Preparation of Metaphase Chromosome from the Bone Marrow of Mice

Aim

To prepare metaphase chromosomes from the bone marrow cells of mice for microscopic observation and karyotypic analysis.

Principle

Bone marrow contains rapidly dividing cells. During mitosis, chromosomes become highly condensed and clearly visible at the metaphase stage.

To accumulate cells at metaphase, colchicine is used, which inhibits spindle fiber formation. Hypotonic treatment causes cell swelling, spreading the chromosomes apart. Cells are then fixed, stained, and observed under a microscope.

The metaphase arrest can be represented as:

$\text{Colchicine} \rightarrow \text{Inhibition of spindle formation} \rightarrow \text{Metaphase arrest}$

Requirements

Apparatus

• Microscope
• Centrifuge
• Syringe
• Slides and cover slips
• Test tubes
• Pasteur pipette
• Water bath
• Forceps and scissors

Chemicals/Reagents

1. Colchicine solution
2. Hypotonic solution (0.075 M KCl)
3. Carnoy’s fixative (Methanol : Acetic acid = 3:1)
4. Giemsa stain
5. Phosphate buffer/distilled water

Procedure

Step 1: Colchicine Treatment

1. Inject colchicine intraperitoneally into the mouse.
2. Wait for about 1–2 hours to arrest dividing cells at metaphase.

Step 2: Collection of Bone Marrow

1. Sacrifice the mouse ethically according to laboratory guidelines.
2. Remove femur bones carefully.
3. Cut the ends of the femur.
4. Flush bone marrow into a centrifuge tube using hypotonic solution.

Step 3: Hypotonic Treatment

1. Incubate the cell suspension in 0.075 M KCl for 15–20 minutes at 37°C.
2. This swells the cells and spreads chromosomes apart.

Step 4: Fixation

1. Centrifuge the suspension.
2. Discard supernatant carefully.
3. Add freshly prepared Carnoy’s fixative slowly.
4. Repeat fixation 2–3 times for proper fixation.

The fixation process may be represented as:

$\text{Methanol : Acetic Acid} = 3:1$

Step 5: Preparation of Slides

1. Drop the fixed cell suspension onto clean slides from a height.
2. Allow slides to air dry.

Step 6: Staining

1. Stain slides with Giemsa stain for 10–15 minutes.
2. Wash gently with distilled water.
3. Air dry the slides.

Step 7: Microscopic Observation

1. Observe the slides under low power and then high power microscope.
2. Identify metaphase plates showing condensed chromosomes.

Observation

• Darkly stained condensed chromosomes are observed.
• Chromosomes appear scattered and distinct at metaphase.
• Metaphase plates suitable for chromosome counting may be visible.

Interpretation

1. Presence of well-spread chromosomes indicates successful metaphase preparation.
2. Condensed chromosomes confirm metaphase arrest due to colchicine.
3. Chromosome number and morphology can be studied from metaphase plates.Labels to Include in Diagram

• Femur bone
• Bone marrow cells
• Metaphase chromosomes
• Centromere
• Chromatid
• Metaphase plate

Result

Metaphase chromosomes were successfully prepared from the bone marrow cells of mice and observed under the microscope.

Precautions

1. Handle laboratory animals ethically.
2. Use freshly prepared fixative.
3. Maintain proper hypotonic treatment time.
4. Avoid overcrowding of cells on slides.
5. Use clean grease-free slides.
6. Handle colchicine carefully as it is toxic.

Viva-Voce Questions

1. 1. Why is colchicine used in chromosome preparation?

   Colchicine is used to stop cell division at metaphase by preventing spindle fiber formation, making chromosomes short, thick, and clearly visible.

   2. What is the function of hypotonic solution?

   Hypotonic solution causes cells to swell, which spreads the chromosomes apart for better observation.

   3. Why is Carnoy’s fixative used?

   Carnoy’s fixative preserves cellular structures, kills the cells quickly, and fixes chromosomes properly for staining and microscopic study.

   4. Why are metaphase chromosomes preferred for study?

   During metaphase, chromosomes are highly condensed, distinct, and easily visible, making them suitable for counting and karyotype analysis.

   5. Which stain is commonly used for chromosome staining?

   Giemsa stain is commonly used for chromosome staining.

   6. What is a metaphase plate?

   A metaphase plate is the arrangement of chromosomes at the equatorial plane of the cell during metaphase.

   7. Why are bone marrow cells selected?

   Bone marrow cells divide rapidly and contain many actively dividing cells, increasing the chance of obtaining metaphase chromosomes.

   8. What is the role of acetic acid in fixation?

   Acetic acid helps preserve chromosomes, softens the cytoplasm, and improves chromosome spreading and staining quality.

Applications

1. Karyotype analysis
2. Cytogenetic studies
3. Detection of chromosomal abnormalities
4. Cancer research
5. Genetic studies
6. Evolutionary studies