- Therefore, it is suggested that the L1 larvae be diluted in sterile
M9 buffer with a density of 6–10 L1 larvae/μL and constant
titer for all downstream procedures [18]. - The larvae must be therefore maintained in sterile conditions in
M9, free of any carcasses or debris that might act as a nutrient
source. - At this step, you should have around 60–100 L1 per plates, do
not hesitate to count following each period of starvation, and
readjust the volume in M9 to maintain the L1 titer. - To simplify the counting, use plates seeded with 0P50 in a “Z”
shape instead of a spot. Larvae will be everywhere on the Z
shape, and this will simplify the counting. - SinceC. elegansis hermaphroditic, it is important to transfer
the mother onto new OP50 plates to prevent self-progeny that
could potentially skew the survivability values. - With prolonged durations of starvation, post-diapause larvae
will recover slowly and will develop with some heterogeneity.
Vulval defects can often be seen at the L4 stage. - L4 larvae are very simple to distinguish by the crescent-like
pattern that appears in the medio-ventral region around the
developing vulva (Christmas tree stage) [19]. The animals are
slightly smaller than adult animals and never possess adult alae
(cuticular ridges on the lateral side visible with a compound
microscope) or oocytes, although later L4 stages may contain
developing sperm. - To simplify the counting, use, as before, NGM plates bacteria
seeded with a “Z” shape. - Brood size can also been determined by visual observation
(Fig.1) and plates recorded as affected (reduced brood size,
<100 progeny) or unaffected (normal brood size, more than
100 progeny). Brood size is a critical measure of fecundity, and
it contributes to overall reproductive fitness if assessed over
generations. - It is important to keep the parental plates, where the P 0 mother
is still alive 3 days post-L4 stage, to be sure that the reduced
brood size phenotype does not derive from somatic defects
(bagging, bursting, etc.). - Water is preferred when washing the L1 larvae because salt
interferes with the adherence of the animals to the poly-L-lysine
coated slides. - At this point, slides may be stored in a labeled box at 80 C
for several days, although they should not be kept more than a
few days at 80 C, as the water will start to evaporate through
sublimation and the larvae will not stain as well. If the slides
were stored at 80 C, let them “warm up” on dry ice for
10 min just before cracking them.
578 Emilie Demoinet and Richard Roy