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Materials
List
-
Day old eggs (at blastoderm stage)
- Sterile Howard's
Ringers solution
(see below)
- 70% Ethanol
- Culture dishes (or cheaper alternative) (see
below)
- Filter paper rings (1-1.5 cm in diameter)
- Fine forceps
- Scissors
- Petri
dishes containing albumen agar
(see below)
- Inner dish, 2mm of albumen agar covered by 1mm
albumen
- 37 °C incubator
- Tapered pipettes, prepare in lab
- Dissecting scope
- Camera
Pre-Laboratory
Set-Up
Agar plate
preparation:
- Autoclave
200 ml beaker with stir bar. Wipe eggs with 70%
ethanol.
- Collect
100 ml albumin by separating egg whites from
approximately 5 eggs into sterile beaker (discard
yolks).
- Stir at
room temperature to break up clumps. Warm to 45°C
in water bath.
- Autoclave
1.3 g agar, 1.3 g glucose and 33 ml Howards' Ringers
solution in 100 ml flask for 15 minutes.
- Cool to
45 °C in water bath.
- Combine
agar and egg whites with stirring. Transfer between
flask and beaker to aid combination.
- Turn
water bath up to 48 °C to keep agar from
solidifying while pouring plates.
- Cut tip
off of transfer pipet. Transfer approximately 3 ml to
35 mm petri dishes. Avoid transferring bubbles.
NB:
If culture dishes are not available, a cheaper
alternative is to use 30mm Petri dish of albumen agar
placed in a 60mm Petri dish of Howard’s ringers.
Special care must be taken with the alternative dishes
to not splash the embryo with ringers once it is
placed on the agar as this can cause the embryo to
slide off the agar into the moat.
Procedure
- Rinse
the egg in EtOH and swirl to release embryo. Lay on
its side so that the blastoderm will float to the
top.
- Fill the
moat of the culture dish with Howard’s Ringers
solution.
- Break
the egg and place the yolk into a sterile Petri dish.
Alternately, use bent forceps to tap a whole in the
shell and remove pieces of the shell until the hole is
large enough to put the doughnut onto the
blastoderm.
- With
sterile forceps, use paper donut to frame blastoderm.
Allow to sit a while, until it looks drier, this will
allow the blastoderm to better adhere to the
doughnut.
- Lift
with tweezers and cut away connective tissue to free
embryo.
- Place
blastoderm, dorsal-side down, onto
albumen “island” in culture
dish.
- Cover,
label, and place the dish in the incubator for 30
min.
- Set up
the microscope and camera.
- Remove
embryo from incubator and photograph it.
- Make a
substantial (large) longitudinal cut in the middle of
the anterior intestinal portal, through the floor of
the foregut with the glass taper cutting from the
bottom portion of the hood covering the developing
brain. Pass the taper through the incision 2 or 3
times. Record the time of the surgery.
- Photograph
the embryo, cover it and return it to the
incubator.
- Observe
the embryo daily until the heart forms and starts to
beat.
- Photograph
the embryo daily and record beating if present. Note
when heart forms and begins to beat.
- If
possible, record video of beating heart.
©Cebra-Thomas,
2000
Last Modified: May 2nd
2004
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