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Upon completion of this activity, you should be able to:
the structure of human chromosomes with reference to size, centromere
position, and presence or absence of satellites.
a metaphase spread using a human tumor cell line known as HeLa
representative chromosomal abnormalities using standard cytogenetic
a human karyotype and determine the number of chromosomes present,
sex of the individual, and the presence or absence of chromosome abnormalities.
the importance of cytogenetics in the study of cancer and
Each somatic cell
in the human body contains 23 pairs of chromosomes. During interphase
of the cell cycle each of these chromosomes is duplicated and consists of two chromatids
joined by a common centromere (spindle attachment region). During mitosis thechromatids separate and become independent chromosomes which move to opposite ends
of the cell. The subsequent division of the cytoplasm results in the formation of two new
daughter cells each containing the same diploid number of chromosomes as the parent cell.
, a plant alkaloid, has the unique property of arresting cells in metaphase of the
mitotic cycle by interfering with the formation of spindle fibers needed for the separation
of chromosomes during anaphase. Since the chromosomes are most coiled and condensedduring metaphase this stage of mitosis is the most convenient to observe. Once treated
with colchicine the cells are then exposed to a hypotonic solution
that causes the cell to
swell and become more fragile. The cells are then fixed
with an acetic acid-methanol
solution which preserves the existing cell components. The cells are then ready to besplattered onto slides and stained. This is followed by a search for ideal chromosome
spreads for the study of chromosome number and structure.
When these techniques were applied by Tijo and Levan (in 1956) to cultured cells of humanlung embryonic tissue, the human chromosome number was established at 2n = 46. This
observation was soon confirmed by other cytogeneticist. The significance of the number of
chromosomes present in humans became apparent in 1959 when Lejeune and hiscoworkers attributed the disorder Down Syndrome to an abnormal chromosome number
(47, +21). All normal
human cells contain identical numbers and types of chromosomes.
Each chromosome pair contains unique physical attributes which distinguish them from all
the others. The three main criteria used to distinguish and identify individual
chromosomes are: 1) length of the chromosome, 2) position of the centromere, and 3)staining/banding pattern of a chromosome when exposed to specific chemicals. Using
these criteria, cytogeneticist have set up a classification system for chromosomes which
labels each pair of autosomes
with a number, or for the sex chromosomes
system of standardization allows for accurate communication among scientists and medicalprofessionals. Many genetics conditions have been associated with a specific change or
- an increase in sets of chromosomes, e.g., 3n
- extra of missing chromosomes, e.g., 2n + 1
or 2n - 1
- attachment of a chromosome or piece of a chromosome to another, e.g.,
• structural alterations
- deletions, duplications, inversions, fragile sites, ring
chromosomes, e.g., 5p-
Most alterations in chromosome number are lethal especially numerical changes involving
extra sets of chromosomes or any of the larger autosomes. Unbalanced chromosome
counts involve thousands of genes and are not compatible with normal growth and
development of the fetus. Sex chromosomal abnormalities are less severe than autosomalabnormalities. Irregular chromosome counts are associated with a constellation of
abnormalities that may include morphological, physiological, and psychological deviations
from normal. Such a complex of symptoms is called a syndrome
. Some examples of
genetic conditions and their respective chromosome aberrations are:
• Down Syndrome
- typically due to an extra chromosome #21 (trisomy 21)
• Cri du chat
- characterized by a deletion of the short arm of chromosome #5.
• Fragile X Syndrome
- characterized by a fragile site or break point on the long arm of
One practical application of karyotype analysis is the early detection of chromosomal
defects through amniocentesis
or chorionic villus sampling (CVS).
some of the amniotic fluid surrounding the fetus is removed by a physician. The fluid
contains fetal cells which will propagate under special laboratory conditions. Once thecells have increased in number a karyotype can be performed on these fetal cells for
prenatal diagnosis of sex and chromosome abnormalities. In karyotyping not involving
fetuses the cell type most often used for analysis are lymphocytes or WBC’s. As with fetal
cells these cells are grown in culture, treated with hypotonic solution, and fixed prior toperforming a karyotype.
