Worm Breeder's Gazette 9(2): 112
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.
I. PUMPING ASSAYS: The basic problems in following pumping are the optical visibility and the mobility of the worms. Visibility is best when the worm's head is not in air, as it is on a dry agar layer. Worms feeding on a fresh (still relatively moist and free of clumps or crystals) bacterial lawn are ideal. Adding liquid and or a cover slip may disturb behavior, but help visibility. Use the highest magnification possible and refocus continuously on the viewed object. Adjusting the light source to a spot or altering the intensity can give more shadowing of the bulbs (but also of the outer surfaces unless the worm is under the liquid-air interface) and hence more apparent contrast (but see below for heat problems). Viewing on agar under the compound microscope without any coverslip works wonderfully except for heating problems. (Don t try this with an oil objective with or without oil!) Here it is important that the agar be thin (for good optics) and uniform to minimize refocusing and avoid touching the agar with the lens. The problem of the objective bumping into the rim of the plastic plate is solved by using a very shallow plate such as the lid of a small plate. J. Sulston's mounting (for postembryonic lineaging) under a coverslip, with a tiny spot of bacteria to minimize wandering, is best for small numbers of animals. Here one can also observe pumping inside the egg if the liquid layer and agar are as thin as possible. Movement out of the field of view can be random and spontaneous, owing to hunger, absence of lawn, overcrowding ( mechanical and chemical stimuli), or taxis due to pH difference, gas ( oxygen or carbon dioxide) or thermal gradients from the open rim of the plate. Viewing can further activate movement, especially repulsion from the warmth produced by the microscope light, so use minimum intensity, infrared filters and a cool, ventilated microscope base. Vibrations from bumping the plate, microscope or table, or from equipment or even noises and talking, as well as wind from breathing or air conditioning are sensed. Removing the lid of the plate enhances all these problems and may set off a shock response if the air is dry or a different temperature from that of the incubator. If conditions are good, visibility is nearly as good with the lid on, especially when plates are viewed inverted. A constant-temperature room for cultures and viewing helps a lot. Movement and pumping will be severely altered if the worms have been anoxic due to centrifugation, etc. or Millipore filtered even momentarily to dryness. The good news is that all these stimuli for movement (and possibly for alteration of pumping) seem to trigger only a quantum of movement, often only for a minute or so. (Niel Croll worked extensively on this problem.) So don t try to score a culture right after it's been put under the microscope and make sure sequential counts agree with each other. Following are the outlines of the 3 types of assay used. Assay 1: Percentage of animals pumping. Scan a prechosen number of animals over a 'typical' field for 5 seconds each. Optimal is to use a small enough lawn and number of animals so that all animals are scored and animals straying from the lawn or up the walls can be quantitatively noted separately. Assay 2: Rate of pumping of individual animals. Optimal is one animal per plate (or per liquid drop with sandwich supported coverslip and air bubble), so that lethargi and molts are known. This is more work but gives more meaningful data. Following the rate requires a clear head, absence of distractions, practice, and preferably a tunable rhythmic sound or light source to compare to. Accurate counting is really only possible with a video camera and slow-motion playback analysis. Assay 3: Uptake or extent of feeding assays. These can be quite useful. I've used uptake of P-32 labelled (washed) bacteria and could count single worms (100 is better) in water without killing them, using the Cerenkov radiation. We played with iron filings and I see from the Gazette (Meeting abstracts 85) that Horvitz's lab has perfected this. A nice semi-quantitative assay of pumping is to add extra fine (grind them dry in a mortar and pestle) carmine particles ( washed and size-settled) to the bacterial lawn before seeding. Other ( soluble) dyes don't seem to work so well. You might try spinning down fresh bacteria and mixing particles with the slurry, rather than letting the lawn grow on the plate a long time. But some growth may be needed to get the proper chemotactic signals. Good luck!