Demonstration of Mitosis in Onion Root Tips (Allium cepa) — Squash Preparation

Aim

To prepare a squash of onion root tip meristem and observe different stages of mitosis under the microscope.

Principle (Why onion roots?)

Onion root tips contain actively dividing meristematic cells. Fixation preserves cell structures, acid hydrolysis softens the cell wall and exposes chromosomes, and basic stains (acetocarmine or aceto-orcein) bind chromatin so chromosomes can be seen clearly in different mitotic stages.

Requirements

Specimen & glassware:
1. Onion bulbs with 1–2 cm long fresh roots
2. Microscope slides & cover slips (clean, grease-free)
3. Forceps, scalpel/razor, dissecting needle
4. Dropper/pasteur pipettes, filter paper/tissue

Reagents (freshly prepared where noted):
1. Fixative: Carnoy’s I (absolute ethanol : glacial acetic acid = 3:1, v/v)
2. Hydrolyzing agent: 1 N HCl (preheated to ~60 °C)
3. Stain: 1% acetocarmine or 2% aceto-orcein in 45% acetic acid
4. Acetic acid (45%) for squashing
5. Mountant (optional for permanent prep): DPX or nail varnish for sealing

Equipment:
1. Compound microscope (10×, 40×; 100× oil immersion optional)
2. Water bath or beaker with hot water (~60 °C)
3. Spirit lamp/Hot plate (optional, for gentle warming)

Safety:
Wear a lab coat, gloves, and goggles. Handle acids and acetic acid in a fume hood. Dispose of chemical waste as per your lab rules.

Step-by-Step Procedure

A. Fixation (5–30 min; or up to overnight at 4 °C)
1. With forceps, cut 5–10 young white root tips (~5 mm from the tip).
2. Immediately transfer to Carnoy’s I fixative.
3. Fix for at least 15 min (good results from 30–60 min; can store up to 24 h at 4 °C).
4. Briefly rinse roots in distilled water.

B. Acid Hydrolysis (cell wall softening; 5–10 min)
5. Place root tips in preheated 1 N HCl at ~60 °C for 5–7 min.
6. Rinse roots thoroughly with distilled water.

C. Staining (5–10 min)
7. Transfer a single soft root tip (~1–2 mm) to a clean slide.
8. Add 1–2 drops of 1% acetocarmine (or 2% aceto-orcein).
9. Warm gently (do not boil) for 1–2 min to intensify staining; allow to stand 5 min.

D. Squash & Mount (2–5 min)
10. Add one drop of 45% acetic acid; tease the root tip with needles.
11. Place a cover slip carefully.
12. Squash vertically with thumb/pencil eraser.
13. Remove excess stain with tissue. Seal cover slip edges if required.

E. Microscopy (10–15 min)
14. Scan under 10× to locate well-spread cells; switch to 40×.
15. If available, use 100× oil immersion for detailed chromosome view.

What to Observe (identify & sketch)

 Interphase: large nucleus, uniform chromatin, distinct nucleolus; no visible chromosomes.
 Prophase: chromosomes condense; nuclear membrane disintegrates.
 Metaphase: chromosomes maximally condensed, aligned at equatorial plate.
 Anaphase: sister chromatids separate and move to opposite poles.
 Telophase: chromosomes decondense; nuclear envelopes reform; cell plate appears.

Precautions

1. Use fresh fixative; fix immediately after cutting tips.

2. Maintain ~60 °C for HCl; overheating causes over-hydrolysis.

3. Warm stain gently, not boiling.

4. Squash vertically to avoid smearing.

5. Choose young white root tips for best results.

6. Clean slides/cover slips to prevent background precipitate.

Viva-Voce / Review Questions

1. Why are onion root tips preferred for mitosis study?
2. Role of Carnoy’s fixative vs. acetic acid during squashing.
3. Why is acid hydrolysis required before staining?
4. How do you distinguish metaphase from anaphase?
5. What is the mitotic index and what does a high MI indicate?
6. Why do plant cells form a cell plate instead of a cleavage furrow?
7. How does colchicine help in metaphase spread preparation?