Several kinds of cancers have been associated with chromosomal abnormalities and many
malignant cells tend to have abnormal chromosome numbers. For example, chronicmyelogenous leukemia (CML) is associated with the so-called Philadelphia chromosome
(Ph). The defective chromosomes in CML occur only in white blood cells and involves a
(exchange of segments) between the long arms of chromosomes 9
and 22. The proper designation for this condition is 46, (XX or XY), [t(9/22)].
Cells grown in tissue culture
are used for most human chromosome studies. In this
exercise, the human tumor cell line, HeLa
, is used for karyotyping. The HeLa cell line
originated in the early 1950’s from the cancerous cervical cells of a women namedHe
cks. Because the cells are of tumor origin they are immortal and have
continued to divide and multiply in tissue culture for over 40 years. Cancer cells result
from uncontrolled mitosis when mutant cells lose cell cycle checkpoints which normally
trigger apoptosis in chromosomally abnormal cells. Many cancer cells are aneuploidscontaining chromosome counts greater than 46 with one, two or even three extra copies of
a particular chromosome. Try to make a chromosome count of your spreads to determine
if the cells have more than 46 chromosomes.
PART I. Preparing A Metaphase Spread Of HeLa Cells
A log phase culture of HeLa cells were incubated with colchicine
, treated with trypsin
, and placed in a hypotonic
solution prior to shipping. The cell suspension will be
stable for a few days but should be refrigerated
when not in use.
Place a new, clean
microscope slide at a 450 angle.
With a pipette, gently resuspend
the cells in the tube provided. Remove a small
sample of cell suspension with the pipette and hold the pipette 2 feet above the slide.
Allow one drop
of cell suspension to "splat” onto the slide about 3/4 inch from the
upper end so that it tumbles down the slide.
Carefully apply 6-8 more drops from various heights
, one drop at a time, onto the
same region of the slide. It is important to release the cell suspension one-drop-at-a-
Do not expel all of your cell suspension in one squirt, or you will obtain poor
Gently blow across the slide for 2-3 seconds. The drying will help "spread” the
Allow the cells to air dry completely
Dip the slide into the tube containing Stain #1 for one second only
. Remove the slide
and dip into Stain #1 again for one second only. Remove the slide and dip into Stain#1 for one second only a third time.
Caution should be taken to avoid carry over of stains. Wipe the bottom of the slide
with a paper towel before transferring.
Drain off the stain and dip the slide into the tube containing Stain #2 for one second
Remove the slide and dip again into Stain #2 for one second, dip a third time
Remove the slide from the stain and thoroughly rinse
with distilled water.
10. Allow the slide to air dry completely
. A stream of warm air or blowing may help
speed up the drying process. Incomplete drying will result in poor resolution when
the mounting medium (Permount) is added.
While your waiting for your slides to dry work on Part II.
Place two drops of Permount as demonstrated by your instructor
on the stained
area of your slide and place a coverslip over the glue. Apply gently pressure to the
coverslip to spread the Permount evenly under the coverslip. You may wish to place 2coverslips side by side so as to allow viewing of the entire microscope slide.
12. Once the Permount has dried
the slide is ready for viewing.
13. Under low power (100 x) scan your spread for cells which appear to have ruptured
and released their chromosomes. Shift to high power (450x) to examine each spreadmore carefully. Low and high dry observations can be made immediately.
Observations with the oil immersion objective should not be done until the Permount
has dried completely (48-72 hours) or should be done very carefully
so as not to
transfer Permount to the objective.
Caution: Only use the oil immersion lens on those areas of the slide containing a coverslip. If
Permount does get onto an objective notify your instructor and try to remove itimmediately using lens paper and xylene.
