Documenting Discoveries of Diversity
A Search for Novel Microbes

Research Approach - A Taxonomic Diversity Survey

1. Isolation of different organisms from sample
   
   1. Collect sample from environment of interest. (avoid samples likely to contain human pathogens).
   2. Dilute, spread bacteria onto petri plates (R2A, TSA), incubate for 48 hrs (avoid 37C).
   3. Select well isolated colonies and streak or patch onto new plates, assigning strain designations.
2. Identification/screening of Isolates.
   1. PCR amplification of 16S rRNA, Send out for sequencing
   2. Search the Type Strain Database at EZTaxon.org
   3. Repatch organisms with Genus/species/strain designations
   4. Prepare Frozen Permanent Stock Cultures
3. The Real Work - Polyphasic Characterization in Parallel with closest relatives - for novel species candidates (those with less than 99% similarity in 16S rRNA sequence during initial screen..
   1. Obtain Complete 16S rRNA Sequence
      
   2. Construct Phylogenetic Tree using RDP II
      
   3. Obtain Type Strain cultures of 2-4 most closely related species for reference and comparison.
   4. Prepare Frozen Permanent Stock Cultures
      
   5. Literature Research on Family, Genus and related species.
      
   6. Repeat all tests from Microbiology course, with novel organism and relatives in parallel.
      1. Streak Plates, Colony Morphology
      2. Cell characteristics (Staining, wet mount, microscopy)
      3. Oxygen & Temperature requirements
      4. pH, NaCl sensitivity
      5. antibiotic sensitivity
      6. carbon metabolism
      7. nitrogen metabolism
      8. differential and selective media
      9. Biolog GenIII plates
      10. Fatty Acid Methyl Ester Analysis (FAME)
          
   7. Isolate genomic DNA
   8. Multi-Locus Sequence Analysis
   9. GC Composition of DNA
   10. Family and Genus-Specific Tests
   11. Integration of Data into Strain Collection Data Sheet

Acknowledgements

This project has been supported by the National Science Foundation Award # DBI-0960114: MRI-R2: Acquisition of Instrumentation for Novel Microbe Characterization by Undergraduate Researchers.