Great question — this is a common source of confusion in C. elegans drug studies!

🧪 Solid NGM Plate Drug Dosing – Two Common Approaches

When applying a drug to solid NGM plates, researchers typically use one of two methods to calculate the final drug concentration:

✅ Method 1: Surface Dose (Applied Volume Only)

Assumes the drug remains mostly on the surface where worms feed and move.

Does not factor in agar volume.

Used when drug diffusion into agar is limited or when the compound acts primarily on surface exposure.

Example Calculation:

You used:

10 mM drug solution

100 µL applied to the plate

This means:

Total drug added = 10 mM × 100 µL = 1 µmol

Plate area = standard 6 cm plate ≈ 28 cm²

This approach doesn’t give a concentration in mol/L, but rather a dose per area (e.g., 1 µmol per 28 cm²).

Best for: antibiotics, hormones, or small molecules acting locally and rapidly; when worms feed from the surface.

✅ Method 2: Whole-Agar Volume Dilution (Bulk Diffusion Model)

Assumes the drug fully diffuses into the agar, thus calculates the final concentration in the agar volume.

Common in longer-term assays (lifespan, reproduction) where diffusion is likely.

Preferred for consistency in many publications.

Your Setup:

Drug solution: 10 mM

Volume added: 100 µL = 0.1 mL

Plate agar volume: 2.5 mL

So:

Final concentration=0.1 mL×10 mM2.5 mL=0.4 mM\text{Final concentration} = \frac{0.1\, \text{mL} \times 10\, \text{mM}}{2.5\, \text{mL}} = 0.4\, \text{mM}

Final concentration in agar = 0.4 mM

🧾 Summary Table Approach Drug Distribution Assumption Calculation Based On When to Use Surface-only Mostly remains on the plate top µmol / cm² (area-based) Short-term, topical exposure, slow-diffusing drugs Whole-agar dilution (your case) Fully diffuses into agar µmol / agar volume (mM) Long-term assays, diffusible drugs, systemic effects ✅ Recommended: Use Whole-Agar Calculation

Since you specified:

Solid NGM

100 µL of 10 mM on 2.5 mL agar

✅ Your final drug concentration in the agar is 0.4 mM, assuming complete diffusion.

This is the most accepted and preferred method in C. elegans drug studies using solid NGM plates.

All the best 👍.

Thankyou for your detailed guidance. Its very helpful.

Hezam Dhaifallah can you comment on this condition of plate? where worms are stucks within OP50. Is this normal or the worms are dying by E.coli infection?

The image shows worms trapped within a dense lawn of E. coli OP50 on an agar plate. Some worms appear immobile or distorted, raising concern about their viability. However, this condition does not necessarily indicate that the worms are dying from a bacterial infection. More likely, the issue is physical entrapment due to an overgrown or excessively sticky OP50 lawn. When OP50 is seeded too heavily or the plates are incubated for too long, the bacteria can form a mucoid layer that physically traps the worms, especially younger or weaker individuals. OP50 is generally a non-pathogenic strain used as a standard food source for C. elegans, and it rarely causes harm unless the worms are immunocompromised (e.g., daf-2 or pmk-1 mutants), or the OP50 culture has mutated or become contaminated. To confirm the cause, you can perform a gentle touch assay to test worm movement, compare behavior on freshly prepared plates with a thinner lawn, or try using heat-killed OP50 to see if worms still get trapped. Unless you observe clear signs of pathology such as body swelling, intestinal transparency, or granulation, this is most likely a physical, not infectious, issue.

Thankyou for detailed answer. 100ul of E.coli on 2.5ml solidified agar in 35mm plate is okay? or i should reduce the quantity of E.coli? and per plate 20 worms are loaded. so, 50ul E.coli will be enough for 20 worms?

Thankyou for detailed answer. 100ul of E.coli on 2.5ml solidified agar in 35mm plate is okay? or i should reduce the quantity of E.coli? and per plate 20 worms are loaded. so, 50ul E.coli will be enough for 20 worms?