and 0.025 M phosphate (Na 2 PO 4 ·H 2 O, pH 7.2).
Formaldehyde is toxic so prepare this and use it in a fume
hood (seeNote 1).- Washing solution (PBST): PBS with 0.1% (v/v) Triton X-100.
- Blocking solution: PBST, 1% (w/v) BSA.
- DAPI nuclear counterstain: DAPI 1μg/mL dissolved in PBST.
- Microscopy essentials: Coplin jar, metal block for freeze crack-
ing, standard rectangular glass microscope slides
(7525 mm/1.1–1.2 mm thick), glass cover slips.
Other materials:- NGM plates (Nematode Growth Medium): mix 3 g NaCl,
17 g agar, and 2.5 g peptone in a 2 L Erlenmeyer flask. Add
975 mL H 2 O. Cover mouth of flask with aluminum foil.
Autoclave to sterilize, and then cool the flask in a 55C
water bath for 15 min. For the following step, use sterile
procedures to proceed: add 1 mL of 1 M CaCl 2 , 1 mL of
5 mg/mL cholesterol (diluted in ethanol), 1 mL of 1 M
MgSO 4 , and 25 mL of 1 M KPO 4 buffer. Swirl to mix well.
Dispense 11–12 mL of NGM solution into 6 cm petri plates
by using a peristaltic pump. Cool plates at room temperature
for 2–3 days before use in order to detect the presence of
contaminants. Plates can be stored in an airtight container at
4 C for several weeks (seeded or unseeded).
11.E. coliOP50 strain: defrost OP50 strain regularly, and start a
culture ofE. colion a LB plate (10 g Bacto tryptone, 5 g Bacto
yeast, 5 g NaCl, 15 g agar, H 2 O to 1 L, pH 7.5). Use a single
colony to start a liquid culture, and let it grow overnight at
37 C (the bottle of liquid culture can be stored at 4C for
several weeks and remains usable). - NGM seeded plates: seed plates using sterile technique with
approximately 50μL of OP50 culture. Spreading bacteria in a
“Z”-shaped lawn will help to visualize and count animals,
since the worms tend to spend most of the time in the
bacteria. Incubate seeded plates overnight at room
temperature.
13.C. elegans N2 and aak-1; aak-2 strains. Strains can be
acquired from the Caenorhabditis Genetics Center (https://
cbs.umn.edu/cgc/strains). Worms were freshly thawed prior
to the experiment and grown at 15C on NGM seeded plates
as previously described [16].
568 Emilie Demoinet and Richard Roy