Is there any way to stain live dauer larvae to distinguish them from non-dauer worms?
You can treat the animals with SDS, which will dissolve or kill non-dauers and leave dauers alive. A glance at a Google search suggests the original reference for SDS treatment of elegans dauers may be Cassada and Russell 1975)
Thank you for the suggestion. However, I want to count the proportion of dauers and non-dauers in the culture, so I would prefer not to dissolve the last ones.
I’m assuming there is some reason you can’t just go by morphology? Or by pharyngeal pumping? In addition to observing for pumping, a variety of dyes and particles have been used to assess pumping and feeding, and dauers do neither.
I found this in a Google search
Sudan black, which stains fat, does not appreciably stain wild-type larvae grown under reproductive growth conditions. However, it darkly stains dauers and Daf-c mutants- from [url=http://dx.doi.org/10.1016/S1534-5807(01)00085-5]Gerisch [i]et al.[/i][/url].
Are you trying to count the dauer proportion from a mixed population? If not, morphology on a synchronized culture is the easiest.
Well, I am planning to count large numbers of worms within a mixed population. Therefore I believe a stain will be the quickest way to tell them apart.
I will look into the Sudan black suggestion.
I encountered a similar problem: As some daf-2 strains form dauer larvae with characteristics of L3 or L4 stage larvae (Labbe et al., PNAS 97, 13233 & my own observations) it’s not useful to distinguish by morphology. Sudan black staining works only upon formaldehyde fixation (thus killing) of specimens (at least I couldn’t find a method in living animals, but these upon fixation: Ogg et al., Molecular Cell 2, 887 & Kimura et al., Science 277, 942).
What would be the best way to analyze the percentage of dauer larvae on a plate? Count the worms, while picking them into 1% SDS and afterwards pipette the 1% SDS solution back on another plate and count the survivors? (Sorry - I don’t have access to Cassada and Russell 1975)
Thank you all for your help.
After thinking about it, I decided to try exactly what mmmchl proposed.
Yes, Sudan Black requires fixation, it stains lipid and triglycerides. Dauers have larger lipid stores, which is why they stain dark.
Another stain you could try that also stains lipid stores is Nile Red. This is a live stain and requires that you add it to the plates, the worms eat it and it stains their lipid deposits as they develop. This way you don’t have to kill your worms and there is less manipulation involved, shifting them in and out of SDS solutuions.
As I understand it, Nile red will stain the bacteria than will be later consumed by the worms, staining them. If I am trying to induce the dauer stage by starvation, so feeding them with Nile red stain bacteria won’t work. Or am I misunderstanding the technique?