physiology - baze university
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PHYSIOLOGY
PRACTICAL SCHEDULE AND WORKBOOK
for the Medical Sciences.
DEPARTMENT OF HUMAN PHYSIOLOGY FACULTY OF BASIC MEDICAL SCIENCES
BAZE UNIVERSITY, ABUJA
NAME___________________________________________________________________
LEVEL _________________________ COURSE _______________________________
MATRIC NUMBER _______________________________________________________
DEPARTMENT OF HUMAN PHYSIOLOGY
FACULTY OF BASIC MEDICAL SCIENCES BAZE UNIVERSITY, ABUJA
200 & 300 LEVEL
TABLE OF CONTENTS Foreword Table of Contents
SECTION: A
1. Introduction to Physiological Laboratory practices
2. Laboratory ethics and precautions 3. Physiological solutions
4. Introduction to Microscopy
5. Hematological Laboratory practices 6. Introduction to Statistic
SECTION B: HEMATOLOGY
1. Determination of the number of Red Blood corpuscle 2. Determination of the number of White Blood Corpuscle
3. Differential White Blood Cell Count
4. Platelet count 5. Estimation of Blood Hemoglobin
6. Packed Cell Volume
7. Normal Blood values and calculations
8. Hemostasis 1: Blood coagulation 9. Hemostasis 2: Prothrombin Time
10. Blood grouping (Typing)
11. Erythrocyte Sedimentation Rate 12. Estimation of Blood volume in Rats
13. Osmotic and Permeability properties of Red Blood Cells
SECTION C: NERVE AND MUSCLES 14. Pitting of Toad
15. Nerve Muscle preparation 16. Recording of simple Isotonic Muscle twitch 17. Demonstration of All or None Law, Tetanus and Fatigue
18. Velocity of Nerve conduction, Effect of Load on Muscle 19. Effect of successive stimulus on muscle
SECTION D: CARDIOVASCULER SYSTEM
20. Perfusion of the Heart
21. Study of cardiovascular vessels, the vein, Jugular venous Pressure etc
22. Determination of Heart Rate and Blood Pressure in Man
23. Assessment of some factors affecting Heart Rate and Blood
Pressure 24. Human Electrocardiography
SECTION E: RESPIRATORY SYSTEM
25. Clinical Examination of the Heart and respiratory system
26. Respiratory Function Test,Spirometry,Vitallography 27. Peak Expiratory Flow Rate
28. Effects of Hyperventilation and Breath Holding Time on respiration
29. Effect of Exercise on Respiratory system
SECTION F: GASTROINTESTINAL SYSTEM
30. Investigation of Digestive activities of Saliva 31. Collection and analysis of Gastric Juice I Rat
32. Intestinal Motility
SECTION G: RENAL PHYSIOLOGY
33. Urine formation in Man
34. Renal regulation of Acid Base Balance 35. Human Diuresis
36. Renal Regulation of ECF
37. Effect of Saline on Urine Tonicity
38. Renal Function Test – Urea 39. Renal Function Test – Creatinine
SECTION H Endocrine Physiology
40. The Thorn’s test
41. Estimation of blood glucose SECTION I
REPRODUCTION
42. A study of estrous cycle in rat by vaginal smear technique 43. Semen Analysis
SECTION J NEUROPHYSIOLOGY II
44. Cutaneous sensation
45. Clinical reflexes
46. Vision I 47. Vision II
48. Hearing and Balance
INTRODUCTION TO LABORATORY PRACTICES
GENERAL NOTES AND INSTRUCTIONS
The objectives of the practical classes are:
1. To enable you to learn some of the practical techniques used in clinical
investigation. This is not necessarily the same as acquiring technical skill,
which comes only with much repetition and practice.
2. To enable you appreciate physiology as an experimental science and as such
requires a scientific and methodological approach.
3. To re-encode the physiological knowledge, you have gained from lectures and
from reading by direct observation.
4. To give you an opportunity of discussing physiological problems with
members of the teaching staff and demonstrators.
HOW TO BENEFIT FROM THE COURSE
1. COME REPARED to all practical classes; this means:
Make sure you know beforehand which experiment you are going to perform
and read it up in the schedule. Go through the apparatus in front of you in
the laboratory before beginning the experiment.
Bring to the laboratory everything you need for the physiology experiment.
These include dissection instruments and the laboratory coat which should be
brought to every class whether or not the experiment indicates the need. It
may sometimes be necessary at the last moment due to a change on the
timetable.
2. Participate as much as possible in the experiments. It may really be possible
for experiments to be performed in pairs (the ideal number for most
experiments) and you will usually find yourself a member of a large group
should do all the work while others are merely spectators or worse still, sit
reading newspapers or magazines. Other active help and/or take it in time to
perform various parts of the experiment.
3. The demonstrators are available to assist when necessary and not to
experiment (unless it is a demonstration). As for their help when you are
genuinely in difficulty and not because you can’t be bothered to read the
schedule.
4. BE PUNCTUAL so that the group introduction to the experiment takes place in
time leaving as much time as possible for your experiment.
5. When experimenting makes sure you obtain ALL NECESSARY data before
leaving the laboratory. Ensure tracings are fully labeled. Such labeling should
include heading, date, and time. Trace arrows indication e. g. addition of
drugsthat should be labeled with the name of the drug, its concentration, and
any relevant information. When you work in groups make sure you have your
own set of data and when only one Kymograph tracing is available, copy or
trace the recordings for your use.
USEFUL HINTS IN WRITING UP EXPERIMENT
SUBMISSION OF WORKBOOK
Your practical schedule/workbook marked after every practical session will
form part of the overall assessment of your practical work.
Successful experimentation consists not only of inaccurate observation and
recording of data but also in the correct interpretation of results. Do not write up
your practice schedule in the laboratory. You can use a rough notebook, loose
writing for making notes in the laboratory during the experiment. Answers to each
experiment should be written up in your practical workbook after the experiment as
soon as possible.
PRACTICAL NOTEBOOK
In addition to the manual and workbook, each student is required to have a
practical notebook. These should large notebooks with soft covers. It is not
necessary to buy the type with alternate graph papers. The write-ups should
normally take the following format;
Title or object of the experiment (with date)
Method: this should not be a repetition of the practice schedule. If the experiment
was performed exactly as in the schedule, then it is sufficient to state that, that was
the case. However, in many experiments you may find you have a minor modification
or it may be useful to add some hints and diagrams,etc, to make the practical sheet
more meaningful.
Result:this should when possible be neatly tabulated, calculations shown in full,
graphs plotted and labeled where necessary, tracings should be cut into small
portions to illustrate relevant points and struck firmly into place.
In expressing your result, be sensible in general, do not put the more significant
figure into a calculated value that is calculated in the measurements themselves.
For example, you may analyze room air and calculate from the result that the
percentage of Oxygen is 21.782%. However, you will know that with the method you
have used coupled with your relative inexperience in handling the apparatus, it will
be unrealistic to assume that you could be as accurate as 22% would, therefore,be
aa more realistic expression of the result obtained.
If you are unlucky and do not obtain any result, you may say so but you may also
find it useful to obtain results from a colleague so that at least you would know how
to handle such results if they are presented to you in an examination.
Conclusion and/or Discussion: Any relevant conclusion should be dated. Also, the
result should be fully discussed including such points as are the result normal? If
not, why not? What physiological mechanisms are involved?
Classsheets: class sheets may be provided for some of the experiments so that all
the results obtained by the group may be collated. This may serve to introduce some
of the concepts of Medical/Biostatistics and to demonstrate how “Normal” your
results are related to the test of the class. The overall class results will be given to
you usually in your next practical class.
NOTES ON INSTRUMENTS
Ideally for physiological experiments, instruments that are usedfor anatomical
dissections should not be used for living tissues. Since it is realized that it is
uneconomical for students to purchase two sets of instruments, your instruments
must be washed very thoroughly after anatomy dissection classes.
The following represents the minimum necessary instruments you will need:
Scissors: two pairs (a) a rough pair of medium size, useful for cutting throughbone,
skin, etc. (b) a fine sharp pair that cut right to the points of the blades
Forceps: two pairs (a) fine-pointed forceps, (b) blunt-ended medium-forceps. A pair
of fine curved forceps may also be used.
Scalpels: will not normally be used for experiments you will perform yourself.
Dividers: a pair of dividers and a ruler is useful for making accuratemeasurements.
CONDUCT IN THE LABORATORY
1. Laboratory coat should normally be worn.
2. All bags etc. should be left outside or near the door of the laboratory.
3. There should be no eating, drinking, or smoking in the laboratory (unless this
form a part of the experiment).
4. Your bench must be left tidy at the end of the class. Alldead animals and tissues
should be put in the bucket provided.
5. Any breakage or faults in the apparatus must be reported to the technical staff.
GENERAL RULES FOR WORKING WITH LIVING TISSUES
In carrying out experiments on any living tissue, certain elementary rules must be
followed:
1. Ensure that the apparatus is completely ready and in working order before
preparing the tissue. Even the most robust "tissue deteriorates after removal
from the animal and the longer it is before experimenting the poorer your
results are likely to be.
2. An important part of preparing the apparatus is to wash your instruments
thoroughly, even if they were washed after last being used, as they should have
been. Care is needed if the instruments have been used in anatomical dissects
of preserved material.
3. Wash your hands thoroughly immediately before beginning dissection and after
any dirty job during the practical, e.g. smoking a drum, or varnishing a tracing
in experiments living tissue a high degree of cleanliness is essential.
4. Living tissues must be kept moist with the appropriate physiological saline. Any
tendency to dry up will lead to irreparable damage and poor results. Handle the
tissue very carefully especially nerves. A nerve damaged by pinching or
stretching will not survive to conduct an impulse well.
5. In carrying out dissections, it is best to sit down at the bench so that the wrist
can be rested on the bench. The hands can then be held much steadier and
better dissection will be attained.
6. Take great care of your instruments. Protect the points of your scissors and
forceps. Keep your fine scissors for fine work; do not attempt to cut bone, skin,
or any other tough tissue with them. At the end of the experiment, wash and
dry instruments thoroughly to avoid corrosion.
7. When pin electrodes are being used they should always be placed carefully in
contact with the tissue to be stimulated, to deform it as little as possible. Make
sure that they are fixed independently of the tissue so as not to drag on it, and
see that they are not submerged in a large pool of saline which will short-circuit
part of the current intended for stimulating the tissue. Do not push pin
electrodes down on top of a nerve. This crushes it.
PREPARATION OF ANIMAL
The actual type of preparation required depends partly on the animal being used
and partly on the type of experiment to be performed.
1. If the experiment in vivo, the suitable anesthetic must be used to ensure that the
animal is unconscious and yet remains alive for the duration of the experiment.
The anesthetic use and its mode of administration are usually given at the
beginning of the appropriate procedure.
2. If the experiment is to be performed to Vivo i.e. outside the animal, then it may
be adequate to kill the animal by a sharp blow on the back of its head, the tissue
then being dissected out from the animal with maximum speed, particularly in
the case of mammalian tissues.
3. In the case of toad experiments, the animal is prepared in various ways
depending on the nature of the experiment to be performed. In all of them, the
FOREBRAIN is DESTROYED so that the animal does not feel pain.
Destruction of the entire nervous system
This is the commonest preparation. Stun a toad striking the back of its head
by placing the lower blade of a strong pair of scissors between the jaws and the other
blade over the dorsal surface of the skull. With one clean out along a line joining the
anterior borders of the tympana, remove the anterior part of the skull containing the
cerebral hemisphere and mid-brain. At this stage, the preparation is decerebrate,
Now, push the blunt seeker down the vertebral canal, thus destroying the rest of the
C.N.S. i.e. the medulla and spinal cord. The entire C.N.S. is now destroyed, all
reflexed are abolished, and the preparation is said to be "pithed". A twisting
movement of the seeker effects the necessary destruction. As the central nervous
system is being destroyed, there is a stimulation of the body and limbs. Make sure
the animal is now without reflexes
Decerebrate Preparation
Destroy the forebrain as already described. After recovery from shock, the
preparation has normal reflexes.
Spinal Preparation
Stun a toad and pass the end of a scalpel handle lightly backward over the
dorsal surface of the head until the occipital-atlentoid joints are reached, this is
recognized as depression just posterior to the slight protrusion formed by the
occipital condyles. Insert the point of the scalpel into the joint and sever the spinal
cord. Then bend the skull forwards and pass a blurt seeker through the opening just
made into the cranium to destroy the brain. Cut off the front of the head as before,
to make certain the forebrain is intact after a short interval, the spinal reflexes can
be readily obtained.
Medulla Preparation
Stun a toad and cut open the occipito-atlantoid joint as for a spinal
preparation. Insert a blunt seeker and pass it downwards destroying the spinal cord.
Now, decrebrate the preparation by cutting off the front of the head. The medulla is
thus left intact.
PHYSIOLOGICAL SOLUTIONS
Isolated organs and tissue will only remain alive outside the body if they are
surrounded by the current physical and chemical environment i.e. if immersed in a
solution containing the correct concentrations of salts, within the permitted range of
temperature and pH and adequate oxygen supply.
Correct in this context means that the solution should as far as possible,
resemble that of its normal environment within the body.
The composition of such, solutions, varies for different animal groups and
different tissues. Some of the common physiological solutions, which you will
encounter during your practical courses, are shown below:
TOAD TISSUES
Normal (i.e. isotonic) saline: 0.6% Nacl (approx. 150 meq/1).
Ringer's solution: NaCl - 0.6%
KCl - 0.042%
CaC12 - 0.024%
NaHC03 - 0.015%
Glucose - 0.1%
MAMMALIAN TISSUE:
Normal (i.e. isotonic) saline - 0.9% NaCl (approx. 150 meq/1)
Heart – Ringer – Locke’s Solution NaCl - 0.9%
KCl - 0.042%
CaC12 0.024%
NaHCO3 - 0.015%
Glucose - 0.1%
Intestine – Tyrode’s Solution NaCl - 0.8%
KCl - 0.02%
CaC12 0.02%
NaHCO3 - 0.01%
NaH2PO4 - 0.005%
Glucose - 0.1%
INTRODUCTION TO MICROSCOPY
A microscope is an optical instrument used for viewing very small objects such
as mineral samples or plant and animal cells typically magnified several hundred
times.
There are three types of microscope presently in use in learning institutions
namely Monocular, Binocular, and Trinocular depending on the number of the
ocular lens present for the viewing of the objects.
PARTS OF MICROSCOPE
The microscope is supported by a U shaped base which is connected to a short
pillar. The extension of the pillar is the arm or handle. Between the pillar and the
arm is the inclination joint.
Attached to the arm is the body tube (draw tube), on top of which is the
eyepiece or ocular. At the posterior end of the tube is the revolving nosepiece. A
compound microscope generally has three objectives connected to the nosepiece, the
low power objective (LPO), high power objectives (HPO), and the oil immersion. The
low power objective is shorter and usually marked X10 or 10X while high power
objectives are marked X10 and X100. The oil immersion is marked X
Just below the objectives attached to the arm is the stage on which the slide
being examined is placed. Beneath the stage is the substage where the condenser
and the iris diaphragm are located. The condenser concentrates light rays on the
object, while the diaphragm regulates the amount of the light passing through the
lens. Underneath the substage attached to the pillar is the light source.
OPERATION OF THE MICROSCOPE
1. Turn the revolving nosepiece until the low power objective (LPO) is in place.
2. Turn on the light source and regulate the amount of light. The entire field of
the microscope should be properly illuminated.
3. Place a slide with the mounted letter (e) in the upright position at the center of
the stage.
4. You may add a drop of water to keep the paper in place.
5. Using a coarse adjustment knob, lower the tube of the microscope until the
objective almost torches the slide.
6. While looking through the eyepiece of the microscope, gradually raise the tube
until the letter comes to view.
Note: if you could not see the letter, it means that the slide has not been
placed at the center of the field.
Never lower the body tube while focusing through the ocular of the
Microscope
7. Use the adjustment knob to bring the specimen to a distinct focus.
8. To increase the magnification, turn the nosepiece until the high power
objective (HPO) is in position.
CARE OF THE MICROSCOPE
The following precautionary measure should always be followed at all times in
handling the microscope:
a. Always carry the microscope with both hands in a vertical position.
b. Place the microscope far from the edge of the table to prevent it from
accidental falls.
c. Before and after use, make certain that all the parts are in place. Report any
missing or damaged parts immediately to the technologist.
d. Always keep the microscope clean. Use lens cleaner in cleaning the lens.
e. Avoid liquid especially chemicals, i. e. acids and alcohol that may come in
contact with the microscope.
VIEWING A LIVING CELL
The structural unit of the creatures is the Cell. It is a small membrane-
bounded compartment filled with an aqueous chemical substance. Cells may exist
as individuals as in certain protozoan (single-cell animal) or maybe intricately
associated as in tissue or many-celled animals as in the case of metazoans.
You may find it difficult to locate all parts of a cell under the light microscope
(LM). A great deal of information about cell structure can be obtained using the
electron microscope (EM),
Procedure
1. Rinse your mouth with water.
2. Using a clean tongue depressor, lightly scrape a few epidermal cells from the
inside of your cheek (mouth cavity)
3. Convey the material to a clean glass slide.
4. Add a drop of water and a small drop of Methylene Blue cover with a cover slip
and observe under the light microscope with both low power (LPO and high
power objectives (HPO).
In your observation, note the following structures:
Nucleus – spherical body located within the cell
Cytoplasm – the materials surrounding the nucleus and extraneous to it is the
cytoplasm. Microbodies and/or organelles suspended in the material, but they are
not usually visible in these cells.
Cell Membrane – the outer boundary of the cell. This is a continuous sheet of liquid
molecules about 4 – 5 nm (nanometer) thick in which various protein molecules
serve as a pump and channels for transporting specific molecules into and out of the
cell.
Exercise
1. Drawa few cheek cells
2. Draw a cheek cell and label the parts
3. Draw a typical animal cell and label the important structures.
INTRODUCTION TO EXPERIMENTS ON BLOOD
Several of the experiments with blood necessitate the use of special blood
pipettes. These pipettes will have been cleaned before they are given to you.
Uncoagulated blood (i.e. blood to which no anticoagulant has been added) will
readily clot inside the pipette. The pipette will then be very difficult to clean. If you
think that the blood you are using is about to clot blow out the blood and start all
over again.
Some of the experiments you will perform will be with your bloodand some will
be that of your colleagues.
(a). Taking a Sample of your Blood
(i) Select the areas from which the sample will be taken. The usual sites are the
pulp or nail bed of the thumb or finger, or the ear-lobe.
(ii) Cleanse the area with a swab soaked in alcohol.
(iii) Quickly prick the area with the sterile lancet provided. Blood should flow freely
with only the slightest application of pressure. Squeezing the area to obtain
more blood may force out tissue fluid, which will dilute the blood and give an
erroneous result. If blood does not flow readily wipe the area and start again.
(iv) When a good size drop of blood has collected, draw the blood into the pipette as
instructed for the experiment you are doing.
(v) Cleanse the area of the wound and stop the bleeding.
(b) Taking a sample of Bank Blood
When blood to which an anticoagulant has been added is left standing on the
bench, the blood cells tend to settle towards the bottom of the container. It is,
therefore, necessary to mix the blood thoroughly, preferably by inverting the
container several times and then quickly taking the sample into the pipette.
EXPERIMENT 1
EXPERIMENT TO DETERMINE THE NUMBER OF RED BLOOD CORPUSCLES PER CUBIC MILLIMETER OF HUMAN BLOOD
Title: Haematology
Aim: To carry out a total red blood cell count of a subject Apparatus and Reagent:
I. Improved Neubauer counting chamber 2. Red cell pipette
3. Red cell diluting fluid
Procedure
1. Draw blood from the chosen site of the subject, or a sample of oxalate blood, into
a red cell pipette, until it is level with the 0.5 marks. This will give a dilution of 1 in 200. If the patient is very anemic, it is advisable to draw the blood up to the 1
mark, to obtain a final dilution of 1 in 100. If blood is drawn above the chosen
mark, the tip of the pipette should be touched with cotton wool to withdraw the excess.
2. Wipe the outside of the pipette with a piece of clean gauze and draw the diluting
fluid up to 101 marks, rotating the pipette during the process.
3. Withdraw the pipette from the diluting fluid and wipe the outside with a piece of clean gauze. Close the tip of the pipette with the thumb, remove the sucker, place
the middle finger over the top and mix well by shaking. Alternatively prepare
1:200 dilution of blood by bulk dilution (by taking 20mm3 (0.02ml) of blood in a hemoglobin pipette and washing it into 4ml of diluting fluid contained in a bijou
bottle). White cells can be similarly diluted by taking 50 mm3 (0.05 ml) of blood
into 0.95ml of white cell diluting fluid to obtain a 1 in 20 dilutions).
4. Thoroughly clean the counting chamber and cover glass, place on a flat horizontal surface, and using a firm pressure slide the coverslip into position on a
counting chamber, obtaining a rainbow effect on both sides (Newton's rings).
5. Mix the suspension well by shaking the pipette for 3 - 4 minutes and discard about a quarter of the mixture.
6. Fill the chamber by holding the pipette at an angle of 45 degrees and lightly
touching the tip against the edge of the coverslip. The fluid mustn't be allowed to overflow into the channels, Should this occur, the chamber should be cleaned and refilled; too much fluid in the resulting in gross errors.
7. Place the chamber on the microscope stage, allow several minutes for the cells to settle. Using a 4mm objective and x10 eye-piece, focus the objective on to the central square millimeter of the counting chamber and count all the cells
contained within 80 of the 400 small squares (five groups of 16 small squares). Cells touching the centerline boarding the top and right-hand side of each group
of 16 square should be included with the count. Those which touch the centerline on the left hand and lower boarder should be disregarded.
Using an ordinary Neubauer counting chamber, cells touching the inside of the
triple lines boarding the top and right-hand side of each large square should be included with the count, those touching the other two sides should be disregarded.
For the final result to be expressed as the number of cells per cubic millimeter, the
following calculation is necessary.
Calculation
Let N equal the number of cells counted in 80 small squares. The area of each small
square is 1/400 sq. mm, and the depth of the chamber is 1/10mm.
The volume of fluid over a small square is therefore
1/400 X 1/10 = 1/4000 c./mm,
If N cells are counted in 80 / 4000 c.mm of diluted blood, then 1c.mm of undiluted blood would contain: N x 4000/80 x 200/1 cells/c.mrn
= N x 10,000 cells/c.mm.
In practice, with a dilution of 1 in 200, and when cells in 80 small squares are counted, four zeros may be added to the number of cells counted.
Do a red cell count of the sample of blood provided. Discuss your results. Note the general formula for calculating cell number in a counting chamber:
counted cells x dilution factor
____________________________________ = cells per ml of blood mm2 of counted area x chamber depths
The experiment should be performed (a) with the blood provided (b) with your blood.
QUESTIONS 1. What is the normal range of R. B. C. count in male and female students in your
practice group?
___________________________________________________________________________
2. List 4 factors that can lead to physiological variations in RBC count.
a. _____________________________ b.__________________________________
c. _____________________________ d. __________________________________
3. Discuss the possible errors of the method
___________________________________________________________________________ ___________________________________________________________________________
4. What are the functions of the different ingredients of the diluting fluid? a.____________________________________________________________________________
b.____________________________________________________________________________
c.____________________________________________________________________________
d.____________________________________________________________________________
e.____________________________________________________________________________
5. Name other methods that are available for the determination of RBC counts ____________________________________________________________________________________________________________________________
______________________________________________________________
__________________________________________________
EXPERIMENT 2
EXPERIMENT TO DETERMINE THE NUMBER OF WHITE BLOOD CORPUSCLES PER CUBIC MILLIMETER OF HUMAN BLOOD
Title: Hematology
Aim: To carry out a total white blood cell count of a subject The estimation of the total number of white cells is relatively a simpler and easier
procedure. It has a definite clinical significance and hence is carried out very
frequently.
Principle: The principle and the method of counting the white blood cells is similar
to that of counting the red cells. The diluting fluid used is different. It contains 2ml
of Glacial acetic acid and a drop of 1% Gentian violet solution and all diluted to 100ml with distilled water. This hypotonic solution very quickly causes complete
lysis of the red cells. The hemoglobin being lost the ghosts of the red cells becomes
invisible under low power (they could be seen under high power).
Though the WBCs also lose their cytoplasm, they show their nuclei prominently
fixed by the acid and stained by the gentian violet. The number of white cells is
counted in a relatively large amount of space and under the low power of the
microscope.
Apparatus and Reagent
1. Improved Neubauer counting chamber 2. White cell pipette (bulb type)
3. White cell diluting fluid, Turk's diluting fluid for white blood cell (give the
composition to the students)
Procedure:
Draw blood to the 0.5 marks on the stem of a white cell pipette, and diluting fluid to
the 11 marks immediately above the bulb. Alternatively prepare a 1/20 dilution of blood as described under the bulk dilution method. Using the improved Neubauer
chamber, count the cells in the 4 corner square millimeters. Apply the same margin
rules as for the erythrocyte count.
Calculation:
Let N = represent the number of cells counted in 4sq.mm.
Since the depth of the chamber is 1/10mm. N cells are counted in 0.4 = 4/10mm3 of
diluted blood.
1mm3 of diluted blood contains N x 10/4 cells. Since blood was diluted 1 in 20.
Therefore, 1mm3 of blood (undiluted) contains N x 10/4 X 20/1 = 50N cells/mm3
In practice, with a dilution of 1 in 20 and when cells in 4-millimeter squares are
counted, the number of White cells may be multiplied by 50, Example: Number of
White cells counted= 200.
Multiplying this by 50 = 10,000 cells per mm3
Note on Technique:
1. When using bulb type pipettes, be sure to expel all the fluid contained within
the stem of the pipette before filling the counting chamber. The stem contains
only diluting fluid, the blood diluents suspension being within the bulb of the
pipette.
2. The pipette should be examined periodically to ensure that the tips are not
chipped or damaged.
