C. elegans Chemotaxis Assay

Summary

A method of quantitatively evaluating the chemotactic response of Caenorhabditis elegans is described. A chemotactic index (CI) was employed as a way to precisely evaluate the response of worms to certain targets, and serve as a platform of comparison between strains and compounds of interest.

Abstract

Many organisms use chemotaxis to seek out food sources, avoid noxious substances, and find mates. Caenorhabditis elegans has impressive chemotaxis behavior.

The premise behind testing the response of the worms to an odorant is to place them in an area and observe the movement evoked in response to an odorant. Even with the many available assays, optimizing worm starting location relative to both the control and test areas, while minimizing the interaction of worms with each other, while maintaining a significant sample size remains a work in progress ^1-10. The method described here aims to address these issues by modifying the assay developed by Bargmann et al.¹. A Petri dish is divided into four quadrants, two opposite quadrants marked "Test" and two are designated "Control". Anesthetic is placed in all test and control sites. The worms are placed in the center of the plate with a circle marked around the origin to ensure that non-motile worms will be ignored. Utilizing a four-quadrant system rather than one 2 or two 1 eliminates bias in the movement of the worms, as they are equidistant from test and control samples, regardless of which side of the origin they began. This circumvents the problem of worms being forced to travel through a cluster of other worms to respond to an odorant, which can delay worms or force them to take a more circuitous route, yielding an incorrect interpretation of their intended path. This method also shows practical advantages by having a larger sample size and allowing the researcher to run the assay unattended and score the worms once the allotted time has expired.

Introduction

Ward first developed the chemotaxis assay in 1973 ⁵, and since then it has had far reaching applications. Neurobiology is one field that has benefitted from using a variety of chemotaxis assays. Olfactory adaptation, a simple form of learning and memory, has been demonstrated in C. elegans using chemotaxis assays ⁶. They have also been used to show that C. elegans can develop ethanol tolerance-a result that not only demonstrates the behavioral plasticity of the worms, but that also shows that the worms can be very useful in the study of alcohol dependence in humans ³. Assays have even been developed to demonstrate....

Protocol

1. Preparing/washing the Worms

1. Synchronize worms to young adult¹¹.
2. Pipette 2 ml of the S Basal onto a 5 cm Chemotaxis plate of staged worms that have just cleared the lawn of OP50 E. coli. Tilt the plate as needed to ensure the worms are washed from the plate surface into the buffer.
3. Pipette 1 ml of the worm-S Basal solution into a microcentrifuge tube.
4. Centrifuge for 10 sec using a PicoFuge at 6,600 rpm.
5. Aspirate the S Basal, leaving the pellet of worms undisturbed.
6. Add 1 ml S Basal solution to the microcentrifuge tube, invert a few times to wash the worms.
7. Rep....

Results

Comparing wild-type (N2) C. elegans to the odr-10(ky10) mutant.

We used diacetyl, a known C. elegans chemoattractant, to compare wildtype worms to that of a mutant that lacks the receptor for diacetyl ^1,12. For wildtype (N2) worms the chemotactic index was 0.100±0.066 to ethanol, and 0.839±0.031 to 0.5% diacetyl. As expected, diacetyl elicits a significant chemoattractive response from wild-type worms (P<0.003). In contrast the odr-10(ky10)

Discussion

Chemotaxis, although controlled by a complex set of neuronal and cellular mechanisms, can be easily and objectively quantified using chemotaxis assays. To obtain the best results from the assays, certain critical steps must be taken. Firstly, staging the worms is essential in yielding consistent experimental results. Worms at different life stages behave differently ¹³; so mixed stage worms may skew experimental results. Secondly, ensuring all E. coli is washed off the worms is crucial, as residua.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
S Basal (- cholesterol) [5.8 g NaCl; 1 M K phosphate buffer, pH 6.0; dH2O to 1 liter.] Autoclave
PicoFuge	Stratagene	400552
Microscopes	Leica
Dissecting	Leica
P1000 Pipette	Gilson
P10 Pipette	Gilson
0.5 M Sodium Azide
Chemotaxis Agar [1.6% BBL-agar (Benton-Dickinson) or 2% Difco-agar. Autoclave. Add 5 mM potassium phosphate, pH 6.0; 1 mM CaCl2, 1 mM MgSO4]
Ethanol/Distilled Water
Test Compounds (eg. 0.5% diacetyl)
Agar	Bio-Rad	166-0600
NH4Cl	Amresco	CA97062-046
MOPS	VWR	CA12001-120
NH4OH	BDH	CABDH8641-2
0.25% Tween 20	Bio-Rad	170-6531