Worm Breeder's Gazette 1(1): 6

These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author.

A Chemotactic Assay Method

C. Butcher

Our lab has developed a method of creating linear gradients of salts 
and other attractants and repellents for testing the chemotaxis 
responses of the nematode.  The basis for this method was originally 
suggested by Larry Soll who was familiar with it from his work with 
Bactoagar, salt and water are mixed to the desired molarity and 
percentage agar (usually 1.5%), autoclaved and cooled to 70 C.  
Approximately 15 ml is poured into a 9-cm petri dish one edge of which 
has been raised 1/4' above a level surface.  In two or three minutes, 
after the agar has solidified, the dish is placed on a level and ~20 
ml of 1.5% agar is poured on top.  The use of the same concentration 
of buffer in the top and bottom agars is feasible when desired.
Plates prepared in this way may be cooled at room temperature or 
chilled for an hour or two.  In any case, they should be allowed to 
equilibrate at the desired test temperature for 1/2 hour before being 
used.  The timing is not critical.  Diffusion calculations show that 
it takes about two hours for the gradient to set-up after the top agar 
is poured but plates can be kept overnight or longer.  All that 
happens is that the gradient flattens slowly and predictably over time.

Water washed worms are generally put down along the middle 1 or 2 
cms of a line drawn across the center of the plate perpendicular to 
the gradient.  Scoring is done by counting the worms that have moved + 
or - at various times after tracking begins.  In some tests it may be 
desirable to avoid scoring individuals close to the edges to minimize 
the effect of the miniscus that forms there.  Counts ranging from the 
proportions of 5 to 1 to 10 to 1 are routinely obtained 20 to 30 
minutes after tracking begins.
These taxis plates will retain a substantially linear gradient which-
is readable by wild type worms for 3 to 4 days after manufacture.  One 
of the more interesting results observed is that counts of wild type 
put down on attractant concentrations of NaCl (.001 to .05 M bottom 
agar) become random 4 to 20 hours after tracking begins, but counts 
made of worms put down on repellent concentrations of NaCl (.5M or 
more) remain 0 to 100 for three or four days.
This observation led to the development of a simple mutant selection 
technique using Falcon 'I' plates.  A steep (45 ) 2.0 or 3.0 M NaCl 
agar barrier is poured along one side of the 'I' followed by a regular 
top poured, with the plate on a level, and deep enough to cover the 
center divider.  Just before these plates are inoculated with 
mutagenized worms a crescent of E.  coli, OP 50, is spread on the side 
opposite the put down area to act as a holding area for mutants that 
wander across the salt barrier.  Initial work with this selection 
technique indicates that it is important to have the worms reasonably 
homogeneous because only certain larval stages of a given mutant may 
cross the barrier.
Once the general technique of using bottom agar as the carrier for 
gradients is appreciated, it becomes obvious that there is virtually 
no limit to the types of gradients that can be set up.  One 
particularly useful combination of gradients is a 'T' plate where a 
shallow, low concentration is placed to attract the worms up the stem 
of the 'T' and a high repellent concentration is placed to repel the 
worms along the line of the top of the 'T'.  Normal behavior is 
accumulation just below the top line of the 'T' and aberrant and/or 
mutant behavior is easily spotted because of lack of response to 
either the attractant or the repellent.
Gradients of dead bacteria of various strains can be made with this 
technique and gradients of the exudate of live bacteria can be made by 
pouring a regular agar on a slope as the bottom layer, cooling it and 
applying a film of live bacteria to it, and then pouring a 40 C top 
layer with the plate on a level.  If the top agar is 1.5% or greater 
and it is poured with care, the worms will tax along the top rather 
than penetrating to the bacterial layer.
Other uses include pouring completion gradients (such as a .01M NaCl 
bottom with a .01M KCl top, and vice versa for control) to see which 
ion the worm prefers.  Similarly the system can further complicate the 
worm's choices by using a Hedgecock-Russell type thermal gradient 
under one or more competing chemical gradients.