PRACTICAL CLASS MANUAL

VETERINARY PHYSIOLOGY
Cardiovascular and Respiratory Physiology
Name ___________________________
Admission No.___________________

Compiled by
Dr. R. Kumar, Professor
Dr. K. B. Sharma, Professor

Department of Veterinary Physiology

College of Veterinary & Animal Sciences
CSK HPKV, Palampur 176 062

2009
2009

PRACTICAL CLASS MANUAL

VETERINARY PHYSIOLOGY
Cardiovascular and Respiratory Physiology

Name_________________________________
Admission No.________________________

Compiled by
Dr. R. Kumar, Professor
Dr. K. B. Sharma, Professor

Department of Veterinary Physiology

College of Veterinary & Animal Sciences
CSK HPKV, Palampur 176 062

2009
2009

TABLE OF CONTENTS
Sr.
No.
1.

Name of Experiment

Page
No.
1

Introduction to haematology

2.

Introduction to anticoagulants

3.

Collection of Blood Samples from various

animals and birds

4.

Coagulation of Blood, Bleeding Time and

Clotting Time
5.

Erythrocyte Sedimentation Rate

10

6.

Determination of Packed Cell volume

13

7.

Estimation of Haemoglobin

16

8.

Erythrocyte Fragility test

20

9.

Blood Grouping

22

10.

Total Erythrocyte Count (TEC)

25

11.

Total Leukocyte Count (TLC)

30

12.

Preparation of Blood Smear and Differential

33

Leukocyte Count (DLC)

13.

Platelet Counting

38

14.

Blood Pressure- Mean Arterial Pressure

41

(MAP) and Central Venous Pressure (CVP)

15.

Recording of Electrocardiogram (E.C.G.)

45

16.

Recording of Respiration rate, Heart rate

50

and Pulse rate

DEMONSTRATION EXERCISES:
17.

Plasma Volume and Blood Volume

54

18.

Cardiac Output

57

Annexure:

Normal

Cardiac,

Respiratory

Hematological Values In Different Animals

And

61

Date

Instructors
Signature

INTRODUCTION TO HAEMATOLOGY
Haematology is the study of blood and is concerned primarily with the study of formed
elements of blood. These include:

Erythrocytes or red blood cells (RBC)

Leucocytes or white blood cells( WBC)

Thrombocytes or platelets (PLT)
Routine haematological examination includes the determination of haemoglobin
concentration, haematocrit (HCT) or packed cell volume (PCV) of cells in circulation and
differential count of leucocytes based on the study of stained blood smear. Besides this,
bleeding time, clotting time, specific gravity of blood, erythrocyte fragility etc are also
determined.
Components of blood and their functions
Blood is the fluid tissue of the body that flows through the vascular channels (arteries,
capillaries and veins) and transports the vital nutrients and waste products of the body.
Other important functions include defending the body against micro-organisms,
homeostasis and maintenance of body temperature.
The blood has two major components- cellular and fluid. The cellular component
consists of erythrocytes, leucocytes and thrombocytes. Thrombocytes are smallest (1 to
4 m in diameter) and the leucocytes are the largest of all the cells with a wide range of
size (9 to 20 m).
Red blood cells constitute the highest number (6.3 106 /l) of formed elements in
circulation followed by platelets (2.5 -5 lakh /l) and white blood cells (5 to12 103 /l).
Note: 1 litre =106 l and 1l =1 cumm. Expression cumm is now obsolete.
Formed elements are suspended in the fluid component of blood called plasma and
are thus able to perform their functions under normal conditions. However, if blood is
removed from the blood vessel, it soon clots. The blood remains fluid as long as it
remains inside the vascular system. On clotting, the clear fluid separates out called as
serum, while the formed elements are trapped in the clot. Composition of serum is similar
to plasma except that the protein fibrinogen, which is present in plasma is absent in
serum as it is converted to fibrin and used in clot formation. Thus, if blood is being
collected for haematological examination, it should not be allowed to clot.

EXERCISE NO. 1: INTRODUCTION TO ANTICOAGULANTS.

These are the chemicals which exert their effect by creating a clotting defect
resembling that of certain clinical diseases, during which there is inhibition of formation of
one or more clotting factors. The various anti-coagulants used are:
1. Ammonium and Potassium Oxalate: 6 parts of Ammonium Oxalate hydrated
1.38 gms, Potassium Oxalate hydrated 0.89 gms and Water 100 ml. 0.1 ml of this is
enough for 1 ml of blood. To prevent deterioration, 1 ml of 40% formalin is added.
The blood collected can be very safely used for haematocrit, haemoglobin
estimation, RBC and WBC counts, prothrombin time and bilirubin concentration etc
except for ESR. If the blood film is to be drawn; it should be made without delay after
the collection of blood with double oxalate.
Let the anti-coagulant (double oxalate) be mixed up with the blood in the collecting
tube depending upon the volume of blood to be collected. For collecting 5 ml of
blood, pipette 0.5 ml of double oxalate solution in the tube and keep in oven at 700 C.
The oxalate will spread along the inner side of the tube uniformly.
2. Sodium Citrate: 3.8% aqueous solution is used. To 9 parts of blood, 1 part of
sodium citrate is added and mixed. One ml of blood requires 2-4 mg of sodium
citrate.
3. Heparin: It is a natural anti-coagulant which aids in maintaining blood in a fluid state.
The main source in the body is mast cells (liver, lungs etc). Heparin inhibits the
reaction between thrombin and fibrinogen and also interferes with the formation of
thromboplastin, which is potentiated by Heparin co-factor normally present in the
blood. Heparin retards agglutination and lyses of platelets. Heparin is used in the
ratio of 0.1 to 0.2 mg per ml of blood. Heparinised blood should not be used for blood
films as it gives faint blue colouration to the back-ground on staining.
4. E.D.T.A. or Versene or Sequestrene: The di-sodium salt of ethylene diamine-tetraacetic acid (EDTA) is a powerful anti-coagulant. It forms highly stable chelates with
divalent metals. It is used in the concentration of 1 to 2 mg per ml of blood. This can
be used in the blood transfusion also. It also prevents blood platelets clumping in
vitro, so can be employed for platelets count.
5. Dicoumarol (Bishydroxycoumarin): It is obtained from spoiled sweet clover. This is
inactive in vitro. The coumarin anti-coagulants affect the transport of vitamin K to its
site of action. Vitamin K is required for normal synthesis of prothrombin and other
clotting factors. It interferes with factor VII and factor X in the liver. It also reduces the
Christmas factor (IX). It reduces the adhesiveness of platelets. It also increases

antithrombin concentration in plasma (the interaction between thrombin and

antithrombin has a destructive nature). It is used to check the intravascular
coagulation effect which is reversed by administration of vitamin K.
6. Hirudin: It is present in the buccal glands of leech. It is a powerful anti-coagulant. It
inhibits the conversion of prothrombin to thrombin.
7. Venoms: The venoms of certain snakes contain a powerful anti-coagulant effect,
which interferes with the action of thromboplastin or they deplete the blood
fibrinogen.
8. Contact and Smoothness of Surface: The clotting of the blood can be prevented
when it comes in contact with the tissue fluid or with the substance which is wet or
has a smooth surface. When the vessel in which the blood is collected is coated with
thin layer of paraffin wax, blood remains in fluid state for half an hour.

EXERCISE NO. 2: COLLECTION OF BLOOD SAMPLES FROM DIFFERENT FARM

ANIMALS.
Blood: It is a connective tissue in a fluid state. Blood consists of cellular elements and
the fluid called the plasma in which are suspended the red cells (Erythrocytes), white
cells (leukocytes) and platelets (Thrombocytes). The mammalian blood contains about
52% plasma and 48% corpuscles by volume. By weight they are 55% and 45%
respectively. The plasma contains a large number of proteins and enzymes. For the
studies on the blood, it is collected from various sites/ routes in different farm animals.
Procedure: The blood can be collected from different blood vessels of the body (artery,
arteriole, capillaries, small veins, large veins and even from heart), but the veins being
superficial in their location and quite easily approachable, the blood is normally collected
from the veins. The venous blood also gives a clear picture of the arterial blood and also
serves well for both haematological examinations and biochemical estimations. The
capillary and the arterial blood can also be used but is difficult to collect.
In case of large animals such as cattle, horses, sheep, goat, camel and buffalo, the
blood is mostly collected from jugular veins (present in the jugular furrow at the neck). In
case of smaller animals e.g. dog and cat, the blood is collected from cephalic vein or
femoral vein.
The blood can easily be colllected with a syringe and needle or with a hypodermic
needle alone. The needle used should be sharp. Before collecting the blood, see that the
syringe and needle are clean and sterile. Hairs from the area over the jugular furrow are
clipped and the part is cleansed and disinfected with 70% alcohol or methylated spirit.
The vein is distended by putting a little pressure at the base of neck at jugular furrow with
the thumb or fingers and needle of appropriate gauge is inserted in the jugular vein. The
gauge/length of the needle and site of blood collection for different animals should be as
follows:
Species
Horse
Camel
Cattle
Dog
Sheep & Goat
Pig

Gauge*
18 - 20
18 - 20
16 - 18
20 - 22
18 - 20
16 - 20

Length of needle in inches Site of blood collection

2-3
Jugular Vein
2-3
Jugular Vein
2-3
Jugular Vein
2-3
Cephalic/Femoral Vein
2-3
Jugular Vein
2-3 & 6 when the blood is to Jugular Vein or Ear Vein
be collected from the ear
Cat
20 - 24 1
Cephalic/Femoral Vein
Poultry
20 - 24 3/4 - 1
Heart or wing vein
Rabbit
18 - 22 1 - 3
Heart
*One gauge =One part of an inch, for example 15 gauge is 1/15th of an inch.

When little blood is required, it can be obtained from the ear vein. Select small vein on
the back of the earlobe, clip the hair, clean the site with methylated spirit and allow it to
dry. Keep the head steady. The vein is punctured by a sharp prick with the help of a
hypodermic needle. If the blood flow is in stasis and comes after squeezing only, then
make a fresh puncture and collect the blood.
Pigs: The blood is collected from tail, ear vein and heart using hypodermic needle of 1420 gauge and 1-6" length depending upon the site of the collection of blood. The neck of
the pig is very thick, so it is difficult to approach the jugular vein. While collecting the
blood from the heart, insert the needle between 2nd and 3rd ribs; push it downward and
forward until it pierces the heart. There will be a gush of blood. But once the heart is
injured, there will be continuous bleeding and that may lead to the death of the animal.
So the collection of blood from the ear and tail is safe.
Poultry: In poultry blood is collected from wing vein and heart. Remove the feathers over
the vein. Apply 70% methanol. Press the vein with the help of thumb and wing vein will
become prominent. Insert hypodermic needle of 20 - 24 gauge and 3/4" - 1" length in
opposite direction to the flow of the blood. A needle of 3" length and 20 - 24 gauge
should be used for collection of blood from heart. Push the needle downward and
between the clavicles or over the window on the left side of tissues formed by ventral
canal and ribs. If the heart is injured more, excessive bleeding will cause the death of the
bird.
Precautions:
1. The needle should always be inserted in opposite direction of the flow of blood.
2. The blood can be collected directly or transferred into a vial or tube containing
anticoagulants. Pressure should be released before the withdrawal of hypodermic
needle from the vein to avoid formation of haematoma.
3. Four to five ml of the blood is sufficient for all haematological examinations.
4. Syringe used must be cleaned and dried and the blood must not be allowed to
coagulate in the syringe. The syringe should be cleaned immediately after the
collection of the blood.

EXERCISE NO. 3: COAGULATION OF BLOOD

It is the conversion of fibrinogen to fibrin which forms meshwork of proteins, in the
interstices of which the formed elements of blood are entangled and the serum, a yellow
coloured fluid oozes out. During coagulation, first of all, there is formation of
thromboplastin which is formed from two sources that is Extrinsic (from tissue) and
Intrinsic (from plasma), which converts prothrombin to thrombin in the presence of
ionized calcium and globulin fractions of normal plasma that is Factor V, Factor VII and
Factor X. The thrombin so formed acts on the two arginyl - Glycine bonds of fibrinogen to
liberate peptide material and expose the donor groups required for subsequent
polymerization of molecules by disulphide bonds to form fibrin. The whole process of
coagulation of blood can be divided into three phases i.e.
First Phase: Antihaemophilic Globulin + Platelets+Plasma thromboplastin Component
(PTC) Intermediate product
Intermediate product + Factor V+ Factor VII + Ca Thromboplastin + Ca
Second Phase: Prothrombin Thrombin
Third Phase: Fibrinogen Fibrin (Clot) + Factor XIII (Firmness of clot or syneresis)
Coagulation Time or Clotting Time: Many methods have been used by the
haematologists for the determination of coagulation time and the results obtained do not
match with one another due to differences in the techniques used and the conditions to
which blood samples are subjected.
1. Drop Method:

Place several drops of blood on a clean glass slide and go on

passing a needle through one drop at a time at one minute interval. When the fibrin
threads stick to the needle and are dragged along it, the coagulation has taken place
and the time is noted from a stop watch from the time of collection of blood to the time
of coagulum formation. In dry warm weather, the slide should be kept on beaker of
warm water to avoid evaporation.
Precaution: The blood should be collected from a sharp deep puncture and the first
few drops should be discarded.
2. Modified drop method: Two glass discs 5mm in diameter are cemented to glass
slide. Blood is placed in these and the slide is kept inverted on beaker of warm water
(40 0C). The coagulation is noted by seeing the shape of the drop while keeping the
slide vertical.
3. Capillary Method: The method is most commonly used and found satisfactory. In
this method, the blood is drawn into the fine capillary tubes. The site is cleaned, dried

and punctured with a sharp needle. First drop is wiped off. The second drop is drawn
into the capillary tube filled by capillary action. Avoid touching of the tube to the skin
of the animal. After one minute place the capillary tube in such a way that index finger
is kept above and thumb below the capillary tube and break a bit of the capillary with
slight pressure from thumb. If there is no thread formation between the two broken
ends when taken apart gently, then break the other one after every 30 seconds
intervals till the gap is bridged. Note the time from the time of receiving the blood till
the bridge formation. This is the coagulation time of that individual.
4. Lee and White Method: Take three clean test tubes rinsed with 0.85% normal saline.
Remove blood from vein with a clean syringe and needle rinsed with saline. Remove
needle and transfer gently about 1 ml of blood to each tube. Place the tube in water
bath at 370C. After about two minutes, gently tilt one of the tubes and observe for
clotting. If the clot has not formed, tilt the second tube followed by third tube. Repeat
the process after every one minute interval till the third tube shows clotting. The time
is noted since the puncturing of vein and the development of firm clot in third tube.
Precautions: The tube used should be small in internal diameter i.e. 8 mm. The vein
should be punctured cleanly with the first attempt. The suction of the blood should be
gentle to avoid drawing of air bubbles into syringe. The tube should be kept close to
the body temperature.
Haemostasis: It is the arrest of bleeding. When a blood vessel is damaged, the platelet
masses aggregate at the site of the injury occluding the flow of blood and prevent
bleeding. The immediate arrest of bleeding is aided by:
1. Constriction of blood vessel walls (by 5-HT liberated from disintegration of
platelets).
2. Formation of blood platelet plug (The platelets first of all agglutinate and then
fuse to form a solid mass). The agglutination of platelets requires the presence
of ADP, traces of thrombin and a protein factor. The action of ADP is calcium
dependent.
A few minutes after the onset of bleeding, the clotting of blood occurs. After about half
an hour when the capillaries reopen due to accumulation of the tissue metabolites, the
area is sealed and the haemostasis is maintained.
Later the clot retracts and is replaced by the fibrous tissue. The fibrin network of the
clot may act as scaffold for the new collagen and new capillaries. The haemostasis will
be normal when there is:
1. Vasoconstriction.

2. Formation of plug by platelets and 3. The coagulation takes

place in normal way. Otherwise haemostasis is deranged e.g. during thrombocytopenia

when the circulating platelets concentration is below 50,000/cu mm and the blood
continues to ooze in small drops (purpura haemorrhagica). The bleeding time is
prolonged due to failure of constriction of minute blood vessels, low concentration of
platelets and due to the defective coagulation.
Bleeding Time: It is the time taken for the formation of clot at the point of puncture,
detected by watching when bleeding stops as ascertained by frequent soaking of the
blood by means of a filter paper. It can be measured by:
Dukes' Method: To determine the bleeding time by Dukes' method, the hair are
clipped from the back of ear lobe. Moisten a piece of cotton with 70% alcohol and
sterilize the middle or tip of the ear of animals and middle of the finger in human being.
Let this dry. Make an incision 4 to 5 mm deep with a sharp scalpel near the tip of the
lobe of ear or in the pulp of finger. Place a half folded filter paper after half minute
interval over the site from where the blood is oozing out of the puncture. The filter paper
should be placed gently over the site. The blood spot on the filter paper become smaller
and smaller with passage of time. This shows that the flow of the blood is gradually
ceasing. When the blood ceases to flow, record the time. Bleeding time is the time
elapsed between the puncture and cessation of bleeding. The normal clotting and
bleeding time in the farm animals and man is as follows:

Animal

Coagulation Time

Bleeding Time

Horse

< 11 min.

1 to 5 minutes in most
domestic animals.

Cattle or Ox

6 min.

Sheep

1 to 2 min

Goat

2 min.

Buffalo

4 to 6 min.

Pig

3 min.

Dog

2 min.

Cat

1 min.

Fowl

4 min.

Rabbit

4 min.

Man

3 to 10 min.

Exercise No. 3: Determination of Clotting Time and Bleeding Time:

Species

Clotting Time

Bleeding Time

Cattle
Buffalo
Sheep
Goat
Dog
Fowl
Human

Date:

(Instructor)

EXERCISE NO. 4: ESTIMATION OF ERYTHROCYTE SEDIMENTATION RATE (ESR)

Erythrocyte Sedimentation Rate (ESR): When citrated or oxalated blood is allowed to
stand undisturbed, the corpuscles aggregate into clumps and start falling down at a
certain rate. The fall of corpuscles during a certain fixed time in the blood is determined.
Normally, this rate is uniform by virtue of the plasma viscosity due to serum globulin,
fibrinogen, lipids, amino acids etc and out of which globulin is probably the most
important factor. The increase in fibrinogen and globulin concentration leads to an
increase in sedimentation rate whereas increase in albumin decreases the sedimentation
rate. The various methods employed for the study are:
1. Linzenmeier method
2. Westergren method
3. Wintrobe method
1.

Linzenmeier method: The time required for the upper level of corpuscles to fall to
a fixed distance is measured.

2.

Westergren method: The fall of the corpuscles which have taken place during the
specified time is noted. This is widely used method and most accurate.

Method: Fill the Westergren tube up to `0' mark and fix it in the stand. While fixing the tip
of the tube, it must be pressed hard against the rubber at the bottom before releasing the
finger; otherwise the blood will flow out. Note the fall of the corpuscles after a fixed time.
3. Wintrobe method:
i)

Collect the venous blood in a tube containing an anti-coagulant.

ii)

Take a clear dry Wintrobe tube and fill the tube up to the 10 cm mark either with
the help of a syringe and needle with polyethylene catheter sufficient to reach to
the bottom of Wintrobe tube or with a Pasteur pipette.

iii)

Adjust the blood present above the mark with the help of a filter paper or cotton.
Place the tube in the stand and keep it undisturbed.

iv)

Note the fall of the corpuscles at intervals of 10, 20, 30, 60 minutes to 24 hours.
The erythrocyte sedimentation rate is always represented as mm/time interval.

Precautions:
i)

The blood collected should be used for determination of sedimentation rate within
two hours of its time of collection. Further delay may be associated with
increased suspension stability of the blood.

ii)

Since the sedimentation rate increases with the increase in temperature, so the
test be carried out at a temperature not less than 220C and not more than 370C. If

10

the blood used has previously been kept in a refrigerator, it should be allowed to
come to the above temperature before being used.
iii)

The haematocrit tubes should be kept vertical as a 30 change in the angle from
the vertical will considerably increase sedimentation rate.

Factors affecting ESR:

i)

Presence of anticoagulants: - An increase in sodium salicylates will lower the

ESR. It also depends on the plasma fibrinogen concentration.

ii)

External temperature: - ESR is increased during increase in temperature i.e. when

the temperature is more than 37.50C and decreased when environmental
temperature is less than 220 C.

iii)

Presence of fibrinogen and globulin in high concentration increases the ESR.

iv)

Presence of albumin in high concentration retards the ESR.

v)

Concentration of RBC: - In anaemia, the ESR is increased (organic diseases) but

under certain conditions there is no effect on ESR e.g during anaemia (functional
disorders) as retardation force is equal to sedimentation rate.

vi)

Addition of lecithin delays the ESR.

vii)

Addition of cholesterol increases the ESR.

viii)

Presence of pneumococcus type III produces an increase in ESR. It is also

increased during pulmonary TB, old age, traumatic pericarditis.

ix)

Position of the tube: - In horizontal condition the retardation force is equal to the
downward force, but when the tube is inclined the retardation force is less than
downward force, so there is an increase in ESR.

x)

The size of the displacing particle i.e. by aggregating the RBC (Rouleaux
formation) causes increased ESR during normal conditions, diseases and during
pregnancy.

xi)

In conditions of inflammation or tissue destruction, the globulin content as also

fibrinogen content go up and consequently there is increase in the sedimentation
rate. A steady rise is an indication of worsening of the condition.

xii)

During chronic infections, globulin and fibrinogen contents are increased i.e.
Pulmonary tuberculosis, Pneumonia, septic infections and pregnancy etc

Erythrocyte Sedimentation Rate in different domestic species of animals:

Horse

: 15-38 mm. /40 Min.

