Protocols in Actinobacterial Research

I. Sample collection and isolation of actinobacteriao Collection and transportation of sampleso Soilo Sedimento Watero Plantso Insectso Fisheso Animalso Earthwormso Processing and pretreatment of sampleso Soilo Sedimento Watero Plantso Animalso Insectso Earthwormso Fisheso Sample pre-treatment for the isolation of actinobacteriao Physical treatmento Chemical treatmento Biological treatmento Isolation and enumeration of actinobacteria -o Standard spread plate methodo Recover and purification of actinobacterial cultures
II. Dereplication and ex situ conservation of actinobacteriao Dereplicationo Colony morphologyo Micromorphologyo FT-IR analysiso RFLP analysiso Ex situ conservationo Slant stocko Glycerol stocko Soil stocko Lyophilisationo Immobilized beads
III. Characterization and identification of actinobacteriao Phenotypic characterizationo Microscopico Culturalo Physiologicalo Chemotaxonomic analysiso Cell wall amino acidso Cell wall sugarso Cell wall lipidso FAME analysiso Menaquinone analysiso Molecular characterizationo DNA isolationo PCR Amplification of 16s rRNA geneo Sequencingo BLAST analysiso GenBank Submissiono Phylogenetic tree construction
IV. Metagenomic analysis of uncultured actinobacterial diversityo Sample collectiono DNA isolationo Amplificationo Clone library constructiono Sequencingo Metagenomic analysiso GenBank submission
V. Screening of actinobacteria for biological activitieso Screening for antimicrobial activityo Cross streak methodo Agar plug methodo Well diffusiono Disc diffusiono Broth dilution method - MTP Assayo MABAo Screening for antiinfective or anti-inflammatory activityo Screening for anti TB activityo Agar dilutiono LRP assayo Broth dilution methodo Bactec MGIT 960o Screening for anticancer activityo MTT assayo Dual stainingo Neutral red uptake assayo Lactic acid dehydrogenase assayo Tryphan blue uptake assayo DNA fragmentationo Screening for antiviral activityo MTT assayo P24 Assayo Plague assayo Screening for antioxidant activityo DPPH assayo Nitric oxide scavenging activityo Metal chelating assayo Total antioxidant assayo Hydrogen peroxide scavenging assayo Screening for pigment productiono Screening for enzyme inhibitorso Screening for mosquitocidal activityo Screening for enzyme productiono Screening for biosurfactant production - please mention different methodsHemolytic assay, bacterial adhesion to hydrocarbons (BATH) assay, drop collapse assay, oilspreading assay, emulsification assay and surface tension measuremento Screening for Quorum Sensing (QSI)o Screening for sideromycins productiono Screening for antibiofilm activity
VI. Production of bioproducts from actinobacteriao Fermentation methodso Agar surface fermentationo Solid state fermentationo Submerged fermentationo Production using immobilized cellso Extraction of bioactive metaboliteso Liquid liquid extractiono Solid liquid extractiono Precipitationo Production optimizationo Classical one-factor-at-a-time methodo Plackett-Burman modelo Response Surface Methodology (RSM)o Kinetics of antibiotic production - add few more kinetic experiments
VII. Eliciting cryptic metabolite productiono OSMAC approacho Inducing cryptic metabolite production using elicitorso Epigenetic modifierso Microparticle enhanced cultivation
VIII. Metabolite profiling of actinobacterial extractso TLCo HPTLCo UV spectral analysiso GC-MS analysiso LC-MS analysis
IX. Bioassay guidedisolation and characterization of metabolites from actinobacteriao Bioassay guided isolationo Preparative TLCo Column chromatographyo Preparative HPLCo Characterization of bioactive metabolites from actinobacteriao Physico-chemical Colour, consistency, solubility Melting point analysis Chemical screening Test for functional groups CHN analysiso Spectral analysis - UV, IR and NMRo XRD analysis
X. Evaluation of actinobacteria for environmental applicationso Heavy metal resistant/accumulationo Pesticide degradation (qualitative & quantitative)o Dye decolourization (qualitative & quantitative)o Plastic degradation(qualitative & quantitative)o Bioleaching of heavy metals from e-wasteo Anticorrosion activityo Antibiofouling activity - antimicrobial, inhibition of mollusc foot adherence, inhibition ofalgal spore germination, field testingo Screening of Co2 sequestration
XI. Evaluation of actinobacteria for agricultural applicationso Pre-experimental screeningo Antagonistic activity against phyto pathogenso PGP properties (IAA production, siderophore production)o Soil fertility enhancement (N2 fixation, P & Zn solubilisation, extracellular enzymeproduction)o Experimental screeningo Non-pathogenic test in plantso Green house experimento Field experiment
XII. Evaluation of actinobacteria for aquaculture applicationso Preexperimental screeningo Antagonistic activityo Acid toleranceo Bile toleranceo Cell adherence propertyo Non haemolytic propertyo pH toleranceo Experimental screeningo Non pathogenic testo Lab scale challenging test for probiotic properties & response measurementXIII.Evaluation of actinobacteria for nanoparticle synthesiso Screening of actinobacteria for nanoparticle synthesis usingo Biomasso Cell free supernatanto Extractso Characterization of nanoparticleso Spectralo Microscopic
XIV. Determination of cytotoxicity of actinobacterial extracts & metaboliteso Cell line based assayso Zebra fish modelo In vivo Animal model