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.
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 bacteria. 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.