THE UNIVERSITY OF ZAMBIA

SCHOOL OF MEDICINE

DEPARTMENT OF PHYSIOLOGICAL SCIENCES

************************************************************************

MEDICAL
PHYSIOLOGY

************************************************************************
MANUAL FOR EXPERIMENTS IN PHYSIOLOGY
******************************************************

©2022
CONTENTS

1. Thermoregulation…………….……………………………………………………4

2. Transport across Cell Membrane...………………………………………………..6

3. Blood I…………………………………………………………………….............8

4. Blood II…………………………………………………………………………..12

5. Introduction to Lab Tutor……..…………………………………………………17

6. Respiratory Air flow and Volume……….………………………………............20

7. CVS I : Arterial Blood Pressure and Pulse…….. ……………………………….23

8. CVS II : ECG and Heart sounds…………..……………………………….........26

9. Renal function tests……………………………………………………………...27

10. Endocrinology Experiments……………………………………………………...29

11. CNS I: Reflexes & Dynamic equilibrium………………………………………..32

12. CNS II: Special Senses…………………………………………………..............34

1
 INSTRUCTIONS TO STUDENTS

Before entering the teaching laboratory make sure that you know which group you
belong to and which experiment you are doing.

The laboratory is neither a shop nor a play group. Therefore, you are required to come to
the lab promptly at the requested time, appropriately dressed for the practical and ready
to participate in the practical procedures. When you get in, sit down QUIETLY and wait
for initial instructions.

Most of the experiments are human being based, thus each student is expected to
volunteer to be the experimental subject. So come to the lab prepared to participate in
whatever capacity.

This manual contains 16 exercises each presented in form of instructions with nothing
else or with minimum background information. This is a deliberate act. It is hoped that
this way, students will make reports which reflect their own synthesis.

The exercises are meant to illustrate or consolidate some of the points discussed during
lectures. Therefore, they are an integral part of the teaching of physiology.

You are expected to study these before you come to the laboratory, so that you will have
a good idea of what is expected of you when you come to the lab and to be familiar with
the phenomenon being investigated. The lab session may be preceded by a short test
(pretest) and/or followed by another test (posttest) according to the prevailing conditions.

After performing the exercise you are required to make a detailed scientific report(s) and
the format to be taken is shown on the next page.

Feel free to interact with the laboratory staff. They are there to facilitate the processes of
your learning but not to do the experiments for you. They play a guidance role but will
have the background knowledge that you may require to fortify your understanding.

The subject lecturer is also available to offer the guidance due and will help show you the
literature to review in your write-up.

Copying of laboratory reports is greatly discouraged as it inhibits your personal learning

and kills the spirit of investigative analysis which is crucial for a clinical scientist that
you are becoming. Plagiarism is a great offense in the scientific world and discipline
starts here.

The assessors usually have a way of detecting plagiarism. Once found, all those involved
get ZERO for their reports.

Diligence is therefore called for as you embark on your career which encourages life-long
learning. Enjoy yourselves!!!!

2
 LABORATORY REPORT FORMAT:

TITLE: All laboratory experiments must have a title. The title is usually indicated for the
particular experiment in the manual provided.

AIM(s)/OBJECTIVE(s): The aim(s)/objective(s) of the experiment must be well

outlined indicating what one hopes to illustrate or achieve by doing the exercise. ( From
the instructions see if you can work out the relevant aim(s) of each exercise). Where there
is more than one aim/objective, clearly identify, outline and give each a prominence
depth of write-up as above.

INTRODUCTION / THEORETICAL BACKGROUND: This should provide relevant

theoretical information of a particular topic involving the experiment.

APPARATUS AND REAGENTS: These should be listed. The names of the apparatus
and reagents to be used can be found in the instructions manual.

METHODS: This should be a concise description of the method used to derive your
results. It must be written in way that is so easy to follow that someone else, by following
your method, would replicate your results.

RESULTS: These should be given in the most concise manner possible. They may be
tabulated and where possible graphs drawn to easily illustrate the findings. There should
be comment(s) on what the results show.

e.g. Relationship of Age to Systolic Blood Pressure

Age Systolic Blood Pressure

1 25 100
2 30 120
3 35 125
4 40 130
5 45 135

COMMENT: Systolic arterial blood pressure increases with age.

DISCUSSION: A concise description of your findings is made in this section. A

comparison is made between these findings and what was expected based on available
literature. Cite the literature and explain the correlations or deviations.

In case of deviations, desist from manipulating your results to suit literature. Negative
findings are also valid as far as you did the right things in your methodology. Do look out
for sources of errors and acknowledge them. Credit is given for the analysis and not for
the “answer”.

CONCLUDING: A summary is made of your findings in relation to your aims and

objectives.

