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.
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
Room temperature should be kept at 25 C. Avoid the sunlight causing temperature change. Low humidity (20 ~ 30%) is preferred for assay.
The small volume (100 ~ 150 L) incubator is preferred to get stable temperature.
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).
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.
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.
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1.
Remove lids of TTX plates and dry the plates for ~ 60 min at
room temperature. |
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2. Place the TTX plates with lids upside-down and mark center and a point of 1.0 ~ 1.5 cm from edge. |
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3.
Take the vials with frozen glacial acetic acid out from cold
room and leave the vials for ~ 10 min at room temperature
(25 Ž¡C) to allow acetic acid starting to melt |
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4.
Put the vial on the center of TTX plate. |
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5.
Leave for 10 ~ 15 min to form stable radial thermal gradient
on the surface of TTX plate. |
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6.
Remove the vial and place an animal on the point of TTX
plate. |
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7. Put the vial on the center of TTX plate again |
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8.
Repeat step 6 and 7 for each TTX plate ¡¡ beginner: 20 min skillful: 10 min expert: 6 min |
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9.
Leave for 50 ~ 60 min. |
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10. Remove the vial and put several drops of chloroform on the lid to stop the animal |
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11. Place the TTX plates at 4 Ž¡C until photograph |
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References
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