HAP I (P) lab manual

Faculty of Pharmaceutical Sciences
Human Anatomy and Physiology – I (P)
I Semester B.Pharm Page 1
Prepared by Dr.Shivalinge Gowda KP Prof. and HOD, Dept of Pharmacology
INDEX
SN Title of the experiment Pages
1 Study of compound microscope. 2
2 Microscopic study of epithelial and connective tissue 5
3 Microscopic study of muscular and nervous tissue 11
4 Identification of axial bones 14
5 Identification of appendicular bones 20
6 Introduction to hemocytometry 26
7 Enumeration of white blood cells (WBC) count. 28
8 Enumeration of total blood corpuscles (RBC) count 31
9 Determination of bleeding time 34
10 Determination of clotting time 35
11 Estimation of hemoglobin content 38
12 Determination of blood group 41
13 Determination of erythrocyte sedimentation rate (ESR). 43
14 Determination of heart rate and pulse rate 46
15 Recording of blood pressure 47

Experiment No 1 Date-
Study of compound microscope
Aim- To study compound microscope using charts and specimen.
Compound microscope- It is an instrument used for magnifying small objects, consisting of a
lens of short focal length for forming an image that is further magnified by a second lens.
Parts of a compound microscope-
A-Course adjuster- It is a knob that makes large adjustments to the focus.
B- Fine adjuster-It is a knob that makes fine adjustments to the focus.
C.Carrying arm-This attaches the eyepiece and body tube to the base.
D- Stage clips/moving clips- Metal clips that hold a slide securely onto the stage.
E-Inclination joint- The arm can swivel at this point.
F- Base- It is made up of hard metal. This supports the microscope. Its other name is horse
shoe base.
G-Eye piece- The speciman kept on the stage can be viewed through eye piece. It contains two
or more lenses. The most common magnification for the eyepiece is 10X. However, they can also
be 2x, 5X and 15X. An eye piece is a removable part that can be interchanged with another
eyepiece.
H- Body tube or barrel- It is the part of the microscope that holds tha eyepiece.
I- Revolving nose piece- The rotating device that holds the objectives (lenses).
J- Low power objective lens- A lens with low magnification power (10X)
Body tube- It is the part of the microscope that holds tha eyepiece.
Arm- The arm connects the body tube to the base. It is the part that a user holds to move the
microscope from one place to another.
Base- As the name suggests, the base is the lowest portion on which the whole structure of the
microscope rests. It is also called as horse shoe base and it is made up of hard metal.
Eye piece- The speciman kept on the stage can be viewed through eye piece. It contains two or
more lenses. The most common magnification for the eyepiece is 10X. However, they can also

be 2x, 5X and 15X. An eye piece is a removable part that can be interchanged with another
eyepiece.
Objective lenses- There are different types of objective lenses. These are the primary lenses of a
compound microscope and can have magnification of 4x, 5x, 10x, 20x, 40x, 50x and 100x. The
magnification values are written on the side of each lens. The objectives are attached to the
nose piece that can be rotated with hand to get placed in the correct position.
Stage-It is the platform below the objective lens on which the object to be viewed is placed.
There is a hole in the stage through which light beam passes and illuminates the specimen that is
to be viewed.
Stage clips-There are two stage clips one on each side of the stage. Once the slide containing the
specimen is placed on the stage, the stage clips are used to hold the slide in place. Moving clips
are fixed in some compound microscopes instead of stage clips.
Condenser- The condenser is a lens that condenses the received light from the illuminator
(mirror).
Iris diaphragm-It is located on the lower surface of the stage. It is used to control the amount of
light that reaches the specimen through the hole in the stage.

Illuminator (mirror)- Simple compound microscopes have a mirror that can be moved to adjust
the amount of light that can be focused on the specimen. However, some advanced types of
compound microscopes have their own light source.
Adjustments- Two adjustment knobs present in the microscope- the fine adjustment knob and
the coarse adjustment knob that helps for the focusing of the lens. The coarse adjustment knob
helps in improving the focus of the low powers whereas the fine adjustment knob helps in
adjusting the focus of the lenses with higher magnification.
Instructions- 1.Carry the microscope by holding the arm with one hand and supporting the base
with the other hand. Place the microscope flat on the table, keeping it away from the edge.
2. Cover the microscope with the dust cover after its use.
Observations & Calculations:
Results:
Signature of the Teacher:
Date:

Expt. No.2 Date-
Microscopic study of epithelial and connective tissue
Aim- To study the microscopic structure of epithelial and connective tissue.
Tissues- A tissue is a group of similar cells that have a common embryonic origin and functions
together to carry out specialized activities.
Four elementary tissues of the body are epithelial, connective, muscular and nervous tissue.
a. Epithelial tissue- An epithelial tissue consists of cells arranged in continuous sheets, in either
single or multiple layers.
Classification- Epithelial tissue classified into simple and stratified epithelium.
i) Simple epithelium- It consists of single layer of cells. On the basis of shape the simple
epithelium is sub classified as below.
Simple squamous epithelium- It consists of single layer of flattened cells with nuclei located at
the center. Location- Alveoli of the lung, blood capillaries, glomerular capillaries.
Simple cuboidal epithelium- - It consists of single layer of cube shaped cells with nuclei located
at the center. Location- Small ducts of many glands, surface of the ovary.
Non-ciliated simple columnar epithelium- It consists of single layer of column like cells with
nuclei near the base of the cells. Goblet cells also present in between epithelial cells. Goblet cells
secrete mucus, which lubricates the surface. Location- Gastrointestinal tract, ducts of many
glands.
Ciliated simple columnar epithelium- It consists of single layer of ciliated column like cells with
nuclei near the base of the cells. They also have few goblet cells. Location- Upper respiratory
tract, central canal of spinal cord, fallopian tubes.
Pseudostratified columnar epithelium- It is not a true stratified epithelium. It is simple
columnar epithelium, but looks like stratified epithelium. Few goblet cells are also present. The
basal surfaces of all cells attached to the basement membrane, but all cells epical surfaces are
not free. Location- Upper respiratory tract, epididymis.

ii) Stratified (compound) epithelium- It consists of more than one layer of cells. On the basis of
shape the cells of superficial layer it is sub classified as below
Stratified squamous epithelium- It consists of several layers of cells. The deeper layer cells have
cuboidal to columnar shape, but the cells of superficial layer have squamous shape. Hence the
name stratified squamous epithelium. If keratin is present then it is keratinized stratified
squamous epithelium (eg.skin). If keratin is not present then it is nonkeratinized stratified
squamous epithelium (eg-mouth, esophagus, vagina).

