Experiment

To Learn the Technique of Staining and

5 Mounting by Preparing Temporary
Mounts of Leaf Epidermal Peel

REQUIREMENTS
Glasswares: Microscopic slides, Cover slips, Petri-plates
Chemicals: Safranin, Acetocarmine, Glycerine
Plant Material: Leaf epidermal peel of Crinum, (monocot), Datura (dicot)
Withania (dicot), Scale leaves of Allium cepa (monocot).
Miscellaneous: Needle, Brush, Forceps, Dropper, Blade, Blotting paper,
Microscope.
PRINCIPLE
The term cell was first used by Robert Hooke (1655), where 'cella' in latin means
cork, cut with
hollow space. His observation was based on a very thin slice of
called cell.
a pen knife which look like a honey comb like structure and hence
1835 which
Modern celltheory was proposed by Schleiden and Schwann in
states that:

1. Cells are the smallest morphological and physiological unit of all organisms.
2. The characteristic features of an organism depends on the property of its
individual cells.
3. All cells arise from pre existing cells.
4. The smallest unit of life is the cell.
Cell within an organism differ in structure and function. Aneasily available
material to observe intact plant cells under the microscope are epidermal cells
Irom the leaves. The epidermal cells differ in their orientation and stomatal
arrangements in the dicot and monocot plants and also contain various appendages
like hairs, glands etc.

PROCEDURE
Peel Preparation
1. Take a small turgid leaf and clean the surface.
2. Using fingers gently fold the leaf from the middle and carefully pull one
portion.
34 Laboratory Manual of CelI Biology

3. Small transparent epidermal peel gets exposed.

4. Using a sharp blade remove small portion of the peel.
Staining
5. Place the peel on the centre of the glass slide using a brush and cover the
materialwith a small drop of stain (safranin/acetocarmine).
6. After one minute remove the excess using a blotting paper.
De-staining
7. This is an important step since the required contrast of the stain is important
to see the cellular details. Over stained material looks dark under the
microscope whereas the under stained material shows no contrast.
8. De-staining is done by carefully washing the material with water using a
brush.
9. Washing is done till the excess stain is removed from the material.
Mounting
10. The peel is then mounted on the slide for microscopic observation using a
proper mounting medium for temporary preparation. Amounting medium is
semiviscous and transparent liquid toprevent drying of the material while
observing.Glycerine (4%) is widely used as a mounting medium.
11. Put a small drop of glycerine on the material and gently place the cover
glass on the mounting medium containing the material using a needle
without trapping any air bubble.
12. Remove excess glycerine using a blotting paper and clean the slide before
keeping it on the stage of the microscope.
13. Observe the material first under the lowpower (LP) and then under the high
power (HP) and draw the cellular details as observed under the microscope.
OBSERVATION
Shape and size of the cells differ widely in different plant materials. As expected
under low power cells are smaller compared to the high power. The size and
position of the nucleus and the distinction between the cell wall and membrane
are clearly observed under the high power. Safranin stains both cytoplasm and
nucleus of all the materials, and the nucleus gets densely stained compared to
cytoplasm since it is a general stain (GS). Acetocarmine on the other hand only
stains the nucleus of the cell and the cytoplasm remains colorless since it is a
specific stain (SS) for nuclear material. In monocot plants cells are generally
rectangular and arranged in a parallel array with the stomatal cells whereas in
the dicot materials cells are polygonal with randomly scattered stomata. These
observations coincide with the parallel and reticulate venation of the monocot
and dicot leaves respectively.
 To Learn the Technique of Staining and Mounting by Preparing Mounts of Leaf.. 35
Crinum Leaf Peel from Lower Epidermis (Fig. 5.1A,B)
1. It is a monocot plant.
2. Cells appear highly elongated and rectangular in shape, which are longer
than broad.
3. The Stomata are seen in parallel rows being amonocot with parallel venation.
Guard cells with starch grains are oriented parallel to the epidermal cells.
4. A prominent densely stained nucleus embedded in a lightly stained
cytoplasm can be seen under high power.

Cell wall
Nucleus
Stomata
Cytoplasm

Nucleus

-Cytoplasm
Stomata Cell wall

Fig. 5.1 Peel Mounts of Crinum: A. As Seen Under HP of the Microscope

B. Schematic Representation

Allium cepa (Onion) Peel from Scale Leaf (Fig. 5.2)

1. Onion is a monocot plant and peel is taken from the scale leaf of the bulb,
hence the peel contains no stomata.
Z. Under low power, rectangular brick like arrangement of thecells containing
nucleus (appear like a dot) in the corner of the cell (acentric) is visible.
3. Under high power cells with distinct cell wall and the underlying cell
membrane with prominent nucleus is clearly visible.
 Nacios

Naciens

E
Fg 52 ee osof Aliu cepe A As Oosenved Under LP of the Microscope
# As Oserves nser HP of the Microscope LP C Schematic
iegresetaoor zs Oosersed Under P of the MicroscopeD. Schemanc
iegreserzoor s Otservec Under mP of the MicrOScope A Stained
wSztrar i Sarec w Acetocamine
 To Learn the Technique of Staining and Mounting by Preparing Mounts of Leaf. 37

Datura Leaf Peel from Upper Epidermis of Leaf (Fig. 5.3 A-D)
1. Epidermalcells of Datura show polygonal cells with wavy margins.
2. Prominent stomata are seen scattered within the epidermal cells.
3. Prominent starch grains are present in the guard cells.
4. The cytoplasm of the cells bears prominent nucleus.
5. Long hairy outgrowths are present in all the epidermal cells.

A B

Hair

Nucleus
-Cell membrane
Cell wall
Stomata

Cytoplasm

C D

FIg. 5.3 Peel Mounts of Datura leaf: A. As Observed Under LP B. As Observed

Under HPC.Schematic Representation of LPD. Schematic Representation
of HP

4. Withania Leaf Peel from Upper Epidermis (Fig. 5.4)

I. The epidermal cells are large polygonal in shape with prominent densely
stained nucleus.
2. Being adicot stomata are seen scattered in the epidermis.
3, Guard cells contain starch grains.
4. Smallmulticellular, branched hairs are present in some cells.
5. Unlike Datura epidermal hairs are thin,small and less in number.
38 Laboratory Manual of Cell Biology

(10 X)
C

-Nucleus

-Cell wall

-Hair

-Cytoplasm
-Stomata

(40 X)

Fig. 5.4 Peel mounts of Withania leaf: (Note the Presence of Multicellular,
Branched Hairs; Arrow) A.AsObserved Under LP B.As Observed UnderHP
C. Schematic Representation of LP D. Schematic Representation of HP
 To Learn the Technique of Staining and Mountingby Preparing Mounts of Leaf.. 39
PRECAUTIONS
. Mounting should be done in the center of the slide.
2, Excess stain should be removed carefully with filter paper without damaging
the peel.
3. Destaining should be done till excess stain is removed from the peel.
4. Avoid trapping any air bubble under the cover glass.
5. Clean the lower and upper surface of the slides before keeping on the stage
for viewing.
Points for Spotting
The slide shows the temporary mount of epidermal peel from a Monocot/Dicot
leaf.
Monocot leaf peel: Elongated rectangular cells arranged in parallel rows and show
parallel arrangement of the stomata. Densely stained nucleus, cell membrane and
the cell wall are observedunder the high power. eg.Crinum
Dicot leaf peel: Polygonal cells with stomata scattered between the epidermal
cells. Epidermal hairs also visible eg. Datura
The slide is stained with a generalised (GS)/specialised stain (SS). GS-eg. safranin
which stains both the nucleus and the cytoplasm. SS-eg. Acetocarmine, which
stains only the nucleus and not the cytoplasm.