School of Pharmacy

Lab Experiments, PHB 103L

Physiology & Anatomy I Lab 1 credit

1. Determination of bleeding and clotting time.

2. Estimation of hemoglobin
3. Determination of blood group
4. Measurement of blood pressure
5. Demonstration of positive feedback mechanism
6. Demonstration of negative feedback mechanism
7. Assessment of human reflexes

Submitted By:
 Index

Exp. No. Name of the Experiment Page No.

Experiment No.: 01

Name of the experiment: Determination of (a) Bleeding and (b) Clotting time.
1 (a): Determination of bleeding time
Objective:
The main objective of the test is to know the bleeding time as well as to identify the defects of
platelets function.
Principle:
The bleeding time test has been used to evaluate the integrity of primary hemostasis, platelet
plug formation and capillary integrity. The bleeding time is dependent upon the capillary
function and the number of blood platelets present and their ability to form a platelet plug and
also include the contribution of blood vessel wall. Factors affecting the bleeding time are the
depth of wound, hyperemia etc.
This test is performed by a small incision on the skin and measuring how much time is needed
for coagulation as well as the interaction between blood vessel walls and platelets. Cessation of
bleeding indicates the formation of hemostatic plugs.
Basically, prolonged bleeding times are generally found when the platelet count is below
50,000/µL, and when there is platelet dysfunction. Occasionally, the bleeding time test will be
ordered on a patient scheduled for surgery and the patient who have a history of long bleeding
time or family history of bleeding disorder.
Normal Range:
The physiological limit of bleeding is 2 to 7 minutes.
Apparatus:
1. Filter paper
2. Disposable sterile lancet
3. Stop watch
4. Cotton
5. 70% ethanol
Procedure:
1. The selected area is rinsed with an antiseptic.
2. Lancing machine is used to prick the selected area of the skin.
3. The stopwatch started immediately as the blood came out.
4. The first drop of blood will be discarded and then the pricked area is touched on the filter
paper every 15 seconds interval and this is continued until the bleeding stops.
Result:

Discussion:

Precaution:
1. Aseptic precautions must be taken during collection of blood.
2. Time must be recorded carefully.
3. The needle should be used only one time.
4. We should be careful in the lab while using any apparatus.
5.
6.
7.
8.
1 (b): Determination of blood coagulation time
Objective:
The main objective of the test is to know the coagulation time to determine the functioning
capability of platelets and to evaluate the presence of sufficient clotting factors.
Principle:
Blood clotting (or hemostasis) and coagulation are essentially synonyms, often used
interchangeably to describe the process of blood changing from liquid to gel to stop bleeding.
While "clotting" refers to the overall process including platelet plug formation. Coagulation
specifically refers to the chemical cascade involving proteins that creates a fibrin mesh to
strengthen that plug.
The coagulation test had been used to measure the clotting ability of blood. For blood to clot, the
enzyme thrombin must be generated from the plasma precursor prothrombin. Thrombin then
converts soluble fibrinogen into insoluble fibrin. Generation of thrombin involves the sequential
activation of a number of other plasmas clotting factors, this process is also being assisted by
Ca2+ and by factors released by platelets and damaged tissues. The time taken for blood to clot
mainly reflects the time required for the generation of thrombin in this manner. If the plasma
concentration of prothrombin or of some of the other factors is low (or if the factor is absent, or
functionally inactive), clotting time will be prolonged. Coagulation tests are also useful in
monitoring people who take medications that affect clotting ability and sometimes recommended
before surgery.
Normal Range:
The physiological limit of coagulation is 6 to 10 minutes.
Apparatus:
1. Capillary tube
2. Disposable sterile lancet
3. Stopwatch
4. Cotton
5. 70% ethanol
6. Filter paper
Procedure:
1. The selected area is rinsed with an antiseptic.
2. Lancing machine is used to prick the selected area of the skin.
3. The blood is then drawn up in the capillary tube.
4. The stopwatch starts immediately.
 5. Then the bottom of the capillary tube is touched on the filter paper at 0 second, then after
2 minutes followed by 1 min and then after every 30 seconds interval until the blood clots
and no spot is visible on the paper.
6. Finally, the capillary tube has been broken gently to see the blood thread.

Result:

Discussion:

Precaution:
1. Aseptic precautions must be taken during collection of blood.
2. Time must be recorded carefully.
3. The needle should be used only one time.
4. We should be careful in the lab while using any apparatus.
5.
6.
7.
Experiment No.: 02
Name of the experiment: Estimation of hemoglobin
Objective:
The main objective of this test is to estimate hemoglobin, which is necessary for diagnosis
purposes.
Principle:
The principle of estimating the hemoglobin by Sahli’s hemoglobinometer is to compare the color
of the acid hematin solution with that of a standard. The hemoglobin (Hb) is converted by
diluting hydrochloric acid into hematin, which in solution is brown in color. The intensity of this
color depends on the amount of acid hematin in solution, which, in turn, depends on Hb
concentration. The color of the solution is matched against brown-tinted glass filter, by direct
vision, which gives the reading in grams per 100ml blood.
Hemoglobin (Hb) is a conjugated protein present in red blood cells. It carries oxygen from the
lungs to the tissues, and carbon dioxide from the tissues to the lungs. It is made up of heme and
globin. The heme group is an iron complex, containing one iron atom. Iron is essential for the
primary function of hemoglobin, the transport of oxygen. When reduced hemoglobin is exposed
to oxygen at increased pressure, oxygen is taken up at the iron atom. The Hb molecule when
fully saturated with oxygen, that is, four oxygen molecules combined with one hemoglobin
molecule, is called oxyhemoglobin. One gram of hemoglobin carries 1.34 ml of oxygen.
Hemoglobin returning carbon dioxide from the tissues is called Carbaminohemoglobin.
 Figure: Hemoglobin
Normal Range
Normal range of Hb concentration in Male: 14-18 g/dl of blood
Normal range of Hb concentration in Female: 12-16 g/dl of blood
Apparatus & Reagents:
1. Sahli’s type hemoglobinometer
2. Micropipette
3. A thin glass rod stirrer
4. Dropper
5. Sterile blood lancets or needle
6. Cotton
7. N/10 HCl
8. 70% ethanol
9. Distilled water

Procedure:
1. 0.1 N HCl is taken in the graduated tube of the hemoglobinometer up to the 20% mark
(red color).
2. A finger prick is done under aseptic condition.
3. Then 20μl blood is drawn using the micropipette.
4. Slowly the blood is transferred into the acid and the mixing tube is placed in
5. the matching box for the formation of acid hematin.
6. Gradually distilled water is added drop by drop stirring the mixture each time until
7. the color of the acid hematin solution exactly matches with the color of the standard.
8. Reading is taken both before and after matching and both in gm percentage.

Results:
Discussion:

Precautions:
1. Aseptic precautions must be taken during collection of blood.
2. Reagents must be added carefully.
3. The needle used only one time.
4. We have to be careful in the lab while using any apparatus.
5.
6.
7.
8.
9.
Experiment No.: 03
Name of the experiment: Determination of blood group

Objective:
The main objective of this test is to determine the blood group of a sample.
Principle:
It was in 1901, that Austrian American immunologist and pathologist Karl Landsteiner
discovered human blood groups. Karl Landsteiner’s work helps to determine blood groups and
thus opened a way for blood transfusions which can be carried out safely. Blood system is the
medium through which nutrients and oxygen are transported to each part of the body and waste
is taken out. Landsteiner divided the blood into four groups A, B, AB and O also discovered
there are two types of chemical component named antigen and antibodies which are present in
our surface of the blood cell causing the blood typing. Blood is often grouped according to the
ABO blood typing system. This system contains four types of categories:

The major concern in blood transfusion procedures is that the cells in the donated blood not
clump due to antibodies in the recipient’s plasma.
The second table includes the outline of determining the Rh group of a person’s blood which is
also one crucial method of blood typing. Rh blood group system, system for classifying blood
groups according to the presence or absence of the Rh antigen, often called the Rh factor, on the
cell membranes of the red blood cells (erythrocytes). If someone has the protein in his RBC then
he is Rh positive and if not, then he is Rh negative.
Apparatus & Reagents:
1. Glass slide
2. Disposable sterile lancet
3. Pasteur pipette/ Dropper
4. Cotton
5. Spirit
6. Anti A serum
7. Anti B serum
8. Anti D serum

Procedure:
1. 3 glass slides are taken.
2. The selected area of the finger is rinsed with the antiseptic.
3. Lancing machine is used to prick the selected area of the skin.
4. Then 3 drops of blood is taken into 3 slides.
5. Then immediately Anti Serum A, B, and D is added 3 different slides and mixed
thoroughly.
6. The blood samples are examined for agglutination for 5 minutes.

Observation should be as follows:

(+) = Presence of agglutination (-) = Absence of agglutination

Results:
Discussion:

Precautions:
1. Aseptic precautions must be taken during collection of blood.
2. Reagents are added carefully.
3. The needle is used only one time.
4. We must be careful while using any apparatus in the lab.
5.
6.
7.
8.
9.
Experiment number: 04
Name of the experiment: Measurement of blood pressure

Principle:
Blood pressure is a measurement of the force applied to the walls of the arteries as the heart
pumps blood through the body. The pressure is determined by the force and amount of blood
pumped, and the size and flexibility of the arteries. Blood pressure is continually changing
depending on activity, temperature, diet, emotional state, posture, physical state, and medication
use. Each time the heart beats, it pumps out blood into the arteries.
Blood pressure is at its highest when the hearts contracts and the pressure present at that instant
is known as systolic pressure. When the heart is at rest between contractions, then blood pressure
falls, and it is called diastolic pressure.
Blood pressure is very important and essential for the flow of blood through the circulatory tree
and, for providing motive force for filtration at the capillary bed, which is essential for tissue
nutrition, formation of urine, formation of lymph. Blood pressure changes during the day. It is
the lowest during sleep and rises when you get up. It also rises during excitation, nervousness, or
any kind of activity.
Blood pressure is always given as two numbers, one systolic and another one is diastolic.
Usually, they are written as for example 120/80 mm Hg, where the first number is for systolic
pressure and the second for diastolic pressure. It is quoted as “120 over 80”.
Purpose:
The main purpose of the test is to know whether the blood pressure is normal or high or low.
Normal Range:
The normal blood pressure for young adults is 120/80 mm Hg on average. The normal range is as
follows:
Systolic pressure: 100-130 mm Hg
Diastolic pressure: 60-90 mm Hg

Apparatus:
1. A sphygmomanometer
2. A stethoscope
Procedure:
1. The BP cuff is wrapped snugly around the arm.
2. The arm is palpated at the ante cubical fossa (crease of the arm) to locate the strongest
pulse sounds. The bell of the stethoscope is placed over the brachial artery at this
location.
3. The cuff bulb is being pumped while we listen to the pulse sounds. When the BP cuff has
inflated enough to stop blood flow, we would stop pumping. Inflation is mostly done up
to 160 - 180 mm Hg.
4. Deflation started afterwards. It is recommended that the pressure should fall at 2 – 3 mm
Hg per second, anything faster may likely result in an inaccurate measurement.
5. The first rhythmic sounds are heard as blood begins to flow through the artery at the
patient’s systolic pressure. This may resemble a tapping noise at first and the gauge
reading is recorded.
6. We will continue to listen as the BP cuff pressure drops, and the sounds fade. The gauge
reading should be noted when the rhythmic sounds stop. This will be the diastolic
reading.

Result:
Discussion:

Precautions:
Experiment No.: 05
Name of the Experiment: Demonstration of positive feedback mechanism

Principle:
Homeostasis is the maintenance of equilibrium in the body's internal environment by constant
interaction of its regulatory mechanisms. One of these regulatory mechanisms is a feedback
system which controls the normal physiological parameters. A feedback system includes a
receptor, a control center and an effector cell/organ as basic components. A receptor recognizes
the changes in the controlled body environment and sends corresponding inputs to the control
center for interpretation. Effector cell/organ receives output from the control center and
accordingly produces the response to balance the altered physiology.
Feedback mechanism can either be positive or negative. Positive feedback mechanism occurs
when change in the parameter triggers a response that causes changes in the same direction
whereas, in negative feedback mechanism, a stimulus causes an opposite output reversing the
initial change to maintain the normal level of the parameter to be regulated.
Positive Feedback Mechanisms: In positive feedback mechanism, the result or response
enhances the original stimulus so that the response is accelerated. This feedback mechanism is
“positive” because the change that results proceeds in the same direction as the initial change,
causing the variable to deviate further and further from its original value or range. Positive
feedback mechanisms usually control infrequent events that do not require continuous
adjustments. Typically, they set off a series of events that may be self-perpetuating and that, once
initiated, have an amplifying effect.
Example:
Blood clotting is a normal response to a break in the wall of a blood vessel and is an excellent
example of an important body function controlled by positive feedback. Basically, once a vessel
has been damaged, blood elements called platelets immediately begin to cling to the injured site
and release chemicals that attract more platelets. This rapidly growing pileup of platelets
temporarily “plugs” the tear and initiates the sequence of events that finally forms a clot.
During hemostasis, positive feedback mechanism works.
The following changes occur when the blood vessel is injured:
1. Aggregation of platelets at the injured site.
2. Release of specific chemicals by platelets to attract more platelets.
3. Initiates the activation of clotting factors.
 4. Formation of fibrin clot i.e. coagulation.

Purpose:
To demonstrate positive feedback mechanism.
Apparatus & Reagents:
1. Cotton swab
2. 70% alcohol or any other suitable antiseptic solution
3. Glass slide
4. Cover slip
5. Scanning electron microscope

Procedure:
Demonstration of positive feedback mechanism:
1. Select a subject and prick his/her ring finger under aseptic condition.
2. Take a blood drop on the clean glass slide.
3. Dip one end of the pricking needle in the blood drop and drag it up.
4. Repeat step no. 3 until the formation of fibrin clot is observed.
5. Observe this process by using light microscope under 40X objective lens and take the
photo.

Observation:
Discussion:

Precautions:
1. Aseptic precautions must be taken during collection of blood.
2. The needle should be used only once.
3. We should be careful in the lab while using any apparatus and reagent.
4.
5.
6.
7.
8.
Experiment No.: 06
Name of the Experiment: Demonstration of Negative Feedback Mechanism

Principle:
Homeostasis is the maintenance of equilibrium in the body's internal environment by constant
interaction of its regulatory mechanisms. One of these regulatory mechanisms is a feedback
system which controls the normal physiological parameters. A feedback system includes a
receptor, a control center and an effector cell/organ as basic components. A receptor recognizes
the changes in the controlled body environment and sends corresponding inputs to the control
center for interpretation. Effector cell/organ receives output from the control center and
accordingly produces the response to balance the altered physiology.
Feedback mechanism can either be positive or negative. In negative feedback mechanism, a
stimulus causes an opposite output reversing the initial change to maintain the normal level of
the parameter to be regulated.
Negative Feedback Mechanisms: Most homeostatic control mechanisms are negative feedback
mechanisms. In these systems, the output shuts off the original effect of the stimulus or reduces
its intensity. These mechanisms cause the variable to change in a direction opposite to that of the
initial change, returning it to its “ideal” value; thus, the name “negative” feedback mechanisms.
All negative feedback mechanisms have the same goal: preventing sudden severe changes within
the body. Example: Regulation of body temperature, pH, water content, electrolyte balance.

When we exercise, negative feedback mechanism works.

The following parameters change during exercise:
1. Increased oxygen demand by muscles.
2. Increased heart rate and pulse rate.
3. Increased blood pressure.
4. Increased body temperature.
5. Change in the pH of blood.

Purpose:
To demonstrate negative feedback mechanism.
Apparatus & Reagents:
1. Clinical thermometer
2. Stopwatch
3. A sphygmomanometer
4. A stethoscope

Procedure:
1. Select a subject and ask him/her to get relaxed for 5 minutes by taking a rest before
starting the experiment.
2. Record pulse rate, heart rate, blood pressure, breathing rate and body temperature
using standard procedures.
3. Record the breathing rate by counting respiration cycles per minute.
4. Prick the ring finger under aseptic condition. Take a drop of blood on pH indicator
paper and check the pH range of blood,
5. Ask the subject to perform any of the following exercises for 3 minutes:
i. running in place with thighs bought up horizontally,
ii. hopping on each foot,
iii. climbing the stairs up and down,
iv. jogging.
6. Record pulse rate, heart rate, blood pressure, body temperature, and breathing rate
immediately after completion of this exercise.
7. Ask the subject to relax for 30 minutes and record all the parameters again.

Observation:
Discussion:

Precautions:
1. We should be careful in the lab while using any apparatus and reagent.
2.
3.
4.
5.
6.
Experiment No.: 07
Name of the Experiment: Assessment of Human Reflexes

Principle:
Reflexes are automatic responses to changes (stimuli) within or outside the body. It is an
immediate response to a stimulus. A reflex is an involuntary (automatic) response to stimulus
that quickly returns the body to homeostasis. There are several kinds of reflexes. Examples are
shivering in response to low core body temperature; or withdrawing your hand from a hot stove
when temperature and pain receptors in your hand register the stimulus. Reflexes also carry out
the automatic actions of swallowing, sneezing, coughing, and vomiting.

A reflex arc refers to the neural pathway that a nerve impulse follows. The reflex arc typically
consists of five components:
1. A receptor, and independent sensory cell, or an ending of a sensory neuron, reacts to a
stimulus (e.g., a stretch receptor).
2. The sensory, or afferent, neuron sends a nerve impulse through an afferent pathway to
the central nervous system.
3. An integration center consists of one or more synapses in the CNS (typically the
spinal cord) where the incoming information and outgoing response are integrated.
4. A motor, or efferent, neuron sends a nerve impulse along an efferent pathway from
the integration center to an effector cell.
5. The effector cell responds to efferent impulses (for example, by contracting, if the
effector is a muscle fiber).
Because integrating center processing may occur at the level of the spinal cord rather than
requiring impulses to travel to the brain, reflex responses have a relatively short path length and,
thus, a quick reaction time compared to voluntary or conscious body movements.
Purpose: The main purpose of the test is to learn about human reflexes.

Apparatus:
1. Reflex Hammer
Procedure:
a. Knee reflex (patellar reflex):
1. Ask the subject to sit on the chair with their legs crossed, with one leg hanging
casually over the other.
2. Gently hit the tendon just below the kneecap on the hanging leg with the reflex
hammer.
Observation:

b. Ankle jerk reflex (Achilles reflex):

1. Ask the subject to put one knee on the chair, with the foot hanging over the edge of
the chair.
2. Gently hit the tendon above the heel with the reflex hammer.
Observation:
c. Blink reflex:
1. Ask the subject to sit comfortably in dim light.
2. Shine a doctor’s penlight towards one eye and observe pupil constriction (direct
response).

Observation:

Discussion:

Precautions:
1. Do not injure the subject, must hit gently with the hammer.
2.
3.
4.
5.