PRELAB: Read about the changes in circulation, heart fusion from
two heart fields, neural control of the heart beat.
This lab will show that during development, the embryo has little
control of its physiology and behaves like a cold-blooded animal.
If the environment is cold, it will grow cold and die, if the
environment is hot, it will get hot and die.
     During early development the heart rate is controled by the
ventricle, then when the atria fuse, by the atrium; then when the
sinus venosus fuses, it becomes the pace maker (at about 72 hr.)

METHOD:
Gently open a chick egg into enough warm saline (less than 40
degrees centigrade) to cover the egg. If the blood vessels break,
the circulation will stop after the shock of bleeding (they have
some clotting ability, but not great). So we must be careful not to
cut the blood vessels with the sharp shell. Therefore, I usually
open the egg under the saline solution and gently let it fall out
of the shell. Fingers are the best instruments for freeing any
parts stuck to the shell or to turn the embryo upright. 
     The heart rate can be observed through a dissection microscope
or can be counted without any magnification. On older embryos where
the amnion covering makes it impossible to see the heart, count the
pulse in a large extrembryonic artery in the allantois or yolk sac.
Record the temperature where you start, and count the heartbeats
for a full minute. Don't try to take shortcuts and multiply, and
you will find out why during the data collection. Slowly change the
temperature, you can just let the solution cool off to room
temperature, then place the dish on some ice to cool down to about
20 degrees. Record two readings every few degrees. Then warm the
embryo back up by adding hot saline, and removing cold saline,
again recording the rate of heart beat on the way up every few
degrees, taking two sets of readings for each. Have different
people count the heart rate and get an average. Take the
temperature up to 42 degrees. Graph your results for both the way
down and the way back up. 
     What conclusions can you make about the relationship of
temperature to heart rate? Did you get the same results for all the
stages of embryos? 










EFFECTS OF CHEMICALS ON HEART RATE
If you have some chemicals try the following:
You want to add only one thing to each embryo, to check out the
result on heartrate. Use the earliest stage of embryo and cut the
whole embryo off the surface of the egg. To cut the embryo off the
egg, insert your forceps just outside the blastodisc (outside the
blood circle) and cut around the entire blastodisc and forceps with
some scissors. Then transfer it using a spoon or spatula under it
to a petri dish of saline with one of the ingredients below. Make
sure they are all warm, at the same temperature. This requires
quite careful students.
     An alternative approach is to break the egg into a petri dish,
cover it with plastic wrap so that it touches the embryo and place
that on a constant temperature pad which has been heated to 50
degrees in an oven or boiling water bath. The pad becomes clear
when the correct temperature, mix the contents of the pad before
placing the petri dishes on it. Place a thermometer on the pad to
be sure the temperature remains the same, since we only want to
vary one independent variable at a time so that when we measure the
dependent variable (the heart rate) we will know the cause of the
change.

Measure the heart rate several times before adding the chemicals,
for each chick. Then you can compare the rate after the addition
and see the change if there is one.

Students can measure the effects due to different things which
interest them. They should decide the day before so they can bring
from home the things they want to test. (Coffee, tobacco extract,
antihistamine, calcium tablets, vitamins, retinA, alcohol (only a
few drops are needed), bicarb. These can be added to the chick
saline before adding a few drops under the plastic wrap. Or: 
     Add vinegar.
     Use a saline made only with KCl- .6M KCl (4.5 g/100ml). This
was the most effective agent I tried- it slows the heart almost to
a stop. The effect can be reversed by adding many drops of regular
saline.
     Add some powdered coffee     
     Add some tea leaves.
     Add a vital stain so the structures become more visible.
     Insecticide.
For observation of chick embryos over several days, they can be
grown in chambers made from plastic drain pipe as shown in the
article: Effects of varying chamber construction and embryo pre-
incubation age on survival and growth of chick embryos in shell-
less culture. BE Dunn, TP Fitzharris, BD Barnett. Anatomical Record
199:33-43. 1981. If embryos become broken or bleed during their
opening, they can still be used to look at under the microscope to
count the somites or to watch the heart beat in saline, look at
differences between different ages. Just grab the embryo with your
forceps and don't let go and get under it with a spoon, transfer to
a petri dish or slide. These are good for doing experiments over
several days.
Graphs can be made of the heart rate versus temperature or age.
DISCUSSIONS: 
     Effects of high K+ concentration: removal of membrane
potential so that it cannot respond.
     Effects of high Ca++: discuss effect of Ca++ on actin and
myosin interaction.
     Many agents that effect the adult heart through the nerves do
not effect the embryo heart because the nervous system has not yet
developed connections to the heart, and the brain control
mechanisms appear very late in development. That is why it is so
important that the hen or the incubator maintains the proper
temperature for the embryo.









































HOW TO BUILD A CHICK INCUBATOR FOR ABOUT $15.00
   (If you have $50 available, you can get a small 50 egg incubator
from the Sears Farm catalog. The same incubator sells for $125 in
scientific catalogs.)

SUPPLIES NEEDED: thermostat for 100 F*, heater coil (Eagle straight
glo-coil)* or light bulb, used computer fan with cord*, porcelain
bulb socket* (all starred items were purchased for $13.50 at Apex
Electronics, a used and surplus store, 8909 San Fernando Rd, Sun
Valley, CA. 818-767-7202), three screw wire connectors, cord with
plug, NON-FLAMABLE CONTAINER like an old aquarium or refrigerator drawer , thermometer, dish to hold water, small sponge for dish (keeps water from
spilling),  holes for air in box. 
     Make holes in the lid  (could be wood) the size of the thermostat and the
porcelain socket and push them through to the inside of the box,
just leaving the wireing and the end sticking outside. Place the
fan at one end (not touching the end) of the box and make a hole
the size of the cord so it can be stuck through to the outside.
     Wiring: take one wire from each of the thermostat, heater
sucket, and fan and screw them together. Take one wire from the
thermostat and screw it together with one wire from the plug-in
cord. Hook the other wire of the plug-cord to the remaining wires
from both the fan and the heater. Now the fan and heater will only
come on after the incubator is plugged in when the temperature is
lower than the thermostat is set. To set the thermostat, make holes
in the lid the size of your thermometer and then five other holes
(I used my 3/8 inch drill bit). Make two holes in each side of the
box, and one in each end. These holes are air holes. Now place your
dish of water with the sponge in it in the box and leave room for
a wire basket of eggs. Replace the lid and plug in the cord. Make
sure the fan cord is pulled up through so it won't touch the heater
or get caught in the fan. Now place the thermometer in the lid and
screw the end of the thermostat clockwise until the fan comes on
(or counterclockwise if you go all the way and nothing happens the
other direction.) Make adjustments with the screw until the proper
temperature is maintained for 24 hours. Now the incubator is ready
for placing in the eggs. If students are going to use this consult your 
electrician so the apparatus is safe.
     Always have the eggs at room temperature before placing them
in. Write the date you place them in on the egg with pencil. Eggs
can be kept for about 2 weeks at 10 degrees C if they are turned
each day. A regular refrigerator kills them. Turn the eggs in the
incubator once a day, you can easily tell when they are turned by
using pencil to make a big X on one side of the egg, keep the water
dish replenished. The moisture is most important at the time of
hatching. Try not to open the lid the last day (and don't turn the
eggs after they pip a hole), as that decreases the humidity and
they will not hatch. You will hear them if they are hatching.
Before hatching you will have to place a barrier in front of the
fan so the chicks don't walk into it, such as chicken wire. I meant
this incubator to be for early development only, but it could be
altered in this way to use to hatch as well. Incorrect temperature
or too low humidity can cause death during development.