Chapter 10: Chromosomes - Endnotes

1. Occasionally, lipids are found within nuclei. It is usually associated with degeneration of the nuclear structure, or with neoplastic alterations of the nucleus.

2. This is a standard means of sealing wet mounts. Melted paraffin is highly flammable, however, and should never be melted with an open flame. The author has found that the slides can be sealed just as well with a coat of nail polish.

3. From: L.M.J. Shaw and R.C.C. Huang. "A Description of Two Procedures Which Avoid the Use of Extreme pH Conditions for the Resolution of Components Isolated from Chromatins Prepared from Pig Cerebellar and Pituitary Nuclei" Biochemistry 9:23 1970. pp 4530-4542.

4. A sample may be taken and fixed for light and electron microscope observation at a later time.

5. Bonner et al.

6. K. Weber and M. Osborn. 1969. "The Reliability of Molecular Weight Determinations by Dodecyl Sulfate Polyacrylamide Gel Electrophoresis" J. Biol. Chem. 244:16, 4406-4412.

7. U.K Laemmli. 1970. "Cleavage of Structural Proteins during the Assembly of the Head of Bacteriophage T4" Nature Vol. 227. pp. 680-685.

8. The procedure is based on one performed at the Centersfor Disease Control, Atlanta, GA on human blood samples. Virtually any animal blood can be substituted, or if cells in culture are available, log phase cultures can be substituted and the process begun with step 5. Blood can be drawn at a local clinic directly into heparanized tubes, and should be disposed of properly.

9. This procedure can be handled as a Level I exercise by purchasing photographs of chromosme spreads (Carolina Biological) and skipping to step 19.

10. Vacutainer Systems, Order #6481. Becton Dickinson, Rutherford, NJ 07070

11. If an inverted phase microscope is available, you can check that the cultures are in a log phase of growth before adding the colcemid.

12. The general process involves swelling of the cells in the hypotonic KCl treatment and dropping them like big water balloons onto the slide. The bigger "splash" they make on impact, the better the chromosomes will spread out as the nuclei burst on contact with the slide. Some investigators actually drop the cells from a distance of five feet. The author has found this to be challenging and fun, but of no real advantage. The chilled slide aids in sticking the chromosomes onto the slide. Siliconized pipettes are necessary to prevent the cells from adhering to the walls of the pipette during transfer. This is the most common reason for not obtaining good chromosome preparations.

13. If only morphology is to be studied, skip steps a-e and begin with step f, but increase the staining to 10-20 minutes.

14. Summarized from H. Macgregor and J. Varley. Working with Animal Chromosomes. John Wiley & Sons, Chichester, 1983. pp. 196-201.

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