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 ?

3. Design Project
Goal:
design an isolated, in vitro C. elegans gonad preparation protocol.
Purpose:
characterizing the molecular mechanisms of heterologous cell-to-cell communication using quantitative microscopy.

4. 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.

5. 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

6. 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.

7. Buffer Recipes

8. 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.

9. 3rd Phase – What's to come optimal dye loading on perfusion chamber
DIC image acquisition
Imaging with argon laser confocal microscope
Tie the process together and formalize protocol