TTX assay protocol


Introduction

Thermotaxis is one of the interesting behaviours in C. elegans. This behaviour, reported first by Hedgecook and Russell (1975), should require some mechanisms for sensing and memorizing temperature, which have been still largely unknown. Thermotaxis is also a behaviour difficult to assay, because this behaviour is influenced sensitively by the environmental conditions such as temperature, population density, and feeding state. However, the critical control of the environmental conditions enable us analyze this challenging behaviour. This protocol describes a series of tips for thermotaxis (TTX) assay.

General remarks

The working bench should be horizontal. There is no source of wind and vibration such as a centrifuge or a draft chamber nearby. Do not use the incubator for TTX assay because it is strong source of wind of fan and vibration from compressor


Temperature and humidity

Room temperature should be kept at 25 C. Avoid the sunlight causing temperature change. Low humidity (20 ~ 30%) is preferred for assay.

Incubator

The small volume (100 ~ 150 L) incubator is preferred to get stable temperature.

6-cm seeded NGM plates

We use a 6-cm plate containing 14 ml of NGM, which is minimal volume required for getting sufficient bacterial lawn. The NGM plates were spread with overnight-cultured E. coli, incubated for a day at room temperature (or for overnight at 37 C), and stored at room temperature. Do not use old seeded NGM plates (over 2 weeks).

C. elegans

Use the healthy adult animals from uncrowded plates. Animals should be maintained by transferring 2 ~ 3 animals to a new NGM plate before starving. Do not use the starved animals or those from a starved agar chunk. We ordinarily transfer ~ 15 of L4 larvae to a fresh and preincubated NGM plate, grow those for 8 ~ 24 hr (depend on cultivation temperature), and use for assay.


Materials

Well fed adult animals (uncrowded)

6-cm seeded NGM plates

9-cm TTX plates

TTX medium consists of 2% agar, 0.3% NaCl and 25 mM potassium phosphate buffer (pH 6.0). Pour 8ml of the medium exactly into a 9-cm plate after autoclaving.

Vials filled with frozen glacial acetic acid (stored at 4 C)

Glass vials are ~ 10-cm long and 2.7-cm diameter. Freeze glacial acetic acid at 20 C and sotre at 4 C.

Procedure

1. Remove lids of TTX plates and dry the plates for ~ 60 min at room temperature.

NOTE: Do not dry TTX plates in the windy place like a clean bench or a draft chamber. Over dry of TTX plates cause the plates surface to become rough. The surface of TTX plates should be smooth as a mirror.




2. Place the TTX plates with lids upside-down and mark center and a point of 1.0 ~ 1.5 cm from edge.




3. Take the vials with frozen glacial acetic acid out from cold room and leave the vials for ~ 10 min at room temperature (25 C) to allow acetic acid starting to melt

NOTE:The melting point of glacial acetic acid is 16.7 C. Do not use the vials just taken out.



4. Put the vial on the center of TTX plate.

NOTE:We use large metal plates for easy handling.




5. Leave for 10 ~ 15 min to form stable radial thermal gradient on the surface of TTX plate.

NOTE: The thermal gradient is kept over 1 hr under the vial with malting glacial acetic acid.Renew the vials when the half of glacial acid is melted.




6. Remove the vial and place an animal on the point of TTX plate.

NOTE: Do not scratch the surface TTX plate. Avoid bringing E. coli onto the TTX plate. For example, move an animal to the place without bacterial lawn in NGM plate (or unseeded NGM plate) and transfer the animal to the TTX plate. Pick the animal up carefully without harm. Do not use the animal which you fail to pick up twice.




7. Put the vial on the center of TTX plate again

8. Repeat step 6 and 7 for each TTX plate

NOTE:It takes 6 ~ 20 min for handling 20 TTX plates.

beginner: 20 min

skillful: 10 min

expert: 6 min




9. Leave for 50 ~ 60 min.

NOTE:Do not walk around during assay to avoid wind and vibration.

10. Remove the vial and put several drops of chloroform on the lid to stop the animal




11. Place the TTX plates at 4 C until photograph

References

  1. Hedgecock E.M. and Russell R. L. (1975) Normal and mutant thermotaxis in the nematode Caenorhabditis elegans . Proc Natl Acad Sci U S A. 72: 4061- 4065.
  2. Mori, I. and Ohshima, Y. (1995) Neural regulation of thermotaxis in Caenorhabditis elegans . Nature 376: 344-348.