Die Embryonalentwicklung der Paradiesschnecke ... - TOBIAS-lib

Kapitel 4
Histology and 3D reconstruction
After nine days, embryos from both PtCl 2 -exposure and control were transferred
into separate glass Petri dishes and, later, small glass bowls depending
on the size. The water was changed every other day and the young snails
were fed with small amounts of Nutrafin Max flakes (Hagen, Germany) and,
occasionally, small pieces of organic carrots. Petri dishes and glass bowls
were cleaned with toothbrushes when necessary. Temperature and light-dark
regime remained the same throughout the whole lifetime of the snails. To
compare the anatomy of the nervous system of adult “sluggish” M. cornuarietis
and normally developed snails, eight weeks after oviposition one animal
from each group was fixed in 2% glutardialdehyde (VWR-Merck) in 0.01 M
cacodylate buffer (VWR-Merck) at pH 7.4. The animal from the control will
be referred as C1 whereas the sluggish snail will be referred to as P1. In addition,
a second adult sluggish snail was investigated. This snail was older than
C1 and P1. It will be referred to as P2. All three individuals were processed
as follows: rinsing in 0.01 M cacodylate buffer at pH 7.6 (3 x 10 min) and
decalcification in a mixture of formic acid and 70% ethanol (1:1) first for 30
minutes and again over night. On the next day, the specimens were rinsed 3
x 10 minutes in 70% ethanol and then dehydrated in a graded ethanol series:
2 x 70% ethanol (30 minutes, 1.5 h), 80% ethanol (1 h), 90% ethanol (1 h),
96% ethanol (1 h), 2 x 100% ethanol (1 h each). They were then immersed in
isopropanol (2 x 1.5 h and 2 h, respectively) and in a mixture of isopropanol
and paraffin (Paraplast, Leica) for 3 h. They were then infiltrated with pure
paraffin, once for 3 h and once for 5 h. Afterwards, they were embedded
in paraffin. Using a microtome (Leica SM 2000R), serial sections of 5 µm
thickness were cut and mounted on slides. The sections were stained with
Mallory’s triple stain (modified after Cason, 1950). All sections were then
viewed by light microscopy and photographed. The pictures were loaded into
Amira 4.1 or 5.3, respectively (Visage Imaging) and 3D models were created.
Pictures of the models were taken. Due to the fact that smaller structures can
get lost in computer based processing, the models were again compared to
the original sections and modified accordingly by hand in Inkscape (depicted
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