A. Counting Chromosomes from Root Tips

1. Sample Collection:

• Cut 1 cm of young, actively growing root tips (preferably in the morning for higher mitotic activity).

2. Fixation:

• Immerse the root tips in a fixative solution (Ethanol:Acetic acid, 3:1 by volume) for 24 hours at room temperature.

3. Hydrolysis:

• Treat the fixed root tips with 1N HCl at 60°C for 10 minutes to soften the tissue and break down cell walls.

4. Staining:

• Place a small piece (about 1 mm) of the root tip on a clean glass slide.
• Add a drop of acetocarmine or aceto-orcein stain.
• Gently heat the slide over a flame for a few seconds to intensify staining (optional).

5. Squash Preparation:

• Cover the stained root tip with a cover slip.
• Gently press vertically (do not twist) to spread the cells into a single layer.

6. Microscopy and Counting:

• Observe under a compound microscope (400x magnification).
• Identify cells in metaphase (chromosomes are most visible).
• Count the chromosomes in at least 5 well-spread cells for accuracy.

B. Counting Chromosomes from Shoot Tips

1. Sample Collection:

• Collect young shoot (apical) meristems from healthy plants.

2. Pretreatment (to arrest cells in metaphase):

• Soak the shoot tips in 0.3% colchicine solution for 6-7 hours in the dark at a cool temperature.

3. Fixation and Enzymatic Digestion:

• Fix the treated shoot tips in Ethanol:Acetic acid (3:1) for 24 hours.
• Optionally, treat with a mixture of cellulase and pectinase enzymes to soften the tissue (especially for woody plants).

4. Staining and Squash Preparation:

• Stain with acetocarmine or aceto-orcein.
• Place a small piece on a slide, cover with a cover slip, and gently squash.

5. Microscopy and Counting:

• Observe under the microscope.
• Look for metaphase plates and count chromosomes as above.

Tips for Best Results

• Timing: Early morning samples yield more dividing cells.
• Hydrolysis: Do not over-hydrolyze, as this can damage chromosomes.
• Squash Technique: Apply vertical pressure only—do not twist the cover slip.
• Verification: Count chromosomes in several cells to confirm accuracy.

Note:

• The root tip method is generally easier and faster for herbaceous plants.
• The shoot tip method is preferred for woody plants where root tips are hard to obtain.

Raghad Mouhamad Thank you so much ma'am. Do I've to put the harvested root tips in colchicine solution also? I've been trying from the root tips but not getting success. I immersed the harvested root tips in colchicine solution for 2-3 hrs first, then into fixative.