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.

Remembrance of Things Passed

R. Cassada

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 
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!