characterization of antibiotic … of...menllnjllkkan kajian lanjlll iintllk menyuling komponen...

t bull f

CHARACTERIZATION OF ANTIBIOTIC-PRODUCING FUNGI FROM UNIMAS RESERVE FOREST AND

THEIR ANTIBIOTICS

Norhafizah Binti Sidek

SF 1S4A NIJ9 Bachelor of Science with Honours lOll (Biotechnology Resource)

2012

Pusat Khidmat Maklumat Akademik bull f UNIVERSm MALAYSIA SARAWAK

PKHIDMAT MAKLUMAT AKADEMIK

111111111 100111111111 1000235680

CHARACTERIZATION OF ANTIBIOTIC-PRODUCING FUNGI FROM UNIMAS RESERVE FOREST AND THEIR ANTIBIOTICS

NORHAFIZAH BINT SIDEK

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 SARA W AK

2012

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

(NORHAFIZAH BINTI SIDEK)

Resource Biotechnology Programme

Department of Molecular Biology


Universiti Malaysia Sarawak

ACKNOWLEDGEMENT

First of all praise to Allah swt 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 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 fiiends especially Olivia Chan Nurulhazwanie Rafiee Mohd

Noorzaifiqrudin and Boo Chie Vi thank you for the support and sharing information To my

dear table partner Rahimah Manan 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

1

Pusat Kbidmat Maklumat Akademik UNIVEltSm MALAYSIA SARAWAK

Table of Contents

Acknowledgement i

Table of Contents ii

List of Abbreviations iv

List of Tables and Figures vii

Abstract 1

10 Introduction 2

20 Literature Review 5

21 Fungi 5

22 Soil as Reservoir of Antibiotic-Producing Fungi 5

23 Antibiotic-Resistance 6

24 Screening Method for Antibiotic Activity 7

241 Agar Overlay Technique 7

242 Antimicrobial Susceptibility Test Using Disc Diffusion Method 8

25 Bioautography _ 9

11

f bull t f

30 Materials and Methods 11

31 Preparation of Media 11

311 Preparation ofPDA Media 11

312 Preparation of NA Media 11

32 Sampling Sites and Collections II

33 Isolation of Microorganisms from Soil 12

331 Isolation and Subculturing 12

332 Subculturing of Bacteria and Fungi Colonies 13

34 Identification and Classification ofFungallsolates 13

341 Macroscopic Examination 13

342 Microscopic Examination 13

35 Molecular Identification 14

351 DNA Extraction 14

352 Polymerase Chain Reaction (PCR) 14

353 Purification of PCR Products 000000 00000000 0000 0015

354 Agarose Gel Electrophoresis 15

36 Antimicrobial Screening of Fungal Isolates 16

361 Preparation of Bacteria 00 00 00 00000000 00 000000 00 00 000000 000000 00 0016

111

t f

362 Preliminary Fungal Colony Screening 16

37 Extraction of Antibiotics 17

38 Minimum Inhibitory Concentration (MIC) of Extracted Antibiotics J8

381 Test Bacteria Preparation J8

382 Disc Diffusion Test of Antibiotic Extracts 18

39 Antifungal Test 19

310 Fractionation of Extracted Antibiotics Using TLC 20

311 Bioautography 21

40 Result 22

41 Isolation and Subculturing of Microorganism from Soil 22

42 Preliminary Colony Screening of Fungi 22

43 Antibiotic Screening for Hexane and DCM Extraction 25


45 Morphological Characterization 29

46 Thin Layer Chromatography (TLC) 33

47 Bioautography 36


IV

t

50 Discussion 39

51 Location ofSoiJ Sampling 39


53 Preliminary Colony Screening 39

54 Antibiotic Screening of Hexane and DCM Extraction 41




58 Bioautography 43


60 Conclusion 45

References 46

v

t t

List of Abbreviation

NA - Nutrient Agar

PDA - Potato Dextrose Agar

DCM - Dichloromethane

MHA - Mueller-Hinton Agar

MHB - Mueller-Hinton Broth

MIC - Minimum Inhibitory Concentration

TLC - Thin Layer Chromatography

PCR - Polymerase Chain Reaction

PBS - Phosphate Buffer Saline

SA Staphylococcuss aureus

ST Salmonella typhi

EC Escherichia coli

EA Enterobacter aerogenes

00 Optical Density

~L - microliter

mm - millimeter

cm - centimeter

mg - milligram

mL - milliliter

vi

List of Tables and Figures

TablelFigure Page

Figure 31 Map on location of soil sampling on UNIMAS reserve forest 12

Figure 32 Diagram representing the arrangement of fungal isolates on test 17 bacteria lawn

