Short lifespan N2 - NGM plates problem?

Model Organism: Worms General Discussion
1

Hi everyone,

after receiving a new batch of N2 (wild type) strain, the lifespan median was 16 days. However, after a few months of maintenance at 20C the median dropped to 13 days.
We wonder now whether this issue arises from the way we prepare the NGM plates. This is a recurrent problem but we don´t see any contamination (fungi, bacteria,…).
We maintain the worms by picking and keeping them at 20C in the dark on OP50 seeded NGM plates.

The freshly prepared NGM (Agar, NaCl, Peptone) is autoclaved. After cooling down we add filter sterilized CaCl2, MgSO4, KPO4 pH6. We don´t check with a thermometer that the NGM is exactly 55C but we estimate it by hands. The plates are seeded with alive OP50 and kept at 20 degree for 2 days, afterwards storage at 4C in the dark.

Thank you in advance for any suggestions.

2

Are you adding cholesterol? Cholesterol starvation has been shown to decrease lifespan. If not this, it could just be random genetic drift, especially if you’re only picking one or two worms for stock maintenance.

3

Could the temperature at which one adds cholesterol to NGM while pouring affect its stability?
How many worms would you recommend to transfer to avoid such genetic drifts?

4

You can add it along with CaCl2, MgSO4, KPO4 when the temperature is around 55C.
Per 1L of NGM you need 1 mL cholesterol (5 mg/mL in ethanol).
To avoid genetic drift maybe pick more than 5 at a time and thaw new ones when needed (eg. we re-thaw our N2s from CGC stock once a year and backcross them to any mutant strains we’re working with)

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