Pharmacognosy:

Microscopic Techniques
Quantitative Microscopy

QUANTITATIVE MICROSCOPY
 The uses of the microscope in the Pharmacognosy have been made since 1847. The microscope is not
only essential in the study of adulterants in powdered plants and animal drugs, but it is also
indispensable in the identification of pure powder drug.
 Organoleptic studies of the many drug plants, the microscopic characters are very prominent. In order
to study these characters (also in order to differentiate it from the common adulterants) a good
laboratory technique is essential.
 This method is used for identification of drugs on cellular level. It is used to determine structure of
organised drugs by their histological characters. It includes examination of whole, certain parts or
powdered crude drugs.
 Histological Characters:
1: Size, shape and relative position of cells and tissues.
2: Chemical nature of cell wall.
3: Fragments of plant cells or tissues
 Importance- It is necessary in:
1: Initial identification of herbs
2: Identification of small fragments of crude or powderd drug.
3: Detection of adulterants (insects, molds, fungi)
 TYPES:
1: Transverse microscopy 2: Powdered microscopy
TRANSVERSE MICROSCOPY
It is used to determine:
1: Size of starch grain
2: Length and width of fibers
3: Size of stomata
4: Diameter of phloem fibers
METHODS OF TRANSVERSE SECTIONING
1: Free hand mounting: It is used for temporary slides. In this method material is cutted with help of blade
and sliced smoothly from upper left towards lower right in a single motion. Keep the specimen and blade
lubricated with water.
Glide mounting:
It is used for solid material sectioning.
It is composed of specimen feed, knife , holder, and specimen orientation.
It’s main quality is that it gives excellent sectioning results.
Cryology Mounting:
This method is used to make slides of fresh and young herb tissues. Cut the sample into small pieces and
embed them with cryomatrix on a crycasste. Freeze them, slice them, mount on glass slide and seal.
Paraffin Mounting
IN this method specimen is embedded in paraffin and then slicing the block. The steps include: Sampling,
Fixation, Dehydration, Vitrification, Slicing , Removing the paraffin, staining with safranin and fast green.
PROCEDURE FOR OBSERVING LEAF CONSTANTS
1.Leaf surfaces are scrapped and peeled off from upper and lower surface.
2: Epidermis is removed carefully.

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 Pharmacognosy:
Microscopic Techniques
Quantitative Microscopy

3: wash it with chloral hydrated solution which is used as a cleaning agent to see the cellular components of
leaf accurately. 4: observe under microscope.
Leaf constants:
1: STOMATAL INDEX:
It is a % which the no. of stomata form to totsal no. of epidermal cells.(each stomata being counted as 1 cell
)
Stomatal index= S/E+S X 100 S= No. of stomata per unit area. E= no. of epidermal cells in the same unit area.
2: VEIN ISLET NUMBER:
It is defined as no. of vein islet per square ml of leaf surface midway b/w midrib and margin.
3: VEIN TERMINATION NUMBER:
It is defined as : No. of veinlet termination per square meter of leaf surface midway b/w midrib of leaf and
its margin.
4: PALISADE RATIO:
It is the average no. of palisade cells beneath each epidermal cells.
Evaluation of drugs
 Evaluation means of confirmation of the purity and identify the quality. The main basis of evaluation of
drugs is to identify quality and determine the purity.
 The identity of the drug is established by actual collection of the drugs from a plant or animal which has
been correctly identified for which “drug gardens” are often established for the authenticity of the plants.
Another method of identification is to compare the drug sample with a published description of drug and
with authentic drug sample.
 The evaluation of drugs includes the following methods:
1. Organoleptic/ Macroscopic
2. Microscopic
3. Biological
4. Chemical
5. Physical
Microscopical method:
 This method allows more detailed examination of drug , and it can be used to identify the organized
drug by their known histological character, it is mostly used for qualitative evaluation of organized
crude drug in entire and powder form.
Leaf constants:
a. Palisade ratio:
Determination of Palisade Ratio
 Palisade ratio is the average number of palisade cells under one epidermal cell. Place leaf fragments
of about 5 × 5 mm in size in a test-tube containing about 5 ml of chloral hydrate solution and heat in
a boiling water-bath for about 15 minutes or until the fragments become transparent.
 Transfer a fragment to a microscopical slide and prepare the mount of the upper epidermis in chloral
hydrate solution and put a small drop of glycerol solution on one side of the cover-glass to prevent
the preparation from drying.
 Examine with a 40x objective and a 6x eye piece, to which a microscopical drawing apparatus is
attached. Trace four adjacent epidermal cells on paper; focus gently downward to bring the palisade