3. After cleaning, new pipettes should be checked for accuracy.
QUESTIONS
1. What is the normal range of W. B. C. count in male and female students in your practice group?
___________________________________________________________________________
2. List 4 factors that can lead to physiological variations in W. B. C. count.
a.______________________________________________________________________________
b______________________________________________________________________________
c______________________________________________________________________________
d. _____________________________________________________________________________
3. What is the ratio of W. B. C to R. B. C.
count?________________________________________
4. Discuss the possible errors of the
method____________________________________________
________________________________________________________________________________
________________________________________________________________________________
____________________________________________________________________
5. What are the functions of the different ingredients of the diluting fluid?
a.______________________________________________________________________________
b.______________________________________________________________________________
c.______________________________________________________________________________
d.______________________________________________________________________________
e.______________________________________________________________________________
6. Discuss the clinical significance of a white cell count. ________________________________________________________________________________
________________________________________________________________________________
____________________________________________________________________
7. Why will this experiment not be effectively carried out with banked blood?
___________________________________________________________________________________
___________________________________________________________________________________
____________________________________________________________
EXPERIMENT 3
DIFFERENTIAL WHITE CELL COUNT
The enumeration of each type of white cell is called the differential count. It is
carried out under an oil immersion lens on a stained film.
The film prepared for this purpose should be made from a small but whole
drop of freely flowing blood.
Preparation of the Slide Film
• Gently touch a fresh drop to one end of a clean grease-free slide. Using a
beveled piece of glass a little narrower than the slide.
• Allow the drop to spread along with it. • Holding the slide and spreader at a suitable angle push the spreader along
the slide, drawing the blood behind it until the whole of the drop has been
smeared. • Do not have too large a drop, or incline the spreader at too great an angle, as
the film will be too thick for satisfactory microscopic examination.
• The count is performed using either the battlement method or the
longitudinal method
Practical
Title: Haematology Experiment: Staining the blood film with Leishman's stain
Aim: To carry out white blood cells differential count of a subject
Apparatus and Materials: Staining rack, petri dish, and buffered diluted water at pH 6.8, Leishman’s stain.
Method:
It is desirable to stain at least two slides at a time.
Place the slide in the petri dish or on the staining rack; with the smear facing you.
Put drop by drop the stain on the film to cover all the film. Count the drops
you add. Note the time; see that all the film is covered by the stain. This
undiluted stain is allowed to act for one and a half minutes. This is done to fix the film in Methyl alcohol (the solvent) which precipitates the proteins and
which sticks firmly to the slide. The omission of this precaution will cause the
loss of the film during washing.
After about one and a half minutes place twice the number of drops distilled water and dilute the stain. A scum appears on the surface.
Mix by rocking and by blowing through an ordinary pipette. The stain should
not be allowed to dry. The film should be allowed to be stained for 10 minutes. Rock the slide frequently to prevent drying.
Wash the excess of the stain with distilled water or.
Now either keep the slide dipped in running tap water or in buffered water pH
6.8 until the film develops a pink appearance.
Drain the excess of water; wipe the backside of the slide with a clean and dry
filter paper. Keep it in a vertical position to drain and dry.
Examine the film to decide whether it is worth investigating or not.
Examine the film under the microscope. A well-stained film shows (1) no precipitate or granules on or between cells (2) the
granulocyte cytoplasm of a slaty blue color (4) the neutrophil cytoplasm granules of
a dull shade of lilac or light purple color (5) the nuclei and the basophilic granules of
blue or purple color and (6) the eosinophil granules of pink color. Certain parts of the cells have an affinity for the basic portion of the stain e.g. the
nuclear chromatin being acid in character takes up basic portion i.e. the methylene
blue and stains deep blue. The basic protoplasmic material takes the acidic - eosin i.e. pink dye: and the neutral material appears violet.
Result of well- stained film show:
1. No precipitate or granules between the cells. 2. The red cells having an orange buff or pink color.
3. The agranulocyte cytoplasm of a slaty blue color
4. The neutrophil cytoplasm granules of a dull shade of lilac or light purple color.
5. The nuclei and the basophilic granules of blue to purple color and 6. The eosinophil granules of pink color
Count 200 cells of all types and discuss their percentages.
Counting:
a. Using The Battlement Method The film is examined systematically, by being transversed 3 fields along the edge, 2
fields up, 2 fields along and 2 fields
b. Using The Longitudinal Method:
The cells are counted in one complete longitudinal strip of the film.
Now shift the slide up [the movement seen under the microscope will be
opposite direction). Bring the lower adjacent field in view. Identity and enter
your observations.
Then go on counting towards the left again till you reach the end. Then shift lower down and go on counting towards the left again till you reach the end.
Then shift lower down and go on counting towards the left again and soon.
In cases where extreme accuracy is required, a minimum of 400 cells should be counted. A multiple counting machine greatly facilities this technique. The number
of each type of cell counted is recorded as well as the total. The absolute number of
a particular type of leucocyte can be calculated from the results of the total WBC count and differential count recorded as a percentage.
Observations
Record the number of cells counted with the features highlighted below
Cell type Staining Reactions
Nucleus Cytoplasm granules
Neutrophils dark blue pale pink reddish lilac
Eosinophils Blue blue red-orange
Basophils purple or dark blue
dark purple almost black
Monocytes (lobated): violet sky blue -
Lymphocytes Violet dark blue -
Sources of Error in Counting Errors in counting may be due to any of the following factors. Low Counts
1. Squeezing the site of the puncture when filling the pipette.
2. Insufficient blood being into the pipette.
3. Too much diluent is drawn into the pipette 4. Insufficient mixing
5. Using the first fluid expelled from the pipette
6. Insufficiently filling the counting chamber 7. Uneven distribution of the cells in the counting chamber
8. Inaccurately calibrated pipette
9. Faulty counting technique
10. Saliva in the mouthpiece increasing the dilution 11. Errors in the calculation.
VARIATIONS An increase in the number of circulating Neutrophils is known as NEUTROPHILIA
and a decrease in NEUTROPENIA.
An increase in the number of circulating lymphocytes is known as LYMPHOCYTOSIS and a decrease as LYMPHOPENIA.
An increase in the number of circulating Eosinophils is known as EOSINOPHILIA
and that of Monocytes is MONOCYTOSIS.
It is important to remember that an increased percentage of Neutrophils for example does not necessarily indicate neutrophilia. This will depend on its ABSOLUTE
VALUE.
Whilst performing differential White cell counts, it usual to examine the red cells for any abnormality. A deficiency of hemoglobin results in a pale or unstained area in
the center of the red cell. This is called HYPOCHROMASIA. Diffuse purplish-grey staining of the cell is called POLYCHROMIA and is a sign of cell immaturity. These cells are usually seen to be reticulocytes when stained with a vital stain.
The term anisochromasia is used to describe varied stain uptake by the cell. Abnormalities in red cell shape referred to as POILKILOCYTOSIS, variation in cell
size is termed ANISOCYTOSIS, occasionally some oval-shaped red cells are seen, this
inherited characteristic is called leukocytosis or elliptocytosis.
Report and discuss our result on the blood specimen supplied.
QUESTIONS
1. What is the normal range of W B C count in male and female students in your
practice group? ___________________________________________________________________________
___________________________________________________________________________
___________________________________________________________________________
___________________________________________________________________________
2. List 4 factors that can lead to physiological variations in W B C count.
___________________________________________________________________________
___________________________________________________________________________
___________________________________________________________________________
___________________________________________________________________________
3. What is the ratio of WBC to RBC count?
___________________________________________________________________________
___________________________________________________________________________
___________________________________________________________________________
___________________________________________________________________________
4. Deviations of different white cell counts from the normal values often indicate a
diseased state.
_____________________________________________________________________________
_____________________________________________________________________________
_____________________________________________________________________________
_____________________________________________________________________________
5. List five conditions which may affect the count of the leucocytes
____________________________________________________________________
____________________________________________________________________
____________________________________________________________________
____________________________________________________________________
____________________________________________________________________
EXPERIMENT 4
PLATELET COUNT
Title: Haematology Experiment: Platelet count Aim: To determine a total platelets count of a subject
Platelets are 2-3 µm in diameter and averaged 250,000 per mm with a range of 150,000 - 300,000/mm3
The tip is nipped off and circulates in the blood as a non-nucleated platelet. They aggregate and break down at the site of injury or when they come in contact
with a foreign surface. To study patients thought to be suffering from blood clotting defects and also ascertain adequate platelet numbers and function, this experiment is essential though not conclusive.
Principle: Platelets are tiny cells that will not be able to be enumerated by physical examination or by use of microscope only. The enumeration can be achieved by diluting it using a special fluid to reduce the numbers and at the end, the dilution
factor and the volume of the counting chambers were used to find the actual platelet
count in such an individual
Apparatus and Materials:
1. Improved Neubauer counting chamber
2. White cell pipette (bulb type) 3. Cotton wool
4. Microscope
5. Coverslip
The Dilution Pipette
White cell pipette is used for the platelets dilution.
The Counting Chamber: See description in the introduction
Platelets Diluting Fluid
1. Boar’s Fluid Sponging B.D.H ….. 0.25 g
Sodium citrate ……. 3.5g
Formalin ……………….. 1.0ml
Brilliant cresyl blue ……. 0.1g Distilled water …….…… 100ml
2. Kristenson's fluid (Lempert's modification)
a. Solution A
Sodium citrate ………….. 1.0 g Mercuric chloride ………. 0.002 g Brilliant cresyl Blue …….. 0.2 g
Distilled water ……………100 ml (added at a temperature of 45°C)
Note: store in the dark place
b. Solution B
Urea (Analar) ………………………. 20 g Buffered distilled water (PH 7.2) … 100 ml
Mix equal volumes of solutions A and B immediately before use. Procedure:
1. Draw diluting fluid to 0.5 marks of the white cell pipette. This helps to prevent
the destruction of platelets as they have direct contact with the glass of the
pipette. The blood which has been anticoagulated with sequestrate may also be used for the test, in which case this precaution is unnecessary and the
blood may be taken up to the 11 marks.
2. Blood is then drawn up to the 0.5 marks (that is the top of the column or diluting fluid is on the 1.0 mark) where a blood sample is provided. In a case
where a blood sample is not given, the demonstrator will explain the technique
of blood collection.
3. Wipe the outside of the pipette with a piece of clean gauze and draw the diluting fluid up to thel1 mark and mix well by rolling the pipette between two
palms.
4. Thoroughly clean the counting chamber and coverslip, place on a flat horizontal surface, and using a firm pressure slide the coverslip into position
on both sides (Newton's rings).
5. Mix the diluted blood well again and blowout at least a third of the content of the blood to remove the unmixed dilution fluid between mark 1 and the tip of the pipette. Alternately prepare 1:20 dilution of blood by taking 50 mm (0.5
ml) of blood into 0.95ml of platelet diluting fluid to obtain a 1 in 20 dilutions. 6. The charging of the counting chamber and counting of the platelets is the
same as for red cells that are in 80 small squares.
Calculation
This is the same as for red cell counting, except that as the dilution is only 1 in 20, the number of platelets per c.mm is a thousand times the number counted in
80 small squares.
Sources of Error in Counting
Low count
1. Inability to draw diluting fluid first before the blood during dilution for blood
not anticoagulated with sequestrate 2. Mechanical hemolysis during mixing of the blood dilution fluid.
See also the sources of error in counting on red and White cell counting enumerated
in experiments. Do platelets count of the sample of blood provided.
Discuss your result.
Questions 1. what is the significant of this experiment?
_______________________________________________________________________
_______________________________________________________________________ _________________________________________________________________________
_______________________________________________________________________
_________________________________________________________________________
EXPERIMENT 5
ESTIMATION OF BLOOD HAEMOGLOBIN
Haemoglobinometry is the measurement of the amount of hemoglobin (Hb) in the blood. The advantage is taken of the following properties of Hb
1. Ability to combine with oxygen 2. The pressure of a definite amount of iron in each molecule of Hb 3. Ability to Hb solution to refract definite wavelengths of light giving typical
absorption bands.
The objective of estimating Hb is to determine the oxygen-carrying capacity of the blood. The results assist in detecting diseases that cause a deficiency or excess
of Hb and in studying changes in the Hb concentration before or after surgery and blood transfusions.
Generally,Hb estimates rely on comparison of color visuals; however, comparisons can lead to inaccuracies and the use of photoelectric absorptiometers is
preferred.
Two methods of Hb determination are described here. The first one Sahli method relies on visual color matching while the second method Oxyhaemoglobin method involves using the absorption meter/spectrophotometer.
Both methods depend upon matching the color produced by the test sample with the color produced by a standard sample of blood. This can be achieved by one of two ways:
(a). By measuring the amount of oxygen contained in the hemoglobin (b). By measuring the amount of iron contained in the Haemoglobin.
A. Acid Haematin Method (Sahli Method)
Hemoglobin (Hb) is converted to acid haematin by the action of hydrochloric acid.
Apparatus and Reagent: 1. Sahli graduated tube and standard
2. 0.1N HCI 3. 0.02ml pipette
Procedure :
1. Fill the graduated tube to the 20 marks with 0.1N HCl.
2. Add 0.02ml of blood, mix well, and leave for 5 to 10 minutes. 3. Add 0.1N HCl drop by drop mixing after each addition until the color matches
the standard.
4. Read the amount of solution in the graduated tube. The calibrations give the hemoglobin concentration as a percentage.
Calculate the hemoglobin content in g. per 100ml of blood as follows:
The standard tube will state the number of grams per 100ml of Hb. equivalent to 100 percent. Some older Sahli standards use 17.2g per 100ml as 100% Hb. using
this apparatus if a Hb. value is 90% the Hb. content in grams per 100 ml is:
90 multiply by 17.2
100 = 15.48g per 100ml If percentage Hb is to be reported, this 15.48g per 100ml would need to be converted
to a percentage regarding 14.6g per 100ml, as being equivalent to 100% 15.48 multiply 100%
14.6
= 106%
B. Spectrophotometric Method
Oxy-hemoglobin released by the addition of distilled water to a specimen of blood and its stability is ensured by adding dilute ammonia. The oxy-hemoglobin is
estimated by using a Spectrophotometer
Apparatus and reagent 1. Spectrophotometer
2. Test tubes
3. 0.004% Ammonia Solution 4. 10 ml pipette
Procedure
1. Place about 4ml of 0.04% ammonia solution into a test tube 2. Add 0.04ml of blood to this solution with the pipette. Oxyhaemoglobin is now
formed.
3. Add more 4ml of the ammonia solution to the oxyhemoglobin (Dilution factor is now 1:200)
4. Using a spectrophotometer, measure the optical density (Note: The
spectrophotometer must be calibrated using distilled water as ‘blank’ at a
wavelength of 540nm) 5. The optical density can then be converted to hemoglobin concentration by
reading off on the calibration curve provided.
Exercise I. You are provided with blood sample A, determine the Hb, content by the Sahli, and
spectrophotometric methods. 2. Compare your results 3. Tabulate the results obtained using both methods and ascertain their accuracies.
4. Compare the Hb. content of samples A & B using a method of your choice. 5. Give your results stating the reasons for the method used.
C. Haemoglobin Card Method
This experiment is based on the principle of color marching between the one formed
with the red blood cells on a prepared paper and that of a standard scale given
Procedure 1. Remove a portion of the demarcated part of the hemoglobin card 2. Drop a drop of blood on the card and allow to stand for 2 minutes
3. Match the color of the blood on the card with the standard scale prepared at the back of the card. This is expressed as a percentage or milligram of Haemoglobin.
Blood Sample
A
Sahli’s
Method
Spectrophotometer
method
Haemoglobin Card
Method
Total
Mean
Standard
deviation
EXPERIMENT 6
DETERMINATION OF PACKED CELL VOLUME
Measurement of the packed cell volume or the Haematocrit value of the blood is the most accurate and simplest of all the methods for determining the presence or
absence of anemia or polycythemia and measuring their degrees. In comparison, the Haemoglobin determination is less accurate and the RBC counting far less accurate.
Hemoglobin measurement and red cell counting should be reserved for finding out the average size (M.C.V) and hemoglobin content (MCH) of the cells.
Principle:
The red blood cells are heavier (sp. Gr. About 1.090) than the plasma (sp. Gr. About 1.030). When the blood is placed in a long tube and is centrifuged the cells settle down and pack themselves because of the centrifugal force. The volume occupied by
the cells is measured and its ratio with the volume of the whole blood is calculated.
Practical Title: Haematology
Experiment: Determination of the Packed Cell volume of a sample of blood of a
subject
Aim: To estimate the packed cell volume of a subject. Apparatus: Capillary tube, or wintrobe tube or Haematocrit tube, long capillary
pipette. Haematocrit centrifuge, Haematocrit reader
Material: The blood is collected by a venepuncture and taken in a 3:2 oxalate mixture with EDTA or Heparin
A. METHOD (WINTROBE TUBE METHOD)
• Rotate the bulb between the palms of the hands to ensure (1) mixing of the
cells and the plasma, evidently wrong results are bound to be obtained this
precaution is not observed and (2) oxygenation of the cells to remove any carbon dioxide. The size of the cells is larger when the PC02 is high.
• Take slightly more than 1 ml of blood in the dry pipette.
• See that there is no air bubble in the capillary stem. • Now insert the tip to the bottom of the wintrobes tube.
• By gentle pressure on the rubber teat, fill the Haematocrit exactly to the zero
marks. • Centrifuge for 30 minutes.
• Read the upper level of the cell layer.
B. THE MICRO - HAEMATOCRIT METHOD
The blood is spun at 10,000 revolutions per minute for 5 minutes. These results in the elimination of all trapped plasma though the method gives results 1-2% lower,
due to complete packing of the red cells. The method has the advantage of speed
simplicity and is suitable for use with capillary blood when a venous sample is not
available.
Method:
1. Fill the special capillary tube either with well-mixed venous blood or directly from a freely flowing capillary puncture (in this ease, use a heparinized
capillary tube). 2. Seal the end of the capillary tube in a Bunsen flame or plasticine. 3. Place the capillary in the micro - Haematocrit centrifuge and screw the safety
cover.
4. Close the lid and spin at 10,000 revolutions per minute for 5 minutes which
time allows even polycythemia blood to be well packed. 5. Place the spun tube into the specially designed scale and read off the PCV as a
percentage.
When the micro - Haematocrit method is used, owing to the elimination of trapped plasma, the apparent range of the mean cell hemoglobin concentration (MCHC) is
extended to 33 - 38% Normal range of the MCH is 32- 36%.
Record the average result from two readings for a male and a female subject. Note the sex difference and state why.
State the normal values and comment on the factors that affect the result. Result
Windrobe Method Micro Hematocrit Method
Blood sample
Mean
The PCV of the blood sample is _________
EXPERIMENT 7
BLOOD INDICES OF ABSOLUTE VALUES MCV, MCHC, MCH, AND CI
Mean Corpuscular Volume or M.CV
Mean Corpuscular Volume or M.CV indicates the mean Volume in cubic
microns of a single red cell. The normal MCV ranges from 79 to 96 cu.
Calculation:
Divide the PCV expressed as cubic microns per cubic millimeter by the RBC count per cubic millimeter
Example:
PCV= 40% RBC count = 5,000,000 per c.mm
There are 103 µ in 1mm and 109 cµ in 1 c.mm
As the PCV = 40% in 100c.mm of blood there are 40c.mm of packed cells The volume of packed cells in 1c.mm = 40/100mm3
There are 5,000,000 RBC in 1c.mm
Volume of 1 cell = 40/100 ÷ 5,000,000/mm3
= _____40 X 109____
100 X 5,000,000
= 80cµ In practice, multiply the PCV by 10 and divide by the number of millions of red cells
per c.mm3.
Mean Cell Haemoglobin Concentration (M.C.H.C)
This refers to the percentage of hemoglobin in 100ml of red blood cells, as
opposed to the percentage of hemoglobin in 100ml of whole blood, giving the concentration of hemoglobin in the cells. So M.C.H.C means 'Mean Corpuscular
Haemoglobin Concentration' and it is expressed as a percentage.
The normal M.CH.C ranges from 32 - 36% M.CH.C is the name of the absolute value that gives the best information regarding the diagnosis of iron deficiency anemia.
Calculation: Divide the hemoglobin content in g. per 100ml by the per 100ml of blood, expressing the result as a percentage.
Example Hb content = 15g / 100ml blood
PCV = 48%
A 100ml of blood contains 15g hemoglobin 48 ml of packed cells contain 15gm of hemoglobin
MCHC = 15x100
48 1
= 31.25 percent
Mean Cell Hemoglobin (MCH)
This expresses the average hemoglobin content (in micro-micrograms) of a single red blood cell.
The normal MCH ranges from 27 - 32 micro-micrograms (i.e. pictogram or pg) per 1 c.mm of blood and divide by the red cell count per 1c.mm. Calculation:
Express the hemoglobin content in micro-micrograms (pictograms) per 1c.mm of
blood and divide by the red cell Count per 1cmm.
Example:
Hb = 14.5g/100ml of blood RBC = 5,000,000 per c.mm
There are 103c.mm in 1ml and 1012 pico-grams in 1gram. As the Hb. content in
100ml = 14.5g.
Therefore, the Hb content in 1ml = 14.5g/100ml and the Hb content in 1c.mm =
14.5g/100 x 1000
That is 14.5/105 or 14.5/105 x 1012 micro-micrograms (mµm) or picograms (pg). There are 5,000,000 (5 X 106) RBC per c.mm
The Hb content of 1 cell = 14.5 x 1012 / 105x 5 x 106 µµg or picograms (pg).
per cell. MCH = 29 micro-micrograms. In practice multiply the Hb content in g per 100ml and divide by the number of
millions of red cells per c.mm.
That is 14.5 x 10/5 = 29 Iµµg or picograms (pg)
Colour Index (C.I)
This expresses the average hemoglobin content of a patient's single red blood
cell, compared with the accepted hemoglobin content of a normal red blood cell. It is calculated by dividing the hemoglobin content (percentage of normal) by the RBC
count (percentage of normal).
Calculation: The values taken as 100 percent are 14.6g of hemoglobin per 100ml of blood and
5,000,000 of red cells per c.mm of blood. In normal blood, the color index would be 1.0. That is Hb = ____14.6 ____ = 100%
RBC 5,000, 000 100
= 1.0
QUESTIONS
1. What cells form the uppermost layer of the packed cell column?
____________________________________________________________________________
2. (a). Do you expect the haematocrit of the blood in capillaries and small vessels of
the body to differ considerably from that in large vessels?
(b). Why?
________________________________________________________________________________
________________________________________________________________________________
________________________________________________________________________________
______________________________________________________________
3. Hb 14.0g/100ml RBC 4,500,000 per c.mm Hb content (% of normal) = 14.0 X 100 = 95.8% (i.e. 14/14.6 x 100 = 95.890)
RBC count (% of normal) = 4,500,000 x 100/ 5,000,000 = 90% (i.e. 4,500,000/5,000,000 x 100/1 = 90%)
Therefore the colour index: 95.8/90.0 = 1.06 Calculate and discuss the absolute values of the blood specimen provided.
_______________________________________________________________________
_______________________________________________________________________
________________________________________________________________________
_______________________________________________________________________
________________________________________________________________________
__________________________________________________________
4. Discuss the significance of the use of the various parameters, which you have
obtained.
_______________________________________________________________________
_______________________________________________________________________
________________________________________________________________________
_______________________________________________________________________
________________________________________________________________________
__________________________________________________________
EXPERIMENT 8
EXPERIMENT ON HEMOSTASIS 1 – BOLLOD COAGULATION
THE ROLE OF VITAMIN K AND THE USE OF DICOUMAROL The prothrombin (PT), factor V and various other factors are synthesized in the
liver. Vitamin K is essential for this synthesis. Dicoumarol has a chemical
configuration similar to vitamin K. When administered it replaces the vitamin Kin
the liver (substrate competition) and the liver cells now cannot synthesize the prothrombin and factor V etc. The coagulation time and the prothrombin time are
then prolonged.
Dicoumarol and other drugs having similar actions are extensively used in cases of thrombosis (coagulation in the vessels to prevent its progress and to allow
the fibrinolysis to occur). Prothrombin time is required to be done very often in this
treatment. Prothrombin is highest in plasma on the 8th day of life (after birth)
WHOLE BLOOD COAGULATION TIME
Title: Haematology Experiment: Determination of the clotting time
Aim: To determine the clotting time of a person using White and Lee
Apparatus and material: capillaries, 4 small tubes, syringes with sterile needles, test tube rack, Water bath, methylated spirit, and cotton wool.
(A) Lee and White Method (DEMONSTRATION) Blood is collected from a vein into a dry syringe with a minimum of trauma.
Only samples of blood collected without technical difficulties are suitable for the test. To minimize the contamination the sample with products of tissue trauma, the first
1 - 2ml of blood into one syringe, removing that needle in the vein, attaching a second syringe into which the test sample of blood is drawn. A stopped clock is
started as soon as the blood appears in the second syringe. As rapidly as possible, exactly 1ml ofblood is placed into each of four clean dry, standard-sized tubes 3 x 3/S in a water bath at 37°C. The tubes aretilted every 30 seconds until they can be
inverted without spilling any blood. - the time from the starting of the stopwatch is
thennoted. The coagulation time for each tube is recorded separately and the
average of the 4 tests is taken as the coagulation time. The normal time at37°C is about 4 minutes but it can vary between 3 and10 minutes.
(B) Using Capillary Blood Obtain a large drop of blood from finger puncture and note the exact time at
which puncture is made. Fill a length of notless than sixinches of the fine capillary
tube provided at half-minute intervals break off about 1 cm from the end of the tube.
Initially, the blood column will break cleanly. At the endpoint, athick strand of a coagulated column will be seen stretching between the broken ends. It isimportant
to break the tube gently without jerking the ends apart or else the movement will
snap the strand before it is observed. Note the time for coagulation to occur.
