Laboratory Protocols & Standard Methods

Obtain the starter culture from the Caenorhabditis Genetics Center (CGC) or recover it from an existing worm plate.

Use a single colony to start the culture. Incubate overnight at 37 °C.

LB agar composition (per liter)

• 10 g Bacto-tryptone
• 5 g Bacto-yeast extract
• 5 g NaCl
• 15 g agar

Adjust the pH to 7.5 with NaOH before pouring and autoclaving.

LB broth composition (per liter)

• 10 g Bacto-tryptone
• 5 g Bacto-yeast extract
• 5 g NaCl

Adjust the pH to 7.0 with 1 M NaOH. Dispense 100 ml into 250 ml screw-cap bottles, autoclave, and store at room temperature for several months.

Incubate at 37 °C overnight. The resulting E. coli OP50 culture is ready for seeding NGM plates.

Store both streak plates and liquid cultures at 4 °C. They remain usable for several months.

Use different plate sizes depending on the application:

• 35 mm: matings or assays using costly reagents
• 60 mm: general strain maintenance
• 100 mm: large cultures or mutant screens

A peristaltic pump such as the Wheaton Unispense is recommended for even dispensing and uniform agar thickness.

Equipment and reagents

• NaCl
• Agar
• Peptone
• 5 mg/ml cholesterol in ethanol (do not autoclave)
• 1 M KPO₄ buffer, pH 6.0
• 108.3 g KH₂PO₄
• 35.6 g K₂HPO₄
• H₂O to 1 L
• 1 M MgSO₄
• 1 M CaCl₂
• Petri plates
• Peristaltic pump

Combine the following into a 2 L Erlenmeyer flask:

• 3 g NaCl
• 17 g agar
• 2.5 g peptone
• 975 ml distilled H₂O

Cover the flask with aluminum foil and autoclave for 50 minutes.

Cool the flask in a 55 °C water bath for 15 minutes. Then add the sterile reagents:

• 1 ml 1 M CaCl₂
• 1 ml 5 mg/ml cholesterol
• 1 ml 1 M MgSO₄
• 25 ml 1 M KPO₄ buffer

Swirl gently to mix.

Dispense aseptically into Petri plates using the peristaltic pump, filling each plate about two-thirds full.

Leave the plates at room temperature for 2–3 days to detect contamination and allow excess moisture to evaporate.

Store plates in an airtight container at room temperature. They remain usable for several weeks.

Use 0.05 ml for small or medium plates or 0.1 ml for large plates.

Optionally spread the drop with a pipette tip or sterile glass rod, keeping the lawn centered to prevent the worms from crawling up the sides and drying out.

Leave the seeded plates at room temperature overnight or incubate them at 37 °C for 8 hours, then cool before use.

Store seeded plates in an airtight container. They remain usable for 2–3 weeks.

Use a 1-inch piece of 32-gauge platinum wire mounted in a Pasteur pipette or loop holder. Flatten or slightly bend the tip into a small hook and avoid sharp points.

Gently touch the side of the worm with the wire tip and lift. Alternatively, pick up a small blob of E. coli OP50 first and let the worm stick to the bacteria.

Touch the picker lightly to the new plate surface and wait for the worm to crawl off. This method is ideal for precision transfers and maintaining genetic stocks.

Use a sterilized scalpel or spatula to cut and move a small chunk of agar containing worms to a fresh NGM plate. The worms will crawl out and spread onto the bacterial lawn. This is convenient for homozygous stocks or starved plates, but not for heterozygous or mating stocks.

Wash worms from 3–6 plates into a 15 ml conical tube with M9 buffer, adjusting the volume to 10–15 ml while avoiding large agar chunks or thick bacterial clumps.

Centrifuge at 1500–2000 rpm for 1–2 minutes, then aspirate the supernatant, leaving about 0.5–1 ml above the pellet without disturbing it.

Prepare fresh bleaching solution, for example 2 parts household bleach, 1 part 5 N NaOH, and 2 parts ddH₂O. Add 5–8 ml to the pellet and invert gently on a rocker for 5–10 minutes until adult bodies dissolve but eggs remain intact.

Pellet the eggs by centrifuging, aspirate the bleach solution, add sterile M9, resuspend, and wash 2–3 times until all traces of bleach are removed.

Resuspend the eggs in 5–10 ml sterile M9 or S medium in a vented tube and incubate at 20 °C with gentle shaking for 12–24 h without food.

Estimate concentration by counting in a small aliquot and transfer the desired number of L1 larvae to OP50-seeded NGM plates or liquid cultures.

Use N2 Bristol or other relevant strains maintained on NGM agar plates seeded with E. coli OP50 at 18–25 °C. Transfer worms regularly to keep healthy gravid adults available for egg isolation.

Prepare agar-only plates (for example 1.7 g agar in 100 ml water) in 35 mm dishes, dry them for 12–24 h, and mark the bottom into four quadrants. Prepare fresh compound stocks in M9 or sterile water with identical solvent percentages across all conditions.

Collect gravid adults, wash them to remove bacteria, expose them to brief alkaline hypochlorite treatment, wash the released eggs thoroughly, and adjust the suspension to roughly 100–140 eggs per 100 µl.

Transfer 500 µl egg stock and 500 µl test solution to each tube, mix gently, and incubate for 4–8 h at 18–22 °C. Keep solvent and exposure time constant across conditions.

Pellet eggs by centrifugation, wash them with M9, resuspend in 240 µl M9, and dispense 80 µl onto each of three pre-labelled plates so that eggs are evenly distributed.

Incubate plates at 16–18 °C for 24 h, image each quadrant with a stereomicroscope and digital camera, and count unhatched eggs and L1 larvae using ImageJ or similar software.

Calculate the fraction of unhatched eggs, pool data from at least three independent experiments, and fit a sigmoidal concentration-response curve to estimate EC50 or EC90 values.

Prepare standard NGM plates and seed them with E. coli OP50. For lifespan assays, supplement with FUDR and ampicillin before pouring, and store Amp/FUDR plates at 4 °C until needed.

Use a timed egg-laying procedure or another synchronization method to produce a population of healthy, well-fed L4 larvae at 20 °C.

Transfer synchronized L4 larvae onto OP50-seeded Amp/FUDR plates, with about 25–30 worms per plate and at least two to three replicate plates per condition. Designate the transfer day as Adult Day 0.

Inspect plates every 24 h and then every 2–3 days thereafter. Transfer worms to fresh Amp/FUDR plates when bacterial lawns deplete, contamination appears, or progeny become excessive.

Tap the plate gently and observe movement. Worms that respond are scored as alive; those that do not respond and fail to move after gentle stimulation are scored as dead.

Record live, dead, and censored worms on each scoring day, then generate Kaplan–Meier curves and compare survival distributions with the log-rank test.

Select disease-associated targets from UniProt or PDB and assess druggability of binding pockets using CASTp or Q-SiteFinder.

Retrieve or model 3D structures with SWISS-MODEL or AlphaFold, then refine them with GROMACS molecular dynamics.

Locate active sites using CASTp, fpocket, or conservation analysis. Gather compounds from ZINC, PubChem, or ChEMBL and filter with Lipinski's Rule of Five in SwissADME.

Screen libraries with AutoDock Vina or Glide, then perform more precise docking on the best hits and analyze using PyMOL.

Improve leads with QSAR or pharmacophore modeling, and evaluate drug-likeness and safety with pkCSM or admetSAR.

Gather disease-related genes from DisGeNET, OMIM, GeneCards, or CTD, and retrieve compounds from TCMSP, PubChem, ChEMBL, or DrugBank. Filter compounds for drug-likeness with Lipinski's Rule of Five or SwissADME.

Use SEA, STITCH, SwissTargetPrediction, or PharmMapper to predict targets and cross-reference them with disease-associated targets.

Construct compound-target and protein-protein interaction networks in STRING or Cytoscape, and analyze hubs or key nodes using degree, betweenness, and closeness metrics.

Perform GO, KEGG, or Reactome enrichment with DAVID, Metascape, or g:Profiler, then visualize the network in Cytoscape or Gephi to identify functional modules.

Prioritize key compounds and targets for experimental validation using qPCR, Western blotting, enzyme assays, or phenotypic assays.