14. An ideal chromosome spread
will contain chromosomes which appear distinct and
do not overlap with adjacent chromosomes. Chromatids should be visible. The hunt
requires careful observation so take your time when scanning the slide. Once you
have found what appears to be a clear and distinct set of chromosomes place a small
drop of immersion oil (if available) on the coverslip over the area and switch to 1000x.Note:
When using the oil immersion lens you will have to increase the amount of light
passing through the specimen. This can be done by increasing the light intensity
adjusting the iris diaphragm
15. Try to identify and locate
the three different types of chromosomes: metacentric,
16. Try to count the number of chromosomes
present in 5 different cells on the slide.
Remember that this cell line is aneuploid and each cell will probably contain adifferent number of chromosomes, each greater than the diploid number (46).
# of chromosomes
Procedure for Preparing a Metaphase Spread
PART II. Morphology Of Chromosomes
On the basis of chromosome length and position of the centromere, normal human
chromosomes have been arranged in seven groups of autosomes
, A to G, and one pair of
sex chromosomes, XX or XY. Chromosome bands are observed with Q- G- and R-staining
methods (Quinacrine, Giemsa and reversed Giemsa bands or R bands). Autosomes arenumbered in order from 1 to 22, and Y and X are listed separately. Short arms of
chromosomes are designated p
and long arms q
. Regions are numbered from 1 (next to
centromere) to the distal end of each arm, and bands are numbered within the regions from
the centromere end to the distal end of each arm according to the 1971 Paris Conference onstandardization in human cytogenetics.
Observe the relative length and centromere location of the chromosomes shown in the
diagram. Draw brackets around and assign the appropriate letter to each of the eightgroups (A-G and sex chromosomes). Note that, in terms of size and centromere position,
the X chromosome is similar to the chromosomes in the C group and the Y chromosome is
How to Identify Human Chromosomes
PART III. Preparing Human Karyotypes
In medical cytogenetics
laboratories 10-50 chromosome spreads for each patient are
examined through the microscope. The best appearing chromosome spreads are
photographed and enlarged. Individual chromosomes are then cut out, arranged in
matched pairs, and glued in proper order on a standardized form. Each karyotypeincludes a complete set of chromosomes for the person or fetus under investigation.
Evaluations for structural and numerical chromosome abnormalities are made form the
karyotype and verified when necessary from direct microscopic observations of the slides.
A karyotype provides a tangible record for comparison, display, and filing for future use.
In recent years most cytogenetics laboratories have begun using an automated, computer-
driven karyotyping system. Although expensive, such a system can be accurate and
Before you begin, place the number found in the upper left hand corner of your
metaphase spread on a blank standard karyotype form. This number identifies thekaryotype to your instructor.
The first step to arranging a karyotype by hand is to count the number of chromosomes to determine deviations in number from the normal d
Carefully cut out each chromosome and attempt to arrange them from large to small.
To identify chromosomes more accurately you must examine the banding patterns.
Use the normal karyotype provided as a guide to chromosome identification.
Use the standard karyotype form to arrange your chromosomes. Be sure to doublecheck your arrangement before permanently pasting or taping any of the
Using standard nomenclature, give the descriptive chromosome symbols, sex, andphenotype or syndrome for each of the karyotypes you prepare.
Human Chromosome Post-Lab Questions
Answer as completely as possible. Turn in with your kayotypes at the beginning of next weeks lab.
Which of the human chromosomes are metacentric
? Answer by giving individual chromosome
Which human chromosomes are submetacentric
chromosomes have centromeres located near one end and satellite regions that represent
the site of ribosomal RNA genes (nucleolar organizing region; NOR). Which human chromosomes are
Give the complete chromosomal description for the following individuals as illustrated in "a”.
An infant male with Patau syndrome 47, XY, +13
A newborn female with Cri du Chat syndrome
Why is it necessary to expose cells to a hypotonic solution when preparing them for karyotyping?
During what stage of mitosis are chromosomes in their most condensed state?
Explain the difference between polyploidy and aneuploidy.
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