COLLECTION AND PRESERVATION OF BLOOD

AIM

To collect the blood and preserve it with various anti-coagulants for various

clinical purposes.

APPARATUS

Lancet/Syringe

Needle

Anticoagulant

Cotton

Spirit.

Blood can be obtainedby following methods:

1. Capillary method by finger puncture

2. Venous method by venous puncture

PROCEDURE

Collection of blood by capillary method:

The usual site used for collection of blood is tip of index finger. Palmer aspect
of thumb, middle or little finger are not used, because the tendon sheath of flexor
muscles, which is close to the tip of finger can have more chance to infection. Margin
of earlobe, great toe and heel in infants are the other areas usually used for collection
of blood.

DEPARTMENT OF ROGA NIDANA, KTGAMC BENGALURU Page 1

Collection method

Area of collection is to be cleaned using cotton soaked in spirit, dry the area,
massage and increase the blood circulation. With sterile needle, gently prick the area
and discard first two drops of blood, as it contains some tissue fluid.Collect the
subsequent drops by capillary tube or pipette.

Collection of blood by venous puncture

The sites of collection are median cubital vein, external jugular vein, great
saphenous vein, superior sagitalsinus, and dorsal venous arch. The last three sites are
used in infants.

Collection method

Blood is collected when the patient is in the lying position or sitting alongthe
side of a table. The puncture area is cleaned by cotton soaked in spirit. A tourniquet is
applied above the cubital fossa. The needle ofsyringe which is sterile is introduced
DEPARTMENT OF ROGA NIDANA, KTGAMC BENGALURU Page 2
into the vein, such a way that thesyringe is parallel to the vein and 450 to the skin
surface. Care should betaken to prevent counter puncture. When the blood appears in
the barrel,withdraw the piston and fill the syringe with required amount of blood.
Tourniquet released, Needle is taken away with the application of firm pressure at the
site ofpuncture. Needle is taken out from the syringe and blood is pushed into
alabeled bottle containing appropriate anti-coagulant and mixed gently.

Precautions

• Avoid the air entering into the vein to prevent air embolism.

• Avoid counter puncture to prevent hematoma formation.

Uses and advantages

Blood collected by this method is used for determination of ESR, PCV

etc.Blood testing using whole blood without anti-coagulant are DC, Clotting time etc.

ANTI-COAGULANTS

Anti-coagulants are substances added to blood in order to prevent

coagulation.Coagulation of blood can be prevented by:

• Removing calcium by addition of oxalate, citrate and EDTA.

• Inactivating thrombin and thromboplastin by the addition of heparin.

• Removing fibrin by defibrination.

Generally anti-coagulants used are Double Oxalate mixture, EDTA, Tri sodium citrate
and Heparin.

i) Double oxalate mixture

This removes calcium by forming insoluble precipitate of calcium oxalate.

Ingredients are Ammonium oxalate (1.2gm), Potassium oxalate (0.8gm), Neutral
Formaldehyde (1ml) and Distilled water (100ml). Ammonium oxalate causes swelling
of RBC and Potassium oxalate causes shrinking of RBC. They have action to balance
each other, so the size of the RBC is unaffected.

DEPARTMENT OF ROGA NIDANA, KTGAMC BENGALURU Page 3

Uses

Tests performed by using double oxalate mixture are Hb% estimation, RBC
count, WBC count, and ESR determination.

Disadvantages

WBC morphology is not preserved well hence it is not used for blood smear.

Oxalate mixture is not recommended for platelet count, as it causes the

platelets to clump.

ii) Ethylene diamine tetra acetic acid (EDTA)

This is calcium chelating compound. Calcium in the blood is found in an un-

ionized form and forms a soluble compound with EDTA. 3mg of EDTA is taken in
dried bottle prevents coagulation of 3-4ml of blood. It is used as Disodium or
Dipotassium salt.

Uses

Tests performed by EDTA are ESR by Wintrobe’s method,Hb %, RBC count,

WBC count, PCV, platelet count etc.

Advantages

• It equates double oxalate mixture in haematocrit studies and issuperior in that, as it

does not produce any artifacts.

• The blood smear can be produced even after 2-4 hrs.

• EDTA is ideal for platelet count, since platelet clumping is inhibited.

Disadvantages

• Excess of EDTA affects RBC, WBC causing shrinkage and degenerative changes.

• Excess of EDTA causes platelets to swell and disintegrate.

DEPARTMENT OF ROGA NIDANA, KTGAMC BENGALURU Page 4

• EDTA is not suitable for using coagulation studies.

iii) Trisodium citrate

This anti-coagulant is used in liquid form. It is mostly widely used in the

determination of the ESR.

Uses

Used in determination of ESR by Westergren’s method and Prothrombin time.

Disadvantage

As it is used as liquid, the blood gets diluted and hence it is unsuitable for
other hematological experiments such as Hb% and cell counting.

iv) Heparin

Act as an inhibitor for thrombin formation. 0.1-0.2mg/dl of heparin is

necessary to prevent coagulation.

Advantages

• It is less toxic and is used for heart surgery and exchange blood transfusion.

• It is used when plasma is urgently required for certain emergency determination,

such as blood sugar, urea and electrolyte.

Disadvantages

• It is expensive.

• It gives a faint blue coloration to the background when films were stained.

DEPARTMENT OF ROGA NIDANA, KTGAMC BENGALURU Page 5

 ESTIMATION OF HAEMOGLOBIN

SAHLI’S ACID HAEMATIN METHOD:

Principle:

Haemoglobin is converted into acid haematin by the action of HCl. The acid
haematin solution is further diluted with distilled water until its color matches with
exactly that of permanent standard of comparator block. The Hb concentration is then
read directly from the calibration tube.

Requirements: HCl solution, Sahli’s Haemoglobinometer, pipette, distilled water.

Procedure:

Fill the Hb caliberated tube upto the mark 20 with 0.1 N HCl by means of a
dropper. Draw blood up to 20µl mark in the Sahli’s pipette. Adjust the blood column
carefully without bubbles. Wipe excess of blood on the sides of the pipette by using a
dry piece of cotton. Blow the blood into the acid in the graduated tube, rinse the
pipette well. Mix the reaction mixtures and allow the mixture to stand at room
temperature for 10 minutes. Dilute the solution by addingdistilled waterdrop by drop
carefully and by mixing the reaction mixture until the color matches the color in the
comparator. The lower meniscus of the fluid is noted and reading is noted in gm/dl.

Normal values:

Male: 14 –18gm%,

Female: 11.5-16.5gm%,

Infants: 16-18gm%.

Result:

The Hb count of the given sample of blood done by Sahli’s acid haematin
method is ………gm%

DEPARTMENT OF ROGA NIDANA, KTGAMC BENGALURU Page 6

Pathological variations:

Hb% is increased in polycythemia, high altitudes, dehydration and burns.

Hb% is decreased in anemia, pneumonia,leukemia, chronic hemorrhage and hook

worm infections, pregnancy, CKD.

DEPARTMENT OF ROGA NIDANA, KTGAMC BENGALURU Page 7

 ENUMERATION OF TOTAL COUNT OF W.B.C

Apparatus:

Improved Neubauer’s double counting chamber

WBC pipette

WBC diluting fluid

Spirit

Needle/Lancet

Microscope.

The WBC counting area on the Neubauer’s double chamber consist of four big
squares in the corner. Each big square is divided into 16 small squares, totally 64
small squares.

WBC pipette is a glass pipette having a narrow stem and bulb. The bulb is
smaller than that of RBC pipette and contains a white bead. There are three markings,
0.5 below, 1 in the middle and 11 above the bulb

DEPARTMENT OF ROGA NIDANA, KTGAMC BENGALURU Page 8

 WBC diluting fluid:

Composition: Glacial acetic acid 2 cc, Gentian violet 1cc, distilled water
100cc. Distilled water causes haemolysis of RBC, glacial acetic acid destroys
haemolysed RBCs and gentian violet stains the nucleus of WBC.

Procedure:

Clean the fingertip with spirit and make a deep prick. Draw blood up to 0.5
mark. Then draw WBC diluting fluid up to the mark 11. Mix the blood thoroughly by
rolling pipette horizontally with the palms. Discard 2-3 drops, clean the counting
chamber and charge the chamber with prepared solution. Air bubbles should be
avoided. Focus the field under low power objective and count the WBC in all the
corner squares, so that the WBC in 64 squares are counted. Knowing this value the
total number of WBC in 1 cubic mm of blood can be calculated.

Result: The number of WBC /mm3 of undiluted blood

=No. of white cells counted x dilution

Areas counted X depth of fluid

DEPARTMENT OF ROGA NIDANA, KTGAMC BENGALURU Page 9

 Dilution =20;

Area counted = 4 x 1 sq. mm = 4 sq.mm;

Depth of fluid = 0.1mm

Value = no. of white cells counted x 50

Result:

Number of white cells/cu mm of whole blood = .

Normal value: The normal range of WBC count is 4500- 11000/mm3 of blood.

Pathological variations:

Increase in number of WBC is called leucocytosis.

Decrease in number of WBC is called leucopenia.

Leucocytosis:

Physiological causes of leucocytosis:

1. After exercise
2. Severe pain
3. Excitation
4. Injection of adrenalin
5. In infants, during pregnancy and during menstruation
6. Due to cold and exposure to ultra violet radiations
7. High temperature

Pathological cause of leucocytosis:

1. Leukemia
2. Hemorrhage
3. Inflammatory conditions,
4. Acute and chronic infections,
5. Allergic conditions

DEPARTMENT OF ROGA NIDANA, KTGAMC BENGALURU Page 10

Leucopenia:1. Typhoid
2. Viral diseases

ENUMERATION OF DIFFERENTIAL COUNT OF W.B.C

Principle:

The polychrome solution is Leishman’s stain containing Methylene blue and

Eosin. These basic and acidic dyes induce multiple colors when applied to the cells.
Methanol act as a fixative agent and also as a solvent. The WBC stained by acidic
dyes is described as Basophilic and acidophilic. The acid component is nucleus that
gets blue to purple shade by the basic dyes and they are called Basophilic. The basic
component of white cells (Cytoplasm) is stained by acidic dye and are called
Acidophilic. The neutral components are stained by both dyes.

Equipments:

Needle/Lancet,

Glass slide

Spreader slide

Leishman’s stain

Microscope.

