Experiment 4: Preparation of a Temporary Mount of Onion Root Tips to Study Mitosis

AIM

To prepare a temporary mount of onion root tips to study mitosis.

PRINCIPLE

Mitosis is a type of equational division where a parent cell divides into two genetically identical daughter cells. It maintains chromosome number and is essential for growth, repair and replacement.

Onion root tip contains actively dividing meristematic tissue with distinct zones like root cap, zone of division, elongation and differentiation. The apical meristem shows rapid mitotic divisions and is ideal material for studying mitosis.

Different stages of mitosis including karyokinesis followed by cytokinesis can be observed under microscope after staining with acetocarmine. Chromosomes become distinct during division phases and interphase is the non-dividing phase.

REQUIREMENTS

• Onion bulbs
• Wide mouthed glass bottle
• Corked tube
• Petri dishes
• Scissors
• Forceps
• Needles
• Methyl alcohol
• Acetic acid
• N/10 HCl
• Acetocarmine stain
• Distilled water
• Spirit lamp
• Compound microscope
• Slides
• Blotting paper
• Cover slips
• Glacial acetic acid and ethanol for fixative
• 70% alcohol
• Paraffin wax and glycerine

PROCEDURE

A. Growing Onion root tips:

1. Select medium onion bulbs and carefully remove the dry roots attached to them.
2. Place them in a bottle filled with water in such a way that only roots touch the water level.
3. Check the water level in the bottle everyday and add few drops of water periodically in order to compensate any evaporation losses.
4. Keep them in position for about 3-6 days. New roots may take 3-6 days to grow.
5. Cut 2-3 cm long freshly grown roots, 2 hrs after the sun rise and keep harvested roots in fixative for 2 hours.
6. Transfer roots from fixative to 70% alcohol.
7. These root tips are ideal material for the study of mitotic cell division.

B. Preparation and study of slide:

1. Take one or two preserved roots and wash them thoroughly in water on a clean slide.
2. Place one drop of N/10 HCl on the root tip followed by 2-3 drops of acetocarmine stain on it. Leave it for 5-10 minutes.
3. Slightly warm the slide on spirit lamp. Care should be taken that the stain is not dried up.
4. Blot the excess stain using blotting paper.
5. Cut the more stained tip portion of root and retain it on slide and discard the remaining portion.
6. After 10-20 seconds, put one or two drops of water and blot them carefully using blotting paper.
7. Gently squash the root by tapping the cover slip with the blunt end of a needle so that meristematic tissue of that root tip is smashed and spread as a layer.
8. There should be no air bubbles under cover slip.
9. Gently warm the slide over a flame and seal the margins of cover slip using paraffin wax.
10. Slide is now ready for the study of mitosis.
11. Observe it in a compound microscope.

OBSERVATION

Under lower magnification of the microscope, rectangular cells with pink nucleus are seen scattered. Cells are mostly in interphase and appear uniform.

Under higher magnification of the microscope different phases of mitosis become distinct. The stages of mitosis can be broadly categorised into two main parts, i.e., karyokinesis followed by cytokinesis.

Those cells which are not in the phases of cell division are considered to be in the interphase. You will observe that most cells in a microscopic field are in interphase.

Interphase

• It is a non-dividing phase of the cell cycle occurring between two successive cell divisions.
• The cells are mostly rectangular, oval or even circular in shape.
• The nucleus is homogenous and looks granular in origin.
• Nuclear envelope is distinct.
• Chromatin fibres appear in the form of an interconnected mass within the nucleus.
• Nucleolus is well observed inside the nucleus.

Stages of Mitosis

a) Prophase

• Nucleus is enlarged and occupies most of the cell volume. Intact nuclear outline is seen.
• Chromatin network gets condensed and appears as long thread-like structures called chromosomes.
• Nuclear membrane may start disappearing.
• Nucleoli may or may not be visible.
• If the cell is in early stage of prophase, then the chromatin fibres are very thin. However, in the cells at late prophase, comparatively thicker chromatin fibres would be visible. Aster and spindle fibres begin to appear.

b) Metaphase

• Chromosomes become shorter and thicker and hence become distinct and clearly visible under the compound microscope.
• Nuclear membrane completely disappears.
• Chromosomes orient themselves towards the equator with their centromeres arranged on an equatorial line forming metaphase plate. Out of two chromatids of each chromosome, one chromatid faces one pole and the other chromatid faces the opposite pole.
• Series of spindle fibres attach the centromeres to the opposite poles.
• Nucleolus is not observed during metaphase.

c) Anaphase

• The two sister chromatids of each chromosome separate due to the splitting of centromere and move towards the opposite poles.
• The daughter chromosomes (separated chromatids) appear in V, J, L and I shapes, depending upon the position of the centromere in the chromosomes.
• Anaphase is designated as early, mid and late stages depending upon the position of moving chromosomes toward the opposite poles.

d) Telophase

Chromosomes reach to the opposite poles, lose their individuality and look like a mass of chromatin again.

• Nuclear membrane reappears to form the nuclei of two future daughter cells.
• Nucleolus gets reconstituted.
• Two daughter nuclei are thus formed and appear to be similar to the parent nucleus both quantitatively and qualitatively.

Cytokinesis

In plant cells, cell plate is formed in the middle after telophase. The plate can be seen to extend outward to ultimately reach the margin of the cell and divide the cell into two equal halves. Such cell plates are characteristic of plant cells.

In animal cell, furrow appears in the membrane at both sides and grows inward to divide cells into two. Daughter cells are formed at the end of cytokinesis.

RESULT

All the stages of mitotic cell division are clearly visible in the slide prepared from onion root tips. Interphase, prophase, metaphase, anaphase and telophase followed by cytokinesis can be distinctly identified under compound microscope.

CONCLUSION

Onion root tip meristem is ideal material for studying mitosis because of active cell division. Mitosis is equational division producing two identical diploid daughter cells and is essential for growth and repair in organisms.

PRECAUTIONS

• Always clean the slide and cover slip thoroughly before and after use.
• Care should be taken that there should be no air bubbles under the cover slip.
• The base of the onion bulb should exactly be in contact with water, while growing the roots.
• Always filter the acetocarmine stain before use.
• Material should be mounted on the centre of slide.
• Also, the material should only be warmed gently, i.e. do not allow solution to boil on slide while warming.