Hap i lab mannual-rdp mam

IICP B. PHARM SEMESTER 1
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Human Anatomy and Physiology-I (108010101)
Aim: Study of Compound Microscope.
Microscope is an optical instrument, used to see the magnified image of very small
objects which cannot be seen with a naked eye. The compound microscope, which
is commonly used in biological experiments, not only, provides high magnification
(enlargement of the image of the objects), but also given fair resolution
(differentiation of neighboring points as separates entities.
Description of the Compound Microscope
It consists of three parts: the stand, the body and the train of optical system
The Stand: The stand has a heavy foot and limb which are joined by hindge joint.
The limbs bear the optical system.
The body: The optical system is held in position by the body which houses coarse
adjustmet and fine adjustment knobs. These are to adjust the height of the tube
such that the objective lens is at its focal length from the object. Just below the
optical system lie stage, sub-stage and the mirror. The stage is a platform which
accommodates a glass microscopic side on which object, to be examined is
mounted. The stage may be of mechanical type. It has two micrometer screws
which move the slide in two planes-from side to side and forward-backward. The
substage consists of a condenser and an iris-diaphragm. The purpose of codensor
is to (1) to focus the parallel rays of light from mirror to the object (2) to hep in
resolving the image. The purpose of iris diaphragm is to control the amount of light
reaching on the object. The mirror is movable and has two surfaces: Plane and
Concave.
The train of optical system: The optical system consists of the external tube and
an internal tube. The external tube has the nose piece which bears the objective
lens of various magnifications: high power, low power and oil immersion lens. The
internal tube carries eye piece at the upper position. In the compound microscope
magnification takes place at two places.
DATE: / /2020
PRACTICAL: 1
COMPOUND MICROSCOPE

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Use and Care of Compound Microscope
 Always keep the microscope clean, dust free and covered.
 Concave mirror is used while using low power lens and the plane mirror is
used while using high power or oil immersion lens. Adjust the mirror such that
the maximum and even illumination is obtained.
 After placing the slide over the stage, bring down the low power lens using
coarse adjustment knob. Bend by side of tube and bring your eyes at the level
of slide while bringing it down the objective with coarse adjustment knob while
looking through the eyepiece of the microscope.
 Slowly but confidently, the objective should be raised, while looking through
the eyepiece of the microscope, using coarse adjustment knob till the object is
seen. It is made clear by using fine adjustment.

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 To use high power lens, raise up the objective again. Change the lens and
bring it down looking from the side. The objective is again raised while looking
through the eyepiece till the object is seen.
 Remove the eyepiece for a while and look into the tube. Adjust the position of
condenser to get better results. It should be racked down while observing
unstained objects or using low power lenses.
 Adjust the iris diaphragm to cut a thin peripheral rim of rays. Iris diaphragm
should be partially closed while observing neubauer counting chamber. After
adjusting condenser and iris, replace the eyepiece and observe again. The
object will be very clear.
 While using oil immersion lens, condenser should be racked up, preferable
having a capillary space between it and the side. A drop of cedarwood oil is
placed on condenser to fill the capillary space and also the slide and it must
touch the objective lens also. The cedarwood oil is preferred to other oils
because its refractive index (1.55) is nearer to that of glass (1.5). If condenser
can not be raised to touch the slide, oil should not be placed on it .The oil on
the condenser and the objective should be removed first with a dry soft cloth
and then with a little Xylol on it. Use of excess Xylol should be avoided.
 Never unscrew any part of microscope. Any difficulty in microscope, if felt,
should be brought to the notice of the teacher in charge. Do not clean any
lens of the microscope with alcohol, as the cementing material for the fixation
of lens is soluble in alcohol. Clean the microscope using fine cloth only.
Self-study Questions
(1) Write functions of Compound Microscope.
(2) Enlist parts of Compound Microscope.
(3) Write functions the iris diaphragm and condenser.
(4) Explain train of optical system of Compound Microscope.

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Observations & Calculations:
Results:
Signature of the Teacher:
Date:

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Aim: Microscopic study of epithelial and connective tissue.
(A) EPITHELIAL TISSUES
Characteristics
 It forms the lining membrane and covers the free surfaces.
 Cells remain close together with minimum amount of intercellular
space.
 If perform the functions of protection, absorption secretion and
excretion.
Types of Epithelial tissue and Characteristics
Squamous epithelium:
It consists of a single layer of cells placed on a thin basement membrane. The cells
are thin and fat, placed like the tiles on the floor. Nucleus is usually placed centrally.
It is found in serous membranes (peritoneum, pleura, etc.), Bowman’s capsule,
Henle’s loop of the nephron, the inner line of heart, blood vessels etc.
Cuboidal or cubical epithelium:
It is composed of a single layer of cubical cells having same dimensions on each
side and placed upon a basement membrane. It found in thyroid follicles, ducts of
various gland germinal epithelium, terminal bronchioles, salivary glands etc.
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PRACTICAL: 2
MICROSCOPY OF EPITHELIAL AND CONNECTIVE TISSUES

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Columnar epithelium:
It consists of a single layer of cubical cells which are taller, prismatic and arranged
on the basement membrane. The nuclei may be placed towards the base of cells.
The free surfaces may show presence of microvilli.
They are found as the inner lining of stomach, small intestine large intestine, free
surfaces of the ovary, and the convoluted part of tubules of the nephron.
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.
Compound epithelium:
Compound epithelium consists of two or more layers of simple epithelium.
Transitional epithelium consists of three to four layers of cells and each layer may
not be of same type. First layer may be made up of quadrilateral shaped cells,
second layer consists of pyriform cells and third layer consists of polyhedral cells.
This type of epithelium is found in the pelvis of kidney, ureter, urinary bladder and
urethra.
Stratified epithelium consists of more than four layers of cells. Depending on the
type of cells they may be
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

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(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.
(B) CONNECTIVE TISSUE
Characteristics
 It is binding structure between two tissues.
 Cells are less in number but intercellular substance called matrix is in
abundant.
 It performs the function of binding support.

