Hi Yamila,

I’m afraid what you have in the last post is a Frankenstein’s monster of a Dauer Assay protocol. It appears to be a composite of sentences taken from various contexts that fit awkwardly together as a general protocol. The inclusion in a ‘general dauer assay protocol’ of dauer counting in daf-22 (daf-d) mutants is unnecessary and also particularly misleading.

So, back to your original two questions;

in temperature treatment:

how many worms per plate do I measure?

Assuming each adult hermaphrodite lays between 4 and 10 eggs per hour, then leaving 4 adults on a 55mm NGM/OP50 plate for 5 hours gives you up to 200 eggs. Obviously for comparing wildtype to a daf-c mutant this would be overkill.

However, in an extreme case, testing two mutants;

e.g. daf-7;hen-1 (0.79 dauer @ 25°C) and daf-7;scd-2 (0.78 dauer @ 25°C)

[http://www.ncbi.nlm.nih.gov/pubmed/18674914]

which look very similar, would need a huge number of worms to be tested: for 80% power @0.05 it’s over 35,000 per mutant!

For the daf-7;scd-2 (0.78) and daf-8 (0.68) the numbers are easier to swallow (power =80%, 0.05, n=309)

Do the math and casual observation of the mutant phenotype before deciding on how many worms you need.

in which larval state do I stress the worms (L1 or L2)?

If you look through articles using some form of dauer assay they often start with eggs at the permissive or restrictive temperature.

crowded/starving:

how do I normalize the results of the differents strains (inicially i will have a plate full of worms it will be impossible to know the number of worm treated)or do I use a small plate with 200 worms (is it crowded?)

To take the first condition last, you can starve 200 worms of a suitable age on an NGM plate without OP50 as easily as you can 2000.

The first condition (overcrowding), then essentially you are sampling from a large population. If you determine that 70 worms in every 100 observed are dauer then the proportion is 0.7. The larger the sample size, the greater the statistical inference that the result is true.

Steve