Cattle

: 0-2 mm. /60 Min.

Dog

: 1-6 mm. /30 Min.

Pig

: 0-6 mm. /30 Min.

Cat

: 1-6 mm. /30

Min.

11

Exercise No. 4. : - Determination of Erythrocyte Sedimentation Rate (ESR) in cattle

and Buffalo blood.
Observations: - For Cattle
Time (Min.)

Fall of Corpuscles (mm)

E. S. R (mm/min.)

Fall of Corpuscles (mm)

E. S. R (mm/min.)

0
5
10
15
30
60
120

For Buffalo:
Time (Min.)
0
5
10
15
30
60
120

Date:

(Instructor)

12

EXERCISE NO. 5: DETERMINATION OF PACKED CELL VOLUME (PCV) OR

HAEMATOCRIT VALUE
The term haematocrit means to separate the blood. By centrifugation, the blood is
separated into three distinct compartments namely; (a) the erythrocyte mass at the
bottom called the packed cell volume or PCV, (b) a white to grey layer of leukocytes and
occurring immediately above the red cell mass and called Buffy coat and (c) the blood
plasma on the top. There are two methods of determining PCV i.e. Wintrobe tube
method and Micro-capillary method.
a) Wintrobe Method:
Procedure:
i)

Take a Wintrobe tube having a uniform 3mm bore and calibrated by a 10 cm scale
with millimetre divisions. The scale on the left side is read from top to bottom for
ESR while scale on the right in the reverse order for PCV.

ii)

Take anticoagulant added blood in a syringe to which a needle is already fixed

with a polyethylene tubing of a 2mm diameter or suck the blood into a Pasteur
pipette with a rubber bulb attached.

iii)

The tip of the polyethylene tubing or Pasteur pipette having a long narrow end is
inserted to the bottom of the haematocrit tube (Wintrobe tube) and the blood is
forced out by pressure on the syringe/rubber bulb. The pipette is slowly withdrawn
simultaneously.

iv)

Fill the Wintrobe tube up to the mark 0, i.e. 1 ml of blood.

v)

Place the tubes in the centrifuge machine and centrifuge at the speed of 3000 rpm
for 30 minutes.

vi)

After thirty minutes, read the packed cell volume from the bottom to top. The
packed cell volume is read in percentage.

b) Micro-capillary Method:
Procedure:
Take micro capillary tube and fill 2/3rd of it with blood (anticoagulant mixed blood
for non-heparinized capillary and fresh blood without anticoagulant for heparinized
capillary). Apply sealing wax/clay on one side and centrifuge at 8000 rpm for 8 minutes.
Place the capillary tube on the reading plate device and PCV in percentage.

13

Precautions:
1.

Use anticoagulant added blood for estimating the PCV with Wintrobe tube method
and while using non-heparinized capillary in Micro-capillary method.

2.

The Wintrobe tube should be filled up to the top i.e. up to 0.00 and avoid formation
of air bubble(s) in the tube.

3.

In case the level of the blood has gone above 0.00 mark, adjust it immediately with
cotton or cloth.

Normal PCV values in % for various domestic species are:

Cattle

35 (24-48)

Cat

37 (24-45)

Horse

42 (32-55)

Sheep

38 (24-50)

Dog

45 (37-55)

Goat

35 (24-48)

14

Exercise No. 5: Determination of Packed Cell Volume (PCV) or Haematocrit value.

Cattle

Buffalo

(i) Wintrobe Method

-----

-----

(ii) Micro-capillary Method

-----

-----

PCV in % as determined by

Date:

(Instructor)

15

EXERCISE NO. 6: ESTIMINATION OF HAEMOGLOBIN

The haemoglobin can be measured by:
1.

Colorimetric procedures.

2.

Gasometric methods: i) Oxygen Capacity ii) Carbon Monoxide Capacity

3.

Physical measurement: i) Sp. Gr. of blood ii) Haematocrit readings

iii) Refractive Index.

4.

Chemical Methods: i) Iron concentration.

1. Colorimetric Procedures: Either by visual means or by photometric methods,

haemoglobin is measured by:
1) Direct method: Tallquist Method
2) Indirect methods: Oxyhemoglobin method or first convert the haemoglobin to
a) Acid hematin

b) Alkali-hematin

c) Cynamethemoglobin

d) Cyanohaematin

e) Pyridine-hemochromogen

1. Tallquist Method: Tallquist in 1900 designed a series of lithographed colours

supposedly representing haemoglobin values in grades of 10 to 100%. The nondiluted blood, collected on a piece of absorbent paper, is compared with these. The
margin of error is between 20 and 50% making it unsuitable for use.
2. Oxyhaemoglobin Method: In this method, blood is diluted with water and matched
by eye with a standard picrocarmine solution or a wedge of glass or variable glass
standards.
a. Acid Haematin Method or Sahli's Method (1895) (Clinical Method).
1. Principle: When haemoglobin reacts with N/10 HCl, it forms Acid Haematin which
is brown in colour.
2. Apparatus: Sahli's haemoglobinometer, N/10 HCl, pipette with adapter.
3. Chemical reaction: Haemoglobin + HCl ------------> Globin + Acid haematin
(Brown colour)
The maximum colour is attained in 40 minutes and after this the colour begins to
fade.
4. Procedure: Take N/10 HCl up to 2 mark into an empty graduated tube. Suck in the
blood up to the 20 l mark of the measuring pipette and add it to the graduated tube
containing 0.1 N HCl. Thoroughly mix it with blood. Keep it aside undisturbed for 710 minutes. After 9 -10 minutes, about 95% of the final brown colour is attained. The
resulting brown fluid is then diluted with distilled water or 0.1 N Hydrochloric Acid until
it matches with the brown glass standard. Read the value of haemoglobin from the

16

scale by noting the height of the column of the diluted acid haematin. The values are
always represented in gm%.
Precautions:
1. Measure the blood accurately up to the mark i.e. 20 l.
2. Wipe off the blood sticking on the outside of the pipette.
3. Air bubbles formation while mixing blood and acid should be avoided.
4. Dilution with water or Acid should be accurate. If the match point is passed, the
whole process must be repeated.
5. The same time interval should be observed on each occasion the instrument is
used. It should be standardized under same conditions.
6. In making comparisons, several rapid readings must be made and the average
of these taken; otherwise the eye will become fatigued and erroneous readings
will be accepted.
b. Alkali-Haematin Method:
Take 0.05 ml of blood in a tube containing 4 ml of N/10 HCl left at room
temperature for 48 minutes and then diluted to 5 ml with 1N NaOH.
OR
Dilute 0.05 ml of blood to 6 ml with N/10 NaOH. Heat in a boiling water bath for 4
to 5 minutes, cool and read against the standard.
Advantage:
1. The addition of excess of alkali produces a true solution of haematin and much better
solution of plasma proteins and fat where as the acid haematin method is affected by
even non-haemoglobin substances (Protein and lipid) in both plasma and cell stroma.
Moreover acid haematin is in colloidal suspension rather than a trace solution and
makes it unsatisfactory for photometry.
2. With alkali haematin, even carboxyhemoglobin, methemoglobin and sulfhemoglobin
forming 2-12% of the total haemoglobin are also converted to haematin, which is not
possible with acid.
Disadvantage: With alkali-haematin method, the blood of the new born and young
infants carrying small amount of foetal haemoglobin is alkali resistant.

17

c. Cynamethemoglobin Method: Ferricyanide converts haemoglobin iron from the

ferrous to the ferric state to form methemoglobin which then combines with Potassium
Cyanide to produce the stable pigment cyanmethemaglobin.
Composition of Drabkin's diluent:
NaHCO3 = 1.0 gm,
Potassium ferricyanide = 200 mg,
Potassium cyanide = 50 mg,
Distilled water = 1000 ml
(Turbid fluid should be discarded)
Keep the solution in a brown bottle in cool place.
Procedure: Measure 0.02 ml of blood and dilute it with 5 ml of Drabkin's diluent. The
optical density of this solution is then compared with that of standard. The colour
intensity is measured in a photometer set at 540 nm band.
d. Cynahematin Method: This method has a high degree of accuracy. Take 0.02 ml of
blood. Add 1.98 ml of N/10 HCl and leave it for 10 minutes for complete transformation of
acid haematin. Then add 2 ml of 2% sodium cyanide and mix.
Standard Solution: Weigh 28.8 mg of pure crystalline haemin (8.57% Iron). Transfer
it to 1 litre volumetric flask. To this, add 200 ml of 5% sodium cyanide. Add distilled water
to make the volume up to the neck of the flask. Keep it at room temperature till the
haemin is dissolved. Then make the volume 1 litre with distilled water & mix. The colour
intensity is then measured in colorimeter or photometer between 530 and 550 nm.
e. Pyridine-Haemochromogen Method: All haeme pigments are converted into
pyridine- hemochromogen and the intensity of absorption of the latter is measured after
calibration against crystalline haemin. But the smell of pyridine is unpleasant and the
technique is exhaustive.
Normal values of Haemoglobin in gm%
Cattle

- 11 (8-15)

Horse

- 14.4 (11-19)

Sheep

- 11.5 (9-15)

Goat

- 10.0 (8-15)

Dog

- 15 (12-18)

Cat

- 12 (8-15)

18

Exercise No. 6: Determine the haemoglobin content in gm% for the given sample.

Haemoglobin content in gm% in

Name of Method Used

Dog

Cattle

Date:

Sheep

Buffalo

(Instructor)

19

Goat

EXERCISE NO 7: ERYTHROCYTE FRAGILITY TEST

Erythrocyte Fragility Test has been devised to measure the resistance of the
erythrocytes to break down (haemolyse) when subjected to varying concentrations of
hypotonic salt solutions i.e. osmotic fragility test. It was first proposed by Hamburger in
1883. It attempts to quantitate the resistance of these cells to haemolysis in decreasing
strengths of hypotonic saline solutions.
Requirement: Stock solution of 10% buffered sodium chloride. It is prepared by
dissolving 90 gms of sodium chloride, dibasic sodium phosphate (Na2HPO4) 13.655 gms
and monobasic sodium phosphate (NaH2PO4 : 2H2O) 2.43 gms and distilled water to 1
Litre. The solution is stored in a glass stoppered tightly closed bottle.
Procedure:
1. Make 1% working solution from the stock solution by taking 1 ml of stock solution and
addition of distilled water of 9 ml to make total volume of 10 ml
2. From the working solution, make the test solutions of 0.25%, 0.50%, 0.90%
concentration of Sodium Chloride. The first two being hypotonic and the last isotonic
solution.
3. The hypertonic solution is made by dissolving and diluting 2 ml of stock solution to 10
ml with distilled water.
4. Take 5 ml of each of these solutions in labelled test tubes and to each of these add
0.1 ml of blood and examine a drop of each under the microscope under high power.
5. In hypotonic solution, the erythrocytes will be swollen due to absorption of fluid
through the membrane and subsequently burst after sometime. The ghost RBC i.e.,
membrane without pigment is visible. The red blood corpuscles will remain unaffected
in isotonic solution while in hypertonic solution the erythrocytes will give shrunken or
crenated appearance due to passage of contents through the semi-permeable
membrane of erythrocytes.

