Demonstration of Mitotic Stages in Allium cepa (Onion) Root Tip by Acetocarmine Squash Technique
Aim: To demonstrate the stages of mitosis in Allium cepa root tip cells by preparing a temporary squash preparation using acetocarmine staining.
Principle: Mitosis is cell division where one parent cell produces two genetically identical daughter cells. It happens in actively dividing tissues. Onion root tip is a classic material for this because the cells at the tip (the meristematic zone) are dividing almost continuously, so you can catch many stages on one slide. The root tip cells are first softened in hydrochloric acid, which breaks down the cell wall. Then acetocarmine stain, which specifically stains chromatin red, is applied. Squashing spreads the cells into a single layer. Under microscope we see cells in different stages: prophase, metaphase, anaphase, telophase, and of course interphase.
Requirements:
Biological material:
- Allium cepa (onion) bulbs, healthy and firm, for growing roots
For root germination:
- Clean glass beakers or plastic cups filled with tap water,
- Toothpicks or wire mesh to hold onion bulb just above water surface
Fixative:
- Carnoy's fixative: Absolute ethanol 750 mL/L and glacial acetic acid 250 mL/L. Prepare fresh. This is a 3:1 mixture by volume. Store in cold.
For staining:
- 1 N Hydrochloric acid (HCl): prepared by diluting concentrated HCl in distilled water carefully (add acid to water, never reverse)
- Acetocarmine staining solution: Carmine powder 1 g/L dissolved in 45% glacial acetic acid prepared in distilled water. Boil under reflux for 1 to 2 hours. Filter and store in amber bottle. Glycerol can be added at 1 mL/10 mL stain to prevent drying of the preparation.
Other reagents:
- 70% ethanol (for washing steps)
- Distilled water
- Canada balsam or DPX mountant (for permanent slide, optional)
Equipment and consumables:
- Compound light microscope with 10X and 40X objectives
- Glass slides and coverslips
- Forceps, razor blade, and dissecting needle
- Watch glass or petri dish
- Dropper or Pasteur pipette
- Water bath set at 60 degrees C
- Spirit lamp or Bunsen burner
- Filter paper pieces
- Permanent marker for labelling slides
Procedure:
Day 1: Setting up onion for root germination
- Take a healthy onion bulb and remove any dry outer scales from the base without damaging the root initials.
- Place the bulb on top of a beaker or glass filled with tap water such that the base just touches the water surface. Use toothpicks on sides to support it if needed.
- Keep this setup in a warm place away from direct sunlight. Change the water every day to prevent fungal growth.
- Roots will start appearing in 2 to 3 days. Wait until roots are approximately 1 to 2 cm long. This usually takes 3 to 4 days.
Day 3 or 4: Fixation of root tips
- Cut root tips of approximately 0.5 to 1 cm length using a clean razor blade. Cut early in the morning (around 8 to 10 AM) if possible, as mitotic activity is highest at this time in Allium cepa.
- Immediately transfer the cut root tips into freshly prepared Carnoy's fixative (3:1 ethanol: glacial acetic acid) in a small vial. Leave for minimum 4 hours or overnight at 4 degrees C. Fixed material can be stored in 70% ethanol at 4 degrees C for days.
Day 4 or 5: Slide preparation
- Transfer a fixed root tip into a watch glass. Wash once with distilled water for 1 to 2 minutes to remove excess fixative.
- Transfer the root tip to 1 N HCl in a watch glass. Place the watch glass in a water bath at 60 degrees C for exactly 8 to 10 minutes. This step is called maceration. It softens cell walls and separates the cells. Do not over-macerate.
- Remove the root tip and wash gently once with distilled water.
- Place the root tip on a clean glass slide. Using razor blade, cut only the meristematic zone, which is the terminal 2 to 3 mm of the root tip. It should look slightly translucent and pale. Discard the rest.
- Add 1 to 2 drops of acetocarmine stain directly onto the root tip piece on the slide.
- Place the slide on a spirit lamp and heat very gently for 30 to 45 seconds, just until small steam wisps rise. Do not boil. This help the stain penetrate properly.
- Place a clean coverslip gently over the stained material.
- Wrap the slide in a filter paper. With your thumb, apply firm and even pressure in a straight downward direction, pressing the coverslip uniformly. Do not rotate or shift the coverslip sideways, or cells will smear and everything will be ruined.
- Observe immediately under microscope. Start with 10X and then shift to 40X objective.
- Look systematically across the slide for cells showing different mitotic stages.
Observation: Under a 45X objective, cells in the meristematic zone appear stained dark red to pink, with nuclei clearly visible. Cells in various mitotic stages (prophase with condensing chromosomes, metaphase with chromosomes aligned at the equatorial plate, anaphase with chromosomes moving to poles, and telophase with two reforming nuclei) are visible, and a large number of cells will be in interphase, showing a well-stained nucleus with chromatin network.
Result: All four stages of mitosis, prophase, metaphase, anaphase, and telophase, were successfully demonstrated in Allium cepa root tip cells using the acetocarmine squash preparation method.
Conclusion: The onion root tip squash is honestly one of the most reliable classical preparations in cell biology, and when done carefully, the stages of mitosis are clearly distinguishable. The meristematic zone gives an excellent material because the cells there are constantly dividing, so one single slide gives you multiple stages at the same time.
Note: This protocol is for educational use in standard teaching laboratories. Small variations may occur depending on lab conditions and handling. Results may vary. biologynotes.in cannot take responsibility for any errors, misuse, or results obtained. Always follow proper lab safety and instructor guidance.