(C) TO DEMONSTRATE THE IMPORTANCE OT CALCIUM IN COAGULATION
A test tube containing 2 ml ofoxalate rat blood is warmed to 37°C and 0.5m1 of 1% CaCl2 is added. Note the coagulation time as before.
(D) TO DEMONSTRATE THE EFFECT OF VARIOUS ANTICOAGULANTS Record your observations when fresh blood (human or animal) is drawn into 5
syringes containing: 1. Nothing _______________________________________________________________
2. Heparin ________________________________________________________________
3. Oxalate_________________________________________________________________
4. Chlorazol fast ink ________________________________________________________
4. Sodium citrate___________________________________________________________
At what stage of the clotting process does each of these substances act?
_________________________________________________________________________
_________________________________________________________________________
_________________________________________________________________________
_________________________________________________________________________
_________________________________________________________________________
QUESTIONS
1. What is Coagulation time
______________________________________________________________________________
______________________________________________________________________________
______________________________________________________________________________
______________________________________________________________________________
______________________________________________________________________________
______________________________________________________________________________
2. On what factors do Coagulation time depend
______________________________________________________________________________
______________________________________________________________________________
______________________________________________________________________________
______________________________________________________________________________
EXPERIMENT 9
EXPERIMENT ON HEMOSTASIS 1 – BLEEDING TIME
Title: Haematology Experiment: Determination of bleeding time Aim: To determine the bleeding time of a subject using IVY's method
Apparatus and material: pieces of filter paper, Sphygmomanometer, blood lancet,
methylated Spirit, cotton wool.
Method:
Choose a suitable site for puncturing (edge of Lobe of the ear in Duke's method; forearm in Ivy's method; but the usual site - tip of left ring finger can also
be used). In Ivy's method, the sphygmomanometer cuff is attached to the upper arm and pressure raised to 40mm Hg to distend the superficial blood vessels of the anterior surface of the forearm to be avoided during pricking or else the bleeding
time may be increased.
• With usual aseptic precautions, inflict a deep cutting wound by a sterile lancet. • Note the time of puncture and the first appearance of the blood.
• With a piece of filter paper gently blot the blood. Do not press or wipe the wound.
Repeat with a fresh piece of filter paper every 10 seconds till no blot appears on the paper.
• Count the number of filter papers which show the blot of blood on them. The
number of blots x 10 seconds will be bleeding time. • Perform a similar experiment upon a healthy person and report the result obtained
as control values.
QUESTIONS 1. Discuss thedifference between bleeding time and coagulation time
______________________________________________________________________________
______________________________________________________________________________ ______________________________________________________________________________
______________________________________________________________________________
______________________________________________________________________________
______________________________________________________________________________
2. On what factors do Bleeding time depend ______________________________________________________________________________ ______________________________________________________________________________
______________________________________________________________________________
______________________________________________________________________________
EXPERIMENT 10
EXPERIMENT ON HEMOSTASIS 2 – PROTHROMBIN TIME
Principle: this test determines the amount of prothrombin in the sample. The speed
of coagulation depends on the concentration of prothrombin. Venous blood is
collected. First, free Ca ions in the blood are eliminated by means of Na-citrate to
prevent clotting in the sample. Then calcium, thromboplastin and phospholipid
containing thrombokinase, needed for coagulation is added in excess to the plasma,
thus clotting only depends on the presence and amount of prothrombin. The
prothrombin time is measured from the timepoint the reagents are added to the
plasma until the fibrin clot develops.
The prothrombin time tests the activities of fibrinogen, prothrombin and factors V,
VII and X. Perfomance: 4.5 ml of venous blood is drawn into a sterile syringe already
containing 0.5 ml of 3.8% Na-citrate. The citrated blood is centrifuged as soon as
possible, max. 2 hours after the blood collection, for 10 min at 1000 g. Then 0.1 ml
plasma is pipetted onto a silicon/ wax covered watch glass and placed in a 37 oC
water bath for 3 min. The thrombokinase reagent is also prewarmed in a test tube at
37 oC. 0.2 ml of prewarmedthrombokinase reagent (Simplastin D®) is added to the
0.1 ml of plasma in the watch glass and the timer is started. Approximately every
second, the tip of an injection needle is pulled through the plasma. The prothrombin
time is recorded when the first fibrin fiber appears at the tip of the needle. The
normal value is: 18-20 second
EXPERIMENT 11
BLOOD GROUPING/TYPING
Recall your lectures on ABO and Rhesus (Rh) blood groups for antigens and antibodies. The practical you are about carrying out is therefore on ABO and Rh grouping techniques.
ABO Grouping TechniqueBlood grouping should always be performed by tube technique unless blood is needed to be transfused urgently when some workers use
tile or slide technique. An essential feature of agglutination is that the red cells (antigens) should be adequately bathed and in contact with the serum (antibodies)
and each other. In tube techniques, tubes with a narrow bore should be used, so
that a longer time is taken for the red cell to sediment (by gravity), thereby keeping them in contact for a longer period ensuring adequate agglutination even when
weakly reacting forms of the group are present.
In the tile method, the distance for the red cells to travel is very much less and they can be left only for a short time since they tend to dry up. This may lead to weakly
reacting forms of group A being missed.
In the tube technique the tube is stood at room temperature for 1/2 - 2 hours before being examined, but in an emergency may be centrifuged after 10 or 15
minutes and the cells examined microscopically.
(+) denotes agglutination (+) denotes no agglutination
Agglutination Your Blood group
is
Your RBCs contain agglutinoge
ns
Your plasma contains agglutinin(s)
Your plasma will agglutinate RBCs of group
Anti-A Serum
Anti-B Serum
+ - A A Anti-B B, AB
- + B B Anti-A A, AB
+ + AB A, B None None
- - O O Both Anti-A and
Anti-B
A, B, AB
Techniques Tube method • Washed suspension of the patients washed red cells.
• 3% suspension of the patients washed red cells
• 3% suspension of washed, pooled, known group A cells. • 30/0 suspension of washed, pooled, known group B cells.
• 30/0 suspension of washed, pooled, known group 0 cells.
Practical Title:Haematology
Experiment: Determination of blood groups Aim: To carry out blood groups in man
Apparatus and Materials: Tiles, slides, Kahn tubes, Racks, pipettes, 0.9% saline,
Anti sera A B and H, glass marking pencil.
Method: • Take three slides and label them A, B and C
• In a small tube take 3ml of 0.9% saline and a drop of fresh capillary blood (from the donor whose
blood group is to be determined). Mix the blood and saline gently but thoroughly and allow the cells to
settle down.
• Label the tube • Pipette out the supernatant fluid (after centrifugation) and replace it by fresh
saline: This washes out
any plasma sticking to the cells and gives a 1 to 2°1o suspension.
Cell suspension is used because:
1. Dilution of cell allows easy detection of clumping when present
2. Plasma factors, likely to interfere with agglutination are eliminated 3. Place type A serum (i.e. Anti-B serum containing the beta agglutinin) on the slide
labeled "A".
4. With a separate clean and dry pipette, place type B serum (i.e. Anti-A serum containing the alpha
agglutinins) on the slide labeled B.
5. With a third separate clean and dry pipette place a drop of 0.9% saline on the slide labeled "C" 6. Now add a drop of the cell suspension of the unknown blood cells to each of the
sera on the two slides and to the saline on the slide "C". The dropper should not touch the serum. 7. Mix the cells and the serum by rocking tilting and tapping or by three separate
glass rods. 8. Allow the two to react for 6 to 10 minutes
9. Examine after 6 minutes and before 10 minutes. The reaction may not be complete before 6 minutes
and drying may occur after 10 minutes. Both these factors may yield false results.
First examine slide C. It should not show any agglutination. Now examine
both A and B, confirm each result under the microscope.
Clumping in both the slides indicates presence of both agglutinogens in the
cells i.e. the cells belong to blood group "AB". Clumping in the slide labeled "A" indicates presence of "B" agglutinogen in the cells anti B in serum/plasma blood
group is "B". Clumping in the slide labeled "B" indicates the presence of "A"
agglutinogen in the cells and anti A (a) in plasma and the blood group is A. Absence of clumping in both slides indicates absence of any of the "A" and "8"agglutinogens
and the blood group is "O"
Methods of Detecting Anti D In order to establish that a serum contains anti-D, it will be tested against D-
positive red cells. D-positive cells that are used will also contain other Rh antigens
so that any positive results must be further investigated to confirm that they are specific for anti - D. Although, reference is made to 0 Rh positive cells; in routine
practice it is preferable to use two types of 0 Rh positive cells. One of the Rh
genotype Cde / Cde (RIRI), and another of the genotype CDE / cde (R2r). Using these
cells, which between them contain all the Rh antigens, it is possible to recognize
every anti-Rh antibody. When testing for antibodies, the saline, albumen and Combs techniques are used, in order that both complete and incomplete antibodies are recognized.
Saline Technique Material Required:
3-5% suspension of washed 0 - Rh+ (D+) red cells
Small precipitation tubes, 50 x 6 mm
Sterile saline Sterile Pasteur pipette
3-5% suspension of washed patients
Cells, patients serum
Technique:
i. Into each of three tubes, place one drop of patients' serum.
ii. To tube 1 add one drop of the 0 – Rh+ve cells. iii. To tube 2 add one drop of the 0 – Rh-vecells.
iv. To tube 3, one drop of the patients red cells.
v. Mix and incubate at 37°C for 2 hrs. VI. Examine under the microscope for agglutination
Tube 2 and 3 are negative controls and should give no agglutination. Tube 1 is the test and agglutination in this tube would indicate the presence of anti-Rh antibody.
Albumen Replacement Technique: Proceed as for the saline technique up to the stage (IV).
v. Incubate for 1½ hrs. vi. Remove the supernatant saline leaving a button of cells in the bottom of the tube
and replace it carefully with 20% bovine albumin. vii. Reincubate for further 30 minutes at 37°C.
viii. Remove from the incubator and read results as for ABO grouping with hand or microscope.
Combs's Test (Indirect Comb's Test on Serum)
Materials Required
Test tube 100 x 8mm 50% suspension of 0 Rh+ve cells
50% suspension of 0 Rh-ve cells
Anti-human globulin serum dilutes to correct titer for use (Comb's reagent). Supply of known incomplete anti-D sera
Patient serum
Sterile Pasteur pipette
Sterile normal saline
Technique:
I. Take three tubes and number them 1, 2 and 3 ii. To tubes 1 & 2 add four drops of patients' serum
iii. To tube 3 add four drops of incomplete anti - D serum
iv. To tubes 1 and 3 add two drops of 50% 0 – Rh+ve cells.
v. To tube 2 add two drops of 50% 0 Rh-ve cells
vi. Incubate at 37°C for 2 hrs vii. Remove from the incubator and wash the cells in saline at least four times. viii. After the final washing remove as much of the saline as possible and add 2
drops of combs reagent to each tube. IX. Mix and transfer the content of the tube on to a flat tile. Mix and observe for
agglutination throughout 10m ins. Tube 3 is a positive control and should give agglutination. Tube 1 is the test and will agglutinate if anti-Rh is present.
NOTE: The optimum titer of the Comb's reagent is determined by the experiment. The combs reagent will be inactivated if the slightest trace of globin is allowed to
come into contact with it.
Determine the blood groups of specimen A, B, C and D provided and discuss your results
QUESTIONS
1. From the results of the slides, identify the blood group of the subject. ________________________________________________________________________________
________________________________________________________________________
2. On blood grouping generally, why are only donor antigens and recipient’s
antibody considered and not vice versa?
____________________________________________________________________________________________________________________________________________________________________________________________________________________________________
____________________________________________________________________________ 3. Is it advisable to give intergroup blood transfusion? Why?
________________________________________________________________________________
________________________________________________________________________________________________________________________________________________________________
____________________________________________________________________________________________________________________________________________
4. What is the other type of blood grouping which has to be done before
transfusion?
Answer - rhesus factor (anti D). What is its significance in blood transfusion?
________________________________________________________________________________
________________________________________________________________________________________________________________________________________________________________
________________________________________________________________
5. What is Landsteiner’s law? Does it apply to all blood group systems?
________________________________________________________________________________
________________________________________________________________________________________________________________________________________________________________
________________________________________________________________
6. What is cross-matching:
___________________________________________________________________________________
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EXPERIMENT 12
ERYTHROCYTE SEDIMENTATION RATE
If anticoagulated blood is allowed to stand undisturbed, the red cells will gradually settle to the bottom of the container leaving a clear layer of plasma. This sedimentation occurs in three phases. First, the cells tend to aggregate and form rouleaux and only fall slightly. In the second phase, the speed of the fall is increased
and as the cells pack, this speed is decreased during the third phase. Physical factors like specific gravity of the cell and the plasma, the temperature, and the viscosity are some factors that determine the rate of settling down. Besides the
physical factors certain other factors that govern the rate of sedimentation. The single important factor affecting the rate of sedimentation is "PILES" or rouleaux formation.
Rouleaux
1. The red blood cells are negatively charged, as they remain suspended in the plasma. These changes tend to keep them separate from one another.
2. Protein molecules in plasma bestow a property of the repulsion caused by the
negative changes. When the cells strike each other because of the random thermodynamic movement they tend to stick to each other and get pile up over
another by forming a rouleaux. Each rouleaux consists of 8 to 12 cells and
when more cells aggregate together the repelling forces cause the rouleaux to
break into two. This limits the size of rouleaux. When rouleauxhave formed the density of its mass is increased and it sinks quickly.
Factors Affecting Rouleaux Formation
a. Different molecules in plasma e.g. protein, cholesterol,etc affect the rate of rouleaux formation. Larger molecules usually increase the rate. Thus
fibrinogen is better than globin which is better than albumin.
b. When the number of erythrocytes is more, the number of piles or rouleaux formed is also more and all of them start settling down. As each mass sinks,
an upward current is created in the plasma by the side of the rouleaux. This
upward flow of the plasma retards the rate of settling down of the adjacent rouleaux. These adjacent rouleaux retard one another rate of sinking. The rate of settling down is therefore slowed down at least for the first hour. As the
rouleaux sink down they strike the bottom and pile over one another. It
requires sometime before the rouleaux get packed. This is another reason why the initial rate observed is always fewer
c. Cell size, shape, and composition. An increase in the M.C.V, a decrease in the
M.C.H, and spherocytosis all retard the rate of rouleaux formation. In certain clinical conditions, many of these changes occur simultaneously and cause a decrease in the ESR. Associated with these changes there may be also an
increased level of bile salts which can reduce the ESR still further.
patients suffering from certain diseases, the red cells sediment more rapidly than the normal subject. This occurs in inflammation, infection, or the production of
abnormal globulins.
There are two main methods of performing the ESR namely Wintrobe's and Westergren's method. For our class practical, we shall describe Westergren's
method.
Practical Title: Measurement of ESR by Westergren's Method
Aim: To determine the ESR of the blood of a human subject
Apparatus: The Westergren pipette or tube. It has a uniform bore on 3mm and a length of about
300mm. It is used with graduation from 0 above to 200 mm. It is open at both ends. The Westergren's stand. There is a rubber cushion at the base on which the lower end of the Westergren pipette is made to rest. At the upper end is a screw cap which fits upon the pipette. It can be screwed down to exert sufficient pressure on the
pipette. This prevents any leakage of blood at the bottom. The stand allows the
pipette to remain exactly in a vertical position. The screw cap may be replaced by a spring clip.
Material: Sample of blood by venepuncture: and mixed with 3.8% sodium citrate solution in a proportion of 4 parts or blood to 1 part of citrate solution.
The minimum amount of blood required is 1.6ml mixed with 0.4ml of citrate
solution. The citrate acts as an anticoagulant and is isotonic with the blood. It is also a diluent.
Method: • Inspect the sample of blood provided and confirm that there is no clot in it.
• See that the tube is clean and dry
• Mix the blood rotating the sample gently between the palms of your hand.
• Suck the blood slowly up to zero marks of the pipette; usually above the mark. Adjust to the exact zero marks.
• With the finger of your hand closing the top of the pipette, press its lower end
on the rubber cushion of the stand taking care that no blood escapes. • Release the finger from the tip while continuing the pressure and fix the
pipette with the cap/clip at the top. The pipette must be fitted exactly
vertically, and the blood level must not change from the zero marks. • Stand in diffused light and away from direct sunlight. • At the end of one hour read the upper level of clear plasma
• Note down the reading as so many mm/hr. Westergren's at the room temperature which you must record.
In cases of extreme anemia or where the ESR is very high the upper level of
the red column is not seen. In such cases read the highest point of maximum
density of the red column.
Observations and Inference
The normal range in Westergren's method at a temperature between 22° and 27°c is 3 to 5 mm an hour in males and 4 - 7 for females for the one hour or 7 to 15mm and
12 to 17mm respectively at the end of the second hour.
Precautions 1. The test must be set up within 3 hours of blood collection or sedimentation
will be retarded.
2. Haemolysed or clotted blood must not be used.
3. Avoid higher temperatures than 27°C as higher temperatures accelerate the sedimentation rate.
4. All the apparatus must be clean and dry
5. There must be no air bubbles in the sedimentation tube 6. The tube must be in the exact vertical position, away from direct sunlight
Determine the ESR of the two fluids provided and discuss your findings.
QUESTIONS 1. The normal human adult male ESR is 2 - 8 mm/hour while in an adult female it
would be between 2-12 mm/hour. Why is the value very often higher in females?
________________________________________________________________________________________________________________________________________________________________
________________________________________________________________________________
________________________________________________________________
____________________________________________________________________________
2. What factors influence the sedimentation of RBC?
________________________________________________________________________________________________________________________________________________________________
________________________________________________________________________________
________________________________________________________________________________ ________________________________________________________________________________
3. List precautions you would take to ensure accurate results. ________________________________________________________________________________________________________________________________________________________________
________________________________________________________________________________________________________________________________________________________________
EXPERIMENT 13
OSMOTIC AND PERMEABILITY PROPERTIES OF RED BLOOD CELLS
I. EXAMINATION OF BLOOD CELLS AND WATER MOVEMENT DUE TO
OSMOSIS
When erythrocytes are placed in solutions, which differ in 'tonicity' (i.e. osmotic
pressure) from the cells, they will either lose or take up water. Either process may eventually lead tothe destruction of the cells and the hemoglobin in the cells being
liberated into solution. This process is referred to as 'hemolysis' or simply lysis'.
When erythrocytes take up water, a critical stage is often reached when the cell
membrane is stretched to such an extent that it then no longer hold the hemoglobin in although the membrane is not broken. At this state, the hemoglobin leaks out
leaving behind the empty membrane or 'red cell ghost'. An isotonic solution is one in which the osmotic pressure exerted by the solute
is the same as that in the plasma and cells and therefore no net movement of water
takes place either into or out of cells placed in such a solution. The tonicity of a solution depends not only on the concentration of the solute but the number of
particles formed from each molecule in solution. Thus the concentration of a non-electrolyte isotonic solute with mammalian tissues is 0.3g moles/1) but for sodium
chloride which produces two ions per molecule, it is 0.15M (0.9%) and for calcium chloride with three ions per molecule but only about 90% ionized it is 0.11M. For other classes of animals, the isotonic concentration is different from that of
mammals. For amphibian tissues, the isotonic concentration of sodium chloride is about 0.6% (see the composition of Frog Ringer solution in preliminary notes on a
physiological solution).
Observation
1. Place 2 - 3 drops of 3% sodium citrate solution in a small tube and add several drops of blood from a finger or ear prick (see instructions on the introduction sheet for obtaining a good blood sample). The sodium citrate solution will
prevent the coagulation of up to seven times its volume of blood. Mix the blood with citrate quickly and thoroughly.
QUESTION
1. Why does citrate prevent blood clotting? ________________________________________________________________________________
________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________
_______________________________________________________________________________
2. Place 1-2 ml of 0.9% sodium chloride in a test tube and transfer to it a small
quantity of the citrated blood. The final mixture should be only pale red. Place
one drop of the diluted blood on a microscope slide cover with a coverslip and examine under the microscope using the first low and then high power.
Observe the appearance of the erythrocytes and note that the thicker rim of
the biconcave disc can be shown by focus up and down. Examine the slide
carefully for leukocytes, which are slightly larger than the erythrocytes and
appear granular on focusing up and down. You will have to search thoroughly because there are approximately 80 erythrocytes to one leucocyte.
3. Now place a drop of distilled water at one edge of the coverslip so that it can
run underneath. To encourage this, hold a piece of filter paper at the opposite edge of the coverslip so that the fluid is drawn through. Examine the slide under the microscope where the distilled water mixes with the cells. Observe and note any change.
________________________________________________________________________________________________________________________________________________________________
_______________________________________________________________________________
4. Make similar observations mixing the cells with 2% NaCl taking a fresh drop of
the diluted blood in 0.9% NaCl for each observation.
________________________________________________________________________________________________________________________________________________________________
________________________________________________________________________________
________________________________________________________________________________
II. FRAGILITY OF RED CELLS
When exposed to hypotonic solutions (i.e. solutions of a concentration less
than isotonic) not all red cells break down at the same concentration, i.e. they vary in 'fragility'). The less fragile the cell, the better the cell membrane resists the distension due to water uptake from hypotonic solutions, and therefore the more
dilute the solution must be before the cell bursts. A rough estimate of the extent to which the red cells have been broken down can be
made by mixing the blood with the solution and allowing any un-lysed cells to settle. Then on examining the tube, the following observations can be made;
(a) If the solution is colorless and cells have settled down the bottom of the
tube, no cells have lysed;
(b) if the solution is colored red and no cells can be observed, all the cells
have lysed;
(c) if the solution is slightly color and there are also cells settled at the bottom of the tube some but not all the cells lave lysed.
Make weak dilutions of the citrated blood in 0.5, 0.4, and 0.3% NaCl as you did with the 0.0% NaCl. Allow the tube to stand for 15 minutes or until the cells
have settled, then observe the appearance of the tubes. If there is a deposit at the
bottom of a tube, shake the tube gently to ascertain if it consists of intact cells or
concentrated hemoglobin from lysed cells. Note the extent of hemolysis in each concentration of NaCl.
QUESTIONS
1. In which tube(s) is the salt solution just slightly colored with hemoglobin?
___________________________________________________________________________
2. In which tube is hemolysis complete
___________________________________________________________________________
III. THE EFFECT OF SUBSTANCES WHICH PENETRATE THE CELL MEMBRANE
The cell membrane of the red cell is not a perfect semi-permeable membrane. It is permeable to water and impermeable to some solutes but permeable to others. It is virtually impermeable to sodium ions and therefore to sodium chloride as the experiments in Section 1 show, but it is highly permeable to urea.
(a) Urea:Mix a small quantity of citrated blood with 1 - 2mls. of 0.3m urea
(18g/l). This is the isotonic concentration for non-electrolytes. Examine the
tube and note whether or not hemolysis has taken place. The reaction is usually too rapid for microscopic examination to be carried out.
(b) Thiourea [SC(NH2)2] penetrates the cell membrane rather more slowly than
urea, (it is slightly larger molecule), and using this it should be possible to observe the changes under the microscope.
Mix a small quantity of citrated blood with 1 - 2mls 0.3m thiourea (22.8g/l). Rapidly transfer one drop of the mixture to a microscope slide. Observe the red
cells under the high power of the microscope and observe the changes taking
place over the next few minutes.
QUESTION
Explain why 'isotonic' solutions of urea and thiourea should cause the red cells to swell and finally haemolysed.
_____________________________________________________________________________
______________________________________________________________________________ ______________________________________________________________________________
______________________________________________________________________________
(c). Hemolytic (Demonstration only)
Blood cells may be haemolysed by substances, which dissolve the lipid cell membrane. These substances are lipid solvents.
To demonstrate their effectiveness, add a drop of each of the following 1%
dilution of blood in 0.9% NaCl in 4 separate tubes shake and note the effect in each test.
1. A drop of ether
2. A drop of Chloroform 3. Bile salt
4. Saponin.
QUESTION
Observe and note what happens in each tube.
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SECTION B
NERVE AND MUSCLES
EXPERIMENT 14
PITTING A TOAD
The frog's muscle tissue is used in experimental work to study the general physiological properties. The frog is a cold-blooded animal and its tissues after
removal from the body, retain their vitality at room temperature for a few hours. The
muscle tissue with its nerve is chosen for the study; its activity is visible can be measured and recorded.
A. Pithing a frog
Pithing is destroying the central nervous system. The organized animal no more exists on pithing, but the individual organs continue to maintain the vitality for
some time. Their activity can only be elicited on direct stimulation. In all the physiological experiments on the tissues of the frog, pithing is preferred to anesthesia because the latter is likely to affect the various systems, thus reducing
the effect of the observations.
Practical
Title: pithing a frog
Aim: To destroy the spinal cord and the brain Apparatus: pithing needle, duster
Material: a frog
Method: (your demonstrator will guide you) - Stun the frog by holding in a duster cloth, both the hind limbs and striking
the dorsum of its head hard against the table. As a result, the brain of the frog
gets a sudden shock and becomes temporarily functionless. - Wrap the stunned frog in the duster cloth and hold with its dorsum towards
you, in the fist of the left hand, leaving your index finger free.
- Bend the head with the index finger on the rim of the first till the skin on the
dorsum of the frog is stretched. - Feel for the depression at the joint of the skull with the vertebral column, with
the pithing needle in the right hand.
- Pierce through the depression to reach the spinal canal. The initial feeling of resistance will later disappear.
- Move the needle from side to side, thus separating the brain from the spinal
cord. - Pass it upwards and rotate it. This will destroy the upper part of the brain. - Now pass it downwards and rotate again. While the cord is being destroyed,
the violent movement of the limbs occurs, followed by stretching of the hind
limbs.
A pithed frog can be used for heart work and the lower part for nerve-muscle
preparation. Your demonstrator will guide you. Record your observation and discuss the results.
EXPERIMENT 15
NERVE MUSCLE PREPARATION
DEMONSTRATION OF ELECTRICAL ACTIVITY TO SHOW SIMPLE TWITCH
In this experiment, a graph of contraction against time under normal condition
and with a single submaximal stimulus is drawn. The trace shows:
1. A period apparent quiescence (latent period) between the moment of stimulation and that of the beginning of contraction.
2. A period of contraction phase.
3. The upward slope of the curve indicating the velocity of the contraction (contraction depolarization) phase.
4. A plateau at the top of the curve indicates the duration of the active state.
5. The period of relaxation (repolarization) phase.
Practical
Title: Skeletal muscle
Aim: To show all-or-none law and simple muscle twitch using the nerve-muscle preparation of a frog.
Apparatus: a student kymograph, frog board, dissecting kit
Material: frog bath, ringer solution, gastrocnemius muscle (nerve-muscle
preparation).
Method;
Get a frog, pith it and dissect out the gastrocnemius muscle-nerve preparation thus, - Lay the pithed frog on the board with its dorsum upwards.
- With the forceps catch, the skin on the back in the middle and with the
scissors make a midline incision on the dorsum. Then cut around the whole
body of the frog/toad. - Hold the animal on the head and shoulders with one hand with forceps on the
other hand grip the loose skin firmly and PULL THE TROUSERS completely.
- Identify the tendon-Achilles just above its attachment to the bone and separate it from the tissue below. Pass a ligature with the help of a needle
between the bone and the tendon and tie it at the lowest round the tendon.
- Cut the tendon below this ligature to separate it from its bony attachment. - Hold the end of the ligature and tear off the muscle from the bones so that it is
now separated up to the knee joint. - Cut the tibia-fibula nearest to the knee joint.
- Separate the joint and the muscle from any remaining attachment. - Now look for a shining line that separates the biceps (iliofibularis) on the
lateral side and the semimembranosus on the medial side.
- Push the sharp end of the scissors in between the two muscle masses to
pierce through the fascia and lowering the end of the scissors upward cut the fascia along the shining line.
- Retract forcefully the adductors medially and the vast muscles laterally by
your two thumbs to expose in the groove between them a big sciatic nerve. DURING DISSECTION THE NERVE SHOULD NEVER BE CUT OR TEASED
WITH THE SCISSORS OR FORCEPS, but with the wooden end of the seeker,
hand gloves.
- Trace the nerve up to the ilio-pubic line and down to the knee joint as far as possible cleaning it with the wooden part of the seeker.
- Now hold the tip of the urostyle (the cartilaginous prolongation of the vertebral
column) with forceps and lift it, snip off its muscular attachments at the lower end.
- With the help of the bone cutting forceps, cut the vertebral column above the seventh and below the ninth vertebra transversely without injuring the nerves.
(Your demonstrator will guide you). The whole preparation must be kept moist
with a frog ringer all through the dissection and the experiment. - Separate the preparation from the body of the frog.
- Mount the preparation in the muscle trough. Attach a suitable weight and
arrange it on the kymograph. - Draw a baseline by starting the drum.
- Now with a minimal stimulus, stimulate the nerve on the electrodes and the
muscle will contract.
- Put the drum in neutral gear and stop kymograph. - Mark the point of stimulation manually (Your demonstrator will be on hand to
guide you).
- Label the points on the trace.
N.B
The latent period is the time required for the impulse to reach the neuromuscular junction, pervade the muscle fiber for the muscle to begin to contract.
Discuss your observation of the trace.
EXPERIMENT 16
RECORDING OF A SIMPLE ISOTONIC MUSCLE TWITCH AND CALCULATION OF
THE WORK DONE BY THE MUSCLE
‘Isotonic’, in this context, refers to- a type of contraction in which the tone of the muscle remains constant while the muscle is allowed to shorten. This is in
contrast to an ‘isometric’ contraction which you will study later.
Most skeletal muscle contractions of the body, however, are a combination of isotonic and isometric, i.e. there is usually shortening accompanied by an increase
in tone.
Procedure
(1) Set up the circuit for single shocks as described in the general instructions at the beginning of this schedule. The drum contacts
should be in the circuit, with the contact arms at 180°.
(2) Prepare the gastrocnemius-sciatic preparation as already described, and place it in position in the bath filled with Ringer's solution. Attach the
thread from the tendon to the muscle lever so that the muscle is just
stretched, and keep it stretched by suspending a small weight (5g) on the long arm of the lever. Screw up the after-loading screw until the
weight is just taken off the muscle. The long arm of the muscle lever
should be horizontal. (3) Lift the nerve out of the bath by raising the vertebra to which it is
attached with forceps and lay it across the electrodes which should be raised above the level of the fluid. Do not touch or stretch the nerve. Turn the drum round until one of the contact arms touches the
electrical contact on the base of the drum. OPEN the shorting switch.
Select a suitable strength of stimulus which just causes the muscle to contract. If using the induction coil method, this should occur on
"break" only.
Complete the circuit making sure that the electrodes are not immersed
in saline, or short-circuited by having a drop of saline between them.
Do not put the nerve on the electrodes until ready to begin and
take it off the electrodes as soon, as the experiment is completed.
(4) Adjust the writing lever so that it moves in a plane tangential to the
surface of the drum, with the lever at right anglesto the radius of the drum at the point of contact. Start the drum revolving at the fastest
speed (640 mm./second) and let drum revolve a few times to get up to
maximum speed.
Push the muscle lever lightly against the drum about one inch from the lower edge, when the drum is rotating steadily OPEN the shorting switch
and record two twitches. Then CLOSE the shorting switch quickly. Stop
the drum with the clutch lever. Leave everything else as it is above all
do not alter the position of the drum on the kymograph a spindle.
(5) To mark the moment of stimulation revolve the drum very slowly by hand in the same direction as before, keeping the switch in the secondary circuit closed till the striker is just about to make contact, then open the switch: very cautiously and steadily continue the
movement of the drum till the muscle contracts so marking the moment
of stimulation with a vertical line. Project the apex of the curve down to the baseline by moving the lever with the finger at the correct position.
Repeat for the other trace which is 180° round the drum from the first.
Remove the nerve from the electrodes and immense in the Ringer solution.
(6) With the drum rotating at maximum speed as before run a time trace
with the tuning fork (100 cycles/ Bee) below the baseline. Keep the preparation especially the nerve, moist with Ringer so that it will be in
good condition for further experiments. Make a note of the bath
temperature. Varnish the tracing if necessary.
(7) Mark a vertical line at the rise of the simple twitch curve. The distance
between this and the point of stimulation is the latent period. The distance between the rise of the curve and the highest peak andthe lowest point of the curve at the far end is the relaxation period. (see
diagram below). Subsequent, smaller-contractions following the main twitch are non-physiological events and are- produced by the mechanical bouncing of the leaver before-coming to rest.
100 cycle/sec
S = point of stimulation
L = Latent period
C = contraction period
R = relaxation period O and O = oscillations of the lever
CALCULATION
1) Using the time trace calculate the following:- a) latent period (msecs) (what causes this?)
b) contraction period (msecs) c) relaxation period (msecs)
2) Measure the height of contraction h 3) from the following measurements:-
a - length of lever (cm)
p - distance of weight from fulcrum (cm) m - weight applied to lever (g)
b - vertical distance from fulcrum to thread
Then i) Actual shortening of muscle (x) =hbcms. a
ii) Mass lifted by muscle (y) = pmg dynes
b
iii) Work done by muscle (w) = pmgh ergs
EXPERIMENT 17
EFFECT OF TEMPERATURE ON THE BEHAVIOUR OF FROG'S MUSCLE
The chemical reactions occurring in the body or living tissues can be
considered as enzymatic reactions. Within limits, all enzymatic reactions are accelerated with a rise in temperature. The activity of the living tissues also shows
acceleration with a rise in temperature and slowing down with a fall in temperature. In hypothermia, besides slowing down other changes in activity occur due to changes in the response to "stimuli" and chemical agents associated with changes in the cell membrane permeability.
Practical
Title: skeletal muscle
Aim: To show the effect of temperature on the contraction process of gastrocnemius muscle-sciatic nerve preparation of a frog
Apparatus: Thermometer, Kymograph, Tissue bath
Material: Hot water (about 70°C), ice and salt mixture
Method:
Arrange the student Kymograph as for simple muscle twitch
• Note the temperature of the isotonic saline at room temperature. • Get ready bowls containing saline in hot water and the ice and salt mixture at
different temperatures.
• Record a simple muscle curve and mark the points of contraction,
relaxation, and latent period. Calculate the times from the time calibration. Your demonstrator will put you through and note the temperature of the
saline. • From the cooled saline, prepare about 100ml having a temperature 5oC less
than the room temperature.
• Drain the saline from the muscle trough. Replace it with the cool saline. Wait for a minute, drain this cool saline also. Replace it with fresh cool saline (same
temperature). Wait for a minute. The muscle will get cooled. Record the temperature.
Record a simple muscle curve with the same strength of stimulus and with the
same point of stimulation.
Label it by the temperature observed on the thermometer
Repeat after lowering the temperature each time by 5oC and then raising it
again by 5oC above the room temperature till the muscle goes into heat rigor.
Label the temperatures of the curves.
Observations
Tabulate the results on the sheet provided
S/N Temperature Latent Contraction Relaxation Height of Any other
Period Period Period Contraction Observation
1
2
3
4
... 5 6
7
8
9
10
11
12
13
14
15
16
Inference and comments:
1. With a rise in temperature, the latent period is reduced, the refractory period is
reduced, the speed of contraction is increased, the duration of active state is reduced and the contraction and relaxation periods are decreased.
2. The capacity of the muscle to perform X work is not changed because of a rise in
ion temperature. The rate of energy output increases. The work obtained in a twitch would be more at a higher temperature. Even the titanic tension developed would be more at a higher temperature but it would be maintained for
a shorter duration.
3. Between the temperatures of 34oC and 45°C. Spontaneous chemical reactions tend to set in and the muscle may contract without being stimulated.
4. Cooling reverses effect; all the processes become slower and prolonged.
5. If kept for a longer time, below zero degrees, the muscle loses its excitability.
The process is reversible on warming.
Discuss your result.
EXPERIMENT 18
DEMONSTRATION OF ALL OR NONE LAW, REFRACTORY PERIOD, TETANUS
AND FATIGUE
1. A STUDY OF THE EFFECT OF STIMULUS STRENGTH ON MUSCLE CONTRACTION
According to the "ALL OR NONE": law if a stimulus to a nerve is above, threshold then it causes a maximal response in the muscle which it supplies. i.e. The -
response is either all or nothing.
To investigate this law in a nerve-muscle preparation increasing strengths of stimulus are used.
Procedure (1) Set up the electrical circuit as in experiment no. 1 but with the drum
excluded, from the circuit,
(2) Set up a nerve-muscle preparation. Place a 5. g weight on the
muscle lever. The lever should touch the drum lightly and be horizontal. Rotate the drum by hand to obtain a base-line,
3) Using a stationary drum applies a very weakstimulus to the nerve
which is not strong enough to make the muscle contract. Move the drum about 1 cm by hand.
(4) Gradually increase the stimulus strength until a small contraction
is obtained. Rotate the drum by a further 1 cm.
(5) Increase the strength of stimulus slightly thus causing further contraction.
(6) Repeat this procedure continuing to increase the strength of the
stimulus turning the drum by hand between each contraction until the maximum height of contraction is obtained.
Indicate on the trace the distance of the secondary coil from the primary for each contraction, or if using a stimulator indicate the strength of the stimulus.
You will notice that the contraction increases graduallyfrom minimum to maximum and then remains at the latter height. How does this fit in with the ‘all or none law’? Why is the
result for skeletal muscle so different from that obtained withcardiac muscle?
See experiment No," 2 on the Cardio-vascularsystem.
EXPERIMENT 19
EFFECT OF DRUG AND IONS ON MUSCULAR CONTRACTION
Title: Smooth Muscle Experiment: Smooth muscle properties Aim: To observe spontaneous contractions, effects of drugs, and ions
on the contractions of a rabbit intestine.
Apparatus: smooth muscle set; isolated organ bath, student physiograph with isotonic complex and its isotonic transducer, petri dish, aerator, Solution: Tyrode solution
Method:
1. A healthy rabbit weighing 200 – 500g is humanely killed by giving a sharp,
firm blow on the neck.
2. The stomach is cut open and a length of the small intestine is removed into the petri dish and the air bubbled in through the aerator.
3. The ileum (small intestine) is severed (about 1cm) and tied to the hook in the
organ bath after carefully washing off the feces (if any) and the suspended
thread is raised to the level for writing on the physiograph paper of the isotonic transducer connected to the isotonic coupler.
Action to be observed 1. Spontaneous contractions
a. Allow the tissue to acclimatize with the new environment for 15 minutes.
b. Put the speed of the physiograph paper (2.5mm/sec) etc and be sure the temperature of the Tyrode’s solution in the organ bath is kept constant at
37oC, record the normal tracing for at least 1 – 2 minutes.
2. Effect of temperature
a. Cold temperature: drain off the solution in the reservoir and the organ bath and fill with cold Tyrode (0 – 10oC)
b. Record the activity of the intestine for 1 – 2 minutes.
c. Do the same with warm Tyrode’s solution (38%) and obtain the tracing. 3. Effect of Drug
a. Adrenaline (0.05%). Wash off the effects of temperature on the tissue and
obtain a normal tracing. Add 1 – 2 drops of adrenaline and obtain a tracing b. Acetylcholine (0.02%). Wash the tissue and obtain normal tracing. Add 1 –
2 drops of Ach and obtain tracing. c. Atropine (0.05%). Wash the tissue and obtain a normal tracing. Add 1 – 2
drops of atropine and obtain tracing. d. Atropine and adrenaline – wash the tissue and obtain a normal tracing.
Add 1 – 2 drops of atropine and 1 – 2 drops of adrenaline and record the
tracing for 1 – 2 minutes.
e. Atropine and acetylcholine: wash the tissue and obtain normal tracing. Add 1 – 2 drops of atropine and 1 – 2 drops of acetylcholine and record the tracing for 1 – 2 minutes.
4. Effect of ions: Barium Chloride: wash the tissue from the effects of drugs and obtain tracing.
Add 1 – 2 drops of 10% Barium Chloride solution and record normal tracing.
Result:
a. Calculate the final concentration of the drugs in the organ bath, take the volume of tyrode solution in the organ bath as 50ml, and the volume of drugs
as 1 drop = 0.05ml. b. Discuss your results
SECTION C
CARDIOVASCULAR SYSTEM
INSTRUMENTS USED IN CARDIOVASCULAR PRACTICALS.
Stethoscope It is a device to hear sounds comfortably without having to place the
ear on the source of a mild sound. The tube is made of material that does not absorb sound energy. The receiver is placed on the source of a sound to be heard. The bell is used to pick high pitched sounds and the diaphragm is used to hear lower frequency sounds.
Sphygmomanometer The sphygmomanometer consists of three parts: a compression cuff, a pressure source and a measuring device.
Cycle ergo-meter It is a stationary cycle conveniently used for the performance of measured amount of work in the laboratory where scientific measurements could be made while performing the exercise. The seat, handle and the pedal resemble ordinary bicycle but wheel is lifted from the round. Varying amount of resistance can
be applied through adjustable belt or suitable brake system. The work therefore can be altered in two ways: speed and load.
EXPERIMENT 20
DETERMINATION OF THE HEART RATE AND BLOOD PRESSURE IN MAN
1. CLINICAL EXAMINATION OF ARTERIAL PULSES
An impulse can be felt over an artery, lying near the surface of the skin. The
impulse is secondary to alternate expansion and contraction of the arterial wall
resulting from the heartbeat. When the heart ejects blood into the aorta, its impact on the elastic walls creates a pressure wave continuing along the arteries. This impact is the pulse. All arteries have a pulse but it is most easily felt where the vessel approaches the body surface. Doctors examine the arterial system to assess
the potency of the arteries and also for information about the function of the heart.
Title: Cardiovascular System Experiment: The study of cardiovascular system in man
Aim: To determine various arterial pulse rates in man. Apparatus/Equipment: Examination Couch, Watch Clock or Stop Watch The presence or absence of the main peripheral pulse: the radial, brachial, carotid,
temporal, popliteal, at the back of the tibia, and dorsalispedis pulses are noted. The
volume of each is compared with the other side .
a. RADIAL PULSE TECHNIQUE
You palpate the radial pulse carefully just above the wrist on the radial side of the wrist using the left four fingertips together. The pulse is usually easily palpable but
may be difficult in situations of low cardiac output or fever disease of the arteries of
the arm.
Palpation of the radial pulse is useful for estimating heart rate and rhythm, and less reliably, the shape of the pulse wave. During palpation of the pulse, the following
data should be recorded;
1. Rate: The rate of the pulse is the number of beats per minute (beats/min). It is counted when any nervousness in the subject has subsided. Count the
beats for 15 - 60 seconds or more.
The normal heart rate is variable, ranging from 50 to 120 beats per minute. The sympathetic and parasympathetic component of the Autonomic Nervous System influences heart rate. The pulse rate is increased by exercise, anxiety,
fever, and overactivity of the thyroid gland. The pulse rate is decreased in
regular physical training and in under activity of the thyroid gland. 2. Rhythm: The normal pulse waves succeed one another at regular intervals.
Ascertain whether the rhythm is regular or irregular. Is the rate regular or
irregular but with rhythm, such as pairs of coupled beats with a compensatory pause between indicating regular ectopic beats? Is it chaotic (irregularly irregular) indicating arterial fibrillation or multiple random ectopic beats?
3. Character: Assess the character or form of the arterial pulse wave. All types or forms of the pulse are best felt at a site closer to the aorta. The sudden, forceful, collapsing pulse of aortic regulation can be felt at the wrist (Corrigan's pulse or water harmer pulse). You can also feel this through the
muscle mass of the forearm by placing the hand around it; the normal pulse
cannot be felt here. The fiat, slowly rising plateau pulse of aortic stenosis, is useful when present. If stenosis and regurgitation occur together the pulse
may have a double peak (bisferiens pulse).
4. Volume: Estimate the volume of the pulse, this gives a rough guide to the pulse pressure. With normal vessels, the pulse volume indicates the stroke volume.
b. CAROTID PULSE TECHNIQUE
The 2 are examined separately and not at the same time to avoid obstruction of blood flow to the brain. The carotid pulse is best felt with thumb or fingers just
lateral to the thyroid cartilage and medial to the anterior head of the sternomastoid
muscle. It lies anteriorly to the jugular venous pulse and is usually visible in the neck as a single sharp pulsation during systole, almost synchronous with the first
heart sound.
Other arterial pulses are readily distinguished at the following locations. a. External maxillary (facial) artery: at the point of crossing the mandible.
b. Temporal artery: at the temple above and to the outer side of the eye.
c. The ulna artery: is palpable at the wrist where it crosses the distal end of the
radius. d. The Brachial artery: is most conveniently felt at the elbow on the inner side
of the biceps.
e. The Subclavian Artery: is felt from behind by pressing downward with the forefinger above the middle of the clavicle, first part of the artery, or from the
front by feeling below the junction of the middle and lateral thirds of the
clavicle. f. The Femoral Pulse at the Groin: lies mid-way between the symphysis pubis
and the anterior superior iliac spine.
g. The Dorsalis Pedis Artery:an anterosuperior aspect of the foot - runs from a point midway between the malleoli to the cleft between the first and second metatarsal bones.
h. The Posterior Tibial Artery: lies one-third of the way along a line between the tip of the medial malleolus and the point of the heel but is easier to feel 2.5cm
higher up where it runs just behind the medial malleolus. i. The Popliteal Pulse: behind the knee.
Practical Assignment Complete the table below:
Locate and determine the above peripheral arterial pulses.
Record the number of pulses per minute, the force and strength of each pulse,
tension offered by the artery to the finger, the interval between the pulses. Discuss your findings.
Artery Number of
pulses per
minute
Force Strength (strong or
weak)
Tension (the extent of
stretch)
Rhythm (regular;
regularly irregular;
irregularly irregular)
Radial
Carotid
Facial or external maxillofacial
Temporal
Ulna
Brachial
Subclavian
Femoral
Dorsalis
Pedis
Posterior Tibial
Popliteal
2. MEASUREMENT OF BLOOD PRESSURE
Title: Cardiovascular system
Experiment: Measurement and recording of blood pressure in man.
Aim: Measurement of blood pressure in a human subject.
Apparatus/ Equipment 1. Stethoscope
2. Sphygmomanometer (Mercury, Aneroid)
There are several methods used for determining blood pressure. Doctors,
Nurses, and Paramedical staff record blood pressure in that treatment decision, and
other investigations are based on these measurements. Of late people are being
trained to record their blood pressure and there are now machines which record blood pressure in some shops and airports.
Preliminary Preparations An adequate explanation of the procedure to the subject is given to allay the
fears of the anxious subject. A person's blood pressure varies from time to time with respiration, emotion, exercises, meals, tobacco, alcohol, temperature, bladder
distension, and pain. The blood pressure is also influenced by changes in circadian rhythms, age, sex, heredity, race, and environment. Furthermore, the subject may have physical characteristics, such as obesity, or diseases which may modify the
blood pressure or make its measurement difficult or inaccurate. The subject should, therefore, avoid exertion and not eat or smoke for thirty
minutes before having their blood pressure measured. The room should be
comfortably warm and quiet, and the subject should be allowed to rest for at least
five minutes before the measurement.
1. Auscultatory Method
Auscultation is a technique based on the appreciation, via a stethoscope, of the quality of audible sound resulting from turbulence generated in a previously occluded blood vessel when the occlusion is released gradually.
The blood pressure is routinely measured indirectly by the use of a device called a
sphygmomanometer. This apparatus consists of an inflatable cuff provided with an inflating bulb and manually operated valve for the occlusion of the vessel in the arm
or leg and a manometer, either a mercury manometer or an aneroid gauge. The steps
employed in determining blood pressure with a sphygmomanometer are the following:
a. With the subject sitting comfortably, rest the right arm on the table at - the level of the heart. The cuff is wrapped securely around the arm about 2.5cm above the elbow.
b. Air is pumped into the cuff with a rubber bulb until the pressure in it is sufficient usually to 200mmHg to stop the flow of blood in the brachial artery. At this point, the brachial pulse disappears. Pressure within the cuff is shown on the sphygmomanometer scale.
c. Place your stethoscope over the brachial artery just below the elbow.
Gradually release the air from within the cuff. Faint tapping sounds corresponding to the heartbeat are heard. When the sound is first heard,
the air pressure from within the cuff is recorded on the scale. This is the
systolic blood pressure and denotes the maximum pressure in the main arteries resulting from the ejection.
d. As the air in the cuff is further released the sounds become progressively
louder. Then the sounds change in quality from loud to soft and finally
disappear. At the point where the sounds change from loud to soft, the manometer reading corresponds to the diastolic pressure.
e.
It denotes the pressure in the main arteries when cardiac ejection has ceased. These sounds are known as the sound of Korotkoff.
These five sounds termed "phases" are as follows:
• Phase I: Sudden appearance of tapping sound (snapping) • Phase II: Sounds become quieter (pulse palpable at most). Sometimes the
sounds seem to disappear during this time (auscultatory gap). This may be
caused by inflation or deflating the cuff too slowly. • Phase III: Sounds become louder and thumping • Phase IV: Muffling sounds
• Phase V: Sounds are absent The basis of these sounds or Korotkoffis attributed to vibrations set up in the wall of
the artery as the contained blood undergoes differing degrees of turbulence in the face of partial occlusion or deformation of the vessel.
2. Palpation Method
Simple palpation of a peripheral pulse may be used to establish that the pulse has
returned during the reduction of the pressure in an occluding limb cuff. Similarly,
the disappearance of the pulse is used to determine the point at which the cuff
pressure exceeds that in the occluded artery. Studies have shown that the method is influenced by the rate of the heart, by the
rate of deflation of the cuff and that the directly measured systolic pressure is
generally underestimated the level of the systolic pressure, however, the method has much to commend it, since no additional apparatus is required.
Procedure: Wrap the right arm of the subject firmly and securely. Palpating the
pulse at the wrist is performed with the seated subjects' arm extended sufficiently to
bring the Wrist close to the level of the heart. The pulse is detected by the palmer tips of the middle or ring fingers, the wrist, such that only the highest pressure is
exerted upon the artery. Raise the pressure in the cuff to about 200mmHg (The
pulse is absent). Gently open the valve of the sphygmomanometer and deflate gradually. When the pulse is felt once more at the Wrist, read the scale. This is the
systolic blood pressure.
Exercise
You are to make a recording of a subject's B.P under the following conditions and tabulate your results (1) while sitting (2) While standing (3) while reclining and (4)
after exercising for 10 minutes. Calculate the subject's pulse pressure (Systolic minus diastolic pressure). Calculate the subject's mean arterial pressure: This is equal to the diastolic pressure plus one - third of the pulse pressure. Mean arterial pressure = diastolic pressure +
1/3 of pulse pressure
Pulse pressure = Systolic minus diastolic pressure. The normal range of pulse pressure is 30 - 60mmHg. S/
N
Position By Palpation By Auscultation
BP in mmHg Systolic BP
in mmHg
Diastolic
BP in mmHg
Pulse
pressure in mmHg
Mean
Arterial Pressure
1. Sitting
2. Standing
3. Reclining or
inclining
4. After 10mins of exercise
Comment on your results on how various positions affect the blood pressure measurements and why?
QUESTIONS 1. Explain the causes of the first and second heart sounds?
________________________________________________________________________________
________________________________________________________________________________________________________________________________________________________________
________________________________________________________________________________
____________________________________________________________
2. What autonomic nerve is responsible for the normal resting heart rate?
________________________________________________________________________________
______________________________________________________________________
3. Does heart rate vary with the respiratory cycle? ____________________________________________________________________________
____________________________________________________________________________ ____________________________________________________________________________
____________________________________________________________________________
4. What are the normal levels of systolic and diastolic blood pressures in man?
________________________________________________________________________________________________________________________________________________________
5. Explain the results of the effects of posture in terms of changes in venous return, cardiac output, blood pressure and baroreceptor activity. ________________________________________________________________________________
____________________________________________________________________________________________________________________________________________________
____________________________________________________________________________ ____________________________________________________________________________
6. Comment on the effect of a moderate exercise on: (i) Systolic pressure, and diastolic pressure
_____________________________________________
______________________________________________________________________________________________________________________________________________________
__________________________________________________________________________
(ii) Why may diastolic pressure fall during exercise?
