Department Sarajevo Medical School

Academic year 2016/2017, 3rd Semester

Course: PHYSIOLOGY 1

STUDENT:

LABORATORY WORK 1

The goal of physiology is to explain the physical and chemical factors that are responsible for
the origin, development and progression of life.

Physiology is an experimental science gaining its knowledge through observations mainly

on living animals, organ preparations and tissue cultures. Observations during medical
procedures on human beings also contribute to this knowledge. Experiments done in the
laboratories should comply with the existing laws and regulations that are based on bioethical
principles. Bioethics is an interdisciplinary science dealing with ethical issues concerning both
biological experiments and medical procedures. During this course different experiments will
be conducted – hematology experiments, human and clinical experiments or examinations;
amphibian and mammalian experiments are replaced with software program simulations.

Human experiments and observations can only be carried out on volunteers after obtaining
their informed consent. Subjects should be aware of the circumstances and possible outcomes
of the experiment. Experiments that might lead to permanent physical or psychological harm
of the subjects are prohibited. Safety precautions should be entirely followed during the
experiments.

In accordance with the law, research using animals is only undertaken when there is no
alternative research technique and the expected benefits outweigh any adverse effects.
Animals are used to gain a knowledge and understanding of some cell structures and
physiological and pathological processes. Whilst it is acknowledged that animal models are
not a perfect model for humans, the use of animals in biomedical research remains essential.
Alternative techniques cannot always reproduce the complexity of a living creature. The use
of animals in medical research has made an important contribution to advances in medicine
and surgery, bringing major improvements to the health of both human beings and animals.
Research using animals will continue to be essential to make progress in developing
treatments for many unsolved medical problems, such as cardiovascular disease, stroke,

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AIDS, other infectious diseases and cancer; and genetic, developmental, neurological and
psychiatric conditions for the foreseeable future.

The three rules of animal experimentation should be observed: the number of animals
included in the experiments should be reduced as much as possible; efforts should be made
to replace animal experiments using tissue cultures or microbiological preparations; and,
experiments should be refined, to ensure minimization of pain and discomfort that an
unanesthetized animal has to endure without the possibility to avoid it.

INTRODUCTION TO LABORATORY WORK IN PHYSIOLOGY

Careful observation is the back bone of scientific method - one of the aims of conducting
experiments is to acquire an aptitude for careful observation. Sometimes this depends simply
upon intelligent use of the sensory organs. More often, observation calls for proficiency in
special techniques, and therefore, some of the experiments will require learning those
techniques. The skill in practical work will grow in the process of learning these techniques, an
it would be valuable in all aspects of clinical practice and research.

Observation yield information only when properly analyzed. Thus the second object of the
training is to learn how to make logical conclusions from observations. All facts learnt in any
science course are conclusions drawn from the results of many experiments. The most
important aim of the Laboratory work in Physiology is to understand how knowledge is acquired
from scientific observations, and to verify certain facts given in the text books. It is not possible
to perform experiments to confirm and verify all the theoretical information that are obtained
from lectures and text books. During Laboratory work in Physiology, a select number of
experiments will be done which will give some understanding of the scientific methods as
applied to different aspects of Physiology.

The practical work in laboratory is only a part of an experiment. An equally important part
is to record it properly. Another aim of the Laboratory work in Physiology is learning proper
documentation of the procedures and observations, and commenting on them. Students are
expected to clarify their own thinking while recording their observations, conclusions and
comments; if done properly, it would benefit them far more than just performing the
experiments. A good record would also be helpful in reviewing the experiments before practical
examinations.

The record is essentially an account of what was done and observed – there is no need for
elaborating on theoretical aspects.

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 THE FORM OF RECORDING

An accepted form of recording is given below via lab record.

Lab record should be finished during the class, after performing experiments, and submited
for correction at the end of the class.

