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Methods
There will be histological cuts made to the compound eye and
ocelli of individual adults of moths, flies, bees and assassin bug
for viewing under the optical microscope.
For the preparations of light and dark adaptation, the following
previous treatment will be performed:
-Adaptation to the
light: the insect will be exposed to the light of a lamp
(approximately 4000 lux) for 24 hours. After that period of
time has passed, the insects bug will be cut in sections. The
obtained piece must contain the eyes and ocelli, and the piece must
be as small as possible; and then it will be immediately submerged
in fixer for viewing under electronic microscope.
-Adaptation to
darkness: the bug will be kept in darkness for 24 hours. After
that period of time has passed, the head will be cut in sections
just like the previous case. This will be done under red light
(wavelength > 695 nm). The obtained piece will be immediately
submerged in fixer for viewing under electronic microscope, always
working under red light.
-Procedure for
histological preparations
The pieces must be as small as possible, containing the eyes and the
ocelli. These will be prepared to be analyzed under the
optical microscope, following the procedure described by Ribi
(1987), which consists of:
a) Fixing: Immediately
submerge the piece in special fixer (view recipes) for about 2
hours.
b) Washing: The pieces
are washed with special buffer (view recipes) for about five minutes
with two changes of buffer.
c) Dehydration: in
ascending series of alcohols: Ethanol 70% 10', Ethanol 80% a0;,
Ethanol 90% 10;, Ethanol 100$ 2 X 10;, mix 1:1 of Ethanol
100%:Acetone 15;.
d) Inclusion: The
piece is taken to acetone (15;). Then it is taken to a 1:1
mixture of acetone and hard resin (hard durcupan, see recipes).
It is left overnight in a closed container with low agitation and in
room temperature.
The container is then
taken (opened) to a stove at 40 C for 1-2 hours with the purpose of
evaporating the acetone.
The piece is then
taken to fresh resin in an adequate mold and it is oriented under a
magnifying glass.
The piece is then
taken to stove at 60 C for 24-48 hours so that resin can be
polymerized.
e) Cuts: The pieces
are removed from stove and left to cool. Perform cuts in
series of 5 micrometers with a glass knife under magnifying glass.
Each of the cuts is to be carefully accommodated with the aid of
fine tweezers in a drop of distilled water on top of a flat surface,
and with the aid of another magnifying glass. The cut is dried
over a hot plate at 60-70 C for some seconds with the purpose of
sterilizing and sticking it to the glass.
f) Colorization: Once
dried, the pieces are covered with a solution of blue metilene (view
recipes). It is then placed on the flat surface on top of a
hot plate at 60-70 C until a yellow ring is observed. It is
then washed with distilled water, dried, and covered with Depex.
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