Practical guide for

PHYSIOLOGY
Compiled by CMT Fourie

Seventeenth edition
2021
North-West University
School for Physiology, Nutrition and Consumer Science
Table of Contents
INFORMATION REGARDING PHYSIOLOGY PRACTICAL LESSONS ........... i

SAFETY GUIDELINES FOR PHYSIOLOGY PRACTICAL LESSONS ........... vi

GOOD LABORATORY PRACTICE FOR PHYSIOLOGY ............................... ix

BLOOD: IMMUNITY AND BLOOD TYPES ...................................................... 1

INFLUENCE OF DIFFERENT NUTRIENTS ON BLOOD GLUCOSE

(Glycaemic index) .......................................................................................... 10

THE CHLORIDE BICARBONATE SHIFT ...................................................... 15

THE ENDOCRINE FUNCTION OF THE PANCREAS ................................... 20

THE QUANTITATIVE EVALUATION OF THE HAEMOLYTIC ACTIVITY OF A

SAPONIN ....................................................................................................... 26

THE MICROSCOPE AND OSMOSIS THROUGH LIVING MEMBRANES .... 30

THE SENSES ................................................................................................ 37

DIFFUSION.................................................................................................... 49

ELECTRICAL EVENTS IN THE CARDIAC MUSCLE (ECG) ......................... 56

RECORDING OF AN ELECTROCARDIOGRAM........................................... 74

FUNCTIONING OF ENZYMES ...................................................................... 86

HISTOLOGY .................................................................................................. 92

A. Histology of the epithelia, connective tissue, cartilage and bone ........... 92

B. Histology of the nervous system .......................................................... 100

C. Histology of muscle tissue ................................................................... 108

D. Histology of the digestive system ......................................................... 115

E. Histology of the urinary, respiratory and reproductive systems ............ 122

METABOLISM: SPIROMETRIC DETERMINATION OF THE METABOLIC

RATE ........................................................................................................... 131

MEASUREMENT OF BLOOD PRESSURE ................................................. 139

SKELETAL MUSCLE ACTIVITY: MECHANICAL FUNCTIONING OF THE

SKELETAL MUSCLE ................................................................................... 145
SPIROMETRY: THE DETERMINATION OF AIRFLOW, LUNG VOLUMES
AND LUNG CAPACITIES. ........................................................................... 157

EXCRETION ................................................................................................ 166

DIGESTION ................................................................................................. 175

ELECTROCARDIOGRAM AND ELECTRO-ENCEPHALOGRAM (EEG) .... 186

DETERMINATION AND REGULATION OF SKIN AND BODY

TEMPERATURE .......................................................................................... 192

LITERATURE USED.................................................................................... 196

 INFORMATION REGARDING PHYSIOLOGY PRACTICAL LESSONS

1. Prescribed book
Practical Guide for Physiology

2. Requisites
White coat—no student will be allowed in the laboratory without a white coat
Watch or cellphone with timer
Safety glasses

3. Purpose of practical lessons

IMPORTANT: Although you have to work together in the practical lessons as a
group and work with the same results, the report must be your own work.

a) The degree of active learning is optimised by understanding the scientific

method of reporting, by writing a report after each experiment. Reports should
contain the following sections:

Purpose: As complete, but as short, as possible.

Introduction & Method: Refer to the guide in your report.
Results: All relevant observations and calculations (i.e. all your results) written
in your own words. Remember that tables have headings and graphs/figures
have subscripts.
Discussion: Summarise the results and discuss it with regards to the
introduction and literature background of the experiment.
Conclusion: Come to a conclusion based on the findings and the discussion
of the experiment.
Bibliography: Reference to literature used, i.e. where did you get the
information from?
There are different styles of references—the University prefers the Harvard
style. The NWU reference guide can be found at
http://library.nwu.ac.za/sites/library.nwu.ac.za/files/files/documents/quoting-
sources.pdf.

i
• Refer to your practical guide as follows:
Fourie CMT. 2021. Practical guide for Physiology. 16th ed. Potchefstroom:
School for Physiology, Nutrition and Consumer Science, North-West University.
pp 67-73.

NB: The page number at the end of the reference are the pages in the practical
guide where the specific experiment is described. Make sure that this is correct.

• For references to the method and apparatus in the text, the reference
method is as follows:
The method used in this experiment can be found in the practical guide
(Fourie, 2021: 67-73).

• Refer to a book as follows:

Widmaier, E.P., Raff, H. & Strang, K.T. 2011. Vander’s human physiology:
The mechanisms of body function. 11th ed. New York: McGraw Hill. 770 p.

• Refer to an article as follows:

Van Rooyen JM, et al. 2002. Cardiovascular reactivity in black South-African
males of different age groups: The Influence of Urbanisation. Ethnicity and
disease, 12:69-75.

• Refer to the internet as follows:

Taher A.T., Weatherall D.J. & Cappanelli M.D. 2017. Thalassaemia.
http://www.thelancet.com/clinical/diseases/thalassaemia. Date of access: 5
February 2021.

b) Acquisition of skills. E.g. communication skills, computer skills, the use of

laboratory equipment.
c) To understand abstract concepts by forming mental images thereof, e.g.
diffusion.
d) To enhance the ability to utilise cognitive skills like creative thinking,
interpretation, judgement, and problem-solving skills.
e) Creating small groups of students and the greater interaction with fellow
students help to strengthen the link between theory and practice. The practical

ii
lessons are focused on physiological processes; the practical work supports
the theory.

4. Timetable for practical lessons

You have to complete all the experiments on the time table and hand in
all the reports on time. Therefore, you must attend all practical lessons. If
you have a valid excuse for missing a practical (e.g. illness or family death), you
must notify with Mr Zaakir within seven [7] days of missing the practical lesson
(Building F12, Office G18; Tel 018 299 2789; email 24222704@nwu.ac.za).
Excuses offered more than seven days after missing a practical lesson
will not be accepted. Remember you still have to arrange for another
practical to attend.

5. Composition of module marks

As set out in the study guide

Practical mark composition for all first year Physiology modules

Practical reports: 60%
Practical tests: 30% 50%
Results: 10% 100% Practical Final
Practical assessment: 50%

6. You are expected to:

• Attend practical classes according to your time tables and groups and to
sign your attendance.
• Hand in a valid excuse to Mrs tina Scholtz beforehand, should you not be
able to attend a practical lesson. If possible, alternative arrangements will
be made for you to catch up on that lesson. In the case of illness, the original
valid doctor’s certificate must be shown to Mr Zaakir within seven days
of your absence.
• Prepare thoroughly for each experiment. At the beginning of each practical
lesson a test will be written.
• Do experiments under guidance of a demonstrator.
• Check your results with the demonstrator.

iii
• Tidy up your area after completion of the experiment.
• Hand in your report on time in the correct box as indicated on the time table
(you will receive your marked report back in the time scheduled on the
practical time table.)
• Make a cover page for your report, furnished with your name, student
number, module code (e.g. FLGX113) and title of the experiment.

Thus, for each practical lesson, you must sign attendance, write the test, show
your results, and write and submit your report for each experiment (practical
lesson). If you did not attend the practical lesson and you hand in a report,
you will not receive a mark for that practical lesson.

7. Marking of reports
• Demonstrators mark reports according to a memorandum.
• If a report is up to a week late without a valid excuse, 20 % of the
report’s marks will be deducted. If your report is more than one week late
without a valid excuse, 40% will be deducted.
• Reports submitted to a wrong box will also lead to a 20 % deduction.
• Every student must write his or her own report. If a report or part of a
report is copied from any source, including other students, it will be
seen as plagiarism. Further actions will be taken.
• It is your responsibility to collect your reports during the scheduled time on
the practical time table and to sort out any problems with the demonstrators
or responsible lecturers. After this opportunity the demonstrators will no
longer be available for questions regarding the practical lessons. It is also
your responsibility to keep your report until you have received your marks
for the module. If any questions do arise, you must be able to show your
marked report.

8. Evaluation
At the end of the module’s practical lessons you will write a practical
assessment which, together with the reports, gives you your final practical
mark. Only one practical assessment is scheduled, and you need a valid

iv
excuse to arrange for another opportunity to write the test. Make these
arrangements as soon as possible.

9. Informed consent for procedures during practical lessons

• You are expected to sign an informed consent form for participation in
all the practical experiment procedures.
• Participation in the practical procedures is voluntary and may be
withdrawn at any time without any negative consequences. Note that this
does not excuse you from attending the practical lesson.
• In order to build a participation mark you have to attend the
practical lessons, write the test and hand in the report, even if you
do not participate in the practical procedures.
• The practical procedures have ethical clearance from the Human
Research Ethics Committee (HREC) of the NWU (NWU-00092-15-A1).

v
 SAFETY GUIDELINES FOR PHYSIOLOGY PRACTICAL LESSONS

Most of the reagents in the physiology laboratory are dangerous or potentially

dangerous. Injuries to yourself or your fellow students can be avoided by
meticulously following prescribed guidelines.

• The safety equipment, which is compulsory, is shown with each experiment

with the following signs:
Eye protection

Gloves

Laboratory coat

Shoes (no open toes)

Be sure that you bring the necessary safety equipment to the practical
lessons.
• Put all bags at the front or back of the laboratory to ensure that that it
is safe to walk in the laboratory between the benches.
• All broken glassware must be disposed of in containers marked broken
glassware immediately. These containers are for glassware only.
• All lancets and injection needles used during practical lessons must be
disposed of in containers marked sharp medical refuse. Papers or cotton soiled
by blood must be disposed of in containers marked medical refuse.
• Treat all chemicals as poisonous. Do not consume any chemicals. Do not
eat or drink in the laboratory. Do not unnecessarily carry reagents around.
If at all possible, try not to move reagent containers. Use a pipette and do not
apply your mouth to a pipette.
• Make sure all reagent bottles are firmly closed when not in use.

vi
• Wash your hands before leaving the laboratory.
• Report any spilt chemicals to a demonstrator. It must be cleaned in a safe
manner.
• Immediately wash reagents from yourself and your clothes by using the
emergency showers.
• Should you get any chemicals in your mouth, thoroughly rinse it with water
and inform a demonstrator. In case you get chemicals in your eyes, the eye
wash bottles must be used, which are available in the laboratory.
• Thoroughly clean all glassware so that no chemicals are forwarded to the
next practical session.
• Keep your work area clean and tidy—it avoids mistakes and keeps your
practical safe and accurate.
• Do not use any equipment if you do not know how to use it. If you are
unsure, ask a demonstrator.
• Handle warm glassware with the tongs designed for it.
• Wear the necessary protective gear when working with dangerous
substances. Gloves must always be worn when working with any tissue liquid
(blood, plasma, serum, urine).

Laboratory safety measures for practical lessons where human blood is

used

When working with blood there is always a risk of transmitting infections from
one person to another. By adhering to the following laboratory safety measures
this risk can be eliminated:
• There is no safety risk for you to prick your finger and use your own blood
for the practical experiments. To cause trouble, microbes must enter your body
tissues, which require crossing an epithelium—for instance the skin, which is
the body’s first line of defence against infections and disease. The skin has
multiple layers and a keratin coating – these barriers provide very effective
protection for underlying tissues. When a person’s skin is chapped, or is
affected by dermatitis and is exposed to large amounts of contaminated blood
for a prolonged time, transmission of microbes may occur. During the practical

vii
lessons you use your own blood and the amounts of blood used are very
small. Cleaning your finger with alcohol prevents any microbes on your skin to
enter your body. Always wear surgical gloves (provided in the laboratory)
if you help somebody during a blood experiment.
• The lancets used during the practical lessons must be thrown in the
containers marked sharp medical waste after use. They are designed to retract
the needle immediately after use; there is therefore no risk of an accidental
pricking with a contaminated lancet. Remember, however, that the risk of HIV
infection after being pricked by an HIV-contaminated hollow-bore needle is
approximately 0.37%. The risk of infection is lower after a prick from a solid
needle. Hepatitis B is transmitted in the same way as HIV, but the risk of
transmission is higher (20–40%). Most people recover from Hepatitis B within
six months and Hepatitis B is preventable through vaccination.
• Infections like HIV can be transmitted if a person is cut by broken glass
containing contaminated blood. Be very careful with the glassware and throw
broken glass apparatus immediately in the containers provided in the
laboratory.
• Although the risk is very low (0.1% or less), infections like HIV can be
transmitted if contaminated blood accidentally splashes into the eyes or mouth.
The amount of blood being used during the practical lessons is very little, but to
be absolutely safe be careful not to splash your blood droplets. If blood does
splash or spill on the workbench, use the laboratory paper and 70 % alcohol
provided in the laboratory to clean the area.
• The HI virus is extremely fragile and dies if it is no longer in body fluid,
especially when it is exposed to oxygen, heat and dryness in the atmosphere.
There is no risk of contracting the virus from somebody else’s blood smear.
• Use basic hygiene principles in the laboratory to prevent transmission of any
infections or diseases: wash all the apparatus with water immediately after use,
keep your working area clean and wash your hands with the antiseptic soap
provided in the laboratory.

viii
 GOOD LABORATORY PRACTICE FOR PHYSIOLOGY

➢ Protective equipment
Laboratory coats must be worn in laboratories at all times.
No student will be allowed in the laboratory without a laboratory coat.
Always wear gloves when you are working with body fluids (such as blood) in a
laboratory. Gloves will be provided in the laboratory.

➢ Biological material
When working with body fluids (like blood, urine, saliva, etc.) in a laboratory,
surgical gloves must be worn at all times.
All material infected with body fluids (like cotton and laboratory paper) must be
discarded immediately in the medical waste bins.

➢ Eating and drinking

Eating, drinking, chewing (including chewing gum and sweets) and smoking are
strictly forbidden in all laboratories. No food or water bottles will be allowed in
the laboratories.

➢ Hygiene
Mouth pipetting is forbidden under all circumstances.
Pipette pumps will be provided, and must always be used. Wash your hands
frequently and always before leaving the laboratory. Keep your working area
clean and tidy—there must be sufficient bench space to allow safe working
procedures.

➢ Sharp objects
All lancets, needles and other sharp items must be discarded immediately in the
sharp medical waste bins provided, and should not be left on the bench.

➢ Broken glass
All broken glassware must be disposed of in containers marked broken
glassware immediately. These containers are for glassware only.

ix
 BLOOD: IMMUNITY AND BLOOD TYPES

The various components of blood serve different physiological functions. The

plasma or fluid portion of the blood provides the major means for distributing
chemicals between organs. For example, it transports food molecules absorbed
through the small intestine and hormones secreted by the endocrine glands.
The plasma also helps to eliminate metabolic waste products by carrying them
to the liver (for excretion in bile) and in the case of carbon dioxide, to the lungs
(for excretion in exhaled air).

In addition to plasma, the blood also contains two major types of cells: red blood
cells (erythrocytes) and white blood cells (leukocytes). Red blood cells
contribute to the respiratory function of the blood by providing transport for
oxygen and to a lesser degree for carbon dioxide. Blood is “typed” based on
the presence or absence of specific molecules (antigens) displayed by red
blood cells (the ABO system and the Rh factor). White blood cells and their
products help to provide immunity from infection by recognising and attacking
foreign molecules and cells. The blood also contains platelets, which are
membrane-bound fragments derived from a bone marrow cell called a
megakariocyte. Platelets, together with plasma proteins, help to maintain the
integrity of blood vessels by forming blood clots.

This practical is divided into two experiments where you will determine a
differential white blood cell count and blood type.

EXPERIMENT 1: DIFFERENTIAL WHITE BLOOD CELL COUNTS

Textbook reference

▪ Martini et al. 2018: 716-724 (Figures 19-9, 19-10 & Table 19-3)
▪ Widmaier et al. 2016:644-646 (Figure 18.1 & Table 18.1)

1
▪ Hall, 2016: 455-464

Objectives

After completion of this experiment you will be able to:

▪ distinguish between the different types of leukocytes by the appearance of
their nuclei and their cytoplasm;
▪ perform a differential white blood cell count and explain the importance of
this information in the diagnosis of diseases; and
▪ explain abnormalities of total and differential white blood cell counts.

Background

Leukocytes (white blood cells)

Leukocytes include all blood cells which do not contain haemoglobin. The total
number of all these cells is known as the leukocyte count. The normal count in
adults varies between 5 000 to 9 000 cells per micro litre.

Leukocytes form in the bone marrow (granulocytes and monocytes) and in the
lymphatic tissue (lymphocytes). The most important function of the white blood
cells is to protect the body against the great variety of pathogenous micro-
organisms, toxins, bacteria and foreign material to which it is exposed daily.

Leukocytres (white blood cells) - lymphocytes, monocytes, neutrophils,

eosinophils and basophils - are all part of the immune system. Lymphocytes
provide immunity against specific antigens, whereas the other leukocytes are
phagocytic. The total white blood cell count and the relative proportion of each
type of white blood cell (differential count) change in a characteristic way in
different disease states.

Abnormalities

Leukocypenia. This is a condition where there is a drastic decrease in the

leukocyte count. It is always a serious condition because the body’s defence
mechanism is then unable to defend itself against bacterial invasion, and
extended infections can result.

2
Leukocytosis. This is a condition of increased leukocyte formation. It can be
very purposeful reaction to protect the body against bacterial invasion.

Leukaemia (blood cancer). This is a condition which has its origin in sustained
heightened leukocyte formation. These leukocytes are differentiated to the
extent that they cannot fulfil their function. The precise cause of leukaemia is
not known. There are clear indications, however, that radio-active radiation can
cause leukaemia.

Differential white blood cell count

Clinically, it is also important to determine the relative quantity (percentage) of

each leukocyte type within a population of white blood cells. This percentage is
obtained by microscopic identification (differentiation) of each leukocyte type
within a total count of 100 white blood cells.

Material

▪ Sterile lancets
▪ 70% alcohol
▪ Cotton wool
▪ Distilled H2O
▪ Immersion oil
▪ Microscope
▪ Coverslips
▪ Stain
▪ Masking tape
▪ Slide rack

Method

Each student will prepare his/her own blood smear and colour it for assessment.

Collecting blood:
▪ Wipe the fingertip with a alcohol cotton wool to disinfect it.
▪ Prick the fingertip with a lancet.
NB: Wear gloves if you assist another person with the procedure!

3
Making a blood smear (Figure 1):
▪ Apply a small drop of blood on one end of a clean and greaseless glass
slide placed flat on the laboratory bench.
▪ Lower a second glass slide at an angle of 30° to the first slide so that it is
lightly touching the first slide in front of the drop of blood (Figure 1a).
▪ Gently pull the second glass slide backwards into the drop of blood,
maintaining the pressure and angle, allowing the blood to spread out along
the edge of the second slide (Figure 1b).
▪ By still keeping the pressure and angle the same, push the second slide
across the first in a rapid, smooth motion (Figure 1c). The blood should now
be spread in a thin film across the first slide. Done correctly, the
concentration of blood in the smear should diminish toward the end,
producing a feathered appearance.

Figure 1: Method for making a blood smear.

Staining of the slide:

▪ Flood the surface of the slide carefully with a staining solution. Gently tilt the
slide back and forth for 1-3 minutes. Be careful not to wash the blood smear
off.
▪ Drip distilled water on top of the stain. Be careful not to wash the stain off.
The mixture of stain with water can be aided by gently blowing on the surface
of the stain. A proper stain is indicated in the presence of a metallic sheen
on the surface of the stain. The diluted stain should be left on the slide for
five minutes.
▪ Wash the diluted stain off the slide with distilled water.
▪ Use masking tape to mark your slide at the one end.

4
▪ Allow the slide to dry completely (for at least five minutes) at an angle.
▪ Drip a droplet immersion oil on the feather like side (lower density cells) of
the blood smear.
▪ Use the oil-immersion objective of the microscope to count the different types
of white blood cells. Start at one point in the feathered-tip (less populated)
area and systematically scan the slide until you have counted a total of 50
leukocytes. Keep a count of the number of neutrophils, eosinophils,
basophils, lymphocytes and monocytes you count and work out the
percentage of each cell type counted.

Criteria for rating a blood smear:

Rating
Very Very
Criteria Poor Adequate Good
poor good Total
1 2 3 4 5
Spreading of blood over slide;
single layer cells is important
(evenness of smear and feather
like appearance, forms a rainbow
in light)
Effective/correct colouring (metal
glow with the naked eye and
colouring of leukocytes as
observed under microscope)
Total: /10

REPORT

Answer the questions below and submit it as a report.

Questions
1. What is the percentage of the different leukocyte types of your own blood?
2. Compare it with normal values and explain any abnormalities.
3. Draw the different types of leukocytes according to their morphology.
4. Describe the different leukocytes as they are classified according to their structure.
5. Write short notes on the following:
a. Neutrophilia

5
 b. Lymphocytosis
c. Monocytosis
d. Eosinophilia
e. Basophilia

EXPERIMENT 2: BLOOD TYPES

Textbook reference

▪ Martini et al. 2018: 706-707, 712-716, 718-719 (figures 19-1, 19-6, 19-7, 19-
8 & table 19-2)
▪ Widmaier et al. 2016:669-670 (table 18.8)
▪ Hall, 2016: 477-481 (table 36.1 & 36.2)

Objectives

After completion of this experiment you will be able to:

▪ explain what is meant by the term ‘blood type’;
▪ explain how agglutination occurs and how agglutination tests can be used
to determine blood types;
▪ explain the dangers of mismatched blood types in blood transfusions; and
▪ discuss the cause of erytrhoblastosis fetalis.

Background

Erythrocytes (red blood cells) have characteristic molecules on the surfaces of

their membranes that can be different between individuals. These genetically
determined membrane molecules could function as antigens – capable of
binding to specific antibodies when exposed to plasma from another person
with a different blood type. This antigen–antibody reaction is characterised by
agglutination or clumping of red blood cells. This agglutination reaction is very
important in determining the safety of transfusions (agglutinated cells can block
small blood vessels). The major blood group antigens are the Rh antigen and
the antigens of the ABO system.

6
Hemolytic disease of the newborn (erythroblastosis fetalis) is characterised by
agglutination and phagocytosis of the fetus’s RBCs. Usually the mother is Rh -
and the father Rh+.

▪ During pregnancy fetal RBC can move across the placenta and may enter
the mother’s blood circulation.
▪ If the baby is also Rh- (Rh+ mother and Rh- baby), there is no problem.
▪ However, when a Rh- mother delivers her first Rh+ child, fetal and maternal
blood mix when the placenta breaks down.
▪ During delivery, the presence of Rh+ blood cells in the maternal blood stream
sensitises the mother, stimulating the mother’s immune system to produce
anti-Rh antibodies.
▪ Problems seldom develop during a first pregnancy, as the mother’s immune
system is not stimulated to produce enough antibodies till after the delivery.
▪ Future pregnancies (Rh- mother, Rh+ baby) may however be a problem – the
mother has anti-Rh antibodies that can cross the placenta, attack the fetal
RBC’s and cause haemolysis.
▪ This condition causes anaemia, even death of the newborn baby, and is
known as haemolytic disease of the newborn.
▪ With anaemia, the fetal RBCs may leave the bone marrow and enter the
blood stream before completing their development. Immature RBCs are
called erythroblasts, thus this condition is also known as erythrobalstosis
fetalis.
▪ If this baby survives, this baby is born anaemic and with a high concentration
of bilirubin (jaundice). The baby then needs a complete blood transfusion to
remove the antibodies produced by the mother. Blood transfusions may also
be given before birth if vital and delivery can be moved earlier to save the
baby.
▪ Production of antibodies by the mother can be prevented by injecting the Rh -
mother with human gamma globulin (Rho-Gam) within 72 hours after the
delivery. This prevents the synthesis of antibodies by the mother.

7
In this experiment you will use the principle of agglutination to determine your
own blood type. Further you will evaluate the blood types of other students in
the class to determine the prevalence of the different blood groups.

Material

▪ 70% alcohol
▪ Sterile lancets
▪ Microscope
▪ Microscope slides
▪ Antiserum A, B and D

Method

▪ Place a drop of antiserum A, B and D a small distance apart from each other
on a microscope slide.
▪ Obtain a drop of blood from your finger as previously described.
▪ Add a small amount of blood to each drop of antiserum. Remember to wipe
your finger after each drop has been applied to avoid contamination.
▪ Tilt the slide carefully to mix antiserum and blood.
▪ Examine each drop for agglutination (thread-like or clumped appearance).
If no visible agglutination is observed after 2 minutes, examine the slide
under low magnification of the microscope.

Questions

Answer the following questions:

1. What is your blood type?

2. Obtain the blood types of all the other students in the class and calculate
the percentage prevalence of each blood type. Compare it with the normal
prevalence as noted in Table 1 on the next page.
3. Discuss transfusion reactions resulting from mismatched blood types in
blood transfusions.
4. Give a full explanation of what erythroblastosis fetalis means and the
physiological cause thereof.

8
Table 1: Distribution of blood groups in South Africa.
ABO group Rh+ Rh- Total
O 38% 7% 45%
A 34% 6% 40%
B 9% 2% 11%
AB 3% 1% 4%
Total 84% 16% 100%

9
 INFLUENCE OF DIFFERENT NUTRIENTS ON BLOOD GLUCOSE
(GLYCAEMIC INDEX)

Aim
To determine the influence of different nutrients on the blood glucose
concentrations.

Introduction
The body needs nutrients and these are absorbed by the digestive tract from
the food we eat. As the level of activity of the cells, tissues and organs change,
the need for nutrients also changes. These changes vary from moment to
moment (active or inactive), hour to hour (sleep or awake) and from year to year
(child and adult).

Food is chemically and mechanically digested and the sugars, amino acids and
lipids are absorbed by the digestive tract and transported by the blood stream
to the cells of the body. Carbohydrates such as sugars and starches must be
broken down into smaller molecules. They are an important source of energy
and almost immediately available. Some of the carbohydrates are already
absorbed by the mucus membranes that line the oral cavity and transported to
the blood stream. Monosaccharides (glucose, galactose and fructose) are
absorbed into the capillary blood in the villi and transported to the liver via the
hepatic portal vein. Lipids (triglycerides) are broken down into fatty acids and
glycerol. Each villus contains a small lymphatic vessel called a lacteal. Fatty
acids and glycerol are coated with proteins and this creates a soluble complex
known as chylomicrons. They are absorbed into the lacteal of the villi and
transported to the systemic circulation via lymph in the thoracic duct. Glycerol
and short-chain fatty acids are absorbed into the capillary blood in the villi and
transported to the liver via the hepatic portal vein. Proteins have very complex
structures, so protein digestion is both complex and time-consuming. The plant

10
cell walls and the connective tissues in animal products have to be broken down
before the enzymes can break the different proteins down into their individual
amino acids. The amino acids are absorbed into the capillary blood in the villi
and transported to the liver via the hepatic portal vein. In the liver, glucose is
stored as glycogen and the liver regulates the blood glucose levels.

Glucose is the most important form in which carbohydrates are transported

through the blood to the various body cells. Glucose is the primary active energy
source of the cells (and also the brain) and for this reason the blood glucose
concentration should be stabilised within reasonably narrow confines. The
hormone insulin is necessary to facilitate the movement of glucose from the
blood into the cells, thus lowering the blood glucose concentration. The
hormone glucagon results in an increased glucose production in the liver, which
dumps glucose in the blood and restores blood glucose concentrations. In the
body cells, energy is extracted from glucose and used in the anabolism of other
nutrients such as glycogen and glycoproteins, or converted into fats
(triacylglycerol). When blood glucose concentrations rise too high
(hyperglycaemia), too much insulin is secreted, which causes blood glucose
concentrations to fall too quickly and too low (hypoglycaemia). Hypoglycaemia
is a dangerous condition for the body, because it means that the brain (which
normally only metabolises glucose for energy) and nerves do not receive
enough glucose.

Normal fasting blood glucose concentrations vary between 3.0 mmol/ℓ and 5.5
mmol/ℓ, and below these values people begin to exhibit symptoms of fatigue,
hunger, trembling, headache, irritation and lack of concentration. When food is
ingested after a period of fasting, the blood glucose concentration will begin to
rise to about 8-9 mmol/ℓ in one hour.

The glycaemic index measures the effects of equal quantities of different

carbohydrates on blood glucose levels. The response of 50 g carbohydrates is
given as a % of the response of 50 g carbohydrates of a standard food (glucose/
white bread – glucose in this experiment). Factors that influence the glycaemic
index are:

11
• Rate of ingestion – higher frequency and smaller amounts lower the
glycaemic index.
• Refinement of the food.
• Size of the grain.
• Cooking and processing.
• Fats and protein.

Refined and processed carbohydrates such as those found in glucose, coke,

cookies, crackers and white bread have a high glycaemic index and their easy
digestion causes rapid elevation in blood glucose and insulin levels. After about
two hours the blood glucose levels decline to more or less the fasting blood
glucose levels. Vegetables and high-fibre whole grains, which have a low
glycaemic index, are slowly absorbed and cause a smaller elevation in blood
glucose levels. Low glycaemic index foods therefore result in stable blood
glucose levels and contribute to overall health. Therefore, judging
carbohydrates by their energy density alone can be misleading.

Apparatus
A glucometer to determine blood glucose.
An automatic lancet with sterile lancets.

Reagents
20% glucose solution (GI – 97) or Coke.
Coke lite.
White bread (GI – 70) with jam or syrup.
Brown bread (GI – 69) with margarine and cheese (GI lowered with fats and
protein).
Test strips.
Alcohol, cotton wool and paper towels.

12
Method
Precautions
1. Test subjects must fast for at least 12 hours before the experiment is
done. During this period only water may be taken. If the fast is not rigorous,
the results of the experiment will be noticeably affected.
2. Although the experiment is best conducted in the morning, it can also be
done in the afternoon. Breakfast must be taken before 08:00 and fat and
protein intake must be limited so that food can move rapidly through the
digestive tract and glucose levels can return to basal levels.
3. The test subjects must not smoke during the procedure and must remain
relaxed and calm throughout.

