Detection and Expression of Biosynthetic Genes in Actinobacteria ...

BERVANAKIS, G.Chapter 2: MATERIALS & METHODScultures were analysed using thin layer chromatography for secondary metaboliteproduction.2.6.1 Actinobacteria agar metabolite extraction: Extraction of metabolitesfrom actinobacterial cultures grown on YME or ½ PDA agar was carried out usingtwo solvents separately, methanol HPLC grade (BDH, Cat. No. 15250) and ethylacetate analytical grade (BDH, Cat. No. 10108). Actinobacterial cultures were grownon ½ strength potato dextrose agar (PDA) which consisted of (g/L) Potato DextroseAgar powder 19.5, Agar 7.5, Cycloheximide 50 mg/ml, Nystatin 50 mg/ml, pH 7.2 orYME for 7 - 10 days. The agar was removed from the petri dish, chopped into smallfragments, and placed into 50 ml centrifuge tubes and immersed in 15 ml of MeOH orEtAc. Tubes were capped and placed onto a orbital mixer for 4 hrs at roomtemperature. Following incubation the extracts were filtered through Whatman® No.1filter paper 70 mm diameter (Cat. No. 1001070) to remove solids. Clear filtrate wascollected and analysed in antimicrobial assays (section 2.7).Culture IsolationFermentation Broth (Variation in media & conditions)Whole BrothExtractionStage 1aStage 1bMycelium (MeOH or EtAc)Extracted Broth SupernatantInactiveBioassaysActivesThin Layer ChromatographyBioautographyLarge scale productionSemi-PurificationPartial Identification LC-MS, UV-Vis SpectroscopyFigure 18. Schematic flow diagram of the isolation of microbial bioactive secondarymetabolites._____________________________________________________________________62