To Prepare a Temporary Mount of Onion Root Tip to Study Mitosis.

Aim:-

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

REQUIRMENT:-

Onion bulbs, corked via/tube, conical flasks/glass bottles, scissors, Petri dishes, forceps, needle, acetic acid, methyl alcohol, hydrochloric acid, acetocarmine, distilled water, spirit lamp, microscope, slides, coverslip, blotting paper, etc.

PROCEDURE:-

1.    Take a medium sized bulb of onion and trim off the old roots from its base by means of sharp blade. 

2.    Place the onion on a conical flask/glass bottle full of water, with its base touching the water.

3.    Keep it for a week to grow the roots.

4.    Cut 5 mm off the tips of roots and put them into a vial containing a mixture of 1 : 3 acetic acid and methanol. deep for one hour. This process is called "fixation".

5.    Remove 2 or 3 root tips and hydrolyses them by warming to 60' C in 1 N HCl acid for 15 minutes.

6.    Remove the root tips and wash them thoroughly in water.

7.    Transfer a drop of acetocarmine on slide. Put one hydrolyses rood tip in a drop and place a coverslip on the root.

8.     Gently squash the root by tapping the coverslip with the blunt end of a pencils or needle until the cells separate and spread out into a very thin layer.

9.    Gently warm the glass slide over a flame for some time.

10.    Observe first under the microscope to locate the dividing cells.

11.    Examine the different stages of mitosis under the microscope. 

OBSERVATIONS:-

Under low power of microscope, rectangular cells with pink nucleus are seen scattered. 

Under high power of microscope following stages become distinct:

1.    Interphase:     

This stage is identified by:

a)     It is the non-dividing phase/stage of the cycle.

b)     Nuclear covered and nucleolus are distinct.

c)     Chromatin fibers appear in the form of network within the nucleus.

2.    Prophase:

a)     Last stage of the prophase when the nucleolus and nuclear membrane disappear. The cellular contents condense into a spindle.

b)     Chromatin material shortens and condenses into thread like structures called Chromosomes.

c)     Each chromosome consists of two chromatids which are jointed at point called centromere.

d)     The centriole divides and shifts to the opposite poles along with astral rays.

3.    Metaphase:

a)    A bipolar, spindle develops in the cell.

b)     Chromosomes become thick and two chromatids of each chromosomes become clear.

c)     Chromosomes become arranged at the equator of the spindle.

d)     Each chromosome gets attached to the spindle fibers at its centromere.

4.    Anaphase:

a)    The two sister chromatids of each chromosome separate from the centromere and move towards the opposite poles.

b)    The daughter chromosomes (separated chromatids) appear V.J.I. and I Shapes, depending upon the position of centromere.

c)     The arms of chromosomes are towards the equator.

d)     Slight depression is seen on the sides of the cell.

5.    Telophase:

a)     The spindle disappears, and the daughters chromosomes uncoil and lengthen to form chromatin fibers at two poles.

b)     Nuclear membrane and nucleolus reappear and two daughter nuclei appear at opposite poles.

c)    Chromosomes become a mass of jumbled network as it was before the non-dividing stage.

6.     Cytokinesis: 

a)     Cell plate formation takes place between the two daughter nuclei.

b)     Two daughter cells are formed in this stage.

PRECAUTIONS:-

1.    Do not touch the slide with objective lens while observation.

2.    The slides are studied under high power: use fine adjustment to focus the slide.

3.    The base of the onion bulb should be in contact of water while growing the roots.

4.    Root tips should be fixed in the morning between 8 to 10 a.m.

5.    The root tips should be washed thoroughly after boiling with dil. HCl. otherwise the cells do not get stained.

6.    The slide should be warmed gently much above the flame of the sprit lamp.

7.    There should be no air bubble in the slide.