CT Group of Institution

Shahpur Campus
Jalandhar

LAB MANUAL
Name of Institution: CT Group of Institution,
Shahpur, Jalandhar

Name of Subject: Human Anatomy and

Physiology

Course: B. Pharmacy

Semester: 1st

Name of Department: CTIPS

Signature of Lab In-charge Signature of HOD Signature of HOI
 List of Experiments
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Sr.No Name of Page No

Experiment
1 Study of compound microscope. 1-4

2 Microscopic study of epithelial and connective tissue. 5-10

3 Microscopic study of muscular and nervous tissue 11-13

4 Identification of axial bones 14-15

5 Identification of appendicular bones 16

6 Introduction to hemocytometry. 17-18

7 Enumeration of white blood cell (WBC) count 19-20

8 Enumeration of total red blood corpuscles (RBC) count 21-22

9 Determination of bleeding time 23

10 Determination of clotting time 24

11 Estimation of hemoglobin content 25

12 Determination of blood group. 26

13 Determination of erythrocyte sedimentation rate (ESR). 27

14 Determination of heart rate and pulse rate. 28

15 Recording of blood pressure. 29

 Experiment No: 1
Experiment Name: Study of compound microscope.

Aim: To study the structure of working procedure for the use of Compound Microscope.

Objectives: The main objective of this study is to understand about various parts of the
microscope, their functioning and uses of the microscope.

Material required: Compound Microscope

Description of Compound Microscope:

The compound microscope has the following main parts:

 The supporting system.

 The focusing system.
 The optical or magnifying system.
 The illumination system including:
 Source of light.
 Mirror.
 Condenser.
The support system: It is a framework to which various functional units are attached. It
consists of the following:

 Base: It is a heavy metallic, ‘U’ shaped or 'horseshoe' shaped base which supports
the microscope on work table and provides maximum stability.

 Pillars: There are two upright pillars that project up from the base and are attached
to the ‘C’ shaped handle. This allows the microscope to be tilted at a suitable angle
for comfortable observation.

 Body tube: It is 16-17 cm long cylindrical tube fitted at the upper end of handle
which is vertical or at an angle through which light passes via the eye piece to the
observer’s eye visualizing the image.

 The stage: The stage is a square platform with an aperture in its centre and fitted to
the limb below the objective lenses. When the slide is placed on it, converging rays
of light emerging from the condenser passes through slide and then objective lens
into the body tube. It can be either the fixed stage or the mechanical stage. The
fixed stage has two clips that hold the slide in position. The mechanical stage has a
calibrated metal frame fitted on right side of stage. It has a spring mounted clip to
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 hold the slide and two screw heads to move the slide from side to side, forward and
backward. The Vernier scale is also attached to indicate degree of movement.

The focusing system: The focusing system consists of coarse and fine adjustment and
screw heads are used for raising and lowering body tube for proper focusing the slide.
The coarse adjustment moves the focusing system up or down through a large distance
via a rack. The fine adjustment works in same way which requires several rotations to
move the tube through a small distance. It is employed for accurate focusing.

The optical or magnifying system: It consists of body tube, eyepiece and the nosepiece.
The body tube is the present between upper end of objective and eyepiece. The eyepiece
fits into top of body tube. They can be 5X, 6X, 8X, 10X or 15X. Each eyepiece has two
lenses; the eye lens at the top and field lens at the bottom. The field lens collects
divergent rays, passes through eye lens to further magnify the image. The nosepiece has
two parts; the fixed nosepiece and the revolving nosepiece. The fixed nosepiece holds
the revolving nosepiece that carries interchangeable objective lenses. The objective
lenses are spring loaded objectives of different magnifying powers. Different types of
objective lenses are low power objective or 10X, high power objective or 45X, oil
immersion objectives or 100X and scanning objective 3X.

Magnification: Magnification is the ability to make small objects seen larger, such as
making a microscopic organism visible. The objective lenses magnify the images as
stated below: Low power objective (10X) = 10 × 10 = 100 times. High power objective
(45X) = 45 × 10 = 450 times. Oil immersion objective (100X) = 100 × 10 = 1000 times.

The illumination system: The microscope will function only when proper illumination or
lightning is provided. The illumination system is to provide uniform, soft and bright
illumination.

The illumination system consists of:

(a) Source of light: It may be external (natural day light, electric lamp or tube light) or
internal (electric in-built light source).

(b) A condenser: It is a system of lenses fitted as short cylinder mounted below the stage.

(c) Mirror: A double sided mirror with one flat side and other concave is located below
condenser and can be rotated in all directions. It focuses light rays into a solid cone of
light onto the material under study and helps in resolving image.

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(d) Iris diaphragm: Iris diaphragm is a thin opaque membranous structure fitted within
condenser with a small lever on the side. The lever can adjust size of aperture of
diaphragm and allows less or more light falling on slide.

