Physiology 1

Practical Notebook
Submitted to:
Dr. Aniza Ishaque
Submitted by:
Momina Nadeem
Rimsha Zareen
Abdullah Najam
Aman Sarfraz
Blood group
RBC count
Bleeding time
Clotting time
 Experiment No. 1
Microscope:

Objective:
To determine the method of setting up a binocular light microscope for visualizing a specimen.

Types of Microscope:
1) Compound Microscope
2) Digital Microscope
3) Multi-Head Microscope
4) Minutia Microscope

Parts of Compound Microscope:

Eyepiece OFF/ON Switch Slide Mirror
Arm Light source Specimen Slides
Stage Fine and Cone adjustment Slide adjustment knot
Objective lens Condenser

Definition:
A microscope is an instrument designed to produce magnified, visual or photographic image
of small objects.

Light Microscope:
It is used to focus light and lenses to magnify a specimen, usually a cell.

Electron Microscope:
A microscope with high magnification and resolution employed electronic beans in phase of
light and using electron lens

Procedure:
i. Focusing the object up to 10x-40x.
ii. Place the specimen. Slide on stage between stage holding clips.
 iii. Not viewing through eye piece, using coarse adjustment knob, raise the stage until
it close to the objective lens.
iv. Not viewing through the eyepiece, lower the stage very slowly, using coarse
adjustment knob until a blurred image of the field appears.
v. Setting up the microscope for critical illumination.
vi. Choose a low power objective.
vii. Focus on a slide, loose the light source to minimum.
viii. Close the iris diaphragm on the condenser to its minimum
ix. Open the light sources until fills the whole field of view remove the field eye
piece, look down the tube and open the iris
x. Adjust the light intensity to suit the personal performance.
 Experiment No. 2
Estimation of Hemoglobin

Hemoglobin:
It is the oxygen carrying, conjugated protein that gives whole body its red color. Each RBR
contain about 280 million hemoglobin molecules.

Structure of Hemoglobin:
A hemoglobin molecule consist of

1) Globin: it is a compound of four polypeptide chains

2) Heme: the heme is bound to each other of four chains of globin. At centre of each heme
ring is a iron ion that combine reversibly with one oxygen molecule allowing each
hemoglobin to bind for oxygen molecule

Function of Hemoglobin:
1) Transport of oxygen:

Each oxygen molecule picked up from the lungs is bound to an iron ion. As blood flows
through the tissue capillaries, the iron oxygen reaction reverses. HB releases oxygen
which diffuses first in interstitial fluid and then into cell

2) Transport of CO2 :

Hemoglobin also transports about 23% of total CO2, and a waste product of
metabolism. as blood flows through the lungs, carbon dioxide is released from
hemoglobin and then exhaled.

3) Regulation of blood flow:

In addition to its key role in transporting oxygen and CO2, hemoglobin also plays an
important role in regulation of blood flow and blood pressure.
4) Act as intracellular buffers:
Hemoglobin acts as a very important intracellular buffer for the the absence of this
buffer system, transport of Co2 becomes very slow
Synthesis of Hemoglobin:
Synthesis of hemoglobin begins in the the pro erythroblast and continuous even into a
rectangular stage of the red blood cells. Therefore, reticulocytes continuous to form minute
quantity e of hemoglobin for another day or so they become mature erythrocytes
 Steps of hemoglobin synthesis:
First: Succcinyl_CoA binds with glycine to form a pyrrole molecule
Second: four parallel molecules combine to form protoporphyrin 1 X
Third: protoporphyrin 1x then combines with iron to form hi molecule.
Finally each heme molecule combine with polypeptide chain named as globulin to form a
subunit of hemoglobin called hemoglobin chain
Characteristic Features:
1. The molecular weight of hemoglobin is 64.440.
2. Hemoglobin forms 341 wet weight and 95% dry eight of RBC.
3. Because each hemoglobin chain has a heme prosthetic group containing an atom of iron and
because there are four globin chains and four heme groups in each heamoglobin molecule, 1
fluid for atoms in each hemoglobin molecule. It contains total amount of oxygen.
4. 1 gram of hemoglobin can carry 1.34 ml of oxygen.
Physiological varieties of hemoglobin:
Different varieties of hemoglobin are due to variation in amino acid composition of polypeptide
chain of globin protein.

Different physiological varieties of hemoglobin are:

 Adult hemoglobin: adult hemoglobin is of two types

 hemoglobin a
 hemoglobin b
 Fetal hemoglobin (HB. F)
 In embryonic/intra_utenine life: it has two types of hemoglobin.
 Gower 1Hb
 GOWER 2 hb

Methods of hemoglobin estimation:

 SAHLI'S Method:
This method is roughly used in physiological laboratories

APPARATUS:

SAHLI'S haemoglobinometer sterilized, lenset, spirit swap, water, HB pipette.

