Experiments on mitotic and meiotic cell division in animals and plants.
Experiment on Mitotic Cell Division:
To prepare a slide of root tip of onion by squash method arid to identify, study and draw the various mitotic stages.
Material required:
Onion root tips fixed in Carnoy fluid (6 parts absolute alc + 3 parts glacial acetic acid + 1 part Chloroform); Aceto carmine stain; slides; cover slip; blotting paper; spirit lamp; watch glass; 1 -N HCI, 45% Acetic acid and filter paper.
Procedure:
Take large sized mature onions about a week before the experiment. Cut all the dried roots from the stem at the base of the bulb. Now place these onions on the mouth of couplin jars filled with water in such a way that the stem portion must be dipped continuously in water.
In about a weeks’ time new roots would develop and would start growing downward in the water. Take the onions and cut the milky white portion of root tips viz., up to 5 mm length. Cut each piece into smaller pieces and fix them in Carnoy fixative for half an hour.
Transfer the material to 90% alcohol and then to 70% alcohol keeping the material for 10 minutes in each. Now preserve the material in 70% alcohol. At the time of proceeding for experiment in the laboratory take few pieces from 70% alcohol in a watch glass in few drops of 1- N HCI and leave for 5 minutes. By this procedure the material would become soft.
Now drain off the HCI and wash the pieces with a little distilled water at least twice or thrice. Now put the root tips on a clear slide on right hand side and pour a few drops of 2% Acetocarmine. Warm the slide gently on sprit lamp for few minutes at least 3-4 times but never let it boil.
Cool and leave for 10 minutes in the stain. Now drain the excess of stain with the help of filter paper and put few drops of 45% acetic acid. Place a cover slip over the material and put above the coverslip a piece of blotting paper or filter paper folded two to three times. Press the coverslip gently with your thumb to break the cell membranes.
The blotting paper or filter paper would save the coverslip from breaking and would also absorb the excess stain which will ooze out from the coverslip. Take care that your thumb may not move sideways while pressing. Seal the coverslip with nail polish if you want to keep the slide for some time.
Fix the slide under low power of microscope and focus. Now change to high power and observe the various stages which would appear as shown in the attached photographs. Draw the various stages and write down comments on each of them.
Experiment on Meiotic Cell Division:
To prepare a slide of meiotic stages through squash method, from testis of Chironomus larva of grass hopper, buds of Tradescantia or spikes of Bajra.
Material:
Buds of Tradescantia or Flax or immature spikelet’s of Bajra or Testes of Grasshopper or Chironomous larva or rat, carnoy fluid; Acetocarmine; needles; slide; watch glass; 45% Acetic acid; coverslip; filter paper or blotting paper; spirit lamp; normal saline; 1% sodium citrate; and 2.2% Trisodium citrate.
Procedure – 1: (for grass hopper testis):
Dissect out testes of grasshopper and keep in a watch glass in saline. Transfer them into another watch glass containing 1.0 ml of fresh sodium citrate solution so that their nuclei may swell up and chromatids may spread over larger area.
Transfer the testes into carnoy fluid for 20 minutes to 1 hr. Dehydrate the testes through 90% and 70% alcohol for 10 minutes each. Now from 70% alcohol transfer the testes to 2% acetocarmine solution for 20 minutes. Drain off the excess stain and pour 45% acetic acid and leave the testes in it for 10 minutes.
Now, place one testicular follicle on a clean slide in 45% acetic acid and put a cover slip over it. Put a piece of filter or blotting paper over the coverslip and put your thumb on the coverslip and gently press the coverslip. The excess fluid shall be blotted off by filter paper. Now seal the coverslip with nail polish and observe under microscope.
Procedure – 2 (For rat testis):
(1) Kill or anesthetize the rat and dissect out its testis. Place it in a 2″ patri dish containing isotonic 2.2% trisodium citrate solution.
(2) Pierce tunica & expose tubules and shake or spin them in isotonic solution to wash away fat.
(3) Transfer these tubules to another dish containing 3 ml of fresh isotonic solution and tease tubules with forceps or needles. Transfer contents of dish gently to a 4 ml centrifuge tube with the help of a pipette. Allow to stand for 10 minutes.
(4) Spin for 5 seconds in centrifuge to sediment larger tubule segments. Transfer supernatant to a clean 4 ml centrifuge tube and discard sediment.
(5) Spin for 5 min at 500 rev/min. Discard supernatant and re-suspend pellet in minimum volume of residual supernatant.
(6) Slowly add 3 ml of hypotonic (1% Trisodium nitrate) solution to the tube. Meanwhile flicking it with forefinger, divide the fluid (solution) between two narrow conical tipped tubes of 2 ml capacity.
(7) Spin for 5 min at 500 rev/min. Pour off supernatant. Let tubes stand for 1 min and then remove, with a fine pipette, the residual fluid that drains down the walls. Make up fixative (4.5 ml abs. ale +1.5 ml acetic acid +0.1 ml chloroform) while the material is in the centrifuge.
(8) Flick the tube repeatedly so as to disperse pellet as a dense suspension. Allow two drops of fixative to fail directly on to cells and then flick tube vigorously Add more fixative until the centrifuge tube is about 3/4 full. Meanwhile continue to flick the tube. Spin for 3 min at 500 rev/min. Change fixative then spin again and change fixative twice more.
(9) The final suspension of cells in fixative should be diluted. Let it stand for 1-3 hrs before making preparation.
(10) Take cell suspension into a fine pipette and put 3 drops at even space on a clean slide. Allow the fluid to spread and then blow it gently to dry. Repeat till there are sufficient cells on the slide.
(11) As soon as dry it may be stained with Lactic-acetic orcein 2 gm. Orcein in 50 ml acetic acid and 50 ml lactic acid. Filter, leave over night and filter again. Dilute with 50% distilled water.
Procedure – 3 (For plant material):
Take young buds of Tradescantia or Flax or immature male spikelet’s of Bajra (Millet) and fix them in freshly prepared Carnoy’s fluid for 10 hrs. After rehydrating in 90% and 70% alcohol store the material in 70% alcohol.
(1) Take a bud of Tradescantia or few smaller anthers from the male spikelet of Bajra in 70% alcohol in a watch glass.
(2) Dissect out the bud or dissect each anther taken from the spikelet with needles & keep them in 45% acetic acid.
(3) Put few anthers on the slide and few drops of Acetocarmlne solution over them.
(4) Warm the slide gently a few times, but never let it boil.
(5) Drain off excess stain and put few drops of 45% acetic acid.
(6) Now put coverslip over the material and cover it with filter paper or blotting paper folded 2 to 3 times.
(7) Press the coverslip with your thumb so as to rupture the anthers.
(8) Observe under the microscope first in low power and then in high power.
Draw the various stages and write down the comments (Fig. 27):
The slide prepared by you might show the following stages:
