Detection and Expression of Biosynthetic Genes in Actinobacteria ...
Detection and Expression of Biosynthetic Genes in Actinobacteria ...
Detection and Expression of Biosynthetic Genes in Actinobacteria ...
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BERVANAKIS, G.Chapter 2: MATERIALS & METHODSadded (BDH, Cat.No. 30223) to breakup the globules. Both the broth supernatant <strong>and</strong>organic layers were tested for antibacterial activity (see section 2.7).2.6.3 Concentration <strong>of</strong> the ExtractClarified organic extract was dispensed <strong>in</strong>to a 1 litre round bottom flask <strong>and</strong> subject toa rotary evaporator. The sett<strong>in</strong>gs <strong>of</strong> the rotary evaporator were speed at 4 m/s <strong>and</strong>temperature set at 45 o C. The organic solvent was evaporated <strong>of</strong>f, a powder wascollected. Filtrate <strong>of</strong> the MeOH or EtAc extract were placed <strong>in</strong> 10 ml glass test tubes<strong>and</strong> concentrated <strong>in</strong> a Savant CENTRIVAP (LABCONCO, Kansas City, Missouri)until the MeOH or EtAc was evaporated <strong>of</strong>f. Follow<strong>in</strong>g concentration the extractswere freeze-dried us<strong>in</strong>g the FTS Systems Maxi-Dry freeze drier hav<strong>in</strong>g the sett<strong>in</strong>gs at300 millitor <strong>and</strong> –70 o C for 6 hr.Section 2.7: Bioassays <strong>of</strong> Secondary Metabolites2.7.1 Plug Type BioassayScreen<strong>in</strong>g the act<strong>in</strong>obacterial cultures for the production <strong>of</strong> antimicrobial substanceswas carried out by extrud<strong>in</strong>g 6-mm plugs from 8 day act<strong>in</strong>obacterial cultures us<strong>in</strong>g asta<strong>in</strong>less steel cyl<strong>in</strong>der cork borer (6-mm <strong>in</strong>ner diameter, 8-mm outer diameter, <strong>and</strong>10-mm length). The plug was transferred to bioassay agar (BA) plates whichconsisted <strong>of</strong> (g/l) Beef extract 4, Peptone 4, Glucose 2.5, Sodium Chloride 3, Agar 15,pH 7.0 seeded with 2% (v/v) <strong>in</strong>oculum <strong>of</strong> test cultures (see table 29), grown <strong>in</strong> TrypticSoy Broth (TSB) culture conta<strong>in</strong><strong>in</strong>g (g/l) tryptic soy broth powder 30 at pH 7.2 to anoptical density <strong>of</strong> 0.1 at 600 nm . Optical measurements were performed on a UV-Visible Spectrophotometer SHIMADZU Model UV-160A. Bioassay agar plates were<strong>in</strong>cubated at 37 o C <strong>and</strong> zones <strong>of</strong> <strong>in</strong>hibition recorded after 2-4 days.Table 27: Test Cultures for BioassaysTest CultureTest activityC<strong>and</strong>ida albicans ATCC10231 AntifungalMicrococcus luteusAnti-bacterial (gram positive)Staphyloccocus aureus Anti-bacterial (gram positive)Bacillus pumilusAnti-bacterial (gram positive)Escherichia coli ATCC25922 Anti-bacterial (gram negative)_____________________________________________________________________64
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