REFERENCES: These are important to validate your source of information. Cite ONLY
the literature you have ACTUALLY read. Penalty is given for falsehood.
3
 1. THERMOREGULATION

Under normal conditions, the core body temperature is relatively constant around 37 oC.
There is a diurnal variation of about 1oC, the maximum occurring in the early evening
and the minimum in the early morning. Heavy or prolonged muscular exercise activity
causes a rise in body temperature.

The value of the body temperature depends in the site on the body at which the
measurement is made, e.g. oral, axillary or rectal. The rectal temperature is regarded as
the most reliable approximation to the temperature of the core of the body.

A. MEASUREMENT OF BODY TEMPERATURE

1. The Clinical Thermometer

(i) Examine the Clinical Thermometer and note the markings. The
glass is shaped to act as a convex lens giving a magnified image of
the mercury column. Since this image is visible only from one
particular angle, the thermometer should be rotated slowly until the
mercury comes into view.

(ii) Before any reading is taken, the thermometer must be shaken

briskly – without banging so that the mercury column runs back
into the bulb. Ensure that in shaking, you hold the end of the
thermometer which is away from the bulb.

2. Oral Temperature

(i) Place the bulb of the thermometer under the tongue and close the
mouth. After exactly half a minute take out the thermometer and
record the reading. Continue in this way, adding an extra half a
minute each time (½, 1, 1½, 2, 2½ )

Plot the results; time against temperature reading. If it is assumed

that the highest reading is the true temperature of the body core,
how long must the thermometer be kept in the mouth?

(ii) Take a mouthful of cold water, move it well around the mouth and
then spit it out. Repeat this several times and then take the mouth
temperature, leaving the thermometer in the mouth for the
optimum length of time determined previously in (i)

How does this reading compare with that recorded in (i)?

What would you expect with a hot drink?

4
 3. Axillary Temperature

Place the thermometer bulb in the axilla and hold it tightly in place for
three minutes. Take at least three readings.

Record the readings and account for any difference between it and the oral
temperature

4. Rectal temperature

This will be determined in your room after the practical class using your
own rectal thermometer.

Insert the bulb end of the thermometer right into the anus for at least 2
minutes. Take it out and without shaking it, take the temperature reading.
Repeat after 5 minutes and record your results.

B. EFFECT OF EXERCISE ON BODY TEMPERATURE

For this study, students should work in pairs.

One member of the pair should have 15 minutes workout (preferably on a bicycle
or treadmill that has provision for adding a workload to it). The other member of
the pair should measure the oral temperature of the subject at the beginning and at
the end of the exercise period.

Explain your results.

C. EFFECT OF HIGH TEMPERATURE

1. Record the room temperature and humidity of the high temperature room
before the fan/heaters are switched on. The fan/heaters will be switched
on for one hour to decrease/raise the temperature of the room. No one
may leave the room during this period, but each student may sit or stand or
read or do whatever he/she likes.

2. Record the onset of sweating.

5. At the end of one hour, record the oral temperature and then the room
temperature and humidity.

How does the change in the core temperature of the body if any, compare with
that of the room temperature, and what is the explanation?

5
 2.THE CELL MEMBRANE

A. Osmotic effects of substances that penetrate or damage cell membranes

(a) Into three clean test tubes place, respectively, 2ml of each of the following
solutions.

(i) 0.15M NaCl

(ii) 0.3M Urea
(iii) 0.15M NaCl plus a drop of soap solution

(b) Working with one tube at a time, add a drop of blood to each solution,
mixing tube contents immediately. Determine the time to complete
haemolysis. If the tube is held in front of a printed page the contents will
become transparent when haemolysed (Haemolysis is a process whereby
haemoglobin, red pigment of the red blood corpuscles, escape from the
cell, due to damage to the surface membrane) and the printing may be read
through it.

Comment on your results and discuss the related processes.

B. Solute molecular size and cell permeability.

The three compounds used in this experiment are very similar in chemical
structure, differing chiefly in molecular size. All three are very water-soluble.
The experiment therefore concerns the “sieving effect” by the membrane pores.

(a) Add two drops of blood to 2 ml of each of the following solutions, mixing
tube contents immediately.

(i) 0.3M Glucose (C6H12O6)

(ii) 0.3M Glycerol (C3H8O3)
(iii) 0.3M Ethylene glycol (C2H6O2)

(b) Note: If the haemolysis time is found to be short, repeat the determination
of time to haemolysis until accurate readings have been obtained, using a
stop clock if necessary. If rapid haemolysis does not occur observe every
5 minutes for 45 minutes.

6
C. Effect of Saline of varying Osmolalities on red blood cell shape.

Investigate the effect on the shape of red blood cells by immersing them in NaCl
solutions of varying concentrations; thus investigate the fragility of red cells when
immersed in NaCl solutions of varying concentrations.

NB: You will need to wear gloves for the handling of blood and blood products.

EQUIPMENT

 One test tube rack containing ten test tubes.

 A microscope with high power objective.
 1% & 3% solutions of NaCl
 Distilled water.
 A dropping pipette with a rubber teat.
 Blood, either obtained by vein-puncture from a volunteer (a sterile needle and
syringe, not previously used, are necessary) or supplied from Blood Bank.