DEPARTMENT OF ROGA NIDANA, KTGAMC BENGALURU Page 11

Procedure:

Place a drop of blood on the slide and make a tongue shaped smear with the
spreader slide and dry it. By using marker write the identification number on the slide.
It is stained by adding 10 – 15 drops of Leishman’s stain and kept for 1 minute. Add
buffer solution and mix the reaction mixture thoroughly by blowing on it through a
pipette. Wash the smear with running tap water and allow it to dry. First examine the
stained blood film under low power microscope. For screening, select an area between
tail and body of the film, where the R.B.C just touches each other without
overlapping. Place a drop of oil in the selected area. Switch to the oil immersion
objective. Identify various white cell, count at least 100 cells and give the % of the
cells seen. 100 squares are made on the paper and write the letter N for Neutrophils, L
for Lymphocytes,M for Monocytes, E for Eosinophils and B for basophils.

AGRANULOCYTES GRANULOCYTES

DEPARTMENT OF ROGA NIDANA, KTGAMC BENGALURU Page 12

Observations:

I) Granulocytes: These are cells with granulated cytoplasm. These

includeNeutrophils, Eosinophils, and Basophils.

1. Neutrophils: Size is 10-12μm. Nucleus - purple colored, eccentric with

heavy thick chromatin masses. It is divided into 2 to 5 lobes, Cytoplasm – abundant
with small pinkish staining dull granules.

2. Eosinophils: Size is 12- 15μm. Nucleus – globed, contains dense chromatin

masses usually nucleusis arranged in spectacle shape, Cytoplasm - the granules are
densely filled, uniform, spherical in size and deep orange pink colored .

3. Basophils: Size is 10-12μm. Nucleus - indented in several planes. It stains

pale purple colour because it contains less chromatin. It is difficult to see the nucleus
because the cytoplasm granules mask it. Cytoplasm - it contains basophilic granules
that overlaps nucleus. They are round dark purple to black color and vary in size.

ii) Agranulocytes:

1) Monocytes: Size is 10-22μm. Nucleus - kidney shaped or horseshoe shaped.

Sometimes it may be round or oval. It stains pale violet and has fine chromatin
granular networks.

2) Lymphocytes: Two forms are observed.

A) Large lymphocytes: Size is 12-16μm in diameter. Nucleus-dense, oval or

slightly indented , Cytoplasm - abundant and pale blue colored.

B) Small lymphocytes: Size is 10-12μm in diameter. Nucleus - dark, round

and sometimes indented, Cytoplasm - it has very little cytoplasm and often more just
around the nucleus.

Normal values:

Neutrophils: 40-75%

Eosinophils: 1-4%

Basophils: 0-1%
DEPARTMENT OF ROGA NIDANA, KTGAMC BENGALURU Page 13
 Lymphocytes: 20-25%

Monocytes: 2-8%

DEPARTMENT OF ROGA NIDANA, KTGAMC BENGALURU Page 14

 ENUMERATION OF TOTAL R.B.C COUNT

Aim:

To determinate the total number of erythrocytes in the given sample of blood.

Principle:

A known volume of blood is diluted and is charged into a chamber of known

dimension and the number of R.B.C s in the distributed blood is calculated.

Apparatus:

Lancet / Syringe

Improved Neubauer’s counting chamber

RBC pipette

R.B.C diluting fluid or Haeyem’s fluid

Microscope

R.B.C diluting fluid contains

1. Trisodiumcitrate:3.13g.
2. Formalin : 1ml
3. Distilled water : 100ml
Formalin acts as a preservative and checks bacterial and fungal growth.
Sodiumcitrate prevents coagulation of blood and provides correct osmotic pressure.
This fluid being isotonic with blood, prevents haemolysis.

Procedure:
RBC Pipette is a glass pipette having a narrow stem and a white bulb. The
bulb contains a red bead. Blood is taken up to 0.5 mark in the R.B.C pipette and
diluting fluid up to 101 mark. The pipette is rotated horizontally during mixing. After
5 minutes,by discarding few drops from the pipette, the counting chamber is charged.
Allow the cells to settle for 2 to 3 minutes. Place the counting chamber on
microscope, switch to low power objective (10x) and locate the large square in the
center with 25 small squares. Now switch to high power objective (45x) The RBC in
DEPARTMENT OF ROGA NIDANA, KTGAMC BENGALURU Page 15
the four corners and in the center square (marked ‘R’ in figure) are counted. R.B.C
count is then calculated.

Total Red blood cells /cumm = No. of red cells counted x Dilution
Area counted x Depth of fluid

= N x 200 = N x 10,000
1/5 x 1/10

Result:

The R.B.C count of the sample of blood =………… million/mm3 of blood.

Normal value:

Male: - 4.5- 6 x106 cells/μ l

Female: - 4 - 4.5 x106 cells/μl.

Pathological conditions

Decrease in the number of circulating erythrocyte indicates anemia and an

increased number of erythrocyte indicates the possibility of polycythemia.

DEPARTMENT OF ROGA NIDANA, KTGAMC BENGALURU Page 16

DEPARTMENT OF ROGA NIDANA, KTGAMC BENGALURU Page 17
 DETERMINATION OF PLATELET COUNT

Requirements:

1) Microscope
2) Improved Neubauer’s counting chamber
3) RBC pipette
4) Platelet diluting fluid.

Specimen:
EDTA anti coagulated blood.

Procedure:

1) By using RBC pipette draw EDTA blood up to 0.5 mark.

2) Wipe excess blood on outside of the pipette.
3) The diluting fluid is drawn up to mark 101(blood is diluted 1:200).
4) Mix the contents in the bulb thoroughly.
5) After 5 minutes, discard the first few drop then transfer a small drop on one
side of the counting chamber.
6) Place the filled mounted counting chamber under a petri dish with a moist
filter paper. Let it stay undisturbed for 15 min.
7) Place the counting chamber carefully on the stage of the microscope, under
low power magnification focus red cell counting area. Move to view the
corner square of the red cell area and change to high power objective.

DEPARTMENT OF ROGA NIDANA, KTGAMC BENGALURU Page 18

 8) Keep the condenser down and reduce the light by adjusting the diaphragm.
The platelets will appear light highly refractile particles.
9) Count platelets in all 25 small squares. The area covered by 25 squares is
equivalent to 1 sq.mm.

Calculation:

Platelets per cu mm= No. of platelets counted x dilution x chamber depth factor

Area of chamber counted

=N x 200x 5 x 10

= N x 10,000.

Normal range:

1,50,000 to 4,50,000 per cu mm.

Clinical significance:

Thrombocytopenia: Aplastic anemia, megaloblastic anemia,hypersplenism, acute

leukemia, Viral fevers, Haemorrhage.

DEPARTMENT OF ROGA NIDANA, KTGAMC BENGALURU Page 19

Thrombocytosis: Polycythemia vera, post splenectomy and chronic myelogenous
leukemia.

DEPARTMENT OF ROGA NIDANA, KTGAMC BENGALURU Page 20

 ESTIMATION OF PACKED CELL VOLUME

Introduction:
The haematocrit (Packed Cell Volume) of a sample of blood is the ratio of the
volume of erythrocytes to that of the whole blood.

Principle:
Packed cell volume (PCV) is the ratio of the volume of formed elements to the
volume of whole blood expressed in %.

Requirements:

i) Instrument: Wintrobe’shaematocrit tube is a special thick walled glass tube 11 cm

long, with an internal diameter of 2.5 mm and a flat inner base. It is calibrated at 1
mm intervals to 105 - 110 mm and holds about 1 ml blood.

ii) Blood sample: Venous blood anticoagulated with EDTA, Wintrobe’s mixture, or
heparin can be used.

Procedure:
 Thoroughly mix the blood in the bottle by repeated inversion or by a
mechanical rotator.
 Draw the blood into a long Pasteur pipette. Fill the Wintrobe’s tube up to the
100 mm mark, starting from the bottom and gradually withdrawing the pipette
as blood is expressed. This is to avoid air bubbles, which can get trapped in the
column of blood.
 Centrifuge the tube at 2000 – 3000 rpm for 30 minutes.

DEPARTMENT OF ROGA NIDANA, KTGAMC BENGALURU Page 21

Reading and interpretation:

RBC

Packed Cell Volume in Wintrobetube:

The height of the column of red cells is taken as the PCV i.e., the volume
occupied by the red cells expressed as a fraction of the total volume of blood. After
centrifugation layers are noted in the Wintrobe tube as:

 Uppermost layer of plasma.

 Thin white layer of platelets.

 Greyish pink layer of leucocytes.

 Lowermost layer of RBCs.

The buffy coat layer is an approximate indication of the number of white cells
and platelets. Normally 0.1 mm of this layer corresponds to about 1000 WBC/cu mm.
When there is marked leukocytosis (more than 30,000 WBC/cu mm) - 0.1 mm then

DEPARTMENT OF ROGA NIDANA, KTGAMC BENGALURU Page 22

corresponds to about 2000 WBC/ cu mm. Occasionally an increase in buffy coat
could be due to marked increase in platelets.

Plasma colour:

 Yellow colour of plasma indicates jaundice.

 Opaque plasma is due to lipaemia.

 Pink colour denotes haemoglobinemia.

Normal Values:

Men: 40 – 54%

Women: 36 – 47 %

At birth: 44 – 62%

Clinical significance:

An individual’s value below the reference range for the age and sex indicates
anaemia and a higher value indicates polycythemia.

Haematocrit reflects the ratio of red cells to plasma and not the total red cell
mass. For example: haematocrit may be low in pregnancy as there is hydremia or
haemodilution in pregnancy. Here the total number of red cell mass is not reduced but
plasma volume is more. The opposite is seen in haem concentration.

DEPARTMENT OF ROGA NIDANA, KTGAMC BENGALURU Page 23

DEPARTMENT OF ROGA NIDANA, KTGAMC BENGALURU Page 24
 ESTIMATION OF ERYTHROCYTE SEDIMENTATION RATE

Principle:

When a normal blood column if kept vertically for some time the RBCs will
settle down to the bottom. E.S.R is the state of measure of the suspension stability of
RBC. If a vertical column of citrated blood is kept, the red cell begins to settle until
they form a packed volume at the bottom of pipette. There are three stages of
sedimentation.

1. Stage of aggregation in the first 10 minutes.

2. Stage of rapid sedimentation taking up to 40 minutes.

3. Stage of steady packing of cells.

The length of the plasma column at the top of blood column at the end of first
hour is measured in mm.

Methods of estimation of E.S.R:

1. Westergren’smethod:Westergren’s pipette is a 30 cm long glass tube having a

diameter of 2.5mm. It is graduated from above downwards from 0 to 200. The anti
coagulant used is 3.8 % sodium citrate or EDTA is also used.

2. Wintrobe’smethod:In this method heamatocrit tube is used. It is a cylindrical tube

open at one end. The total length is 11cm. The tube is uniform and has an internal
diameter of 2.5mm. It is graduated from 0 to 10. The anti coagulant used is sodium
citrate, EDTA or double oxalate.

Procedure:
DEPARTMENT OF ROGA NIDANA, KTGAMC BENGALURU Page 25
Westergren’smethod: Blood is collected and mixed with anti coagulant in the ratio
1:4. The temperature should be 25-280C. Blood is drawn up to the mark 0. The tube is
kept exactly vertical for 1 hour in a stand. The height of plasma column after one hour
is noted.

Result: E.S.R of the given sample of blood by Westergren’s method is……mm/after 1

st
hr.

Normal values:

1. Westerngren’smethod :

Male: 0 - 15 mm/after 1sthr.

Female: 0 - 20 mm/after 1sthr.

2. Wintrobe’smethod :

Male: 0 - 9 mm/after 1sthr.

Female: 0 - 20 mm/after 1st hr.

Factors influencing E.S.R:

1. Number of cells: high cell count- decreases ESR, low cell countincreasesESR.

DEPARTMENT OF ROGA NIDANA, KTGAMC BENGALURU Page 26

2. Shape of cells: tendency towards spherical form – decreasesESR.

3. Size of cells: increased size- increases ESR.

4. Rouleaux formation: increases ESR.

5. Plasma protein: fibrinogen and globulin - increases ESR (but albumin retards
sedimentation).

6. Viscosity in plasma: increases ESR.

7. Specific gravity of plasma: less - increases ESR.

8. A rise of temperature above 200C : increases ESR.

9. Position of tube, in standing position ESR is more.

10. ESR increases if the tube is shorter or narrow.

Variations:

1. Physiological:-

a. New born: ESR is low.

b. In old age: it is increased.

c. ESR is increased during menstruation, pregnancy.

2. Pathological: ESR is decreased in :

a. Polycythemia vera

b. Sickle cell anemia

c. Congestive heart failure

d. Whooping cough

e. Dehydration.

DEPARTMENT OF ROGA NIDANA, KTGAMC BENGALURU Page 27

ESR is increased in:

a. Anemia except sickle cell anemia

b. Acute myocardial infection

c. Carcinoma

d. Tuberculosis

e. Acute gout.

ESR is rapidly raised in:

a. Temporal arteritis

b. Rheumatoid arthritis

c. Kala azar

d. Multiple myeloma

e. Leukemia

f. Chronic renal disease.

ESR in diagnosis:

1. To distinguish functional from organic diseases.

2. In acute rheumatoid arthritis, acute gout and infective arthritis it is markedly raised,
while in osteoarthritis it remains normal.

3. In myocardial infarction, it is raised, while in angina it is not.

4. It is raised in pelvic inflammation, and not in unruptured ectopicgestation.

DEPARTMENT OF ROGA NIDANA, KTGAMC BENGALURU Page 28

 ESTIMATION OF BLEEDING TIME

Principle:

Bleeding time is the time taken between the appearance of blood to the
cessation of blood.

Apparatus:

Needle, blotting paper, stop watch.

Procedure:

Clean the fingertip with spirit and allow it to dry. Make a deep prick. Blood
will start flowing. Note the time. With the blotting paper touch the blood coming after
each ½ minutes. A fresh part of blotting paper should be used each time. Note that the
blood spot on the filter paper gets thinner, till it disappears and the bleeding stop. Note
the time. This time, in interval gives the bleeding time.

Result:- Bleeding time of the given sample of blood is …….. minutes.

Normal value: - 2-4 minutes.

DEPARTMENT OF ROGA NIDANA, KTGAMC BENGALURU Page 29

Pathological conditions:-

Bleeding time is increased in

1. Thrombocytopenia purpura.

2. Hemophilia.

3. Vitamin K deficiency.

Bleeding time is decreased, when platelet extract is administrated intravenously.

DEPARTMENT OF ROGA NIDANA, KTGAMC BENGALURU Page 30

 ESTIMATION OF CLOTTING TIME

Principle:

Clotting time is the time interval between appearance of blood and appearance
of clot.

Apparatus:

Capillary tube (10cm length) stop watch needle etc.

Procedure:

A deep prick is made on the finger tip. Flow the blood into the capillary tube.
Start the stop watch simultaneously. When capillary tube is filled, place it for 1-2
minutes. After this, start breaking the tube at ½ minutes interval until the fiber in
thread is seen between the broken pieces. When the fibrin thread appears, time is
noted. The time taken gives clotting time.

Result: Clotting time of given sample of blood is …………. minutes.

Normal value: 5-10 minutes.

Factors preventing coagulation.

1. By adding substances of biological origin such as prothrombin, heparin etc.

2. By avoiding contact with water – wettable surface.

3. Removal of Ca2+ ions.

4. Precipitation of fibrinogen.

DEPARTMENT OF ROGA NIDANA, KTGAMC BENGALURU Page 31

Factors that fasten coagulation

1. Contact with water wet table surface.

2. Addition of foreign bodies.

3. Addition of thrombin and thrompoplastin,VitK, CaCl2 etc.

Variations:

Clotting time is prolonged in hemophilia due to the absence of anti hemophilic

factor. But bleeding time is normal.

DEPARTMENT OF ROGA NIDANA, KTGAMC BENGALURU Page 32

 BLOOD GLUCOSE DETERMINATION

Name of the method: Orthotoluidine method (monostep).

Requirements:
1) Burette or a dispenser
2) Test tubes (15 x 25mm)
3) Push button pipette
4) Glucose reagent
5) Glucose standard:100mg/dl
6) Bunsen burner or hotplate
7) Water bath
8) Stopwatch
9) Centrifuge
10) Photometer

Test principle:

Glucose reacts with the ortho-toluidine in hot acidic medium to form a green
colored complex. The intensity of the final color produced is measured by using a
photometer at 620nm – 660 nm. The measured colour intensity is directly proportional
to the concentration of glucose in the specimen.

Normal values:

Serum/plasma (fasting) - 70-110 mg/dl

Serum/plasma (postprandial) two hours after lunch –up to 130 mg / dl

Specimen collection:

Patient should fast for 12 – 16 hours.

Fasting sample (F): collect 2-3 ml of blood in fluoride bulb.

Post prandial sample (PP): 2-3ml of blood to be collected after 2 hours after taking
food.

DEPARTMENT OF ROGA NIDANA, KTGAMC BENGALURU Page 33

Sample material: Plasma.

Preparation and stability of the reagent:

1) Orthotoluidine reagent: It contains 940 ml of glacial acetic acid, 60 ml of

orthotoluidine and 1.5 gm of thiourea. The reagent is corrosive and should not
be pipetted by mouth.
2) Glucose standard, 100mg /dl, in saturated benzoic acid.
3) Additional- 5 gm /dl trichloroacetic acid - this reagent is stable at room
temperature.

Procedure:
By using a burette or a dispenser, pipette in the tubes labeled as follows-

Test Standard Blank

1) Glucose reagent (ml) 5.0 5.0 5.0
2) Serum/plasma,ml 0.05 - -
3) Glucose standard:100mg/dl,ml - 0.05 -
4) Distilled water , ml - - 0.05

Mix thoroughly and place the tubes in the boiling water bath for exactly ten
minutes. By using tap water, cool the tubes to room temperature. Measure the optical
densities of test and standard against blank at 640 nm (red filter, 620-660 nm).

Calculations:
Serum/plasma glucose,mg/dl = O.D.test X 100
O.D.std

DEPARTMENT OF ROGA NIDANA, KTGAMC BENGALURU Page 34

Clinical significance:
Increased blood glucose levels – diabetes mellitus, hyperthyroidism,
hyperpituitarism, adrenocortical hyperactivity and occasionally in hepatic disorders.

Low fasting blood glucose values are associated with hypothyroidism,

hypopituitarism and hypo adrenalism.

GLUCOSE TOLERANCE TEST

After drawing blood in the fasting period, the patient should be administered
with 75 gms of glucose (1.75gms/kg body wt) and blood collected after 2 hours and
investigated.

DEPARTMENT OF ROGA NIDANA, KTGAMC BENGALURU Page 35

 DETECTION OF MALARIAL PARASITE
(THIN AND THICK MP SMEARS)

Principle : Two blood smears are made (1) thin and (2) thick.

1. Thin blood smear is stained by using Leishman’s stain (Wright’s stain). The stained
blood gives an idea of the morphology of blood cells smear and helps to identify the stage
of malarial parasite in its life cycle.

2. The thick smear is stained by Field’s stain, without fixing with methanol. This leaves
behind the imprints, pink parasite dots and other structures.

Requirements : Glass slides grease free, spreader slide and microscope.

Specimen : EDTA anticoagulated or capillary blood.

Procedure :

1. Prepare thin and thick blood smear.

2. Stain thin smear in Leishman’s stain or Wright’s stain.

3. Stain thick smear (without fixing in methanol) in field‘s stain A and B.

4. Scan the smears under the high power objective (40 x) and examine closely
under oil immersion objective.

Observations : Various stages of parasites seen are

(1) Trophozoite (immature and mature),
(2) Schizont and
(3) Gametocyte.

STAGE APPEARANCE
Immature trophozoite Undivided nucleated cells with blue coloured cytoplasm or
ring of cytoplasm within the red cells.
Mature trophozoite Compact cytoplasm. Enlarged amoeboid shape, no ring
structure.
Schizont Individual nucleated cells distributed throughout the red
cell in circle form.
Gametocyte One compact, round or elongated gametocyte (male or
female) filling entire red cell.

DEPARTMENT OF ROGA NIDANA, KTGAMC BENGALURU Page 36

 WIDAL TEST
(SLIDE AGGLUTINATION METHOD)

Principle :

Serum sample of the patient is tested for ‘O’ and ‘H’ antibodies by using
antigenic suspensions, Salmonella typhi ‘O’ and Salmonella typhi ‘H’ respectively. In
typhoid fever an increase in the agglutinations (titer above 240) is observed. For
paratyphoid testing, the antigen suspensions S. paratyphoid ‘AH’ and S. paratyphoid
‘BH’ are used.

Specimen : Fresh fasting serum.

Requirements : Glass plates with ceramic rings, test tubes(100x10mm), droppers,

Widal (slide test) kit, timer, applicator sticks (match sticks).

Procedure :

 In each of the 4 rings of the slide, labeled as ‘O’, ‘H’, ‘AH’ and ‘BH’ add 1
drop of the serum.

 Add corresponding antigen drop (one) in each ring of the glass plate, mix the
antigen suspension and the diluted serum drop in each ring using separate
applicators.

 Slowly rock and tilt the glass plate and observe for 3 minutes.

 Record the degree of agglutination as 4+, 3+, 2+, 1+ or negative.

DEPARTMENT OF ROGA NIDANA, KTGAMC BENGALURU Page 37

 WIDAL TEST
(TUBE AGGLUTINATION METHOD)

Introduction: The test commonly includes agglutination of ‘O’ and/or ‘H’ Salmonella
antigens for group A, B, C or D.

Specimen : Serum.

Requirements : Same as above.

Additional requirements: 36 widal tubes (5x50 mm), 0.1 and 1 ml serological

pipettes, test tube rack.

Procedure :

 For each serum sample under test arrange four rows (labeled as ‘O’, ‘H’, ‘AH’
and ‘BH’) of 6 tubes each in a rack .
 Take 5 tubes in another rack and prepare dilutions as follows :
1) Place 7 ml of the normal saline in first tube and 3.5 ml in remaining
four tubes.

2) Add 0.5 ml of serum in the first tube, mix well.

3) Transfer 3.5 ml of the mixture from the first tube to the second tube
and continue successive transfer of 3.5 ml quantities till the last tube.

4) The dilutions obtained are 1:30, 1:60, 1:120, 1:240 and 1:480.

 Transfer 0.5 ml quantities from the master dilution tubes to each of the
corresponding vertical row in the test tubes placed in first rack.
 Place 0.5 ml of normal saline in the 6th tube (from each row).
 Add 0.5 ml saline and 1 drop of concentrated S. TYPHI ‘O’ antigen to the test
tube row labeled ‘O’.
 To each of the 6 tubes in the second (H) third (AH) and fourth (BH) horizontal
rows add 0.5 ml of S. typhi ‘H’, S. paratyphi A (H) and S. paratyphi B (H)
antigens respectively.
 Shake the rack well and keep at 370C overnight (16-20 hours).
 Observe the tubes next day and note the highest dilution in which
DEPARTMENT OF ROGA NIDANA, KTGAMC BENGALURU Page 38
 agglutination is observed by naked eyes (use hand lens if necessary). The last
tube (6th) containing only normal saline and the antigen should be used as a
reference tube for the comparison with the positive findings.

Interpretations :

 Serum from normal individuals may agglutinate the antigens upto the dilutions
1:60.

 A moderate rise in titer of all the three ‘H’ agglutinins (simultaneously) (H,
AH and BH) against all ‘H’ antigens is suggestive of recent TAB vaccination.

 A rise of titer (O and H) above 240 is typical of enteric fever.

 A rise in titer is also prevented by antibiotic treatment.

 Positive results should be correlated with clinical findings and previous history
of immunization.

DEPARTMENT OF ROGA NIDANA, KTGAMC BENGALURU Page 39

 PHYSICAL EXAMINATION OF URINE

Volume

the arrange output of urine is 2.25 L/day. Volume may be increased by excessive
water in take, increased salt in take and in diabetes mellitus. Volume may be
decreased due to dehydration, low BP and shock.

Appearance

Normal urine is clear and transparent. It may become turbid when exposed for a long
time due to conversion of urea to (NH4)2CO2 (Ammonium carbonate). In decreased
condition, urine may be cloudy due to presence of WBC.

Color

Normally urine is straw colored due to the presence of Hemochromes. Bright red
color indicate large amount of fresh blood. Brownish yellow or green indicate the
presence of bile pigments.

Odor

Normally urine is aromatic due the presence of small amount of uric acid. When urine
is allowed to stand still, it given an ammoiacal odor because of decomposition of urea
with the liberation of NH3.presence of ketone bodies produce fruity smell.

pH

Normal pH is 6-8 .to measure the pH of urine, pH paper are available.

DEPARTMENT OF ROGA NIDANA, KTGAMC BENGALURU Page 40

Specific gravity

It is define as the ratio of weight of fixed volume of solution to that at same volume of
water at specified temperature. The urino-meter method of measuring specific gravity
of urine is based on the principle of buoyancy. Procedure:-mix well the urine and is
allowed to attain the room temperature. Urine is produced in the cylindrical tube to
that it is nearly full. If there are any air bubble, or froth, they are removed using filter
paper. Float the urinometer in urine and due that the instrument does not touch the
sides of they cylinder. Note the reading. Normal value:- 1-010-1.025 Pathological
conditions:- Specific gravity of urine is decreased in excessive in diabetes and in
diabetes insipidus and excessive perspiration.FDFGG

DEPARTMENT OF ROGA NIDANA, KTGAMC BENGALURU Page 41

 CHEMICAL EXAMINATION OF URINE

Protien-Heat Or Acetic Acid Test

This test is based on the principle that protein coagulate on heating, (addition of few
drops of 3% CH3OOH which eliminate the phosphate and carbon).

Procedure

Tell a clean dry test tube with 2/3rd of urine, which is faintly acidic. Heat the upper
part of the test tube till urine boils. If protein or phosphate or carbonate are present, a
white loud like color will appear on the heated portion. Add 2-

3 drops of 3% CH3COOH the cloudiness disappear. Due to the presence of urine

proteins, cloudiness presents.

Observation:- cloudiness presents after the addition of CH3COOH

Result:-Protein is present/Absent in the given sample of urine.

Pathological conditions

Protein urine:-Nephrosis, tuberculosis, cancer, nephritis, polycystic kidney, fever,

toxemia, trauma, severe anemia, aspirin.

DEPARTMENT OF ROGA NIDANA, KTGAMC BENGALURU Page 42

SUGAR - BENEDICT’S TEST

Aim

To determine the presence of sugar in the given sample of urine

Principle

Sugar present in urine contain aldehyde group which will reduce the blue colored
CuSO4 to CuO and the color of the reagent.

Procedure

Take 5 ml of benedicts qualitative reagent in a test tube and add & drops of urine to it.
Then toil it for 2-3 minutes allow to cool and note the color.

Observation

The blue color of the solution, gradually changes to green and later on green colored
solution with yellow precipitate was obtained.

Color and range:-

Change of color in solution and precipitate formed.

Clean blue or green with no precipitate : 0%

Green with yellow precipitate : 0.5%

Green to yellow with yellow precipitate : 1%

orange solution with yellow precipitate : 1.5%

Orange to red precipitate : 2% above

Result

The give sample of urine contain …………….. of sugar.

Pathological conditions: increased glucose in urine is found in diabetes

mellitus, brain injury & myocardial infraction.

DEPARTMENT OF ROGA NIDANA, KTGAMC BENGALURU Page 43

Interfering factors

1. Pregnancy and lactation may cause false +ve.

2. Ascorbic acid, creatinine, streptomycin etc may cause false +ve

3. Stress, excitement, testing after heavy meal etc may cause false +ve

KETONE BODIES - ROTHERA’S TEST

DEPARTMENT OF ROGA NIDANA, KTGAMC BENGALURU Page 44

Aim

To determine the presence of ketone bodies in the given sample of urine.

Principle

Action of auto acidic acid with sodium nitropruside in the presence of an alkali
produce a purple color.

Procedure

Take 5ml of urine in a test tube. Saturate it with ammonium salphate and then add
crystals of sodium nitropruside and mix well then gently add 1ml of liquor NH3
through the sides of the test tube so that it former a layer on the top of urine. If the
ketone present a purple (permanganate) color will form at the function of 2 layer.

Observation

A purple colored ring is formed at the junction of the two liquid.

Result

Ketone bodies are present in the given sample of urine.

Pathological condition

1. Ketone and ketone urea may occur whenever increased amount of fats are
metabolized carbohydrate in take is restricted or diet is rich in fat.

2. Ketone bodies in urine appear first then in blood.

3. Ketonuria is associated with fever, gastrointestinal disturbances prolonged

vomiting, anorexia, fasting, starvation etc.

4. Drugs that may cause false + ve are levodopa, insulin, pyridine,ether, paraldehyde
etc.

BLOOD - BENZIDINE TEST

Aim

To determine the presence of blood in the given sample of urine.

DEPARTMENT OF ROGA NIDANA, KTGAMC BENGALURU Page 45

Principle

Peroxides present in the hemoglobin decomposer H2O2 and liberate nascent O2 .it
oxides benzidine to form a blue colored complex.

Reagents

• Saturated solution of benzydine in glacial acetic acid (48%)

• 3% H2O2.

Procedure

Mix equal volume of benzidine solution and H2O2 in a test tube. Take two ml of urine
in another test tube. Bal it and cool. To it then add equal volume of benzidine. A blue
color indicator the presence of blood.

Observation:- a blue colored complex is formed.

Result:- blood is present in the given sample of urine.

Pathological condition:-

1. Hemoglobin urea is found in extensive burns and crushing injuries; transfusion

reaction compatible blood, fibril intoxification, malaria, hemolytic anemia etc

2. Drugs which may cause + ve result- bautracin, caumarin, aspirin etc.

3. Interfering factors:-

4. 1.high doses of vitamin C may give false negative results.

3. Drugs causing positive results are bromide, copper, iodine and

oxidizing agents.

BILE SALTS – HAY’S TEST

Aim

To determine the presence of tile salts in the given sample of urine.

Principle

DEPARTMENT OF ROGA NIDANA, KTGAMC BENGALURU Page 46

Bile salts when present in the urine lower the surface tension of urine. When sulphur
powder is sprinkled on the surface of urine, it sinks to the bottom of the test tube. So
in case of normal urine, sample, thesulphur powder floats on the surface of urine.

Procedure

Like about 5ml of urine in a test tube, sprinkle a little dry sulphur powder on the
surface of urine. Observe the sulphur powder, whether it sinks or not. If it sinks down
the bile salts is present. It not bile salts is absent.

Observation:-sulphur sinks to the bottom

Result:- bile salts are present in the given sample of urine.

DEPARTMENT OF ROGA NIDANA, KTGAMC BENGALURU Page 47

BILE PIGMENTS- FOUCHET’S TEST

Aim

To determine the presence of bile pigments in the given sample of urine.

Principle

When soluble BaCl2 is added to the urine, Ba2+ combine with SO4 2- in the urine to
form BaSO4. Any bile pigments if present may adhere to the precipitate and detected
by oxidation of bilirubin (yellow) to biliverdin (green) and treatment which FeCl2 in
the presence of trichloro acetic acid (TCA) Blue color is given by tile cyanin.

Procedure

Take 5 ml of urine in test tube and add a few drops of MgSO4 and BaCl2. Mix well
and filter paper, on the top of another filter paper, to dry. In the precipitate on the filter
paper, add 2.3 drops of Fouchet’s reagent. Development of a blue or green color
indicates the presence of bilirubin.

Observation:- a green color is developed.

Result:- bile pigment is present in the given sample of urine. Pathological

conditions:-hepatitis, Cirrhosis, Cholecystitis, lymphoma.

DEPARTMENT OF ROGA NIDANA, KTGAMC BENGALURU Page 48

MICROSCOPIC EXAMINATION OF URINE

Aim

To identify the microscopic elements present in the given sample of urine.

Procedure

Place 10ml of urine in a conical –u shaped centrifugal tube and centrifuge for 1500
rpm for 5 minutes. Pour out supernatant liquid. Mix the sediments remaining in the
tube. Transfer one drops into a slide. Apply cover slip and examine under a low power
microscopic with reduced light. The urine sediments are divided into 2 broad classes
organized and unorganized sediments. Organized sediments: include

1. R.B.C:-when large number of R.B.C are present in urine it is pathological. Cells

may be large one appearance crenated R.B.C should be qualified by reporting the
number of present/ hpf.

2. Epithelial cells:- Normally a few of there are present in a urine of marked increase
indicates desquamate of surface tissue of urinary tract.

3. Pus cells:- When they are present in large number (more than 5/hpt). They include
inflammation or infection of urinary trail.

4. Cast

a. Hyaline casts:- They are colorless semitransparent broad straight and varying in
length. They consists of coagulated protein material. They are present in large number
in various kidney diseases.

b. Granular casts:- They are cast containing granules. They are coagulated protein in
which numerous proteins are embedded. The granular are due to disintegration of
WBC or epithelial cells which presence of there cells indicate renal diseases.

c. Blood cell casts:-There are clots with visible red cells in coagulated protein and
commonly seen in acute nephritis.

d. Pus cell casts:- There are seen in poly nephrites, where there is inflammation and
formation of pus in kidney. Pus cells are

DEPARTMENT OF ROGA NIDANA, KTGAMC BENGALURU Page 49

embedded in protein matrix.

e. Epithelial cell casts:- There consists of coagulated protein in which epithetical cells
from renal tubules are embedded. It indicates renal diseases.

f. Fatty casts:- There casts contain numerous fat globules, which indicate renal
diseases.

g. Waxy casts:- There resemble hyaline cast, but are much more rustle, and opaque
neither than transparent with a duel waxy appearance. They are found in later stages
of nephritis Unconjugated sediments:-

A. Crystals found in acid urine:

1. Calcium oxalate:- colorless envelope shaped crystals with infective corners. More
rarely appears a oval shape or biconcave discs.

2. Lewine crystals:- They are slightly long or oblong resembling spleen. flat globules
with delicate striations.

3. Crystine crystals:- Colorless, highly refractile, thick hexagonal plate

4. Thyroxine crystals:- Colorless fire needle cluster crossing at various angles.

Clusters may appear as blade in centre.

5. Uric acid crystals:- Usually yellowish brown rheumatic crystals may be colorless.

6. Sulphur crystals:- There various considerably in shape, most of them are like,
sheath of needle. It may be clear or brown, in color usually appears with concentric
finding.

7. Cholesterol crystals:-- Large flat plate with one or more corners cut off. They may
occur in nephritis or lipid nephrosis.

B. Crystals found in alkaline urine:-

1. Ammonium magnesium phosphate:

2. They are colorless and appear like feathers or leaf

3. Calcium carbonate:- They are small dumb ball shaped and dissolved in 10% acetic

DEPARTMENT OF ROGA NIDANA, KTGAMC BENGALURU Page 50

acid

4. Ammonium carbonate:- They are yellow spherical bodies usually with radical and
concentric striations

5. Calcium phosphate:- Often form large irregular usually granular colorless plates.
Small cells may be mistaken for squamous epithelial cells.

DEPARTMENT OF ROGA NIDANA, KTGAMC BENGALURU Page 51

 ROUTINE EXAMINATION OF SPUTUM

Mucous that is coughed up from the lower airways is called Sputum.

Normal sputum is colourless, watery and odourless tracheobronchial secretion. It is an

inconstant mixture of plasma, mucin, electrolytes and water.

Specimen Collection:
 Specimen – preferred is early morning or whole day specimen in a sterile wide mouth
glass or plastic container with screw cap (50 – 60 ml capacity).
 Instructions given to the patient
 The mouth should be rinsed well by using water.
 The sputum must be coughed up from the lungs or bronchi directly into the
container.

PHYSICAL EXAMINATION
a) Quantity
b) Colour
c) Consistency and appearance
d) Odour
e) Layer formation

QUANTITY

Normal Value:

 Morning specimen – 2 to 5 ml
 24 hrs specimen – about 100 ml

Pathological Variations:

 Over 100 ml ( 24 hrs collection ) – Pulmonary oedema, Luna abscess,

Bronchiectasis, Pulmonary haemorrhage, advanced pulmonary TB.
 Over 500 ml – Ruptured amoebic abscess of liver in lungs.

DEPARTMENT OF ROGA NIDANA, KTGAMC BENGALURU Page 52

 COLOUR

Normal: Clear and colourless.

Pathological variations:
 Yellow – Pneumonic process
 Greenish – Pseudomonas infection, rupture of liver abscess in lungs
 Rust coloured – Pneumococcal pneumonia, Pulmonary gangrene
 Bright Red – Recent haemorrhage which can follow acute cardiac infarction or
pulmonary infarction and rupture of vessels, Pulmonary Tuberculosis, Ca Lung,
Pulmonary Embolism.
 Black – Due to inhalation of dust and coal dust.

CONSISTENCY AND APPEARANCE

Normal: Colourless, watery and opalescent.

Pathological variations:

 Serous – Pulmonary oedema

 Mucoid – Acute bronchitis, Asthma, Whooping cough.
 Purulent – Ruptured empyema, Bronchiectasis.
 Tenacious – Lobar pneumonia, Bronchomoniliasis, Bronchial Asthma.
 Mucopurulent – Lung cavitation,Bronchomoniliasis.
 Bloody – Mitral stenosis, Pulmonary infarction, Pulmonary Tuberculosis,
Carcinoma of lungs.

ODOUR

Normal: Odourless.

Pathological:

DEPARTMENT OF ROGA NIDANA, KTGAMC BENGALURU Page 53

 Putrid – Lungabscess, Bronchiectasis, Gangrene of lungs.
 Sweetish – Pulmonary TB, Bronchiectasis.
 Cheesy – Necrosis of malignant tumours of the lungs, prforatingempyemas.

LAYER FORMATION

Procedure: Specimen is placed in a test tube for 2 – 3 hrs.

Normal:No formation of layer.

Pathological variations:
Formation of 2 – 3 layers
Top – Frothy mucous
Middle – Opaque water material
Sediment – Pus, tissue, bacteria etc.
 Seen in Bronchiectasis, Gangrene, Lung abscess.

DEPARTMENT OF ROGA NIDANA, KTGAMC BENGALURU Page 54

 MINUTE MACROSCOPIC EXAMINATION

Procedure: A portion of the sputum is observed by placing in a petri dish (thin layer
of sputum ).

Observations:

a) Cheesy masses – Pulmonary TB and Pulmonary gangrene.

b) Bronchial Casts (Present as a white branching tree like casts) –Fibrinous
bronchitis and Consolidation of pneumonia.
c) Dittrich’s plugs (Yellowish white plugs, size ranging from pin head to a bean) -
Bronchial asthma, Putrid bronchitis and Bronchiectasis.
d) Lung stones – Calcified tuberculosis material.
e) Sulphur granules (Yellow coloured) – Actinomycosis of the lungs.

DEPARTMENT OF ROGA NIDANA, KTGAMC BENGALURU Page 55

 MICROSCOPIC EXAMINATION OF UNSTAINED SPUTUM

Procedure: Place a drop of well mixed sputum on glass slide and place a coverslip
on it. Observe first under low power and afterwards under high power objective.

Observation:

1) Pus cells (many) – Inflammation in the respiratory tract.

2) RBC’s (many) - Inflammation in the respiratory tract, malignant tumor.
3) Heart failure cells (siderophages which contains haemosiderin) – Passive
pulmonary congestion due to mitral stenosis, Pulmonary infarction, Pulmonary
haemorrhage.
4) Carbon laden cells – Anthracosis (those who inhale large amount of tobacco
smoke or live in a smoky atmosphere).
5) Curschmann’s spirals – Bronchial asthma.
6) Elastic fibres – Breaking down of the lung parenchyma.
7) Charcot Leyden crystals – Bronchial asthama
8) Fatty acid – Chronic tuberculosis, Putrid bronchitis,Gangrene
9) Cholesterol – Empyema, Chronic tuberculosis, Chronic lung Abscess
10) EntamoebaHistolytica – Ruptured amoebic abscess of liver in lungs.

GRAM’S STAINING METHOD

Requirements :
1)Sputum specimen
2)Glass slides and cedar wood oil
3)Nichrome loop and Bunsen burner
4)Microscope
5) Reagents:
i. Crystal Violet stain
ii. Gram’s iodine
iii. Alcohol acetone or Rectified spirit
iv. Distilled water
Procedure:
a) Smear preparation :
DEPARTMENT OF ROGA NIDANA, KTGAMC BENGALURU Page 56
 Take a grease free dry slide and make an oval shaped mark at the center by using
a glass marker.
 Sterilize the inoculating (nichrome) loop on the flame of a Bunsen burner.
 Transfer a loopful of sputum by the sterile nichrome loop and make a smear in
the premarked area on the slide (smear should not be very thin as well as very
thick)
 Allow the smear to dry in the air.
 Fix the dry smear by passing the slide 3 to 4 times through the flame quickly with
the smear side facing up.
b) Gram staining :
 Place the slide on the staining glass rods.
 Cover the smear with crystal violet stain and leave for 1 min.
 Wash carefully under running tap water.
 Flood the smear with Gram’s iodine solution and wait for one min.
 Drain off the iodine.
 Decolorize the smear with alcohol acetone (or Rectified spirit) for 20 to 30
seconds. Continue till purple stain just stops coming on the slide .
 Gently wash the slide under running tap water and drain completely.
 Counterstain the smear with safranin for 10 seconds or with dilute (1:20) basic
fuchin for about 1 min.
 Drain the staining solution and allow the stained smear to dry in air.
 First observe for uniform stained area under low power objective, afterwards
high power objective and finally under oil immersion.

Results

The staining results of gram stain are as follows:

1. Gram negative bacteria: pale to dark red

2. Gram positive bacteria: dark purple
3. Yeast cells: dark purple
4. Nuclei of pus cells: red

DEPARTMENT OF ROGA NIDANA, KTGAMC BENGALURU Page 57

5. Epithelial cells: pale red

DEPARTMENT OF ROGA NIDANA, KTGAMC BENGALURU Page 58

Staphylococcus aureus, Gram-positive cocci, in purple and Escherichia coli (Escherichia coli Gram-negative bacilli, in red.

ACID FAST STAINING:

Requirements
1) Sputum specimen
2) Glass slides and cedar wood oil
3) Nichrome loop and Bunsen burner
4) Microscope
5) Reagents:
i. Methylene blue
ii. Carbol-fuchsin stain
iii. 20% sulphuric acid
iv. Distilled water

Procedure:

1. Prepare smear from the sputum specimen on a glass slide and fix it by heating
on bunsen burner flame .
2. Staining: place the heat fixed slide on the staining rack or rods and flood the
smear with working carbolfuchsin stain.
3. Heat gently by Bunsen burner flame, until steam rises. Avoid boiling and
continue heating for about five minutes. Do not aloe to dry the stain on the slide.
Add more stain if necessary.

DEPARTMENT OF ROGA NIDANA, KTGAMC BENGALURU Page 59

4. Wash the stain off the slide with water and continue rinsing until the water runs
off is colourless.
5. Decolourisation: cover the slide with 20% sulphuric acid for about one minute.
6. Counterstaining: cover the slide with methyline blue stain for about one minute.
7. Wash with tap water, allow water to drain, and allow it to dry in air or blot
carefully.
8. Microscopic examination: observe the slide under low power objective and then
examine under oil immersion object.

Results:

Acid fast organisms: bright red bacilli on blue background.

WRIGHT STAINING METHOD

Procedure:

1. Make a smear of the sputum, dry at room temperature and fix by gentle heating
on a flame.
2. Stain the smear using Wright’s stain, observe the stained smear under oil
immersion lens for the differential leukocyte determination.
OBSERVATIONS:

DETECTION NORMAL ABNORMAL CLINICAL CONDITION

OF FINDINGS FINDINGS
Neutrophills Present, few Increased Pyogenic infection

Lymphocytes Present, few Increased Early or mild cases of

tuberculosis

Eosinophills Absent Increased Asthma and eosinophilic

lung

Erythrocytes Absent Present Haemorrhage or

inflammatory condition

DEPARTMENT OF ROGA NIDANA, KTGAMC BENGALURU Page 60

 ESTIMATION OF SERUM BILIRUBIN

Principle:
The method is based on the van den Bergh reaction. When Bilirubin reacts
with diazo reagent, purple colored azobilirubin is formed. Methanol is used as
reaction accelerator. Since total Bilirubin is soluble in it. By using only distilled water,
direct Bilirubin is determined. The difference between total Bilirubin and direct
Bilirubin gives measure of indirect Bilirubin. The optical densities of total test and
direct test are measured against respective blanks at 540 nm.

Metod :Malloy and evelyn method

Requirements:

1. Test tubes :15X125mm

2. 5.0,0.2ml serological pipettes
3. Stop watch
4. Test tube stand
5. Photometer

Reagents:

1. DiazoA : it is prepared by mixing 0.1 gm of sulfanilic acid in 100 ml of 1.5 %

hydrochloric acid .
2. Diazo B: it is prepared by mixing 0.5 gm of sodium nitrate in 100ml of
distilled water.
3. Diazo blank reagent : ( 1.5% Hcl): it is prepared by adding 1.5 ml of conc.
Hcl to about 90 ml of distilled water in a 100 ml of volumetric flask. Distilled
water added upto the mark.
4. Methanol
5. 10 mg/dl artificial bilirubin std
It is prepared as follows –
a) Stock standard: it is prepared by mixing 0.29 gm of methyl red in
100 ml of glacial acetic acid.
b) Working standard: 0.1 ml of stock std, 0.5 ml of glacial acetic acid
DEPARTMENT OF ROGA NIDANA, KTGAMC BENGALURU Page 61
 and 1.44 gm of sodium acetate are mixed in distilled water and
diluted to 100 ml by adding distilled water.

Test procedure:

Prepare fresh Diazo mixture by mixing 5 ml of Diazo A and 0.15 ml of

Diazo B. this mixture is stable only for a day.

Pipette in the tubes labeled as follows-

Total test Total blank Direct test Direct blank

Distilled water,ml 1.8 1.8 1.8 1.8
Serum ,ml 0.2 0.2 0.2 0.2
Diazomixture,ml 0.5 - 0.5 -
Diazo blank reagent,ml - 0.5 - 0.5
Methanol,ml 2.5 2.5 - -
Distilled water ,ml - - 2.5 2.5

Keep in dark for 30 minutes. Read the intensities at 540 nm ( green filter).

Read O.D. of the artificial bilirubin standard (undiluted) by transferring the

standard solution in a dry cuvette at 540 nm.

Calculations:

O.D.of total bilirubin= O.D. of total test-O.D. of total blank.

O.D.of direct bilirubin= O.D. of direct test-O.D. of direct blank.

Total bilirubin mg/dl = O.D.of total bilirubin X 10

O.D.ofstd

Directbilirubin ,mg/dl = O.D.of total bilirubin X 10

O.D.ofstd
Normal value:

DEPARTMENT OF ROGA NIDANA, KTGAMC BENGALURU Page 62

Total bilirubin –up to 1mg/dl

Conjugated bilirubin –up to 0.5 mg/dl

Unconjugated bilirubin – up to 0.5 mg/dl

Interpretation:

Unconjugated bilirubin increases in hemolytic jaundice, conjugated Bilirubin

increase in obstructive jaundice and total bilirubin in hepatocellular jaundice.

DEPARTMENT OF ROGA NIDANA, KTGAMC BENGALURU Page 63

DEPARTMENT OF ROGA NIDANA, KTGAMC BENGALURU Page 64
 TOTAL SERUM PROTEIN DETERMINATION

Principle:

Proteins react with cupric ions in alkaline medium to form violet colored
complex. The intensity of the color produced is directly proportional to proteins
present in the specimen and can be measured on a photometer at 530 nm.

Normal range: 6-8gms/dl

Name of the method: Biuret method

Requirements:

1. Test tubes : 15X125 mm,

2. Serological pipette 5 ml
3. Test tube stand
4. Push button pipette of 0.05ml or serological pipette of 0.1 ml
5. Photometer

Specimen:Serum

Preparation of the reagents:

Stock Biuret reagent: dissolve 45 gm of Rochelle salt I about 400 ml of 0.2 N

sodium hydroxide and add 15 gm of copper sulphate by stirring continuously until the
solution is complete. Add 5 gm of potassium iodide and make up to a litre with 0.2 N
sodium hydroxide.

1) Protein reagent ( ready to use): (working Biuret reagent)

Dilute 200 ml of stock reagent to a litre with 0.2 N sodium hydroxide
which contains potassium iodide /litre .
2) Protein standard :6.0gm/dl:6 gm of bovine albumin dissolved in 100 ml of
normal saline, containing 0.1 g /dl, sodium azide
3) Sample blank reagent: 9.0g of Rochelle salt & 5 gm of potassium iodide
dissolved in one liter of 0.3N sodium hydroxide.

DEPARTMENT OF ROGA NIDANA, KTGAMC BENGALURU Page 65

Procedure:Mono-step method

Pipette in three- tubes labeled as follows

Test Std Blank

Protein reagent,ml 5.0 5.0 5.0
Serum,ml 0.05 - -
Protein Std ,6 g/dl,ml - 0.05 -
Distilled water,ml - - 0.05

Mix thoroughly and keep at room temperature (250c+/- 50c) for exactly 10 min.
Measure the test and standard by setting blank at 100 % T,by using 530 nm.

Calculations:

Serumproteins g/dl = O.D. test X6

O.D.Std
Clinical significance

Total serum protein values decrease below normal range in different clinical
conditions associated with nephritic syndromes, malnutrition, cirrhosis of liver and
other liver disease in which liver cells are severely damaged. Increased total protein
values may be found in multiple myeloma and conditions associated with high
globulin concentration.

DEPARTMENT OF ROGA NIDANA, KTGAMC BENGALURU Page 66

DEPARTMENT OF ROGA NIDANA, KTGAMC BENGALURU Page 67
 DETERMINATION OF SERUM TRIGLYCERIDES

Principle:
In presence of lipoprotein lipase, triglycerides are split into glycerol and fatty acids.
In presence of ATP and Glycerol kinase Glycerol is converted into Glycerol-3-Phosphate and
ADP. Glycerol Phosphate oxidase dissociates Glycerol-3-Phosphate to Dihydroxy acetone
phosphate and Hydrogen peroxide. In presence of peroxidase Hydrogen peroxide reacts with
4- amino- phenazone and p-chlorophenol to form colored complex. It is measured at 520
nm(green filter).

Reagents:
1. Buffer/enzymes/chromogen: it contains
a) lipoprotein lipase – 30 units,
b) Glycerol kinase-10 units,
c) Glycerol Phosphate oxidase- 5 unit
d) Peroxidase 5 units
e) Glyceol – phosphate in 100 ml of phosphate buffer , pH :7.2 ( 50mmol/l)
2. p-cholorophenol reagent : 30mg /dl

Preparation of working reagent:

It is prepared fresh by mixing two parts of reagent 1 & one part of reagent 2.

Procedure:
Pipette in the tubes are labeled as follows

Test Standard Blank

1)working reagent,ml 1.0 1.0 1.0
2) serum , ml 0.01 - -
3) standard,ml - 0.01 -
4) distilled water - - 0.01
Mix well. Keep at 370 c for fifteen minutes. Read absorbance of test and standard
against blank.

Calculations :
Serum triglycerides, mg/dl = O.D.Test X100
O.D. std

DETERMINATION OF SERUM HDL CHOLESTEROL

DEPARTMENT OF ROGA NIDANA, KTGAMC BENGALURU Page 68
Principle :
In the presence of phosphotungstic acid and magnesium chloride, LDL, VLDL
& chylomicrons are precipitated. Centrifugation leaves only the HDL in the
supernatant. Cholesterol in the HDL fraction can be tested by the usual methods.

Method: Colorimetric method (Watson)

Sample material : Serum (fasting)

Requirements:
a) Test tubes (15X125mm)
b) 0.2ml pipettes
c) Dispensers
d) Stop watch
e) Push button pipette
f) Photometer
g) Centrifuge tubes
h) 0.1ml, 1 ml graduated pipettes
i) Centrifuge

Preparation of reagents:
1. Cholesterol reagent 1 – It is prepared by mixing 5.6gms of 2,5-di methyl
benzene sulphonic acid in 200ml of Glacial acetic acid and 300ml of
Acetic anhydride. This reagent is stable in an amber coloured bottle at
room temperature (25°C±5°C) for 1 year.
2. Cholesterol reagent 2 – Concentrated Sulphuric acid.
3. Phosphotungstic acid reagent – it is dissolving prepared by 2.25 g of
phosphotungstic acid in 8.0 ml of IN sodium hydroxide and 42.0 ml of
distilled water.
4. Magnesium chloride reagent: 20.34 g of magnesium chloride in distilled
water. It is diluted to 50ml.
5. Cholesterol standard: 100mg/dl ( it is prepared in glacial acetic acid).

Stability of the reagents :

All the reagents are stable at room temperature for several months.

DEPARTMENT OF ROGA NIDANA, KTGAMC BENGALURU Page 69

Procedure:

Pipette in centrifuge tubes labelled as follows:

Test
1) Serum, ml 0.5
2) P.T.A.reagent ,ml 0.05
3) MgCl2 reagent , ml 0.02

Mix well. Centrifuge at 3000 R.P.M for 20minutes. Separate the supernatant
by using a Pasteur pipette. Now pipette in the tubes labelled as follows:

Test Std Blank

1. Cholesterol reagent,1 ml 2.5 2.5 2.5
2. Supernatant ,ml - - -
3. Cholesterol std, 100mg/dl ml - 0.1 -
4. Distilled water, ml - - 0.1
Mix well; keep in a water bath at room temperature for 5 minutes. Afterwards add
5. Cholesterol reagent 2, ml 0.5 0.5 0.5

Mix thoroughly, cool the tubes to room temperature by dipping in a water- bath ( at
room temperature ).

Read absorbance of test and standard against blank at 575 nm. (Yellow – green filter:
520-580 nm )

Normal range :

Men: 30-60mg/dl

Women: 40-70 mg /dl

Calculations :

Serum HDL cholesterol, mg/ dl = O.D.test X 114

O.D. Std

DEPARTMENT OF ROGA NIDANA, KTGAMC BENGALURU Page 70

DEPARTMENT OF ROGA NIDANA, KTGAMC BENGALURU Page 71
 ROUTINE EXAMINATION OF SEMEN

The various important purposes of routine semen analysis are –

1) Evaluation of infertility.
2) Routine follow up of patients who have underwent vasectomy.
3) Artificial insemination.
4) Examination of stored semen specimen.
5) For men whose future fertility is threatened may be by the need for radiotherapy or
chemotherapy in the treatment of cancer.

SPECIMEN COLLECTION
 Length of abstinence
2 to 3 days is an adequate length of abstinence for the proper assessment of semen
quality.
 Site of production of the semen specimen
The patient must be allowed to choose where he wishes to produce the specimen. It
may be at home or in a room of laboratory or hospital which is quiet, secluded and
which will guarantee total privacy.
 Time of production of semen specimen
A semen specimen is best produced in the morning for an assessment of motility 6 hrs
after production. The presence of any ‘deadline’ for semen production should be
avoided.

STORAGE AND TRANSPORT OF THE SPECIMEN

 Sperms are easily damaged by either excessive heat or excessive cold.
 Semen specimen should not be left for any length of time in direct sunlight.
 The semen specimen should be brought to the laboratory at closed to body
temperature.
DEPARTMENT OF ROGA NIDANA, KTGAMC BENGALURU Page 72
 It is best but not essential that the semen specimen is delivered to the laboratory
within 2 hrs of production.

DEPARTMENT OF ROGA NIDANA, KTGAMC BENGALURU Page 73

 PHYSICAL EXAMINATION OF THE SEMEN

1) Colour
2) Volume
3) Viscosity
4) Formation of coagulum and the liquefaction time

COLOUR

Semen is normally grey – (pale) – yellow opalescent fluid. The colour of semen may
change due to the following reactions.

 Presence of urine
Urine may occasionally contaminate semen and this may occur in disturbances of
bladder neck function and ejaculation.
The presence of urine can be detected by the consistency of semen and by uriniferous
odour of semen. A high urea content of the sample may confirm the presence of urine.
 Presence of blood
Traces of fresh blood will colour semen pink. Larger amounts of blood may impart
brighter colour to semen.
 Presence of bilirubin
Presence of bilirubin may impart yellow colour to semen.

VOLUME
 Semen volume can be measured by using a 10 ml graduated measuring cylinder or a 10
ml graduated centrifuged tube.
 Normal semen volume = 2 - 6 ml
[ Volumes as low as 1 ml and as high as 10 ml may be regarded as normal ]
 To express this aspect, following are the related terms used –
 Aspermia: Means the total absence of ejaculate.
 Azoospermia: Absence of Spermatozoa in semen.
 Oligospermia/Hypospermia: A reduction in the volume of the ejaculate.
 Hyperspermia: Increase in semen volume.

DEPARTMENT OF ROGA NIDANA, KTGAMC BENGALURU Page 74

VISCOSITY
Normal viscosity is defined as that which will allow semen to be poured drop by drop
out of a container.
This is observed by taking the specimen in a Pasteur Pipette or Capillary Tube of 10
cm in length and which contains 0.1 ml of semen and by allowing it to pour drop by
drop. Viscosity can be quantified by measuring the time it will take for 1 drop of
semen to leave a standard pipette.

DEPARTMENT OF ROGA NIDANA, KTGAMC BENGALURU Page 75

 FORMATION OF COAGULUM AND LIQUEFACTION TIME

In the humans, semen is ejaculated in a liquid form and it forms a gel like clot ( after
ejaculation ) within 5 to 20 minutes.

The coagulation of semen is dependent on the presence of a fibrinogen like substance

manufactured by the seminal vesicle which is acted upon by the enzyme vesiculase
produced by the prostate.

Determination of Liquefaction:

It is usually assessed visually.

1) Unliquefied Semen – forms a gel like coagulum.

2) Partially Liquefied Semen – contain many small gel like clots.
3) Fully Liquefied Semen – no such clots are seen and the semen appears completely
liquid.

DEPARTMENT OF ROGA NIDANA, KTGAMC BENGALURU Page 76

 CHEMICAL EXAMINATION OF SEMEN

1) Determination of pH
2) Determination of Fructose

DETERMINATION OF pH

The pH of normal fresh Semen lies between 7.9 and 8.1. It may slowly fall as the
specimen ages ( after storage ).

It is measured by using pH paper, with a range of 6 to 9.

Clinical Significance:Inflammatory conditions, particularly of the prostate or seminal

vesicles may alter the pH of the semen.

DETERMINATION OF FRUCTOSE

Procedure:

 Pipette 5 ml of resorcinol reagent in a test tube.

 Add 0.5 ml of semen specimen.
 Mix and place in a boiling water bath for 5 minutes, or heat on a gas burneror spirit
lamp .

Observation:

1) No change in colour: Fructose absent.

2) Red coloured precipitate forms within 30 seconds: Fructose present.

Normal Value: 150 – 300 mg/dl

Clinical Significance:

Reduction in fructose concentration:

 Disorders of seminal vesicles

 Polyzoospermia

DEPARTMENT OF ROGA NIDANA, KTGAMC BENGALURU Page 77

 MICROSCOPIC EXAMINATION OF SEMEN

1) Study of motility of sperms

2) Assessment of morphology of spermatozoa
3) Determination of sperm count

STUDY OF MOTILITY OF SPERMS

Procedure:
 Place a small drop of liquefied semen on a glass slide and cover it with a coverslip.
 Examine the coverslip preparation under the high power objective with reduced
illumination.
 Count the chamber actively motile sperms out of the total count of 200.
 Calculate the percentage of the sperm showing actual progressive motion.
 Examine the slide after 2 hrs, 3 hrs, 6 hrs( to prevent the drying effect the coverslip,
preparation should be placed under a petri dish with a wet filter paper ).

ASSESSMENT OF MORPHOLOGY OF SPERMATOZOA

The terms commonly used in relation to abnormalities in the morphology of the

spermatozoa are –

 Teratozoospermia: Means the presence of an increase in the numbers of

morphologically abnormal sperms in semen.
 Teratoasthenozoospermia: Describes the abnormal sperm showing poor or absent
motility.

Procedure:

Smear preparation: A drop of well mixed undiluted semen is placed on one end of a
clean glass slide and a smear is made exactly the same way as far a blood film. The
smear is then air dried and fixed.

DEPARTMENT OF ROGA NIDANA, KTGAMC BENGALURU Page 78

 For staining the smear Leishman’s staining method can be used.

