Worm Viability Assay

## Protocol

*Time in parentheses indicates the # of hours after the initial L4 were picked that the steps that day should be completed.*

**Day 1:**

* Pick a set number of L4s for each genotype to individual plates. I generally do 11 worms per genotype (11 plates). Brood size for N2 is 200-300 worms, generating 2000-3000 data points—you can scale the assay depending on the size of dataset you desire. This initial step should be completed EARLY in the morning or LATE in the evening as the next step needs to be done as close to 36 hours after your initial picking as possible. If you do it midday, you’ll end up in lab in the middle of the night all week.

**Day 2:**

* Chill out. Your days will be filled with counting soon enough.

**Day 3 (36 hours):**

* Move original worms to new plates, labeled as day 2. Worms start laying eggs ~12 hours post L4 so your day 1 plate is really 24 hours of egg laying—the same each day of the assay. This allows you to account for changes in viability as the worms fecundity decreases with age if you desire.
* Immediately count eggs and oocytes on original plates (Day 1).

**Day 4 (60 hours):**

* Move original worms to new plates, labeled as day 3.
* Immediately count eggs and oocytes on day 2 plates.

**Day 5 (84 hours):**

* Move original worms to new plates, labeled as day 4.
* Immediately count eggs and oocytes on day 3 plates.
* Count progeny and males on day 1 plates. This is 2 days after they were laid, so they should be around the L4 stage. You need to put the plates at 4C for at least 30 minutes to inhibit movement. *Note that the progeny counts don’t need to occur as immediate as the egg/oocyte counts—just don’t wait too long, or you’ll start getting F2s on your plates which is no fun*

**Day 6 (108 hours):**

* Kill original worms by picking from day 4 plates into flame. At this point, the parental worms are essentially done laying eggs so I just murder them. If your genotype is delayed in egg laying, you may want to continue moving them every 24 hours for days 5&6.
* Immediately count eggs and oocytes on day 4 plates
* Count progeny and males on Day 2 plates.

**Day 7:**

* Count progeny and males on Day 3 plates.

**Day 8:**

* Count progeny and males on Day 4 plates.
* Enter data into excel (I actually do this as I go along). Calculate viability (progeny/eggs\*100), brood size (total progeny), and Him phenotype (males/progeny\*100). Note that you can count oocytes, but they don’t factor into viability counts.

## Important Notes

1) Labeling your plates well is really important. You need to label the genotype, the day #, and the plate # for that genotype. Again, I usually do 11 plates per genotype. If something is likely to be WT (i.e. null allele of non-essential gene), I'll just do 6 plates per genotype.

2) Try to use plates that were just spread or with really thin lawns. The hardest part of this assay is counting the eggs/oocytes and they are much easier to see if there is less bacteria on each plate. You don't need much food on each plate since just 1 parent will be laying eggs.

3) To count worms, I draw a grid using a sharpie on a worm petri dish top. This way you can count the # of worms in each small square, making it easier to keep track of which worms/eggs you've already counted and those still to count.

4) There will always be some L1s on the plates when you count eggs/oocytes, be sure to include these in your counts as eggs.

5) When counting progeny, worms should be put at 4C for at least 30 minutes to stop movement. It's impossible to get accurate counts if they are moving so this is absolutely necessary. I usually take 2-4 plates out from the fridge at a time to count.

6) Remember, you are going to need A LOT of plates for this assay. If you are doing 11 plates for 6 genotypes, you would need (11\*6\*4) or 264 plates. You may want to spread a box for yourself if we are low.

Updated: March 2016 by Christian Nelson