Laboratory Methods and Protocols
I share standardized experimental protocols for C. elegans studies and Maintenance.
Preparation of Growth Media
1. Preparation of Bacterial Food Source
Although C. elegans can be maintained axenically, this method is difficult and results in slow growth. In most laboratories, C. elegans is maintained monoxenically using E. coli strain OP50 as a food source (Brenner, 1974).
OP50 is a uracil auxotroph, which limits its growth on NGM plates. A thin bacterial lawn is ideal for clear observation and efficient mating.
Preparation Steps:
OP50 is a uracil auxotroph, which limits its growth on NGM plates. A thin bacterial lawn is ideal for clear observation and efficient mating.
Preparation Steps:
- Obtain a starter culture of E. coli OP50 from the CGC or recover it from existing worm plates.
- Streak the culture onto an LB agar plate:
-
LB Agar Composition (per liter):
- 10 g Bacto-tryptone
- 5 g Bacto-yeast extract
- 5 g NaCl
- 15 g agar
- Adjust pH to 7.5 with NaOH
- Incubate overnight at 37 °C.
-
LB Agar Composition (per liter):
- Using a single colony, aseptically inoculate L Broth:
-
L Broth Composition (per liter):
- 10 g Bacto-tryptone
- 5 g Bacto-yeast extract
- 5 g NaCl
- Adjust 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.
-
L Broth Composition (per liter):
- Incubate the inoculated broth overnight at 37 °C.
- The resulting E. coli OP50 culture is now ready for seeding NGM plates.
- Store both streak plates and liquid cultures at 4 °C; they remain usable for several months.
2. Preparation of NGM Petri Plates
C. elegans is routinely maintained on Nematode Growth Medium (NGM) agar (Brenner, 1974), poured aseptically into sterile petri plates.
Plate Size and Use:
Plate | Size Diameter | Common Use
Small | 35 mm | Matings or assays using costly reagents
Medium | 60 mm | General strain maintenance
Large | 100 mm | Large cultures or mutant screens
A peristaltic pump (e.g., Wheaton Unispense) is recommended for even dispensing, ensuring uniform agar thickness across plates.
If needed, drugs such as levamisole, streptomycin, or nystatin may be added to the molten NGM just before pouring (Caldicott et al., 1994).
Small | 35 mm | Matings or assays using costly reagents
Medium | 60 mm | General strain maintenance
Large | 100 mm | Large cultures or mutant screens
A peristaltic pump (e.g., Wheaton Unispense) is recommended for even dispensing, ensuring uniform agar thickness across plates.
If needed, drugs such as levamisole, streptomycin, or nystatin may be added to the molten NGM just before pouring (Caldicott et al., 1994).
1. Preparation of NGM Plates
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
Procedure:
- Combine in a 2 L Erlenmeyer flask:
- 3 g NaCl
- 17 g agar
- 2.5 g peptone
- 975 ml distilled H₂O
Cover with aluminum foil and autoclave for 50 minutes.
- Cool flask in a 55 °C water bath for 15 minutes.
- Add (sterile):
- 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 (fill plates about ⅔ full).
- Leave plates at room temperature for 2–3 days to check for contamination and allow moisture to evaporate.
- Store plates in an air-tight container at room temperature; they remain usable for several weeks.
3. Seeding NGM Plates
Using sterile technique, apply E. coli OP50 culture onto solidified NGM plates.
Procedure:
Procedure:
- Pipette:
- 0.05 ml OP50 culture for small or medium plates, or
- 0.1 ml for large plates.
- Optionally, spread the drop using a pipette tip or sterile glass rod to create a uniform lawn (avoid edges).
- Keeping the lawn centered prevents worms from crawling up the plate sides and drying out.
- Allow plates to dry overnight at room temperature or incubate at 37 °C for 8 hours (then cool before use).
- Store seeded plates in an air-tight container; they remain usable for 2–3 weeks.
Culturing C. elegans on Petri Plates
Transferring Worms Grown on NGM Plates
C. elegans is transparent and can be visualized using a dissecting stereomicroscope with a transmitted light source. Common models include the Wild Leitz M5A and Zeiss SV6, equipped with 10× eyepieces and objectives ranging from 0.6× to 5× (total magnification of 6×–50×).
Several methods are commonly used to transfer worms between plates:
Several methods are commonly used to transfer worms between plates:
1. Picking Individual Worms
For precision transfers or when maintaining genetic stocks.
Equipment:
Equipment:
- Worm picker made from a 1-inch piece of 32-gauge platinum wire, mounted in a Pasteur pipette or loop holder.
- The wire heats and cools quickly, allowing frequent flaming for sterilization.
Tips:
- Flatten or slightly bend the wire tip to form a small hook—avoid sharp points.
- To pick a worm, gently touch the side of the worm with the wire tip and lift.
- Alternatively, pick up a small blob of E. coli OP50 on the wire first; worms will stick to the bacteria.
- To transfer, gently touch the picker to the new plate’s surface and wait for the worm to crawl off.
2. Chunking Method
A quick and convenient way to transfer large numbers of worms.
Procedure:
Procedure:
- Use a sterilized scalpel or spatula to cut and move a small chunk of agar (containing worms) from an old plate to a fresh NGM plate.
- The worms will crawl out of the agar chunk and spread onto the new bacterial lawn.
Notes:
- Ideal for homozygous stocks or starved plates.
- Not recommended for heterozygous or mating stocks, as mixed genotypes can be transferred.