Procedure:

 Examine the permanent slide/blood film/specimen first with naked eye.

 Place the microscope on working table in an upright position, and raise the body
tube approximately 7-8 cm above the stage.

 Put the slide on the stage and using the mechanical stage, bring the specimen
over the central aperture. Select the low magnification objective (10X).

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 Select and adjust the mirror (plane or concave) so that the light shines on the
specimen.

 Adjust the condenser well down, and partly close the diaphragm to cut down
excess light.

 Looking from the side, and using the coarse adjustment, brings the body tube
down so that the low power lens is about 1 cm above the slide. Look into eyepiece
and gently raise the tube till the slide comes into focus.

 Then choose the area of interest for viewing it under higher magnifications.

 For focusing under high magnification, simply rotate the nosepiece so that the
high magnification objective (45X) ‘clicks’ into position.

 Raise the condenser to mid position and open the diaphragm to admit enough
light. Use fine adjustment as required.

 For focusing under oil immersion objective (100X), raise the body tube 8-10 cm
above the slide. Place a drop of cedar wood oil, paraffin or glycerin on the slide.

 Looking from the side bring down the objective till it just enters the oil drop. Use
other adjustments as required.

Reference:

 A Practical Book of Human Anatomy and Physiology by Dr. Shilpa, Nirali

Prakashan.

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Experiment number: 2

Experiment name: Microscopic study of epithelial and connective tissue.

Aim: To study the microscopic structure of epithelial and connective tissue.

Objectives: The main objective of this study is to gain the knowledge about various types
of tissues, epithelial and connective, along with the site of their presence and functions.

Requirements: A compound microscope and permanent tissue slides.

Epithelial tissue: Epithelial tissue or epithelium forms the outer covering of the skin and
also lines the body cavity.

 Simple Epithelia: The cells are arranged in a single layer, forming one cell thick
epithelium. Simple epithelia are further divisible as follows:

 Simple Squamous Epithelium: It consists of only one layer of flat, scale

like cells, usually polygonal cells which are closely fitted together like the
tiles on a floor. It is also known as pavement epithelium.

 This epithelium is present in the wall of the Bowman’s capsule

and descending loop of Henle of the nephrons of kidneys,
terminal bronchioles and alveoli of the lungs, membranous
labyrinth (internal ear), blood vessels, lymph vessels.

 Function: Protection, excretion, gas exchange and secretion

 Simple Cuboidal Epithelium: The simple cuboidal epithelium is

composed of one layer of cuboidal or squarish shaped cells resting on a
basement membrane. The nuclei are rounded and situated centrally. The
cells of cuboidal epithelium often form microvilli on their free surface
border called brush bordered cuboidal epithelium.

 This epithelium is present in the proximal and distal convoluted

tubules of the nephrons of kidneys, ovaries,seminiferous tubules
of testes, small salivary and pancreatic ducts and ciliary bodies,
choroid and iris of eyes.
 Function: Protection, secretion, absorption, excretion, gamete
formation.

 Simple Columnar Epithelium: It consists of a single layer of elongated

cells placed side by side, many of which have modified structure. Three
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 common modifications are goblet, cilia and microvilli. In the intestine
plasma membranes of many columnar cells extend out in hundreds and
hundreds of microscopic finger like microvilli, to increase the absorptive
surface area and is called brush bordered columnar epithelium.

 This epithelium lines the stomach, intestine, gall bladder and bile
duct. It also forms the gastric glands, intestinal glands.

 Function: Protection, secretion, and absorption.

 Simple Ciliated Epithelium: It bears numerous delicate hair like

outgrowths called cilia arising from basal granules which help to create
a current to transport the materials. Mucus secreting goblet cells also
occur in the ciliated epithelium.

 Ciliated columnar epithelium: It lines respiratory tract, fallopian

tubes, ventricles of brain, central canal of spinal cord, tympanic
acvity and auditory tube.

 Ciliated cuboidal epithelium: It occurs in certain parts of

nephrons of the kidneys.

 Function: The main function of ciliary epithelium is to maintain

flow of mucus or liquid or suspended particles or bodies
constantly in one direction. In the respiratory tract the cilia helps
to push mucus towards the throat. In the oviducts the cilia help to
move the egg towards the uterus. In nephrons of kidney, cilia
keeps the urine moving.

 Pseudo-stratified Epithelium: The cells presnt in this epithelium are

columnar shaped but unequal in size. The long cells extend upto the free
surface whereas the short cells do not reach the outer surface.

 Pseudostratified columnar epithelium: It consists of columnar

cells and occurs in the parotid salivary glands and urethra of the
human male.

 Pseudostratified columnar ciliated epithelium: It consists of

columnar cells in which the long cells bear cilia at their free
surface. It occurs in trachea and large bronchi.

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  Function: Protection, secretion, movement of secretions.