Procedure
1. Switch the glucometer on by inserting the test strip into the meter with the
arrow in the direction of the meter. The meter automatically displays the
code number. If the code number on the meter and the code number on the
test strip bottle do not match, press the arrows on the meter until the code
numbers match and then press OK.
1. Blood is drawn by using a lancet to prick a fingertip, which must be
disinfected with alcohol beforehand.
2. NB: Wear gloves if you assist another test subject with the procedure.
3. Keep the end of the test strip (which is in the meter) against the drop of
blood. The strip automatically draws enough blood.
4. The reading will show on the screen.
5. Measure the fasting blood glucose concentration.
6. The test subjects are divided into four groups. The carbohydrate intake of
the groups is as follows:
Group 1 – 1 g of glucose per kg body mass (250 ml 20% glucose solution
/ person of 50 kg) or Coke (10% sucrose solution) 5 ml / kg body mass.
Group 2 – 1 g of carbohydrates (white bread) per kg body mass (± 70 g /
person of 50 kg) with jam or syrup.
Group 3 – 1 g of carbohydrates (brown bread) per kg body mass (± 70 g /
person of 50 kg) with margarine and cheese or peanut butter.
Group 4 - Coke Lite (no glucose/sucrose) 5 ml / kg body mass.

13
7. The carbohydrates must be taken immediately after the fasting blood
glucose measurements.
8. Measure the blood glucose concentrations at precisely 15, 30, 45, 60, 90
and 120 minutes after ingestion of glucose or carbohydrates.

Assignment
1. The results of the four groups must be tabulated. Draw a graph of the results
by plotting the time in minutes on the X axis and the blood glucose
concentrations in mmol/ℓ on the Y axis. All three groups’ results must be
plotted on one graph.
2. Give a discussion to explain the differences in blood glucose observed for
the different interventions.

14
 Where applicable

THE CHLORIDE BICARBONATE SHIFT

Carbon dioxide formed through cellular metabolism in cells diffuses from cells
into blood, where it participates in several chemical reactions, contributing to
the transport of carbon dioxide to the lungs to be excreted (exhaled).

In a resting state, 10% of carbon dioxide is transported (in blood) in a dissolved

state, 30% bound to haemoglobin (carboxyhaemoglobin – HHbCO2) and 60%
as bicarbonate ions.

Carbon dioxide from cells dissolves in blood plasma and diffuses into red blood
cells where most of it reacts with water to form carbonic acid:
carbonic anhydrase

CO2 + H2O  H2CO3

Carbonic acid is converted to hydrogen and bicarbonate ions through the
presence of the enzyme carbonic anhydrase (accelerate reaction 5000 times):
H2CO3 H+ + HCO3-
In absence of this enzyme, only 0.1% of carbonic acid will be converted to
hydrogen and bicarbonate ions. The formed hydrogen ions combine with
haemoglobin (pH buffering effect of haemoglobin), while bicarbonate ions move
from a high concentration in the red blood cell to the blood plasma via a carrier
protein. To counteract the misdistribution of negative charges, chlorine ions
(most abundant negative ions in blood plasma) are transported via a carrier
protein into the red blood cell. The net effect is an increase in the bicarbonate
ion and a decrease in the chloride ion concentration of the plasma.

With gas exchange in the lungs, the opposite occurs (see Figure 1). Oxygen
drives carbon dioxide and hydrogen ions from haemoglobin. As a result the
hydrogen ion concentration increases in the red blood cell. Bicarbonate ions
are transported back into the red blood cell, while chloride ions are transported

15
to the plasma. Bicarbonate ions combine with hydrogen ions and carbonic
anhydrase catalyses the formation of water and carbon dioxide to be exhaled.

In this experiment the conditions to which blood is exposed in the tissues and
lungs are simulated by bubbling carbon dioxide and oxygen through blood,
terminating in the determination of chloride concentration by titration.

plasma alveolar air

# carbonic anhydrase

HHbCO2
CO2 CO2 CO2
H2CO3 #
H2O H2O

HHb H+ HCO3– HCO3–

Cl– Cl–
HbO2 Hb–
O2 O2 O2

Figure 1: Chloride bicarbonate shift in the lungs

Objectives
After completion of the practical experiment you will be able to:
▪ Explain the transport of oxygen and carbon dioxide in blood; and
▪ calculate the shift of chloride across erythrocyte membranes as a result of
gas exchange in the lungs and tissues.

16
Textbook reference
See Guyton and Hall (2011), Chapter 40: Transport of oxygen and carbon
dioxide in blood and body fluids under the following headings:
▪ Transport of oxygen in blood, pp 495-502.
▪ Transport of carbon dioxide in blood, pp 502-504.

Materials
▪ Ox or sheep blood with 2-3 mg oxalate per ml blood (oxalate prevents blood
clotting).
▪ Pure oxygen and carbon dioxide.
▪ Two conical flasks (250 ml).
▪ Four centrifuge tubes.
▪ Table centrifuge.
▪ Mineral oil.
▪ Pasteur pipettes.
▪ Pipettes (1. 2. 7 and 15 ml).
▪ Burette.
▪ Glass beaker (50 ml).
▪ 0.66 M sulphuric acid.
▪ 10% sodium tungstate solution.
▪ 0.005 M mercury nitrate solution.
▪ 0.5% diphenylcarbazon indicator.

Method
▪ Pipette 15 ml of oxalate blood into a 250 ml conical flask (2x).
▪ Allow carbon dioxide to bubble through the blood in one flask for five
minutes and oxygen through the blood in the other flask for the same time.
▪ Immediately thereafter pour each sample of blood in a centrifuge tube and
cover the blood with a thin layer of mineral oil (prevent oxygen/carbon
dioxide from escaping from blood).
▪ Centrifuge both tubes for 25 minutes at maximum rpm. of the table
centrifuge.

17
▪ After centrifugation you will notice three layers in each centrifuge tube,
namely a thin mineral oil layer at the top, an almost colourless middle layer
(plasma) and a red coloured bottom layer (blood cells).
▪ Separate the blood plasma of both centrifuge tubes by means of a Pasteur
pipette.
▪ Treat each plasma sample in the following way:
1. Add 1 ml plasma to a centrifuge tube.
2. Add 7 ml of distilled water.
3. Add 1 ml of the 10% sodium tungstate solution.
4. Add 1 ml of the 0.66 M sulphuric acid.
▪ Mix the contents of the centrifuge tube thoroughly (if mixed correctly it
should have a milky appearance as a result of precipitation of all the plasma
proteins).
▪ Centrifuge both centrifuge tubes for five minutes at maximum rpm. of the
table centrifuge.
▪ After centrifugation you will notice two layers in each centrifuge tube. The
top layer is protein-free supernatant.
▪ Pipette 2 ml of protein-free supernatant in a 50 ml glass beaker.
▪ Add 0.5 ml of diphenylcarbazon indicator to the glass beaker.
▪ Titrate the contents of the glass beaker with the 0.005 M mercury nitrate in
the burette. The colour change will be from colourless to a permanent
bluish purple.
▪ Repeat the titration for each blood sample three times to obtain a mean
titration value. Use this mean value to calculate the amount of chloride that
moves across the red blood cell membrane in 100 ml blood as a result of
oxygen uptake by using the following calculation:

Titration reaction: 2NaCl + Hg(NO3)2→ 2NaNO3 + HgCl2

To calculate the concentration of Hg(NO3)2 that is titrated with:

1 M Hg(NO3)2 contains 324.6 mg Hg(NO3)2/ml,
but 0.005 M Hg(NO3)2 is used,
means the concentration of Hg(NO3)2 used is 324.6 x 0.005 = 1.623 mg/ml.

18
To calculate the amount of chloride in the plasma:
From the titration reaction, 324.6 mg Hg(NO3)2 will react with 117 mg (58.5 mg
× 2) NaCl. The amount of chloride in the titration reaction is therefore = (117
mg × X × 1.623 mg/ml)/324.6 mg, where X = the mean titration difference
between oxygen and carbon dioxide blood.
Because of diluting the plasma, the answer is Y mg chloride in 0.2 ml plasma.
If the haematocrit of blood is 45%, it means that 55% is plasma.
If 0.2 ml plasma contains Y mg chloride, then 55 ml of plasma (100 ml blood)
contains Y mg x 55ml /0.2 ml chloride.

Questions
1. Calculate the amount of chloride ions that moves across the red blood cell
membrane in 100 ml blood as a result of oxygen uptake. Discuss your
answer.
2. Why is it important that carbonic anhydrase catalyses the reaction in both
directions?
3. Briefly explain the transport of oxygen in blood.

19
 THE ENDOCRINE FUNCTION OF THE PANCREAS

Objective
When you have completed this assignment you should be able to determine
the glucose tolerance of a participant, draw a glucose toleration curve and
observe the functioning of insulin and glucagon from it.

Blood glucose concentrations

The pancreas consists of an exocrine and endocrine glandular section. The
pancreas acini constitute the exocrinal section and the secretion originates from
a duct in the small intestine. The Islets of Langerhans constitute the endocrinal
part and histologically appear like islands among the acini. The endocrine
(ductless) glands secrete hormones directly into the blood stream which then
transports them to the target organs where they perform their function. The
island tissue consists mainly of alpha and beta cells, where the alpha cells
secrete glucagon and the beta cells secrete insulin.

Glucose is normally the most important factor that controls insulin secretion,
although amino acids and fatty acids also influence insulin secretion.
Hypoglycaemia stimulates the secretion of glucagon, which can be regarded as
the insulin antagonist. Glucose is the primary active energy source of the cells,
and for this reason the blood glucose concentration should be stabilised within
reasonably narrow confines.

The carbohydrates (polysaccharides: starch and sugar), which occur in the diet,
are broken down in the digestive system into monosaccharides (glucose,
fructose and galactose). Absorbed galactose is changed to glucose in the body
and fructose is metabolised like glucose. Glucose is therefore the most
important form in which carbohydrates are transported through the blood to the
various body cells. In the body cells, energy is extracted from glucose and used

20
in the anabolism of other nutrients such as glycogen and glycoproteins, or
converted into fats (triglycerides).

Glucose has to be transported across the cell membrane for these processes
to occur in the cell. Because glucose is a large fat-insoluble molecule, it has to
be carried across the cell membrane by glucose carriers. These carriers are
stored in fat and muscle cells in an intracellular membrane pool and move to
the surface of the cells as soon as the hormone insulin has bound to its
membrane receptor. Insulin is therefore necessary to facilitate the movement
of glucose from the blood into the cells. The beta cells secrete insulin in the
blood as soon as the blood glucose concentration starts to rise after a meal.
When blood glucose concentrations rise too high (hyperglycaemia), too much
insulin is secreted, which causes blood glucose concentrations to fall too
quickly and too low (hypoglycaemia). Hypoglycaemia is a dangerous condition
for the body, because it means that the brain (which normally metabolises
glucose for energy at a rate of about six grams per hour) and nerves do not
receive enough glucose for energy. This might lead to irreversible brain damage
and even death. Hypoglycaemia stimulates sympathetic activity and the release
of the hormones adrenalin, glucagon, growth hormone and glucocorticoids. The
result of this is an increased production of glucose in the liver, which deposits
glucose in the blood and restores blood glucose concentrations.

Normal fasting blood glucose concentrations vary between 3.0 mmol/ℓ and 5.5
mmol/ℓ. Below these values people begin to exhibit symptoms of fatigue,
hunger, trembling, lack of concentration and others. Values below 1.9 mmol/ℓ
are life threatening. After a period of fasting, the blood glucose concentration
should be between 3.0 mmol/ℓ and 5.5 mmol/ℓ. When food is then ingested,
the blood glucose concentration will begin to rise to about 8-9 mmol/ℓ within one
hour, however, normally no higher than 11.1 mmol/ℓ. The latter value is the
threshold value of the kidneys for the reabsorption of glucose. We call this
increase in blood glucose concentration the hyperglycaemic response. After
about two hours, the fasting value is once again reached. As a result of an over-
secretion of insulin, the blood glucose concentration will fall slightly below the
normal fasting value. This is called the hypoglycaemic response. The pancreas

21
now secretes glucagon. This glucagon causes mainly liver glycogen to be
converted into glucose and to be released into the blood stream. The blood
glucose concentration then returns to the normal fasting value.

When we plot the blood glucose concentration on the Y-axis and the time on
the X-axis, we obtain a glucose tolerance curve.

Abnormalities
Hypo-insulinism: diabetes mellitus
Hypo-insulinism, also called relative insulin deficiency, causes a clinical
syndrome which is called diabetes mellitus. As a result of an insulin deficiency,
the blood glucose rises and glucose is excreted in the urine because the
glucose threshold (11.1 mmol/ℓ) of the kidney is exceeded. This condition is
called glycosuria. Polyuria also develops because of the loss of osmotically
active glucose molecules (osmotic diuresis). Continuous heightened blood
glucose leads to microvascular complications, hypotension, ketoacidosis,
decreased protein synthesis and net calcium and sodium loss.

When we carry out the glucose tolerance test on persons with hypo-insulinism,
we usually find a much higher fasting value, a sharp increase in blood glucose
concentration. We also find that glycosuria occurs, because blood glucose rises
above the kidney’s threshold value and the blood glucose takes longer to return
to normal.

Hyperinsulinism
Hypoglycaemia (because of too much insulin) can be the result of excessive
insulin secretion by the  cells. As the brain is dependent on glucose for its
energy needs, hypoglycaemia is a serious condition. In this case the fasting
glucose values are very low; they do not rise sharply during glucose intake and
return to fasting values fairly quickly.

22
Apparatus
A glucometer to determine blood glucose.
An automatic lancet with sterile needles.

Reagents

20% glucose solution.

Test strips.
Alcohol.
Cotton wool.
Paper towel.

Method
Precautions

1. Test subjects must fast for 12 hours before the experiment. During this
period, only water may be ingested.

NB: Adherence to the above instructions is essential for the success of the
experiment. If the participants do not fast completely, it will be noticed in the
results.

Although the experiment is best conducted in the morning, it can also be

done in the afternoon with certain adjustments:

Breakfast must be taken before 8:00. Fat and protein intake must be limited
so that the food can move rapidly through the digestive tract and blood
glucose levels can return to basal values. This can be achieved by eating
one of the following standardised breakfasts:

(a) 2 slices of brown or whole-wheat bread or 10 Provitas (eaten with 1

teaspoonful of spread/margarine, 1 teaspoonful of jam and 15 g of
cheese or 1 wedge of cheese.
Half a glass of milk
1 apple

(b) 1 Weetbix or 1 cup of All Bran/maize porridge/oats (eaten with ½ cup of

milk and 2 teaspoonful of sugar)

23
 1 glass of milk
1 apple

(c) 2 medium-sized brown or buttermilk rusks

1 glass of milk
1 apple

2. The test subject may not smoke during the tolerance test, must remain
relaxed and calm throughout and may not eat or drink.

Procedure
2. Switch the glucometer on by inserting the test strip into the meter (with the
arrow in the direction of the meter). The meter automatically displays the
code number. If the code number on the meter and the code number on the
test strip bottle do not match, press the arrows on the meter until the code
numbers match and then press OK.
3. Blood is collected from the fingertip, which must be disinfected with alcohol
beforehand, by pricking it with a lancet.
NB: Wear gloves if you assist another test subject with the procedure!
4. As soon as there is a drop of blood on the fingertip, hold the other end of
the test strip (which is still in the meter) against the drop of blood. The strip
automatically draws enough blood through capillary force. Do not place the
drop of blood on top of the strip.
5. A reading will show on the screen. Do not remove the test strip before the
reading appeared.
6. Note the fasting blood glucose concentration.
7. A glucose solution (1 g glucose per kg body mass, thus 5X body mass) is
taken directly after the fasting blood sample was taken (at time 0). A
person that weights 50 kg will, for example, ingest 5 x 50 = 250 ml
glucose.

8. As soon as the glucose solution has been drunk, the time is taken.
Thereafter the blood glucose concentration is measured at precisely 15,
30. 45, 60. 90 and 120 minutes (as described in steps 1-5).

24
Assignment

The results must be tabulated neatly.

Display it graphically by plotting the time in minutes on the X-axis and the blood
glucose concentrations in mmol/ℓ on the Y-axis.

Compare your glucose tolerance curve with that of a normal curve.

Questions

1. Define hyperglycaemia and name two negative effects thereof.

2. Define hypoglycaemia and mention one negative effect, as well as four

symptoms thereof.

3. On another graph, plot the respective glucose tolerance curves obtained for
an untreated diabetes mellitus patient and a patient suffering from
hyperinsulinism. How do these curves differ from the normal curve?

25
 Where applicable

THE QUANTITATIVE EVALUATION OF THE HAEMOLYTIC ACTIVITY OF

A SAPONIN

Saponins are steroid or triterpenoid glycosides that are generally found in a

large number of plants and plant products. In plants they perform a protective
or “immunological” function as antimicrobial compounds (destroying micro-
organisms that attempt to infect the plant). Saponins’ name originated from its
ability to form stabile, soapy foams in watery solutions. Traditionally saponins
were used by natives of South America to kill fish and by Europeans as soap.

To date a wide variety of different biological functions of saponins have been

described, with the damage of erythrocyte membranes (haemolysis) being the
most well-known. The mechanism through which saponins cause haemolysis
may be described as follows: Saponins have a high affinity for cholesterol that
occurs with phospholipids in cell membranes. When saponins come into
contact with cholesterol in cell membranes, an insoluble and irreversible bond
is formed between the saponin and cholesterol molecule. As other saponin-
cholesterol bonds form, movement of saponin-cholesterol molecules occurs in
the level of the membrane and several saponin-cholesterol molecules
aggregate to form a complex. The cholesterol and saponin in the complex are
rearranged with the cholesterol molecules facing outward and saponin
molecules facing inward. This rearrangement gives rise to the formation of one
large central pore, with a diameter of approximately 4-5 nm, between the
saponin molecules. At the same time several identical pores are formed in the
erythrocyte membrane. Extra cellular fluid rushes into the erythrocyte through
the formed pores (due to an osmotic gradient) and with time the erythrocyte will
swell and burst open to release its intra cellular content, including haemoglobin,
in the extra cellular environment.

26
Haemoglobin is a pigment and at a wavelength of 540 nm the quantity of light
that is absorbed by haemoglobin can be measured or quantified by making use
of a spectrophotometer.

By adding different concentrations of saponin to erythrocytes, the influence of

concentration saponin on haemolysis can be investigated. This enables
drawing a cumulative dose-response curve (y-axis: biological effect and x-axis:
concentration) and determining the HD50 concentration of the saponin. The
HD50 concentration is the concentration saponin that is required to cause 50%
haemolysis. The HD50 concentration makes it possible to compare the toxicity
of different saponins with each other. The smaller the HD50 concentration, the
more toxic the saponin is considered to be.

Aim:
During this practical you will quantify the haemolytic activity of a saponin
isolated from the plant Gypsophyla paniculata.

Material and Methods:

You will not collect blood and obtain erythrocytes, as described below, on your
own. The product of this (x% erythrocyte solution in 0.9% NaCl) will be made
available to you to determine the haemolytic activity of a saponin.

Collection of blood and obtaining of erythrocytes:

▪ The blood is collected from the abattoir.
▪ Gently transfer approximately 13 ml blood to a centrifugable test tube and
centrifuged for five minutes at 2000 rpm.
▪ Blood plasma is removed with a Pasteur pipette/micro pipette.
▪ Approximately 7 ml 0.9% NaCl solution is added to the test tube, followed
by five minutes of centrifuging at 2000 rpm.
▪ The last two steps are repeated twice (to remove plasma, plasma proteins
and leukocytes).
▪ The obtained pellet only contains erythrocytes.

27
▪ A 1% erythrocyte solution in 0.9% NaCl is made. 1% erythrocyte solution
= (volume erythrocytes (ml) / volume 0.9% NaCl solution (ml)) x 100 – 5
ml cells (pellet) + 450 ml 0.9% NaCl.

Haemolytic determination:
▪ Fill one test tube with 5 ml 0.9% NaCl solution (blank).
▪ Fill six test tubes with 5 ml x% erythrocyte solution and label the test tubes
with the following concentrations: 0. 20. 40. 60. 80. 100 g/ml saponin.
▪ Fill one test tube with 5 ml dH2O.
▪ Label a cuvette correspondingly for each test tube.

The following steps must follow in quick succession to guarantee usable results:
▪ First add 50 µl of the pellet to the distilled water.
▪ Add 50 l x% erythrocyte solution to the 0 g/ml test tube (negative
control) and note the time.
▪ Add 50 l of the different concentrations saponin to each of the
corresponding test tubes and note the time.
▪ Add 50 l of the erythrocyte pellet to the test tube filled with 5 ml dH2O
and note the time (positive control).
▪ Tilt test tubes carefully to mix solutions.
▪ After 20 minutes the test tubes (seven in total, except the blank) are
centrifuged for five minutes at 2000 rpm.

Spectrophotometry:
▪ Remove approximately 3 ml of the fluid (supernatant) from each of the test
tubes and place in cuvettes.
▪ Place the cuvettes in the 5 spectrophotometer and measure the
absorbancy of light at a wavelength of 540 nm.
▪ Measure the absorbancy of light of the blank.
▪ Measure and note the absorbancy of light for each of the other cuvettes.
▪ % Haemolysis = absorbance of cuvette / absorbance of dH2O x 100.

28
Questions:
1. What is a negative and positive control and what did you use in this
experiment as these controls?
2. Make use of graph paper to draw a cumulative dose-response curve of
your group’s data.
3. Make use of the curve (graph) to determine the HD50 concentration of the
saponin.
4. A saponin, named saponin 123. has a HD50 = 150 µg/ml. Is this saponin
used by you more or less haemolytic? Motivate your answer.

Reference:
▪ Francis, G., Kerem, Z., Makkar, H.P.S., Becker, K. 2002. The biological
action of saponins in animal systems: a review. British Journal of
Nutrition. 88: 587-605.

29
 Where applicable

THE MICROSCOPE AND OSMOSIS THROUGH LIVING MEMBRANES

Objective
To know the microscope as research instrument, to examine diffusion through
living membranes and to show the influence of isotonic, hypotonic and
hypertonic solutions.

A microscope is one of the most common pieces of equipment used by

scientists. It is therefore crucial that you can identify all the different parts and
know their functions.

Outcome
1. To identify all the different parts and list the function of each.
2. To be able to use the microscope effectively.

The different parts of a microscope

1. Base: The part that supports the microscope.
2. Sub-stage light: located in the base. Supplies the light that passes through
the subject from below.
3. Light control: To control the intensity of the light.
4. Condenser: Concentrate the light on the subject. The height of most
condensers can be adjusted and the best position is usually close to the
subject/stage.
5. Iris diaphragm: Located at the bottom of the condenser. Controls the
amount of light that travels through the condenser and is used to adjust the
contrast.
6. Stage: The platform on which the slide rests.
7. X, Y – adjustment knob: Allows movement of the stage.

30
8. Objective: The lenses that are attached to the nosepiece. Usually three
different lenses, a low-power lens (4 x), a medium-power lens (10 x), a high-
power lens (40 x) and an oil immersion lens (100 x).
9. Adjustable nosepiece: An adjustable ring, which carries the objective lenses
and permits the sequential positioning of these lenses over the light passing
through the hole in the stage.
10. Coarse adjustment focus knob: Used to focus the object.
11. Fine adjustment focus knob: Used for precise focusing once coarse focusing
has been completed.
12. Arm: Used to carry the microscope and supports the head of the
microscope.
13. Head: Supports the ocular/eyepiece and the nosepiece.
14. Ocular/eyepiece: Lenses at the end of the head through which observations
are made. Usually 10 x magnifications.

Rules when using the microscope

1. When carrying the microscope it should be in an upright position with one
hand on its arm and the other supporting its base.
2. Use grit-free lens paper to clean the lenses. Clean all lenses before and
after use.
3. Always begin the focusing process with the lowest power objective lens in
position.
4. Never use the coarse adjustment knob with the oil immersion or high-power
lens.
5. Always use a coverslip when working with temporary (wet-mount)
preparations.
6. Always remove the microscope slide from the microscope with the low-
power lens in position before putting the microscope away.

31
 Working of a microscope

Eye

Ocular lens

Real image

Objective lens
Object
Virtual image

Iris
diaphragm
Condenser

Light

Fig 1: The optical system of the microscope

OSMOSIS THROUGH LIVING MEMBRANES

Osmosis is the movement of water across a selectively permeable membrane

from one solution to another solution that contains a higher solute
concentration. Osmosis can only take place if the membrane is fully permeable
for water, but only partly or not at all permeable for solutes. The osmotic
pressure of a solution is an indication of the force of the water movement into
that solution as a result of its solute concentration.

The total solute concentration in an aqueous solution is the solution’s osmolarity

or osmotic concentration. Osmolarity determines the movement of water
between the extra cellular and intracellular fluid compartments of the body. The
nature of the solutes, however, is often as important as the total osmolarity. For
example, consider a solution that has the same osmolarity intracellular fluid but
a higher concentration of one or more individual ions. If any of those ions can
diffuse into the cell, the osmolarity of the intracellular fluid will increase and that

32
of the extra cellular solution will decrease. Osmosis will then occur, moving
water into the cell. The term tonicity is sometimes used instead of osmolarity.
If a solution does not cause an osmotic flow of water into or out of a cell, the
solution is called isotonic.

Blood consists of plasma (water, plasma proteins and other solutes) and formed
elements (red blood cells, white blood cells and platelets). The red blood cells
have the same osmotic pressure as plasma has. Red blood cells can therefore
be used as osmometers to determine the osmolarity of solutions, since red
blood cells will neither expand nor shrink in an isotonic solution, keeping their
normal appearance. When placed in a hypertonic (higher osmolarity) solution,
a red blood cell will shrink (a process called crenation) as a result of the net
efflux of water. When a red blood cell is placed in a hypotonic (lower osmolarity)
solution, it will expand or perhaps even burst (a process called haemolysis) as
a result of the net influx of water, extruding its haemoglobin into the solution.
Haemoglobin is a protein which is responsible for the red colour of red blood
cells and gives red blood cells the ability to transport oxygen in the blood. When
haemolysis occurs the haemoglobin will colour the solution of the cells red and
the colour change is clearly visible after centrifuging.

33
 (a) (b) (c)

a) Isotonic solution: Because the red blood cells are immersed in an isotonic
saline solution, no osmotic flow occurs and the cells appear normal.
b) Hypotonic solution: Immersion in a hypotonic saline solution results in
osmotic flow of water into the cells. The swelling may continue until the
cell membrane ruptures, or lyses.
c) Hypertonic solution: Water moves out of the cells. The red blood cells
shrivel and become crenated.

Apparatus
Microscope.
Microscope slides.
Coverslips.
Blood.
Pasteur pipette.
Test tubes.

Reagents
Isotonic solution of 0.9% NaCl (physiological solution).
Hypertonic solution of 1.5% NaCl.

34
Hypotonic solution of 0.2% NaCl.
Distilled water.

Method
A. To determine haemolysis:
1. Take four test tubes and place 5 ml of 0.2% NaCl in test tube 1. 0.9% NaCl
in test tube 2. 1.5% NaCl in test tube 3 and 5 ml distilled water in test tube
4. Mark each test tube clearly.
2. Place three drops of blood in each test tube with the different solutions.
3. Mix the blood and the solutions carefully.
4. Leave the test tubes for 30 minutes. In the meantime examine the red blood
cells under the microscope in the different solutions (method B).
5. Centrifuge the solutions in the test tubes after 30 minutes for five minutes at
2000 rpm.
6. Measure the absorbancy against distilled water with the aid of a photometer
at the 550 nm wavelength.
7. Note the results.

B. Examine the red blood cells as follows under the microscope:

1. Place a big drop of 0.9% NaCl on a slide with a Pasteur pipette.
2. Add a very small drop of animal blood to the solution on the slide.
3. Tilt the slide to mix and cover with a coverslip.
4. Immediately examine the preparation under the microscope. Note the
shape of the red blood cells in the different solutions.
5. Repeat steps 1 - 4 with 1.5%, 0.2% NaCl and with distilled water.
6. Complete method A.

Assignment
1. Draw the shapes of the red blood cells in the different solutions in your
assignment.
2. Tabulate the absorbance of the different solutions and red blood cells
and give the % of haemolysis and draw a graph of the results. 100%
haemolysis is obtained by distilled water and red blood cells.
3. Note all the results precisely and discuss them in your assignment.

35
Questions
1. Intravenous medication should be in an isotonic solution. Why?
2. Distinguish between extra and intracellular fluid.
3. Make a drawing to show the influence of hyper and hypotonic extra
cellular fluids on red blood cells.
4. Name the different parts of the microscope and the function of each part.
5. Name the rules when using the microscope.

36
 THE SENSES

Aim
The aim of this session is for students to familiarise themselves with their
senses, sensory and related phenomena and some sensory illusions.

Background
To be aware of our external environment, the body needs a communication
system. This communication system works via afferent and efferent nerve
impulses from and to the brain, which enable us to function normally and to
protect us against daily physical danger. Conventionally, there are five senses:
sight, hearing, taste, smell and touch (visual, auditory, gustatory, olfactory and
tactile senses respectively). This is clearly an oversimplification. Additional
sensory modalities include temperature, pain, vibration, joint position and
proprioception.

The materials needed for this experiment include:

Pins and paperclips.

White paper and a black, fibre-tipped pen.

Large beakers (three per station).

Hot, cold, and lukewarm water.

Some apple, raw potato, and raw onion.

Sugar (15 g/50 ml), table salt (5 g/50 ml) and citric acid (5 g/50 ml).

Some cotton buds.

A swivel chair.

Ruler or measuring tape.

Sellotape.

37
Sight
The eyes are extremely sophisticated visual instruments – more versatile and
adaptable than the most expensive cameras, yet more compact and durable.
The wall of the eye contains three distinct layers: (1) an outer fibrous layer, (2)
an intermediate vascular layer and (3) a deep inner layer. The visual receptors
or photoreceptors are located in the inner layer. (See pp 625 - 644 of Martini et
al, 2014 for more information regarding the anatomy and physiology of the eye.)

