human metaphase chromosomes. mazen zaharna molecular biology 1/2009 experiment experiment objectives...

Human Metaphase Human Metaphase ChromosomesChromosomes

Mazen Zaharna Molecular Biology 1/2009

Experiment Experiment Objectives

• Preparing, staining and observing Preparing, staining and observing human metaphase chromosomes.human metaphase chromosomes.


Chromosome Chromosome MorphologyMorphology

• Chromosomes are not visible under the light Chromosomes are not visible under the light microscope in non-dividing cells microscope in non-dividing cells (interphase cells).

• As the cell begins to divide, the threads of chromatin As the cell begins to divide, the threads of chromatin (DNA-protein complex) in the nucleus begin to (DNA-protein complex) in the nucleus begin to condense into multiple levels of coiled structures condense into multiple levels of coiled structures recognizable as chromosomes. recognizable as chromosomes.

• There are two modes of cell division: There are two modes of cell division: – mitosis and meiosis. Mitosis is responsible for the mitosis and meiosis. Mitosis is responsible for the

proliferation of body (somatic) cells, proliferation of body (somatic) cells, – whereas meiosis is responsible for the production of whereas meiosis is responsible for the production of

gametes. gametes.

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


Cell divisionCell division

• Cell division can be divided into:Cell division can be divided into: Interphase, Interphase, MitosisMitosis

Prophase,Prophase,Metaphase, Metaphase, Anaphase, Anaphase, Telophase.Telophase.

CytokinesisCytokinesis


Metaphase Metaphase

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

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


Chromosome AnalysisChromosome Analysis

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

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

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


Chromosome AnalysisChromosome Analysis

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

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


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

1.1. Collection of bloodCollection of blood2.2. Cell cultureCell culture3.3. Stopping the cell division at Stopping the cell division at

MetaphaseMetaphase4.4. Hypotonic treatment of red & white Hypotonic treatment of red & white

blood cellsblood cells5.5. FixationFixation6.6. Slide preparationSlide preparation7.7. Staining Staining


1- Collection of blood1- Collection of blood

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


2- Cell Culture2- Cell Culture

• Sterile technique must be used throughout Sterile technique must be used throughout the cell culture preparation, because it is the cell culture preparation, because it is possible to cause major contamination possible to cause major contamination during this procedureduring 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



MediumMedium• Pipette 10 ml Pipette 10 ml RPMI 1640 medium RPMI 1640 medium with L-with L-

Glutamine into a 15 ml labeled sterile culture Glutamine into a 15 ml labeled sterile culture tubetube

• Supplement the medium with the following:Supplement the medium with the following:

Penicillin-Penicillin-Streptomycin Stock Streptomycin Stock solutionsolution

10 µl10 µl (100000 u (100000 u penicillin/ml – 100 penicillin/ml – 100 mg/ml mg/ml Streptomycin Streptomycin

PhytohemagglutiniPhytohemagglutininn

0.3 ml0.3 ml 20 µg/ml20 µg/ml

Fetal bovine Serum Fetal bovine Serum 20%20%

2 ml2 ml



IncubationIncubation• Add 1 ml of whole heparinized blood Add 1 ml of whole heparinized blood

into the tube containing the into the tube containing the supplemented mediumsupplemented medium

• Mix contents of tube with gentle Mix contents of tube with gentle inversioninversion

• Incubate in 5% COIncubate in 5% CO22 incubator at incubator at 3737ooC for 72 hoursC for 72 hours


3- Stopping cell division at 3- Stopping cell division at MetaphaseMetaphase

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

• Add 25 µl of pre-warmed Colchicine Add 25 µl of pre-warmed Colchicine to the cultureto the culture

• Mix gently and incubate at 37Mix gently and incubate at 37ooC for C for 30-60 minutes30-60 minutes


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

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

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

(0.075 M KCl) at 37(0.075 M KCl) at 37ooCC• Mix and then add 9 ml of hypotonic solutionMix and then add 9 ml of hypotonic solution• Mix well by Pasteur pipetteMix well by Pasteur pipette• Incubate at 37Incubate at 37ooC incubator for 17 minutesC incubator for 17 minutes• hypotonic solution should not be in contact hypotonic solution should not be in contact

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


5- Fixation5- Fixation

• Fixative must be prepared freshFixative must be prepared fresh• Add 3 parts of chilled absolute Add 3 parts of chilled absolute

methanol: 1 part glacial acetic acidmethanol: 1 part glacial acetic acid


5- Fixation5- Fixation

• Centrifuge for 10 minutes at 1000 – Centrifuge for 10 minutes at 1000 – 1500 rpm1500 rpm

• Remove supernatant leaving about Remove supernatant leaving about 0.5 ml of fluid on top of cells0.5 ml of fluid on top of cells

• At this time there is probably a small At this time there is probably a small whitish or reddish film at the bottom whitish or reddish film at the bottom of the tubeof the tube

• The film contain red blood cell debris The film contain red blood cell debris and enlarged WBCsand enlarged WBCs


5- Fixation5- Fixation• Add 5 ml of fixative to the tubeAdd 5 ml of fixative to the tube• Mix with a Pasteur pipette 3-4 timesMix with a Pasteur pipette 3-4 times• Place in refrigerator for 30 minutesPlace in refrigerator for 30 minutes• Centrifuge the tube for 10 minutes at 1000-Centrifuge the tube for 10 minutes at 1000-

1500 rpm1500 rpm• Remove supernatant and add another 6 ml of Remove supernatant and add another 6 ml of

cold fixative, & mix wellcold fixative, & mix well• Centrifuge the tube for 10 minutes at 1000-Centrifuge the tube for 10 minutes at 1000-

1500 rpm1500 rpm• Repeat the last two stepsRepeat the last two steps• Remove the supernatant leaving 1 ml of fluid at Remove the supernatant leaving 1 ml of fluid at

the bottomthe bottom• The remaining material will be used to make The remaining material will be used to make

the slides the slides


6- Slides Preparation6- Slides Preparation

• The slide must be exceptionally The slide must be exceptionally cleanclean

• Lay slides on a paper towelLay slides on a paper towel• Withdraw a few drops of cell Withdraw a few drops of cell

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

drops of fluid on each slidedrops of fluid on each slide• Allow the slides to dryAllow the slides to dry


7- Staining 7- Staining

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

• Remove slides from stain & rinse in Remove slides from stain & rinse in distilled water distilled water

• Observe under microscope X40 then Observe under microscope X40 then under oil immersionunder oil immersion


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

• http://www.youtube.com/watch?http://www.youtube.com/watch?v=E0WkZr819UUv=E0WkZr819UU

human metaphase chromosomes. mazen zaharna molecular biology 1/2009 experiment experiment objectives...

Documents

typical metaphase chromosome

cell cultureincubationadd

cell divisioncell division

modes of cell division

case of blood cell culture

chromosome analysisthe

chromosome analysisoften

chromosome preparations