 Compound Epithelia: It is complex in structure and basically made up of two or

more than two layers of cells. The compound epithelia may be stratified and
transitional.
 Stratified Epithelium: This epithelia comprises of many layers of cells in
which the deepest layer is made up of columnar cell or cuboidal cells.
 Stratified keratinized squamous epithelium: The cells in the
deepest layer are columnar or cuboidal with oval nuclei called the
germinative layer. The middle layer, called the intermediate layer
consists of polyhedral cells with rounded nuclei.
 Location: Epidermis of the skin of land vertebrates
 Stratified squamous non keratinized epithelium: its free surface
is moist, and the outer epithelial cells, unlike those found in the
skin, do not contain keratin. This type of epithelium serves as a
protective function.
 Location: It is found lining the oral cavity, pharynx, oesophagus,
anal canal, lower part of urethra, vocal cords, vagina, cervix and
conjunctiva of eyes.
 Stratified cuboidal epithelium: It consists of two or more rows of
low cuboidal shaped cells which are arranged randomly over a
basement membrane.
 Location: It is found in the sweat gland ducts, larger salivary and
pancreatic ducts.
 Stratified columnar epithelium: It is protective epithelium and has
multiple layers of columnar cells. Only the most superficial cells
are truly columnar in appearance. Epithelium of this type is rare.
 Location: It is found in male urethra and in the mucous layer near
the anus. It also lines mammary gland ducts and epiglottis.
 Transitional Epithelium: This is a multi-layered epithelium and is 4-6
cells thick. It differs from stratified squamous epithelium in that the
cells at the surface are not squamous. The deepest cells are columnar
or cuboidal. The middle layers are made up of polyhedral or pear-shaped
cells. The cells of the surface layers are large and often shaped like an
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umbrella. Because of its distribution in the urinary system, it is also
called urothelium.
 Location: This epithelium is found in the renal pelvis and calyses,
the ureter, the urinary bladder and part of the urethra.

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CONNECTIVE TISSUE:
Tissue that supports, protects, and gives structure to other tissues and organs in the
body.
 Connective Tissue Proper: Its encompasses all organs and body cavities
connecting one part with another and, equally important, separating one group of
cells from another. This is a very large and diverse group of tissues and includes
adipose tissue (fat), areolar (loose) tissue, and dense regular tissue.
 Areolar (Loose) Connective Tissue: The fibres of areolar connective tissue
are arranged in no particular pattern but run in all directions and form a
loose network in the intercellular material. Collagen (collagenous) fibres are
predominant. They usually appear as broad pink bands. Some elastic fibres,
which appear as thin, dark fibres are also present.
 It is present under the skin as subcutaneous tissue in between and
around muscles, nerve and blood vessels, in submucosa of gastro-
intestinal tract and respiratory tract, in the bone marrow etc
 Function: It is used to attach the skin to the underlying tissue. It also
fills the spaces between various organs and thus holds them in place
as well as cushions and protects them. It also surrounds and

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 supports the blood vessels.

 Adipose Connective Tissue: It is fat storing connective tissue. The cells of

adipose (fat) tissue called adiposites or fat cells are characterized by a
large internal fat droplet, which distends the cell so that the cytoplasm is
reduced to a thin layer and the nucleus is displaced to the edge of the cell.
 These tissues are found in the subcutaneous tissue, around the heart,
kidney, eyeballs,mesenteries and omenta where fat is stored.
 Adipose tissue is chiefly a food reserve. Adipose tissue, also forms
shock absorbing cushion around and protects certain organs (eye
balls & kidney) and regions of the body. As well, it forms an
insulating layer under the skin which helps regulate body
temperature.
 Dense (Fibrous) Regular Connective Tissue: Dense connective tissue is
characterized by an abundance of parallel bundles of fibres fibres with
fewer cells, as compared to the loose connective tissue.
It is divided into two types: White fibrous connective tissue and yellow
elastic connective tissue.
 Specialized Connective Tissues: This group includes cartilage, bone, and blood.
Cartilage and bone form the skeletal framework of the body while blood is the
vascular (transport) tissue of animals.
 Cartilage (Gristle): Cartilage is a somewhat elastic, pliable, compact type of
connective tissue. It is also skeletal tissue. Cartilage is a non-vascular
tissue. As such, the cartilage cells or chondrocytes rely on blood vessels in
the tissue surrounding the cartilage for nutrient supply and waste removal.
 Bone: Bone is the hardest tissue in the body and supports various organs. A
typical bone comprises of periosteum, matrix, endoosteum and bone
marrow. The entire outer surface of bone is covered by a thick and tough
sheath called the periosteum. Periosteum contains blood vessels. The
periosteum also has osteoblast which produce new bone material. The
matrix is composed of a protein called ossein. The main salts found in
matrix are calcium carbonate, calcium phosphate, sodium chloride, and
magnesium phosphate. The matrix of bone occurs as layers called

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 lamellae.The Haversian canals present in the matrix is a characteristic
feature of the mammalian bone. Each haversian canal contains an artery, a
vein, a lymph vessel, a nerve, some bone cells, all packed in with connective
tissue. The haversian canals are connected by transverse channels called
the volkmann’s canals.

