Simple and Accessible Egg-Hatching Assays in Caenorhabditis elegans: Adaptable Formats for Drug Screening

Summary

This study presents a flexible and standardized egg-hatching assay platform in Caenorhabditis elegans that integrates multiple experimental formats. The approach enhances reproducibility, scalability, and accessibility, enabling robust evaluation of drug effects and developmental processes, while remaining cost-effective and easily adaptable for both research applications and educational settings.

Abstract

Egg-hatching assays (EHA) are widely used to evaluate the effects of chemical compounds on early developmental stages of nematodes, particularly in anthelmintic research. However, existing methodologies vary substantially in format, complexity, and reproducibility, and are often presented as fixed protocols rather than adaptable experimental frameworks. In this study, we establish a simple, accessible, and flexible platform for egg-hatching assays in Caenorhabditis elegans by integrating and directly comparing three experimental configurations: (i) controlled incubation in microcentrifuge tubes followed by seeding on agar plates, (ii) direct exposure on drug-containing agar plates, and (iii) multiwell-based liquid assays. Two representative compounds were used to illustrate the assay: one that inhibits egg hatching and one that has no detectable effect. Each configuration provides distinct advantages in terms of experimental control, throughput, and accessibility. The microcentrifuge tube-based approach enables precise control of drug exposure and supports quantitative pharmacological analyses. Agar-based assays offer a simplified and low-cost alternative suitable for continuous exposure and educational settings. Multiwell formats enable scalable, standardized screening consistent with modern drug discovery pipelines. By directly comparing these formats, we show how methodological choices influence assay reproducibility, drug exposure dynamics, and biological interpretation, particularly in relation to stage-specific factors such as the protective role of the eggshell and developmental variation in molecular target expression. Overall, this work establishes egg-hatching assays in C. elegans as a versatile and scalable platform adaptable to diverse applications, including anthelmintic screening, toxicological assessment, developmental studies, and teaching laboratories, thereby facilitating broader and more rational use of this methodology.

Introduction

Egg-hatching assays (EHA) have long been established as a cornerstone methodology in nematology for evaluating ovicidal activity and early developmental effects of chemical compounds. Originally developed for parasitic nematodes, these assays quantify the inhibition of embryonation or larval emergence under controlled in vitro conditions, providing robust and biologically relevant endpoints such as hatching percentage or concentration–response analysis (e.g., EC₅₀ or IC₅₀)^{1,2,3,4,5,6,7}.

Classical egg-based approaches in parasitic nematodes such as Haemonchus contortus and Trichostrongylus colubriformis have been widely used to evaluate the efficacy of synthetic drugs and natural products, as well as to monitor anthelmintic resistance^1,2,6,8,9,10. These methodologies encompass a range of readouts, from direct assessment of egg viability and hatching to the evaluation of larval development following fecal culture. Collectively, they have played a central role in linking drug exposure to parasite reproductive output and transmission potential. However, these approaches often require complex sample preparation from infected hosts, extended incubation periods, and specialized infrastructure, which can limit their accessibility, scalability, and inter-laboratory reproducibility.

The use of Caenorhabditis elegans as a model organism has emerged as a powerful strategy to overcome several limitations associated with parasitic nematodes. Its short life cycle, genetic tractability, ease of culture, and the conservation of key molecular targets shared with parasitic species make it particularly suitable for pharmacological, toxicological, and developmental studies. Importantly, embryogenesis in C. elegans is rapid and highly reproducible, allowing egg viability and hatching assays to be used as quantitative and reliable readouts of early developmental processes and their disruption by xenobiotics^{11,12,13,14,15,16,17}.

In addition, the availability of well-characterized mutant strains has enabled mechanistic insights into drug action and resistance. Previous studies have shown that resistance to bioactive compounds can emerge in a stage-dependent manner, with embryonic and larval stages displaying differential sensitivity. For example, alterations in glutamatergic signaling have been associated with resistant phenotypes in larvae, while egg hatching remained unaffected^14,18,19,20. This highlights the stage-specific nature of drug sensitivity and suggests that embryonic stages may involve distinct targets or differential accessibility compared to later developmental stages.

