Physiology Practical procedures

1. 1
SOPs: BOTANY PRACTICAL EXERCISES OF UNDERGRADUATE
STUDENTS
(DBT Life Sciences Star College Scheme, Ministry of Science &
Technology, Govt. of India)
Reaccredited with ‘A’ grade by NAAC
Compiled by
Ms. Prabhjyoti
Department of Botany
DOABA COLLEGE, JALANDHAR – 144 00 PUNJAB

2. 2
SOPS
of
B.Sc. Medical (Semester-V)
Subject : Botany
Paper: Plant Physiology, Biochemistry and Plant Biotechnology

3. 3
CONTENTS
1. To study the permeability of Plasma membrane using different concentrations of organic
solvents.
2. To study the effect of Temperature on permeability of Plasma membrane.
3. Determining the osmotic potential of vacuolar sap by plasmolytic method.
4. Determining the water potential of any tuber (Potato tuber).
5. To demonstrate the ascent of sap using a dye.
6. To demonstrate the Transpiration pull by mercury method.
7. Demonstration of Osmosis by Potato osmoscope.
8. Comparison of loss of water from two surfaces of leaf by CoCl2 method/ four leaf method.
9. Demonstration of imbibition pressure by plaster of Paris method.
10. Demonstration of O2 is evolved during photosynthesis in an aquatic plant.
11. Demonstration of Phototropism movements.
12. To demonstrate the measurements of growth by arc auxanometer.
13. To study the activity of enzyme catalase as influenced by PH & temperature.
14. To study the activity of enzyme peroxidase as influenced by PH & temperature.
15. Seperation of chloroplast pigments by solvent method.
16. Seperation of pigments by Paper chromatography.
17. Seperation of amino acids in a mixtures by paper chromatography & their identification by
comparison with standards.
18. Write down the precautions observed during aseptic manipulation of Tissue culture
laboratory.
19. Write down the equipments commonly used in Tissue culture laboratory.

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Experiment 1: To study the permeability of Plasma membrane using different
concentrations of organic solvents.
Requirements: Fresh Beet root, test tubes, cork borer, beaker, blade or cutter, measuring
cylinder, alcohol, formalene, benzene & tap water.
Procedure:
1. Take a large fresh beet root, wash it thoroughly under tap water.
2. With the help of cork borer , cut uniform cylinders of beet root.
3. With the help of sharp blade cut cylinders into equal size disc and edges of the disc
should be uniform.
4. Wash the discs repeatedly with distilled water till the pigments start diffusing into water.
5. Put 3 discs in each of the test tube labeled as A,B,C,D.
6. Added 5ml,10ml,15ml of 100%,80% & 50% alcohol in each of test tubes.
7. Take another set of 3 test tubes labeled as X,Y,Z & added 5ml,10ml& 15ml of 40%,30%
&20% formalene in each test tube.
8. Added 3 discs of the beet root in each tet tube.
9. Take another set of test tubes labeled as 1,2,3 & added 5ml ,10ml& 15ml benzene
respectively.
10. Added 3 beet root discs in each test tube containing benzene.
11. Keep all the sets of test tubes at room temperature for 15 mins.
12. After 15 or 20 mins. Shake them & observe the leaching of pigments in the solution.
Precautions:
1. Beet root should be fresh & large in size.
2. Beet root discs should be washed thoroughly
3. Edges of the disc should be of same size.
4. Number of discs in each test tube should be same.
5. Test tubes should be shaken thoroughly.

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Experiment 2: To study the effect of Temperature on permeability of Plasma membrane.
Requirements: Fresh Beet root, test tubes, cork borer, measuring cylinder, boiled water, ice
cubes, spirit lamp, tripod stand & test tube stand.
Procedure:
1. Take a large fresh beet root, wash it thoroughly under tap water.
2. With the help of cork borer , cut uniform cylinders of beet root.
3. With the help of sharp blade cut cylinders into equal size disc and edges of the disc
should be uniform.
4. Wash the discs repeatedly with distilled water till the pigments start diffusing into water.
5. Put 3 discs in each of the test tubes in 3 different water samples.
6. In the 1st
test tube the ice cold water is added.
7. In the 2nd
test tube add water that should be at room temperature.
8. In the 3rd
test tube boiling water is added.
9. Put these test tubes aside and observe after 20 mins.
10. Note the result & effect of temperature on the permeability of plasma membrane.
Precautions:
1. Beet root should be fresh & large in size.
2. Beet root discs should be washed thoroughly
3. Edges of the disc should be of same size.
4. Number of discs in each test tube should be same.
5. Test tubes should be shaken thoroughly.

