SCREENING FOR AMYLASE PRODUCING MICROORGANISMS FROM

THE SOIL AND FROM SPOILT CAKE SAMPLES

TAIWO, ATOYEBI BABAJIDE

MATRIC NO- 04/0369

A RESEARCH PROJECT

SUBMITTED TO

THE DEPARTMENT OF MICROBIOLOGY

COLLEGE OF NATURAL SCIENCES (COLNAS)

UNIVERSITY OF AGRICULTURE, ABEOKUT A,

OGUN STATE.

IN PARTIAL FULFILLMENT OF THE REQUIREMENT FOR THE AWARD

OF THE BACHELOR OF SCIENCE DEGREE (B.Se)

IN MICROBIOLOGY.

This project report is dedicated to God Almighty for His grace and faithfulness over my life.

Also to my folks for their unwavering support over the years.

I hereby acknowledge the creator of Heaven and Earth for the enablement to carry out this

research as part of the concluding part of my course synopsis. I also want to thank my one and

only supervisor in the person of Professor Inyang Akpan for his constructive criticism and hand

I also want to appreciate my mum for her unending support ever since my birth. Your display of

Studies were carried out to screen amylase producing microorganism from soil and spoilt cake.

Screening procedures adopted were plating of decimal dilutions of soil and cake on Sabouraud

dextrose agar with incubation at 30°c, isolation of pure cultures and flooding of isolate grown on

2% starch agar with iodine. Amylase positive isolate was detected by the formation of a hallo

against blue black background.

A total of 15 pure isolate of black mould (Aspergillus spp) was isolated and using the above

procedure, however none ofthe isolate was positive.

Titlepage

Certification 11

Dedication III

Acknowledgement IV

Abstract V

List of tables vi

Tableof content vii

CHAPTERONE

1.0 INTRODUCTION 1

1.1 AIMS AND OBJECTIVES 3

CHAPTERTWO

2.0 LITERATUREREVIEW 4

2.1 ENZYMES 4

2.1.1 PRODUCTIONOF ENZYME USING SOLID STATE

FERMENTATION(SSF). 4

2.1.1.0 MICROORGANISMSUSED FOR THE

PRODUCTIONOF ENZYME IN SSF SYSTEMS. 5

2.1.1.1 SUBSTRATESUSED FOR THE PRODUCTIONOF ENZYMES IN SSF. 6

2.1.1.2 FACTORS AFFECTINGENZYME PRODUCTIONIN SSF SYSTEMS. 8

2.2 ENZYMESPRODUCEDBY SSF PROCESS. 8

2.3 AMYLASES 9

2.4 SCREENINGFOR AMYLASEPRODUCINGMICROORGANISMS. 10

2.4.1 STARCHAGAR METHOD. 10

2.5 STARCH HYDROLYSIS BY AMYLASES. 12

2.6 MODE OF ACTION AND DISTRIBUTION OF ALPHA-AMYLASE. 12

2.7 LEVEL OF AMYLASE OCCURRING IN NATURE. 13

CHAPTER THREE

3.0 MATERIALS AND METHODS.

3.1 MATERIALS.

3.1.1 MEDIA.

3.1.2 CHEMICALS.

3.1.3 EQUIPMENT.

3.1.4 SUBSTRATES.

3.1.5 MICROORGANISMS.

3.2 METHODS

3.2.1 ISOLATION OF Aspergillus spp.

3.2.2 SCREENING FOR AMYLASE PRODUCING ORGANISMS.

CHAPTER FOUR

4.0 RESULTS AND DISCUSSIONS.

4.1.0 RESULTS.

4.1.1 TEST FOR AMYLASE PRODUCTION.

4.2 DISCUSSION.

CHAPTER FIVE

5.0 CONCLUSION AND RECOMMENDATIONS

5.1 CONCLUSION

5.2 RECOMMENDATIONS.

REFERENCES

APPENDIX

1.0 INTRODUCTION

Among a large number of non-pathogenic microorganisms capable of producing useful enzymes,

Aspergilli are particularly interesting due to their cultivation and high production of extracellular

enzymes. These enzymes are applied in various industries like detergents, starch, drinks, food,

textile, animal feed, baking, pulp and paper, leather, chemical and biomedical products (Pandey

et a/., 2000). Amylases constitute a class of industrial enzyme having approximately 25% share

of the enzyme market (Rao et al., 1998). The microbial amylases meet industrial demands with a

large number of them available commercially and have almost completely replaced chemical

hydrolysis of starch in starch processing industry (Pandey et al., 2000). The major advantages of

using microorganisms for production of amylases are the ability to produce in bulk and ease at

which it can be manipulated for desired products (Lonsane and Ramesh, 1990).

Also most of the reactions in living organism are catalysed by protein molecules called enzymes

(Schmid et ai, 2001). Enzymes can rightly be called the catalytic machinery of living organism.

(Rosenberg and Cohen, 1985) define enzymes as organic biological catalysts produced by living

cells

Amylase is of very wide occurrence in living organism ranging from human saliva to several

species of fungi and bacteria (Reed, 1996). Amylase is a very important commercial enzyme

having found use in the conversion of starch to varied product (Forgarty and Kelly, 1979).

Amylases are important enzymes employed in the starch processing industries for the hydrolysis

of polysaccharides such as starch into simple sugar constituents (Akpan et al., 1999; Omemu et

aI., 2004). Starch degrading enzymes like amylase have received great deal of attention because

of their perceived technological significance and economic benefits.

Among the microorganisms, many fungi had been found to be good sources of amylolytic

enzymes (Omemu et al., 2004).

Solid-state fermentation has been used in the production of industrial enzymes like amylase and

it has great potentials in the developing countries due to its simplicity of operations, low capital

cost and high volume productivity (Akpan et al., 1999; Omemu et al., 2004).

There is a continuing search for amylase with specific qualities especially with regards to

thermo stability, thermodurability, and thermoactivity and pH optimum. In communities which

are searching for and adopting non barley starchy grains for industrial production of beer, there is

increased demand for amylase as such grains may contain inadequate content of seedling

amylase (Jayatissa et aI., 1980; Ekot, 1992).

1. Isolation of amylase producers from soil and spoilt cake.

2. Screening of the amylase producers using 2% starch agar.

2.0 LITERATURE REVIEW

2.1 ENZYMES

Enzymes are among the most important products obtained for human needs through microbial

sources. A large number of industrial processes in the area of industrial, environmental and food

biotechnology utilize enzyme at some stage or the other. In addition to the conventional

application in food and fermentation industries, microbial enzymes have attained significant role

in biotransformation involving organic solvent media, mainly for bioactive compounds. Current

developments in biotechnology are yielding new applications for enzymes (Ashok et al., 2000).

Solid state fermentation (SSF) holds tremendous potential for the production of enzymes. It can

be of special interest in those processes where the crude fermented product may be used directly

as the enzyme source (Tengerdy, 1998; Ashok et aI., 2000). This system offers numerous

advantage over submerged fermentation (SMF) system, including high volumetric productivity,

relatively higher concentration of the products, less effiuent generation, requirement for simple

fermentation equipment, etc. (Chahal and Moo-young, 1981; Doelle et al., Pandey, 1994).

A large number of microorganisms including bacteria, yeast and fungi produce different groups

of enzymes. Selection of a particular strain however remains a tedious task, especially when

commercially competent enzyme yield are to be achieved. The selection of a suitable strain for

the required purpose depends upon a number of factors in particular upon the nature of the

substrate and environmental conditions (Ashok et al., 2000). Generally hydrolytic enzymes, e.g

cellulases, xylanases, pectinases etc are produced by fungal cultures, since such enzymes are

used in nature by fungi for their growth. Trichoderma spp and Aspergillus spp have most widely

been used for these enzymes. Amylolytic enzymes too are commonly produced by filamentous

fungi and the preferred strains belong to the species of Aspergillus and Rhizopus (Ashok et al.,

2000).

Although commercial production of amylases is carried out using both fungal and bacterial

cultures, bacterial amylase is generally preferred for starch liquefaction due to its high

temperature stability. In order to achieve high productivity with less production cost, apparently

genetically modified strains would hold the key to enzyme production (Ashok et al., 2000)

EJr.zyme Prua:ss

FunpI ceIIuIases ••• hcmic:dlulases EnzJmes nsiskd msiling

FunpI ceIIuIasa md hcmic:dlulases Bioplocasing of crops msidue

FunpI pcdill.,es cdlulases and hemicdIuIases Fibre pocess (Jdting)

Amylases" putl:ases" IipIses" c:dIuIaKs" Feed supplement

J-IemicdIuJases

Xylases Biopulping

IIJdmIJtic CIIZJ1De Dila:ted Wluposite

Lucases" Ii~ Soil bioranediaIioo

Tridllodeuua hazWnmJ ceIIuImes Post-banestn:sidue.~

T. baziMum cdIuIme helper fimdion Biopesticide

2.1.Ll SllWraas •••• fiJr tile •••••••••• of CllLJrm ill sgT.