Beyond parasitology, egg viability assays in C. elegans have become increasingly relevant in toxicology and early drug discovery. These assays have been applied not only to assess anthelmintic activity but also to evaluate developmental toxicity and drug-induced embryonic defects, with outcomes that can correlate with higher-organism toxicity profiles^15,16. At the same time, the presence of the eggshell introduces important biological constraints, as it acts as a protective and selective permeability barrier surrounding the embryo^21,22. As a result, some bioactive molecules may fail to reach the embryo despite showing activity at later developmental stages^14,19,23,24. Access to the embryo is therefore likely to depend on the physicochemical properties of each compound, including molecular size and polarity. This may contribute to differences between the activity observed in egg-hatching assays and that in later developmental stages. This distinction underscores the importance of clearly defining both the experimental context and the biological endpoint being measured.

Methodologically, egg-hatching assays in Caenorhabditis elegans have been implemented using a broad range of experimental formats, including liquid incubation of synchronized eggs, direct exposure to drug-containing agar plates, and multiwell-based assays adapted for medium- to high-throughput screening^{11,13,14,16,19,23,24}.

Liquid-based and microplate formats have been widely adopted in pharmacological screening studies, as they provide homogeneous exposure conditions, facilitate precise control of drug concentrations, and allow quantitative analysis of concentration–response relationships. In parallel, comparable multiwell-based strategies have been extensively developed for parasitic nematodes, ranging from conventional 96-well plate assays to ultra-high-throughput platforms using 384-well formats^4,5. Together, these approaches highlight the versatility and scalability of egg-hatching assays across different nematode species and their adaptability to diverse experimental settings, from small-scale mechanistic studies to large-scale compound screening.

Each format presents distinct advantages and limitations in terms of experimental control, reproducibility, throughput, and accessibility. Liquid-based assays provide homogeneous exposure conditions and are well-suited for quantitative pharmacological analyses, whereas agar-based assays allow continuous exposure throughout development but introduce variability related to drug diffusion and bioavailability. Multiwell formats, in turn, enable parallelization and reduced reagent consumption, aligning with modern screening strategies.

Despite widespread use, egg-hatching assays are often presented as fixed protocols rather than as a flexible methodological framework. Direct comparisons between formats are scarce, and guidance on selecting the most appropriate configuration for specific applications, ranging from mechanistic studies to high-throughput screening or teaching environments, remains limited.

In this study, we propose a simple, accessible, and adaptable platform for egg-hatching assays in C. elegans, integrating and comparing three experimental configurations: (i) controlled incubation in microcentrifuge tubes followed by seeding on agar plates, (ii) direct exposure on drug-containing agar plates, and (iii) multiwell-based liquid assays.

By systematically evaluating these configurations, we aim to establish a practical framework that highlights the trade-offs between experimental control, simplicity, and scalability. This integrated perspective bridges classical parasitological methodologies with modern model-based screening approaches, positioning egg-hatching assays not as a single standardized technique, but as a versatile platform adaptable to diverse research contexts, including anthelmintic discovery, toxicological assessment, developmental biology, and educational applications.