______________________________________________________________________________________________________________________________________________________________________
___________________________________________________________________________________
_______________________________________
EXPERIMENT 21
TO INVESTIGATE SOME FACTORS WHICH AFFECT PULSE RATE AND BLOOD
PRESSURE
Before beginning, this experiment makes sure that you are fully conversant with the methods described in Experiment 3 so that every member of the group can
quickly and accurately measure pulse rate and blood pressure. The practice is the only way this can be achieved
PROCEDURE
In each experiment, first, take three resting (basal) readings of pulse rate and
systolic and diastolic blood pressure. If they are constant proceed with the experiment. If they are not, take further readings until three successive readings are
obtained which do not vary by more than four beats/minute for pulse rate and
5mm.Hg. for blood pressure. Allow the subject to perform the appropriate change of posture, exercise, etc. and try to obtain at least one reading of pulse rate and blood
pressure during the maneuver. Obtain reading of each immediately after the change
of position or exercise and further readings at 30 seconds intervals thereafter until
constant readings are again obtained. The experiments will normally be carried out with groups of at least five students
per group as follows:
A - Acts as a subject throughout the experiment B - Takes blood pressure (this should be carried out simultaneously)
C - Takes pulse rate and compute within seconds
D - Acts as a timer E - Acts as recorder
N.B:Each member of the group must continue with his own, allocated job throughout the experiment so that meaningful and comparative results may be obtained for the set of conditions. If time allows repeating the experiments after
reshuffling the members of some.
FACTORS TO BE STUDIED
(a) The Influence of Posture
Determine the influence of the following postures on pulse rate and blood pressure of the following bodily positions:
i. Lying at rest ii. Sitting upright iii. Standing
iv. Strap the subject firmly to the tilt-table and study his responses in the
head down
position v. Repeat (iv) with the subject in the foot-down position
vi. Repeat (iv) and (v) several times increasing the rate at which the table is
tilted.
(b) The Effect of Exercise
(i) Determine the effect of exercise on blood pressure and pulse rate. The exercise consists of stepping up onto and down from the one-foot high stool
provided, 30 times a minute. Using a bicycle ergometer step up at one second, down at two up at three, and so on. Exercise for two minutes. (ii) Repeat using a more severe form of exercise e.g. exercising with a load of approximately half your body weight on your back.
(c) The Effect of Temperature
Determine the effect on pulse rate and blood pressure on sudden immersion of
both feet in:
i. Ice cold water
ii. The warm water of 450C.
(d) The Effect of Respiration
Determine the effect on pulse rate and blood pressure of:
i. Voluntary breath-holding for as long as possible in
ii. maximum inspiration iii. maximum expiration
iv. maximum inspiration with the face immersed in water at room temperature
(to demonstrate the “selective Ischaemia” reflex). v. Valsalva maneuver (i.e. forced expiration against a closed glottis - as, for
example, straining during defaecation)
(e) The Effect of Pressure on the Carotid Sinus; The subject must lie completely relaxed on his back. Feel for the pulsation of the
common carotid artery deep to the anterior edge of the sternomastoid muscle. The
bifurcation of the carotid artery and the carotid sinus is at the level of the upper border of the thyroid cartilage. Compress the artery firmly against the vertebral
bodies for two seconds only. See if a change in pulse rate occurs.
Do not compress both carotid arteries simultaneously.
(I) The Effect of Drugs
(1) Amyl This should not be performed by anyone who has had any form of ‘heart trouble’. The subject must be in a supine position.
Record normal pulse rate and blood pressure.
Ask the subject, to inhale deeply through a piece of cotton-wool on which is
a few drops, of amyl, have been placed.
In addition to the pulse rate and blood pressure readings ask the subject to
describe his subjective sensations.
(2) Intravenous Atropine A medically qualified member of staff will give the subject a suitable dose of atropine intravenously.
In this experiment note not only the effects on pulse rate and blood pressure but
also other autonomic effects such as respiration, pupillary diameter, salivary secretion, gastrointestinal activity (by listening to intestinal “rumblings” with a
stethoscope on the abdomen).
QUESTION Record the results of the experiments graphically where possible. Explain the
physiological mechanisms of the observation you have made. _________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________
___________________________________________________________________________________
______________________________________________________________________________________________________________________________________________________________________
___________________________________________________________________________________
______________________________________________________________________________________________________________________________________________________________________
___________________________________________________________________________________
___________________________________________________________________________________
______________________________________________________________________________________________________________________________________________________________________
EXPERIMENT 22
HUMAN ELECTROCARDIOGRAPGY
Title: The Electrocardiography (CVS) Experiment: The normal electrocardiogram in man Aim: To demonstrate and record ECG in man Equipment/Apparatus: Electrocardiograph, ECG recording paper, Electrode
plates, Rubber straps, Electrolyte paste Introduction:
Diagnosis of disorders from the electrocardiogram entails familiarity with the
normal state and pattern recognition and is enhanced by an understanding of the basic feature of electrocardiography. The electrocardiogram (ECG) is a record of the electrical activity of the heart at the skin surface. It consists of three bipolar leads (I,
II, III); the three augmented unipolar limb leads, designated aVR, aVL, and aVF
respectively and the six unipolar precordial (chest) leads, as the V1 - V6. The 12 lead ECG is thus classified as consisting of the six standard limb leads (I, II, III, aVR, aVL, and aVF) and the six precordial leads V1 - V6. The six standard leads are
considered to be looking at the heart in the frontal or vertical plane. Leads I, II, and
aVL look at the left lateral surface of the heart, III, and aVF look at the inferior aspect and aVF views the right atrium. Leads V1 - 2 look at the right ventricle. Leads
V3 - 4 looks at the interventricular septum. Leads V5 - 6 looks at the left ventricle.
Some factors influencing the variability of the electrocardiogram include age, sex, race, height, weight, physical fitness, heart position, phase of respiration, heart
rate, recent ingestion of food, etc.
The Electrocardiograph Grid Electrocardiographic paper is a graph in which horizontal and vertical lines
are present at 1mm intervals. A heavier line is present every 5mm.
Time is measured along the horizontal lines 1mm=0.04sec, 5mm = 0.2seconds. Voltage is measured along the vertical lines and is expressed as mm (10mm = 1mV).
In usual calibration is a 1mV signal which produces a 10mm deflection.
Electrocardiographic Leads The electrocardiographic lead is defined as a pair of terminals with designated
polarity, each connected either directly or via a passive and active network to recording electrodes. A pair of electrodes constitutes an electrocardiographic lead in its simplest form. The commonly used 12 - lead ECG consists of three bipolar limb leads, three
augmented unipolar limb leads, and six unipolar precordial leads.
Bipolar Limb Leads
The three standard bipolar limb leads introduced by Einthoven (I, II, III) record
the difference in potential between two extremities when each is connected to one of the input terminals of the recording device.
The bipolar leads represent a difference in the electrical voltage between 2
selected sites:
Lead I = difference of voltage between the left arm and the right arm (LA - RA) Lead II = difference of voltage between the left leg and the right arm (LL - RA)
Lead III = difference of voltage between the left leg and the left arm (LL - LA)
The sites RA - Right Arm, LA - Left Arm and LL - Left Leg was chosen by Einthoven, a pioneer in electrocardiography - he postulated the Einthoven triangle hypothesis which states, that the heart can be considered to be at the center of an equilateral
triangle, the angles of which are at the right arm (RA),left arm (LA) and left Leg (Ll.). The sides of the triangle from the standard limb leads I, II, III.
The Einthoven triangle hypothesis is based on the notion that the thorax rs a
homogenous conductor. This view is no longer tenable.
The relation between the three leads is expressed algebraically by the Einthoven equation: Lead II = Lead I + Lead III.
This is based on Kirchoff's law, which states that the algebraic sum of all the potential differences in a closed circuit equals zero.
Unipolar Augmented Limb Leads In the unipolar recording, the ECG may be recorded by using an active or exploring
electrode connected to an indifferent electrode at zero potential. By an elective
arrangement, potentials recorded by one electrode (indifferent electrode) are
rendered negligible and so only the electrical activity of the exploring electrode is recorded.
The indifference electrode terminal is RA + LA + LL and equals zero. When the
exploring electrode is placed on the right arm, a voltage difference between RA and the indifferent electrode is recorded. Because of the negligible potential of the
indifferent electrode, it is assumed to be zero. Hence the potential recorded is the
actual potential of RA. In like manner, the actual potential of LA and LL are recorded
by placement of the exploring electrode over the left arm and left leg respectively. These three leads are designated VR, VL, and VF.
Leads aVR, aVL, and aVF are called augmented leads because the ECG
enhances the small waveforms that would normally appear from these unipolar leads. By a minor change in the techniques, the voltage by these leads can be increased to 50%, and therefore these leads are called augmented leads aVR, aVL, and aVF or the Goldberger lead.
Unipolar Precordial (Chest) Lead- The Wilson leads The precordial leads consist of six electrodes at specified sites on the anterior and
left lateral thorax and central terminal to which right and left arm and left leg
electrodes are all connected. The common precordial positions used are as follows: V1 = fourth intercostal space at the right sternal border
V2 = fourth intercostal space at the left of the sternum
V3 = midway between V2 and V4 V4 = fifth intercostal space at the midclavicular line
V5 = placed at the fifth intercostal space at the anterior to the axillary line
V6 = the last of the precordial leads is placed at the fifth intercostal space at the midaxillary line.
These precordial leads are useful in monitoring ventricular arrhythmias, ST-segment
changes, and bundle branch blocks.
COMPONENTS OF THE NORMAL ECG
1. P Wave The P wave consists of an ascending part, a peak, and a descending limb. It is due to
atrial depolarization. It is seldom taller than 2.Smm. Its width should not exceed
0.01 seconds. The P wave duration is measured from the beginning to the end of the P Wave. The amplitude of the P Wave is measured using the baseline level at the end
of the TP of UP internal as the level of reference from which its voltage is measured.
2. QRS Complex The complex comprises of Q, R and S waves. Sometimes, besides, there are R1 and
S1 waves. The QRS complex is produced by ventricular depolarization. The QRS interval is measured from the onset of the Q wave, or of the R wave, if no Q is written, to the end of the S wave, or of the R wave, if no S is written. The normal
range of the QRS complex is 0.08 - 0.12 seconds. 3. T Wave
The T Wave is produced by ventricular repolarisation. The width and amplitude of the T wave are measured using the level at the terminal of the TP or UP interval as
the level of reference for measurements. This is normally upright except in lead AVR.
4. U Wave
The U wave is attributed to the repolarisation of the Purkinje fibers, the papillary muscles, or the ventricular system. It follows the T wave and precedes the P wave of
the next cycle. The duration and amplitude of the U wave is measured using the termination of the TP or UP interval as the level of reference for the measurements. U waves are best seen in leads V2 and V3.
5. P-P and R- R Interval
When there is sinus rhythm, the P - P and R - R interval are equal. They are used to
calculate the heart rate. The P - P internal denotes the atrial rate and the R - R interval denotes the ventricular rate.
Determination of Cardiac Rate (Heart Rate)
The cardiac rate can be determined from the electrocardiogram. 1. Since one small square is 0.04 sec, wide, the number of small squares
equal to 1 minute is 60/0.04 or 1,500. Therefore, the number of small
squares in one R -R interval divided into 1,500 will give the heart rate per
minute (the R - R interval is measured from the peak one R wave to the peak of the succeeding R wave)
2. If a large square is 0.04 x 5, or 0.2 second wide, the number of large
squares equal to one minute is 60 ÷ 0.2, or 300. Therefore, the number of large squares in one R -R interval divided into 300 gives the heart rate per
minute.
PR interval
The PR interval is measured from the onset of the P Wave to the onset of the
QRS complex hence the term PQ interval is more accurate. It represents the time interval between arterial and ventricular depolarization and hence includes the time taken for atrial depolarization, atrial repolarization, and the delay of excitation in the
AV node. The normal P - R interval ranges from 0.12 - 0.20 Second.
QT Interval The QT interval is the summation of ventricular depolarization and
repolarization and represents the duration of ventricular systole. It is measured from the beginning of the Q wave to the end of the T wave. The Q - T interval is related to cardiac rate, age and sex, Q - T interval corrected for the heart rate is called Q - TC
interval.
Q – TC = Actual Q - T Interval
R - R Interval The normal range for Q - T interval: 0.35 - 0.45 seconds,
ST-Segment The interval physiologically represents the early phase of ventricular
repolarization. The ST sequence is measured from the ST function to the beginning
of the T Wave. It is usually isoelectric but maybe slightly depressed (0.5mm) or
elevated (0.2mm) in precordia Leads. If elevation occurs, suspect acute myocardial infraction (in such cases the ST elevation is convex-shaped), or a normal variant
often seen in Africans. If the ST segment is depressed, this may be due to ischemic
heart disease or non-specific myocardial disorders.
Assignment
With the help of your instructors and technical staff, make an ECG tracing and
interpret your findings.
QUESTIONS
1. What are the normal values for the PR interval, QRS duration, and ST intervals?
PR Interval______________________________________________
QRS duration____________________________________________
ST interval ______________________________________________
2. What do prolongations of the PR interval and QRS duration denote respectively?
________________________________________________________________________________
________________________________________________________________________________
____________________________________________________________________
3. How can you explain differences in the appearance of the QRS complex from V1
to V6 in terms of the mean electrical axis of the heart? ________________________________________________________________________________
________________________________________________________________________________
________________________________________________________________________________
______________________________________________________________
4. What is the normal range for the mean electrical axis of the heart?
____________________________________________________________________________
5. What do right and left axis deviations indicate?
___________________________________________________________________________________
___________________________________________________________________________
SECTION E
RESPIRATORY SYSTEM
EXPERIMENT 23
CLINICAL EXAMINATION OF THE HEART AND RESPIRATORY SYSTEM
EXAMINATION OF THE HEART (a) Inspection
Inspect the front of the subject’s chest and look for the apex beat of the heart.
This is a pulsation occurring in the fifth intercostal space about the mid-clavicular line. It is most easily seen in a thin person. Incline the subject at 45° and notice if
there is the filling of the jugular veins. Determine the vertical height of the vein from the sternal angle.
(b) Palpation
This is done with the subject lying down. Place your warm palm over the region of
the heart and note the point where cardiac pulsation thrusts maximally against a palpating finger. Determine its location. This is the Apex beat.
(c) Percussion
With the subject supine, place the left palm flat on the chest and tap firmly on the 2nd phalanx of the left middle finger with the tip of the right middle finger. Note the character and pitch of the sound produced. Carry out the procedure over the
anterior-chest wall, working from the sides towards the midline and note the change in pitch as the finger passes from the resonant lung area to the area of cardiac
dullness. The anatomical outline of the heart corresponds to this “area of relative
cardiac dullness”. Map out this area on the chest.
(d) Auscultation
Using the stethoscope, listen to the following areas:
Mitral Area Corresponding to the apex beat Tricuspid area: Lying just left of the lower end of the sternum
Aortic area: Right of the sternum at the second intercostal space
Pulmonary area: Left of the sternum at the second intercostal space
Note that these positions are where the sounds are best heard and not projections to the surface of the anatomical sites of the valves. The first heart sound is heard as
(LUB) while the second is clear short and high in pitch (DUB). The second is followed
by a pause.
STETHOGRAPHY
Stethography means recording of the movements of the chest occurring during respiration. It shows a large member of facts about respiratory physiology such as:
1. Respiration rate which at rest is 12 to 16 times per minute. 2. Respiration cycle: inspiration and expiration phases and pause.
A stethograph can be used to determine the rate and respiratory cycle. Other
respiratory ventilation parameters can be measured using a spirometer and such
measurements except residual volume are: 1. Tidal air as volume 500ml (TV)
2. Pulmonary ventilation: Tidal volume (500ml or 0.5L) x rate of 12 respiration
gives the pulmonary ventilation per minute = 600ml or 6L/min 3. Inspiratory capacity (IC) = TV + IRV = 0.5L + 2.5L = 3L
4. Vital capacity (VC) = TV + IRV + ERV or IC + ERV = 4 - 5L
5. Inspiratory Reserve Volume (IRV) (2,500ml or 2.5L)
6. Expiratory reserve volume (ERV) (1,000ml or 1L) 7. Alveolar ventilation per minute (TV - Dead space vol.) xf Respiration Rate
(500 - 150) = 350 x 12 = 4,200ml
8. Functional residual capacity FRC = ERV + RV= 1,000 + 1,500 = 2.5L 9. Total lung capacity TLC = FRC + IC (2.5L + 3L = 5.5L)
Example; A man with a normal dead space breathes in 500 cc room air twelve (12) times in a minute. He can add an extra 2,500 IRV of air during maximal inspiration and can expel 1,000 cc ERV after his normal expiration. The volume of air still left in
his lungs is 1,200cc. Calculate the above parameters from these values.
In stethography, a stethograph is tied around the chest of a subject above the
nipples and is seated comfortably on a stool in front of the apparatus and backing it. One end of the stethograph that is open is connected with a hollow rubber tubing
and connected to the opening base of the marry's tambour. Pressure changes caused by movements of the chest cause air pressure changes in the tambour which will
cause movement of the liver with a stylus mounted on the tambour and adjusted to write on the drum of a kymograph. The chest movement changes according to respiratory phases are then recorded on the drum.
Alternatively, electronic stethograph also known as respiration transducer can
be connected to a respiration coupler of a physiograph for recording such movement
changes of the chest on the recording paper of the physiograph. The following breathing movement changes of the chest can be obtained e.g.
Normal inspiration followed by normal expiration and when the subject is carrying
out acts such as whispering, loud speech, deglutition, or swallowing, breath-holding, voluntary hyperventilation, violent exercise, emotion e.g. laughing. After each event
wait until the record returns to the resting pattern.
Record a quiet inspiratory movement on fast speed. Record the inspiration as a
downstroke and expiration as an upstroke. All the upstrokes reach a constant level while the downstrokes vary slightly in
their lengths, indicating that the end-expiratory position of the chest is the normal
resting position. The inspiration starts from this level and the expiration ends at this level.
Lung Volumes and capacities (Spirometry).
Lung Volumes:
1. Inspiratory Reserve Volume (IRV) = 2.5L to 3.5L 2. Tidal volume (TV) = 400 to 500ml 3. Expiratory Reserve Volume (ERV) = 1L to 1.5L
4. Residual volume (RV) = 1.5L Lung capacities:
1. Vital capacity (VC) = IRV + TV + ERV = 4 to 5L
2. Total lung capacity (TLC) = VC + RV = 6L
3. Functional Residual Capacity = FRC = 2.5L 4. Inspiratory Capacity = IC = IRV + TV = 2.5 to 3.5 L
Definitions Lung volumes
1. IRV: Maximum volume of air which can be inspired after completing a normal
tidal inspiration (i.e. from normal to inspiratory position).
2. TV: Volume of air breathed in or out during quiet respiration (i.e. at rest). 3. ERV: Maximum volume of air which can be expired after completing a normal
tidal expiration (i.e. expiration from normal end to expiratory position)
4. RV: Volume of air that remains in the lungs after a maximal expiration.
Lung capacities:
1. VC: Maximum volume of air that can be expelled from the lungs by a
maximum effort following a maximal inspiration. Normal values of 4 to SL will fall in paralysis of respiratory muscles. Examples- in spinal cord injury or
poliomyelitis (values can decrease to 500 - 1000ml while as high as 6- 8L in
well-built athletes. Other examples in fall are chronic asthma, lung cancer, and pulmonary congestions.
2. TLC: Total volume of air contained in the lungs after a maximal inspiration.
3. FRC: Volume of air remaining in the lungs at the resting expiratory level or the end of normal expiration.
4. IC: Maximum of air which can be inspired from the resting expiratory level. Note: Of these volumes and capacities, all except TLC, FRC, and RV can be
measured by the use of a recording spirometer. TLC, FRC, and RV cannot be measured directly and require special techniques.
QUESTIONS
1. Explain the causes of the first and second heart sounds? ________________________________________________________________________________
________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________
EXPERIMENT 24
RESPIRATORY FUNCTION TESTS
INTRODUCTION
Respiratory function tests are simple measurements of lung volumes, capacities, and
flow-rates. They provide a good deal of information on respiratory mechanics and
provide sufficient data to assess the ventilatory functions of the lung. If those functions are abnormal. The defects can further be classified into three types.
a) Obstructive ventilatory defects - reduce primarily the flow-rates while vital
capacity remains normal e.g. Bronchitis, Asthma, Chronic Bronchitis, COPD e.g Pulmonary, Fibrosis, Black lungs.
b) Restrictive ventilatory defects - reduce vital capacity while flow-rates are normal
c) Combined restrictive/obstructive defects. In our laboratory, these tests will be performed on two types of apparatus.
1) Spirometer - It is used to measure the static lung volumes
ii) Vitalograph - It is used to measure the dynamic lung volumes and airflows
A. SPIROMETRY (STATIC LUNG VOLUMES)
1. Spirometer with pen recorder 2. Recording drum with chart
3. Three-way stop-cock 4. Two-way respiratory valve
5. Nose clip 6. Mouthpiece.
Procedure
1. Fill the spirometer with room air so that the writing pen is adjusted at about 2.5 liters mark on the chart.
2. Attach the three-way stop-cock and a mouth-piece.
3. Apply the nose-clip to subject and ask the subject to breathe through a three-way stop-cock connected to the atmosphere.
4. When the person has adjusted to the apparatus and is breathing normally,
connect the stop-cock to the spirometer.
5. Put the recording drum in gear marked 0.2 (2 mm/sec.) 6. Record 4 or 5 normal respiratory movements.
7. At the end of normal expiration, ask the subject to make maximum effort to
breathe in and then expire maximally. 8. If possible again take a few records of normal breathing.
9. Take the tracing standing and supine to see the effect of posture on various
volumes and capacities.
Paste Recording From the tracing thus obtained one can calculate the following. Insert results in table
Standing Term
(Symbol)
Definition Reading
from the record
Tidal volume (TV) The air volume of a single breath expired or inspired
Expiratory Reserve
Volume (ERV)
Maximum volume of air which can be
exhaled after normal expiration i.e. from
the resting respiratory level
Inspiratory Capacity
(IC)
Maximum volume of air which can be
inhaled after a normal expiration, i.e. from the resting respiratory level.
Vital Capacity(VC) Maximum volume of air which can be
exhaled after maximum inspiration without regard to the speed of expiration
Residual volume (RV) Air volume remaining in the lungs after maximum expiration
* Functional Residual
Capacity (FRC)
Air volume contained in the lungs after
normal expiration (ERV + RV)
Total Lung Capacity
(TLC)
Air volume contained in the lungs after a
maximum inspiration
NB. The speed of the drum is 2 mm/sec. Calculate the rate of Breathing = *Can be estimated using the Nitrogen Dilution Method.
B. VITALOGRAPHY
Vital capacity or forced vital capacity (FVC) is the maximum volume of air that
can be expired following maximum inspiration and can be timed and this is referred to as the timed vital capacity (TVC) or forced expiratory volume FEV for clinical
purposes. The spirometer used for this measurement is known as Vitalograph.
That is the largest volume of air that can be expired in a given time. In one second a normal subject can expire 75% or slightly more of this VC. This can be described clinically as a lung function index or test.
Forced Vital Capacity (FVC) - Gas volume expired after maximal inspiration with expiration being as rapidly forceful and as complete as possible. For men FVC
is 3.92L and for women it is 2.91L.
Forced Expiratory Volume - (FEV1) - This is the forced expiratory volume in one second (1st second). This is reduced in some forms of lung diseases. FEV1 for males is 3.32L and 2.54L/second for females. FEV1 is decreased in the following
circumstances: (1) The absolute reduction in the functioning of lung tissue (2) Limitation of chest expansion (3) Limitation of diaphragm distension due to the limitation of lung expansion.
Significance of lung function index Test or Pulmonary function
In airways, obstructive diseases such as chronic bronchitis, Asthma, the lung
function test is less than 75% i.e., FEV1 × 100% = < 75%
FVC 1
That is to say that the FEV1 or FEV1% is less than normal. This condition of
obstructive disease IS characterized by difficulty in expiration (i.e. any value 69% or less). However in airway restrictive diseases such as diffuse interstitial fibrosis.
Asbestosis forced vital capacity or vital capacity is reduced and the lung function
test result is greater than ( > ) 75% FEV1 × 100% = < 75%
FVC 1
(E.g. 88-90% or more)
In this case, the FVC is less than normal but the FEV1 may be normal. The restrictive disease is characterized by difficulty in inspiration. MVV means Maximum Voluntary Ventilation = FEV1 x 3.75 L/ min.
Normal range 150 - 170 L/min
Total Pulmonary Ventilation: This is the product of tidal volume × respiration rate (breaths /minute)
A fraction of each tidal volume occupies parts of the respiratory tract that act only as air passageways. These airways called anatomical dead space, occupy about 150ml, and do not permit gas exchange with the blood.
Tidal Volume - Dead Space indicates the volume of fresh air entering the lungs.
Therefore the more physiologically meaningful alveolar ventilation would be:
Alveolar Ventilation = Respiratory rate x (Tidal Volume - Dead Space Volume)
The control of alveolar ventilation, rate, and depth of breathing is by the respiratory centers in the medulla and pons of the brain. Here the sensory information from
mechanoreceptor and chemoreceptors is integrated and rhythmic motor commands
are sent to respiratory muscle.