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Experiment 3: Determining the osmotic potential of vacuolar sap by plasmolytic method.
Requirements: Leaves of Rhoeo discolor, Tradescantia leaves, different concentrations of sugar
solutions, slides, microscope, coverslips etc.
Theory:
Plasmolysis: Plasmolysis is defined as the shrinkage of protoplasm of the cell from the cell wall
when the cell is emersed in the solution which has a higher solute concentration
than the cell sap.
Osmotic potential: Osmotic potential is defined as the excessive hydrostatic pressure which
must be applied to it in order to make its water potential equal to that of pure
water.
O.P=C.R.T
Where,
O.P= Osmotic potential
C = molar concentrations
R = gas constant
T = absolute temperature
Procedure:
1. Peel off a small segment from lower surface of Leaf bytearing the leaf obliquely with
single jerk by scrapping it with safety blade.
2. Mounted the peel in a drop of water on slide and then placed a coverslip and observe
under microscope.
3. Make different molar concentrations of Sugar solution such as: 2.0M, 4.0M, 6.0M,8.0M
etc.
4. Take another peel and similarly mounted the peel of Tradescantia in a drop of Sugar
solution of different concentrations on different slides.
5. After 30 minutes observe each preparation under microscope.
Conclusion:
1. This appear to be a normal condition where cell sap presses the protoplasm against
the cell wall which is slightly inflated.
2. The withdrawn of cell content is due to the loss of water from the cell causing
vacuole to shrink. The small space between cell wall & cell content indicate the
beginning of plasmolysis.
3. With increasing the concentration of Sugar solution the space between cell wall &
cell contents widens.
4. Finally cell contents collected on one side & the cell is called plasmolysed.

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Precautions:
1. Use only fresh leaves.
2. Do not allow the material to dry up.
3. The strip should be ultra thin preferably made up of single layer of cells.
4. Dip the strip in solution completely.
5. Use brushes for transfer of strips.
6. Dry the brush before use in different solution.

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Experiment4: Determining the water potential of any tuber (Potato tuber).
Requirements: Peeled potato, Sugar solution of different molar concentrations (1.0M, 2.0M,
3.0M ,4.0M, 5.0M), Methylene blue, test tubes, pipette, dropper, forcep, cork
borer, test tube stand etc.
Water potential: Water potential is the difference in the chemical potential & free energyper
unit molal volume of water in a system (µw) and that of pure water(µwº) at
the same temperature.
Procedure:
1. Weigh 34.2 gm of sugar & dissolve in 100ml of distilled water.
2. Take 10 test tubes and make different molar concentrations of Sugar solution in them.
3. Then cut small cylindrical pieces of Potato of about 2cm in size. Dried these Potato
pieces between folds of filter paper.
4. Among two sets of test tubes with molar concentrations, in one set put small pieces of
Potato tuber.
5. Now, add some drops of Methylene blue till the solution becomes sufficiently blue.
6. Then place these test tubes undisturbed for about 20 minutes.
7. Put a drop of coloured solution into the test tube of corresponding molar concentration.
8. Now observe the movement of drop in Sugar solution whether it moves up & down or
simply diffuses.
Observations:
1. If a drop rises, the test tube solution has decrease in density over its initial
concentration.
2. If a drop falls down, it means density of test tube solution has increased.
3. If a drop diffuse out, it means the test tube solution has not change its density &
record your reading.

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Experiment 5: To demonstrate the ascent of sap using a dye.
Requirements: Two fresh leafy shoots cut under water, razor or sharp edged knife, a needle, two
beakers, stand, water, Eosine solution, plane slide, coverslip, Microscope etc.
Ascent of sap: The upward movement of water and the dissolved substances from roots to the all
aerial plant parts through xylem is called Ascent of sap.
Procedure1:
1. Take a leafy shoot freshly cut under water & dip the cut end inEosine solution taken in a
beaker.
2. Adjust the leafy shoot in the stand& keep it for 30-40 minutes.
3. Cut a transverse section of the leafy shoot.
4. Mount the section in a drop of glycerine, put a coverslip & observe under the microscope.
Observations: Red coloured xylem elements will indicate the path of coloured solution.
Conclusion: The ascent of sap takes place through xylem.
Procedure 2:
1. Cut the leafy shoots under water with the help of a sharp edged blade.
2. Remove pith of one ugly shout with the help of a needle.
3. Remove xylem in the middle from the 2nd
leafy shoot.
4. Adjust both of these shoots in stand so that their ends dip in water taken in two beakers.
5. Observe the shoots after 3-4 hours.
Observations: The leaves of the 1st
leafy shoot remain turgid but those of the 2nd
shoot would
get wilted.
Conclusion: The turgidity of leaves in the1st case is due to the continuity of water column in the
intact xylem but as this xylem is continued the 2nd
shoot, the leaves wilt due to non
availability of water.

