generation also

contribute to more studies and urged to produce natural-based product of med icine.

2.2 Taxonomy of Neolamarckia macrophylla.

Neolamarckia macrophylla has other synonym names such as Nauclea macrophylla

(Wall.) Spreng., Nauclea macrophylla Roxb., and Anlhocepha/us macrophyllus (Roxb.)

Havil. (Porcher, 2015). It is included in the family of Rubiaceae. N. macrophylla derived

fro m tropical monsoon climate regions and can be found in Indonesia, Malaysia, Vietnam

and Philippine.

5

In Malaysia, two common species of Neolamarckia species that can be found are N.

macrophylla and N. cadamba. These two species may be considered synonymous but

differences in appearances and growth can be recognized by both foresters and farmers

(Krisnawati et al., 2011). The local name for N. macrophylla species is known as "Red

Kelampayan" as it possess red colour stem as shown in Figure 2.1 . N. macrophylla tree has

its own characteristics such as a fast growing species and are able to abort the twigs and

leaves naturally or ' self-pruning'.

Figure 2.1 : Red Kelampayan at the age of7 to 8 month.

(Source: rambangnews.wordpress.com/tag/jabon/, Retrieved on 15 December 2015)

The tree capable to reach until 40 to 45 meter height with a round rod and

perpendicular to 70-80% with a trunk circumference reaches more than 150 cm. It IS a

strong and durable wood therefore it has high economical value. It has subtle texture of

wood and straight wood fibres appearance. Therefore, they are mainly used as plywood

supply, furniture, home accessories and others. In addition, this timber species also can be

used as a buffer zone for conservation purposes due to its deep root (Halawane et al.,

2012). It is also mentioned that this wood is resistant to pests and diseases. Halawane

6

compared the Neofamarckia species with Afbasia fafcata species which is also a fast

growing t imber tree. Apparently, Neofamarckia species are able to withstand the infections

ofa disease cause by a fungus, Uromycladium tepperianum.

2.3 Medicinal use of Neofamarckia species.

Accord ing Useful Tropical Plants (2015), N. macrophylla barks and leaves were used in

traditional medication. The dried barks are believed to relieve fever and can be produce as

a tonic meanwhile the leaves serves as a mouth gargle. Other than wood product, N.

macrophylla also benefit in traditional medicine uses that commonly practiced by society

in Halmahera Tengah, Maluku Utara. It is commonly use as stamina enhancer, reduce

fatigues and cholesterol. (Halawane et aI. , 20 II).

Another common species related to N. macrophylla is Neofamarckia cadamba

which also come from the same family genus. Traditionally N. cadamba is widely used to

treat skin disease by a tribe in Western Ghats in the form of paste. A research done by

Umachigi et af. (2007) later proved that the leaf extract ofN. cadamba has shown potential

in wound healing and have good antioxidant properties. Another study showed that leaf

extract exhibit strong activity towards tested microorganism when extracted with

chloroform and acetone (Chandrashekar and Prasanna, 2009). Previous study also reported

about the ant ifungal activity of this species and its active chemical constituents would be

helpful in treat ing various kinds of diseases (Patel et af., 20 II). As mentioned above, there

are several researches on N. cadamba leaf extract to determine the function in medicinal

areas but so far there is no scientific research done to N. macrophylla.

7

2.4 Evaluation of antibacterial activity of N. macrophylla leaf extract.

Disc diffusion method or known as Kirby Bauer method has been a common practice to

detect the pre ence of antibacterial activity since Bauer et aI., first described this technique

back in the 1966. This method involves placing the disc contains the sample extract

directly on the surface of agar which has been inoculated with bacterial cultures. The

approach using this technique is based on the measurement of sizes of zone that believes is

rational in theory and it can correlate better with results of dilution methods, minimal

inhibitory concentration (MIC). According to Bauer, this method has been found to be

easily performed, reproducible and of great value as a guide to therapy (Bauer et aI., 1966).

8

3.0 Materials and Methods

An overview of the process that has been conducted throughout this study was illustrated

through a flow chart below (Figure 3.1).

I Samples Collection I

I Sample Extractions I

! l I Maceration I I Soxhlet I I Direct Use I

I I

Disc diffusion method II

Figure 3.1: An overview of the process for evaluation of antibacterial activity of N. macrophylla.

3.1 Materials

The fre h leaves of Neolamarckia macrophylla were collected from Laboratory of Forest

Genomics and Informatics, Department of Molecular Biology in UNIMAS. Meanwhile,

the bacteria used for this study were three Gram-positive and four Gram-negative bacteria;

Bacillus cereus, Staphylococcus aureus, Listeria monocytogenes, Pseudomonas

aeruginosa, Escherichia coli, Salmonella typhimurium and Klebsiella sp. which were

obtained from Laboratory of Yiro logy, Department of Molecular Biology in UNIMAS.

9

...- ---~

3.2 Preparation of leaf powder

The preparat ions of leaves were done according to Ilodibia et aI., 2015 with slight

modifications on the temperature used. First, the leaves were thoroughly washed under

running tap water and rinsed with distilled water. Next, sterile with ethanol and finally the

leaves were dried using oven at constant temperature, 40°C for at least 12 hours. After

dried, the leaves were grounded using mechanical grinder and the powder products were

kept in a ir-tight container which stored in dry and cool place until extraction (Patel et aI. ,

201 I).

3.3 Crude extraction

3.3.1 Maceration

3.3.1.1 Ethanol Extraction

The method for both aqueous and ethanol extraction were followed the procedure as

described from I10dibia et aI., (2015) with slight modifications. For ethanol extraction,S

gram of leaves powder soaked in 100 ml 80% of ethanol. The mixture then mixed

thoroughly, left under rotary shaker at 120 rpm for at least 3 days under room temperature.

Next, the mixtures were filtered using sterile filter paper. The marc was press to obtained

full filtrate from the mixtures. The filtrate obtained were dried using oven at constant

temperature 40°C. The crude extract obtained was weighed and dissolved in 10% DMSO

(Ankad el aI., 2016). Then, serial dilution were prepared; 100 mg/ml, 50 mg/ml, 25 mg/ml

and 12.5 mg/ml. The 10% DMSO and antibiotic (Gentamicin) were used as negative and

positive contro I respective Iy.

10

3.3.1.2 Aqueous Extraction

For aqueou extraction, 5 gram of leaves powder soaked In 100 ml of sterile distilled

water. The mixture then mixed thoroughly, left under rotary shaker at 120 rpm for at least

3 days under room temperature. Next, the mixtures were filtered using sterile filter paper.

The marc was press to obtained full filtrate from the mixtures. The fi Itrate obtained were

dried using oven at constant temperature, 40 °e. The crude extract obtained was weighed

and dissolved in 10% DMSO. Then, serial dilution were prepared; 100 mg/ml, 50 mg/ml,

25 mg/ml and 12.5 mg/ml. The 10% DMSO and antibiotic (Gentamicin) were used as

negative and positive control respectively. For the direct use of aqueous extraction, before

the filtrate collected were dried, 5-10 ml of filtrate were separated in sealed container and

kept in coo l place (4 0q. This mixture was used directly in the disc diffusion assay without

addition of 10% DMSO.

3.3.2 Hot Continuous Extraction (Soxhlet)

The solvent used for hot continuous extraction was 80% ethanol. In this method, 8 g of

plant powder was placed in 'thimble ' made of thick filter paper. The thimble was then

placed in the main chamber of Soxhlet apparatus and exposed continually with the solvent

(Nayak et ai., 2015). After 5 hours, the collected fluid was dried using oven at constant

temperature, 40°C.

II

3.3.3 Direct Use of Aqueous Extraction

For direct use of aqueous extraction, 5 gram of leaves powder soaked in 100 ml of sterile

distilled water. The mixture then mixed thoroughly, left under rotary shaker 120 rpm for at

least 3 days under room temperature. Next, the plant mixture undergoes filtration process

and filtrate obtained was collected. An amount of the filtrate, approximately 5 ml were

separated and tested directly in the bioassay. The rest of the filtrate was allowed to dried

then dissolved in OMSO (10%) and also tested for the antibacterial activity.

3.4 Preparation of culture media

Mueller-Hinton agar were prepared by dissolving 38 g Mueller-Hinton powder in 1000 mL

ofdistilled water and were boiled for complete dissolve. Then, it was autoclaved at 121°C

for 15 minutes for sterilization. Next, the mixture were poured onto petri d ish for

approximately 5-6 mm thickness and allowed to solidify (Mishra et aI., 2013; Mhatre et

al.,2014).

3.S Preparation of bacterial culture

In sterile condition, the single colony of bacteria used were cultured in 10 ml of fresh

Mueller-Hinton broth and incubated at 37°C overnight. Then, the next day standardised

optical density (00) at 600 nm of 0.60 was attained (Sim et aI., 2013). The inoculums then

spread on the Muller-Hinton agar by using sterile cotton swabs and let it dried for few

minutes before implemented disc on top of the agar.

12

3.6 Antibacterial Assay

The antibacteria l activity of N. macrophylla leaf extract was evaluated by usmg disc

diffusion method (Bauer et aI., 1966). A sterile cotton swab was inserted into the bacterial

suspension, rotated, and then compressed against wall of the test tube to eliminate any

excess fluid. The swab then streaked on the surface of Mueller Hinton agar plate. The swab

was streaked three times over the entire plate surface to ensure a uniform and confluent

growth of bacteria (Mohammed and Orner, 2015). Each extract (aqueous and ethanol) at

100 mg/ml, 50 mg/ml, 25 mg/ml and 12.5 mg/ml concentration were impregnated into 6

mm blank antimicrobial susceptibility disc and were laid on top of the agar. Commercial

Gentamicin disc (10 !!g) and blank paper disc impregnated with 10% DMSO were used as

the positive and negative control respectively. After 24 hours incubation at 37°C, the

potential antibacterial activities were recorded by measuring the diameter of zone

inhibition growth (Lalnundanga et aI., 2015). Tests were performed triplicate. The

overview explaining methodology of antibacterial assay is shown in Figure 3.2. Antibiotic

Penicillin G, Tetracycline and Kanamycin for comparison purposes were conducted with

same method used in the antibacterial assay.

13

Bactelial culhues Impregnation of inoculated on leaf extract onto

Mullet Hinton agar J disc

,---------.,'.

Disc placed on I bacterial lawn

Incubated for 24 hours at 3 / C

Measurement of zone inhibitions

Figure 3.2: An overview represents the methodology of antibacterial assay.

3.6.1 Preparation of Disc for the Assay

Blank antimicrobial susceptibility paper disc of6 mm diameter were used and sterilized by

autoclaved at 121°C for 15 minutes before applying the extracts. Then 1 0 ~ I of the leaves

extract in different concentrations were applied to the sterilized paper disc after which the

disc will be allowed to air-dry (Lalnundanga et al.. 2015). In order to make a precise

impregnation of extract onto the disc, 5 ~I of extract of each concentration were applied on

both sides of the disc and allowed to dry. Then another 5 ~I were applied in same way as

before. All the disc must be in fully dried before impregnated onto bacterial lawn.

(Razmavar et al .. 2014). The position of disc is illustrated in Figure 3.3.

14

I

Figure 3.3: The position of disc that impregnated onto each bacterial lawn.

3.7 Positive and Negative control

Antibiotic (Gentamicin, 10 Ilg) were used as a positive control. The antibiotic disc was

placed on streaked Mueller-Hinton agar plate surface in sterile conditions. The

antibacterial activity was detected by measuring zones of inhibition (Mohammed and

Orner, 2015). For negative control, 10% of OMSO was used. Similarly, disc was placed on

streaked Mue ller-Hinton agar plate surface in sterile conditions. Negative control should

not exhibit any zone of inhibition. Then, the plates were incubated in incubator at 37 DC for

24 hours. Any observations were recorded.

15