Legume seeds (cowpea, adzuki bean or mung bean) with
approximately two-day old Callosobruchus eggs were placed in
solid watch glasses containing colchicine solution (0.1% colchicine
solution in buffered insect saline (0.75% sodium chloride made up
in distilled water buffered to pH 6.8 with Sörensen’s phosphate
buffer)). Under a binocular microscope the eggs were carefully
dislodged from the seeds with fine pins. Damage to the eggs was
minimal, but each egg must have a small perforation made in its
ventral surface to allow penetration of solutions. The eggs were left
in the colchicine solution for 12 min, then carefully transferred to
half-isotonic potassium chloride (hypotonic (half-isotonic) potas-
sium chloride e 0.48% potassium chloride in distilled water buff-
ered to pH 6.8 with Sörensen’s buffer) in a fresh watch glass, using
a Pasteur pipette. After a further 12 min the eggs were pipetted
into a watch glass of fixative (3 parts absolute ethanol to one part
glacial acetic acid). The fixative was pipetted off and replaced four
times, to remove all water. The eggs were then covered and left
for 30e60 min. For slide preparation, individual eggs were picked
up with fine forceps and placed on clean microscope slides