The
surgery involved in this procedure is delicate and
requires a large measure of manual dexterity. These
skills should be honed before relevant data is
collected. In this experiment, surgery was practiced
on embryos of appropriate age a week before data was
attained.
All eggs
were sterilized with 70% ethanol and allowed to dry
before opening. Blastoderm stage chicks were harvested
from 1-day old eggs using two techniques. In the
first, bent forceps were used to tap a whole in the
shell. Pieces of the shell were removed until the hole
was large enough to allow a filter paper ring to be
placed onto the yolk so that the ring framed the
embryo (Figure 1). Alternately, the egg was swirled to
dislodge the embryo and allowed to sit on its side so
that the yolk would rotate and place the embryo on the
upper side of the egg. The egg was cracked on the side
opposite the embryo and the yolk and albumen were
carefully placed in a large petri dish (figure 2).
Filter paper was again used to frame the embryo. Many
embryos were lost at the point when the yolk was
broken and the embryo sank into the yolk, out of view.
The procedure was the same for both techniques after
this point. Sterile albumen from the egg was pipetted
onto the albumen agar of the culture dish to form a
thin nutrient layer for the embryo. Sterile Howard’s
Ringers solution was pipetted into the moat of the
culture dish. The amniotic membrane covering the
embryo was allowed to adhere to the ring for one
minute. The yolk was then punctured with scissors and
the ring surrounding the embryo held with forceps
while the chorion and other connective membranes were
cut. The membrane around the embryo was cut and the
ring was placed on the albumen agar such that the
embryo was dorsal side down, exposing the heart on the
ventral side. Drops of sterile Ringers solution were
used to clear off any stray yolk that had accidentally
been transferred with the ring and embryo onto the
albumen agar. All embryos were staged and the culture
dishes covered, labelled, and placed in a 37°C
incubator for half an hour or until the embryo reached
stage eight of development (Figure 3).
Glass
pipette tips were flamed and pulled until they broke
and formed extremely thin operating tools. Multiple
attempts were required to make a straight taper
because hot glass tends to curl after it breaks.
Embryos at the Hamburger and Hamilton stage eight of
development were operated under an Olympus DP12
microscope camera. The embryo was carefully moistened
before the procedure with drops of Ringer’s
solution. The drops were placed beside the embryo and
not directly on or above it, in order to prevent
damage to the embryo. The straightest, most thin glass
taper was used to cut a slit down the midline of the
embryo from the midway point of the embryo to the
bottom half of the hood of the gut portal (Figure 5).
Care was taken to avoid completely severing the upper
half of the embryo (Figure 6). Development continues
more normally if the brain is not completely bisected
(J. Cebra-Thomas, personal communication, April 2004).
Control embryos were not operated.
All embryos were returned to the 37°C incubator
between observations. Photographs were taken daily.
Video footage was recorded once the hearts formed and
started to beat.
©Cebra-Thomas,
2000
Last Modified: May 2nd
2004
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