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Experiment 6 : To demonstrate the Transpiration pull by mercury method.
Requirements: A leafy twig, cork, a long narrow glass tube wax, mercury water, beaker ,stand
etc.
Transpiration pull: A strain or tension developed in the water column in the xylem elements
due to transpiration is called Transpiration pull.
Procedure:
1. Cut a leafy shoot & fix it in the one end of a narrow glass tube filled with water with the
help of a cork.
2. Apply wax to seal the joint.
3. Cover the other end of the glass tube with your finger & adjust it upright in a petridish
containing mercury with the help of a stand.
4. Set the apparatus in bright sunlight.
5. Observe the rise of mercury in the glass tube.
Observations:The length of water column in the glass tube decreases & mercury rises in the
glass tube.
Conclusion: The water is lost to the atmosphere due to Transpiration by a leafy shoot & suction
so created in the water column results in the rise of mercury in the narrow glass
tube. This is called Transpiration pull.
Precautions:
1. Take the leafy shoot from a vigorously transpiring plant.
2. Set the apparatus in bright sunlight.
3. Check properly the joint should be air tight.

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Experiment 7: Demonstration of Osmosis by Potato osmoscope.
Requirements: Potato tuber, water, Sugar solution, beakers, petriplates, common pins, cork
borer, filter paper, razor, scalpel etc.
Osmosis: Osmosis is the phenomenon of movement of solvent molecules from their higher
concentration to lower concentration or from a solution of low concentration to high
concentration through semipermeable membrane.
A semipermeable membrane is a membrane which allows only solvent molecules to pass
through it but not the solute molecules.
Osmosis is of two types: 1. Exosmosis 2. Endosmosis
1. Exosmosis: It is the movement of solvent molecues from a
plant cell , when it is placed in a hypertonic solution.
2. Endosmosis: It is the movement of solvent molecules into a
plant cell, when it is placed in a hypotonic solution.
Procedure:
1. Take a large potato tuber & peel off its outer skin with the help of a scalpel.
2. Cut its one end flat & make a cavity in the potato tuber almost up to the bottom with the
help of a cork borer.
3. Put some sugar solution to the half of the cavity of the potato tuber & mark the level with
the help of a common pin.
4. Put the potato osmometer-1 in a petriplate containing water & keep it undisturbed for
sometime.
5. Similarly prepare another potato osmometer-2 & pour water to the half of its cavity &
mark the level with the help of common pin.
6. Place this osmometer in a petriplate with sugar solution & keep it as such for sometime.
Observations: In the 1st
potato osmometer, the level of sugar solution will rise up, where as in
the 2nd
potato osmometer, the lev el of water will fall down.
Conclusion:
1. The level of sugar solution in the 1st
potato osmometer rises up due to the process of
Endosmosis.
2. The level of water in the 2nd
potato osmometer falls down due to Exosmosis.
Precautions:
1. Potato tuber should be peeled off because its periderm is impermeable to water.

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2. Bottom of the potato should be flat.
3. The cavity should be deep.
4. The sugar solution should be concentrated.
5. Mark the initial level carefully.

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Experiment 8: Comparison of loss of water from two surfaces of leaf by CoCl2
method/ four leaf method.
I. CoCl2 method
Requirements: Potted plant, Cobalt chloride solution, filter paper, beakers,
desiccators,glass slides, thread etc.
Procedure:
1. Prepare 3% cobalt chloride solution in a beaker.
2. Dip filter paper strips in it, squeeze out excess of solution.
3. Dry the strips & keep them in the desiccators.
4. Take a potted plant & select a healthy leaf.
5. Place dry cobalt chloride treated filter strips on both surfaces of this leaf &
immediately cover them with two glass slides.
6. Tie the slides firmly with thread.
7. Note the time taken for the change of colour of strip on both sides.
Observations: The blue cobalt strip of lower epidermis turns pink first.
CoCl2 + H2O → CoCl2 .2H2O OR CoCl2 .4H2O
Unhydrated cobalt chloride Hydrated cobalt chloride
Blue in colour Pink in colour
Conclusions: The time taken for the change of colour of CoCl2 strip on the lower
epidermal surface is less as compare to the upper surface because of
more number of stomata and hence high rate of transpiration.
Precautions:
1. Keep the dried CoCl2 strips in a dessicator.
2. Select a healthy & dry leaf for experiment.
3. Bind the slides immediately after placing strips on the sides of leaf.

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II. Four leaf method
Requirements: Four leaves of Ficus religiosa (Peepal), Vaseline, thread, iron
stands, physical balance, forceps etc.
Theory: The rate of transpiration is directly perportional to the number of stomata
present on the leaf surface.
Procedure:
1. Take four healthy& young Peepal leaves of almost equal size.
2. Apply Vaseline to their cut ends.
3. Tie all these four leaves with a help of thread & label them as A, B, C & D.
4. Apply Vaseline to the upper surface of leaf ‘A’, lower surface of leaf ‘B’, both
surfaces of leaf ‘C’ & apply no Vaseline on leaf ‘D’.
5. Note the weight of each of these four leaves one by one.
6. Hang these leaves with the help of iron stands & keep them in sunlight for few
hours.
7. Observe the leaves carefully one by one & note down their final weight.
Observations & Results:
Sr.No. Name of Leaf Initial
weight(gm)
Final
weight(gm)
1 Leaf ‘A’
2 Leaf ‘B’
3 Leaf ‘C’
4 Leaf ‘D’
i. The leaf ‘D’ wilts first followed by leaf ‘A’ & leaf ‘B’.
ii. The maximum loss of weight is observed in leaf ‘D’ followed by leaf ‘A’.
iii.The change in weight is almost nil in case of leaf ‘C’& is negligible in leaf ‘B’.
Conclusion:
i. The no. of stomata are more on the lowersurface of leaf as compare to
that of upper surface & so rate of transpiration is higher in leaf ‘A’ as
compare to that of leaf ‘B’.
ii. The Vaseline blocks the stomata in leaf ‘C’ that does not show change in
weight or wilting.
iii. The leaf ‘D’ shows maximum loss due to transpiration fom both the
surfaces.
Precautions:
i. Apply Vaseline smoothly & thoroughly on the assigned leaf surface.
ii. Block the cut end of the petiole with Vaseline.
iii. Keep the leaves in bright sunlight.
Experiment 9 : Demonstration of imbibition pressure by plaster of Paris
method.

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Requirements: Seeds of Gram or Pea, water, petridish, beaker, plaster of Paris
(Calcium sulphate), funnel, filter paper etc.
Theory: The pressure developed due to the adsorption of water by an imbibant
submerged in pure imbibing solvent is called imbibition pressure.
Procedure:
1. Weigh 100 gm of plaster of Paris & add 40ml of distilled water to make its paste.
2. Fix a cone of filter paper into a funnel & pour the paste in it.
3. Place few seeds of Gram or Pea over it & add more paste to cover all the seeds
properly.
4. Keep the funnel undisturbed till cone of plaster of Paris hardness.
5. Pour water to fill the half of petridish & keep the hardened cone in it.
6. Observe the change after about 1 hour.
Observations: Cracks develop in the cone of plaster of Paris due to imbibitional
pressure developed by the swollen seeds.
Precautions:
1. Always prepare fresh paste before starting the experiment.
2. Take dry & viable seeds.
3. Allow the plaster of Paris to set properly before putting it in water.
Experiment 10 : Demonstration of O2 is evolved during photosynthesis in an aquatic plant.

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Requirements: Fresh Hydrilla plant, beaker,funnel, water, test tube, Sodium bicarbonate,
match box etc.
Theory: Oxygen gas helps in burning.
Procedure:
1. Place a fresh Hydrilla plant in a funnel so that the cut end of the plant points upwards in
its stem.
2. Take a beaker & place the funnel with plant at its bottom.
3. Fill the beaker with water to cover the stem of the funnel.
4. Add some Sodium carbonate in water.
5. Invert a test tube full of water over the funnel & place the apparatus in bright sunlight.
Observations:
The gas bubbles are seen collecting in the test tube. When sufficient gas has been
collected, remove the test tube & introduce a burning match stick which will burn
brightly to show that the gas collected in tube is oxygen.
Conclusion : Oxygen gas is evolved during photosynthesis.
chlorophyll
6CO2 + 6H2O →→→ C6H12O6 + O2 ↑
Sunlight
Precautions:
1. Dip the stem of the funnel completely in water.
2. Take fresh Hydrilla plant.
3. Set the apparatus in bright sunlight.
4. The cut end of the plant should point upwards in the stem of the funnel.
Experiment 11 : Demonstration of Phototropism movements.