Exercise 1: Convergence of gaze (p 644 Martini et al, 2014)

Binocular vision requires that the separate images in the right and left eyes be
“fused” to give a single view. Fusion of the images of an object is possible only
if the images fall on corresponding parts of the right and left retinae. If they do
not, a double view of the object results.

1. Hold one arm outstretched, with the index finger upright and in line with
some distant object (for example, a clock on a far wall). Look at your
finger (keep it in focus), but concentrate your attention on the distant
object. Note that the distant object is seen doubled: there are two
images, side by side.
2. Cover the right eye. Note that the right image of the distant object
disappears.
3. With both eyes open, look at the distant object. Note that your finger is
seen doubled.
4. Cover the right eye. Note that the left image of your finger disappears.
5. Ask a volunteer to look at a distant object and then at an object held
close-up (15 cm from the face). Note that the volunteer’s eyes are turned
inwards when looking at a close object. Why?

Exercise 2: Accommodation (focusing) (p 635 Martini et al, 2014)

The eye can accommodate (change focus) for far or near vision by varying the
shape of the lens. The lens is held in place by suspensory ligaments that
originate at the ciliary body. Smooth muscle fibres in the ciliary body act like
sphincter muscles. When the ciliary muscle contracts, the ciliary body moves
toward the lens, thereby reducing the tension in the suspensory ligaments. The

38
lens is then pulled into a more spherical shape that increases the refractive
power of the lens. This enables it to bring light from nearby objects into focus
on the retina. When the ciliary muscle relaxes, the suspensory ligaments pull at
the circumference of the lens, making the lens flatter. The greatest amount of
refraction is required to view objects that are very close to the lens. If light
passing through the cornea and the lens is not refracted properly, the visual
image will be distorted. Therefore, accommodation is the automatic adjustment
of the eye to give us clear vision. During accommodation, the lens becomes
rounder to focus the image of a nearby object on the retina and the lens flattens
when we focus on a distant object.

1. Cover or close one eye and hold a pin about 15 cm in front of the other
eye in line with some distant object.
2. Look at the distant object and note that the pin appears blurred and dim:
it is out of focus.
3. Now look at the pin. Note that the distant object becomes dim and
indistinct. Note also that accommodation for the near object (the pin) is
accompanied by a feeling of effort.
4. Cover one eye and hold the pin at arm’s length. While looking at the point
of the pin, slowly bring it towards the face until it becomes blurred. The
shortest distance at which the pin can keep in focus is the ‘near point’.
5. Let a follow student measure and note the distance.

Exercise 3: The blind spot (p 631 Martini et al, 2014)

The visual field for each eye includes a blind spot, representing the optic disc –
a part of the retina with no photoreceptors. Axons from an estimated 1 million
ganglion cells converge on the optic disc and penetrate the wall of the eye in
order to transport visual signals to the optic centre of the brain. The central
retinal artery and the central retinal vein, which supply the retina with blood,
pass through the optic nerve and emerge on the surface of the optic disc. The
optic disc does not have any photoreceptors or other structures typical of the
rest of the retina. Because light striking the area of the optic disc goes
unnoticed, the optic disc is commonly called the blind spot. You do not notice

39
a blank spot in your field of vision because involuntary eye movements keep
the visual image moving and allow your brain to fill in the missing information.

1. Obtain a pen that writes with black ink but has a white barrel.
Alternatively, wrap some white paper around the barrel of a black fibre-
tipped pen, leaving only the black writing tip exposed.
2. Mark a small cross on a piece of white paper. Close the left eye and look
steadily at the cross at a distance of about 25 cm. For the rest of this
exercise, keep your head completely still and continue to look at the
cross.
3. Move the pen out (to the right) from the cross. At a certain distance the
tip will become invisible. Mark this place with a spot on the paper.
4. Carry the pen further to the right until it becomes visible again. Mark this
place with another spot.
5. Similarly, mark the upper and lower limits of the blind spot.

With care and patience, the entire outline of the blind spot can be traced. It is
oval in shape with irregular margins. The irregularities represent places where
the retinal blood vessels emerge from the optic disc.

Exercise 4: Mechanical stimulation of the retina

The eye has properties similar to those of a camera in that the image formed
on the retina is inverted. Light falling on the retina on one side of the eye gives
a visual response in the opposite side of the visual field. Mechanical stimulation
of the retina, by pressure on the eyeball, also gives a visual response that is
inverted.

1. Turn your gaze to the left and shut both eyes. Keep looking to the left.
2. With a finger-tip, press gently on the right side of your right eyeball at the
corner of the eye. Note the visual effect.
3. Slide your finger up and down and note the direction of movement of the
visual response.
4. Turn your gaze to the right and similarly press on the left side of your
right eyeball, at the corner of the eye. Again note the visual effect. You

40
 should find that the main visual response to stimulation is a bright circle
or disc on the opposite side of the visual field from the site of stimulation.
Stimulation of the retina on the right side of the eye gives a response on
the left, and vice versa.

Exercise 5: The positive after-image (pp 639 - 642 Martini et al, 2014)
Rhodopsin is a visual pigment that is found in photoreceptors and consists of a
protein called opsin, which is bound to a pigment called retinal. During the
activation of rhodopsin by a photon (light), the retinal molecule changes
conformation that leads to the visual stimulus being generated. Retinal
photoreceptors have a surprisingly long and slow response to light. The reason
being that after the photoreceptor cell has absorbed a photon, the conformed
rhodopsin molecule must first be broken down and reassembled in a process
called “bleaching” in order for the signal to be sent to the brain that the visual
stimulus has ended. A brief but extreme visual stimulus therefore gives rise to
a response that outlasts the stimulus long enough to give an after-image. For
example, following intense exposure to light similar to seeing the flash from a
camera, a photoreceptor cell cannot respond to further stimulation until its
rhodopsin molecules have been regenerated. As a result, a “ghost” image
remains on the retina after the flash from the camera has ended.

1. Face a bright and contrast scene such as a sunlit window or a strongly

illuminated bench-top.
2. Close both eyes and cover them with your hands. Wait for 30 seconds.
3. Remove your hands and open your eyes for the shortest possible time,
then close them again.
4. Note the after-image. Bright features of the scene remain visible for an
appreciable time (a substantial fraction of a second).

Exercise 6: The negative after-image (pp 639 - 642 Martini et al, 2014)
The sensitivity of retinal photoreceptors decreases gradually while they are
being stimulated by light and increases while they are not. This adaptation to
light and dark allows visual function over a very wide range of light intensities.
However, it has the side effect of giving rise to negative after-images. After 30

41
minutes in the dark, almost all the visual pigments of the photoreceptors will
have recovered from bleaching and will be highly sensitive and ready to be
stimulated by photons (adapted to the dark). If the lights are then switched on,
they at first seem unbearably bright but as the sensitivity of the photoreceptors
decrease the light seems more bearable. The receptor will therefore become
adapted to the light. The size of the iris also plays a part in light or dark
adaptation, as it lets in more light when it is dark and it lets in less light when
the person is exposed to bright light.
If you stare at a red image for 60 seconds, the photoreceptors that are excited
by the red light will temporarily become less sensitive. If you then look at a white
paper (white light stimulates photoreceptors for all colours), the image will
appear green because the red photoreceptors are temporarily “worn out”.

1. Place a black object on a piece of white paper, or draw a black square

on the paper.
2. Look fixedly at the black object for 30 seconds. You may blink, but should
take care to keep your gaze fixed.
3. Shift your gaze to a piece of plain white paper and note the after-image
of the black object. The image lasts for many seconds. The image is
inverted in contrast (the black object gives a bright after-image), hence
the name “negative after-image”.
4. Repeat with a coloured object and note the colour change in the after-
image. For example, a red object gives a green after-image.

Touch

Tactile receptors provide the closely related sensations of touch, pressure and
vibration. Touch sensations provide information about shape and texture,
whereas pressure sensations indicate the degree of mechanical distortion and
vibration sensations indicate pulsing pressure. (See pp 561 - 562 of Martini et
al, 2014 for more information regarding tactile receptors.)

Exercise 7: Two-point discrimination (pp 561. 562 and 564 Martini et al,
2014)

42
Fine touch and pressure receptors provide detailed information about a source
of stimulation, including its exact location, shape, size, texture and movement.
The receptors are extremely sensitive and have narrow receptive fields. Crude
touch and pressure receptors provide poor localisation and because they have
large receptive fields, give little information about the stimulus.
The density of tactile receptors in the skin differs greatly in different parts of the
body. The two-point discrimination test can provide a detailed map of the
distribution of tactile receptors on the surface of the skin. Typically a normal
individual loses two-point discrimination at 1 mm on the surface of the tongue,
at 2-3 mm on the lips, at 3-5 mm on the backs of the hands and feet, and at 4-
7 mm over the general body surface.

1. Take a metal paperclip and unfold it. Bend it into a U shape, with the wire
points about 10 mm apart.
2. Touch the two points gently on the palm of a volunteer’s outstretched
hand and ask if one point or two are felt. With a separation of 10 mm,
the double stimulus from the two points can be easily felt.
3. Ask the volunteer to close both eyes. Bend the paperclip so as to bring
the points closer together. By repeated trials with different point
separations, find the smallest separation that the volunteer can
distinguish as two points. You can test the truthfulness of the volunteer’s
responses, from time to time, by turning the paperclip slightly and
pressing only one of the points down.
4. Measure the separation of the points with a ruler.
5. Repeat steps 3 and 4 with trials on different parts of the body (for
example, a fingertip, the back of the hand and the back of the forearm).

Exercise 8: A tactile illusion

1. Cross two adjacent fingers over so that the fingernails lie side by side, but in
a position reserved from the normal. Most people find it easiest to cross the
middle finger over the index finger.

43
2. Place a small object such as a pen in the V-shaped gap between the two
fingernails and gently move it back and forth. There should be a striking illusion
that two objects are being felt.

Temperature

Temperature sensors, or thermo-receptors, are free nerve endings located in

the dermis, in skeletal muscles, in the liver and in the hypothalamus. (See p
561 of Martini et al, 2014 for more information regarding the temperature
receptors.)

Exercise 9: A thermal illusion (pp 559 and 561 Martini et al, 2014)
Many sensory systems show adaptation: a declining response to a continued
steady stimulus. Thermo-receptors in the skin are phasic receptors because
they are very active when the temperature is changing, but they quickly adapt
to a stable temperature. Thermo-receptors adapt to conditions where the
temperature is stable and thermal sensations of warmth or cold are determined
more by changes in temperature than by the temperature itself. For instance,
when you enter an air-conditioned classroom on a hot summer day, the
temperature change seems extreme at first but you quickly become comfortable
as adaptation occurs. Similarly, when you leave your hand in cold water for 30
seconds and you put it in warm water, the warm water feels hotter than it
actually is because the temperature change from the cold to the warm water is
larger than if your hand moved from room temperature water to warm water.

1. Obtain three containers (small buckets or large beakers). Fill one

container with hot, but not painfully hot water. Fill another with cold water
and fill the third with lukewarm water.
2. Place one hand in hot water and the other hand in cold water. Leave
them there for 30 seconds.
3. Now place both hands in lukewarm water. The water feels warm to one
hand and cold to the other.

44
Taste

Taste receptors are distributed over the superior surface of the tongue and
adjacent portions of the pharynx and larynx. Taste receptors and specialised
epithelial cells form sensory structures called taste buds. An adult has about 5
000 taste buds. (See pp 621 - 624 of Martini et al, 2014 for more information
regarding taste.)

Exercise 10: Taste and smell (pp 621 - 624 Martini et al, 2014)
Much of the sensation that is commonly called “taste” is really smell. The level
of stimulation from the olfactory (smell) receptors plays an overwhelming role
in taste perception. It is for that reason that you are several thousand times
more sensitive to tastes when your olfactory organs are fully functional. On the
other hand, when you have a cold and your nose is blocked, airborne molecules
cannot reach your olfactory receptors, so food tastes dull and unappealing.

1. Ask a volunteer to close his or her eyes and to pinch the nostrils together,
preventing air flow through the nose.
2. Place a small previously prepared piece of apple in the volunteer’s
mouth and ask him or her to try to identify it by taste.
3. Repeat with a piece of raw potato and then with a piece of raw onion.
Identification is extremely difficult.
4. Repeat steps 2 and 3. but this time allow the volunteer to breathe through
the nose. Identification is now easy.

Exercise 11: Taste bud distribution (pp 621 - 624 Martini et al, 2014)
Taste receptors (“taste buds”) are found principally on the tongue, but also on
the palate and pharynx. Four kinds of taste buds are recognised: sweet, sour,
salt and bitter. Each kind of taste bud has a characteristic spatial distribution on
the tongue. The anterior (front) part of the tongue has a greater sensitivity to a
salty-sweet taste and the posterior (back) part of the tongue has a greater
sensitivity to a bitter-sour taste. However, there are no differences in the
structure of the taste buds, and taste buds in all portions of the tongue can
provide all four primary taste sensations.

45
 1. Prepare or obtain small beakers of the following solutions: Sucrose
(sugar), sodium chloride (table salt) and citric acid.
2. Dip a small piece of clean cotton wool or the end of a cotton bud in the
sucrose solution and then shake off the excess solution. Apply the cotton
wool or bud to the back of a volunteer’s tongue, and ask the volunteer to
report the sensation. Discard the cotton wool or bud. Using a fresh piece
of cotton wool or new cotton bud, test the sensitivity of one side of the
tongue. Similarly, test the tip of the tongue. You should find that the tip
of the tongue gives the most intense sweet sensation.
3. Repeat step 2. but with the salt solution. Note the different distribution of
salt sensitivity.
4. Repeat step 2. but with the citric acid solution. Note the different
distribution of sour sensitivity.

Joint position

Proprioceptors monitor the position of joints, the tension in tendons and

ligaments, and the state of muscular contraction. (See pp 563 - 564 of Martini
et al, 2014 for more information regarding proprioceptors.)

Exercise 12: The “joint position” sense (p 563 Martini et al, 2014)
The capsule and ligaments of a joint receive sensory innervations that are able
to detect changes in joint position. These proprioceptors are located in joints,
tendons, ligaments and muscles and do not adapt to constant stimulation, so
each receptor is constantly sending information to the central nervous system.
A small proportion of the arriving proprioceptive information reaches your
awareness, although most proprioceptive information is processed at
subconscious levels.
The effectiveness of this little-known sense is easily demonstrated.

1. Ask a volunteer to hold out one hand with the palm facing up and the
fingers stretched out.

46
 2. Hold the volunteer’s index finger by placing your thumb on one side and
your index finger on the other. Do not hold the volunteer’s finger by the
front and back; that could give cues about movements, derived from the
force of lifting or pulling down.
3. Bend the volunteer’s finger up, while saying “This is up”. Then pull the
finger down to the original extended position, while saying “This is down”.
4. With the volunteer’s eyes shut, test his or her ability to identify the
direction of a series of finger movements. Try both large and small
movements.

Equilibrium

The semi-circular canals are liquid-filled canals in the temporal bone of the skull
and form part of the inner ear. Receptors inside these semi-circular canals
convey information regarding the rotational movements of the head and
equilibrium to the brain. (See pp 647 - 651 of Martini et al, 2014 for more
information regarding the inner ear and equilibrium.)

Exercise 13: The semi-circular canals (pp 647 - 651 Martini et al, 2014)
There are three semi-circular canals, the anterior, posterior and lateral semi-
circular canal. They detect rotary movements of the head on three axes by
means of the receptors (hair cells) located in the semi-circular canals, which
are stimulated by the rotation of the head.

The exercise requires a swivel chair (one that can be rotated smoothly on its
vertical axis). Many chairs used in offices or for computer work are of this kind.

1. The volunteer should sit on the swivel chair, with both feet in the air, and
close both eyes.
2. Ask the volunteer to indicate when a rotation is detected and to indicate
in which direction. Test the volunteer’s ability to sense rotary motion by
rotating the chair at various speeds and for various durations. You
should find that even very slight movements are reliably detected.

47
The semi-circular canals detect rotation but do not signal the body’s position.
The reason for this is that the hair cells in the semi-circular canals are active
during a movement but they are quiet when the body is motionless. Movement
of the head, therefore, causes the fluid in the semi-circular canals to push
against the hair cells, activating them while if the head is still, the fluid in the
canals do not move and the hair cells are not activated.
You can test this by showing that the volunteer’s idea of which direction he or
she is facing becomes unreliable after a sequence of rotations, such as quarter-
turn in one direction and then a half-turn in the other. However, the test is not
straightforward to do, because the volunteer will make use of other directional
cues. The ambient light intensity may change with rotation and this can be
sensed even through closed eyelids. Also, acoustic cues are likely to be present
in many laboratories.

Assignment
1. Discuss the following topics in your assignment (it is important to
remember that these mechanisms could be asked in the practical
examination):
1.1 Accommodation of the eye.
1.2 Adaptation of the eye.
1.3 Two-point discrimination.
1.4 Balance – sense of posture and movements.
1.5 Smell sensation.

2. The following must be included in your assignment of each topic:

2.1 Title
2.2 Aim.
2.3 Literature study or background – mechanism discussion with references
Martini et al, 2014.
2.4 Method.
2.5 Results.
2.6 Conclusion.
2.7 Literature list.

48
 Where applicable

DIFFUSION

Objective
To determine the influence of the differences in concentration and the molecular
mass on the diffusion rate.

The molecular mass

Theoretical background
Diffusion is the movement of molecules from a high to a low concentration. The
movement in this direction will always take place unless there is an obstruction
or different force that is stronger than the diffusion force driving it in another
direction. Such forces can be electrical, mechanical or chemical.

All substances that move from the extra cellular to the intracellular fluid, or vice
versa, have to pass through a cell membrane. The cell membrane therefore
forms an obstruction with regard to free diffusion. This is a special obstruction,
however, because it is selectively permeable. In other words, only certain
substances can move through the cell membrane according to the laws of
simple diffusion, while others cannot.

Other methods of transport through the cell membrane are:

1. Facilitated diffusion by means of linkage to a carrier molecule.
2. Active transport in which a molecule can be transported against a
concentration gradient because of the release of chemical energy.
3. Endocytosis and exocytosis.

We call the diffusion of water, which takes place freely through the cell
membrane, osmosis. A volume of water equal to 100 x the total volume of the

49
cell moves through the cell every second. Osmotic pressure develops if there
is a water concentration gradient between two compartments.

Diffusion is a process that is essential for the maintenance of homeostasis and

is the mechanism in the following:
1. The reabsorption of urea, uric acid, etc. in the kidney.
2. The absorption of fatty acids from the digestive tract, CO2 and O2
movement across alveolar and capillary membranes.

We can determine the diffusion rate (changes in the concentration of the

dissolved substances per time unit) by factors such as:
• Concentration – the greater the differences in concentration of the
dissolved substances, the higher the diffusion rate – a direct proportion.
• Contact surface – the greater the contact surface between the two fluids,
the greater the diffusion rate – a direct proportion.
• Temperature – the higher the temperature, the higher the diffusion rate –
a direct proportion.
• Molecular mass – the greater the molecular mass, the lower the diffusion
rate – an inverse proportion.
• Distance – the greater the distance the molecule has to move, the lower
the diffusion rate – an inverse proportion.

We can therefore state that

DR = Kd x C x S x T
M x D

Where DR = diffusion rate

Kd = diffusion constant (unique for each substance)
S = contact surface
T = temperature
M = molecular mass
D = distance
C = concentration

50
In this experiment we will indicate how differences of concentration and
molecular mass may affect the diffusion rate. We will use the apparatus
illustrated in the figure. The membrane which is used is, just like a cell
membrane, permeable to certain substances.

Beaker

Fluid
compartment
A

Elastic band

Membrane
B
Magnet
Magnetic
stirrer

Two fluid compartments, A and B, are separated from each other by a

membrane. Compartment A contains a dissolved acid, while B contains no
acid. Because of this difference in concentration, the acid will diffuse effectively
from A to B. However, B contains a low concentration of alkali with an added
indicator. As soon as the acid from A lands in B, a neutralising action takes
place, for example:
HCl + NaOH → H2O + NaCl
Hydrochloric acid sodium hydroxide water sodium chloride
(acid) (alkali) (salt)
As soon as all the alkali present in B has reacted with the acid which entered
by diffusion, the indicator changes in colour. Note the time from when the two
solutions came into contact to the point of neutralisation. When there are
different concentration ratios between A and B, or when acids of different
molecular masses, but the same concentration, diffuse from A to B, we can
obtain an indication of the influence of concentration and molecular weight on
the rate of diffusion.

51
Apparatus
Magnetic stirrer.
Magnet.
Dialysis membrane.
Special liquid container.
Elastic bands.

Reagents
Hydrochloric acid solutions: 0.5 N, 0.4 N, 0.3 N, 0.2 N and 0.1 N (36.5 g/mol).
Sodium hydroxide solution: 0.01 N (with added indicator).
0.5 N acetic acid (60 g/mol)
0.5 N sulphuric acid (98 g/mol)
0.5 N oxalic acid (126 g/mol)
0.5 N citric acid (210 g/mol)
0.1 N Ethylenediaminetetraacetic acid (EDTA) solution (0.1 N)

Method
A The influence of differences in concentration on the rate of diffusion
1. Place the membrane in 50 ml boiling EDTA solution in a 100 ml glass
beaker for three minutes. Rinse with distilled water and cut through
lengthways without damaging or tearing it.
2. Pull the membrane tightly across the bottom large opening of the inner
compartment (A in the figure) and fasten with an elastic band. See to it
that the membrane is taut and smooth, but make sure that it does not
tear. Add about 50 ml water to compartment A to check that neither the
membrane nor the compartment is leaking. Pour out the water when all
is in order.
3. Put 50 ml 0.01 N sodium hydroxide solution and a small magnet in a 250
ml beaker and place this on the magnetic stirrer. This is compartment
B.
4. Add 50 ml 0.5 N hydrochloric acid to compartment A. See that there is
no acid on the outside edge of compartment A. This could have a big
influence on the result.

52
 5. Place compartment A into compartment B so that A rests on the edge of
the glass beaker. Note the time immediately and at the same time switch
on the magnetic stirrer. After a certain period of time, the pink colour will
completely disappear. Measure the period of time (in seconds) and note
this in a table.
6. Pour the acid out of compartment A and rinse the compartment
thoroughly with tap water until not even the slightest amount of acid
remains.
7. Repeat steps 3 - 6 twice until all the acids are tested. In each instance
note the time in the table.
B The influence of the molecular mass on the diffusion rate
1. Repeat steps 3 - 6 with the acids with a different molecular mass. Note
the time lapse in a table.
2. Duplicate each experiment.

Instruction
Calculation of the diffusion rate
• You must calculate the diffusion rate in each instance.
As time passes, the concentration of the acid in compartment B rises as a
result of diffusion from A to compartment B. Represent this graphically as,
for example, curve I in the figure.

53
 (I)

0.02
Increase in the
(II)
normality of acid in
compartment
0.01

00 20 40 60
t1

t2
Time (seconds)

If the concentration in A is lower, you will have curve II of the figure. Seeing
that the concentration of alkali in compartment B is 0.01 N sodium hydroxide
and you note the time lapse to the point of neutralisation, the concentration
of the acid in compartment B, after diffusion, will also change to 0.01 N. Per
definition and from the figure the diffusion rates in the two cases are as
follows:

0.01
For curve I Diffusion rate =
t1 ∆ normality / second

0.01
For curve II Diffusion rate =
t2 ∆ normality / second

It is clear that the diffusion rate of curve I is higher than that of curve II, as
could be expected because of the differences in concentration between I
and II.
1. Now calculate the rate of diffusion in the same way for all the experiments
and fill in the results, together with all the other required information, in
tables. Indicate all your calculations.

54
2. Plot on graph I, the diffusion rates (Y-axis) against the hydrochloric acid
normality (X-axis).
3. Plot on graph II, the diffusion rates (Y-axis) against the inverse of the
molecular mass (X-axis).
Questions
1. Give a detailed discussion of the results of graph I and II.
2. Distinguish between osmosis and diffusion.
3. What is the influence of “distance” on the reaction rate in the experiment?
4. Define the following:
• isotonic solution
• hypotonic solution
• hypertonic solution
• haemolysis
• crenation
• pinocytosis
• phagocytosis.

55
 ELECTRICAL EVENTS IN THE CARDIAC MUSCLE (ECG)

Objective
This practical assignment teaches you to set up various ECG leads in order to
examine the flow of electrical currents in the heart. You will learn how to
simulate myocardial infarction to study the changes in ECG pattern.

The origin of an action potential

The permeability characteristics of the cell membrane result in a higher
concentration of sodium ions outside the cell than inside, while the reverse is
true of potassium ions. Because of this difference in concentration, there is a
difference in potentiality of -70 mV across the cell membrane. The inside of the
membrane is negative in regard to the outside.
When a cardiac muscle receives an effective stimulus, the following happens:
1) The membrane becomes permeable for sodium ions.
2) Sodium ions flow to the inside of the cell because of the concentration
gradient.
3) The net gain of positive charges on the inside of the membrane
changes the membrane polarity to +30 mV (depolarisation).
4) The sodium ion permeability is maintained for 150 ms.
5) Calcium ions flow into the heart during this period.
6) The sodium ion permeability diminishes.
7) The potassium ion permeability of the cell membrane increases.
8) Potassium ions flow to the outside (net gain of positive charges on
the outside of the membrane).
9) The polarity of the cell membrane returns to the normal value
(repolarisation).

The differences in potential that the cell membrane undergoes as a result

of the administration of a positive stimulus are called the action potential.

56
The modified cells
We find the following three types of modified cells:
1) P-cells.
2) Transitional cells.
3) Perkunje cells.

The P-cells are arranged in such a way that they form the Sinoatrial (SA) and
atrio-ventricular (AV) nodes. The P-cells can develop action potential (auto-
rhythmicity) because their membranes continually let through sodium ions.
They are the pacemakers of the heart.
The transition cells are between the P-cells and the contractile cells and serve
as linkage. The Perkunje cells are long and are grouped together to form the
Perkunje tissue which distributes the actions potential co-ordinately through the
myocardium. The upper part of the Perkunje tissue is the Hiss bundle and is
the conduction link between the atria and the ventricles.
The action potential that develops in the SA node spreads through the entire
atrial wall. In the AV node this is held up for 60-120 ms. This interval gives the
atria a chance to complete their pumping action and complete the ventricular
filling. From the AV node, the impulse quickly moves along the Hiss bundle,
the cluster branches and the Perkunje tissue through the ventricular wall.

The ECG Registration

The body serves as a volume conductor of electrical current (action potential)
generated by the heart. You can pick this current up from the skin surface with
electrodes and register it.

This registration is the ECG or electrocardiogram and consists of the following:

1) P-wave. This is a monophasic wave which represents the depolarisation
of the atria.
2) PR segment. This is the distance between the end of the P-wave and
beginning of the Q-wave. It is caused by the depolarisation of a part of
the AV node, the Hiss bundle and the bundle branches. No deflection is

57
 obtained because the electrical activity that develops in this tissue is very
small.

An action potential in a cardiac muscle

The electrocardiogram: the naming of the various waves and segments

58
 3) QRS-complex. This complex represents depolarisation of the ventricles
and lasts approximately 0.06 seconds. The subsequent positive
deflection is the R-wave and the next negative wave is the S-wave. If
the QRS-complex begins with a positive deflection, it is called the R-
wave. The QRS-complex has no Q-wave.
4) ST-segment and J-point. The spot where the QRS-complex goes over
into the ST-segment is called the J-point. The ST-segment stretches
from the J-point to the beginning of the T-wave and is normally
isopotential (reveals no deflection).
5) T-wave. This represents the ventricular repolarisation. Atrial
repolarisation cannot be distinguished, because it concurs with the QRS-
complex. The T-wave is a sensitive indicator of myocardial deviations.
6) U-wave. This is normally not visible. It becomes observable, however,
in the case of hypokalaemia and can be the result of retarded and
uneven repolarisation.
7) PR-interval. This has to be measured beginning with the P-wave up to
the beginning of the QRS-complex to the end of the T-wave. It reflects
the duration of the electrical systole of the ventricles. The duration of the
Q-pause changes in inverse proportion to the calcium concentration.
8) QT-interval. This stretches from the beginning of the QRS-complex to
the end of the T-wave. It reflects the duration of the electrical systole of
the ventricles. The duration of the Q-pause changes in inverse
proportion to the calcium concentration.

The electrocardiograph
An electrocardiograph is basically a transistorised amplified galvanometer
which can render changes in potential permanently on paper or temporarily on
the screen of an oscilloscope. The changes in potential which occur as a result
of electrical changes in the myocardium are registered on the body surface with
the aid of plate electrodes and are transported to the apparatus by protected
cables.
To measure the changes in electrical potential, the electrocardiograph has to
be connected to the body by a positive and a negative electrode. A complete
conduction track then exists between the electrocardiogram and the body.

59
Standard electrocardiogram registration
By making use of 12 different standard electrode positions, we can investigate
the electrical events in the heart. Each position is known as a lead. Three of
the 12 leads are bipolar, that is, both electrodes, positive and negative, are
subjected to a change in potential. The others are monopolar, that is, only the
positive electrode is subjected to a change in potential.

The electrode placing during registration of the bipolar (standard)

electrocardiogram registrations

In this description of the leads, imagine that the heart is surrounded by an

equilateral triangle of which the angles are constituted by the bases of the right
arm, the left arm and the left leg.
The electrodes are connected to the wrists and the ankle because the limbs are
regarded as being part of the electrode.
The bipolar leads
With lead I we measure the change in potential between the two arms with the
negative electrode on the right arm.
With lead II we measure the change in potential between the right arm and the
left leg with the negative electrode on the right arm.
With lead III we measure the change in potential between the left arm and the
left leg with the negative electrode on the left arm.
If a negative electrode is in a negative electrical field, we will obtain a positive
deviation. If a negative electrode is situated in a positive electrical field, we will
obtain a negative deviation. If a positive electrode is situated in a positive

60
electrical field, we will find a positive deviation and if a positive electrode is
situated in a negative electrical field, we will find a negative deviation.