20

Exercise No. 7: Erythrocyte Fragility Test.

Observations:-

Date:

(Instructor)

21

EXPERIMENT NO. 8: BLOOD GROUPING

Landsteiner in 1901 demonstrated that human beings could be classified into 4
groups depending on whether their red cells contain A or B agglutinogen or both AB or
neither i.e., O. He also showed that there are antibodies (agglutinins) to A and B
agglutinogen named Anti-A and Anti-B and that a person's serum does not contain the
antibody for the antigen present in his own red cells but carries antibodies against the
agglutinogens he doesnt possess. So the ABO blood group system is as follows:
Group

Agglutinogen/Antigen

Agglutinin/Antibody in Serum

Anti-B

Anti-A

AB

A and B

None

None

Anti-A

So far, in human beings 14 blood group systems have been identified.

Requirement: 1. Blood samples. 2. Anti-Serum for A and B.
Procedure:
1. The suspension of RBC is obtained by placing one drop of blood from the finger into
3 ml of normal saline solution. OR 0.5 ml of blood is expelled into a test tube
containing about 10 ml of Normal Saline Solution, the tube is gently shaken to wash
the cells, the supernatant fluid is poured off after centrifugation and fresh saline is
added to make a 1% or 2% suspension.
2. One drop of anti-serum-B is placed on left side of a glass slide and 1 drop of antiserum-A on the right side. One drop of unknown cell suspension is mixed with each
of the antisera followed by tilting of the slide back and forth for 3-5 minutes.
3. Cover the drop with a cover slip and examine under the microscope. On the basis of
clumping seen, the samples are classified into various groups as follows :

No Clumping

- Group-O.

Clumping with Antiserum - A

- Group - A.

Clumping with Antiserum - B

- Group - B.

Clumping with both Antisera

- Group - AB.

22

Slide testing method for ABO and Rhesus D:

Blood group O positive: neither anti-A nor

anti-B have agglutinated, but anti-Rh has

Result: Blood group B negative: anti-A and

anti-Rh have not agglutinated but anti-B has

In the slide testing method shown above, three drops of blood are placed on a cavity
glass slide with liquid reagents. Agglutination indicates the presence of blood group
antigens in the blood.

ABO blood group antigens present

on red blood cells and IgM
antibodies present in the serum

Blood Group Systems: - The blood group system in different species is given in the
table below:
Species

Blood Group
Systems

Blood Group
Factors

Technique used for blood

typing

1. Human being

14

100+

Agglutination

2. Cattle

12

80+

Haemolytic

3. Sheep

30+

Haemolytic Agglutination

4. Horses

30+

5. Pigs

15

65+

6. Dogs

11

15+

7. Cats

2+

8. Chicken

12

30+

-doAgglutination Haemolytic Antiglobulin

-doHaemolytic Agglutination
Agglutination

23

Exercise No. 8: Determine the Blood Group.

S. No. Name of the student

Blood group

-----------------------------------------------------------

-----------

-----------------------------------------------------------

-----------

-----------------------------------------------------------

-----------

-----------------------------------------------------------

-----------

-----------------------------------------------------------

-----------

-----------------------------------------------------------

-----------

-----------------------------------------------------------

-----------

-----------------------------------------------------------

-----------

-----------------------------------------------------------

-----------

-----------------------------------------------------------

-----------

-----------------------------------------------------------

-----------

-----------------------------------------------------------

-----------

-----------------------------------------------------------

-----------

-----------------------------------------------------------

-----------

-----------------------------------------------------------

-----------

Date:

(Instructor)

24

EXERCISE NO. 9: TO DETERMINE TOTAL ERYTHROCYTE COUNT (TEC)

Principle: It consists of an accurate dilution of a measured quantity of blood with a fluid
which is Isotonic with the blood and prevents coagulation.
Apparatus:
1.

A Thoma RBC diluting pipette: It consists of a capillary tube graduated in tenths,

which opens into a bulb containing a red coloured glass bead.

2.

Diluting Solutions: Any of the following diluting solutions can be used for diluting

the blood for TEC.

I

Hayem's Solution:
Sodium Sulphate

-2.5 gm

Sodium chloride

-0.5 gm

Mercuric chloride

-0.25 gm

Distilled water

-100 ml

II Toisson's Fluid
Sodium Chloride

-1 gm

Sodium Sulphate

-8 gm

Methyl Violet

-0.025 gm

Neutral Glycerine

-30 ml

Distilled water

-180 ml

Note: The Toisson's fluid has certain disadvantages as:

a) The specific gravity is very high so the Red Corpuscles do not settle out in it readily.
b) The leukocytes are also stained blue and the counting of White Blood Corpuscles can
also be done but the dilution for red corpuscles is too much to afford for leukocyte
enumeration.
c) The fungi grow in it rapidly, therefore it requires frequent filtering
III Normal Saline: 0.9%
IV Ringer's Solution:

Sodium Chloride

-0.8 gm

Potassium Chloride

-0.0075 gm

Calcium Chloride

-0.01gm

Sodium Bi-carbonate

-0.01gm

DW-

-to make 100 ml

Locke's Solution:
Sodium Chloride

-0.9 gm

Potassium Chloride

-0.042 gm

25

Calcium Chloride

-0.024 gm

Sodium Bicarbonate

-0.02 gm

Glucose

-0.2 gm

DW-

-to make 100 ml

Hayem's solution is the best but it should not be more than two or three weeks old.
3.

Counting Chamber:

It is a heavy glass slide in the centre of which are two ruled

platforms. These are separated from each other by a central moat and from the elevated
bars on each side by transverse moats. These lateral bars are so ground that a cover
slip resting on them lies exactly 0.1 mm above the ruled platforms. On each platform is
engraved a ruled area 3 mm on each side (9 sq mm). This area is divided into 9 large
squares (Primary squares) which are again sub divided, the 8 outside squares being
divided into 16 medium small squares (Secondary squares) and the central one into 400
smallest squares (Tertiary squares).
Each of the largest squares is 1 mm on the side or 1 sq mm in area. Each of the
medium squares is 0.25 mm on side. One smallest square in the central largest square
is 0.05 mm on side or 0.0025 mm2 in area. In the improved Neubauer's chamber, the
400 smallest squares in the central square are arranged into 25 groups of 16 squares
separated by "split" boundary lines (double line).
Method:

Draw blood by suction into a Thoma red cell pipette up to 0.5 mark or if

anaemic to mark 1. Wipe off the blood sticking on the outer surface of the pipette. In
case the blood has slightly passed 0.5 mark, it should be adjusted by touching the tip of
the pipette against cloth or cotton. The diluent is then drawn up to 101 mark. While
drawing the diluent, the pipette is then rotated between the finger and thumb in order to
mix the blood thoroughly with the diluent. Hold the pipette horizontally and mix for half a
minute.
For uniform mixing, hold the pipette loosely in one hand and rotate the attached
rubber tube with thumb and index finger of the other hand. Rotation in one direction
should be avoided. Mixing should be repeated each time before expelling a drop for
examination. Keep the Neubauers chamber on a smooth surface. The cover glass is
then placed on the counting chamber.
Several drops of fluid are expelled from the pipette and discarded. This is done to
expel the Hayems fluid in the capillary which is not mixed up with the blood. A small
drop of mixed fluid is then placed between the cover glass and the ruled platform. The
fluid will run under the cover glass by the capillary action. The fluid should completely
cover the chamber and none should run over the sides.

26

Allow the blood to settle for few minutes and examine for the uniform distribution of
the red corpuscles in the chamber under low power. Then place under the high power
objective of microscope and count the erythrocytes in the central small squares. The
number of cells in the four corner groups of 16 squares and one central one is recorded.
Count in L (inverted or upright) form and omitting the cells lying on or outside the
opposite line.
3mm

3mm

1mm

1mm

1mm

Counting Area in Neubauer's Chamber

Calculations:
Total Erythrocyte count = N x 10,000
N

= Total count of RBC in 5 squares

If the dilution is 1 to 200 (blood drawn to the 0.5 mark), then the total of cells found in
the 5 groups of 16 squares is multiplied by 10,000 in order to give the number of cells per
cu mm of blood. If the dilution is 1 to 100, then total number of cells is multiplied by
5,000.
The length of the side arm of smallest square is =0.05 mm
The area of the smallest squares is = 0.0025 sq. mm. i.e. (0.05 x 0.05 = 0.0025)
The depth
Total volume

= 0.1 mm
= 0.1 x 0.0025 = 0.00025 cumm

Since 80 such squares are counted the area covered is =0.00025x80=0.02 cumm
or the number of cells counted per cumm is = 1/0.02 or 50
as the dilution was 1 to 200.
The multiplication factor is = 50 x 200 = 10,000

27

Precautions:
1. Neubauer chamber should be free from dust or grease
2. Wash the counting chamber with lukewarm water or wipe with alcohol.
3. Wash the pipette three times each with water/alcohol and ether of equal volume
4. After washing dry the pipette.
5. A horse hair (never a wire) should be used in removing blood which has clotted in
the pipette. It is extremely important to avoid making even the slightest crack in the
bore of the pipette.
6. Always use pipette, counting and cover glass certified by the US Bureau of
Standards. If it is expensive, then one such set in the laboratory can be used as a
basis of comparison.
7.

In case Hayem's fluid is used and coarse particles are observed which could be
produced by
a. clumping of red corpuscles due to cold hemagglutinin, can be prevented by
warming the Hayems fluid before use.
b. The coarse particles may also be produced by precipitation of globulins by heavy
metals in the diluting fluid.
c. In cirrhosis of liver and a typical pneumonia, cold hemagglutinin may be formed.
Such pseudo agglutination can be prevented by using Gower's solution i.e.:
Sodium Sulphate

-12.5 gm

Acetic Acid

-33.3 gm

Distilled water

-100 ml
or

By adding 0.01 gm gelatine to100 ml of the Hayem's solution.

The red blood corpuscles count in different animals under normal conditions is as
follows:
Species

Species

Cattle

RBC Count
(millions/ l)
6-8

Sheep

8-15 (10-13)

Goat

13-20

Horse

6-9 - 12.0

Dogs

5.5 - 8.8

Poultry

1.6 - 1.5

Rabbit

4.8 - 8.5

Buffalo

RBC Count
(millions/ l)
6-8

Cat

6-8

Man

5-6

Woman

4 -5

Elephant

2.4

Camel

5.4

28

Exercise No. 9: To Determine Total Erythrocyte Count (TEC):

Species

Total Erythrocyte Count

(Millions/
l)

Cattle

-------------------------

Buffalo

-------------------------

Dog

------------------------

Poultry

-------------------------

Date:

(Instructor)

29

EXERCISE NO. 10: TO DETERMINE TOTAL LEUKOCYTE COUNT (TLC)

Principle: It is carried out on the same principle as the erythrocyte count. The only
differences are:
1. The dilution of the blood is less as their number is less.
2. The diluent contains 3% acetic acid which destroys the erythrocyte. The presence of
colouring material gentian violet stains the leukocytes and makes them more visible.
3. The count is made in 4 large (1 mm sq) squares of the Neubauer counting chamber.
Apparatus:
1. Neubauer counting chamber

2. Cover slip

3. Thoma WBC diluting pipette containing white bead in the bulb.

4. Turk's fluid:
Glacial acetic acid -

3 ml

Gentian Violet -

1 ml

Distilled water to make the volume 100 ml

Method: Draw the blood by sucking into a thoma white cell pipette containing white
bead to the mark 0.5. Wipe off the excessive blood sticking to the outer side of the
pipette. The blood in excess than the mark 0.5 may be drawn out by applying against the
cotton or cloth and draw Turks fluid into diluting pipette upto mark 11. Keep the pipette
horizontal on the palm of the hand and mix thoroughly by rotation with the other hand.
Mixing with Turk's fluid causes haemolysis of RBC (Acetic acid) and stains WBC
(Gentian violet). After mixing for 1 minute expel several drops of fluid from the stem of
the pipette containing only the Turks fluid. A small drop of mixed fluid is then placed
between cover slip and the ruled chamber. Allow it to settle for some time and examine
under 10X power of the microscope in four corner group of sixteen squares. The
counting is made in L form or up and down omitting the cells lying on the other lines.
This is done to remove the error of recounting.
Calculation:
Total Leukocyte count (T.L.C) = X x 50
X = Total number of cells counted in all four squares
50 = Multiplication factor

30

Multiplication factor =

_____20_____
1x 1 x 0.1 x 4

= 50

where
20 =

dilution

1x1 =

area of one large square

0.1 =

depth between the cover slip and counting chamber

No. of large squares in which counting is done.

Note:
1. Uniform distribution is ascertained only when the variation between the 4 squares
is not more than 8 cells during the normal count.
2. During leukaemia the dilution should be made in erythrocyte pipette and the
correction applied, accordingly.
Normal value of WBC
Cattle - 5000 - 12000/l
Buffalo- 9000 - 15000/l

31

Exercise No. 10: Enumeration of Total Leukocyte Count (TLC).

Results:
Species

Total Leukocyte Count

(Thousands/l)

Cattle

--------------

Buffalo

--------------

Dog

-------------

Poultry

-------------

Date:

(Instructor)

32

EXERCISE NO. 11: PREPARATION OF BLOOD SMEAR FOR DIFFERENTIAL

LEUKOCYTE COUNT (DLC)
The blood smear can be made either on cover glass or glass slide.
1.

Cover Glass Technique:

Though it is difficult but this is preferred to the use of

slides because of their thinner preparations and more even distribution of the leukocytes.
The cover glass in the right hand is held just over a freshly drawn drop of blood. It
will spread over the glass by capillary action. The drop should be about two to three mm
in diameter. Avoid touching of the cover slips to the skin. This cover glass is held in the
left hand in the cross wise fashion. The blood will spread quickly and uniformly between
the two surfaces. The two cover glasses are separated by drawing apart the cover
glasses horizontally in the opposite direction rather than vertically, before the blood
spreads completely. If the separation is not done immediately the clotting will take place
and it will be difficult to separate. If the preparation is successful, the blood will be spread
evenly and the RBC will neither overlap nor Rouleaux.
2. Slide Technique: A drop of blood larger than that used for the cover glass method is
placed on the surface of the slide near one end. The slide is then held between two
fingers and steadied with the little finger of the left hand. The blood may be spread by
means of another glass slide known as spreader which is held in right hand. The
spreader is held just to the left of the drop of blood and then it is pulled back to edge of
the drop. The blood will spread out behind the spreader which is then pushed to the left.
The movement must be quick and steady.
The smear will be thin or thick according to the movement of the spreader i.e.
slow or rapid and depending on the angle at which the spreader slide is held. The angle
should be about 30 degree. A good smear should be smooth, homogeneous and without
serrations, have even edges and should occupy approximately the middle third of the
slide. The slide is good for RBC examination, parasites but a differential count is not
satisfactory since the polymorphs are pushed to the edges and the lymphocytes remain
scattered throughout the smear.
Fixation of Smears:
The blood is allowed to dry on the glass in the open air. With most stains, fixation
is brought about in the first minute when the undiluted stains which contain alcohol is
applied to the smear for 5 minutes. Some stains do not require prefixing as the fixation is
done by the stain itself because these contain alcohol e.g. Leishman's stain.

33

Stains and Staining of Blood Films:

The aniline dyes which commenced in 1877 were first used by Ehrlich for the
staining of blood films. Most recently, the modifications of Ramanowasky stain have
been commonly used. The stains used are:
I. Leishman's Stain: Dissolve 0.15 gms of Leishman's powder in 100 ml of pure acetone
free methyl alcohol. Place the Leishman's stain in a clean and dry glass pestle mortar
and dissolve in little alcohol. Repeat the process till whole of the powder is dissolved.
The stain is then filtered and ready for use or use readymade Leishman's stain.
Technique:
1.

Place the freshly prepared, air dried film, with its smeared surface upward on a
staining rack.

2.

Put on undiluted Leishmans stain for one to two minutes to fix the smears.

3.

Add double the quantity of neutral distilled water or buffer water with the help of a
dropper and mix the fluids by sucking and expelling them with the dropper.

4.

Let it remain for 10 minutes to stain the slide.

5.

Wash and dry in the air after blotting gently with a filter paper.

6.

Examine under oil immersion lens.

II. GIEMSA STAIN (1902)

Composition:

Azur II

Eosin

= 3 gms.

Azur II

= 0.8 gms.

Glycerine chemically pure = 250 ml

Dissolve chemically pure Azur II - Eosin and Azur II in pure anhydrous glycerine at
0

60 C. Then methyl alcohol is added and mixture is allowed to stand overnight. It is

filtered and kept in stoppered bottle.
Rapid Technique:
1. Fix the blood film in Methyl Alcohol for three to five minutes.
2. Dilute the stock solution 1 in 10 with buffer (pH 7.0)
3. Put in the fixed smears in diluted stain for half to one hour
4. Wash with buffer water.
5. Wash, blot dry and examine under oil immersion lens.
6. This method is good for trypanosomes examination.
Slow Technique:
1. Dilute the stain 1 in 20 with buffer water.
2. Place the fixed slide in the diluted stain for 16 to 24 hours. Wash, blot, dry and
examine under oil immersion lens. The slow method is used for staining of
spirochetes etc. It is good method for differential leukocyte count.

34

III. Wright's Stain (1902). Similar to Leishman's stain technique. It is prepared by

heating methylene blue with sodium bicarbonate. Then mixture is mixed with Eosin or
use ready made solution.
Technique:
1. Fresh smear is placed either in racks or on two glass rods placed parallel fixed in
wooden blocks. The width between the two glass rods should be less than the length
of the slides.
2. Stain the smear and cover the smear with stain.
3. Keep it for one minute. Fixation is complete during this phase.
4. Dilute the stain with distilled water approximately using the same volume of water as
that of stain. A greenish metallic scum comes up and the margin shows a reddish
tinge.
5. Wash the stain after 3 or 4 minute until the film is yellowish or pink and examine
under oil immersion lens.
Precautions:
1. The stain should not dry on the slide.
2. Water should not run over the edges of the cover glass or slide.
3. The stain should be floated off the slide by adding water.
Examination of stained blood smears:
The stained blood smears are examined for parasites, protozoa, bacteria and
Differential Leukocyte Count and many more purposes.
Differential Leukocyte Count (DLC): It is used for the clinical examination. A good
slide, better staining is a good index for accurate DLC. The smear should be prepared
from a free flowing drop of blood and fixed and stained immediately.
Technique:
1. Draw a smear, fix, stain and examine under oil immersion lens.
2. Count different leukocytes and record with the help of mechanical cell counter and
find out the percentage.
The four field counting method is the best. In this 50 cells in each field are counted
by moving the field from edge to the centre and back. Repeat the process till 100 cells
are counted.
Counting is done with the help of blood cell counter which has a separate key for
each type of blood cell and the total is attained automatically. After counting 100 cells
the counter gives a bell. Thus, the percentage of each cell can be read directly. In case

35

the counter is not available then it can be done with the help of Tally counting as given
below:
Neutrophils

33

35

Monocytes

IIII IIII IIII IIII =

20

Eosinophils

IIII IIII

10

Lymphocytes

IIII IIII IIII IIII IIII IIII III

IIII

Basophils

IIII IIII IIII IIII IIII

II

2
________
100

Neutrophil

Eosinophil

Basophil

Granulocytes

Monocyte

Large and small lymphocytes

Agranulocytes

36

Exercise No. 11: Determine the Differential Leukocyte count (%) in different
species of animals.

Species

Neutrophil

Lymphocyte

Monocyte

Eosinophil

Cattle

Buffalo

Sheep

Goat

Dog

Poultry

Date:

(Instructor)

37

Basophil

EXERCISE NO. 12: THROMBOCYTE (PLATELET) COUNT

A. Collection of blood
1. Venous blood is preferred to capillary blood, and it should be drawn with a plastic
syringe and placed in a siliconized test tube containing 2.5 to 5 mg di-sodium EDTA/2
ml of whole blood to eliminate clumping.
B.

Accuracy of Count

1. At best, thrombocyte counts are often unsatisfactory; the thrombocytes have their
own effective characteristics that allow them to avoid being counted, or they are
confused with artefacts. Their small size, irregular distribution, rapid disintegration,
ready agglutination and adhesion make accurate, reproducible counting difficult.
2. The thickness of the haemocytometer does not allow the oil immersion objective to be
used with the direct method, and difficulty is encountered in distinguishing
thrombocytes from other particles such as dust and debris.
3. The phase microscope with appropriate equipment provides the best method but is
usually not available
C.

Indirect method It is the simplest and most practical for the average person

1. A stained blood smear is examined and the number of thrombocytes is noted in

several representative fields and averaged.
a. Any of the routine blood stains, including new methylene blue can be used.
b. Finding 3 or less thrombocytes per oil immersion field suggests a thrombocytopenia.
In some cases of thrombocytopenia, it is not unusual to find only a few or no
thrombocytes on the entire slide.
c. The number of thrombocytes can be compared to the number of leukocytes in the
smear, and this relative number may be transposed into an absolute number by the
following calculation:
Number of thrombocytes/ microliter =

No. of thrombocytes
--------------------------X total WBC count/microliter
100 leukocytes

D.

Direct method:

1.

