One of the many advantage of working with C. elegans is the ability to make frozen stocks from which viable animals can be recovered as needed. While we often brag about this to our fly colleagues, we all know that the truth is that worms really don’t freeze all that well. When thawing an aliquot of frozen stock, we are happy if we can recover more than just a few viable larvae amongst the mass of dead worms. Hoping to develop a better method for freezing worms, I turned to the literature on freezing plant cells which are difficult to freeze as cryoprotectants are often unable to penetrate the thick cell walls. Since the cuticle of nematodes might also block the penetration of some cryoprotectants, I decided to try a cryoprotectant that works on plant cells.The following is a protocol that I have modified for use in worms and have employed for the past 5 years. I have found that it is consistently far superior to the traditional glycerol-based method and allows the recovery of a sizable recovery of the frozen worms including all larval stages and adults.
Freezing Worms with Trehalose-DMSO
- Grow worms on 1-3 100 mm plates until freshly starved. Generally the more worms the better.
- Wash worms off plates in M9 buffer and collect by centrifugation (5000 rpms for 3 minutes is sufficient.
- Wash once in 4 ml room temperature Trehalose-DMSO freezing buffer.
- Re-suspend worms in 4 ml room temperature Trehalose-DMSO freezing buffer. Dispense 1ml per tube in 4 cryogenic vials.
- Incubate at room temperature for 30 minutes. This sometimes increases the rate of survival.
- Put vials into Nalgene Cryo 1 C Freezing Container (Cat. No. 5100-0001). Put container in -80°C freezer.
- 1 week later, test thaw one of the vials. Many animals should survive.
Trehalose-DMSO freezing buffer (Good for six months when stored at room temperature):
Add 15.1 g Trehalose (Sigma T0167) and 17.7 ml DMSO (14.13M) to enough M9 buffer so total volume equals 500ml. Filter-sterilize and store at room temperature.
Final concentration: 0.08M Trehalose, 0.5M DMSO.