EXPERIMENTS FOR Biol241. M.L. SPARLING

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.