Laboratory Methods and Protocols

I share standardized experimental protocols for C. elegans studies and microalgae research, ensuring accuracy and reproducibility. Each method includes step-by-step instructions, required reagents, and troubleshooting tips for wet lab research.

Protocol 01: Cryopreservation and Recovery Protocol for Caenorhabditis elegans

Cryopreservation is an essential method for maintaining C. elegans strains over long periods. Freezing nematodes properly ensures their viability upon thawing, allowing for reliable revival and continued research. The following protocol outlines step-by-step procedures for freezing and thawing C. elegans.

Materials Required

C. elegans cultures
Nematode Growth Medium (NGM) plates seeded with E. coli OP50
M9 buffer (Composition: NaCl, KH₂PO₄, Na₂HPO₄, H₂O)
Freezing solution: Soft Agar Freezing Solution (see below)
Cryovials (1.5–2 ml)
Ice bucket
Sterilized metal spatula
-80°C freezer or liquid nitrogen storage

Preparation of Soft Agar Freezing Solution

Prepare the freezing solution as follows:

0.58 g NaCl
0.68 g KH₂PO₄
30 g glycerol (acts as a cryoprotectant)
0.56 ml 1 M NaOH
0.4 g agar
Distilled water to 100 ml

Autoclave the solution to sterilize. Before use, melt the solution and maintain it at 37°C to prevent solidification (WormBook, NCBI).

Cryopreservation Protocol

1. Culturing Worms

Grow C. elegans on NGM plates with E. coli OP50.
Incubate at 20°C until worms reach the just-starved L1-L2 stage, as this stage has the highest survival rate during freezing (Hayden et al., 2021).

2. Harvesting Worms

Wash each plate with 1.5 ml of M9 buffer to collect worms.
Transfer suspensions to a 15 ml conical tube.
Allow worms to settle and remove excess buffer, leaving about 3 ml (protocols.io).

3. Cooling and Mixing

Place the tube on ice for 15 minutes.
Add an equal volume (3 ml) of the pre-warmed Soft Agar Freezing Solution.
Mix gently by inverting several times (WormBook, 2006).

4. Aliquoting and Freezing

Transfer 1.5 ml of the mixture into labeled cryovials.
Store vials in a styrofoam box at -80°C for slow cooling.
After 24 hours, transfer vials to liquid nitrogen or permanent -80°C storage (Andersen Lab, 2023).

Recovery Procedure

1. Thawing

Retrieve a cryovial from storage.
Thaw rapidly at room temperature by swirling the vial.
2. Plating and Incubation
Transfer thawed suspension onto a seeded NGM plate.
Incubate at 20°C for 48–72 hours.
Confirm survival by observing gravid adults and eggs (JoVE).

Key Considerations and Troubleshooting

Use of Glycerol: Reduces ice crystal formation, preventing cellular damage during freezing (ScienceDirect).
Cooling Rate: A slow cooling rate is essential for worm viability; rapid freezing results in poor survival (NCBI).
Optimal Worm Stage: L1-L2 stage worms have the highest post-thaw survival rate (WormBook, 2006).
Labeling: Always label cryovials with strain information and date.

References

Protocols.io (2020). Freezing worms protocol. https://www.protocols.io/view/freezing-worms-bhwfj7bn
Hayden, J., et al. (2021). Cryopreservation of dehydrated Caenorhabditis elegans. PMC. https://pmc.ncbi.nlm.nih.gov/articles/PMC8546488/
Stiernagle, T. (2006). Maintenance of C. elegans - WormBook, NCBI. https://www.ncbi.nlm.nih.gov/books/NBK19649/
Andersen Lab (2023). Soft Agar Freezing & Thawing Protocol. https://andersenlab.org/Protocols/SoftAgarFreezing%26Thawing.pdf
JoVE Video Protocols (2012). Maintenance of C. elegans. https://app.jove.com/v/5104/maintenance-of-caenorhabditis-elegans
ScienceDirect (2023). Cryopreservation of nematodes using vitrification. https://www.sciencedirect.com/science/article/abs/pii/S0011224023000846

Protocol 02: Bleaching C. elegans to Obtain Eggs and L1s

Bleach solution will kill worms but will not destroy eggs because they contain a protective eggshell. The eggs retained in a worm are within ~2 hours of fertilization, so bleaching can be used to synchronize populations of animals.

CAUTION: While handling bleach, wear gloves, goggles, and a lab coat.

