How long should I let the OP50 culture grow?

Inoculate fresh LB with a single colony from a streak plate, shake at 37 °C overnight (~16 h). The culture should be visibly turbid with OD600 of 1.0–1.5. Avoid culturing past 24 h — overgrown cultures form clumps that seed unevenly.

Why do NGM plates need 2–3 days to dry before seeding?

Freshly poured NGM has a wet surface that causes the OP50 lawn to spread to the rim and produce uneven growth. Two to three days at room temperature gives the surface enough time to dry without cracking the agar. If you need plates faster, leave them in a 37 °C incubator for 4–6 hours.

What's the standard seeding volume for each plate size?

35 mm assay plates: 10 µL spot. 60 mm maintenance plates: 100 µL lawn. 100 mm bulk-prep plates: 250 µL spread. The defaults assume a saturated overnight culture (OD600 ~1.5) — scale up for less concentrated cultures.

When should I use HT115 instead of OP50?

For RNAi feeding experiments. HT115 is an RNase III–deficient E. coli strain that lets you express dsRNA from a feeding vector. Grow with ampicillin + tetracycline + IPTG. Standard maintenance and unrelated assays should always use OP50 — HT115 is metabolically slower and slightly toxic over multiple passages.

How many plates does one 250 mL flask of LB make?

A 250 mL flask with 150 mL working volume yields about 100 mL of saturated OP50. That seeds ~1,000 standard 60 mm plates (100 µL each) or ~400 standard 100 mm plates (250 µL each). Scale your flask count to your seeding volume, not your plate count.

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C. elegans OP50 Bacterial Lawn Prep Calculator.

Compute OP50 culture and LB volumes for any combination of 35/60/100 mm NGM plates with custom seeding volumes and HT115 RNAi feeder substitution.

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Plate Counts & Seeding Volumes

Plate size	Plates	Seeding (µL/plate)	Notes
35mm			Small assay plates — 10 µL spot.
60mm			Standard maintenance plates — 100 µL lawn.
100mm			Bulk-prep plates — 250 µL spread.

Use HT115 RNAi feeder strain instead of OP50Switches the antibiotic note to ampicillin + tetracycline + IPTG induction

Culture Plan

OP50 culture

6 mL

total seeding × 1.2 overage

LB inoculation

9 mL

OP50 × 1.5

Flasks

× 250 mL flasks

Total plates

ready after seeding

Antibiotic / induction: OP50 is grown antibiotic-free for standard C. elegans maintenance.

Prep Protocol

Pour NGM plates 2–3 days before seeding so the surface is dry.
Inoculate 9 mL LB with a single OP50 colony (or HT115 if using RNAi).
Shake at 37 °C for 16 h until OD600 reaches ~1.5 (saturated culture).
Pipette the standard seeding volume onto each NGM plate.
Let lawns dry 1–2 days at room temperature before adding worms.

When to use

Planning a weekly OP50 prep sprint for stock maintenance
Scaling LB volume to a multi-experiment plate batch
Coordinating NGM pour timing with seeding day
Switching between OP50 and HT115 RNAi feeder workflows
Onboarding new lab members to standard plate prep

Do not use for

For non-NGM plate types (worm picks, CV plates) — adjust seeding volumes
For specialty bacterial diets (Comamonas DA1877, Bacillus megaterium)
For automated liquid culture systems with their own concentration math

Always pour plates before you grow OP50

NGM needs 2–3 days to dry before seeding. If you grow OP50 first, the culture sits in the fridge degrading while you wait for plates.

Don't over-grow the OP50 culture

Cultures past 24 h form clumps that seed unevenly and contaminate readings. 16 h overnight is enough.

Seed in volumes you can actually pipette

A 10 µL drop on a 35 mm plate is reproducible. A 5 µL drop is not — pipetting errors dominate the lawn variance.

Store seeded plates upside down at 4 °C

Right-side-up plates collect condensation that washes the lawn into the rim. Inverted storage at 4 °C gives 2 weeks of usable life.

Method

Total OP50 culture (mL) = sum(plates $\times$ seeding volume per size) $\times$ 1.2 overage / 1000. LB inoculation volume = OP50 $\times$ 1.5 (concentration/loss step). Flasks needed = ceiling(LB / 150 mL working volume per 250 mL flask), minimum 1. Default seeding volumes: 35 mm = 10 µL, 60 mm = 100 µL, 100 mm = 250 µL. HT115 toggle replaces antibiotic-free OP50 note with ampicillin + tetracycline + IPTG.

Validated

Last validated 2026-04-06. Calculations are designed for planning and documentation support; verify procurement decisions against manufacturer specifications or institutional SOPs.

How to cite

How to Cite

ConductScience C. elegans OP50 Bacterial Lawn Prep Calculator (v0.99.0). ConductScience, Inc. 2026. Available at: https://conductscience.com/tools/celegans-op50-prep-calculator

Stiernagle T. Maintenance of C. elegans. WormBook. 2006. doi/10.1895/wormbook.1.101.1

Brenner S. The genetics of Caenorhabditis elegans. Genetics. 1974;77(1):71-94.

Kamath RS, Ahringer J. Genome-wide RNAi screening in Caenorhabditis elegans. Methods. 2003;30(4):313-21.

NGM Plate Prep Timing

NGM plate prep is a multi-day workflow — plan backward from when you need worms.

Day 1: Pour NGM plates

Make NGM agar (3 g/L NaCl, 17 g/L agar, 2.5 g/L peptone), autoclave, cool to 55 °C, add cholesterol/CaCl₂/MgSO₄/K-phosphate buffer, pour into Petri dishes. Pour at room temperature in a clean hood.

Days 2–3: Dry the plates

Stack plates upside-down at room temperature for 2–3 days. The surface should be dry and slightly indented but not cracked. Label batches with pour date.

Day 4: Inoculate OP50 LB

Pick a single colony from a streak plate into LB, shake at 37 °C overnight (~16 h).

Day 5: Seed plates

Pipette the standard volume onto each plate. Let dry on the bench for 30 min before stacking and incubating.

Day 6: Lawn establishment

Lawns are ready for worms 18–24 h after seeding. Plates can be stored at 4 °C for up to 2 weeks.

HT115 RNAi Feeder Substitution

For RNAi by feeding, OP50 is replaced with HT115 carrying a dsRNA expression vector.

Why HT115?

HT115 is RNase III–deficient, so dsRNA accumulates in the bacteria instead of being degraded. Worms ingest the bacteria, the dsRNA enters the gut epithelium, and the systemic RNAi machinery silences the target gene throughout the worm.

Growth conditions

LB + 100 µg/mL ampicillin (selects for the dsRNA vector)
LB + 12.5 µg/mL tetracycline (selects for HT115 itself)
1 mM IPTG in LB and on the plate (induces dsRNA expression)
Otherwise grow exactly like OP50: 37 °C overnight

Plate prep differences

Use NGM + 1 mM IPTG (instead of plain NGM) for HT115 plates. Seed with 100 µL HT115 culture. The lawn establishes overnight at room temperature. Use within 1 week — HT115 lawns degrade faster than OP50.

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