Blood is drawn into an erythrocyte-diluting pipette to the 0.5 mark and the diluting

fluid is drawn to the 101 mark similar to the erythrocyte count. Diluents that may be used
include the following:
a. Rees-Ecker:
Sodium citrate

-3.8 g

Formaldehyde 40%

-0.2 ml

38

Brilliant cresyl blue

-0.1 g

Distilled water

-100 ml

Keep stoppered and in refrigerator. Filter before use.

b. 1% ammonium oxalate:
Ammonium oxalate 1H2O

-1.1 g

Distilled Water

-q.s. to100 ml

Store in refrigerator to avoid bacterial growth

c. 1 or 2% disodium EDTA in 0.85% sodium chloride. A crystal or two of brilliant cresyl
blue can be added.
2. Mix thoroughly for 5 min, preferably in a mechanical rotator.
3. Expel and discard the first 4 drops before filling both chambers of the
haemocytometer. To prevent evaporation, place the top of a Petri dish containing a wet
filter paper or a wet pledget of cotton over the haemocytometer.
4. Allow 15 min for cells to settle.
5.

With the illumination partially reduced, count the thrombocytes in the entire central

ruled area on each side of the haemocytometer.

a. The central ruled area is the one usually used for erythrocytes.
b. 2x25 small squares or 50 groups of 16 very small squares are counted for a total of 2
sq. mm.
c.

Use the fine adjustment to focus on each cell to identify the thrombocytes as lilac

coloured, oval, rod or comma shaped, and considerably smaller in size than the
leukocytes that are also visible.
Calculation:
No. of thrombocytes/ microliter = No. of thrombocytes x 1000
The results obtained should be checked by making a survey of the blood smear to
determine if there is good correlation between the two methods.

39

Exercise No. 12: Enumeration of Thrombocytes (Platelet count).

Results:
Species

Thrombocytes/Platelet Count
(Lakhs/l)

Cattle

-------------------

Buffalo

-------------------

Dog

-------------------

Poultry

-------------------

Date:

(Instructor)

40

EXERCISE NO. 13: DETERMINATION OF BLOOD PRESSURE.

Blood Pressure
It is the lateral pressure exerted by the blood against the wall of the blood vessel.
It is a normal terminology, because without blood pressure the circulation of blood would
cease. It is the difference in the pressure at the two ends of the vessels which keeps the
flow of blood from the arterial to venous blood vessels. The blood pressure is high in the
arteries and the flow is high and pulsatile and in vein the pressure is less or almost zero
and without any pulsation.
Determination of Blood Pressure
Direct - Method
Procedure:

The blood pressure is determined by catheterization of any of the artery

i.e. Carotid, Brachial or Femoral.

Carotid Artery Catheterization:
1. Secure the animal and tie it properly without any restraint.
2. Clean and shave about 2 sq inches area of the skin over the jugular furrow.
3. Inject I/M, local anaesthesia i.e 2% procaine hydrochloride solution in the area
and isolate the carotid artery.
4. Pass a polyethylene catheter (P.E. 200 I.D; 0.14 cm length 90 cm) filled with
heparinized saline into the carotid artery.
5. Tie the catheter with the thread along with the artery.
6. Connect the catheter with the U shaped mercury manometer through rubber
tubing and a three way cannula. (Three way cannula is used to flush away the
coagulated blood in the catheter if it is present).

Before making connections

between carotid artery and the manometer always keep slightly higher pressure in
manometer than the normal systolic pressure of the animal so that the blood may
not rush into catheter filled with heparinized saline solution, when the connection
is restored, and the blood should stay only up to the tip of the catheter.
7. On restoring the connections between the catheter and the manometer, the
mercury column will rise on the opposite column of U shaped mercury manometer
having a pointer floating on the mercury.
8. The column of mercury shows upward and downward deflections.
9. Record the deflection by a pointer on a kymograph.
10. Two types of deflections are recorded, one of low amplitude (B.P.) and other of
higher amplitude building up slowly and steadily (Respiration), record to stability.

41

11. Disconnect the three way cannula and bring the open end of the catheter at the
level of the heart or aorta of the animal. The column of the mercury will fall along
the pointer. Mark the line where the pointer is stabilised.
12. Measure the maximum and minimum height from the base line and multiply with 2
to determine the systolic and diastolic pressures.

(The height in mm Hg is

multiplied by 2 as the mercury column is deflected in double column).

13. Determine the pulse pressure by subtracting the diastolic pressure from the
systolic pressure.
14. Determine the mean arterial pressure by adding 1/3 of the pulse pressure to the
diastolic pressure.
Indirect Method:
Auscultation / Palpation Method:
The term auscultation means the detection and study of sounds. The palpation
means the feeling of pulse. The blood pressure in indirect method is determined by the
use of Sphygmomanometer.
Sphygmomanometer Method:
The sphygmomanometer consists of a cuff.

The cuff encloses a rubber bag

wrapped by a cloth. The rubber bag has two attachments, one for the manometer and
the other for the bulb which has the arrangement for inlet and outlet of air, regulated by
screw knob.
1. Wrap the cuff round the arm above the elbow.
2. Close the outlet on the bulb and make the connections between bulb and
manometer.
3. Inflate the rubber bag in the cuff until it exceeds the arterial systolic pressure (2030 mm Hg above the normal systolic pressure).
4. Place the stethoscope on the cubital fossa on the elbow joint or feel the radial
pulse towards the side of the thumb.
5. Start releasing the pressure by opening the outlet on the bulb and hear the sound
or feel the pulse.
6. When there is no flow of blood no sound is heard/no pulse is felt. As the pressure
in the cuff falls just below the systolic pressure the flow of blood will produce
turbulence and a sound is heard or a pulse will be felt with the index finger. Note
the pressure. Because this is the peak pressure during systole of the ventricle so
it is called SYSTOLIC PRESSURE, which is greater enough to force the blood
under the inflated cuff.

42

7. Then allow the pressure in the inflated cuff to fall further. The intensity of the
sound increases and finally the sound muffles or disappears. Note the pressure.
It is the minimum pressure in the artery during the diastole of the ventricle. So it is
called DIASTOLIC PRESSURE.
8. Deflate the cuff more, the murmur finally disappears completely. The sounds
disappear because after the cuff pressure is below diastolic pressure the blood
flows throughout the cardiac cycle without any turbulence so there is no sound.
Central Venous Pressure (CVP): It is measured with the help of apparatus called CVP
Saline manometer set. It carries a three way cannula connected on one side with a
scale and on other side with a bottle which carries the normal saline solution. The third
end is connected with the help of a catheter to jugular vein of patient. To record the CVP,
the scale is kept at the level of heart. The saline manometer is filled with saline expelling
air-bubbles from the tubes on all sides. One end is connected to jugular vein of the
patient through a catheter or a hypodermic needle. By clamping the tubing from where
fluid comes, it is made air tight. The connections are made between the vein and the
scale by moving three way cannula and direction is indicated by various arrows.
Immediately after connection, there is fall in saline column and wherever it stabilizes, that
observation indicates the central venous pressure i.e. CVP. It is expressed in centimetre
(cm) saline column.

43

Exercise No. 13: To measure the blood pressure by direct and indirect method.
Observations: By Indirect Method
Name of Method used:
S. No.

----------------------------------------------

Name of Student

B.P (mm of Hg)

Systolic/Diastolic

--------

----------------------------------------------------------

---------------------

--------

----------------------------------------------------------

---------------------

--------

----------------------------------------------------------

---------------------

--------

----------------------------------------------------------

---------------------

--------

----------------------------------------------------------

---------------------

--------

----------------------------------------------------------

---------------------

--------

----------------------------------------------------------

---------------------

--------

----------------------------------------------------------

---------------------

--------

----------------------------------------------------------

---------------------

--------

----------------------------------------------------------

---------------------

--------

----------------------------------------------------------

---------------------

--------

----------------------------------------------------------

---------------------

--------

----------------------------------------------------------

---------------------

Date:

(Instructor)

44

EXERCISE NO. 14: ELECTROCARDIOGRAPHY

Electrocardiography (E.C.G): The study of electrical activities of the cardiac muscle is
termed as Electro-cardiography. The instrument used to record the cardiac potentials
(electrocardiogram) is called electrocardiograph.
Since the body is a good conductor of electricity, the electric field generated inside
the heart spreads over the different parts of the body which can be detected by
connecting two suitable points on the body surface in a circuit with the help of a sensitive
device called E.C.G. The electrocardiogram is normally recorded by the use of three
types of lead systems.
1. Standard Lead System
2. Augmented Lead System
3. Chest Lead System.
I. Standard Lead System or Bipolar Lead System:
When two poles of the body are connected, they form a lead. These are standard
leads. Lead I, Lead II and Lead III. They are called standard as they were first used for
ECG and are used as standard for other tracings of ECG.
Lead I

= Left arm - Right arm (the negative terminal of ECG is connected to the

electrode on the right arm and positive to left arm i.e. when right arm is -ve to the left arm
there is +ve or upward deflection).
Lead II= Left Leg - Right arm (the negative terminal is connected to right arm and
positive to left leg).
Lead III= Left Leg - Left arm (the -ve terminal is connected to left arm and the positive
terminal to left leg).
The fourth electrode is applied to the right leg. This does not enter into the
electrocardiographic picture but serves to ground the machine for all leads and thus
helps to eliminate alternating current. Three standard leads are bipolar leads because
they show the activity of the heart as viewed simultaneously from two different locations
or poles i.e. double exposure view. They help in diagnosis of abnormal rhythms as
double exposure characteristic exaggerates the amplitude of P waves and makes them
more easily recognizable and may be confusing some times as waves in Lead III
resemble a cardiac abnormality and yet may result from cardiac position and are normal.

