LAB 2

Objectives:

A. To introduce several types of scientific methodology:

dissection,

microscopy,

image analysis,

data recording,

statistics, and

pH recording.

B. To explore properties of a large cell:

anatomy

physiology

pH gradients, and

solvent / solute movement.

C. To establish parameters of report writing.

General Procedures:

A. Establish the general parameters of chicken egg anatomy.

B. Using a microscope.

C. Perform microscopy of the hard boiled egg.

D. Compare a deshelled chicken egg with a shelled egg.

E. Modeling osmosis with a deshelled egg.

F. Prepare a brief report of the lab experience.

References:

Campbell 5^th: Chapter 8 Membrane Structure and Function &emdash; pp 136 &emdash; 144.

Purves 5^th: pp 105-109.

For an advanced look at osmosis and diffusion, try the following WEB site:

<http://cweb.middlebury.edu/cr/labbook>

A. Establish the general parameters of chicken egg anatomy:

Begin this section by considering the following question.

Do shell weight, albumen weight, and yolk weight vary proportionally with the changing weight of the egg?

Please consider the sense of the question using the following analogy.

If both a 300 pound woman and a 150 pound woman gave birth to babies after normal pregnancies, would the 300 pound woman have a 14 pound baby and the 150 pound woman a 7 pound baby?

To view the question more specifically, the mean weight of a chicken egg is 58 grams and the mean weight of a chicken hatchling is about 38 grams. According to Romanoff and Romanoff (page 115) "the hen's egg is roughly six parts albumen, three parts yolk, and one part shell."

Do these ratios hold whether the initial egg weight is 70 grams or 50 grams?

To extend the question to the original analogy: Does the brain of the 300 pound woman weigh twice as much as the brain of the 150 pound woman?

To address this question, you will take a hard boiled chicken egg and consider both its external (gross) and internal (dissected) anatomy. Start the process by selecting an egg and recording the egg weight in the space below and then read for further directions.

%

1) egg weight ___________________ 100 .

2) shell weight (with shell membranes) ___________________ ________

3) albumen weight ___________________ ________

4) yolk weight ___________________ ________

Dissection represents the disassembly of some anatomical entity. Once a biological object is taken or cut apart, it is generally impossible to put it back together. Dissection is far more complex a process than most people realize. Positional geometry can often reveal a great deal about how something works or how it is created. The chicken egg can be used to reveal elements of critical dissection.

Begin the dissection by examining the gross anatomy of the hard boiled egg. The egg has a blunt end and a sharp end and thus can be said to exhibit polarity. This anatomical polarity is functionally important.

• The blunt end has more surface area. An internal air space, which enlarges during incubation, is found beneath the blunt end of the shell.

• This geometry also makes the shell stronger and less likely to break.

Romanoff and Romanoff (page 105) provide the following measurements for the "ideal" chicken egg:

long circumference, 15.6 cm; _________________________

short circumference, 13.5 cm; _________________________

surface area, 68 cm2 _________________________

length, 5.7 cm; and _________________________

width, 4.2 cm. _________________________

 

Validate these measurements by measuring your own egg and recording the values in the space above.

Begin the dissection by invading the egg from the blunt end and take advantage of the air space for entering the egg. Peel the shell and associated underlying shell membranes with the intention of keeping all the parts and pieces. As you take the shell apart, consider this question: Is it possible that the shell will exhibit anatomical differences reflecting the general polarity of the egg? To restate: are there regional variations in the shell anatomy that accommodate the physiology of development?

Carefully cut the cooked albumen to reveal the internal yolk sphere. Again, perform the dissection is such a way that positional information is not lost. Consider what is top or bottom and what is right or left. (Not an easy task.)

Record the weight for the egg components listed earlier and calculate the per cent that each provides to the total egg weight.

Is your egg "typical"? ________________________________________________________

Stop for just a moment…. What does typcial mean? How will you define the term? Should your egg values be within 10% of the accepted? What should the class accept as typical?

Did you obtain the 6 to 3 to 1 ratio that Romanoff and Romanoff suggest? _______________

As you may know, eggs are graded and sized by weight. For the labs this week we obtained eggs of different sizes. The weight ranges for the weight grading (medium, large, extra large, and jumbo) are listed on the board. By taking collective data from all the labs, we may be able to answer the "300 pound woman" question: Are the egg components proportional through the weight ranges? In the space below record your observations and comments.

B. Microscopy of the Hard Boiled Egg.

You are going to investigate the microscopic structure of the shell, albumen, and yolk. If done correctly something of the diurnal nature of egg manufacture and the exchange of gas should become apparent. We will begin with the albumen compartment.

Albumen Compartment:

Romanoff and Romanoff (pages 137-139 and 319-320) report that in life, the albumen compartment has four components arranged as concentric layers: chalaziferous (3%), outer liquid (23%), middle dense (57%), and inner liquid (17%). The viscosity of the layers is regulated by the number of fibers in the layer. In eggs laid during hot weather, the middle dense layer is diminished. The mean value of water in albumen is 88% and 10.5% of the substance is protein.

You are going to make a "wet mount" of the albumen. It is obvious that the boiling of the egg transformed the protein of the albumen region. Obtain a microscope slide and place several drops of water on the slide. Place a piece of "cooked" albumen no larger than a drop of water into the several drops of water and macerate with a toothpick the albumen. Place a coverslip over the wet preparation and observe with a compound microscope. Contrast can be given the mount by bleeding a drop of methylene blue stain at the edge of the coverslip.

