Lab Manual PDF

Human Anatomy & Physiology I (13PH0101)
Marwadi Education Foundation’s Group of Institutions

Vision:
Our vision is to address the challenges faced by our society and planet through
education that builds capacity of our students and empowers them through their
innovative thinking, practice and character building. This, in turn, would boost their
creativity while making them responsible towards the utilization of the limited natural
resources to face the challenges of the 21st century.
Mission:
• To produce creative, responsible and informed professionals
• To produce individuals who are digital-age literates, inventive thinkers, effective
communicators and highly productive
• To deliver cost-effective quality education
• To offer world-class, cross-disciplinary education in strategic sectors of economy
through well devised and synchronized delivery structure and system
• To provide a conducive environment that will enable students to experience higher
level of learning acquired through constant immersion leading to the development of
character, virtues, values & technical skills.
Faculty of Pharmacy
Vision:
Our vision is to be a globally recognized premier educational and research centre with
world-class facilities, adopting international best practices, focused on the integration of
science and technology in the areas of drug discovery, drug delivery, and healthcare
products.
Mission:
• To strive and achieve the best in pedagogy and research, through the creation of a
dedicated team of faculty and state-of-art research facility
• To develop skilled human power and modern cost-effective technology to support
national healthcare programmes.
Programme Education Objectives (PEO)

1. To create trained undergraduates with in-depth knowledge in pharmaceutical
sciences in a futuristic, state-of-the-art set-up to facilitate research and leadership.
2. To enhance industry-institute interaction for industry-oriented education and
research.
3. To promote professionalism, team spirit, social and ethical commitment to boost
leadership, assisting improvement in the healthcare.
4. To create pharmacy professionals who would spread across the country and the
world in various areas including education, research, industry and government.
Programme Specific Outcome (PSO)

After completing their graduation from Marwadi University’s Faculty of Pharmacy, a
student will:
1. Have expertise and industry-relevant knowledge in the field of pharmacy.
FoP, Marwadi University, Rajkot | B. Pharm. Sem.-I (2020-2021) Page | 1

2. Be an expert in the use of modern methods, tools, and resources related to pharmacy.
3. Be a pharmaceutical professional who understands their role as educators for the
promotion of good healthcare in society.
4. Be an ethical pharmacy professional with honourable personal ethical and moral
principles and understand the responsibility in their actions.
5. Be a stakeholder in contributing to the national healthcare with ample knowledge to
assess societal, health, safety and legal issues.
6. Be an effective communicator who can connect with society at large and prepare
detailed, effective reports.
7. Have analytical, logical and scientific ability to evaluate pharmacy related problems
in order to take effective decisions.
8. Have leadership skills, understand human behaviour, facilitate team building and
provide motivation as important factors for the society’s development.
9. Understand the need and values of environment protection with sustainable
development in the context of the pharmacy profession.
10. Have a passion for lifelong learning to adapt to new technological changes.

INDEX FOR EXPERIMENTS PERFORMED
B. Pharmacy, Semester-I Human Anatomy & Physiology -I (13PH0101)
Sr. Page
Title of experiment Date Grade Sign
1. Study of compound microscope.
2. Microscopic study of epithelial and

connective tissue.
3. Microscopic study of muscular and nervous
tissue. No. No.
4. Identification of axial bones.
5. Identification of appendicular bones.
6. Introduction to haemocytometry.
7. Enumeration of white blood cell (WBC)

count.
8. Enumeration of total red blood corpuscles
(RBC) count.
9. Determination of bleeding time.
10. Determination of clotting time.
11. Estimation of haemoglobin content.
12. Determination of blood group.
13. Demonstration of erythrocyte

sedimentation rate (ESR).
14. Determination of heart rate and pulse rate.
15. Recording of blood pressure.

PHARMACIST’S OATH
I swear by the code of Ethics of Pharmacy Council of
India in relation to the community and shall act as an
integral part of health care team.
I shall uphold the laws and standards governing my

profession.
I shall strive to perfect and enlarge my knowledge to

contribute to the advancement of pharmacy and
public health.
I shall follow the system, which I consider best for

pharmaceutical care and counselling of patients.
I shall endeavour to discover and manufacture drugs

of quality to alleviate sufferings of humanity.
I shall hold in confidence the knowledge gained

about the patients in connection with professional
practice and never divulge unless compelled to do so
by the law.
I shall associate with organizations having their

objectives for betterment of the profession of
Pharmacy and make contribution to carry out the
work of those organisations.
While I continue to keep this Oath in violated, may it

be granted to me to enjoy life and the practice of
pharmacy respected by all, at all times!
Should I trespass and violate this oath, may the

reverse be my lot!
PHARMACY COUNCIL OF INDIA

INSTRUCTIONS FOR STUDENT

Students shall read the points given below for understanding the theoretical concepts
and practical application.
❖ Listen carefully to the lecture given by teacher about importance of subject,

curriculum, graphical structure, and skills to be developed, information about
equipment, instruments, procedure, method of continuous assessment, tentative
plan of work in laboratory and total amount of work to be done in a year.
❖ Students shall undergo study visit to the laboratory for types of chemicals,
equipment, instruments before performing experiments.
❖ Read the write up of each experiment to be performed a day in advance.
❖ Organize the work in the group whenever suggested and make a record of
suggestions made by teacher wherever possible.
❖ Understand the purpose of experiment and its practical implications.
❖ Write the answers of the questions allotted by the teacher during practical hours if
possible or afterwards, but immediately.
❖ Students should not hesitate to ask any difficulty faced during conduct of practical
exercise.
❖ Student shall visit the recommended industry or hospital or retail pharmacy and
should study the knowhow of the shop door practices and the operations of
machines.
❖ Student shall develop maintenances skills as expected by the industries.
❖ Student shall develop the habit of group discussion related to the experiments /
exercises so that exchange of knowledge / skills should take place.
❖ Student shall attempt to develop related hands-on-skills and gain conﬁdence.
❖ Student shall focus on development of skills rather than theoretical knowledge.
❖ Student shall visit the nearby medical stores, industries, laboratories, technical
exhibitions; trade fair even if not included in the Lab Manual. In short, students
should have exposure to the area of work right in the student hood.
❖ Student shall insist for the completion of recommended laboratory work, visits,
answers to the given questions, etc.
❖ Student shall develop the habit of evolving more ideas, innovations, skills, etc. than
included in the scope of the manual.

❖ Student shall refer technical magazines, proceedings of the Seminars, refer websites
related to the scope of the subject and update their knowledge and skills.
❖ Student should develop the habit of not to depend totally on teachers but to develop
self-learning techniques.
❖ Student should develop habit to submit the practical exercise continuously and
progressively on the scheduled dates and should get the assessment done.
List of requirements for Human Anatomy & physiology I laboratory

❖ Neat and clean apron
❖ Complete Journal
❖ Record book
❖ Plastic sheet
❖ Glass Slide
❖ Cover Slip
❖ Napkin
❖ Detergent soap
❖ Hand sanitizer
❖ Calculator
❖ Pricking needle or Lancet
❖ Thomas coverslip – 2
❖ Glass Dropper
❖ Watch glass

EXPERIMENT NO.:1
Aim: Study of compound Microscope
Reference: Dr. R. K. Goyal, Dr. N. M. Patel
Practical Anatomy and Physiology
13th Ed.2009
B. S. Shah Prakashan, Ahmedabad.
Page No.:3-4
Theory:
Zaccharias Janssen and his father Hans lippershey had invented the
compound microscope (which is a microscope that uses two or more lenses).
Microscope is an optical instrument, used to see the magnified images of very
small objects which cannot be seen with a naked eye. Microscopes are employed as a
basic tool to observe and study the structural details of the cells. Nowadays, highly
sophisticated microscopes such as phase contrast, dark field and electron microscopes
are available but simple and compound microscopes remain as a basic tool in routine
research work.
The compound microscope which is commonly used in biological experiments,
not only provides high magnification (enlargement of the image of the object), but also
gives fair resolution (differentiation of the neighbouring points as separate entities).In
order to achieve higher magnification power, combination of lenses is used as objective
lens and ocular lens in components are grouped into two categories:
I. Structural components
II. Optical components
I. Structural components: three structural components of microscope are head, arm
and base. Head contains the optical parts in the upper part of the microscope. Base
supports it and contains eliminator (condenser). The arm is fitted with the coarse and
fine adjustment knobs and help in easy handling of microscope. The stand of
microscope has a heavy foot and limb which are joined by a hinge joint. The limb bears
the optical system.
II. Optical components: there are 2 types of optical structures:
a. Eye piece/ocular: it is a cylinder containing 2 or more lenses. Its function is to bring
the image into focus for the eye. Typical magnification values for the ocular lens is 2x,
10x and 50x.
b. Objective lens: objective lens is usually a cylinder containing lens attached to the
circular disk with the movable head of the microscope. Nose piece has objective lens of
various magnifications: high power, low power and oil immersion lens. Magnification of
the objective lens ranges from 10x, 40x to100x. Three to four objective lenses are
attached to the nose piece.
c. Stage: The stage is the flat platform which accommodates a glass microscope slide on
which object, to be examined, is mounted. The stage may be of mechanical type. An
eliminator is provided under the stage to provide the sufficient light which passes
through the holes on the stage for focussing the contents of the slide. It has two
micrometre screws which move the slide in two planes – from side to side and forward
– backward.
d. Condenser and iris-diaphragm:
The sub-stage consists of a condenser and an iris-diaphragm.
The purpose of condenser is

(1) To focus the parallel rays of light from mirror to the object
(2) To help in resolving the image.
The purpose of iris is to control the amount of light reaching on the object.
The mirror is movable and has two surfaces: Plane and Concave.
Working:
The objective lens is placed close to the object to be viewed and ocular lens is
placed closed to the eyes. The primary enlargement of the object is produced by
objective lens. The image produced thus is transmitted to the ocular where the final
enlargement occurs. Therefore, the magnifying capacities of the compound microscope
are the product of magnification of the objective and the ocular lens. For e.g.; using an
objective lens of 40x and ocular lens of 10x will produce a magnification of 400x. The
primary image produced by the objective lens is real and inverted image.
COMPOUND MICROSCOPE
USE AND THE CARE OF MICROSCOPE
(1) Always keep the microscope clean, dust free and covered.
(2) 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.
(3) After placing the slide over the stage, bring down the low poser lens using coarse
adjustment knob. Bend by the side of tube and bring your eyes at the level of
slide while bringing it down. Never bring down the objective with coarse
adjustment knob while looking through the eye-piece of the microscope. Bring it
down to the extent that it is just near to (but not touching) the slide.
(4) Slowly but confidently, the objective should be raised, while looking through the
eye piece of the microscope, using coarse adjustment knob till the object is seen.
It is made clear by using fine adjustment.
(5) To use high power lens, raise up the objective again. Change the lens and then
bring it down looking from the side. The objective is again raised while looking
through the eyepiece till the object is seen.
(6) Remove the eye piece 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.

(7) Adjust the iris-diaphragm to cut a thin peripheral rim of rays. Iris diaphragm
should be partially closed while observing Neubauer’s counting chamber. After
adjusting condenser and iris, replace the eye piece and observe again. The object
will be very clear.
(8) While using oil immersion lens, condenser should be racked up, preferably having a
capillary space between it and the slide. A drop of cedar wood oil is placed on
condenser to fill the capillary space, and also on the slide and it must touch the
objective lens also. The cedar wood oil is preferred to other oils because its
refractive index (1.55) is nearer to that of glass (1.5).
If condenser cannot be raised to touch the slide, oil should not be placed on it.
The oil on the condenser and 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.
(9) 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.
PROPER USE OF MICROSCOPES:

o To avoid breaking a cover slip and/or microscope slide while focusing (more
importantly scratching a lens), first locate the specimen using the low-power
objective, and then switch to the higher power objective.
o Never focus the high power objective with the coarse adjustment knob, and
never use these lenses when examining thick specimens or whole mounts of
specimens.
o To avoid dropping the microscope or banging it against a laboratory bench, carry
the microscope in an upright position using both hands.
o When carrying the microscope, place one hand on the base and the other hand
around the arm.
o Do not place the microscope in an upside down position. Pieces will fall out.
o Keep microscope away from the edge of the bench, particularly when not in use.
o Make sure power cords are out of the way.
o Never force the microscope parts to work.
o Never dismantle the microscope.
o Use lens cleaners and paper both before and after use.
o Take all slides off the stage prior to storage.
o Always store the microscope in low power objective.
Teacher’s sign:

EXPERIMENT NO.:2
Aim: Microscopic study of epithelial and connective tissue.
13th Ed.2009
Page No.:101-106
Introduction:
Tissue: A tissue is a group of cells having a common embryonic origin that function
together to carry out specialized activities. Tissue may be hard (bone), Semisolid (fat)
or liquid (blood) in their consistency. Tissue may vary with respect to the types of
cells present, their arrangement and type of fibres present.
Histology: Histology [Gr. histos =web, logos =study of] is the study of the structure
and function of cells, tissues, and organs of the body at the microscopic level. Hence, it
is often referred to as Cell, Tissue & Organ Biology. In the past, it has also been called
microscopic anatomy.
Basic Types of Body Tissues:

1) Epithelial tissue
2) Connective tissue
3) Muscular tissue
4) Nervous tissue
Epithelial Tissue:
Definition:
An epithelium is a tissue composed of one or more layers of cells covering the external
and internal surfaces of various body parts. Epithelial tissue also forms glands. The
term “epithelium” (sing, of epithelia) was given by a Dutch anatomist Ruysch (1638-
1731) to refer to the fact that epithelial (Gr. epi- upon, thelio- grows) tissues grow
upon other tissues.
Location:
The epithelial tissues occur on external and internal exposed surfaces of the body parts
where they form protective covering.
Origin:
Epithelial tissues evolved first and are also formed first in the embryo. The epithelial
tissues arise from all the three primary germ layers: ectoderm, mesoderm and
endoderm, of the embryo. For example the epidermis of the skin from the ectoderm,
coelomic epithelium from the mesoderm and epithelial lining of alimentary canal (= gut)
from the endoderm.
Features:
Epithelial tissues consist of variously shaped cells closely arranged in one or more
layers. There is little intercellular material between the cells. The cells are held together

by intercellular junctions. The epithelial tissues usually rest on a thin non-cellular

basement membrane.
Usually blood vessels are absent in epithelial tissues. However the underlying
connective tissues are generally well supplied with blood vessels. Nutrients enter
epithelial tissues from the underlying connective tissues by diffusing through the
basement membrane.
Nerve endings may penetrate the epithelial tissues. The epithelial tissues have a good
power of repair (regeneration) after injury. A specialized epithelium, the stria vascularis
of the cochlea of internal ear has blood capillaries within the thickness of the
epithelium.
Basement Membrane:
The epithelial tissues usually lie on the non-cellular basement membrane which
consists of two layers:
(i) Basal Lamina:
It is outer thin layer (near the epithelial cells), composed of mucopolysaccharides and
glycoproteins, both secreted by epithelial cells. It is visible only with the electron
microscope.
(ii) Fibrous or Reticular Lamina:

It is inner thick layer, composed of collagen or reticular fibres of the underlying
connective tissue. It is visible with light microscope. The basement membrane provides
elastic support. It also allows selective chemical exchange between epithelial tissues
and surrounding blood vessels.
Types of Epithelia Tissue:

On the basis of arrangement and shape of the cells, the epithelia are classified into two
groups: simple epithelia and compound epithelia.
A. Simple Epithelia (Uni-layered Epithelia):

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

1. Simple Squamous Epithelium (Pavement Epithelium):

Structure:
It is composed of large flat cells whose edges fit closely together like the tiles in a floor;
hence it is also called pavement epithelium. The cells rest on a thin basement
membrane. If the cells are seen from the surface, they seem to be polygonal in shape.
The nuclei of the cells are flattened and often lie at the centre of the cells and cause
bulging’s of cell surface (Fried-egg appearance).
Location:
This epithelium is present in the terminal bronchioles and alveoli of the lungs, wall of
the Bowman’s capsules and descending limbs of loops of Henle of the nephrons of the
kidneys, membranous labyrinth (internal ear), blood vessels, lymph vessels, heart,
coelomic cavities, and rete testis of the testis.
In the blood vessels, lymph vessels and heart it is called endothelium. In the coelom, it is
called mesothelium. The cells of endothelium and mesothelium may become wavy;
hence these epithelia are called tessellated. The skin castings of frog are cast off in single
layers, hence are treated as squamous epithelium.
Function:
Protection, excretion, gas exchange and secretion of coelomic fluid.
2. Simple Cuboidal Epithelium (cubical epithelium):

Structure:
As the name indicates the cells are cubical and more or less square in shape. In surface
view, the cells look like polygonal. The nuclei are rounded and lie in the centre of the
cells. The cells of cuboidal epithelium often form microvilli on their free surface.
This gives a brush-like appearance to their free border called brush bordered cuboidal
epithelium. Microvilli increase absorptive surface area. In ovaries and seminiferous
tubules of the testes it is called germinal epithelium because it produces gametes (ova
and sperms respectively).
Location:
The cuboidal epithelium is present in the small salivary and pancreatic ducts, thyroid
follicles, parts of membranous labyrinth, proximal and distal convoluted tubules of the
nephrons of kidneys, ovaries, seminiferous tubules of testes, and ciliary bodies, choroid
and iris of eyes, other sites of cuboidal epithelium are the inner surface of the lens, the
pigment cell layer of the retina of the eye and sweat glands of mammalian skin.
Function: Protection, secretion, absorption, excretion, gamete formation.
3. Simple Columnar Epithelium:

Structure:
The cells are elongated and placed side by side like column. The outer free surface of
each cell is slightly broader. The nuclei are somewhat elongated along the long axis of
the cells. Nuclei lie near the bases of the cells. Certain cells of this epithelium contain
mucus (a slimy substance) and are called goblet (or mucous) cells as they look like
goblet.

The epithelium containing mucus secreting cells, along with the under lying supporting
connective tissue is called mucosa or mucous membrane. The latter is present in the
stomach and intestine. The intestinal mucosa (= mucous membrane) has microvilli to
increase the absorptive surface area and is called brush-bordered columnar epithelium
which is highly absorptive.
Location:
It lines the stomach, intestine, gall bladder and bile duct. It also forms the gastric glands,
intestinal glands and pancreatic lobules (present in the pancreas) where it has secretory
role and is called glandular epithelium.
Function: Protection, secretion and absorption.
4. Simple Ciliated Epithelium:

Structure:
The cells bear numerous delicate hair like outgrowths, the cilia, arising from basal
granules. Mucus secreting goblet cells also occur in the ciliated epithelium. The cilia
remain in rhythmic motion and create a current to transport the materials which come
in contact with them.
The ciliated epithelium is of two types:

(i) Ciliated Columnar Epithelium:
It comprises columnar cells which have cilia on the free surface. They consists of about
15 – 25 hair like structures on the cell which are known as cilia. In some cells, cilia are
not present and they are known as non-ciliated. This epithelium lines most of the
respiratory tract (trachea and bronchioles) and Fallopian tubes (oviducts).
It also lines the ventricles of the brain and the central canal of the spinal cord. It is also
present in tympanic cavity of middle ear and auditory tube (Eustachian tube). Ciliary
movement helps in the flow of mucus, suspended particles and bacteria.
(ii) Ciliated Cuboidal Epithelium:

It consists of cubical cells which have cilia on the free surface. It occurs in certain parts
of nephrons of the kidneys.
Functions of Ciliated Epithelium:

The main function of ciliary movement is to maintain a flow of mucus or liquid or
suspended particles or bodies constantly in one direction. In the respiratory tract the
cilia help to push mucus towards the pharynx (throat). In the oviducts the cilia help to
move an egg towards the uterus.
The cilia of the ventricles (cavities) of the brain and central canal of the spinal cord help
to maintain the circulation of cerebrospinal fluid present there. In the nephrons of the
kidneys, cilia keep the urine moving.
5. Pseudo-stratified Epithelium:
Structure:
The cells are columnar, but unequal in size. The long cells extend up to free surface. The
short cells do not reach the outer free surface. The long cells have oval nuclei; however,
short cells have rounded nuclei. Mucus secreting goblet cells also occur in this
epithelium.

Although epithelium is one cell thick, yet it appears to be multi-layered which is due to
the fact that the nuclei lie at different levels in different cells. Hence, it is called pseudo
stratified epithelium.
It is of two types:
(i) Pseudo stratified Columnar Epithelium:
It consists of columnar cells. It occurs in the large ducts of certain glands such as parotid
salivary glands and the urethra of the human male. It is also present in the olfactory
mucosa.
(ii) Pseudo stratified Columnar Ciliated Epithelium:

It consists of columnar cells. The long cells have cilia at their free surface; however, the
short cells are without cilia. This epithelium occurs in the trachea and large bronchi. The
movements of the cilia propel the mucus and foreign particles towards the larynx.
Functions of Pseudo stratified Epithelium: Protection, secretion, movement of secre-
tions from glands, urine and semen in the male urethra and mucus loaded with dust
particles and bacteria from the trachea towards the larynx.
B. Compound Epithelia (Multi-layered Epithelia):

These are made up of more than one layer of cells. The compound epithelia may be
stratified and transitional.
1. Stratified Epithelium:
It has many layers of epithelial cells in which the deepest layer is made up of columnar
or cuboidal cells. This epithelium is classified on the basis of the shape of the cells
present in the superficial layers.
It is of four types:
(i) Stratified Squamous Epithelium:
The cells in the deepest (= basal) layer are columnar or cuboidal with oval nuclei. It is
called germinative layer (= stratum germinativum or stratum Malpighi). The cells of this
layer divide by mitosis to form new cells. The new cells gradually shift outward.
In the middle layers the cells become polyhedral with rounded nuclei. These are called
intermediate layers. The superficial layers are flat with transversely elongated nuclei.
These layers are called squamous layers.
It is of two types:
(a) Keratinized Stratified Squamous Epithelium:
In the outer few layers, the cells replace their cytoplasm with a hard, water proof
protein, the keratin. The process is called keratinization. These layers of dead cells are
called stratum corneum or horny layer. The deeper layers have living polygonal cells.
The keratin is impermeable to water and is also resistant to mechanical abrasion
(scraping). The horny layer is shed at intervals due to friction. This epithelium occurs in
the epidermis of the skin of land vertebrates.
(b) Non-keratinized Stratified Squamous Epithelium:

As the name indicates, it does not have keratin. It is unable to check water loss and
provides only moderate protection against abrasion (scraping). This epithelium occurs
in the oral cavity (buccal cavity), tongue, pharynx, oesophagus, middle part of anal

canal, lower parts of urethra, vocal cords, vagina, cervix (lower part of uterus),
conjunctiva, inner surface of eye lids and cornea of eye.
(ii) Stratified Cuboidal Epithelium:

It has outer layer of cuboidal cells and basal layer of columnar cells. It forms the
epidermis of fishes and many urodels (tailed amphibians such as salamanders). It also
lines the sweat gland ducts and larger salivary and pancreatic ducts.
(iii) Stratified Columnar Epithelium:

It has columnar cells in both superficial and basal layers. It covers the epiglottis and
lines mammary gland ducts and parts of urethra.
(iv) Stratified Ciliated Columnar Epithelium:

Its outer layer consists of ciliated columnar cells and basal layer of columnar cells. It
lines the larynx and upper part of the soft palate.
2. Transitional Epithelium (= Urothelium):

Structure:
This epithelium consists of 4 to 6 layers of cells. The cells of deepest (= basal) layer are
columnar or cuboidal. The cells of middle layer are polyhedral or pear shaped. The cells
of the surface layer are large and globular or umbrella shaped. When this epithelium is
stretched all the cells become flattened.
Location:
This epithelium is found in the renal calyces, renal pelvis, ureters, urinary bladder and
part of the urethra. Because of its distribution, transitional epithelium is also called
Urothelium (epithelium present in the urinary system).
Function:
It permits distension. The transitional epithelium of the urinary bladder can be
stretched considerably without being damaged. When stretched it appears to be thinner
and the cells become flattened or rounded. Thus this epithelium is stretched or relaxed.
It is also protective in function. The cells of the basal layer of the transitional epithelium
show occasional mitosis, but it is much less frequent than that of stratified squamous
epithelium, as there is normally little erosion of the surface.
Glandular Epithelium:
A gland may consist of a single cell or a group of cells.
They are specialised cells that secrete substances into ducts.
The glands are classified into endocrine or exocrine glands on the basis of their
secretions.
Functions of Epithelial Tissues:

(i) Protection:
They protect the underlying tissues from:
(a) Mechanical injury
(b) Entry of germs (infection)
(c) Harmful chemicals and

(d) Drying up.
(ii) Selective Barriers:

Epithelia check the absorption of harmful or unnecessary materials.
(iii) Absorption:
Epithelium of uriniferous tubules (nephrons), stomach and intestine is absorptive.
(iv)Conduction:
Ciliated epithelia (e.g., those of respiratory and genital tracts) serve to conduct mucus or
other fluids in the ducts they line.
(v) Excretion:
The epithelium of uriniferous tubules is specialized for urine formation for excretion.
(vi) Sensation:
Sensory epithelia of sense organs (e.g., olfactory epithelium, etc.) help to receive various
stimuli from the atmosphere and convey them to the brain.
(vii) Regeneration:
When epithelia are injured, they regenerate more rapidly than other tissues, and thus
facilitate rapid healing of wounds.
(viii) Exchange of Gases:

Epithelium of alveoli of the lungs brings about exchange of gases between blood and air.
(ix) Pigmentation:
Pigmented epithelium of the retina darkens the cavity of eyeball.
(x) Secretion:
Epithelium also forms glands that secrete secretions such as mucus, gastric juice and
intestinal juice.
(xi) Reproduction:
Germinal epithelium of the ovaries and somniferous tubules of the testes produce ova
and sperms respectively.
(xii) Exoskeleton:
Epithelium also produces exo-skeletal structures such as scales, feathers, hair, nails,
claws, horns and hoofs.
Connective Tissue
Connective Tissues: Most abundant, widespread and varied of all tissue types in the
body. Responsible for providing and maintaining form in the body.
Histogenesis: Mesodermal in origin
General Characteristics: Unlike epithelium, connective tissues do not have a free

surface. However, on a microscope slide you may see open spaces around the
connective tissues. They are made up of fewer cells that are set far apart.

Connective tissue generally consists of a considerable amount of extracellular

material called matrix (supported by abundant intercellular substance). Usually there is
more extracellular matrix than cellular material. The matrix is composed of varying
types of fluids and proteins and fibers. They also contain connective tissue fibers. The
different contents of intercellular substance, Connective Tissue cells and fibers account
for the difference in appearance of the various connective tissues. Examples of cells
found in various connective tissues. Cartilage cells or “Chondrocytes” Bone Cells or
“Osteocytes” Tendon cells or “Fibroblasts”.
General Functions
o Provide a matrix that serves to connect and bind the cells and organs
o Give mechanical support to the body
o Storage of fat and certain minerals like calcium in the bones
o Exchange of metabolites between blood and tissues
o Significant role in the repair and healing of wounds
o For protection against infection
Classification of Connective tissue
Composition of Connective Tissues

Connective tissue cells Connective
tissue fibers Intercellular or
Ground substance
Blood vessels – except in Mucous Connective Tissue and in cartilage
Loose (Areolar) Connective Tissue

This type of connective tissue cushions organs and underlies epithelia. It is
characterized by having cells that produce fibers (fibroblasts) and macrophages as well
as collagen, reticular, and elastic fibers in the matrix.
Dense Connective Tissue

There are two types of dense connective tissue proper. They are dense regular
connective tissue and dense irregular connective tissue. Both types contain an
extensive matrix full of collagen fibers. These fibers are produced by fibroblasts in the
tissue. The major difference between the two tissues, besides where they are found, is
the direction in which the fibers run.

1. Dense Regular Connective Tissue -- Found in tendons and ligaments and it is

dense with collagen fibers that run parallel to one another (thus the name). This
design allows the tissue to resist tension pulling in one direction.
a) White fibrous tissue: made up of white shining thin fibres that are non-
branching. Fibres are present in bundles. This tissue is made up of protein
known as collagen.
Location: tendon, ligaments of limbs.
b) Yellow elastic tissue: made up of fibres that are thicker, branched & yellow in
colour. It helps in binding various parts together & also helps in movement of the
organ.
Location: Bronchi, larynx, lungs, arteries
2. Dense Irregular Connective Tissue -- Found in joint capsules, the dermis of the
skin and in hollow organs on the digestive tract. It is dense with collagen fibers
that run in many different directions. This design allows the tissue to resist
tension pulling from numerous directions.
Cartilage
Cartilage is a form of connective tissue that has an extensive matrix surrounding
the cartilage producing cells (chondroblasts or chondrocytes). These cells are trapped
inside of small spaces called lacunae (little lakes).
The major difference in identifying the different cartilage types is found in the
visible components of the matrix. Hyaline cartilage lacks any visible fibers in the matrix,
where elastic cartilage looks similar to hyaline cartilage, but it has elastic fibers in the
matrix. Fibrocartilage is very dense with thick collagen fibers throughout the matrix.
1. Elastic Cartilage
Provide support with flexibility
Yellow in colour & contains many elastic fibres. It differs from hyaline cartilage only for
the presence of its enormous elastic fibres in the matrix. The matrix in addition to the
elastic fibres contains collagen fibres.
Rigid but elastic
Location: External ears (pinna), Eustachian tube, epiglottis & in some laryngeal
cartilages.
2. Hyaline cartilage
Hyaline means glass and in fresh condition it appears as a translucent bluish-white
mass. Cartilage cells or chondrocytes occupy small empty spaces called lacunae in the
matrix. Matrix is solid and smooth.
The nucleus is large and it’s provided with two or more nucleoli. When the cells are
arranged in group of two, four etc. The cytoplasm is very rich in glycogen.
Location: on the surface of the parts of the bones which forms joints.Forms the costal
cartilages which attach the ribs to the sternum.Forms parts of larynx, trachea and
bronchi.
Function: covers and protects joints of body, Involved in growth that increase bone
length. Strong support & some flexibility.
3. Fibro cartilage
Cells are large, arranged in groups and placed inside lacunae.
The collagen fibres are more than that found in hyaline cartilage.
Location: in intervertebral discs, menisci of knee joint, mandibular joints, pubic
symphysis.

Bone
Bone is perhaps one of the easiest connective tissues to identify. The matrix in bone is
made up of calcium salts that have been deposited on collagen fibers. The result of this
deposition in dense bone is the formation of layers of matrix often in concentric rings
(part of a structure called the osteon). Like cartilage, bone has lacunae. In bone,
however, the lacunae (small dark structures at high magnification) contain osteocytes.
Blood
It would seem that blood should not be connective tissue, but it has the same
characteristics as the other types of connective tissue: it comes from the same
embryonic tissue and it contains considerable extracellular matrix (blood plasma). The
most abundant cells in blood are erythrocytes (red blood cells) and the darker cells on
the slide are leukocytes (white blood cells).
Adipose tissue
Adipose is the tissue that stores lipids. Commonly known as fat. It is characterized by a
large internal fat droplet, which distends the cell so that the cytoplasm is reduced to a
thin layer and the nucleus is displaced to the edge of the cell. These cells may appear
singly but are more often present in groups. When they accumulate in large numbers,
they become the predominant cell type and form adipose (fat) tissue.
Function: storage site, regulate body temperature, protect certain organs & regions of
the body.
Lymphoid tissue
Contains lymph, lymph glands, lymphatic vessels etc.
Distribution: Peyer’s patches in small intestine (ileum), lymph glands, pharyngeal
tonsils etc.
Reticular tissue
Cells of this tissue form a part of the reticulo-endothelial system.
Distribution: found in spleen, liver, lymph glands, bone marrow etc.
Teacher’s sign: _

EXPERIMENT NO.:3
Aim: Microscopic study of muscular and nervous tissue.
Reference:
1. Dr. R. K. Goyal, Dr. N. M. Patel
13th Ed.2009
Page No.:106-108
2. Dr. M.Prashad, Dr. Kumar Jha
Practical Book of Human Anatomy & Physiology I
Nirali Prakashan
Page No.:3.1-3.4
Theory:
Muscle
Origin:
Most of muscle tissue develops from mesoderm that gives rise to mesenchymal cells.
Skeletal muscle develops from paraxial mesoderm, organized into myotomes in
somites. Muscles of the head develop from mesenchyme of brachial arches. Cardiac
muscle develops from cardiogenic mesoderm. Smooth muscle develops from splanchnic
mesoderm - except of iris where smooth muscle arises from neuroectoderm.
Muscle function:
1. Contraction for locomotion and skeletal movement
2. Contraction for propulsion
3. Contraction for pressure regulation
Muscle classification:
Muscle tissue is excitable and contractile (it shortens forcefully).muscle tissue may be
classified according to a morphological classification or a functional classification.
Morphological classification (based on structure):

There are two types of muscle based on the morphological classification system:
1. Striated
2. Non striated or smooth.
Functional classification:
There are two types of muscle based on a functional classification system:
1. Voluntary
2. Involuntary.
Types of muscle:
There are generally considered to be three types of muscle in the human body.
Skeletal muscle: striated and voluntary
Cardiac muscle: striated and involuntary
Smooth muscle: non-striated and involuntary
Skeletal Muscle:
A typical muscle cell is a highly modified, giant, multi-nucleated cell.

Characteristics of skeletal muscle:

The cells (running vertically) are long, straight, striated and multinucleated.
Skeletal muscle cells are elongated or tubular (cylindrical). The individual cells can be
as long as the muscle itself. As the muscle length increases, the individual cells get ready
to divide, double their cytoplasmic contents including nucleus, but they do not complete
cytokinesis. They have multiple nuclei and these nuclei are located on the periphery of
the cell, situated just below the sarcolemma.
Skeletal muscle has an alternating pattern of light and darks band results from
the arrangement of myofibrils, small protein contractile units embedded in the
sarcoplasm which gives “cross-striped” or striated appearance to the cell.
Function:
Contraction - voluntary and rapid body movement, muscle tissue in the tongue
(speech, mixing of food), breathing, voice
Cardiac Muscle:
The cells are much more branching than skeletal muscle, but are still
striated. The key feature is the dark lines that run across the tissue between cells. These
are intercalated discs and are only found in cardiac muscle.
Contraction - involuntary; rapid and rhythmic- in the heart (myocardium)
Characteristics:
Cardiac muscle cells are not as long as skeletal muscles cells and often are
branched cells. Cardiac muscle cells may be mononucleated or binucleated. In either
case the nuclei are located centrally in the cell. Cardiac muscle is also striated. In
addition cardiac muscle contains intercalated discs (connecting adjacent cardiac muscle
cells are specialized cellular junctions called intercalated discs which are darker pink
than the striations seen throughout the cytoplasm of the cardiac muscle cell).
Cardiac muscle cells – 3 types
Contractile cells
Impulse generating and conducting cells (initiate heart beat)
Myoendocrine cells (production of hormone for regulation of: Na+, K + & water balance)
Smooth Muscle:
The striations are not present (thus the name) and that the cells are flattened
and stretched out (spindle shaped).
Function:
Contraction is involuntary; weak and slow - in the wall of hollow organs
(stomach, small intestine).
Characteristics of Smooth muscle:
Smooth muscle cell are described as spindle shaped. That is they are wide in the
middle and narrow to almost a point at both ends. Smooth muscle cells are not nearly as
long as skeletal muscle fibers (cells). Smooth muscle cells have a single centrally located
nucleus. Oval or rod shaped nuclei in the centre. Nuclei stained darker within individual
smooth muscle cell cytoplasm.
Smooth muscle cells (cytoplasm) do not have visible striations although they do
contain the same contractile proteins as skeletal and cardiac muscle, these proteins are
just laid out in a different pattern.
Muscle terminology
myofibre or myocyte: a muscle cell

Sarcolemma: the plasma membrane of a muscle cell

Sarcoplasm: the cytoplasm of the muscle cell
Sarcoplasmic reticulum: the endoplasmic reticulum of a muscle cell
Sarcosome: the mitochondria of a muscle cell
Sarcomere: the contractile or functional unit of muscle
Nervous Tissue
Nervous tissue is composed of neurons and their supporting cells (glial cells or
neuroglia). Neurons (Nerve cells) are specialised cells that conduct electrical impulses
called an action potential across its length.
All neurons have the same basic structure:
Dendrites extend from the cell body (dendron - Greek for tree). These are fairly
short, with lots of branches, and they are the points at which nerve impulses
are received by the cell.
The cell body(perikaryon): includes cytoplasm & nucleus. Most of the cell bodies
of neurons are in the central nervous system (brain and spinal cord), or in the
ganglia (which lie just outside the spinal cord) of the peripheral nervous system.
Nucleus: The nuclei are large, highly visible, & often you will find the nucleolus within
the nucleus that looks like an ‘owl’s eye’.
The axon: a single nerve 'fibre' which transmits impulses to the distal end. Axons can be
very long - around 1 metre, and vary in diameter from 0.2 to 20 µm. It is the largest
process attached to the neuron’s cell body.
Axons in the periphery are never "naked." They always have some form of
covering, which is the product of the activities of the Schwann cell (or, in the central
nervous system, the oligodendrocyte).Two forms of this "insulation" exist. The first type
is a very elaborate multi-layered covering of plasma membrane, a thick and efficient
barrier to charge leakage, easily seen with the light microscope. This is a myelin
sheath. The myelin sheath is made by Schwann cells in the peripheral nervous system
and by oligodendrocytes in the central nervous system. Axons with such a covering are
referred to as myelinated. In axons of equal size the rate of conduction of a signal is
much higher when there's a myelin sheath. Myelination prevents leakage of membrane
charge into the surrounding intercellular space.
People often use the terms "neuron" and "nerve" interchangeably, but this is
incorrect. A "nerve" is a grossly visible anatomic structure, and is a bundle of axonal
processes from many different neurons, wrapped in a connective tissue sheath. A
"neuron" is a cell, one part of which is included in a nerve.
There are three basic shapes to the neurons:

Bipolar (single axon and single dendrite) - special neurons in the sensory pathways for
sight, smell and balance.
Pseudo-unipolar (single axon and dendrite arise from a common stem) - the primary
general sensory neurons are usually pseudo-unipolar.
Multipolar (the commonest) - most motor neurons are multipolar.
Teacher’s sign:

EXPERIMENT NO.:4
Aim: Identification of axial bones.
13th Ed.2009
Page No.:68-72
SKELETAL SYSTEM
The human skeleton consists of approximately 206 individual bones. The whole
skeleton can be divided into two parts:
Axial skeleton: which includes skull, vertebral column, ribs and sternum.
Appendicular skeleton: which includes the bones of upper and lower limbs.
THE AXIAL SKELETON
The skull: It is a large bony structure consisting of the cranium and facial bones
attached to the cranium.

The cranium: The cranium is a large and hollow bony case, formed by fusion of
different bones by zigzag edges. It accommodates the brain and its meninges, cerebral
vessels.
The cranium (the brain box) consists of eight bones:

Frontal (1)
Occipital (1)
Parietals (2)
Temporal (2)
Sphenoid (1)
Ethmoid (1)
(Mostly flat bones Meant for protection of brain)
They are joined together by means of irregular, and saw like edges called sutures.
The occipital bone forms the back and a part of the base of the skull. Its lower
part contains a large circular opening known as ‘Foramen Magnum’ through which
spinal cord continues downward. The upper edge of the occipital bone is joined with the
parietal bones. Parietal bones form the side walls and also the greater part of the roof of
the cranium. The frontal bone is in the front central portion of the cranium. It is joined
with two parietal bones. There are two cavities known as ‘frontal sinuses’ in the frontal
bone. Sagittal suture separates two parietal bones. Coronal suture separates frontal
bone from parietal bone. Lambdoid suture separates parietal bone from occipital bone.
Skull also consist the orbits which lodges eye-balls in it. The roofs of the orbits
are formed by the frontal bone. The temporal bones are situated in the lateral sides,
attached to the sphenoid bone in front, the parietal bones above and the occipital bone
behind. At the base of the skull, in front of the occipital bone, there is sphenoid bone.
The Ethmoid bone is cubical in shape. It fills the space between the orbits.

The facial bones:

They form the lower part of the front of the skull.
There are fourteen facial bones which include:
Maxillae (2)
Mandible (1)
Nasal (2)
Palatine (2)
Zygomatic (2)
Lacrimal (2)
Vomer (1)
Inferior turbinate (2)
(Mostly irregular bones give shape to the face)
The maxilla forms the upper jaw and the major portion of the palate. The upper
set of teeth are fixed in it. Vomer is a thin vertical bone which separates the nasal cavity.
The zygomatic bones or cheek bones are two most prominent bones of the cheeks.
Mandible or the lower jaw is the largest bone of the face which fixes in its respective
sockets, the lower set of teeth. It is the only movable bone of the skull.

The vertebral column (the back bone):

It consists of series of vertebrae and forms the axis to which other bones of the
skeleton are connected. There are 33 vertebrae amongst which upper 24 are movable.
All bones are irregular shaped; intervertebral discs are present between each vertebra.
The first 7 vertebrae of the neck are known as ‘Cervical vertebrae’. The next 12 of the
chest region are known as ‘Thoracic vertebrae’. The remaining 5 vertebrae of the loin
region (below rib cage, above pelvic region) are known as ‘’Lumbar vertebrae’’. The
lowest 5 fused vertebrae form a wedge shape structure known as ‘’sacrum’’. The end
portion of the vertebral column is known as ‘’coccyx’’ in which 4 vertebrae are fused
together. The coccyx represents remnant of the tail of the monkey in man. This indicates
that man has evolved from the monkey.
Functions of vertebral column:

1. It effectively protects the spinal cord from shock, accidents, jerks and injuries.
2. Pairs of spinal nerves emerge from the spinal cord through the vertebral column.
3. It gives attachment to ribs, and a large group of muscles of the neck, chest and back.
Bones of thorax
The thorax is formed behind by the thoracic or dorsal vertebrae, anteriorly by the
sternum and costal cartilages and the remainder of the circumference by the ribs.

The Ribs: There are twelve pairs of ribs bilaterally situated. They are curved, flat, strip
shaped bones joined at the back with the thoracic vertebrae. Ribs move with every
breath of respiration. Thoracic cage protects the organs of the chest, especially the heart
and the lungs.
The last five pairs are called ‘’false ribs’’ and of these, the eleventh and the
twelfth pairs are termed as ‘’floating ribs’’. During respiration, maximum movement is
found in the lower part of the thorax.
Diaphragm also serves as the musculotendinous partition between the thorax
and abdomen which contracts during inspiration and relaxes during expiration.
Teacher’s sign:

EXPERIMENT NO.:5
Aim: Identification of appendicular bones.
13th Ed.2009
Page No.:73-76
THE APPENDICULAR SKELETON
The appendicular skeleton consists of bones of two upper limbs and two lower limbs.
The upper limb: It consist of bones of shoulder, upper arm, fore arm, wrist and fingers.
Clavicles and scapula form the pectoral or shoulder girdle. Humerus is the bone of the
upper arm. Ulna and radius are two parallel bones of the fore-arm. There are eight

carpals or wrist bones. They are followed by five metacarpal bones. There are
fourteen phalanges. Metacarpal bones are the bones of the palm.
The Lower limb: The bones forming the lower limb are the hip bones, femur (the
thigh bone), patella (the knee-cap), tibia (the skin bone), fibula (the splint bone),
tarsals (the ankle bones), metatarsals (the instep bones) and phalanges (the bones of
toes)
The Femur: It is the thigh bone which resembles somewhat with the humerus of the
upper arm. Femur is the longest and the strongest bone of the body.
The Appendicular Skeleton

• Pectoral girdle attaches the upper limbs to the trunk.
• Pelvic girdle attaches the lower limbs to the trunk.
• Upper and lower limbs differ in function – share the same structural plan.
The Pectoral Girdle

• Consists of the clavicle and the scapula

• Pectoral girdles do not quite encircle the body completely
• Medial end of each clavicle articulates with the manubrium and first rib
• Laterally – the ends of the clavicles join the scapulae
• Scapulae do not join each other or the axial skeleton
• Provides attachment for many muscles that move the upper limb
• Girdle is very light and upper limbs are mobile
• Only clavicle articulates with the axial skeleton
• Socket of the shoulder joint (glenoid cavity) is shallow
• Good for flexibility – bad for stability
Articulated Pectoral Girdle

Clavicles
• Extend horizontally across the superior thorax
• Sternal end articulates with the manubrium
• Acromial end articulates with scapula
• Provide attachment for muscles
• Hold the scapulae and arms laterally
• Transmit compression forces from the upper limbs to the axial skeleton
Scapulae
• Lie on the dorsal surface of the rib cage
• Located between ribs 2-7
• Have three borders
• Superior, medial (vertebral), and lateral (axillary)
• Have three angles
• Lateral, superior, and inferior
Forearm
• Radius and ulna articulate with each other
• At the proximal and distal radioulnar joints
• Interconnected by a ligament – the interosseous membrane
• In anatomical position, the radius is lateral and the ulna is medial
Ulna
• Main bone responsible for forming the elbow joint with the humerus
• Hinge joint allows forearm to bend on arm
• Distal end is separated from carpals by fibrocartilage
• Plays little to no role in hand movement
Radius
• Superior surface of the head of the radius articulates with the capitulum
• Medially – the head of the radius articulates with the radial notch of the ulna
• Contributes heavily to the wrist joint
• Distal radius articulates with carpal bones
• When radius moves, the hand moves with it
Hand
• Includes the following bones

• Carpus – wrist
• Forms the true wrist – the proximal region of the hand
• Gliding movements occur between carpals
• Composed of eight marble-sized bones
• Metacarpals – palm
• Phalanges – fingers
Pelvic Girdle
• Attaches lower limbs to the spine
• Supports visceral organs
• Attaches to the axial skeleton by strong ligaments
• Acetabulum is a deep cup that holds the head of the femur
• Lower limbs have less freedom of movement
• Are more stable than the arm
• Consists of paired hip bones (coxal bones)
• Hip bones unite anteriorly with each other
• Articulates posteriorly with the sacrum
Bony Pelvis
• A deep, basin-like structure
• Formed by coxal bones, sacrum, and coccyx
True and False Pelvis • Bony pelvis is divided into two regions
• False (greater) pelvis – bounded by alae of the iliac bones
• True (lesser) pelvis – inferior to pelvic brim
• Forms a bowl containing the pelvic organs
Pelvic Structures and Childbearing

• Major differences between male and female pelvis
• Female pelvis is adapted for childbearing
• Pelvis is lighter, wider, and shallower than in the male
• Provides more room in the true pelvis
Thigh
• The region of the lower limb between the hip and the knee
• Femur – the single bone of the thigh
• Longest and strongest bone of the body
• Ball-shaped head articulates with the acetabulum
Patella
• Triangular sesamoid bone
• Imbedded in the tendon that secures the quadriceps muscles
• Protects the knee anteriorly
• Improves leverage of the thigh muscles across the knee
The Foot
• Foot is composed of: Tarsus, metatarsus, and the phalanges
• Important functions
• Supports body weight
• Acts as a lever to propel body forward when walking

• Segmentation makes foot pliable and adapted to uneven ground
Tarsus
• Makes up the posterior half of the foot
• Contains seven bones called tarsals
• Body weight is primarily borne by the talus and calcaneus
Metatarsus
• Consists of five small long bones called metatarsals
• Numbered 1–5 beginning with the hallux (great toe)
• First metatarsal supports body weight
Phalanges of the Toes

• 14 phalanges of the toes
• Smaller and less nimble than those of the fingers
• Structure and arrangement are similar to phalanges of fingers
• Except for the great toe, each toe has three phalanges: Proximal, middle & distal
Arches of the Foot

• Foot has three important arches
• Medial and lateral longitudinal arch
• Transverse arch
• Arches are maintained by:
• Interlocking shapes of tarsals
• Ligaments and tendons
Disorders of the Appendicular Skeleton

• Bone fractures
• Hip dysplasia – head of the femur slips out of acetabulum
• Clubfoot – soles of the feet turn medially
The Appendicular Skeleton throughout Life

• Growth of the appendicular skeleton
-Increases height
-Changes body proportions
-Upper-lower body ratio changes with age
-At birth head and trunk are 1.5 times as long as lower limbs
-Lower limbs grow faster than the trunk
-Upper-lower body ratio of 1 to 1 by about age 10
Teacher’s sign:

EXPERIMENT NO.: 6
Aim: Introduction to haemocytometry.
13th Ed.2009
Page No.:6-10
Theory:
The counting of cells (Red blood corpuscles or White blood cells) in blood using
haemocytometer 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.
Haemocytometer set which is used for counting the number of cells in blood
consist of (1) dilution pipettes, (2) counting chamber (named as Thomas or Neubauer’s
counting chamber), and (3) special coverslip (also known as Thomas coverslip).
The dilution pipette:-

It consists of four 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 long stem is divided into 10 equal parts from the tip to the
mark ‘1’ just near the bulb. The fifth division is heavily marked and labelled 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 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 white or
red bead which helps in uniform mixing of blood with dilution fluid. The bulb ends in a
short stem which have the 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 a rubber or polythene tube. At the end of the tube there is the
plastic mouth piece.
The differentiating points of the RBC and WBC dilution pipettes have been
illustrated in fig.
The counting chamber:-

It is a single piece thick glass slide having two counting areas in the central part.
The two counting areas are separated from each other and the other part of the slide by
an ‘H’ shaped groove. On the either side of these areas are the platforms which are
raised by 0.1 mm above the counting areas. On these platforms rests special coverslip –
Thomas coverslip.
The ruling areas of chamber are the sharp contrast of bright lines against the
darker metallised background. A thin semi-transparent layer of the metal has been
deposited on the glass counting areas and the lines are ruled out through the metal
surface. Just as one cannot see the stars during the day, the ruling cannot be seen if
bright light is focused on the chamber. To adjust the chamber one must partially close the
diaphragm of the microscope.

The total ruling area of each side is 3 mm in length and 3 mm in breadth. It is

divided into nine equal squares of 1 sq. mm area (Fig.). 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 ¼ x ¼ = 1/16 sq. mm.
Each RBC square is divided by triple lines into 25 equal small RBC squares, and each of
these 25 small RBC squares are further divided into 16 smallest squares by single lining.
Thus whole central square is divided into 400 smallest squares, the area of each is 1/20
x 1/20 sq. mm.
The dilution fluids:

Various dilution fluids are used in haemocytometry 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, fixation
etc. Table 1 and 2 give the most commonly used dilution fluids for RBC and WBC count
with the importance of each constituent.

Table 1 (Hayem’s RBC dilution fluid)

Substance Amount Purpose
Sodium chloride (NaCI) 1.0 gm. Provides isotonicity,
Prevents haemolysis
Sodium sulphate (Na2SO4) 5.5 gm. Provides isotonicity,
Prevents rouleax formation
Mercuric 0.5 gm. Causes fixation of the cells,
chloride(HgCI2) Prevents bacterial growth
Water (H2O) Up to 100 ml Diluent
Table 2 (WBC dilution fluid)

Glacial acetic acid 2.0 ml Destroys RBCs
Gentian or Methyl violet 1.0 ml Stains nuclei of WBCs
(1%)
Water Up to 100 ml Diluent
SOME USEFUL HINTS:

(1) Always clean and adjust the counting chamber, coverslip, microscope and the
pipettes before pricking the finger for blood collection.
(2) 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.
(3) A knot may be given to the sucker tube after sucking the dilution fluid upto the
particular mark. The care should be taken that fluid does not come out from
either end. One end may be closed by putting finger at the tip of stem while
putting knot.
(4) Never forget to discard a few drops of fluid in the stem of the pipette because it
does not contain blood.
(5) The fluid should not enter into the grooves of slide. It it happens, due to capillary
action, it will draw cells thus giving error in counting. It is 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.
(6) The cells must be allowed to settle for a period of 2-3 minutes before starting the
actual count. For counting the number of cells, prepare a table consisting of
columns and enter the number of cells in each column.
(7) The cells touching two boundaries (preferably left and top) are to be counted
while cells touching other two boundaries (right and the bottom) not to be
counted. (Fig.)

Teacher’s sign:

EXPERIMENT NO.:7
Aim: Enumeration of white blood cells (WBC) count.
13th Ed.2009
Page No.:11-13
Requirements:
(a) Haemocytometer set which consists of Neubauer’s counting chamber and a WBC
dilution pipette (b) Thomas coverslip (c) Microscope (d) WBC dilution fluid (e) Pricking
needle (f) Absorbent cotton (g) 70% alcohol (h) Xylol to clean glass slide and coverslip.
Composition of dilution fluid: WBC dilution fluid

Glacial acetic acid 2.0 ml Destroys RBCs
Gentian or Methyl violet 1.0 ml Stains nuclei of WBCs
(1%)
Water Up to 100 ml Diluent
Principle:
WBC present in plasma takes part in body defence against invading micro-
organisms. They are produced from the pluripotent stem cell in the bone marrow in
adults. In case of foetus haemopoiesis occurs in liver and spleen.
A sample of whole blood is mixed with a weak acid solution that lyses non-
nucleated red blood cells. Following adequate mixing, the specimen is introduced into a
counting chamber where the WBCs in a diluted volume are counted. WBCs, cells should
be stained and fixed with the Turk solution, diluting the sample10 times. The diluted
blood is placed in a capillary space of known capacity in between a special ruled slide
(counting chamber) and a coverslip. The cells thus spread out in a single layer in this
space and the number of cells can be counted under low 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:
(1) The Thomas coverslip, counting chamber and lenses of microscope are cleaned with
the help of xylol and then absorbent cotton.
(2) The counting chamber is adjusted and observed under low power of microscope
keeping the Thomas coverslip resting on the platforms of the slide.
(3) WBC 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.
(4) The first drop of blood is discarded and the second drop is sucked in the WBC
pipette up to the mark 0.5. Immediately the dilution fluid sucked exactly up to mark
11.0. When it is filled up to the mark, pipette is brought to a horizontal position and the
finger is placed over the tip of pipette. The simple knot is given to rubber tube.
(5) The pipette is rolled between the palms to mix the blood with dilution fluid.

(6) Few drops (2-3) are discarded and then the pipette is held at an angle of 45º to the
surface of counting chamber and tip is applied to narrow slit between the counting
chamber and the coverslip. A drop is allowed to come out 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.
(7) The fluid is allowed to settle for three minutes on the stage of microscope.
(8) The WBC chamber is located and WBCs in 16 smallest squares of WBC are counted.
They are counted in 4 squares chambers such.
Normal ranges:
Age Normal WBC Count (Cells/mm3 of
Blood)
At birth 10,000-25,000
Infant 8,000-15,000
4-7 years 6,000-15,000
8-18 years 4,500-13,500
Adults 4,000-10,000
Pregnancy 12,000-15,000
Clinical significance of total leukocyte count:

Increase in total leukocyte count of more than 11,000/cu mm (µl) is known as
leucocytosis and decrease of less than 4000 cu mm (µl) as leukopenia.
Leukaemia is malignant (cancerous) disease in which total WBC count goes beyond
100000/ cu mm. premature cells are increased in blood in large number. These cells are
functionless.
Causes of leucocytosis:
I. It is common for a transient period in infections(bacterial, protozoal(malaria), or
parasitic)
II. Leucytosis is also observed in severe haemorrhage and in leukaemia
III. High temperature
IV. Severe pain
V. Accidental brain damage
Cause of Leucopenia:
i. Certain viral(hepatitis, influenza, measles, etc.) & bacterial (typhoid, paratyphoid,
tuberculosis, etc.) infections
ii. Primary bone marrow depression (aplastic anaemia)
iii. Secondary bone marrow depression (due to drugs, radiation etc)
iv. Anaemia (iron deficiency megaloblastic etc.)
Calculation:
Total number of WBC in 4 small squares (64 smallest WBC squares) = N
Length of the small WBC square = 1 mm
Breadth of the small WBC square = 1 mm
Therefore, Area of the small WBC square = 1 x 1 = 1 sq. mm.
Volume of fluid in small WBC square = 1 x 1/10 = 0.1 cmm
Volume of fluid in 4 small squares = 4 x 0.1 cmm = 0.4 cmm
0.4 cmm of diluted blood contains N WBC

0.1 cmm of diluted blood contains N x ¼

1 cmm of diluted blood contains N x ¼ x 10
Dilution is 0.5 in 10 or 1 in 20
Therefore total number of WBC in undiluted blood = N x ¼ x 10 x 20
= N x 50 / cmm
Result:
Conclusion:
Teacher’s sign:

EXPERIMENT NO.: 8
Aim: Enumeration of total red blood corpuscles (RBC) count.
Reference:
1) Dr. R. K. Goyal, Dr. N. M. Patel
13th Ed.2009
Page No.:22-25
2) Dr. S.A.Deshpande, Dr. N.S.Vyaswahare
A Practical book of Anatomy and Physiology
Nirali Prakashan
Page No.:24-26
Requirements:
(a) Haemocytometer set which consists of Neubauer’s counting chamber and a RBC
dilution pipette, (b) Thomas coverslip, (c) Microscope, (d) RBC dilution fluid, (e)
Pricking needle, (f) Absorbent cotton, (g) 70% alcohol, (h) Xylol to clean glass slide and
coverslip.
Composition of dilution fluid: Hayem’s RBC dilution fluid

Sodium chloride (NaCI) 1.0 gm. Provides isotonicity, Prevents haemolysis
Sodium sulphate 5.5 gm. Provides isotonicity, Prevents rouleax
(Na2SO4) formation(anti-coagulant)
Mercuric 0.5 gm. Causes fixation of the cells, Prevents
chloride(HgCI2) bacterial growth(anti-microbial)
Water (H2O) Up to 100 ml Diluent
Principle:
RBC, Red blood corpuscles (also called as erythrocytes) are major blood cells
which contain the haemoglobin carry oxygen & CO2.The life span of normal RBC is 120
days. The production of RBC occurs in red bone marrow. The numbers of red blood cells
in blood are too many and the size of the cells is too small. It is, therefore, 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. RBCs should be diluted 100 times
with the dilution fluid, called Hayem’s solution. This is a clear liquid as due to their high
haemoglobin content, RBCs can be visualised in the light microscope without staining.
The diluted blood is placed in a capillary space of known capacity in between a special
ruled slide (counting chamber) and a coverslip. 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 the number with dilution factor
and are reported as cells per cubic mm of blood.
Procedure:
(1) The Thomas coverslip, counting chamber and lenses of microscope are cleaned with
the help of Xylol and then absorbent cotton.
(2) The counting chamber is adjusted and observed for RBC squares under low power
of microscope keeping the Thomas coverslip resting on the platform of the slide.

(3) 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.
(3) 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.
(4) The first drop of blood is discarded and the second drop is sucked in the RBC pipette
up to the mark 0.5. Immediately the dilution fluid sucked exactly up to mark 101. When
it is filled up to the mark, pipette is brought to a horizontal position and the finger is
placed over the tip of pipette. The simple knot is given to rubber tube.
(5) The pipette is rolled between the palms to mix the blood with dilution fluid for one
minute.
(6) Few drops (2-3) are discarded and then the pipette is held at an angle of 45º to the
surface of counting chamber and tip is applied to narrow slit between the counting
chamber and the coverslip. A drop is allowed to come out 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.
(7) The fluid is allowed to settle for three minutes on the stage of microscope.
(8) The RBC chamber is located and RBCs are counted in 16 smallest squares. This is
repeated in another four such chamber.
Tips
First prepare the required dilution fluid.
When it is ready, disinfect and punch the middle finger, wipe off the first blood drop
then gently massage the finger to draw a larger drop of blood on the surface of the
finger.
Immerse a disposable sterile pipette tip attached to the corresponding pipette into the
drop of blood make sure that the pipette tip is not pressed to the skin, otherwise even
sucking of the blood is not possible.
Take care to avoid air bubbles inside the pipette tips!
Once pipetted, blood should be immediately mixed with the already prepared dilution
fluid.
Normal ranges:
Male: about 5.4 million/cmm of blood
Female: about 4.8 million/cmm of blood
Clinical significance:
The decreased count of RBC indicates several conditions like iron deficiency anaemia,
megaloblastic anaemia, pernicious anaemia, thalassemia, sickle cell anaemia, aplastic
anaemia, chronic renal failure, haemorrhage, in bone marrow suppression etc.
Increased RBC production (Polycythaemia vera) occurs in burn, cardiac hypertrophy,
congenital heart disease, lack of oxygen during rock climbing, Emphysema (respiratory
disorders) etc., when the viscosity of blood increases.
Calculations:
Total number of RBC in 80 smallest squares = N
Length of one smallest square = 1/5 x ¼ = 1/20 mm
Breadth of one smallest square = 1/20 mm
Therefore, Area of one smallest square = 1/20 x 1/20 = 1/400 sq. mm.
Volume of fluid in smallest RBC square = 1/400 x 1/10 = 1/4000 cmm
If 80 smallest squares contain N RBC in diluted blood

1 smallest square contains N/80 RBC in diluted blood OR

1/4000 cmm of diluted blood contains N/80 RBCs
1 cmm of diluted blood contains N/80 x 4000 RBCs
Dilution factor is 200
1 cmm undiluted blood contains N/80 x 4000 x 200 RBCs
= RBCs / cmm
Result:
Conclusion:
Teacher’s sign:

EXPERIMENT NO.:9
Aim: Determination of bleeding time.
13th Ed.2009
Page No.:32-34
Requirements: pieces of filter paper, sterile pricking needle, 70% alcohol, stop watch.
Principle:
Haemostasis or hemostasis is a complex process which causes the bleeding
process to stop. It refers to the process of keeping blood within a damaged blood vessel.
It prevents excess loss of blood and thereby avoids further complications. This process
involves multiple processes like vasoconstriction, platelet plug formation and blood
clotting.
Bleeding time is the time interval between the onset of bleeding and
spontaneous unassisted stoppage of bleeding. The blood flow stops chiefly because of
platelet function. Bleeding time is usually be measured by Duke’s method or Ivy method.
Since, platelets are an important regulator of bleeding time. Alteration in platelet
count is clinically significant. Bleeding time: provides assessment of platelet count and
function. The bleeding time indicate the conditions of the blood clotting mechanisms. It
shows the mutual effects of blood and injured tissue.
The usual bleeding time is 1 - 3 min.
When is it ordered?
 When a patient presents with unexplained bleeding or bruising
 It may be ordered as part of a pre-surgical evaluation for bleeding tendencies
 When a patient is on intravenous (IV) or injection heparin therapy, the APTT is
ordered at regular intervals to monitor the degree of anticoagulation.
The bleeding time is dependent upon

 The efficiency of tissue fluid in accelerating the coagulation process
 Capillary function
 The number of blood platelets present and their ability to form a platelet plug.
Prolonged bleeding times are generally found when

 The platelet count is below 50,000/µL
 When there is platelet dysfunction.
Purpura is the condition associated with platelet deficiency and is characterized by the
appearance of red spots on skin and prolonged bleeding time.
Factor deficiencies
Inherited:
1. Hemophilia A
2. Hemophilia B
3. Von willebr’s disease (missing or defective clotting protein)

4. Glanzmann’s thrombasthenia (deficient fibrinogen receptor for platelets)

Acquired:
1. Anticoagulant therapy
2. Liver diseases
3. DIC -Disseminated intravascular coagulation (blood clots in small blood
vessels)
Four procedures are currently in use for determining the bleeding time:
 The Duke method.( This test method is the easiest to perform, but is the least
standardized and has the less precision and accuracy)
⚫ The Ivy Method.
⚫ The Mielke Method.
⚫ The Simplate or Surgicutt Method.
Procedure (Duke’s Method):

1) The tip of ring finger is sterilized with 70% alcohol and a bold prick is given so that
blood flows freely. The stopwatch is immediately started.
2) The blood is soaked on a filter paper (while soaking, the filter should not touch the
skin of finger). This is repeated every 10 sec. till no blot appears on the paper.
3) The time from the first appearance of the blood to the cessation of bleeding is the
bleeding time.
Precaution:
The prick is about 3–4 mm deep.
No repeat testing is allowed due to space.
The test causes nervousness in the patient.
Time should be noted properly.
Needle should be sterilized.
Pricked with a special needle or lancet, preferably on the earlobe or fingertip
Result:
Conclusion:
Teacher’s sign:

EXPERIMENT NO.:10
Aim: Determination of clotting or coagulation time.
13th Ed.2009
Page No.:32
Requirements: Dry glass Capillary tubes(narrow diameter 1 top 2 mm, minimum 10

cm long) or slide,70% v/v ethyl alcohol, Spirit wetted cotton swab, absorbent cotton,
Sterile disposable pricking needle or lancet, Stop watch
Principle:
During coagulation sol form of the blood is changed to gel form. The time elapsed
between the moment of escape of blood outside the vessel and the observation of
physical change is taken as clotting time. It is to be noted that coagulation is the
property of plasma alone. The red and white cells do not take part in it. They only
become caught up in the meshed of the clot and are thereby removed. It is due to this
fact that the clot has a red colour, and the serum is a clear non-cellular fluid. Blood
platelets take part in the process.
Coagulation Tests
1. Tests of the Vascular Platelet Phase of Haemostasis:
Bleeding Time (BT)
2. Tests of the Coagulation Cascade:
Clotting Time (CT) or Coagulation time
Activated Partial Thromboplastic time (APTT)
Prothrombin Time (PT).
3. Tests of Fibrinolysis and the Mechanisms That Control Hemostasis:
Fibrin Degradation Products (FDP)
The clotting process is very important as it prevents excessive blood loss irrespective of
reason of bleeding. The process occurs in three stages:
• Formation of Prothrombin activator or prothrombinase (combination of
activated clotting factors V and X) by intrinsic and extrinsic pathway (an injury
to blood vessel and tissue respectively).
• Conversation of Prothrombin (clotting factor II) into the enzyme thrombin by
prothrombinase.
• Conversation of soluble fibrinogen (clotting factor I) into insoluble fibrin by
thrombin; fibrin forms the threads of the clot.
Although the mechanism of clotting appears to be simple, there are no direct evidences
for above reactions expect that of the formation of thrombin. Various factors involved in
the clotting have been isolated independently by various workers. Following are the
names of the factors accepted universally:
Clotting factors:

Procedure:
Lee and White’s method: About 4 cc of blood is obtained through a venepuncture and
1 cc is placed in each of four small test tubes. As soon as blood enters the barrel of
syringe, stop watch is started. The test tubes are placed in water bath at 37oC. The test
tubes are tilted at every 30 sec. The time when blood does not move on tilting 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 stop watch is started and a capillary tube is filled up to three-
fourth of its total length. After every 30 sec. 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. The time is noted and the difference between the time when blood comes out
from the finger and the unbroken coagulum seen is the coagulation time. This method is
usually adopted as a bed side procedure. The surface of the glass cappilary initiates the
clotting process. This test is sensitive to the factors involved in the intrinsic pathway.
Duke’s method or Glass Slide method: The finger is sterilized and a drop of blood
approximately 5 mm diameter is placed on a special glass slide. The slide is held vertical at
every 30 sec. The time is noted when no change in the shape of the drop is seen. This gives
the clotting time. Normal value: 3 – 5 minutes.
Glass Tube Method: The finger is pricked and the blood is made to flow into a glass
tube about 15 cm (6 inches) long. The period between the appearance of blood in the
finger and the formation of this thread is taken as the coagulation time. The average
time, by this method, is 3-4 minutes.
Capillary blood clotting time method (Wright’s method)

Glass slide method

Clinical significance:
Clotting time decreased:
Indications: Haemorrhage, Malnutrition and
parturition.
Clotting time increased:

Indications/ factors responsible: Thrombosis,
Haemophilia, Hypoprothrombinemia,
Fibrinogenemia, Heparinemia
Discussion:
The time between the moment of escape of blood
outside the vessel and the development of fibrin is defined as the clotting time.
The usual clotting time is 4 – 10 min.
Haemophilia is the condition caused by the absence of any one of the factor required
for clotting of blood. It causes prolongation of the clotting time.
Result:
Conclusion:
Teacher’s sign:

EXPERIMENT NO.:11
Aim: Estimation of haemoglobin content.
13th Ed.2009
Page No.:18-22
Requirements: Sahli’s haemoglobinometer or hemometer 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 millimetre (cmm) capacity, pricking needle, N/10
HCI, distilled water, 70% alcohol and absorbent cotton.
Sahli’s Haemoglobinometer consists of:

• Comparator: It is a rectangular plastic box with a slot in middle to accommodate
the calibrated haemoglobin tube. The golden brown tinted glass rods are fitted
on each side of slot for matching the colour. An opaque white glass is fitted
behind the glass rod to provide uniform illumination.
• Haemoglobin tube: It is a calibrated glass tube with marking of g Hb % i.e. of Hb
in 100 ml of blood (2-24 g.Hb%) in yellow colour on one side and %Hb (20-
140%) in red colour on other side. The brush is provided to clean the tube.
• Haemoglobin pipette: It is 8-10 inches long glass capillary pipette with only a
single calibration mark of 20µl or 0.02 ml.
• Stirrer: It is a thin glass rod with a flattened end which is used for stirring and
mixing the blood, acid and water.
• Pasteur pipette: It is 8-10 inches long glass tube with a long thin nozzle with a
rubber teat.
Structure of haemoglobin:
The Haemoglobin molecule is a tetramer consisting of two pairs of similar
polypeptide chains called globin chains. To each of the four chains is attached haem
which is a complex of iron in ferrous form and protoporphyrin.
The major (96%) type of haemoglobin present in adults is called HbA and it has 2 alpha
globin chains and 2 beta globin chains (α2β2). The gene that codes for the formation of
α globin chains is located on chromosome 16 and that which codes for the formation of
β globin chains is on chromosome 11. In adults, a minor amount of HbA2 (α2β2) is also
present and constitutes less than 3.5%.
The structure and time of occurrence of various haemoglobins is shown in the table
below.
Name Structure Present in
Adult Hb (HbA) α2β2 Adults
Fetal Hb (HbF) α2γ2 Fetal life
Hb Portland ζ2γ2 Embryonic life
Gower-1 ζ2ε2 ,,
Gower-2 α2ε2 ,,
Function of Haemoglobin:

Heam has the ability to bind oxygen reversibly and carry it to tissues. It also facilitates
the exchange of carbon dioxide between the lungs and tissues. Thus, haemoglobin
functions as the primary medium of exchange of oxygen and carbon dioxide.
Various methods are available for estimation of haemoglobin in the laboratory.

I. Methods based on development of colour.
These are
1. Sahli’s or acid hematin method
2. Drabkin’s method or Cyanmethemoglobin method
3. Oxyhemoglobin method
4. Alkaline hematin method
II. Measurement of oxygen combining capacity
III. Measurement of iron content
Principle: (Sahli’s method)

When blood is mixed with N/10 HCI, RBCs are haemolysed and Hb is liberated.
This Hb is converted in to acid hematin which is reddish brown in colour. The intensity
of colour produced depends upon concentration of acid hematin which in turn depends
upon the concentration of Hb. The solution is diluted with distilled water till it matches
with the standard glass (comparison) tubes. The Hb% can directly be read from the
graduated tube. The readings are obtained in g% (gram per cent). The levels of Hb are
significant to establish conditions like anaemia, leukaemia, chronic infections, certain
cancers, etc.
Advantages:
Easy to perform
Quick
Inexpensive
Can be used as a bedside procedure
Does not require technical expertise
Does not need any sophisticated apparatus
Most widely accepted
Disadvantages:
i) Subjective visual colour comparison
ii) Need for accurate pipetting of 20 microliter of blood
iii) Estimation of only acid hematin formed
iv) Fading of comparator on prolonged use
v) Poor sensitivity and reliability.
Average range of Hb is as follows:

Males: 14.5 g% (13.5-18.0 g%)
Female: 12.5 g% (11.5-16 g%)
Clinical significance of Haemoglobin estimation:

1) More than normal: Polycythaemia, Increased viscosity of blood.
2) Less than normal: All types of anaemia
Procedure:

(1) The graduated diluting tube and the micropipette are cleaned thoroughly and
dried.
(2) The graduated diluting tube is filled with N/10 HCI up to mark 2 gm. 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 the mark
20 cmm.
(4) The blood is immediately deposited at the bottom of the graduated tube. The
pipette is rinsed two to three times in HCI.
(5) The blood is mixed with the help of stirrer and then solution is allowed to stand
for 10-15 minutes so that all Hb is converted into acid hematin.
(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
obtained.
(7) When the matching is complete, stirrer is taken out from the diluting tube and
the scale is read on the side of tube.
Some points of consideration:

• 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. For
example, in Sahli’s method 100% reading corresponds to 14.5%, in Haldane’s
method 100% reading corresponds to 13.8 gm.%.
• We cannot use sulphuric acid (H2SO4) because
(a) There is every danger of the appearance of the tinge of acid in the
solution.
(b) H2SO4 and distilled water are not easily miscible as compared to HCI
and distilled water.
(c) The standard tubes with which the colour is being matched are
available with the dilution of hydrochloric acid.
• The disadvantage with HNO3 is similar to those of H2SO4. In addition the fumes of
nitrous oxide may be evolved in the experiment. Hydrofluoric acid (HF) has still
additional disadvantage that it may destroy the calibrations of the tube.
• NaOH if used instead of HCI, forms alkaline hematin which is neither stable as
acid hematin, nor it has identical colour as acid hematin.
• If more acid is used instead of distilled water it does not alter the observation,
however if less acid is used, formation of acid hematin may not be complete and
chances of errors are there.
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 HCI.
(4) While observing for colour match, stirrer should be kept out of solution (but
not out of the tube), and the calibrated side should not come in view. The
observation must be done while facing it against uniform intensity of light.

Observations and Calculations

Observed Hb gm. % = gm. %
Observed Hb% = %
International value of Hb is 14.5gm% = 100%

Calculated Hb% = gm. % ÷14.5 x 100
Oxygen carrying capacity

Amount of O2 in cc carried by 100 ml of blood during one pulmonary circulation.
Hb gm. % x 1.34 cc
1 gm. of Hb contains 1.34cc oxygen
Therefore, N gm. of Hb contains = N x 1.34 cc oxygen

= cc oxygen%
Result:
Conclusion:
Teacher’s sign:

EXPERIMENT NO.:12
Aim: Determination of blood group.
13th Ed.2009
Page No.:26-28
Requirements:
Microscopic slides, glass marking pencil, pricking needle, 70% alcohol, microscope &
reagents (antisera A, antisera B, and antisera D).
Principle:
According to ‘ABO’ system of blood grouping, there can be four blood groups
‘A’,‘B’,‘AB’ and ‘O’. This classification is based on the presence of agglutinogen A, B, both
A and B or none of them respectively. As Landsteiner stated, if particular agglutinogen
is present in the blood, then the corresponding agglutinin is always absent and if
particular type of agglutinogen is absent in the blood then the corresponding
agglutinin is always present.
The reagent antisera ‘A’ consists of agglutinin alpha and it causes clumping of
RBCs of blood containing agglutinogen A. The reagent antisera‘B’ consists of
agglutinin beta and it causes clumping of RBCs of blood containing agglutinogen B.
Hence blood group is determined as follows:
1. Clumping in antisera ‘A’ →blood group ‘A
2. Clumping in antisera ‘B’ →blood group ‘B’
3. Clumping in both antisera ‘A’ and ‘B’ →blood group ‘AB’
4. No clumping in all the three sera →blood group ‘O’
The blood group are assigned as + ve (or) – ve, according to the Rh system. If ‘Rh’
factor (a type of agglutinogen) is present, the blood group is assigned as positive (+ ve)
and if Rh factor is absent, the blood group is assigned as negative (– ve). The reagent
antisera ‘D’ contains antibodies against ‘Rh’ factor, hence if blood clumps in antisera ‘D’,
blood group is assigned as positive (+ ve) and if no clump, blood group is assigned as
negative (– ve).
Procedure:
1. Take three slides and mark them A, B and D.
2. Place antisera A on slide A, antisera B on slide B and antisera D on slide D.
3. Sterilize the finger and prick it boldly. Place one drop of blood on each of
the sera.
4. Mix the blood drop and the reagents with the help of another clean slide.
5. Allow it to react for 5 minutes.
6. Examine after 5 minutes but not later than 10 minutes (the reaction may
not be complete before 5 minutes and drying may occur after 10 minutes.
Both these factors may yield false results).

Observation Table:
Slide No. Anti-serum Clumping (Yes/No)

1. (A) antiserum A
2. (B) antiserum B
3. (D) antiserum D
Result:
Conclusion:
Teacher’s sign:

EXPERIMENT NO.:13
Aim: Demonstration of erythrocyte sedimentation rate (ESR).
13th Ed.2009
Page No.:34-37
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 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’s stand
(2) Oxalate bulb
(3) Sodium citrate solution (3.8%)
(4) Sterilized syringe and needle to collect the blood by venepuncture.
Westergren’s pipette: It has a bore of 3 mm and length of 300 mm. It is marked with
graduations from 0 to 200. It is open from both the ends.
Westergren’s Stand: It can accommodate six pipettes. There is a base containing
groove each containing a rubber cushion to prevent leakage of blood from Westergren’s
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 3 ml) is obtained by a venepuncture and is mixed with
3.8% sodium citrate solution in proportion of four parts 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’s stand, taking care that no blood
escapes. 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).
WINTROBE’S METHOD
Requirements:
(1) Wintrobe’s pipette
(2) Stand for pipette
(3) Long capillary pipette
(4) Sodium citrate solution (3.8%)
(5) Sterilized syringe and needle to collect the blood 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 tube. Each main division represents 1 cm

and each smaller division represents 1 mm. The mouth cap to prevent loss of fluid of
evaporation.
Long Capillary pipette: It consists of 100 mm long glass capillary and a broad stem
which can hold about 2 ml of blood. It is fitted with a rubber teat.
Procedure:
(1) A sample of blood (about 3 ml) 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 1-3 ml 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
teat, 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 hours, the upper level of red cell column is read. It
will indicate mm of clear plasma (ESR).
Discussion:
ESR is defined as mm of clear plasma formed at the top of the vertical column in
one hour. The normal range of ESR is as follow:
Method At the end of one hour At the end of two hours

Male Female Male Female
Westergren’s 3-5 4-7 7-15 12-17
Wintrobe’s 0-7 0-7 7-15 7-15
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 heavier than
plasma (specific 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.
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 stick to each other
and get piled over one another to form Rouleaux. Each rouleaux consists of 8 to 12 cells
and when more cells aggregate together, the repelling forces cause 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 concentration
(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 in all acute general
infections like septicaemia (100 mm/hr.), pulmonary tuberculosis (65 mm/hr.),

anaemia, sickle shape anaemia, jaundice, malignant tumour and Inflammatory

conditions. The ESR decreases in allergy and protein shock.
Teacher’s sign:

EXPERIMENT NO.:14
Aim: Determination of heart rate and pulse rate.
Aim: To determine the pulse rate.

Reference: Dr. S.A.Deshpande, Dr. N.S.Vyaswahare
Nirali Prakashan
Page No.:35-36
Requirement: Stopwatch
Theory:
The pulse is the surge of blood that is pushed through the arteries when the
heart beats. The pulse rate is how many times one can feel a pulse every minute. The
pulse rate is a vital sign that can tell a lot about a victim's medical condition.
The pulse rate changes with exercise, so healthcare providers like to compare
resting pulse rates, which should always be between 60-90 beats per minute. Extremely
fast pulses -- more than 150 beats per minute -- or slow pulses of less than 50 per
minute can indicate problems with the heart. An extremely slow pulse combined with
dizziness can indicate shock and help identify internal bleeding. Pulse is examined to
diagnose the conditions of heart, arteries and blood pressure.Besides the pulse rate,
other indicators of how a person is doing come from the regularity and strength of the
pulse.
The pulse is often confused with the heart rate but refers instead to how many
times per minute the arteries expand and contract in response to the pumping action of
the heart. Taking the pulse is, therefore, a direct measure of heart rate.
Fast facts about pulse:

• As the heart pumps, the arteries expand and contract. This is the pulse.
• The pulse is easiest to find on the wrist or neck.
• A healthy pulse is between 60 and 100 beats per minute (bpm).
What is the pulse?

The pulse is the expansion of the arteries. This expansion is caused by an
increase in blood pressure pushing against the elastic walls of the arteries each time the
heart beats. These expansions rise and fall in time with the heart as it pumps the blood
and then rests as it refills. The pulsations are felt at certain points on the body where
larger arteries run closer to the skin. Most common places are radial, carotid, brachial,
and femoral arteries.
This is the pulse running through one of the carotid arteries. These are the main
arteries that run from the heart to the head.Pulse rate above the normal is called as
Tachycardia and decrease in pulse rate than normal is called as Bradycardia.
NOTE: Many things-such as anxiety, pain and fever-can raise the patient's pulse (heart
rate) and certain medications such as beta blockers or digoxin can lower it; all of these
reasons should be considered when assessing and recording the patient's pulse. If you
are taking repeat measurements of the same patient, try to measure the pulse under the
same conditions each time.

Steps to Determine the Pulse Rate
1. Stay Safe.
2. Locate the pulse.
3. Count the beats.
4. Calculate the pulse rate.
Procedure:
1. Locate the radial artery at the own wrist level.
2. Palpate the radial artery by pressing them with finger against the underlying
bones (the pulse will be felt).
3. Record the pulse for 1 minute.
4. Take three readings at the interval of 5 minutes and calculate the mean pulse
rate.
A normal pulse is regular and strong. Pulse rates (number of beats per minute)
change with age and can vary between individuals of the same age.
Normal pulse rate range, by age
Pulse rate
Age
(beats per minute)
New born (resting) 100-180
Infant (resting) 80-150
Child 2-6 years 75-120

Child 6-12 years 70-110
Adolescent-adult 60-90
Tips
1. Never use your thumb to take a pulse. In most people, there is a pulse in the thumb
that can interfere with the one you're trying to feel in the patient, and thumbs aren't
as sensitive as the other fingers.
2. The rate of the pulse is only part of the story. The quality of the pulse is also
important. When taking a pulse rate, make a note of the strength of the pulse and
whether it is regular or erratic. An irregular or weak pulse can tell medical providers
important information about a patient's condition.

3. The pulse in the wrist is called the radial pulse, but pulses can also be felt in the
neck, upper arm, groin, ankle and foot.
Observation table:
Sr. No. Observation No. Pulse rate (pulse/min)
1 1 reading
st
2 2nd reading
3 3rd reading
4 Mean
Result:
Conclusion:

Aim: To determine the heart rate.

Reference: Dr. S.A.Deshpande, Dr. N.S.Vyaswahare
Nirali Prakashan
Page No.:37-38
Requirements: Stethoscope
Stethoscope is an instrument used for listening of sound produced inside the
body with amplification. The commonly used stethoscope consists of:
• Ear frame consists of two curved metallic tubes joined together with a flat ‘U’
shaped spring which keeps them pull together.
• Conducting tubes are simple flexible and soft pressure tubes of rubber or latex
material.
• Chest piece consists of a bell and flat diaphragm which causes an amplification
of the body sound.
Principle:
The number of heart beats recorded per minute is called as heart rate. The heart
beat is normally initiated by impulses generated in SA node. The rhythm is determined
by route of impulse transmission through the conducting system. It is usually measured
by stethoscope but more specifically determined by electrocardiogram (ECG).
However, a normal heart rate depends on the individual, age, body size, heart
conditions, whether the person is sitting or moving, medication use and even air
temperature. Emotions can affect heart rate; for example, getting excited or scared can
increase the heart rate. Most importantly, getting fitter lowers the heart rate, by making
heart muscles work more efficiently. A well-trained athlete may have a resting heart
rate of 40 to 60 beats per minute, according to the American Heart
Association (AHA).Knowledge about your heart rate can help you monitor your fitness

level, and it may help you spot developing health problems if you are experiencing other
symptoms. But a heart rate lowers than 60 don’t necessarily indicate a medical problem.
It could be the result of taking a drug such as a beta blocker. A lower heart rate is also
common for people who get a lot of physical activity or are very athletic. Active people
often have lower heart rates because their heart muscle is in better condition and
doesn’t need to work as hard to maintain a steady beat.
How Other Factors Affect Heart Rate

• Air temperature: When temperatures (and the humidity) soar, the heart pumps
a little more blood, so your pulse rate may increase, but usually no more than
five to 10 beats a minute.
• Body position: Resting, sitting or standing, your pulse is usually the same.
Sometimes as you stand for the first 15 to 20 seconds, your pulse may go up a
little bit, but after a couple of minutes it should settle down.
• Emotions: If you’re stressed, anxious or “extraordinarily happy or sad” your
emotions can raise your pulse.
• Body size: Body size usually doesn’t change pulse. If you’re very obese, you
might see a higher resting pulse than normal, but usually not more than 100.
• Medication use: Meds that block your adrenaline (beta blockers) tend to slow
your pulse, while too much thyroid medication or too high of a dosage will raise
it.
Five conditions are considered as most influential: (a) resting period before
measurement; (b) posture of the person; (c) environmental conditions such as
temperature or visual and acoustic stimuli; (d) method used to record heart rate; and
(e) data analysis, i.e., derivation from raw data.
Measurement
Electronic measurement
A more precise method of determining heart rate involves the use of an
electrocardiograph, or ECG (also abbreviated EKG). An ECG generates a pattern based
on electrical activity of the heart, which closely follows heart function. Continuous ECG
monitoring is routinely done in many clinical settings, especially in critical care
medicine. On the ECG, instantaneous heart rate is calculated using the R wave-to-R wave
(RR) interval and multiplying/dividing in order to derive heart rate in heartbeats/min.
However, it is normal for the heart rate to vary in response to movement, activity,
exercise, anxiety, excitement, and fear.
If you feel that your heart is beating out of rhythm or at an unhealthy speed of
less than 40 b.p.m. or over 120 b.p.m. and this can be felt when taking a pulse, discuss
this with a doctor. You might also feel that your heart has missed or "skipped" a beat, or
there has been an extra beat. An extra beat is called an ectopic beat. Ectopic beats are
very common, are usually harmless, and do not need any treatment. If there are
concerns about palpitations or ectopic beats, however, visit a doctor.
Blood Pressure vs. Heart Rate (Pulse):
Heart rate and blood pressure do not necessarily increase at the same rate. A
rising heart rate does not cause your blood pressure to increase at the same rate. Even
though your heart is beating more times a minute, healthy blood vessels dilate (get
larger) to allow more blood to flow through more easily. When you exercise, your heart
speeds up so more blood can reach your muscles. It may be possible for your heart rate

to double safely, while your blood pressure may respond by only increasing a modest
amount.
Procedure:
1. Select the subject and make him to sit comfortably on the chair.
2. Place the chest piece of stethoscope against thoracic wall.
3. Record the heart beat for the period of 1 minute.
4. Take three readings at the interval of 5 minutes and calculate the mean heart rate.
Note:
Note values of heart rate/minute:
1. Foetus: 140-160 beats.
2. Children: 140 beats.
3. Adults Males: 64-74 beats.
4. Adults Females: 72-80 beats.
Observation table:
Sr. No. Observation No. Heart rate (beats/min)
1 1 reading
st
2 2nd reading
3 3rd reading
4 Mean
Result:
Conclusion:
Teacher’s sign:

EXPERIMENT NO.:15
Aim: Recording of Blood pressure.
13th Ed.2009
Page No.:39-42
Definition: Blood pressure is the force exerted by your blood against the wall of blood
vessels. As your heart pumps, it forces blood out through arteries that carry the blood
throughout your body. The arteries keep tapering off in size until they become tiny
vessels, called capillaries. At the capillary level, oxygen and nutrients are released from
your blood and delivered to the organs.
Types of Blood Pressure: There are two types of blood pressure: Systolic blood
pressure refers to the pressure inside your arteries when your heart is pumping;
diastolic pressure is the pressure inside your arteries when your heart is resting
between beats.
When your arteries are healthy and dilated, blood flows easily and your heart doesn't
have to work too hard. But when your arteries are too narrow or stiff, blood pressure
rises, the heart gets overworked, and arteries can become damaged.
Measuring Blood Pressure: There are many other types of machines for recording
blood pressure, such as electronic devices, but these may not be readily available. They
can also be difficult to maintain and therefore may give inaccurate readings.
Blood pressure is measured with an instrument called a sphygmomanometer.

First, a cuff is placed around your arm and inflated with a pump until the circulation is
cut off. A small valve slowly deflates the cuff, and the person measuring blood pressure
uses a stethoscope, placed over your arm, to listen for the sound of blood pulsing
through the arteries. That first sound of rushing blood refers to the systolic blood
pressure; once the sound fades, the second number indicates the diastolic pressure, the
blood pressure of your heart at rest.
Blood pressure is measured in millimetres of mercury (mm Hg) and recorded with the
systolic number first, followed by the diastolic number. For example, a normal blood
pressure would be recorded as something under 120/80 mm Hg.
Blood pressure readings can be affected by factors like:

• Smoking
• Coffee or other caffeinated drinks
• A full bladder
• Recent physical activity
Blood pressure is also affected by your emotional state and the time of day. Since so
many factors can affect blood pressure readings, you should have your blood pressure
taken several times to get an accurate measurement.

What Is Normal Blood Pressure?

Experts consider normal blood pressure to be less than 120/80 mm Hg. At one point,
blood pressure at or above 120/80 and less than 140/90 was considered normal to
high; these numbers are now considered pre-hypertensive. Blood pressure consistently
at or above 140/90 is considered high blood pressure or hypertension.
Blood pressure normally rises as you age and grow. Normal blood pressure readings for
children are lower than for adults, while blood pressure measurements for adults and
older teenagers are similar.
Blood pressure can also be too low, a condition called hypotension. Hypotension refers
to blood pressure lower than 90/60. Symptoms of hypotension include dizziness,
fainting, and sometimes shock.
We record systolic pressure first (on the top) and the diastolic pressure second
(below). For example, if the systolic pressure is 120 mmHg (millimetres of mercury)
and the diastolic pressure is 80 mmHg, we would describe the blood pressure as ‘120
over 80’, written 120/80.
All patients must be assessed for fitness before they undergo surgery. As part of this
assessment, it important to measure and record the patient's blood pressure. There are
two reasons for this:
1. It provides an initial recording (a ‘baseline’). If the blood pressure falls suddenly

below this baseline after surgery, we are alerted to the fact that the patient may
be experiencing complications.
2. It allows us to confirm that the patient is fit enough to undergo surgery. A high
blood pressure reading, or indeed a very low blood pressure reading, could
suggest that the patient has other medical problems, e.g. an undiagnosed heart
condition. He or she may need further medical tests and possibly medication to
stabilise the blood pressure before undergoing surgery.
When measuring the blood pressure, some factors can affect the reading and possibly
give a false reading, which could lead to unnecessary medical investigations. These
factors include:
• blood pressure cuff is too small or is placed over clothing

• the subject has recently exercised
• the subject is cold or otherwise uncomfortable
• the subject has consumed alcohol or caffeine less than 30 minutes before the
reading
Blood pressure may vary according to whether the person is lying down, sitting or
standing. It is normally recorded with the patient sitting.
Clinical Significance:
Blood pressure Indication/Factors responsible

Increased • Obesity, Ageing, Emotional stress, Exercise

• Hypertension
• Atherosclerosis
• Heart diseases
• Kidney diseases
• Diabetes
Decreased • Hypotension
• Severe blood loss
• Severe water and electrolyte imbalance
Requirements:
• sphygmomanometer
• blood pressure cuffs: small, medium, large
• stethoscope
• chair
• observation chart
• alcohol wipe
Preparation
• Ask the person to sit down. The person should have rested for 3–5 minutes
before starting the procedure.
• Explain to the patient what you are going to do. This will help reduce their
anxiety.
• Explain the sensation of the cuff tightening on their arm and reassure them that
this is safe.
Method
• Ask the subject to loosen any tight clothing or remove long-sleeved garments so
that it is possible to access the upper arm. Do not use an arm that may have a
medical problem.
• Place the cuff around the upper arm and secure.
• Connect the cuff tubing to the sphygmomanometer tubing and secure.
• Rest the subject's arm on a surface that is at the level of the heart.
• Uncover the arm up to shoulder & tie the cuff around the arm, neither too tight
nor too loose.

• Record the blood pressure first with palpatory method followed by auscultatory
method:
Palpatory method:
• Palpate the radial artery at wrist and feel the pulse.
• Tighten the screw of leak valve and inflate the cuff slowly with air pump until the
pulse disappear and note the reading.
Auscultatory method:
• Place the stethoscope over the bifurcation of brachial artery (in the bend of the
elbow) and listen to the pulse.
• Pump up the cuff slowly and listen for when the pulse disappears. This is an
indication to stop inflating the cuff.
• Start to deflate the cuff very slowly whilst watching the mercury level in the
sphygmomanometer.
• Note the sphygmomanometer reading (the number the mercury has reached)
when the pulse reappears: record this as the systolic pressure.
• Deflate the cuff further until the pulse disappears: record this reading as the
diastolic pressure.
• Record these two measurements, first the systolic and then the diastolic (e.g.,
120/80).
• Take three readings at an interval of 30 minutes with the auscultatory method
and find out the average and express the result as systolic/diastolic blood
pressure.
• Disinfect the stethoscope drum and ear pieces with the alcohol wipe.
• Wash and dry your hands.
• Report an extremely low or high reading to the clinically qualified person.
Fall in
Phase Description of sound
pressure
I 10-12 mm Hg Clear sharp tapping sound due to rushing of blood
through the constricted artery
II 10-15 mm Hg Murmurish sound
III 12-24 mm Hg Clear knocking sound
IV 4-5 mm Hg Muffled and faint and disappears as the blood flow
becomes streamline.
Observation table
Sr. No. Systolic BP Diastolic BP Average BP
1
2
3
Result:
Conclusion:
Teacher’s sign:

Human Anatomy & Physiology- I (13PH0101)

(QUESTION BANK)
Exp.1 Compound Microscope
1. Enlist different parts of microscope.
2. What is the utility of microscope?
3. What are the components of optical magnifying system?
4. Differentiate between fixed stage & mechanical stage.
5. What is oil-immersion lens?
Exp.2 Epithelial & connective tissue
6. Define: Gross Anatomy, Histology, tissue.
7. What are different types of epithelial tissues and describe them in detail.
8. Describe functions of epithelial tissue.
9. Give detailed account on connective tissue.
10. Describe dense and loose connective tissue in detail.
Exp.3 muscular and nervous tissue
11. Differentiate between :Skeletal and Cardiac muscle
12. Describe in detail about Nervous tissue.
13. Write short note on Cardiac muscles.
14. Define: Neuroglia, axon, dendrite.
15. Draw neat and clean diagram of nerve cell.
Exp.4 Axial skeletal
16. Explain bones of skull.
17. Describe vertebra in detail.
18. What do you mean by floating ribs?
19. “Cheek” of the face is formed by which bone?
20. Axial skeleton is structured from how many bones?
Exp.5 Appendicular skeletal
21. Which bones are included in appendicular skeletal?
22. How many bones form the skeleton of upper limb?
23. What are the characteristics of patella?
24. Name the bones which are present in the palm.
25. Number of wrist bones.
Exp.6Intro. to haemocytometry
26. Name the components of hemocytometer.
27. Give differentiating features of RBCs and WBCs diluting pipettes.
28. Describe pattern of counting grid of Neubauer’s chamber.
29. Justify the dilution of blood with diluting fluid before cell counting.
30. Why bulb of RBC pipette is larger than that of WBC pipette?
Exp.7WBC count
31. Give the functions of WBCs.
32. Write composition of WBC diluting fluid.
33. Give role of each component of WBC diluting fluid.
34. What is normal level of WBC in blood?
35. What do you mean by Haemocytometry?
Exp.8 RBC count
36. Give functions of RBCs.
37. Give composition of RBC diluting fluid.
38. Give role of each component of RBC diluting fluid.
39. What is normal level of RBC in blood?

40. Explain: Polycythemia.

Exp.9Bleeding time
41. What do you mean by Hemostasis?
42. Define bleeding time & what is normal range?
43. Name two methods for determination of bleeding time.
44. Give clinical significance of bleeding time determination.
45. Explain disorders associated with abnormal bleeding time.
Exp.10Clotting time
46. Define clotting of blood and give its normal range.
47. Name two mechanisms of blood clotting and give steps in blood clotting.
48. Differentiate between bleeding time & clotting time.
49. Why clotting time is more than bleeding time?
50. In which disorders clotting time is altered?
Exp.11 Hb content
51. What is the normal value of Hemoglobin in blood?
52. Name & explain components of Sahli’s Haemoglobinometer.
53. Explain pathological conditions with low hemoglobin.
54. Explain principle of Sahli’s method of hemoglobin determination.
55. How much oxygen is carried by one hemoglobin molecule?
Exp.12 Blood group determination
56. Explain ABO blood grouping system.
57. What are agglutinogen and agglutinin?
58. What is Rh factor?
59. Explain principle behind blood group determination.
60. Which blood group is considered as universal donor?
Exp.13 ESR
61. Define ESR & give values of ESR
62. Explain the process of RBCs sedimentation.
63. What do you mean by Rouleaux formation?
64. Write the factors affecting ESR.
65. Give clinical significance of ESR
Exp.14 Heart rate & pulse rate
66. Define pulse.
67. What is normal pulse rate?
68. How the pulse is measured?
69. What is the significance of increased or decreased pulse rate?
70. Which arteries are palpated for measuring pulse rate?
71. Define heart rate.
72. What is normal heart rate?
73. Define: Bradycardia, Tachycardia
74. Give the role of SA node.
75. What is clinical significance of increased or decreased heart rate?
Exp.15 Blood pressure
76. Define blood pressure. What is systolic and diastolic blood pressure?
77. Name the instrument used for recording of blood pressure & explain its
components.
78. Describe the sounds produced during recording of blood pressure.
79. Name & explain the methods of recording of blood pressure.
80. What is the normal level of blood pressure? Give its clinical significance.

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Human Anatomy & Physiology I (13PH0101)

Marwadi Education Foundation’s Group of Institutions

Programme Education Objectives (PEO)

Programme Specific Outcome (PSO)

FoP, Marwadi University, Rajkot | B. Pharm. Sem.-I (2020-2021) Page | 1

FoP, Marwadi University, Rajkot | B. Pharm. Sem.-I (2020-2021) Page | 1

INDEX FOR EXPERIMENTS PERFORMED

B. Pharmacy, Semester-I Human Anatomy & Physiology -I (13PH0101)

2. Microscopic study of epithelial and

5. Identification of appendicular bones.

7. Enumeration of white blood cell (WBC)

10. Determination of clotting time.

11. Estimation of haemoglobin content.

12. Determination of blood group.

13. Demonstration of erythrocyte

15. Recording of blood pressure.

FoP, Marwadi University, Rajkot | B. Pharm. Sem.-I (2020-2021) Page | 1

I shall uphold the laws and standards governing my

I shall strive to perfect and enlarge my knowledge to

I shall follow the system, which I consider best for

I shall endeavour to discover and manufacture drugs

I shall hold in confidence the knowledge gained

I shall associate with organizations having their

While I continue to keep this Oath in violated, may it

Should I trespass and violate this oath, may the

PHARMACY COUNCIL OF INDIA

FoP, Marwadi University, Rajkot | B. Pharm. Sem.-I (2020-2021) Page | 2

INSTRUCTIONS FOR STUDENT

❖ Listen carefully to the lecture given by teacher about importance of subject,

❖ Read the write up of each experiment to be performed a day in advance.

❖ Understand the purpose of experiment and its practical implications.

❖ Student shall develop maintenances skills as expected by the industries.

❖ Student shall attempt to develop related hands-on-skills and gain conﬁdence.

❖ Student shall focus on development of skills rather than theoretical knowledge.

FoP, Marwadi University, Rajkot | B. Pharm. Sem.-I (2020-2021) Page | 3

List of requirements for Human Anatomy & physiology I laboratory

FoP, Marwadi University, Rajkot | B. Pharm. Sem.-I (2020-2021) Page | 4

FoP, Marwadi University, Rajkot | B. Pharm. Sem.-I (2020-2021) Page | 5

FoP, Marwadi University, Rajkot | B. Pharm. Sem.-I (2020-2021) Page | 6

PROPER USE OF MICROSCOPES:

FoP, Marwadi University, Rajkot | B. Pharm. Sem.-I (2020-2021) Page | 7

Basic Types of Body Tissues:

FoP, Marwadi University, Rajkot | B. Pharm. Sem.-I (2020-2021) Page | 8

by intercellular junctions. The epithelial tissues usually rest on a thin non-cellular

(ii) Fibrous or Reticular Lamina:

Types of Epithelia Tissue:

A. Simple Epithelia (Uni-layered Epithelia):

Simple epithelia are further divisible as follows:

FoP, Marwadi University, Rajkot | B. Pharm. Sem.-I (2020-2021) Page | 9

1. Simple Squamous Epithelium (Pavement Epithelium):

2. Simple Cuboidal Epithelium (cubical epithelium):

Function: Protection, secretion, absorption, excretion, gamete formation.

3. Simple Columnar Epithelium:

FoP, Marwadi University, Rajkot | B. Pharm. Sem.-I (2020-2021) Page | 10

Function: Protection, secretion and absorption.

4. Simple Ciliated Epithelium:

The ciliated epithelium is of two types:

(ii) Ciliated Cuboidal Epithelium: