Study of Mitosis in Onion root tips.

All living organisms grow, differentiate and reproduce by cell division. There are two types of cell division
namely, mitosis and meiosis.
Mitosis occur in somatic cells and is known as duplication division. It results in 2 daughter cells with same
number of chromosomes.
Each mitotic cell division has two events.
1. Karyokinesis- Division of the nucleus.
2. Cytokinesis- Division of the cytoplasm.
The events that occur during karyokinesis are
1. Prophase- Appearance of chromatids and disappearance of nuclear membrane.
2. Metaphase- Spindle fibres are prominent, holding the chromatids at the
equatorial plane.
3. Anaphase- Movement of chromatids towards poles by splitting at the centromere.
4. Telophase- Formation of daughter nuclei.
The meristematic cells of plants provide suitable material for studying mitosis.
The root apices of onion is used in the experiment.

Aim: To prepare a stained squash of the onion root tip.

Materials required: Onion bulbs, Beaker, water, tooth pick or pins, specimen tubes, a pair of scissors, forceps,
mounting needle, methyl alcohol, acetic acid, hydrochloric acid acetocarmine, distilled water, spirit lamp,
slides, coverslip, blotting paper, compound microscope.

Procedure: Take medium sized onion bulbs and trim off the old roots from its base with a sharp pair of scissors.
Place the bulbs on the beakers filled with water with the base touching the water. Leave it undisturbed for the
new roots to develop. When the roots start growing, cut 5mm of the tips of roots and put them in a fixative
made up of 1:3 glacial acetic acid and ethanol. This will help preservation of the dividing tissues. The cutting
should be done in the morning between 7 and 8 AM.
Remove the root tips from the fixative and soften them by heating with 1NHCl at 60oC for 15 minutes.
Remove the tips and wash them in distilled water.
Place a drop of freshly prepared aceto carmine on the slide and place the root tip on it. Gently warm the stain on
a spirit lamp by holding the slide much above the flame.
Place a coverslip on the specimen and with the backside of the needle prepare a squash by gently running the
needle in one direction only. Observe the specimen under low and under high power and record your
observation.

Observation: Under low power, dividing cells are seen scattered. Under high power, the following stages appear
distinctly.
Interphase: The non-dividing phase where the chromatin appear as a network. Nuclear envelope is distinct.
Prophase: Chromatin condense into chromosomes as chromatids attached by centromere. Nuclear membrane
disintegrates.
Metaphase: Bipolar spindle develops and the chromatids appear at the central region of the cell.
Anaphase: The sister chromatids are pulled apart by the receeding spindle and appear as V, J, L or I shaped.
Telophase: The spindle disappears and the chromosomes assemble at the polar region. Nuclear membrane
reappears.
Cytokinesis: The two nucleated cells with cell plate at centers are observed.

Precautions:
1. Base of onion bulbs to be in contact with water while growing roots.
2. Root tips should be fixed in the morning.
3. The slide must not touch the flame while warming.
4. The rolling of needle should be done gently in one direction.
 Study of the action of salivary amylase on starch

Enzymes are biocatalysts required for metabolic activities in living organisms. The digestive enzymes belong to
the group namely, Hydrolases.
Saliva is secreted by the salivary glands. It is viscous, colourless with a specific gravity of 1.003 and the pH
ranges from 6.2 to 7.4.
It contains the enzyme salivary amylase or ptyalin that acts on starch and converts it into simple disaccharide
like maltose.
salivary
Starch maltose, isomaltose,limit dextrins.
amylase
Aim: To study the effect of salivary amylase on starch.
Materials required: Boiling tube, test tubes, test tube stand, test tube holder, measuring cylinder, dimpled tile,
pipette, funnel, beakers, droppers, cotton wool, burner, thermometer, water bath, iodine solution, soluble starch,
sodium chloride, Benedicts reagent, Fehlings A and B reagent, distilled water, warm water, labels and dimple
tile.
Procedure:
Repeat the same every 2 minutes and observe the colour change of the iodine solution.
Note the time when the colour change does not occur and record the observations.
This is known as the achromic point.
Observation:
The iodine in the dimple will change to blue-black initially.
The reaction may not occur after 6 or 8 minutes in the experimental
Preparation of 1% starch solution:
Take 1 gm of soluble starch in a beaker and add 10 ml distilled water. Stir well to make a paste. Add 90 ml of
hot distilled water and stir it. Boil the solution to completely dissolve the starch. Cool and filter the solution.

Preparation of 1% sodium chloride solution:

Take 10 gm of NaCL and make 100 ml of the solution with distilled water to get 1% NaCl solution. Na+ is the
cofactor of enzyme amylase.

Collection of saliva:
Rinse the mouth clean with water. Sip about 20 ml of luke-warm water and keep it in the mouth for 5 to 10
seconds. Gently collect the same in a boiling tube. This gives the correct dilution of the saliva. Label the tube.

Preparation of substrate tubes:

Take 2 clean test tubes and label A-Experimental and B-Control. Take 1 ml of starch solution and I ml of NaCl
solution in both the test tubes A and B. Place both in a hot water bath that is maintained at 37oC for about 10 to
15 minutes. Add 1 ml of saliva solution to A and 1ml of distilled water to B.

Take a dimple tile with 6 dimples. Label the dimples in linear order A- 0 minutes, A-2min , A-3 min.. and B-
0min, B-2 min, B-3 min and so on.
Add 2 drops of iodine solution to the labelled dimples.
Take 2 drops each from A and B and add to the dimples labelled A-0 min and B-0min respectively. Record the
colour turning blue black in a tabular column.
mixture.
The control will remain the same.
Note the time of the achromic point.
Confirm the reaction.by confirmatory tests.
Confirmatory Test:
To 2 ml of the enzyme-substrate mixture A, add 2 ml of Benedicts solution. Boil and observe the colour
change.
Repeat the test with Fehlings A and B solution.
Repeat the tests with the content in B.
Record your observation.
Inference:
The mixture when tested with the reagents change from green to yellow to orange to brick red confirming the
breakdown of starch to simple sugars.
The starch is broken down to maltose at the ________th minute.
The achromic point is _____________.

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Draw a tabular column with three columns, titled- time, experimental tube and control on the left page and
enter your recordings.
Study the effect of different temperature on the action of salivary amylase on starch

Enzymes typically operate best in a relatively narrow range of environmental conditions.

Many of the enzymes in our bodies work best at body temperature. At significantly lower temperatures the
substrate molecules do not have enough kinetic energy for the reaction to take place even in the presence of the
enzyme.

At body temperatures significantly higher than normal, the enzyme will not work well because

Hydrogen bonds are easily disrupted by increasing temperature. This, in turn, may disrupt the shape of
the enzyme so that its affinity for its substrate diminishes.
The kinetic energy from the molecules in the solution containing the enzyme is so high, that the
enzyme's shape is pulled apart to the point that the enzyme is not able to properly function.

Human enzymes generally work best at our body temperature-37oC.

Aim: To study the effect of different temperature on the action of salivary amylase on starch.
Materials required: Boiling tube, test tubes, test tube stand, test tube holder, measuring cylinder, pipette, funnel,
beakers, droppers, cotton wool, burner, thermometer, water bath, iodine solution, soluble starch, sodium
chloride, Benedicts reagent, Fehlings A and B reagent, distilled water, warm water, ice cubes, labels and
dimple tile.
Preparation of substrate tubes:
Take 6 clean test tubes and label A1, A2, A3-Experimental and B1, B2, B3-Control.
Take 1 ml of starch solution and I ml of NaCl solution in all the 6 test tubes.
Prepare three water bath- 80oC, 370C and one with ice cubes.
Place A1 and B1in water bath 80oC, A2 and B2 in 370C and A3 and B3 on ice cubes for about 10 to 15 minutes.
Add 1 ml of saliva solution to A series and 1ml of distilled water to B series.
Procedure:
Take a dimple tile with 6 dimples.
Label them in linear order A1, A2, A3 and B1, B2, B3.
Add 2 drops of iodine solution to the labelled dimples.
Take 2 drops each from A and B and add to the dimples labelled A1 and B1 respectively. Note the time as 0
minute.
Clean the dimple tiles and repeat the same every 3 or 4 minutes and observe the colour change of the iodine
solution.
Note the time when the colour change does not occur and record the observations.
This is known as the achromic point.
Observation:
The iodine in the dimple will change to blue-black initially.
The reaction may not occur after 6 or 8 minutes in the experimental mixture A2.
The A1, A3 and the control B serieswill remain the same.
Note the time of the achromic point.
Confirm the reaction.
Inference:
The mixture when tested with the reagents change from green to yellow to orange to brick red confirming the
breakdown of starch to simple sugars.
The breakdown of starch to educing sugar occurred only in A2 as it provides the optimum temperature for the
reaction.
High temperature-80oC, will denature the enzyme and low temperature will inhibit the enzyme action.
The optimum temperature for salivary amylase is 370C.
Study the effect of different pH on the action of salivary amylase on starch

The activity of enzymes is strongly affected by changes in pH.

Each enzyme works best at a certain pH, its activity decreasing at values above and below that point.
Examples:
the protease pepsin works best as a pH of 12 (found in the stomach) while
the protease trypsin is inactive at such a low pH but very active at a pH of 8 (found in the small
intestine as the bicarbonate of the pancreatic fluid neutralizes the arriving stomach contents).

Changes in pH alter the state of ionization of charged amino acids that may play a crucial role in substrate
binding and/or the catalytic action itself.

Aim: To study the effect of different pH on the action of salivary amylase on starch.
Materials required: Boiling tube, test tubes, test tube stand, test tube holder, measuring cylinder, pipette, funnel,
beakers, droppers, cotton wool, burner, thermometer, water bath, iodine solution, soluble starch, sodium
chloride, Benedicts reagent, Fehlings A and B reagent, distilled water, warm water, pH tablets, labels and
dimple tile.
Preparation of substrate tubes:
Take 6 clean test tubes and label A1, A2, A3-Experimental and B1, B2, B3-Control.
Take 1 ml of starch solution and I ml of NaCl solution in all the 6 test tubes.
Place all the test tubes in a hot water bath that is maintained at 37oC for about 10 to 15 minutes.
Add pH2 (acidic) to A1 and B1, pH7 (neutral) to A2 and B2, pH10 (alkaline) to A3 and B3
Add 1 ml of saliva solution to A series and 1ml of distilled water to B series.
Procedure:
Take a dimple tile with 6 dimples.
Label them in linear order A1, A2, A3 and B1, B2, B3.
Add 2 drops of iodine solution to the labelled dimples.
Take 2 drops each from A and B and add to the dimples labelled A1 and B1 respectively. Note the time as 0
minute.
Clean the dimple tiles and repeat the same every 3 or 4 minutes and observe the colour change of the iodine
solution.
Note the time when the colour change does not occur and record the observations.
This is known as the achromic point.
Observation:
The iodine in the dimple will change to blue-black initially.
The reaction may not occur after 6 or 8 minutes in the experimental mixture A2.
The A1, A3 and the control B series will remain the same.
Note the time of the achromic point.
Confirm the reaction.
Inference:
The mixture when tested with the reagents change from green to yellow to orange to brick red confirming the
breakdown of starch to simple sugars.
The breakdown of starch to reducing sugar occurred only in A2 as it provides the optimum pH for the reaction.
The optimum pH for salivary amylase is pH 7 (neutral).