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Requirements: A potted plant, Phototrophic chamber.
Theory: Phototropism is the movement of plant organs towards the stimulus of light. Stem
shows positive phototropic response & root shows negative.
Phototropic chamber: The phototropic chamber is an ordinary wooden box with removable top.
It has a hole on one side for the supply of unidirectional light. The inside
of the chamber is painted black to prevent the internal reflection of light.
Procedure:
1. Take a potted plant.
2. Water it properly.
3. Place the plant in phototropic chamber.
4. Keep the chamber near a window or provide an artificial light.
5. Observe after few days.
Observations:The stem has bent towards the hole & leaves got oriented at right angles to the
light.
Conclusion:
1. The stem is positively phototrophic.
2. The leaves are diaphototrophic.
Precautions:
1. Water the plant properly before keeping it in the chamber.
2. Place the box correctly so that plant receives proper sunlight through the hole.
Experiment 12 : To demonstrate the measurements of growth by arc auxanometer.

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Requirements: An arc auxanometer, a potted plant etc.
Procedure:
In arc auxanometer, an indicator is fixed to a wheel around which passes a cord. One end of the
cord is tied, gummed or taped to the apex of the stem. The cord is now passed over a small wheel
to which an indicator is fixed. The other end of the cord carries a weight.
Results: The arc indicator moves along the arc.
Calculations & Conclusions:
As the growth takes place, the stem increases in length. The wheel slowly rotates, due to the
movement of indicator along the arc scale. The growth of the plant is thus recorded. From the
records, actual growth is obtained. The magnification of growth given by the indicator is first
made known. For example: if the size of the pulley is 4 inches & needle 20 inches from the
centre of the disc, the magnification is 10 times. The actual growth during this period shall be:
5/20cm=0.25 or 2.5mm/20=0.1m/hour.
During elongation of growth of stem, the following processes take place:
1. Active cell division in the apical region of the shoot.
2. The newly added cells undergo elongation & consequently increase in the length of stem
occurs.
Experiment 13: To demonstrate the Bioassay of Auxin, Cytikinin, Gibberalic acid ,
ABA & Ethylene by using appropriate plant material.

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Bioassay of Auxin
I. Avena coleoptile test:
Requirements: Germinated seeds of Avena sativa (oat/javi), sharp blade, glass
slide, three sets of petridishes, Auxin solution of appropriate
concentration.
Procedure:
1. Seeds of Avena sativa are germinated on filter paper soaked with distilled water in
about dark at 25ºC with red light exposure of short period (5-10 minutes are given
during day time).
2. After 2 days of germination when coleoptiles takes approximately 2-3cm long
then tips are removed by sharp blade & their first leaves are removed.
3. Now the section of about 5mm from subapical region of several coleoptiles are
taken out by section culture.
4. These sections are floated in petridishes containing :
i.water ii. sucrose solution iii. sucrose & auxin olution
5. More than 5 coleoptile sections are placed in each petridish after about 24 hours
in dark.
6. The size of section is measured.
II. Split Pea Test
Requirements: Germinated Pea seeds, sharp blade, glass slides,Auxin solution of
appropriate concentration, sucrose solution, two sets of
petridishes, water etc.
Procedure:
1. Pea seeds are germinated in dark & allow to grow for 8 days.
2. They are exposed to red light for 3 hours everyday.
3. A 20mm long piece of stem is taken between 2nd
& 3rd
node.
4. It splits into lengthwise.
5. In water the two parts curved outwardly to 120ºC due to greater absorption of
water by inner cortical cells.
6. In auxin solution they start bending towards inner sides within 2-3 hours.
This curvature is measured after about 24 hours, it is proportional to the strength of
auxin used.
Bioassay of Cytokinin
I. Cotyledon expansion test

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Requirements: Germinated Radish seeds, glass slides, two sets of petridishes,
cytokinin solution of appropriate concentration.
Procedure:
1. The seeds of Radish are germinated on a wet filter paper in dark.
2. Seedling about 2-3mm of hypocotyl are selected & then seed coat are
removed under green safe light.
3. The two cotyledons are separated by a sharp blade & each cotyledon is floated
on petridish containing : (a) water (b) kinetin solution.
4. More than 5 cotyledons are floated in each petridish. Precaution should be
taken that their abaxial surface touches the solution. Petridishes then placed in
continuous dark for 3 days.
5. After 3 days each cotyledon is measured by placing them on a graph paper.
6. Marked expansion should be observed in cotyledon which are treated with
kinetin solution.
II. Leaf senescence test
Requirements: Wheat leaves, sharp blade, glass slides, water, two sets of
petridishes, kinetin solution .
Procedure:
1. Prepare a solution having appropriate concentration of kinetin hormone in it
i.e 0.001-10mg/lt.
2. Now cut the pieces of upper part of wheat leaf i.e one piece from each leaf
with the help of sharp blade. Precautions should be taken that cut pieces
should not be yellow already.
3. Now take 2-10 ml of water in one petridish, 5-10ml of kinetin solution in
other. Place 2 pieces of wheat leaf in each petridish & cover them & place in
dark for 48-72 hours. Then examin the colour of leaves that is whether the
chlorophyll is present or not.
Bioassay of Gibberellin
I. Lettuce hypocotyl test