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Types of Connective tissue and Characteristics
Loose CT
Areolar connective tissue:
It is type of connective tissue, found
(I) in the subcutaneous, sub mucous and sub serous tissue,
(II) Between muscles and nerves,
(III) In the interior of organs binding different parts.
It consists of ground substance called matrix. Within matrix lie two kinds of fibers:
(i) Yellow or elastic fibers and (ii) white or collagenous fibres. These fibers cross and
intercross, making a network. The other space is occupied by various types of cells.
These are fibroblasts, histocytes, basophils, plasma cells, pigment cells, mast cells,
lymphocytes, monocytes etc.
Yellow elastic fiber:
It is a type of fibrous connective tissue distributed in the areolar connective tissue,
hollow viscera, ligaments, in the walls of bronchi and larynx uniting the cartilages.
It is made up of yellowish, thick, branched fibers running singly and in loose
bundles. The bundles are not wavy however when they are torn, the broken ends
quickly curl up due to recoil. Chemically they are composed of protein elastin.
White fibrous tissue:
It is made up of white, thin, unbranched fibers running in bundles. It is found in
tendons, ligaments etc. the fibers contain protein known as collagen.
Adipose tissue:
This tissue is characterized by presence of fat globules inside fat cells. These cells
are supported by loose areolar tissue. It is found in subcutaneous tissue, mammary
gland etc.
Reticular Tissue:
It consists of network of interlacing reticular fibers and reticular cells.
LocationStroma (frame work) of liver, spleen and lymph node.

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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.
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
Hyaline cartilage:
It is made up of a homogenous, translucent, hard ground substance matrix.
Chemically it is chondrion. Collagen fibers are not visible. It consists of number of
cartilage cells or chondrocytes which are occupied in small empty spaces called
Lacunae. The cells are usually arranged in the groups of two, three, or four.
It is found in articular ends of bones, between epiphysis and the diaphysis of the
growing long bones, and the anterior ends of ribs. The cartilage of nose, external
meatus, larynx trachea and bronchial tubes also belong to this class.
Fibrous cartilage:
It consists of cells which are large, arranged in groups and placed inside lacunae.
The collagen fibers are more that found in hyaline cartilage.
It is found in intervertebral discs, menisci of knee joint mandicular joints, pubic
symphysis etc.
Elastic cartilage:
It is present in pinna, Eustachian tube, and epiglottis and in some of the laryngeal
cartilages. It also consists of numbers of cell in lacunae. Elastic Cartilage differs
from hyaline cartilage only for the presence of enormous elastic fibers in the matrix.

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Bone:
It is the hardest of all the tissues in the body. It consists of bone cells and the
intercellular ground substance. There are three types of bone cells osteoblast,
osteocyte and osteoclast the intracellular part consists of collagen fibers.
Osteocollagenous fibres. Mucopolysaccharide cementing material (ossein) and
various inorganic salts, calcium being the most important part.
Blood:
It is a type of fluid connective tissus. It is distributed in blood vessels-arteries, veins,
arterioles, venules and capillaries. The ground substance is watery fluid called
plasma. Suspended in plasma are two types of cells: white blood cells (4,000-
11,000cmm) and red blood cells (4.5million/cmm).Red blood cells (erythrocytes) are
nucleated and more or less oval shaped in frog while, they are non-nucleated, round
and biconcave in human. Various types of white blood cells (leucocytes) such as
neutrophils, acidophils, basophils, monocytes and lymphocytes may be seen. Apart
from these cells platelets or thrombocytes are also present in blood.
Lymph:
It is the modified tissue fluid containing 94% water and 6% solids. Only lymphocytes
(500-7500/cmm) are present platelets and RBC are absent.
(1) Write characteristics of Epithelial tissue.
(2) Enlist types of Epithelial tissue.
(3) Write characteristics of Connective tissue.
(4) Enlist types of Connective tissue.
Date:

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1.
Aim: Microscopic study of muscular and nervous tissue
(A) THE MUSCULAR TISSUE
Characteristics
 It consists of large elongated cells with minimum intercellular spaces.
 Presence of contractile proteins in the characteristic of the muscular
tissue.
 Muscular tissue performs the function of locomotion and movement of
the body.
Types of Muscular tissue and Characteristics
Skeletal muscles:
They are usually attached to bone and hence known as skeletal muscles. They are
under the contral of will and so they are known as voluntary muscle fibers. Fibers
are long, cylindrical shaped showing distinct striations (hence Also known as
striated fibers).Each fiber is surrounded by a thin membrane sarcolemma. Each
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PRACTICAL: 3
MICROSCOPY OF MUSCULAR AND NERVOUS TISSUE

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fiber consists of number of nuclei. The myofibrils are embedded in the sarcoplasm.
Fibres are unbranched.
Visceral muscles:
It is the type of muscular tissue found in heart visceral organs (hence known as
visceral muscles). They are under the control of autonomic nervous system and
hence are involuntary in nature.
Each fiber is a long, spindle shaped and unbranched with single centrally placed
nucleus. The striations are not seen (hence known as smooth muscles). The
nucleus is surrounded by small amount of cytoplasm-sarcoplasm and the fibers are
not surrounded by any membrane, but several of them are joined together by loose
connective tissue.
Cardiac muscles:
It is the type of muscular tissue found in heart (and hence known as cardiac
muscles). They are also not under the control of will and hence are involuntary in
nature.
Fibers are long, cylindrical and branches join with other fibers ultimately forming
three dimensional networks. Each fiber is surrounded by sarcolemma and has
longitudinal myofibrils. At intervals there are thick dark intercalated discs which
represent the cell membranes of adjacent cardiac muscles cells. Each muscles cell
has a distinct nucleus.
(B) NERVOUS TISSUE
Characteristics
 It consists of nerve cells, its processes called dendrites, axons and
neuralgia cells.
 Tissue has highly developed properties of irritability and conductivity.
They are specialized for reception and discharging stimuli and their
transmission.