 Blood: Blood is a mobile connective tissue. It is the softest tissues of the

body. It is a slightly alkaline fluid having pH 7.4. Blood is composed of a
watery fluid called plasma and floating bodies termed formed elements
(blood corpuscles). Plasma is a alkaline non-living intercellular substance.

References:
 Structural Organisation In Animals-Animal Tissues, Trueman’s Elementary Biology,
K.N.Bhatia et a., Edition 2016, pp. 168-213
 PV’s Human Anatomy And Physiology-I, S.S. Randhawa et al., Edition 2017
 Bones, Blood , Epithelial tissue, Textbook Of Human Histology, Second Edition, pp.
66-137.

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Experiment number: 3
Experiment name: Microscopic study of muscular and nervous tissue.
Aim: To study muscular and nervous tissues with the help of permanent slides.
Objective: the main objective of this study is to know about various muscular and
nervous tissues, their functions and location in the body.
Material required: Microscope, permanent slides.
MUSCULAR TISSUE:
The muscle tissue originates from embryonic mesoderm. The muscle cell is called
myocyte. They are of 3 types:
 Skeletal Muscle
 Smooth Muscle.
 Cardiac Muscle.
Skeletal Muscle:
 It’s also called striated muscle.
 Shapes cylindrical, Multinucleate.
 Striations (alternate light and dark bands) are present.
 Sarcoplasmic reticulum is well developed .
 It’s voluntary in function and gets fatigue soon.
 Intercalated discs are absent.
 They are innervated by motor nerves.
 Blood supply is abundant.
 Location: Limbs, biceps and body wall.
Smooth Muscle:
 It’s also called visceral and involuntary muscle.
 Shape: Spindle, uninucleate, nucleus at the centre striations is absent.
 Sarcoplasmic reticulum is less developed.
 Involuntary in function and don’t get fatigue soon.
 They contract slowly for a long time.
 Location: Hollow visceral organs like GIT, blood vessels urinary bladder, biliary
body, respiratory system etc.
Cardiac Muscle:
 This tissue forms 3-D network.
 Shape-short, cylindrical and branched.
 Uninucleate –nucleus at the centre.

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Location: Heart

NERVOUS TISSUE:
 Nerve cells are called Neurons.
 It’s structural and functional unit of nervous system.
 Cell body (Cyton) consists of neuroplasm,nucleus, mitochondria and Golgi bodies.

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The cell process is two types:
 Dendron’s (Dendrites): These are much branched process for receiving
impulses.
 Axon: A single long cylindrical process for conducting impulses away from
cyton.

 The nerve fibres are of two types i.e., Myelinated and Non-Myelinated.
 The Myelinated nerve fibre has nodes of Ranvier which helps in rapid
transmission of nerve impulses.
 The neurilemma consists of Schwann’s cells which produce myelin
sheath around the neurons.
 Neuroglia: It is supporting and packing cells found in brain, spinal cord
and Ganglia.
Functions: To receive, discharge and transmit impulses.
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References:
 Structural Organisation In Animals-Animal Tissues, Trueman’s Elementary Biology,
K.N.Bhatia et a., Edition 2016
 PV’s Human Anatomy And Physiology-I, S.S. Randhawa et al., Edition 2017

