Basic Techniques in Isolating, Purifying, Screening, and Identifying Microorganisms PDF

Basic Techniques in isolating, purifying, screening, and identifying microorganisms Overview Basic Isolation and Cultivation Techniques Types of Isolation A. SHOTGUN any natural or man-made environment B. OBJECTIVE specific sites where microorganisms with desired characteristics were present in the...

Basic Techniques in isolating, purifying, screening, and identifying microorganisms Overview Basic Isolation and Cultivation Techniques Types of Isolation A. SHOTGUN any natural or man-made environment B. OBJECTIVE specific sites where microorganisms with desired characteristics were present in the natural microflora Culture-dependent Methods Solid Media (Plating) SPREAD PLATING selective media and inhibitors For in situ isolation Not detrimental to heat-sensitive microorganisms Cons: Formation of coalesced colonies. POUR PLATING no pre-drying of agar needed small and subsurface colonies might be overlooked Liquid media (Broth culture) -selective media, inhibitors or enriched media -used widely for cultivation Types of Media Selective Allows growth of desired microorganisms but inhibits the growth of some microorganisms Differential Allows the desired group of microorganisms to be visually distinguished from other microorganisms Enriched Supplemented with nutritious materials that encourages the growth of desired group of microorganisms Serial Dilution Method -to reduce the number of microbial species for plating techniques -decide on the best dilution concentration and solvent to use -detrimental to rare species Cell Spray - liquid cell suspension is atomized with sterile air leading to the scattering of cells onto an agar plate. Filtration - separate particulate contents according to size and shape -membrane filters: 1. Filters from intertwined organic filters 2. Polycarbonate filter Centrifugation - separation according to size and density -for concentration of larger cells Optical Tweezers -Optical trapping - a focused light beam attracts the medium towards the center of the beam. - isolate cells using micropipette - commonly used for photo physiological studies of phytoplankton Micropipette - for single cell isolation -avoids cell damage and clean isolation Phototaxis - for isolation of flagellates that exhibit phototaxis Principle: A focused light source at one end of the apparatus will be used to attract a phototactic cell allowing isolation of the cell/cells Flow cytometry - to count and analyze optical properties of cells -Sorting of cells Fluorescence-activated cell sorting (FACS) - Sorts cells based on specific light scattering and fluorescent characteristics of cells. Isolation and cultivation methods for novel microorganisms Soil Substrate Membrane System (SSMS) Principle: micro-cultivation of soil bacteria using polycarbonate as growth support and soil extract as substrate Hollow-fiber membrane chamber (HFMC) Principle: Cultivation of microbial cells in the chambers is made possible through diffusion of nutrients from the environment/ liquid media to the chambers -in situ cultivation -encourage microbial growth requiring interspecific/intraspecific interactions Diffusion chamber Principle: Incubation of diffusion chamber in situ to allow diffusion of nutrient from environment -inoculation of mix environmental cells iChip -similar with diffusion chamber -contains hundreds of chambers to accommodate one cell each -to produce pure cultures Live-Fluorescent in situ hybridization (FISH) Principle: Labelled DNA probes target rRNA of specific taxonomic groups thru natural or chemical transformation or electroporation. -commonly combined with fluorescenceactivated cell sorting (FACS) cell sorting Reverse Genomics Principle: Utilization of engineered genome-informed antibody to capture specific microorganisms from complex microbial communities. Antibody-isolated cells would be cultivated in high-throughput media selections. Things to consider during isolation Purification of Isolates Plating Moist Chamber Culture Separation by Size (Microalgae) Filtration Differential Centrifugation Sonication and Vortexing Dilution Agar Plating Micropipette Checking of Purified Isolates Gross Morphological observation Microscopy: Gram Staining Microscopy: Lactophenol Blue Staining simple staining Phenol -kill microorganism Lactic acid -preserve fungal structure Cotton blue -stain chitin cell walls Screening of Microorganisms with Bioactivity General Process Assay Development Primary Screening High throughput Screening Secondary Screening Indirect Assays Automated equipment Direct Assays Indirect Assays Cell based assays based on cellular enzymes and proteins Dye exclusion assays Colony formation assay Detection of apoptosis indicators -cytosolic calcium concentration, apoptosis-related genes and proteins, caspases, DNA fragmentation/damage Indirect Assays Diffusion methods ⚬ Agar disk-diffusion method ⚬ Agar well diffusion method Dilution methods ⚬ determination of minimum inhibitory concentration (MIC) values ⚬ Broth dilution methods, agar dilution methods Time-kill test/ Time-kill curve ⚬ determination of bactericidal or fungicidal effect ⚬ time-dependent vs. concentration dependent effect Indirect Assays Absorbance Spectroscopy ⚬ measure reactant or product based on absorbance Diffusion assay ⚬ uses modified or selective media that shows enzymatic activities such as clearings. Immunochemical analysis ⚬ to quantify enzymes via immunoreactivity. ⚬ Western blotting Direct Assays Direct Assays Identification of microorganisms CHROMOGENIC MEDIA MICROSCOPY TECHNIQUES BIOCHEMICAL TECHNIQUES MOLECULAR TECHNIQUES Chromogenic Media culture-dependent identification thru production of certain metabolites such as lactic acid Microscopy analysis of morphology and motion of microorganisms dyes: easier and more specific visualization Biochemical Analytical Methods Biochemical Analytical Methods Mass Spectrometry-based methods ⚬ HPLC-based methods ￭ analysis of non-volatile or thermally labile high molecular compounds ⚬ GC-MS ￭ lipid elements ⚬ MALDI-TOF ￭ ionization of microbial cells with laser impulses ￭ molecular fingerprinting Biochemical Analytical Methods Spectroscopic Methods ⚬ analysis of molecular functional groups of the sample Electrokinetic Separation methods ⚬ analysis of microbial composition via migration patterns Microfluidic Chips ⚬ analysis of microbial constituents via movement of fluids ⚬ platforms with microchannels, pressure systems, and detection system Molecular Methods Molecular Methods Molecular Methods PCR ⚬ molecular markers: 16S rDNA (bacteria), 18s rDNA/ITS (Fungi) ⚬ rapid amplification of nucleic acids ⚬ Primers- for specific identification of gene of interests ⚬ RT-PCR ￭ higher sensitivity and accuracy ￭ for quantification of DNA concentration ⚬ Random amplification of polymorphic DNA-RAPD-PCR ￭ amplification of random, repetitive DNA regions ￭ to identify diverse range of bacterial species Gel Electrophoresis Molecular Methods RLFP ⚬ unique fingerprints based on polymorphisms in homologous DNA sequences ⚬ restriction enzymes are used to fragment genomic DNA in different lengths AFLP ⚬ ligated adaptors to amplify restriction fragments Ribotyping ⚬ rRNA based phylogenetic analysis ⚬ employs cutting of gene of interest using restriction enzymes then gel electrophoresis and hybridization of probes followed by analysis of ribotype RFLP bands