LIST OF EXPERIMENTS

EXPERIMENT 1. Study of Anatomical Planes and regions

EXPERIMENT 2. The Skeleton System – Axial system

EXPERIMENT 3. The Skeleton System – Appendicular system

EXPERIMENT 4. Determination of bleeding time

EXPERIMENT 5. Determination of clotting time

EXPERIMENT 6. Detection of Blood group

EXPERIMENT 7. R.B.C. Counting

EXPERIMENT 8. Total W.B.C. Counting

EXPERIMENT 9. Detection of Haemoglobin

EXPERIMENT 10. Preparation of blood film & staining

EXPERIMENT 11. Measurement of Blood Pressure

Safety Procedures

1. No open food or drink is permitted at any time, whether a lab is in progress or not.
No eating, drinking, candy, cough drops, chewing gum or tobacco is permitted. All
beverage and food containers must be put away in a backpack/bag/purse or left
outside of the lab. There are shelves outside in the hallway to store food and
beverages during lab.

2. Know the locations of the eye and hand wash, as may be required in case of
accidents.

3. Safety instructions are given at the beginning of each lab period. Always arrive
on time so that you know what you are supposed to do and are informed of any
specific safety concerns or safety equipment associated with the day’s lab activity.

4. Stash book bags safely so that they won’t trip people.

5. Report all illnesses, injuries, breakages, or spills to your laboratory instructor

immediately.

6. Bring clean and complete record notebook as well observation notebook in each
lab session.

7. Before issuing or returning of any lab material don’t forget to write down in
register.

8. Do not play with bones, skeletal system or microscope.

9. Chemical are costly and useful items, take precaution while using that. Wastage
of chemical are not allowed.

10. without knowledge don’t taste or touch the chemicals with bare hand as it may
be harmful.

11. Inform immediately about breaking of any glass materials (which includes glass
slides, cover-slip, test-tube etc.)

12. Clean broken glass (glass that is not contaminated with any chemical reagents,
blood, or bacteria) can be swept up using the dust pan and placed in the broken glass
container. If the glass is contaminated in any way, keep the area clear to prevent
tripping or laceration hazards, and consult your instructor for proper disposal
guidance.

13. All the experiments which include blood, it is advised to the students that blood
affected materials like glass slides, cover-slip, test-tube or any other thing should be
touched and cleaned by only the blood donor person. Other students are not allowed
to touch the blood by bare hand.

14. Do not copy the experiment from other students, students are instructed to do it
by themselves. Notify your instructor if any of the equipment is faulty.

15. Clean up your entire work area before leaving. Put away all equipment and
supplies in their original places and dispose of reagents and infectious materials in
the designated receptacles. Disinfect your work surface if the lab activity involved
any infectious materials. Otherwise, wipe the entire work surface down with a clean,
wet paper towel (no soap).

16. Use the appropriate waste containers provided for any infectious or hazardous
materials used in lab. Wash your hands before leaving the lab room. Keep silence
in the laboratory while performing experiment. Have patients while performing the
experiments.
 Date:
EXPERIMENT 1

Aim: Study of Anatomical Planes and regions

BASIC ANATOMICAL TERMINOLOGY : -

Scientists and health-care professionals use a common language of special
terms when referring to body structures and their function.
Body Positions :-
Anatomical position the subject stands erect facing the observe , with the
head level and the eyes facing directly forward .The feet are flat on the floor
and directed forward, and the upper limbs are at the sides with the palms
turned forward. Two terms describe a reclining body. If the body is lying
face down, it is in the prone position .If the body is lying face up, it is in the
position.
Regional Names :-
Anterior view -A
CEPHALIC HEAD
Cranial Skull
Facial face
Frontal Forehead
Temporal Temple
Orbital or ocular eye
Otic Ear
Buccal Cheek
Nasal Nose
Oral mouth
Mental Chin
CERVICAL NECK
Axillary armpit
Brachial Arm
Antecubital Front of elbow
Antebrachial Forearm
Carpal Wrist
Palmar or volar Palm
Digital or phalangeal Fingers
Pollex thumb
Manual Hand
Sternal breastbone
Thoracic Chest
Mammary Breast
Abdominal Abdomen
Umbilical Naval
Coxal Hip
Pelvic Pelvis
Inguinal Groin
Pubic Pubis
Femoral Thigh
Patellar Anterior surface of knee
Crural Lag
Pedal Foot
Tarsal Ankle
Digital or phalangeal toes
Dorsum Top of foot
Hallux Great toe

B-posterior view
Occipital Base of skull
Acromial shoulder
Scapular Shoulder blade
Vertebral Spinal Column
Olecranal or cubital Back of elbow
Lumber loin
Sacral Between hips
Coccygeal tailbone
Gulteal buttock
Perineal Region between anus and
external genitals
Popliteal Hollow behind knee
Sural calf
Plantar Sole
Calcaneal heel
Directional Terms:
Words that describe the position of one body port relative to another.
DIRECTIONAL DEFINITION
TERM
Superior( Soo’-PER-e –or ) Toward the head, or the upper part of
(cephalic or cranial ) a structure.
Inferior (in’-FĒR-ē-or) (caudal) Away from the head. Or the lower
Part of a structure.
Anterior (an-TĒR -ē-or) (ventral)* Nearer to or at the front of the body.
Posterior ( pos-TĒR-ē-or)(dorsal) Nearer to or at the bock of the body.
Medical (MĒ-dē -al) Nearer to the midline.
Lateral (LAT-er-al Father from the midline.
Intermediate (in‘-ter-MĒ-dē-at) Between two structure
Ipsilateral (ip-si-LAT-er-al On the same side of the body as
another structure.
Contralateral (CON-tra-er-al) On the opposite side of the body from
another structure.
Proximal (PROK-si-mal) Nearer to the attachment of a limb to
the trunk; nearer to the origination of
a structure.
Distal (DIS- tal) Father from the attachment of a limb
to the trunk; father from the
origination of a structure.
Superficial (soo’-per-FISH-al) Toward or on the surface of the body.
(external)
Deep (internal) Away from the surface of the body.

Planets and Sections:-

Study parts of the body relative to planes, imaginary flat surfaces that pass through
the body parts. A sagittal plant (SAJ –i -tal; sagitt- =arrow) is a vertical plane that
divides the body or an organ in to right and left sides.
More specifically, when such a plane passes through the midline of the body or an
organ and divides it into equal right and left sides, it is called a midsagittal plane or
a median plane.
A frontal or coronal plane (kō-KŌ-nal; corona crown) divides the body or an organ
into anterior (front) and posterior (back) portions. A transverse plane divides the
body or an organ into superior (upper) and inferior (lower) portions. Other names
for a transverse plane are a cross-sectional or horizontal plane. Sagittal, frontal, and
transverse planes are all at right angles to one another. An oblique plane (ō-BLĒK),
by contrast, passes through the body or an organ at an oblique angle (any angle other
than a 90-degree angle.

Body Cavities:-
Body Cavities are spaces within the body that help protect, separate, and support
internal organs.
 CAVITY COMMENTS

Cranial cavity formed by cranial bones and contains brain

Vertebral canal Formed by vertebral column and contains spinal cord and
The beginnings of spinal nerves.

Thoracic cavity Chest cavity; contains pleural and pericardial cavities

and
Mediastinum.

Pleural cavity Each Surrounds a lung; the serous membrane of each

pleural
Cavity is the pleura.

Pericardial cavity Surrounds the heart; the serous membrane of the

pericardial
Cavity is the pericardium.

Mediastinum Central portion of thoracic cavity between the lungs;

extends
From sternum to vertebral column and from first rib to
Diaphragm; contains heart, thymus, esophagus,
trachea, and several large blood vessels.

Abdominopelvic Cavity Subdivided into abdominal and pelvic cavities.

Abdominal cavity Contains stomach, spleen, liver, gallbladder, small

intestine;
And most of large intestine; the serous membrane of the
Abdominal cavity is the peritoneum.

Pelvic cavity Contains urinary bladder, portions of large intestine, and

internal
Organs of reproduction.
 Date:
EXPERIMENT 2

Aim: Study of Skeleton System – Bone

THE SKELETAL SYSTEM BONE TISSUE

The entire framework of bones and their cartilages, along with ligaments and
tendons, constitutes the skeletal system.
FUNCTIONS OF SKELETAL SYSTEM:-
The skeletal system performs several basic functions:-
1. Support: the skeleton serves as the structural framework for the body by
supporting soft tissues and providing attachment points for the tendons of
mask skeletal muscles.
2. Protection: The skeleton protects the most important internal organ from
injury.
3. Assistance in movement: Most skeletal muscles attach to bones; when they
pull on bones to produce movement.
4. Mineral homeostasis (storage and release):- Bone tissue stores several
minerals, especially calcium and phosphorus, which contribute to the
strength of bone. Bone tissue stores about 99% of the body’s calcium.
5. Blood cell production. Within certain bones ,a connective tissue called red
bone marrow produces red blood cells , white blood cells , and platelets , a
process called hemopoiesis .red bone marrow consists of developing blood
cells, adipocytes, fibroblasts, and macrophages within a network of reticular
fibers. It is present in developing bones of the fetus and in some adult bones,
such as the hip (pelvic) bones, ribs, sternum (breastbone), vertebrae
(backbone), skull, and ends of the bones of the humerus ( arm bone) and
femur (thigh bone). In a new-born, all bone marrow is red and is involved
in hemopoiesis. With increasing age, much of the bone marrow changes
from red to yellow.
6. Triglyceride storage. Yellow bone marrow:- consists mainly of adipose
cells, which store triglycerides. The stored triglycerides are a potential
chemical energy reserve.
STRUCTURE OF BONE

Macroscopic bone structure consists of the following parts:

1. The diaphysis (dī -AF- i-sis = growing between) is the bones shaft or
body –the long, cylindrical, main portion of the bone.
2. The epiphyses (e-PIF-i-sēz = growing over, singular is epiphysis) are
the proximal and distal end the bone.
3. The metaphyses (me TAF-i-sēz; meta-= between; singular is
metaphysis) are the regions between the diaphysis and the epiphyses.
In a growing bone, each metaphysis contains an epiphyseal (growth)
plate (ep’-i-FIZ-ē-al), a layer of hyaline cartilage that allows the
diaphysis of the bone to grow in length (described later in the
chapter).When a bone ceases to grow in length at about ages 18-21,
the cartilage in the epiphyseal plate is replaced by bone; the resulting
bony structure is known as the epiphyseal line.
4. The articular cartilage is a thin layer of hyaline cartilage covering the
part of the epiphysis where the bone forms an articulation (joint) with
another bone. Articular cartilage reduces friction and absorbs shock
at freely movable joints. Because articular cartilage lacks a
perichondrium and lacks blood vessels, repair of damage is limited.
5. The periosteum (per-ē-OS-tē-um; peri- = around)is a tough
connective tissue sheath and its associated blood supply that
surround the bone surface wherever it is not covered by articular
cartilage . It is composed of an outer fibrous layer of dense irregular
connective tissue and an inner osteogenic layer that consists of cells.
Some of the cells enable bone to grow in thickness, but not in length.
The periosteum also protects the bone, assists in fracture repair, helps
nourish bone tissue, and serves as an attachment point for ligaments
and tendons. The periosteum is attached to the underlying bone by
perforating (Sharpey’s) fibers, thick bundles of collagen that extend
from the periosteum into the bone extracellular matrix.
6. The medullary cavity (MED-ū- lar -ē; medulla- = marrow, pith), or
marrow cavity, is a hallow, bone marrow and numerous blood
 vessels in adults. This cavity minimizes the weight of the bone by
reducing the dense bony material where it is least needed. The long
bones’ tubular design provides maximum strength with minimum
weight.
7. The endosteum (end-OS-tē-um; endo- = within) is a thin membrane
that lines the medullary cavity. It contains a single layer of bone-
forming cells and a small amount of connective tissue.

Structure of long bone

HISTOLOGY OF BONE TISSUE: -
at the microscopic level. Like other connective tissues, bone or osseous tissue
(OS-ē-us), contains an abundant extracellular matrix that surrounds widely
separated cells. The extracellular matrix is about 15% water, 30% collagen fibers,
and 55% crystallized mineral salts.
The most abundant mineral salts is calcium phosphate [Ca3 (PO4)2], calcium
hydroxide [Ca (OH) 2], hydroxyapatite [Ca10 (PO4)6 (OH) 2] .Calcium carbonate
(CaCO3), and ions such as magnesium, fluoride, potassium, and sulphate. As these
mineral salts are deposited in the framework formed by the collagen fiver of the
extracellular matrix, they crystallize and the tissue hardens. This process, called
calcification (kal’- si- fi- KĀ-shun), is initiated by bone building cells called
osteoblasts (described shortly).
Although a bone’s hardness depends on the crystallized inorganic mineral salts, a
bone’s flexibility depends on its collagen fibers.
Four types of cells are present in bone tissue: osteogenic cells, osteoblasts,
osteocytes, and osteoclasts.
1. Osteogenic cells (os’-tē-ō-JEN-ik; -genic =producing) are unspecialized
bone stem cells derived from mesenchyme, the tissue from which almost all
connective tissues are formed. They are the only bone cells to undergo cell
division; the resulting cells develop into osteoblasts. Osteogenic cells are
found along the inner portion of the periosteum, in the endosteum, and in
the canals within bone that contain blood vessels.
2. Osteoblasts (OS-tē-ō-blasts’; -blasts = buds or sprouts) are bone bone-
building cells. They synthesize and secrete collagen fibers and other organic
components needed to build the extracellular matrix of bone tissue, and they
initiate calcification (described shortly). As osteoblasts surround themselves
with extracellular matrix, they become trapped in their secretion and become
osteocytes.
3. Osteocytes (cytes =cells), mature bone cells, are the main cells in bone tissue
and maintain its daily metabolism, such as the exchange of nutrients and
wastes with the blood. Like osteobasts, osteocytes do not undergo cell
division.
4. Osteoclasts (Os-tē-ō-klasts’; -clast = break) are hung cells derived from the
fusion of as many as 50 monocytes (a type of white blood cells) and are
concentrated in the endosteum. On the side of the cell that faces the bone
suface, the osteoclast’s plasma membrane is deeply folded into a ruffed
border. Here the cell releases powerful lysosomal enzymes and acids that
digest the protein and mineral components of the underlying extracellular
bone matrix. This breakdown of bone extracellular matrix, termed
resorption (rē-SORP-shun), is part of the normal development,
maintenance, and repair of bone.

Bone is not completely solid but has many small spaces between its cells and
extracellular matrix components. Some spaces serve as channels for blood
vessels that supply bone cells with nutrients. Other spaces act as storage areas
for red bone marrow. Depending on the size and distribution of the spaces, the
regions of a bone may be categorized as compact or spongy
 Types of cells in bone tissue

Compact Bone Tissue:-

Compact bone tissue Contain few spaces (Figure 6.3a) and is the strongest fore of
bone tissue. It is found beneath the periosteum of all bone and makes up the bulk
of the diaphyses of long bones. Compact bone tissue provides protection and
support and resists the stresses produced by weight and movement.
Compact bone tissue is composed of repeating structural units called osteons, or
haversian systems (ha-VER-Shan). Each osteon consists of concentric lamellae
arranged around a central (haversian) canal.
The concentric lamellae (LA- mel –ē) are circular plates of mineralized
extracellular matrix of increasing diameter, surrounding a small network of blood
vessels, lymphatic, and nerves located in the central canal(Figure 6.3a).These
tube-like units of bone generally form a series of parallel cylinders that, in long
bone. Between the concentric lamellae are small spaces called lacunae (la-KOO-
nē= little lakes; singular singular is lacuna), which contain osteocytes. Radiating
in all direction from the lacunae are tiny canaliculi (kan-a LIK-ū-lī = small
canaliculi are slender finger like processes of osteocytes (see inset at right of
(Figure 6.3a). Neighboring osteocytes communicate via gap junctions (see
Section 4.2). The canaliculi connect lacunae with one another and with the central
canals, forming an intricate, miniature system of interconnected canals throughout
the bone. This system provides many routes for nutrients and oxygen to reach the
osteocytes and for the removal of wastes.
Osteons in compact bone tissue are aligned in the same direction and are parallel
to the length of the diaphysis. As a result, the shaft of a long bone resists bending
or fracturing even when considerable force is applied from either end. Compact
bone tissue tends to be thickest in those parts of a bone where stresses are applied
in relatively few directions.
The areas between neighboring osteons contain lamellae celled interstitial
lamellae (i’n’-ter-STISH-al), which also have lacunae with osteocytes and
canaliculi. Interstitial lamellae are fragments of older osteons that have been
partially destroyed during bone rebuilding or growth.
Blood vessels, lymphatic vessels, and nerves from the periosteum penetrate the
compact bone through transverse perforating canals or Volkmann’s canals
(FOLK-mans).The vessels and nerves of the perforating canals connect with those
of the medullary cavity, periosteum, and central canals.
Arranged around the entire outer and inner circumference of the shaft of a long
bone are lamellae celled circumferential lamellae (ser’-kum-fer-EN-shē-al). They
develop during initial bone formation. The circumferential lamellae directly deep
to the piriosteum are celled outer circumferential lamellae. They are connected to
the periosteum by perforating (Sharpey’s) fibers. The circumferential lamellae
that line the medullary cavity are called inner circumferential lamellae.

Spongy Bone Tissue:-

In Contrast to compact bone tissue, spongy bone tissue, also referred to as
trabecular or cancellous bone tissue, does not contain osteons (figure
6.3b,c),spongy bone tissue is always located in the interior of a bone, protected by
a covering of compact bone. It consists of lamellae that are arranged in an irregular
pattern of thin columns called trabeculae (tra-BEK-ū-lē = little beams; singular is
trabecula). Between the trabeculae are spaces that are visible to the unaided eye.
These macroscopic spaces are filed with red bone marrow in bones that produce
blood cells, and yellow bone marrow (adipose tissue) in other bones. Both types
of bone marrow contain numerous small blood vessels that provide nourishment
to the osteocytes. Each trabecula consists of concentric lamellae. Osteocytes that
lie in lacunae, and canaliculi that radiate outward from the lacunae.
Spongy bone tissue makes up most of the interior bone tissue of short, flat,
sesamoid, and irregularly shaped bones.
Spongy bone tissue tends to be located where bones are not heavily stressed or
where stresses are applied from many directions. The trabeculae do not achieve
their final arrangement until locomotion is completely learned.
Spongy bone tissue is different from compact bone tissue in two respects. First,
spongy bone tissue is light, which reduces the overall weight of a bone. This
reduction in weight allows the bone to move more readily when pulled by a
skeletal muscle. Second, the trabeculae of spongy bone tissue support and protect
the red bone marrow. spongy bone in the hip bones, ribs, sternum (breastbone),
vertebrae, and the proximal end of the humerus and femur is the only site where
red bone marrow is stored and, thus, the site where hemopoiesis (blood cell
production)occurs in adults.
 Date:
EXPERIMENT 3

Aim: Classification of human bones – Axial and Appendicular system

Adult skeletal system contains 206 named bones, most of which are paired. Bones

of adult skeleton are grouped into two principal divisions: axial skeleton and

appendicular skeleton. Axial system consist of 80 bones whereas appendicular

contain 126 bones, as shown below:

 Diagram of
Skeletal system

Diagram of hyoid bone

Skull consist of 8 cranial bones and 14 facial bones

Diagram of Skull
Diagram of pelvic griddle

Diagram of vertebral column

Diagram of hand and feet
 Date:
EXPERIMENT 4

Aim: Perform an experiment to determine the bleeding time of a subject with the
help of Duke’s method and draw an appropriate inference.
Theory:
The time required for complete stopping of blood flow from the punctured blood
vessels called the bleeding time. Bleeding time is the interval between the moment
when bleeding starts and the moment when bleeding stops. Normally it is 1-3
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. Bleeding
time is prolonged in purpuras, but normal in coagulation disorders like haemophilia.
Purpuras can be due to

1. Platelet defects - Thrombocytopenic purpura.

1. Primary (Idiopathic) - Thrombocytopenic purpura
2. Secondary - Thrombocytopenic purpura
2. Vascular defects - Senile purpura

Clinical significance

It plays a significant role

i) To study the haemorrhagic disorders.
ii) To study the coagulation defects
iii) To have an idea about the platelets count of the patient. Bleeding time is
prolonged in few disorders like: vascular lesions, platelet defect, severe liver
disease, uraemia and anti-coagulant drug administration.

Materials Required

Sterilized needle, filter paper, cotton, Ethanol, and stop watch.

Procedure (Duke's method)

 1. Sterilize the fingertip using ethanol and allow it to dry.
2. Make a sufficiently deep prick using a sterile lancet, so that blood comes out
freely without squeezing.
3. Note the time (start the stop-watch) when bleeding starts.
4. Mop the blood by touching the fingertip with a filter paper.
5. This is repeated every 30 seconds, each time using a fresh portion of the
filter paper, till bleeding stops.
6. Note the time (stop the stop-watch). It is seen that the blood stains on the
filter paper get smaller to disappear finally when bleeding stops. The time of
no stain has come is noted properly; it is the bleeding time of the subject.

Results

Name of person:
Bleeding time: ......... Minutes

Precaution

Following precautions should be enforced

i) Needle should be sterilized.
ii) Stain of blood should not be avoided.
iii) Time should be noted properly.
 Date:
EXPERIMENT 5

Aim: Determination of clotting time with the help of Wrighting's method

Theory

Normally blood remains in the blood vessels in liquid form. When it is drawn from
the body it becomes into semisolid gel. The mechanism of formation of gel is called
blood clotting or coagulation. Blood does not clot inside vessels due to presence of
heparin and anti-thrombin III. A blood clot is a gel that contains the formed elements
of the blood, arrested in the stable fibrin mesh.
The factors, involved in blood coagulation are:
Fibrinogen (I),
Prothrombin (II),
Thromboplastin (III),
Calcium (Ca++, IV),
Labile Factor (V),
Stable Factor (VII),
Anti-Hemophilic Factor A (VIII),
Anti-Hemophilic Factor B (IX),
Stuart Power Factor (X),
Anti-Haemophilic Factor C (XI),
Hageman Factor (XII),
Fibrin Stabilizing Factor (XIII).
Blood plasma without clotting factors is called serum. Factors III, IV and V are
synthesized in the platelets of plasma. Factors I, II, VII, VIII, IX, X, XI, and XII are
synthesized in liver. Factor XII can be synthesized in liver and platelets. Factor IV
is also obtained from diet and bones.
Clotting time is the time taken to coagulate the blood after rupture of blood vessels.
Normally it is 3-8 minutes for a normal human's blood depending upon platelets
count (200000 to 350000 /cubic mm) and other clotting factors inside the plasma.

Clinical significance

When a blood vessel is damaged, clotting is required to stop the loss of blood
(haemostasis) and healing of the vessel, in which platelets play a vital part. Now it
is clear that every factor is equally important for the clotting of blood and the process
is impossible in absence of a single factor as the activation of one factor is done by
another factor.
 a) Therefore, if there is haemorrhage (i.e. clotting time is more than the normal
range) we can guess few possibilities:

Pathway of blood coagulation: Extrinsic pathway (Tissue damage)

Intrinsic pathway (contact with glass, tin etc.)

Thrombocytopenia: Blood platelets count is below 150000/ cubic mm of blood.

Vitamin K deficiency: Vitamin K is essential for synthesis of factor II, VII, IX, and
X. Syntheses of these factors are impaired and haemorrhagic disease is progressed
due to Vit K deficiency.
Hamophilia: Bleeding disease that causes excessive bleeding especially in men.
Hemophilia A: Factor VIII is abnormal and less biologically active. About 1 in
10000 men suffers from classical haemophilia in United State.
Hemophilia B or Christmas disease: Factor IX is deficient.
Both of these factors are transmitted genetically from female chromosomes.
Therefore, women do not have chance of haemophilia due to deficiency of these
factors.
Von Willebrand's disease
Factor VIII has two parts, a large component and a small component. The smaller
component is more important in intrinsic pathway of blood clotting and loss of this
component causes the classical haemophilia. Sometimes haemophilia is seen due to
deficiency of the large component, called Von Willebrand's disease.

b) When the clotting time is lower than the normal range we can guess there
might be thromboembolic condition inside the tissue:
Thrombus: An abnormal blood clot developed inside the blood vessels.
Emboli: Tiny parts of breakdown thrombi are called emboli.
Causes of thromboembolic condition: i. Rough endothelial surface caused due to
atherosclerosis, infection or trauma. ii. Slow blood flow

Consequences of thromboembolic condition

Femoral venous thrombosis: When the blood flow is blocked for many hours in
any blood vessel due to immobility of the person. Generally during sitting for a
long time or sleeping the blood clot in the leg vein (femoral part), occasionally in
the common iliac and inferior vena cava.
Massive pulmonary embolism: The movement of the clot (1 in 10) is then towards
the right side of heart followed by the pulmonary arteries and thus cause massive
blockage of pulmonary artery, called the Massive pulmonary embolism.
Disseminated intra vascular coagulation: The condition can be raised due to
presence of large amount of dying or traumatized tissue those are capable of
releasing the tissue factors in the blood. Generally, when some bacteria or bacterial
toxins are present in the blood vessels for long time, they provoke the activation of
platelets and formation of thrombus. In long term, the thrombus are broken into
tiny particles and spread up all over the blood vessels. This condition prevents the
supply the oxygen and nutrition to the tissue due to plugging of blood capillaries
by the emboli followed by exacerbates circulatory shock. This is the consequence
of septic shock in case of 85% patients.

Materials Required:

Sterilized needle, Sabraze's capillary tube (length 15 cm. & internal diameter 0.8-
1.0 mm.), cotton, spirit, stop watch.

Procedure

Wrighting's method
1. Sterilize the fingertip using spirit and allow it to dry.
2. Make a sufficiently deep prick using a sterile lancet, so that blood comes out
freely without squeezing.
3. Time of pricking is noted.
4. Sufficient blood is made to flow inside the capillary tube.
5. Touch the blood drop at the fingertip using one end of the capillary tube kept
tilted downwards. The tube gets easily filled by capillary action.
6. After about two minutes start snapping off small lengths of the tube, at
intervals of 20 seconds, each time noting whether the fibrin thread is formed
between the snapped ends.
7. The time to appear the thread is noted; it is the clotting time of the subject.

Results

Name of person:
Clotting time: ......... Minutes

Precautions

i. Needle should be sterilized

ii. Capillary should be touched properly in finger
iii. Time should be noted properly.

Discussion:

Clotting time is the interval between the moment when bleeding starts and the
moment when the fibrin thread is first seen.
Normal value is 3to 10 minutes.

Bleeding time and clotting time are not the same. Bleeding time depends on the
integrity of platelets and vessel walls, whereas clotting time depends on the
availability of coagulation factors. In coagulation disorders like haemophilia,
clotting time is prolonged but bleeding time remains normal.

Clotting time is also prolonged in conditions like vitamin K deficiency, liver

diseases, disseminated intravascular coagulation, over dosage of anticoagulants etc.
 Date:
Experiment No 6
Aim: To determine the blood group of given sample using ABO system.
Materials required: Cotton, ethanol, pricking needle, 2 glass slides and blood
grouping anti-sera.
Theory:
Determination of ABO blood groups depends upon the immunological reaction
between antigen and antibody. Landsteiner found two antigens on the surface of
RBCs and named them as A antigen and B antigen. These antigens are also called
agglutinogens because of their capacity to cause agglutination of RBCs. He noticed
the corresponding antibodies or agglutinins in the plasma and named them anti-A or
α-antibody and anti-B or β-antibody. However, a particular agglutinogen and the
corresponding agglutinin cannot be present together. If present, it causes clumping
of the blood. Based on the presence or absence of antigen A and antigen B, blood is
divided into four groups:
1. ‘A’ group
2. ‘B’ group
3. ‘AB’ group
4. ‘O’ group.
Blood having antigen A belongs to ‘A’ group. This blood has β-antibody in the
serum. Blood with antigen B and α-antibody belongs to ‘B’ group. If both the
antigens are present, blood group is called ‘AB’ group and serum of this group does
not contain any antibody. If both antigens are absent, the blood group is called ‘O’
group and both α and β antibodies are present in the serum. Blood grouping is an
important step during blood transfusion.

Rh factor is an antigen present in RBC. This antigen was discovered by Landsteiner

and Wiener. It was first discovered in Rhesus monkey and hence the name ‘Rh
factor’. There are many Rh antigens but only the D antigen is more antigenic in
human. The persons having D antigen are called ‘Rh positive’ and those without D
antigen are called ‘Rh negative’. Rh group system is different from ABO group
system because, the antigen D does not have corresponding natural antibody (anti-
D). However, if Rh positive blood is transfused to a Rh negative person anti-D is
developed in that person. On the other hand, there is no risk of complications if the
Rh positive person receives Rh negative blood.
Procedures:
1. First clean the index finger using ethanol dipped cotton swab, let it dry.
2. Use the sterilized need to prick the finger and put three drops of blood in
separate region of a glass slide. Mark each region as A, B and D respectively.
3. Take the anti-sera and put one drop of each sera in their corresponding region.
4. Wait for 1 minutes and observe the appearance of agglutination and write the
result.

Anti-Sera A Anti-Sera B Anti-Sera D Result

Result: Given blood sample was found to be ……. Blood group.

Precaution:
1. Always use sterilized needle for pricking. Don’t prick with too much force.
2. Don’t touch the blood of other person.
3. Don’t allow the blood to mix with different regions (like mixing of A to B or
other).
 Date:
Experiment No 7

Aim: Red blood cell count using Haemocytometer/Neubauer’s Chamber.

Requirements: Cotton, pricking needle, Ethanol, Hemocytometer/ Neubauer’s

chamber, RBC diluting pipette, RBC diluting fluid, coverslip and microscope.

Theory: Very large number of red blood cells are present in the blood specimen.
RBCs are round shaped biconcave disc present in the blood that helps in
transportation of gases (especially O2). Normal range of RBCs count in healthy male
adult is 4.7 to 6.1 million cells/µl, whereas in female it ranges from 4.2 to 5.4 million
cells/µl. These number can vary with diseases like polycythaemia (increase in RBC
count above 6.5 million/mm3) and Erythropenia (decrease in RBC count below 3.5
million/mm3). Practically counting these numbers of RBCs directly under
microscope is highly impossible. So the RBCs are counted by using a special type
of chambers designed for the counting of blood cells in the specimen, known as
Haemocytometer/ Neubauer’s chamber.

Neubauer’s chamber is a thick glass plate with the size of a glass slide (30x70x4mm).
The counting region consists of two square shaped ruled areas. There are depressions
or the moats on either side or in between the areas on which the squares are marked
thus giving an “H” shape.
The ruled area is 3mm2 divided into 9 large squares each with a 1 mm2 area. The
large central square (which can be seen in it’s entirely with the 10X objective), is
divided into 25 medium squares with double or triple lines. Each of these 25
squares are is again divided into 16 small squares with single lines, so that each of
the smallest squares has an area of 1/400 mm2.

The glass cover is a squared glass of width 22 mm. The glass cover is placed on the
top of the Neubauer’s chamber, covering the central area. The ruled area is 0.1 mm
lower than the rest of the chamber. So that when a cover slip is kept on the counting
region, there is a gap of 0.1 mm (1/10mm) between the cover slip and the ruled area.
RBC Counting Area
The large centre square is used for RBC counts. As already stated, this area is
subdivided into 25 medium squares, which in turn are each divided into 16 squares.
Of the 25 medium squares, only the four corner squares and the centre square
within the large centre square are used to perform RBC counts.

Neubauer’s chamber

Area of blood cell count

Procedure:
1. Sterilize the fingertip (the index- or ring finger) using spirit and allow it to
dry. If you have been pricked before on that finger, use another finger.
2. Make a sufficiently deep prick using a sterile lancet, so that blood comes out
freely without squeezing.
3. Bring a cleaned RBC dilution pipette tip close to the droplet of oozing blood.
Using the bulb allow (by capillarity and pressure) blood to enter up to the 0.5
mark. Wipe out the pipette externally to avoid false reading.
4. Using a small tube of RBC diluting fluid, additionally aspirate this solution to
reach the mark 101. When blood is sucked up to 0.5 mark and the diluting
fluid up to 101 mark, it gives the 1:200 dilution of blood.
5. Be cautious there should be no air bubble in the pipette.
6. Mix the blood and diluting fluid by rotating the pipette (horizontally) between
your palms.
7. Clean the Neubauer’s chamber with a swab. Similarly clean the coverslip.
8. Put the glass cover on the Neubauer’s chamber central area. Use a flat surface
to place the chamber, like a table or a workbench.
9. Place pipette tip close to the glass cover edge, right at the centre of the
Neubauer’s chamber.
10. Release the plunger slowly watching how the liquid enters the chamber
uniformly, being absorbed by capillarity action. Do not overcharge the
chamber and there should be no air bubble in the chamber.
11.In case of the appearance of bubbles, or that the glass cover has moved, repeat
the operation.
12.After charging wait for 3-5 minutes so that the cell settle down in the chamber
and then focus the chamber under microscope for counting.

Microscope set up and focus.

1. Place the Neubauer’s chamber on the microscope stage. If the microscope has
a fixing clamp, fix the Neubauer’s chamber.
2. Turn on the microscope light.
3. Focus the microscope (using 10X) until you can see a sharp image of the cells
looking through the eyepiece and adjusting the stage.
4. Look for the first counting grid square where the cell count will start. In case
of RBC, 5 big squares from the centre square will be counted using 40X objective
lens.
5Start counting the cells in the first square.
6. Count the cells which are lying on the right and lower lines of the 5 small
squares but not the opposite line.
7. In case of marginal cell, count the cells on ‘L’ line that is right and lower line
or left and upper lines. Write down the amount of cells counted in the first square.
 Direction for counting blood cells in Hemocytometer

Indication for correct counting of marginally aligned cells

8. Repeat the process for the remaining squares, writing down the counting results
from all of them. The higher the number of cells counted, the higher the accuracy
of the measurement.

Calculation for RBC count

Total RBC=Nx10000 per mm3

N= total number of RBC counts in all 5 small squares.

Result:
Total red blood cell present in given specimen is …… million/mm3
 Precautions:
1. Which sucking the blood or diluting fluid make precaution.
2.Accurately measure the amount of specimen and diluting fluid to avoid error in
result.
3.Mix the specimen and diluting fluid appropriately by gently rotating between
your palms.
4.Carefully charge the Hemocytometer it should not be overcharge and no air
bubble should present.
5.In case of air bubble present redo the whole experiment.
Experiment No 8 Date:

Aim: To determine whether there is adequate number of White blood cell present in
circulation by using Haemocytometer/Neubauer’s Chamber.

Requirements: Cotton, pricking needle, Ethanol, Hemocytometer/ Neubauer’s

chamber, WBC diluting pipette (Thoma pipette), WBC diluting fluid, coverslip and
microscope.

Theory: White blood cells play an important role in your body’s immune system,
searching the blood for invading viruses, bacteria, and fungi. When a foreign virus
or bacteria enters your blood, the white blood cell, or leukocyte, recognizes and
destroys the invading particle before it can cause disease. White blood cells originate
in the bone marrow but circulate throughout the bloodstream. There are several
different types of white blood cells, each with their own function and the blood
usually contains a percentage of each type. Some directly destroy the foreign
bacteria, while others attack cells that are infected by viruses. Other types of white
blood cells can even play a role in allergic reactions. The five major types of white
blood cells are: neutrophils, lymphocytes, eosinophil, monocytes and basophils.

A white blood cell (WBC) count is a test that measures the number of white blood
cells in the body. As the white blood cell count can fall or rise out in the healthy
range. Having a higher or lower number of WBCs than normal may indicate an
underlying condition. Two common reasons include: Leukopenia (is the medical
term used to describe a low WBC count) Leucocytosis is the medical term used to
describe a high WBC count.

Neubauer’s chamber is a thick glass plate with the size of a glass slide (30x70x4mm).
The counting region consists of two square shaped ruled areas. There are depressions
or the moats on either side or in between the areas on which the squares are marked
thus giving an “H” shape.
The ruled area is 3mm2 divided into 9 large squares each with a 1 mm2 area. The
large central square (which can be seen in it’s entirely with the 10X objective), is
divided into 25 medium squares with double or triple lines. Each of these 25
squares are is again divided into 16 small squares with single lines, so that each of
the smallest squares has an area of 1/400 mm2.
The glass cover is a squared glass of width 22 mm. The glass cover is placed on the
top of the Neubauer’s chamber, covering the central area. The ruled area is 0.1 mm
lower than the rest of the chamber. So that when a cover slip is kept on the counting
region, there is a gap of 0.1 mm (1/10mm) between the cover slip and the ruled area.
WBC Counting Area
The large 4 corner primary squares are used for WBC counts. As already stated,
each of these big square area is subdivided into 16 medium squares. All the 16
medium squares from each corner primary big squares are used to perform WBC
counts.

Neubauer’s chamber

Area of blood cell count

Procedure:

1. Sterilize the fingertip (the index- or ring finger) using spirit and allow it to dry.
If you have been pricked before on that finger, use another finger.
2. Make a sufficiently deep prick using a sterile lancet, so that blood comes out
freely without squeezing.
3. Bring a cleaned WBC dilution pipette tip close to the droplet of oozing blood.
Using the bulb allow (by capillarity and pressure) blood to enter up to the 0.5
mark. Wipe out the pipette externally to avoid false reading.
4. Using a small tube of WBC diluting fluid, additionally aspirate this solution to
reach the mark 11. When blood is sucked up to 0.5 mark and the diluting fluid
up to 11 mark, it gives the 1:20 dilution of blood.
5. Be cautious there should be no air bubble in the pipette.
6. Mix the blood and diluting fluid by rotating the pipette (horizontally) between
your palms.
7. Clean the Neubauer’s chamber with a swab. Similarly clean the coverslip.
8. Put the glass cover on the Neubauer’s chamber central area. Use a flat surface
to place the chamber, like a table or a workbench.
9. Place pipette tip close to the glass cover edge, right at the centre of the
Neubauer’s chamber.
10. Release the plunger slowly watching how the liquid enters the chamber
uniformly, being absorbed by capillarity action. Do not overcharge the chamber
and there should be no air bubble in the chamber.
11. In case of the appearance of bubbles, or that the glass cover has moved, repeat
the operation.
12. After charging wait for 3-5 minutes so that the cell settle down in the chamber
and then focus the chamber under microscope for counting.

Microscope set up and focus.

1. Place the Neubauer’s chamber on the microscope stage. If the microscope has
a fixing clamp, fix the Neubauer’s chamber.
2. Turn on the microscope light.
3. Focus the microscope (using 10X) until you can see a sharp image of the cells
looking through the eyepiece and adjusting the stage.
4. Look for the first counting grid square where the cell count will start. In case
of WBC, 4 big squares from extreme end will be counted using 40X objective
lens.
5Start counting the cells in the first square.
6. Count the cells which are lying on the right and lower lines of the 16 small
squares but not the opposite line.
7. In case of marginal cell, count the cells on ‘L’ line that is right and lower line
or left and upper lines. Write down the amount of cells counted in the first square.

Direction for counting blood cells in Hemocytometer

Indication for correct counting of marginally aligned cells

8. Repeat the process for the remaining 3 squares, writing down the counting
results from all of them. The higher the number of cells counted, the higher
the accuracy of the measurement.
 Calculation for RBC count

Average of cells x Correction for dilution

WBC/  L =
No . of squares counted x Volume of one square

Average of cells = the average of the total number of cells counted in the four
large squares on BOTH sides of the Hemocytometer
Correction for dilution = the dilution factor (which is the reciprocal of the
blood dilution). The dilution when using the WBC Unopettes is 1/100, so the
dilution factor is 100.
Number of squares counted = four (4)
Volume of one large square = 0.1 μL
EXAMPLE:
Number of cells counted in side 1 = 27
Number of cells counted in side 2 = 29
Average Number = 28
WBC/µL = 28 x 100 = 7.0 x 103/µL
4 x 0.1
Result:
Total White blood cell present in given specimen is …… million/mm3

Precautions:
1.Which sucking the blood or diluting fluid make precaution.
2.Accurately measure the amount of specimen and diluting fluid to avoid error in
result.
3.Mix the specimen and diluting fluid appropriately by gently rotating between
your palms.
4.Carefully charge the Hemocytometer it should not be overcharge and no air
bubble should present.
5.In case of air bubble present redo the whole experiment.
 Date:
Experiment No 9

Aim: Estimation of Haemoglobin with the help of Sahli's Haemoglobinometer.

Requirements: Sahli's Haemoglobinometer, Hydrochloric acid, distilled water,

pricking needle, ethanol, and cotton.

Theory

Haemoglobin is iron containing red pigments of the blood. It is the tetrapyrrole

porphyrin ring, consisting of four heme moiety and four globin chain (96%). Heme
is iron (present in ferrous Fe++Fe++ state) containing pigments. Globin is a
histone type protein. Haemoglobin carries the O2 from lung to tissue and the CO2
from tissue to lungs.
After the normal life span is over, the red blood cells are destroyed by the
macrophages in liver, spleen or red bone marrow into heme moiety (biliverdin and
iron) and globin protein. The iron and globin are recycled to reuse and the
biliverdin is converted into the bilirubin which is further converted into the
stercobilin to be removed in the feces. The colour of feces is yellow because of the
presence of the stercobilin.

Types of Haemoglobin (Hb):

i. Adult Haemoglobin:
a. Hb A: It consists of two αα chain and two ββ chains in globin
protein, α2β2α2β2. 97% of adult Hb is of this type.
b. Hb A2 (α2δ2α2δ2): Minor Hb A2 present in adult, the concentration of Hb
increases in some types of anaemia.
ii. Fetal haemoglobin:
a. Hb F (α2γ2α2γ2): Hb F accounts for 70-90% of haemoglobin at term, 25% in
one month, and 5% in six months. Hb F concentration in adults increases in some
types of anemia, haemoglobinopathies, and some time in leukemia.
b. Haemoglobin Bart's(γ4γ4): It's concentration increases in fetal life in
thalassemia.
iii. Embryonic haemoglobin:
Hb Gower 1 (η2ϵ2η2ϵ2), Hb Gower 2 (α2ϵ2α2ϵ2), and Hb Portland (α2γ2α2γ2).
iv. Abnormal haemoglobin:
a. There are four clinically important abnormal haemoglobins: Hb S, Hb C, Hb D,
and Hb E, present in different hereditary haemoglobinopathies. The most
commonly encountered hemoglobin is Hb S (α2β2α2β2) where in the beta chain
valine is substituted by glutamic acid at the sixth position. Hb S is present in sickle
cell anemia.
b. Unstable haemoglobin are haemoglobin variants that undergo denaturation and
precipitate in the red cells at Heinz bodies. These are present in a type of
congenital nonspherocytic hemolytic anemia.

Clinical significance of haemoglobin count

Each RBC contains about 280 million of Hb, which is responsible for the
transportation of oxygen and carbon dioxide (23%). The percentage of RBC in the
blood is called the haemacrit which is clinically important. The normal range of
haemacrit for male is 40-54% and it is 38-46% in case of female.

Normal range of Haemoglobin:

Male: 14-17 gm/dl of blood, Female: 12-14 gm/ dl of blood

Clinical significance of Haemoglobin estimation

i. Anaemia: Haemoglobin estimation below normal level indicates that the patient
is anaemic. In this condition there is not enough Hb available to carry sufficient
oxygen from the lungs to tissue.
Types of anaemia:
A. Based on haemoglobin estimation: if haemoglobin level is below 10gms/dl it is
called mild anaemia. If haemoglobin level goes below 8gms/dl, and 6gms/dl, the
conditions are called moderate and severe anaemia respectively.
B. Based on etiology of anaemia: Deficiency of iron in diet (iron deficiency
anaemia), Vitamin B12 and Folic acid are deficient in diet or folic acid is destroyed
due to drug's action (Megaloblastic anaemia), Due to impaired absorption of
Vitamin B12 from the small intestine, as the intrinsic factor is not synthesized by
stomach or destroyed due to gastric surgery (Pernicious anaemia), Synthesis of Hb
is impaired due to bone marrow failure (Hypoplastic or aplastic anaemia),
destruction of Hb in circulation due to effect of drugs, free radicals or other toxic
chemicals (Haemolytic anaemia). Haemoglobin A is replaced by Hb S, Hb C, Hb
D, and Hb E (hereditary haemoglobinopathies, Sickle cell anaemia etc.),
Haemoglobin Batrs (consists of four gamma chains) is present instead of normal
Haemoglobin (Thalassemia).
ii. Polycythemia: When RBC count is elevated sometimes in disease condition
like haemoconcentration due to burns, cholera, chronic heart disease, conditions of
decreased lung function such as emphysema or due to climbing (as the oxygen
level is less in the high altitude).

Methods of Haemoglobin count:

1. Visual colour comparison method

i. Sahil's method ii. Dares method iii. Hadens method iv. Wintrobes method v.
Haldanes method vi. Tallquists method
2. Gasometric method
Van Slyke method
3. Spectrophotometric method
i. Oxyhaemoglobin method ii. Cyanmethemoblobin method
4. Automated haemoglobinometry
5. Non-automated haemoglobinometry
6. Other methods:
i. Alkaline-haematin method ii. Specific gravity method iii. Comparator method.

Hb count by Sahli's Haemoglobinometer (acid haematin method):

Principle: Anticoagulated blood is added to the 0.1 N HCl and kept for 5-7 minutes
to form acid haematin. The colour of this acid haematin should be matched with
the solution, present in the calibration tube. Distilled water is added to the acid
haematin until the colour matches and the final reading is directly noted from the
graduation in the calibration tube. [Please note that 100 percent on the scale
corresponds to 14.5gm % to 15gm %].

Procedure:
1. Place N/10 HCL in diluting tube up to the mark 0.2.
2. Take blood in the haemoglobin pipette up to 20-cubic-mm-mark and blow it
into diluting tube and rinse well.
 3. After waiting for 10 minutes add distilled water in drops and mix the tube
until it has exactly the same colour as the comparison standards.
4. Note the reading, which indicates the percentage of haemoglobin.

Result

The Hb estimation of the given sample is..... g/100 ml of blood/.....g/dl of

blood/.....G%.

Precautions

i. Pipetting of blood should be done cautiously

ii. Mix the blood properly with HCl by using stirrer
iii. Match the colour cautiously.
 Date:
Experiment No 10
Aim: Prepare two thin blood smears which are even, smooth and have an acceptable
feathered edge using wedge method. Stain the prepared blood smears with Wright’s
stain so that all formed elements are readily identifiable.

Requirements:

1. 2 Glass slides
2. Pricking needle
3. Ethanol
4. Cotton

Procedure

1. Tale ethanol dipped cotton to clean the index finger.

2. Take one glass slide and place on a flat surface.
3. Use a pricking needle and place a drop of blood, about 2 microliter at one end
of the glass slide (at approximately 1 cm distance from edge)
4. With your right hand, place the smooth clean edge of a second (spreader) slide
on the specimen slide, just in front of the blood drop.
5. Hold the spreader slide at a 30 angle, and draw it back against the drop of
blood.
6. Allow the blood to spread almost to the edges of the slide.
7. Push the spread forward with one light, smooth, and fluid motion. A thin film
of blood in the shape of a bullet with a feathered edge will remain on the slide.
8. Allow the blood film to air-dry completely before staining. (Do not blow to
dry. The moisture from your breath will cause RBC artifact.)

Precautions during smear preparation

1. A good blood film preparation will be thick at the drop end and thin at the
opposite end.
2. As soon as the drop of blood is placed on the glass slide, the smear should be
made without delay. Any delay results in an abnormal distribution of the
white blood cells, with many of the large white cells accumulating at the thin
edge of the smear.
3. The blood smear should occupy the central portion of the slide and should not
touch the edges.
4. The thickness of the spread when pulling the smear is determined by the 1)
angle of the spreader slide (the greater the angle, the thicker and shorter the
smear), 2) size of the blood drop and 3) speed of spreading.
a. If the hematocrit is increased, the angle of the spreader slide should be
decreased.
b. If the hematocrit is decreased, the angle of the spreader slide should be
increased.
5. Common causes of a poor blood smear:
a. Drop of blood too large or too small.
b. Spreader slide pushed across the slide in a jerky manner.
c. Failure to keep the entire edge of the spreader slide against the slide
while making the smear.
d. Failure to keep the spreader slide at a 30 angle with the slide.
e. Failure to push the spreader slide completely across the slide.
6. Although this is the easiest and most popular methods for producing a blood
smear, it does not produce a quality smear. The WBCs are unevenly
distributed and RBC distortion is seen at the edges. Smaller WBCs such as
lymphocytes tend to reside in the middle of the feathered edge. Large cells
such as monocytes, immature cells and abnormal cells can be found in the
outer limits of this area. Spun smears produce the most uniform distribution
of blood cells.
7. Selection of appropriate segment of smear for looking of cells. The density of
cell varies across the smear. Cells will be “heaped and piled” close to the point
where drop of blood was placed on the site. White blood cells are shrunken,
and some types are difficult to distinguish from each other. On the other hand
at the tip, very few cells are presents. In this region, white blood cells are
sometimes damaged and erythrocytes may be deformed. The best area to look
at is between these two regions. Where it is located exactly and how wide it
is will depend on the smear, but middle of the smear is a good starting point.
8.Formation of correct thin film smear.
9.Unacceptable thin film smears are:
Biologic causes of a poor smear

1. Cold agglutinin - RBCs will clump together. Warm the blood at 37º C for 5
minutes, then remake the smear.
2. Lipemia - holes will appear in the smear. There is nothing you can do to
correct this.
3. Rouleaux - RBC’s will form into stacks resembling coins. There is nothing
you can do to correct this.

Staining:
Blood cells have structures that are acidophilic and some that are basophilic, so they
vary in their reactions (pH). The nuclei are basophilic and stain blue. The highly
basophilic (acidic) basophil granules also stain blue. Hemoglobin (being basic)
stains acidophilic or red. Stains that are made up of combinations of acid and basic
dyes are called Romanowsky Stains and its various modifications are available such
as Wright’s, Leishman’s, Giemsa’s and Jenner’s stains. Most use Methylene Blue as
the basic stain, though Toluidine Blue is used by some. Most use Eosin as the acid
stain though Azure I and Azure II are also used. Leishman’s Stain is used for
visualizing protozoa in blood films. Giemsa’s Stain is used for demonstrating
protozoa and spirochaetes in blood. Wright’s Stain is used to demonstrate protozoa
and helminthic parasites in blood
Principle of wright’s stain
Wright’s Stain
The Wright’s stain is a Romanowsky stain. A Romanowsky stain is any stain
combination consisting of eosin Y or eosin B with methylene blue and/or any of its
oxidations products. Such stains produce the typical purple coloration of leukocyte
nuclei and neutrophilic granules as well as the numerous blues and pinks found in
other cell types. Methyl alcohol is used as both a solvent and fixative in this
procedure.
Reagents and equipment

2. Commercial Wright’s stain (1mg Wright’s powder into 400 mg absolute

methanol)
3. Deionized water
4. 3 Coplin jars
5. Clothes pin or forceps for holding the slide
6. Paper towels

Procedure

1. Attach a clothes pin (or use forceps) to the thick edge of the blood smear.
2. Place the slide in the Coplin jar with Wright’s stain. Allow to stand 5-10
seconds.
3. Raise the slide out of the stain and allow the majority of the stain to run off
the slide.
4. Place the slide in the first jar containing deionized water. Allow to stand 10-
20 seconds.
5. Remove the slide carefully and dip several times in the second jar containing
deionized water to rinse off the excess stain.
6. Wipe off excess fluid from the back of the slide. Place the slide upright on a
paper towel with the feathered edge up and allow to air dry.
7. When completely dry, examine the smear with the microscope as follows:

Low power (10x) scan

Determine the overall staining quality of the blood smear.
a. Stain should not be too dark or too pale.
b. There should be no stain precipitate present on smear.
c. RBCs should be appropriate color of reddish pink.
d. Lymphocytes have dark purple nuclei with varying shades of blue
cytoplasm
e. Neutrophils have dark purple nuclei with reddish, granular cytoplasm.
f. Monocytes have a lighter purple nucleus with a gray-blue cytoplasm.
g. Eosinophils have bright red/orange granules
h. Basophils have dark purple nuclei and granules.
Determine if there is a good distribution of the cells on the smear.
a. Scan the edges and center of the slide to be sure there are no clumps
of RBCs, WBCs or platelets.
b. Scan the edges for abnormal cells.

High power (40x) scan

Find an optimal area for the detailed examination and enumerations of cells.

a. The RBCs should not quite touch each other.

b. There should be no area containing large amounts of broken cells or
precipitated stain.
c. The RBCs should have a graduated central pallor.
d. Nuclei and cytoplasm of WBCs should be the proper color.
e. Platelets should be clearly visible.

Diseases: Malaria, Leishnamiasis (Kala Azar), Filariasis

 Appearance of formed cells under microscope

Precautions:

i.Methanol is poisonous, so make sure not take in mouth or sensory organs.

ii.Wright’s solution is flammable, so keep in air tight brown container and open
only when used.
iii.A serious concern with wedge preparations is the uneven distribution of
different cell types.22 Monocytes (and other large leukocytes) are pushed to the
end of the spreader film (the feathered edge) and to the sides. This leads to a 5%
to 10% underestimation of monocyte presence when compared with monoclonal
antibody-based flow cytometry differential counts (upper normal limit in
proportional count is 11% in normal individuals, not 10% as measured by
microscopy)
Microscope Parts:

Eyepiece Lens: the lens at the top of the microscope that you look through. The
eyepiece is usually 10x or 15x power.

Tube: Connects the eyepiece to the objective lenses.

Arm: Supports the tube and connects it to the base of the microscope.

Base: The bottom of the microscope, used for support.

Illuminator: A steady light source (110v) used in place of a mirror. If your

microscope has a mirror, it is used to reflect light from an external light source
up through the bottom of the stage.

Stage: The flat platform where you place your slides. Stage clips hold the slides
in place. If your microscope has a mechanical stage, you will be able to move the
slide around by turning two knobs. One moves it left and right, the other moves
it forward and back.

Removing Nosepiece or Turret: This is the part of the microscope that holds
two or more objective lenses and can be rotated to easily change power
(magnification).

Objective Lenses: Usually you will find 3 or 4 objective lenses on a microscope.

They almost always consist of 4x, 10x, 40x and 100x powers. When coupled with
a 10x (most common) eyepiece lens, we get total magnification of 40x (4x times
10x), 100x, 400x, and 1000x. To have good resolution at 1000x, you will need a
relatively sophisticated microscope with an Abbe condenser. The shortest lens is
the lowest power, the longest one is the lens with the greatest power.

Rack Stop: This is an adjustment that determines how close the objective lens
can get to the slide.

Condenser Lens: The purpose of the condenser lens is to focus the light onto the
specimen. Condenser lenses are most useful at the highest powers (400x and
above). Microscopes with a stage condenser lens render a sharper image than
those with no lens (at 400x).
Diaphragm or Iris: Many microscopes have a rotating disk under the stage. This
diaphragm has different sized holes and is used to vary the intensity and size of
the cone of light that is projected upward into the slide.
How to Focus Your Microscope: The proper way to focus a microscope is to
start with the lowest power objective lens first and while looking from the side,
crank the lens down as close to the specimen as possible without touching it.
Now, look through the eyepiece lens and focus upward only until the image is
sharp. If you can't get it in focus, repeat the process again. Once the image is
sharp with the low power lens, you should be able to simply click in the next
power lens and do minor adjustments with the focus knob. If your microscope
has a fine focus adjustment, turning it a bit should be all that is necessary.
Continue with subsequent objective lenses and fine focus each time.
 Date:
Experiment No 11
Aim:
1. To measure the blood pressure of given subject using Sphygmomanometer
and get the reading of systolic, diastolic, pulse pressure and mean arterial
pressure.
2.Study the variation in blood pressure with following conditions:
i. Determine the difference in blood pressure reading between left and right
arm.
ii. Determine the difference in blood pressure reading between hanging and
horizontal position.
iii. Determine the difference in blood pressure reading in supine relax, supine
cross legged, sitting, immediate standing and standing after 3 minutes
relax.
3. Determine the ankle brachial index using automatic blood pressure
measuring device.

Apparatus Required:
1. Sphygmomanometer (A sphygmomanometer consists of an inflatable bag
inside a covering called a cuff, an inflating bulb, a manometer from which
blood pressure can be read, and a valve that is used for deflation.)
2. Stethoscope
3. Chair
4. Table or other surface to support arm

Theory: Blood pressure is a measurement of the force applied to the walls of the
arteries as the heart pumps blood through the body. The pressure is determined
by the force and amount of blood pumped, and the size and flexibility of the
arteries. Blood pressure is continually changing depending on activity,
temperature, diet, emotional state, posture, physical state, and medication use.
The ventricles of heart have two states: systole (contraction) and diastole
(relaxation). During diastole blood fills the ventricles and during systole the blood
is pushed out of the heart into the arteries. The auricles contract anti-phase to the
ventricles and chiefly serve to optimally fill the ventricles with blood. The
corresponding pressure related to these states are referred to as systolic pressure
and diastolic pressure .The range of systolic pressure can be from 90 mm of Hg
to 140 mm of Hg with the average being 120 mm of Hg. The diastolic pressure
typically varies from 60mm of Hg to 90 mm of Hg and the average being 80 mm
of Hg.
The upper arm is wrapped with the cuff belt connected to a mercury pressure
gauge and air is pumped with a rubber ball to increase cuff pressure about 30
mmHg higher than the systolic blood pressure to block the artery and stop blood
flow downstream. Then, the cuff pressure is slowly lowered. The artery opens at
the instant when the cuff pressure decreases below the systolic blood pressure and
blood begins to flow on and off in synchrony with pulses causing the opening and
closing of the artery. The sound emitted by the pulses is named Korotkoff's and
continues until the cuff pressure decreases below the systolic blood pressure and
the artery ceases the opening and closing. The stethoscope placed closely to the
artery downstream of the cuff is used to hear Korotkoff's sound; the blood
pressures are measured. Cuff pressure when Korotkoff's sound begins to be heard
is defined as the highest blood pressure and that when the sound disappears is
defined as the lowest pressure.
Blood pressure is a vital parameter used in day to day activity but it is also very
variable, as it changes with position, food and other activity. It is very important
to know the correct method of its measurement and study its variation in sitting,
lying or standing position.
Procedure for first objective:
1. The subject should sit comfortably, with the arm slightly flexed, palm up, and
the forearm supported at heart level on a table or other smooth surface. If such a
surface isn’t available, provide support to the subject’s forearm while taking the
measurements.
2. Place the deflated cuff on the subject’s upper arm, with the lower edge of the
cuff about 1 inch above the inner elbow crease. The inflatable bag should rest on
the brachial artery, which is on the inner part of the upper arm. The inflatable bag
should encircle at least 80% of the arm; if it does not, use a larger
sphygmomanometer.
3. Apply the stethoscope lightly to the arm, just at the inner elbow crease. Make
sure the stethoscope doesn’t touch the cuff or any of the tubing from the
sphygmomanometer.
4. While watching the manometer and listening for pulse sounds through the
stethoscope, inflate the bag about 30 mm Hg above the point at which pulse
sounds disappear. (Inflating the bag closes off the blood flow in the brachial
artery, causing the pulse sounds to stop.)
5. Slowly deflate the bag at a rate of about 3 mm Hg per second (or per heartbeat).
As you release the pressure, pulse sounds will become audible, go through several
changes in clarity and intensity, and then disappear again. You must listen
carefully to the pulse sounds while you watch the readings on the manometer.
• Systolic pressure is the point at which pulse sounds first become audible. You
should hear faint but clear tapping sounds.
• Diastolic pressure is the point at which the pulse sounds disappear.
7.Note down both the readings and remove the cuff.
8.Take another reading by following steps 1-6.
9.Take the average of both readings.
10.Calculate the pulse pressure and mean arterial pressure by using the formula:
Pulse pressure= Systolic pressure – diastolic pressure
Mean arterial pressure = diastolic pressure + one-third of pulse pressure
Procedure for second (i) objective:
Repeat the above procedure first for left hand than for right hand and note the
reading.
(ii) : Obtained the blood pressure reading for left hand in sitting position with
hand I horizontal (heart’s level)as described for first objective than repeat for
same hand in hanging position(when hand is below heart level parallel to body
axis)
(iii) First, the subject was instructed to lie in supine position and relax for 3
minutes. After that take the blood pressure reading for left hand.
Next, the subject was allowed to cross the limbs in the same supine position
for 3 minutes and note down the left hand’s blood pressure.
Told the subject to sit and relax for another 3 minutes and take blood
pressure reading.
Immediately after measure the BP reading once the subject stands.
Finally allow the subject to stand for 3 minutes and take another reading.
Procedure for third objective:
i.First the subject has to rest in supine position for 5 minutes.
ii. Than take an automatic BP measuring device and take the BP reading from
both hand and legs in following order: right arm, right leg, left leg and then left
arm. Blood pressure of hand was take at brachial region whereas in legs it was
taken in just above the ankle region.
Calculate the ABI value for each side as:
ABI left leg = systolic pressure of left leg divided by systolic pressure of left arm.
ABI right leg = systolic pressure of right leg divided by systolic pressure of right
arm.

Results:
1. First objective
 Systolic blood pressure:
 Diastolic blood pressure:
 Pulse pressure:
 Mean arterial pressure:
2.Second objective
I. Blood pressure reading of left hand:
Blood pressure reading of right hand:

II. Blood pressure reading of left hand at heart’s level:

Blood pressure reading of left hand at hanging level:

III.
BP supine Supine sitting Immediate After 3
reading cross leg after minutes of
standing standing
(in mm
Hg)

3.Third objective:
Left arm Left leg Right arm Right leg
(Systolic pressure in mm Hg)

ABI left leg = ABI right leg =

Precautions:
1.Always keep the sphygmomanometer and person’s hand at heart’s level.
2.In sitting position both the feet should comfortably touch the ground.
3.Always take two reading for each hand and note the average.
4.Always select the correct size of cuff for BP reading.