Materials

Bleach
Sodium hydroxide (NaOH)
1× M9 buffer (Dilute 10× M9 1:10 before using; sterile, 2 to 3 L may be needed for washes)
Adult gravid hermaphrodites (several plates recommended)
100-mm seeded plates for recovery
15-ml conical tubes
Standard benchtop centrifuge
Pasteur pipettes (for removing excess liquids)
Rotator or shaker
Compound microscope and dissecting microscope
Roller platform
Coverslips

Protocol

Step 1: Prepare Fresh Bleach/NaOH Solution

(Do not use if older than 1 week.)

Volume Needed | 5 ml | 10 ml | 50 ml
Sterile H₂O | 3.35 ml | 6.7 ml | 33.5 ml
8.25% bleach | 1.45 ml | 2.9 ml | 14.5 ml
50% (w/w) NaOH | 200 μl | 400 μl | 2.0 ml

Note: Commercial bleach (from grocery stores) is usually about 8.25%, but different brands vary in concentration and effectiveness. Cheaper brands without scent-masking additives work better.

Step 2: Harvest Gravid Worms

Wash gravid worms from plates with 1× M9 buffer into a 15-ml conical tube.
Spin in a benchtop centrifuge at 3000 rpm for 1 min (set brakes to HIGH).
Remove excess liquid using a Pasteur pipette, leaving about 1 ml in the tube.
Wash 1-2 additional times with 1× M9 until the solution is no longer cloudy with food.
Aspirate the last wash down to 3 ml worms in M9.

Step 3: Bleaching Treatment

Add 3 ml of bleach/NaOH mix from step 1. Shake vigorously by hand and monitor adult lysis under a microscope.
- Adults should break open and dissolve within ~7 min.
- Over-bleaching will damage the eggs, while under-bleaching will cause bacterial contamination.
As soon as adult bodies are broken open and starting to dissolve, add 6-8 ml of 1× M9.

Step 4: Egg Recovery

Immediately spin for no more than 1 min at 3000 rpm, then brake rapidly.
Aspirate as much liquid as possible without disturbing the egg pellet at the bottom of the tube.
Resuspend the pellet in 1× M9, then shake until the pellet is entirely resuspended.
Spin for 1 min at 3000 rpm.
Repeat the wash and spin steps twice more.

Step 5: Embryo Preparation and Culturing

Suspend embryos in 1× M9 to 1-3 ml, depending on the number of animals. Shake hard to separate embryos sticking together.
Examine eggs under a microscope. A good preparation will show shiny, smooth eggshells.
Place the 15-ml tube on a roller platform overnight.
The next day, dispense 5-10 µl of the preparation onto a coverslip and count L1 larvae under a dissecting microscope to estimate survival, if desired.
Dispense animals onto larger growth plates or into liquid culture for growth.

References

Porta-de-la-Riva M, Fontrodona L, Villanueva A, Cerós M. Basic Caenorhabditis elegans Methods: Synchronization and Observation. PMC3607348. Available here.
Al-Asmar A. Worm Synchronization Protocol (Bleaching). Protocols.io. Available here.
ResearchGate. Bleach Synchronisation of C. elegans v1. Available here.
WormBook. Methods for Studying C. elegans. Available here.
Stiernagle T. Maintenance of C. elegans. WormBook, 2006. Available here.
Sulston JE, Brenner S. The Genetics of Caenorhabditis elegans. Genetics, 1974.

Protocol 03: Generating Males by Heat Shock

Males arise spontaneously from hermaphrodite self-fertilization at about 1 in 500 eggs laid, which is often too infrequent for performing genetic crosses. The frequency of self-progeny males can be increased by employing heat shock (EPFL).

Materials

Six or more plates with at least five L4 hermaphrodites of the desired genotype on each
Incubator at 30°C

Procedure

Incubate the plates with the L4 hermaphrodites for 4 to 6 hours at 30°C. Longer times can be used but may result in fewer progeny (WormBook).
Return the plates to 20°C.
After 3 days, screen the plates for males. These can be recognized by the distinctive morphology of the tail and their behavior. There will likely be many unhatched eggs, but a few males per plate should appear in the F1 generation (PMC).
To establish a culture with many males for genetic crosses, cross the newly arisen males to hermaphrodites of the desired genotype. Since males generated from heat shock often have low fertility, it is helpful to use an excess of these males (as many as 8 to 10) with a few L4 hermaphrodites of the same genotype (NCBI).
The next generation should contain many males arising from cross-fertilization. These males can be used for subsequent genetic crosses or for establishing a strain that routinely produces a high number of males (ScienceDirect).