PRACTICAL-1:

STUDY OF PLANT CELLS

Cell: The cell (from Latin cella, meaning "small room") is the basic structural, functional,
and biological unit of all known living organisms. A cell is the smallest unit of life. Cells are
often called the "building blocks of life". The study of cells is called cell biology.

Cells are not only the basic building units of living organisms. They are also the
physiological units of a living being. Since there is a close relationship between structure and
function it is necessary to understand cell structure in order to understand its physiological
functions. In the following experiments, the basic structure of a plant cell as seen under the
light and electron microscope will be described.

Fig. 1. Schematic diagram of a typical young plant cell. Ch = chloroplast. D = dictyosome,

ER = endo¬plasmic reticulum, ERmR = endoplasmic reticulum with ribosomes, Li = Lipid
bodies, Ly = Lysosomes. M = mitochondria, Mt = microtubule N = nucleus, Pd =
plasmodesmata, PI = plasmalemma, R = ribosomes, T= tonoplast, V = vacuole, Zw = Cell
wall. (After Urbach era/. 1976)

Several parts of a plant cell can easily be observed under a light microscope, e.g., cell wall,
plasma membrane, vacuole, plastids and nucleus (Fig. 1). Due to their relatively smaller size,
mitochondria cannot easily be seen with a light microscope but they can be made visible
under light microscope by staining with special chemicals.

A) LIGHT MICROSCOPY MATERIALS:

Leaves of Funaria hygrometrica, Elodea canadensis, Zebrina pendula, Rhoea discolor.

Vallisnaria sp., Tradescantia sp., Urtica dioca, Stelleria media, Cucurbita pepo (cucumber),
Zea mays (maize), Spinacia oleracea (spinach), scales of onion bulb (Allium cepa), carrot
(Daucus carota), tomato fruit (Lycopersicum esculentum), flowers of roses (Rosa sp.), pansy
(Viola tricolor), potato tuber (Solanum tuberosum).
Aceto-carmine solution (boil 4-5 g carmine in 100 ml of 50% acetic acid and filter after
cooling), cone, sulphuric acid, iodine solution (dissolve 0.5-1.0 g of potassium iodide (KI) in
100 ml of water. Then dissolve 1 g of iodine crystals), carmine-alum solution (dissolve l g
carmine + 10 g alum (K2SO4 . Al2(S04)2. 24 H2O) in 200 ml distilled water by warming. Filter
the solution. Add 1 ml of formaldehyde to preserve the stain), neutral-red solution (5 mg in
100 ml water), extremely dilute solution of gentian-violet (the solution should be just lightly
coloured), rhodamine B solution (dissolve 0.10 mg rhodamine B in 100 ml of tap water), 2 M
sucrose solution
Ordinary compound microscopes and phase contrast microscopes

PROCEDURE:

i) Protoplasm movement: Remove few hair cells from the leaves, leaf petioles or other
plant organs of one or more of the following plants: Cucumber, Elodea, Vallisneria,
Tradescantia, Urtica dioca or Stelleria media and mount them on a glass slide. Put a drop
of water and place a coverslip over the material. First observe under the low power of a
microscope and select a most suitable cell. Observe under the high power the movement of
protoplasm. Depending upon the plant material, it may be seen that along with the
protoplasm also the cell inclusions in the protoplasm are seen moving along with the
protoplasm.
ii) Cell wall: Cut an onion bulb into four pieces. Cut out a small piece from the interior side
of the bulb and peel up carefully an epidermal layer. Place the stripe of epidermal cells on
a glass slide and mount on a drop of carmine-alum stain. Allow to stain for about 15 min,.
Wash away excess of stain with dilute acids. The cell wall stains red. In the same way
stain an another stripe of epidermis with a drop of neutral-red solution. The middle lamella
will be stained red.
iii) Nucleus: Nucleus can distinctly be seen in an epidermal cell of an onion scale as prepared
above. Nucleus can be stained by applying a drop of aceto-carmine solution. If carefully
observed at high power, one can even see nucleioli which are not or only very faintly
stained by the stain. In addition to epidermal cells of onion scale, very distinct and big
nuclei can also be seen in the hairs (trichome cells) of a cucumber leaf or the epidermal
cells of young leaves of Tradescantia.
iv) Plastids: Chloroplasts: Mount thin sections of green leaves of maize, spinach or intact
leaves of a moss plant (Funaria sp.) or an aquatic plant (Elodea sp.) on a microscopic
slide. Place a cover slip over the material. First observe under the low power and then
under high power of a microscope. In maize leaf transverse section, one can observe
chloroplast dimorphism, i.e., two different kinds of chloroplasts. Observe the chloroplasts
in the meosphyll cells and the bundle sheath cells. Mark the differences between the two
types of chloroplasts. Chromoplasts: Chromoplasts are plastids which contain no
chlorophyll pigments. They contain yellow (carotenes, xanthophylls) or red (anthocyanins)
pigments and are commonly found in fruits (tomato) and flowers. Make thin sections of
the yellow flower petals (e.g. pansy, roses), red tomato-fruit skins and carrot. Mount them
with a drop of water on a glass slide, cover with a coverslip and observe carefully, first
under low power and then under high power. Yellow and red coloured round bodies can
be distinctly be seen. Leucoplasts: They are colourless plastids and are found in roots,
rhizomes and different storage organs. Peel off lower epidermis from the leaves of Rhoea
discolor and mount it in a drop of water on a glass" slide. Cover with a cover slip. Focus
 under the high power a distinct nucleus. The nucleus is surrounded by a number of small
round bodies, the leucoplasts. Replace water on the glass slide with a drop of gentian-
voilet solution for 10-20 seconds. Leucoplasts will appear as voilet-coloured bodies.

v) Mitochondria: Prepare few stripe of epidermis from onion scales as described above.
Place them in a bottle containing water. Apply suction using an aspirator. Take out the
stripes, place them on a glass slide and treat with few drops of rhodamine-B solution for
about 30 min. Wash away the excess of stain with tap water. Observe under a phase
contrast microscope. Under green light, mitochondria appear as black rounded structures.

vi) Plama-membrane and vacuole: Plasma-membrane and vacuole can best be observed in
epidermal cells containing coloured cell sap. Peel off an epidermal layer from the leaves of
Zebrina pendula or Rhoea discolor and mount it a drop of 2 M sucrose solution. Observe
under microscope. Due to concentrated sugar solution, the cells will show the sign of
plasmolysis. Plasma-membrane becomes distinct and the vacuole is seen full of red
pigments, the anthocyanin.

B. ELECTRON MICROSCOPY

MATERIALS
Electron micrographs showing ultra structure of a plant cell

PROCEDURE:

With the help of electron micrographs, identify different cell components and their internal
structures. Identify cell inclusions such as ribosomes, endoplasmic reticullum, golgi bodies,
peroxisomes, etc, which are not visible under a light microscope.

RESULTS:

Draw diagrams of a plant cell showing its different parts and their ultrastructure.