Agro-industriaIlaiclllCS .egeoaaIly considen:d the best subsltrates fOr SSF procrss md the use

of SSF fOr die production of mzyme is no exa:plioo 10 1bat. A IIIIIIIbcr of suda suhstnfes have

been cmpIoJUI fOr the cuIImIOOn of IIIic:moIpJisms 10 produce die host of CIIIZ)IIIeS. Some of

the substJates 1bal have been used include \\fJeat bran" rice Inn" maize bran" wheat s1IaW~ria:

maw~ rice bu.*" soy bull" grapem.e trimminp dust" saw dust" aJIIIICCJbs,.••••• waste" tea

wBI; ~. waste" palm oil mill waste" SlIp" bed pulp" S\\a:t SOfEbum pulp" apple fICJIIB";

peanut meal,. GIpCSllXdcab; c:ooonut oil cab; mll'SWd oil cab; ~ ••••. ~wheat ••••. " com

ftour" saeamed ria;, steam pm-tmded willow" stan:b ( Pandey el ol~ 1994; TcngmIy" 1996;

selvakumar et al., 1998).the selection of a substrate for enzyme production in SSF process

depends upon several factors, mainly related with cost and availability of the substrate, and thus

may involve screening of several agro-industrial residues. In a SSF process, the solid substrate

not only supplies the nutrients to the microbial culture growing in it but also serves as anchorage

for the cells. The substrate that provides the entire needed nutrient to the microorganisms

growing in it should be considered the ideal substrate. However, some of these nutrients may be

available in sub-optimal concentrations, or even absent in the substrate. In such cases, it would

become necessary to supplement them externally (Ashok et al., 2000).

Among the several factors that are necessary for microbial growth and enzyme production using

a particular substrate, particle size and moisture level/water activity are the most critical (Pandey,

1992; Pandey, 1994).

Generally, smaller substrate particles provide larger surface area for microbial attack and, thus a

desirable factor. However, too small a substrate particle may result in substrate agumulation,

which may interfere with microbial respiration/aeration efficiency (due to increased inter-particle

space), but provide limited surface for microbial attack. This necessitates a compromised particle

size for a particular process (Nigam and Singh, 1994).

SSF processes are distinct from submerged fermentation (SMF) culturing, since microbial

growth and product formation occurs at or near the surface of the solid substrate particle having

low moisture content. It also makes it far easier to separate the chemical or enzyme of

importance from the medium and is therefore far more cost-effective (Padmanabhan et al.,

1992). Recovery of the enzymes from the fermented matter is an important factor that affects the

cost effectiveness of the overall process. It can be of special interest in those processes where the

crude fermented products may be used directly as enzyme sources (Padmanabhan et al., 1992).

The major factors that affect the microbial synthesis of enzymes include: selection of a suitable

substrate and microorganism; pre-treatment of the substrate; particle size (inter-particle space

and surface area) of the substrate; water content and water activity of the substrate; relative

humidity; type and size of innoculum; control of temperature of fermenting matter or removal of

metabolic heat; period of cultivation; maintenance of uniformity in the environment of SSF

system and the gaseous atmosphere, Le. oxygen consumption rate and carbon dioxide evolution

rate (Ashok et al., 2000).

Ideally, almost all the known microbial enzymes can be produced under SSF systems. Literature

survey reveals that much work has been carried out on the production of enzymes of industrial

importance like proteases, cellulases, lignases, xylanases, pectinases, amylases, glucoamylases

etc. and attempts are been made to study SSF processes for the production of inulinases,

phytases, phenolic acid esterases, microbial rennets, aryl-alcohol oxidases, tannin acyl

hydrolase, etc (Ashok et al., 2000).

The soil is known to be a repository of amylase producers (Jaeyoung et aI., 1989). Amylases are

hydrolytic enzymes, which degrade starch into simple sugar (Akpan, 1996). Amylase and

glucoamylase are characterized based on their individual actions on polysaccharides (Wood,

1991).

The family of the enzymes has been well characterized through the study of vanous

microorganisms. Presence of two major classes of starch degrading enzymes has been identified

in the microorganisms, a-amylase (endo-l, 4 - a-D-glucan glucohydrolase, EC-3.2.1.1) which

randomly cleaves the 1, 4-a-D-glucosidic linkage between the adjacent glucose units in linear

amylase chain, and glucoamylase (synonym amyloglucosidase also referred to as glucogenic

enzyme, starch glucogenase, gamma amylase; exo-l,4-a-D-glucan glucanohydrolase, EC-

3.2.1.3) which hydrolyses single glucose units from the non reducing ends of amylase and

amylopectin in a stepwise manner. Unlike a-amylse, most glucoamylases are also able to

hydrolyse the 1, 6-a-linkages at the branching point of amylopectin, although at a slower rate

than 1, 4-linkage (Ashok et al., 2000). The optimum pH of a-amylases ranges from 4.5-7.0

depending on the source from which the enzyme is extracted (Reed and Thorn, 1971). The

optimum temperature also ranges from 55-80°c (Reed, 1975). The product of starch hydrolysis

by a-amylase are maltose, glucose or maltotriose and a-limit dextrins (Forgarty and Kelly,

1979). The optimum pH of glucoamylase ranges from 4.0-5.0 and exhibits a temperature

optimum in the range of 50-60°c (Reed, 1975). Amylases and glucoamylases are produced by

various microorganisms including bacteria, fungi and yeast but a single strain can produce both

of these enzymes as well. These enzymes have found applications in processed food industry,

fermentation technology, textile and paper industries etc (Selvakumar et al., 1996; Ashok et ai,

2000).

Beta amylase is exo-acting and hydrolysis starch into beta maltose and beta limit dextrin

(Fogarty and Kelly, 1979). Beta amylase cannot cleave alpha-I, 6 branch linkage of starch

(Schmid et ai., 2001).

Glucoamylase is capable of hydrolyzing alpha-I, 6 as well as alpha-I, 4 glucosidase linkages

(Forgarty and Kelly, 1979).

Alpha amylase in contrast with beta amylase and glucoamylase is an endohydrolytic enzyme that

attack in chain linkages within starch component, having three or more linear alpha amylase

attack on starch which are limiting dextrins, maltose, glucose and maltotriose (Fogarty and

Kelly, 1979). The most important amylase in starch processing industries is alpha amylase and

glucoamylase (Aiyer, 2005).

Amylase production by microorganisms is usually detected by growing the organisms on solid

media containing starch and testing for starch hydrolysis (Forgarty and Kelly, 1979). Starch

hydrolysis can be detected as a clear zone surrounding the colony (Iverson and Millis, 1974).

The amylose fraction of starch produces blue-black colouration on flooding with iodine solution,

therefore the clear zones can be rendered more obvious by flooding starch agar plates with iodine

solution (Forgarty and Kelly, 1979).

Suh!i.tra Miaootpamns E'.nzJmes

Wheat ••• ~Imwdrii a -aIIIJIase

Wheal ••• Bodlbt.s lic_"'wis a-fllllYlme

Wheat ••• BodIIas mt.IglI.ions a-.nylase

Ricelnn ~";ger. la~~Il~oryaJf!; I

Wheat ••• AspBgilhts ,,;ger GIucmmyIase

Rice ...,. soyabean mcaI ~1IigeT GIucoaInyIase

Rice •••• -11- ~ sUIdt -11- AspBgilhts :species <iIucoaInyIze

ricehullsII

Depending on the relative location ofthe bond under the attack by amylases, the products ofthis

digestive process are dextrin, maltotriose, maltose and glucose etc. Dextrins are shorter, broken

starch segments that form as a result of the random hydrolysis of internal glucosidic bonds. A

molecule of maltotriose is formed if the third bond from the end of the starch molecule is

cleaved, a molecule of maltose is formed if the point of attack is the second bond; a molecule of

glucose results if the bond being cleaved is the terminal one, and so on. The breakdown of large

particles drastically reduces the viscosity of gelatinized starch solution resulting in a process

called liquefaction because of the thinning of the solution. The final stages of depolymerization

are mainly the formation of mono-, di- and tri-saccharides. This process is called

saccharification, due to the formation of saccharides (Bailey and Ollis, 1986; Nam Sun Wang,

2008).

Alpha amylase (Alpha-I, 4-D-glucan glucanohydrolase) is distributed widely in microorganisms.

It hydrolysis alpha 1, 4 glucosidic bonds in amylose, amylopectin and glycogen in an endo

fashion, but alpha 1,6 glycosidic linkages in branched polymers are not attacked. The properties

and mechanisms of action of alpha amylase depend on the sources of enzymes (Nam Sun Wang,

2008). They are all endo acting enzyme and therefore effect a rapid decrease in iodine staining

power and a simultaneous rapid decrease in the viscosity of starch action. Hydrolysis of amylose

by alpha amylase causes its conversion into maltose and maltriose, initially. Hydrolysis of

maltriose, which is a poor substrate, follows in a second stage reaction (Schmid et aI., 2001).

Hydrolysis of amylopectin by alpha-amylase also yields glucose and maltose in addition to a

series of branched alpha-limit dextrins. The dextrins of four or more glucose residues contain all

the alpha-I, 6 glucosidic linkages of the original structure. With amylopectin or glycogen, the

second stage of alpha amylase degradation involves slow hydrolysis of maltotriose as well as

slow hydrolysis of specific bonds near the branched points of the alpha limit dextrins for

example, the hydrolysis of 6-alpha maltotriosyl maltotetraose to 6-alpha glucosyl maltotetraose

and maltose. Different alpha amylase produces different alpha limit dextrin (Ashok et aI., 2000).

In addition to singly branched dextrin iso-maltose is not formed in these reactions because alpha

I, 6- linkages are resistant to alpha-amylases and they confer some stability on certain alpha-I, 4

linkages near the branch points. Formation of enzyme substrate complexes appears to be

restricted by the presence of these branch (alpha-I, 6) linkages.

Millers and Bakers found that flours from mechanically harvested wheat had little or no alpha

amylase activity, although considerable and adequate amylase activity increases during

germination and for both wheat and barley the increase is about 1000 fold. The low level of

alpha amylase in ungerminated wheat requires supplement of the flour with the amylase for

proper functioning in the production of baked goods. The alpha amylase serves two functions:

It provides continued formation of fermentable sugars for yeast activity and continued

vigorous starch production.

It affects dough properties and improves the structure and the keeping quality of bread.

(Gerald, 2000).

3.0 MATERIALS AND METHODS

3.1 MATERIALS

Sabouraud Dextrose Agar (Oxoid Ltd., D.K); Nutrient Agar (Biotec media, U.S.A); 2% Starch

Agar.

Chemicals used were manufactured by BDH chemicals Ltd., Poole, England. These are; Sodium

acetate, Acetic acid, Monobasic sodium phosphate, Dibasic sodium phosphate, potassium iodide

and hydrochloric acid.

The following equipment were used in the course of this work; Incubator (Gallenkamp, U.S.A),

Water bath (Clifton, U.K), Hot air oven (Gallenkamp, U.S.A), Bucket autoclave (Clifton, U.K),

Electronic weighing machine (Jenway 3015, Germany), Thermometer (Pyrex, England).

Rice bran, Soya-bean flour was obtained from a local market in Abeokuta, Ogun State. Starch

was obtained also in local market within the metropolis.

Aspergillus strain was isolated from spoilt cake samples and soil using the pour plate method as

demonstrated below.

The pour plate method was used to isolate Aspergillus sp from soil sample and spoilt cake

sample in an aseptic working environment achieved by swabbing the work bench with 75%

alcohol and using a spirit lamp containing alcohol. Ig of soil and spoilt cake was diluted serially

in 6 bottles each containing 9ml of distilled water each. Iml each of the 10-2, 10-4 and 10-6was

pippetted into 6 sterile Petri dishes, and then still liquid Sabouraud Dextrose Agar medium was

poured and gently swirled in the Petri dishes. These plates were allowed to set and incubate at

30°c for 48 hours to allow growth of the organism.

This was carried out on starch nutrient agar which was prepared by adding 2% starch (w/v) into

nutrient agar. This was then sterilized by autoclaving at 121°c for 15 minutes in an autoclave and

then allowed to cool. The glass Petri-dishes were placed in a canister and sterilized in a hot air

oven at 160°c for 2 hours, the work bench was swabbed with 75% alcohol. After cooling, it was

poured into sterile labeled plates over flame and was allowed to set. After setting, a stab

inoculating wire was flamed red hot and was used to point inoculate the spore of the isolated

Aspergillus strain making two stabs opposite each other to get discrete colony and the plates

were incubated at 30°c for 48 hours. After 48 hours of incubation, the plates were flooded with

iodine solution (Appendix III) and were observed for zone of hydrolysis i.e. clear zones.

CHAPTER FOUR

4.0 RESUL TS AND DISCUSSIONS.

It was observed that the Aspergillus strain isolated tested amylase negative as it did not

demonstrate a region of clear zone of hydrolysis around the colonies when flooded with iodine

solution. The negative colonies showed no haUo around the colonies against a blue-black

colouration on 2% starch agar.

The use of starch nutrient agar and iodine for detecting amylase (hydrolytic enzyme) producing

microorganisms have been reported by Forgarty and Kelly, (1979) and also by Iverson and

Millis, (1974) that starch hydrolysis can be detected on plates as a clear zone surrounding a

colony. This procedure employed showed a positive result for the Aspergillus strain isolated. The

mechanism of clear zone observed was due to the fact that the amylase produced during the

growth of the organisms has hydrolysed the starch around the colony, thereby testing negative

when flooded with iodine. The un-hydrolysed part of the plate tested positive to the presence of

starch (amylose), hence the blue-black appearance.

According to Akpan et ol. (1999) screening for amylase producing microorganism by the

method described above is time consuming and inconven~nt for direct isolation of intact cells,

as the cells die after flooding with iodine, therefore a rapid screening method such as Remazol

Brilliant Blue (R.B.B) will be more effective.

The former method was adopted for this research which involves screening through the use of

starch agar and iodine solution. The formation of clear zones indicated that the organisms test

positive but out of 15 isolates screened none tested positive.

In conclusion, work done and critical analysis of literature, shows that production of industrial

enzymes by solid state fermentation offers several advantages, some of which are high

productivity per unit volume of reaction, non septic condition, use of raw materials as substrate,

reduced energy requirement, low waste water output, low capital investment, etc. It is hoped that

our country which is endowed with raw materials needed will develop this promising technology

for industrial enzyme production, composting, moulds ripened cheese production and mycotoxin

production.

Nigeria is a country blessed with abundant resources needed for the production of these

enzymes. On this basis I recommend that;

.:. Scale-up strategies for the extraction of enzymes from microorganisms through Solid

State fermentation;

.:. Strain development strategies should be looked into for detection of high fermenting and

hydrolyzing strains.

Amylase by Aspergillus niger in cheap solid medium using rice bran and agricultural

materials. Journal of Tropical Science, 39:77-79.

Production of industrial enzymes. Journal of Science and Industrial Research, 55:443-

449

Bailey, J.E. and Ollis, D.F. (1986). Biochemical Engineering Fundamentals. McGraw-Hill, 2nd

Jaeyoung, K., Takashi, N. and Rya, S. (1999). Amylase synthesis by Aspergillus spp. Journal of

20

state fermentation: a potential tool for achieving economy in enzyme production and

starch hydrolysis. Advances in Appl. Microbiol. 35: 54-56.

by amylase of Aspergillus niger AM07 isolated from soil. African Journal of

Biotechnology, 4: 19-25.

Reed, G. and Thorn, J. (1971). Wheat chemistry and Technology. American Association of

Cereal Chemist Journal, 2:453-491.

Schmid, A., Dordick, J.S., Haver, B., Kiener, A., Withalt, B. (2001). Industrial biocatalysis today

and tomorrow. 409: 258-268.

Tengerdy, R. (1998). in Advances in Biotechnology (ed. Pandey, A.), Educational Publishers and

Distributors, New Delphi, pp. 13-16.

Woods, L.F. (1991). Enzymes in starch processing, Van Nostond and Reinhold (U.K). pp. 212-

215

Appendices

Suspend 65g in lL of distilled water. Bring to the boil to dissolve completely and for proper

homogenization. Sterilize by autoclaving at 121°c for 15 min.

2g of starch was added to 2.8g of nutrient agar and was suspended in lOOml of distilled water.

This was homogenized by heating and was sterilized by autoclaving at 121°c for 15 min.

6g of iodine crystals was mixed with 9g of potassium iodide. The mixture was dissolved in lL of

distilled water.