Methods to Study Microbial Biodiversity

I.  Inventorying and Monitoring Microorganisms
     A.  Environmental Matrices and Sampling Methods			
          1.  Air
          2.  Water										
          3.  Sediment										
          4.  Soil											
          5.  Higher organisms - plants and animals

     B.  Estimation of Numbers and Biomass
          1.  Cultivation
               a.  Typical abundances of bacteria
               b.  Advantages
               c.  Drawbacks
               d.  Media selection 
               e.  Cultivation conditions
                    - aerobic methods
                    - anaerobic methods
               f.  "Uncultureds" vs. "Unculturables"
          2.  Direct Examination (microscopy) 						
               a.  Typical abundances of bacteria 
               b.  Advantages
               c.  Drawbacks
               d.  Light microscopy
                    - Phase contrast and Nomarski differenential interference contrast
                    - Epifluorescence microscopy
               e.  Scanning electron microscopy
               f.  Particle counters and Flow Cytometry
          3.  Indirect Methods
               a.  Advantages
               b.  Drawbacks
               c.  Biochemical Methods
                    - Compounds in all microorganisms
                    - ATP
                    - Total DNA
                    - Protein
                    - Cell wall components 
                         + muramic acid
                         + lipopolysaccharide
                         + diaminopimelic acid
                         + teichoic acids
                         + chitin
                     - Membrane and storage lipids
                         + fatty acid composition
                         + sterols
                     - Photosynthetic pigments
                    - ELISA - enzyme linked immunosorbant assays
               d.  Physiological Methods
                    - Utilization of organic substrates 
                    - Chloroform fumigation method
               e.  Genetic Methods
                    - Colony hybridizations
                    - Membrane-based hybridizations				
                    - Genetic probes

II.  Cellular Activity and Viability
  A.  Viable but Not Culturable Concept 
     B.  Cellular Activity Methods
          1.  Adenylate energy
          2.  Direct viable count method
          3.  Electron transport system methods 					
          4.  Microautoradiography
           5.  Enzyme assays

III.  Detection of Microbial Populations / Community Identification
  A.  Biochemical Methods
          1.  Fatty acid profiles (FAME method)
          2.  Carotenoid pigments - different algae have different compositions
     B.  Physiological Methods
          1.  Substrate utilization patterns (Biolog microplates)			
     C.  Genetic Methods
          1.  %G+C Content - Bisbenzimide Method				
          2.  DNA-DNA Hybridizations					
          3.  PCR detection
          4.  Genetic Fingerprinting  

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