Determining Leaf Constants and Microscopic Features of Medicinal Plant Samples

Department of Pharmcognosy, L. M. College of Pharmacy, Ahmedabad 2021
Practical Manual prepared by Dr. Preeti Verma Page 1
PRACTICAL NO. 2 DATE: _________
QUANTITATIVE MICROSCOPY-I
AIM: Determination of Leaf constants.
A) Vein islet no. and Vein termination no. of Datura leaf.
B) Stomatal number and Stomatal index of Vinca leaf (lower surface).
Requirements:
Leaf drug sample, chloral hydrate or KOH solution, dil. HCl, lactophenol or
glycerin, black chart sheet, drawing board, glass marking pencil, blade, brush,
glass slides, coverslip, test-tube holder, Compound microscope, Camera lucida,
Stage micrometer slide.
Leaf constants:
These are quantitative parameters which are constant for a given species of the
plant and are used as a characteristic for identification of the plant and
differentiation of allied or closely related species of the same genus in air-dried
as well as fresh condition (important diagnostic characters of Dicot leaves). E.g.
Indian and Alexandrian Senna can be easily differentiated by their leaf
constants.
A) Determination of Vein islet no. and Vein termination no. of Vasaka leaf:
Vein-islet number:
Definition: The vein-islet number is the average number of vein-islets per
square millimeter of the leaf surface, midway between midrib of the leaf and its
margin.
The mesophyll of the leaf in dicot leaves is divided into small portions by
branching of the veins throughout the tissues. The small areas of the green
tissue surrounded by the veinlets are termed as vein-islets.
Vein termination number:
Definition: It is the number of veinlet termination per sq. mm of the leaf
surface, midway between the midrib and the margin.
A vein termination is the ultimate free termination of veinlet.

Procedure for determination of vein-islet number:
1. Surface preparation of the leaf (epidermis): Cut 4 mm x 4 mm piece from
leaf surface (from the central portion, midway between the midrib and the
margin). Clear the piece of the leaf by boiling in chloral hydrate/KOH
solution. Wash the cleared piece with water, treat with dil. HCl to remove
calcium oxalate crystals. Then mount it on a glass slide with lactophenol or
glycerine. Put coverslip and observe under microscope (in low power i.e.
10X).
This figure is used for demonstration purpose only

2. Arrange camera lucida and drawing board with black chart sheet for making
the drawings.
3. Place stage micrometer slide on the microscope stage. Now by viewing into
the microscope, simultaneously draw a line equivalent to 1 mm on the chart
sheet.

4. Construct a square on this line. (that is equivalent to 1 sq. mm. area)

1 mm of stage
micrometer
= 10 cm on chart
sheet
1 mm2
square area

5. Now, remove the stage micrometer slide and place the slide of Surface
Preparation of given leaf on the stage.
6. Move the chart sheet so that the square is seen in the eye piece, in the centre
of the field.

7. Trace off the veins and veinlets which are included within the square
(covering a little area outside too), by looking through the microscope when
a superimposed image of the leaf portion and chart sheet are seen at the same
time on the chart sheet. Complete the outlines of those islets which overlap
two adjacent sides of the square.
8. Count the number of vein islets in the square millimeter. Where the islets are
intersected by the sides of the square, include those on two adjacent sides
and exclude those islets on the other sides.
9. Repeat this procedure on 3 more squares and report the average value as
final value for one sq. mm.

Procedure for determination of vein termination number:
Same as for the Determination of vein-islet number. Count the number of
veinlet terminations present within the square. Find the average number of
veinlet termination number from the four adjoining squares to get the final value
for one sq. mm.
Results:
Vein-islet number for given Datura leaf is _______________.
Vein-termination number for given Datura leaf is _____________.