Stratified cuboidal epithelium- It consists of two or more layer. The cells in the superficial
(apical) layer are cube shaped. Location- Ducts of sweat glands, male urethra.
Stratified columnar epithelium- It consists of several layers of irregular cells, but the cells in the
superficial (apical) layer are column shaped. Location- Conjunctiva of the eye.
Transitional epithelium-It consists of 3-4 layers of cells. The shape cells of the apical layer are
variable. When stretched cells are squamous shape, when relaxed cuboidal shape. Location-
Urinary bladder, ureter, urethra.
b. Connective Tissue- (CT). It is one of the four elementary tissue that support, connect, or
separate different types of tissues and organs in the body.
Classification- I.Embryonic CT- Mesenchymal and mucosal CT.

II.Mature CT- a.Loose CT- Areolar, adipose and reticular CT.b.Dense CT- Dense regular, dense
irregular, elastic CT. c.Cartilage- Hyaline, fibro, elastic CT d. Hard CT- Bone e.Fluid CT- Blood,
lymph.
I.Embryonic CT-
Mesenchyal CT- It consists of irregular shaped mucosal cells with semi fluid matrix and fine
reticular fibers. Location- Developing bones of embryo.
Mucosal CT- It consists of star shaped cells with viscous jelly matrix and collagen fibers.
Location- Umbilical cord of fetus.
II Mature CT-
a.Areolar CT- It consists of fibers (collagen, elastic and reticular ) and several kinds of cells
(fibroblasts, macrophages, plasma cells, adipocytes and mast cells) and semi fluid matrix.
Location- Subcutaneous layer, around blood vessels, nerves and organs.
Adipose CT- It consists of oval shaped fat cells. The cytoplasm is loaded with fat (triglycerides)
and the nucleus is located in the pheriphery. Location- Eye, heart, kidney and intestine.
Reticular CT- It consists of network of interlacing reticular fibers and reticular cells. Location-
Stroma (frame work) of liver, spleen and lymph node.

b.Dense CT-
Dense CT regular- (White fibrous tissue-regular)- It consists of parallel bundles of collagen fibers.
The collagen fibers are unbranched. The fibroblasts (fiber cells) are present in between bundles.
Location- Tendons (connect muscle to bone) and ligaments (connect bone to bone).
Dense irregular CT (White fibrous tissue-irregular)- It consists of irregularly or randomly
arranged collagen fibers with few fibroblasts. Location- Periosteum of bone, joint capsule.
Elastic CT-It consists of branched elastic fibers with fibroblasts. The elastic fibers are not
arranged in parallel bundles. Location- Lung tissue, elastic artery.
c. Cartilage- It is a type of dense CT. It is less hard than bone. Cartilage consists of bluish white
semisolid matrix known as chondrin. The chondrocytes (cartilage cells) are present within spaces
known as lacunae. Each lacuna consists of 3-4 chondrocytes. If fibers are not present then it is
hyaline cartilage (eg. trachea, nose, ends of long bone). If collagen fibers are present then it if

fibrocartilage (intervertibral discs, symphysis pubis). If elastic fibers are present then it is elastic
cartilage (eg. ear auricle, epiglottis).
d.Hard CT- Bone is the hard connective tissue. The bone is covered by periosteum. The compact
bone and spongy bone are the other layers. Medullary cavity is present in the long bone and is
filled by red bone marrow. As age advances the red bone marrow is replaced by yellow bone
marrow.
Microscopy of bone- Bone composed of numerous haversian systems. Each haversian system is
composed of a central canal known as haversian canal. The bone is deposited around this canal
in concentric circles known as lamella. The space between two lamellae is the lacuna. The bone
cells (osteocytes) are located in the lacuna. The canaliculi are the thread like structures which
give connections between haversian canal and bone cells.
Observations
Date:

Expt. No 3 Date-
Microscopic study of muscular and nervous tissue.
c. Muscular Tissue-
The three types of muscular tissues are - skeletal, smooth and cardiac muscles.
Skeletal muscle- Skeletal muscle fibers are cylindrical, unbranched, multinucleated (present at
the edge), striated and voluntary control. The cell membrane is sarcolemma and the cytoplasm
is sarcoplasm. Its endoplasmic reticulum is known as sarcoplasmic reticulum.
A single skeletal muscle fiber is very long (up to 30-40cm). Another feature is its striations,
alternating dark and light striations within the fiber that are visible under a light microscope.
Each fiber also has minute thread like structures known as myofibrils. Examples- facial muscles,
diaphragm, intercostals, biceps and triceps.
Smooth muscles- Smooth muscle fibers are spindle shaped, uninucleated (present at the
center), nonstriated and involuntary control. The cell membrane is sarcolemma and the
cytoplasm is sarcoplasm. Its endoplasmic reticulum is known as sarcoplasmic reticulum. The
striations are absent in smooth muscles. Examples- Stomach, intestine, urinary bladder.

Cardiac muscles- Skeletal muscle fibers are cylindrical shaped, branched, uninucleated (present
at the center), striated and involuntary control. Intercalated discs are present in cardiac muscles.
Example- Heart muscle.
d.Nervous tissue-
Neuron is the structural and function unit of the nervous system. A neuron consists of cell body,
dendrites and axon.
i) Cell body (cyton)- It is the factory of the neuron. It produces proteins required for the
neuronal activity. The organelles present in the cell body are nucleus, mitochondria, Golgi
apparatus, endoplasmic reticulum, ribosomes, etc. The nucleus synthesizes mRNA required for
the synthesis of proteins in the cytosol. The nucleolus involved in the synthesis of RNA. The ER
synthesizes proteins. The Golgi apparatus is involved in the modification of proteins synthesized
by the ER. Mitochodria synthesizes ATP by utilizing oxygen and glucose. The brain consumes
large amount of ATP. Nissl bodies are the modified form of ER found in the cell body and are the
sites of protein synthesis. Lipofusion pigments (products of lysosomal digestion) are also present
in the cell body.
ii) Dendrites- These are the branched projections of a neuron. They receive the impulses from
the surrounding neurons. They receive thousands of synaptic inputs from other neurons.They
function as an ‘antennae’ of the neuron.
iii) Axon-It is a long process of a neuron and its length ranges from 0.1mm to 2m. Many neurons
do not have axons; such neurons are termed amacrine neurons. The axons conduct electrical
impulses from cell body into nerve endings. Axon is surrounded by myelin sheath. Such neurons

are myelinated neurons. This sheath is produced by Schwann cells. The node of Ranvier is the
gap of myalin sheath between adjacent Schwann cells. The axon is covered by neurolemma and
axolemma. Axoplasm is the cytoplasm of the axon. The axonal nerve endings which consisting of
synaptic vesicles which store neurotransmitters. The important neurotransmitters are
acetylcholine, adrenaline, glutamate, glycine, histamine, etc.
Observations
Date:

Experiment No-4 Date-
Identification of axial bones-
Aim- To study bones of the axial skeletal system using specimen charts.
Bones of the skull- Bones of the cranium and face.(8+14= 22 bones)
Cranial bones-
Frontal bone- It forms forehead. It forms the roof for the orbits. It gives protection to the eye
and front part of the brain.
Parietal bones- Two parietal bones joined by suture (fibrous joint). They form posterior roof and
side of the skull. It has four borders- anterior, posterior, medial and lateral border.
Temporal bones- It forms the lower lateral side of the skull. The main parts of the temporal
bone are external auditory meatus, styloid process and zygomatic process. The zygomatic
process articulates with zygomatic bones of the face.
Occipital bone- It is a trapezoidal shaped bone found at lower back of the skull. It has a large
opening called Foramen Magnus. The spinal cord passes through Foramen Magnus. The occipital
bone articulates with atlas.
Sphenoid bone- It is situated at the base of the skull. It resembles a bat with its wings. It
protects the brain.
Ethmoid bone- It is located in front of sphenoid bone. It consists of two masses, one on each
side of the nasal cavity. Both are connected by a thin cribriform plate.
Facial bones-

Nasal bones (2)- These are two small oblong bones that form the bridge and roof of the mouth.
They contribute to the shape of the face.
Lacrimal bones (2)- It is the smallest bone of the skull located in the eye socket. Lacrimal glands
are located in the lacrimal bones.
Vomer (1) – It is located in the nasal cavity. It articulates with six bones- sphenoid, ethmoid, two
palatine and two maxillae bones.
Inferior nasal conchae (turbinates) (2)- These bones are located in the nasal cavity. These bones
help in moistens and warms the inspired air.
.
Palatine bones (2)- These bones forms the roof of the mouth cavity.
Maxilla (maxillae) (2)- They form the upper jaw. The upper teeth are attached to its sockets.
Zygomatic bones (cheekbones)- They articulates with the maxilla, the temporal bone, the
sphenoid bone and the frontal bones
Mandible (Lower jaw)- This is only movable bone in the skull. It is the largest, strongest and
lowest bones in the face. The lower teeth are attached to its sockets. The important parts of the
mandible are condyle, coronoid process, body, ramus, angle, mental nerve, mental foramina,
etc.

Bones of the vertebral column- 33 bones form vertebral column. (24 separated and 9 fused
vertebrae), Separated vertebrae - Cervical (7), thoracic (12) and lumbar (5).
Cervical vertebrae (7)- Atlas (C1)- It is the first cervical vertebra. It has no body or spine. It has
larger spinal canal. On its transverse process foramen (opening) for vertebral artery is present. It
articulates with occipital bone above and atlas below.
Axis (C2)- It is the second cervical vertebra. Its body is odontoid process (densa). It has small
bifid spine. It also has transverse foramen and larger spinal canal.
Typical cervical vertebra (C3-C7)- It has body, bifid spine, spinal canal, and foramen for the
vertebral artery. All cervical vertebrae have three openings.

Thoracic vertebra
Thoracic vertebra (T1 to T12)- Twelve in number. It has heart shaped body. Large spine is
present and it is facing downwards. Spinal canal is present. All thoracic vertebrae have one
opening. Each vertebra has facets (joining surfaces) for the ribs. A pair of ribs is attached to the
thoracic vertebra on either side.
Lumbar Vertebra (T1 to T5)- Five in number. It has single opening. It has large kidney shaped
body. Its body is flat and facing straight. It has smaller spinal canal.

Fused vertebrae (9)- It consists of sacrum (5) and coccyx (4)
Sacrum- It is made up of 5 fused vertebrae (S1 to S5). It is triangular in shape. Its anterior surface
is concave and its posterior surface is irregular. On its sides ridges of transverse process is
present. It has transverse foramina for the passage of sacral nerves. On its posterior surface the
ridges of spine also present. The sacral canal also present in the sacrum. Sacrum forms the back
of the pelvic cavity.
Bones of the ribs- (12 pairs)- Three types- True ribs- (first 7 pairs) directly attached to the
sternum by means of coastal cartilage. False ribs- the 8th, 9th and 10th ribs are attached to the
sternum indirectly. The last two pairs (11th and 12th) are unattached.

Typical rib-It is a long curved bone. It has two ends- sternal end and vertebral end. Through
sterna end it joins with sternum. Through vertebral end it joins with thoracic vertebra. In the
vertebral end it has head, neck, tubercle are present. In its shaft body, costal groove and angle
are present.
Sternum- It is a flat bone. It has three parts- manubrium, body and xiphoid process. The clavicle
joins with clavicular notch of the sternum. On the superior surface jugular notch is present. The
first pair rib joins with manubrium. The second pair rib joins at the junction between manubrium
and body. This point is known as sternal angle.
Observations
Date:

Identification of appendicular bones
Aim- To study bones of the appendicular skeletal system using specimen charts.
Bones of the appendicular skeletal system divided into- bones of the shoulder girdle with upper
limb and bones of the pelvic girdle with lower limb.
Bones of the shoulder girdle- Clavicle and scapula
Clavicle (collar bone)- It is a long curved bone. It has two extremities (ends)- acromial extremity
and sternal extremity. The acromial extremity articulates with acromium of the sacrum
(acromio-clavicular joint). The sternal extremity articulates with manubrium of the sternum
(sterno-clavicular joint). It has four borders (surfaces)- superior, inferior, anterior and posterior
borders.
Scapula- It is a flat and triangular bone. It has three angles- superior, medial and lateral. It has
three borders- superior, medial and lateral. In the lateral side there is a cavity known as glenoid
cavity. The head of the humerus bone joins with glenoid cavity (ball and socket joint- shoulder
joint). Near the glenoid cavity coracoid process is present. It has two fossae (surfaces)- anterior
and posterior. The anterior fossa is known as sub-scapularis fossa. Subscapularis muscle is
attached to this fossa. The posterior fossa is divided into supraspinatous and infraspinatous
fossa. In its posterior surface spine and acromium are present. Near the superior border
suprascapular notch is present.

Bones of the upper limb - Humerus, radius and ulna, carpals, metacarpals and phalangeals.

Humerus- It is a long bone. It has two extremities- upper and lower. The parts of the upper
extremity are head, neck, greater tubercle, lesser tubercle, intertubercular groove. Deltoid
tuberosity is present in the shaft. The parts of the lower extremity are olecranon fossa, coronoid
fossa, capitulum, trochlea, medial epicondyle and lateral epicondyle.
Radius and ulna-
Radius- It is a long bone. It has two extremities and a central shaft. The parts of the upper
extremity are button shaped head, neck, radial tuberosity. Styloid process is present in the lower
extremity.
Ulna- It is a long bone. It has two extremities and a central shaft. The parts of the upper
extremity are olecranon process, coronoid process. Head and styloid process are present in the
lower extremity.
Carpals, metacarpals and phalanges.- These are short bones. Carpal bones are eight in each
wrist.

Bones of the pelvic girdle with lower limb-
Pelvic girdle- Two pelvic bone (innominate bones) together with sacrum forms pelvic girdle.
Pelvic bone (innominate bone)- It has three parts – ileum, pubis and ischium. The important
identifying characters are obturator foramen, pubic symphysis, ischial spine, acetabulum (a cup
like cavity). The head of the femur joins with acetabulum to form hip joint. The ileum joins with
sacrum to form ileo-sacral joint.
Pelvic girdle

Bones of the lower limb- Femur It is a long bone. It has upper and lower extremities. The parte
in the upper extremity- head, anatomical neck, greater trochanter, lesser trochanter, inter
trochanter. The middle part is the shaft and the important parts of the lower extremity are
medial condyle, lateral condyle and patellar surface.
Patella (kneecap) – It is a thick, circular-triangular bone which articulates with the femur and
covers and protects the articular surface of the knee joint.
Tibia and fibula- Tibia is a long bone. The parts of the upper extremity are medial condyle,
lateral condyle, intercondylar eminence, tibial tuberosity. Medial malleolus is present in the
lower extremity. The fibula is also a long bone. The head is present in its upper extremity and
lateral malleolus is present in the lower extremity. Upper and lower tibiofibular joints are
present between tibia and fibula.

Bones of the foot-
Observations
Date:

Experiment No- 6 Date-
Introduction to hemocytometry (hemocytometer)
Aim- To study Hemocytometer or Neubauer chamber
The Neubauer chamber is a thick crystal slide with the size of a glass slide (30 x 70 mm and 4 mm
thickness). In a simple counting chamber, the central area is where the cell counts are
performed. The chamber has three parts: (1) the central part, where the counting grid has been
set on the glass, and (2) double chambers/two counting areas that can be loaded independently.
Neubauer chamber's counting grid is 3 mm x 3 mm in size. The grid has 9 square subdivisions of
width 1mm. In case of blood cell counting, the squares placed at the corners are used for white
cell counting. Since their concentration is lower than red blood cells a larger area is required to
perform the cell count. The central square is used for platelets and red cells. This square is split
in 25 squares of width 0.2 mm (200 µm). Each one of the 25 central squares is subdivided in 16
small squares. Therefore, the central square is made of 400 small squares.

Glass cover
The glass cover is a squared glass of width 22 mm. The glass cover is placed on the top of the
Neubauer chamber, covering the central area. The glass cover leaves room for the cell
concentration between the bottom of the chamber and the cover itself. The chamber is
designed so that the distance between the bottom of the chamber and the cover is 0.1 mm. It is
not uncommon that the glass cover remains slightly lifted when we introduce more liquid than
necessary in the chamber. To avoid this, some counting chambers have two special clamps to
avoid the cover glass to avoid edge-lift. If the glass cover is lifted, the distance between the
chamber and the cover will be higher than 0.1 mm, and the cell counts will not be accurate.
Observations
Date:

Expt No 7 Date-
Determination of WBC count (Total leukocyte count-TLC)
Aim- To determine the WBC count of your blood and report
Principle: The blood specimen is diluted 1:20 in a WBC pipette with the diluting fluid (water:
glacial acetic acid: gentian violet = 97:2:1) and the cells are counted under low power of the
microscope (10X) by using a counting chamber. The glacial acetic acid lyses the red cells while
the gentian violet slightly stains the nuclei of the leukocytes to locate the WBC under
microscope.
Theory: White blood cells, present in plasma take part in body defense against invading micro-
organisms. They are produced from the pluripotent stem cell in the bone marrow in adults. In
case of foetus haemopoiesis occurs in liver and spleen.
Normal ranges:
Age Normal WBC count (cells/cmm of blood
At birth 10000-25000
Infants 8000- 15000
4-7 y 6000- 15000
8-18y 4500-15000
adult 4000-10000
Pregnancy 12000- 15000
Clinical Significances of total leukocyte count: Increase in total leukocyte count of more than
10,000/cu mm is known as leukocytosis and decrease of less than 4 000 cu mm as leukopenia.
Causes of leukocytosis: a. Infections (bacterial, malaria or parasitic), b. In severe hemorrhage
and in leukemia, c. High temperature d. Severe pain e. Accidental brain damage.
Causes of Leucopenia: a. Certain viral (hepatitis, influenza, measles, etc.), and bacterial (typhoid,
paratyphoid, tuberculosis, etc) infections b. Bone marrow depression, c. anaemia

Requirements: Compound microscope, Improved Neubauer's Chamber, WBC pipette, WBC
diluting fluid [It is prepared as follows: a) Glacial acetic acid: 2.0 ml, b) 1 % (w/v) gentian violet:
1.0 ml, c) Distilled water: 97 ml. This solution is stable at room temperature (25°C ± 5°C). A pinch
of thymol may be added as preservative, blood.
Procedure- With all aseptic precautions a drop of blood was obtained by puncturing the finger
tip with a sterile lancet. The blood was drawn into the WBC pipette up to the mark 0.5. The
excess blood was wiped off using filter paper. WBC dilution fluid (Turk’s fluid) was withdrawn
into the WBC pipette up to the mark 11. The pipette was held horizontally and rolled in between
the palms so that the blood mixes with the fluid. The first 2-3 drops discarded from the pipette
as the fluid not containing blood cells. The Neubauer’s counting chamber with a cover slip was
focused under low power (10X) objective. Then the counting chamber was removed from the
stage without disturbing the focus and kept on the table for charging. The tip of the pipette was
brought to the edge of the cover slip and a small drop of diluted blood was placed on the
chamber. The chamber fills slowly by capillary action. After charging the chamber was allowed
for about 2-3min for the cells to settle down on the chamber.
The chamber was placed on the stage and cells in all four large WBC squares were counted
under the low power objective. Each large square contains 16 medium squares making a total of
64 medium squares. WBC recognized as round faintly dark (purplish) under low power. To avoid
counting twice, cells touching the left and lower margin of square should be omitted and those
touching the upper and right margin should be counted.
Calculations-
Let the total No of cells counted – N
Total No of squares counted- 64
1 square contains N/64 cells
Volume of fluid in each small WBC square = ¼ x ¼ x 1/10 = 1/160 cumm (cubic millimeter)
1/10 is the thickness of the counting chamber
160 cumm of diluted blood contains N/64 cells
Dilution is 1 in 20
1/160 cumm undiluted contains N/64x 20 cells

1 cumm of undiluted blood contains N/64 x 20 x 160 = 50 x N
Observations and Results-
Report- The WBC count (TLC) of my blood was found to be---------------------------- cells/ cumm
Date:

Expt No 8 Date-
Determination of RBC count
Aim - To determine the RBC count of your blood sample and report.
Requirements- Hemocytometer set (Neubaur’s Counting Chamber, RBC dilution pipette), cover
slip, compound microscope, RBC dilution fluid, Pricking needle, cotton, 70% Alcohol (spirit).
Principle- The RBCs are flexible and oval biconcave discs. They lack a cell nucleus and most
organelles, in order to accommodate maximum space for haemoglobin. Approximately 2.4
million new erythrocytes are produced per second in human adults. Its life span is 120 days. The
aged RBCs are destroyed by the macrophages. The cell membrane is composed of proteins and
lipids. The size of RBC is 7-8 µm. Normal RBC count is 4.5 to 5 million cells/cmm of blood.
Causes for decreased RBC count- Anaemia, bone marrow depression, erythropoietin deficiency,
internal or external bleeding, pregnancy, nutritional deficiency.
Causes for increased RBC count- Cigarette smoke, kidney cancer, dehydration, pulmonary
fibrosis, polycythemia vera ( due to genetic mutation), etc.
Red blood cell count is performed to determine the number of circulating red blood cells per
cubic millimetre (mm3), of blood. Red blood cells carry oxygen to all tissues; thus a drastic
reduction in the red cell count will cause immediate reduction in available oxygen.
A decrease in red blood cells can result in anemia. When the red blood cell count is increased
above normal limits, the condition is called polycythemia.
A hemocytometer is used to count the RBC or WBC in a blood sample. The blood specimen is
diluted (usually 200 times) with red cell diluting fluid (Hayme’s fluid).
Ingredients of RBC dilution fluid
a. Sodium chloride- (provides isotonicity)- 0.5gm
b. Sodium sulphate (prevents roulex formation)- 2.5gm
c. Mercuric chloride (act as preservative- antibacterial and antifungal)- 0.25gm
d. Distilled water (solvent) - 200cc
Procedure- With all aseptic precautions a drop of blood was obtained by puncturing the finger
with a sterile lancet. The blood was drawn into the RBC pipette up to the mark 0.5. The excess

blood was wiped off using filter paper. Hayem’s fluid was withdrawn into the RBC pipette up to
the mark 101. The pipette was held horizontally and rolled in between the palms to mix the
blood with the fluid. The first 2-3 drops present in the stem of the pipette discarded, as this fluid
not containing the blood cells. The Neubauer’s counting chamber with a cover slip was focused
under low power objective. Then the chamber was removed from the stage without disturbing
the focus and kept on the table for charging. The tip of the pipette was brought to the edge of
the cover slip and a small drop of diluted blood was placed on the chamber. Then the slide was
kept for about 2-3 min to settle down the cells on the chamber. Then the chamber was placed
on the stage and focused on the high power. The RBCs were counted in five medium squares
(out of 25), ie R1, R2, R3, R4 and R5. Each medium square contains 16 small squares. Hence RBCs
counted in 80 small squares. To avoid counting twice, cells touching the left and lower margin of
square should be omitted and those touching the upper and right margin should be counted.
Calculations-
Let the No of cells counted in 80 small squares be- N
1 square contains N/80 cells
Volume of fluid in each small RBC square = 1/20 x 1/20 x 1/ 10 = 1/4000 cubic mm
1/4000 cubic mm diluted blood contains N/80 cells
Dilution factor is 1 in 200
1/4000 cubic mm of undiluted blood contains N/80 x 200.
1 cubic mm of undiluted blood contains N/80 x 200 x 4000 = N x 10000 ( million = one lakh )

Observations
Calculations-
Report- RBC count of my blood was found to be -------- million cells/ cubic mm of blood.
(Normal Values - Males- 4.5 – 6 million cells / cubic mm of blood, Females- 4 to 5.5 million cells /
cubic mm of blood, New born- 6 to 8 million cells / cubic mm of blood.)
Date:

Expt No-9 Date-
Determination bleeding time
Aim - To determine the bleeding time of your blood sample (Duck’s method)
Theory- The time required for complete stopping of blood flow from the punctured blood
vessels called the bleeding time. Normally it is 1-4 minutes for a normal human's blood. Normal
clotting time and bleeding time values differ because bleeding time is the time for stopping
bleeding by the formation of fibrin network on the surface of punctured skin; that is it is the
surface phenomenon. But the clotting time is the time for clotting the whole blood, collected in
the capillary tube; therefore it is a volume phenomenon. For this reason clotting time is more
than the bleeding time, when determining by conventional methods.
Clinical significance- Bleeding time is prolonged in defects like haemophilia, platelet deficiency
(thrombocytopenic purpura), severe liver disease, etc.
Materials -Sterilized needle, filter paper, cotton, spirit, and stop watch.
Procedure (Duke's method) - Sterilized the finger tip with rectified spirit and allowed to dry. The
finger tip was pricked with sterilized needle. Time of pricking was noted using stop watch. Mop
the blood by touching finger tip on a filter paper. This was continued every 15sec till the
bleeding stops. The time of no stain has come was noted and recorded as the bleeding time.
Precaution - Following precautions were enforced during the measurement
i) Needle should be sterilized.
ii) Time should be noted properly.
Observations-
Report- The bleeding time of my blood was found to be- sec (- min). (Normal-1-4min)
Date:

Expt No-10 Date-
Determination of clotting time
Aim- To determine the clotting time of my own blood
Theory- Normally, blood remains in its liquid form as long as it stays within its vessels. When
blood is shed it loses its fluidity in a few minutes and sets into a semisolid jelly. This
phenomenon is called coagulation or clotting. The normal clotting time is 3-8 min. The clotting
involves series of chemical reactions that results in formation of fibrin threads. Clotting involves
several substances known as clotting factors. Most clotting factors are identified by Roman
numerals that indicate the order of their discovery.
Clotting factors-
Numbers Names
I Fibrinogen
II Prothrombin
III Tissue factor (thromboplastin)
IV Calcium ions
V Proaccelerin, labile factor
VII Stable factor, proconvertin
VIII Antihemophilic factor A
IX Christmas factor, or antihemophilic factor B
X Stuart - Prower factor
XI Plasma thromboplastin antecedent (PTA) or antihemophilic factor C
XII Hageman factor, glass factor, contact factor, or antihemophilic factor
D
XIII Fibrin stabilizing factor (FSF)
Mechanism of blood clotting- Clotting can be divided into three stages.
1. Two pathways, called the extrinsic pathway and the intrinsic pathway, lead to the formation
of prothrombinase. The next two stages are referred as the common pathway.

a) Extrinsic pathway- It has fewer steps than the intrinsic pathway and occurs rapidly. A tissue
protein called tissue factor (TF), also known as thromboplastin (factor III), leaks into the blood
from the surfaces of damaged tissue cells and initiates the formation of prothrombinase. TF is a
complex mixture of lipoproteins and phospholipids. In presence of Ca2+, TF begins a series of
reactions that ultimately activates clotting factor X. Once factor X is activated (Xa) it combines
with factor V (labile or proaccelerin) in the presence of Ca2+ to form the active enzyme
prothrombinase.
Clotting pathway
b) Intrinsic pathway- It is more complex than extrinsic pathway and it occurs more slowly
usually requiring several minutes. In this pathway outside tissue damage is not required. If
endothelial cells become roughened or damaged, blood can come in contact with collagen fibers
in the connective tissue around the endothelium of the blood vessel. In addition, injury to
endothelial cells causes damage to platelets, resulting in the release of phospholipids by the
platelets. When blood contact with collagen fibers, activates clotting factor XII (contact factor or
glass factor or Hageman factor). The activated XII (XIIa) in presence of Ca2+ activates clotting
factor X. The activated X factor (Xa), combine with factor V (proaccelerin) in presence of Ca2+ to

form prothrombinase. The phospholipids released from the damaged platelets can also
participate in the activation of clotting factor X.
The common pathways-
2. Formation of thrombin- The activated prothrombinase in presence of Ca2+ converts
prothrombin (clotting factor II) into the enzyme thrombin. The prothrombin is a plasma protein
formed by the liver.
3. Formation of fibrin threads- Thrombin converts soluble fibrinogen (clotting factor I) into
insoluble fibrin (loose fibrin threads). Fibrinogen is another plasma protein formed by the liver).
Thrombin also activates the clotting factor XIII (fibrin stabilizing factor). Activated XIII (XIIIa)
converts loose fibrin threads into strengthened fibrin threads.
Requirements- Fine capillary tubes, cotton swab, rectified spirit, blood lancet, stop watch.
Procedure: Capillary tube method: (Wright’s method)
Under sterile precautions a sufficiently deep prick was made on the finger tip. The time was
noted when bleeding starts (start the stop watch). The blood drop at the finger tip touched using
one end of the capillary tube kept tilted downwards. The tube gets easily filled by capillary
action. After about two minutes a small length of the tube was broken was repeated once in 15
min noting whether the fibrin thread is formed between the snapped ends. The time was noted
(stop the stop watch) when the fibrin thread is first seen.
Report- The clotting time of my blood was found to be------------- min. (normal 4 to 8 min).
Date:

Expt No-11 Date-
Estimation of hemoglobin
Aim- To determine the hemoglobin content of my blood by Sahli’s method.
Theory- Hemoglobin is the protein molecule in red blood cells that carries oxygen from the lungs
to the tissues and returns carbon dioxide from the tissues back to the lungs. It is abbreviated as
Hb, or Hgb. Hemoglobin is made up of four protein molecules (globulin chains) that are
connected together. The normal adult Hb molecule contains two alpha-chains and two beta-
globulin chains.
Each globulin chain contains iron containing porphyrin compound termed heme. Iron atom is
located within this heme. Each heme binds to one oxygen molecule (HbO2-oxyhemoglobin).
Hence Hb molecule can bind four oxygen molecules (HbO8). The heme part of the Hb is also
responsible for the red color of the blood.
Normal value- The normal range for hemoglobin is: in male-13 to 18 gm/dl, in female-12 to 16
gm/dl.
Formation of hemoglobin- Formation of hemoglobin begins in the proerythroblast stage during
the synthesis of erythrocytes. The following are the stages of hemoglobin synthesis.
1.The succinyl CoA formed in the Krebs metabolic stage, binds with glycine to form a pyrrole
molecule.
2. Four pyrroles combine to form protoporphyrin IX.
3. Protoporphyrin IX combines with ferrous iron (Fe++) to form the heme molecule.
4. Each heme molecule combines with a long polypeptide chain, a globin synthesized by
ribosomes, forming a subunit of hemoglobin called a hemoglobin chain.
5.Each chain has a molecular weight of 16,000, four of these chains bind together to form whole
hemoglobin molecule.
Functions of hemoglobin- The hemoglobin present in the blood carries oxygen molecules in the
form of oxy-hemoglobin and delivers the oxygen to the tissue cells and carrying the carbon
dioxide (metabolic waste) to the alveoli of the lung. The carbon dioxide eliminated from the lung
through expiration.

Principle- When blood is mixed with 0.1N HCl and kept for 5-7 minutes, the hemoglobin is
converts into the yellowish brown coloured acid hematin. The acid-hematin is then diluted with
distilled water till the color of the acid hematin matches that of the yellowish brown solution
present in the calibration tube. The hemoglobin is estimated by reading the value directly from
the scale. (Please note that 100 percent on the scale corresponds to 14.5 to 15 gm %). Fetal
hemoglobin is not converted to acid hematin and therefore this technique is not used for small
infants.
Apparatus and accessories.
1.Sahli’s hemoglobinometer (comparator)- This consists of yellowish brown standard tubes.
Opaque white glass is present at the back to provide uniform illumination.
2.Sahli’s hemoglobin tube which is marked in grams percent g% (2-24) and percentage % (10-
140)
3,Hemoglobin pipette or Sahlis’ pipette (marked at 0.02 or 20µl) attached to a flexible rubber
tube with white sucker, no bulb.
4. Stirrer- Thin glass rod.
5.N/10 HCl or 0.1N HCl.
6.Distilled water
7.Spirit swabs.

8.Blood lancets.
Procedure- N/10 HCl (0.1NHCl) was placed in to the Sahli’s graduated tube the mark 2 grams.
With the help of Sahli’s pipette blood was pipette out exactly up to 20µl mark. The blood
adhering to the outer part of the pipette was wiped off with the help of cotton. The collected
blood sample was added to the N/10 HCl present in the Sahli’s tube. The contents mixed with
the help of stirrer, and allowed the tube for 10 minutes. Then distilled water added drop in to
the tube till the color of the solution matches with the standard yellowish brown color. The
reading of the lower meniscus was noted in grams and percentage.
Observations and report-
1. The hemoglobin content of my blood is ------------- gm% ----------- %.
2. The oxygen carrying capacity of my blood = ---------- gm x 1.34 ml = ml%
3. The iron content of my blood is ------------- gm % x 3.35 mg = ------------- mg iron %.
Date:

Expt No- 12 Date-
Blood group estimation
Aim- To determine my blood group using slide agglutination method.
Theory- When more than 40% of blood is lost within few hours by hemorrhage; it has to be
restored by intravenous infusion. This is known as blood transfusion. Donor is the person who
donates the blood. Recipient is the person who receives the blood.
According to ABO system of blood grouping there are 4 types of blood groups- A, B, AB and O.
This classification is based on the presence of antigen (agglutinogen).
Blood group O is known as universal donor because it does not contain any agglutinogen and
hence can be donated to any group.
Blood group B is known as universal recipient because it does not contain any agglutinin and
hence can receive blood of any group.
Agglutination- (Clumping of RBCs) - If unmatched blood is transfused, it results in agglutination.
For example- if a person with blood group A receives blood from a person with blood group B.
The recipient’s blood contains A- antigens on the red blood cells and beta antibodies in the
blood plasma. The donor’s blood (group B) contains B antigens and alpha antibodies. In this
situation two things can happen
a) The alpha antibodies in the recipient’s plasma can bind to the B antigen on the donor’s
erythrocytes, causing agglutination and haemolysis of the red blood cells.
b)The beta antibodies in the donor’s plasma can bind to the A antigens on the recipient’s red
blood cells cause agglutination.
Blood
group
Agglutinogen
( antigen)
Agglutinin
(antibody)
Can be donor to Can be recipient for
A A beta A and AB A and O
B B alpha B and AB B and O
O None alpha and beta All O only
AB A and B None AB only All

Agglutination is the reaction between antigen (agglutinogen) and antibody (agglutinin).
Rh blood group- The Rh blood group is so named because the antigen was discovered in the
blood of the Rhesus monkey. People whose RBCs have Rh antigens are designated as Rh+
( Rh positive- A+, B+, AB+ and O+) ;those who lack Rh antigens are designated as Rh – (Rh
negative- A-, B-, AB- and O-). If an Rh – person receives blood from a person with Rh + group,
immune system start to make anti-Rh antibodies that will remain in the blood. But if the same
person receives the Rh + blood, then agglutination takes place.
Complications of agglutination- Clumping of RBCs leads to anemia. The clumps may become
moving clots (embolus), they may lodge on the small blood vessels, and this disturbs the blood
flow. The complications caused by this are- myocardial infarction, renal failure, convulsions,
coma and death.
Procedure- A, B letters marked near each cavity of a cavity slide. On another cavity slide D letter
was marked. After proper sterile precautions, a bold prick was made using a disposable needle.
The blood was transferred to all the cavities labeled A, B, and D. One drop of each (Anti-A, Anti-
B, and Anti-D) added to side A, B and D. Blood and anti sera were mixed using separate edge of
another slide and observed for agglutinations.
Anti-A- This contains alpha agglutinins (antibodies)- forms agglutination with A-antigens.
Anti-B- This contains beta agglutinins (antibodies)- forms agglutination with B-antigens.
Anti-D- This contains anti Rh-antibodies- forms agglutination with Rh factor-antigens.
Report- The blood group of my blood sample was found to be-
Date:

