AO staining protocol

Use 5µl of AO (acridine orange) stock (10mg/ml) per ml M9 buffer

Add 500µl of this solution to a plate of unstarved worms (~20 24hour adults on plate)

Rotate plates to be sure that solution is spread throughout the surface of the plate then store plates in the dark at RT for 1 hour

Wash worms –

Add 1.5 ml of M9 buffer, transfer to an eppendorf tube and wash three times by spinning for 2 sec at 1000 rpm

-or- transfer worms to small watchglass containing M9 buffer and repeat transfer to fresh M9 buffer two more times

Replate washed worms on a new plate (-or- leave in a small watch glass with M9 buffer) and place in the dark at RT for 45 min

On a 3% agarose gel pad place 5µl of tetramisile (1:5 dilution of 10mg/ml stock) and mount worms on pad. Cover slide and seal with clear nail polish

Look at worm under flourescent scope at an absorbance wavelength of 489nm