JAYA SAKTHI ENGINEERING COLLEGE

(Approved By AICTE, New Delhi & Affiliated to Anna University,Chennai)

St.Mary’s Nagar, Thiruninravur (Near Avadi), Chennai – 602024.

DEPARTMENT OF BIOMEDICAL ENGINEERING

BM3351 – ANATOMY AND HUMAN PHYSIOLOGY

III SEMESTER

YEAR – 2023 -2024

NAME :

REGISTER NO :

YEAR :

SEMESTER :
 JAYA SAKTHI ENGINEERING COLLEGE
(Approved By AICTE, New Delhi & Affiliated to Anna University,Chennai)

St.Mary’s Nagar, Thiruninravur (Near Avadi), Chennai – 602024

BONAFIDE CERTIFICATE
Name: ………………….……………………………………………………

Year: II/ Semester: III Branch: BIOMEDICAL ENGINEERING

University Register No:

Certified that this is the bonafide record of work done by the above student in

BM3351 – ANATOMY AND HUMAN PHYSIOLOGY during the academic year 2023-2024

Signature of Head of the Department Signature of Lab In charge

Submitted for the University Practical Examination held on ………………...

Signature of Examiners:

INTERNAL EXTERNAL
 BM3351 – ANATOMY AND HUMAN PHYSIOLOGY

TABLE OF CONTENTS

SL. PAGE
DATE NAME OF THE CONTENT SIGN of faculty
NO NO.

1. COLLECTION OF BLOOD SAMPLES

2. IDENTIFICATION OF BLOOD
GROUPS
3. BLEEDING TIME AND CLOTTING
TIME
4. ESTIMATION OF HEMOGLOBIN

5. TOTAL RBC COUNT

6. TOTAL WBC COUNT

7. DIFFERENTIAL COUNT OF BLOOD

CELLS.

8. ESTIMATION OF ESR

9. ESTIMATION OF
PCV,MCH,MCV,MCHC
10. HEARING TEST- TUNING FORK

11. VISUAL ACTIVITY-SNELLEN’S

CHART & JAEGER’S CHART

EXERCISES COMPLETED / INCOMPLETE / LATE SUBMISSION

………………………………
Signature of the Lab Incharge
 EXP. NO. 1

COLLECTION OF BLOOD SAMPLES

DATE:

Aim:
To know about the collection of blood samples and types of the vacutainers.
Theory:
A Vacutainer blood collection tube is a sterile glass tube with a coloured rubber stoppercreating
a vacuum seal inside of the tube facilitating the drawing of a predetermined volume of liquid.
Vacutainer tubes may contain additives designed to stabilize and preserve the specimen prior to
analytical testing. Tubes are available with a safety-engineered closure (rubber stopper), with a variety
of labelling options and stopper colours as well as a range of draw volumes.Vacutainer needle
are double ended, with one side being encased in a thin rubber coatingfor safety. When the needle is
screwed into the translucent plastic needle holder, the rubber needle is inside the holder;the exposed
needle will be inserted into the vein.
When a vacutainer tube is inserted into the holder, its rubber cap is punctured by the inner
needle and the vacuum in the tube pulls blood through the needle and into the tube. The filled tube is
then removed and another can be inserted and filled the same way. The amount of air evacuated from
the tube predetermines how much blood will fill the tube before blood stops flowing.Each tube is
topped with a color-coded plastic or rubber cap. Tubes often include additives that mix with the blood
when collected and the color of each tube's plastic cap indicates which additives it contains.

Types of tubes:
Blood collection tubes expire because over time the vacuum is lost and blood will not be drawn
into the tube when the needle punctures the cap.Vacutainer tubes may contain additional substances
that preserve blood for processing in a medical laboratory. Using the wrong tube may make the blood
sample unusable for the intended purpose. These additives are typically thin film coating applied
using an ultrasonic nozzle.The additives may
include anticoagulants (EDTA, sodium citrate, heparin) or a gel with density between those of blood
cells and blood plasma. Additionally, some tubes contain additives that preserve certain components of
or substances within the blood, such as glucose. When a tube is centrifuged, the materials within are
separated by density, with the blood cells sinking to the bottom and the plasma or serumaccumulating
at the top. Tubes containing gel can be easily handled and transported after centrifugation without the
blood cells and serum mixing.

1
Vacutainer blood collection tubes:

Vacutainer system

2
Containers containing coagulants:
 Gold or "tiger" red/black top: clot activator and gel for serum separation
 Red top (plastic, not glass): clot activator but no serum separation gel
 Orange or grey/yellow "tiger" Top: thrombin, a rapid clot activator, for stat serum testing

Containers containing anticoagulants:

 Green: sodium heparin or lithium heparin used for plasma determinations in clinical
chemistry (e.g. Urea and electrolyte determination). Sodium heparin collection tubes are the
classically preferred tube for peripheral blood or bone marrow for cytogenetic studies. Lithium
heparin is considered suboptimal for cytogenetics.
 Light green or green or gray: For plasma determinations.
 Purple or lavender: K2 EDTA. This is a strong anticoagulant and these tubes are usually used
for complete blood counts (CBC). Lavender top tubes are generally used when whole blood is
needed for analysis. Can also be used for some blood bank procedures such as blood type and
screen. EDTA tubes are preferred by most molecular genetics laboratories for molecular genetic
studies (DNA or RNA).
 Grey: sodium fluoride and oxalate. Fluoride prevents enzymes in the blood from working, by
preventing glycolysis so a substrate such as glucose will not be gradually used up during storage.
Oxalate is an anticoagulant.
 Light blue: sodium citrate. Citrate is a reversible anticoagulant, and these tubes are used
for coagulation assays.
 Dark Blue: EDTA.These tubes are used for trace metal analysis.
 Black: Used for Erythrocyte Sedimentation Rate (ESR).

Other coloured containers:

 Red (glass): Contains no additives.
 Light yellow: Contains sodium polyanetholsulfonate (SPS). Used for blood culture specimens
or acid-citrate-dextrose (ACD), used for blood bank studies, HLA phenotyping, and paternity
testing.
 Tan (glass or plastic): Contains either sodium heparin (glass) or K2EDTA (plastic). Used for
lead determinations. These tubes are certified to contain no lead.

Result:
Thus the Collection of blood samples and types of the vacationers have been identified.

3
 Procedure:

Directions: Front Grouping

Step Action
1 Prepare a 3-5% suspension of red blood cells to be tested in isotonic saline.(Washed
or unwashed cells may be used)
2 Place 1 drop of Anti-A and Anti-B respectively, in two small, properly labeled test
tubes
3 Add one drop of rbc suspension into the tube and mix.

4 Centrifuge the test tube for appropriate centrifuge time.*

5 Completely resuspend cells and examine macroscopically for agglutination.

Note: Hemolysis may be a consequence of bacterial contamination and should notbe

interpreted as a positive result.
6 Grade and record results

Directions: Reverse Grouping – performed on individuals greater than 4 months old

Step Action
1 Label a test tube for each rbc reagent to be tested. (A1 or B).

2 Place two (2) drops of the serum/plasma into each labeled tube.

3 Add one (1) drop each of A1 cells and B cells to the appropriate tube.

4 Mix gently. Centrifuge the test tubes for appropriate centrifuge time.*

5 Completely resuspend cells and examine macroscopically for agglutination.

6 Grade and record results

4
 EXP. NO. 2
IDENTIFICATION OF BLOOD GROUPS
DATE:

Aim:
To determine the blood group using both forward and reverse method.

Principle:
Testing with both Anti-A and Anti-B is necessary to determine if red blood cells possess or lack A
and/or B blood group antigens. Absence of agglutination is a negative test result, which indicates the
corresponding antigen is not demonstrable. Agglutination of red blood cells with a given reagent is a
positive test result, which indicates the presence of the corresponding antigen on the red blood cells.
(Forward Type)

Direct agglutination of A1 or B reagent red cells with the patient serum/plasma indicates the presenceof
the appropriate ABO antibody. (Reverse type)Forward and reverse typing will be performed on
samples for all patients greater than four monthsof age to allow for discovery of subtypes as well as to
confirm typing. Individuals less than four months old may not have developed sufficient antibody to
allow for detection.

Specimen:
No special preparation of the patient is required prior to specimen collection. The specimen should be
tested as soon as possible after collection, but EDTA and clotted specimens may be stored at 2 to 8 Cfor
up to 10 days if there is a delay in testing. (Note: Storage may result in weaker-than-normal reactions)
Bacterial contamination of the specimen may cause false test results.

Result:
Thus the blood group of the given blood sample have been identified.

5
Pathway of blood coagulation: Extrinsic pathway (Tissue damage)

6
 EXP. NO.

BLEEDING TIME AND CLOTTING TIME

3 DATE:

Determination of clotting time with the help of Wrighting's

method

Aim:
To determine the clotting time of the given blood sample.

Theory:
Normally blood remains in the blood vessels in liquid form. When it is drawn from the body it becomes
into semisolid gel. The mechanism of formation of gel is called blood clotting or coagulation. Blood does
not clot inside vessels due to presence of heparin and anti thrombin III. A blood clot is a gel that
contains the formed elements of the blood, arrested in the stable fibrin mesh.

The factors, involved in blood coagulation are: Fibrinogen (I), Prothrombin (II), Thromboplastin (III),
Calcium (Ca++, IV), Labile Factor (V), Stable Factor (VII), Anti-Hemophilic Factor A (VIII), Anti-
Hemophilic Factor B (IX), Stuart Power Factor (X), Anti-Hemophilic Factor C (XI), Hageman Factor
(XII), Fibrin Stabilizing Factor (XIII). Blood plasma without clotting factors is called serum.

Factors III, IV and V are synthesized in the platelets of plasma. Factors I, II, VII, VIII, IX, X, XI, and
XII are synthesized in liver. Factor XII can be synthesized in liver and platelets. Factor IV is also
obtained from diet and bones.

Clotting time is the time taken to coagulate the blood after rupture of blood vessels. Normally it is 3- 8
minutes for a normal human's blood depending upon platelets count (200000 to 350000 /cubic mm) and
other clotting factors inside the plasma.

Clinical significance
When a blood vessel is damaged, clotting is required to stop the loss of blood (haemostasis) and healing
of the vessel, in which platelets play a vital part. Now it is clear that every factor is equally important
for the clotting of blood and the process is impossible in absence of a single factor as the activation of
one factor is done by another factor.

Therefore, if there is haemorrhage (i.e. clotting time is more than the normal range) we can guess few
possibilities:

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

7
Vitamin K deficiency
Vitamin K is essential for synthesis of factor II, VII, IX, and X. Syntheses of these factors are
impaired and hemorrhagic disease is progressed due to Vit K deficiency.

Hamophilia
Bleeding disease that causes excessive bleeding especially in men.

Hemophilia A
Factor VIII is abnormal and less biologically active. About 1 in 10000 men suffers from classical
haemophilia in United State.

Hemophilia B or Christmas disease

Factor IX is deficient.Both of these factors are transmitted genetically from female
chromosomes. Therefore, women do not have chance of haemophilia due to deficiency of these factors.

Von Willebrand's disease

Factor VIII has two parts, a large component and a small component. The smaller component is
more important in intrinsic pathway of blood clotting and loss of this component causes the classical
haemophilia. Sometimes haemophilia is seen due to deficiency of the large component, called Von
Willebrand's disease.

When the clotting time is lower than the normal range we can guess there might be thromboembolic
condition inside the tissue.

Procedure
The clotting time can be determined by either 'Modified Dale's method' or 'Wrighting's method' andthe
normal rage depends on the method, used.

Wrighting's method
Finger of a subject is sterilized with spirit and pricked with sterilized needle. Time of pricking is noted.
Sufficient blood is made to flow inside the capillary tube. The tube is broken and thread formation is
noted in 20 seconds interval till a jelly like thread is come. The time to appear the thread is noted; it is
the clotting time of the subject.

Result:
Thus the Bleeding Time and Clotting Time for the given sample have been identified.

8
 Determination of bleeding time (Duke's method)

Aim:
To determine the bleeding time of a patient.

Theory:
The time required for complete stopping of blood flow from the punctured blood vessels called the
bleeding time. Normally it is 1-3 minutes for a normal human's blood. Normal clotting time and
bleeding time values differ because bleeding time is the time for stopping bleeding by the formation of
fibrin network on the surface of punctured skin; that is it is the surface phenomenon. But the clotting
time is the time for clotting the whole blood, collected in the capillary tube; therefore it is a volume
phenomenon. For this reason clotting time is more than the bleeding time, when determining by
conventional methods.

Clinical significance:
It plays a significant role

i) To study the haemorrhagic disorders.

ii) To study the coagulation defects

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

Procedure:
Finger of a subject is sterilized with spirit and pricked with sterilized needle. Time of pricking is noted.
Take the stain of the punctured point on a filter paper on 30 second and keep taking stain of blood in
20 second intervals until the bleeding stops. The time of no stain has come is notedproperly; it
is the bleeding time of the subject.

Result:

Normal range of Haemoglobin:

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

9
 EXP. NO.

ESTIMATION OF HAEMOGLOBIN
4 DATE:

Aim:
To estimate the amount of haemoglobin present in the given blood sample.

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

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

Types of Haemoglobin (Hb):

i. Adult Haemoglobin:
a. Hb A: It consists of two αα chain and two ββ chains in globin protein, α2β2α2β2. 97% of adult Hb
is of this type.

b. Hb A2 (α2δ2α2δ2): Minor Hb A2 present in adult, the concentration of Hb increases in some types

of anaemia.

ii. Fetal haemoglobin:

a. Hb F (α2γ2α2γ2): Hb F accounts for 70-90% of haemoglobin at term, 25% in one month, and 5%
in six months. Hb F concentration in adults increases in some types of anemia, haemoglobinopathies,
and some time in leukemia.
b. Haemoglobin Bart's(γ4γ4): It's concentration increases in fetal life in thalassemia.

10
 iii. Embryonic haemoglobin:
Hb Gower 1 (η2ϵ2η2ϵ2), Hb Gower 2 (α2ϵ2α2ϵ2), and Hb Portland (α2γ2α2γ2).

iv. Abnormal haemoglobin:

a. There are four clinically important abnormal haemoglobins: Hb S, Hb C, Hb D, and Hb E,
present in different hereditary haemoglobinopathies. The most commonly encountered hemoglobin is
Hb S (α2β2α2β2) where in the beta chain valine is substituted by glutamic acid at the sixth position.
Hb S is present in sickle cell anemia.

b. Unstable haemoglobin are haemoglobin variants that undergo denaturation and precipitate in the
red cells at Heinz bodies. These are present in a type of congenital nonspherocytichemolyticanemia.

Clinical significance of haemoglobin count:

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

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

Result:
Thus the Hemoglobin level has been identified.

11
 Steps of Erythropoiesis

Improved Neubauer's chamber

12
 EXP. NO.

5 DATE: TOTAL ERYTHROCYTE COUNT

Aim:
To estimate the totalerythrocytescount in the given blood sample.

Theory:
Erythrocytes, also known as red blood corpuscles, contain the haemoglobin (Hb) which carries the
oxygen to cells and tissues, is responsible for the red colour of blood. The life span of normal RBC is120
days. The RBC is produced in red bone marrow in adult. Before birth, the production of RBC
(erythropoiesis) first occurs in the yolk sac of the embryo and later in the liver, spleen, thymus, and
lymph nodes of a fetus up to seven months. Thereafter, production of RBC occurs in red bone marrow.
The agents, required for the synthesis of blood cells are called haematinic agents (iron, folic acid,
vitamin B12, erythropoietin, colony stimulating factors).

Normal ranges:
Male: about 5.4 million per µl or cubic mm of blood

Female: about 4.8 million per µl or cubic mm of

blood

Clinical significances: 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 etc. Increased RBC production (Polycythemiavera) occurs in burn, lack
of oxygen during rock climbing, respiratory disorders etc., when the viscosity of the blood increases.

Procedure:
1. Make a 1:200 Dilution using EDTA Whole blood and saline
2. Put one drop of the solution in the Neubauer's chamber, using a pipette
3. Count the center square (R marked) of the chamber.
Calculation: Cells/ cubic mm of blood = Number of cell counted x dilution factor x 5 /depth of fluid (0.1)
i.e. 10,000 times no of cells counted

Result:
Thus the total Erythrocyte count have been studied.

13
 Improved Neubauer's chamber

Normal ranges:
Table 1. Normal ranges of total leukocyte count:

Table 2. Differential leukocyte count:

14
 EXP. NO.

6 DATE: TOTAL LEUKOCYTE COUNT

Aim:
To estimate the total leukocyte count for the given blood sample.

Principle:
The blood specimen is diluted 1:20 in a WBC pipette with the diluting fluid (water: glacial acetic acid:
gentian violet = 97:2:1) and the cells are counted under low power of the microscope (10X) by using a
counting chamber. The glacial acetic acid lyses the red cells while the gentian violet slightly stains the
nuclei of the leukocytes to locate the WBC under microscope.

Theory:
White blood cells, present in plasma take part in body defense against invading micro-organisms. They
are produced from the pluripotent stem cell in the bone marrow in adults. In case of foetus
haemopoiesis occurs in liver and spleen.

Clinical Significances of total leukocyte count: Increase in total leukocyte count of more than
10,000/cu mm (µl) is known as leukocytosis and decrease of less than 4 000 cu mm (µl) as
leukopenia.
Causes of leukocytosis: i. it is common for a transient period in infections (bacterial, protozoal
(malaria), or parasitic), ii. leukocytosis is also observed in severe hemorrhage and in leukemia ii.
high temperature iii. severe pain iv. accidental brain damage.

Causes of Leucopenia: i. certain viral (hepatitis, influenza, measles, etc.), and 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).

15
Clinical significances of differential leukocyte count:

16
 Procedure:
1. Make a 1:20 dilution of blood.
2. Cork the tube tightly and mix the suspension by rotating in a cell-suspension mixer for at
least 1 minute.
3. Fill the Neubauer counting chamber by means of a Pasteur pipette or glass capillary (the
depth of the fluid should be 0.1 mm).
4. Focus on one of the 'W' marked areas (each having 16 small squares) by turning objective to
low power (10 X).
5. Count cells in all four W marked corners.
Calculations: Number of white cells/cu mm (µl) of whole blood = (number of white cells counted *
dilution/ 16) / (depth of fluid * 1/4x 1/4). [Dilution = 20; Depth of fluid = 0.1 mm (constant)]

Result:
Thus the total leukocyte count for the given blood sample have been identified and studied.

17
EXP. NO.

7 DATE: DIFFERENTIAL COUNT OF BLOOD

CELLS

Aim:
To study the differential count of the given blood sample.

Principle:
The polychromic staining solution (Leishman stain) contains methylene blue and eosin. These basic and
acidic dyes induce multiple colours when applied to cells. Methanol acts a fixative and also as a solvent.
The fixative does not make them adhere to the glass slide. The basic components of white blood cells
(cytoplasm) are stained by acidic dye and they are describing as eosinophilic or acidophilic. The acidic
components (nucleus & nucleic acid) of the cells take blue to purple shadesby basic dye and they are
called basophilic. The neutral components of the cell are stained by the dyes.

Morphology and function of leukocytes:

Neutrophil
Neutrophils are round shape, 10 – 15µ in diameter. The cell contains cytoplasm and nucleus. The
nucleus shows variable numbers of lobes, 2 – 7 lobes hence called polymorphonuclear leukocytes. The
nucleus stain purple blue and the chromatin are coarse and ropy. The cytoplasm contains two types of
granules – Primary granules and Secondary granules. When the cell stained with Leishman stain, only
secondary granules are stained and these are violet colour granules, which are amphophilic. The
cytoplasm takes pink colour.Neutrophils are called first line defenses as they move first to fight the
invading micro-organisms (bacteria etc.). Neutrophil with their enzymatic armoryare superb killers.
The activated neutrophils engulf the bacteria (phagocytosis) and released different enzymes into the
phagocytic vesicles, which killed the bacteria and then digest it.

Eosinophil
Eosinophils are round shaped, 10 – 15µ in diameter. The cell contain cytoplasm packed with coarse
brick red colour granules which takes acidic stain (eosin) hence called eosinophil. And the nucleus ofthe
cell consists of 2 – 3 lobes. The nucleus stains purple blue colour and chromatin is course and
ropy.Eosinophil contains a Major Basic Protein (MBP) which damages the larvae of the parasites.
There is one eosinophilic cation protein which probably neutralizes heparin anticoagulant. They havea
property that prevents anaphylaxis (anti allergic action). They are motile and phagocytic. Chemotoxis is
also shown by the eosinophils.

18
 NORMAL VALUES

Neutrophils : 40 – 75 % ( mean : 57 %

) Band Formed : 2 – 6 % ( mean : 3 % )

Segmented : 50 – 70 % ( mean : 54 %

) Eosinophils : 1 – 4 % ( mean : 2 % )

Basophils :0–1%

Lymphocytes : 20 – 45 % (mean : 37 % )

Monocytes : 2 – 8 % (mean : 6 % )

19
 Basophils
Basophils are round shape, 10 – 15µ in diameter occasionally seen in the blood smear from a healthy
person. The cell contain „bilobed‟ or „S‟ shaped nucleus stains purple blue and chromatin is coarse
and ropy. The cytoplasm of the cell packed with course blue basophilic granules. On an average all
granules are of equal size, which obscured the nucleus.Basophils release the histamine resulting in
immediate hypersensitivity reaction and also have role in inflammation. They contain heparin,protease
and other mediators of inflammation. They are motile and phagocytic.

Monocyte
Monocytes are large, round, 10 – 20µ in diameter The cell contains a large kidney shaped, light purple
blue stained nucleus and pale blue colour cytoplasm contain no granules. Some time few fine purple
granules and vacuoles are seen in the cytoplasm.The monokines secreted by monocytes stimulated T-
cells, take part in inflammation, act as pyrogen and stimulate formation of acute phase proteins. They
converted into macrophases and phagocytosis the microorganism, dead tissues etc.

Lymphocyte
In the blood circulation two types of lymphocyte are found, these are small lymphocyte and large
lymphocyte. The small lymphocytes are believed to be the resting phase and when active they become
large.They are involved in the very important defense mechanism, called immunity. The B-
lymphocytes are responsible for Humoral immunity and T-lymphocytes are responsible for Cell
mediated immunity.

Requirements
1. Microscopic slide and a glass Spreader slide.
2. Cedar wood oil (immersion oil)
3. Leishman stain
 Leishman powder : 0.15 gm
 Acetone free methanol : 1.0 liter
Preparation of stain: 0.15 gm of Leishman powder is dissolved in 100 ml of Acetone free methanol and
the mixture is warmed at 50°C for 15 minutes. It is then filtrated and dye is ripened by keeping the
filtrate in incubator at 37°C for 7 days. The stain is routinely used for staining the blood smear.
4. Buffer solution (PH : 7.0)
 Sodium Dihydrogen Phosphate (NaH2PO4) : 3.76 gm
 Potassium Dihydrogen Phosphate (KH2PO4) : 2.10 gm
 Distilled water: 1.0 liter
5. Staining rack.
6. Cotton and Tissue paper.
7. Pipette

20
 8. Timer
9. Microscope with 10X and 100X objectives.
10. Cell counter.

Procedure:
1. A thin blood film is prepared by spreading a drop of well mixed blood evenly on a clean and
dry glass slide and dries the smear at room temperature. Adequate drying is essential to
preserve the quality of the blood film.
2. Write the Identification number on the slide by using a lead pencil or a Marker pen.
3. Dried blood film prepared slide is placed on the staining rack.
4. Covered the smear with the Leishman staining solution by adding 10 – 15 drops on the smear
by using a Pasteur pipette. Wait for exactly 1½ minutes.
5. Added equal amount of buffer solution on the slide. Mixed the reaction mixture adequately
by blowing that contain on it through a pipette. Wait for 10 minutes.
6. Washed the smear by using running tap water.
7. Stand the slide in the draining rack or on the laboratory counter to dry the smear.
8. Examine the stained smear under the low power objective (10X) in the microscope for
screening purpose and Chooses the proper portion of the smear. Placed one drop of Cedar
wood oil (immersion oil) on the smear. Switch to the oil immersion objective (100X) and
increase the light by opening the iris diaphragm. Examine the field by moving from one field
to next field systematically. Record the types of leukocytes seen in each field. Count at least
a total of 100 leukocytes and give the result as percentage (%) of the cells.

Result:
Normal range
Male: 0-15 mm after 1st hour.
Female: 0-20 mm after 1st hour.

21
 EXPT NO: 8
DATE:
ESTIMATION OF
ERYTHROCYTE
SEDIMENTATION RATE

Aim:
To determine the erythrocyte sedimentation rate for the given sample.

Principle:
The distance(mm)the erythrocyte falls during placing the anticoagulated blood in the ESR tube in a
vertical position, is measured after a certain time (1/2 hour).

Theory:
When anticoagulated blood is allowed to stand undisturbed for a period of time, the erythrocytes tendto
sink to the bottom. Two layers are formed, the upper plasma layer and the lower one of red blood cells.
The rate at which the red cells fall is known as the erythrocyte sedimentation rate. The first is the stage
of aggregation when the red cells form rouleaux (RBCs cling together like coins in pile). This is followed
by the stage of sedimentation in which the falling of the red cells takes place. The rate of falling of
erythrocyte is directly proportional to the aggregation in first stage.

Clinical significance
ESR is increased in all conditions where there is tissue breakdown or where there is entry of foreign
proteins in the blood, except for localized mild infections. The determination is useful to check the
progress of the infectious disease. If the patient is improving the ESR tends to fall. If the patient's
condition is getting worse the ESR tends to rise. The ESR increases high in some chronic bacterial
diseases like tuberculosis, typhoid, rheumatic diseases etc.

Factors affecting ESR:

i. The changed levels of plasma proteins such as fibrinogen and globulins tend to increase rouleaux
formation. ESR is therefore increased in any condition causing an increase in fibrinogen (any cause
of tissue breakdown such as tuberculosis and other chronic infections) or globulins (rheumatic fever,
myeloma, kala-azar, etc.)
ii. Albumin retards sedimentation.
iv. ESR is greater in women than in men.

v. During pregnancy ESR gradually increases after 3rd month and returns to normal in about 3 to 4
weeks after delivery.

22
 iii. High blood count however, tend to lower the sedimentation rate, while low blood counts tend to
accelerate the rate of fall.

vi. ESR is low in infants and gradually increases up to puberty.

vii. The Laboratory factors which influence ESR are as follows:

a. Time: The test should be performed as early as possible after the collection of fasting specimen.
There is progressive decrease in sedimentation in first four hours and after that there is a rapid
decrease in sedimentation.
b. The length of the ESR tube: ESR is greater with longer tubes (Westergren's tube) than with shorter
tube (Wintrobe's tube). To ensure reliable results the column of blood should be as high as possible.
The internal diameter of the tube should be more than 2.5 mm. The tubes should be kept in vertical
position. Deviation of the tubes from the vertical position increases the ESR.
c. Temperature: The red cell sedimentation is increased at higher temperature.

Procedure:
1. Fill the Westergren's tube exactly up to zero mark by means of a rubber bulb (avoid air bubbles).

2. Place the tube upright in the stand. It should fit evenly into the groove of the stand.

3. Note the time. Allow the tube to stand for exactly one hour.

4. Exactly after one hour, note the level to which the red cell column has fallen.

5. Report the result in terms of mm/after 1st hour.

Result:
Thus the Erythrocyte Sedimentation Rate of the given blood sample have been studied.

23
NORMAL VALUE:
Male : 41 – 50 % (Average 47 %)

Female: 36- 44 % (Average 40 %)

24
 EXP. NO. 9

DATE: DETERMINATION OF BLOOD INDICES

A
To determine the indices of the given blood sample.
i
P mCV
:
Principle:
When anti coagulated blood is centrifuged the RBC‟s which are heavier than the WBC‟s and
platelets settle at the bottom.This red blood cells column is called haematocrit.

Clinical significance:
Determination of PCV helps in diagnosis and treatment of anemia, polycythemia, dehydration
and blood transfusion recovery. It is increased in dengue fever and decreased in pregnancy and
cirrhosis of liver.

Theory:
Haemoglobin is a red pigment present in all walls of RBC. It is a respiratory filament that combines
with O2 to form oxyhaemoglobin, that carries oxygen to the tissues. Decreased concentration below
normal value leads to anaemia. Haemoglobin values are lower in women and children, than in men. The
value also gets decreased during pregnancy. Increased haemoglobin concentration can occur due to
thalassemia.

Procedure:
1. Fill the wintrobes haematocrit tube (rinsed with potassium oxaloacetate) upto 100 mark.
Wintrobe tube is a small graduated tube of uniform bore.
2. Centrifuge the tube for 30 minutes at 3000 rpm till the packing is complete.
3. The RBC packed at the bottom is the packed cell volume and the clear plasma remains
above this.
4. It is between the RBCs and the plasma, there is a white buffy coat, which is informed by
white blood cells and the platelets.
5. Read the packed cell volume directly from the graduated given on the tube & express the
result in percentage.

25
 MCV, MCH, MCHC
Aim:
To determine blood indices like MCV – Mean Corpuscular Volume, MCH – Mean Corpuscular
Hemoglobin, MCHC – Mean Corpuscular Hemoglobin Concentration.

Clinical significance:
Blood indices are simply meant for erythrocytes. The number, shape, volume and the colourof
the RBC indicate the quality of blood. So, these features are named as blood indices.

Importance of blood indices:

Blood indices have got diagnostic value in determining the type of anemia.

Different blood indices:

Following are the various blood indices.
1. Mean Corpuscular Volume
2. Mean Corpuscular Hemoglobin
3. Mean Corpuscular Hemoglobin Concentration
4. Colour Index

MEAN CORPUSCULAR VOLUME (MCV):

MCV is the average value of a single RBC and it is expressed in cubic microns. The normal MCV is
90 cu mm. When MCV increases, the cell is known as macrocyte and when it decreases, thecell is called
microcytic. MCV is mone in pernicious anemia and megaloblastic anemia in which the RBCs are
macrocytic in nature. MCV is less in microcytic anemia.

For example,
PCV-36%

RBC- 4.5 million cells/cubic litre

MCV = PCV/RBC*10
= 36/4.5*10

= 80 cu. Micron

26
CALCULATION:
Mean Corpuscle Volume:

Volume of packed cell in ml per 1000 ml of blood

MCV  RBCs in millions per cu.mm of blood
PCV in 1000 ml or in 100 ml  100
 RBC count in millions / cu mm
30  10  75 cu.mm
 4
NORMAL VALUE:

If MCV is less than 78 then it is microcytic anemia. If the value is more than 94, it is macrocytic
anemia

Mean Corpuscle Hemoglobin

Hemoglobin in grams per 1000 ml of blood
MCH  RBC count in millions / cu.mm
80
  20 pg
pg
4
NORMAL VALUE:

A figure less than 27 picogram indicates hyperchronic anaemia

Mean Corpuscular Hemoglobin Concentration

Hemoglobin in grams /100 ml of blood
MCHC  PCV in100 ml of blood 100
8 800
  100   26.67%
30 30
NORMAL VALUE:

MCV– Mean Corpuscular Volume = 78 to 90 cu

MCH – Mean corpuscular Hemoglobin = 27 to 32

pg MCHC – Mean Corpuscular Hemoglobin Concentration = 30 to 38%

27
 MEAN CORPUSCULAR HAEMOGLOBIN (MCH):
It is the quantity or amount of hemoglobin present in one RBC. It is expressed in micro gram or
pictogram. The normal value of MCH is 30 pg. It decreases or remains normal inpreniciousanemia and
megaloblastic anemia, in which RBCs are macrocytic and normochromic or hypochromic.It decreases
in hypochromic anemia when MCH is norma, it is called normochromic state.

For example,

Hb = 12.5 g/dl

RBC = 4.8 million cells/ cubic litre

MCH = Hb/RBC*10
= 12.5/4.8 * 10

= 25 Pg

MEAN CORPUSCULAR HAEMOGLOBIN

CONCENTRATION (MCHC):
It is an expression of the average the concentration per unit volume of packed red cell. This is the
concentration of hemoglobin in one RBC. It is the amount of hemoglobin expressed in relation to the
volume of one RBC. So, the unit of expression is percent. This is the most important absolute value in
the diagnosis of anemic. The normal value of MCHC is 30%. It decreases in iron deficiency anemic in
which, RBC‟s are microcytic and hypochromic. It is expressed in g/dl or in %

For example,

Hb = 12.5g/dl

PCV = 44 %

MCHC = Hb/PCV * 100

= 12.5/44 * 100 = 28.57 %

Result:
Thus the values of PCV, MCV, MCH, MCHC of the blood sample have been studied.

28
 EXP. NO.
HEARING TEST – TUNING FORK

10 DATE:
Aim:
To estimate the hearing ability of the ear.
Principle:
The Rinne testis a hearing test, primarily for evaluating loss of hearing in one ear (unilateral hearing
loss). It compares perception of sounds transmitted by air conduction to those transmitted by bone
conduction through the mastoid. Thus, one can quickly screen for the presence of conductive hearing
loss.
A Rinne test should always be accompanied by a Weber test to also detect sensorineural hearing
loss and thus confirm the nature of hearing loss.
The Rinne test was named after German otologist Heinrich Adolf Rinne (1819–1868)the Weber test
was named after Ernst Heinrich Weber (1795–1878).

Procedure
The Rinne test is performed by placing a 512 Hz vibrating tuning fork against the patient's mastoid
bone and asking the patient to tell you when the sound is no longer heard. Once the patient signals they
can't hear it, the still vibrating tuning fork is then placed 1–2 cm from the auditory canal. The patient is
then asked again to indicate when they are no longer able to hear the tuning fork.

Normal hearing
 Air conduction should be greater than bone conduction and so the patient should be able to hear
the tuning fork next to the pinna (outer ear) after they can no longer hear it when held against the
mastoid.

Abnormal hearing
 If the patient is not able to hear the tuning fork after it is moved from the mastoid to the pinna, it
means that their bone conduction is greater than their air conduction. This indicates there is
something inhibiting the passage of sound waves from the ear canal, through the middle
ear apparatus and into the cochlea (i.e., there is a conductive hearing loss).

29
  In sensorineural hearing loss the ability to sense the tuning fork by both bone and air conduction
is equally diminished, implying they will hear the tuning fork by air conduction after they can no
longer hear it through bone conduction. This pattern is the same to what is found in people with
normal hearing, but patients with sensorineural hearing loss will indicate that the sound has
stopped much earlier. This can be revealed by the person administering the test (with normal
hearing) placing the fork close to their own ear after the patient indicates that the sound has
subsided, noting that the sound from the fork is still noticeable to a normal ear.

Air vs. bone conductive hearing loss

Air conduction uses the apparatus of the middle ear (pinna, eardrum and ossicles) to amplify and direct
the sound to the cochlea, whereas bone conduction bypasses some or all of these and allows the sound
to be transmitted directly to the inner ear albeit at a reduced volume, or via the bones ofthe skull to
the opposite ear.

Limitations
This test and its complement, the Weber test, are quick screening tests and are not a replacement for
formal audiometry. Recently, its value as a screening test has been questioned.
The Rinne test is not reliable in distinguishing sensorineural and conductive loss cases of severe
unilateral or total sensorineural loss. In such cases, bone conduction to the contralateral normal ear will
be better than air conduction, resulting in a false negative. In such a case, the Weber test will, however,
show signs of lateralization, implying some kind of pathology. Formal audiometry testing would be
required if any abnormal result is presented.

Result:
1.Air Conduction and 2, Bone Conduction values have been noted down.

30
 EXP. NO.
VISUAL ACUITY
11 DATE:

Aim:
To determine the defects in the eye and correcting it.

Theory:
An eye chart is a chart used to measure visual acuity. Eye charts are often used by health care
professionals, such as optometrists, physicians or nurses, to screen persons for vision impairment.
Ophthalmologists, physicians who specialize in the eye, also use eye charts to monitor the visual acuity
of their patients in response to various therapies such as medications or surgery.
The chart is placed at a standardized distance away from the person whose vision is being tested. The
person then attempts to identify the symbols on the chart, starting with the larger symbols and
continuing with progressively smaller symbols until the person cannot identify the symbols. The
smallest symbols that can be reliably identified is considered the person's visual acuity.
The Snellen chart is the most widely used. Alternative types of eye charts include the logMARchart,
Landolt C, E chart, Lea test, Golovin–Sivtsev table, the Rosenbaum chart, and the Jaeger chart.
Charts display several rows of optotypes, which are standardized symbols for testing vision. Optotypes
are usually letters, numbers, or geometric symbols. Each row of the chart depicts optotypes of a
different size. Typically, the largest optotypes are in the top row. The optotypes become progressively
smaller towards the bottom of the chart.
The person removes any glasses or contact lenses and stands or sits a standardized distance from the
chart (e.g., 20 feet for the Snellen chart). The person is then asked to identify the optotypes on the chart,
starting with large rows and continuing to smaller rows until the optotypes cannot be reliably identified
any more. The row in which the person can reliably identify symbols defines the visual acuity.
One eye is tested at a time. Practically, this is accomplished by covering the other eye with a hand, piece
of paper, or a small paddle. After testing without glasses or contact lenses, testing is repeated while the
person wears them, if applicable. Often, the use of such refractive lenses will correct visual acuity to
normal. Refractive error can be corrected using a pinhole occluder. If the visual acuity improves with
the use of pinholes, refractive lenses can be utilized to improve visual acuity. Squinting can
achieve the same effect as a pinhole occluder.

31
SNELLEN‟S CHART TUMBLING E CHART

EYE TEST RINGS

JAEGER‟S CHART

32
With the Snellen chart, the visual acuity is recorded as a fraction with 20 in the numerator (top
number) and values ranging from 10 to 600 in the denominator (bottom number). The denominator
indicates the distance in feet at which a person with normal vision could stand to correctly identify
the same symbols identified by the person tested. For example, a visual acuity of 20/20 is considered
normal.

The Snellen Eye Chart

Many different variations of the Snellen eye chart are used, but generally they display 11 rows of capital
letters with the top row containing one letter, usually the capital letter “E”. Subsequent rows contain
letters that get gradually smaller.During your eye exam, your eye doctor will ask you to find the
smallest line of letters that you can see and ask you to read it. If you can read the bottom row of letters,
your eye sight is very good.

Tumbling E Charts
The tumbling E chart is often used to test the visual acuity of young children and others who can‟t read
letters aloud. Other times it is used when the person being tested is illiterate or has another handicap
that makes it impossible for him to recognize letters or read them aloud.
This chart works the same way as a standard Snellen eye chart, but all of the characters on the chart
are capital letter “E” in different spatial alignments. That is, they are rotated in increments of 90
degrees and facing either right, left, up or down.
The person being tested is asked to use extended fingers to show which direction the “fingers” of the E
are pointing. It has been shown that visual acuity measurements using a tumbling E chart are
essentially the same as those obtained from testing with a standard eye chart.

Jaeger Eye Charts

Your eye doctor may use a small hand-held card called a Jaeger eye chart to test your near vision. This
chart consists of short blocks of text in various type sizes. The type scale ranges from J1 to J11 or
larger, with J1 being the smallest type. J2 is considered the equivalent of 20/20 distance vision at the
reading distance indicated on the card, which is usually 12 to 14 inches from your eyes.

Depending on what your eye doctor is trying to measure, the Jaeger chart can be used in two
different ways.
 The chart is held at a certain reading distance and you are asked to read the smallest type you
can see.
 The chart is moved forward and back until you are able to read a specific type size.

33
 What 20/20 Vision Means
In most cases an eye chart is hung on a wall for 20 feet away from your eyes. If the doctor‟s office isn‟t
20 feet long, the eye chart may be hung behind the patient‟s chair and mirrors used to make it appear
in front of them, simulating a distance of 20 feet.

If you can read a letter at 20 feet that most human beings should be able to read at 20 feet, you have
20/20 vision. This is considered “normal” vision.
If you can read the big E at the top but nothing below it, your vision is considered 20/200, which is very
poor. It means you can read a letter at 20 feet that people with “normal” vision can read at 200 feet.

Result:
Thus the Visual Activity of an individual of been studied.

34
 AUGMENTED
EXPERIMENTS

35
EXP. NO. 12

PREPARATION OF SOLUTIONS

DATE:

Aim:
To know the preparation of percentage, molar and normal solutions.

Principle:
 The substance which is dissolved is called a solute.
 The substance in which the solute is dissolved is called a solvent.
 A homogenous mixture consisting of two or more substances that forms a single phase is called
solution.
Characteristics of a solution are identically distributed through it. A solution is stable in given
conditions. Solution is on one phase means that the whole of it is either gaseous, liquid or solid.

Percentage solution:
“Percentage solution” is used to describe a solution with the unit %. It is very common to
express the concentration of solution in terms of percentages. Percent means per 100 parts, where for
solutions, part refers to a measure of mass (μg, mg, g, kg, etc.) Or volume (μl, ml, L, etc). In percent
solutions, the amount (weight or volume) of a solute is expressed as a percentage of the total solution
weight or volume. Percent solutions can take the form of weight/volume % (wt/vol % or w/v
%), weight/weight % (wt/wt % or w/w %), or volume/volume % (vol/vol % or v/v %). In each case, the
percentage concentration is calculated as the fraction of weight or volume of the solute related to the
total weight or volume of solution.

36
 As noted above, weight refers to mass (i.e., measured on a balance). When examining the
equation for each of the percent solutions above, it is very important to note that in all cases the
denominator refers to the solution mass or volume and not just the solvent mass or volume. Thus,
solution mass is the combined mass of solute and solvent, and solution volume is the combined volume
of solute and solvent.

Molarity:
Molarity (M) is defined as the number of moles of solute per litre of solution.

Molarity = moles of solute/litres of solution

A molar solution is an aqueous solution that contains 1 mole (gram-molecular weight) of solute
in 1 litre of the solution. This is the method most frequently used by chemists to express concentration.
Molar concentration (molarity) is not same as molar solution.

Molar concentration, also called molarity, amount concentration or substance concentration is a

measure of the concentration of a solute in a solution, or of any chemical species, in terms of
amount of substance per unit volume. A commonly used unit for molar concentration in
chemistry is the molar which is defined as the number of moles per litre(unit symbol: mol/L or M). A
solution with a concentration of 1 mol/L is equivalent to 1 molar (1 M). Molarity, also known as molar
concentration, measures the number of moles of a substance present in per litre of solution. Molarity is
denoted with a capital M. Morality and normality are two methods of measuring concentration. These
two concepts play an important part in breaking down the mixtures and solutions. These two
measurements help us determine the amount of substance dissolved in another substance.

Normality:
Normality is basically a measure of concentration that is equal to the gram equivalent weight
per litre of solution. An equivalent is the number of moles of reactive units in a compound. Gram
equivalent weight is a measure of the reactive capacity of a molecule. The normality of a solution is the
concentration expressed as the number of equivalent weights (equivalents) of solute per litre of solution.
Normality is denoted with a capital N. Normality of an acid is how many H+ ions it has that can be
donated to a solution. Similarly, in a base, normality is determined by how many OH- ions does the
compound have to negate the hydrogen ions in the solution.

The normality of a solution is determined by the solution‟s role in the reaction. Normality is
indicated by Eq/L and mol/L. Normality is commonly used in acid-base reactions to express the
concentration of protons (H+) or hydroxide ions (OH−) in a solution. An example of normality: one
mole of hydrogen ions is equal to one equivalent of hydrogen ions. Every substance may be assigned
an equivalent weight. The equivalent weight may be equal to the formula weight (molecular weight,
mole weight) of the substance or equal to an integral fraction of the formula weight.

37
The normality of a compound can be determined by the number of H+ and OH- ions present in
thesolution.

38
39
40