Prepare a temporary mount of onion root tip to study mitosis

Aim: Prepare a temporary mount of onion root tip to study mitosis

Material / Apparatus Required:

• Onion bulbs
• Needles
• Acetocarmine stain
• Glass slide
• Conical flask to germinate onion root tips
• Brush
• Ethanol
• Blotting paper
• Cover slip
• Forceps
• Compound microscope
• Fixative
• Blade
• Dropper
• Corked vial/tube
• Watch glass
• N/10 HCL
• Burner
• Tile

Principle:

A single cell divides to make two cells and these two cells then divide to make four cells, and so on. We call this process "cell division" and "cell reproduction", because new cells are formed when old cell divides. The ability of cells to divide is unique for living organisms.

Cells divide for many reasons. For example, when the skin of our knee, cells divide to replace old, dead or damaged cells. Cells also divide to help living organism grow. Organisms only grow because cell divides to produce more and more cells. In cell division, the cells that is dividing is called the "parent cells". The parent cell divides into two "daughter cells". The process than repeats to form a cell cycle.

Depending on the type of the cell, there are two ways in which cell divide-mitosis and meiosis. Each of these methods of cell division has special characters. One of the key differences in mitosis is a single cell divides into two cells that are replicas of each other and have the same number of chromosomes. This type of cell division is good basic growth, maintenance and repair. In meiosis, a cell divides into cells that have half the number of chromosomes.

Mitosis cell division: Mitosis is how somatic cell divides. Somatic cells make up the most of our body tissue and organs including skin, muscles, lungs, gut, and hairs cells. In mitosis, each daughter cells have same chromosomes and DNA as parent cells. The daughter cells from mitosis are called diploid cells having two complete pair of chromosomes. Since the daughter cell have exact copies of their parent cells DNA, no genetic diversity is created through mitosis in normal healthy cells. Mitosis cell division creates two genetically identical daughter diploid cells.

Mitosis cell cycle: Before a cell start dividing, it is in the "Interphase". It seems that cell must be constantly dividing, but each cell actually spends most of its time in the interphase. Interphase is the period where cell prepare itself to divide and start the cell cycle. During this time, cell gather nutrients and energy. The parent cell also makes a copy of its DNA to share equally between the two daughter cells. The mitosis division process has several steps or phases of the cell cycle- Interphase, prophase, prometaphase, metaphase, anaphase, telophase, and cytokinesis- to successfully make the new diploid cells. The mitosis cell cycle includes several phases that result in two new diploid cells.

Procedure:

1. Take an onion and place it on the tile.
2. Carefully remove the dry roots present using sharp blade.
3. Grow root tips by placing the bulbs in a conical flask filled with water.
4. New roots may take 3-6 day to grow.
5. Cut off 2-3 cm of freshly grown roots and let them drop into a water glass.
6. Using a forecep, transfer them to vial containing freshly prepared fixative of aceto-alcohol (1: 3: glacial acetic acid: ethanol)
7. Keep the root tips in the fixative for 24 hours.
8. Using a forecep, take one root and place it on glass slide.
9. Using a dropper, place one drop of N/10 HCL on the root tips following 2-3 drops of acetocarmine stain.
10. Warm it slightly on burner. Care should be taken that the stain is not dried up.
11. Carefully blot the excess stain using blotting paper.
12. Using a blade, cut the comparatively more stained portion of the root, retain it on the slide and discard remaining portion.
13. After that, put one drop of water on the root tip.
14. Mount a coverslip on it.
15. Now slowly tap the coverslip slowly so that meristematic tissues of the root tip below the coverslip is properly squashed and spread as a thin layer of cells.
16. This prepared slide is now ready for the study of mitosis.
17. Place the slide under the compound microscope and observe the different stages of mitosis.
18. Various stages of mitosis are prophase, metaphase, anaphase and telophase.

Observations:

• Interphase: The nucleus is metabolically active during this stage and takes part in DNA replication, formation of macromolecules and other energy rich compounds. It is also referred as invisible phase of mitosis since in this stage chromosomes prepare themselves for equitable distribution later on.
• Karyokinesis: It is stage of division which includes following phases: -
1. Prophase: Coiling of chromatin occur, nucleolus gradually degenerates from early prophase to late prophase and nuclear envelope start disappearing in late prophase.
2. Metaphase: Nuclear membrane disappears completely, chromatids acquire a specific shape and size, moreover chromosomes are attached to poles by spindle fibers through centromere, at equatorial plates.
3. Anaphase: Centromere of each chromosome divides into two chromosomes split along longitudinal axis. Two chromatids with its own centromere start moving towards opposite poles. Chromosomes appear V-, L-, J- and I- shaped based upon the position of the centromere.
4. Telophase: It is reverse prophase. Chromosomes reorganize in nuclei and elongate to form chromatin. Spindle fiber disappears and nuclear envelope reappears.
• Cytokinesis: it is the division of protoplasm following the nuclear division. Normally, it start during middle anaphase and is completed simultaneously with telophase.

Result:

All the stages of mitotic cell division are clearly visible in the slide prepared from onion root tips.

Precautions:

1. The base of the onion should exactly be in contact with water, while growing the roots.
2. Filter the acetocarmine stain before use.
3. Clean the slide and coverslip thoroughly before use.
4. Avoid air bubble in the slide.