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Nervous tissue
Nervous tissue consists of nerve cell and its processes:
The processes of nerve cells are dendrites (which receive the impulses) and axons
(which discharge the impulses). Axons form nerve fibers which may be myelinated
or non-myelinated.
Myelinated fibers show central axis cylinder, surrounded by myelin sheath. It is
interrupted at regular intervals by contriction known as nodes of Ranvier. Outside
myelin sheath lie nucleus and other cell components. The whole fiber is surrounded
by a membrane neurolemma or neurolemma. Non-myelineted fibres differ from
myelinated fibres in not having any myelin sheath or nodes of Ranvier.
(1) Write characteristics of Muscular tissue.
(2) Enlist types of Epithelial tissue.
(3) Write characteristics of Nervous tissue.
Date:

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Aim: Identification of axial bones
The axial skeleton is the part of the skeleton that consists of the bones of the
head and trunk of a vertebrate. In the human skeleton, it consists of 80 bones and is
composed of six parts; the skull bones, the ossicles of the middle ear, the hyoid
bone, the rib cage, sternum and the vertebral column. The axial skeleton together
with the appendicular skeleton form the complete skeleton.
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PRACTICAL: 4
AXIAL SKELETAL SYSTEM

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The skull (Bones of head)
Cranium (Large and hollow bony
case accommodate brain)
Facial Bones ( Bones of face give shape
to face)
Bone Position Bone Position
Frontal (1) Front and Central Maxilla (2) Forms upper jaw with
teeth
Parietal (2) Side Walls and Roofs Mandible (2) Forms lower jaws,
Largest facial bone
Occipital (1) Back and base with
an opening foramen
magnum
Nasal (2) Small bone in the
middle, attached to
frontal bones
Temporal (2) Lateral and base Palatine (3) Small bone
Sphenoid (1) Like the bird ‘bat’ two
wings. Lies at the
base and in the front
of temporal bone.
Zygomatic(2)
Lachrimal(2)
Cheek bones
Smallest and fragile

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The Vertebral Column
Each vertebrae consists of
Body: Anterior circular body with two neual arches. Two arches form bony ring for
spinal cord.
Spinous Processes: Projection from the back of bony ring.
Transverse Processes: Two projections one on each side from the bony ring.
Different types of vertebrae can be differentiated on the basis of characteristics of
body, spinal and transverse processes.
Ethmoid Cubical shaped, lies
in the space between
the orbits.
Vomer (2)
Turbinate (2)
Thin, vertical separate
nasal cavity
Spongy and Curved

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Types of
Vertebrae
Body Spinous process Transverse
process
Atlas (1st
Cervical)
Absent Absent Small
Axis (2nd Cervical) Odontoid (tooth
like process from
the body)
Large and strong Very small
Typical Cervical Small and Oblong
shaped
Long and bifid Large and contains
a well marked
foramen
Thoracic Small, flat with
coastal facets on
the body
Long, markedly
downward sloping
Large with a facet
for rib
Lumbar Kidney shaped Strong,
Quadrangular
broad and flat
almost horizontal
First four are thin a
Fifth is massive
Bones of the Thorax (Chest Cavity)
Consist of sternum and ribs attached to vertebral column

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Sternum: Flat, dragger shaped, slight convex anteriorly and concave posteriorly.
Sternum has three parts
Manubrium: Triangular, attached to it are clavicle, the first and second ribs.
Body : Attached to it are 3rd and 6th ribs.
Xiphoid process: Cartilagenous in Children, ossified in adults.
Ribs: 12 pairs.
They are ‘C’ shaped, flat bones, ship like 1 to 6th ribs are attached to sternum
(manubrium and body)
7th to 10th ribs are joined to one cartilage whih is finally joined to body at the
sternum.
11th and 12th ribs are ‘Floating ribs’ joined only to vertebral column.
(1) Enlist bones of skull.
(2) Enlist vertebrae of vertebral column.
(3) How many ribs are in thoracic cage.
(4) Enlist facial bones. Which is largest facial bone?
Results:
Date:

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Aim: Identification of appendicular bones
The appendicular skeleton is the portion of the skeleton of vertebratesconsisting of
the bones or cartilage that support the appendages. The appendicular skeleton
includes the skeletal elements within the limbs, as well as
supporting pectoral and pelvic girdles. Of the 206 bones in the human skeleton, the
appendicular skeleton comprises 126.
The appendicular skeleton forms during development from cartilage, by the process
of endochondral ossification.
The appendicular skeleton is divided into six major regions:
1. Pectoral girdles (4 bones) - Left and right clavicle (2) and scapula (2).
The Clavicle bone is long bone and has two curves.The Scapula is large flat
bone, anteriorly concave and posteriorly convex. Upper portion has coracoids
process and acromion. Lateral upper part has glegoid cavity in which head of
the humerousus fits in.
2. Arms and forearms (6 bones) - Left and right humerus (2) (arm), ulna(2)
and radius (2) (forearm).
Humerus: bone of upper arm, consists of head but no neck, head is ball like,
a long cylindrical shaft.
Radius: Upper end has smooth round head a neck and occipital tuberosity.
Other end is broader with styloid process and has a long shaft.
Ulna: Upper end expanded strong hook like structure in with a large ‘c’
shaped cavity (trochlear notch). Othe end has styloid process. Shaft is
broader and becomes thinner downwards.
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PRACTICAL: 5
APPENDICULAR SKELETAL SYSTEM

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3. Hands (54 bones) - Left and right carpals (16)
(wrist), metacarpals (10),proximal phalanges (10), intermediate
phalanges (8) and distal phalanges (10).
Carpals: Small, square bones-scaphod, lunar, triquetral pisiform, trapezium,
trapezoid capitates and hamate.
Metacarpals: small but long bones with a small shaft and two heads.
Phalanges: Like metacarpals but some are short.
4. Pelvis (2 bones) - Left and right hip bone (2).
The pelvis consists of sacrum and hip bones.
Sacrum-fusion of 5 sacral bones
Hip bones consists of Ilium, Ischium and Pubis. Pubis has acetabulum and
obturator foramen.
5. Thighs and legs (8 bones) - Left and right femur (2) (thigh), patella (2)
(knee), tibia (2) and fibula (2) (leg).
Femur: Bone of thigh, consists of head and neck, head is ball like, a long
cylindrical shaft.
Tibia: Long, strong,cylindrical shaft, upper end is flat and femur rests on it.
Strongest bone.
Fibula: Long, weak, thinner shaft, upper end is little round, lower end is
haves lateral mammeous, muscles are attached to fibula.
Patella is a mobile small disk of bone forming knee cap.
6. Feet and ankles (52 bones) - Left and right tarsals (14)
(ankle), metatarsals (10), proximal phalanges (10),intermediate
phalanges (8) and distal phalanges (10).
Tarsals: small, but stout bones situated in two rows.