1. Observations – this is the most important part of the write up. Always have an open
and critical mind. Describe fully what you have observed? If possible, tabulate your
observations in order. Give diagrams when desirable, with adequate labels.
2. Calculations – if any.
3. Discussion – here you can write the conclusions from your observations. Also make
any relevant comments on the limitations of the experimental techniques and any
alterations or additions that you would like to make. Specially discuss any unusual
observation of your findings. Avoid extensive theoretical discussions. It is a good
practice to make your discussion no longer than the account of your observations.

COLLECTION OF BLOOD SAMPLES

Since blood is confined within the cardiovascular system, the skin has to be punctured
before blood can be obtained. There are two common sources of blood for routine laboratory
tests: blood from a superficial vein by puncturing it with a needle and syringe, or from skin
capillaries by skin-prick. Arterial blood and blood from cardiac chambers may be required for
special tests. None of these samples can be called a representative sample because there are
minor variations in their composition. But for routine hematological tests, however, these
differences can safely be ignored. The differences between these two sources of blood are
given in Table 1.

Table 1. Sources and differences between Venous blood and Capillary blood

Venous blood Capilary blood

1. It is obtained from a superficial vein by
venepuncture.
2. A clean venepuncture provides blood without
any contamination with tissue fluid.
3. There is less risk of contamination since
sterile syringe and needle are used.
4. Cell counts, Hb, and PCV values are
generally higher.
5. Venous blood is preferable when normal
blood standards are to be established, or when
1. It is obtained from a skin puncture, usually
over a finger, ear lobe/or the heal of a foot.
2. Blood from a skin prick comes from punctured
capillaries and from smallest arterioles and
venules.
3. There is greater risk of contamination and
transmission of disease as one may be careless
about sterilization since skin prick is considered
a harmless procedure.
4. These values are likely to be on the lower
side since some tissue fluid is bound to dilute
the blood even when it is free-flowing.
5. Capillary blood is not suitable for these
purposes.

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 two samples from the same person are to be
compared at different times.

ASEPSIS. The term asepsis refers to the condition of being free from septic or infectious
material—bacteria, viruses, etc. The skin is a formidable barrier to the entry of foreign invaders
and the first line of defence against bacteria and other disease-causing microorganisms which
are present in abundance on the skin and in the air. Therefore, puncturing the skin always
poses the danger of infection In order to achieve asepsis, the following aspects need to be
kept in mind:

 Sterilization of Equipment. Irradiated and sealed, single-use syringes, needles, lancets and
blades are now freely available and are in common use. Otherwise all the instruments to
be used for collecting blood—syringes, needles, lancets, and cotton and gauze swabs—
should preferably be sterilized in an autoclave. The old practice of boiling glass syringes
and needles in tap water is now obsolete.
 Cleaning/Sterilization of Skin. Though it is impossible to completely sterilize the selected
site for skin puncture, every aseptic precaution must be exercised. The selected area need
not be washed and scrubbed unless grossly dirty. If washed, the area should be allowed
to dry before applying the antiseptics because these agents do not act well on wet skin. At
least 2–3 sterile cotton/gauze swabs soaked in 70% alcohol, methylated spirit, or ether
should be used to clean and scrub the area. Cotton swabs are likely to leave fibers sticking
to the skin and provide an undesirable contact, or they may appear as artifacts in a blood
film. But if they are used, the final cleaning should be done with gauze swab. Note: After
cleaning the skin, allow the alcohol to dry by evaporation (do not blow on it), because
sterilization with alcohol is effective only after it has dried.
 Prevention of Contamination. Any material used for skin puncture, or the operator’s hands
may cause contamination. Therefore, once the site has been cleaned and dried, it should
not be touched again. Care must be taken to prevent contamination until the puncture
wound has effectively closed/healed.

THE BLOOD SAMPLE. The term “blood sample” refers to the small amount of blood—a few
drops or a few milliliters—obtained from a person for the purpose of testing or investigations.
These tests are carried out for aiding in diagnosis and/or prognosis of the disease or disorder.

Containers for Blood Sample. A container is a receptacle into which blood is transferred from
the syringe before sending it to the laboratory. Clean and dry 10 ml glass test tubes, collection
bottles such as clean and dry 10 ml discarded medicine vials, glass bulbs, etc are the usual
ones in use. A container may or may not contain an anticoagulant depending on whether a
sample of blood/plasma, or serum is required.

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 For a sample of whole blood or plasma (plasma = blood minus all the blood cells). The
blood is transferred to a container containing a suitable anticoagulant. This is to prevent
clotting of blood.
 For a sample of serum (serum = plasma minus fibrinogen and all the clotting factors). No
anticoagulant is used. The blood is allowed to clot in the container and serum is collected
as described later.
 Obviously, capillary blood does not require a container or anticoagulant.

COMMONLY USED ANTICOAGULANTS. Anticoagulants are substances employed to delay,

suppress, or prevent clotting of blood. They are classified into 2 groups: the in vitro (outside
the body) anticoagulants, and the in vivo (in the body) anticoagulants. The commonly used in
vitro anticoagulants include: EDTA, trisodium citrate, double oxalate, sodium fluoride, heparin,
and ACD and CPD-A mixtures. The use of fluoride and heparin is limited to pH, blood glucose
and gas analysis. The in vivo anticoagulants include: heparin and dicoumarol derivatives
(warfarin, dicoumarin). Thus, heparin is both an in vivo and an in vitro anticoagulant.

COLLECTION OF VENOUS BLOOD. Caution: The blood sample from a vein must be
collected by a medically qualified staff member who should screen the volunteer for any
communicable diseases/especially viral hepatitis, and AIDS. Do not touch blood other than
your own.

Note: Two types of blood samples are not suitable for hematological tests:

 Clotted samples. Even tiny clots in the anticoagulated blood can negate the results.
 Hemolysed samples. The red cells may be damaged and ruptured during collection or
handling of blood. The released Hb tinges the plasma or serum red, rendering the sample
unfit for tests.

COLLECTION OF CAPILLARY BLOOD (SKIN-PRICK METHOD). Note: Remember that one

deep puncture, which will give you free-flowing blood, is less painful than 3 or 4 superficial
stabs.

Capillary blood is also called “peripheral blood” as it comes out of the peripheral vessels
(capillaries) in contrast to venous blood. In adults and older children, capillary blood is generally
obtained from a skin puncture made on the tip of the middle or ring finger, or on the lobe of the
ear. In infants and young children in whom the fingers are too small for a prick, the medial or
lateral side of the pad of the big toe or heel is used. The site for skin-prick should be clean and
free from edema, infection, skin disease, callus, or circulatory defects.

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Important: The thumb and little finger are never pricked because the underlying palmar fasciae
(venous bursae) from these digits are continuous with those of the forearms. Any accidental
injury to these fasciae may cause the infection to spread into the forearm.

APPARATUS. Note: Since our aim is to get free-flowing blood drops with only a single prick,
without squeezing the skin around the puncture, choosing a suitable instrument is very
important, whether a blood lancet, an injection needle, or a pricking gun.

1. Blood Lancet/Pricking needle. Disposable, sterile, one-time use, blood lancets (flat, thin
metal pieces with 3–4 mm deep penetrating sharp points) are commercially available and
should be preferred. Ordinary, narrow-bore injection needles are useless since they only make
shallow cuts rather than deep punctures. However, wide-bore (22 gauge) needles may be used
in an emergency or if blood lancets are not available. A cutting needle with 3-sided cutting
point (used by surgeons) can serve the purpose well. Pricking gun. A spring-loaded pricking
gun that has a disposable, 3-sided sharp point, and a loading and releasing mechanism, is
ideal because the depth of the puncture can be preselected. After pulling back the release
lever, and thus “loading” the gun, it is placed on the ball of the finger and the release lever
pressed. The subject does not see the sharp point and the pain is thus minimized.

2. Sterile gauze/cotton, moist with 70% alcohol/ methylated spirit.

3. Glass slides, pipettes, etc. according to requirements.

Note: Lancets may be reused 2–3 times, if required, after passing their points through a flame.
Too much heating, however, is likely to blunt the pricking points.

PROCEDURES. All aseptic precautions must be taken. The person giving the prick should
wash his/her hands with soap and water, and wear gloves if possible. Note: Keep all the
equipment ready before getting a prick. If the finger to be pricked appears cold and bloodless,
especially in winter, immerse it in warm water for 2–3 minutes.

1. Clean and vigorously rub the ball of the finger with the spirit swab, followed by a final cleaning
with dry gauze. (Scrubbing increases local blood flow). Allow the alcohol to dry by evaporation
for the following reasons:

 Sterilization with alcohol/spirit is effective only after it has dried by evaporation.

 The thin film of alcohol can cause the blood drop to spread sideways along with alcohol so
that it will not form a satisfactory round drop.
 The alcohol may cause hemolysis of blood.

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2. Steadying the finger to be pricked in your left hand, apply a gentle pressure on the sides of
the ball of the finger with your thumb and forefinger to raise a thick, broad ridge of skin. (Do
not touch the pricking area).

3. Hold the lancet between the thumb and fingers of your right hand, and keeping it directed
along the axis of the finger, but slightly “off” center so as to miss the tip of the phalanx (i.e. not
too far down or too far near the top of the nail bed), prick the skin with a sharp and quick vertical
stab to a depth of 3–4 mm and release the pressure. The blood should start to flow slowly,
spontaneously and freely (without any squeezing)—if a good prick has been given.

Important: Do not squeeze or press the finger as the tissue fluid squeezed out will dilute the
blood and give false low values. The squeeze also tends to close the wound edges. You may
exert a slight tension on either side of the puncture with your thumbs in order to open up the
wound more widely (a plug of epithelial cells tends to block the puncture especially if the wound
is shallow, as often happens if a narrow-bore injection needle is used). The forearm or the
hand may be squeezed or milked towards the fingers to facilitate blood flow. If all efforts fail, a
fresh prick may be required.

4. Wipe away the first 2 drops of blood with dry, sterile gauze as it may be contaminated not
only with tissue fluid, but also with epithelial and endothelial cells which will appear as artifacts
in the blood film.

5. Allow a fresh drop of blood of sufficiently large size (about 3–4 mm diameter) to well up from
the wound, and make a blood smear, or fill a pipette as the case may be.

6. Clean the area of the prick with a fresh swab and ask the subject to keep the swab pressed
on the wound with his/her thumb till the bleeding stops, which occurs in a minute or so.

Ear-lobe Prick. After selecting the site, rub the lobe of the ear between your thumb and finger
until warm. Clean the skin with alcohol and give a 2 mm deep prick. (The skin here is usually
thinner than at the fingertip). Wipe away the first drop and allow a new one to form.

Pricking the Heel. In infants and young children, blood can be collected from the cleaned and
warmed medial or lateral areas of the heel. The central plantar and the posterior curvature
areas of the heel should be avoided as the prick may cause injury to the underlying tarsal
bones which lie near the surface.

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PRECAUTIONS

1. Keep the equipment for the test ready before getting/giving a finger prick

2. The selected site should be clean, free from infection, edema, or skin disease

3. The site should be vigorously cleaned and scrubbed with sterile gauze and alcohol.
Scrubbing increases local blood flow

4. The lancet/needle should be sterile, and if it is to be reused, it should be passed through a

flame

5. The puncture should be deep enough to give free-flowing blood but not so very deep that it
takes inordinately long time for the bleeding to stop.

6. Do not press or squeeze the finger to increase the blood flow from the skin-prick, though the
arm or the hand may be milked towards the fingers.

Date: Assistant: