BIOS.113.Lab.04.Chromosome.Staining.S2025 (1)

BIOS 113 Spring 2025 Lab #4: Chromosome Staining and Karyotyping

Background

• Purpose: Assess cells based on their chromosomal content for cell biology research and medical tests.

• Source of Cells: Large testes of the male European Cricket (Acheta domesticus).

  • These cells are rapidly dividing during meiosis to produce sperm.

  • Cells will be harvested and artificially halted in meiosis for viewing.

• Colchicine Treatment: Used to inhibit the polymerization of microtubules and spindle formation, leading to stalling of cells in various meiotic stages.

  • Safety Note: Colchicine is corrosive; gloves and goggles must be worn.

Materials

• Male Acheta domesticus (large adult crickets)

• Pressurized CO2 gas tank and regulator

• Tubing for gas transfer

• Anesthetizing gas chamber

• 1 ml medical syringes

• 0.1% Colchicine in RO/DI water

• Dissecting kit (scalpel, scissors, pins)

• Centrifuge tubes

• Transfer pipettes

• Petri dishes

• Slide warmer

• Compound microscope

• Microscope slides and glass coverslips

Procedure

Part A: Dissecting the Cricket Testes

1. Gather necessary equipment: dissecting tray, scalpel, scissors, pins, centrifuge tube, anesthetized male cricket.

2. Place paper towel over dissecting tray and position cricket belly-side up.

3. Pin the cricket through the head to kill it.

4. Make transverse incision in abdomen.

5. Incise down midline anteriorly, avoiding the head.

6. Pin back body wall with dissecting pins. Testes will be visible.

7. Locate the two large, white testes situated laterally in the anterior abdomen.

8. If obstructed, reposition the gut to expose the testes.

9. Use tweezers to carefully remove the testes and place in labeled centrifuge tube.

10. Fixing Testes: Add 500μl of 3:1 Ethanol: Acetic Acid to the tube and fix for 10 minutes.

Part C: Preparing the Chromosomal Spreads

1. Collect materials: petri dishes, dissecting scalpel, acetic acid, chilled PBS, Giemsa stain, Giemsa buffer.

2. Warm up slide warmer to 45°C and prepare a clean microscope slide.

3. Remove fixed testes from centrifuge tube, place in petri dish, and add 50 μl of 50% acetic acid.

4. Mince tissue thoroughly with a scalpel to create cell suspension.

5. Drop cell suspension onto warmed slide to mechanically lyse cells.

6. Return slide to warmer to air dry.

7. Digest proteins: cover slide spots with 0.025% trypsin for 2 minutes at room temperature.

8. Rinse the slide with chilled PBS for 10 minutes to wash away proteins.

9. Stain with 5% Giemsa stain (pH 6.8) for 15 minutes.

10. Rinse slide twice with Giemsa buffer and wash with RO/DI water for 30 seconds.

11. Your instructor applies permount, and you cover it with a cover slip, waiting 5 minutes for it to harden.

Part D: Viewing Chromosomal Spreads

1. Examine the slide under a compound microscope starting at low magnification, moving to higher objectives.

2. Draw chromosomes viewed at 100X and 400X.

3. For viewing at 1000X, use immersion oil between the objective lens and cover slip.

4. Focus on good spreads for analysis and photograph using your instructor's microscope with a camera.

Analysis

• Discuss chromosome spreads, counting identifiable chromosomes and noting distinguishable pairs by length and banding patterns.