BIOLOGY PRACTICALS: TERM- II

EXPERIMENT.1:

Aim: To distinguish and study the various pigments present in plants through the
process of paper chromatography.

Theory: Plants carry out the process of photosynthesis during which light energy
from the sun is converted into chemical food energy. The capturing of light energy is
carried out by molecules known as pigments present in plants. These are chemical
compounds which are able to reflect only a particular range of wavelengths of visible
light. Leaves of plants primarily contain four pigments –
Pigment Colour
Chlorophyll A Dark green
Chlorophyll B Yellowish-green
Xanthophylls Yellow
Carotenoids Orange
In order to view and distinguish the primary four plant pigments, a simple technique
known as chromatography can be used.

Chromatography: It is a technique that is used to distinguish between different

molecules. This differentiation is based on these attributes-shape, size, charge, mass,
adsorption and solubility. There are four types of chromatography: Column
chromatography, Paper chromatography, Partition chromatography, Thin layer
chromatography

Mechanism of Paper Chromatography:

In this technique, the interaction between three components is involved – solid
phase, mixture to be separated and a solvent.
The mixture is spotted onto the paper and is dried. The solvent is made to flow
through the capillary attraction. While the solvent moves through the paper, the
various components of the mixture differentiate into varied coloured spots.
Paper is dried, the position of various compounds is viewed. The substance that is
most soluble moves further on the paper as compared to the other substances that
are less soluble.

Material Required: Petri dish, Spinach leaves, Mortar and pestle, Scissors, Ether
acetone solvent, Acetone, Brush, Filter paper disc, Cotton Wick

Procedure:

1. In this experiment, spinach leaves are used to separate different pigments,

pluck a few fresh leaves.
2. Cut out small pieces of spinach using scissors. Add them to the mortar
3. Accurately measure 5ml acetone using a measuring cylinder and add it into
the mortar
4. With the help of mortar and pestle, grind the spinach
5. Shift the paste of spinach with the help of a spatula into the watch glass
6. Place a filter paper disc with a hole at the centre in which we need to fix a
small cotton wick.
7. Using a brush, load a ring of leaf extract slightly apart from centre of the filter
paper disc.
 8. Let the drop dry. Repeat the same process of adding a ring over it again and
allowing it to dry for 4-5 times.
9. In the petri dish, pour the ether acetone solvent and put the paper disc with
wick. Only wick must be dipped in the solvent.
10. Let the set-up remain uninterrupted for a while
11. We notice that the solvent passes along the paper scattering various pigments
of the blend to different distances
12. Once the solvent forms 3-4 rings around the loaded ring on the paper disc,
carefully take the paper disc off and allow it to dry

Observation: The dried paper disc displays 4 different rings or bands. Discrete
pigments can be distinguished with the help of colours.

Conclusion: The Carotene pigment is observed at the outermost as an orange-yellow

band of pigments distinctively. Just inside this band, a yellowish band appears
which indicates the pigment xanthophyll. The third ring appearing dark green
indicates chlorophyll a pigment. The yellowish-green band present at the centre is
the chlorophyll b pigment.

Precautions:

1. The leaves that are picked should be green and fresh spinach leaves
2. From the centre of disc, the loading ring needs to be .5 cm apart
3. While suspending the filter paper strips in the chamber, one needs to ensure
that the loading spot needs to be set up above 1cm from the level of the
solvent.

(Diagram of Experimental set-up)

EXPERIMENT.2:
Aim: To study the pattern and distribution of stomata in both the upper and lower
leaf surfaces.
Theory: Stomata are tiny openings that are located in the young shoots of plants and
epidermis of the leaves. They govern the gas exchange process in plants. The
structure of the stomata includes a pair of specialized cells that are found girdling
around the opening. These cells are termed as guard cells and are responsible to
check and regulate the size of the closing and opening of the stomata.
Through the process of transpiration, water escapes from the stomata into the
atmosphere in the form of water vapor. Along with this, carbon dioxide and oxygen
too are exchanged in the leaf through these openings.
In a dicot leaf, in comparison with the upper surface, the lower surface has a higher
distribution of stomata whereas in a monocot leaf, usually, the upper and the lower
surfaces usually see an equal distribution of stomata.
Material Required: Compound microscope, Four O’clock/ Beetle leaf/ Tradescantia
plant, Glycerine, Safranin solution, Forceps, Needle and brush, Distilled water,
Dropper, Blade, Watch glass, Glass Slide, Coverslip
Procedure:
• One fresh leaf from above mentioned plant is used in this experiment
• On two watch glasses, add some distilled water
• Slit the leaf in an oblique manner
• With the help of forceps, peel a section from the upper surface of the leaf.
• Set this section into one of the watch glass holding water
• With the help of forceps, peel another section from the lower surface of the leaf
• Set this section on another watch glass which is also holding water
• With the help of a dropper, add few drops of safranin solution into both the watch
glasses
• Place cleared glass slides on each of the peels one at a time with the help of a
brush
• From each of the peels, cut a square or a rectangular piece with the help of a
blade
• With the help of a dropper, add one drop of glycerine on each of the slides
• With the help of a needle, gently place a coverslip on the peel
• Examine each of the glass slides under the microscope
• Notice and count the occurrence of stomata in each of the peels of both the lower
and upper epidermis of the leaf.
Observation And Conclusion:
The section of leaf plucked from the four-o’clock plant shows that the number of
stomata is much more in the lower epidermis while a few are found in the upper
epidermis of the leaf.
Precautions:
• Avoid leaf curling
• Gently place the coverslip on the slide to avoid air bubbles
• Transferring peel to the slide from the watch glass should always be done
using a brush