45

II. Augmented Lead or Unipolar Lead System:

It is used to eliminate double exposure of standard limb lead and obtain a picture
of the electrical activity as seen from one extremity only. This is brought about by
applying electrode to the desired limb (exploratory electrode applied either to right arm,
left arm or the left leg as desired) and completing the circuit through an indifferent
electrode whose voltage is approximately zero. The indifferent electrode is prepared by
applying three electrodes to the extremities as usual, one to the left arm, the second to
the right arm and the third to the left leg. The wires from these three electrodes are then
connected to each other and this three tailed electrode is connected to machine as if it
were a single electrode.
The electrical activities of the heart as seen from these three points simultaneously
neutralize each other and the result is a three part indifferent electrode whose voltage is
always approximately zero.
For obtaining the Unipolar electro-cardiogram as seen from the left arm only, the
exploring electrode is applied to the left and indifferent electrode is applied to the left and
right arm and to the left leg. Thus left arm tracing equals:
Left arm = (Left arm + Right arm + Left leg i.e. ZERO)
So the left arm potentials will be equal to left arm -ZERO
The leads applied to all the three extremeties are called Unipolar leads or Vector
leads. Vector is used to indicate the direction and magnitude of the single force which
results when several forces of different magnitudes act simultaneously in different
directions on a single point.
The unipolar limb leads can be expressed as:
VL = L - (L + R + F)

VR= R - (L + R + F)

VF = F - (L + R + F)

Unfortunately, the unipolar limb leads are low in amplitude and difficult to study, so
increase or augmenting their amplitude is required. Disconnecting lead from the three
tailed indifferent electrode from that extremity to which the exploring electrode is already
applied, the amplitude of the complexes will be increased by 50%. Thus aVL=L-(R+F),
aVR=R-(L+F), aVF=F-(L+R).
III. Chest Leads:
An indifferent electrode is formed by uniting all of the standard electrodes i.e. from
left arm, right arm and left leg, through a resistance of 5,000 ohms each to a central
terminal and this is connected to the negative terminal of the electrocardiograph. The
potential at the central terminal remains at Zero. The positive terminal is connected to the
electrode placed on the various areas of the chest. There are six standard positions on

46

the chest which are referred as V1, V2, V3, V4, V5 and V6. The placement of these
chest leads is:
V1= 4th intercostal space to right of sternum
V2= 4th intercostal space to left of sternum
V3= Mid way between left sternal border and mid clavicular line in a line joining position
2 and 4.
V4= 5th intercostal space in mid clavicular line.
V5= 5th Intercostal space in left anterior axillary line
V6= 5th intercostal space on left mid axillary line.
The electrical potentials in the auricle and ventricle appear in the form of
deflections called P wave, QRS and T wave. The cardiac potentials are recorded on
ECG paper which is ruled horizontally and vertically, and the paper is allowed to move at
a speed of 25mm/sec. Before making a record, the machine is to be calibrated.
Evaluation of E.C.G. Tracings:
The different waves recorded are - P wave, QRS and T wave.
P WAVE: It denotes the electrical activity over the atria i.e. wave of atrial depolarization.
It is mostly positive in all the three leads. The potential in standard lead system is 0.20.3 mV and the duration varies between 0.08 - 0.12 sec. Atrial repolarization is absent
because ventricular depolarization superimposes it. Atrial repolarization is called atrial T
wave. When ventricle is prevented from depolarization and also the gain of amplifier is
increased considerably. The T wave will appear as a low voltage with downward
deflection.
QRS Complex (Ventricular depolarization): It is the resultant of the left and right
bundles, Purkinje fibres and left and right ventricular cardiac muscle electrical activity. Its
direction, amplitude and shape depend upon the position of the heart. Q denotes the
potential on the ventricular septum. R at the apex and S at the base of the ventricle. The
base of the ventricle is the last part to be depolarized. The QRS is normally 1mV and
lasts for 0.06 to 0.10 sec.
T Wave: (Ventricular repolarization): The amplitude is normally about 0.3 mV and lasts
for 0.16 sec.
PR-Interval:

Extends from origin of P wave to origin of QRS complex. As Q wave is

mostly absent, the interval is called PR rather than PQ. The time elapsing between the

47

activation of the SA node and the activation of AV node (Auriculo - ventricular conduction
time) normally measures about 0.16 and not more than 0.20 sec.
Q-T Interval: Extends from origin of QRS to the termination of T wave. It is also known
as intraventricular conduction time or electrical systole. It is affected by the beat of the
heart. At heart rate of 70 per minute the Q-T interval is about 0.36 sec. and at 120/min, it
is 0.28 sec.
S-T Segment: Extends from termination of QRS to the beginning of T Wave. It is also
called isoelectric phase and normally it is at the base line level. It gets elevated during
the cardiac abnormalities.
S-T Interval: Extends from the termination of QRS to the termination of T wave.
R-R Interval: Extending between two RR of two QRS complexes. It denotes total time
taken per beat. To evaluate the heart rate per minute divide 60 by R-R interval (60/R-R
interval) where 60 is the factor to convert seconds to minutes.

48

Exercise No. 14: Evaluate the ECG tracing recorded.

Date:

(Instructor)

49

EXERCISE NO. 15: TO RECORD RESPIRATION RATE, HEART RATE AND PULSE
RATE IN DOMESTIC ANIMALS
Respiration Rate: Normally, two terms are used i.e. respiration and breathing. The
term respiration and breathing are not synonyms.

In fact, breathing is a part of

respiration. Breathing begins with the movement of chest and the subsequent effect on
the movement of air into and out of the lungs. Respiration includes i) movement of air
into the lungs (i) oxygenation of pulmonary blood (iii) transportation of oxygenated blood
from lungs to tissues (iv) utilization of O2 and production of CO2 at tissue level (v)
transportation of CO2 by blood from tissue to lungs (vi) movements of CO2 from lungs
into the atmosphere.

The respiratory mechanism/respiration rate is divided into two

parts.
(A) Inspiration and (B) Expiration.
The rate and the amplitude of the respiratory movement in the different animals
can be recorded by the use of following methods.
Physical methods:
1. Paper method: The respiration rate of animals can be recorded by placing a paper
strip close to the nostrils. The paper will move towards the nostrils during inspiration
and pushed away during expiration. The pull and push of the paper will constitute
one respiration. To record the respiration rate count the number of times the paper is
pushed per minute.
2. Abdominal method/Flank method:

The respiration rate in the animal is mostly

recorded by standing at the back of the animal and making the observations on the
rise and fall of the abdominal flank (Differentiate it from the rise and fall of the left
abdominal flank at the time of ruminal contraction). The respiration rate of the animal
will be the number of times the flank rises per minute.
Precautions:

Always stand at the back of the animal and observe the fall and rise of

the flank unnoticed by the animal otherwise the animal once disturbed will not give the
normal respiration rate as during disturbance the respiration rate gets increased.
3. Auscultation method:

The respiration rate is recorded by placing a stethoscope

over the chest and recording the respiratory sounds during inspiration and expiration.
Experimental Methods:
1.

Cannula method:

Insert a cannula or catheter into the pleural cavity between two

ribs with the care to prevent the entrance of more than a small bubble of air through
the opening. Connect the catheter with a tambour/ manometer fitted with a pointer to
write on a kymograph. During inspiration, with the fall in pressure the pointer will

50

show downward deflection and during expiration due to increase in pressure in the
catheter, the pointer will show upward deflection. Take the upward and downward
deflection of the writing pen as one respiration. Count the number of respirations per
minute.
2. Tracheal cannula: Insert a tracheal cannula in the tracheal tube and connect it to a
tambour with recorder. Inspiration will decrease the pressure in the endotracheal
tube, tambour and recorder showing a downward movement of the writing lever,
expiration will cause an upward movement of writing point.
3. Pneumograph or Stethograph method: The pneumograph /stethograph is tied
around the chest. The pneumograph is connected to a recorder. During inspiration
the capacity of the pheumograph is increased and the writing lever move lower and
expiration showing upward movement.
The stethograph, a metallic cylinder is closed at both the ends (as compared to
pneumograph which a rubber coil is enclosing a rubber tubing open at one end) and
has a rubber diaphragm. The string is tied to the centre of each membrane and the
apparatus tied around the thorax. A tambour is connected by rubber tubing to a side
tube of the stethograph. Inspiration pulls up the rubber diaphragm which creates a
negative pressure in stethograph tube and tambour. Expiration produces the
opposite effect.
4. Chest Plethysmograph:

The plethysmograph is connected to a spirometer. The

sheath and inflated rubber bag of the plethysmograph are tied over the thorax of
animal. During inspiration the air from the inflated bag goes into the spirometer,
whose inner cylinder is raised which has a writing pen. During expiration the inner
cylinder again returns to the same level.
5. Body Plethysmograph: It is used for measuring respiration rate and the respiratory
volumes.
6. Transducers:

Recently electronic device has been employed for recording the

respiration rate. The thermister transducer is placed before the nostrils i.e. in the
respiratory air stream just below the nasal passage and above the lips. The
transducer is held in position by adhesive tape. The transducer is connected to a
preamplifier of polyrite physiograph which magnifies pressure changes many times
and feed it to the writing pen through a motor. During inspiration the pen gives
downward movement and during expiration upward movement.
7. Benodict-Roth respiration apparatus: This apparatus is used for the metabolic
studies. The metabolic apparatus also imparts the information about the respiratory
frequency of the animal.

51

Determination of Heart Rate/Pulse Rate/Cardiac Sounds:

Heart rate is the number of times the heart beats per minute. The heart rate can be
determined by using either of these methods i.e. by auscultation, by palpation, by E.C.G.
or by heart rate meter.
Auscultation method: For the record of the heart rate/minute, place a stethoscope on
the left side of the chest/thorax on the 4th/5th intercostal space and record the cardiac
sounds i.e. lub and dub. The lub and dub forms one heart beat or count the total number
of sounds per minute and divide them by two to get heart beat/heart rate per minute.
Palpation method: For recording the pulse. In cattle, the record of pulse is made from
the coccygeal artery; dogs- femoral artery; man- radial artery.
Electrocardiographic technique: The E.C.G. is recorded by the application of different
standard leads. The time elapsing between the two R-R intervals is recorded. It is the
time taken by one heart beat. To evaluate the heart rate per min, divide 60 by RR
interval.
Phonocardiographic technique: The heart sounds are recorded by phonotransducer
with phonocardiogram preamplifier.

The phonotransducer is placed over the 4th/5th

intercostal space on the left side of the thorax. After calibration, the heart sounds are
recorded from the region of maximum amplitude. Normally, two cardiac sounds are
recorded. The 1st which is of higher amplitude and lasts for a longer duration than the
second which is of lower amplitude and lasts for shorter duration. Some times 3rd and 4th
cardiac sounds are also recorded.

52

EXERCISE No. 15: Determine the heart rate / pulse rate and respiration rate in the
following:

Cattle Buffalo Sheep

Goat

Dog

Poultry Man

Heart Rate

Pulse Rate
Respiration
Rate
Tidal
Volume
Inspiratory
capacity
Expiratory
capacity

Date:

(Instructor)

53

EXERCISE NO. 16: TO DETERMINE BLOOD VOLUME AND PLASMA VOLUME IN

DOMESTIC ANIMALS.
Blood Volume: It is the volume of blood flowing in the blood vessels which constitutes
1/13 or 7.7 percent of the total body weight. It represents the composite picture of blood
plasma and haematocrit. The plasma volume is about 5% of the total body weight. All
animal cells from the free living protozoan to the specialised tissue cell of the higher
forms of the animal life require water to carry on their functions. In the more complex
forms of animal life, the blood serves the purpose of supplying each cell with the required
water, oxygen, electrolytes, nutrients and hormones and the blood receives the waste
end products of metabolism for transport to the organs of excretion.
The total volume of circulating blood is a function of lean body weight. The volume of
blood is so important to the dynamics of circulation that it is kept remarkably constant
despite
1. Periodic intake of water

2. Metabolic water 3. Water loss via skin, lungs,

kidneys, mammary glands and alimentary tract. 4. Haemorrhage

5. Blood infusion.

The earliest method for estimating blood volume consisted of bleeding the animal
to death followed by washing out of the blood vessels and adding the blood contained in
the washing to that collected during bleeding which is a very tedious and cumbersome
method. Now-a-days blood volume and plasma volume is determined by dye-dilution
technique or by tracer technique. The different dyes used for the determination are T1824, Bengal blue, Vital red, RISA, Cr51, P32, and Fe39. Out of all these, T-1824 and
RISA have been employed successfully for the determination of blood volume and
plasma volume.
Determination of Plasma and Blood Volume by Dye Method
Principle: When a known volume of a substance, a non-toxic azodye, Evan's blue is
injected into circulation, it is uniformly distributed after some time (normally 10 minutes).
The extent of dilution in the circulating plasma is determined by measuring the
concentration of dye in the plasma. The total volume of the fluid is determined from the
equation;
V = A/C,

where

V = Volume of plasma

A = amount of dye injected

C = average concentration of dye.
Procedure: The dye dilution method of Fisher and Dalton 1961 using T-1824 (Evan's
blue) is employed for the determination of blood volume and plasma volume.