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Metatarsals: small longer bones, one end thicker, navicular, cune form.
Phalanges : Identical to phalanges of arm but are smaller.
1. Enlist bones of upper limbs.
2. Enlist bones of lower limbs
3. How many bones form Appendicular skeleton?
4. Which are strongest and longest bones of upper limb and lower limb?
Results:
Date:

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Aim: Introduction to hemocytometry
GENERAL CONSIDERATION:
The counting of cells (Red blood corpuscles or white blood cells) in blood using
haemocytometry set is called haemocytometry. The numbers of cells in blood are
too many and the size of cell is too small. The cells will appear in clusters if seen
through microscope. This difficulty is partly overcome by diluting blood to a known
degree. Haemocytometry set which is used for counting the number of cells in blood
consists of
1) Dilution pipettes
2) Counting chamber (named as thomas or neubauer’s counting chamber)
3) Special cover slip (also known as thomas cover slip)
1) The Dilution pipette:
It consists of 4 parts-the long stem. The bulb, the short stem and the sucker. The
long stem has a uniform capillary bore extending from a well ground conical tip and
merging in the bulb. The fifth division is heavily marked and labeled as 0.5. This part
is to measure exact amount of blood taken for counting. The fluid in this part does
not take part in the dilution. And hence this quantity must be deducted while
calculating the dilution factor.
The bulb is the part where dilution of blood takes place. It consists of red head,
which helps, in uniform mixing of blood with dilution fluid. The bulb ends in short
stem, which have mark ‘11’ (in WBC pipette) or ‘101’ (in RBC pipette). The short
stem also consists of a uniform capillary bore. The sucker is fitted to the end of short
stem. The sucker is the rubber or polythene tube. At the end of the tube there is the
plastic mouthpiece.
The differentiating points of the RBC and WBC dilution pipettes have been
illustrated in the figure.
2) The counting chamber:
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PRACTICAL: 6
HEMOCYTOMETRY

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It is a single piece thick glass slide having two counting areas in the central part. An
‘H’ shaped groove separates the two counting areas from each other and the other
part of the slide. On the either side of these areas are the platforms, which are
raised by 0.1 mm above the counting areas. On this platform rests special cover
slip–Thomas cover slip.
The ruling areas of a chamber are the sharp contrast of bright lines against the
darker metalised background. A thin semi-transparent layer of the metal has been
deposited on the glass counting areas and the lines are ruled out through metal
surface. Just as one cannot see the stars during the day; the ruling cannot seen if
bright light is focused on the chamber. To adjust the one most partially close the
diaphragm of the microscope.
The total ruling of each side is 3mm in length and 3mm in breadth. It is divided in
nine equal squares of 1sq, mm area. The boundary lines of these squares are triple
linings. Four squares of the corners are used for WBC counting while the central
square is for RBC counting. Each WBC square is further divided into 16 equal
squares, by single lining. The area of each square is 1/4 X 1/4 =1/16sq.mm.
Summery of Neubauer Counting Chamber
Area Volume of fluid
One small WBC square 1 sq. mm 0.1 cmm
One smallest WBC
square
1/16 sq. mm 1/160 cmm
One small RBC square
(16 smallest RBC
squares)
1/25 sq. mm 1/250 cmm
One smallest RBC square 1/ 400 sq. mm 1/4000 cmm
The dilution fluids:
Various dilution fluids are used in haemocytomety but the basic criteria for preparing
the dilution fluid is that it should be isotonic to blood plasma. The composition of
dilution fluid depends on other requirements such as staining; fixing etc. Table 1 and
2 give the most commonly used dilution fluids for RBC and WBC count with the
importance of each constituent.

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Hayem’s RBC Dilution Fluid
Substance Amount Purpose
Sodium Chloride (NaCl) 1.0 g Provides isotonicity, prevents hemolysis
Sodium Sulphate
(Na2SO4)
5.5 g Provides isotonicity, prevents rouleax
formation
Mercuric Chloride (HgCl2) 0.5 g Causes fixation of the cells
Prevent bacterial growth
Water (H2O) Up to 100ml Diluent
WBC Dilution Fluid
Substance Amount Purpose
Glacial acetic acid 2.0 ml Destroys RBCs
Gentian or Methyl violet (1%) 1.0 ml Stains nuclei of WBCs
Water Up to 100ml Diluent
1) State the difference between
A) RBC and WBC dilution pipettes
B) Ordinary and Thomas cover slip.
2) Draw Neubauer’s counting chamber and give a summery of its counting.
3) Give the composition of WBC and RBC dilution fluids.

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Results:
Date:

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Aim: Enumeration of white blood cell (WBC) count of own blood.
(A)To find out Total Leucocytes or White blood cells (WBC) count of own
blood.
Requirements
Microscope Neubauer’s counting
chamner
Absorbent cotton
70% alcohol WBC dilution Pipette Thomas cover slip
Xylol WBC dilution fluid Pricking needle
Composition of Dilution Fluid
Substance AMOUNT PURPOSE
Glacial acetic acid 2.0 ml Destroy RBCs
Gentian or methyl
Violet (1%)
1.0 ml Stains nuclei of WBCs
Water Up to 100 ml Diluent
Principle
The number of white blood cells in blood is too many and the size of cells is too
small. It is impossible to count the cells even under high power. This difficulty is
partially overcome by diluting the blood with a suitable fluid to a known degree. The
diluted blood is placed in a capillary space of known capacity in between a special
ruled slide (counting chamber) and a cover slip. The cells thus spread out in a single
layer in this space and the number of cells can be calculated by multiplying with
dilution factor and are reported as cells mm of blood.
Procedure
 The Thomas cover slip, counting chamber and lenses of microscope are clean
with the help of Xylol and then absorbent fine cloth.
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PRACTICAL: 7
WBC COUNTS

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 The counting chamber is adjusted and observed under low power of microscope
keeping the Thomas cover slip resting on the slide.
 WBC pipette is cleaned and dried. The ring finger is sterilized with 70% alcohol
and it is pricked boldly with the help of pricking needle.
 The first drop of blood is discarded and second drop of blood is sucked in the
WBC pipette up to mark 0.5. Immediately the dilution fluid sucked exactly up to
the mark 11.0. When it is filled up to the mark, pipette is brought to horizontal
position and the finger is placed over the tip of pipette. A simple knot is given to
rubber tube.
 The pipette is rolled between the plasma to mix the blood with dilution fluid.
 Few drops (2-3) are discarded and then the pipette is held at an angle of 45˚ to
the surface of counting chamber and tip is applied to the narrow slit between the
counting chamber and the cover slip. A drop is allowed to come out from the
pipette. The fluid is will run into the capillary space because of capillary action
and it is filled. The drop should not flow into the moat.
 The fluid is allowed to settle for three min. on the stage of microscope.
 The located WBC chamber is focused and WBCs in 16 smallest squares of WBC
are counted. They are counted in 4 such squares chambers.
Experimental hints to be followed:
 Always clean and adjust the counting chamber, cover slip, microscope and the
pipette before pricking the finger for blood relation.
 Suck the blood and the dilution fluid slowly, uniformly and exactly to the mark.
After sucking the blood up to the required mark, remove the extra amount of
blood from the tip of the pipette by cotton gently and carefully.
 A knot may be given to the sucker tube after sucking the dilution fluid up to the
particular mark. Care should be taken that fluid does not come out from the either
end.
 Discard a few drops of fluid in the stem of thw pipette because it does not contain
blood.
 The fluid should not enter into the grooves of the neubauer’s chamber. If it
happens, due to capillary action, it will draw cells thus giving error in counting. It is