(Diagram: Structure of Stomata and Distribution of stomata in a leaf peel )

SPOTTING 1: (ANIMAL TISSUES)
1.1: Squamous Epithelium
• The covering or protective tissues in the animal body are epithelial tissues
• Simple squamous epithelial cells are extremely thin and flat and form a
delicate lining.
• They are tightly packed and have prominent nucleus.
• The oesophagus and the lining of the mouth are also covered with squamous
epithelium.
• The skin, which protects the body, is also made of squamous epithelium
1.2: Smooth Muscle fibers
• These are found in the alimentary canal, blood vessels, iris of the eye, in
ureters and in the bronchi of the lungs and other internal organs.
• We can not move them as per our wish so they are called as involuntary
muscles.
• The cells are long with pointed ends (spindle-shaped) and uninucleate (having
a single nucleus).
• They are also called smooth or unstriated muscles as they lack striations.
1.3: Skeletal Muscle fibers
• These are located in limbs.
• These muscles can move as per our desire,such muscles are called voluntary
muscles.
• These muscles are also called skeletal muscles as they are mostly attached to
bones and help in body movement.
• Under the microscope, these muscles show alternate light and dark bands or
striations when stained appropriately, so they are also called striated muscles.
• The cells of this tissue are long, cylindrical, unbranched and multinucleate.
• They show fatigue.
1.4: Cardiac Muscle fibers
• These muscles are found in heart.
• They show rhythmic contraction and relaxation throughout life.
• Heart muscle cells are cylindrical, branched and uninucleate.
• They are striated and unvoluntary in action.
• They are immune to fatigue.
1.5: Mammalian blood smear

• RBCs or Red blood cells are oval biconcave disc-shaped cells, it contains
haemoglobin protein (iron-containing pigment) which helps in carrying oxygen.
• WBCs or White blood cells (neutrophils) are also known as leukocytes, it helps
to fight infections by attacking different microbes that enter the body. The most
common WBCs in blood is neutrophils which have a multilobed nucleus.
• Platelets or thrombocytes are biconvex discoid structures in the blood whose
main function is to prevent bleeding by initiating blood clotting (with other
coagulating factors).
• Blood plasma is a pale-yellow liquid which consists of all the blood cells along
with salts and enzymes.

(Diagrams of all 5 tissues)

SPOTTING 2: (MITOSIS)
2.1. Interphase
• A cell spends a period of its growth in interphase before entering mitosis. When
in interphase, it goes through the three phases mentioned below.
• G1 Phase: This is the period preceding DNA synthesis.
• S Phase: This is the phase in which DNA synthesis occurs.
• G2 Phase: The period between the end of DNA synthesis and the start of
prophase.
• During this phase chromosomes are not clearly visible but only chromatin
network is seen.

2.2. Prophase

• The process of mitosis is initiated at this stage wherein coiling and thickening
of the chromosomes occurs
• Shrinking and hence the disappearance of the nucleolus and nuclear
membrane takes place
• The stage reaches its final state when a cluster of fibres organize to form the
spindle fibres.
2.3. Metaphase

• Chromosomes turn thick in this phase. The two chromatids from each of the
chromosomes appear distinct
• Each of the chromosomes is fastened to the spindle fibres located at its
centromere
• Chromosomes align at the centre line of the cell
2.4. Anaphase

• Each of the chromatid pair detaches from the centromere and approaches the
other end of the cell through the spindle fibre
• At this stage, compressing of the cell membrane at the centre takes place
2.5. Telophase

• Chromatids have reached the other end of the cell

• The disappearance of the spindles
• Chromatin fibres are formed as a result of uncoiling of daughter chromosomes
• The appearance of two daughter nuclei at the opposing ends due to the
reformation of the nucleolus and nuclear membrane
• At this phase, splitting of the cell or cytokinesis may also occur.

(Diagrams of all 5 stages of Mitosis)