Article | Published:

High-content behavioral analysis of Caenorhabditis elegans in precise spatiotemporal chemical environments

Nature Methods volume 8, pages 599605 (2011) | Download Citation


To quantitatively understand chemosensory behaviors, it is desirable to present many animals with repeatable, well-defined chemical stimuli. To that end, we describe a microfluidic system to analyze Caenorhabditis elegans behavior in defined temporal and spatial stimulus patterns. A 2 cm × 2 cm structured arena allowed C. elegans to perform crawling locomotion in a controlled liquid environment. We characterized behavioral responses to attractive odors with three stimulus patterns: temporal pulses, spatial stripes and a linear concentration gradient, all delivered in the fluid phase to eliminate variability associated with air-fluid transitions. Different stimulus configurations preferentially revealed turning dynamics in a biased random walk, directed orientation into an odor stripe and speed regulation by odor. We identified both expected and unexpected responses in wild-type worms and sensory mutants by quantifying dozens of behavioral parameters. The devices are inexpensive, easy to fabricate, reusable and suitable for delivering any liquid-borne stimulus.

Access optionsAccess options

Rent or Buy article

Get time limited or full article access on ReadCube.


All prices are NET prices.


  1. 1.

    The use of control systems analysis in the neurophysiology of eye movements. Annu. Rev. Neurosci. 4, 463–503 (1981).

  2. 2.

    , , , & High-throughput ethomics in large groups of Drosophila. Nat. Methods 6, 451–457 (2009).

  3. 3.

    Chemotaxis by the nematode Caenorhabditis elegans: identification of attractants and analysis of the response by use of mutants. Proc. Natl. Acad. Sci. USA 70, 817–821 (1973).

  4. 4.

    , & Odorant-selective genes and neurons mediate olfaction in C. elegans. Cell 74, 515–527 (1993).

  5. 5.

    et al. Dissecting a circuit for olfactory behaviour in Caenorhabditis elegans. Nature 450, 63–70 (2007).

  6. 6.

    , , , & Olfactory behavior of swimming C. elegans analyzed by measuring motile responses to temporal variations of odorants. J. Neurophysiol. 99, 2617–2625 (2008).

  7. 7.

    , , , & Step-response analysis of chemotaxis in Caenorhabditis elegans. J. Neurosci. 25, 3369–3378 (2005).

  8. 8.

    et al. Subcellular positioning of small molecules. Nature 411, 1016 (2001).

  9. 9.

    et al. Neutrophil chemotaxis in linear and complex gradients of interleukin-8 formed in a microfabricated device. Nat. Biotechnol. 20, 826–830 (2002).

  10. 10.

    et al. Metabolic gene regulation in a dynamically changing environment. Nature 454, 1119–1122 (2008).

  11. 11.

    Worm chips: microtools for C. elegans biology. Lab Chip 10, 432–437 (2010).

  12. 12.

    , & Microfluidics for the analysis of behavior, nerve regeneration, and neural cell biology in C. elegans. Curr. Opin. Neurobiol. 19, 561–567 (2009).

  13. 13.

    , & Microfluidics for in vivo imaging of neuronal and behavioral activity in Caenorhabditis elegans. Nat. Methods 4, 727–731 (2007).

  14. 14.

    et al. Artificial dirt: microfluidic substrates for nematode neurobiology and behavior. J. Neurophysiol. 99, 3136–3143 (2008).

  15. 15.

    et al. Enhanced Caenorhabditis elegans locomotion in a structured microfluidic environment. PLoS ONE 3, e2550 (2008).

  16. 16.

    Behavioural analysis of nematode movement. Adv. Parasitol. 13, 71–122 (1975).

  17. 17.

    , & The fundamental role of pirouettes in Caenorhabditis elegans chemotaxis. J. Neurosci. 19, 9557–9569 (1999).

  18. 18.

    , & Neurons regulating the duration of forward locomotion in Caenorhabditis elegans. Neurosci. Res. 50, 103–111 (2004).

  19. 19.

    , & A circuit for navigation in Caenorhabditis elegans. Proc. Natl. Acad. Sci. USA 102, 3184–3191 (2005).

  20. 20.

    , & Dopamine and glutamate control area-restricted search behavior in Caenorhabditis elegans. J. Neurosci. 24, 1217–1225 (2004).

  21. 21.

    , & A behavioral switch: cGMP and PKC signaling in olfactory neurons reverses odor preference in C. elegans. Neuron 59, 959–971 (2008).

  22. 22.

    & Parallel use of two behavioral mechanisms for chemotaxis in Caenorhabditis elegans. J. Neurosci. 29, 5370–5380 (2009).

  23. 23.

    et al. Neuropeptide feedback modifies odor-evoked dynamics in Caenorhabditis elegans olfactory neurons. Nat. Neurosci. 13, 615–621 (2010).

  24. 24.

    , & Automated on-chip rapid microscopy, phenotyping and sorting of C. elegans. Nat. Methods 5, 637–643 (2008).

  25. 25.

    , , & High-throughput study of synaptic transmission at the neuromuscular junction enabled by optogenetics and microfluidics. J. Neurosci. Methods 191, 90–93 (2010).

  26. 26.

    The genetics of Caenorhabditis elegans. Genetics 77, 71–94 (1974).

  27. 27.

    et al. Fabrication of microfluidic systems in poly(dimethylsiloxane). Electrophoresis 21, 27–40 (2000).

  28. 28.

    , & Plastic masters-rigid templates for soft lithography. Lab Chip 9, 1631–1637 (2009).

  29. 29.

    , , , & The parallel worm tracker: a platform for measuring average speed and drug-induced paralysis in nematodes. PLoS ONE 3, e2208 (2008).

Download references


We thank S. Leibler for the use of his clean-room facility, J. Larsch for control experiments and assistance with manual scoring of behavior, and members of the Bargmann laboratory for critical help, insight and advice. This work was supported by the Howard Hughes Medical Institute and by the G. Harold and Leila Y. Mathers Foundation. C.I.B. is supported by the Howard Hughes Medical Institute. D.R.A. is supported by a Career Award at the Scientific Interface from the Burroughs Wellcome Fund.

Author information


  1. Howard Hughes Medical Institute and Laboratory of Neural Circuits and Behavior, The Rockefeller University, New York, New York, USA.

    • Dirk R Albrecht
    •  & Cornelia I Bargmann


  1. Search for Dirk R Albrecht in:

  2. Search for Cornelia I Bargmann in:


D.R.A. designed and fabricated the devices, wrote the analysis code, performed the experiments and analyzed data. D.R.A. and C.I.B. designed the experiments and wrote the paper.

Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to Dirk R Albrecht.

Supplementary information

PDF files

  1. 1.

    Supplementary Text and Figures

    Supplementary Figures 1–12, Supplementary Table 1, Supplementary Notes 1–4

  2. 2.

    Supplementary Data 1

    Microfluidic channel pattern file.


  1. 1.

    Supplementary Video 1

    Worm locomotion in the structured microfluidic environment. Real-time video shows a wild-type young adult worm crawling forward through the arena, pausing briefly (at 8 s), then performing two pirouettes: long reversals (at 12 s and 20 s) followed by a sharp turns (at 17 s and 25 s). Video shows a 2.3 mm × 1.7 mm region. All videos are encoded with the Xvid codec (available at

  2. 2.

    Supplementary Video 2

    Loading worms into a device. Video shows 18 wild-type (right arena) and 22 tax-4 (left arena) worms loaded simultaneously into a single device containing two 16 mm A 15 mm arenas. Wild-type worms paused and reversed frequently for about 20 min, whereas tax-4 mutants quickly dispersed. tax-4 worms did not respond to the central stripe containing 0.92 μM isoamyl alcohol (IAA) odor and dye for visualization. Video is accelerated 15×.

  3. 3.

    Supplementary Video 3

    Annotated video of an odor pulse assay. Video shows tracking and analysis of 23 wild-type worms responding to repeated 0.92 μM IAA odor stimulation according to the pulse sequence in Figure 3a. Each worm is numbered in the arena and labeled with its current centroid (circle and green +) and a line indicating 30-s of centroid history. Line color indicates behavioral history according to the legend, for example, white indicates forward movement and red indicates the pirouette state. The ethogram (right) shows a scrolling 45-s window of worm behavior, time-adjusted such that each worm's response lines up with the odor pulse pattern. Green arrowheads on the ethogram indicate the position of each worm at the current video frame. Tick marks represent 15 s. Video is accelerated 15×.

  4. 4.

    Supplementary Video 4

    Dynamic odor stripe assay. Video shows the locomotor responses of 25 worms to two attractive odor stripes (0.92 μM and 1.84 μM IAA, top to bottom) separated by odor-free buffer. Flow is left to right, and odor stripes contain dye for visualization. Worms experience a sharp odor gradient at the stripe edges but no mechanical or flow rate discontinuity. Near the end of the video, odor stripes are turned off by closing external valves. Video is accelerated 15×.

  5. 5.

    Supplementary Video 5

    Example behavioral responses upon odor stripe exit. Odor (0.92 μM IAA) is present in the lower half of the video (gray). Three outward encounters of the odor stripe edge are shown; each response is shown twice. The first worm responds with a correct forward turn, the second with a correct pirouette and the third with an incorrect pirouette. 'Correct' refers to the worm remaining in the attractive odor stripe after the response. Video shows a 3.9 mm × 3.3 mm region and is accelerated 15×.

Zip files

  1. 1.

    Supplementary Software 1

    Software to identify instantaneous behavioral states from video, and to summarize behavior and speed data over space and time.

About this article

Publication history





Further reading