Figure 33 Diagram represented the arrangement of antibiotic extracts according to 19 concentra tion

Figure 34 Diagram represented the arrangement of selected fungal isolate for 20 antifungal test

Figure 41 Zone of inhibition formed by eight fungi isolates against E aerogene 24

Figure 42 Inhibition zone demonstrated by hexane extract of F5 isolate against EA 25 The MIC obtained was 025 mgml

Figure 43 Formation of concave and non concave inhibition zones between fungal 28 isolates and test fungus isolate Fusarium sp

Figure 44 Pure culture of fungal isolate F5 for the determination of macroscopic 30 characteristics

Figure 45 Fungal isolates F5 and F6 under compound microscope A Fungal isolate F5 30 B Fungal isolate F6

Figure 46 Spots observed under UV light demonstrated by DCM extracts of F7 isolate 36

Figure 47 Activity presented of fractionated hexane extracts of fungal isolates towards SA 38

Table 41 Preliminary colony screening for identifying of antibiotic-producing fungi 23

Table 42 Inhibition zones demonstrated by hexane extracts of F4-F8 isolate against 26 test bacteria

vii

I

Table 43 Inhibition zones demonstrated by OCM extracts of F4-F8 isolate against 27 test bacteria

Table 44 Presence of antifungal property in various fungal isolates 30

Table 45a Macroscopic characteristics of isolated antibiotic-producing fungi 31

Table 45b Microscopic characteristics of isolated antibiotic-producing fungi 32

Table 46a Rr value of fungal isolates for hexane extraction 34

Table 46b Rr value of fungal isolates for dichloromethane (OCM) extraction 35

Table 47a Rr value of fractionation hexane extraction on test bacteria ST 37

Table 47b Rr value of fractionation hexane extraction on test bacteria SA 37

VJll

t

Characterization of Antibiotic-Producing Fungi from UNIMAS Reserve Forest and Their Antibiotics


Resource Biotechnology Programme Faculty of Resource Science and Technology


ABSTRACT

A study was conducted to isolate antibiotic-producing fungal from soil samples collected from UNIMAS forest reserve Kota Samarahan Sarawak The soil was suspended and homogenized in phosphate buffer saline (PBS) and then was inoculated on potato dextrose agar (POA) or nutrient agar (NA) by spread-plate method Antibiotic-producing microorganisms were identified through preliminary screening of the isolated fungi by using agar overlay technique Out of the eight fungi isolated five were selected namely F4 F5 F6 F7 and F8 were selected for extraction with hexane and dichloromethane (OCM) This procedure involved drying 14 days old fungal culture followed by immersion of the dried agar in hexane and OCM The extracts were dried and subjected to disc diffusion assay against StaphylococclIs alreliS (SA) Salmonella typhi (ST) Escherichia coli (EC) and Enterobacter aerogenes (EA) Result showed that one or both the hexane and OCM extracts of all the five fungi isolates showed inhibitory activity against at least one of the test bacteria but at different concentration The strongest activity was shown by hexane extracts of F5 on EA with the MIC value of 025 mgfmL thin layer chromatography (TLC) and bioautography of all the extracts revealed three to six spots per chromatogram when subjected to Bioautography against the test bacteria only ST and SA were found to be inhibited This showed that further studies to fractionate and purity the antibiotic components could be carried out in the future In addition preliminary colony antifungal assay revealed that six of the eight fungal isolate showed inhibition of test fungus whic h is Fusarillm sp

Keywords Antibiotic antibiotic-producing fungal soil sample

ABSTRAK

Kajian dijalankan IIntllk memencilkan klliat penghasil antibiotik daripada sampel tanah yang diambil dari kawasan hutan sillpan UNMAS Kota Samarahan Sarawak Sampeltanah yang diambil dilarutkan bersama lanttall phosphate buffer saline (PBS) dan diiklti dengan homogenasi Kemlldian sedikit daripada lanttan tanah yang dihomogen diinokulasi pada permllkaan agar potato dextrose (PDA) dan agar nlllrient (NA) melalui kaedah piring sebaran (spread-plale) Penyaringan lInllIk mengenal pasti tindakan penghasilan antibiotic dijalankan menenlsi saringan awal mennggllnakan teknik pelapisan agar Daripada lapan jenis klliat yang diasingkan lima daripadanya iaitll F4 F5 F6 F7 dan F8 dipilih IIntllk diekstrak menggunakan hexane dan dichloromethane (DCM) Ekstrak dikeringkan dall diteruskan dengan penyaringan antibiotic dan diuji ke atas bacteria StaphylococclIs allreus (SA) Salmonella typhi (ST) Escherichia coli (EC) and Enterobacter aerogenes (EA) Hasil kajian IIendapati saw mahllplln kedlla esktrak hexane dan DCM daripada kesemlla lima klliat yang diasingkan menllnjllkkan aktiliti perencatan keatas sekurangnya satll bacteria pengllj i dalam kepekatan yang berbeza Aktiliti perencatan dapat dilihat daripada ekstrak hexane bagi klliat F5 keatas EA dengan nilai kepekatan perencatan minimllm ()25 mgmL kesemlla lima ekstrak kulat yang dillji dengan thin layer chromatography (TLC) dan bioatograji menunjukkan kesemua ekstrak mempunyai tiga hingga lima tlNk per kromatogram tetapi hasil kajian bioalltograji mendapati hanya bacteria SA dan ST yang dapat direncatkan Hasil menllnjllkkan kajian lanjlll IIntllk menyuling komponen antibiotic boleh dijalankan Disamping itll saringan awal koloni anti-klliat menllnjllkkan enam daripada lapan klliat yang diasingkan dapat merencatkan pembesaran klilat pengllji iaitu Fusarium sJl

Kata kunci Antibiotik klliat penghasil antibiotic sampeltanah

1

10 INTRODUCTION

Antibiotics are secondary metabolites produced by microbes or chemically synthesized

substances with the ability to kill other microorganism or inhibit their growth (Denyer et

01 2004) The first antibiotic to be commercially manufactured in the world was penicillin

which is produced by the fungus Penicillium nota tum (Silver amp Bostian 1993) Penicillin

became widely used in the late 1940s but its effectiveness was hampered by the

emergence of penicillin-resistant Staphylococuss aurells These Penicillium resistance

Staphylococcus aureus emerged just a few years after the first introduction of penicillin in

1950s (Frisvad et 01 1990)

Since the discovery of penicillin more than 100 antibiotics have been deve10ped by

the pharmaceutical industry and only some are commonly used in the health care

management until now (Borregaard 2001) Newly introduced antibiotics are effective in

the control of bacteria and infection including respiratory infection pneumonia and

bacterial throat infections treatment of typhoid fever and other intestinal infections like

dysentery tuberculosis sexually transmitted diseases like gonorrhoea and syphilis The

introduction of these antibiotics have proven that they are remarkably effective in the

control of bacterial and fungal infections (Eileen et 01 2010)

However antibiotic-resistant strains of bacteria have emerged and this resistance is

rapidly transmitted to other bacterial strains and species (Hughes amp Anderson 2001) In

recent years The Center for Disease Control and Prevention (CDC) USA estimates that

each year nearly 2 million people in the United States have acquired an infection while in

a hospital resulting in 90000 deaths More than 70 percent of the bacteria that cause these

2

(

middot

infections are resistant to at least one of the antibiotics commonly used to treat them

(Aureden et al 2010)

Despite the high expectation of finding synthetic molecules with effective

antimicrobial properties this alternative method has not been on the promising Hence

naturally occurring antibiotics are still needed in the programme to curb the problems of

ineffectiveness of existing antibiotics for the control of newly emerging antibiotic-resistant

bacterial strains This need has led to the increased interest in screening for strains of

microorganisms which have the potential of producing novel antibiotic

Soil has been used as a source for screening of antibiotic producing microorganism

for several decades Recent record have shown that screening of soil for antimicrobial

activity are still carried out in many parts of the world (Makut amp Owolewa 2011) One of

the target of soil screening is to find Penicillium sp which are able to produce nonshy

penicillin antibiotics This filamentous fungi of the genus Penicillium that belong to

ascomycetes are known to be easily found in the environment (Peberdy 1987)

Sarawak have been known as the large centre of biodiversity with large amount of

lands still not being used as a source for finding antibiotic-producing microorganism

Previously unexplored environments have their own ecosphere interactions and evolution

that might contain new producer organisms Those facts motivated the development of a

research program which aims to perform the isolation of antimicrobial substances from

UNIMAS soil fungi that might give any prospect to contribute in therapeutic industry

Based on the abundance of penicillin resistance bacteria and the ability of Penicillium sp

3

to produce non penicillin antibiotic a research study on discovering non penicillin

antibiotic by Penicillium sp is plausible

OBJECTIVESshy

1 To isolate antibiotic-producing fungi from soil UNIMASs reserve forest with

potential antibiotic

2 To characterize and identifY the isolates using macroscopic and microscopic

examination

3 To fractionate crude extract and identify the presence of antibiotics in the fraction

by using Bioautography

Ii

4

usat Khidmat Maldumat Akademik UNIVERSm MALAYSIA SARAWAK

20 LITERATURE REVIEW

21 Fungi

Fungi are microscopic plant-like cells that grow in long threadlike structures or hyphae that

make a mass called mycelium The mycelium absorbs nutrients from the roots it has

colonised surface organic matter or the soil (Tabasso 2006) Fungi is a group of simple

plants that have no chlorophyll There are some species of fungi that are single celled

organisms and there are other kinds of fungi that are multi-cellular organisms It produces

special hyphae that create the reproductive spores Fungi have many different structures

but they can act in similar ways Since the discovery of penicillin a number of antibiotics

have been discovered mostly from soil inhabiting fungi (Vaughan amp Barnes 2009)

A number of metabolites were isolated from fungi which found their way into

medical applications as natural products starting material for pharmaceuticals or as lead

structures for the development of pharmaceutical products (Kavanagh 2011) For example

nowadays research has been improved in order to isolates a novel active metabolites

which produce by fungi This fact together with the knowledge about the great potentiaL of

microscopic fungi for production of bioactive metabolites (eg penicillin from Penicillium

notatum ergotamin from Claviceps purpurea cidosporin from Tolypocladium inflatum)

(Tabasso 2006)

22 Soil as Reservoir of Antibiotic-Producing Fungi

Soil is commonly used as medium of finding a new antibiotic as it rich in diversity of

microorganism including fungi and bacteria Soil has capacity to supply adequate nutrients

5

I

to meet the needs of growing microorganism Fungi are an important component of the soil

microbiota typically constituting more of the soil biomass than bacteria depending on soil

depth and nutrient conditions The role of fungi in the soil is an extremely complex and is

fundamental to the soil ecosystem They perform ecological services that strongly impact

the quality of human life and have enormous potential for providing economic benefits

eg the isolation and identification of the soil fungus Penicillium leading to a large

pharmaceutical industry of antibiotics (Takashi et al 2008)

As fungi playa major role in soil ecosystems along with bacteria protists small

invertebrates and plants through complex trophic interactions Most soil fungi are

regarded as saprobes decomposing organic matter and contributing to nutrient cyding

while several species form mycorrhizal associations with plants or are plant pathogens

(Pfenning amp Abreu 2006) Also recognized as prolific secondary metabolite producers

fungi have provided several bioactive compounds and chemical models currently used as

pharmaceuticals and soils are traditionally the main source of fungal genetic resources for

bioprospection programs (Adrio amp Demain 2003) Despite that the biodiversity and

biotechnological potential of the soil mycobiota in many tropical regions is still poorly

studied This environment is thought to select species with adapted metabolism and good

potential for delivering new bioactive metabolites

23 Antibiotic-Resistance

Antibiotic resistance occurs when pathogenic microorganism are capable to inactivate

antibiotics or survive under the selective pressure of antibiotics (Gillespie 200 I)

Resistance is a condition in which the antibiotic fails to harm the pathogen enough to cure

6

shy

diseases Emergence of resistance often begins with a large pathogen population in which a

tiny fraction is naturally resistance genes from other microbes (Drlica amp Perlin 2004)The

emergence of antibiotic resistance in bacteria has been mainly attributed to the overuse of

antibiotic and indiscriminate antibiotic treatment are often the main cause that contribute to

the emergence of bacterial resistance to antibiotics (Eileen et aI 20 10)

