On February 19, 2001, our group started a project that would possibly yield a karyotype of our own chromosomes. Today, I can say that we were very successful in accomplishing this goal. Although we ended up with successful results, the manner in which we got them was long and tedious, but with some luck and innovation we succeeded.
At first we had trouble getting enough blood. It was something we just had to compensate by enduring a little more pain and a few more pricks of our fingers. We eventually drew enough blood, incubated the sample and prepared the sample for the sides. Our first innovative idea came as a result of some difficulty dropping our sample two feet on to a small slide. We came up with the solution you will find on our web page at http://karyotyping.tripod.com/preparation.html . This solution involved placing a pipette in a test tube clamp attached to a ring stand and raising it above the slide that was fixed in a similar fashion on another ring stand. Due it accuracy, this idea proved to be very successful and saved us a lot of grief with missed dropping.
The next problem we encountered was actually finding a karyotype on the slides we had made stained with giemsa stain. I guess at this point we did not know what to look. After much searching and almost giving up we found some chromosomes. These chromosomes can be viewed in the first chromosome picture on our web site at http://karyotyping.tripod.com/giemsa.html . We knew what to look for at this point and we were able to find many more chromosomes and even a whole karyotype soon after. The problem we had at this point was that the chromosomes seemed to be condensed into one area sometimes overlapping each other. To solve this problem we used a cold side technique. This technique required that we place the prepared sides into a -75°C freezer for 20 minutes. The slides were then taken out and the above procedure was used to drop the sample on to the slide. This time we blew across the slide after the drop landed on the slide. This technique worked well at dispersing the chromosomes in the karyotype. The cold side would be analogous to sticking a wet finger to an ice cube. The cell membranes sticking to the slide are ripped open on impact and are further spread by the blowing process.
Our success at giemsa staining motivated us to move on to more adventurous karyotyping procedures such as T banding. Following the procedure in the book proved to be unsuccessful and modifications would have to be made to our procedure. The slides we made using the procedure from the book were not very clear and distinct. We felt that the trypsin used in this procedure was the variable and this factor would have to be changed. Different techniques were tried in order to achieve a better T banding pattern. The first variation we did was to vary the length of time the T banding stain was on the side. Again, this proved to be unsuccessful. Next we tried placing trypsin directly on the slide for a short length of time and then adding the stain. The trypsin was too concentrated in this procedure and seemed to totally ruin the protein in the chromosomes. The next variation that we tried was to use the trypsin with a PBS solution since the concentration of trypsin was so high in the previous method. Again, the results were not as good as we had hoped, but they were better. The last procedure was the one that worked the best out of all. This technique involved placing the trypsin in the refrigerator for 20 minutes before applying it to the slide. This slowed down the activity of the trypsin and yielded better results. After this success we moved on to procedures that involved fluorescent staining techniques that also yielded great results.
Over all the procedure we used and have outlined in our lab book and web page along with these variations was very successful in yielding a karyotype of our chromosomes. The only problem we had from this point was the adjustment of focus on the camera used. We had tried several techniques to compensate for the camera being out of focus but were only partly successful. The picture can be adjusted only slightly on Adobe Photoshop, but the rest of the adjustment has to be made on the camera itself. Possibly these adjustment can be made in the future by a professional.
My last recommendation would be to remember to shrink the photos obtained in Adobe Photoshop if you are going to use them on a web site. The shrinking should be proportional to the size of the picture you are going to use on the web page.
I would highly recommend using our procedure and variations to reproduce the result we obtained. This was truly a learning experience in many ways.