1. Scoop the animals off of a culture plate in a coating of E. coli using a pick
2. Place the animals and bacteria into metal hat until interior compartment is full to the brim
3. Place a blank hat on top to seal the compartment and load into HPF machine to freeze
4. There should be no air space surrounding the sample within the metal hat [to obtain maximum pressure]
Freeze Substitution
5. Hold frozen sample at –90 C for 3 days in 2.0% OsO4, 0.1% Uranyl Acetate in acetone fixative
6.
Warm sample to –74 C in same fixative and then to –60 C over 14 hour period
7.
Warm sample to – 30 C in same fixative over 14 hour period
8.
Warm sample to 0 C in same fixative over 1 hour period
9.
Transfer sample to 100% acetone three times at room temp, 20 min total
Infiltration
First change is in 1 part resin to 1 part acetone, allow to sit 30 min
Second change is in 2 parts resin to 1 part acetone, allow to sit for 30 min
Third change is into pure resin plus accelerator and hold one hour
Fourth change is into pure resin plus accelerator and hold overnight
The next day, place samples in fresh resin plus accelerator at room temp and place infiltrated worms in eppendorf tubes or gelatin capsules
Cure samples under UV irradiation overnight or until hard; keep at –20 C while curing in Pelco Cryobox
If desired, cut best worms out of embedment and re-embed in resin at desired orientation and re-cure in sealed flat mold
Resin formula
LR Gold resin plus 0.5% BME accelerator
Special Equipment needed
High Pressure Freeze apparatus; a very expensive item, but they are available at several national facilities including those in Madison, Minneapolis, Berkeley, and Albany. We used the machine run by Dr. Stan Erlandsen, in Minneapolis. Ya Chen operated the new Balzer’s instrument there, and others in that lab conducted the freeze substitution.
Freeze substitution apparatus; can be home-built or purchased
