1. Development of an Isolated, in Vitro C. elegans Gonad Preparation
Adam Broslat
Advisor: Dr. Kevin Strange
Professor of Anesthesiology and Pharmacology

2. What are C. elegans ? ~1 mm dwells in the soil
completely sequenced genome
simple body plan

3. Design Project
Goal:
design an isolated, in vitro C. elegans gonad preparation protocol.

4. Purposes
for the purpose of characterizing the molecular mechanisms of heterologous cell-to-cell communication using quantitative microscopy
This includes: Voltage sensitive dyes, pH sensitive dyes, Ca+ sensitive dyes, etc.

5. 1st Phase – Micro-dissection procedure
The nematode's gonad will be isolated in such a way not to harm the physiology of the gonad. Gonad operates independently of the worm
Problems:
The intestines “cloud” the view of gonad
after dissection and gonad does not completely
remove itself without manipulation.

6. Solutions to Date
Incisions made with a modified injection needle (red line)
Gonad is half extracted through depressurization
The other half is forced by suction using micropipettes

7. 2nd Phase – Functional Buffer
The worm and/or gonad must be placed in a buffer solution that promotes normal gonad function
Problems:
Worms are extremely active in buffer.
Buffer allows floating and movement.

8. Buffer Recipes

9. Worm Response to Buffer
Any of the previous listed buffers would sustain function to a point.
The worm gonad needs the the salts to mimic the interstitial fluid of the worm.
The gonad must also have an energy source this is where the glucose comes in.

10. Solutions to Date
.1% Tricaine and .01% Tetramasole anesthetic was added to chilled buffer for stabilization.
Veterinary glue was used on the glass of perfusion chamber to secure gonad.

11. 3rd Phase - Imaging optimal dye loading on perfusion chamber
DIC image acquisition
Imaging with argon laser confocal microscope
Tie the process together and formalize protocol