This emergence of resistance in bacteria towards antibiotics has posed a big

challenge in the treatment of infectious diseases worldwide (Eileen et ai 20 I 0) The

resistance has arisen not only against both natural and semi-synthetc antibiotics but also

towards pure synthetic chemical compounds Some of the bacteria even become multidrug

resistant where resistance has been developed against several drugs

The antibiotic resistance property can be transmitted to the next generation through

vertical transmission of inherited mutations or by horizontally exchange of genetic

materials (Gillespie 200 I) Occasionally spontaneous mutations which occur in receptor

or binding site of the bacteria often leads to the change that renders antibiotics to become

ineffective (FDA 2009) Besides transferring of mutant resistance-causing genes from one

bacterium to another can also cause a bacterium to become resistant rapidly For example

horizontal transfer of mecA gene from Staphylococcus sciuri results in the emergence of

MRSA (Carlos amp Cuevas 1996)

24 Screening Method for Antibiotic Activity

241 Agar Overlay Technique

Agar overlay technique is used for preliminary and secondary antimicrobial activity The

strength of an antimicrobial agent can be detennined by agar overlay technique

7

(Frankhauser 20 I 0) In this technique a melted agar with test bacteria is poured evenly on

the top of an agar plate and allowed to solidify Reduction in the turbidity of the test

bacterial lawn around the extracted colonies shows the zone of inhibition Zone of

inhibition produced because of the antibacterial properties of the colony The size of the

zone of inhibition formed around it reflected the strength of the antimicrobial agent A

wider zone of inhibition will be observed for a greater strength of antimicrobial agent

(Hosttetmann 1999)

242 Antimicrobial Susceptibility Using Disc Diffusion Method

For the discovery of new antimicrobial drugs that are effective against an infection it is

crucial to determine the susceptibility of the pathogen Several techniques had been

developed for the test such as Ditch Plate Broth Dilution method standard tube dilution

minimum inhibitory concentrations standardize antibiotic sensitivity testing (AST) method

and others Kirby-Bauer disc diffusion method is commonly used as antibiotic

susceptibility test to determine whether a bacterium is susceptible to a specific antibiotic

(Jacobs et al 1979) The MIC value is determined to evaluate the minimum concentration

ofdrug that will inhibit the bacterial growth

During incubation the antibiotic diffuses outward into the medium and inhibits the

growth of test microorganism at certain concentration The effectiveness of antibiotics

towards test bacteria is analyzed by the formation of inhibition zone around the paper disc

The diameter of the zone of inhibition is proportional to the degree of susceptibility of the

test bacteria toward the antibiotic The organism is characterized as susceptible

8

middot

intennediate or resistant based on the diameter of the zone of inhibition formed (Mcdougal

amp Thornsberry 2000)

The paper discs which are impregnated with different concentration of antibiotic

are placed on the surface of an agar plate that has been inoculated with test bacteria Kirby

Bauer disk diffusion method employs the principle of the correlation between the size of

zone of inhibition around the antibiotic disc and sensitivity of bacteria to the antibiotic

(Drew el al 2001) The zone size is directly proportional to the sensitivity of the bacteria

to the antibiotic Antibiotic sensitivity is expressed in the term of MIC in which the

minimum concentration of drug that will inhibit the growth of bacteria was evaluated

25 Bioautography

Bioautography is a technique that combines thin layer chromatography (TLC) with

bioassay in situ (Shittu et al 2006) It can be used for the screening of separated

components of natural product extracts

Bioautography is often the only way to detect antibiotics that have been separated

on paper or thin layer chromatograms It is based on the biological effects of the substances

to be detected In general these effects can be the inhibition or promotion of growth of the

organism that is exposed to the zone that was separated on paper chromatography

(Touchstone 1992) Zones of inhibition in paper chromatography and zones of growth in

thin layer chromatograms make possible the detection of antibiotics Several hundred

antibiotics have been discovered in the past 30 years Due to the wide variations in

chemical properties of these compounds bioautography is the only general method for

their detection on thin layer chromatograms

9

In the flrst steps in the investigation of unknown antibiotics bioautography

becomes a method for classiflcation 0 crude antibiotics by TLC when it is not yet available

in pure form The method was developed for identifying the antibiotics contained in a

crude mixture during the early stages of isolation of these compounds The method was

used as a screening method for the present of antimicrobial activities in isolates ( Karen et

al 1996)

10

30 MATERIALS AND METHODS

31 Preparation of Media

311 Preparation of PDA Media

A total of 156 g of PDA powder was used to prepare 400 ml media The PDA powder was

transfer into a 400 ml bottle and added with distilled water Then the mixture was stirred

thoroughly until the powder completely dissolved Sterilization of the media was achieved

by autoclaving at 121degC for 2 hours Subsequently the PDA media was poured into petri

dish for preparation of PDA plates use for culturing the sterilize fungi samples About 13

ml of the media was aseptically poured into each plate Then the plates were allowed to

cool and solidify in the laminar flow hood before store at 4degC for future use

312 Preparation of NA Media

Similarly 112 g of NA powder were prepared for 400 ml media After autoclaving the

NA media was dispersed into petri dish to an average depth 4-5 nun After that the plates

were allowed to cool and solidify in the laminar flow hood before store at 4degC for future

use

32 Sampling Sites and Collections

The soil sample was collected in UNIMAS reserve forest areas (Figure 31) The sample

collected from two different sites with depths from 0-10 cm The samples then were

brought into laboratory and let to dry at room temperature

11

Figure 31 Map on location of soil sampling on UNIMAS reserve forest

33 Isolation of Microorganisms from Soil

331 Isolation and Subculturing

For each sample 19 of soil sample was transferred into a Falcon tube The soil Was then

suspended with 10 ml of PBS and homogenized with a Vortex mixer The tube was let to

stand at room temperature for 1 hour after which two ten-fold serial dilution with 9 ml PBS

100 11 of each dilution was dispensed into PDA or NA and spread with hockey stick

There were three replicates of NA and three replicates of PDA prepared Then the plates

were incubated at room temperature for 5 days

12

332 Subculturing of Bacteria and Fungi Colonies

After 5 days of incubation fungal colonies that showed inhibition towards the growth of

other microorganism were isolated and subcultured onto new agar media to obtain the pure

culture Then the plates were incubated for another 3 days at room temperature

34 Identification and Classification of Fungal Isolates

The morphologies of the fungal isolates were identified through macroscopic and

microscopic observations

341 Macroscopic Examination

The cultures were observed and physical characteristics were identified such as top and

reverse color parameter growth behavior mycelia mat and changes of medium

342 Microscopic Examination

The slide culture of the fungal isolates was prepared A small sample of fungus and agar

were cut out from the fungal culture and were transferred onto microscope slide The

sample was covered with a cover slip supported by plasticins Subsequently the culture

slide was placed into petri dish which was then sealed with parafilm After 5 days of

incubation at room temperature examination of the slide culture was carried out by

Binocular Compound Microscope (NIKON Corporation Japan) Microscopic

characteristics such as mycelial end branching structure of hypha and presence of spore

were observed using light microscope

13

Pusat Khidmat Maklumat Akademik bull f UNIVERSm MALAYSIA SARAWAK

PKHIDMAT MAKLUMAT AKADEMIK

111111111 100111111111 1000235680

CHARACTERIZATION OF ANTIBIOTIC-PRODUCING FUNGI FROM UNIMAS RESERVE FOREST AND THEIR ANTIBIOTICS

NORHAFIZAH BINT SIDEK

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

Bachelor of Science with Honours

(Resource Biotechnology)


UNIVERSITI MALAYSIA SARA W AK

2012

DECLARATION



higher learning






ACKNOWLEDGEMENT












when time needed

1


Table of Contents

Acknowledgement i




Abstract 1

10 Introduction 2


21 Fungi 5







11

f bull t f



















111

t f









40 Result 22







47 Bioautography 36


IV

t

50 Discussion 39








58 Bioautography 43


60 Conclusion 45

References 46

v

t t


NA - Nutrient Agar










ST Salmonella typhi

EC Escherichia coli


00 Optical Density

~L - microliter

mm - millimeter

cm - centimeter

mg - milligram

mL - milliliter

vi


TablelFigure Page














vii

I









VJll

t





ABSTRACT



ABSTRAK



1

10 INTRODUCTION








1950s (Frisvad et 01 1990)














2

(

middot






















3



OBJECTIVESshy




examination



Ii

4



21 Fungi
















(Tabasso 2006)




5

I























6

shy























7







(Hosttetmann 1999)















8

middot










25 Bioautography













9






al 1996)

