Chapter Outline: Assessment of prokaryotic biological activity, at the single cell level, by combining microautoradiography with fluorescence in situ hybridization (FISH)

Chapter Outline: Assessment of prokaryotic biological activity, at the single cell level, by combining microautoradiography with fluorescence in situ hybridization (FISH)

Proposed title: Assessment of prokaryotic biological activity, at the single cell level, by combining microautoradiography and fluorescence in situ hybridization (FISH) 

Chapter no.: _________

Author(s):      Cleber C. Ouverney (PhD)

                        Department of Biological Sciences

                        San Jose State University

                        San Jose, CA 95192-0100

                        Phone:             +1(408) 924-4806

Fax:                 +1(408) 924-4840

                        E-mail:             cleber.ouverney@sjsu.edu

Notes:

·       Some materials in this chapter may overlay with those covered in the Molecular Detection of Target Organisms and Sequences as well as chapters covering Physiological Detection of microorganisms.

Proposed topics

1)    A culture-independent method to render function of the 99% microbes in most environmental sites.

2)    A brief overview of in situ detection of biological activity by environmental microbes.

3)    How do MAR-FISH, STARFISH, Micro-FISH methods work to assess microbial metabolic activity in situ at the single cell level.

4)    Advantages to the approach, specifically the ability to simultaneously detect specific microbes using fluorescently labeled oligonucleotide probes and detect the capacity of microbes to uptake specific dissolved nutrient.

5)    What are the pros and contras of such approach?

6)    How do the microautoradiography-FISH methods compare to similar approaches?

7)    What substrates are used to render what type of metabolic activity?

8)    Suggestions on how to increase fluorescence signal to detect slow growing organisms.

9)    Will such laborious methods withstand the era of fast genomic sequencing?

10) Broad implications from studies applying such methods in the understanding of biogeochemical cycling of dissolved organic and inorganic nutrients in aquatic systems.

11) What other discoveries in the microbial world have been made from these techniques?

Chapter Highlights

The following concepts will be conveyed in this chapter:

1. How metabolic activity can be assessed from uncultured microbes in natural microbial communities.

2. Is Autoradiography-FISH technique right for my application? What do I need to know to make an intelligent decision?

3. Can genomic sequence data be tested/validated using this approach? How?