21. 21
Requirements: Lettuce seeds, petridishes, filter papers, water, gibberalic acid
solution.
Procedure:
1. Lettuce seeds are germinated in light for 2 days at 12-25ºC.
2. They are transferred to petridish having moisture filter paper with test solution
& water in another petridish.
3. Cut the hypocotyl & place in the gibberalic acid solution & water.
II. Cereal Endosperm digestion
Requirements: Barley seeds, petridishes, filter papers, water, gibberalic acid
solution.
Procedure:
1. In this test endosperm is detached from embryo of Barley.
2. Endosperm is sterilized & allow to remain in 10ml of test solution i.e CaCl2
buffer for 1-2 days.
3. After this endosperm is tested for quantity of sugar which is proportional to
quantity of gibberellin.
Experiment 13 : To study the activity of enzyme catalase as influenced by PH &
temperature.

22. 22
Requirements: Potato tuber, knife, petridish, test tube, test tube stand, test tube holder, cork
borer, spirit lamp, Hydrogen peroxide etc.
Procedure:
1. Cut small & thin pieces of potato tuber.
2. Place the pieces in petridish filled with water.
3. Take few small pieces & put them in two separate test tubes & mark them as A & B.
4. Fill both the test tubes with appropriate amount of water.
5. Leave the test tube A in the stand.
6. Boil the potato tuber pieces in test tube B.
7. Drain out water from both the test tubes.
8. Now add Hydrogen peroxide solution (30 parts of water & 1 part of H2O2), so as to
completely immerse the pieces of potato tuber.
9. Note the changes occurring in both the test tubes.
Result:
1. Evolution of bubbles seen in test tube A.
2. There is no evolution of bubbles in test tube B, because enzyme is destroyed/ denatured
when potato pieces are boiled at high temperature hence no enzyme activity.
Conclusion: Catalase brings about decomposition of H2O2 into H2O & O2.
Therefore, O2 evolution is an indication of activity of enzyme catalase.
2H2O2 → 2H2O + O2 ↑.
Experiment 14 : To study the activity of enzyme peroxidase as influenced by PH &
temperature.

23. 23
Requirements: Potato tuber, knife, petridish, test tube, test tube stand, test tube holder, cork
borer, spirit lamp, Hydrogen peroxide, beaker, 2% alcoholic solution of
Benzidine etc.
Procedure:
1. Cut small & thin pieces of potato tuber.
2. Place the pieces in petridish filled with water.
3. Take few small pieces & put them in two separate test tubes & mark them as A & B.
4. Fill both the test tubes with appropriate amount of water.
5. Leave the test tube A in the stand.
6. Boil the potato tuber pieces in test tube B.
7. Drain out water from both the test tubes.
8. Add 2% alcoholic solution of Benzidine in both test tubes so that potato pieces are
completely immersed.
9. Allow the test tube to be undisturbed for10-15 minutes.
10. Remove Benzidine solution from both the test tubes.
11. Add dilute solution of H2O2 to both the test tubes & observe the changes.
Result:
1. Potato pieces in test tube A changes to blue colour very rapidly.
2. No change in colour in test tube B because enzyme peroxidase is denatured due to
high temperature.
Conclusion:
Peroxidases are of wide occurrence in plant tissue & oxidized to various substrates (phenols,
amine etc.) in the presence of H2O2 as electron acceptor.
Experiment 15 : Seperation of chloroplast pigments by solvent method.

24. 24
Requirements: Pestle & mortar, Spinach leaves, petroleum ether, acetone, methyl
alcohol,diethyl ether, distilled water, potassium hydroxide, separating
finnel, conical flask & measuring cylinder.
Procedure:
1. Take 100 gm of fresh spinach leaves in pestle & mortar.
2. Crush them with 40ml of 80% acetone & added a pinch of CaCO3 & again crushed.
3. Acetone dissolves out the pigments & deep green solution is obtained.
4. Solution is filtered in Buchner flask.
5. Filterate contains acetone extract of chlorophyll-a, chlorophyll-b, carotene &
xanthophyll in addition to other acetone soluble compound in leaves.
6. Take 40ml of acetone extract in separating funnel & add 60ml of petroleum ether in it
& shake the funnel gently.
7. Pour 75ml of distilled water in to the separating funnel & again shake the funnel. The
mixture of extract separated into two layers. The lower layer containing acetone &
water, which is drained out by opening the stopper of separating funnel.
8. The upper layer having ether & pigments, is washed again with water, the lower layer
again discarded.
9. Take upper layer(petroleum-ether layer), add 40ml of 92% methyl alcohol into it.
Shake the funnel & allow the two layers to seperate.
10. Upper layer is petroleum ether layer & lower layer is methyl alcohol layer.
Seperation of chlorophyll-a & carotene:
1. In upper petroleum ether layer gently add 50ml of 30% ethyl alcohol-potassium
hydroxide solution (CH2OH-KOH).
2. Now add 30ml of distilled water & shake the funnel .The two layers will be
separated.
3. Upper Blue Green layer i.e; Methanolic-KOH layer indicates the presence
Chlorophyll-a.
4. Lower Orange layer i.e; ethyl-ether layer indicates the presence of Carotene.
Seperation of chlorophyll-b & xanthophyll:
1. Take lower layer & gently added 50ml of diethyl ether into it & shake the funnel.
2. Add 5ml of water to wash the mixture. Wash many times & allow the two layers
to separate.
3. Upper layer ethyl-ether layer & lower layer methyl-alcohol layer. Discard the
lower layer.
4. In the upper layer added 15ml of Methanolic-Potassium hydroxide (CH3OH-
KOH) solution. Shake the funnel & add 30ml of water. Again shake the mixture
& allow the two layers to separate.

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5. The upper Olive Green i.e; Methanolic-KOH layer indicate the presence of
Chlorophyll-b & lower Yellow-Green i.e; ethyl-ether layer indicates
Xanthophyll.
Experiment 16 : Seperation of pigments by Paper chromatography.

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Requirements : Spinach leaves, whatman filter paper No.1, pestle & mortar, beakers,
acetone, petroleum ether, jar with cork having central hole, glass rod
with hook, capillary tube, Calcium carbonate, acid washed sand,
cooling apparatus etc.
Theory: Paper chromatography is a technique of separation of mixtures into their
constitutions by preferential adsorption by a solid on strip of filter paper.
Resolution front or RF value is the distance travelled by an organic substance divided by
the distance travelled by solvent from origin.
RF value for
Carotene = 0.95
Xanthophyll = 0.71
Chlorophyll a = 0.65
Chlorophyll b = 0.45
Procedure:
I. Preparation of paper
1. Take a whatman filter paper No.1.
2. Cut it into columnar strips of the size that would fit easily in the
chromatography jar.
3. Draw a line 3cm above the bottom with pencil.
II. Preparation of extract
1. Macerate 10gm of fresh spinach leaves in 50ml of precooled 80% acetone in a
pestle & mortar.
2. Add acid washed sand & calcium carbonate & grind it further.
3. Filter the crushed material & evaporate it to concentrate.
III. Preparation of solvent
1. Solvent is prepared by mixing 25ml of petroleum ether & 3ml of acetone.
2. Pour it into a glass jar & tight cork having a central hole.
3. In the hole of cork, fit a glass rod with a small hook.
IV. Loading of extract: Take the paper & apply a spot of concentrated acetone
extract with a capillary tube in the middle of the line.
V. Suspend the paper strip with leading support carefully into the jar with the help of
hook so that the spot is not dipped in the solvent.
VI. Make the apparatus air tight & keep it for few hours.
VII. Take out the strip when solvent reaches upto upper level.
VIII. Dry the chromatographic strip with drier & observe.
IX. Identify the pigments with the help of their colours.

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Observations & Results:
The photosynthetic pigments are separated in the form of spots of different
colours on the chromatographic filter paper strip.
Conclusion:
Sr. No. Colour of the spot Pigment isolated
1 Orange-yellow Carotene
2 Yellow Xanthophylls
3 Blue green Chlorophyll a
4 Yellow green Chlorophyll b
Precautions:
1. Use fresh spinach leaves.
2. Place the chromatographic strip carefully in the jar so that loaded spot should not
dipped in solvent.
3. Dry the strip.
Experiment 17 : Seperation of amino acids in a mixtures by paper chromatography & their
identification by comparison with standards.

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Requirements: Water imbibed seeds (germinating seeds), ethyl alcohol, butanol, glacial acetic
acid, water, phenol, ninhydrin as spray agent, chromatography jar, capillary
tubes, stapler, whattman’s filter paper, pestle & mortar etc.
Theory: Chromatography is the separation of chemical substances by partitioning of two
media. 1st
media may be solid or liquid & 2nd
media may be liquid or gas.
Chromatography provides a means for the separation of compounds that are very similar. Hence,
difficult or impossible to analyse by oher method. The Russian Botanist Michael Tswett in 1872
to 1919 invented the technique of chromatography. Paper chrpmatography is a liquid-liquid
partition.
Procedure:
I. Amino acid extract:
1. Grind about 10gm of imbibed germinating seeds in about 50ml of 80% ethyl
alcohol.
2. Filter and evaporate the filterate to use the near dryness.
3. Redissolve the solid in distilled water to make up the volume of extract 2ml.
(A mixture of standard can also be usedlike glycine, aspartic acid, phenylalanine,
tryptophan, valine etc.)
II. Chromatography chamber:
1. Use suitable container (according to the size of the paper to be used). Use proper
lids so that container should be air tight.
2. Of the two chambers fill phenol in one while n-butanol, glacial acetic acid &
water in 3:1:1.Level of solvent should be 1 inch from the bottom.
III.Application and Development:
1. Cut the chromatography whattman paper of suitable size depending upon the size
of the container.
2. Mark a dot with a pencil about 1 inch from bottom & 2 inch from left hand edge
& deposit a small amount of extract gradually on pencil dot.
3. Hang the paper along glass rod with dot at base after spot of extract is dried.
4. Allow the paper edge to dip in the solvent but keep the pencil mark above the
solvent level.
5. Close the chamber airtight & permit to stand for 16 -18 hours to develop the
chromatograph.
6. Remove the paper & allow it to dry & mark the position of the solvent.
7. Now turn the paper at 90 in the chamber & close the chamber airtight.
8. Allow to stand for 10-12 hours & remove paper before solvent reaches its tip.
9. Dry the paper & spray uniform ninhydrin (0.1gm in 100ml of water) & saturated
n-butanol.
10. Heat the paper at 90 for 5 sec. Outline the spots with pencil.

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Result: Ninhydrin reacts with all the amino acids & give different colours. Other
compounds also react with ninhydrin, if present & these include primary &
secondary amines& non aromatic heterocyclic compounds.
 Tryptophan gives olive green colour.
 Proline & hydroxy proline gives Yellow colour.
 Asparagine, cystine gives Brown colour.
 Phenylalanine, tryptophan & aspartic acid give Blue colour.
Experiment 18: Write down the precautions observed during aseptic manipulation of
Tissue culture laboratory.
Precautions :

30. 30
1. Enter in the lab without shoes, but wearing coat.
2. Avoid handling alcohol around flames.
3. Never pipette out chemicals by mouth.
4. Handle strong acids & alkalies by extreme precautions.
5. Wash the hands (preferable with alcohl) & bandage all cuts immediately.
6. Ensure before opening autoclave that pressure is reduced to zero & temperature below
100ºC.
7. Switch off the electrical appliance For example: PH meter, inoculation cabinet, weighing
balance when not in use.
8. Cover all equipments to avoid contact with dust & other contaminants.
9. Clean benches & work surfaces regularly.
10. Take care that dirty glassware or other items are away while work is in progress.
11. Separated space should be allocated for storage of chemicals , equipments & solutions.
12. Mop the floor in lab & culture with an improved disinfectant.
Experiment 19: Write down the equipments commonly used in Tissue culture laboratory.
I. Requirements for media preparation
1. Gas, water & electrical appliances.

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2. Provision for compressed air & vaccume line.
3. Water heater or small stove.
4. Hot plate with magnetic stirrer.
5. Glass or stainless steel container for heating & dissolving media.
6. Autoclave or pressure team sterilizer.
7. PH meter , balances sensitive to mg quantities.
8. Graduated measuring cylinder, flasks, beakers, petridishes & pipettes.
9. Culture tubes, bottles & other glasswares with suitable closures such as cotton
plugs etc.
10. Small transfer instruments such as spatula, scalpels, forceps or dissecting needles.
11. Hot air oven for rapid heating of media & agar mixtures.
12. Double distilled water units.
13. Chemicals for preparation of culture media, growth hormones & other
constituents.
II. Requirements for isolation of culture
1. Laminar air flow cabinet.
2. Spirit lamp in the inoculation cabinet.
3. Ethyl alcohol for sterilization & flaming of all metal instruments.
4. Tiles or glass plates for use during sterile cuttings.
5. Hypochlorite solution for sterilization of plant material.
III.Requirements for culture room
1. Temperature control 17-27ºC.
2. Electricity supply essential for lighting, cooling & heating.
3. Shelves for culture racks.
4. Fluorescent tubes for lightening.
5. Times for regulate in day length.
6. Rack for culture vials.
7. Observation table.
IV.General equipments:
1. Refrigerator for storing stock solutions & chemicals that degrade atroom
temperature.
2. Markers, labels for wrapping culture vessels, glasswares & other lab ware.
3. Acid proof bath for cleansing glasswares.
4. Dispensing devices (trolleys with trays , wire-mesh, baskets & metal racks for
holding test tubes).
5. Lab should be equipped with fire extinguisher.