Marine Microbiology Group - College of Marine Science - University of South Florida
Marine Microbiology Group - College of Marine Science - University of South Florida Marine Microbiology Group - College of Marine Science - University of South Florida Marine Microbiology Group - College of Marine Science - University of South FloridaMarine Microbiology Group - College of Marine Science - University of South Florida
Marine Microbiology Group - College of Marine Science - University of South Florida
Marine Microbiology Group - College of Marine Science - University of South Florida Marine Microbiology Group - College of Marine Science - University of South Florida Marine Microbiology Group - College of Marine Science - University of South Florida Marine Microbiology Group - College of Marine Science - University of South Florida
Marine Microbiology Group - College of Marine Science - University of South Florida

EDUCATIONAL OUTREACH

Fluorescence Lab for HS as part of the Biocomplexity Project

Concepts:
  1. What is the visual spectrum of light?
  2. What is fluorescence?
  3. How does Stokes Law dictate the possibilities for fluorescence emission?
  4. How is fluorescence used to detect biomolecules and microbes?
  5. How does a Molecular Beacon work, to detect specific biological targets?

    1. Skill I. The Excitation and emission of fluorescent compounds

      Using a darkened room and a white light source, split white light into its spectral components using a prism. With a poster of the spectrum on the wall or using the light spectrum, introduce the concept of light energy and wavelength. Then, introduce Stokes Law (only a longer wavelength can be emitted during fluorescence compared to the excitation wavelength).

      Using three fluorochromes in tubes with differing emission, display the "reflected" colored light using white light, and then using a blacklight, the emitted fluorescence light- Choices of compound_= fluorescein (emits 519), rhodamine (emits 576), Texas Red (emits 615)-You can use others based on cost and availability. Discuss the concepts of reflected, absorbed, and emitted light.


    2. How is fluorescence used as a tool in Biology?

      The idea here is to demonstrate the potential to detect biomolecules and microbes by using fluorescence technology.

      Have students illuminate test tube with 10-5 M Hoechst 3328 in 1X SSC with black light. Then, add a dropful of 0.1 mg/ml Salmon sperm DNA.

      Using a fresh tube of Hoechst, add 1 ml culture of bacteria, maybe +/- formalin (increases permeability and enables staining).

      Using the Fluorobacter - this is a fluorescent bacterial analogue to detect efficiency of hand washing - Ask Theresa about this as some arrived for her the other day.


    3. Detection of Amplified targets with Molecular Beacons

      We don't know if this will work so a trial is necessary. I was envisioning using a 200 ul optical tube inside a glass test tube that had water in it. We would need to have the beacon at 100 nm final concentration, perhaps in 100 ul of NASBA-like buffer (correct KCl, Tris, etc). To get started, use the fluorescence spectrophotometer in the dark lab, and see if you can see fluorescence viewed from the top. We could use either brevibeacon or the poliovirus beacon. Then, add some amplified target (Note this requires doing a NASBA first without beacon). See if you get a big increase in fluorescence. Try this also with a black light. With black light and a hot plate, see if you get thermal increase in visual fluorescence (heat up to 65o°C or maybe 70)-thrust in ice and see if you get decrease in fluorescence).


Marine Microbiology Group - College of Marine Science - University of South Florida Marine Microbiology Group - College of Marine Science - University of South Florida Marine Microbiology Group - College of Marine Science - University of South Florida Marine Microbiology Group - College of Marine Science - University of South Florida Marine Microbiology Group - College of Marine Science - University of South Florida Marine Microbiology Group - College of Marine Science - University of South Florida Marine Microbiology Group - College of Marine Science - University of South Florida
Marine Microbiology Group - College of Marine Science - University of South Florida Marine Microbiology Group - College of Marine Science - University of South Florida Marine Microbiology Group - College of Marine Science - University of South Florida Marine Microbiology Group - College of Marine Science - University of South Florida
Marine Microbiology Group - College of Marine Science - University of South Florida Marine Microbiology Group - College of Marine Science - University of South Florida Marine Microbiology Group - College of Marine Science - University of South Florida
Marine Microbiology Group - College of Marine Science - University of South Florida Copyright © 2008, University of South Florida, College of Marine Science 140 7th Avenue S., St. Petersburg, FL 33701 Marine Microbiology Group - College of Marine Science - University of South Florida
Marine Microbiology Group - College of Marine Science - University of South Florida Marine Microbiology Group - College of Marine Science - University of South Florida Marine Microbiology Group - College of Marine Science - University of South Florida Marine Microbiology Group - College of Marine Science - University of South Florida Marine Microbiology Group - College of Marine Science - University of South Florida