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Notes to Instructor Developmental and Physiological Aspects of the Chicken Embryonic Heart

Penn State Lehigh Valley / Muhlenberg College

General Information

Chicken Eggs

Solutions

Temperature

Tools

Techniques for Heart Removal

Recommended Atlas

General Information

The lab that we have provided for this ABLE workshop is actually the second of a two-part laboratory experience. During the first three hour lab the students learn the fundamentals of 48 and 72-hour old chicken embryo anatomy (identification of the brain vesicles, somites, eyes, otic pit, pharyngeal pouches and clefts, limb buds, and heart), as well as two of the techniques they use in the following lab; thus, by the end of the first lab, they can window an egg, allowing them to observe the living embryo, and explant the live embryo into a dish of warm saline and maintain that embryo for at least an hour.

It is possible to teach them these two techniques and complete the heart lab all in one 3 hour period. We believe, however, the students are more likely to appreciate the beauty and dynamic nature of embryogenesis if they can concentrate on general anatomy and techniques in the first lab and the development of the heart in the following.

The four techniques the students utilize in this lab are: windowing a chicken egg, explantation of the living embryo into a dish of warmed saline, removal of the beating heart, and isolation of the heart chambers. The first two of these are very straightforward and the students can learn by observing our demonstrations. The latter two techniques, however, require us to demonstrate them while we are looking through a dissecting microscope and the students observe our manipulations. If you have a dissecting microscope with a camera attachment, the students can easily watch the monitor instead of you. Another approach would be to create a video of the procedures (using a camera attached to the microscope) and use this as the teaching tool. Currently, we are planning to produce such a video which we would then make available to any instructor.

Chicken Eggs

As for the stars of the show, the chicken eggs, they can be bought from a local chicken farm or an agricultural school. It is easiest to get them at the correct age on the day that you need them; this way you do not have to worry about incubating them for a long period.

With regard to egg incubation, you can purchase a Styrofoam egg incubator from Carolina Biological Supply (about $44 US). The incubator will keep the eggs warm throughout the lab period and can also be used to raise the embryos over a long period of time, if needed. An automatic egg turner from Carolina Biological Supply (about $49 US) will simulate the natural egg rotation necessary for proper development.

Normally, about five eggs per student or student pair will suffice. It is recommended that you order at least 25% more eggs than you plan to use since many are infertile or abnormal.

Solutions

There are only two or three solutions you need to make and the most crucial is the chick saline (a.k.a. Howard's Ringers). The caffeine and alcohol stock solutions are made with chick saline.

Recipe for Chick Saline

Materials:
  1. 7.20 grams NaCl
  2. 0.23 grams CaCl2 H2O
  3. 0.37 grams KCl
  4. 1 liter of distilled water
Combine all the above ingredients and adjust the pH to 7.2 - 7.3.

Recipe for Alcohol Stock Solution

Materials for 25% Gin Stock Solution:
  1. 200 ml of chick saline
  2. 50 ml of pure gin (94.6 proof or 47.3%)
Combine the above ingredients.
100% Ethanol may be substituted.
Dilutions should be made with chick saline.

Temperature

Have you ever heard the one about the three most important factors in choosing a house to buy? For this lab, the three most important factors are: temperature, temperature, and temperature.

In order for the heart rate to stay normal and constant (about 100 beats per minute), the embryos must be maintained at 37 C. This can be easily controlled by having a steady supply of warm chick saline. To ensure that it is warm enough, we keep about six 200 ml bottles of the chick saline in a 45 C water bath. This temperature is a little higher than the normal body temperature (37 C) but since the bottles are continually being removed from the water bath, it probably all evens out. We encourage the students to use the saline and then promptly return it to the water bath. They probably shouldn't keep a bottle on their bench for more than five minutes.

A test tube rack should also be placed in the water bath. It will be used to keep the test tubes of the students' drugs warm; they can store or remove their tubes as they perform their experiments.

gooseneck lamp The other important way in which to keep an embryo at optimal temperature is to place a lit gooseneck lamp close to, if not over, the embryo. Last year, one of us even discovered that the wattage of the bulb can make a huge difference; 60 watts was too low and 100 watts seemed ideal.

The only drawback to this method is the in vivo windowed embryos will quickly dry out under the hot lamp unless the students periodically add drops of chick saline. Fortunately, the lab doesn't require the embryos to stay in this state for very long and most of the work is done in vitro in a dish of warm chick saline. Unfortunately, the heat created by the lamp could burn the students hands. We have not found this to be a problem, nonetheless students should be warned to be extra careful.

Tools

The most challenging procedure is removal of the heart from the live explanted embryo. With the correct tools and some simple rules, anyone can do it. The two tools that we have used for heart explantation are iris microdissecting scissors from Carolina Biological Supply (about $20 US a piece) and microknives (about $0.02 US a piece). Both have proven to be effective but some students prefer one over the other. If you prefer the microknives, you are welcome to take one of them home with you when you leave. If you decide to make them yourself, you can follow Tyler's (1994) directions which are similar to the following:

Directions For Making Microknives (modified from Tyler, 1994)

Materials:

  1. fresh single edge razor blades
  2. wooden applicator sticks (about 2 mm diameter and 100-150 mm long)
  3. super glue
  4. pair of pliers or tin snips
Break the razor blade into small fragments with the pliers or tin snips, making sure that each fragment has the cutting edge on it. Avoid damaging this edge. The cutting edge should be up to 2 mm long and the fragment should be between 5 and 10 mm long.

Soak one end of the wooden applicator stick in water for about 5 minutes; this will soften the wood so that the tip can be split.

Make a 5 mm split down the center of the moistened end of the stick with a fresh razor blade. Do not splay out the two sides of the split; just create a crack in the wood large enough for the razor blade fragment to fit into.

Carefully insert the rough edge of the razor blade fragment into the split. You can't use the pliers to do this because they will damage the cutting edge. Thus, you must use your fingers - be careful! The cutting edge of the blade should not be embedded in the wood at all and should be oriented about 45 degrees to the axis of the stick. You should be able to comfortably hold the stick like a pencil and have the razor's edge almost flat with a desk surface. Let the wood completely dry.

Paint the split edges with super glue so the razor blade fragment will be held in place.

Store the microknives so their edges are protected. One method is to stick Styrofoam peanuts on the razor edge.

Note: They can only be used for one lab period - they dull quickly.

Another useful tool to have for emergencies is a small plastic ice cream spoon that has holes in it. These are great for rescuing an embryo that has sunk down into the yolk during explantation. Like the microknives, these are cheap (and often free) to make. Most ice cream stores will willingly donate a few in the name of science.

Directions For Making an Embryo Spoon (Tyler, 1994)

Materials:

  1. 1 small, sample size, plastic (not wooden) ice cream spoon
  2. insect or dissecting pin
  3. Bunsen burner or alcohol flame
  4. 220 and 400 grit sandpaper
Warm the pin over the flame and use it to melt a small hole in the back of the end of the spoon.

Repeat this process so there are about a dozen holes.

Use the sandpaper to remove the sharp plastic edges.

One of the techniques that is used to explant chicken embryos requires filter paper donuts.

Directions For Making Filter Paper Donuts

Materials:

  1. Whatman #1 filter paper (3.2 cm)
  2. fine scissors
Fold paper in half, cut out a semi-circle.

Cut out a smaller semi-circle in center of larger one.

The end result should be an oval shaped donut.

paper donut

Techniques for Heart Removal

The prospect of surgically manipulating such a tiny embryo usually strikes great doubt in the students - "There is no way that I can cut out that heart AND keep it beating!" Fortunately, it is not as hard as it seems because the chicken embryo is a very resilient subject; you can put considerable tension on its tissues and it will not be destroyed.

There are a couple of tips that make this procedure easier. First, you need the correct lighting; each student should spend some time changing the angle of the light to achieve maximum clarity under their microscope. Secondly, the fact that the heart is beating makes it very easy to keep track of, even when it is tangled up with the extraembryonic membranes and vitelline envelop. This, again, reveals why it is so important to keep the in vitro embryo's heart beating at a normally high rate.

There are two general methods for removing the heart using the iris microdissecting scissors or microknife. One is to simply cut the heart out of the embryo's chest without removing any other tissues beforehand. The other method is to remove the head and then the trunk below the heart; you are left with the spine right behind the heart and the heart itself. Then, you can hold on to the spine and cut out the heart.

In either case, you are usually left with a heart that needs to be separated from the various membranes. This is where the students need some patience. They need to unwrap the membranes from around the heart using forceps (two pair helps the process), and then use their cutting tool to cut away these membranes. It is OK to stretch the tissues, the embryos can handle it.

Recommended Atlas

Mathews, W. W., and G. C. Schoenwolf. 1998. Atlas of descriptive embryology. ..... Fifth edition. Prentice-Hall, Upper Saddle River, New Jersey, 266 pages. ..... [ISBN 0-13-593740-X] [book]

This page was last modified May 27, 1999.       Send questions or comments to jxm57@psu.edu

Copyright© 1999 Dr. Jacqueline McLaughlin and Dr. Elizabeth R. McCain   All Rights Reserved
This material may not be reproduced without expressed written permission from the authors.

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