Other
Dissection.
Wednesday, September 17th, 2008
Today was, as expected, a very busy day. The band went to Holy Cross. The football team won. It rained. A lot. It was fun. Aside from that, I wrote a lab report. Sorry, no image provided.
Chick Embryo Lab
I.Introduction
In this experiment, our goal is to successfully remove the embryo of a chick from an egg, isolate it, and take a picture of it. We will then stain it and take another picture. After, we will appropriately label the picture and attempt to accurately determine its age. We will do so using a plethora of tools, including scissors, tweezers, needles, spoons, plastic bowls, glass petri dishes, avian ringer, and microscopes. The image itself will be taken with a standard video camera using Motic image software. In order to determine the correct age of the embryo, we will refer to various sources provided to us.
II.Methods
During the experiment, we employed a specific technique in order to get through the hard shell that we named the “chisel-pick method.” The chisel pick method, developed in order to effectively remove the shell in a clean fashion and without causing harm to the embryo, involves using the needle and the handle-end of a pair of scissors. We slowly “chiseled” at the egg until we had achieved perforation and created a very small hole. This kept the process very clean and controlled. Then, using the scissors, we cut a circle out of the shell, giving us a “window” into the egg. To our luck, we almost immediately spotted the embryo as we were pouring the contents out of the window and into the bowl. If we had not experienced such great luck in locating the embryo, the process would have been much more tedious—having to sift through the yolk to find the embryo. Upon first glance, our embryo was relatively large—perhaps a quarter of an inch in diameter.
Our next step was to isolate the embryo from the other contents of the egg. We achieved this by dumping all contents into one bowl, spooning the embryo out and placing it in a second container (catching as little liquid as possible), and repeating the process until almost no liquid remained. When this was achieved, we transferred our embryo to the glass petri dish and added avian ringer. The avian ringer is a clear, water-like substance and it is used to prevent the embryo from drying out. We made sure to only use an adequate, but not excessive amount of ringer. The reason for this will be described later.
We then moved the embryo to the microscope, where we used tweezers and the needle to remove the membrane surrounding it. This was done to make staining more effective, and proved to be the most difficult step—comparable to trying to remove skin—which took at least fifteen minutes. We encountered an issue when the membrane would not detach from the embryo near the allantois and the wing bud. However, we could not be careless of forceful with our subject, or we may risk puncture. Instead we were only more careful and gentle—this time using scissors with which we cut the membrane as close to the actual body as we could. One of the great observations during this step was only seen under the microscope—the embryo’s heart was still beating, and its body twitching somewhat in response to each beat. We then brought the embryo over to the camera.
The process of taking the picture was a little difficult, as the avian ringer caused a great deal of reflection from the lights. This also caused the contrast of the whole picture to change, and some of the embryo’s characteristics to be more hidden. The video camera used was operated using Motic Imaging Software. One of the two lights was placed horizontal to the bottom of the dish, illuminating the bottom portion of the embryo, and the other came from above, illuminating the top.
After the original pre-stained picture was taken, it was necessary to stain the embryo and take another picture. Before the stain, toluidine-blue, was added, it was necessary to remove as much avian ringer as possible. This was done to allow for less dilution during the staining process. Earlier we had anticipated this, however, and added only as much ringer as was necessary. Using a paper towel, we soaked up as much excess ringer as possible, and added two drops of stain directly above the embryo. We then allowed the stain to soak and settle for about ten minutes (per instructions).
In the post-staining picture, taken by me personally, I went to great lengths to eliminate as much light reflection as possible, and to allow for the greatest definition of each part of the embryo. For this reason, I only used one of the lights, and directed it at about a sixty-five degree angle, about five inches from the embryo. The dimmer lighting allowed for a much better accentuation of the important features, and it was ultimately this image that was kept.
III.Results (refer to image)
The final product was a very clean, clear image of the embryo (though some of that sharpness was compromised in the printing). The eight parts labelled were the midbrain, eye, allantois, leg bud, wing bud, amnion, heart, and ear. The leg bud, the little lump near the “tail end” of the embryo, will develop into the lower portion of the chick. In the printed image, the wing bud and the allantois are a bit deceiving, but it helps to remember that the image is representative of a three-dimensional model. The wing bud, therefore, is foremost (close to the side of the embryo in this picture), and the allantois is more near the center. The amnion surrounds the embryo, but there is much less visible in the picture due to the staining. It appears almost as a “film” in this image. The heart, seen here as a small red speck, was actually nearly thrice the size represented, and still beating at the time the picture was taken. The ear can be seen vaguely as a darker portion between the eye and midbrain. The “spine”, though not labelled, can be seen along the bottom of the embryo, reaching around to the “tail”.
IV.Discussion
In comparing the image taken to the images of the various stages of embryo development (as listed and described in A Series of Normal Stages in the Development of a Chick Embryo by Viktor Hamburger and Howard L. Hamilton), my conclusion was that the embryo was currently in stage nineteen of development.
It’s amazing how quickly the embryo can grow from just one cell into a full-fledged fledgling. Just two days earlier, the embryo would have been in about the sixth stage, and would have looked something like a near-microscopic worm with no discernible features. Two days later, however, and you have an alienesque embryo (stage 26) that is beginning to take the shape of a chick. Its limb buds are now “jointed” appendages; its allantois has grown variably. Furthermore, the blood vessels, ear, and brain, all of which have not even begun to develop past a “spot” on our subject embryo, have developed into definite structures. The eye has shrunk considerably in relation to the rest of the body, and the visceral arches have developed into many complex parts. Although the embryo grows considerably in size, it is using the nutrients inside the egg to grow. Therefore, as the embryo grows, the contents of the egg lessen. It is for this reason that I believe that no changes in weight will occur.
My conclusion is based mainly on comparison, noting a few very important attributes present in our embryo. The similarities are especially clear in the tail. The leg bud, wing bud, allantois, and ear are of the same proportion to the body as in the stage 19 image provided in the text. In our embryo, however, the eye appears to be much larger, but I believe this is due to the staining method used in the text. A careful look reveals an almost three-dimensional circle surrounding the dark spot which is the eye. I believe that this is also part of the eye, and simply did not appear so evident in a non-color picture. The midbrains of each are equal in proportion, but there appears to be a cleft behind that of the one in the text. This I again attribute to the difference in imaging technique.
The positive side of photographic imaging is that you get a very clear (hopefully) image. It is much easier to take a picture than to draw a sketch. However, one rarely gets a sample that falls perfectly into one category or one stage of a process, especially in development. For this reason, sketches may be preferred.
One of the most important factors to note is the variance that can occur in development. As Hamilton and Hamburger admitted, “The shortcomings of a classification based on chronological age are obvious to every worker in this field, for enormous variations may occur in embryos even though all eggs in a setting are placed in the incubator at the same time.” (1) They go on to say that many environmental factors can affect the exact physical appearance of the embryo. It is with this in mind that I discuss the final deviance between the text and our embryo: the tail. In the image, the tail has remained straight (and does so until two stages later). However, our embryo’s tail has already taken on a curve. The source of this could come from the difference in incubation of the eggs used in the Hamburger and Hamilton experiments and those used in ours. Unfortunately, the specifics of incubation are unknown to me. For further analysis, I have included the criteria for assessing the current stage of the embryo, as described in the Hamburger and Hamilton experiments (figure 1.2). My final conclusion, therefore, is that our embryo was in stage nineteen of development, and was 68-72 hours old at the time of imaging.
Works Cited
1.Hamburger, Viktor, and Howard L. Hamilton. “A Series of Normal Stages in the Development of the Chick Embryo.” Journal of Morphology 88 (1956): 231-259.