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 Pharmacognosy:
Microscopic Techniques
Quantitative Microscopy

into view and trace sufficient palisade cells to cover the area of the outlines of the four epidermal cells.
Count the palisade cells under the four epidermal cells.
 Where a cell is intersected, include it in the count only when more than half of it is within the area of
the epidermal cells. Calculate the average number of palisade cells beneath one epidermal cell,
dividing the count by 4; this is the “Palisade ratio” (See Fig 1).

Figure 1: Palisade ratio

For each sample of leaf make not fewer than ten determinations and calculate the average number.
Palisade ratio =18.4/4=4.5
Examples
Species Palisade ratio

Atropa belladonna 6 to 10

Cassia angustifolia 5.1 to 7.5

Datura stramonium 4 to 7

Datura tatula 4 to 7
b. Stomatal no. It is average no. of stomata per sq. mm of the epidermis of the leaf ,it is effected by various
factor like age of plant, size of leaf, enviourmental condition etc
Determination of Stomatal Number
 Place leaf fragments of about 5x5 mm in size in a test tube containing about 5 ml of chloral hydrate
solution and heat in a boiling water-bath for about 15 minutes or until the fragments become
transparent. Transfer a fragments to a microscopic slide andprepare the mount the lower epidermis
uppermost, in chloral hydrate solution and put a small drop of glycerol-ethanol solution on one side
of the cover glass to prevent the preparation from drying.
 Examine with a 40 x objective and a 6x eye piece, to which a microscopical drawing apparatus is
attached. Mark on the drawing paper a cross (x) for each stomata and calculate the average number
of stomata per square millimeter for each surface of the leaf.
Table 8: Determination of Stomatal Number

Species number of stomata per sq.mm

upper surface lower surface

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 Pharmacognosy:
Microscopic Techniques
Quantitative Microscopy

atropa belldona 7.5 to 10 to 17.5 77.5 to 113 to176

cassia angustifolia 180 to 200 to 195 to 220 to 257

223
b. Stomatal index:
Determination of Stomatal Index
 The stomatal index is the percentage of the number of stomata formed by the total number of
epidermal cells, including the stomata, each stoma being counted as one cell. Place leaf fragments
of about 5 × 5 mm in size in a test tube containing about 5 ml of chloral hydrate solution and heat
in a boiling water-bath for about 15 minutes or until the fragments become transparent. Transfer
a fragment to a microscopic slide and prepare the mount, the lower epidermis uppermost, in
chloral hydrate solution and put a small drop of glycerol-ethanol solution on one side of the cover-
glass to prevents the preparation from drying.
 Examine with a 40x objective and a 6x eye piece, to which a microscopical drawing apparatus is
attached. Mark on the drawing paper a cross (x)for each epidermal cell and a circle (o) for each
stoma. Calculate the result as follows:
 Stomatal index = s*100/E+S Where S = the number of stomata in a given area of leaf ; and E = the
number of epidermal cells (including trichomes) in the same area of leaf.
 For each sample of leaf make not fewer than ten determinations and calculate the average index.
Examples: Stomatal Index

Species Stomatal index

upper lower surface
surface
atropa belldona 2.3 to 3.9 to 20.2 to 21.7 to 23.0
10.5
cassia angustifolia 17.1 to 19.0 17.0 to 18.3 to 19.3
to 20.7
c. Vein islet number:
Determination of Vein-Islet Number
 The mesophyll of a leaf is divided into small portions of photosynthetic tissue by anastomosis of the
veins and veinlets; such small portions or areas are termed “Vein- Islets”. The number of vein-islets
per square millimeter is termed the “Vein-Islet number”.
 This value has been shown to be constant for any given species and, for full-grown leaves, to be
unaffected by the age of the plant or the size of the leaves. The vein-islet number has proved useful
for the critical distinction of certain nearly related species.
 The determination is carried out as follows:
 For Whole or Cut leaves–- Take pieces of leaf lamina with an area of not less than 4 square
millimeters from the central portion of the lamina and excluding the midrib and the margin of the leaf.
Clear the pieces of lamina by heating in a test tube containing chloral hydrate solution on a boiling
water-bath for 30 to 60 minutes or until clear and prepare a mount in glycerol-solution or, if desired,
stain with safranin solution and prepare the mount in Canada Balsam. Place the stage micrometer on
the microscope stage and examine with 4x objective and a 6x eye piece.
 Draw a line representing 2 mm on a sheet of paper by means of a microscopical drawing apparatus
and construct a square on the line representing an area of 4 square millimeters. Move the paper so
that the square is seen in the centre of the field of the eyepiece. Place the slide with the cleared leaf
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 Pharmacognosy:
Microscopic Techniques
Quantitative Microscopy

piece on the microscope stage and draw in the veins and veinlets included within the square,
completing the outlines of those vein-islets which overlap two adjacent sides of the square. Count the
number of vein-islets within the square including those overlapping on two adjacent sides and
excluding those intersected by the other two sides. The result obtained is the number of vein-islets in
4 square millimeters. For each sample of leaf make not fewer than three determinations and calculate
the average number of vein-islets per square millimeter.
 For Leaf Fragments having an area less than 4 square millimeters– Take fragments of leaf lamina
each with an area of not less than 1 square millimeter, excluding the midrib and the margin of the leaf.
Clear and prepare a mount as stated above. Use a 10x objective and a 6x eyepiece and draw a line
representing 1 mm on a sheet of paper by means of a microscopial drawing apparatus and construct
a square on this line representing an area of 1 square millimetre. Carry out the rest of the procedure
as stated above. The result obtained is the number of vein-islets in 1 square millimetre. For each
sample of leaf make no less than 12 determinations and calculate the average number.
Quantitative microscopy
Lycopodium spore method:
 It is an important technique for powdered drug, especially when chemical &other method fail as
accurate measure of quality. lycopodium is composed of spores of lycopodium elavatum.
 Each spore is tetrahedral in shape, the base is rounded and the three side wall makes the three well
marked covering ridge, which join one other at filled with fixed oil.
 The spore are exceptional uniform in size(25µm) and the shape tetrahedral so that one can always
know that a definite no. of spore present in particular weight of lycopodium.on an average 94000
spores per mg of powdered lycopodium are present.using this figure one can calculate theweight of
any number of spores under any condition under the microscope.
 A powdered drug is evaluated by this technique ,if its contains:
 Well defined particles may be count ed e.g. starch grain or pollen grain.
 Single layered cells or tissue,the area of which may be tracedunder suitable magnification.
 The object of uniform thickness ,the length of which can be measured under suitable magnification
and actual area can be calculated
Procedure:
 Determine the loss on drying of powdered sample material of both sample &lycopodium powder at
105ºc.
Mix about 100 mg powdered sample drug and 50 mg of lycopodium powder using a small flexible
spatula on a glass plate ,with a little suspending fluid.
 In this mixture incoroporate a sufficient quantity of suspending fluid (glycerin:mucilage of
tragacanth:water::2:1:2 or an oil) until a smooth line paste results .transfer it to stoppered tube by
washing with excess of suspending fluid .adjust the final volume so that about 15 to 20 spore are
observed in a field using a 4 mm of objective in microscope.
 Oscillate the stopered container gently in order to obtain uniformity of the suspension.place one
drop of suspension on each of two slide ,spread with a thin glass rod or needle ,apply the cover slip
and leave aside for few minutes on the table in order to allow the fluid mixture to settle eventely.
 Count the starch chacterstics structureof sample and lycopodium spores under microscope in each
of 25 different field selected for observation.
 Calculate the %purity of powdered drug by using the following equation:
% purity of crude drug=n*w*94000*100/s*m*p
 N=number of charactersic structure of sample in 25 field
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 Pharmacognosy:
Microscopic Techniques
Quantitative Microscopy

 W=weight in mg of lycopodium 94000=number of lycopodium spore mer mg

 S= number of lycopodium spore in same 25 field
 M=weight in mg of sample, calculated on the basis of sample dried at 105ºc
 P=number of charcterstic sturucture of sample in 1 mg (p is 2,86000 in case of ginger starch grain
powder)

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