• This activity has two different approaches to observing chick embryogenesis. The first part is a modification of Dunn's embryological research in which a chick is grown "shell-less" or outside of its egg shell. A growth chamber is constructed using a 4" PVC plastic pipe, HandiWrap, and a sterile petri dish as a cover for student observation of chick embryogenesis. Part two is an extended activity in which students study the effects of various "recreational" and/or "over-the-counter drugs," e.g., caffeine, aspirin, ethanol, nicotine, etc., on embryonic development This is done by injecting small concentrations of these drugs into 72 hour chick eggs.

Many of the students we teachers come in contact with come from diverse backgrounds and experiences. Students are beginning to develop their own points of view that may differ from their parents, teachers, and other adults. This is a time of experimentation in their lives, e.g., alcohol, smoking, and "recreational drugs." Many believe that they are "indestructible" and that they have a full life ahead of them. All to often tragedy strikes and they face the reality that we all are truly mortal. Also, a number of students are becoming sexually active and are unaware of what consequences their actions have upon future generations. The goal of these of activities are to help form, modify, and possibly change student behavior.

In the first part of the activity, we are using the chick embryo as a model of human development. In the first 24 hours of chick development several critical systems "come on line." These critical systems include the nervous, circulatory, gastrointestinal, and urogential. In humans the same systems develop in the first six to eight weeks. It is these systems, especially brain, spinal cord, and heart development, that we want students to focus upon.

In the second part of the activity, we are using the chick embryo to demonstrate that even "over-the-counter" and/ or "recreational" drugs have an effect on the development of critical systems. The objective is to reinforce the concept of taking "care of your baby before it is born."

Some students may strongly object to the use of vertebrate animals for experimentation. See the end of this activity for further information on Bioethics

• General to Advanced Biology students
• Grade Level: 10⁺

Required of students

• Readings and research on chick and human embryology. Familiarity with early development of nervous, circulatory, gastrointestinal, and urogential systems.

1 1/2 to 2 hours are needed to construct the "shell-less" chambers. Get materials from a local hardware store. Construct enough chambers for small groups of two (or four) students.

Make arrangements with a local hatchery for at least one fertilized egg per group of students; plus a couple of extra per class (accidents happen and sometimes eggs are infertile). If  collecting eggs from several sources, e.g., student's home, etc., have them store the eggs in refrigerator, about 18 degrees C,  immediately upon collecting from the hens until you have sufficient numbers. The eggs can remain in the refrigerator for up to one to two weeks. On the day of the experiment let all the eggs warm to room temperature at the same time.

Fertile chick eggs can be ordered from Carolina Biological Supply

Equilibrate the incubator(s) to 37.5 - 38.5 degrees C for at least one day.  Make sure a large bowl of water is in the incubator.  The water will prevent the eggs from dehydration which is one of the causes of death.

Rotate the eggs 180 degrees twice a day during incubation.  This rotates the embryo within the shell and prevents adhesion of embryonic membranes to the inside of the shell.  Furthermore, rotating the eggs stimulates extra-embryonic membranes and promotes fluid redistribution among various parts of the egg during incubation.

Class time needed


Initial set-up will take two 45 minute or one 90 minute period.
Follow-up sessions of 5 to 10 minutes per day for recording observations for the duration of the activity.

• Hazards/Precautions for Shell-less environment
• Chick mortality is high - 50% by 18.5 days, few hatch in 21 days
• Egg needs to be rotated - try rocking it at 30 degree angles 2 or 3 times daily
• 1% CO₂ environment - use Alkaseltzer tablet(s) in water w/in enclosure
• 80 % humidity - put apparatus w/in enclosure w/ water
• Ca⁺⁺ replacement, esp. by day 14 - 18 -- can use shell fragments  w/ intact shell membrane
• Infections: use 70% Ethanol to "sterilize" HandiWrap and egg shell, before cracking open. Sterilize chamber and petri plate in pressure canner or autoclave.
• Chick embryos before 72 hours have a low survival rate (trade-off for early development observations
• Suggested Classes of 1% Stock Solutions for Testing Common Drugs:  Caffeine, Nicotine, Alcohol, Aspirin (order from Carolina Biological Supply)

1.  Chick Embryo Ringer’s Solution

Add the following to make up to 1 Liter in glass using distilled water:
 

7.20  g  NaCl	0.37 g   KCl
0.17 g   CaCl	0.02 g   Phenol Red Indicator

(Source:  Mary S. Tyler, Developmental Biology:  A Guide For Experimental Study, p. ?)
 