Electrode placing during registration of the unipolar electrocardiogram leads

Repolarisation begins on the left-hand side of the atria (at the SA node) and
runs in the direction of the apex. From the sketch you can see that the
arrangement of electrodes for the registration of the bipolar deductions is
always such that the negative electrodes are in a negative field and the positive
electrodes in a positive field. The leads therefore always render positive
registrations.

Unipolar leads
We obtain the leads by connecting the negative electrodes with two of the limbs
by means of similar resistances (indifferent electrode) and by using the positive
electrode as an investigator electrode. The unipolar leads are called the
following:
1) AvL if the examining electrode is placed on the left arm.
2) AvR if the examining electrode is placed on the right arm.

61
 3) AvF if the examining electrode is placed on the left leg.
In the case of the unipolar electrode, the following holds:
If the electrical current moves in the direction of the electrode, a positive
deviation will be obtained and if the current moves away from the electrode, a
negative deviation will be obtained.

The chest leads

With the chest leads, we place the examination electrode (positive) on six
specific positions on the chest, while we connect the other (negative) electrode
by means of similar resistances to the ends of the Eindhoven triangle (three
relevant limbs). In contrast to the situation with the bipolar leads, the direction
of deviation with unipolar leads is not always positive.

Naming the direction of a vector in terms of degrees

A vector indicates the zero degree direction if it is horizontal and pointing to the
left side of an object. From the zero reference point the scale of vectors rotates
clockwise (see sketch). If there is movement in an anticlockwise direction, the
direction is accompanied by a negative.

The axes of the standard and unipolar leads

Each lead is actually a few electrodes that have been placed on either side of
the heart on the body. The direction from the negative to the positive is called
the axis of the lead.
Lead I connects the left and the right arms. It is therefore connected horizontally
to the positive electrode on the left. The axis of the lead is then 0° degrees.
Lead II connects the right arm and the left leg. The connecting points of the
arm and the leg are respectively right and left on the trunk. The connecting line
between the connecting points constitutes an angle of about 60° with the 0° line
(horizontal level). The axis of lead II is then 60°.
In the same way we can determine that the axis of lead III is 120°.

62
The naming of the direction of a vector in terms of degrees

Determination of the mean electrical axis of the ventricles

During the electrical cycle of the ventricles (vector cardiogram), the main
direction of the flow of the electrical current is in the direction of the apex. This
main direction of the current flow is called the mean electrical axis of the
ventricles. The mean electrical axis of normal ventricles is 59°. In certain
pathological conditions there can be a pronounced shift of the electrical axis.

Method for determining the mean electrical axis of the heart from the
standard leads
1) Measure the amplitudes (deviations from the base line/isoelectric line) of
the QRS-complexes of leads I and III in a positive and a negative
direction.
2) Subtract the two values from each other. Use the calibration of the y-
axis to express the net deflection in mV for lead I and III separately.
Select a suitable scale and plot these on the axial system of the standard
deviations as indicated in the sketch.

63
3) Use the cut-off point of the axes as the basic point. If the amplitudes
were positive, plot it in a positive direction. If they were negative, plot it
in a negative direction.
4) Draw perpendicular lines on the axes from the points of the vectors of
leads I and III.

The axes of the standard electrocardiogram leads

5) Connect the intersecting points of the two perpendicular lines with the
intersecting point of the axes of leads I and III. Measure the angle with
regard to lead I and use the scale to calculate the size of the electrical
axis. This is the mean electrical axis of the heart.
NB: The mean electrical axis of the heart is a vector with size and direction.

Abnormalities
Abnormalities in rhythm
We can calculate the heart rate by counting the number of R-R intervals
which occur in a specific period of time. We determine the heart rate and
rhythm by the SA node, only the discharge rate is faster than that of the
other potential pacemaker (e.g. Perkunje cells).

64
 The further the potential pacemaker is situated from the SA node, the slower
the inherent discharge rate. The duration of the R-R intervals can change.
Then we are dealing with rhythm abnormalities (arrhythmias).

Determination of the mean electrical axis of the heart

Arrhythmias can occur as a result of the following:

1) A too rapid impulse production.
2) A too slow impulse production.
3) Irregular impulse discharge.
4) Blocking of the impulse conduction.
The causes can lie in the SA node, the atria, the AV node or in the ventricles.
Certain arrhythmias can be created as a result of the changes in activity of the
vagus nerve which serves the SA node.

Abnormalities of the ECG form

These abnormalities are usually caused by ischaemia, an injury or necrosis of
the myocardium.
Ischaemia is a condition of an oxygen shortage. An injury indicates that too
little energy is available to ensure the normal maintenance of the cardiac

65
muscle and especially of the cell membrane. Necrosis indicates that part of the
myocardium has died off to create an infarct.

Myocardial infarction
Infarction of the myocardium is usually the result of the closing off of one or
more of the coronary arteries (atherosclerosis). This is caused by exposure to
certain risk factors, such as the following:
1) Too high fat (cholesterol) ingestion.
2) Smoking.
3) Stress.
4) Physical inactivity.
5) Inherited factors (high cholesterol, etc.).

These risk factors can lead to the depositing of fat in the intima (innermost layer)
of the coronary artery (among others). The invasion of connective tissue and
later, calcification of the fatty deposits lead to the formation of hard plates, which
finally constrict the coronary blood vessel so that the blood supply to a part of
the myocardium is reduced or even completely occluded (cut off). This is a
gradual process which occurs with the passage of time.
Acute coronary occlusion can occur if the hard atherosclerotic plate breaks
through the intima and creates a blood clot which then suddenly cuts off the
blood supply to a certain part of the myocardium. If a small blood clot breaks
away and occludes the artery some distance further on, it is called an embolus.
Sometimes a spasm of the coronary artery as a result of irritation of the wall
can cause acute occlusion of the coronary artery.
Infarction begins to develop if less than 1.3 ml oxygen per 100 g tissue is being
supplied to the cardiac muscle.
A vicious circle usually develops following the occlusion of a blood vessel. A
part of the myocardium becomes ineffective as a result of the decrease of the
perfusion in it.
This causes the pumping action of the heart to decrease. The undamaged part
of the heart has to work harder to maintain the pumping function, with the result
that more oxygen and thus better perfusion is needed, which in turn cannot be
supplied by the weakened heart. Less oxygen reaches the infarcted area

66
because it is being used by the more active cardiac muscle, with the result that
tissue damage increases in the heart. This leads to a decrease in the efficient
working of the heart, which once again leads to a poorer oxygen supply, etc.
The following are the usual causes of death:
1) A decrease in cardiac output.
2) Pulmonary congestion (oedema of the lungs).
3) Tearing of the infarct, with the result that blood collects in the pericardial
space (in the pericardium or cardiac sac). (The heart is compressed so
that it does not receive blood, and thus cannot pump it out [cardiac
tamponade].)
4) Fibrillation of the ventricles. (This is the spontaneous formation of action
potentials through the damaged tissues, which renders the contraction
of the heart uncoordinated to the extent that the pumping function is lost.)

Electrocardiographic effects of myocardial ischaemia

Ischaemia of the cardiac muscle is represented by an inverted T-wave. It is a
non-specific finding that can be associated with a whole number of other normal
and abnormal conditions. An ischaemic T-wave usually has, in contrast to other
cases, a sharp peak and is also symmetrical.

Electrocardiographic effects of a myocardial injury

A myocardial injury as a result of infarction usually occurs only in the inner or
outer muscular layer of the ventricle wall.
The injured section depolarises irreversibly. It therefore remains negative with
regard to the normal tissue. As a result of the charge differences, there is a
constant flow of electrical current between the normal and the injured area. We
call these injury currents. This means that there is always a negative current
from the damaged surface of the heart (1) and a positive current from the
opposite surface of the heart (2).
If we place an electrode (A) across such an injured surface, the negative current
will register as a base line that has been transferred to the bottom (negative
current to positive electrode). If the ECG electrode is oriented in such a way
with regard to the area of injury as electrode B in the figure, the P-QRS waves
deviate from this shifted base line.

67
Immediately after polarisation the normal tissue is also negative, just like the
area of injury, with the result that there is no longer any current flow from the
injury currents. The ECG returns to its normal base line which is now situated
above the new base line for electrode A, with the result that it seems as if the
ST-segment has been replaced to the upper area.

The reversal of the T-wave of the electrocardiogram as a result of ischaemia

If the ECG electrode, however, is oriented in such a way with regard to the area
of injury as electrode B in the figure, then the normal base line will be replaced
to above (positive current to positive electrode). The P-QRS waves therefore
deviate from the shifted base line. Because no injury currents flow directly after
depolarisation, the ECG will return to its original base line with the result that it
will seem as if the ST-segment has been transferred to the bottom. Therefore,
if the injury area is on the same side as the positive electrode, the ST-transfer
is to the upper area and if the electrode is on the opposite side, the ST-transfer
or shift is to the bottom.

68
The electrical effects of myocardial injury (infarction)

69
The electrocardiographic effects of myocardial necrosis
Necrotic cardiac muscles are muscles that have died and are therefore
electrically inactive. If the necrotic area fills the whole thickness of the
myocardium, it is electrically and functionally a hole or window in the
myocardium. When we place an electrode on or near the window, the ECG will
reflect the electrical events in the myocardium which is situated across the
window. Seeing that the normal depolarisation wave spreads from the
endocardium (inside of the cardiac muscle) to the pericardium (outside of the
cardiac muscle), the next movement of current is away from the electrode, with
the result that a negative Q-wave will be registered. Necrosis of the cardiac
muscle is therefore reflected in the ECG as a broad negative Q-wave.

The composite ECG pattern of a fully developed myocardial infarction

During fully developed myocardial infarction the infarcted area consists of (1) a
central area of necrotic tissue and (2) is circled by an area of damaged tissue,
which in turn is circled by an area of ischaemic tissue.
A conventional electrode cannot be placed on the heart but only on the body
surface. As a result of the distance of the electrode from the heart, it registers
electrical changes in normal, ischaemic, injured and necrotic tissue. The ECG
is therefore a composition of all the deviating patterns.

70
The electrocardiographic effects of myocardial infarction

71
ECG changes in pattern during myocardial infarction

Apparatus

Laboratory apparatus:
1) Computerised physiology workstation with printer/ECG monitor.
2) ECG electrodes.
3) Shielded cables.
4) Electrode jelly.

Method
ECG in humans
1) The plate electrodes must be firmly attached to their limbs and there
must be electrode jelly under the electrodes.

72
2) Depending on the lead made, the polarities must be placed as indicated.
3) Make a registration.

Instructions
1) Register leads I, II and III on your own. Connect the electrodes correctly.
Register leads I, II and III again, but reverse the polarities. Explain the
differences between the normal ECGs and those registered when the
polarities were reversed. Also calculate the mean electrical axis of the
heart.

73
 RECORDING OF AN ELECTROCARDIOGRAM

Objective
The recording of a good electrocardiogram (ECG) and the recognition of
abnormalities of the ECG.

Electrocardiogram
An electrocardiogram is a general non-invasive test that records the electrical
activity of the heart. ECG is used to measure the rate and regularity of
heartbeats, the presence of any damage to the heart, and the effects of drugs
or devices used to regulate the heart (such as a pacemaker).

The ECG Registration

Each time the heart beats a wave of depolarisation radiates through the atria,
reaches the AV node, travels down the interventricular septum to the apex,
turns and spreads through the ventricular myocardium towards the base of the
heart.
The body serves as a volume conductor of electrical current (action potential)
generated by the heart. You can pick up this current from the skin surface with
electrodes and register it.

This registration is the ECG or electrocardiogram and consists of the following:

74
 9) P-wave. This is a monophasic wave which represents the depolarisation
of the atria.
10) PR-segment. This is the distance between the end of the P-wave and
beginning of the Q-wave. It is caused by the depolarisation of a part of
the AV node, the Hiss bundle and the bundle branches. No deflection is
obtained because the electrical activity which develops in this tissue is
very small.
11) QRS-complex. This complex represents depolarisation of the ventricles
and lasts approximately 0.06 seconds. The subsequent positive
deflection is the R-wave and the next negative wave is the S-wave. If
the QRS-complex begins with a positive deflection, it is called the R-
wave. The QRS-complex has no Q-wave.
12) ST-segment and J-point. The spot where the QRS-complex goes over
into the ST-segment is called the J-point. The ST-segment stretches
from the J-point to the beginning of the T-wave and is normally
isopotential (reveals no deflection).
13) T-wave. This represents the ventricular repolarisation. Atrial
repolarisation cannot be distinguished, because it concurs with the QRS-
complex. The T-wave is a sensitive indicator of myocardial damage.
14) PR-interval. This has to be measured from the beginning of the P-wave
to the beginning of the QRS-complex. It reflects the duration of the
impulse to spread from the SA node across the atrial surface, the AV
node, along the intraventricular septum and the bundle branches to the
Purkinje fibres and the ventricles.

The electrocardiograph
An electrocardiograph is basically a transistorised amplified galvanometer that
can render changes in potential permanently on paper or temporarily on the
screen of an oscilloscope. The changes in potential that occur as a result of
electrical changes in the myocardium are registered on the body surface with
the aid of plate electrodes and are transported to the apparatus by protected
cables.

75
To measure the changes in electrical potential, the electrocardiograph has to
be connected to the body by a positive and a negative electrode. A complete
conduction track then exists between the electrocardiograph and the body.

Standard electrocardiogram registration

By making use of 12 different standard electrode positions, we can investigate
the electrical events in the heart. Each position is known as a lead. Three of
the 12 leads are bipolar, that is, both electrodes, positive and negative, are
subjected to a change in potential. The others are monopolar, that is, only the
positive electrode is subjected to a change in potential.

In this description of the leads, imagine that the heart is surrounded by an

equilateral triangle of which the angles are constituted by the bases of the right
arm, the left arm and the left leg.
The electrodes are connected to the wrists and the ankle because the limbs are
regarded as being part of the electrode.

The bipolar leads

With lead I we measure the change in potential between the two arms with the
negative electrode on the right arm.
With lead II we measure the change in potential between the right arm and the
left leg with the negative electrode on the right arm.
With lead III we measure the change in potential between the left arm and the
left leg with the negative electrode on the left arm.

76
Unipolar leads
We obtain the leads by connecting the negative electrodes with two of the limbs
by means of similar resistances (indifferent electrode) and by using the positive
electrode as an investigator electrode. The unipolar leads are called the
following:
4) AvL if the examining electrode is placed on the left arm.
5) AvR if the examining electrode is placed on the right arm.
6) AvF if the examining electrode is placed on the left leg.

In the case of the unipolar electrode, the following holds:

If the electrical current moves in the direction of the electrode, a positive
deviation will be obtained and if the current moves away from the electrode, a
negative deviation will be obtained.

77
The chest leads
With the chest leads, we place the examination electrode (positive) on six
specific positions on the chest, while we connect the other (negative) electrode
by means of similar resistances to the ends of the Eindhoven triangle (three
relevant limbs). In contrast to the situation with the bipolar leads, the direction
of deviation with unipolar leads is not always positive.

Abnormalities
Abnormalities in rhythm
We can calculate the heart rate by counting the number of R-R intervals that
occur in a specific period of time. We determine the heart rate and rhythm
by the SA node; only the discharge rate is faster than that of the other
potential pacemaker (e.g. Perkunje cells).
The further the potential pacemaker is situated from the SA node, the slower
the inherent discharge rate. The duration of the R-R intervals can change.
Then we are dealing with rhythm abnormalities (arrhythmias).
Arrhythmias can occur as a result of the following:
5) A too rapid impulse production – sinus tachycardia;
• Rapid heart rate, more than 100 beats per minute, calculated by
counting the number of R-R intervals.
• Causes – increased body temperature (fever), sympathetic
stimulation of the heart or toxic condition of the heart.

78
6) A too slow impulse production – bradycardia.
• A slow heart rate, below 50 bpm.
• Causes – trained athletes have an increased cardiac output per beat
and vagus stimulation with the inhibitory effect of parasympathetic
stimulation.

79
 7) Irregular impulse discharge, for instance, an extra systole.

8) Blocking of the impulse conduction.

The causes can lie in the SA node, the atria, the AV node or in the ventricles.
Certain arrhythmias can be created as a result of the changes in activity of the
vagus nerve which serves the SA node.

Abnormalities of the ECG form

These abnormalities are usually caused by ischaemia, an injury or necrosis of
the myocardium.
Ischaemia is a condition of an oxygen shortage. An injury indicates that too
little energy is available to ensure the normal maintenance of the cardiac
muscle and especially of the cell membrane. Necrosis indicates that part of the
myocardium has died off to create an infarct.

Myocardial infarction
Infarction of the myocardium is usually the result of the closing off of one or
more of the coronary arteries (atherosclerosis). This is caused by exposure to
certain risk factors, such as the following:
6) Too high fat (cholesterol) ingestion.
7) Smoking.
8) Stress.
9) Physical inactivity.
10) Inherited factors (high cholesterol, etc.).

These risk factors can lead to the depositing of fat in the intima (innermost layer)
of the coronary artery (among others). The invasion of connective tissue and

80
later, calcification of the fatty deposits leads to the formation of hard plaques
which finally constrict the coronary blood vessel so that the blood supply to a
part of the myocardium is reduced or even completely occluded (cut off). This
is a gradual process that occurs with the passage of time.
Acute coronary occlusion can occur if the hard atherosclerotic plaques break
through the intima and create a blood clot which then suddenly cuts off the
blood supply to a certain part of the myocardium. If a small blood clot breaks
away and occludes the artery some distance further on, it is called an embolus.
Sometimes a spasm of the coronary artery as a result of irritation of the wall
can cause acute occlusion of the coronary artery.
Infarction begins to develop if less than 1.3 ml oxygen per 100 g tissue is being
supplied to the cardiac muscle.
A vicious circle usually develops following the occlusion of a blood vessel. A
part of the myocardium becomes ineffective as a result of the decrease of the
perfusion in it.
This causes the pumping action of the heart to decrease. The undamaged part
of the heart has to work harder to maintain the pumping function, with the result
that more oxygen and thus better perfusion is needed, which in turn cannot be
supplied by the weakened heart. Less oxygen reaches the infarcted area
because it is being used by the more active cardiac muscle, with the result that
tissue damage increases in the heart. This leads to a decrease in the efficient
working of the heart, which once again leads to a poorer oxygen supply.
The following are the usual causes of death:
5) A decrease in cardiac output.
6) Pulmonary congestion (oedema of the lungs).
7) Tearing of the infarct, with the result that blood collects in the pericardial
space (in the pericardium or cardiac sac). (The heart is compressed so
that it does not receive blood, and thus cannot pump it out [cardiac
tamponade].)

81
8) Fibrillation of the ventricles. (This is the spontaneous formation of action
potentials through the damaged tissues, which renders the contraction
of the heart uncoordinated to the extent that the pumping function is lost.)

Electrocardiographic effects of myocardial ischaemia

Ischaemia of the cardiac muscle is represented by an inverted T-wave. It is a
non-specific finding that can be associated with a whole number of other normal
and abnormal conditions. An ischaemic T-wave usually has, in contrast to other
cases, a sharp peak and is also symmetrical.

The reversal of the T-wave of the electrocardiogram as a result of ischaemia

Electrocardiographic effects of a myocardial injury

A myocardial injury as a result of infarction usually occurs only in the inner or
outer muscular layer of the ventricle wall.

82
1st Frame Normal ECG.
2nd Frame Hours after an infarction, the ST-segment has become
elevated.
3rd Frame Hours or days after an infarction, the T-wave is inverting and
the Q-wave is becoming larger.
4th Frame Days or weeks after an infarction, the ST-wave is almost back
to normal, but the T-wave remains inverted.
5th Frame Weeks to months after an infarction, the T-wave has become
upright again, and the only residual of a myocardial infarction
may be an abnormally large Q-wave.

Method
Recording an ECG
• Explain to the person what you propose to do.
• Ensure that the person rests for about 15 minutes before you undertake the
ECG recording.
• Ask the person to undress sufficiently for you to apply the limb and chest
leads.
• He or she should lie on a couch in a warm room in a comfortable supine
position.
• Necklaces, bracelets and the patient’s wristwatch should be removed.
• A hairy chest will need a shave.
• The person must be relaxed during the procedure.

83
Placing of the electrodes
• Before placing the limb electrodes, rub some jelly on the person’s skin
on the inner aspects of the wrists and ankles.
• Attach the limb leads on the electrodes.
• Now go to the chest leads. The six electrodes should be located in the
intercostal spaces. In women, the chest leads should always go below
the breast. Before positioning the electrodes, apply some jelly to the
patient’s chest. Use a small amount at each electrode position and rub
well.
• Place chest leads as follows:

1. V1 – right edge of sternum, 4th interspace

2. V2 – left edge of sternum, 4th interspace
3. V3 – halfway between V2 and V4
4. V4 – midclavicular line, 5th interspace
5. V5 – anterior axillary line, at the level of V4
6. V6 – midaxillary line, at the level of V4
• Record the ECG.
• Remove the electrodes and clean off any gel remaining on the
person’s skin.

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Answer the following questions:
1. Describe the recording of a good ECG.
2. Sketch and describe a normal ECG registration.
3. Discuss the following arrhythmias according to the changes seen on the
ECG:
a. sinus bradycardia
b. sinus tachycardia
4. Discuss the changes on the ECG registration as seen with a myocardial
infarction and discuss the causes and risk factors of this disease.

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 Where applicable

FUNCTIONING OF ENZYMES

Objective
When you have completed this assignment you should be able to determine
the influence of the following on reaction rates of an enzyme catalysed reaction:
Substrate concentration.
Enzyme concentration.
Time.
Temperature.

The enzyme used is catalase prepared from blood. The substrate is hydrogen
peroxide.

Enzymes
Enzymes are biological catalysts which influence the chemical processes in
living cells. They determine the speed of biochemical reactions, direct and
select the metabolic directions.

Information about the extra cellular environment is transferred to the cells by

hormones which react through a change in the activity or concentration of a
relevant enzyme. Through linkage with the hormonal system, the cellular
enzyme plays an important role in the maintenance of homeostasis.

Enzyme structure
Enzymes are protein molecules. Many enzymes are inactive in the absence of
small amounts of other substances known as cofactors. In some cases, the
cofactor is a trace metal, such as magnesium, iron, zinc, or copper. Binding of
one of the metals to an enzyme alters the enzyme’s conformation so that it can
interact with the substrate (reactant in the enzyme-mediated reaction). In other

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cases, the cofactor is an organic molecule that directly participates as one of
the substrates in the reaction, in which case the cofactor is termed a coenzyme.
A single coenzyme molecule can be used over and over again to transfer
molecular fragments from one reaction to another. Coenzymes are derived
from vitamins, for example, NAD+ and FAD derived from the B-vitamins niacin
and riboflavin respectively.

Multi-enzyme reactions
Metabolic pathway is the sequence of enzyme-mediated reactions leading to
the formation of a particular product. Each reaction produces only a small
change in the structure of the substrate. By such a sequence of small steps, a
complex chemical structure, such as glucose, can be transformed to the
relatively simple molecular structures of carbon dioxide and water. Since
different enzymes have different concentrations and activities, it would be
extremely unlikely that the reaction rates of all these steps will be exactly the
same. Therefore, one step is likely to be slower than all the others. This step
is known as the rate-limiting reaction. By regulating the concentration or activity
of the rate-limiting enzyme, the rate of flow through the whole pathway can be
increased or decreased.

Enzyme functioning
The rate of an enzyme-mediated reaction depends on substrate concentration
and on the concentration and activity of the enzyme that catalyses the reaction.
Biological reactions can also occur in the absence of enzymes, but then only at
a very slow rate. Enzymes accelerate biochemical reactions by binding with
the substrate. The formed enzyme substrate complex decreases the activation
energy of the reaction so that it can take place more easily.

The product is formed without the enzyme changing.

E + S → ES → E + P

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Factors that influence enzyme activity
The rate of enzyme functioning is influenced by a number of factors.
Substrate concentration
If the temperature and pH are held optimally, an increase in substrate
concentration will increase the rate of the enzyme-catalysed reaction. As soon
as enough substrate is present to form a complex with all the enzyme
molecules, the reaction rate will reach a plateau and remain constant. The rate
of reaction is then independent of the substrate concentration.
Enzyme concentration
At optimal concentration and pH an increase in enzyme concentration will
create a rectilinear increase in reaction rate until the substrate has compounded
with an enzyme molecule. Then the reaction rate will remain constant.
Product concentration
In some enzyme-catalysed reactions an increase in the concentration of the
product can inhibit the enzymatic activity. The reaction rate will therefore
decrease. A product can therefore have the ability to control its own synthesis.
This is mediated through covalent or allosteric modulation.
pH
Each enzyme has a specific pH at which its catalytic function is maximal. This
optimal pH for most enzymes is neutral or slightly acid. It can be as low as 1.5–
2.0 (pepsin) or as high as 8– 11 (trypsin).
Temperature
In mammals the optimal temperature for enzyme activity is about 40°C. An
increase of 10°C usually doubles the activity of an enzyme. Because enzymes
are usually proteins, temperatures higher than 40°C cause denaturing of these,
with a subsequent decrease in activity.

Catalase
The enzyme catalase which will be used in this experiment is a composite
enzyme (iron-porphyrine compound). It catalyses the breakdown of hydrogen
peroxide into water and oxygen.

2H2O2 catalase 2H2O + ½ O2

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One molecule of catalase is able to break down 5 000 000 molecules of
hydrogen peroxide in one minute at a temperature of 0°C. Catalase is found
together with other enzymes in intracellular organelles (peroxisomes). It occurs
in all cells of the body.

Apparatus
Latex gloves
Lancet
Sali pipette
50 ml measuring cylinder
Cotton wool
Alcohol
Glass pipettes 6 - 8
Water baths: 37° and 50°
Bucket with ice
50 ml burette
Erlenmeyer flask
Glass beakers: 1 X 100 ml

Reagents
Catalase
0.24% hydrogen peroxide, pH 7
2 N sulphuric acid
0.05 N potassium permanganate

Preparation of catalyse of human blood

Cleanse the fingertip with pure alcohol and prick with a lancet. As soon as the
drop of blood is large enough, suck 0.04 ml into a blood pipette and bring to the
mark with a piece of filter paper. Transfer to 50 ml ice-cold water and rinse the
pipette three or four times. Make sure that the blood is well mixed.

Method
In this experiment we determine the concentration of hydrogen peroxide by a
titration with potassium permanganate. The titration value of the control tube

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gives an indication of the peroxide concentration at the beginning of the
experiment. The titration value of the test tubes is once again an indication of
the amount of peroxide that remains at the end of the experiment, while the
difference between the control and the experiment indicates the concentration
that has been destroyed.

The influence of the enzyme concentration

Pipette 5 ml hydrogen peroxide into each of six test tubes. With intervals of 30
seconds, place 0.1. 0.2. 0.4 and 0.8 ml enzyme into the first four tubes
respectively and shake well. Add 5 ml sulphuric acid to the experimental and
the control tubes, exactly five minutes after the addition of the enzyme. The
sulphuric acid destroys the enzyme and thus ends the reaction.

Titrate one of the control tubes with potassium permanganate to a light pink
colour. Titrate the four test tubes and the control tube to the same pink colour
and note the titration figure.

The influence of the substrate concentration

Take six test tubes and mark them 1 - 6. Pipette 5 ml hydrogen peroxide into
test tubes 1 and 2; 2.5 ml into tubes 3 and 4 and 1.25 ml into tubes 5 and 6.
Add distilled water until the volume in tubes 3 to 6 is also 5 ml. Tubes 1. 3 and
5 serve as controls. With intervals of 30 seconds, add 0.5 ml enzyme to tubes
2. 4 and 6. After exactly five minutes, add 5 ml sulphuric acid to all six tubes.
Titrate with potassium permanganate.

The influence of time

Pipette 5 ml hydrogen peroxide into five separate test tubes. To the first tube
add 5 ml sulphuric acid followed by 0.5 ml enzyme. This tube serves as the
zero time control. Now add to the other tubes 0.5 ml enzyme and terminate the
reaction by the addition of 5 ml sulphuric acid after 1. 3. 5 and 10 minutes
respectively. Titrate the tubes with potassium permanganate.

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The influence of temperature
Place 5 ml hydrogen peroxide in six separate test tubes. Two tubes serve as
control and four as experimental tubes. Place all tubes (experimental and
control) in the water bath at the relevant temperatures (4°C, 22°C, 37°C and
50°C) for six minutes. Thereafter, place 0.5 ml enzyme in the four experimental
tubes. No enzyme is added to the control tubes which can be incubated at any
two different temperatures. Place all test tubes again at the relevant
temperatures and, after exactly five minutes, add 5 ml sulphuric acid to both the
test and the control tubes, and shake thoroughly. Titrate all tubes with
potassium permanganate until a light pink colour appears and note the
beginning and end titration reading of each tube.

Instruction
Tabulate your results and draw the following graphs:

1. Enzyme concentration against titration figures (enzyme activity).

2. Substrate concentration against titration figure.
3. Reaction rate against titration figure.
4. Temperature against titration figure.

Explain all the results. Compare your results with those of your classmates and
describe how they compare.

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 HISTOLOGY

A. Histology of the epithelia, connective tissue, cartilage and bone

Not one cell contains all the metabolic organelle to fulfil all the bodily functions;
therefore through the process of differentiation, cells develop certain
characteristic properties. There are trillions of cells in the body but
approximately only 200 types. Cells combine to form tissue. Histology is the
study of tissue.

Four basic tissue types exist:

• Epithelial tissue.
• Connective tissue.
• Muscular tissue.
• Nerve tissue.

