Experiment ObjectivesExperiment Objectives

• Preparing

• Staining and observing human metaphase chromosomes.

Chromosome MorphologyChromosome Morphology• Chromosomes are not visible under the light microscope

in non-dividing cells (interphase cells).• As the cell begins to divide, the threads of chromatin

(DNA-protein complex) in the nucleus begin to condense into multiple levels of coiled structures recognizable as chromosomes.

• There are two modes of cell division: • Mitosis: is responsible for the proliferation of body

(somatic) cells, • Meiosis: is responsible for the production of gametes.

• Because mitotic cells are easy to obtain, morphological studies are generally based on mitotic metaphase chromosomes.

Cell divisionCell division

• Cell division can be divided into:• Interphase• Mitosis

ProphaseMetaphaseAnaphaseTelophase

• Cytokinesis

MetaphaseMetaphase

• At metaphase the chromosomes are at their most condensed state,

• Spindle fibers attaching to the area of the centromere called the kinetochore, forming pole-chromosome fibers.

• Anaphase begins with the division of the centromere and the separation of chromatids.

• Once separated, each chromatid is known as a chromosome.

• The kinetochore: is the protein structure on chromatids where the spindle fibers attach during cell division to pull sister chromatids apart.

Chromosome AnalysisChromosome Analysis

• The best mitotic stage for chromosome analysis is pro-metaphase or metaphase.

• A typical metaphase chromosome consists of two arms separated by a primary constriction or centromere.

• Each of the two sister-chromatids contains a highly coiled double helix of DNA.

• Often the sister chromatids are so close to each other that the whole chromosome appears as a single rod-like structure

• A chromosome may be characterized by its total length and the position of its centromere.

Chromosome NumberChromosome Number

• The diploid chromosome number is the number of chromosomes in the somatic cell and is designated by the symbol 2N.

• The gametes, which have one half the diploid number, have the haploid number N.

• Thus, there are 23 pairs of chromosomes in human cells.

• Of these, 22 pairs are not directly involved in sex determination, and are known as autosomes.

• The remaining chromosome pair consists of the sex chromosomes, and is directly involved in sex determination.

• In females the two sex chromosom es are identical (XX), whereas in males the two sex chromosomes are not identical (XY).

Types of TissueTypes of Tissue

• A variety of tissue types can be used to obtain chromosome preparations.

• Some examples include peripheral blood, bone marrow, amniotic fluid and products of conception.

• In the case of blood cell culture only cells that are actively dividing can be used for cytogenetic studies.

• Normally only white blood cells are used for cytogenetic analysis.

• Specific techniques differ according to the type of tissue used.

Overview of ProcedureOverview of Procedure

1. Collection of blood

2. Cell culture

3. Harvesting: stopping the cell division at metaphase

4. Hypotonic treatment of red & white blood cells

5. Fixation

6. Slide preparation

7. Staining

1- Collection of blood1- Collection of blood

• Draw 5 ml of venous blood into a sterile heparinized tube containing 0.1 ml of sodium heparin (500 units/ml).

2- Cell Culture2- Cell Culture• Sterile technique must be used throughout the

cell culture preparation, because it is possible to cause major contamination during this procedure,

• 70% of the problems are due to a lack of good sterile technique.

• Antibiotics do not eliminate problems of gross contamination which result from poor sterile technique or antibiotic-resistant mutants.

• Autoclaving renders pipettes, glassware, and solutions sterile.

2- Cell Culture2- Cell CultureMedium• Pipette 10 ml RPMI 1640 medium with L-

Glutamine into a 15 ml labeled sterile culture tube• Supplement the medium with the following:

Penicillin-Streptomycin Stock solution

10 µl (100000 u penicillin/ml – 100 mg/ml Streptomycin

Phytohemagglutinin 0.3 ml 20 µg/ml

Fetal bovine Serum 20% 2 ml

2- Cell Culture2- Cell Culture

Incubation• Add 1 ml of whole heparinized blood into the

tube containing the supplemented medium• Mix contents of tube with gentle inversion

• Incubate in 5% CO2 incubator at 37oC for 72 hours

3- Harvesting3- Harvesting• Harvesting: mitotic spindle formation is blocked: usually

by adding colcemide to the culture, and the cell division is stopped at the metaphase level.

• Pre-warm the Colchicine (0.04 mg/ml) in incubator at 37oC.

• Add 25 µl of pre-warmed Colchicine to the culture. • Mix gently and incubate at 37oC for 30-60 minutes.

• Note: Colchicine inhibits microtubule polymerization by binding to tubulin, one of the main constituents of microtubules

4- Hypotonic treatment of red & white blood cells4- Hypotonic treatment of red & white blood cells

• Centrifuge for 10 minutes at 2000 rpm.• Discard supernatant without disturbing the cells

leaving 0.5 ml of fluid.• Add 1 ml of pre-warmed hypotonic solution

(0.075 M KCl) at 37oC.• Mix and then add 9 ml of hypotonic solution.• Mix well by Pasteur pipette.• Incubate at 37oC incubator for 17 minutes,• hypotonic solution should not be in contact with

cells more than 27 minutes (may cause rupture of WBCs).

5- Fixation5- Fixation

• Fixative must be prepared fresh• Add 3 parts of chilled absolute methanol:1 part

glacial acetic acid.• Centrifuge for 10 minutes at 1000 – 1500 rpm.• Remove supernatant leaving about 0.5 ml of fluid

on top of cells.• At this time there is probably a small whitish or

reddish film at the bottom of the tube.• The film contain red blood cell debris and

enlarged WBCs.

5- Fixation5- Fixation• Add 5 ml of fixative to the tube.• Mix with a Pasteur pipette 3-4 times.• Place in refrigerator for 30 minutes.• Centrifuge the tube for 10 minutes at 1000-1500 rpm.• Remove supernatant and add another 6 ml of cold

fixative, & mix well.• Centrifuge the tube for 10 minutes at 1000-1500 rpm.• Repeat the last two steps.• Remove the supernatant leaving 1 ml of fluid at the

bottom.• The remaining material will be used to make the slides.

6- Slides Preparation6- Slides Preparation

• The slide must be exceptionally clean• Lay slides on a paper towel• Withdraw a few drops of cell suspension into a

pipette • From a height of 20 cm, drop 2 or 3 drops of

fluid on each slide• Allow the slides to dry

7- Staining 7- Staining

• Stain the slides by immersion in fresh Giemsa stain for 7-10 minutes

• Remove slides from stain & rinse in distilled water

• Observe under microscope 40X then under oil immersion

• http://www.biology.arizona.edu/human_bio/activities/karyotyping/patient_a/patient_a.html

• http://www.youtube.com/watch?v=E0WkZr819UUhttp://www.youtube.com/watch?v=E0WkZr819UU