Antibacterial Activity of Biosurfactant

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CHAPTER 3
MATERIALS AND METHODS
3.1Materials
All the reagents used were analytical grade unless otherwise specified. SurfactinManufacturer
Surfactin standardSigma

Solvent Manufacturer
Acetonitrile, ACNMerck
Methanol Merck

Chemicals Manufacturer
Ammonium nitrate, NH4NO3Systerm
Disodium hydrogen phosphate, Na2HPO4Merck
Potassium dihydrogen phosphate, KH2PO4Bendosen
Calcium chloride, CaCl2Merck
Hydrous iron (II) sulfate, FeSO4.7H2OMerck
Ethylenediaminetetraacetic acid, EDTAMerck
Magnesium sulfate, MgSO4Merck
Glucose, C6H12O6Scharlau
Trifluoroacetic acid, TFASigma
Hydrochloric acid, HClFischer Scientific
Sodium hydroxide, NaOHMerck
3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT)Sigma

Pre-culture mediaManufacturer
Nutrient agarOxoid
Nutrient brothOxoid

Standard antibioticManufacturer
Streptomycin sulfateCalbiochem

Equipment Manufacturer
BiophotometerEppendorf
High performance liquid chromatography (HPLC)Agilent
MinicentrifugeEppendorf
CentrifugeBeckman
pH meterMetler Toledo
ELISA readerBiotex
Electronic balanceSartorius
Micropipette Eppendorf
Horizontal shaker and incubatorSastec

Microorganism Supplier
Bacillus subtilis ATCC21332

Shigella dysentriae ATCC 11311
Serratia marcescens ATCC 13830
Bacillus cereus ATCC 13061
Streptococcus faecalis ATCC 29212
Staphylococcus epidermidis ATCC 12228
Salmonella typhimurium ATCC 13311
Klebsiella pneumonia ATCC 13883

3.2 Method
3.2.1 Production of surfactin by microorganism
3.2.1.1 Microorganisms
In the production of surfactin from Bacillus sp., two types of Bacillus subtilis strains were used in isolating the wanted lipopeptides; surfactin fot this study. Bacillus subtilis ATCC 21332 and Bacillus subtilis 3M was selected to produce surfactin provided by Faculty of Sciences and Technology, Universiti Sains Islam Malaysia. 3.2.1.2 Culture Media

Both Bacillus subtilis ATCC 21332 and Bacillus subtilis 3M was first subcultured individually on nutrient agar preparing by diluting the nutrient agar powder with distilled water; 28g producing 1 litre or nutrient agar. The quantity of nutrient agar produced was according to the packaging of the manufacturing. For the first and second stages of the fermentation, the seed cultured from the nutrient agar were transferred into a defined mineral salts medium (MSM) developed by Cooper et al, 1981 known as Cooper`s media. The media consists of 0.03M KH2PO4, 0.04M Na2HPO4, 0.05M NH4NO3, 7.0 x 10-6 M CaCl2, 4.0 x 10-6M FeSO4.7H20, 4.0 x 10-6M EDTA, 8.0 x 10-4M MgSO4 and 4%(w/v) glucose. All chemicals were diluted with distilled water and sterilized in autoclave at 121ºC for 15 minutes except for 4.0 x 10-6M FeSO4.7H20 which after diluting with distilled water was sterilized using syringe filter. 3.2.1.3 Inoculum and culture conditions

Culture of Bacillus subtilis ATCC 21332 and Bacillus subtilis 3M were taken from -80ºC frozen stock and agar slant stock respectively for precultured onto the nutrient agar. The nutrient agar streaked with the Bacillus subtilis ATCC 21332 and Bacillus subtilis 3M were incubated at 37ºC for overnight. Two loops of cells were then inoculated into 100mL of Cooper`s media in 250mL Erlenmeyer flask and incubated in a rotator shaker at 37ºC with 200rpm agitation for 24hours as the first stage of fermentation or for the preparation of seed culture. The second stage of the fermentation was done by transferring 5mL of seed culture into 245mL of Cooper`s media in 500mL Erlenmeyer flask and further incubated in a rotator shaker at 37ºC for 144hours with 200rpm agitation. For every 24hours, samples were collected for further analysis of bacterial growth determination and surfactin concentration. 3.2.2 Analytical measurement

3.2.2.1 Measurement of Bacterial Growth
Each of the samples taken during the time interval was used to determine the bacterial growth during the cultivation period of...
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