B) Determination of Stomatal number and Stomatal index of Vinca leaf
(lower surface):
Stomatal number:
Definition: It is defined as the average number of stomata per square mm area
of the leaf surface (epidermis).
Stomatal index:
Definition: It is the percentage which the number of stomata form to the total
number of epidermal cells, each stoma being counted as one cell.
Stomatal index can be calculated by using following formula:
I = S x 100
E+S
where, I = Stomatal index
S = No. of stomata per unit area
E = No. of epidermal cells in the same unit area
NOTE: Stomatal number may vary for the leaves of same plant grown in
different environment or under different climatic conditions, or with the age of
the plant, etc. However, the ratio of the number of stomata to the total number
of epidermal cells in a given area of epidermis is fairly constant for any age of
the plant and under different climatic conditions. Hence, Stomatal Index is not
much affected by these factors and is relatively constant. That is why, stomatal
index is more significant in the evaluation of a leaf drug and is considered as
Official Parameter.
Procedure:
I. Surface preparation of the leaf (epidermis): By Peeling method
Peel out the lower epidermis (from the central portion of leaf, midway
between the midrib and the margin). Clear it slightly by treating with chloral
hydrate/KOH solution. Then mount with lactophenol or glycerine. Put
coverslip and observe under microscope (first in in low power, then in high
power i.e 40X or 45X).

II. For stomatal Index:
1. Arrange camera lucida and drawing board with black chart sheet.
2. Place the surface preparation slide of lower surface of vinca leaf on the
stage of microscope.
3. Trace epidermal cells and stomata (in round) in high power.
4. Count the number of the stomata and the epidermal cells (in the round).
5. Calculate the stomatal index from its formula.
III. For stomatal number:
1. Arrange camera lucida and drawing board with black chart sheet.
2. Observe stage micrometer slide (with 1 mm scale) in high power and
draw a square using 20 divisions (to make 0.2 x 0.2 mm2
i.e 0.04 mm2
area).
3. Now, place the surface preparation slide of lower surface of vinca leaf on
the stage of microscope.
4. Trace epidermal cells and stomata in high power (within the visible round

area)
(Note: the area of square should lie within the area of visible round)
5. Count the number of stomata in square area (0.04 mm2
area) of leaf
surface.
6. Calculate mathematically the number of stomata in 1 mm2
area.
Note: The results of ten fields are recorded and average is calculated as final
results.
Results:
Stomatal number for given Vinca leaf is ________ for lower epidermis.
Stomatal index for given Vinca leaf is ________ for lower epidermis.
Reference: Practical Pharmacognosy, Dr. C. K. Kokate.

1 mm scale
20 divisions taken, so area = 0.2 x 0.2 mm2
= 0.04 mm2
area
In high power (45X)

PRACTICE EXERCISE
(to be witten on plain side of the journal only)
A) Vein islet no. and Vein termination no. of Datura leaf.
Observations:
1. Vein islet number = _____________
2. Vein termination number = _____________

B) Stomatal number and Stomatal index of Vinca leaf
(lower surface).
FOR STOMATAL INDEX (Consider the Round in the
Photograph taken)
Observations:
In round, S = ___________, E = ___________
Calculation:
I = S x 100 = ____________
E+S
Final Result

FOR STOMATAL NUMBER (Consider the Square)
Observations:
In square (0.04 mm2
): S = __________
Calculation:
100 divisions of stage micrometer slide = 1 mm
So, 1 division of stage micrometer slide = 1 mm = 0.01 mm
100

Divisions considered (for drawing the square) = 20 divisions
So, length of 1 side of square drawn = 20 x 0.01 mm = 0.2 mm
Hence, AREA of the square drawn = 0.2 x 0.2 mm2
= 0.04 mm2
Since, we want final stomatal number in 1 mm2
area of leaf surface,
We have to extrapolate our result to 1 mm2
area:
In square (0.04 mm2
): S = X (from observation)
So, 1 mm2
area = X x 1 = _________
0.04
Final Result

Determining Leaf Constants and Microscopic Features of Medicinal Plant Samples

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