28
advisable to form a drop at the tip of pipette and the drop should simply touch the
slide. The fluid will spread automatically due to capillary action.
 The cells must be allowed to settle for a period of 2-3 min. before starting the
actual count. For counting the number cells, prepare a table consisting of
columns and enter the number or cell in each square.
 The cells touching two boundaries (preferable left and top) are to be counted
while cells touching other two boundaries (right and bottom) are not to be
counted.
(B) Aim: To count number of various types of White blood cells (WBC) and to
obtain differential WBC count of Own blood.
Apparatus
Microscopic slides, 70% alcohol, Pricking needle, Leishman’s stain, Microscope with
oil immersion lens, Mechanical stage and cedarwood oil
Procedure:
 The microscope (neat and clean with lenses and the light adjusted) is kept
already. Two slides are cleaned properly with xylol.
 The fingertip is sterilized as usual and a bold prick is given to obtain a small drop
of blood. It is then touched to the slide at one end in the center at least 1cm. away
from the narrow border.
 The slide is placed on the smooth surface of the table such a way that the drop of
the blood is front. The spreader slide is held along its long edge to form an acute
angle of 30°-40° on the right side.
 The spreader slide is pushed ahead towards the left and with quick and uniform
motion and a light even pressure. The blood flows along with the spreader slide to
form a film.
 The film is dried in air and Leishman’s stain is poured on the film drop-wise to
cover the film. The stain is allowed to act for one minute.
 Double the amount of water is placed on this slide with stain on it. The stain is
mixed thoroughly with water by blowing. This is now allowed to act for three min.
(care should be taken such that the stain is not getting dried up).

29
 The stain is washed in running tap water for 5 min. till slide gets pinkish
appearance.
 Water is drained out and slide is allowed to dry.
 A drop of cedarwood oil is placed on the portion of film which is apparently good.
The slide is examined under oil immersion lens.
 An observation chart having 100 squares is prepared, and after identifying each
cell, it is entered by writing latter say N (Europhiles), E(Eosinophiles), B
(Basophil), M (monocyte) and L (Lymphocyte). All the cells in the view are
identified without ignoring any cell. The slide is moved in definite direction and
cells are identified till it reaches 100.
 The different WBC count is obtained directly from the observation chart by
summing up total number of different WBCs.
1 State your observation and give identification profile.
2 Classify WBC and give characteristics of each type.
3 Give the composition of Leishman’s stain.
4 State various disorders associated with abnormal differential WBC counts.
5 State your observation and calculate total WBC count of your blood.
6 State the characteristics and functions of blood.
7 What is normal WBC count and which are the various disorders related to it?
8 During which pathological condition, total WBC is performed.

30
Results:
Date:

31
Aim: Enumeration of Total Red Blood Corpuscles (RBC) count
Requirements
Microscope Neubaur’s counting chamber Absorbent cotton
70% alcohol RBC dilution pipette Thomas cover slip
Xylol RBC dilution fluid Pricking needle
Principle
The number of red blood cells in blood is too many and the size of cells is too small.
It is impossible to count the cells even under high power. This difficulty is partially
overcome by diluting the blood with a suitable fluid to the known degree. The diluted
blood is placed in a capillary space of known capacity in between a special ruled
slide (counting chamber) and a cover slip. The cells thus spread out in a single layer
in this space and the number of cells can be counted under high power of a
microscope. The count can be calculated by multiplying with dilution factor and are
reported as cells per cubic mm of blood.
Procedure
 The thomas cover slip, counting chamber and lenses of microscope are clean
with the help of xylol and then fine cloth.
 The counting chamber is adjusted and observed for RBC squares under low
power of microscope, keeping the Thomas cover slip resting on the platforms of
the slide.
 The objective of microscope is raised and then it is adjusted for high power and
the chamber of RBC square is adjusted under high power.
 RBC pipette is cleaned and dried. The ring finger is sterilized with the 70%
alcohol and it is pricked boldly with the help of pricking needle.
DATE: / /2021
PRACTICAL: 8
TOTAL RBC COUNTS

32
 The first drop of blood is discarded and the second drop is sucked in the RBC
pipette up to mark 0.5 immediately the dilution fluid sucked exactly up to the
mark 101.When it is filled up to the mark ,pipette is brought to horizontal position
and the finger is placed over the tip of pipette. A simple knot is given to rubber
tube.
 The pipette is rotated between the palms to mix the blood with dilution fluid.
 Few drops (2-3) are discarded and then the pipette is held at an angle of 45˚to
the surface of counting chamber and tip is applied to the narrow slit between the
counting chamber and the cover slip. A drop is allowed to come out from the
pipette the fluid will run into the capillary space because of capillary action and it
is filled. The drop should not flow into the moat.
 The fluid is allowed to settle for three min. on the stage of microscope.
 The located RBC chamber is focused and RBCs are counted in 16 smallest
squares. This is repeated in another four such chamber.
1) State your observation and calculate total RBC count of your blood.
2) Write a note on RBC.
3) Describe the various disorders associated with abnormal RBC count.
4) Explain different types of anemia.
5) Give the full form of RBC and justify it.
6) WBC are counted under low power while RBC under high power-Explain.

33
Results:
Date:

34
Aim: Determination of bleeding time of own blood.
Requirements
Piece of filter paper, prickling needle, 70% alcohol.
Procedure
 The tip of finger is sterilized with 70% alcohol and a blod prick is given so that
blood flows freely. The stopwatch is immediately started.
 The blood is soaked on a filter paper (while soaking, the fillter should not touch
the skin of finger). This is repeated every 10 seconds till no blot appears on the
papers.
 The time from the first appearance of the blood to the cessation of bleeding is the
bleeding time.
SELF STUDY QUESTIONS:
1. State your bleeding time.
2. Define bleeding time, state factor affecting it.
3. Explain the terms: thrombocytopenia, and purpura.
Results:
Date:
DATE: / /2021
PRACTICAL: 9
BLEEDING TIME

35
Aim: Determination of clotting time of own blood.
Requirements
Capillaries or small test tubes or slide, 70% alcohol, cotton, pricking needle or
syringes with needle.
Procedure
LEE AND WHITE’S METHOD
About 4cc of blood is obtained through a vein puncture and 1cc is placed in each of
small test tubes. As soon as blood enters the barrels of syringe, stopwatch is
started. The test tubes are placed in water bath at 30˚C. The test tubes are filled at
every 30 sec. The time when blood does not move on titling is being noted down.
WRIGHT’S METHOD
The ring finger is sterilized and pricked boldly with the needle. As soon as the blood
comes out, the stopwatch is started and a capillaries tube is filled up to three-fourth
of its total length. After every 30sec. a portion of capillary tube (about 1/10th of the
tube) is broken until a thin line of unbroken coagulum is seen stretched between the
two broken ends. This time is noted and the difference between the time when blood
comes out from the finger and the unbroken coagulum seen is noted as coagulation
time.
DUCK’S METHOD
The finger is sterilized and a drop of blood approximately 5mm diameter is placed
on a special glass slide. The slide is held vertically at every 30sec. The time is noted
when no change in the shape of the drop is seen. This gives the clotting time.
1. State your clotting time.
2. Define clotting time, state factor affecting it?
3. Given in detail the mechanism of clotting.
4. Explain the terms: Hemophilia
DATE: / /2021
PRACTICAL: 10
CLOTTING TIME

36
Results:
Date:

37
Aim: To find out Hemoglobin content, Oxygen carrying capacity and color
Index of own blood.
Requirements
Sahli’s haemoglobinometer or haemometer which consists of two sealed
comparison tubes fixed in rack, a specially graduated diluting tube, a thin glass rod
(stirrer) and micropipette of 20 cubic millimeter (cmm) capacity, pricking needle,
N/10 HCL, distilled water, 70%alcohol and absorbent cotton.
Principle (Sahli’s method)
When blood is mixed with N/10 HCl, RBCs are haemolyzed and Hb is liberated. This
Hb is converted in to acid hematin which is reddish brown in colour. The solution is
diluted with distilled water till it matches with the standard glass (comparision) tubes.
The Hb% can directly be read from the graduated tube.
Procedure
1. The graduated diluting tube and the micropipette are cleaned thoroughly and
dried.
2. The graduated diluting tube is filled with N/10 HCl up to the mark 4gm or till the
micropipette touches the level of acid in the tube.
3. The finger is cleaned with 70% alcohol and it is pricked to obtain a drop of
blood. First drop is wiped out. Second drop is sucked in the micropipette up to
mark 20 cmm. While filling the micropipette entry of air bubble should be avoided.
It is advisable to collect blood and discarding the excess.
4. The blood is immediately deposited at the bottom of the graduated tube. The
pipette is rinsed two or three times in HCl. Care should be taken so that blood
does not get deposited on the sides of the tube.
5. The blood is mixed with the help of stirrer and then solution is allowed to stand
for 10-15min. so that all HB is converted into acid haematin.
6. The mixture is then diluted with distilled water. Distilled water is added drop by
drop and every time it is stirred till the exact match with standard glass tubes is
DATE: / /2021
PRACTICAL: 11
ESTIMATION OF HAEMOGLOBIN CONTENT

38
obtained. While observing the color match, stirrer should be kept out of solution
and the calibrated side should not come in view. The observation must be done
while facing it against uniform intensity of light.
7. When the matching is complete, stirrer is taken out from the diluting tube and
the scale is read on the side of the tube.
Precautions
1. While filling the micropipette entry of air bubbles should be avoided. It is
advisable to fill up the micropipette more than 20 cmm mark. The excess of
blood in the micropipette may be removed by touching the tip of the pipette on
palm.
2. While depositing blood into graduated tube, one should not blow it forcibly so
that blood sticks to the side of pipette. Rinsing should also be done slowly.
3. Never take less quantity of N/10 HCl.
4. While observing for colour match, stirrer should be kept out of solution (but
not out of the tube), and the caliberated side should not come in view. The
observation must be done while facing it against uniform intensity of light.
Some points of consideration
1. It is better to express the Hb value in terms of gm% instead of percentage of
standard because percentage varies according to different methods used.
2. We cannot use sulfuric acid because
3. There is every dange of the appearance of the tinge of acid in the solution.
4. H2SO4 and distilled water are not easily miscible as compared to HCl and
distilled water.
5. The standared side tubes with which the coloru is being matched are
available with the dilution of HCl.
6. The disadvantages with HNO3 are similar to those of H2SO4. In addition the
fumes of nitrous oxide may be evolved in the experiment. Hydrofluric acid has
still additional disadvantage that it may destroy the caliberation of the tube.
7. NaOH if used instead of HCl forms alkaline hematin which is neither as stable
as acid haematin, nor it has identical colour as acid haematin.
8. If more acid is used instead of distilled water it does not alter the observation
however if less acid is used, formation of acid haematin may not be complete
and chances of errors are there.

39
1) State your observation as
(a) Observed HB gm% (d) Mean HB%
(b) Observed HB % (e) Color index
(c) Calculated% (f) Oxygen carrying capacity
2) Draw the apparatus used in Sahli’s haemoglobinometer.
3) Give the principle underlying Hb estimation by Sahli’s method.
4) Give other methods for estimation of Hb..
5) Define: Color index, oxygen carrying capacity.
6) Explain the following: anemia, Haemolysis.
7) Write about chemical structure of haemoglobin.
8) Can H2SO4 or NaOH be used instead of N/10HCl in sahli’s method, why?
Results:
Date:

40
Aim: To evaluate case study of Congestive Heart failure ( minimum 2 cases)
Aim: Determination of blood group.
Requirements
Microscopic slides, glass-marking pencil, Antiserum A, Antiserum B and Antiserum
D(Rh), pricking needle.70% alcohol and microscope.
Procedure
 Take three slides and mark them A, B and C.
 Place a drop of type A serum (AntiserumA) on slide A and Antiserum B on
slide B on third slide Antisrum D.
 Sterilize the finger and prick it boldly. Place one drop of blood on each of the
sera.
 Mix the cells and serum with the help of another slide. (Use separate slides or
separates edges for mixing each serum.)
 Allow it to react for 5 minutes.
 Examine after 5 minutes, but not latter than 10 minutes. Both these factors
may yield false results.
1. State your observation and conclusion.
2. Given the principle underlying your observation.
3. Write a note on ABO system.
4. Given the importance of “RH factor”.
9 Explain the following terms: Blood transfusion, Universal donor, Universal
recipient, Agglutinins, Agglutinatination, Erythoroblastosis foetalis.
DATE: / /2021
PRACTICAL: 12
DETERMINATION OF BLOOD GROUP

41
Results:
Date:

42
Aim: Determination of erythrocyte sedimentation rate (ESR).
Principle: Erythrocytes of blood have a tendency to settle down because of their
greater density than plasma and rouleaux formation. Thus if the blood, mixed with
anti-coagulant, placed in a long vertical tube, the sedimentation of erythrocytes
occurs leaving a small clear plasma at the top. ESR is mm. of clear plasma at the
top in a vertical column in one hour.
WESTERGREN METHOD:
Requirements:
1) Westergren’s pipette and westergren stand
2) Oxalate bulb
3) Sodium citrate solution (3.8%)
4) Sterilized syringe and needle to collect the blood by vein puncture.
Werstergren’s stand:
It can accommodate six pipettes. There is base containing six grooves each
containing a rubber cushion to prevent leakage of blood from westergren pipette. At
the top there are six screw caps. These are screwed down to exert sufficient
pressure on the pipette. The stand allows the pipettes to remain exactly in a vertical
position.
Procedure:
1) A sample of blood (about 3ml) is obtained by a venepuncture and is mixed with
3.8%sodium citrate solution in proportion of four part blood to one part of citrate
solution. The mixing of blood is done by rotating the sample gently between the
palms of hands.
2) The blood is sucked slowly up to the mark zero in the westergren’s tube.
3) The tube is set up right in the westergren stand, taking care that no blood escape.
The tube is fixed with the help of screws cap.
4) At the end of one hour and two hours. The upper level of red cell column is read.
It will indicate mm of clear plasma (ESR).
DATE: / /2021
PRACTICAL: 13
DETERMINATION OF ERYTHROCYTE SEDIMENTATION RATE

43
WINTROBE’S METHOD:
Requirements:
1) Wintrobe’s pipette
2) Stand for pipette
3) Long capillary pipette
4) Sterilized syringe and needle to collect the blood by venepuncture
Wintrobe’s pipette:
It is a heavy cylindrical tube of uniform bore and flat bottom. It is graduated on the
left hand from the top of the tune. Each main division represents 1 cm. and each
smaller division represents 1mm. the mouth cap to prevent loss of fluid by
evaporation.
Long capillary pipette:
It consists of 100mm long glass capillary and a broad stem which can hold about
2ml. of blood. It is fitted with a rubber teat.
Procedure:
1) A sample of blood (about 3ml) is obtained by venepuncture and is mixed with
3.8% sodium citrate solution in proportion of four part of blood to one part of
citrate solution.
2) About 1-3ml. of blood is taken in a dry capillary pipette. The capillary pipette is
inserted into wintrobe’s tube and by applying gentle pressure on the rubber
tear, the tube is filled exactly up to zero mark.
3) The tube is set up right in the stand.
4) At the end of one or two hour, the upper level of red cell column is read. It will
indicate mm of clear plasma (ESR).
Discussion:
ESR is defined as mm of plasma formed at the top of the vertical column in one
hour. The normal range of ESR is as follows:
Method At the end of one hour
Male Female
At the end of two hour
Male female
Westergren’s 3-5 4-7 7-15 12-17
Wintrobe’s 0-7 0-7 7-15 7-15

44
Normally RBCs remain suspended in the plasma. This ability of RBCs to remain in
the suspension form is known as suspension stability. However the cells are heaver
than plasma (sp.gravity of RBC is 1.090 and that of plasma is 1.030). Thus when
the blood is taken out and allowed to stand in a tube, the cells tend to settle down.
The actual rate of sedimentation is more rapid than the one accountable by physical
factors such as specific gravity, viscosity of plasma and temperature. This is mainly
because of another property of RBCs i.e. Rouleaux formation
Rouleaux formation:
The RBCs are negatively charged as they remain suspended in the plasma. These
charges tend to keep them separate from one another. However, protein molecule
in plasma have a property of cohesiveness to RBCs and it overcomes the repulsion
caused by negative charge and when the cells strike each other, they tend to each
other and get piled over one another to form Rouleaux. Each rouleaux consists of 8
to 12 cells and when the cells aggregate together, the repelling forces the Rouleaux
to break into two. Rouleaux are heavier and tend to sink down quickly.
Various factors increasing the Rouleaux formations are:
1) Large protein molecules
2) Globulin cocentration
3) Increase in number of RBCs
4) Increase in the size of RBC.
ESR is of prognostic value (indicating the progress of disease rather than diagnostic
(indicating the nature of disease). It is increased all acute general infection like
septicemia (100mm/hr), pulmonary tuberculosis (65mm/hr), anemia, and sickle
shape anemia. Jaundice, malignant tumor and inflammatory conditions. The ESR
decreases in allergy and protein shock.
Study Question:
1. Mention the different components of blood?
2. Define ESR.
3. Enlist methods for determination of erythrocyte sedimentation rate
4. Explain Rauleax formation.

45
Results:
Date:

46
Aim: Determination of heart rate and pulse rate.
ARTERIAL PULSE AND HEART RATE
The arterial pulse is wave of increased pressure propagated centrifugally, with
each ventricular ejection at an increasing velocity (4-10mg/sec). The arterial pulse
does not occur synchronously in all arteries. The extent of rise at pressure is not the
same everywhere. However, examination of radial pulse is one of the important
steps in clinical examination. Much information can be gained from the rate and
quality of the pulse regarding cardiovascular diseases. This is all the more important
in a country like “India” were most population lives in villages.
‘Radial pulse ‘is chosen since it is easily accessible lies on the bone.
AIM: To examine the radial pulse.
APPARATUS: A wrist-watch with seconds arm.
METHOD:
(1) The subject is asked to be seated comfortably and fully relaxed.
(2) The right fore arm of the subject is help in semi-prone position (i.e. keeping thumb
up wards and little finger touching the table).
(3) The radial pulse is felt with the middle three fingers of the examiner’s right hand
and the characteristics are noted down as follow:
Observation and discussion
(1) Rate per mintue:
(For this, record three observation at the interval of 5 min. in between two reading
using wrist-watch) One can count 30 sec or 15 sec but it may give erroneous
results. Hence, one should count for at least one min. If the rate goes on changing
the conclusion is that the person is not resting. Pulse varies with age as follows
DATE: / /2021
PRACTICAL: 14
DETERMINATION OF HEART RATE AND PULSE RATE

47
Athletes show decreased rate (50/min) but if is in normal person it may be called as
slow pulse rate or ‘bradycardia’. The pulse rate is the measure of heart rate.
Define Bradycardia and Tachycardia.
Results:
Date:
STAGE RATE PER MIN
Newly born infant 130
<5years 90
<10years 80
<15 and above 70-75

48
Aim: Recording of blood pressure
Apparatus: Stethoscope and a mercury sphygmomanometer.
STETHOSCOPE
It is an instrument used for measuring rate or heart pulse. The commonly used
stethoscope is binaural stethoscope.
Construction:
Stethoscope consist of
1. Ear-frame
2. Conducting tube
3. Chest piece
1. The Ear-frame: It consists of two metallic tube jointed by a flat curved strip,
which acts as a spring to pull together the upper end of the two tubes.Each
tubes has an upper horizontal limb, which runs medilly, downward and forward,
giving a curveture suitable to the fitted in the external auditory meatur. The tip of
each tube entering the meatus is coverd with a smooth plastic ring to protect the
wall of the meatus against injury.
2. Conducting tubes: They are simple flexible and a soft pressure tube of rubber
or latex materials. They are about 35 cm in length and 3mm in the inner
diameter. These tube connect the chest piece with the ear-frame and may be
(1)only one tube from the chest piece and then bifurcated to the two ears by a
‘Y’ shaped joint or (2)two separate tubes leading directly from the chest piece to
the ears.
3. Chest piece: It consists of a metallic tambour about 3cm in diameter and having
an air chamber of 3-5 mm in depth. The diaphragm, which is made up of
cellulose material, covers the air chamber. The diaphragm caused an
amplification of the sounds, which are heard much louder then original.
SPHYGMOMANOMETER
It is an instrument used to measure the blood pressure of human being.
DATE: / /2021
PRACTICAL: 15
RECORDING OF BLOOD PRESSURE

49
CONSTRUCTION:
It consists of
(1) A mercury manometer
(2) A rubber bag covered with a linen cuff
(3) A rubber pump with a valve
1 Mercury manometer consists of two limbs. One limb is long and graduated from
‘0’ to ‘250’ at the top. The other limb is short and broad, forming a sort of mercury
well. The long limb is open to the atmosphere at the upper end ,and is filled with
the valve, which prevent spilling of mercury .The short limb is covered with an
airtight lid containing a valve, which allow free communication of air but prevents
spilling of the mercury even when it is laid horizontal.
2 Rubber bag, which is covered by a linen cuff is soft and elastic and is fitted with
two –pressure rubber tubing. One tube is fitted with a metal adapter which is an
airtight joint with the short limb of the manometer. The other tube is joined to the
airtight. The linen cuff has broad but short extension on one side and along
tapering tail on the other side which is provide with a metallic hook
3 Rubber pump is a simple thick walled rubber bulb of an oval shape and a size
that fist in the fits it has two opening which are fitted with valves the end of pump
through which air comes out is fitted with metal piece. This is connected to the
rubber tubing of the bag it is provide with a screw which when loosened the air to
escape out side.
Principle
The blood usually flows through a large sized artery is obstructed by means of air
pressure exerted through a rubber bag wrapped around the limb the pressure is
slowly released and the entry of blood through the obstruction is studied by
1 Feeling of the pulse palpatory method
2 Observation of oscillation of the mercury level oscillation method
3 Hearing with the stethoscope the sound produced in the segment of the artery
distal to obstruction (auscultotary method)
The blood flow stops when the pressure transmitted to the artery through the rubber
bag is equal to or more than the blood pressure the first entry of blood through
obstruction indicates the blood pressure. Usually the arm or thigh is used because

50
there is only one big vessel which runs superficially in each of this part of the
extremities.
Procedure:
 The subject is asked to sit or lie down on a couch in the supine position the
garment round the arm are place on the table and the position is adjusted such
that it is at the level of the heart
 The cuff is tide around the arm it should be neither too tight to cause any
discomfort to the subject nor too loose to allow its movement around the arm
 The artery is felt and its course is marked in the cubital fossa also the radial
pulse is felt and marked at the place where it felt well
 The manometer is placed by the side of the subject between subject’s arm and
body. The screw the rubber pump is tightened and the pump is pressed to inflate
to raise the mercury to 200mmlevel or 20-25mm Hg higher after the
disappearance of pulse.
 Keeping the eyes fixed at mercury level and the finger at the pulse pressure is
slowly released by unscrewing the valve of the rubber pump the readings of the
level of the mercury when the pulse reappears give the systolic pressure
recorded by this method is about 5-10mm lower than the actual systolic pressure
When the pulse appears it is also noted that mercury starts oscillating. The first
oscillation level gives systolic pressure. On continuation of deflation the oscillations
start increasing in magnitude and they slowly diminish the level at which the
oscillations are maximum is taken as the diastolic pressure. This is oscillatory
method. In this method the deflation has to be carried out slowly. The systolic
pressure should be read within fifteen seconds and the diastolic pressure within
thirty seconds.
In the auscultatory method after inflation as usual the chest piece of stethoscope is
placed over the brachial artery in the cubital fossa and deflation is started by slowly
releasing the pressure the level at which a sudden tap is heard is the systolic blood
pressure. The sound suddenly gets muffled and disappears. The level at which the
sound muffles is the diastolic pressure. The sounds heard are korokorr’s sounds.
These are due to interrupted flow of blood.

51
In many instances when the systolic blood pressure is very high. The first sound is a
faint tap and is then missed as the pressure in the bag is reduced the sound
produced becomes louder and audible at much lower level this reading is then
erroneously taken as the systolic blood pressure reading. The difference between
this reading and the true systolic reading is described as the auscultatory gap.
SELF STUDY QUESTIONS:
1 Draws a stethoscope and a sphygmomanometer.
2 Define B.P and explain the factors affecting it.
3 Explain the following terms: Diastolic B.P, Systolic B.P, Mean B.P, Hypertension
and Hypotension.
4 State alternate techniques for measuring B.P.
Results:
Date:

Hap i lab mannual-rdp mam

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