1

ANATOMY AND PHYSIOLOGY PRACTICAL

MANNUAL

AQSA INSTITUTE OF MEDICAL SCIENCES, SARGODHA.

ANATOMY & PHYSIOLOGY PRACTICAL MANNUAL
PREPARAD BY MISS. MARIA REHMAN
2

EXPERIMENT NO 1

DETERMINATION OF COAGULATION (CLOTTING) TIME (CT)

REQUIREMENT
Lancet, stopwatch, capillary tube, cotton swab and spirit.
INTRODUCTION
The coagulation tiome is the time required for a sample of blood removed from the blood vessel
to clot.
Normal value
2-6 min (by capillary tube method)
Capillary tube method
PROCEDURE
1. Clean the finger tip with spirit swab and allow to it air dry. Puncture it with lancet and
discard the first drop of blood
2. Hold the capillary tube horizontally and dip its one end into oozing of the blood. Allow
the tube to fill by capillary action
3. After exactly one minute, carefully break off a small piece (about 1 cm) of the capillary
tube and determine whether a thread of coagulated blood is visible between the two
pieces of tubing.
4. Repeat the step 3 after every 30 seconds until such a thread is obtained.
5. Record the coagulation time.
PRECAUTION
1. Avoid air bubble in the capillary tube.
2. Keep the other end of the capillary tube open while filling it with drop of blood.
3. The method should be repeated for 3 cappilary tubs to record accurate timing.

AQSA INSTITUTE OF MEDICAL SCIENCES, SARGODHA.

ANATOMY & PHYSIOLOGY PRACTICAL MANNUAL
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EXPERIMENT NO 2

DETERMINATIONs OF BLEEDING TIME (BT) BY DUKE’S METHOD.

INTRODUCTION
The Bleeding Time is taken as the time for a small sharp incision to stop Bleeding.This test help
in the diagnosis of various hemorrhagic disorders of vascular or platelets origin.
REQUIREMENT
Lancet, stopwatch, filter paper, cotton swab and spirit.
Normal value
1-6 min (by duke’s method)
PROCEDURE
1. Clean the fingertip or ear lobule with spirit swab and allow it to air dry.
2. Puncture the fingertip or ear lobule with lancet about 3mm deep.
3. Set stop watch and start it from the moment bleeding starts from the puncture.
4. Apply the edge of filter paper on blood drop and take this time as zero reference.
5. At 15 seconds intervals, wipe the blood drop away completely with the filter paper.
Continue this until no more blood stains are picked up on the filter paper.
6. Record the difference of time between the starting and stoppage of bleeding
PRECAUTIONS
1. Puncture should be done under aseptic measure.
2. Do not touch finger tip or ear lobule when wiping the blood away.

AQSA INSTITUTE OF MEDICAL SCIENCES, SARGODHA.

ANATOMY & PHYSIOLOGY PRACTICAL MANNUAL
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EXPERIMENT NO 3

DETERMINATION OF BLOOD GROUP

INTRODUCTION
There are various methods for the determining of blood group but OAB systm and Rh system is
the most important one that assures safe blood transfusion.
APPARATUS
Anti-serum A, B, D, glass slide cotton swab, spirit, lancet
PROCEDURE
1. One drop of ant-serum A was placed on one end of glass slide one drop of Anti-serum B
and one drop of Anti-serum D on the one end of another slide.
2. One drop of blood was dropped on each slide which are labelled A, B and C.
3. One drop of RBC suspension was mixed with Anti-serum.
4. The presence of agglutination is confirmed by the presence of thick masses of RBCs.
5. The absence of agglutination was confirmed by the clear mixture with dispersed RBSs.
6. For determination of Rh Factor Anti-serum D was mixed with RBCs suspension. The
presence of Rh factor was confirmed by the presence of thick masses of RBCs.
INTERPRETATION
1. If agglutination occur with Anti-serum A the Anti-serum A contains A antibody. The
agglutination occurs if the RBCs contain A antigen so the blood group is A.
2. If the agglutination occurs anti serum- B the blood group is B.
If the agglutination occurs with both anti – serum A and B, the blood group is AB
3. If the agglutination does not occur with both anti-serum A and B the blood group Is O.
4. For the determination of Rh factor (if the agglutination occurs with anti-serum D, the
blood group is +ive and if the agglutination does not occur with anti-serum D the blood
group is -ive). Agglutination

AQSA INSTITUTE OF MEDICAL SCIENCES, SARGODHA.

ANATOMY & PHYSIOLOGY PRACTICAL MANNUAL
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EXPERIMENT NO 4

MEASUREMENT OF BLOOD PRESSURE

INTRODUCTION
“Blood Pressure (B.P) is defined as the blood pressure exerted by the flowing blood against the
walls of arteries.”
Arterial blood pressure has two important components;
1. Systolic blood pressure
2. Diastolic blood pressure
Systolic blood pressure:
The highest pressure exerted by the blood on the walls of arteries produced by the ventricular
contraction.
It ranges between 100-140 mmHg in normal healthy adult.
Diasytolic blood pressure:s
The lowest blood pressure exerted by the blood on the walls of arteries during ventricular
relaxation.
Its normal value 80 mmHg.
APPARATUS
Stethoscope, sphygmomanometer, Mercury manometer.
PROCEDURE
1. Cuff is tightly placed around arm and chestpiece of stethoscope is placed below the cuff
over the antecubital fossa.
2. Pressure is raised in cuff so that the brachial artery is occluded due to compression.
3. After that arm cuff is deflated, while doing so series of sound heared through stethoscope.
4. Appearance of clear tapping sound during first phase indicated systolic blood pressure
and disappearance of sound is in 5th phase show diastolic blood pressure.

AQSA INSTITUTE OF MEDICAL SCIENCES, SARGODHA.

ANATOMY & PHYSIOLOGY PRACTICAL MANNUAL
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EXPERIMENT NO 5

DETERMINATION OF ERYTHROCYTE SEDIMENTATION RATE

INTRODUCTION
Erythrocyte Sedimentation Rate (ESR) is a rate at which Erythrocytes settle down.
Normal the Erythrocytes remain suspended uniformly in circulation. This is called suspension
stability of RBCs.
DETERMINATION OF ESR:
There are 2 methods to determine ESR.
1. Westergren method
2. Wintrobe mrthod
APPARATUS
Spirit swab, sringe, anticoagulant (3.8 % sodium citrate), gloves, Westergren tube, Westergren
stand.
PROCEDURE
1. Drew the blood fromm the vein of subject with the help of syringe.
2. 1.6 ml of blood is mixed with 0.4ml of 3.8 ml of anticoagulant and filled in westergren
tube.
3. The tube is filled to the stand vertically and left undisturbed.
4. The reading is taken at the end of 1 hour.
Normal values of ESR:
In males 3-7 mm/hour
In female 5-9 mm/hour
In infant 0-2 mm/hour
SIGNIFICANCE OF DETERMINED ESR
1. ESR helps in diagnosis as well as prognosis. ESR is increased in inflammation, anemia,
rheumatic fever, some kidney diseases and some cancer.
AQSA INSTITUTE OF MEDICAL SCIENCES, SARGODHA.
ANATOMY & PHYSIOLOGY PRACTICAL MANNUAL
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The ESR decrease in Sickle cell anemia, leukemia and congestive heart failure.

EXPERIMENT NO 6

TESTING OF VISUAL ACTIVITY

REQUIREMENTS
Snellen’s chart and subject.
INTRODUCTION
“Visual acuity is the degree to which the details and contours of the objects are perceived.”
Usually defined in term of the minimum separable by which 2 lines can be separated and still be
perceived 2 lines.S
Clinically, visual acuity is often determined by use of the familiar Snellen’s letter chart viewed at
distance of 20cft (6 meter) of letter of vary sizes constructed that top letter is visible at 60 meters
and subsequent lines at 36, 24, 18, 12, 9, 6 and5. The patient reads down the chart as far as he
can.
PROCEDURE
For Distant Vision
1. Subject being tested is 20 feet away from Snellen’s chart and reads smallest line
distinguishable loudly.
2. Snellen’s charts are designed so that a normal individual can read, the letters in the
smaller (7th) line at 20 feet or 6 m. (subtends a visual angle of 5 min).
3. A person visual acuity is started as V=d/D in which “d” is the distance at which the
patient can read the letters and D is the distance at which a normal eye can read the
letters. Normal visual acuity 20/20 or 6/6.
4. If vision is recorded 10/20 it is subnormal because one must approach to within 10 feet to
read letters that one readable at 20 feet by the normal eye.
For Near Vision
Visual acuity at the ordinary reading distance is assessed by using reading test types of vary
sizes, the notation being based on the printer’s point system. The smallest print used is N5. The
smallest print is N5. The near vision is recorded as the smallest type which is the patient can read
comfortably.

AQSA INSTITUTE OF MEDICAL SCIENCES, SARGODHA.

ANATOMY & PHYSIOLOGY PRACTICAL MANNUAL
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PRECAUTIONS
1. Examiner must have 6/6 (with or without glasses) vision and no color blindness.
2. Each eye should be tested separately.
3. One eye should remain close while the other is being examined.
EXPERIMENT NO 7

RECORDING AND INTERPRETATION OF ECG

INTRODUCTON
The electrocardiogram is a graphical recording of the action potential of the heart.
It is recorded with an electrocardiograph and the study of this cardiac electrical activity is
called Electrocardiography (ECG).

REQUIREMENTs
ECG machine, Electrode gel and subject.

PROCEDURE
1. Connect all the limbs and chest electrodes properly using contact gel. Conventionally
all the electrodes are marked with codes and have specific colors. Make sur that the
subject is lying comfortably having no metallic object on.
2. Check the amplitude on QRS complex in lead I, II and III. According to the
Einthoven’s law.
3. Examine the initial strip of the record and check that there should be no artifact.
4. Record in sequence, first for standard limb leads followed by augmented limb leads
and finally for chest leads.
5. After having recorded the ECG, calculate the heart rate first and then observe the
various components of ECG carefully.
There are many different ways to calculate heart rate, a simple way is to divide 300
by the number of large squares between 2 consecutive beats (R-R interval) if the
rhythm is regular.
NORMAL DURATION/ VOLTAGE

COMPONENTS DURATION (Sec) VOLTAGE (mv)

P wave 0.08 – 0.1 0.2 – 0.3

QRS complex 0.08 – 0.12 1–3

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T wave 0.16 – 0.27 0.3 – 0.4

PRECAUTIONS
1. Relax the patient before taking a record.
2. Use proper voltage and handle the electrical recorder gently.
3. There should be no metallic objects on the subject.
4. Contact of the electrodes with the skin should be made with electrolyte gel.

AQSA INSTITUTE OF MEDICAL SCIENCES, SARGODHA.

ANATOMY & PHYSIOLOGY PRACTICAL MANNUAL
PREPARAD BY MISS. MARIA REHMAN
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EXPERIMENT NO 8

DETERMINATION OF RED BLOOD CELLS (RBCs) COUNT

INTRODUCTION
The red blood cells, one of the formed elements of the blood, transport oxygen and carbon
dioxide to and from the body tissues. The number of cells in a small volume of accurately diluted
blood is counted in a glass counting chamber (hemocytometer).
Anemia often result from an abnormal decrease in the number of erythrocytes, so that
insufficient oxygen is carried to the tissues and they become oxygen starved.
For accurate diagnosis of the cause of anemia, the complete status of the RBCs must be
examined i.e. hematocrit, hemoglobin, RBCs count, cell size, hemoglobin in each cell and other
factors.
Normal Values (in Adults):
Males 4.6 – 6.0 million /mm3
Female 4.0 – 5.2 million / mm3
REQUIEMENTS
RBCs pipette, improved hemocytometer microscope, blood lancet, cover slip, spirit swab and
Heyem’s fluid comprising of:
Nacl = 1 gm
Na2SO4 = 5gm
Distilled water = 200ml
PROCEDURE
1. Focus Neubauer’s chamber (hemocytometer) under the microscope, first add low
power and then use high power to focus the small squares.

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2. Before placing hemocytometer on the stage, clean it with distill water and dry it with
soft tissue paper.
3. Clean the tip of your finger with spirit and allow it to dry. Then pierce the finger to
obtain a drop of blood. Discard the first drop and with the help of clean and dry
pipette try to suck the oozing blood up to the mark 0.5 exactly. If excess blood is
obtained, touch the pipette, tip to a tissue to draw the blood back to the 0.5 mark.
4. Now place the pipette tip in Heyem’s fluid and draw diluting fluid up to the mark
101.
5. Securing the 2 ends of pipette such as tying the rubber tubbing well, mix and shake
the contents thoroughly for about 2 – 3 min.
6. Discard about 3 – 5 drops to remove the diluting fluid from the stem of the pipette.
Place the tip of pipette to the junction of the cover slip present over the counting area
of hemocytometer. The diluted cells flow in by capillary action to charge the
chamber. Allow 2 min for cells to settle before the beginning the RBCs count.
7. Focus on the central square of the counting area using high power. Count the number
of red cells in five of the 1/25 mm2 squares and take their average. Using the 4-outer
square and middle one are counted. While counting apply Thomas rule which suggest
that while counting the cells which lies on the left and lower lines of square are
considered to be outside of that square.
8. Calculate the number of RBCs /mm2 of blood by taking into account the following
multiplication factors:
 The blood was diluted 200 times in the pipette, therefore, multiply the average
number of RBCs per square by 200.
 The depth of counting chamber is 0.1 mm. therefore, multiply the number of
cells by 10.
 The square counted was in the 1/25 size of the area of the center
square(1mm2). Therefore, multiply the RBCs by 25.
 Thus, the whole multiplication factor:
200*10*25=50,000
For example, if you count an average of 120 RBCs per square then the RBCs
count will be 120*50000=6,000,000/mm3
PRECAUTION
1. Select the correct portion of Neubauer’s chambers.
2. Filling of the pipette with blood a then with diluting fluid should not have bubbles of
air.
3. For a good distribution, the mixing and charging of the chamber should be very
careful and thorough.
4. While counting the RBCs use proper illumination, magnification and follow the
Thomas rule.
5. There should be no overflowing of fluid in the H groove of chamber.
AQSA INSTITUTE OF MEDICAL SCIENCES, SARGODHA.
ANATOMY & PHYSIOLOGY PRACTICAL MANNUAL
PREPARAD BY MISS. MARIA REHMAN
12

EXPERIMENT NO 9

DETERMINATION OF TOTAL LEUCOCYTE COUNT (TLC)

INTRODUCTION
The white blood cells (WBCs) or leucocytes are one of the three specialize classes of the blood
cells. White blood cells combat inflammatory process and invading organism. WBCs must be
present in a sufficient number to carry out these function properly but no in an abnormal excess.
Thus, counting of total number of WBCs is an important clinical measurement which help to
establish the capacity of blood for performing their defensive function. In this technique, the
pipette used contain white beat and dilute the cells 20 times.
Normal value (in adult):
4000 – 11000/mm3 (4-11 *109/L)
REQUIREMENTS
WBCs pipette, improved hemocytometer microscope, blood lancet, cover slip, spirit swab and
Turk’s fluid comprising of:
Glacial acetic acid = 1ml
1% aqueous gentian violet solution = 1ml
Distilled water = 100 ml
PROCEDURE
1. Focus Neubauer’s chamber (hemocytometer) under the microscope, first add low
power and then use high power to focus the small squares.
AQSA INSTITUTE OF MEDICAL SCIENCES, SARGODHA.
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2. Before placing hemocytometer on the stage, clean it with distill water and dry it with
soft tissue paper.
3. Clean the tip of your finger with spirit and allow it to dry. Then pierce the finger to
obtain a drop of blood. Discard the first drop and with the help of clean and dry
pipette try to suck the oozing blood up to the mark 0.5 exactly. If excess blood is
obtained, touch the pipette, tip to a tissue to draw the blood back to the 0.5 mark.
4. Now place the pipette tip in Turk’s fluid and draw diluting fluid up to the mark 11.
5. Securing the 2 ends of pipette such as tying the rubber tubbing well, mix and shake
the contents thoroughly for about 2 – 3 min.
6. Discard about 3 – 5 drops to remove the diluting fluid from the stem of the pipette.
Place the tip of pipette to the junction of the cover slip present over the counting area
of hemocytometer. The diluted cells flow in by capillary action to charge the
chamber. Allow 2 min for cells to settle before the beginning the WBCs count.
7. Count the number of WBCs in the four corner squares (64 small squares) and take
their average. While counting apply Thomas rule which suggest that while counting
the cells which lies on the left and lower lines of square are considered to be outside
of that square.
8. Calculate the number of WBCs /mm2 of blood by taking into account the following
multiplication factors:
 The blood was diluted 20 times in the pipette, therefore, multiply the average
number of WBCs per square by 20.
 The depth of counting chamber is 0.1 mm.
 Volume of one large square is equal to 1*1*1/10 =1/10 mm3
 Supposed number of WBCs in 1 large square = 43.
 Total number of WBCs in one mm3 of diluted blood = 43*10
 Total number WBCs in whole blood = 43*10*20 = 86,000/mm3
PRECAUTIONS
1. Select the correct portion of Neubauer’s chambers.
2. Filling of the pipette with blood a then with diluting fluid should not have bubbles of
air.
3. An appropriate WBCs pipette (with white beat) must be selected.
4. Care should be taken to suck the blood exactly up to 0.5 mark in the WBCs pipette.
5. The hemocytometer must be cleaned only with distill water
6. The WBCs pipette must be cleaned before and immediately after using it with proper
solutions.
7. While counting the RBCs use proper illumination, magnification and follow the
Thomas rule.

AQSA INSTITUTE OF MEDICAL SCIENCES, SARGODHA.

ANATOMY & PHYSIOLOGY PRACTICAL MANNUAL
PREPARAD BY MISS. MARIA REHMAN
14

EXPERIMENT NO 10

DETERMINATION OF HEMOGLOBIN (HB) IN THE BLOOD

INTRODUCTION
Hemoglobin, the red oxygen carrying pigment, is the main component of red blood cells. The
amount of Hemoglobin in the blood is measured by destroying the red cell membrane
(hemolysis) so that the pigment is released into the plasma and can be estimated calorimetrically.
Determination of Hemoglobin in blood is an important clinical measurement which helps in the
diagnosis of anemia.
Normal values
Male: 13.0 - 18.0 g/dl
Female: 11.5 – 16.5 g/dl
Sahli’s Method
REQUIREMENTS
Sahli’s Haemoglobinometer, lancet, diluting tube, Hb pipette (20 microliter) and N/10 HCl
solution.
PROCEDURE
1. Place 5 drops of 0.1 N HCl in the bottom of graduated sahli’s tube. This amount should
fill the tube to the mark 20 on the scale. HCl converts blood Hb to Brownish haematin
compound.

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2. Pierce your finger with help of lancet to obtained the drop of blood under aseptic
measures. Place tip of sahli’s pipette on the drop a gently suck a solid column of blood
into the pipette up to 20 marks (0.02ml). while sucking, use the mouth piece and rubber
tubing attach to the pipette. When too much blood is drawn, touch the pipette tip to a
filter paper or a tissue paper to remove the excess blood. Do not allow air bubbles to enter
the pipette as the blood will coagulate and will block the bore. To clean it flush the
pipette repeatedly in the washing solution containing water and alcohol. Coagulation of
blood in pipette can be avoided by drawing a heparin solution (1:1000) in and out of
pipette prior to filling it with blood.
3. Insert the tip of the pipette beneath the surface of HCl In the Sahli’s tube and gently blow
out the blood. Rinse the pipette if any blood is left in by drawing the solution in and out
of pipette 2 times.
4. Mix the blood and HCl by stirring with a glass rod, and then let the tube stand for 10 min.
5. Place the tube in the comparator block and hold it in front of strong light. At distilled
water drop by drop to the haermatin solution (stir after each addition). Until it color
matches with the color of standard plates on the comparator.
6. Read the scale on the Sahli’s tube to obtained the percentage of Hb and grams of
Hb/100ml of blood. Note that the Hb standard used in the calibration made differ from
tube to tube. The standard for describing Hb percentage is imprinted on each type of tube
for specific use.
PRECAUTIONS
1. Discard the first drop of blood.
2. There should no air bubble present in the pipette.
3. The matching of color should be seen in good light.

AQSA INSTITUTE OF MEDICAL SCIENCES, SARGODHA.

ANATOMY & PHYSIOLOGY PRACTICAL MANNUAL
PREPARAD BY MISS. MARIA REHMAN