2. Tyrode Solution
 

8.0 g  NaCl	0.2 g  KCl
0.2 g  CaCl2	0.1 g  MgCl2 · 6H20
0.05 g  NaHP04 · 2H20	1.0 g NaHC03
1.0 g Glucose	1.0 L H20

Source:  M.V Tyrode (1910) Arch. Int. Phar., 20:205-223.
 
3. Hanks Solution
 

8.0 g NaCl	0.4 g KCl
0.14 g CaCl2	0.2 g MgSO4 7H20
0.06 g KH P04 2H20	0.35 g NaHC03
1.0 g Glucose	0.02 g Phenol Red Indicator
1 L of Distilled H20

Source:  J.H Hanks and R.E Wallace (1949)  Proc. Soc. Exp. Biol. Med., 71:196-200.

• Yolk Sac
• Albumen
• Allantois
• Chalaza
• Blastoderm
• Somites
• Ectoderm
• Mesoderm
• Endoderm
• Chorion
• Vitelline membrane
• Amnion
• Primitive Streak
• Neural Groove
• Hensen's node

Experiment #1:

Observations of Early Embryogenesis in Chicks in a "Shell-less" Environment (modified from "Chicken Little" - Kristal Watts, Sam Barlow H.S. Gresham, OR, USA and based upon research by Bruce E. Dunn)

Materials:
 

Incubator	Sterile petri dish TOP
Shell-less chamber w/ clamp	70% Alcohol swab
6" square of plastic HandiWrap	fertilized egg
Nile Blue Sulfate	Stain Neutral Red Stain

Construction of PVC "Shell-less" Chick Embryo Chamber

Setting up the chamber:

1. Place the HandiWrap square into the shell-less chamber. Make a depression with the center so that the bottom is just off of the table surface.

2. Secure the HandiWrap with clamp below the top rim by ½" to leave room for the petri dish TOP.

3. Swab plastic wrap with 70% alcohol and let air dry completely.

1. Working in small groups get one fertilized chicken egg. Crack the shell with a sharp blow on the table’s edge. Put the contents of the egg into the depression by separating the two halves of the shell much as one would place an egg into a frying pan for "sunny-side-up" eggs. Under ordinary circumstances, the yolk will orient by gravity, with the embryonic disc uppermost. The embryo is now ready to be observed.
 
Note: since cooling will adversely affect development, keep the embryo out of the incubator for no longer than necessary.  If observations exceeds 2 minutes position a lamp above the chamber. The heat will keep the embryo warm until you return it to the incubator.  Keep the chamber covered with the petri dish to avoid contamination.
 
2. Add a drop or two of 0.01% Neutral Red or Nile Blue Sulfate directly over the blastoderm.  Gradually during a period of five to ten minutes, the embryonic cells will concentrate the dye.  In so doing the thicker parts of the embryo will become darker than the thinner cellular areas.  Hensen's node, the primitive streak, neural folds, brain vesicles, etc., will gradually become visible.

3. Observe daily for five to seven days. Write and illustrate a journal of your observations. Identify and label the following structures: blastoderm, yolk sac, allantois, chalaza, somites, albumen, Hensen’s node, primitive streak, neural folds and brain vesicles.
 

---

The Effects of "Recreational Drugs" on the Development of Chick Embryogenesis

Introduction:

This experiment involves the injection of living eggs with various "recreational drugs" including caffeine, alcohol, nicotine, and aspirin to see their possible effect on embryonic development.  In fact, this is one of the standard industrial tests for the biological activity and permissible dosage of pharmaceuticals before they are released for human use.
 
Materials:
 

Incubator	4 -- 72-hour eggs
Lamp	1 bottle of Ringer's Solution
Candling Box	1 bottle of 70% alcohol
27 gauge, .5 " Tuberculin/Insulin Syringe	Adhesive Plastic Tape
Scissors	Black Felt Pen
Stock Solutions of either Caffeine, Alcohol, Nicotine or Aspirin

 Procedures:

1. Select four 72-hour eggs from the incubator. Candle each egg before opening it.
 How to Assemble the Candling Box

2.  Swab each egg with 70% alcohol to limit bacterial contamination. Allow the egg to remain in a horizontal position for a minute or two to dry and allow the embryo to rotate to the top.

3.  Sterilize the scissors blades by dipping them in 70 % alcohol and pass it through an open flame.

4.  Hold the egg with one hand and hold one point of the scissors near its tip in the other.  With a twisting movement, drill a small hole through the blunt end of the egg.  The hole should be large enough to admit a hypodermic needle.

5.  Place the egg in a egg carton with the hole on top and (put a drop of 70% alcohol on the hole to keep it sterile while) preparing the remaining eggs and solutions for injection.

6. Using a felt tip marking pen, label each egg A, B, C, or D. This indicates what concentration of your assigned drug is in each egg. Egg "D" is the control.

7. Your group will do a serial dilution of your assigned drug. Using your syringe, make a dilution of 1 unit of your drug to 9 units of Ringer's solution into a sterile container—this is 0.1%. Repeat the dilution by taking the 0.1% solution and make another 1:9 dilution—this is 0.01%.   Repeat the series for solutions "B" and "C."

8. Using a sterile syringe draw out 0.5 ml of a solution and inject into the egg. The needle should just pierce the vitelline membrane.
 
9.  Seal the hole in the shell with a piece of adhesive tape.

10. Incubate the eggs for 5 to 7 days at 37.5 to 38 degrees C--- and then open to examine the embryo.  Evaluate the experimental results by recording them on the white board for class results.

11.  Record the chemical name of the compound tested and evaluate the effects at all dilutions on a 5-point scale as follows:
 
++  Development strongly enhanced as compared with control egg D

+   Development slightly enhanced as compared with control egg D

0   No detectable effect

-   Development slightly impaired as compared with control egg D

--  Development stopped or embryo killed at time of injection

Experiment #2:  Students will compare and contrast normal embryological development to eggs that have been exposed to different concentrations of "recreational drugs." Comparisons will be observed, described, sketched, and measured in their journals.

• Experiment #2 could be done in the "shell-less" environment and the effects of the "recreational" drugs could be observed at all stages of early development. The risk is chick higher mortality.
• Consider using Zebrafish embryos  http://zfish.uoregon.edu/

1.  Animal Models in Biomedical Research:
     Poultry http://netvet.wustl.edu/org/awic/BIB/srb92-17.htm

2. Deeming, C. and  Ferguson, M.  1991, "Egg incubation:  its effects on  embryonic
     development in birds and reptiles", Cambridge University Press,  New York.

3.  Dunn, Bruce E., 1991 "Egg Incubation: Its effects on embryonic development in birds and
     reptiles," Demming and Ferguson. Cambridge University Press, Cambridge.

4.  Hanks J.H., and  Wallace R.E.,  (1949) Proc. Soc. Exp. Biol. Med., 71:196-200.

5.  Marthy H.J. 1990, "Experimental Embryology in Aquatic Plants and Animals",  Plenum
     Press,  New York.

6.  Rugh, R., 1962,  "Experimental Embryology - Techniques and Procedures"   Burgess
     Publishing Company,  Minneapolis.

7.  Tyrode M.V., (1910) Arch. Int. Phar., 20:205-223.

8.  Zagris, N., Duprat, A.M., and Durston, A., 1995 "Organization of the Early Vertebrate
     Embryo,"  Plenum Press, New York.

• Some people have moral and ethical objections to the use of vertebrate models for biological research. This is an issue that you as the teacher have to be proactive and comfortable addressing. You will also need the support of your administrator(s) and parents. You will also need to not only provide justification for this lab; but also alternatives in which students can receive similar information.
• For further information on bioethics go to the 1992 Woodrow Wilson Institute Earth Ethics home page or Animal Use Case Studies.
• For further information on the use of poultry as biomedical models go to the Animal Models in Biomedical Research

• Cut 3" off of a 3" diameter PVC plastic pipe for your stand and cut another 1" off for the clamp. Split the 1" piece open into a "C" shape.


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A candling box can be prepared most simply by cutting an oval hole two-thirds the diameter of an egg in the top of a paper box of sufficient size to hold an ordinary desk lamp inside.  The lamp should have the shade removed or turned so light can shine up.  Since the effectiveness of the candling box is dependent upon having a maximum amount of light pass through the egg against a black background, the hole in the box should be lined with soft black felt against which the egg can be pressed snugly.  The box should be placed in a shaded corner or dark room for maximum effective use. Look for the shadow of the embryo, the vitelline veins and the sinus terminalis.  Use only candled eggs with a proven embryo in them.
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Each dilution is to be 1/10^th or 1.0 x 10^-1 less concentrated than the previous. For students unfamiliar with this process it is best to let them practice with well plates or small test tubes with food coloring. The following is an activity that demonstrates this:

1. Using an eye dropper place one drop of concentrated food coloring into the first well. Add nine drops of water. This gives you a 1 to 10 or 10^-1 dilution from the original.
2. Take one drop of the new dilution and place it into the next well and add nine drops of water. This gives you a 1 to 100 or 10^-2 dilution from the original.
3. Repeat step 2 until you get the desired final dilution.

Pricing was based on 1997 Science/ Math Catalogue No. 66. Phone: 1 (800) 334-5551
 

Part No.	Item	Unit Price	Additional Info.
K3-l1726	Fertile chick eggs	$16.70/dozen	order two weeks before use; highest fertility -- Feb.-June p.74
K3-85-1570	Caffeine	$20.95/ 100 gm	p. 629
K3-87-7270	Nicotine	$63.25/ 100 ml	p. 636
K3-86-1261	70% Ethanol	$6.55/ 500 ml	p. 631
K3-88-7070	Salicylic Acid	$15.96/ 500 gm	p. 638
K3-87-6853	1 % Neutral Red	$8.65/ 120 ml	p. 635
K3-87-7431	Nile Blue Sulfate	$13.65/ 25 ml	p. 636
K3-69-7765	1cc syringe	$7.75/ 25 pcs	p. 1064
K3-69-7810	27 G, 1/2" needles	$13.25/ 50 pcs	p. 1064

note: may use substitutes from local pharmacies or other local sources, e.g., Caffeine = No-Doze, Nicotine = soaked cigarettes, Salicylic Acid = aspirin
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Jim Hashimoto has been teaching for 6 years at Franklin High School.  He received his B.S. in Biology from Western Oregon State College in 1991 and his currently working on his M.S.T at Portland State University.  His areas of scientific interest include animal physiology, aquatic invertebrates and natural resource conservation.  He is a member of  NSTA, OSTA, NABT, and The Nature Conservancy.  He has been involved in several research and professional development activities:  ISMTE Project, 1991-1993; DOE TRAC program, Richland, WA in 1993; Green City Data Project, 1994; Access Excellence Program, San Francisco, CA in 1995 and is currently participating in the Woodrow Wilson National Fellowship Program in Princeton, N.J.    For further information contact Jim at  jhash@teleport.com

John S. Colvin teaches high school biology and integrated biology to sophomores at Sam Barlow High School in Gresham, OR. Lately he has been involved in the Partners in Science Program, Research Corp. Tucson, AZ doing medical research on sperm motility at the Oregon Regional Primate Research Center. For further information contact John at jcolvin@gresham.k12.or.us
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