Record your observations, by word and / or drawing. You may wish to confirm whether there are different anatomical regions in the albumen (although the cooking will make this difficult to do).

Yolk Compartment:

Romanoff and Romanoff (page 315-318 and 132-134) report that the yolk has two distinct parts: white yolk (5%) and yellow yolk (95%). The white yolk is 86% water, 5% protein, and 4% lipid. The yellow yolk is 45% water, 15% protein, and 36% lipid. The white yolk is slowly deposited during the late of night. The yellow yolk is added to the developing ovum during the active periods of the day. Although an oocyte may wait several years before initiating development, the yolk is produced in about a week and consequently there are about six layers of white yolk interspersed between the much larger yellow yolk layers. Physiological phenomena which demonstrate alternating periods of activity that correspond to light and dark daily cycles are said to be diurnal.

Digression: Underfed hens have more white yolk in their eggs. The yellow yolk is a function of an active, eating chicken. During the day the hen eats and makes yellow yolk and by night roosts and makes a little white yolk. In humans, do periods of activity influence how food moves through our body as it does in a hen?

Obtain a microscope slide and label one area "W" and label another area "Y". Turn your attention to the yolk sphere for which you recorded the "polarity". Break the yolk sphere open along its polar axis (blunt to sharp end) using a single edge razor blade or a scalpel. Examine the diagram posted in the lab for help.

At the center of the yolk sphere the latebra (white yolk) should be detected. If no other white yolk can be detected, use this material as an example of white yolk. Take a small amount of latebra and of yellow yolk and mix each with several water drops. Coverslip each and examine through the compound microscope. Bleed some methylene blue for contrast, if necessary. Yellow yolk has globules that are from 25 to 150 microns in diameter. White yolk has spherules that are 4 to 75 microns in diameter.

Can you confirm the dimorphic appearance of yolk? ____________________________

Shell:

The shell is quite porous. There are over 10,000 holes over the surface. The holes allow air to transfer in and out of the shell. These holes also allow water to escape from the egg (6 or more grams of water loss during incubation). Although these holes allow air and water to move through, the contents of the egg stay sterile because of the double shell membranes.

Once again obtain a microscope slide. Place one piece of shell with the outer side facing up and place another piece of shell with the inner side facing up. You may have to displace the shell membranes in order to study the inner surface of the shell. Don't overlook the relationship of the membranes to the shell. Please characterize each surface and speculate why the holes through the shell look different depending which side one examines.

C. Comparing a deshelled chicken egg with a shelled egg:

Remember that the yolk compartment represents a single very large cell. The yolk membrane is the plasmalemma of that cell. The albumen is an accessory layer on the outside of the cell. The egg shell and the two shell membranes then wrap the two compartments together: the yolky egg cell and the accessory layer albumen. The analogy would be an M&M peanut chocolate candy. The peanut is the yolk; the chocolate is the albumen; and the candy coating is the egg shell and its membranes. Each compartment (the yolk and albumen) have very distinct and different roles to play in the physiology of the developing chicken embryo. We are going to explore a few of the properties of these compartments.

Take a raw egg and carefully separate the albumen from the yolk. Take the pH of each compartment:

albumen pH = ____________________ yolk pH = ________________________

Now take the pH of a 5% solution of acetic acid (vinegar). __________________

If an egg is soaked in vinegar for two or so days, the calcium shell is dissolved leaving behind intact shell membranes with an exposed albumen and yolk compartment.

Obtain and weigh a deshelled egg. _____________________

How different is the weight between the raw, hard boiled and deshelled egg? What accounts for this weight difference? This is a very important question.

Break open the deshelled egg and measure the pH of the albumen and the yolk.

albumen pH = ___________________ yolk pH = _____________________

Observe the computer which has been set up to monitor the pH change of the albumen compartment during the deshelling process. Characterize the pH transition during deshelling.

Given the pH change and the weight change, what is happening?

D. Modeling Osmosis with a deshelled egg:

Working as an extended class group, one table will follow the deshelled egg response to distilled water; one table the deshelled egg response to 1 Molar Glucose; and one table the deshelled egg response to 2 Molar Glucose.

Each table will acquire three labeled beakers with the respective soaking solution. Number each beaker. Each table will adopt 3 deshelled eggs. Blot dry the eggs on paper toweling and weigh each and record the weight. Place each egg into a labeled beaker of solution and record the time. At 15 minutes intervals, remove the egg, blot dry, record the weight, and return to the soaking solution. Complete the data matrix below.

	0 min.	15 min.	30 min.	45 min. op.	60 min. op.
#1 0.00 M
#2 0.00 M
#3 0.00 M
#1 1.0 M
#2 1.0 M
#3 1.0 M
#1 2.0 M
#2 2.0 M
#3 2.0 M

What can you conclude about the movement of solute / solvent through the deshelled egg? Please refer to Campbell 5th, pages 136 &emdash;137 or Purves 5^th, pages 105 &emdash; 109.

E. Prepare a Brief Report

Your lab instructor will provide you with details of how to prepare a report dealing with your lab experience.

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Please clean up your area. Dispose of the egg parts as directed. Take the time to get the sticky albumen off the counter surfaces. Replace the microscope to the proper place. Place the disposable microscope slides in the proper glass refuse container. Be sure to wash your hands. And when you are ready to leave, please return the lab stool under the bench space.

Thank you for your cooperation.

References:

Romanoff, A.L. and A.J. Romanoff, 1949. The Avian Egg. Wiley: NY. 918 pp.

Carlson, B.M. 1988. Pattern's Foundation of Embryology, 5th edition. McGraw Hill: NY. 750 pp.

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