Chapter 10: Chromosomes - Endnotes
Occasionally, lipids are found within nuclei. It is usually associated
with degeneration of the nuclear structure, or with neoplastic alterations
of the nucleus.
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.
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.
A sample may be taken and fixed for light and electron microscope
observation at a later time.
Bonner et al.
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.
U.K Laemmli. 1970. "Cleavage of Structural Proteins during the Assembly
of the Head of Bacteriophage T4" Nature Vol. 227. pp.
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
This procedure can be handled as a Level I exercise by purchasing
photographs of chromosme spreads (Carolina Biological) and skipping to step
Vacutainer Systems, Order #6481. Becton Dickinson, Rutherford, NJ 07070
If an inverted phase microscope is available, you can
check that the cultures are in a log phase of growth before
adding the colcemid.
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
If only morphology is to be studied, skip steps a-e and begin with step
f, but increase the staining to 10-20 minutes.
Summarized from H. Macgregor and J. Varley. Working with Animal
Chromosomes. John Wiley & Sons, Chichester, 1983. pp. 196-201.
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Cell Biology Laboratory Manual
Dr. William H. Heidcamp, Biology Department, Gustavus Adolphus College,
St. Peter, MN 56082 -- email@example.com