10















use





11










12




















13

DECLARATION



higher learning






ACKNOWLEDGEMENT












when time needed

1


Table of Contents

Acknowledgement i




Abstract 1

10 Introduction 2


21 Fungi 5







11

f bull t f



















111

t f









40 Result 22







47 Bioautography 36


IV

t

50 Discussion 39








58 Bioautography 43


60 Conclusion 45

References 46

v

t t


NA - Nutrient Agar










ST Salmonella typhi

EC Escherichia coli


00 Optical Density

~L - microliter

mm - millimeter

cm - centimeter

mg - milligram

mL - milliliter

vi


TablelFigure Page














vii

I









VJll

t





ABSTRACT



ABSTRAK



1

10 INTRODUCTION








1950s (Frisvad et 01 1990)














2

(

middot






















3



OBJECTIVESshy




examination



Ii

4



21 Fungi
















(Tabasso 2006)




5

I























6

shy























7







(Hosttetmann 1999)















8

middot










25 Bioautography













9






al 1996)

10















use





11










12




















13

ACKNOWLEDGEMENT












when time needed

1


Table of Contents

Acknowledgement i




Abstract 1

10 Introduction 2


21 Fungi 5







11

f bull t f



















111

t f









40 Result 22







47 Bioautography 36


IV

t

50 Discussion 39








58 Bioautography 43


60 Conclusion 45

References 46

v

t t


NA - Nutrient Agar










ST Salmonella typhi

EC Escherichia coli


00 Optical Density

~L - microliter

mm - millimeter

cm - centimeter

mg - milligram

mL - milliliter

vi


TablelFigure Page














vii

I









VJll

t





ABSTRACT



ABSTRAK



1

10 INTRODUCTION








1950s (Frisvad et 01 1990)














2

(

middot






















3



OBJECTIVESshy




examination



Ii

4



21 Fungi
















(Tabasso 2006)




5

I























6

shy























7







(Hosttetmann 1999)















8

middot










25 Bioautography













9






al 1996)

10















use





11










12




















13


Table of Contents

Acknowledgement i




Abstract 1

10 Introduction 2


21 Fungi 5







11

f bull t f



















111

t f









40 Result 22







47 Bioautography 36


IV

t

50 Discussion 39








58 Bioautography 43


60 Conclusion 45

References 46

v

t t


NA - Nutrient Agar










ST Salmonella typhi

EC Escherichia coli


00 Optical Density

~L - microliter

mm - millimeter

cm - centimeter

mg - milligram

mL - milliliter

vi


TablelFigure Page














vii

I









VJll

t





ABSTRACT



ABSTRAK



1

10 INTRODUCTION








1950s (Frisvad et 01 1990)














2

(

middot






















3



OBJECTIVESshy




examination



Ii

4



21 Fungi
















(Tabasso 2006)




5

I























6

shy























7







(Hosttetmann 1999)















8

middot










25 Bioautography













9






al 1996)

10















use





11










12




















13

f bull t f



















111

t f









40 Result 22







47 Bioautography 36


IV

t

50 Discussion 39








58 Bioautography 43


60 Conclusion 45

References 46

v

t t


NA - Nutrient Agar










ST Salmonella typhi

EC Escherichia coli


00 Optical Density

~L - microliter

mm - millimeter

cm - centimeter

mg - milligram

mL - milliliter

vi


TablelFigure Page














vii

I









VJll

t





ABSTRACT



ABSTRAK



1

10 INTRODUCTION








1950s (Frisvad et 01 1990)














2

(

middot






















3



OBJECTIVESshy




examination



Ii

4



21 Fungi
















(Tabasso 2006)




5

I























6

shy























7







(Hosttetmann 1999)















8

middot










25 Bioautography













9






al 1996)

10















use





11










12




















13

t f









40 Result 22







47 Bioautography 36


IV

t

50 Discussion 39








58 Bioautography 43


60 Conclusion 45

References 46

v

t t


NA - Nutrient Agar










ST Salmonella typhi

EC Escherichia coli


00 Optical Density

~L - microliter

mm - millimeter

cm - centimeter

mg - milligram

mL - milliliter

vi


TablelFigure Page














vii

I









VJll

t





ABSTRACT



ABSTRAK



1

10 INTRODUCTION








1950s (Frisvad et 01 1990)














2

(

middot






















3



OBJECTIVESshy




examination



Ii

4



21 Fungi
















(Tabasso 2006)




5

I























6

shy























7







(Hosttetmann 1999)















8

middot










25 Bioautography













9






al 1996)

10















use





11










12




















13

t

50 Discussion 39








58 Bioautography 43


60 Conclusion 45

References 46

v

t t


NA - Nutrient Agar










ST Salmonella typhi

EC Escherichia coli


00 Optical Density

~L - microliter

mm - millimeter

cm - centimeter

mg - milligram

mL - milliliter

vi


TablelFigure Page














vii

I









VJll

t





ABSTRACT



ABSTRAK



1

10 INTRODUCTION








1950s (Frisvad et 01 1990)














2

(

middot






















3



OBJECTIVESshy




examination



Ii

4



21 Fungi
















(Tabasso 2006)




5

I























6

shy























7







(Hosttetmann 1999)















8

middot










25 Bioautography













9






al 1996)

10















use





11










12




















13

t t


NA - Nutrient Agar










ST Salmonella typhi

EC Escherichia coli


00 Optical Density

~L - microliter

mm - millimeter

cm - centimeter

mg - milligram

mL - milliliter

vi


TablelFigure Page














vii

I









VJll

t





ABSTRACT



ABSTRAK



1

10 INTRODUCTION








1950s (Frisvad et 01 1990)














2

(

middot






















3



OBJECTIVESshy




examination



Ii

4



21 Fungi
















(Tabasso 2006)




5

I























6

shy























7







(Hosttetmann 1999)















8

middot










25 Bioautography













9






al 1996)

10















use





11










12




















13


TablelFigure Page














vii

I









VJll

t





ABSTRACT



ABSTRAK



1

10 INTRODUCTION








1950s (Frisvad et 01 1990)














2

(

middot






















3



OBJECTIVESshy




examination



Ii

4



21 Fungi
















(Tabasso 2006)




5

I























6

shy























7







(Hosttetmann 1999)















8

middot










25 Bioautography













9






al 1996)

10















use





11










12




















13

I









VJll

t





ABSTRACT



ABSTRAK



1

10 INTRODUCTION








1950s (Frisvad et 01 1990)














2

(

middot






















3



OBJECTIVESshy




examination



Ii

4



21 Fungi
















(Tabasso 2006)




5

I























6

shy























7







(Hosttetmann 1999)















8

middot










25 Bioautography













9






al 1996)

10















use





11










12




















13

t





ABSTRACT



ABSTRAK



1

10 INTRODUCTION








1950s (Frisvad et 01 1990)














2

(

middot






















3



OBJECTIVESshy




examination



Ii

4



21 Fungi
















(Tabasso 2006)




5

I























6

shy























7







(Hosttetmann 1999)















8

middot










25 Bioautography













9






al 1996)

10















use





11










12




















13

10 INTRODUCTION








1950s (Frisvad et 01 1990)














2

(

middot






















3



OBJECTIVESshy




examination



Ii

4



21 Fungi
















(Tabasso 2006)




5

I























6

shy























7







(Hosttetmann 1999)















8

middot










25 Bioautography













9






al 1996)

10















use





11










12




















13

middot






















3



OBJECTIVESshy




examination



Ii

4



21 Fungi
















(Tabasso 2006)




5

I























6

shy























7







(Hosttetmann 1999)















8

middot










25 Bioautography













9






al 1996)

10















use





11










12




















13



OBJECTIVESshy




examination



Ii

4



21 Fungi
















(Tabasso 2006)




5

I























6

shy























7







(Hosttetmann 1999)















8

middot










25 Bioautography













9






al 1996)

10















use





11










12




















13



21 Fungi
















(Tabasso 2006)




5

I























6

shy























7







(Hosttetmann 1999)















8

middot










25 Bioautography













9






al 1996)

10















use





11










12




















13

I























6

shy























7







(Hosttetmann 1999)















8

middot










25 Bioautography













9






al 1996)

10















use





11










12




















13























7







(Hosttetmann 1999)















8

middot










25 Bioautography













9






al 1996)

10















use





11










12




















13







(Hosttetmann 1999)















8

middot










25 Bioautography













9






al 1996)

10















use





11










12




















13

middot










25 Bioautography













9






al 1996)

10















use





11










12




















13






al 1996)

10















use





11










12




















13















use





11










12




















13










12




















13




















13

characterization of antibiotic … of...menllnjllkkan kajian lanjlll iintllk menyuling komponen...

Documents