Better worm freezing survival with low hydration and trehalose

I made a series of observations that led me to test whether trehalose could improve worm survival in freezing. I don’t really have time to keep testing conditions, but it be something that others who make a lot of worm stocks could try.

  1. I cultured worms on the bottom of plastic petri dishes in NGM salts (no nutrient, no cholesterol) with cholesterol added. Worms actually did fine in this media even without trace nutrients for up to 9 generations.
  2. When a few plates accidentally dried out I observed that adding distilled water would rehydrate worms and that dauers survived similar to previous observations on worm anhydrobiosis.

Since anhydrobiosis in worms probably depends in part on trehalose synthesis I tested whether trehalose could improve survival if present in dehydrated conditions and if trehalose could improve survival for worms that are frozen.

  1. I tested whether an approximately equal number of freshly starved N2 L1s/dauer mix washed and resuspended in NGM salts could survive in the following conditions. Note worms frozen in plastic petri dish instead of in a cryotube.

a) Freeze@-80°C only in NGM-salts
b) Freeze@-80°C after mixing 1:1 with 2x [] worm freezing buffer
c) Freeze@-80°C after mixing 1:1 with 2x [] worm freezing buffer and 15% trehaolse
d) same solution as a) except dehydrate overnight (25°C on benchtop) then Freeze@-80°C
e) same solution as b) except dehydrate overnight then Freeze@-80°C
f) same solution as c) except dehydrate overnight then Freeze@-80°C

Worms were only frozen for 24 (d,e,f) or 48 hours(a,b,c) hours then thawed and survivors counted the next day. For dehydrated worms that were frozen (d,e,f) water was just added to the dehydrated pad or crystal by pipetting water into the petri dish, no agitation was done, animals were just allowed to rehydrate for 1 hour. After 1 hour of thaw worms were pipetted onto NGM plates with food.

-Found that c) where trehalose was present had better survival rates than a & b.
-Mainpulation f) where worms were dehydrated then frozen survived better than all 5 other conditions. Condition f) had about double the # of survivors than b) which is the most similar condition to standard freezing protocol.

Confirmed that for f) both dauers and L1s/L2s survive.
4) another freeze experiment is just to confirm that trehalose allows L1s/L2s to survive the dehydration conditions in f) by using daf-16(mu86) worms which do not form dauers.

g) Same conditions as b - Survival OK
h) Same conditions as f - Survival better than g), and suggests the improved survival may not just be because more dauers survive freezing.

It would be nice if someone wanted to confirm whether presence of the best condition (dehydrating) where both glycerol and trehalose are present can be reproduced since it might help in freezing difficult strains. The lower water content of a dehydrated sample (with unknown amounts of residual water activity) might reduce ice crystal damage and it’s also possible trehalose stabilizes membranes as proposed by Erkut et al., 2011.