Observations:

Normal:

Normal Observation Colour

1) Spermtozoa head caps Light blue

2) Nuclear posterior Dark blue
3) Bodies and tails Red or pink
4) Spermatozoa size 50 – 70 µ
5) Head size 3 – 6µ x 2 – 3 µ

Pathological:

Morphological abnormalities that may be present in sperm are –

 Head abnormalities –
 Marked reduction in head size
 Marked increase in head size
 Presence of vacuoles within the head
 Tapering of pyriform head
 Tail abnormalities –
 Coiled tails
 Eccentrically inserted tails
 Hair pin deformities
 Short stubby tails
 Duplicate or triplicate tails
 Mid piece defects
 Presence cytoplasmic droplets

 Other microscopic abnormalities

DEPARTMENT OF ROGA NIDANA, KTGAMC BENGALURU Page 79
  White Blood Cells: The white cells should be expressed as the number
per 100 spermatozoa. From this number of WBC’s per ml of semen can
be calculated.

Clinical Significance:
o Normal – Presence of few WBC’s
o Inflammatory condition – Presence of large number of WBC’s
 Epithelial Cells: These arise from the lining of the genital tract and are
always present in semen. Even the presence of large number of
epithelial cells doesn’t show any pathological condition.
 RBC’s:
o Normal – Absence of RBC’s
o Infection or possibility of malignant disease within the genital tract –
Presence of RBC’s
 Germinal Cells: These represents preformed sperm and these are
deemed usual in normal semen. These are expressed per 100 ml of
spermatozoa.
 Protozoa and Bacteria: The presence o protozoa or bacteria in semen
clearly indicate an infection. The most common protozoan is
Trichomonas, visible bacteriae are Stephylococcusepidermidis and
Streptococcus viridians.

DEPARTMENT OF ROGA NIDANA, KTGAMC BENGALURU Page 80

 DETERMINATION OF SPERM COUNT

Procedure:
 After liquefaction, gently mix the specimen.
 Draw semen to the 0.5 mark of a WBC pipette.
 Draw in the semen diluting fluid 11 mark and mix well.
 Load the Improved Neubauercounting chamber and allow the sperms to settle for
about 5 minutes.
 Count sperm in the 4 corner squares ( as in WBC count ).

Calculation:

Sperms/ml of semen = Sperms counted in 4 squares x 10 x 20 x 1000

Normal Sperm count: 20 million – 110 million/ml

DEPARTMENT OF ROGA NIDANA, KTGAMC BENGALURU Page 81

 ROUTINE EXAMINATION OF FAECES

Collection of the stools specimen:

1. Collection of sufficient quantity: morning specimen (at least 5 to 6ml capacity)
is collected in a 50ml clean and dry container. Cover the container.
2. Label the container.
3. Examined within one hour of collection.
4. Warm stools are best for detecting ova and parasites. Do not refrigerate for ova
and parasites.

Preservation:

Mix 3 parts of 10% formal saline with one part of stool.

Peptone water – in cholera stools.

Low viscosity poly vinyl alcohol for cysts, protozoan

Physical examination
1. Consistency
2. Colour
3. Mucus
4. Blood
5. Parts of parasite
6. Adult parasite

Chemical examination
1. Reaction
2. pH
3. occult blood

DEPARTMENT OF ROGA NIDANA, KTGAMC BENGALURU Page 82

Microscopic examination:

DEPARTMENT OF ROGA NIDANA, KTGAMC BENGALURU Page 83

 1. Protozoans
2. Nematonelminths
3. Platyhelminths
4. Plants cells and fibres
5. Meat fibres
6. Crystals
7. Fat globules
8. Yeast cells
9. Bacteria
10. Erythrocytes
11. Pus cells

DEPARTMENT OF ROGA NIDANA, KTGAMC BENGALURU Page 84

 GROSS EXAMINATION
Inspection of feces may be helpful in -
Diagnosis of parasitic infection,
Obstructive jaundice,
Diarrhea,
Malabsorption,
Rectosigmoidal obstruction,
Dysentery or ulcerative colitis,
Gastrointestinal bleeding.

Consistency:
Normal : well formed

BRISTOL’s stool chart

1.Separate hard lumps, like nuts (hard to pass).

2.Sausage-shaped but lumpy

3.Like a sausage but with cracks on the surface

4.Like a sausage or snake, smooth and soft

5.Soft blobs with clear-cut edges

6.Fluffy pieces with ragged edges.

7.Watery, no solid pieces. Entirely Liquid

1,2 constipation

3,4 –optimal especially 4 is easiest to pass

5,6,7 –are associated with increased tendency for diarrhea or urgency.

Abnormal consistency Expected reasons

DEPARTMENT OF PG STUDIES IN ROGA NIDANA, GAMC BENGALURU Page 85

Pale, bulky, frothy Steatorrhea
Hard Constipation
Flattened and ribbon like Obstruction in the lumen of bowel
Watery stools Infection, purgatives
Rice water stools Cholera
Colour:
NORMAL COLOUR: light to dark brown

Abnormal colour Possible reasons

Black Bleeding in the upper GIT,
Iron supplementation, charcoal and bismuth,
foods such as cherries, high proportion of meat in the
diet.
Bright red Bleeding at the lower GIT,
bleeding piles,
tumours of the colon,
contamination with menstrual blood.
Fresh blood with Amoebic dysentery
mucus
Clay coloured Post-hepatic jaundice,
obstruction to the flow of bile to the intestine
White After barium meal
Green Chlorophyll rich vegetables
Calomel intake
Bright yellow Giardiasis
Drug induced colour changes are given below

Black Iron salts, bismuth salts, charcoal

Green Mercurous chloride, indomethacin, calomel.
Green to blue Dithiazine
Brown staining Anthraquinones

DEPARTMENT OF PG STUDIES IN ROGA NIDANA, GAMC BENGALURU Page 86

Red Phenolphthalein, pyruviniumpamote,tetracyclines
Yellow Santonin
Yellow to brown Senna
Whitish discoloration Antacids
Orange red Phenazopyrinidine
Pink to red black Anticoagulants salicylates causing internal bleeding

Mucus in stool
Even the slightest quantity is abnormal
Remarks Causes
Translucent gelatinous mucus clinging to Spastic constipation,
the surface of the formed stool mucous colitis
Bloody mucous clinging to stool mass Neoplasm,
inflammation of rectal canal
Mucus with pus and blood Ulcerative colitis,
bacillary dysentery,
ulcerating carcinoma of the colon , acute
diverticulitis
Copious mass , up to3-4 liters of mucus Villious adenoma of the colon
per day

Presence of adult worms or their parts

 Large round worms
 Pin worms
 Whip worms
 Hook worms
 Tape worms

DEPARTMENT OF PG STUDIES IN ROGA NIDANA, GAMC BENGALURU Page 87

 CHEMICAL EXAMINATION

Determination of the pH of stools:

Normal: 5.8-7.5.

Procedure: Dip the pH paper in small quantity of the fecal material. Observe for color
change, compare with color chart.

Clinical significances:
Strongly acidic stools (pH below 5.5)
 Non pathologic: excess of carbohydrate in diet
 Pathologic: fermentation may be due to lactose intolerance.

Strongly alkaline stools (pH more than 7.5)

 Non pathologic: excess of protein in diet

Occult blood:
Name of the method: BENZIDINE TEST

Requirements:
1) Glass slides
2) Applicator stick or wire loop
3) Glacial acetic acid
4) Hydrogen peroxide
5) Benzidine powder

DEPARTMENT OF PG STUDIES IN ROGA NIDANA, GAMC BENGALURU Page 88

 Procedure:
1) Take pinch of Benzidine powder in a small test tube.
2) Acidify it with 2 to 3 drops of glacial acetic acid and mix well.
3) Add about 1 ml of hydrogen peroxide and mixed well.
4) Place small quantity of stool specimen on a clean and dry glass slide.
5) Place 1 or 2 drops ofbenzidine-glacial acetic acid- hydrogen peroxide mixture on
the stool specimen on the glasss slide.
6) Observe change in color.

Observations:

1) No change in color: Occult blood absent

2) Color changes to green to blue: occult blood present

DEPARTMENT OF PG STUDIES IN ROGA NIDANA, GAMC BENGALURU Page 89

 MICROSCOPIC EXAMINATION
Requirements:
1) Glass slides
2) Coverslips
3) Normal saline
4) Lugol’s Iodine solution
5) Saturated saline solution
6) Small bottles

Procedure:
a) Saline specimen preparation
1) Place a drop of saline on a glass slide.
2) Take a little fecal material by using an applicator stick and mix with a drop of
normal saline.
3) Place a coverslip over it. Avoid formation of air bubbles below the coverslip.

b) Iodine specimen preparation

1) Place a drop of Lugol’s iodine on the other side of the slide.
2) Mix little fecal matter with a drop of iodine solution.
3) Place a coverslip on it.
Observations:
 Cells
1) Pus cells: Ulcerative colitis
2) Epithelial cells: Inflammation of the bowel
3) Macrophages : Bacillary dysentery, Ulcerative colitis
4) Erythrocytes: Lesion in the colon, rectum or anus

 Crystals
1) Triple phosphate and calcium oxalate: Non pathological (present due to
ingestion of certain food i.e. spinach, berries, tomato.)
2) Charcot–Leyden crystals (pointed needle like): Ulcerative condition.
3) Hematoidin crystals: Intestinal haemorrhage.

DEPARTMENT OF PG STUDIES IN ROGA NIDANA, GAMC BENGALURU Page 90

  Protozoa(cysts)
1) Entaemoebahistolytica

2) Giardia lamblia

DEPARTMENT OF PG STUDIES IN ROGA NIDANA, GAMC BENGALURU Page 91

  Worms
1) Round worm egg

2) Pin worm and its eggs

DEPARTMENT OF PG STUDIES IN ROGA NIDANA, GAMC BENGALURU Page 92

 3) Tape worm

DEPARTMENT OF PG STUDIES IN ROGA NIDANA, GAMC BENGALURU Page 93

 4) Hook worm

5) Whip worm

DEPARTMENT OF PG STUDIES IN ROGA NIDANA, GAMC BENGALURU Page 94

 HANGING DROP TEST
•Place a drop stool in the centre
of a coverslip.
•Place a drop of water / vaseline
at each corner of the coverslip.
•Invert a slide with a central
depression over the coverslip.
•The coverslip will stick to the
slide and when the slide is
inverted the drop of bacterial
culture will be suspended in the
central depression of the slide.
•Examine microscopically (X100)
for motile organisms.

DEPARTMENT OF PG STUDIES IN ROGA NIDANA, GAMC BENGALURU Page 95

DEPARTMENT OF PG STUDIES IN ROGA NIDANA, GAMC BENGALURU Page 96