Formulae for predicted values: FVC
Male = 27.63 - (0.112 x age) x ht (cm)
Female = 21.78 - (0.101 x age) x ht (cm)
EXPERIMENT 25
PEAK EXPIRATORY FLOW RATE
The Peak Flow Rate Meter (Peak Flow Meter)
This measures the maximum flow rate in a single forced expiration known as the Peak Expiratory Flow (P.E.F) and expressed as liters per unit time which is usually per minute and hence P.E.F.R.
The P.E.F reading is influenced by sex, age, musculature (Torso), and the body build of the individual subject.
The following regression equation is used to predict the normal value. Formulae
Male: P.E.F.R (L/min.) = 331.47 - 0.96 x age + 4.66 x ht in inches
Female: P.E.F.R (t/rnln.) = 460.68 - 2.46 x age + 1.62 x ht inches To avoid unnecessary calculation, consult its Nomogram.
Usage of Machine (Peak Flow Meter) It is held vertically with handle or horizontally without the handle type.
The subject takes in a breath as deep as he can and then places the mouthpiece in
his mouth, gripping it tightly with his/her teeth and sealing it with his/her lips. He then blows out as hard as possible in a short, sharp blast, using all the muscular
forces of his chest to do so. An adequate period of rest between each attempt is
advisable.
Peak Expiratory Flow, therefore, is the maximum velocity of the airflow that
can be produced during a single forced expiration. For males, the range is 510 – 560
± 100L/min and for females 400 - 480 ± 80L/min. Low values are obtained when
expiratory muscle activity is weak or when there is an obstruction in airflow passage e.g. as In Asthma.
MVV means Maximum Voluntary Ventilation as liters/min = FEV1 x 3.75
L/min since Peak Flow Rate is a measure of the Peak or Maximum Flow of expired air, it becomes a sensitive test for the presence of obstructive disease. Patients with
a low FEFR would have to be further evaluated for obstructive pathologies.
There are several systems which physicians use to determine the severity of the disease. Here is one way that is very commonly used:
• Normal PEFR (i.e. PFT) outcome > 85% of predicted values • Mild Disease > 65% but < 85% of predicted values
• Moderate Disease > 50% but < 65% of predicted values
• Severe Disease < 50% of predicted values
In most good spirometers in the market today, there is a set of the normal
table (sometimes multiple sets of tables) which can be chosen as you perform the
PFT. Also, there are interpretive microchips in the PFT machines which will tell you
what the diagnosis is for a particular patient. These two features make it easy for the
Clinician to immediately see what the predicted values (normal table values) are for a specific patient and whether or not the PFT has a normally observed outcome.
Normal values (Caucasians) are related to the patient's height as follows:
Height
(cm)
PEFR
(L/min)*
120 215
130 260
140 300
150 350
160 400
170 450
180 500
* mean; 2 SD = ±100
An easy to remember approximation is: PEFR (L/min) = [Height (cm) - 80] x 5
Exercise
To compare the Peak expiratory flow rate in fifteen male students and fifteen
female students in your practice group.
Apparatus
- Wright Peak Flowmeter or any other brand available
- Disposable mouthpiece Direction
1. The flow meter should be held in a vertical plane, the calibrated dial facing to the right. It should be held firmly in both hands taking due care that vent holes are
not obstructed. 2. The subject takes as deep a breath as he can, and then places the mouthpiece in
his mouth, gripping it lightly with his teeth and sealing it with his lips. He then
blows out as hard as possible in SHORT SHARP BLAST using all the muscular forces of his chest to do so. Three readings of PEF are made and if the test is performed properly, reading should differ very little from each other. Take the
average.
Normal range - Male (510 - 560 l/m) ± 100: Female (400 - 480 l/m,) ± 80
Predicted values
Obtained by the equation derived by Femi.Pearse in Nigeria Males - = (3.27 x Ht) - 1.59 x Age) - 14.63, ± 79; Females = (1.09 x Ht) -(3.99 x
Age) + 673.17 ± 567 Ht. in cm., Age in years. Record your observed values and compare them with the Predicted values.
Subject
Number
MALE FEMALE
Height Weight PEFR Height Weight PEFR
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
Mean
SD
S.E.M
EXPERIMENT 27
ARTIFICAL RESPIRATION AND CARDIAC RESUSCITATION
When the heart or lungs fail for unnatural causes from which they can be revived, and the person is detected within five minutes so that there is no brain death, there is a good chance of reviving the person. As resuscitation in time makes the difference between life and death, every medical student should be skilled in
delivering artificial respiration and cardiac massage. Artificial respiration and / or cardiac massage should be delivered only if the organ concerned has completely failed. A model, Resusci Anne, is provided to practice these manoeuvres with an
electrical system for guidance. A hand out will be given at the session and trained tutors will demonstrate the procedure.
Exercise
a) Explain why mouth to mouth method is the best of all types of artificial respiration available
------------------------------------------------------------------------------------------------
------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------
--------------------------------------------------------
b) Describe the risks of this method to the victim and to the operator_____________________________________________________________________
_____________________________________________________________________________
__________________________________________________________________________
c) Explain the importance of correct hand position during cardiac massage____________________________________________________________________
_____________________________________________________________________________
________________________________________________________________________________
SECTION F
GASTROINTESTINAL PHYSIOLOGY
EXPERIMENT 27
COLLECTION AND ANALYSIS OF GASTRIC JUICE IN RAT
INTRODUCTION Gastric juice should be obtained from rats that have been fasted for 24 hours.
However, water should be taken during the fasting period.
In this test, the secretory response of the rat stomach is investigated on basal acid secretion as well as the maximal (histamine - stimulated) acid secretion. Basal
secretion is the amount of gastric juice obtained in the absence of any stimulation. The maximum secretion of gastric juice is the amount obtained after an
augmented dose of a substance that stimulates gastric secretion has been administered. Such a substance is histamine.
PROCEDURE Basal secretion - Normal saline solution is used to perfuse the stomach. The rate of perfusion is regulated such that l0ml of gastric contents are collected through the
stomach cannula in 15 mm. Collect l0ml of gastric contents every 15 minutes for 1
hr. in labeled measuring cylinders. Maximum secretion - Inject a dose of 0.1 mg/g body wt. Histamine acid phosphate (1ml of given solution) subcutaneously. After 5 minutes collect l0ml of gastric
contents again every 15 minutes for 1 hr. in labeled measuring cylinders.
EXAMINATION OF SAMPLES
pH - determine the pH of the fluid using the pH meter
Total acidity - pipette exactly 5m1. of the sample into a porcelain evaporating dish.
Add 1 drop of 1% Phenolphthalein and titrate with 0.0025N NaOH from a burette. The endpoint is a pink color (pH6) which lasts a few minutes. Take the burette
reading.
CALCULATION OF ACID CONCENTRATION (ACIDITY) (f) Calculation of acidity
The reaction between gastric acid and the titrating base can be represented by
the equation: NaoH + Hcl - (Nacl + H2O ........................ (i)
At the end-point of titration NaoH + Hcl -Nacl + H20
NAVA = NB VB i.e. NA = NB VB
VA ................. (ii)
But normality (N) = Concentration (C) in gm/litre
Gm. Eq. Wt. (G) i.e. C = N x G
(Acid concentration is gm/litre (C) = NA x G Substituting for NA in (ii), C = NB.VB.G gm/litre VA
C = NB.VB.G x 100 mg/100ml. ................ (iii)
VA But acid concentration is Meq/litre - Concentration in mg/litre
(m. Eq. Wt. (G)
Therefore, Acid concentration in Meq/litre = NB.VB. G x 100 .................... (iv)
VA.G
But NB = 1/400
Substituting for NB in (3.3) Acid concentration in Meq/litre = 1/400 x V 1000 = 5/2 VB
VA ......... (v)
Since the gastric effluent sample titrated = VA = 5ml. The acid concentration in titrated sample (Meq/litre) = VB........... (vi)
2
But the 5ml is only 1/2 of the total effluent sample. Therefore, total acidity for each 10ml sample = VB x 2
2
= VB INTERPRETATION OF RESULT8 Express your results in the form of a table and graph. Plot the time (minutes) along
the abscissa, mark the scale of pH along the right-hand ordinate, and the acid concentration (meq/liter) along the left-hand ordinate.
EXPERIMENT 28
INTESTINAL MOTILITY
INTRODUCTION
Intestinal smooth, muscle differs from skeletal muscle in: (i) having an autonomic nervous control (ii) spontaneous activity
(iii) the slowness of its contraction and relaxation
(iv) by its power of propagating waves of contraction from fiber to fiber. (v) And by its ability, even when isolated to show tone and give spontaneous
contractions.
Most smooth muscles, when isolated from the body, and suspended in a solution of saline isotonic with the blood, will exhibit spontaneous contractions and alterations
in tonus, when the external conditions e.g. temperature are varied. The saline
solution contains other' salts besides sodium chloride - in particular calcium and
potassium chlorides, which are careful, balanced, one against the other. Mammalian smooth muscle must be kept at 37°C and the solution must be oxygenated, under
these conditions it will live for many hours.
The spontaneous activity is modified by some endogenous (Acetylcholine, Adrenaline, Histamine, 5-HT, Bradykinin, Prostaglandins, etc.) and exogenous
(Nicotine, Barium, etc.) substances.
This is the experiment which demonstrates the normal spontaneous contractions, the effect of temperature and the modification of the spontaneous activity by a variety of substances Furthermore, this experiment also demonstrates the fact that
different substances act on small intestine by occupying different receptors and these actions can selectively be prevented by pretreatment of the tissue with specific antagonists e.g.
APPARATUS
The muscle bath (filled with warm, aerated tyrode solution) is surrounded by a water-bath, which also contains a spiral glass tube. This spiral glass tube connects above with a bottle of Tyrode solution and below with the lower part of the muscle.
Tyrode solution is composed of: Na, K, Ca, HCo3,HPo4and Glucose in the
Compounds following.
eceptor Agonist Effect Antagonist
Muscarinic Cholinergic
Acetylcholine Stimulation (contraction)
Atropine
Nicotinic
(ganglia)
Cholinergic
Nicotine Stimulation Hexamethonium
Hi Histaminergic Histamine Stimulation Mapyramine
α and β
adrenergic
Adrenaline Relaxation α - Blocker
Phenoxybenzamine β - Blocker
Propranolol
5HT Serotonergic
5-HT Stimulation Cyproheptidine
NaCl - 0.8 g KC1- 0.02 g NaHCO3- 0.01 g NaH2 P04 - 0.005 g CaCI20.02 g Glucose - 0.1 g
Distilled water to make up to 100 ml. When it is necessary to change the solution in the muscle bath, a solution from the bottle (warmed in passing through the spiral) is allowed to-run in slowly and displace the used solution, which overflows into the
water bath. When necessary siphon off some of the water from the water bath and throw it away. The temperature of the water bath should be kept at about 37°C. The
glass tube inside the muscle bath ends below in a hook to which one end of the piece of the gut is tied. (It also serves to bring the air supply.
PREPARATION
Euthanize the rabbit by a blow on its head. As soon as possible after the death, open the abdomen and remove a piece of proximal ileum freed from the mesentery near
the duodenum, as it shows the greater spontaneous activity as compared to the
distal ileum. Immerse it in ice-cold saline and cut into small segments ( 3 - 5 cms long). If the intestine is full of the faecal matter this can be washed out by gently
blowing saline through a pipette held gently at one end of the segment. The muscle
must not be stretched nor touched with anything but scissors or forceps.
PROCEDURE:
Using a threaded needle, attached a piece of thread to each end of the segment. At
one end make the thread into a small loop and attach it to the hook in the organ
bath. The other piece of thread should be attached to the writing lever with plasticine. If necessary, add more plasticine to the lever until it is almost
counterbalanced. When recording, the writing point should swing freely. The of the
inner bath should be made to bubble through the solution in a gentle stream. If the preparation is satisfactory, it will show spontaneous contractions within about 10
minutes of being set up. If the muscle does not show spontaneous contractions
do not interfere with it, you will only make it worse. Sometimes an inactive
muscle is encountered and this cannot be helped; it will probably show responses to drugs quite satisfactorily. Observe and record the following:
1. Spontaneous Contraction: Record the spontaneous activity on a slowly moving drum (Drum as slow as possible). Note the character and frequency of
the contractions. Often they slowly wax and wane and sometimes there are slow changes of the tone.
2. Effect of Temperature: Having obtained a tracing of the spontaneous activity
at 37°C. stop the drum and add cold water to the outer bath until the temp, of
the inner bath (muscle bath), is 25° to 30° C. Record the contractions at this temp, for a short period then stop the drum and wait for the temp, of the inner
bath to increase by 4o - 50°C. Take a further recording at this temperature
and repeat at intervals until the Tyrode solution is at 37° again. Note the
effects of temperature on the contractions and tone.
3. Effect of Drugs: Record a length of normal tracing. Note the level of fluid in
the organ bath and ensure that the fluid is kept at this level.
The sequence of drugs used and their dosages:
Adrenaline - 0.01-0.02 ug/ml - Note the effect, wash
Adrenaline - 0.01-0.02 ug/ml - Note the effect, wash
Acetylcholine - 0.5 - 5 ug/ml - Note the effect, wash Nicotine - 0.01 - 0.2 µg/ml - Note the effect, wash
Histamine - 50 - 400 µg/ml - Note the effect, wash
Hexamethonium - leave it for 3 to 5 minutes. Now without washing and nicotine and
note its effect. Retest also the response to acetylcholine, histamine, and adrenaline.
Wash two or three times.
Atropine - 0.1 ug/ml - add atropine, wait, and now add acetylcholine. Note the effect. Retest the response to histamine and barium chloride. Wash two or three
times.
Mepyramine- 0.01 - 0.0 ug/ml - add the drug, wait, and now add histamine and note its effect. Retest the response to Barium chloride and acetylcholine.
Discuss your results from your knowledge of the pharmacological actions of the
drugs used. If all the drugs are not available, you can study their effects from the accompanying schematic representations.
PLEASE NOTE
In all your experiments, a continuous tracing should be obtained showing the state of the intestine before the experiment, the effect of the stimulus, and
the effect of removing the stimulus. Points of application and removal of the
stimulus must be marked on the tracing at the time and not put on later.
Wash- means wash out the drug used and run fresh Tyrodes's solution into the
bath.
QUESTIONS:
1. Describe the movement observed in the isolated rabbit ileum under normal
conditions. What is the function of the movement?
________________________________________________________________________________
________________________________________________________________________________________________________________________________________________________________
________________________________________________________________________________
____________________________________________________________
2. What is the effect of different drugs used and mechanism of their action?
________________________________________________________________________________________________________________________________________________________________
________________________________________________________________________________
________________________________________________________________________________
____________________________________________________________
3. What is the main difference between Ringer, Ringer-locke's and Tyrode solution?
________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________
________________________________________________________________________________
____________________________________________________________
4. Describe the types of movements in the small intestine.
________________________________________________________________________________
________________________________________________________________________________
________________________________________________________________________________
____________________________________________________________________________________________________________________________________________
____________________________________________________________________________
____________________________________________________________________________
5. List the differences between intestinal smooth muscle and skeletal smooth
muscle.
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SECTION G
RENAL PHYSIOLOGY
EXPERIMENT 29
URINE FORMATION IN MAN
WATER BALANCE When water is drunk it is rapidly absorbed by the intestine. The resulting
dilution of blood is sensed by osmoreceptors and commands are sent to reduce the
output of A.D.H. from the posterior pituitary gland. The kidneys respond to the lower levels of hormones by increasing the rate of production of urine. This has low
specific gravity. If however, a saline solution (isotonic with blood) is drunk instead of water, dilution of blood, does not follow. In the following experiments, as far as
possible, each subject is approximately under the same conditions i.e. at the same time of the day and following a standard meal. Only one cup of water should be taken with meals.
Test subject No. A
Will act as ‘Control’ to establish the rate of urine secretion before the test. On
entering the laboratory he empties his bladder and this urine is discarded. Now he
drinks 1200ml of water. The bladder is emptied at thirty minutes interval. Volume, specific gravity, and chloride content of each sample of urine are measured and the results are expressed on a graph. How long does it take to excrete the quantity that
he drank i.e. restore water balance?
Test subject No. B
After emptying the bladder and discarding urine, drinks normal saline (0.9%
NaCl solution) at the same rate as Subject A. Bladder is emptied at thirty minutes. Volume, specific
gravity, and chloride content of each sample are measured and the results are
expressed on the same -graph as subject A. Comment on the response.
Effect of A.D. H. Injection (intramuscularly) Test subject No. C.
After emptying his bladder, drinks water as subject A. The first thirty minutes urinary sample is collected and its volume, specific gravity, and chloride content are
measured. A.D.H. (Dose 1 unit of pitressin/ kg of body weight) is now injected
subcutaneously and the volumes and S.G. values of thirty minutes samples of urine are recorded on the sample graph as subject A. Comment on the response. What is
water intoxication?
Effect of Previous dehydration: Test Subject D: The subject is deprived of drinking water with his meals for 24hours. After
emptying his bladder, drinks water as Subject A. Collect and analyze 30 minutes
samples of urine and graph your results.
Effect of smoking cigarettes; Test Subject No. E
The procedure is repeated as for subject A. Except that instead of A.D.H.
injection this subject smokes a cigarette. Comment or the response. What is the site of action of Nicotine?
Analytical Methods
Analysis of Urine for Chloride
NaCl Solution: 585mg standard sodium chloride is weighed out and dissolved in 1 liter of distilled water in a volumetric flask (10 mEq per liter).
ASSIGNMENT OF STUDENT ACTIVITIES IN EACH GROUP
Time subject
A Control
water
diuresis
B
Saline diuresis
C
Water and
pitressin
D
Effect of
previous dehydration
E
Effect of smoking a Cigarette
The day before the laboratory period, meals
as usual, fluid as specified
Water and
fluids as
usual
Water and fluids as
usual
Water and fluids as
usual
No water or drinks after
2.p.m lunch
As for Subject A
The night before the lab. 9.30p.m. each subject voids and discards urine
From 9.30p.m
night before 9.30a.m day of lab.
after 9.30 p.m. each student is to save all urine including final
voiding at 9.30 a.m on the day of the lab. The urine of each subject is pooled to provide a 12-hour sample. The volume,
specific gravity, and chloride content of each pooled samples
are measured as specified under procedures. Each subject
may have breakfast, but no fluids except as specified.
9.30a.m
Drink
1200 ml
of water
Drink 1200 ml
of 0.9% NaCl
solution
Drink 1200
ml of water and take pitressin
injection
Drink 1200 ml of water
From 9.30 a.m to 12.30p.m
collect urine at 30 minutes intervals, more often if necessary.
On each sample, record time, measure volume-specific gravity, and determine chloride.
12.30pm.
Complete all analysis
Before leaving
Lab
Enter results in appropriate spaces in large sheet(s) of paper
which will reflect individual and group results.
Mercuric Nitrate: l.7gm of crystalline mercuric nitrate are dissolved in a few hundred ml of distilled water with the addition of 20 ml 2N Nitric acid and made up
1 liter with distilled water. The solution is titrated against the above standard sodium chloride solution, using 4 drops of the freshly prepared indicator (diphenyl-Carbazone) such that 5 ml of the mercuric nitrate will neutralize 5 ml of sodium chloride.
Procedure:
Take (0.5 ml. – 2ml) of urine depending on the concentration of the urine into a
porcelain dish. Add 4 drops of the indicator solution titrate with mercuric nitrate until the clear solution turns violet-blue This (end-point) does not fade
Calculation
- Vol. of mercuric nitrate x 8.54meg x total volume of urine Volume taken
= CI. mEq/L for urine passed in each period.
Express your results in CI excretion/hour
RESULTS
Urine
formation
Subject A Subject B Subject C Subject D Subject E
Control
period
30’
60’
90’
120’
150’
Graph your results and prepare similar tables and graphs for specific gravity of urine and chloride excretion in all urine samples.
QUESTIONS 1. What % of the water of saline load was excreted by each subject during the
experimental period?
2. Comment on the observed differences 3. Do you have evidence to support the view that pressing is an antidiuretic
hormone?
4. What is its mode of action?
5. What are the effects of this hormone in subject C compared with the effects of previous dehydration in subject D on urine formation?
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EXPERIMENT 30
RENAL REGULATION OF ACID-BASE BALANCE
The kidneys play an important role in Homeostasis (now appropriately referred to as homoeokinesis) in the regulation of the constancy of the composition of the internal environment (E.C.F.). One of the major functions of the kidney is to regulate the hydrogen ion concentration of body fluids. It does this simply by secreting extra
H+ ions into the urine when these ions are in excess and by the conservation of
base.
This experiment is designed to illustrate the various mechanisms involved in
the renal secretion of hydrogen ions. The experiment starts at least 24 hours before the day of the practical classes. Thus all students involved must report to the
lecturer in charge of the FRIDAY before the Monday practical session. Each
volunteer will be provided with a 2'/2 liter Winchester bottle containing 3 ml. of
Toluene as a preservative. At exactly 7 a.m. on Sunday each student volunteer empties his or her bladder by voiding urine. This urine specimen is discarded. From
7 a.m. on Sunday from 7 a.m on Monday, all specimens of urine passed must be
voided into the large Winchechester bottle. It takes a lot of self-denial and sacrifice to collect 24-hour samples of urine. At 7 a.m. on Monday, the bladder is again
completely emptied but this time, the urine so voided is COLLECTED into the SAME
WINCHESTER bottle. This constitutes a 24-hour sample of urine. These specimens should be brought to the Monday practical classes.
There shall be different groups of volunteer students in each group taking test
substances. SUBJECT A
Will take 5 gm of ammonium chloride with a glass of orange squash at 6. p.m on the day before the laboratory. This should be followed by a Tight dinner and urine
collection should proceed as stated above. Ammonium chloride produces a mild acidosis since it is broken down into Ammonia and HCL The ammonia is converted
to urea by the liver whilst the acid is first buffered by the body fluids before its final excretion by the kidneys.
SUBJECT B:
Will take four tablets of acetazolamide (Diamox) (250 mg. each). One tablet at
bedtime on Saturday and one with every meal on Sunday. Acetazolamide is a carbonic anhydrase inhibitor and since this enzyme plays a major role in the
secretion of hydrogen ions by the renal tubules, renal excretion of hydrogen ions
falls and the urine is alkaline.
SUBJECT C:
Will act as control subjects, i.e. they will collect 24-hour urine specimens as
described above without the ingestion of any of the above drugs.
Experimental Procedure
(1) Measure the total 24-hour urine volume accurately (2) Determine the pH of each sample with a direct reading pH meter
(3) Determine the urinary ammonia as well as the titratable acid excretion and calculate the total concentrations of ammonia and titratable acidity in the 24-hour sample. Tabulate your results and compare the results obtained by the THREE subjects.
Reagents and Materials 1. KOH 40%
2. H2SO4, 0.01N
3. Petroleum jelly 4. Conway dishes with covers
5. 1 % Boric Acid solution in alcohol and water with a combined indicator
(Bromeresol green and methyl red).
6. 0.04% phenol red 7. O.lNNaOH
8. P04 Buffer 0.1M, pH 7.4
9. Methyl red 0.1%
Analyses
1. Ammonia determine: (This will be demonstrated for the group by an instructor)
2. (a) Add 1 ml of boric acid buffer solution to the center well.
(b) Place 0.2 ml of urine on the right side of the outer ring; attempt to prevent it from spreading. Place a greased coverslip on the dish leaving the portion of the left side outer ring uncovered, providing an opening
for adding the KOH (c) Place 1 ml. of 40% KOH solution the opening into the left side of the
outer ring; seal the dish quickly by sliding the cover over without moving the porcelain dish itself.
(d) When all urine samples have been treated in this way, carefully tilt and rotate the dish so that the urine mixes with the carbonate solution in the outer ring. Let stand for 90 minutes.
(e) Using a tuberculin syringe and No. 20 needle, titrate the boric acid
buffer in the center with 0.01 N H2SO„ to the colour of a water blank
which was treated in the same way as the urine samples.
2. Titratable acid:
Preparation of the standard color for end-point comparison; Take.20 ml phosphate buffer (0.1M, pH 7.4). To it add enough methyl red to color
the solution the same place yellow tint as the diluted urine sample. Add 1 ml of
phenol red (0.04%). This provides the colored solution which you are to match in
determining the acidity of the urine.
Titration of urine:
To 20ml. urine add 1 ml phenol red (0.045). Titrate this with 0. IN NaOH until the color matches that of the standard.
Express the results in terms of milliequivalents of ammonia and titratable acidity
excreted per hour, and present them in the form of bar diagrams or a line graph
against time.
Calculations
1. Titratable Acidity (meq) = Volume of NaOH x 0.1 x volume of urine passed (meq)
Vol. used in titration 2. Ammonia = volume of H2S04 x 0.01 x vol. of urine (meq.)
0.2 ml.
QUESTIONS
1. With ammonium chloride ingestion, compare urine volume, ammonia a
Titratable acidity with the control subjects. The amount for the changes in ammonia and Titratable acidity.
2. In the subjects who took acetazolamide, compare the excretion of ammonia
and titrable acidity with (1) above, and explain the differences.
3. What is the mechanism of acetazolamide action? What was the effect of this drug issubjects B and C on urinary pH, ammonia, and Titratable Acidity?
4. How are the rate of NH3 and titratable acid excretion related in the control
and experimental urine samples?
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EXPERIMENT 31
HUMAN DIURESIS
The main variations in the volume of water excreted are due to variations in the facultative reabsorption of water in the distal convoluted tubule and the collecting duct of the kidney. This facultative re-absorption is controlled by antidiuretic hormone (ADH) secreted from the posterior pituitary gland. The intake of
a large volume of water reduces the facultative re-absorption resulting in a marked
increase in urine flow and the production of a very dilute urine i.e. 'water diuresis'. This diuresis continues until most of the water load has been excreted.
In this experiment, you will follow the cause of a water diuresis by measuring
(1) the volume of urine produced in half-hour periods, (2) the specific gravity of the urine which gives a reasonably good measure of the total solute concentration
provided that no protein is present
Preparation for the Experiment As many students as possible should act as subjects. Some will act as controls
taking 1 liter of water others will take water with a diuretic of water in the injection
of ADH or saline. You should start the experiment in a moderately dehydrated state. For a
morning class restrict your intake from making to 2 tea-cupfuls (=2/3 pint = 379ml)
and avoid strong tea or coffee as these act as diuretics. For an afternoon class takes no fluid after 11 am. Except sips of water if necessary to allay thirst.
ONE TO TWO HOURS BEFORE COMING TO THE CLASS EMPTY YOUR BLADDER
DISCARDING THE URINE BUT NOTING THE TIME EXACTLY. This is the start of the collection period for measuring the basal urine flow.
Procedure At the beginning of the class empty the bladder again, this time collecting the
urine and again noting the time. Measure the volume of the urine and calculate the
rate of urine production in ml/min. This is the basal urine flow rate. Save the sample (accurately labeled) for specific gravity measurement and chloride estimation.
The demonstrator in charge of the class will divide you into groups. The following experiments will be performed.
Water Diuresis: Drink 1 liter of water (or as much as you can manage) and
measure the urinary column and specific gravity at half-hourly intervals. This
constitutes the "dilution test" for assessing the combined function of the two kidneys in removing excess water from the blood.
Construct histogram type diagrams to show how each of the following varies with time (1) Urine flow rate (2) Specific gravity.
Also, calculate the percentage of the water load excreted in the period of observation.
This is done by calculating the total volume of urine which would have been excreted in the urine flow had remained at the basal rate for the whole period following the
drinking of water and subtracting this from the total volume of urine excreted in the
same period.
Anti-diuretic Hormone: The subject should drink 1 liter of water and immediately afterward should receive a subcutaneous injection of vasopressin. The experiments
should be continued as for the water diuresis. N.B. Subjects receiving ADH should not drink any more fluid until they have had a diuresis otherwise they may suffer from the effects of overhydration.
Effect of Saline of varying Tonicity: This group is divided into three sub-groups.
One third takes 0.51 water; one third 0.51 of hypertonic saline (1.5% and one-third of 0.51 of normal (0.9%) saline. Empty the bladder every 30 minutes and after this
explain the differences obtained in the volume SG curves and chloride concentration
and excretion rates.
Urea Concentration Test: Ingest 100ml of 15% urea. Empty the bladder hourly and
read the volume and SG or if possible, analyze the urine samples to determine the
urea concentration. Graph volume and SG against time and notice that the latter increases to a peak at
2 - 3 hours (a SpG of 1,030 corresponds in normal individuals to a urea
concentration of over 2%). There is a limit to which the tubules can prevent back diffusion into the bloodstream and this is a urinary urea concentration of about 4%.
The power of the kidneys to concentrate urine is an important indication of their
functional ability. Effect of Various Substances: Measure volume, specific gravity as described
previously. Collate all the results obtained for the various conditions used. QUESTIONS
1. What % of the water or saline load was excreted by each subject during the experimental period? Comment on the observed differences.
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2. Do you have evidence to support the view that vasopressin is an antidiuretic hormone? What is its mode of action
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3. Discuss the mechanism involved in the control of water excretion and the mode
of action of the various substances used in the experiment.
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EXPERIMENT 32
RENAL REGULATION OF ECF
Title: Regulation of ECF Aim: To study the role of the kidney in control of ECF by changes in urine output
(volume) and specific gravity
Introduction/principle: Water is absorbed into the body from the food and drinks taken by humans.
The absorption takes place in both the small and large intestines into the
circulation. The osmolality of circulating blood is kept within the physiological range with the help of hypothalamic osmoreceptors. The osmolality of body fluids is a reflection of the concentration of dissolved osmotically active substances, chiefly
sodium ions (Na+). An increase or decrease in osmolality is resisted by physiological
mechanisms involving the release of hormones such as vasopressin from the posterior pituitary gland and aldosterone from the adrenal cortex respectively. The release of vasopressin is affected by certain exogenous substances such as cigarettes
and ethanol in alcoholic drinks.
The control of water balance and thus the ECF volume is part of homeostasis. The intracellular and transcellular fluid volumes are in a state of dynamic
equilibrium.
Introduction of hypertonic solution tends to increase the osmolality of blood thus triggering homeostasis mechanism to restore normality of blood by modification
of urine volume and specific gravity and conversely the same holds for the
administration of the hypotonic solution. 0.9% NaCl is considered isotonic to body
fluid and the effect of its administration can be predicted and actually determined in this experiment.
Material and method: measuring cylinder, urine trough, urinometer
First subject:
He/she is the experimental control and serves as a baseline on which to
determine changes. Ask him/her to completely void urine to obtain an empty bladder. This emptying of the bladder would be subsequently referred to as presetting of the bladder. The volume of hourly urine voided is collected and the
volume/specific gravity is measured.
Second subject:
The subject bladder condition is preset as in the first and he/she drinks water
at the rate of 12ml/kg body weight, urine is voided after every 20 minutes. The volume and specific gravity are then measured and the values represented on a linear graph. Using the graph and extrapolation, estimate the time taken to excrete
the volume of water the subject consumed early in the experiment.
Third subject: Repeat procedures as above using 0.9% (Normal saline) instead of water.
Fourth subject: The subject bladder condition is preset as done previously. The urine samples
are collected every 20 minutes and the volume and specific measured. 1µg/ml/kg
body weight of vasopressin is administered parentally by the intramuscular route. 20 minutes of urine is collected for volume and specific gravity measurement. These values measured are then plotted on the graph for 2nd subject and the
purpose of comparison. Describe and compare the lines on the graph. Discuss briefly the physiological adjustments to fluid overload/water intoxication.
Changes in urine volume and specific gravity after smoking cigarette
Fifth subject:
The bladder of the subject is preset and fluid is taken at a rate of 12ml/kg body weight. The subject then smokes cigarettes and urine is collected subsequently
after every 20 minutes for volume and specific gravity measurement.
Urine concentration and dilution test:
As fluid crosses the various segments of the nephrons, selective re-absorption takes place and water re-absorption can be modulated at the distal convoluted tubule and collecting duct. Damage to renal tubule results in failure of re-absorption
of water and thus loss of power of urine concentration by the kidneys. Non
functional tubules in a diseased kidney are capable of decreasing the specific gravity (1.001 – 1.003) below that of glomerular filtrate. This implies the failure of Na+ and Cl-.
Dilution test: For this next line of tests, 3 subjects are required and are labeled A – C.
Subject A: the condition of the bladder is preset by urine voiding on commencement
of the experiment. Water is taken at 15ml/kg body and half hourly (30 minutes), urine is collected for volume and specific gravity measurements. 5 specimens could
be collected in 2hours 30minutes.
Analysis of result:
In interpreting the findings, note the relationship between volume and specific
gravity. Note also the range of specific gravity 1.002 – 1.010 at the beginning and the end of the experiment.
Concentration test:
Subject B: the bladder is preset by emptying and vasopressin is administered at a
dose of 1µg/kg body weight intramuscularly. Hourly, urine samples are collected for
measurement of volume and specific gravity.
Analysis of result:
A specific gravity below 1.020 may be suggestive of renal insufficiency and at least
one of the urine specimen collected may have a specific gravity above 1.030. Use this
information to comment on your results.
Urea concentration test:
Subject C By slow intravenous infusion, administer 100ml of 15% urea after presetting
the bladder. Collect hourly urine and determine the volume and specific gravity. Plot
the values obtained on a different graph sheet. It should be noted that the specific
gravity of the urine peak at about 3 hours (upto 1.030 is considered normal for a healthy person taking 2% urea) of the onset of this experiment. When urine flow is low, more urea leaves the renal tubule and only 10 – 20% of filtered urine is
excreted. At higher urine flow, 50 – 60% urea is excreted.
Note: in measurement of specific gravity, hydrometer are checked by floating them in distilled water (specific gravity = 1). Deviation from zero is converted by addition
or subtraction.
Specific gravity = 1 + reading on hydrometer
100
Remember to adjust the volume of urine to an adequate level and use the
dilution factor to multiply the final apparent reading to obtain the real specific gravity (perspiration with loss of water). Make all readings specifying the current
room temperature.
Tabulated experimental results
30 min. 60 mins. 90 mins. 120 mins.
Subject All subjects to empty
bladder before the
experiment
Vol. (ml)
Sp.Gr. Vol. (ml)
Sp.Gr. Vol. (ml)
Sp.Gr. Vol. (ml)
Sp.Gr.
A Nothing
was taken (control)
B Water took at
12ml/kg body
weight
C Normal
saline taken at
12ml/kg body weight
D Water
taken at 12ml/kg body
weight + 2 sticks of
cigarette
1. Plot the results on one graph paper for the subjects A, B, C and D as Sp. Gr.
Vs Time and another as urine volume vs. Time
2. Comment on your graph results and draw conclusion
SECTION H
ENDOCRINE PHYSIOLOGY
EXPERIMENT 33
THE THORN TEST (PRINCIPLE)
Glucocorticoid hormones (cortisol) a hormone of the adrenal cortex induces a
decrease in lymphocytes and eosinophil granulocytes counts in the peripheral blood. In this experiment,Adrenocorticotrophic hormone (ACTH) is used to mobilize cortisol
and this experiment is used to demonstrates decreasing effects cortisol has on granulocytes counts. The control is venous blood from a rabbit ear. The blood is sucked into a melangeur-pipette (with the white pearl), and diluted 10-fold with Türk
solution and the total leukocyte count is calculated (in the practice general leukocyte count is defined). This is followed by subcutaneous administration of 40 units of ACTH, and the blood count is repeated hourly under the intervals of 3 hours. Note
that in this experiment, the total leukocyte count is calculated.
The decrease is due to the decrease of the eosinophils and the lymphocytes, however, in the original Thorn test, the number of eosinophils has to be counted.
Affected by ACTH, the leukocyte number (eosinophil granulocyte, lymphocyte) will decrease to half. Later it will increase again but usually, it does not return to the
initial value even by the
EXERCISE Calculate the total number of lymphocytes, granulocytes, and eosinophils before and
after the administration of ACTH
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EXPERIMENT 34
ESTIMATION OF GLUCOSE LEVEL IN HUMAN BLOOD
A glucose tolerance test is a medical test in which glucose is given and blood samples were taken afterward to determine how quickly it is cleared from the blood. The test is usually used to test for diabetes, insulin resistance, and sometimes reactive hypoglycemia and acromegaly, or rarer disorders of carbohydrate
metabolism. A variant is often used in pregnancy to screen for gestational diabetes. In the most commonly performed version of the test, an oral glucose tolerance test (OGTT), a standard dose of glucose is ingested by mouth, and blood levels are
checked two hours later. Procedure: 1. A zero time (baseline) blood sample is drawn. 2. The patient is then given a measured dose of glucose solution (75 g glucose in 0,5 L water) to drink within a 5-minute time frame. 3. Blood is drawn at 60 minutes and
120-minute intervals for measurement of glucose (blood sugar), and sometimes
insulin levels. A 2-hour OGTT glucose level below 7.8 mmol/L (140 mg/dL) is normal, whereas higher glucose levels indicate hyperglycemia. Blood plasma glucose between 7.8 mmol/L (140 mg/dL) and 11.1 mmol/L (200 mg/dL) indicate "impaired
glucose tolerance", and levels above 11.1 mmol/L (200 mg/dL) at 2 hours confirms a
diagnosis of diabetes.
EXERCISE
SECTION I
REPRODUCTION
EXPERIMENT 35
A STUDY OF ESTROUS CYCLE IN RAT BY VAGINAL
SMEAR TECHNIQUE
INTRODUCTION
Mammals other than primates do not menstruate and their sexual cycle is
called an estrous cycle'. It is named for the conspicuous period of "heat" estrous at the time of ovulation, normally the only time during which the sexual interest of the female is aroused. In spontaneously ovulating species such as the rat, the
underlying endocrine events are essentially the same as those in the menstrual cycle, although the days of the cycle are numbered from the day of estrus.
Although the main physiological events of the estrous cycle occur in the
ovaries, these events are reflected in the changes that take place in the vagina under
the influence of the ovarian hormones estrogen and progesterone.
Estrous Cycle - The cycle makes its appearance at puberty at the age of 2-3 months
and the whole cycle lasts for about 5 days is divided into (i) Diestrous which lasts for 2½ days and the vaginal smear consists preponderantly of leucocytes with an
occasional presence of cornified epithelial cells (ii) Proestrous – during which there is
a nearly complete disappearance of leucocytes and their replacement by large
numbers of round nucleated epithelial cells about 3 times as large as the leucocytes; at this time and 12 hours afterward the animals will breed but at no other time, this
is an estrous phase (iii), (iv), Metestrous – In this phase desquamation of the
epithelial cells occurs. White cheesy masses of disintegrating squamous cells are found in the smear, leucocytes now again make their appearance and the cycle is
repeated.
Materials: (1) Adult Female rats (2) Pasteur pipettes
(3) Cotton
(4) Normal saline (5) Slides (6) Microscope
Method of Taking Vaginal Smear: Pasteur pipettes are filled with a few drops of saline, gently inserted in the
vagina, and used to flush around.
Cotton swabs made with toothpicks or thin sticks moistened with saline are gently inserted and slightly rotated within the vagina. The swab is then pressed in a drop of saline on the slide and examined under a microscope. The saline is then
drained back into the pipette and few drops are pressed.
Observations: Identify the different phases of the Estrous cycle and draw diagrams
to illustrate vaginal cytology in the different phases.
QUESTIONS 1. What is the significance of taking vaginal smears in humans and their clinical
relevance? ___________________________________________________________________________ ___________________________________________________________________________ ___________________________________________________________________________
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2. What are different phases of estrous cycle?
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3. What do you mean by reflex ovulation?
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4. Draw a diagram showing timing of estrous cycle and menstrual cycle. ___________________________________________________________________________
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5. What is the significance of detecting ovulation time
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6. What is safe period?
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EXPERIMENT 36
SEMEN ANALYSIS
A. SPERM MOTILITY
INTRODUCTION
An Examination of Human Semen.
Volume: 2 – 5ml / ejaculate Colour: Whitsh / light gray Liquefaction – 20min pH – 7.2
There are several types of sperm motility but two types are common.
(1) Directional progressive motility in which the sperm moves forward in one
direction.
(2) Vibratory motility, in which the sperm moves its tail, but does not progress in a forward direction
Various factors influence sperm motility e.g. biochemical and biophysical states of the epididymal fluid, seminal plasma, cervical mucus, endometrial fluid, oviductal fluid, and peritoneal fluid.
In this experiment, you will be doing a microscopic examination of the sperm cells and you will also get some rough idea about their motility too.
METHOD:
1. Euthanize a mature rat and remove the epididymis from one side.
2. Place a few drops of physiological saline (0.85% NaCl) on a slide. 3. Cut through the epididymis and, while holding a piece of the tissue with
forceps, place the cut edge of the tissue on a slide and move the tissue back
and forth a few times through the saline to transfer sperm to the slide.
4. Discard the tissue, place a coverslip on the slide and examine the slide under a microscope. Spermatozoa are examined for motility under high power.
5. Both motile and immotile cells are calculated from the mean value. The value
is then adjusted to the closest five. 6. Mean quality of sperm motility (progressive motility) can be graded from 1 to 4
(0 = no motility, 4 = excellent motility).
B. COUNTING OF SPERMATOZOA (1) Diluting Fluid: 1% Phenol:
4% NaHC03 - or NaHC03 – 5g Formalin (35%) - 1ml.
Distilled water to make up to 100 ml. (2) Dilution: 1:20; 1:50; 1:100; 1:200
(3) Counting: is done in a hemocytometer. Preferably under a high power objective lens.
The difference between the two determinations should not exceed 10% at low or 20% at high density.
MORPHOLOGY OF SPERMATOZOA
(i) The technique of making a seminal smear is principally similar to that for
making blood smears.
(ii) A smear is fixed by air drying
(iii) Giemsa stain or Leishman’s stain is used (iv) The smear is examined under oil immersion lens
QUESTIONS: 1. What are morphological characteristics of the sperm? ___________________________________________________________________________ ___________________________________________________________________________
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2. What factors influence their motility? ___________________________________________________________________________
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3. Mention a few morphological abnormalities of sperm and their effect on fertility?
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4.What is normal sperm count per ejaculate? ___________________________________________________________________________
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5.What are the functions of the epididymis? ___________________________________________________________________________ ___________________________________________________________________________
___________________________________________________________________________ __________________________________________________________________________
6. What is difference between spermatogenesis and spermiogenesis?
___________________________________________________________________________ ___________________________________________________________________________ ___________________________________________________________________________
__________________________________________________________________________
7.How does variation in temperature affect spermatogenesis? ___________________________________________________________________________ ___________________________________________________________________________
___________________________________________________________________________ ____________________________________________________________________________
8.What happen when the vasa differentia are tied?
___________________________________________________________________________ ___________________________________________________________________________
___________________________________________________________________________
SECTION J
NEUROPHYSIOLOGY II
EXPERIMENT 37 CUTANEOUS SENSATION
The experiments described in these notes are designed to illustrate some of the main features and especially the limitations of your cutaneous touch pressure,
thermal, and pain systems. Some of these experiments are used as clinical tests, so this class also serves as an introduction to the sensory testing which forms part of a
neurological examination of a patient.
PRECAUTIONS
1. When testing any subject, it is very important not to suggest the response to the subject either verbally or by letting him/her see the test stimulus. Even the best-intentioned subject will consciously or unconsciously use clues or
information gained from seeing the stimulus. Therefore, always blindfold your
subject. 2. Test at least five spots in each small square of the grid.
3. Because these tests require the co-operation of the subject, attention will
wander. Check attention regularly, for example with a dummy or negative test. 4. Always choose areas that are free of hair or shave the area concern.
A. TOUCH/PRESSURE SYSTEM
DENSITY OF ‘SENSITIVE SPOTS’
The organization of cutaneous sensibility has been described as non-uniform, that is, arranged in sensitive areas or spots. You should test this point and describe
the density of spots for the following different regions.
1. Apply the small rubber stamp grid to the palm at the base of the thumb, forearm, and the back of the neck. Choose areas that are free from hairs, or shave the area concerned.
2. Blindfold the subject.
3. Starting with the grid on the neck, test for touch-sensitive spots within each small square. The grid is only for spatial location, not to indicate spot size. Superficial touch/pressure is tested using Von Frey hairs. These provide a
reasonably standardized pressure stimulus because as increasing pressure is applied to them, they bend. Mostly the finest will be used. The ‘hairs’ used in the class consist of nylon fibers mounted in hypodermic needles.
4. Record (using some convenient code) the positions of touch-sensitive and touch-
insensitive spots within each stamped small square. Also, in any area where you find touch spots to be sparse, mark their positions accurately.
II. TWO-POINT DISCRIMINATION
The subject’s ability to distinguish two separate stimuli is an important
property of a sensory system. This
is usually tested by determining the minimum separation of two points which can be distinguished as being two rather than one.
1. Use the hand forearm and the back of the neck as in experiment I
2. Blindfold the subject 3. Set the dividers (aesthesiometer) to some separation and then briefly touch the
skin lightly with both points simultaneously. The subject should report “one”, “two” or “not sure”
4. Retest after varying the separation of the points (occasionally check by
stimulating with one point) 5. For each site, record the minimum separation distinguished with certainty
III LOCALISATION The ability to localize a cutaneous sensation means that the subject can
indicate accurately the size of stimulation on the skin.
1. Blindfold the subject
2. Select and mark a spot for stimulation in each of three different regions, e.g. palm, forearm, and back of the neck.
3. The experimenter should briefly and lightly touch the subject skin with a ball-
point pen or felt pen at one of these spots. 4. The subject should then try to place a second pen tip of a different color on the
stimulated spot. To reduce errors from motor performance, the subject should
use his writing hand to do the locating and is permitted to “search” until satisfied with his localization.
5. Continue testing by stimulating the same spot at least 5 times. (You may want
to randomize the presentations by stimulating the different spots in rotation). 6. Examine the resulting distributions of responses and note any difference
between the different regions.
ADAPTATION
One important property of neurons is adaptation. During a prolonged stimulus, the response declines after the initial transient at the onset of the
stimulus. Adaptation can be illustrated in intact nervous systems as well as in single neurons.
1. Blindfold the subject. Each student should act as a subject for this
experiment.
2. Place a cork or paper on the subject’s forearm. The subject should concentrate
on the sensations evoked during the next 15 seconds and report on the following:
(a) The sensation changes after placement of the cork?
(b) Did the sensation persist throughout the 15 seconds? 3. After allowing the cork to rest on the subject’s forearm for 15 seconds, quickly
but carefully lift the cork off the skin. What does the subject experience?
4. Repeat steps 2 and 3 using the heavier weight.
B. COLD AND WARM SYSTEMS
1. COLD SPOTS
1. Blindfold the subject 2. For the grids on the hand, forearm, and back of the neck map out spots as
for touch, using the rod packed in ice. Dry the rod carefully before use and
use it once before recooling it in ice.
NOTE: The subject should report when the stimulus elicits a sensation of coldness, NOT when the stimulus can be detected by its pressure. Insert appropriate symbols of the responses directly into the small squares. II. WARM SPOTS 1. Blindfold the subject 2. Map out warm spots using the warmed rod, in the same areas tested for cold.
III ANALYSIS OF THERMAL SENSATION (I) Thermal sensations are often called temperature sensations implying that they
indicate the temperatures of objects, but do they?
1. Prepare three beakers of water at temperatures of about 40°C, 30°C, and 20°C. 2. Place the forefinger in the 40°C water and describe your thermal sensation. Then
place the other forefinger in the 20°C water and describe that sensation.
3. After 2 minutes, compare the current sensations with the initial ones. Has
adaptation to the thermal stimuli occurred? 4. Now transfer the finger in 20°C water to the 30°C water and describe your
sensation. Then transfer the finger in 40°C water to the 30°C water and describe
that sensation. Are these two the same?
IV ANALYSIS OF THERMAL SENSATIONS (II)
From the results of steps 3 and 4 above it might be thought that one’s thermal sense indicates relative temperature, i.e. the difference between the temperatures of
one’s skin. The following experiment is a test of this idea. 1. Compare the thermal sensations produced by a brass rod and a wooden pencil,
both at room temperature, placed against the cheeks.
2. Report the thermal sensation evoked.
C. PAIN SYSTEM PAIN SPOTS
This experiment is concerned with fast, first, or pricking pain. Very little pressure is required to elicit this pain from uncallused skin.
1. Blindfold the subject
2. Using a lancet to apply brief, light stimuli, map out pain-sensitive areas within
the grids used for mapping tough spots.
3. Check for adaptation by continuous application of painful stimulus for 15 sec. and 2 min.
NOTE: The subject should report when the stimulus elicits a sensation of pain. NOT WHEN THE stimulus can be detected by its pressure.
OBSERVATIONS
CODE
Touch
Cold 0
Warm
Pain A ∆
In case of insensitive point put (-) on top of the relevant code.
QUESTIONS 1. In which region is the sensitivity of spots greatest for touch, pain, and temperature? What determine this?
________________________________________________________________________________________________________________________________________________________________
_________________________________________________________________
2. Does two-point discrimination vary with the region? If so how? ________________________________________________________________________________
________________________________________________________________________________
_________________________________________________________________
3. What is the relationship between two point discrimination and density of sensitive
spots?
________________________________________________________________________________________________________________________________________________________________
_________________________________________________________________
4. Does error of localization vary with the region? If so how?
________________________________________________________________________________
_________________________________________________________________________________________________________________________________________________
5. Is the ability to localize related to the density of spots in that region? If so how?
_________________________________________________________________________________________________________________________________________________________________________________________________________________________________
6. According to the results of experiments give the sequence of adaptation of different sensations.
________________________________________________________________________________
_________________________________________________________________________________________________________________________________________________
7. Does the thermal sensation indicate absolute temperature or relative
temperature? ________________________________________________________________________________
________________________________________________________________________________
_________________________________________________________________
8. Is there any other aspect of thermal stimulus involved in thermal sensation? ________________________________________________________________________________________________________________________________________________________________
_________________________________________________________________
9. Are there pain spots and analgesic areas of skin tested? Do pain, cold, warm and touch spots
coincide?
________________________________________________________________________________________________________________________________________________________________
_________________________________________________________________
EXPERIMENT 38
CLINICAL REFLEXES
INTRODUCTION
A reflex is a stereotyped effector response to a stimulus. The reflexes
commonly tested in clinical practice are:
A. Stretch reflexes (= tendon jerks myotatic reflexes) B. Flexor reflexes (= withdrawal reflexes)
C. Visual reflexes, particularly pupillary reflexes D. Vestibular reflexes.
Reflexes A and B are examined in this class; reflexes C and D are examined in other
classes.
A. Stretch Reflexes
The adequate stimulus for the reflex is stretch of a muscle. The response is the contraction of the same muscle. Stretch reflexes are examples of mono-synaptic
reflexes.
Pathways
Receptors are muscle spindles (intrafusal fibers). Afferent fibers are large,
myelinated group Ia from primary (annual spiral) endings. The synapse is in the
spinal cord (or brainstem) at different levels between the afferent and the motor neuron. Efferent fibers innervate extrafusal fibers of the same muscle. Any damage
to the afferent or efferent nerves or the synapse will result in a diminution or
absence of the reflex.
Influences on the reflex pathways
(a) On the spindle: The efferents influence the tension (and sensitivity) of the
intrafusal fibers and thus indirectly the extrafusal fibers. (b) On the motor neuron: Pyramidal and extrapyramidal pathways influence
the excitability of the motor neuron, and thus extrafusal fibers.
B. Flexor Reflexes
The adequate stimulus for the reflex is nociceptive, although weak responses can occur to mechanical stimulus. The response is the contraction of a group of
flexor muscles. Flexor reflexes are multisynaptic reflexes. Pathways
Receptors are nociceptors in skin and subcutaneous tissue. Afferents are small myelinated (and perhaps unmyelinated) fibers. Synapses (at least three) occur
in the spinal cord (or brainstem), involving interneurons and motor neurons of several different muscles. Efferent fibers innervate groups of flexor muscles.
A. Stretch Reflexes
1. Ankle Jerk Gastrocnemius muscle; spinal cord levels Ankle at 900 or less;
preferably ask the subject to kneel with feet dangling. Tap Achilles
(gastrocnemius) tendon behind the ankle joint. The response is plantar flexion of the foot.
2. Knee Jerk Quadriceps femoris muscle; level L
The subject sits with legs dangling clear of the floor. Tap on patellar ligament just below the knee joint. The response is an extension of the knee joint. Observe the effects on the reflex if you ask the subject to:
(a) Voluntarily suppress it,
(b) Try to separate his hands clasped together in a “monkey grip”
3. Triceps Jerk Level C
Elbow flexed (about 90°), forearm supported. Tap triceps tendon just above the elbow joint (posteriorly). Because this is a relatively broader tendon, the response is
often slight. The response is a twitch of the triceps or extension of the elbow joint.
4. Biceps Jerk Level C Elbow flexed about 90°, hand supinated (palm up), forearm supported. Place
your thumb on the tendon in the elbow and tap your thumb. The response is a
twitch of the biceps or flexion of the elbow joint (lifting of the forearm).
5. Brachioradialis Jerk (“supinator” Jerk). Level C
Elbow flexed to about 90°, forearm semi-supinated (thumb side upwards). Tap tendon about 4 cm. above the wrist joint. The response is a twitch of brachioradialis or flexion of the elbow.
6. Jaw Jerk Masseter muscle; mesencephalic and motor nucleus of Trigeminal (V) nerve. Level of midbrain and pons.
The subject sits with jaw relaxed, mouth slightly open. Place your thumb on the point of the chin and tap your thumb. The response is a brief twitch of the
masseter muscles or a brief upward movement of the lower jaw. It is not always easy to elicit.
B. Flexor Reflexes 1. Planter Reflex Levels
Subject kneels on the bench, feet dangling. Using a key, or the handle of the
hammer, firmly scratch the outer border of the foot starting at the ankle and
running across the ball of the foot. The response is normally plantar flexion of the big toe; movement may be slight. In the presence of a pyramidal tract lesion, the toe
extends and sometimes the other toes also extend and fan out.
Observations Abnormal Reflex
Normal
Root value
1. Ankle jerk Right Left 2. Knee Jerk Right Right 3. Triceps Jerk Right Left 4. Biceps Jek Right Left
5. Supinator Jerk Right Left
6. Jaw Jerk Right Left 7. Planter reflex Right Left
8.Abdominal
reflexes: (a) Upper right quadrant
(b) Lower right quadrant
(c) Upper left
quadrant (d) Lower left
quadrant
Conclusion and comments
2. Abdominal Reflexes Level T 5-12 If subject volunteers to bare his abdomen, he should lie on the bench and relax. Firmly draw a key or hammer handle across the upper right quadrant of the abdomen. Also, test at the level of the umbilicus on the right and in the lower
right quadrant. Repeat on the left side. The response is a contraction of the abdominal muscles underlying the skin which was stimulated.
QUESTIONS
1. Describe the reflex pathways for knee jerk.
________________________________________________________________________________________________________________________________________________________________
____________________________________________________________________________________________________________________________________________ ___________________________________________________________________________
2. Describe the reflex pathways for planter reflex
________________________________________________________________________________________________________________________________________________________________
_________________________________________________________________
___________________________________________________________________________ ________________________________________________________________________________
________________________________________________________________________________
_________________________________________________________________
________________________________________________________________________________________________________________________________________________________________
_________________________________________________________________
3. One knee jerk was found to be absent in a patient while other reflexes were normal. (a) What is the possible site of lesions?
(b) What other tests would be perform to confirm your suggestions? _________________________________________________________________________________________________________________________________________________________________________________________________________________________________
________________________________________________________________________________
_________________________________________________________________________________________________________________________________________________
________________________________________________________________________________
_________________________________________________________________________________________________________________________________________________
EXPERIMENT 39
VISION 1
(Visual acuity, blind spot, inversion of the retinal image, the near point,
focusing, Schemer’s experiment, Sanson’s experiment, Sanson’s image, the retinal vessels, and stereoscopic vision)
METHOD (a) Visual acuity
Distance vision is tested by the use of Snellen’s types, cards on which graded
rows of letters are printed, the larger letters at the top. The rows of letters are marked with figures, indicating the distance in meters at which the thickness of the line of the individual letters subtends an angle of one minute and the height of the
letters five minutes at the eye. At that distance, the normal eye will comfortably
distinguish the letters of that particular row. The test is carried out at a distance of six meters, at which a normal eye will read the row of the letters marked with figure 6.
Place the card in good daylight and stand or sit directly in front of it at six
meters distance. Use one eye at a time, keeping the other covered with an opaque disc in a spectacle frame or with an eyeshade. Visual acuity is expressed by a
fraction, of which the numerator represents the test distance in meters and the
denominator the smallest row of letters which can be read at that distance. Thus normal vision is 6/6; vision in which only the top letter fractionis the reciprocal of
the visual angle (in minutes).
If the subject is hypermetropic he will be able to read the same line with and without
a positive (convex) lens in front of his eye; find the largest positive lens with which this can be
done. If; on the other hand, he is myopic, a negative (concave) lens will markedly
improve his acuity. (b) Blindspot
1. Make two black circles about 1/8 in. diameter about 4 ins apart. Hold the paper up in front of the right eye at ann’s length; close the left eye. Fix the right eye on the left-hand mark. Bring the paper slowly towards the face. The
right-hand mark will disappear and then reappear as the paper is brought nearer.
2. Rest the subject’s chin on support (e.g a book) about 10 ins, above the bench. He gazes steadily (left eye closed) at a small cross in the center of a piece of
white paper below his right eye. The observer prepares a long strip of white
paper with a large (1/8 in) black dot at one end, holding the other and makes the black dot travel over the paper from the right towards the cross. When the
dot becomes invisible to the subject and when it reappears, the observer
makes a mark through the dot on to the paper below. This procedure is repeated for vertical and oblique movements of the dot across the blind area.
Finally, the points are joined up to outline the projected image of the blind
spot. The largest diameter is measured and the actual size of the blind spot is
calculated by the method of similar triangles, given that the distance between the nodal point of the eye and the retina is 15 mm. Calculate also the distance
of the blind spot from the visual axis -i.e. the fovea centralis. Approximately
how many times the diameter of the blind spot is this?
(c) Inversion of the retinal image Prick a hole in a piece of black paper, hold it in the left hand about 3 ins, from
one eye, close the other eye. Look through the hole in the sky. Hold a pen in the right hand so that its head is close to the eye between the paper and the eye. It appears to be upside down. The light coming through the hole in the paper casts a direct shadow of the pin’s head on the retina and this shadow is the same way up as
the pin itself.
(d) Near point
Hold an open book in front of the eye and bring it nearer until the point can
no longer be seen clearly. Measure the distance to the eye. Do this again without spectacles if these are worn. Repeat this experiment at home on people of various
ages. What influence does age have on the near point?
(e) Accommodation Process Accommodation takes place almost instantaneously, enabling man to shift his
gaze from near to far objects without a focussing problem. It must be remembered
however, that man can only focus on one thing at a time. This can be demonstrated with a simple experiment. If an open book is placed about two feet from the eyes and
the viewer look at it through a screen of mosquito netting held some six inches from
the eyes, he can see either the mesh of the screen or the letters in the book with clarity, but not both at the same time. When the mesh is in sharp focus, the letters are blurred, and vice versa. In this particular example - looking at two objects that
both happen to be very close to the eye - focusing requires a conscious effort. However, in looking around us at things across the room or the street, the lens
makes the fine adjustment from near to middle to far distances with such ease and
rapidity that man is unaware that his mind is directing a change of focus. Not even the most skilled photographer with the finest equipment can shift his camera’s focus
with anything like the speed of the eye.
(f) Focusing
Hold a pencil between one eye and the corner of the room. Keep the other eye closed. Attempt to focus both the corner of the room and the pencil at the same
time. Illustrate the sensation using diagrams.
(g) Schemer’s Experiment Make two pinholes in a card about 1 to 2 mm apart (less than the diameter of
the pupil). Place the card close to one eye, the other being closed and look through
the holes at a distant object - e.g. the cross piece of a window. Hold a pencil with its point about 10 ins, in front of the eye so that it comes into the field of vision. While
looking at the distant object the pencil-point will appear double; carefully slide along
an opaque card to cover one only of the pinholes and focus on the pencil point; the
window cross-piece will now appear double. Again slide in a card to obscure the same pinhole as before and notice which of the double images disappears.
Draw diagrams for each case to show the paths of rays from the two objects to
the retina.
(h) Pulfrich Effect
A piece of string with a weight at the end is swung from side to side like a
pendulum in front of the eyes. The viewer looks at this pendulum with both eyes,
but with one eye covered by a piece of dark glass or a piece of exposed photographic film, anything that is fairly dark but that can still be seen through. When viewed in this fashion the pendulum no longer seems to be swinging strictly to and fro, but
appears to swing in an ellipse. The explanation is complicated but it depends chiefly on the fact that the dark glass causes the covered eye to become dark-adapted, which makes a very slight delay in the message reaching the brain from this eye. So the uncovered eye sees the weight immediately while the dark-adapted one sees it
slightly in the past tense. As the weight speeds up in the middle of its pendulum
swing, the filtered eye sees it farther and farther behind its real position, and when the two versions are put together binocularly, the result is an apparent elliptical
motion.
If the grey ring on the right is viewed with a ruler laid along the top of the rectangle, the lower half seems brighter than the top because of contrast with the
dark background.
(i) Retinal vessels
Look at the sky through a pinhole in a piece of black paper held close to the eye. Close the other eye. Move the paper up and down and from side to side.
Shadows of the retinal vessels will be seen - the group of blood vessels seen depends
on the direction of movement. The vessels lie in front of the retina and in this experiment a clear-cut shadow is cast by the light from the small aperture on to the sensitive layer of the retina. When the paper is held stationary the vessels are not
seen because the retina becomes adapted. This accounts for the fact that the vessels are not seen in informal circumstances. Also, light usually enters the eye from the whole of the lens uncovered by the iris, and reach the rods and cones. Only a diffuse
shadow of vessels can in these circumstances be cast on the light-sensitive layer.
(j) Stereoscopic vision Thread a needle, note how you do it and how long it takes. Close one eye and
repeat the experiment. How does this affect your performance?. You must not allow your hands to touch one another during this test; otherwise, tactile clues will reduce the effect of closing one eye. Note if there is any difference in the ease with which the
experiment is completed when one eye alone is used if the hand holding the thread
and the hand holding the needle approach one another (a) along the visual axis and
(b) at a right angle to the visual axis.
RESULTS
Give an account of your results and explain them from your knowledge of the
physiology of vision.
___________________________________________________________________________ ___________________________________________________________________________
___________________________________________________________________________
___________________________________________________________________________ ___________________________________________________________________________
___________________________________________________________________________
___________________________________________________________________________
___________________________________________________________________________
EXPERIMENT 40
VISION 2
FIELD OF VISION, COLOUR VISION, AND THE USE OF OPHTHALMOSCOPE
(a) Field of vision Close the eye and press the fingertip lightly on to the sclerotic through the
eyelid, preferably on the outer and lower part of the eyeball. By this means the retina is mechanically stimulated, and a bright ring-like image (a phosphene) is seen. Note that this appears at the point diametrically opposite to the site of stimulation. Similarly, images of objects outside the eye fall on opposite areas of the retina and
are subconsciously by experience, translated in the brain so that the individual
obtains a true knowledge of the position of the objects in space.
1. Approximate field of vision:
The charting of the field of vision is often of great practical value in the localization of lesions in the brain. For clinical purposes here is a simple
procedure that may discover gross changes. The subject stands facing the
examiner with his back to the light and at a distance of about 2 ft. Each eye must
be examined separately; while the examiner closes the eye opposite the subject’s closed eye. The subject should look fixed at the examiner’s open eye, while the
latter, holding his hand midway between himself and the subject, moves the
outstretched fore-finger from the periphery towards the center of the visual field. The subject is asked to say when he sees the movement of the finger. Both the
examiner and the subject should see the movement of the finger at the same
moment provided that in both the field of vision is normal. The movements of the hand are repeated in different meridians of the field until it has all been explored. Thus the examiner’s field is compared with the subject’s and as the examiner is
constantly watching the subject’s eye any wandering from the point of fixation is quickly observed and corrected.
2. The Perimeter
(a) A more accurate examination can be made by the use of the perimeter. This
consists of a metal quadrant rotatable about one end to describe a hemisphere; the fixed end is mounted on a stand in front of a large black disc. At the center
of the black disk, there is a small white disc or a small plane mirror used as a
point of fixation. The quadrant is marked off in degrees so that the position of the rider on it can be read off. There may be a frame behind the black disc to
carry the perimeter chart which rotates with the quadrant. At the opposite end
of the instrument is an adjustable chin rest on a pillar. The subject places his
chin on this and the height is adjusted till the eye to be examined is on the same level as the fixation point and is directly above the forked end of the pillar.
The subject should sit with his back to the light and close the other eye or have
it covered with a shade.
If a frame is provided then make a large pinhole through the center of
the appropriate perimeter chart (right or left) and put it vertically in the frame
while the quadrant is horizontal (at three O’clock to the subject). Bring up the hinged shelf engraved in degrees against the chart. The position of the shelf now
indicates on the chart the position of the quadrant concerning the subject.
Cover the disc on the rider with white chalk and take it to the end of the
quadrant; bring the rider inwards until it just comes within the field of vision of the eye of the subject who is gazing steadily at the fixation point. Read off the angle on the quadrant, lay a pencil point on the shelf at the position indicating
this angle, and push against the chart. Repeat this procedure with different positions of the quadrant. Finally, join up the dots on the chart at the boundary of the field of vision and compare it with the field already printed on it. Try to account for the peculiar shape of the field.
(b) Map out the blind spot by bringing the rider along the quadrant in several
meridians close to the horizontal on the temporal side of the field. (c) Repeat the observations for colored test objects; it will be found that the fields
are smaller for yellow, blue, red, and green (in this descending order) than for
white.
(b) Colour Vision
1. Chromatic aberration of the eye
Look at a bright circle of light through a piece of cobalt glass, which transmits only red and blue rays. A halo of one color will be seen around the light because
the eye cannot focus simultaneously long and short wave-lengths. If the eye is
astigmatic the halo will be elliptical. 2. Ishihara’s tests for color blindness
Test the color vision of your partner using the Ishihara Book. Instructions are
provided with the book. Do not leave the plates exposed to the light for too long. (c) The use of Ophthalmoscope
The ophthalmoscope is the instrument used to examine the interior of the eye.
Light is reflected into the eye by a mirror on the front of the instrument, and the observer, placing his eye behind the hole through the mirror, looks directly into the subject’s eye along the beam of light. In the body of the instrument are
several graded positive and negative lenses that can be placed at will in front of the observer’s eye; and the whole is provided with a handle.
If you wear glasses remove them. Practice first on the artificial eye provided.
Rotate the knurled disc which controls the chain of lenses until there is no lens opposite the eye-hole. Switch on the light of the instrument, put your eye against the
eye-hole and shine the beam of light through the aperture of the artificial eye bringing it closer to you until the vessels at the back of the eye are seen. Identify a
white circular area which is the nerve head, r disc. It will be found near the center of
the eye.
Now practice on your partner. Both subject and observer must relax their
accommodation by “looking into the distance”. Use the right eye to look at the
subject’s right eye, and the left eye to look at his left eye. Try and identify the vessels and disc.
Use a plus 20 lens in the ophthalmoscope to examine the corneal surface, plus
12 to examine the exterior surface of the lens and a plus 8 to examine the posterior surface of the lens.
RESULTS
Give an account of your results and explain the physiological principles involved. ___________________________________________________________________________
___________________________________________________________________________
___________________________________________________________________________
EXPERIMENT 41
HEARING AND BALANCE
A. HEARING
Auditory Acuity Cover one ear, slowly bring up a watch behind the subject’s head, and notice
the distance at which the ticking is first received. Repeat with the other ear covered.
Comparison of air and bone conduction of sound
a. Webers Test: Rest the base of a vibrating tuning fork on the vertex and describe
the sensation. Close one ear and then the other with the finger and record the sensation.
b. Rinnes Test: Hold the vibrating fork with its base firmly on the mastoid
pressure; when it is no longer heard, transfer it to a position with the plugs near
the outer ear, when it will be heard once more.
The effect of blocking the external auditory meatus
Block one auditory meatus with a piece of wet cotton wool and repeat the test
for auditory acuity of Weber’s Test and Rinne’s Test. To show that the latter two tests depend on the making effect of room noise, remove the cotton wool place the
fork on the right mastoid process, and at the moment when it becomes inaudible,
insert a finger in the right ear. Whereupon the sound will once more be heard for a few seconds.
Localization of sound
The subject closes his eyes. The observer makes clicking noises with the forceps behind the subject and asks him to locate the position. Record the results in
tabular form.
Masking of sound
Ask the subject to read from a hook. After a few sentences make a rattling
noise, use a tin- box with some stones, etc. near his ear. The intensity of the voice will be raised. This does not occur in a deaf person.
Limits of Audibility
The audiometer will be used for this experiment and is used as follows: 1. Put the headphones as the subject. Note that one is gray and maroon. 2. Connect the audiometer to the male. Turn the ‘power switch’ to the “ON”
position. If the circuit is working, a small red light at the top of the right-hand
side of the box will light up. 3. Turn the ‘output’ selector to the position ‘grey’ or ‘maroon’. This sends the
impulse to the grey or maroon headphone respectively.
4. Put the switch labeled ‘II Reverse- in the ‘on’ position. The input switch should be left on tone’. The masking control in the off position.
5. Put the ‘Hearing ions indicator with the mark (50) opposite the black line on
the panel. For the frequency regulator to 1,000 cycles/sec. The machine gives
an interrupted signal, turn up the hearing lose indicator. The push-button attached to the headboard can be pressed by the subject to indicate that he
can hear the tone and light on the left side of the panel will appear.
6. When you are satisfied that the subject understands the signals he is meant to hear, find the position if the hearing loses indicator for each frequency in which he can just hear the signal when you have done this, on similar tests on
the other ear by altering the position of the output selector. The hearing loss is given in decibels.
What to do
Plot a graph of hearing loss against frequency using a different symbol for the ear.
Screening Audiometer
2.5 Data transfer to a printer The audiometer is designed for use with a designated portable thermal printer
for printing air conduction results, (see 3.7) You must use the designated Amplivox
printer cable supplied with the audiometer to enable the audiometer to transfer data to the printer. Upon receipt of the printer, it is recommended that the printer is initially charged for a minimum of 8 hours before use.
3. Using the Audiometer 3.1
Switching the audiometer on and off
Press the ON switch situated on the back panel and labeled ON. The "ON" light
illuminates.
Note: If using batteries, the instrument will automatically switch off approximately
90 seconds after the last key was pressed to save battery power. Any test results will be automatically saved.
To switch off, press the MENU and YES keys together and release them.
3.2 Testing the patient response switch
Press the patient response switch and the light labeled RESPONSE (to the
right of the display) will illuminate.
3.3 Audiometer display
On start-up the display will show the following setting:- SIGNAL dBHL FREQUENCY Hz
30 1k
LEFT ear - Green light on
This indicates that when the PRESENT key is pressed, the tone presented will be at
30dBHL at a frequency of 1kHz (1000 Hz) to the left ear. Please note that on startup, the audiometer defaults to the left ear.
• Check the printer is ready for printing and press the PRINT key. The
audiogram will then print. Upon initial receipt of the printer, it is recommended that the printer is charged for a
minimum of 8 hours.
Safety Note:
To comply with EU law as defined in the Medical Device Directive and specifically EN60601-1:1990 for safety, the patient must be 1.5 meters from the printer. - The printer cable is specifically designed for use with Amplivox diagnostic
audiometers. 4. The sequence of Operation and Suggested Test Procedure
(air conduction measurements)
Pre-test (1) Switch the audiometer on.
(2) Perform a listening check.
(3) Decide whether to use the Threshold Retention Function or an audiogram card to record the patient's hearing thresholds for each test frequency.
(4) Position the audiometer so that the patient cannot see the control panel or any
movement of the operator's hands.
(5) Give instructions to the patient to acknowledge any tone presented either by signaling with the patient response switch or by raising or lowering the finger
if the patient response switch is not being used. Give the following
instructions to the patient. "As soon as you hear the tone, press the switch. When you no longer hear the
tone release the switch". (6) Fit the headset to the patient. Select the better hearing ear (according to the
patient) by pressing either the LEFT or RIGHT key and start the familiarisation session.
Familiarisation (7) Present the tone 30dB at 1kHz for between 1 and 2 seconds. If there is no
response at 30dB, increase the attenuation level in 10dB steps until the patient responds. When the patient responds, wait for 1 to 2 seconds and
present the frequency again at the same level. (8) If the responses are consistent with the pattern of tone presentation, proceed
to step 9 and start measuring the patient's hearing thresholds. If not, repeat the familiarisation session in step 7.
Test
(9) Present the first test tone at 30dB at 1 kHz. (10) if the patient responds, reduce the signal level in 10dB steps until they no
longer respond. Then increase the signal level in 5dB steps until the patient responds.
(11 ) if they fail to hear the first tone, increase the signal level in 5dB steps • until they do respond and then continue with step 10.
(12) Repeat the test by reducing the signal level in 10dB steps until the patient no
longer responds. Then increase the signal level in 5dB steps until they do respond and note this level.
(13) Repeat step 10 until the patient responds three out of a maximum of five times
at the same signal level. This indicates the patient's hearing threshold level for that frequency. Either mark the threshold on an audiogram card or press the
appropriate ear key once to activate the Threshold Retention Function and
save the threshold level on the screen.
(14) Proceed to the next test frequency. It is common to practise to test the frequencies in the following order: 1k, 2k, 3k, 4k, 6k, 8k, and 500 Hz.
(15) Repeat steps 9 to 15 for the other ear.
B. BALANCE (VESTIBULAR FUNCTION)
Labyrinthine functions are concerned with a sense of position movement and
equilibrium. The otolith apparatus (utricle and saccule) gives information about the position of the head in -space; the semicircular canals give of the head in space; the
semicircular canals give information regarding movement. The vestibular division of
the VIIIth nerve is the sensory nerve concerned.
Stimulation of the semi-circular canals
Two methods may be used to stimulate the semi-circular canals - rotation and
warm or cold douches. a. Rotation
The subject sits in a swivel chair with his head bent or forward so that a line
drawn from the external auditory meatus to the eye is horizontal. The chair is rotated in a clockwise direction, two times in twenty seconds. The rotation is stopped. The subject should describe his sensations during and after rotation.
Observe the subject’s eyes, watch for nystagmus, and note the direction of the fast component of the movement. To show nystagmus well, ask the subject to look about 45o to the side opposite to that towards which rotation has occurred. The subject should now stand upright, placing he heals together. The observer
must stand with one hand in front of the subject and one hand behind, ready to
prevent him from falling. The subject closes his eyes. Observe any tendency to fall. Note the direction. The examiner now faces the subject (with his eyes open) touches
the index finger, keeping his arm straight. The subject now closes his eyes, raises
his arm to the vertical and still keeping the arm straight, and tries to touch the examiner's finger again. Note carefully what happens. Note the direction of the error
- “past pointing”, asking the subject to walk straight forward and observe any
deviation.
Which of the canals has been affected by these tests?
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‘Repeat the rotation with the subject’s head bent forward” at an angle of 1200 - the bead can return to the vertical when the rotation has ceased. Compare the nystagmus with the observer above. Repeat the rotation with the head bent forward
on the left shoulder. Spin anticlockwise, then repeat the experiment - again with the
head on the left shoulder spinning the chair clockwise.
In each case, describe the nystagmus produced, name the semicircular canals
affected, and locate the nerve pathways involved. __________________________________________________________________________________
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b. Barany’s Test
The roof of the external auditory meatus may be stimulated with hot or cold
water. The heat or cold is transmitted to the endolymph in the underlying semicircular canal, altering its specific gravity so that a movement of fluid occurs.
Thus, cold douches will produce a downward movement of fluid, hot douches an
onward movement. This test can be applied unilaterally and by suitably positioning the individual groups of semi-circular canals can be stimulated. Great care should be exercised during these tests which should only be carried
out with a demonstrator present. The WARM DOUCHE SHOULD NOT BE BEVI’ER THAN 45°C and the cold douche not colder than 200C. Even as some degree of pauses and perhaps vomiting may occur. When using the ear syringe, remember
that the ear is a delicate organ and too much force may injure the tympanic membrane. Before syringing examine the eardrum to confirm that it is intact and
that the meatus is not blocked with wax. Draw the circle upwards and backward and steady the puzzle of fluid is directed along the roof of the meatus so that the
fluid flow follows the contour of the tympanic membrane with being driven forcibly against it. When the head is normally vertical, the horizontal canal slopes backward at an
angle of 300 from the true horizontal. The other canals are roughly perpendicular to
the slope of the horizontal canal, with the head in the normal sagittal plane, till it
forward 50o so that the lateral and superior canals are vertical, the eyes should be fixed on an object in front.
Irrigate for one minute with hot or cold water. When the irrigation is finished,
note the nystagmus produced the past pointing and spontaneous deviation - as in the rotation tests. Compare the effects of hot and cold water.
Now, tilt the head back 60o so that the horizontal canal is vertical. The subject
looks up at the ceiling. Note the nystagmus. Finally, ask the subject to stand as
before with one hand in front and one behind. Observe what happens when the subject closes his eyes.
QUESTION Give a full account of your results and the physiological principles involved.
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