Procedure
1) Place 5 to 8 drops of N/10HCL in a graduated HB tube up to the mark 20%
2) Get a finger prick under espetic condition and always the first to 2 drops
3) a large drop of free floating blood has form again then place the HP of HB pipette on drop of
blood.
4) When a large drop free floating blood has found again, then place the HP of HB fit on drop
of blood.
5) Gently blood to the mark 20 withdraw the prepared from the table, touching at the side of the
tube does and sure that no mixture carries out of the tube.
6) Mixer blood with acid solution with flat and of the stirrer.
7) Folder comparator at eye level away from your face, against draught but diffused light, read
the scale at lower ministers of the fluid in the HB tube.
8) Record the reading.
Observation and Results:
Report your result as:
Normal Value:

In males 13-16 gm/dl

In females 12-15 gm/dl
In children 14-19gm/dl
 Experiment No

Determination of Erythrocyte Sedimentation Rate (ESR)

Method for determination of ESR

  Westergen’s Method
 Winyrob’s Method

Westergen’s Method

Apparatus:
Westergen’s tube, petri dish, 3.8% sodium citrate, spiritswab and disposable syringe.

Procedure:
 Introduce yourself, take consent and explain what you are going to do.
 Add 3.8% sodium citrate in a petri dish.
 Draw 1.6cc/ml of venous blood under aseptic measure and transfer this blood to petri
dish and mix content gently
 Draw blood in the westergen’s tube exactly upto the zero mark after placing finger tip
over the top westergen’s tube
 Place the tube vertically in the ESR stand by firmly pressing its lower and into rubber
caushion while keeping your finger over the westergen’s tube rack.
 Leave westergen’s tube undisturbed for one hour and than note the length of the column
of the clear plasma at the top of RBSs at the end of first hour.

Precautions:

 The tube should be absolutely verticle, otherwise slight deviation may result in false
reading.
 Avoid air bubbles in the column of blood.
 Avoid clotting of blood while mixing of anticoagulant.
 The westergen’s tube must be filled up to mark zero
Wintrobe’s Method
Apparatus:

Disposable syringe and needle. Sterilized cotton swap. Container with double oxalate
mixture, Pasteur pipette with thin long nozzle and wintrobe’s tube

Procedure:

 Draw 2.0ml of venous blood under aseptic technique and transfer it to a container contain
anticoagulant.
 Mix the content gently, but well by inverting the container a few time or by swirling it
 Do not shake a well cause frothing.
 Using the pasture pipette, fill the wintrobe’s tube from below upward.
 Ensure that there are not bubbles.
 Transfer the tube to it stand and adjust the screw so that it will remain verticle.
 Leave the tube undisturbed in this position for one hour
 At the end of witch the mm of clear plasma above the red cells.

Normal values:

Male: 2-8mm after 1st hour

Female: 4-11mm after 1st hour

 Experiment No 4

Determination of Hematocrit (HCT) or Paked cell volume (PCV)

Material:

Microhematocrit centrifuge, sterilized lancet, Microhematocrit reader, spirit or alcohol

swab, haparanized capillary tube and Plasticine

Procedure:

 Pick your finger with a sterile lancent to obtain a drop of blood under aseptic measure.
 Discard the first drop onto an alcohol swab and dispose of this properly in a designated.
 Obtain a heparinized capillary tube (heparin is an anticoagulant).
 Notes that one end of the tube is marked with a red blood.
 Allowing blood to enter the tube by capillary action and gravity
 The tube does not have to be completely fill (half fill or more is adequate) and air bubble
are not important
 Seal the red-banded end of the capillary tube by gently pushing it upright into the clay
capillary relant.
 Care fully rotate and remove the tube.
 Place the sealed capillary tube in a numbered slot of the micro capillary the plugged end
of the capillary tube facing outward against the rubber gas kept.
 Screw to top plate onto the centrifuge head and centrifuge for 3-5 mints.
 Determine the hematocrit reader provided at the end of the centrifugation and enter this
value in your work book

Precautions:

 Avoid air bubbles in capillary tube.

 The closed end of the capillary tube must face the outer margin of base.
 The lid should be properly light.
 Experiment no. 6
Determination of RBC cell Count
Principle:
The blood is distilled 200 times in a red cell pipette and the cells are counted in the counting
chamber knowing The dissolution employed, their number in undiluted blood can be easily
calculated.
Apparatus:
RBC pipette, improved neubouer's chamber, cover slip, microscope, dispersible blood lancet,
sterile cotton. 70% alcohol and hymns fluid
Procedure:
1) Place about 2 ml of hayem's fluid in a watch glass.
2) Adjust and focus Neubouer's chamber with cover slip centered on it under low
magnification
3) Bring chamber to your work table for charging it with distilled blood
4) Get a figure freak prick after cleaning fingertip
5) Suck hayem's fluid to the mark 10 ml and mix the contents for 2 to 3 minutes
6) Discard first 2 to 3 drop from RBC pipette to remove the diluting fluid and place the
tip of the RBC pipette at the corner of cover slip
7) Wait for 3 to 4 minute for the cells to settle down, because they cannot be counted
when they are moving and changing their position due to current in fluid.
8) Counting of RBC: switch over the high magnification.
Rules of counting:
a) Count RBC in five small squares
b) Follow Thomas rule during counting i.e leaf lower and left.
c) Calculate the number of RBC/ mm3 in undiluted blood according to the following:
Calculation of size and volume:
Size of one small square= 1/5 X 1/4 =1/20mm
Area of one small square=1/20 X 1/20= 1/40mm
Depth of one small square=1/10mm
Volume of one small square=1/400 X 1/10= 1/400mm3
Calculation of RBC count:
Number of red cells in 80 small squares= X RBCs
Number of red cells in 01 small square= X/80 RBCs
Number of RBC in 1 mm3 in diluted blood =X/8
Diluted Footer=200 Times
Number of RBCs in 1mm3 in undiluted blood= Y X 200 cell/mm3
Result:
Express your results= million/mm3
The average cell counts are
Males: 5 million/mm3 (4.75-6million/mm3)
Female: 4.5 million/ mm3(4.00-5.5 million/mm3)
 Experiment No. 7
BLEEDING TIME
AIM:

To determine the bleeding time of a subject.

Requirements:

 Sterile lancets
 Cotton
 Rectified spirit
 Filter paper
 Stop watch

Procedure:

Duke’s method: Sterilize the finger tip using rectified spirit and allow to dry. Make a
sufficiently deep prick using a sterile lancet, so that blood comes out freely without squeezing.
Note the time (start the stop-watch) when bleeding starts. Mop the blood by touching the finger
tip with a filter paper. This is repeated every 15 seconds, each time using a fresh portion of the
filter paper, till bleeding stops. Note the time (stop the stop-watch). It is seen that the blood stains
on the filter paper get smaller to disappear finally when bleeding stops.

Discussion:

Bleeding time is the interval between the moment when bleeding starts and the moment when
bleeding stops. Normal bleeding time (Duke’s method) is into 4 minutes. Bleeding time is
prolonged in purpuras, but normal in coagulation disorders like hemophilia. Purpuras can be due
to:

1. Platelet defects - Thrombocytopenic purpura.

1. Primary (Idiopathic) - Thrombocytopenic purpura

2. Secondary - Thrombocytopenic purpura

2. Vascular defects - Senile purpura

Henoch Schonlein purpura

Platelets are important in preventing small vessel bleeding by causing vaso constriction and
platelet plug formation.

Other method:
Ivy’s method: Apply the sphygmomanometer cuff to the arm. Raise the cuff pressure and
maintain at 40 mm of Hg. Under sterile conditions make a deep prick on the forearm just below
the elbow. Bleeding time is noted as in Duke’s method.

Normal value is 2 to 7 minutes

Result:
 Experiment No. 8:
CLOTTING TIME
AIM

To determine the clotting time of a subject.

Requirements:

Fine capillary glass tubes of about 10 mm length, cotton, rectified spirit, lancet, stop watch.

Procedure:

Capillary tube method: (Wright’s method)

Under sterile precautions make a sufficiently deep prick in the finger tip. Note the time when
bleeding starts (start the stop watch). Touch the blood drop at the finger tip using one end of the
capillary tube kept tilted downwards. The tube gets easily filled by capillary action. After about
two minutes start snapping off small lengths of the tube, at intervals of 15 seconds, each time
noting whether the fibrin thread is formed between the snapped ends. Note the time (stop the
stop watch) when the fibrin thread is first seen.

Discussion:

Clotting time is the interval between the moment when bleeding starts and the moment when the
fibrin thread is first seen.

Normal value is 3to 10 minutes.

Bleeding time and clotting time are not the same. Bleeding time depends on the integrity of
platelets and vessel walls, whereas clotting time depends on the availability of coagulation
factors. In coagulation disorders like haemophilia, clotting time is prolonged but bleeding time
remains normal.

Clotting time is also prolonged in conditions like vitamin K deficiency, liver diseases,
disseminated intravascular coagulation, overdosage of anticoagulants etc.

Other method:

Modified Lee and White method:

 Under aseptic precaution venepuncture is done and one ml. of blood is collected in each 3 small
test tubes. Note the time when blood is taken. Keep the test tube in a water bath maintained at
37°c. Tilt the tubes every 30 seconds and see whether the blood is flowing. Repeat this till the
tube can be inverted without the blood flowing out. Nore the time. Average value of the results
in the 3 test tubes gives the clotting time.

Normal value is 2 to 7 minutes

RESULT: