softened
by incubation in a 2 mg/l pronase
solution (protease from Streptomyces
griseus, Westerfield, 2000; Sigma-
Aldrich) with an activity of 4 units per
mg in dilution water for 1 ± 0.5 min at
28.5°C and mechanically disrupted by
means of two very fine pairs of forceps or
dissection (insect) needles. Control experiments
were carried out to ensure that
pronase treatment at the given concentrations
did not have any effect on the further
development of the embryos. Since
the optimal incubation time depends on
the developmental stage, the optimal
duration should be checked in rangefinding
experiments. Without enzymatic
digestion, the mechanical stress resulted
in destruction of the embryo. Disruption
of the chorion without enzymatic
softening of the egg shell turned out to be
possible only in embryos older than 48 h.
Dechorionated embryos were incubated
in 24-well microtiter plates and
exposed to potassium chromate (hydrophilic),
4-chloroaniline (moderately
lipophilic) and lindane (lipophilic; all
p.a. quality; Fluka-Sigma-Aldrich) and
compared to non-dechorionated embryos
exposed under similar conditions. In order
to avoid excessive adsorption to the
microtiter plates, the wells were incubated
with toxicant 24 h prior to addition of
the eggs.