Protocol

1. Caenorhabditis elegans strains and culture

1. Use the wild-type N2 (Bristol) strain and/or the transgenic strain PD4251 (ccIs4251; dpy-20(e1282)) to develop and validate the methodology^25,26. PD4251 expresses GFP, facilitating visualization and potential imaging applications when required, while showing egg-hatching and developmental responses indistinguishable from those observed in the wild-type N2 strain under the experimental conditions used in this study²⁷.
2. Obtain strains (e.g., wild-type N2 strain) from the Caenorhabditis Genetic Center (CGC), supported by the National Institutes of Health Office of Research Infrastructure Programs (P40 OD010440).
3. Maintain worms under standard laboratory conditions at 18–25 °C on freshly prepared Nematode Growth Medium (NGM) agar plates seeded with Escherichia coli OP50 as a food source. Prepare NGM by dissolving 16 g agar, 3 g NaCl, and 2.5 g bactopeptone in 975 mL distilled or ultrapure water. Sterilize by autoclaving. After cooling the medium to 55 °C, add sterile solutions of potassium phosphate buffer (25 mL, 1 M, pH 6.0), CaCl₂ (1 mL, 1 M), MgSO₄ (1 mL, 1 M), and cholesterol (1 mL, 5 mg/mL in absolute ethanol). Mix thoroughly and pour plates under sterile conditions.
4. Perform routine handling, propagation, and maintenance following established procedures as described in WormBook and related studies^24,26,28,29. Maintain worms under non-starvation conditions by ensuring continuous bacterial food availability and avoiding overcrowded plates. Keep cultures within the recommended temperature range (18–25 °C) and avoid excessively dry conditions, as these may accelerate agar dehydration.
5. General considerations before starting the assay
   1. Define all experimental parameters in advance, including temperature (18–25 °C), incubation time (4–8 h), humidity  (50–70%), and handling procedures.
   2. Prepare all materials prior to starting the assay to ensure proper timing and workflow coordination.
   3. Maintain consistent experimental conditions across all replicates to minimize variability.

2. Preparation of test compounds and controls

1. Freshly prepare all compounds on the day of the experiment. Dissolve stocks in M9 buffer (22 mM KH₂PO₄, 42 mM Na₂HPO₄, 86 mM NaCl, 1 mM MgSO₄) or sterile distilled water, depending on the compound's compatibility and assay conditions^30,31.
   NOTE: The choice of aqueous medium can influence compound activity in C. elegans assays. For example, compounds such as serotonin may display different effective concentrations when prepared in sterile distilled water versus M9 buffer. Therefore, both vehicle compatibility and biological response should be empirically evaluated during assay optimization. Also, for compounds with low aqueous solubility, appropriate vehicles such as dimethyl sulfoxide (DMSO) or ethanol can be used. Final solvent concentrations should remain low (typically below 0.2% v/v) to avoid toxicity. Previous studies in C. elegans reported that DMSO concentrations up to 0.5% produce minimal effects under standard laboratory conditions¹⁷.
2. Prepare at least five concentrations per compound to allow construction of concentration–response curves and estimation of IC₅₀ or IC₉₀ values. Use a serial dilution with 2- to 3-fold concentration steps as a starting point, and adjust the final concentration range according to the expected potency, solubility, and experimental response of each compound.
   NOTE: If the test compounds are sensitive to light, all preparation steps should be performed under dim light to prevent degradation. This includes preparing stock solutions, dilutions, and drug-containing agar plates. Whenever possible, plates should be covered with aluminum foil and stored in the dark. Similarly, multiwell plates should be kept covered (e.g., with foil or opaque lids) during incubation. Using closed containers or humid chambers that limit light exposure is recommended to maintain compound stability throughout the experiment.
3. Include the following controls in every experiment: buffer-only control (M9 or water) and vehicle control (e.g., DMSO or ethanol at the same concentration used in treatments).
   NOTE: The choice of solvent is critical. DMSO and ethanol are commonly used because they dissolve hydrophobic compounds while remaining biocompatible at low concentrations. Alternative approaches, such as Tween 20 or cyclodextrins, may be considered to improve solubility. Drug solutions should be used immediately after preparation, as stability over time may vary and could affect reproducibility.

3. Obtaining synchronized C. elegans eggs

NOTE: The isolation of C. elegans eggs has been extensively described in previous studies^29,32. Here, the main steps are summarized, incorporating minor modifications to optimize the recovery of clean, debris-free eggs suitable for egg-hatching assays.

1. Collection of gravid worms
   1. Start with 3–5 NGM plates containing confluent cultures of gravid adults^25,26. Prefer plates containing large numbers of gravid worms and visible eggs, indicating active reproduction, while avoiding overcrowded or starved cultures. Exact worm counts are typically not determined at this stage, as egg isolation protocols in C. elegans commonly rely on confluent reproductive cultures rather than precise animal counts. As a general reference, each 60 mm plate should contain several hundred gravid adults.
   2.  Add distilled water to each plate and gently wash worms into a 15 mL conical tube. Avoid scraping the agar surface to minimize debris.
   3. Centrifuge at 266 × g to 3 min (18–25 °C) to pellet the worms. Remove the supernatant containing bacteria.
   4. Remove the supernatant and repeat the washing step three times. Resuspend the pellet gently each time to improve the removal of residual bacteria.
2. Egg isolation by bleaching
   1. Resuspend the worm pellet in a freshly prepared bleaching solution (1.2% NaOCl and 0.5 M NaOH; final volume 5 mL). Prepare the solution immediately before use to ensure activity.
      NOTE: Correctly bleached samples were defined as preparations in which adult carcasses were disrupted/digested while intact eggs remained morphologically normal and viable. Over-bleached samples were identified by damaged or lysed eggs, abnormal egg morphology, reduced hatching rates, or the absence of viable larvae after incubation²⁵.
   2. Vigorously agitate the tube to promote dissolution of adult bodies and release of eggs. Mix continuously during incubation to ensure uniform exposure.
   3. Monitor the reaction closely and do not exceed 5 min. Check visually: adult bodies should be mostly dissolved while eggs remain intact.
   4. Monitor the reaction closely and stop it at 5 min by adding cold sterile distilled water (preferably 10 °C) up to a final volume of 14 mL. Do not allow the incubation to exceed this time to prevent egg damage.
   5. Cap the tube and gently invert several times to rapidly dilute the bleaching solution. Perform this step without delay to preserve egg viability.
   6. Centrifuge at 266 × g for 3 min (18–25 °C) and carefully discard the supernatant.
   7. Wash the egg pellet three times with sterile distilled water to completely remove residual bleach. Ensure thorough resuspension between washes. Resuspend the eggs in 5 mL of M9 buffer or sterile water.
      NOTE: Bleaching gravid hermaphrodites yields a sterile egg preparation suitable for synchronized assays. In the absence of food, hatched larvae enter L1 developmental arrest and resume development synchronously upon feeding.
3. Purification step (sucrose flotation)
   1. Perform this step if worm debris is visible after bleaching.
   2. Prepare a 30% sucrose solution by diluting a 60% sterile sucrose stock solution (e.g., mix equal volumes: 5 mL of 60% sucrose with 5 mL of distilled water).
   3. Add the sucrose solution to the pellet and mix gently by inversion. Avoid vortexing.
      NOTE: Because this purification step is brief and followed by immediate washing steps to remove residual sucrose, no detectable effects on egg viability were observed under the conditions used in this study. In contrast, removal of worm debris substantially improves egg visualization, counting accuracy, and the uniformity of drug exposure across samples.
   4. Centrifuge at 266 × g for 5 min (18–25 °C).
   5. Identify the egg layer as a thin white ring at the top of the solution. Immediately collect 4 mL of the supernatant containing eggs by carefully moving the pipette tip along the ring.
   6. Perform this step rapidly, as eggs begin to sink shortly after centrifugation due to gravity.
   7. Transfer the collected supernatant to a clean tube containing sterile distilled water. Wash the eggs twice with cold sterile water to remove residual sucrose.
      NOTE: This step is optional but strongly recommended. Removing debris improves egg visualization and counting accuracy, and ensures more uniform exposure to test compounds by preventing eggs from becoming trapped within residual worm material.
4. Preparation of egg stock solution
   1. Mix the egg suspension gently before sampling, as eggs rapidly sediment.
   2. Place three 20 µL drops on a slide and count eggs under a stereomicroscope.
   3. Calculate the average and adjust the concentration to 50–100 eggs per 100 µL using M9 buffer. Mix again before aliquoting to maintain uniform distribution.
   4. Aliquot the required volume of egg suspension for each experimental condition according to the selected protocol.

4. Egg-hatching assay formats

1. Short-term egg exposure in microcentrifuge tubes (high-control format)
   NOTE: This experimental configuration is based on previously described procedures²⁵, with adaptations to improve control over exposure conditions and reproducibility.
   1. Preparation of agar plates for egg-hatching assays
      1. Prepare drug-free agar plates using agar and distilled water only (1.7 g agar in 100 mL water). Do not add nutrients or bacteria, as the aim is to evaluate egg hatching without interference from food sources. Pour approximately 3 mL of agar per 35 mm Petri dish to maintain consistent agar depth across experiments.
      2. Prepare plates 12–24 h before use. Avoid exceeding this time window, as excessive drying reduces egg viability and affects larval movement.
      3. Divide each plate into four quadrants after solidification to facilitate counting.
      4. Store plates at 8–10 °C in sealed conditions to prevent dehydration. Allow plates to equilibrate to room temperature before use.
   2. Transfer 500 µL of egg suspension into a 1.5 mL microcentrifuge tube for each condition. Mix gently between pipetting steps to prevent egg sedimentation, as eggs rapidly settle by gravity. Maintaining a homogeneous suspension is critical to ensure comparable egg numbers across conditions.
   3. Add 500 µL of treatment solution or corresponding control to the 500 µL egg suspension. Incubate at 22 °C for 4–8 h. Define the incubation time in advance and keep it constant across experiments (e.g., 6 h), as variations in this time directly affect drug exposure.
      NOTE: Prepare treatment solutions at 2x the desired final concentration so that dilution after mixing with the egg suspension yields the intended final working concentration.
   4. Maintain continuous gentle agitation during incubation using a tube rotator (e.g., 5–20 rpm vertical rotation) or rocking platform. Ensure that tubes remain in constant motion to prevent egg sedimentation and to promote uniform exposure. This step is critical, as static conditions lead to egg accumulation and heterogeneous drug exposure.
   5. Centrifuge the tubes at 3,420 × g for 3 min (18–25 °C) after incubation. Identify the pellet, which appears as a small white deposit on the tube wall. Orient tubes consistently (e.g., mark one side) before centrifugation to facilitate rapid pellet localization and prevent accidental loss during supernatant removal.
   6. Remove the supernatant carefully without disturbing the pellet. Wash the eggs three times with M9 buffer to eliminate residual compound. Resuspend the final pellet in 240 µL of M9 buffer.
   7. Plate the suspension onto agar plates by dispensing 20 µL per quadrant. Use three plates per condition. Allow drops to dry completely before moving or inverting plates to prevent egg displacement across the surface.
      NOTE: Start with a larger total volume of egg suspension than strictly required to compensate for egg loss during washing steps and ensure sufficient final sample size for plating and counting. To simplify the procedure and reduce agar plate use, particularly in teaching settings, transfer the resuspended eggs directly into a multiwell plate rather than onto agar plates. Adjust the final volume to 200 µL per well and fill unused wells with sterile water to minimize evaporation.
2. Direct egg exposure on drug-containing agar plates
   1. Prepare an agar solution containing only agar and sterile distilled water (1.7 g agar in 100 mL). Heat until fully dissolved and maintain the solution in liquid form. Allow the agar to cool to 50–55 °C before adding any compound, and monitor the temperature using a laboratory thermometer. This step is critical to preserve compound stability and ensure homogeneous incorporation, as previously described²⁸. Prepare at least five concentrations per compound to enable concentration–response analysis and IC₅₀ estimation.
      NOTE: Do not add bacterial food sources. In the absence of food, hatched larvae enter synchronized L1 developmental arrest, preventing progression to later larval stages during the assay period^26,33.
   2. Add the appropriate volume of drug solution or vehicle control to the cooled agar. Mix gently by slow inversion to ensure homogeneous distribution. Avoid vortexing or vigorous shaking to prevent bubble formation.
   3. Immediately pour approximately 3 mL of agar into 35 mm Petri dishes. Work steadily to maintain consistent temperature and plate thickness. Prepare at least 3–5 plates per condition.
   4. Storage and handling of plates as follows: Allow plates to solidify at room temperature on a flat surface. Store plates inverted to prevent condensation from reaching the agar surface.
   5. Set up the experiment: Seed 50–100 eggs directly onto the agar plates. Maintain constant experimental parameters, including temperature, incubation time, humidity, and plate preparation timing. Use at least five concentrations for concentration–response analysis.
      NOTE: Prepare plates 12–24 h before use. Avoid over-drying, as it may affect egg viability and larval movement. Wrap plates to reduce dehydration and protect from light when working with light-sensitive compounds. Store plates in a clean, preferably humidified environment (e.g., a sealed container with moist paper) to minimize variability.
3. Egg-hatching assay in multiwell plates (medium- to high-throughput format)
   NOTE: This format is widely used in both C. elegans and parasitic nematode studies for medium- to high-throughput screening, enabling parallel evaluation of multiple compounds and concentrations under standardized conditions.
   1. Well plate preparation and experimental design: Use 96-well plates (or an alternative format depending on throughput needs). Prepare the plate layout in advance and define drug concentrations (arranged across columns), replicates (across rows), and controls, including buffer-only (M9 or water) and vehicle controls (e.g., DMSO or ethanol at the same final concentration used in treatments).
      NOTE: Use transparent, flat-bottom multiwell plates to allow direct visualization and counting in each well under transmitted LED illumination with a stereomicroscope. Eggs and hatched L1 larvae are quantified directly in the wells without transfer steps, minimizing sample loss and handling artifacts.
   2. Preload each well with the appropriate volume of drug solution or control. Use equal volumes across all wells.
   3. Addition of egg suspension
      1. Prepare a well-homogenized egg suspension with a defined concentration (see Section 2).
      2. Add an equal volume of egg suspension to each well containing the drug solution. Use a final volume of 200 µL per well.
      3. Adjust the egg density to at least 30 eggs per well (ideally 30–100). Do not exceed 100 eggs per well, as overcrowding substantially complicates manual quantification. Individual eggs become difficult to distinguish, while motile L1 larvae actively swim throughout the well, reducing counting accuracy and increasing experimental variability.
      4. Gently mix the egg suspension before dispensing to ensure uniform distribution.
   4. Load the plate carefully. Avoid introducing air bubbles. Pipette consistently and change tips between conditions to prevent cross-contamination. Gently tap the plate to ensure that all contents settle at the bottom of the wells.
   5. Incubation conditions: Incubate the plate at 20–22 °C under controlled and humid conditions for a total of 24 h from egg isolation.
      NOTE: Minimize variability by avoiding the use of outer wells or filling them with buffer to reduce evaporation. Incubate plates in a humidified chamber (e.g., a sealed container with moist paper) and protect from light when working with light-sensitive compounds. Avoid unnecessary movement during incubation. Maintain consistent timing of egg preparation and drug exposure across all replicates, as variations may affect developmental stage and data interpretation.

5. Evaluation and counting

1. Evaluate egg hatching 24 h after egg isolation, ensuring that control conditions have reached complete hatching under the defined temperature. Calculate this time point from the moment of egg collection to allow sufficient embryonic development. Consider any eggs that remain unhatched at this stage as affected by the treatment.
2. Examine samples under a stereomicroscope. For each condition (agar plates or multiwell plates), quantify the number of unhatched eggs and the number of hatched L1 larvae.
3. Scan the entire observation area, including plate edges and well boundaries, as motile L1 larvae may distribute unevenly and accumulate in these regions.
   NOTE: Perform direct counting under a stereomicroscope for rapid evaluation without additional equipment. Maintain consistent egg density across conditions, use at least 3–5 technical replicates per condition, and perform a minimum of three independent experiments using different egg preparations to ensure reliable comparisons.

6. Image acquisition and documentation

1. Acquire images using a stereomicroscope equipped with a digital camera. For agar plates, record each quadrant; for multiwell plates, record each individual well. Ensure that all areas are captured, including plate edges and well boundaries, where larvae may accumulate.
   NOTE: Standard 10x eyepieces and objectives, which range from 0.6x to 5x (total magnification of 6x to 50x), are widely used and allow clear visualization of both eggs and L1 larvae within the counting area. Use transmitted LED illumination. Maintain constant camera acquisition settings (brightness, exposure, and contrast) across all experimental conditions to ensure consistent image quality and reliable comparison between samples.
2. Use appropriate acquisition software to capture images. Process and analyze images using image analysis software. Maintain identical analysis parameters across all experimental conditions, including brightness, contrast, magnification, the same acquisition area/field, and counting criteria.
3. Quantify the number of unhatched eggs and L1 larvae from the acquired images. Consider larvae as hatched only when completely emerged from the eggshell. Eggs containing partially emerged larvae should be classified as unhatched. Apply identical classification criteria across all experimental conditions.
4. Incubate plates or multiwell plates for an additional 12–24 h at 16–18 °C to assess whether the observed inhibition of egg hatching is reversible. Under the defined experimental conditions, eggs remaining unhatched at this stage are considered affected by the treatment.

7. Data analysis

1. Calculate egg-hatching inhibition as:
   Fraction of unhatched eggs = (Number of unhatched eggs) / (total number of eggs + larvae)
2. Perform at least 3–5 independent experiments using different egg batches. Express data as mean ± standard deviation (mean ± SD).
3. Generate concentration–response curves using nonlinear regression and determine IC₅₀ values.
4. Perform statistical comparisons between conditions using Student’s t-test and define statistical significance at p < 0.05.
5. Analyze data using appropriate statistical software.

8. Application of Egg-Hatching Assays Using Trans-cinnamaldehyde and Linalyl Acetate

NOTE: This study is not centered on a specific compound, but rather uses representative agents to illustrate how each experimental format can be implemented, controlled, and interpreted under comparable conditions. Trans-cinnamaldehyde (TCA) was selected as an illustrative compound because it has been previously characterized and consistently produces robust, reproducible inhibition of egg hatching across the three assay configurations presented here. Importantly, the purpose of including TCA was to demonstrate protocol implementation and representative concentration–response behavior, rather than to establish relative potency or comparative performance between formats.

1. Experimental setup and compound preparation
   1. Prepare agar plates (3 mL per 35 mm dish) 12–24 h in advance using agar-only medium (1.7% in distilled water). Label the plates, divide them into four quadrants to facilitate counting, store them under sealed conditions to prevent dehydration, and equilibrate them to room temperature before use.
   2. Prepare fresh trans-cinnamaldehyde (TCA) stock solutions in DMSO and dilute them in sterile water or agar to obtain eight final concentrations (0.05–0.7 mM). Maintain the final DMSO concentration below 0.2% in all conditions. Include water, agar, and/or DMSO as negative and vehicle controls, respectively.
   3. Test linalyl acetate at 5 mM as a negative compound control to evaluate the ability of the assay to discriminate between active and inactive compounds under identical experimental conditions.
   4. Prepare synchronized egg suspensions as described above and adjust the density to 50–100 eggs per 100 µL. Use equal volumes and comparable egg numbers across all formats to ensure consistency between conditions.
2. Application of experimental formats using a reference compound
   1. Short-term exposure in microcentrifuge tubes (high-control format)
      1. Mix 500 µL of egg suspension with 500 µL of treatment solution containing 0.05–0.7 mM TCA. Incubate the tubes for 6 h at 18–20 °C and maintain the incubation time consistently across all experiments.
      2. Keep the tubes under continuous gentle agitation using a rotator during incubation.
      3. Centrifuge the tubes at 3,420 × g for 3 min (18–25 °C) after exposure and carefully remove the supernatant. Wash the eggs three times with distilled water to remove residual compounds and limit exposure during the defined incubation period.
      4. Resuspend the final pellet in 240 µL distilled water and plate the suspension onto agar plates at 20 µL per quadrant using three plates per condition. Incubate the plates at 16–18 °C for 24 h after egg isolation.
   2. Direct exposure to drug-containing agar plates
      1. Prepare drug-containing agar plates by adding TCA solutions to agar cooled to 50–55 °C and mixing gently to ensure homogeneous distribution. Pour approximately 3 mL of agar per plate and prepare the plates 12–24 h before use.
      2. Seed eggs directly onto the surface of the plates at densities comparable to those used in other formats (50–100 eggs per 100 µL). Do not perform washing steps in this configuration.
      3. Incubate the plates at 16–18 °C until 24 h after egg isolation.
   3. Multiwell plate assay (medium- to high-throughput format)
      1. Preload 96-well plates with drug solutions and controls. Add egg suspensions to obtain a final volume of 200 µL per well and a density of 50–100 eggs per well.
      2. Ensure homogeneous distribution during loading by avoiding bubbles and maintaining consistent volumes across wells. Incubate the plates at 20–22 °C under humid conditions for 24 h after egg isolation.
3. Image acquisition and quantification
   1. Evaluate egg hatching 24 h after egg isolation, once hatching is complete under control conditions. Count the number of unhatched eggs and L1 larvae under a stereomicroscope for each condition. Examine plate edges and well boundaries carefully, as larvae may accumulate in these regions.
   2. Acquire images using a stereomicroscope equipped with a digital camera for documentation purposes. Record each quadrant for agar plates and systematically image each well for multiwell plates. Use image analysis software when required to support quantification.
   3. Incubate the plates for an additional 12–24 h after image acquisition to assess whether the observed inhibition of hatching is reversible.
   4. Perform all experiments using at least three independent biological replicates.

Results

TCA produced clear concentration-dependent inhibition of egg hatching across all experimental formats, as shown in Figure 1. Representative concentration–response curves are presented for each assay configuration: microcentrifuge tubes (Figure 1A), direct exposure on agar plates (Figure 1B), and multiwell plates (Figure 1C).

The estimated IC₅₀ values were 0.19 ± 0.0262 mM in ...

Discussion

This study presents a simple and adaptable platform for egg-hatching assays in Caenorhabditis elegans, structured around three complementary experimental formats that differ in control, exposure dynamics, and scalability. Rather than defining a single optimal protocol, our results support the view that egg-hatching assays should be considered a flexible methodological framework adaptable to specific experimental objectives.

The microcentrifuge tube-based format provides a highly contr...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Bacteriological Agar	Britania	B0101406	should be prepared fresh (1.7 gr in 100 mL H2O)
Bleach solution	N/A	N/A	1.2 % (v/v) NaOH and 0.5 M NaOH
Centrifuge	ThermoScientific.	Sorvall ST16R.
Centrifuge	BiofugePICO to eppendorf tube	Heraeus 75003235
Digital camera	ToupCam	TP605100A	Industrial digital camera. Adjustabe focal length USB 2.0
DMSO	BioBasic	RAS#7-68-5	MW: 78,13
Linalil Acetate	SIGMA	No.CAS: 115-95-7	should be prepared fresh
Lysogeny broth (LB) medium	N/A	N/A	10 g Bacto-tryptone, 5 g yeast extract, 10 g NaCl to 1 L destilled water
M9 Buffer	N/A	N/A	3 g KH2PO4, 6 g Na2HPO4, 5 g  NaCl, 0,25 g MgSO4 to 1 L destilled water
Mini Rotator	BioSan	Bio RS-24
Nematode Growth Medium (NGM)	N/A	N/A	3 g NaCl, 17 g agar, 2,5 g  Bacto-Peptone to 1 L destilled water. Sterilize by autoclaving. Supplemented 1 mL cholesterol (5 mg/mL),  1 mL CaCl2 (1 M) , 25 mL potassium phosphate buffer (1 M, pH 6.0) and 1 mL  MgSO4 (1 M)
Petri Dish (3 - 5 mm)	ThermoScientific. Nunclon DeltaSurface	153066
Petri Dish (90 mm x 15 mm)	ExtraGene	1026160305	For laboratory use only, Polystyrene.
Pipette	Fisherbrand	N/A
SigmaPlot 12.0	Systat Software	N/A	Version 12.0
Stereomicroscope	ARCANO	ZTX-T 1:4 LED	Trinocular stereomicroscope (10x–40x total magnification; LED incident and transmitted illumination)
Sucrose	Laboratorio Cicarelli	Art 841	60 % (v/v) sucrose
Tissue culture plate 96 well	Biofil	TCP 012096	Polystyrene, Low Evaporation Lid.
ToupView	ToupTek Photonics	N/A
Transcinnamaldehyde (TCA)	Santa Cruz Biotechnology	J1712	Should be prepared fresh