Epithelial tissue
A layer of cells that form borders or demarcations with specific properties:
• Covering all surface areas belonging to exposed bodies, e.g. skin surfaces.
• Fulfils respiratory, reproductive, urinary and digestive functions as well as
internal cavity, e.g. chest cavity, ventricles of the brain, eyes, inner ear and
inner surface of blood vessels.
• Classified by number and amount of cells and the form of the exposed cells,
namely:
Single layered – very thin epithelium, not renowned for protection but is
characteristic in areas where absorption and secretion take place. It can
be further divided into three types, namely:
• Squamous epithelial cells.
• Cubical epithelial cells.
• Cylindrical epithelial cells.
Multi-layered – contains numerous layers of cells covering the basement
membrane and are suited for protective, mechanical and chemical
functions. Three different types exist, namely:

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 • Multi-layered paving epithelium.
• Multi-layered ciliated epithelium.
• Multi-layered cubical epithelium.

Properties
• Consists of cells.
• Has basilar surface where it attaches to underlying tissue.
• Contains basement membrane.
• Contains no blood vessels as it receives nutrition by means of
diffusion/absorption.
• Damaged or lost epithelial cells are replaceable.

Functions
• Offers protection against dehydration.
• Controls permeability by means of selective absorption or secretion, which
in turn is modified and regulated by hormones.
• Offers sensation due to it containing numerous sensory neurons.
• Offers specialised secretions – from glandular cells, namely exocrine and
endocrine secretions termed as hormones.
• Contains cilia (outgrowths) enlarging surface area as in the trachea where
coordinated movements cause particles, e.g. dust, pollen, mucous,
pathogens to move upwards and be coughed out.

1) Simple squamous epithelium

Cells are hexagonally shaped, resembling fried eggs. The nucleus is small and
located centrally. Cells are very thin therefore cells are very fragile. A single
layer of cells cannot provide much mechanical protection, and they are found
only in protected areas inside the body. They line internal compartments and
passageways, including the ventral body cavities, the chambers of the heart
and all blood vessels. The simple squamous epithelium reduces friction,
controls permeability and is necessary for absorption and secretion.

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2) Stratified squamous epithelium
It has several layers of cells covering the basement membrane. Stratified
epithelia are generally found in areas subject to mechanical or chemical
stresses, such as the surface of the skin and the lining of the mouth. Cells are
small and flattened in the direction of the free surface. Cells on the basement
membrane are more cubical in form/shape. These cells can be found in the
mouth cavity and oesophagus and offer physical protection against abrasions,
pathogens and chemical substances. Has protein keratin that provides support
and water resistance to upper surface.

3) Simple cuboidal epithelium

Singular cell layer with the nuclei near the centre of each cell. These nuclei are
big and round. These cells provide limited protection and occur in regions
where secretions or absorption take place. In the pancreas and salivary glands,
simple cuboidal epithelia secrete enzymes and buffers and line portions of the
ducts that discharge those secretions. The thyroid gland contains chambers
called thyroid follicles that are lined by a cubical secretory epithelium. Thyroid
hormones accumulate within the follicles before being released into the blood
stream.

4) Stratified cuboidal epithelium

These cells are relatively rare. They are located along the ducts of sweat
glands and in the larger ducts of the mammary glands. They tolerate
considerable stretching, are situated in regions of the urinary system, such as
the bladder, where large changes in volume occur.

5) Simple columnar epithelium

Cells are longer than they are wide. The nuclei are crowded into a narrow band
close to the basement membrane. The height of the epithelium is several times
the distance between two nuclei. A singular columnar epithelium typically
occurs where absorption or secretion is underway, such as inside the small
intestine. Inside the stomach and large intestine, the secretions of singular
cylindrical epithelium provide protection from chemical stresses.

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6) Pseudo stratified columnar epithelium
Portions of the respiratory tract contain a columnar epithelium that includes
several different cell types with varying shapes and functions. Because the
distances between the cell nuclei and the surface vary, the epithelium appears
to be layered or stratified. It is not truly stratified because all the epithelial cells
contact the basement membrane. Pseudo stratified columnar epithelial cells
typically possess cilia. This type of epithelium lines most of the nasal cavity,
the trachea, bronchi and portions of the male reproductive tract.

7) Stratified columnar epithelium

These cells are relatively rare, providing protection along portions of the
pharynx, epiglottis, urethra and anus as well as along a few large excretory
ducts. The epithelium may have either two layers or multiple layers. When
multiple layers exist, only the superficial cells are columnar.

Epithelium cells are examined for a variety of reasons – to indicate cancer or to

identify the pathogens involved in an infection. Cells taken from the tip of the
cervix are used in a Pap test (named after Dr George Papanicolau) to test for
cervical cancer. Epithelium cells from amniotic fluid (the fluid that surrounds
the developing foetus) are tested to determine if the foetus has a genetic
abnormality, such as Down syndrome, that affects the number and structure of
chromosomes.

CONNECTIVE TISSUES
Connective tissues include tissues such as bone, fat and blood, which are quite
different in appearance and function. Nevertheless, all connective tissues have
three basic components:
1. Specialised cells.
2. Extracellular protein fibres.
3. A fluid known as the ground substance.

The extracellular fibres and ground substance constitute the matrix that
surrounds the cells. Whereas cells make up the bulk of epithelial tissue, the

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extracellular matrix typically accounts for most of the volume of connective
tissues.

Connective tissues are found throughout the body but are never exposed to the
environment outside the body. Many connective tissues are highly vascular
and contain sensory receptors that provide pain, pressure, temperature and
other sensations. The functions of connective tissue include:
• Establishing a structural framework for the body.
• Transporting fluids and dissolved materials from one region of the body to
another.
• Protecting delicate organs.
• Supporting, surrounding and interconnecting other tissue types.
• Storing energy reserves, especially in the form of lipids.
• Defending the body from invading microorganisms.

Three basic types of fibres are found in connective tissue:

• Reticular fibres.
• Collagen fibres.
• Elastic fibres.

1) Reticular connective tissue

Contains the same protein subunits as collagen fibres, but they are arranged
differently. These fibres are thinner than collagen fibres and they form a
branching, interwoven framework that is tough but flexible. Because they form
a network rather than share a common alignment, reticular fibres can resist
forces applied from many different directions. This interwoven network, called
a stroma, stabilises the relative positions of the functional cells. It can be found
in the liver, spleen, bone marrow and lymph nodes. It contains macrophage
and fibroblast cells.

2) Collagen connective tissue

Collagen fibres are long, straight and unbranched. These are the most common
fibres in connective tissue proper. Each collagen fibre consists of a bundle of

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fibrous protein subunits wound together like the strands of a rope. Like a rope,
a collagen fibre is flexible, but it is stronger than steel when pulled from either
end. Tendons, which connect skeletal muscles to bones, consist almost
entirely of collagen fibres. Typically ligaments are similar to tendons, but they
connect one bone to another. Tendons and ligaments can withstand
tremendous forces. Uncontrolled muscle contractions or skeletal movements
are more likely to break a bone than to snap a tendon or ligament. Collagen
fibre provides firm attachment, withstands high tension, stabilises relative
position of muscles and reduces friction between muscles.

3) Elastic connective tissue

Elastic fibres contain the protein elastin. Elastic fibres are branched and wavy.
After stretching, they will return to their original length. Elastic ligaments are
dominated by elastic fibres. They are relatively rare but have important
functions, such as interconnecting the vertebrae. Elastic fibres form a network
and fat cells are present between the fibres. Elastic connective tissue can be
found in the walls of blood vessels, respiratory tract and between the vertebrae.

CARTILAGE
Cartilage is also called supporting connective tissue, because it provides the
framework that supports the rest of the body. It contains no blood vessels
therefore nutrition takes place by diffusion through the matrix. The matrix is a
firm gel that contains the cartilage cells, namely chondrocytes. Chondrocytes
are contained within small pockets called lacunae (lacus means pool).

Three types of cartilage occur:

• Hyaline.
• Elastic.
• Fibrous.

1) Hyaline cartilage
The most common type of cartilage is hyaline. The matrix of hyaline cartilage
contains closely packed collagen fibres, making it tough but somewhat flexible.

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Because the fibres do not stain well, they are not always apparent in light
microscopy. Examples of this type of cartilage in the adult body include the
connections between the ribs and the sternum, the nasal cartilage and the
supporting cartilage along the conducting passageways of the respiratory tract.
The articular cartilage covers opposing bone surfaces within many joints, such
as the elbow and knee. It provides sturdy and slightly flexible support. It
reduces friction between bony surfaces.

2) Elastic cartilage
Contains numerous elastic fibres that make it extremely resilient and flexible.
Elastic cartilage forms the external flap of the outer ear, the epiglottis and airway
to the middle ear cavity and small cartilage in the larynx. Elastic cartilage
provides support and resists bending well, making it elastic.

3) Fibrous cartilage
Has little ground substance and its matrix is dominated by collagen fibres. The
collagen fibres are densely interwoven, making this tissue extremely durable
and tough. Fibro-cartilaginous pads lie between the spinal vertebrae, between
the pubic bones of the pelvis and around or within a few joints and tendons. In
these positions, fibrocartilage resists compression, absorbs shocks and
prevents damaging bone to bone contact. Cartilages in general heal poorly and
damaged fibrocartilages in joints such as the knee can interfere with normal
movements.

Fibrocartilages and joint injuries

Several complex joints, including the knee, contain both hyaline cartilage and
fibrocartilage. The hyaline cartilage covers bony surfaces, and fibrocartilage
pads in the joint prevent bone contact during movement. Injuries to these joints
can produce tears in the fibrocartilage pads, and these tears do not heal.
Eventually, joint mobility is severely reduced. Surgery generally produces only
a temporary or incomplete repair.
Recent advances in tissue culture have enabled researchers to grow
fibrocartilages in the laboratory, which can be inserted into damaged joints. In
the future, this technique may be used to treat severe joint injuries in humans.

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BONE
Approximately a third of the matrix consists of collagen fibres. The remaining
matter is a mixture of calcium salts, phosphates and carbonates. Minerals are
organised around the collagen fibres. It is strong, slightly flexible and resistant
against crumbling. Bone cells (osteocytes) appear in the lacunae of the matrix.
Lacunae occur typically around the blood vessels. Canaliculi (little canals) form
a network for the exchange of material between blood vessels and the
osteocytes. Bone surface is surrounded by the periosteum, which consists of
an outer fibrous and inner cellular layer. The periosteum helps with the
attachment of the bone to surrounding tissues and associated tendons and
ligaments. Cellular layer helps with bone growth and recovery. Bone
undergoes changes throughout life, namely growing stronger and thicker with
exercise whilst becoming thin and brittle with inactivity (paralysis). Bone stores
calcium and serves as support.

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B. Histology of the nervous system

NEURONS

Neural tissue contains two basic types of cells:

1. Neurons which are responsible for the propagation of electrical impulses
from one part of the body to another. These are the basic information
and process units of the nervous system.
2. Neuroglia (glial cells) perform functions such as providing a supporting
framework for neural tissue and help supply nutrients to neurons.

Neurons are fully differentiated and they have a very limited ability to repair
themselves after injury. (Incapable of dividing under normal circumstances.)

A typical neuron consists of:

- A cell body or soma.
- A large nucleus with a prominent nucleolus.
- Dendrites – various branching processes (projections or outgrowths)
extending from the soma. Dendrites receive information, typically from
other neurons.
- Axons conduct impulses to synaptic terminals (away from the cell
body/soma).
- Axons end in synaptic terminals which form a synapse with the dendrites
of the next neuron, muscle tissue or endocrine tissue.
- They play an important role in impulse conduction.
- The cytoplasm includes mitochondria, Golgi apparatus and rough
endoplasmic reticulum.

Function
Impulse conduction.

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NEUROGLIA
There are four types of neuroglia (neuro=nerve, glia=glue) in the central
nervous system, each with its own functions and distinctive features.

1. Astrocyte
Distinctive features:
• Star shaped cells with numerous outgrowths.
Functions:
• Maintaining the blood-brain barrier.
• Provides structural support.
• Repairing damaged neural tissue.
• Nutritional function.

2 Oligodendrocytes
Distinctive features:
• Possess slender cytoplasmic extensions.
• Cell bodies are smaller and they have fewer processes than astrocytes
have.
Functions:
• Myelinate CNS axons.
• Provide structural framework.

3. Microglia
Distinctive features:
• Small cells with cytoplasmic processes that have many fine branches.
• They are capable of migrating through neural tissue.
Functions:
• Remove cell debris, wastes and pathogens by phagocytosis.

4. Ependymal cells
Distinctive features:
• Single layer epithelial cells, cuboidal to columnar in shape.
• Cilia at the luminal surface.

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 • Do not rest on basement membrane, unlike other epithelia.
Functions:
• Line ventricles (brain) and central canal (spinal cord).
• Assist in production, circulation and monitoring of cerebrospinal fluid.

The two types of neuroglia in the peripheral nervous system are:

1. Satellite cells.
• Surround the neuron cell bodies in peripheral ganglia.
2. Schwann cells.
• Form a sheath around every peripheral axon.
• Responsible for myelination of some peripheral axons.
• Participate in repair process after injury.

THE CEREBRAL CORTEX

Distinctive features:
• Neurons of the cerebral cortex consist of five different cell types which
are found in different layers.
• Pyramidal cells.

Pyramid-shaped cell bodies

Apex of cell points to the surface of the cortex and a thick branched dendrite
protrudes to the surface of the cortex.
Thin axon from bottom of cell to adjacent white matter.
• Stellate (granule cells)
Small neurons with short vertical axons and different short branched
dendrites (star shaped cells).

• Cells of Martinotti
Small cells with only a few short dendrites.
Axons that lead to the surface where they split and lie horizontally with
the surface.

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• Fusiform cells
Spindle cells that lie rectangular to the surface of the cerebral cortex originate
axons at the cell body side and stretch to the surface.

Dendrites originate on every side of the cell body and branch in deeper and
more to the surface layers.
• Horizontal cells of Cajal
Small spill-formed cells which lie parallel to the surface.
Found in most superficial layers.
Axons progress laterally to form a synapse with dendrites of the pyramidal
cells.
Cortex also consists of neuroglia cells (astrocytes, oligodendroglia and
microglia).
Cerebral cortex consists of a further six layers where integration of impulses
takes place.

Functions:
The main intellectual integration area of the brain.

CEREBELLUM
Distinctive features:
• White matter on the inside (myelinated fibres).
• Myelinated fibres branch from the nucleus of the cerebellar folds.
• These fibres are incoming fibres to the cortex and efferent fibres from
the cortex.
• The cortex is divided into three layers:
o Outer molecular layer with relatively few cells and very few fibres
which lie horizontally.
o Inner granular layer with many small cells.
o Middle layer, which consists of Perkunje cells.
• Dendrites of Perkunje cells go up to the molecular layer.

Functions:
• Plays an important role in motor reactions.

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 • Balance.

THE SPINAL CORD

Distinctive features:
• Integrated with the brain.
• The anterior median fissure and the posterior median sulcus mark the
partition between the left and right side of the spinal cord.
• White matter has a considerable amount of myelinated and
unmyelinated axons.
• Grey matter consists mainly of neuronal cell bodies, glial cells and
demyelinated axons, surrounds the central canal and has an “H” or
butterfly shape.
• Branches of grey matter to the outside are called roots.
Functions:
• Integration area for reflex movement.
• Controls sensory stimuli to the brain and motor stimuli from the brain to
the rest of the body.

THE ENDOCRINE SYSTEM

Hypophysis (Pituitary gland)
Properties:
➢ The anterior pituitary (adenohypophysis) can be subdivided into three
regions:

- Pars distalis.
- Pars tuberalis.
- Pars intermedia.

• The pars distalis

- Largest portion of entire pituitary gland.
- Contains two cell types:
- Chromophobe and chromophile cells.
- Chromophile cells are divided in alpha (acidophilic) and beta
(basophilic).

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 - Chromophobe cells are the inactive forms of acidophilic and basophilic
cells.
• Pars tuberalis
- Wraps around the adjacent portion of the infundibulum.
• Pars intermedia
- Forms a narrow band adjacent to the posterior pituitary.

➢ The posterior pituitary (neurohypophysis or pars nervosa):

- Contains the axons of hypothalamic neurons.
➢ The pituitary gland secretes nine important peptide hormones:
• Anterior pituitary
- Pars digitalis.
- TSH, ACTH, FSH, LH, PRL, GH.
- Pars tubularis and pars intermedia.
- MSH (melanocyte stimulating hormone).
• Posterior pituitary
- ADH and oxytocin.

THYROID GLAND
Properties:
• The thyroid gland contains large numbers of thyroid follicles (functional
unit): a round structure which consists of a single layer of cuboidal
epithelium cells which are bound to a basal membrane.
• Follicles vary in size.
• Follicles are filled with colloid (liquid with much protein).
Functions:
Synthesis, storage and secretion of thyroid hormones.

ADRENAL GLAND
Divided into two parts:
A superficial adrenal cortex and inner adrenal medulla.

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The cortex consists of three distinct regions:
• Zona Glomerulosa
Produces mineralocorticoids – steroid hormones that affect the
electrolyte composition of body fluids.
Aldosterone regulates sodium ion balance.
• Zona Fasciculata
Produces glucocorticoids – plays an important role in glucose
metabolism.
Cortisol is secreted under the influence of ACTH, which is secreted by
the anterior pituitary gland.
• Zona Reticularis
Produces small quantities of androgens (sex hormones).

The adrenal medulla secretes catecholamines, adrenaline and noradrenaline

under direct influence of the central nervous system.

THE PANCREAS
The pancreas consists of the exocrine and endocrine pancreas.
• The exocrine pancreas, roughly 99% of the pancreatic volume, consists
of clusters of glandular cells (called pancreatic acini) and their ducts.
Together, the glandular and duct cells secrete large quantities of an
alkaline, enzyme-rich fluid. This secretion reaches the lumen of the
digestive tract by travelling along a network of secretory ducts.
• The endocrine pancreas consists of small groups of cells scattered
among the exocrine cells. The endocrine clusters are known as
pancreatic islets, or Islets of Langerhans. The Islets of Langerhans
account for only about 1% of all cells in the pancreas. Nevertheless, a
typical pancreas contains roughly 2 million islets.
• Like other endocrine tissue, the pancreatic islets are surrounded by an
extensive, fenestrated capillary network that carries its hormones into
the blood stream.
• Each islet contains four cell types:

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o Alpha cells – produce the hormone glucagon. Glucagon
raises the blood glucose levels by increasing the rate of glycogen
breakdown and glucose release by the liver.
o Beta cells – produce the hormone insulin. Insulin lowers
blood glucose levels by increasing the rate of glucose uptake and
utilisation by most body cells and increasing glycogen synthesis
in skeletal muscles and the liver.
o Delta cells – produce a peptide hormone identical to
growth hormone – inhibiting hormone. This hormone suppresses
the release of glucagon and insulin by other islet cells and slows
the rates of food absorption and enzyme secretion in the digestive
tract.
o F cells – produce the hormone pancreatic polypeptide. It
inhibits gallbladder contractions and regulates the production of
some pancreatic enzymes.

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C. Histology of muscle tissue

Muscle tissue is specialised to enable movement of the body. Muscle cells are
specialised to contract, and to do that effectively, muscle cells are long and are
therefore called fibres rather than cells. Each cell in skeletal muscle tissue is a
single muscle fibre.

Muscle tissue has its own terminology. Cytoplasm is called sarcoplasm, cell
membrane is called sarcolemma and the endoplasmic reticulum is called the
sarcoplasmic reticulum. Contractile units are called sarcomeres; interactions
between the myofilaments of the sarcomeres are responsible for muscle
contraction. Skeletal tissue forms skeletal muscles, organs that contain
connective tissues, nerves and blood vessels. Without muscle tissues, nothing
in the body would move and no body movement could occur.

Three types of muscle tissue exist:

Skeletal muscle – striated voluntary muscle.
Cardiac muscle – striated involuntary muscle.
Smooth muscle – non-striated involuntary muscle.

SKELETAL MUSCLE
Types:
Slow-oxidative (red) muscle fibres
• Primary source of ATP production is oxidative phosphorylation.
• Cross-sectional diameter is small.
• Contraction speed is slow.
• Fatigue resistance is high, thus the rate of fatigue is slow.
• Contain red pigment myoglobin; myoglobin content is high.
• Extensive network of capillaries.
• Contain many mitochondria.
• Glycolytic enzyme concentration in sarcoplasm is low.
• Substrates used for ATP generation during contraction are lipids,
carbohydrates and amino acids (aerobic).

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• Myosin-ATPase activity is low.
• Motor unit size and size of innervating fibre are small.

Fast-oxidative-Glycolytic (intermediate) muscle fibres

• Primary source of ATP production is oxidative phosphorylation.
• Cross-sectional diameter is intermediate.
• Contraction speed is fast.
• Fatigue resistance is intermediate, thus the rate of fatigue is intermediate.
• Contain red pigment myoglobin; myoglobin content is high (myoglobin
content is low according to Martini).
• Intermediate network of capillaries.
• Contain intermediate mitochondria.
• Glycolytic enzyme concentration in sarcoplasm is intermediate to high.
• Substrates used for ATP generation during contraction are primarily
carbohydrates (anaerobic).
• Myosin-ATPase activity is high.
• Motor unit size and size of innervating fibre are intermediate.

Fast-Glycolytic (white) muscle fibres

• Primary source of ATP production is glycolysis.
• Cross-sectional diameter is large.
• Contraction speed is fast.
• Fatigue resistance is low, thus the rate of fatigue is fast.
• Contain white skeletal muscle, myoglobin content is low.
• Limited network of capillaries.
• Contain few mitochondria.
• Glycolytic enzyme concentration in sarcoplasm is high.
• Substrates used for ATP generation during contraction are carbohydrates
(anaerobic).
• Myosin-ATPase activity is high.
• Motor unit size and size of innervating fibre are large.

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The organisation of skeletal muscle
Connective tissue components
• The entire muscle is surrounded by the epimysium, a dense layer of
collagen fibres.
• Skeletal muscle is divided into a series of compartments, each containing a
bundle of muscle fibres called a fascicle.
• Perimysium surrounds the fascicle and contains blood vessels and nerves
that maintain blood flow and innervates the fascicles.
• Within a fascicle, the delicate connective tissue of the endomysium
surrounds the individual skeletal muscle fibres and interconnects adjacent
muscle fibres.
• Endomysium consists of reticular connective tissue, collagen and blood
vessels.
• Amount of connective tissue varies between muscles.
• Inside the muscle fibre, branches of the transverse tubules encircle
cylindrical structures called myofibrils. Myofibrils consist of bundles of
myofilaments, protein filaments composed primarily of thin and thick
filaments. These myofilaments are organised into repeating functional units
called sarcomeres.

Structure
• Sarcoplasm contains hundreds of nuclei, just internal to the cell membrane.
• Filaments are organised in repeating groups. Difference in size, density and
distribution of thick filaments and thin filaments account for the banded
appearance.
• Sarcomere organisation:
o There are dark bands (A bands) – actin and myosin.
o Light bands (I bands) – actin.
o I band has dark lines in the middle – Z line (Zwichenscheibe, -actin).
o A band has a lighter region – H zone (Hensen’s band; myosin). The
central portion of each thick filament is connected to its neighbours
by proteins of the M line (Mittelscheibe).

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• Functional unit of contraction – sarcomere (sarcos, flesh or muscle + meros,
part or small). Z line marks the boundary between adjacent sarcomeres.
Sarcomeres consist of A band and part of I band (till Z line) on each side.

Myofibril
• Each skeletal muscle fibre contains hundreds to thousands of myofibrils –
nuclei just beneath sarcolemma, Golgi apparatus and mitochondria near the
nuclei.
• Myofibrils are bundles of thin and thick filaments. Myofibrils are organised
into repeating functional units called sarcomeres. Each sarcomere has a
resting length of 1.6-2.6 µm.
• Myofibrils consist of bundles of myofilaments (±100), protein filaments
composed primarily of actin and myosin. Actin – thin filament is 5-6 nm in
diameter and 1 µm in length. Actin contains four subunits (proteins) and is
a twisted strand composed of two rows of individual molecules. Two other
proteins, tropomyosin and troponin form the troponin-tropomyosin complex.
A tropomyosin molecule is a double-stranded protein that covers seven
active sites. It is bound to one molecule of troponin midway along its length.
• At either end of the sarcomere, the thin filaments are attached to the Z line.
The I band is formed by actin filaments.
• Myosin – thick filaments are 10-12 nm in diameter and 1.6 µm long. Each
myosin molecule consists of a pair of myosin subunits twisted around one
another. The long attached tail is bound to other myosin molecules in the
thick filament. The free head, which projects outward towards the nearest
thin filament, consists of two globular protein subunits. All myosin molecules
are arranged with their tails pointing toward the M line.

Sarcolemma
• Transverse tubules or t tubules are narrow tubes that are continuous with
the sarcolemma and surround the myofibrils.
• At each end of the skeletal muscle fibre, the myofibrils are anchored to the
inner surface of the sarcolemma.

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Blood vessels
• The connective tissue of the epimysium and perimysium contains the blood
vessels and nerves that supply the muscle fibres.
• Muscle contraction requires tremendous quantities of energy. An extensive
vascular network delivers the necessary oxygen and nutrients and carries
away the metabolic wastes generated by active skeletal muscles.
• The blood vessels and the nerve supply generally enter the muscle together
and follow the same branching pattern through the perimysium.
• Within the endomysium, arterioles supply blood to a capillary network that
surrounds each individual muscle fibre.

CARDIAC MUSCLE TISSUE

• Cardiac muscle tissue consists of a branching network of interconnected
muscle cells.
• Prominent striations resemble those of skeletal muscle; the actin and
myosin filaments are arranged in the same way in both cell types – cardiac
muscle is involuntary.
• Cardiac muscle cells do not rely on nerve activity to start contraction.

Structure
• Connections between cardiac cells occur at specialised regions known as
intercalated discs.
• Cardiac muscle cells are 50-100 µm long and have a diameter of 10-20 µm.
• Cardiac muscle is striated and has A, I, H bands, as well as Z and M lines
as in skeletal muscle.
• A typical cardiac muscle cell has one centrally positioned nucleus, but some
have as many as five.
• Intercalated discs – dark lines, and cross Z line.
• Mitochondria are abundant in sarcoplasm, between myofibrils.

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Intercalated discs
• At an intercalated disc the cell membrane of two adjacent cardiac muscle
cells is extensively intertwined and bound together by gap junctions and
desmosomes.
• Desmosomes attach one cell to another, are very strong and they can resist
stretching and twisting.
• At a gap junction, two cells are held together by an interlocking of membrane
proteins. These are channel proteins and allow ions and small molecules
to move from one cell to another. Therefore, an action potential can travel
across an intercalated disc, moving quickly from one cardiac cell (myocyte)
to another.
• Myofibrils in the two interlocking muscle cells are firmly anchored in the
intercalated disc.
• Because their myofibrils are locked together, two muscle cells can “pull
together” with maximum efficiency.
• Cardiac muscle cells are mechanically, chemically and electrically
connected to each other and the entire tissue resembles a single enormous
muscle cell.

SMOOTH MUSCLE TISSUE

Structure
• Cell is a small, spindle-shaped cell with tapering ends. Ranges from 5-10
µm in diameter and 30-200 µm in length.
• Single, centrally located nucleus.
• When a contraction occurs, the muscle cell twists like a corkscrew.
• Size of smooth muscle cells varies.
• Sarcoplasm contain mitochondria, free ribosomes, Golgi apparatus en some
rough endoplasmic reticulum.
• Myofilaments scattered throughout sarcoplasm – no Z lines or sarcomeres.
• Thin filaments consist of actin and tropomyosin and thick filaments of
myosin. The filaments are organised differently, and smooth muscle tissue
has no striations. Smooth muscle cells have more cross bridges per thick
filament.

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• Smooth muscles around blood vessels in the integumentary system
regulate the flow of blood to the superficial dermis.
• In the digestive system, smooth muscles are arranged in an inner, circular
layer and an outer longitudinal layer. These layers play an essential role in
moving materials along the tract.
• In the cardiovascular system, smooth muscle encircling blood vessels
control the distribution of blood and help regulate blood pressure.

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D. Histology of the digestive system

1. Salivary glands
• Saliva is produced by three pairs of salivary glands, namely parotid salivary
gland, sublingual salivary gland and submandibular salivary gland.
• Saliva watery secretion containing mucus, enzymes (salivary amylase –
breaks down starches), antibodies and an assortment of ions.
• Two types of secretory cells are found in the salivary glands: serous cells
and mucous cells.

Parotid gland
• Consists mainly of serous cells – produces thick serous secretion containing
salivary amylase and antibodies.
• Divided in numerous lobules by connective tissue.
• The parotid gland consists mainly of serous units.
• The secretions are drained by a parotid duct (simple to stratified columnar
epithelium), which empties into the vestibule at the level of the second upper
molar.

Sublingual salivary gland

• Consists mainly of mucous cells – produces a thick watery mucous
secretion that acts as a buffer and lubricant.
• Numerous sublingual ducts open in the oral cavity.
• Interlobular excretory duct is lined by stratified cuboidal epithelium.

Submandibular salivary glands

• Consist of a mixture of serous and mucous secretary units, but mainly
serous.
• Secrete a mixture of buffers, glycoproteins and salivary amylase.
• Mixed units are typical mucous units surrounded by one or more groups of
serous cells.
• Supporting tissue radiates between lobules.
• Contain occasional adipocytes.

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Functions
• Lubricating the mouth.
• Moistening and lubricating materials in the mouth.
• Dissolving chemicals that can stimulate the taste buds and provide sensory
information about material.
• Initiating the digestion of complex carbohydrates before the material is
swallowed.

