Faculty of Resource Science and Technology

Antibiotic Screening of Soil Microorganisms Isolated from Remote Village in Nanga Merit,

Sarawak

Nurul Saiyidah Binti Abdul Rahim (19633)

Bachelor of Science with Honours

(Resource Biotechnology)

2010

DECLARATION

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

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

higher learning.

............................................................

(NURUL SAIYIDAH BINTI ABDUL RAHIM)

Resource Biotechnology Programme

Department of Molecular Biology

Faculty of Resource Science and Technology

Universiti Malaysia Sarawak.

ii

ACKNOWLEDGEMENT

First of all, praise to Allah s.w.t, the Mighty One, for His bless in giving me the strength

and good health to complete this study.

Most appreciation to Prof. Dr. Ismail bin Ahmad for being such a dedicated and

responsible supervisor. Thank you for the knowledge and guidance that has been given

throughout conducting the study. This appreciation is also dedicated to Lab Assistant, Mr.

Zaidi and Mr. Iskandar for providing the equipments and laboratory needs, and thank you

to all MASTER students of Virology Lab for the guidance and advice.

To my dear colleagues and friends, especially Fatin Fatanah Ramle, Munirah Abdul Talip,

Aisha Izam, Nurhamizah Merali, Joan Alicia Joseph Blandoi, Nurulain Mustafa Udin, and

Azyyati Zatil Iman Muhammad, thank you for the support and sharing of information. To

my dear table partner, Chai Sin Lin, lots of thanks for always helping and sharing

knowledge with me. Not to forget, to my beloved family, thank you for the pray and for

always being there when time needed.

iii

Table of Contents

Acknowledgement …………………………………………………………….. ii

Table of Contents ………………………………………………………………iii

List of Abbreviations ………………………………………………………….. v

List of Tables and Figures …………………………………………………….. vi

Abstract ………………………………………………………………………... 1

1.0 Introduction ………………………………………………………………... 3

2.0 Literature Review ………………………………………………………….. 5

2.1 Antibiotics …………………………………………………………. 5

2.2 Antibiotics-Resistant Bacteria ……………………………………... 7

2.2.1 Staphylococcus aureus……………………………………. 8

2.2.2 Methicillin-Resistant Staphylococcus aureus (MRSA) ….. 8

2.3 Antibiotic-Producing Microorganisms …………………………….. 9

2.4 Isolation of Soil Microorganisms ………………………………….. 10

3.0 Material and Methods ………………………………………………………11

3.1 Media Preparation …………………………………………………. 11

3.2 Sample preparation …………………………………………………11

3.3 Isolation of Microorganisms from Soil ...………………………….. 11

3.3.1 Isolation and Subculturing ……………...…………………11

3.3.2 Subculturing of Bacteria and Fungi Colonies ..…………...12

3.4 Preliminary Test of Antibiotic-Producing Bacteria and Fungi …..….12

3.4.1 Preliminary Screening …..………………………………...12

3.4.1 Secondary Screening ……………………………………...13

iv

3.5 Antibiotic Extraction ………………………………………………..14

3.6 Antibiotic Screening for Methanol Extraction ………….………… 14

3.7 Antifungal Test ……………………………………………………..15

3.8 Morphological Characterization ……………………………………16

3.8.1 Macroscopic Observation ……………………………….. 16

3.8.2 Microscopic Observation ………………………………... 16

4.0 Result ……….………………………………………………………………17

4.1 Isolation and Subculturing of Microorganisms from soil ..………... 17

4.2 Preliminary Test of Antibiotic-Producing Bacteria and Fungi ……..18

4.2.1 Preliminary Screening .…...…………………………........ 18

4.2.2 Secondary Screening ……………………………………...19

4.3 Antibiotic Screening for Methanol Extraction ………………………24

4.4 Antifungal Test ……………………………………………………... 27

4.5 Morphological Characterization ……………………………………. 27

5.0 Discussion …………………………………………………………………... 29

5.1 Location of Soil Sampling ………………………………………….. 30

5.2 Isolation and Subculturing of Microorganisms from Soil ………….. 30

5.3 Peliminary and Secondary Screening ………………………………. 32

5.4 Antibiotic Screening of Methanol Extraction ………………………. 33

5.5 Morphological Characterization ……………………………………. 33

6.0 Conclusion and Recommendation ………………………………………….. 35

References …………………………………………………………………….… 36

v

LIST OF ABBREVIATIONS:

NA - Nutrient Agar

NB - Nutrient Broth

PDA - Potato Dextrose Agar

MHA - Mueller-Hinton Agar

MHB - Mueller-Hinton Broth

MIC - Minimum Inhibitory Concentration

MIT - Massachusetts Institute of Technology

PBS - Phosphate Buffer Saline

SA - Staphylococcus aureus

ST - Salmonella typhi

EC - Escherichia coli

EA - Enterobacter aerogenes

MRSA - Methicillin-Resistant Staphylococcus aureus

OD - Optical Density

µL - microliter

mm - millimeter

cm - centimeter

nm - nanometer

vi

LIST OF TABLES AND FIGURES:

Table/Figure Page

Figure 2.0 6

Figure 3.0: Diagram represented the arrangement of bacterial isolates through 13

spot-inoculation technique

Figure 3.1: Diagram represented the arrangement of filter disc for antibiotic 15

screening

Figure 3.2: Diagram represented the arrangement of selected fungal isolates for 16

antifungal test

Figure 4.2: Colonies of Bacterial and Fungal Isolates 21

Figure 4.3: Methanol extraction of fungal isolates 25

Figure 4.4: Fungal isolates showing antifungal property 27

Figure 5.0: Various colonies within the zone of inhibition 32

Table 4.1: Average of Total Colonies Counted of Microorganisms Isolated 18

Table 4.2: Primary Screening of Antibiotic-Producing Bacteria/Fungi 20

Table 4.3a: Secondary Screening of Antibiotic-Producing Bacteria 22

Table 4.3b: Secondary Screening of Antibiotic-Producing Fungi 23

Table 4.3: Antibiotic Screening 26

Table 4.4a: Macroscopic Characteristics of Isolated Antibiotic-Producing Fungi 28

Table 4.4b: Microscopic Characteristics of Isolated Antibiotic-Producing Fungi 29

ANTIBIOTIC SCREENING OF SOIL MICROORGANISMS ISOLATED FROM

REMOTE VILLAGE AREA IN NANGA MERIT, KAPIT, SARAWAK

NURUL SAIYIDAH BINTI ABDUL RAHIM

This project is submitted in partial fulfillment of the requirement for the degree of

Bachelor of Science with Honours

(Resource Biotechnology)

Faculty of Resource Science and Technology

UNIVERSITI MALAYSIA SARAWAK

2010

1

Antibiotic Screening of Soil Microorganisms Isolated from Remote Village Area in Nanga

Merit, Kapit, Sarawak

Nurul Saiyidah Binti Abdul Rahim

Resource Biotechnology Programme Faculty of Resource Science and Technology

University Malaysia Sarawak

ABSTRACT

A study was conducted to isolate antibiotic-producing microorganisms from soil samples

collected from a remote village in Nanga Merit Kapit, Sarawak. The soil was suspended

and homogenized with phosphate buffer saline (PBS), and then was inoculated on potato

dextrose agar (PDA) or nutrient agar (NA) by spread-plate method. Screening for the

antibiotic-producing activity was accomplished through preliminary and secondary

screening, by using agar overlay technique. These procedures were followed with

antibiotic screening using disc diffusion test. Through preliminary screening, 16 bacterial

and 8 fungi isolates that showed zone of inhibition were selected for secondary screening.

After secondary screening, only six of the fungal isolates were selected for further

experiment which involves extraction of antibiotic by methanol. This procedure involved

drying of agar containing the selected isolates, followed by immersion of the dried agar in

10ml of absolute methanol. Antibiotic screening was then performed to screen antibiotic

activity for the methanol extractions. Result showed that two types of fungal isolates,

respectively coded as L10.1.F3 and L12.1.F1 carried the potential of producing antibiotic

with strong zone of inhibition against test bacteria. Another four fungal isolates, coded as

L11.2.F1, L11.2.F3, L11.2.F4, and L12.1.F1, only showed zone of inhibition on primary

and secondary screening. Probabilities of having insufficient amount of antibiotic extracts

may be the reason for these fungi to showed negative activity throughout the antibiotic

screening. Even so, these finding suggest that soil from a remote village in Nanga Merit

Kapit, Sarawak does contain with antibiotic-producing microorganisms. Isolation of novel

strains of antibiotic-producing microorganisms from this location seems to be possible.

Key words: Antibiotic, antibiotic-producing microorganisms, soil sample

2

ABSTRAK

Kajian untuk memencilkan mikroorganisma penghasil antibiotik daripada sampel tanah

yang diambil dari kawasan pedalaman sebuah perkampungan di Nanga Merit, Kapit,

Sarawak, telah dijalankan. Sampel tanah yang diambil dilarut bersama larutan ‘phosphate

buffer saline’ (PBS) dan diikuti dengan homogenisasi. Kemudian, sedikit daripada larutan

tanah yang dihomogen diinokulasi pada permukaan agar ‘potato dextrose’ (PDA) dan

agar nutrien (NA) melalui kaedah piring sebaran (spread-plate). Penyaringan untuk

mengenal pasti tindakan penghasilan antibiotik dijalankan menerusi saringan awal dan

saringan sekunder dengan menggunakan teknik pelapisan agar. Kemudian, diikuti dengan

saringan antibiotik menerusi ujian penyerapan disk. Sebanyak 16 jenis bakteria dan 8

jenis kulat yang menghasilkan zon perencatan telah dipilih daripada saringan awal untuk

melalui saringan sekunder. Hanya 6 daripada kulat yg dipencil telah dipilih daripada

saring sekunder untuk melalui eksperimen seterusnya yang mana melibatkan proses

pengekstrakan antibiotik menggunakan metanol. Prosedur ini memerlukan agar yang

mengandungi kulat yang dipilih, dikeringkan terlebih dahulu sebelum direndam bersama

10ml 100% metanol. Procedur penyaringan antibiotik dijalankan untuk menyaring

tindakan antibiotik daripada ekstrak metanol. Hasil kajian menunjukkan dua jenis kulat,

masing-masing dilabel sebagai L10.1.F3 dan L12.1.F1, berupaya menghasilkan antibiotik

yang kuat dan mampu merencat tindakan bakteria penguji. Hasil ujian daripada empat

kulat pencilan yang lain masing-masing dilabel dengan L11.2.F1, L11.2.F3, L11.2.F4, dan

L12.1.F2 hanya menunjukkan zon perecatan pada saringan awal dan sekunder. Hal ini

berkemungkinan disebabkan oleh kandungan antibiotik yang diekstrak keluar adalah

kurang. Meski pun begitu, secara keseluruhannya kajian ini menunjukkan bahawa tanah

dari kawasan pedalaman sebuah perkampungan di Nanga Merit, Kapit, Sarawak

mengadungi mikroorganisma penghasil antibiotik, dan tidak mustahil mikroorganisma

jenis baru dan berupaya menghasilkan antibiotik dapat dipencil daripada lokasi ini.

Kata kunci: Antibiotik, mikrooganisma penghasil antibiotik, sampel tanah

3

1.0 INTRODUCTION

The term antibiotic refers to chemical substances that provide the capacity to inhibit the

growth or even destroy bacteria and other microorganisms that cause infectious diseases in

human and animal (Hairston et al., 2000). The discovery of sulfonamide drug and later the

penicillins have led to other discoveries of different antibiotics. Since then, more than 100

varieties of these drugs have been developed by the pharmaceutical industry and are

broadly used until now (Bertrand et al., 2004). Bacteria begin to evolve and gained the

ability to resist the antibiotics‟ effect. Accordingly, a steady decline in the number of

effective antibiotics each year has been reported (Bertrand et al., 2004). Therefore, new

antibiotics are required to be developed. The antibiotics should be not only susceptible to

the resistant strain, but should also be able to replace some effective antibiotics such as

streptomycin which has been abandoned due to its negative side effects (Hairston et al.,

2000).

Staphylococcus aureus are pathogenic bacteria that can infect various tissues in the

body and leading to development of varieties of diseases. The difficulty in controlling S.

aureus infection is related to its ability to acquire resistant to antibiotic as soon as it is

exposed towards those antibiotics (García-Lara, 2005). It is therefore not surprising that the

number of methicillin-resistant S. aureus (MRSA) in hospital acquired infections, has

increased in recent years by 10-15% in countries such as Germany, United Kingdom, and

the United States (García-Lara, 2005). Its impact on health care problem is so serious, that

the antibiotic resistance and virulence in S. aureus is claimed to be one of depressing

evolutionary progression (Deresinski, 2005). This is due to the ability of this strain to

become resistant not only towards methicillin and β-lactams, but also to drugs of last resort

such as vancomycin, linezolid, and daptomycin (Memmi et al., 2008).

4

Earlier studies had found that three unrelated groups of microbes were mostly

involved in the production of natural antibiotics used in medicine. Those are the

Penicillium and Cephalosporium molds, the actinomycetes and the Bacillus species (Todar,

2009). Actinomycetes for example, have become the mainstay of the antibiotics industry.

This group of bacteria species has produces most of the clinically-useful antibiotics that are

not beta-lactams such as tetracyclines, streptomycin, and erythromycin (Todar, 2009). As a

matter of fact, these groups of microbes naturally inhabiting the soil. Therefore the soil

microorganisms from remote village in Nanga Merit, Kapit, Sarawak might provide with

possibilities of isolating novel antibiotics to which the resistant bacteria are susceptible

(Todar, 2009).

OBJECTIVES:-

1. To isolate microorganisms from the soil sample collected from a remote Village in

Nanga Merit, Kapit, Sarawak.

2. To extract antibiotics from the selected microorganism isolates.

3. To determine the susceptibility of the selected test bacteria towards the antibiotic

extracts.

5

2.0 LITERATURE REVIEW

2.1 Antibiotics

Antibiotic is derived from the word anti, which means „against‟ and bios which is referring

to „life‟- since a bacterium is a life forms (Nordqvist, 2009). Therefore, antibiotic was

originally defined as a substance produced by a microorganism, which has the ability to

inhibit the growth of other microorganisms (Russell, 1992). However, due to the

introduction of synthetic methods, the definition of antibiotic has undergone some

modifications. Currently, the definition of antibiotic is referred to a substance produced by

a microorganism, or a chemically similar substance that will either inhibit the growth or

destroy the other pathogenic microorganisms when applied at low concentration (Russel,

1992; Romanowski, 2007).

According to Todar (2009), most of microbiologists have distinguished two groups

of antimicrobial agents that are used for treated infectious diseases. Those that involve

natural substances produced by certain group of microorganisms are known as antibiotics,

while those that are chemically synthesized are referred to chemotherapeutic agents. In

addition to these groups, there is another group which is known as semisynthetic antibiotic.

This group of antibiotic contains molecular part produced by fermentation process using

the appropriate microorganisms and the product is then further modified through a

chemical process (Russell, 1992). Amoxicillin is one of the semisynthetic antibiotics in

which the main part of the molecule, 6-aminopenicillanic acid produced by a fungus, is

chemically modified by the addition of side chains (Figure 2) (Todar, 2009).

The mechanism of action of an antibiotic is described via the characteristic of the

antibiotics, either bactericidal or bacteriostatic (Nordqvist, 2009). Bactericidal antibiotic

kills the pathogenic bacteria by interfere with the formation of the bacteria cell wall or the

6

cell contents in the cytoplasm. Whereas, bacteriostatic antibiotic will inhibits the

multiplication without killing the bacteria (Todar, 2009; Nordqvist, 2009). The activity of

antibiotic can either be classified as broad or narrow spectrum. This is depends on its

specific action towards different groups of microorganisms. Todar (2009) stated that broad

spectrum antibiotics are referring to antibiotic that are effective for killing or inhibit a wide

range of prokaryotes, whereas if the antibiotics are mainly effective against either Gram-

positive or Gram-negative bacteria, they are said to be narrow spectrum. Nevertheless, if

they are restricted against a single organism or disease, they are referred as limited

spectrum.

One of the remaining medical challenges is the treatment of intracellular infections.

The problem arises from the inability of many antibiotics to penetrate and act in the

intracellular milieu (Lemaire et al., 2005). This phenomenon is typically related to

infections caused by Listeria monocytogenes or S. aureus. These symptoms are probably

due to the persistence of intracellular forms of these bacteria in both phagocytic and non-

phagocytic cells (Lemaire et al., 2005). As such, these organisms are difficult to eradicate

even after sustained antibiotic therapy.

Penicillin G Amoxicillin

Figure 2: Molecular structure of antibiotics

7

2.2 Antibiotic-Resistant Bacteria

The possibility on the development of antibiotic resistant bacteria had already been

expected by Dr. Alexander Fleming since the time he published his discovery of penicillin

(Roberts, 1998). The increase in usage of antibiotics has brought about the emergence of

bacteria that are resistant to currently used antibiotics. As a result, the numbers of

antibiotics that are effective have begun to decline (Bertrand et al., 2004). The

development of penicillin resistance was indicated through an increase in the MIC value of

the antibiotic. Such observation is related to the alterations in the enzymatic targets of β-

lactam antibiotics which is the penicillin-binding proteins (PBPs) (Tomasz, 1988).

Hughes and Datta, cited by Roberts (1998), had examined a collection of

Escherichia coli isolated at the beginning of the 20th century at which time, antibiotic

therapy was not yet available. Through the examination, they had found that these early

bacteria were not antibiotic resistant; hence they postulated a hypothesis claiming that the

antibiotic resistant E. coli was developed in response to antibiotic use in the last 50 years.

Therefore, it is believed that if disease-causing bacteria are continuously exposed to

antibiotics, there is a high probability that the bacteria will accelerate their adaptation to

these chemicals through evolutionary mutation (Hairston, 2000). This also being supported

by Tomasz (1988), as he stated that “ The prompt emergence of bacterial strains resistant

to these drugs (antibiotics) show that the ingenuity of clinical chemists is more than

matched by the resourcefulness of bacteria in finding counter measures that allow them to

evade the inhibitory effects of these antibiotics”.

8

2.2.1 Staphylococcus aureus

Staphylococcus aureus is a commonly occurring bacterium that resides on the skin and in

the nose of healthy persons (The Federal Bureau of Prison, 2005). This bacterium has the

ability to grow comparatively well under conditions of high osmotic pressure and low

moisture (Tortoro, 2004). Amongst all the Staphylococcus groups, S. aureus is the one that

causes most of infections (Stöppler, 2009). The bacteria gained their pathogenicity via

production of many toxins that increases the ability to invade the body or damage the

tissue (Tortoro, 2004; Stöppler, 2009).

In hospital, the surgical wound infection by S. aureus is a common problem.

However, its ability to quickly develop resistance toward antibiotics such as penicillin has

become a threat to patients in hospital environments (Tortoro, n.d.; García-Lara et al.,

2005). Moreover, the formation of intercellular aggregates and biofilms in which increases

evasion of host defenses and antibiotics tolerance have provide an explanation regarding

the ability of S. aureus to adhere to inert surfaces like medical devices and colonize living

tissue such as cartilage and heart valves (García-Lara et al., 2005).

2.2.2 Methicillin-Resistant Staphylococcus aureus (MRSA)

Methicillin-Resistant Staphylococcus aureus (MRSA) is a strain of S. aureus that is

resistant to methicillin and other β-lactam antibiotics including penicillin, ampicillin,

amoxicillin, and oxacillin (Federal Bureau of Prison, 2005; Stöppler, 2009). As stated by

Stöppler (2009), MRSA infections are usually mild superficial infections of the skin that

can be treated successfully with proper skin care and antibiotics. Nevertheless, due to

insufficient effective antibiotics available, MRSA can be difficult to treat and can progress

to life-threatening blood or bone infections.

9

Currently, bacteria known as vancomycin-intermediate resistant S. aureus (VISA)

and vancomycin-resistant S. aureus (VRSA) have been identified. These bacteria have

developed the strains that are resistant towards antibiotics vancomycin, eventhough this

type of antibiotics is normally effective against Staph infections (Stöppler, 2009).

2.3 Antibiotic-Producing Microorganisms

Acknowledge with the ability of certain fungi and bacteria to produce chemical substances

which inhibits or destroy pathogenic organisms (Waksman, 1952) has supported the fact

that, these unrelated groups of microbes have involved in most of the natural antibiotics

production (Todar, 2009). These substances are hypothesized to confer a selective

advantage to the producer when competition is significant to microbial fitness (Kinkel,

2006). A fungus such as Penicillium chrysogenum is an important industrial organism due

to its ability to produce several types of β-lactam antibiotics. Different antibacterial

properties could be obtained through the substitution of R-group substituent of the

penicillin nucleus (Onyegeme-Okerenta, 2009).

In September 1943, American microbiologist, Selman Waksman and associates

had managed to isolate the first streptomycin-producing microorganism from the soil

(Waksman, 1952). These were then followed with the isolation of other potential different

antibiotics, produced by the same species of Streptomyces such as actinomycin,

streptothrin, and neomycin (Romanowski, 2007). The specialized feature of Streptomyces

which are able to produce more than one antibiotic within a single strain (Hopwood, 1988)

might be the reason for actinomycetes to be the organisms that are recently significant to

antibiotics industry.

10

2.4 Isolation of Soil Microorganisms

As different microbes require different needs as well as different environmental conditions,

therefore it is impossible to devise only a single isolation technique that can be applied on

every type of microbes (Parkinson et al., 1971). One technique involves the isolation from

the soil suspension, by which the soil sample is suspended in medium or diluting fluid

(Parkinson et al., 1971). Besides, there is also method known as soil plate method.

Through this method, it allows the isolation of fungi from a large number of separate soil

samples, without the need of using plenty amount of dilution blanks.

Recently, study involves the isolation of soil-dwelling type bacteria named as

Rhodococcus is being conducted. These bacteria use to be known as non-antibiotics

producer. However, it has currently been discovered to gain the ability of producing

antibiotic that can be utilized for treatment of Helicobacter pylori, which causes stomach

ulcers in humans (Trafton, 2008). Due to this new discovery, it is believes to enhance the

development of strategies for finding other new antibiotics mainly from the soil.

11

3.0 MATERIALS AND METHODS

3.1 Media Preparation

In this study, PDA and NA media were used for growing and culturing the soil

microorganisms. The media were prepared according to the directions of the manufacturer,

DifcoTM

(USA) and OXIOD, CM0003 (England), respectively.

3.2 Sample Preparation

Soil samples were provided by UNIMAS Soil Laboratory. The soil samples were

originally collected from a remote village in Nanga Merit, Kapit, Sarawak. The samples

were obtained from seven different sites with two different depths, which are 0-20 cm and

20-40 cm. The samples were then brought to the laboratory and were air dried at room

temperature. From the original sample, about 10g was randomly sampled and put into

smaller plastic bag. These samples were then stored in the refrigerator at 4°C.

3.3 Isolation of Microorganisms from Soil

3.3.1 Isolation and Subculturing

Firstly, 1g of soil sample was transferred into a Falcon tube. Then the sample was

suspended with 10ml of PBS. After that, the suspension was homogenized by a vortex

machine and was left to stand for 1 hour in order to allow the large particles to settle.

Culturing was done by spread-plate method, in which 100µL of the aliquot was pipetted

onto the petri plates and was then spread by using sterilized cotton swab. For each sample,

there were four replicates of NA and four replicates of PDA. Then, the plates were

incubated for 5 days at room temperature.

12

3.3.2 Subculturing of Bacteria and Fungi Colonies

After 5 days of incubation, bacterial or fungal colonies that showed inhibition towards the

growth of other microorganisms were isolated and subcultured on new agar media to

obtain the pure culture. The plates were then incubated for another 3 days at room

temperature while the rest of the petri plates were transferred into a refrigerator at 4°C

before being subjected to preliminary screening of antibiotic activity.

3.4 Preliminary Test of Antibiotic-Producing Bacteria and Fungi

The preliminary test was conducted in two phases, which involves preliminary screening

and then followed by secondary screening. The agar overlay technique (Nkanga &

Hagedorn, 1978) was applied in both phases. This technique involves the preparation of

homogeneous bacterial lawn within a thin layer of agar across the surface of a plate

(Fankhauser, 2005). The bacterial lawn was prepared by adding appropriate concentration

of bacteria into soft NA (0.75%), followed by homogenizing it. In this study, three Gram

negative bacteria which were E. coli, Salmonella typhi, and Enterobacter aerogenes were

used as the test bacteria, whilst S. aureus was used as Gram positive test bacterium.

3.4.1 Preliminary Screening

Sample plates with the growth of bacterial and fungal isolates were took out from the

refrigerator and allowed to adapt to room temperature. Four replicates of each sample were

labelled as EC, EA, SA, and ST, respectively. Aliquots of 3 ml of both NB and soft NA

were prepared in the bijou bottles. The desired test bacteria were inoculated into NB and

were left at 37°C for an overnight incubation. After 18 hours of incubation, optical density

(OD) of the incubated test bacteria culture was measured and then adjusted to 0.6 OD at

13

520nm. Bijou bottles containing hot melted soft agar (NA), were placed inside water bath

to allow them to cool to 45°C, and the temperature was maintained.

Aliquots of 100µl of test bacteria were pipetted into the melted agar followed by

homogenizing using the vortex machine. Then, the agar mixture was immediately poured

onto the agar plate of sample. The plate was tilted back and forth, and shook gently to

ensure an even distribution. After the agar mixture had solidified, the plate was inverted,

and then incubated overnight at room temperature (28 °C). After the incubation, colonies

producing inhibition zone were observed and subcultured. The selected bacterial and

fungal isolates with same physical morphology were eliminated.

3.4.2 Secondary Screening

Fresh agar plates (NA for isolated bacteria, and PDA for isolate fungi) were prepared and

labelled. Pure selected fungal isolates were inoculated, and the plates were then incubated

for 4 days at room temperature (28°C). For bacterial isolates, spot-inoculation technique

(Huck et al., 1991) was applied to inoculate pure culture of the selected bacterial isolates

(Figure 3.0). The plates also were incubated at room temperature for 2 days. After the days

of incubation, same agar overlay technique as applied in the preliminary screening was

conducted. Isolates that produced zone of inhibition were once again observed. The pure

cultures of positive activity of bacterial and fungal isolates were cultured on slant agar

media for storage.

14

Figure 3.0: Diagram represented the arrangement of bacterial isolates through spot-inoculation

technique

3.5 Antibiotic Extraction

Selected bacterial and fungal isolates that showed the zone of inhibition on the preliminary

test were cultured on fresh agar media for 3 to 7 days. Then the agar was left to dry at

room temperature (28 °C) by slightly opening the cover of the petri plates. After period of

agar drying has completed, the dried agar containing the cultured antibiotic-producing

bacteria and fungi were immersed in 10ml of absolute methanol for 3 to 4 days. Then, the

methanol suspension was filtered to remove excess agar. The filtered methanol suspension

was evaporated to dryness at room temperature (28 °C). After that, the remaining residue

was suspended with 500µL of sterile distilled water. The suspension was transferred into

the eppendorf tube and stored inside the refrigerator.

Bacterial

isolates

Agar

media

15

3.6 Antibiotic Screening for Methanol Extraction

The test was performed to reconfirm and screen the susceptibility of the bacteria towards

the extracted antibiotic. Disk diffusion technique (McDougal & Thornsberry, 1983) was

applied to conduct this analysis. Suspension of each of the test bacteria was prepared to a

particular McFarland standard (0.168 OD at 510 nm). Then, an appropriate amount of each

suspension was accordingly swabbed over the surface of each Mueller-Hinton Agar

(MHA) media labelled SA, ST, EA, and EC. The agar media plates were then allowed to

dry for a few minutes. After that, filter paper disc with diameter 6mm were arranged onto

the surface of agar media (Figure 3.1). Aliquots of 10µL of each of the previously

extracted antibiotics were dispersed onto the filter disc. The plates were incubated for 24

hours at 37°C. After incubation, antibiotic extract producing the zone of inhibition were

observed, and the diameter was measured and recorded.

Figure 3.1: Diagram represented the arrangement of filter disc for antibiotic screening

3.7 Antifungal Test

The analysis of antibiotic-producing fungi was furthered with antifungal test in which

Fusarium sp. was used as the test fungal. In this test, four antibiotic-producing fungi were

first cultured on PDA media at appropriates position (Figure 3.2). Then, a test fungus

Negative

control

Positive

control

Methanol

extract

16

culture was placed at the centre of the PDA plate media. The plate was incubated at room

temperature for 5 days. Antibiotic-producing fungi that able to inhibit the growth of test

fungi were observed and the result was recorded.

Figure 3.2: Diagram represented the arrangement of selected fungal isolates for antifungal test

3.8 Morphological Characterization

The morphologies of the antibiotic-producing fungi were identified through macroscopic

and microscopic observation.

3.8.1 Macroscopic Observation

Physical characteristics of the selected fungal isolates were observed. Features of the

fungal isolates such as top and reverse colour, parameter, growth behaviour, mycelia mat,

and changes of medium colour were identified and recorded.

3.8.2 Microscopic Observation

Slide cultures of the fungal isolates were prepared. Microscopic characteristics such as

mycelium end, branching, structure of hypha, and presence of spore were observed using

light microscope.

Fungal

isolates

Test fungus