Objective. To prepare temporary acetocarmine stained mount of onion roo! tip to study various stages of mitosis.#EQUIREMENTS Onion bulbs, conical flasks/glass bottles, corked vial/tube, petridishes, scissors, forceps, needles, methyl alcohol, acetic acid, hydrochloric acid, acetocarmine, distilled water, spirit amp, microscope, slides, coverslips, blotting paper ete. 1. Take a medium sized bulb of onion and trim off the old roots from its base by means of a sharp blade. 2, Place the onion on a conical flask/glass bottle full of water, with its base touching the water. Keep it for a week to grow the roots. 3. Cut 5 mm off the tips of roots and put them into a vial containing a mixture of 1 : 3 acetic acid and methanol. Keep for one hour. This process is called fixation. (Cutting of root tips should be done in the morning between 7.00 a.m. to 8.00 a.m. during the summer and between 9.30 a.m. to 11.30 a.m. during the winter). 4, Remove 2 or 3 root tips and hydrolyse them by warming to 60°C in 1 N hydrochloric acid for 15 minutes. Fig. 6.1. Method of growing onion root tips. 5. Remove the root tips and wash them thoroughly in water. 6. Place a drop of acetocarmine on a slide. Put one hydrolysed root tip in a drop and place a coverslip on the root. 7. Gently squash the root by tapping the coverslip with the blunt end of a pencil or needle until the cells separate and spread out into a very thin layer. Make sure that there are no air bubbles under the coverslip. 8 Gently warm the slide over a flame for a few seconds. 9. Observe first under the low power of the microscope to locate the dividing cells. Exam- ine the different stages of mitosis under the high power of the microscope. ~~ OBSERVATIONS Under low power of the microscope, rectangular cells with pink nucleus are seen ssttored. Under high power of the microscope following stages become distinet: (Figs. 6.2 6.3)44 Maturation zone (HY WE HI Mi Prophase Zono of elongation Photaphase Moristomatic ono Tolopte Anaphass Intorphiase not tip. Fig. 6.2. Different stages of mitonin in the onion 10 1. Interphase (i) 11 is a non-dividing phase of the cell eycle between two fue (i) Chromatin fibres appear in the form of a network within U (iii) Nuclear envelope and nucleolus are distinct. y nucleus, 2, Prophase (® Chromatin material shortens and condenses into thread like structures called chromosomes. at a point called centromere. (ii) Each chromosome consists of two chromatids, jointed (iii) Nuclear membrane and nucleolus start disintegration and disappear at the end of prophase. 3. Metaphase (i) A bipolar spindle develops in the cell. Chromosomes become thick and two chromatids of each chromosome become clear. (ii) Chromosomes become arranged at the equator of the spindle. (ii) Each chromosome get attached to the spindle fibres at its centromere. 4, Anaphase (i) The two sister chromatids of each chromosome separate from the centromere and move towards the opposite poles. (ii) The daughter chromosomes (separated chromatids) appear V, J, L and I shapes, depending upon the position of centromere. 5. Telophase (i) The spindle disappears and the daughter chromosomes uncoil to form chromatin fibres at the two poles. (i) Nuclear membrane and nucleolus reappears and two daughter nuclei ap t pear opposite poles. ) Cytokinesis occurs by cell plate formation between the two daughter nuclei (iii:Interphase stage [__cell wall P Nuclear membrane | — Nucleolus | —— Chromosomes Spindle fibres | —— Chromatids Cell wall Chromosomes (Chromatids) Spindle fibres Late anaphase fig. pe Nuclear |_— membrane Chromatin fibres -— Nucleolus Cell membrane Nuclear membrane |__ Disappearing nucleolus, |—— Chromosomes |— Cell wall Late prophase Me. A, Daughter As ‘chromosomes, OND | NU | Spindle fibres aye Early anaphase Daughter cells membrane Telophase stage 6.3. Various stages of mitosis in onion root tip cells.