Experiment No 13 Date-
Determination of Erythrocyte Sedimentation Rate (ESR)
Aim- To determine the ESR of the given blood sample.
Theory- The erythrocyte sedimentation rate (ESR) is a common haematological test for
nonspecific detection of inflammation that may be caused by infection, some cancers and
certain autoimmune diseases. ESR is defined as the rate at which RBCs sediment in a period of
one hour.
Principle-When anticoagulated blood is allowed to stand in a narrow vertical glass tube,
undisturbed for a period of time, the RBCs- under the influence of gravity- settle out from the
plasma. The rate at which they settle is measured as the number of millimetres of clear plasma
present at the top of the column after one hour (mm/hr).
Mechanism of ESR- This involves three stages:
1.Stage of aggregation- It is the initial stage in which pilling up of RBCs takes place. This is known
as Rouleaux formation. It occurs in the first 10-15min.
2. Stage of sedimentation- In this stage falling of RBCs occurs at constant rate. This occurs in 30-
40min out of one hour.
3.Stage of packing- This is the final stage and is also known as stationary phase. In this stage,
there is slower rate of falling of RBCs and packing occurs. It occurs in final 10min out of one
hour.
Methods of ESR determination- There are two methods-
Wintrobe’s method and Westergren’s method.
Wintrobe’s method- This method uses Wintrobe’s tube, a narrow glass tube closed at the lower
end only. It has a length of 11 cm and internal diameter of 2.5cm. It contains 0.7 ml to 1ml
blood. The marker is 0 at the top and 10 at the bottom for ESR.
Requirements- Anticoagulated blood (EDTA, double oxalate), Pauster pipette, timer, Wintrobe’s
tube, Wintrobe’s stand.

Procedure- The blood was mixed with an anticoagulant. Using Pauster pipette blood was
transferred in to Wintrobe’s tube. The tube was kept in Wintrobe’s stand undisturbed for a
period of one hour. The ESR was recorded at the end of one hour.
Wintrobe’s tube Pasteur pipette
Wintrobe's stand

Normal Value- For males- 0- 9 mm/hr, females- 0-20mm/hr
Westergren’s method- It is better than Wintrobe’s method. The reading obtain is magnified as
the column is lengheir. The Westregren tube is open at both ends. It is 30cm in length and 2.5
mm in diameter. The lower cm is marked with 0 at the top and 200 at the bottom. It contains
about 2 ml of blood.
Normal Value- Males- 0-10 mm/hr, females- 0- 15mm/hr.

Clinical significance of ESR-
1. ESR increases in all types of anaemia except sickle cell anaemia.
2.ESR increases in HIV, tuberculosis (TB), arthritis.
3.ESR decreases in polycythemia, dehydration, Dengue fever.
Observations-
Date:

Determination of pulse rate and heart rate
Aim- To determine the pulse rate and heart rate of the own and report.
Theory- Pulse is the mechanical pulse of blood flow through the capillaries caused by the
contractions of the heart per minute. The pulse can be felt with the fingers at different pulse
pressure points throughout the body. The pulse rate is equal to heart rate. The normal range is
60 to 100beats /min. Heart rate is the number of times per minute that the heart contracts - the
number of heart beats per minute (bpm). It is usually equal or close to the pulse measured at
any peripheral point.
Significance of pulse- In an emergency situation, the pulse rate can help to find the pumping
ability of the heart. Measurement of pulse rate helps to detect the abnormalities like
palpitations (rapid heartbeat), dizziness, chest pain, etc. Help to check the blood flow after an
injury. The pulse rate increases in exercise, anemia, fever, hyperthyroidism, stress, cigarette
smoking, etc.
Pulse rate decreases in hypothyroidism, some heart diseases, etc.
Procedure- Gently placed the 2 fingers of other hand on the radial artery located near wrist
joint. The thumb was not used because it has its own pulse that may feel. Counted the beats
for 30 seconds, and then doubled the result to get the number of beats per minute.
The heart rate was measured in sitting position in a quiet place. The heart rate was measured
after keeping the stethoscope chest piece against the skin of the diaphragm over the chest wall.
Observations-
Report-
Date:

Expt No-15 Date-
Recording of blood pressure
Aim- To determine the blood pressure of the own by palpatory and auscultatory methods.
Blood pressure is the lateral pressure (hydrostatic pressure) exerted by the blood on the arterial
wall, while it is flowing through it.
Precautions before measuring BP-
1. Subject should be in calm state of mind, rested for 5min.
2. The heart and the Sphygmomanometer should be on the same level.
3. Standard width of the cuff is 12cm. In obese or very lean persons the cuff width should be
changed.
4. Repeated readings on the same limb at short interval give false high valves.
5. While tying the cuff on the cloth.
6. The lower border of the cuff should be tied at least 1 inch above the cubital fossa.
7. The cuff should not be too tight or too loose.
8.The rubber tubing should come to the medial side of the arm while the cuffs are tied/should
not obstruct the placement of stethoscope.
Apparatus- Sphygmomanometer, Stethoscope.
Sphygmomanometer

Stethoscope
Measurement of BP - Palpatory method
Procedure- The cuff was tied on the arm. The radial pulse palpated using three fingers. The
pressure (air pressure) was raised using the inflator of the Sphygmomanometer. At one point of
pressure was noted when the pulse is obliterated (no pulse). The pressure was raised to
30mmHG more than this. Gradually the air pressure was lowered by releasing the air using the
knob of the inflator. The pressure was noted when the pulse is again felt. This marks the systolic
BP.
Advantages and disadvantages- Simple procedure, but only systolic BP can be measured.
Auscultator method- The cuff was tied on the arm. The radial pulse palpated using three fingers.
The pressure (air pressure) was raised using the inflator of the Sphygmomanometer. At one
point of pressure was noted when the pulse is obliterated (no pulse). The stethoscope was
placed lightly over the brachial artery of the cubital fossa. Gradually air pressure was lowered by
releasing the air using the knob of the inflator. The pressure was noted (systolic BP) when the
commencement of some heart sounds (korotkoff sounds) osculated. The pressure was noted
again (diastolic BP) immediately when the disappearance of these sounds takes place.
Observations-
Report- The systolic BP of my own was found to be --------------- mm Hg (Palpatory method).
The systolic and diastolic BP of my own was found to be --------------- mm Hg (Auscultator
method).
Date:

HAP I (P) lab manual

Recommended

Recommended

More Related Content

What's hot

What's hot (20)

Similar to HAP I (P) lab manual

Similar to HAP I (P) lab manual (20)

More from Dr.Shivalinge Gowda KP

More from Dr.Shivalinge Gowda KP (20)

Recently uploaded

Recently uploaded (20)

HAP I (P) lab manual