2. The oesophagus
The wall of the oesophagus contains mucosal, submucosal and muscularis
layers.
• Mucosa contains non-keratinised, stratified squamous epithelium similar to
that of the pharynx and oral cavity.
• Mucosa and submucosa are thrown into large folds that extend the length
of the oesophagus. These folds allow for expansion during the passage of
a large bolus; except when you swallow, muscle tone in the walls keeps the
lumen closed.
• The muscularis mucosae consist of an irregular layer of smooth muscle.
• The submucosa contains scattered oesophageal glands, which produce a
mucous secretion that reduces friction between the bolus and the
oesophageal lining.
• The muscularis externa has inner circular and outer longitudinal muscle
layers.
• There is no serosa (serous membrane), but an adventia of connective tissue
outside the muscularis externa anchors the oesophagus in position against
the dorsal body wall.
• Lamina propria – the loose connective tissue that underlies the mucous
epithelium and forms part of the mucous membrane.
Functions
To carry solid food and liquids to the stomach.

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Stomach
Cardia
• The surface is covered by a single layer of columnar epithelial cells. The
epithelium is a secretory sheet, which produces a carpet of mucus that
covers the interior surfaces of the stomach.
• Included in this epithelial layer are exocrine cells that secrete mucus. The
alkaline mucous layer protects epithelial cells against the acid and enzymes
in the gastric lumen.
• Lamina propria contains lymphatic ducts.
• Lamina propria has shallow depressions called gastric pits with mucus cells
at the base.
Fundus
• The wall of the fundus contains mucosa, submucosa and muscularis
externa and serosa.
• Each gastric pit communicates with several gastric glands, which extend
deep into the underlying lamina propria.
• Muscularis mucosa and muscularis externa of the stomach contain extra
layers of smooth muscle cells in addition to the usual circular and
longitudinal layers.
• The muscular externa has an inner oblique layer of smooth muscle. The
extra layer of smooth muscle strengthens the stomach wall and assists in
the mixing and churning activities essential to the formation of chyme.
• The muscularis mucosae generally contain an outer circular layer of muscle
cells.
• Neck of gastric pit contains mucous cells.
• Gastric glands are dominated by two types of secretory cells: parietal cells
and chief cells.
• Smooth muscle cells of the muscularis mucosae extend through the lamina
propria between the gastric glands.
Functions
• The bulk storage of ingested food.

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• The mechanical breakdown of ingested food and the disruption of chemical
bonds in food material through the action of acids and enzymes to form
chyme.
• Production of intrinsic factor, a glycoprotein whose presence in the digestive
tract is required for the absorption of vitamin B12.
• Parietal cells secrete intrinsic factor and HCl, while chief cells secrete
pepsinogen, an inactive proenzyme.

The small intestine

Duodenum
• This portion of the small intestine is the “mixing bowl” that receives chyme
from the stomach and digestive secretions from the pancreas and liver.
• The wall of the duodenum contains the same layers as those of the fundus
of the stomach.
• The intestinal lining bears a series of transverse folds called plicae (plicae
circularis). Unlike the rugae in the stomach, each plica is a permanent
feature that does not disappear when the small intestine fills.
• The mucosa of the small intestine is thrown into a series of fingerlike
projections, the intestinal villi.
• The intestinal villi are covered by a simple columnar epithelium that is
carpeted with microvilli. Because the microvilli project from the epithelium
like bristles on a brush, these cells are said to have a brush border.
• The lamina propria of each villus contains an extensive network of
capillaries.
• Each villus contains a lymphatic capillary called a lacteal which transports
material that is unable to enter blood capillaries.
• Between the columnar epithelial cells, goblet cells eject mucins (responsible
for the lubricating properties of mucus) onto the intestinal surfaces.
• At the base of the villi are the intestinal glands or crypts of Luberkuhn which
extend deep into the underlying lamina propria.
• The submucosa contains submucosal glands, or Brunner’s glands, which
produce mucus when the chyme arrives.

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Functions
• Digestion and absorption of most substances; mixing and propulsion of
contents.
• Food (protein, carbohydrates and fat) digestion by enzymes.
• Lubrication by mucus.

The large intestine

• Can be divided into three parts – cecum, colon and rectum.
• Colon can be subdivided into the ascending colon, transverse colon,
descending colon and sigmoid colon.
• Although the diameter of the colon is roughly three times that of the small
intestine, its wall is much thinner. The wall has the same layers as the small
intestine.
• The major characteristics of the colon are the lack of villi, the abundance of
goblet cells and the presence of distinctive intestinal glands.
• The mucosa of the large intestine does not produce enzymes; any digestion
that occurs results from enzymes introduced in the small intestine or from
bacterial action.
• Large lymphoid nodules are scattered throughout the lamina propria and
submucosa.
• The muscularis externa of the large intestine is unusual, because the
longitudinal layer has been reduced to the muscular bands of the taenia coli.

Functions
• The mucus provides lubrication as the faecal material becomes less moist
and more compact.
• Absorption of salt and water.
• Storage and concentration of undigested matter before defecation.
• Absorption of vitamins generated by bacteria.

The liver
• The liver can be divided into a right and left lobe.

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• Each lobe is divided by connective tissue into approximately 100 000 liver
lobules.
• Adjacent lobules are separated from each other by an interlobular septum.
• Each lobule consists of hepatocytes that form a series of irregular plates
arranged like spokes of a wheel.
• Within a lobule, sinusoids between adjacent plates empty into the central
vein.
• In addition to typical endothelial cells, the sinusoidal lining includes a large
number of Kupffer cells which engulf pathogens, cell debris and damaged
blood cells.
• Blood enters the sinusoids from small branches of the hepatic portal vein
and hepatic artery proper. Blood then leaves the sinusoids and enters the
central vein of the lobule. The central vein ultimately merges to form the
hepatic veins, which empty into the inferior vena cava.
• Liver secretes bile into bile canaliculi (network of narrow channels) which
extend outward, away from the central vein.
• Bile canaliculi connect with bile ductules which carry duct to bile ducts.
These ducts unite to form the common hepatic duct which leaves the liver.
Functions
• Three major functions: metabolic regulation, haematological regulation and
production of bile.

The pancreas
• Partitions of connective tissue divide the interior of the pancreas into distinct
lobules.
• In each lobule, the ducts branch repeatedly before ending in blind pockets
called pancreas acini.
• Each pancreatic acinus is lined by a simple cuboidal epithelium.
• Pancreatic islets (Islets of Langerhans), the endocrine tissue of the
pancreas, are scattered among the acini. The islets account for 1% of the
cellular population and secrete insulin and glucagon.

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• The exocrine cells include the acinar cells and the epithelial cells that line
the duct system. Together they secrete pancreatic juice, an alkaline mixture
of digestive enzymes, water and ions into the small intestine.
Functions
• Enzymes help with the digestion of carbohydrates, fats, proteins and nucleic
acids.
• Insulin and glucagon help with the regulation of blood glucose levels.
• Alkaline secretions neutralise HCL entering the small intestine from the
stomach.

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E. Histology of the urinary, respiratory and reproductive systems

URINARY SYSTEM
The ureters
Characteristics
• Wall consists of three layers:
o An inner mucosa, consisting of a transitional epithelium and the
surrounding lamina propria.
o A middle muscular layer made up of longitudinal and circular bands
of smooth muscle.
o An outer connective tissue layer that is continuous with the fibrous
renal capsule and peritoneum.
• Has convoluted lumen due to longitudinal mucosal folds.
• Lumen wall consists of a mucosa, muscularis and adventia (fibrosa).
Function
About every 30 seconds a peristaltic contraction sweeps along the ureter
forcing urine toward the bladder.

Urethra
Characteristics
• Consists of a stratified epithelium that varies from transitional at the neck of
the urinary bladder, through stratified columnar at the midpoint, to stratified
squamous near the external urethral meatus.
• Lamina propria is thick and elastic and the mucous membrane is thrown into
longitudinal folds.
Function
Urination is possible through relaxation of the external urethral sphincters.

Bowman’s capsule
Characteristics
• Forms the outer wall of the renal corpuscle and surrounds the glomerulus.

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• The outer wall of the capsule is lined by a simple squamous parietal
epithelium. This layer is continuous with the visceral epithelium, which
covers the glomerular capillaries.
• The capsular space separates the parietal and visceral epithelia.
• The visceral epithelium consists of podocytes (complex processes or feet).
Materials passing out of the blood at the glomerulus must be small enough
to pass between the narrow gaps, filtration slits, between adjacent
podocytes.
• Space opens into the proximal convoluted tubule.
Function
Filtration of plasma – blood pressure forces water and small solutes across the
membrane into the capsular space.

Nephron
Characteristics
• Consists of glomerulus, Bowman’s capsule, proximal and distal convoluted
tubule.
Function
Functional unit of the kidney.

REPRODUCTIVE SYSTEM

Female reproduction system

Histology of the vagina

Characteristics
• Glycogen is a prominent component of the cells in the vaginal epithelium,
reaching its maximum just before ovulation.
• Consists of: epithelium layer, mucosa lamina propria, muscularis mucosa
and fibrosa (adventitia).
• The lining epithelium is non-keratinised stratified squamous whose cells are
rich in glycogen.
• Mucosa has transverse folds.

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• Small blood vessels are numerous throughout the lamina propria.
• The broad lamina propria is moderately dense irregular connective tissue
with excessive elastic fibre.
• The muscularis is made up predominantly of longitudinal and obliquely
arranged bundles of muscle fibre.
• In the adventia are numerous veins – part of a large venous plexus.
Function
Part of birth canal.

Ovaries
Characteristics
• Surface epithelium composed of cuboidal cells, covers the ovary.
• Cortex occupies the greater part of the ovary, contains stroma – type of
connective tissue, in which spindle-shaped fibroblasts predominate.
• The stroma of the medulla is typical dense irregular connective tissue.
• Numerous follicles in various stages of development are embedded in
stroma of the cortex.
• Various parts of follicle:
o Thecae – surrounding follicle.
o Membrane granulosa.
o Large antrum filled with follicular fluid.
o Cumulus oophorus in which is embedded the primary oocyte.
Function
Important for reproduction.

Corpus Luteum
Characteristics
• Endocrine structure, named for its yellow colour (lutea, yellow).
• Wall consists of theca lutein cells.
• The stroma surrounding the corpus luteum is highly vascular.

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Function
Lipids contained in the corpus luteum are used to manufacture steroid
hormones known as progestins, principally the steroid progesterone.

Corpus Albicans
Characteristics
• Also known as white body.
• Pale scar tissue due to degeneration of follicles, especially those that
ovulated.
• Results from proliferation of collagen fibres.

Cervix
Characteristics
• The mucosa of endocervix (cervix uteri) is lined with tall mucus-secreting
columnar epithelium.
• Cervical glands extend deep into the wide lamina propria.
• In the lower part of the cervix, at the os cervics or opening of the cervical
canal into the vaginal canal, the columnar epithelium changes to stratified
squamous.
• Vaginal portion of the cervix – portio vaginalis.
• The muscularis is not as compact as in the body of the uterus.
• Both muscularis and lamina propria are well vascularised.
Functions
• Lower part of the uterus.
• Cervical smear is taken to diagnose cervical cancer.

Mammary gland
Characteristics
• Consists of 15-20 lobes, each of which is an individual compound alveolar
gland with its own lactiferous duct opening onto the surface of the nipple.
• The interlobar stroma is dense collagenous connective tissue and varying
amounts of fat.

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• A lobule contains groups of small tubules lined with cuboidal or columnar
epithelium.
• The tubules are surrounded by loose fine-fibred vascular connective tissue,
with numerous fibroblasts, some lymphocytes, plasma cells and
eosinophils.
Function
Milk production or lactation.

Male reproductive system

Seminiferous tubules and testes

Characteristics
• Septa subdivides testis into series of lobules.
• Seminiferous tubules are distributed among the lobules.
• Each tubule averages about 80 cm in length, and a typical testis contains
nearly a half a mile of seminiferous tubules.
• Each tubule is surrounded by a capsule, and areolar tissue fills the spaces
between the tubules.
• Within the spaces are numerous blood vessels and large interstitial cells
(cells of Leydig).
Functions
• Spermatozoa are produced here.
• Takes place through a process of spermatogenesis – involves mitosis,
meiosis and spermiogenesis.
• Interstitial cells are responsible for the production of androgens, the
dominant sex hormones in males.
• Testosterone is the most important androgen.

Prostate gland
Characteristics
• Alveoli vary in size.
• Lumens are large and typically irregular in contour because of epithelial-
covered folds.

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• The epithelium is simple columnar – with variation, epithelium may be
squamous or cuboidal.
• The fibro-muscular stroma is a distinctive feature of the prostate.
• Smooth muscle fibres and connective tissue can sometimes be
distinguished.
Functions
Produces prostatic fluid that contributes 20-30% of the volume of semen.

Spermatozoa
Characteristics
• Have three distinct regions – head, neck (middle piece) and tail.
• The head is a flattened ellipse containing a nucleus with densely packed
chromosomes.
• At the tip of the head is the acrosomal cap; the middle piece contains
mitochondria.
• The tail is the only flagellum in the human body.

Functions
• The acrosomal cap contains enzymes essential for fertilisation.
• Mitochondrial activity provides the ATP that is needed to move the tail.
• The flagellum moves the cell from one place to another.

RESPIRATORY SYSTEM

Respiratory epithelium
Characteristics
• Pseudo stratified ciliated columnar epithelium with numerous goblet cells
lines the nasal cavity and superior portion of the pharynx.
• The epithelium lining in the inferior portions of the pharynx is a stratified
squamous epithelium similar to that of the oral cavity.
• Lamina propria is the underlying layer that supports the respiratory
epithelium – contains mucous glands.
• Contains goblet cells, stem cells and basement membrane.

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Functions
• This portion of the pharynx, which conducts air to the larynx, also conveys
food to the oesophagus.
• The pharyngeal epithelium protects against abrasion and chemical attack.
• In the nasal cavity, cilia sweep mucus and any trapped debris or
microorganisms toward the pharynx, where it will be swallowed and
exposed to the acids and enzymes of the stomach.

Trachea
Characteristics
• The trachea is a tough, flexible tube with a diameter of about 2.5 cm and a
length of about 11 cm.
• It begins anterior to vertebra C6 and ends at the level of T5, where it
branches to form the right and left primary bronchi.
• The mucosa resembles that of the nasal cavity.
• The submucosa, a thick layer of connective tissue, surrounds the mucosa
and contains mucous glands.
• Mucous glands communicate with the epithelial surface through a number
of secretory ducts.
• Each trachea contains 15 - 20 C-shaped tracheal cartilages.
• Each tracheal cartilage is bound to neighbouring cartilages by elastic
annular ligaments.
Functions
• The tracheal cartilages stiffen the tracheal walls and protect the airway.
• They also prevent its collapse or overexpansion as pressures change in the
respiratory system.
• The open portion of the C-shaped cartilages faces posterior – the tracheal
wall can easily distort when you swallow, permitting large masses of food to
pass through the oesophagus.
• An elastic ligament and the trachealis muscle, a band of smooth muscle,
alter the diameter of the trachea.

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Bronchioles
Characteristics
• Each tertiary bronchus branches several times, giving rise to the finest
conducting branches, terminal bronchioles.
• The lumen of each terminal bronchiole has a diameter of 0.3-0.5 mm.
• The walls of bronchioles, which lack cartilaginous support, are dominated
by smooth muscle tissue.
Functions
• The autonomic nervous system regulates the activity in the smooth muscle
layer and thereby controls the diameter of the bronchioles. Sympathetic
activation – bronchodilatation; parasympathetic stimulation –
bronchoconstriction.
• Bronchioli are responsible for the flow of air to and from the respiratory
exchange surfaces.

Alveolar organisation
Characteristics
• Respiratory bronchioles are connected to alveoli along regions called
alveolar ducts.
• Alveolar ducts end at alveolar sacs.
• Each lung contains about 150 million alveoli, and their abundance gives the
lung an open, spongy appearance.
• An extensive network of capillaries is associated with each alveolus; the
capillaries are surrounded by a network of elastic fibres.
• The alveolar epithelium consists primarily of simple squamous epithelium.
• The squamous epithelium cells, called type 1 cells, are usually thin and
delicate.
• Roaming alveolar macrophages patrol the epithelial surface, phagocytising
any particulate matter that has eluded other respiratory defences and
reached the alveolar surfaces.
• Septal cells, also called type II cells, are scattered among the epithelium
cells.

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• These large cells produce surfactant, an oily secretion containing a mixture
of phospholipids and proteins.
• Surfactant is secreted onto the alveolar surfaces, where it forms a superficial
coating over a thin layer of water.
• Surfactant reduces the surface tension in the liquid coating of the alveolar
surface.
• Alveolar walls are very delicate – rather like air bubbles; without surfactant,
the surface tension would be so high that the alveoli would collapse.
Functions
• The elastic tissue helps maintain the relative positions of the alveoli and
respiratory bronchioles.
• Recoil of these fibres during exhalation reduces the size of the alveoli and
helps push air out of the lungs.

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 METABOLISM: SPIROMETRIC DETERMINATION OF THE METABOLIC
RATE

Objective
When you have completed this assignment you should be able to determine
the metabolic rate (MR) under different circumstances from oxygen
consumption by using a spirometer.

The metabolic rate

During respiration the oxygen present in the alveoli diffuses through the alveolar
wall, combines with the haemoglobin in the red blood corpuscles and is
distributed through the body with the aid of the circulatory system. In the
tissues, the oxygen diffuses into the cells and oxidises the hydrogen produced
during the catabolism of nutritive substances in the mitochondria to form energy
and water. Of the total amount of energy, 38% is captured in ATP (oxidative
phosphorylation). The rest is released as heat.
The above processes take place continually in all body tissues. The basal
metabolic rate is the amount of energy needed to maintain the processes
essential to life, such as, for example, respiration, circulation and body
temperature.
To determine the inherent metabolic activity of the body tissues (in the absence
of any change in these factors), we measure the metabolic speed under basal
conditions. This makes the comparison of different people’s metabolic rates
possible. The basal conditions are as follows:
1) The individual should have fasted for 12 hours to prevent the specific
dynamic action of the food from having any influence on the metabolism
rate.
2) The night prior to the determinations the person should have slept well.
This lowers the activity of the sympathetic nervous system and other
metabolic stimulators to a minimum.

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3) No exercise should be done after the night’s sleep prior to the tests.
4) The subject has to lie down and rest for 30 minutes prior to the test. This
condition is very important and should be adhered to scrupulously.
5) All physical and psychological factors which could excite the subject
should be eliminated.
6) The environmental temperature has to be comfortable, that is, about
20°C to 26°C; the sympathetic nervous system becomes progressively
more active in an effort to maintain body temperature. Above 26°C the
metabolic rate is increased by discomfort, sweat and other factors.
7) Body temperature must be normal (37°C). Fever increases the
metabolic rate.
The BMR (Basal Metabolic Rate) is determined by the following factors:
1) Age. Decreases with age.
2) Sex. Lower in women than in men.
3) Height, mass and body surface. The basal metabolic rate is
proportional to the body mass to the power 0.74. The proportion
holds true for animals from mice to elephants. From the
proportion it emerges that smaller types of animal have a greater
metabolic activity per body mass unit than larger animals have.
In proportion to its weight, a mouse should therefore eat 50 times
as much as a horse to maintain its basic bodily activity.
4) Growth stage. BMR decreases with age.
5) Menstruation, pregnancy and breastfeeding.
The basal metabolic rate varies cyclically with the menstrual
cycle. About 13 days prior to menstruation the basal metabolic
rate is normal. In the subsequent days it increases until about
three days prior to menstruation when a decrease of 10% is
manifested. Then there is a decline until about three days after
menstruation when normal levels are reached.
From 22 weeks after the onset of pregnancy there is a gradual
increase in the basal metabolic rate of about 15%. Two weeks
prior to birth the BMR reaches a climax, after which it remains
constant or declines somewhat. Just after birth there is a
dramatic decrease, after which it stabilises. We can ascribe the

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 increase in the BMR to the increase in the secretion of hormones
such thyroxine, adrenal corticoids and sex hormones.
6) Infection and other disease conditions. These will change the
BMR.
7) Body temperature. For each degree Celsius elevation the BMR
increases by 12%.
8) Time since the last meal. After a meal the metabolic rate is 10%
to 30% higher (specific dynamic action [SDA]). The additional
heat comes from the increase in the metabolic activity of the liver.
9) Long-term fasting. BMR decreases because of the decrease in
body mass.
10) Muscular activity. Intense muscular activity can increase the
metabolic rate 20 - 50 times.
11) Emotional conditions. BMR increases as a result of an increase
in muscle tone and the secretion of hormones.
12) Sleep. BMR decreases as a result of the muscle tone that
decreases.
13) Environmental temperature. If temperature decreases, the
muscle tone increases and trembling begins, which makes the
BMR increase.
14) Hormonal levels, especially of epinephrine and thyroxin. An
increase in the hormonal levels of the above hormones increases
the BMR.
The mean (normal) BMR values (Calories/hour/m2) are as
follows:
Age Men Women
18 42.9 37.3
19 42.1 37.2
20-24 41.0 36.9

We regard a 10% variation in BMR as normal. This variation is

the reason why some people are thinner or fatter than other
people who eat just as much and who are just as active. A 10%
higher BMR than normal means an additional energy

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 consumption of 375 MJ (90 000 cal). This is equivalent to the
metabolism of ten kilograms of fat per year.

Apparatus
Spirometer.

Method
Preparation of the subject
1) The subject should fast for at least 12 hours.
2) The subject should rest for 30 minutes prior to the start of the experiment.
3) During the experiment the person should rest physically and emotionally.
4) The environmental temperature has to be comfortable (20° - 26°C).
5) For at least three days prior to the experiment, the subject should follow
a diet that does not contain too much protein because of its high specific
dynamic activity.

The experiment
1) Weigh the subject and determine his or her height.
2) Fill the spirometer with oxygen.
3) Close all die spigots. Place the mouthpiece in the subject’s mouth. The
spirometer valve has to be set in such a way that the subject still
breathes in atmospheric air.
4) Clamp the subject’s nose with a nose clamp so that he or she cannot
breathe through the nose.
5) Let the subject breathe atmospheric air for about one minute to become
used to the mouthpiece.
6) In the meantime see to it that the cursor on the computer screen is in the
correct position.
7) Adjust the spigot so that the subject breathes oxygen from the
spirometer. This has to be done without the subject being aware of it.
The measurement begins at the moment of adjustment. After five
minutes the spigot is turned so that the subject will be breathing
atmospheric air again. Note that the subject does not experience
difficulty in breathing.

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8) Determine the subject’s pulse rate at the beginning of the experiment
and again after two minutes. The pulse rate has to be constant otherwise
you will need to repeat the experiment after a rest period.
9) Repeat the procedure three times.
10) Correct the volume, read from the screen in terms of temperature and
barometer pressure (converted to volume at 0°C and 760 mm mercury
[STPD] with the aid of the tables provided). Correct the volume with the
calibration factor of the apparatus as well.

Questions
1) Determine the body surface of your subject from the nomograms
provided (see Chapter 4).
2) Calculate the BMR of your subject.
3) Calculate the MR of the subject before and after physical activity (do a
recording of at least three minutes’ duration after he or she has exercised
on a bicycle ergometer.
4) Calculate the percentage difference of the measured MR values (normal
and after exercise).
5) Describe and discuss the results in detail.
6) Discuss in detail the influence of the hormones epinephrine and thyroxin
on metabolic rate.
7) Discuss and explain the requirements that must be met before you can
measure the BMR.
8) Discuss the physiological and pathological factors that may influence the
BMR.

135
DUBOIS Body surface Chart

136
Correction of Gas Volumes

137
Deviate from normal BMI

138
 MEASUREMENT OF BLOOD PRESSURE

Objective
When you have completed this assignment you should be able to measure
systolic and diastolic blood pressure in a subject by means of a
sphygmomanometer (auscultatory method) and a stethoscope.

Blood pressure
The left ventricle forces a large amount of blood into the blood vessels in a short
space of time. This means that there is always more blood in the blood vessels
than they can transport. The result is that the flexible blood vessels are always
under pressure because the stretched vessels continually exert an elastic back
pressure on the blood. This blood pressure is essential for the maintenance of
sufficient perfusion of the tissues. Blood pressure can vary because of the
following:
1) Changes in the activity of the heart.
2) Changes in the circumference of the blood vessels.

The rhythmic pressure pulses observed in the arteries are the result of the
rhythmic contractions of the heart and are called arterial pulses. When the
ventricle contracts, a quantity of blood is suddenly pumped into the already full
aorta, with the result that the pressure will rise in the first segment of the aorta.
The rise in pressure stretches these segments of the aorta. The stretched
segment pushes backward and forces the blood into the next segment of the
aorta, which is then stretched in turn. This arterial pulse goes on throughout
the entire arterial system.
When the heart is contracting (diastolic blood pressure), the pressure in the
blood vessels is on average 80 mm Hg, and when the heart is contracting
(systolic blood pressure), it is an average of 120 mm Hg. We usually state (and
write) blood pressure as systolic pressure/diastolic pressure: 120/80 mm Hg.

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Physiological factors
The following physiological factors influence blood pressure:
1) Age. Directly after birth the blood pressure is 80/45 mm Hg. At the age
of four, the blood pressure is 100/85 mm Hg. In an adult it is 120/80 mm
Hg. With an increase in age there is a gradual increase because the
elasticity of the blood vessels decreases.
2) Sex. Blood pressure is lower from puberty to the clomacteric
(climacterium) for women than for men, after which it is slightly higher for
women than for men.
3) Body build. Hypertension occurs more in people who tend to be
overweight.
4) Body posture. Blood pressure increases when one changes from a
supine to an upright position.
5) Emotional stress. Fear, anger, excitement and other emotions increase
the blood pressure as a result of sympathetic nervous stimulation via
adrenalin and noradrenalin excretion.
6) Physical exertion. Systolic pressure increases with an increase in labour
loads. Diastolic pressure increases less than systolic pressure.
7) Race. The blood pressure of Eastern people is generally lower than that
of those from the West. The difference is especially noticeable after the
fiftieth year. Salt intake possibly plays a role. The blood pressure of
Bushmen, Australian Aborigines and some Red Indian tribes who eat
little salt is also lower than that of Europeans.
8) Other. Smoking, medication, diet, etcetera.

Abnormalities
Hypertension
Hypertension is a sustained condition of increased systemic arterial blood
pressure. Blood pressure values of 130/85 mm Hg are generally accepted as
the upper normal level. A blood pressure of 140/90 is recognised as
hypertensive. Hypertension is responsible for 10% of all deaths over the age
of 50.
There can be an increase in the systolic or the diastolic, or both pressures.

140
Clinically the interest is mainly in the diastolic blood pressure because this is a
reflection of the sustained working load of the heart and the tension in the walls
of the blood vessels. When we talk of hypertension or high blood pressure, we
usually mean increased systolic and diastolic blood pressure.
In some persons older than 60 years of age, the systolic blood pressure will
increase and the diastolic blood pressure will decrease because the blood
vessels now react as solid tubes. Increased heart activity is almost never the
cause of chronic hypertension. The cause of hypertension is usually always
increased peripheral resistance (constricted surface flood vessels). In about
10% of patients with increased blood pressure, the increased pressure is the
result of one or other specific illness. Therefore damage to the kidneys results
in a decrease in sodium excretion, which leads to an increase in blood volume
and also in blood pressure. We also call this type of increased blood pressure
secondary hypertension.
In the other 90% of cases, hypertension itself is probably the primary disease.
In this case we refer to primary or essential hypertension, that is, hypertension
of unknown origin.
The increased stress on the heart usually leads to hypertrophy (thickening) of
the cardiac muscle. This can later lead to heart failure and also atherosclerosis
of the coronary vessels, which inhibits the myocardial blood flow and causes
infarction (damage) of the cardiac muscle.
Hypertension can also lead to vascular damage of the brain. The bursting of a
blood vessel in the brain can lead to a local increase in pressure as well as
ischaemia of the relevant brain tissue. A stroke (apoplexy) which occurs in this
way shows symptoms of acute neurology, such as, for example, paralysis of
certain parts of the body.

Hypotension
Hypotension is not considered to be an illness, although serious degrees of
hypotension, that is, blood pressure of lower than 75/45, can lead to loss of
consciousness. The decrease in perfusion as a result of hypotension causes a
decrease in an individual’s ability to work. Regular exercise is one way of
improving a hypotensive condition.

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Apparatus
Sphygmomanometer.
Stethoscope.
We measure blood pressure with a sphygmomanometer. This consists of the
following:
1) A band-shaped rubber cuff provided with two tubes and covered with a
sturdy cloth bag.
2) A cloth bag extends so that it can be used as a tourniquet to keep the
rubber bag in position.
3) A calibrated manometer filled with mercury and linked to one of the
rubber tubes.
4) A rubber balloon linked to the other tube which is used to pump air into
the rubber bag.

Method
1) Fasten the rubber cuff to the upper arm with the aid of the cloth covering.
2) Ensure that the rubber cuff has been fitted neatly over the inner surface
of the upper arm.
3) Force air into the rubber bag until the pressure is high enough to cut off
the brachial artery which runs down the inside of the upper arm.
4) When no sound caused by turbulent flow of blood in the compressed
artery can be heard any longer in the artery running across the fossa
cubiti, the brachial artery has been cut off.
5) Let the air gradually out of the rubber cuff (the pressure decreases) while
the flow sounds in the brachial artery and the fossa cubiti are observed
by means of a stethoscope.
6) The gradual decrease in pressure will allow the pressure on the artery to
be less. When the point is reached where the pressure on the artery is
slightly lower than the pressure in the artery, blood will be pumped past
the pressure point. The turbulent blood flow in the artery lessens. When
the pressure on the artery has decreased to such an extent that the blood
flow can take place absolutely unimpeded, the sounds disappear. These
sounds are known as the Korotkoff sounds, because they were first
described by Korotkoff in 1905.

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7) The pressure at which the sounds first appear is called the systolic
pressure.
8) The pressure at which the sounds finally disappear is called the diastolic
pressure.

Please note the following precautions

1) The rubber cuff should not shift on the arm and should be applied in the
right position.
2) The subject has to be relaxed during the taking of blood pressure
readings. Muscular tension has to be reduced.
3) The rubber bag should be fully deflated following each reading.
4) You should always take the average value of three readings.

Other measuring apparatus of blood pressure are the OMRON (oscillometric)

and the Finapres (finger arterial pressure) (continuous measurement of blood
pressure).

Questions
1) Determine systolic pressure, diastolic pressure, pulse rate and pulse
pressure while a person
a) is lying down;
b) is sitting down;
c) is standing still;
d) has just finished gentle exercise; and
e) has just finished strenuous exercise.

Processing of results
Remember to take three measurements and tabulate your results.
Calculate the arithmetical mean in each case.
Tabulate the results of your classmates.
Calculate the standard deviation.
Discuss and explain your results.

2) Define blood pressure.

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3) Explain the origin of blood pressure.
4) Explain the changes in blood pressure as a result of exercise.
Study of the cardiovascular events during the Valsalva manoeuvre
(baroreceptor reflex).

To do the Valsalva manoeuvre the following procedure can be followed:

• Record the pulse wave and heart rate with the aid of the Finometer
apparatus.
• Blow for 15 seconds in a mercury manometer with the aid of a syringe
placed in the test subject’s mouth at a pressure of 40 mmHg.
• Record the Valsalva manoeuvre with the aid of the Beatscope Easy
software.
• Interpret and analyse the manoeuvre.

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 SKELETAL MUSCLE ACTIVITY: MECHANICAL FUNCTIONING OF THE
SKELETAL MUSCLE

Objective
In this section you will study the following characteristics of skeletal muscle
contraction in humans:

Voluntary change in contractile force

Muscle fatigue
You will also study the influence of loading on skeletal muscle contraction

Skeletal muscle
The skeleton provides support and articulation for the body. Bones act as
support structures and joints function as pivot points. Skeletal/striated muscles
are connected to bones either directly or by tendons, which are strong bundles
of collagen fibers. Skeletal muscle is composed of long, multinucleate cells
called fibers grouped into fascicles (Figure 1). Two or more muscles usually
work antagonistically. In this arrangement, the contraction of one muscle
stretches the other.

Figure 1. Skeletal Muscle Organization

Each individual fiber is innervated by a branch of a motor axon. Under normal

circumstances, a neuronal action potential activates all of the muscle fibers
innervated by the motor neuron and its axonal branches. The motor neuron,
together with all of the individual muscle fibers that it innervates, is termed a

145
motor unit (Figure 2). This activation process involves the initiation of an action
potential, either voluntarily or as a result of electrical stimulation of a peripheral
nerve, conduction of the action potential along the nerve fiber, release of
neurotransmitter at the neuromuscular junction, and depolarization of the
muscle membrane with resultant contraction of the muscle fibers.

Figure 2. The components of a motor unit

The muscle action potential causes a brief increase in the intracellular

concentration of calcium ions, [Ca2+], and activates the contractile molecular
machinery inside the fiber. This requires the use of intracellular supplies of
adenosine triphosphate (ATP) as the energy source. The result is a brief
contraction called a twitch.

A whole muscle is controlled by the firing of up to hundreds of motor axons.

These motor nerves control movement in a variety of ways. One way in which
the nervous system controls a muscle is by adjusting the number of motor
axons firing, thus controlling the number of twitching muscle fibers. This
process is called recruitment. A second way the nervous system controls a
muscle contraction is to vary the frequency of action potentials in the motor
axons. At stimulation intervals greater than 200 ms, intracellular [Ca 2+] is
restored to baseline levels between action potentials, and the contraction
consists of separate twitches. At stimulation intervals between 200 and 75 ms,
[Ca2+] in the muscle is still above baseline levels when the next action potential
arrives. The muscle fiber, therefore, has not completely relaxed and the next
contraction is stronger than normal. This additive effect is called summation. At

146
even higher stimulation frequencies, the muscle has no time to relax between
successive stimuli. The result is a smooth contraction many times stronger than
a single twitch, called a tetanic contraction. The muscle is now in a state of
tetanus.

Figure 3.Simple muscle curve and the influence of different loads

The simple muscle curve

A single depolarisation of the skeletal muscle system gives rise to a brief
contraction (twitch contraction), which is followed by a relaxation phase. We call
this isotonic contraction the simple muscular curve.
A simple muscle contraction curve is divided into the following three main
phases:

1) The latent period. This is the period of time from the moment of
stimulation (A) of the nerve fiber, which conducts the impulse to the
muscle, to the moment that the contraction begins (B). This is during the
period when the processes associated with the excitation-contraction
coupling take place. The more strenuously the muscle is loaded, the
longer the latent period lasts.
2) The contraction phase. This is the duration from the beginning of the
muscular contraction (B) to the maximum of the contraction. This is the
time period between B and C in Figure 3. The duration of the contraction

147
 phase differs in different muscles, for example in the eye muscle this
lasts about 5 ms (milliseconds). The contraction or shortening rate
(gradient of the contracting phase) remains constant for about 70% of
the contraction period. With increasing loading the contraction rate and
the height decreases.
3) The relaxation phase. This is the period of time from the beginning of the
relaxation (C) until the muscle has relaxed completely (D). It is important
to note that muscular relaxation is not simply a reaction on muscular
contraction, but by itself a normal physiological process with its own
characteristic mechanism. The registrations which deviate from the
baseline in the area D to E are only after-oscillations because of the
experimental set-up.

Electromyogram
During a contraction there is synchronous activity in a number of fibers in the
same muscle. The electrical signal recorded from a contracting muscle is called
an electromyogram, or EMG. The EMG provides a depiction of the timing and
pattern of muscle activity during complex movements. The raw surface EMG
signal reflects the electrical activity of the muscle fibers active at that time. Motor
units fire asynchronously and it is sometimes possible, with exceedingly weak
contractions, to detect the contributions of individual motor units to the EMG
signal. As the strength of the muscular contraction increases, however, the
density of action potentials increases and the raw signal at any time may
represent the electrical activity of perhaps thousands of individual fibers.
The raw EMG signal during voluntary contractions may be processed in various
ways to indicate the intensity of EMG activity. In the method used here, known
as the root mean square (RMS), the negative-going portions of the EMG are
inverted by squaring the whole signal, and then the whole signal is averaged
and the square root calculated. This process smooths out individual spikes and
makes the time course of changing activity much clearer. In this experiment you
will examine co-activation, a phenomenon in which contraction of a muscle
leads to more minor activity in the antagonist muscle. The physiological
significance of this is not entirely clear, but it has been suggested that it helps
to stabilize the joint.

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Nerve and muscle disorders cause the muscles to react in abnormal ways.
Measuring the electrical activity in muscles and nerves can help detect the
presence, location and extent of diseases that damage muscle tissue (such as
muscular dystrophy) or nerves (such as amyotrophic lateral sclerosis: Lou
Gehrig's disease). In the case of nerve injury, the actual site of nerve damage
can often be located. In a clinical setting, EMG and nerve conduction studies
are usually conducted together.

Apparatus
Own apparatus:
Protective laboratory coat.
Laboratory apparatus:
1. Shielded Lead Wires (5 Snap-on)
2. Dry Earth Strap
3. Disposable ECG Electrodes
4. Alcohol Swabs
5. Four books or objects of similar weight (about 1 kg each)
6. Hand Dynamometer

Procedures
Electromyography
Equipment Setup and Electrode Attachment
Lead placement on arm:

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Figure 4. Equipment Setup for PowerLab 26T

1. Channel 1 will lead to the biceps, Channel 2 will lead to the triceps, and the
Earth will be connected to the Dry Earth Strap. Attach the Disposable
Electrodes to the end of the Channel 1 and Channel 2 wires and the Dry Earth
Strap to the end of the Earth wire. Refer to Figure 4 for proper placement, but
do not attach them to the volunteer. Follow the colour scheme on the Bio Amp
Cable.
2. Remove any jewellery from the volunteer’s hand and arm. Use the ballpoint
pen to mark two small crosses 2-3 cm apart on the skin above the biceps
muscle and triceps muscle. Use Figures 4 and 5 as a guide.

Figure 5. Skeletal muscle structure

5.Clean the area with an Alcohol Swab to remove the dead skin cells. Wait for
the skin to dry, and stick the Disposable Electrodes to the skin (Figure 3). Put

150
the Dry Earth Strap around the volunteer’s wrist, with the fuzzy side against the
skin.
6. Check that all four electrodes and the Dry Earth Strap are properly connected
to the volunteer and the Bio Amp Cable before proceeding.

Exercise 1: Voluntary Change in Contractile Force

In this exercise, you will examine changes in voluntary muscle contraction and
how contractile force changes with increasing demand.

Note: Channels 1 and 2 are the RMS activity of the biceps and triceps muscles.
RMS activity is commonly used in the assessment of muscle function because
it is easier to quantify. Use these two channels when completing your analysis.
1. Have the volunteer sit in a relaxed position with his/her elbow bent 90 o and
palm facing upward. Make sure the volunteer’s elbow is not on the table. The
volunteer’s other hand should grasp the wrist of the recorded arm. Make sure
the volunteer is facing away from the monitor.
2. Have the volunteer make a strong contraction of the biceps muscle. This is
done by bending the recorded arm further while resisting this movement with
the other arm. Observe the signal and adjust the range in the dialog so that the
maximal electrical response occupies about one half to two-thirds of the full
scale.
3. Repeat step 2 for the triceps signal. A strong contraction of the triceps muscle
is made by trying to straighten the recorded arm while resisting this movement
with the other arm.
4. Start recording. Add a comment with the volunteer’s name. Have the
volunteer make a strong contraction of the biceps and then the triceps. Add a
comment at the start of each contraction. Stop recording.
5. Have the volunteer return to their original relaxed position. Start recording.
The blue line in Chart View will help you indicate the change in procedure.
6. Prepare a comment with “one book.” After a few seconds, add the comment
and place one book on the hand of the subject. Leave it on for three seconds
and remove it. Repeat this process with two books, then three, and then four
books to give a series of increasing weights. Add a comment each time you add
books. Save your data.

151
Exercise 2: Muscle Fatigue
In this exercise, you will observe the decline in maximal force during a
sustained contraction and will examine some properties of muscle fatigue.
First, you will calibrate the Hand Dynamometer with respect to the volunteer’s
maximal grip strength.

Equipment Setup and Calibration

Figure 6. Equipment Setup for PowerLab 26T

1. Have the volunteer loosely grip the Hand Dynamometer in the fist of their
dominant hand, as shown in Figure 6.
2. Start recording. Have the volunteer squeeze the Hand Dynamometer as
hard as possible for a second or two, and then relax their grip. After
recording for a few seconds, have the volunteer repeat the maximum grip
and then relax. Stop recording.
3. Click-and-drag over the largest response to select a range of data that
includes both the relaxed and maximum force signals (Figure 7).

152
Figure 7. Data Selection for Units Conversion

4. In the Units Conversion dialog, select Units: %. Then select part of the
trace where the force is zero, and click the Point 1 arrow. Enter 0 %. Then
select part of the trace at the peak (Figure 8), click the Point 2 arrow and
enter 100 %.

Figure 8. Units Conversion Dialog

5. Select Set units for: All and new data, then click OK to close the dialog.

Procedure
1. Adjust the scale for Channel 1 to show -20 to 120%.
2. Allow the volunteer to view the monitor. Start recording. Ask the volunteer
to maintain 20% maximal grip strength while watching the recorded trace.
The Range/Amplitude display for Channel 1 shows the percentage force
applied. Add a comment with “20%.”
3. After 20 seconds, tell the volunteer to relax. Stop recording.

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4. Wait for 30 seconds to allow recovery of muscle function, and repeat steps
2-3 for contractions of 40%, 60%, 80%, and 100% of maximal grip
strength. Allow the volunteer to rest for 30 seconds in between each
contraction. Add a comment with the maximal grip strength percentage
each time.
5. Have the volunteer rest for two minutes. Then have the volunteer turn
away from the monitor so they cannot see the data trace.
6. Start recording. Ask the volunteer to produce a sustained maximal
contraction. After 10 seconds, or when the force has obviously declined,
instruct them to try harder. After another 10 seconds, repeat the
encouragement. After five more seconds, tell the volunteer to relax. Stop
recording. Allow the volunteer to rest briefly.

Note: Most volunteers can produce temporary increases in muscle force

during a fatiguing contraction, when sufficiently motivated by verbal
encouragement.

7. Start recording again. Ask the volunteer to produce a sustained maximal

contraction as before. Every 10 seconds, allow the volunteer to relax very
briefly for ½ second, and then have them return to maximal contraction.
Stop recording after 30 to 40 seconds.

Note: Even brief periods of relaxation allow substantial recovery from fatigue,
but the recovery is only temporary (Figure 9).

Figure 9. Fatiguing Contraction

154
8. Turn the volunteer so they can see the monitor again. Start recording. Ask
the volunteer to produce a 40% contraction while watching the data trace.
After 10 seconds, add a blank comment to denote the time.
9. Have the volunteer close their eyes and attempt to maintain exactly the
same contraction force for the next 30 seconds.
10. After the elapsed time, have the volunteer open their eyes and adjust the
contraction force back to 40%. Stop recording.

Note: Almost all volunteers will show a declining force while their eyes are
shut, which is very similar to fatigue. This is referred to as pseudo-fatigue.
This is not true fatigue because the full 40% can be exerted easily, as can be
seen when the volunteer’s eyes are opened again.

Analysis

Exercise 1: Voluntary Change in Contractile Force

1. Examine the data in the Chart View. Autoscale, if necessary. Note the
changes in activity in the “Biceps” channel. Note also that placing weights
on the hand gives rise to little or no activity in the triceps muscle.

2. Note the relationship between the “Biceps” channel and the “RMS Biceps”
channel. The height of the RMS trace reflects the overall activity of the raw
EMG signal and gives a simpler view of the muscle’s electrical activity.
Note the changes in the RMS trace as books were added and removed.

3. Select data points from “RMS Biceps” when books were added. Enter
these values in Table 1 of the Data Notebook. The height of the trace
correlates with the force produced by the muscle.

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Data Notebooks

Table 1. Force Produced by Adding Books

RMS Biceps
Amplitude
(mV)
One Book

Two Books

Three Books

Four Books

Exercise 2: Muscle Fatigue

1. Examine the data in the Chart View, and Autoscale, if necessary.
2. Print out graph and indicate the following: relaxation, muscle fatique and
maximal contraction (see example Figure 9).

Report

Study Questions

1. Unlike the discrete waveform from an electrocardiogram, the

electromyogram waveform is irregular. Why do you suppose this is?

2. What happened to the biceps EMG trace when you added weights to your
arm? Was this expected?

3. Explain the phenomenon of coactivation in your own words.

4. What are the two ways by which the nervous system can control the force
generated by a muscle?

5. What explanations can you think of for pseudo-fatigue?

156
 SPIROMETRY: THE DETERMINATION OF AIRFLOW, LUNG VOLUMES
AND LUNG CAPACITIES.

Objectives
Upon completion of this experiment, you should be able to:
▪ Determine lung volumes and capacities with a spirometer and interpret the
results;
▪ determine and interpret the forced expiratory vital capacity;
▪ compare your results of the pulmonary function test with those of a typical
person and a person with an obstructive lung disease;
▪ present your results in a scientific manner.

Textbook reference
See relevant section in your textbook.

Background
Gas exchange occurs between the pulmonary blood and alveolar air in the
lungs occurs in the alveolar air sacs (alveoli). The efficiency of gas exchange is
dependent on ventilation. Cyclical breathing movements’ leads to air moving in
and out of the alveoli (see Figure 1). Inspiration provides the alveoli with
atmospheric air and expiration removes some of the stale air, which has
reduced oxygen and increased carbon dioxide concentrations.

157
 Figure 1. A schematic diagram of the human respiratory system.

Spirometry

Spirometry is a method used to measure the volume of air moving into and out
of the lungs during each inspiration and expiration. A spirogram depicting the
different lung volumes and capacities can be seen in Figure 2. The use of
spirometry is becoming more important due to a worldwide increase in
respiratory diseases. Spirometry is the most suitable method for the rapid and
reliable screening of patients that could possibly be suffering from an
obstructive pulmonary disease such as Chronic Obstructive Pulmonary
Disease (COPD) and asthma or a restrictive lung disease such as tuberculosis.
Most COPD cases are completely avoidable with the majority of cases caused
by tobacco smoking.

158
 Figure 2. Lung volumes and capacities.

Many important aspects of lung function can be determined by measuring

airflow and the corresponding changes in lung volume. In the past, this was
commonly done by breathing into a bell spirometer, in which the level of a
floating bell tank gave a measure of changes in lung volume. Flow, F, was then
calculated from the slope (rate of change) of the volume, V:
𝒅𝑽
𝑭= Equation 1
𝒅𝒕

Airflow can be measured easily and directly with a pneumotachometer (from

Greek roots meaning “breathe speed measuring device”). The PowerLab
pneumotachometer arrangement is shown in Figure 3.

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 Figure 3. The PowerLab pneumotachometer.

Different types of flow measuring devices are available and each type has
advantages and disadvantages. The flow head that will be used in this practical
is a “Lilly” type. This type of pneumotachometer measures the difference in
pressure on either side of a membrane with a known resistance. This resistance
causes a small pressure difference that is proportional to the flow rate. Two
plastic tubes transmit this pressure difference to the Spirometer Pod, where a
converter will convert the pressure signal into an electrical signal that will be
recorded by the PowerLab system and be displayed in LabTutor. The volume,
V, is then calculated as the integral of flow:

𝑉 = ∫ 𝐹 𝑑𝑡 Equation 2

This integration represents a summation of values over time; the volume

recordings that you will see in LabTutor during the experiment are obtained by
adding successive values of the flow signal that is added together in order to
determine the amount thereof. The integral resets to a zero value each time a
new recording starts.

A variable that must be considered when measuring volume by using the

above-mentioned method is the change in temperature of the air of which the
volume is measured. The volume of a gas increases when it is warmer. The air
temperature at the Spirometer Pod (at room temperature) and the air exhaled

160
from the lungs (at body temperature) will differ. Therefore, the volume of air
exhaled from the lungs will be slightly larger than that of what was inhaled.

Due to this factor, a volume recording will – as calculated by the integration of

airflow – measure the expiration value higher than it actually is. In order to
compensate for this, the flow should be integrated separately during inhalation
and exhalation through the correction factor (body temperature, atmospheric
pressure, saturated with water pressure (BTPS)).

Spirometry allows many components of pulmonary function (see Figure 2) to

be visualized, measured and calculated. Respiration consists of repeated
cycles of inspiration followed by expiration.

If we only take the lung volume and not the changes in volume into account, we
refer to static lung volumes and capacities. If we take the volume changes
during respiration into account, we refer to dynamic lung volumes.

Static lung volumes

Tidal volume (VT) is the amount of air that is breathed in or out during normal
conditions of rest.

Inspiratory reserve volume (IRV) is the extra volume of air that can be
inspired over and above the normal tidal volume.

Expiratory reserve volume (ERV) is the extra volume of air that can be expired
by forceful expiration over and above the normal tidal volume.

Residual volume (RV) is the volume of air which remains in the lungs following
maximal expiration.

Lung capacities
Total lung capacity (TLC) is the volume of air that is present in the lungs
following maximal inspiration. It is the sum of IRV, VT, ERV and RV.

Vital capacity (VC) is the maximal volume of air which can be breathed in. It is
the sum of ERV, VT and IRV. The vital capacity might be restricted when normal

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expansion of the chest or the lungs is restricted (as in restrictive lung diseases).
This can be the result of the following:
- An increase in the tightness of the lungs, for example, as a result of lung
fibrosis.
- Chest compression such as, for example, tight bandages, obesity and
kyphoscoliosis (convex scoliosis of the vertebral column, or hunchback).
- Lung compression during pleural effusion.

Inspiratory capacity (IC) is the volume of air which can be breathed in

maximally following an unhurried expiration. This is the sum of VT and IRV.

Functional residual capacity (FRC) is the volume of air which remains in the
lungs after an unhurried normal expiration. It equals the sum of ERV and RV.

Dynamic lung volumes

This is the measurement of the change in volume per time unit. These
measurements provide more clinical information than the static lung volumes
and are often used as lung function tests.

When the vital capacity is exhaled as quickly as possible following a maximal

inspiration, the forced vital capacity curve (FVC) is obtained. The largest part
of the vital capacity is exhaled in one second (the steep part of the curve).
Through measuring the volume which can be exhaled in one second, we obtain
the forced expired volume in one second (FEV1). The percentage that the
FEV1-value constitutes of the VC (normal 70 – 80%) gives an indication of the
efficacy of the breathing process.

The FEV1/VC is used to distinguish between restrictive and obstructive lung

diseases. With restriction, the FEV1 and the VC are restricted equally and the
FEV1/VC will appear normal. FEV1 will, however, be notably smaller than
normal. In the case of obstructive lung diseases, the expiration flow is always
restricted without the VC being restricted. The gradient of the FVC curve always
decreases with obstructive bronchial conditions. The FEV 1/VC ratio will then
become smaller. The whole VC will be breathed out in about five seconds,
which means that the FEV5 and the VC will be about the same.

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In normal ventilation, the breathing frequency (ƒ), also called the respiratory
rate (RR), is approximately 15 respiratory cycles per minute. This value varies
with the level of activity. The product of ƒ and VT is the expired (respiratory)
minute volume (VE), the amount of air exhaled in one minute of breathing. This
parameter also changes according to the level of activity.

Most lung volumes and variables show a normal type of population distribution.
Ninety five percent of the population falls within the limits of two standard
deviations (SD) from the mean. If your values are less that the predicted value
(nomogram value -2 x SD), there is a 95% possibility that they are abnormal, in
other words, indicating the presence of a lung disease or a very inaccurate
measurement.

Apparatus
▪ Spirometer
▪ Bacterial filter
▪ Nose clip
▪ Computer work station

Method
Preparation
1. Log in to LabTutor with the log in details provided by the
demonstrator/lecturer.
2. Make sure the spirometer is set up correctly and in working order.
3. Collect a clean (cleansed with 70% ethanol) mouthpiece and sealed
bacterial filter from the demonstrator/lecturer.
4. Note the subject’s height and age. The subject must be healthy with no
known respiratory conditions.
5. The subject must be in a standing position for all measurements, with his/her
back facing the computer screen.

Registration of static and dynamic lung volumes and capacities

During this practical experiment, the LabTutor program, specifically the
Respiratory Air Flow and Volume package, will be used. Carefully read through

163
the instructions and follow the steps to complete the following tasks (you will
only need to follow steps 1 – 10 of 13):
i. Become familiar with the equipment: In this exercise, you will learn the
principles of spirometry, and how integration of the flow signal gives a
volume.
Make sure that you know how to calibrate (Zero Pod) the spirometer.
Read carefully through the procedure in the LabTutor program and
follow this procedure precisely, before the recording of every
measurement (every time you see the zero pod button).
ii. Lung volumes and capacities: Here you will study the respiratory cycle
and measure changes in flow and volume.
iii. Pulmonary function tests: Here you will measure parameters of forced
expiration that are used in evaluating pulmonary function.
iv. Simulating an airway obstruction: In this exercise, you will simulate an
airway restriction.

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Complete the table below as you go through the prescribed steps:

Table 1: Lung volumes, lung capacities and pulmonary function.

Abbreviation,
Term Symbol & Results Unit
Formula
Breathing rate
Respiratory Rate RR breaths/min
Lung Volumes
Tidal Volume VT L
Expired Minute Volume VE = RR x VT L/min
Inspiratory Reserve Volume IRV L
Expiratory Reserve Volume ERV L
Residual Volume RV (predicted) L
Lung Capacities
Inspiratory Capacity IC = VT + IRV L
EC = VT +
Expiratory Capacity L
ERV
VC = IRV +
Vital Capacity L
ERV + VT
FRC = ERV +
Functional Residual Capacity L
RV
TLC = VC +
Total Lung Capacity L
RV
Pulmonary function tests (Normal)
Peak Inspiratory Flow PIF L/min
Peak Expiratory Flow PEF L/min
Forced Vital Capacity FVC L
Forced Expired Volume in 1 sec FEV1 L
FEV1/FVC x
% FVC expired in 1 sec %
100
Pulmonary function tests (Airway obstruction)
Peak Inspiratory Flow PIF L/min
Peak Expiratory Flow PEF L/min
Forced Vital Capacity FVC L
Forced Expired Volume in 1 sec FEV1 L
FEV1/FVC x
% FVC expired in 1 sec %
100

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 EXCRETION

Objective
When you have completed this assignment you should be familiar with and able
to use some of the best known methods for the analysis of urine.

Urine formation by the kidneys

The kidneys are the most important excretory organs. During the passage of
blood through the kidneys, substances are removed from the kidneys to a
varying degree so that urine composition actually represents the momentary
homeostatic imbalance. Urine analysis can therefore give an indication of
homeostatic abnormalities caused by disease conditions and can thus lead to
the diagnosis of such disease conditions.
The functional unit of the kidney is the nephron. The nephron consists of
various parts. In the glomerulus (a cluster of blood vessels surrounded by the
Bowman capsule), the blood filters as a result of a hydrostatic pressure gradient
(higher than colloid-osmotic pressure) between the glomerulus and the
Bowman cavity. The filtrate moves from the Bowman cavity to the proximal
duct from where about 80% of the reabsorption takes place. The peritubular
capillary network which surrounds the proximal duct removes the reabsorbed
substances. Reabsorption takes place through passive diffusion as a result of
an electrochemical (concentration) gradient or through active transport.
Secretion (movement of substances from the peritubular blood to the filtrate)
also takes place by a passive or active process. With the movement of the
filtrate through the Henlé arch and the distal ducts, the further 20% of
substances are absorbed and the filtrate gradually changes into urine, which
finally goes to the bladder via the collecting ducts to the kidney, pelvis and the
ureter.

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Here we direct our attention to qualitative analysis because quantitative
analyses demand a 24-hour urine sample and thus preservation. The scope of
the practical makes the obtaining of enough 24-hour samples more difficult.
We will deal with a different method of urine analysis in each assignment of this
section.

DETERMINATION OF THE SPECIFIC GRAVITY (SG) OF URINE

Specific gravity of urine

The definition of a liquid’s specific gravity (SG) is the density (kg/cubic metre)
of the liquid divided by the density of pure water at 4°C. Specific gravity is a
ratio and therefore has no unit.
The SG of urine is dependent on the concentration of substances dissolved in
the urine. The normal SG varies between the values 1.003 to 1.030. Low
values of 1.001 to 1.003 are possibly an indication of damage (infection) of the
renal duct epithelium (the kidney uses the ability to concentrate urine) or of
diabetes insipidus. This is an inability of the distal ducts to respond to ADH
(antidiuretic hormone) or a deficient secretion of ADH (a lesion which affects
the supraoptic nuclei, hypothalamus-pituitary or neuro-pituitary). The excretion
of large amounts of diluted urine is called polyuria.
The presence of sugars, proteins and excretory products of the X-ray contrast
median in urine increases the SG (higher than 1.030). Glycosuria and
proteinuria begin as a result of kidney lesions. We also encounter a high uric
SG when there has been excessive loss of water through, for example,
perspiration, fever, vomiting and diarrhoea.

Apparatus
Urinometer.

Method
1) Fill a clean glass cylinder with fresh urine up to about 2 cm from the
top.
2) Remove the froth with a piece of filter paper.

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 3) Let the urinometer float on the urine without touching the sides of the
cylinder.
4) Read the SG on the lower side of the meniscus.
5) Correct the reading for temperature influence (the SG is 0.001 higher
for each three degrees above 15°C).
6) Rinse the urinometer and dry it before taking the next reading.
7) Determine the SG of all the urine samples.
8) Wash the urinometer and cylinder thoroughly after the experiment.

Questions
Tabulate your results and make a conclusion.

THE STUDY OF URINE WITH THE AID OF LABSTIX

Labstix
Labstix are commercial testing strips which make possible the qualitative
testing of pH, protein, glucose, ketones and blood in the urine. There are five
absorbent surfaces on the tips of the test strips.

Determination of pH
The absorbent surface has been treated with methyl red and bromothymol blue.
This can indicate, within half a pH unit, pH changes from 5 to 9.
The pH of urine is generally dependent on diet. A protein-rich diet leads to a
low urine-pH, while a vegetarian diet leads to a high urine pH.

Determination of proteins
The surface responsible for the testing of proteins has been treated with tetra-
bromophenol in a buffered acid medium.
This area is yellow in the absence of protein but changes to green in the
presence of proteins. The type and the concentration of proteins in the urine
will determine the intensity of the green colour.

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Determination of glucose
This testing area has been treated with glucose-oxidase, which acts as a
catalyst and reacts with glucose in the presence of atmospheric oxygen to form
glucuronic acid and hydrogen peroxide. The hydrogen peroxide and
peroxidase oxidise the colour to produce a shade ranging from red to purple.
About 0.1 g glucose per 100 ml urine is detectable. The sensitivity of the test
can be influenced, however, by the temperature and pH of the urine or vitamin
C medication. This includes remedies using vitamin C as a reduction agent, for
example, tetracyclines.
The strip does not react at all with any other compound which may occur in the
urine. It also does not react with other sugars such as lactose, galactose and
fructose.
Interpret the colour results as follows:
 Light colour: 0.25 g and less glucose/100 ml urine present.
 Medium colour: glucose present.
 Dark colour: 0.5 and more glucose/100 ml urine present.

Ketones
Ketones are organic compounds which contain a ketone group (C = 0).
Ketones which can be present in urine are acetone, acetic acid and betahydroxy
butyric acid. The presence of these in the urine is known as ketonuria.
These compounds occur in the urine, especially during the acidotic condition of
a patient with diabetes. The concentrations are related to the seriousness of
the patient’s condition. Acetone is also found in the urine, however, during
hunger acidosis and when there are abnormalities in fat metabolism. Acetic
acid normally accounts for about 60% of the ketones present in urine.
The testing area for the determination of ketones has been treated with sodium
nitroprusside, glycine and a buffer. It reacts with acetic acid and acetone in
urine, but not with betahydroxy butyric acid. A positive colour reaction results
also from excessively large amounts of phenyl ketones, 8-hydroxi-quinoline and
following the ingestion of ptyalin complexes.

169
Blood
The testing area on the strip has been treated with a buffered mixture of organic
peroxide and ortholydine. The test is more sensitive for free haemoglobin
(haemoglobinuria) and myoglobin than for intact red blood corpuscles
(haematuria).
Haemorrhagic (bleeding) nephritis, tuberculosis of the kidney, malignant
papilloma (tumour in kidney papillae), chronic infection and kidney stones are
some of the causes of haematuria.
Haemoglobinuria can be the result of malaria, haemolytic toxins and other
haemolytic conditions.
High concentrations of ascorbic acid (vitamin C) influence the sensitivity of the
test. Some oxidising contaminants, for example, hypo-chlorides, can give an
incorrect positive result.
In clinical practice Labstix are not used as a diagnostic tool. Always follow up
positive results with specialised tests.

Apparatus
Labstix test strip.

Method
1) Dip the test strip in fresh urine so that all test areas are wet.
2) Run the edge of the strip against the rim of the container to remove
excess urine.
3) Compare the test areas with the colour chart on the label of the Labstix
container by holding the test strip against it immediately after you remove
it from the urine.

Question
Describe and discuss your observations.

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TESTING FOR PHENYLKETONURIA: THE FERRICCHLORIDE TEST

Phenylalanine
Phenylalanine is an essential amino acid which is normally broken down in the
liver to thyroxin through the enzyme phenyl-alanine-4-mono-oxygenase
(phenylalanine hydroxylase). This enzyme is absent in about one out of every
10 000 people because of recessive mutant genes. (The mutant genes are
inherited from both parents.) In the absence of phenylalanine hydroxylase, a
different phenylalanine metabolic route is followed that is normally not used at
all, or very seldom. In this metabolic route, phenylalanine is changed into
phenylpyruvic acid which accumulates in the blood and is secreted by the
kidneys. Excess phenyl pyruvate circulating in the blood leads to mental
retardation in children, as there is a decrease in protein syntheses in the brain
cells. A decrease in the formation of serotonin in the brain also contributes to
retardation. The development of mental retardation can be prevented by letting
the child follow a diet free of phenylalanine.

The ferric chloride test

The ferric chloride test is not specifically intended for the detection of
phenylketonuria, but this is one of its most important uses. The forming of a
dark brown colour that bleaches to yellow between the ferric chloride and the
urine, which contains the abnormal metabolite, is the result of the formation of
complexes between ferric ions and the metabolite (phenyl pyruvic acid).
The ferric chloride test gives positive reactions in the presence of different
congenital metabolic defects and some non-congenital metabolic defects.
The congenital metabolic defects are histidynemia, tyrosynemia with hepato-
renal effect, tyrosynosis (Mede’s syndrome), alcaptonuria, oasthouse disease
and forminimotransferase defect syndrome. The non-congenital metabolic
defects are tyrosynemia, cirrhosis of the liver, icterus, diabetes acidosis,
melanoma, and pheochromocytoma.
Research shows that the urine of patients who take a number of different
medications can lead to a positive ferric chloride test. Therefore, salicylates
give a stable purple colour, Phenothiazine produces a purple colour, antipyrine
a red colour, L-dopa metabolites a yellowish-green to brownish-purple colour,

171
isonicotine acid hydrozide a green colour and iodochlorohydrox-quinine
containing medicines a blue-green colour.

Apparatus
Glass apparatus in your cupboard.

Reagents
10% ferric chloride solution. (Dissolve 10 g ferric chloride solution in a small
amount of distilled water and dilute with more distilled water to 100 ml.)

Runuart method
1) Place 1-2 ml of each fresh urine sample in a separate clean test tube.
2) Add some of the 10% ferric chloride solution (drop by drop).
3) Observe the colour development.

Question
Describe and discuss your observation.

THE BENEDICT TEST

The Benedict test

A condition of glycosuria which develops in the course of diabetes and other
pathological conditions can be detected by means of the Benedict test. The
test rests on the fact that glucose in a warm alkaline environment reduces
cuprous salts to copper oxide.

Apparatus
Glass apparatus in your cupboard.

Reagents: Benedict’s reagent.

Copper (II) sulphate 5 hydrate 1.73 g.
Tri-sodium citrate 2-hydrate 17.3 g.
Sodium carbonate (water-free) 27.0 g.
Distilled water to 100 ml.

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Dissolve the citrate and carbonate in 70 ml warm distilled water. Dissolve the
copper (II) sulphate in 10 ml warm distilled water and add to other. After cooling
up to 100 ml and filtrate.

Method
1) Place 5 ml Benedict’s reagent in a number of test tubes, depending on
the number of urine samples provided.
2) Add eight to 10 drops of urine to each.
3) Place the mixture in boiling water for five minutes.
Observation
With a urine-glucose concentration of higher than 300 mg/100 ml, a red
(orange-brown) precipitate forms, depending on the glucose concentration. If
the glucose concentration in the urine is lower than 300 mg/100 ml, a precipitate
only forms if the test tube is left standing for a period of time.

Question
Describe and discuss your observation.

FOUCHET’S REACTION

Fouchet’s reaction
We can determine the presence of bilirubin in the urine with Fouchet’s reaction.
The breakdown of haemoglobin leads to the formation of bile pigment. In the
process converted and bilirubin (decomposition products of haemoglobin) are
converted into bilirubin (bile pigment) by the liver. Bilirubin binds with glucuronic
acid and is excreted in the gall (bile). In the intestine the bilirubin diglucoronide
is turned into various types of bilogenes which are then excreted in the faeces.

An accumulation of bilirubin may occur in the blood. This then precipitates into
the skin and the sclera of the eye and is excreted through the kidneys. The
condition is known as jaundice (icterus). The reason for the accumulation of
bilirubin can be the result of the following:
1) Large scale haemolysis of red blood corpuscles as a result of, for
example, malaria infestation, toxic conditions or spherocytosis.

173
 2) Damage to liver parenchymatous cells, together with an obstruction of
the gall capillaries in the liver, as is the case with toxic or virus hepatitis.
3) An obstruction of the larger bile ducts, mainly outside the liver, by, for
example, gall stones or carcinoma of the pancreas or other surrounding
structures.

Apparatus
Glass apparatus in your cupboard.

Reagents
Fouchet’s reagent: Dissolve 25 g trichloroacetic acid and 1 g ferric chloride in
100 ml water.
10% barium chloride solution.

Method
1) Add 10 ml urine to 5 ml 10% barium chloride solution.
2) Mix and filter. The precipitate is stained yellow in the presence of bile
fluids.
3) Fold open the filter paper and dry the precipitate slightly with another
filter paper.
4) Place one to two drops of Fouchet’s reagent on the precipitate of
bilirubin absorbed into the barium chloride.
5) Repeat the procedure with each urine sample.

Observation
If bilirubin is present, a green or blue-green hue will develop.

Question
Describe and discuss your observation.

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 Where applicable

DIGESTION

Objective
To investigate the actions of the enzymes involved in the digestion of proteins,
carbohydrates and fats.

Introduction
A constant supply of nourishing substances is an essential requirement for all
living organisms. Through digestive functions, materials taken into the body
are degraded, dissimulated and liquefied. In the process food substances lose
all semblance of their former state and are reduced to a very fine consistency
so that it can be absorbed and metabolised by the body. In this way digestion
and assimilation help to prepare the body’s own substance.

Digestion begins in the mouth. It is here that food is mechanically broken down
into smaller particles and ensalivated prior to swallowing. The digestive
enzyme salivary amylase acts to decompose starch. Another component of
saliva is maltase, an enzyme catalysing the catabolism of the disaccharide
maltose. Assimilation also begins in the mouth, where the mucus membranes
lining the oral cavity absorb sugars and transmit them directly to the blood.

Enzymes are large protein molecules produced by body cells. They are
biological catalysts, which increase the rate of a chemical reaction without
themselves becoming part of the product. Digestive enzymes are hydrolytic
enzymes (hydrolyses) which break down organic food molecules by adding
water to the molecular bonds, thus cleaving the bonds between the subunits
(monomeres). Each enzyme hydrolyses only one or a small group of substrate
molecules and requires very specific environmental conditions to function
optimally. Digestive enzymes actually function outside the body cells, in the
digestive tract, and their hydrolytic activity can therefore be studied in test tubes.

175
After food has been thoroughly chewed and lubricated by the salivary
secretions, the process of swallowing is initiated. Involuntary peristaltic
contractions transport the food along the oesophagus to the stomach. Although
the stomach is closed off at the anterior end by the cardiac (lower oesophageal)
sphincter, this valve opens as food materials approach and food enters the
stomach.

Gastric digestion takes place in the pylorus, a highly active region in the
stomach where vigorous churning mixes the food with gastric secretions. The
stomach contents (chyme) are prevented to leave the organ by the pyloric
sphincter and the content remains within the stomach for several hours. The
pyloric sphincter opens and allows small portions of chyme to enter the small
intestine where further digestion takes place. One of the most important
functions of the stomach is thus storage. Gastrectomised patients can
therefore lead fairly normal lives, but they must ingest more frequent and
smaller meals.

Gastric juice is produced by many tiny glands lining the stomach wall and
contains several digestive elements. Pepsinogen, an inactive form of the
enzyme pepsin, is secreted into the stomach where it is converted to an active
form by the highly acidic stomach contents. This enzyme helps to degrade
proteins. The parietal cells secrete HCl (hydrochloric acid) which forms a large
component of the stomach secretions.

The small intestine is divided into three parts: the duodenum, the jejunum and
the ileum. The stomach contents first move through the pyloric sphincter to the
duodenum. It is here that ulcers are often found, possibly because of the highly
acidic nature of gastric fluids. Within the duodenum, the semifluid mass of
partially digested food mixes with secretions from the pancreas and the gall
bladder. The alkaline nature of the pancreatic juice helps to maintain the normal
duodenal pH.

The flow of pancreatic secretions is regulated by the hormone secretin. The

production of secretin is induced by the presence of the acid chyme within the

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small intestine. Pancreatic secretions also contain many digestive enzymes,
including lipase, amylase, trypsin and chymotrypsin. The latter two enzymes
are both secreted in an inactive form, are activated in the small intestine and
play a role in the catabolism of proteins.

Bile is a digestive fluid that enters the duodenum from the gall bladder by way
of the common bile duct. Although bile contains no digestive enzymes, it plays
a highly important role in the emulsification of fatty substances. Emulsified fats
are more easily acted upon by pancreatic lipase. The flow of materials from the
gall bladder is regulated by the sphincter of Oddi, which is under hormonal
control. The presence of fats in the duodenum induces the production of
cholecystokinin (CCK), a hormone that relaxes the sphincter and induces
contraction of the muscular walls of the gall bladder.

As the chyme continues through the jejunum, it mixes with secretions from the
small intestinal wall. These secretions contain mucus and various catabolic
enzymes such as peptidases, disaccharidases and lipase. These enzymes
complete the digestive process.

Assimilation is the chief function of the small intestine. The small intestine
contains finger-like processes, called villi, which increase the surface area and
promote absorption. The villi are surrounded by capillaries and, through the
process of diffusion and active transport, nutrients are taken up into the blood.
Each villus also contains a tiny lymphatic vessel called a lacteal. Amino acids
and sugars pass into the capillaries of the villi, while fatty acids, glycerol,
glycerides and lipid soluble vitamins are absorbed into the lacteal.

At the juncture of the small intestine with the large intestine, the ileocaecal valve
prevents material from backing up into the small intestine. Within the large
intestine, which is subdivided into ascending, transverse and descending
colons, there exist teeming numbers of micro-organisms. This intestinal flora
further degrades undigested materials and helps to provide essential vitamins
(such as vitamin K and some B vitamins) to the body. The absorption of large

177
 amounts of water also occurs here. The residue passes into the rectum and is
eliminated. Digestion can be summarised as follows:

FOODSTUFF ENZYMES SITE OF ACTION

Starch
 Salivary amylase Mouth
Carbohydrate Disaccharides
digestion  Pancreatic amylase Small intestine
Lactose maltose sucrose
 Enzymes in small
Galactose glucose fructose intestine – lactase, Small intestine
maltase en sucrase

Proteins
 Pepsin (stomach glands)
In the presence HCl Stomach
Proteases, peptones
 Pancreatic enzymes
Protein (trypsin, chymotrypsin Small intestine
digestion en carboxypeptidases)
Polypeptides, dipeptides
 Aminopeptidases and
dipeptidases Small intestine
Amino acids

Unemulsified fats
Emulsified by detergent
 action of bile salts Small intestine
Fat digestion
 Pancreatic lipases
Monoglycerides Glycerol
and fatty acids and fatty
acids

178
Reagents
PROTEIN digestion
5% pepsin solution.
0.5 N HCl.
Egg white.
Distilled water.

CARBOHYDRATE digestion
1% soluble starch
Lugol’s iodine solution.
1% glucose solution for positive Benedict test.
Benedict’s solution.
0.5 N HCl.

FAT digestion
1.5% pancreatin.
Phenol red pH indicator.
0.2% NaOH.
0.5 N HCl.
Vegetable oil.
2% bile solution.

Apparatus
Markers.
Test tubes and test tube rack.
Pipettes (2 ml and 5 ml).
Pasteur pipettes.
Parafilm and beakers for saliva amylase.
Pipet plungers (different colours).
Jug with ice.
Water bath set at 37°C.
Hot plate and pot with boiling water.

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Method
1. PROTEIN digestion by PEPSIN
Pepsin, produced by the chief cells of the stomach glands, hydrolyses proteins
to smaller fragments (peptides and polypeptides).
• Mark all the test tubes clearly.
• The disappearance of the egg white indicates complete protein digestion.
• Prepare the experimental test tubes as follows:
REMEMBER TO SHAKE THE TUBES AT REGULAR INTERVALS
DURING INCUBATION TIME!
Test tube 1
▪ 4 ml pepsin solution
▪ 2 ml 0.5 N HCl
▪ Egg white
▪ Incubate at 37°C for 1.5 hours
Test tube 2
▪ 4 ml pepsin solution
▪ 2 ml 0.5 N HCl
▪ Egg white
▪ Incubate at 0°C (ice) for 1.5 hours
Test tube 3
▪ 4 ml pepsin solution
▪ 2 ml distilled water
▪ Four drops 20% NaOH
▪ Egg white
▪ Incubate at 37°C for 1.5 hours
Test tube 4
▪ 4 ml distilled water
▪ 2 ml 0.5 N HCl
▪ Egg white
▪ Incubate at 37°C for 1.5 hours

• Record the results.

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2. STARCH digestion by SALIVARY AMYLASE
• Salivary amylase (the enzyme secreted by the salivary glands into the
mouth) hydrolyses starch to maltose. Salivary amylase thus causes a
decrease in the starch content and an increase in the sugar content.
• Before you can start with the experiment, saliva should first be collected.
Induce salivation by chewing on a parafilm square and collect 15 - 20 ml of
saliva in a beaker.
• First prepare the experimental test tubes (b) and then, during the incubation
time, the control test tubes (a).
• Mark all test tubes clearly.
a) Prepare the control test tubes as follows:
Test tube 1
▪ Place 3 ml of starch solution in the test tube and add three drops of
▪ Lugol’s iodine solution.
▪ The presence of a blue-black colour when the solution is added indicates
the presence of starch.
▪ As the starch is progressively hydrolysed, the colour of the blue-black
solution will change to blue-red and light red.
▪ If all the starch has been digested the colour will disappear.
Test tube 2
▪ A negative starch test is obtained by placing 3 ml of distilled water in a test
tube and adding three drops of Lugol’s solution.
Test tube 3
▪ A positive sugar test is obtained by placing 3 ml glucose solution in a test
tube and performing the Benedict test.
Benedict test:
➢ Add 15-20 drops of Benedict’s solution to the solution in the test tube.
➢ Mix the solution well and place the test tube in boiling water for five
minutes (the solution HAS TO boil for five minutes).
➢ A yellow-brown colour change indicates the presence of sugar.
➢ A colour change to green also indicates the presence of sugar, but in
smaller amounts.

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 Test tube 4
▪ A negative sugar test is obtained by placing 3 ml distilled water in a test
tube and performing the Benedict test.
▪ The colour of the solution will remain unchanged.

b) Prepare the experimental test tubes as follows:

REMEMBER TO SHAKE (STIR) THE TEST TUBES REGULARLY
DURING INCUBATION TIME!
Before the preparation of the test tubes are done: First place 5 ml saliva in
a test tube, place it in boiling water for five minutes and let it cool down.
Test tube 5
▪ Three drops Lugol’s solution
▪ 1 ml starch solution
▪ 2 ml saliva
▪ Incubate at 37°C for 1 hour
Test tube 6
▪ Three drops Lugol’s solution
▪ 1 ml starch solution
▪ 2 ml saliva
▪ Incubate at 0°C (ice) for 1 hour
Test tube 7
▪ 3 drops Lugol’s solution
▪ 1 ml starch solution
▪ 2 ml saliva (which has previously been boiled and cooled)
▪ Incubate at 37°C for 1 hour
Test tube 8
▪ Three drops Lugol’s solution
▪ 1 ml starch solution
▪ 2 ml saliva
▪ Two drops 0.5 N HCl (decrease pH to ± 3)
▪ Incubate at 37°C for 1 hour

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• Record the results after one hour incubation period on a table.
• Perform the Benedict’s test on test tubes 5 - 8, after the colour changes have
been recorded, and also indicate these results on the table.

3. PANCREATIC LIPASE digestion of FATS and the action of BILE

• Fat digestion requires two reactions:
1. Fats/oils are first emulsified to small droplets by bile.
2. Fats/oil droplets are broken down by lipase to monoglycerides and fatty
acids.
• Some of the end products of fat digestion (fatty acids) are organic acids that
decrease the pH. This provides an easy way to recognise that digestion is
ongoing or completed.
• The pH indicator, called phenol red (which you will use), changes from red
to yellow as the test tubes contents become acid.
• First prepare the experimental test tubes (c) and then, during the incubation
time, the control test tubes (a + b).
• Mark all test tubes clearly.

a) Prepare the control test tube as follows:

▪ Add 3 ml pancreatin solution, 3 ml bile solution and three drops phenol
red indicator to a test tube marked “control”.
▪ Add 10 drops of 0.2% NaOH and mix continuously until the tube content
turns to pink.
▪ Add five drops of vegetable oil.
▪ Add 2 drops 0.5 N HCl.
▪ The liquid will now turn yellow – this indicates that the test tube contains
an acidic product and will identify the tubes in which fat hydrolysis has
occurred.

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b) Bile has an emulsifying action of fats and thereby increases the surface
area so that enzymatic digestion can take place. To demonstrate the
action of bile on fats:
▪ Mark two test tubes A and B.
▪ Add 6 ml water, five drops of vegetable oil and one drop of phenol red
to test tube A.
▪ Add 3 ml water, 3 ml bile solution, five drops of vegetable oil and one
drop of phenol red to test tube B.
▪ Shake each tube vigorously and allow the tubes to stand at room
temperature for 15 minutes.
▪ If emulsification has occurred, the fat droplets will be suspended
throughout the water (forming an emulsion). If not, the oil will be floating
on the surface of the water.

c) Prepare the experimental test tubes as follows:

REMEMBER TO SHAKE THE TUBES AT REGULAR INTERVALS
DURING INCUBATION TIME!
Test tube 1
▪ 3 ml pancreatin
▪ 3 ml distilled water
▪ Three drops phenol red
▪ 10 drops 0.2% NaOH (colours pink)
▪ Five drops of vegetable oil
▪ Incubate at 37°C for 1 hour
Test tube 2
▪ 3 ml pancreatin
▪ 3 ml bile solution
▪ Three drops phenol red
▪ 10 drops 0.2% NaOH (colours pink)
▪ Five drops of vegetable oil
▪ Incubate at 37°C for 1 hour

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 Test tube 3
▪ 3 ml pancreatin
▪ 3 ml bile solution
▪ Three drops phenol red
▪ 10 drops 0.2% NaOH (colours pink)
▪ Five drops of vegetable oil
▪ Incubate at 0°C (ice) for 1 hour
Test tube 4
▪ 3 ml distilled water
▪ 3 ml bile solution
▪ Three drops phenol red
▪ 10 drops 0.2% NaOH (colours pink)
▪ Five drops of vegetable oil
▪ Incubate at 37°C for 1 hour

• Tabulate the results.

ASSIGNMENT
• Tabulate the results of the digestion of proteins, carbohydrates and fats in
your report.
• Explain the different results thoroughly in your report.

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 ELECTROCARDIOGRAM AND ELECTRO-ENCEPHALOGRAM (EEG)

Aim
Determining cardiovascular and electrocardio-graphic reactivity after the
administration of a laboratory stressor namely the colour-word-conflict /
SROOP test.

Introduction
Cinciripini (1986) describes cardiovascular reactivity as the differences in heart
frequency, blood pressure and other cardiovascular functioning measurements
that are observed during periods of rest and during the administration of a
stressor. The cardiovascular reaction to stress refers to the cardiovascular
component of a behavior reaction to stressful incidents.
The behavior reactions to a psychological stressor includes somato motor,
neuro endocrine and cardiovascular components.
a) Somatomotor reactions entail actively managing a stressor that offers a
threat or challenge.
b) The neuro-endocrine reactions entail a combination of the hypothalamus
adrenal cortex (HAC) and the sympathetic adrenal medulla (SAM) secretions,
e.g. cortisol and catecholamine (Henry et al., 1986). Stimulation of the alpha
and beta receptors is implied here.
c) The cardiovascular reactions include the following: An increase in the heart
frequency and myocardial contractility, skeletal muscle vasodilation, splanchnic
vasoconstriction, renal vasoconstriction, and a decrease in the renal excretion
of natrium

One laboratory stressor, namely color-word conflict test (CWT) is used to

bring about cardiovascular reactions.

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Color- word conflict test: Simulation of everyday situations with cognitive,
active control must/can be exercised over stress situations (stimulation of the α
and β receptors).
The management of these stresses can be influenced by:
• Previous experiences and perceptions that can influence the behavior
reaction.
• the person's perception of the stressor as a challenge of threat is
important and will influence the management of this stressor, namely
active (challenge) or passive (threat) (Henry et al., 1986) ;
• Physical and psychological components; and
• individual control over the environment and stressful incidents lower the
feelings of anxiety, as well as the circulated reactions in urinary excretion
of catecholamine and cortisol.

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188
Method
With the aid of a Finometer and the B-Alert X10 systems cardiovascular,
electrocardiogram and electro-encephalogram recordings will be
simultaneously obtained during resting and a one-minute exposure to a mental
stressor.

Finometer
• A resting baseline value (one minute recording _will be taken when the
subject is resting and relaxed. This means that the blood pressure must
be constant and not fluctuate much.
• Blood pressure values after administration of the color-word conflict test
are taken directly after the recording of the baseline values.
• Let the patient rest approximately 5 to 10 minutes after administering the
color would conflict test before continuing with the next stressor. Now do
the cold pressor test/hand in ice test. And place it in lukewarm water,
when dry it off.
• Silence must be maintained to obtain reliable results.

1. Note the following blood pressure results:

a. resting baseline values
b. blood can use during the administration of a stressor.
2. Calculate
a. average systolic blood pressure
b. average diastolic blood pressure
c. average heart frequency
To obtain optimal values, the entire one-minute recording should be
chosen to calculate an average value. because the values are
unstable and very considerably, a section of the one-minute
recording must be chosen, also called a window.
Therefore, first choose a window in the relevant minutes recording
from the printout of the four-channel writer.
the window size indicating the stable or plateau values in a minute I
found in a window consisting of approximately 20 to 30 more
seconds. nice table plateau values must then be used to calculate
the average blood pressure value.

189
 The window must therefore include 20 to 30 seconds of blood
pressure readings where the three highest values distributed over the
20 to 30 second window chosen, and an average value is calculated.
The window choice during the stress recordings will probably include
the last 20 to 30 seconds, is a person will only show his or her
strongest reaction to the stressor in that time frame.

3. Reactivity values: method of calculation

The changes in the blood pressure parameters while the stressors
are administered are printed out as percentage changes from the
baseline values and are calculated in the following way:
(X-Y) x (100/Y) = percentage change in the variable
Where X-Y= the difference between the baseline and stressor value
and
Y = baseline value
4. Graphical representation
you must present the average values of the reactivity blood pressure
values using radar diagrams *. For examination purposes, it is
important to note that you must also be able to represent the values
using histograms.
*radar diagram: Example Fig. 9.1
5. Typical questions that you must be able to answer in the exam and
not part of the report
a) Discuss color would conflict test on the cardiovascular, ECG, EEG
and limbic system.
b) Genetic and environmental factors influence the management of
stress?
c) Explain what is meant by stress management.
d) Define a resting blood pressure value.

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191
 DETERMINATION AND REGULATION OF SKIN AND BODY
TEMPERATURE

Objective
The aim is to observe the mechanisms that the body use to control body
temperature, and the influence of work on core temperature. The work will be
done using a step-up test to determine the effect on body temperature.

Introduction
The temperature in deeper tissue of the body is called core temperature, and
this temperature stays relatively constant within 0.6˚C except if a person
develops a disease. A person that is exposed to extremely high temperatures
in dry air can still maintain his temperature. The skin temperature is opposite of
core temperature, as skin temperature rises and falls with the temperature of
the environment. If we refer to the skin’s ability to lose heat to the environment,
it is the skin temperature that we refer to.

There is no single normal core temperature, as temperature differs between 36

and 37.5˚C if measured in different people. The average normal core
temperature is therefore between 36.7˚C and 37˚C measured orally, and 0.5˚C
higher if measured rectally. (Guyton & Hall, 2000:822)

Body temperature is controlled by balancing heat production with heat loss. If

the rate of heat production in the body is higher than the rate of heat loss, the
temperature will rise. This is also true for the opposite where body temperature
will fall if heat loss is greater than heat production.

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Apparatus
• Inner-ear thermometer.
• Oral thermometer.
• Skin thermometer.
• Step.

Method
• Thermo-connectors are attached to the person’s upper arm, chest, thigh
and calf.
• The subject is weighed.
• A table is used to ensure 75 W - 80 W of work during the step-up test. This
table gives the height of the step that must be used according to the
subject’s weight.
• During the step-up test the subject will drink 200 ml of water every 30
minutes (the experiment will last 90 minutes, i.e. the subject will drink water
twice).
• Every 10 minutes the following will be measured and noted in a table:
Body temperature at the four different parts of the body (°C):
• skin temperature;
• oral temperature;
• inner-ear temperature; and
• heart rate.
• After the 90 minutes the subject will be weighed again. (400 ml water
weighs approximately 400 g; this is added to the weight before the
experiment.)

193
 Table

Temperature measurements

ARM CHEST THIGH CALF Average skin ORAL EAR HEART

TIME temp temp (CORE) RATE
temp
ºC ºC ºC ºC ºC ºC ºC Beats/min

T0

T10

T20

T30

T40

T50

T60

T70

T80

T90

o Calculate the average skin temperature by using the following equation:

T AVERAGE: 0.3TARM + 0.3TCHEST + 0.2TTHIGH + 0.2TCALF

o Calculate the weight loss of the subject after the 90 minute step-up test.

194
 Weight before the experiment:
+ 0.4 kg H20

- Weight after the experiment:

= Weight loss

Graphs
▪ Draw a graph with Time (min) on the x-axis and Temperature (ºC) on the y-
axis. Indicate oral temperature, ear temperature and the average skin
temperature on the same graph (on the graph paper supplied).
▪ Hand this graph in the following day before 16:00 at Office 108. These marks
will form a part of the participation mark.

▪ Draw another graph with Time (min) on the x-axis and Heart rate (beats/ min)
on the y-axis (on the graph paper supplied).

After completion of this practical you must be able to:

• Draw a graph of the fluctuations of oral, ear and average skin temperature
against time;
• draw a graph of the fluctuations of heart rate against time;
• discuss the different mechanisms of heat loss;
• discuss the results and graphs in full according to the literature (text book);
• calculate the weight loss of the subject after the 90 minute step-up test; and
discuss the reason for this.

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LITERATURE USED

1. AMENTA, P.S. 1991. Histology and Human microanatomy. 6th ed. Italy:
Piccin. 660 p.
2. COETZEE, H.L., LOOTS, G.P. & MEIRING, J.H. 1997. Menslike histologie.
2nd edition. JL van Schaik. Akademies. 417p.
3. DI FIORE, M.S.H. 1981. Atlas of Human Histology. 5th ed. Philadelphia:
Lea & Febiger. 267 p.
4. FOX, S.I. 2002. Laboratory guide for Human Physiology. 9th ed. New
York: McGraw Hill. 418 p.
5. GUYTON, A.C. & HALL, J.E. 2011. Textbook of medical Physiology.
International edition. Pennsylvania: Elsevier Saunders. 1091 p.
6. LAUBCHER, P.J. & HUISMAN, H.W. 1996. Praktiese Fisiologie. 1st
edition. Unisa Publishers. 150 p.
7. MACLABSYSTEM, ADINSTRUMENTS. 2005. Physiology Experiments Manual.
Australia. [CD-ROM.]
8. MARTINI, F.H. 2015. Fundamentals of Anatomy & Physiology. 10th ed.
New Jersey: Prentice Hall. 1262 p.
9. HAMMERSEN, F. 1985. Sabotta/Hammersen Histology. 3rd ed. Munich:
Urban & Schwarzenberg. 260 p.
10. WIDMAIER, E.P., RAFF, H. & STRANG, K.T. 2012. Vander’s Human
Physiology: The mechanisms of body function. 13th ed. New York: McGraw
Hill. 708 p.
11. WISE, E. 2003. Laboratory manual to accompany Anatomy and
Physiology. 6th ed. New York: McGraw Hill. 544 p.
12. YOUNG, B. & HEATH, J.W. 2000. Wheater’s Functional Histology. 4th ed.
International: Churchill Livingston. 413 p.

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