54

1. Collect 10 ml of blood in a heparinized test tube to be used for determination of

haematocrit and as a blank.
2. Dissolve Evan's blue in normal saline and prepare a stock solution of 5%
concentration.
3. Weigh the animal and inject the dye Evan's blue @ 0.25-0.40 mg/kg body weight in
to the left jugular vein.
4. After 10 minutes of injection of the dye, collect the venous blood from opposite
jugular vein.
5. Spin the blood without dye (1) and with dye (4) and separate the plasma.
6. Take blood plasma without dye as blank and determine the optical density of
unknown sample collected after 10 minutes of injection of dye.
7. Determine the concentration of dye in blood plasma by comparing the optical density
of sample with that of standard curve.
8. Determine the haematocrit of the blood by Wintrobe tube method or micro capillary
method.
9. Determine the plasma volume by using the equation V=A/C
10. Determine the blood volume by using the equation:
Blood Volume: =

Plasma Volume
1 Ht

Where Ht= Haematocrit

Standard Curve: Prepare the standard curve same way as prepared for measuring the
concentration in determination of cardiac output.
Precautions: Before selecting the dye for the determination of blood volume, the dye
used should have the following characters:
1. The dye should be non-toxic
2. It should not diffuse into the tissues or from one compartment to another.
3. It should have a uniform rate of elimination i.e. neither excreted rapidly nor slowly.
4. It should disperse easily in the fluid.
5. It should not be lost during its 1st circulation.
6. It should not be influenced by changes in oxyhaemoglobin concentration.

55

Exercise No.16: Determine the blood volume and plasma volume in the following
species of animals.

Cattle

Buffalo

Sheep

Plasma volume ------

-------

Blood volume

-------

------

Goat

Dog

-----

------

-----

-------

-----

------

-----

-------

Date:

Fowl

(Instructor)

56

EXERCISE NO. 17: TO DETERMINE THE CARDIAC OUTPUT IN DOMESTIC

ANIMALS
Cardiac Output
The volume of blood pumped out by the heart per minute is known as cardiac
output or minute volume. The cardiac output is determined by:
1. Direct Fick method or oxygen consumption method.
2. Dye dilution method.

3. Pulse pressure method.

Determination of Cardiac Output by Dye Dilution Method

Principle: Based on the Stewart principle that the volume of any fluid can be determined
if you know the total amount of dye and the average concentration of dye in a sample of
the fluid. The volume (V) will be = A/C
Amount of dye injected (A)
Volume of Plasma = ---------------------------------------------Average concentration of dye in the sample(C)
Procedure: Cardiac catheterization:
1.

For cardiac catheterization (Right ventricle) keep the animal off feed and water for 12
hours and note temperature, pulse, respiration and body weight.

2.

Clean and shave about 2 sq. inches area of skin over the left jugular vein.

3.

Inject I/M local anaesthesia 2% procaine hydrochloride solution in the area and
perform the venipuncture with 4-1/2 inch 14 gauge needle and pass a polyethylene
catheter (PE 200 ID 0.14 cm length 90 cm) filled with heparinized saline into the right
ventricle. Ascertain the presence of catheter in the right ventricle from the ventricular
pressure.

4.

Remove the hypodermic needle carefully and keep the catheter in the position.

5.

Similarly isolate the carotid artery and catheterize on the other side of the neck.

Dye dilution technique: The dye dilution method of Fisher and Dalton (1961), Stowe
and Good (1960) using T-1821 (Evan's Blue) for the determination of cardiac output is as
follows:
1.

Collect about 10 ml of blood in a heparinized tube to be used as a blank and

determination of hematocrit.

2.

Dissolve Evan's blue in normal saline and prepare a stock solution of 5%.

3.

Inject the Evan's blue dye @ 0.25-0.40 mg/kg body weight through jugular catheter
into right ventricle.

57

4.

Immediately flush with the normal saline the remains of the dye in the syringe, or
flush the dye by drawing the blood into the syringe and re-injecting it into the
catheter i.e. in the right ventricle.

5.

Immediately after injecting the dye, start collecting arterial blood samples from the
carotid catheter in the heparinized tubes at an interval of 1-2 sec. Maintain the
constant time interval between the consecutive samples collected.

6.

Collect fourteen such blood samples from the carotid catheter for the determination
of average concentration of dye circulated.

7.

Spin the blood samples and separate the blood plasma.

8.

Take blood plasma without dye (from the blood collected before injecting the dye T1824) through the jugular catheter as No.1) as blank, determine the optical
densities of the unknown 14 samples collected from the carotid catheter, in
Spectronic 20 at 620 nm.

9.

Determine the concentration of the dye in the 14 unknown samples by comparing

their optical densities with that of standard curve.

10. Plot the concentration of dye in blood plasma (mg/ml) against time (sec) on a log
paper and draw a concentration curve.
11. Extrapolate the concentration curve to the base line and measure the total
concentration under the curve.
12. From the area under the curve, the average concentration of the dye (mg/ml) is
determined.
13. Determine the hematocrit value (%) by the Wintrobe method by centrifuging the
Wintrobe tubes at 3000 revolution per minute for 30 minutes or by microcapillary
method. The true hematocrit value is obtained by multiplying the hematocrit with a
correction factor which varies in different animals (CF. sheep- 0.95, goat- 0.81, and
buffalo calf- 0.87).
14. Determine the cardiac output by using the equation
F = __A60___
Ct (1-Ht)
Where F= Cardiac output litres/min.
A

=Total amount of dye injected.

=Average concentration of dye T-1824 in mg

=Average time in seconds.

Ht =Haematocrit value %
60 =Time factor for conversion of seconds to minute.

58

Preparation of Standard curve

a) Prepare a stock solution of Evan's blue by dissolving 500 mg in 100 ml of distilled
water.
b) Make different graded concentrations like 0.1, 0.2, 0.4, 0.8, 1, 2, 5, 10 mg/lit from
the stock solution.
c) Take distilled water as a blank and set O.D. or 100% transmittance at 620 nm of
the Spectronic-20.
d) Determine the optical density of the known graded concentration.
e) Plot the standard curve on a graph by plotting the concentration against optical
density. Plot concentration along X-axis and OD along Y axis.
f)

Draw the best fitted line on the graph.

Precautions:
1. The animal should not be disturbed during the collection of blood samples.
2. The dye should be completely flushed in to the venous circulation. The process of
re-flushing the dye should be completed in 1-2 seconds.
Factors affecting cardiac output:
1. Heart rate

2. Stroke volume

3. Anaemia

4. Anoxia and hypercapnia

5. Sleep

6. Cardiac abnormalities

7. Hyperthyroidism

8. Exercise

9. Temperature

10. Starvation

11. Emotions

59

Exercise No. 17: Determine the cardiac output in the following species of animal
and derive the various Cardiac Indices:

Cardiac
Output

Cattle

Buffalo

------

-------

Sheep

Goat

Dog

Fowl

-------

------

-----

-----

Date:

(Instructor)

60

Annexure
NORMAL CARDIAC,
CARDIAC, RESPIRATORY AND HEMATOLOGICAL
HEMATOLOGICAL VALUES IN DIFFERENT ANIMALS
Horse

Cattle

Buffalo

Camel

Sheep

Goat

Pig

Dog

Cat

Rabbit

Fowl

Man

8-16

10-30

18-30

12-20

12-20

12-20

08-18

10-30

20-30

20-30

15-30

12-20

32-44

60-70

40-60

30-40

70-80

70-80

60-80

70-120

110-130

200

200-400

60-90

99.7

101.5

101.5

102.3

103.8

102.5

102

101.5

103.1

107.1

98.4

15-38/20 2-6/24

60-65/2

1/

0.00/

0.00/

0-6/

5-25/

7-10/

4-5/24

7/2

1.6/ hour

minute

hours

hours

hour

hour

hour

30 min

60 min

hour

hour

hour

PCV (%)

33

40

32

28

32

34

41

45

40

35-57

41

44

Hb (gm/dl)

10.0

14.0

11.6

13.1

12.4

10.9

12.0

13.0

10.5

12.6

12.0

14.5

Sp. gravity

1.042/

1.046/

1.040/

1.030/

1.038/

1.042/

1.055/

1.054/

1.050/

1.050/

1.043/

1.052/

of blood

1.060

1.058

1.045

1.045

1.065

1.062

1.059

1.062

1.054

1.063

1.063

Clotting

11.5

6.5

4-6

1.5-2.5

2.5

3.5

2.5

1.5

4.5

3.5-10.0

5.5

4.5

1-4

1-4

1-4

1-4

1-4

1-4

1-4

1-4

2-6

6.9

6.3

6.8

8.2

8.1

13.9

11.4

6.2

7.2

5.9

3.2

5.4

Resp. Rate
/minute
Heart
rate/min
Rectal
Temp. oF
ESR

Time (min)
Bleeding
Time (min)
TEC
millions/ l

61

Horse
WBC Thousand/l 5-11

Cattle

Buffalo

Camel

Sheep

Goat

Pig

Dog

Cat

Rabbit

Fowl

Man

5-12

8.9-15.2

18-20

4-10

5-14

7-20

8-18

9-24

8.2

16-40

30-65

25-45

22-32

30-60

10-50

30-48

28-47

60-70

35-75

42

13

60-65

25-70

45-75

45-66

33-58

40-75

50-70

39-62

12-30

20-55

52

75

15-30

1-7

2-7

8-9

1.5-6.0

0-6

0-4

2-10

3-10

1-5

5.7

3-7

0-11

2-20

8-22

2-17.5

0-10

1-8

1-11

2-10

2-12

2.5

1-4

0-3

0-2

0-0.2

0-0.5

0-3

0-1

0-2

2.4

0.5-1

163

125-166

125

110

120

169

150

140-170

100

135

100

68

59-85

78

58-74

70

58-95

92

70

60-75

40-50

70

43-63

37-54

50-55

46-53

53

36-50

43-58

46-48

40-55

46

50

75

99-113

119

107-114

129

138

69

121

70

850

580

230

53

14

72

D
L
C

Blood Pressure
(mmHg) mean
Blood Volume
(ml/kg)
Plasma Volume
(mm/Kg)
Cardiac Output
(ml/kg/min)
Stroke
Volume(ml/beat)

(Calf)

62