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Experiment number: 4
Experiment name: Identification of axial bones
Aim: To study and identify axial bones.
Objective: the main objective of this study is to gain knowledge about various types of
axial bones, their location and special functions.
Human skeleton:
The human skeleton is the internal framework of the body. It is composed of around 270
bones at birth – this total decreases to around 206 bones by adulthood after some bones
get fused together. The human skeleton can be divided into the axial skeleton and the
appendicular skeleton.
The axial skeleton is formed by the vertebral column, the rib cage, the skull and other
associated bones.
The appendicular skeleton, which is attached to the axial skeleton, is formed by the
shoulder girdle, the pelvic girdle and the bones of the upper and lower limbs.
Axial skeleton:
The axial skeleton (80 bones) is formed by the vertebral column (32–34 bones; the
number of the vertebrae differs from human to human as the lower 2 parts, sacral and
coccyx geal bone may vary in length), a part of the rib cage (12 pairs of ribs and the
sternum), and the skull (22 bones and 7 associated bones).
 Skull: It is also called as cranium. It forms a hallow structure in which the brain is
present and protected. It consists of 22 bones in which they are classified as
cranial and facial bones.
 Cranial bones: they are 8 in number
 Spheboid bone -1
 Parietal bone-2
 Osscipetal-1
 Temporal bone-2
 Frontal bone-1
 Ethmoid bone-1
 Facial bones: they are 14 in number including frontal lobe
 Nasal-2
 Maxilla -1
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  Zygotmaic-2
 Lacrimal -2
 Palatine-2
 Inferior nasal chonchoe-2
 Vomer-1
 Mandible-1
 Hyoid bone-1
 Vertebral column: it is also called the back bone which gives support for the body
to perform any physical activity. It has 33 bones called vertebrae. Each individual
bone of vertebral column is called vertebrae.
 Cervical vertebrae-7
 Thoracic vertebrae-12
 Lumbar-5
 Sacral-1(5 fused vertebrae)
 Coccyx-1(4 fused)
 The first vertebrae is called atlas on which the brain rest. it has two facets on which
the skull is placed. it brings about the nodding movement of the head
 The second vertebrae is called axis. It is adjacent to the atlas and has two facts.
One for the atlas and other for adjacent vertebrae. It helps in side to side movement
of neck
 Thoracic vertebrae present in thoracic region. They are rigid and movement is
limited. It aids in the movement of the entire vertebral column.
 Lumbar vertebrae are found in back portion of abdomen.
 The sacrum is fused wedge shaped bone which adjoins with the pelvic girdle
forming the ileo-sacral joint. It maintains the weight of the body. Hence, it is fused.
 Coccyx is the tail of the vertebral column and ends in the pelvic girdle.
Reference:
 Structural Organisation In Animals-Animal Tissues, Trueman’s Elementary Biology,
K.N.Bhatia et a., Edition 2016
 PV’s Human Anatomy And Physiology-I, S.S. Randhawa et al., Edition 2017 .

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Experiment number: 5
Experiment name: Identification of appendicular bones.
Aim: To study and identify appendicular bones.
Objectives: The main objective of this study is to know about various bones and
functions of appendicular skeleton.
Appendicular skeleton:
The parts of the appendicular skeletal system are pectoral girdle, pelvic girdle, bones of
upper and lower limb.
 Pectoral girdle: Each of the two bodies. Pectoral girdle consists of clavicle and
scapula .each pectoral girdle attacks on upper line to the axial skeleton.
 Bones of upper limb: each of two upper limbs contains 30 bones. The bone of
each upper limb includes humerous
 ulna (1)
 radial (1)
 carpals (8)
 metacarpals (5)
 phalanges (14).
 Pelvic girdle: the pelvic girdle consists of two hip bones which are ileum, ischium,
and pubic. The hip bones contain sacrum and pubic symphasis from the pelvic
bone. Support the vertebral column. the pelvic viscera attaches the lower limbs to
the axial skeletal.
 Bones of lower limb: each of the two limb consists 32 bones, they include femur
(thigh bone ), patella, tibia(1), fibula(1), tarsal(7), metatarsals(5), phalanges(14).the
bones of the foot are arranged in two arches i.e. the longitudinal arch and
transverse arch to provide the movement.

References:
 Structural Organisation In Animals-Animal Tissues, Trueman’s Elementary Biology,
K.N.Bhatia et a., Edition 2016
 PV’s Human Anatomy And Physiology-I, S.S. Randhawa et al., Edition 2017 .

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 Experiment number: 6

Experiment name: Introduction to hemocytometry.

Aim: To study about hemocytometer.
Objective: The main objective of this study is to know about hemocytometer, its parts,
and functioning.
HAEMOCYTOMETRY:
It is a technique used to enumerate the total cell count in the blood or other biological
body fluids. This can be done either by using haemocytometer or by electronic cell
counter.
PURPOSE:
In certain pathological conditions the value of different type of cells may have the
variation. Thus, by counting the cells in the blood or body fluids, it can be find out if an
individual is normal or not.
Broadly,the cell count is done mainly:
 To find out normal and abnormal count of the cells
 To support and confirm clinical diagnosis of the patient
 To find out the response of the patient to the treatment
HAEMOCYTOMETER:
 This is an instrument used for counting the cells in blood or fluid.
 It consists of a special instrument called counting chamber, cover glass, pipette
for diluting the blood rubber tube with plastic mouth piece for drawing blood or
fluid in pipette.

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 Counting Chamber: It is a thick glass slide with two identical ruled areas
separated by empty space and having two elevated ridges on their both sides.
Either of the ruled areas is used for counting the cells. There are different types
of counting chamber viz. old neubauer counting chamber, improved neubauer
counting chamber, burker counting chamber and fuch’s rosenthal counting
chamber.
 Cover Glass: A special cover glass is used which has a very smooth, flattened
surface and even thickness. Different thickness are: 0.3mm, 0.4mm (most
common), 0.5mm. two sizes are common: 16 × 22 sq.mm and 22 × 23 sq.mm.
 Diluting Pipette: It is a glass tube pipette with rubber sucking arrangement. The
tubular part of the pipette is graduated from 0 to 1 with the division of 0.1 units.
The bulb portion can accommodate 100 units of volume graduated from 1 to
101 on both sides of the bulb. Bulb serves as diluting and mixing chamber for
blood. The red bead in it, aids in mixing and for identification of red cell pipette

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 from white cell pipette.
Reference:
1. Haematology, Practical Human Anatomy And Physiology, S.R. Kale et al., Nirali
Prakashan, Eight Edition, 2002, pp. 11-12

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Experiment number: 7

Experiment name: Enumeration of white blood cell (WBC) count.

Aim: To find out total WBC count in given sample.
Requirements: WBC diluting fluid, WBC pipette, watch glass.
Objective: The main objective of this study is to know about how the WBC count in the
blood if measured along with the actual range of WBC and their functions.
Procedure:
 Take WBC diluting fluid (Turk’s Fluid) in a watch glass.
 After pricking finger, suck the second drop of blood into the WBC pipette exactly
up to 0.5 mark and dilute it with WBC diluting fluid by sucking the fluid up to
11mark ( dilution 1 in 20 ) .
 Gently rotate the pipette at least 3-4 minutes in the palm of the hand to ensure the
proper mixing of the blood and the fluid.
 Discard first few drops of WBC fluid in the stem of the pipette, charge the counting
chamber (a small drop of fluid is allowed to form at the tip of the pipette and
gently brought into contact with the edge of the cover slip that is already placed on
the chamber) and allow time for settling of the cells.
 Under low power objective, identify and check the distribution of WBCs in the 4
corner squares.
 Recharge the chamber if distribution is not uniform (WBCs are seen as regular
nucleated, rounded bodies with a clear refractivity around them).
 Count the number of WBCs in each WBC square preferably under low power
objective.
 Count the WBCs in 4 corner WBC squares and enter your observations in the
corresponding squares.
Calculations:
 Calculation of diluting factor Dilution factor:
Final volume achieved (10 parts) Original volume taken (0.5 parts)
 Calculation of volume fluid examined:
Area of 4 WBC squares = 4 X 1mm X 1mm = 4 sqmm Depth of the chamber =

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 0.1mm

 Calculation of Total Leukocyte count:

Let ‘N’ be the total number of WBCs in 4 WBC squares i.e. in 0.4 cu.mm of diluted
blood
Then total number of WBCs in 1 cu.mm of undiluted blood :
N x Dilution factor (20) 0.4 = N x 50

Reference:
 Structural Organisation In Animals-Animal Tissues, Trueman’s Elementary Biology,
K.N.Bhatia et a., Edition 2016
 PV’s Human Anatomy And Physiology-I, S.S. Randhawa et al., Edition 2017 .

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Experiment number: 8

Experiment Name: Enumeration of total red blood corpuscles (RBC) count.

Aim: To find out the number of red blood cells in one cubic millimeter of blood.
Objective: The main objective of this study is to find the method to count total RBC count
of the blood along with their importance.
Material required: Hemocytometer, RBC diluting fluid, compound microscope, sterile
lancet, watchglass, cotton, rectified spirit.
HAEMOCYTOMETER: This includes a counting chamber, a special cover slip, and RBC
diluting pipette and a WBC diluting pipette.
Procedure:
 Clean and dry the counting chamber and put on the special cover slip provided.
Focus under the high power objective and identify the RBC counting area.
 Clean the RBC pipette first with distilled water, then with absolute alcohol and
finally with ether and keep it dry.
 Take a small quantity of diluting fluid in a watch glass and keep aside. Clean the
finger tip using rectified spirit and make a deep prick with a sterile lancet, so that
blood comes out freely without squeezing.
 Wipe off the first drop which may contain tissue fluid also. Allow a good sized
blood drop to form hanging drop and keep the pointed tip of the pipette touching
the drop. Suck in blood upto the 0.5 mark carefully, without any air bubble.
 Excess blood at the tip of the pipette is removed using a bloating paper or piece of
cotton. Immediately, diluting fluid from the watch glass is sucked in upto the 101
mark without any air bubble by keeping the pipette in vertical position.
 Then thoroughly mix the blood and diluting fluid in the pipette by gently rolling the
pipette held horizontally between the palms and keep aside.
 Mixing takes place only in the bulb of the pipette. The column of diluting fluid
contained in the stem of the pipette does not enter into the dilution (i.e. 101-1 =
100).
 So that the blood sucked upto 0.5 mark will have a dilution of 0.5 in 100 or 1 in 200.
Now take out the counting chamber for charging discard first few drops from the
pipette, as the stem contains only diluting fluid.
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  Bring one small drop of diluted blood at the tip of the pipette, to the edge of the
cover slip on the counting chamber at an angle of about 450.
 The fluid enters by capillary action under the cover slip and fills the counting
chamber. Both areas are filled.
 Focus the RBC counting area under high power.
 Keep the counting chamber undisturbed about 3 minutes for the cells to settle
down in the counting area, and start counting.
 At least 5 squares, each having 16 smallest squares (preferably 4 corner and 1
central) should be counted to obtain a satisfactory average and a better dispersal
value.
 While counting each small square, cells touching the top and left margin of each
square should be omitted and cells touching bottom and right margin of each
square should be counted.
 Draw a chart of the counting squares in the record and enter the number of cells in
each square and when counted.
Calculation:
 Let the number of cells counted in (5x16) 80 smallest squares be “N”
 Number of cells in 1 smallest square is N/80
 Side of 1 square = 1/20mm
 Area of 1 square = 1/400mm2
 Depth of fluid film in counting chamber is 1/10mm
 Volume of diluted blood in 1 square=1/400x1/10=1/4000mm3
 Number of cells in 1/4000mm3 diluted blood = N 80
 Number of cells in 1 mm3 of diluted blood N 80x1/4000 = Nx4000 80 The dilution
factor is 1 in 200 (Total diluted volume in bulb of the pipette is 100 parts, out of
which 0.5 is blood. So dilution is 0.5 in 100 i.e.1 in 200)
 So number of cells in 1 mm3 of undiluted blood =Nx 4000x200 = Nxl0000 80
Reference:
 Structural Organisation In Animals-Animal Tissues, Trueman’s Elementary Biology,
K.N.Bhatia et a., Edition 2016
 PV’s Human Anatomy And Physiology-I, S.S. Randhawa et al., Edition 2017 .

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Experiment number: 9
Experiment name: Determination of bleeding time.
Aim: To determine bleeding time of the subject.
Objectives: The main objective of this study is to find out bleeding time of the subject.
Material required: Sterilized needle, filter paper, cotton, spirit, and stop watch.
Procedure:
 Finger of a subject is sterilized with spirit and pricked with sterilized needle.
 Time of pricking is noted.
 Take the stain of the punctured point on a filter paper on 30 second and keep
taking stain of blood in 20 second intervals until the bleeding stops.
 The time of no stain has come is noted properly; it is the bleeding time of the
subject.
Precautions:
 Needle should be sterilized.
 A fain stain of blood should not be avoided.
 Time should be noted properly
References:
 Structural Organisation In Animals-Animal Tissues, Trueman’s Elementary Biology,
K.N.Bhatia et a., Edition 2016
 PV’s Human Anatomy And Physiology-I, S.S. Randhawa et al., Edition 2017

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Experiment number: 10
Experiment name: Determination of clotting time.
Aim: To determine the clotting time of a subject.
Objectives: The main objective of this study is to find out the clotting time of the subject.
Material required: Fine capillary glass tubes of about 10 mm length, cotton, rectified
spirit, lancet, stop watch.
Procedure:
 Capillary tube method: (Wright’s method)
 Under sterile precautions make a sufficiently deep prick in the finger tip.
 Note the time when bleeding starts (start the stop watch).
 Touch the blood drop at the finger tip using one end of the capillary tube
kept tilted downwards.
 The tube gets easily filled by capillary action.
 After about two minutes start snapping off small lengths of the tube, at
intervals of 15 seconds, each time noting whether the fibrin thread is
formed between the snapped ends.
 Note the time (stop the stop watch) when the fibrin thread is first seen.
References:
 Structural Organisation In Animals-Animal Tissues, Trueman’s Elementary Biology,
K.N.Bhatia et a., Edition 2016
 PV’s Human Anatomy And Physiology-I, S.S. Randhawa et al., Edition 2017

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Experiment number: 11
Experiment name: Estimation of hemoglobin content.
Aim: To determine the hemoglobin content in 20µl of blood sample.
Objective: The main objective of this study is to find out the hemoglobin content of the
subject along with the normal ranges for that.
Material required: Hemometer, Single mark pipette, Distilled water, Needle, Spirit, Cotton,
HCl.
Procedure:
 Take 1/10 HCl in the Hb tube upto the lowest mark ‘2’.
 Prick the finger with needle and collect 20µl of blood sample with single mark
pipette.
 Place the Hb tube on working table for five minutes for the formation of hemin
crystals.
 Place the Hb tube in the compater/hemometer and add drop by drop of distilled
water into it until the colour of the solution in the Hb tube coincides with the glass
plates of the compater.
 If the colour coincides with the glass plates of the compater, observe the reading
in the Hb tube.
 The percentage of Hb can be calculated from the reading.
Data analysis:
Hb content in grams X 100 / 14.5
NORMAL VALUES:
 Males = 14 to 18 grams
 Females = 13 to 14 grams
 Children = 10 to 13 grams
References:
 PV’s Human Anatomy And Physiology-I, S.S. Randhawa et al., Edition 2017

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Experiment number: 12
Experiment name: Determination of blood group.
Aim: To determine the blood group of the subject.
Objectives: The main objective of this study is to know about various blood groups along
with various antigen and antibody concept in the blood groups.
Material required: Toothpicks, Blood sample, Alcohol Swabs, Lancet, Clean glass slide,
Sterile cotton balls, Biohazard disposal container, Monoclonal Antibodies (Anti-A, B, and
D).
Procedure:

 Take a clean glass slide and draw three circles on it.

 Unpack the Monoclonal Antibodies (MAB) kit. In the first circle add Anti-A, to the
second circle add Anti-B and to the third circle add Anti-D with the help of a
dropper.

 Keep the slide aside safely without disturbing.

 Now wipe the ring finger with the alcohol swabs and rub gently near the fingertip,
where the blood sample will be collected.

 Prick the ring fingertip with the lancet and wipe off the first drop of the blood.

 As blood starts oozing out, allow it to fall on the three circles of the glass slide by
gently pressing the fingertip.

 Apply pressure on the site where it was pricked and to stop blood flow. Use the
cotton ball if required.

 Mix the blood sample gently with the help of a toothpick and wait for a minute to
observe the result.

Reference:

 https://byjus.com/biology/blood-group-test/

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Experiment number: 13

Experiment name: Determination of erythrocyte sedimentation rate (ESR).

Aim: To determine erythrocyte sedimentation rate.

Objectives: The main objective of this study is to determine the erythrocyte

sedimentation rate.

Material required: Spirit, cotton, syringe, Westergren’s ESR pipette

Procedure: The ESR is estimated by three different methods :

1) Westergren’s Method

2) Wintrobe’s Method

3) Cutler’s Method

Westergren’s Method

 Westergren’s pipette (open at both ends) is about 30 cm long with a bore diameter
of about 2.5mm.

 The lower 20cm are marked from 0 (top) to 200 (bottom).

 Anticoagulant used is 3.8% trisodium citrate solution. One part of anticoagulant is

added to four parts of blood.

 The pipette accepts about 1ml of blood. Fill the pipette by sucking till the 0 mark
and clamp it vertically in the Westergren rack.

 Read the upper level of red cells exactly after 1 hour.

References:

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 Haematology, Practical Human Anatomy And Physiology, S.R. Kale et al., Nirali
Prakashan, Eight Edition, 2002, pp. 28

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Experiment number: 14

Experiment name: Determination of heart rate and pulse rate.

Aim: To determine the heart rate and pulse rate of the subject.

Objectives: The main objective of this study is to examine the heart rate and pulse rate of
the subject along with the knowledge of the normal ranges of them.

Material required: Stethoscope & Stop clock.

Heart rate: It refers to the number of times your heart beats per minute. It is a measure of
how efficiently your heart is pumping blood throughout your body. A normal resting heart
rate for adults typically ranges from 60 to 100 beats per minute, although this can vary
depending on age, fitness level, and other factors.

Pulse rate: It is the number of times a person’s artery expands and contracts per minute
in response to the heart pumping blood. The normal range lies between 75-80 per
minutes. It may vary with different age groups.

Procedure:

 Allow the subject to sit or lie in a calm environment.

 Place the chest piece of the stethoscope against the thoracic wall of the subject.

 Record the heart beat for 1 minute.

 The radial artery is located on the wrist (on the side opposite the back of the hand)
just below the base of the thumb.

 You can find it taking two fingers and placing them in this area: you should easily
feel a rhythmic pulse.

 Count the pulse for at least 30 seconds and then multiply times by two; the result
will be the heart rate.

 Do not use your thumb to count the pulse. Many people have a strong pulse in
their thumbs and this can interfere with accurately feeling someone’s pulse.

References:

Page | 33
 Physiological Parameters, Practical Human Anatomy And Physiology, S.R. Kale et
al., Nirali Prakashan, Eight Edition, 2002, pp. 44

Page | 34
Experiment number:15
Experiment name: Recording of blood pressure.
Aim: To determine the blood pressure of the subject.
Objectives: The main objectives of this study are to determine the method to find out
blood pressure. It also give knowledge about the systolic and diastolic blood pressure.
Material required: Sphygmomanometer, stethoscope.
Procedure:
Blood pressure is the force created by the blood circulation through the circulatory
system.

 Allow the patient to relax for 15 to 20 minutes before taking their readings.

 Wrap the blood pressure cuff evenly around the patient’s arm above the
antecubital fossa for an accurate reading.

 Now place the bell of the stethoscope over the brachial artery at this location to
get the strongest pulse sounds.

 Once, after everything is set, start pumping the cuff bulb gradually and listen to the
pulse sounds. Keep on checking the reading in the Sphygmomanometer.

 Continue to expand the cuff up to the point where the pulse sound is no longer felt.
This reading is recorded as the diastolic pressure.

 Now slowly reduce the cuff until the pulse sounds are felt. This reading is recorded
as the systolic pressure.
References:
 https://byjus.com/biology/blood-pressure-test/

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