Documenting Discoveries of Diversity
A Search for Novel Microbes

Course Approach - The Unknown Microbe ID Lab

1. Advanced Prep by Instructor
   
   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 onto new plates as unknowns for students.
2. Initial Phenotypic Characterization
   (schedule for Bio321W Microbiology - Lab portion of the course)
   1. Week 1 Tues - Lab Safety (print 2 copies), Aseptic Technique, Inoculation, Streak Plate Technique.
   2. Week 1 Thurs - Collect Colony Morphology Results, Photograph plates, Prepare pure cultures for stains, permanents. Discuss Lab Documentation on iGoogle page and Data Management.
   3. Week 2 Tues - Gram Stain, Endospore Stain, Wet Mount, Light Microscopy, Prepare Frozen Permanents.
   4. Week 2 Thurs - Media Preparation.
   5. Week 3 - Control of microbial growth by oxygen, temperature, ionic strength and antibiotics.
   6. Week 4 - Carbohydrate and nitrogen metabolism biochemical tests
   7. Week 5 - Exoenzyme tests, differential and selective medium
   8. Week 6 - Introduction to Bergey's Manuals. Identification of Bacteria using Biolog GenIII plates
   9. Week 7 - PCR amplification of 16S rRNA gene, gel electrophoresis, quantification of PCR product, sequencing.
   10. Week 8 - Fatty Acid Methyl Ester Analysis (FAME)
   11. Week 9 - Analysis of 16S rRNA Sequence Data, Construction of Phylogenetic Trees
   12. Week 10 - Biomerieux API Test Strips
       
       
3. The Real Work - Polyphasic Characterization in Parallel with closest relatives (by research students) - 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.
      
   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.