Principles & Protocols

Protocols - Plunge freezing

Put on goggles!
Put on gloves!
Raise humidity in tent to 70% or higher
- turn on compressed air with bubblers
- spray water on inside walls of tent
Fill pool with liquid nitrogen
Use ethane to blow liquid nitrogen out of specimen area
Adjust the speed of the gas to fill pool with liquid ethane
Keep the tip of the hose submerged in ethane until full
Turn down ethane to remove hose, but do not turn gas off until out of pool
Align pool and plug in heater to prevent ethane form freezing
Test tweezers to make sure everything is lined up
Secure grid holey carbon grid (carbon side up) in tweezers with o-ring
Place sample on grid with pipette. Spread sample around grid with pipette tip.
Mount tweezers in plunge mechanism
Blot sample
- Put filter paper in contact with sample
- You will be able to see the grid through the paper
- An air bubble will form obscuring the grid
- Wait one second
Initiate Plunge with foot pedal
Keep foot on pedal
Keep tweezers in ethane
Raise plunger (can rest left hand to stabilize sample)
Raise o-ring on tweezers keeping sample submerged in ethane
Quickly splash grid into LN2 to remove ethane
Move grid into box
Top off pool with LN2
Adjust grid box in preparation of next sample

-- DavidStokes - 18 Mar 2005