PROCEDURE

Label tubes from 1 to 10 in sequence. Into tube 1 put 40 drops of 3% NaCl. Into tube 2
put 32 drops of 1% NaCl and 8 drops of distilled water.

Similarly into test tubes 3 to 10 in sequence put 28, 24, 22, 20, 18, 16, 14, & 12 drops of
1% NaCl and 12, 16, 18, 20, 22, 24, 26 and 28 drops of distilled water. To each test tube
add one drop of blood and thoroughly mix it with the salt solution. Leave the test tubes
standing for 1 hour and then inspect them. If no haemolysis has occurred red cells will
settle at the bottom of the test tube and there will be clear saline above them. Partial
haemolysis will leave cells at the bottom of the test tube and pink fluid above, and
complete haemolysis will result in pink saline with no red cell sediment. Shake the test
tubes and observe the red cells or their remnants in fluid under the microscope. Look for
changes in red cell shape. Take note of the concentration of NaCl at which haemolysis
begins and at which it is complete.

Try making these observations on blood from a subject who has completed a long
distance run (30 minutes run on the treadmill).

7
 3. BLOOD I

BLOOD CELL COUNTS

The methods in general use are based on the estimation of the number of cells in a small
volume of diluted blood. The counting is carried out in a glass counting chamber. The
volume of the fluid over each square is calculated from the area of the square and the
depth of the fluid layer over it.

The average number of cells lying in one square is found from the counts of a series of
squares. The product of this average number and the dilution gives an average of the
cells in undiluted blood. From this the number of cells per cubic milliliter of the
undiluted blood is easily found.

Material and Apparatus

1. The haemocytometer: This is a thick glass slide on which have been formed two
counting areas separated by an H-shaped trough. Various types of “ruling” have
been used. The improved Neubauer ruling will be used in this class. Running
right across the slide are two raised shoulders, which support a cover slip exactly
1mm above the counting areas. These areas look shiny by reflected light and dark
by transmitted light because a thin layer of metal has been deposited on the
surface of the glass. The ruling has been scratched into the surface with a very
fine diamond. Since it cuts through the metalized layer the scratch appears as a
very bright line, which is easy to see.

Examine the ruling under the low power of the microscope and compare it with
the diagram supplied. The ruled area is divided into nine large squares, 1 mm x 1
mm, each. The squares in the four corners are further subdivided into 16 medium
squares, but the centre square is subdivided into 25 small squares, each of which
is divided into 16 smaller squares. Important boundaries are emphasized by
ruling three lines close together, but it is always the middle line which determines
the actual boundary of the square.

Red blood cell counting is done on the centre square from which 5 randomly
selected small squares are used for cell counting. Use the diagramme provided as
a guide.

3. Diluting Fluids: For red blood cells, Hayem’s fluid is used [HgCl 2 (0.25g),
Na2SO2 (2.5g), NaCl (0.5g), Water (100 ml)] Hayem’s fluid fixes the red blood
cells and protects them from haemolysis and distortion.

4. Microscopy:
8
A. RED BLOOD CELL COUNT

Procedure: Make a 1:200 dilution of blood. Collect 20μL of blood taken into a
micropipette into 4ml of diluting fluid contained in glass or plastic tube/ bijou
bottle. After sealing the tube/bottle with a lid, mix the diluted blood by hand for at
least two (2) minutes by tilting the tube through an angle of about 120° combined
with rotation, thus allowing the air bubbles to mix the suspension.

Get a clean improved Neubauer counting chamber gently breathe over it and fix a
cover slip immediately on top of the ruled area so that a series of concentrically
arranged rings (Newton’s rings) are seen. Then fill it with the sample without
delay. This is simply accomplished with the aid of a Pasteur pipette. Take the
fresh gently shaken diluted blood sample using a pasture pipette. Place the tip of
the pipette on the counting area of the haemocytometer at the edge of the cover
slip and release just enough sample to fill the counting area completely.

Care should be taken that the counting chamber is filled in one action and that no
fluid flows into the surrounding moat.

Counting: Place the haemocytometer on the microscope stage, which must be

kept in a horizontal position. Examine the whole field under the 2/3 objectives to
make sure that the cells are reasonably uniformly distributed. Only the centre 1
mm square is used. Cells on its four corner 0.2 mm squares and on the centre 0.2
squares are counted using the 1/6 objective. Where cells actually touch the
margin of a square there is a danger that they may be counted twice. To avoid
this, count only those cells, which touch the upper and the left hand margin of
the square.

Calculation

You have counted the cells in 5 medium squares A, B, C, D and E.

Total count = N cells
Area of each medium square = 0.2 x 0.2 sq mm.
Volume of each medium square = 0.2 x 0.2 x 0.1 cu mm. = 0.004 cu mm.
Volume of the five squares: 0.004 x 5 = 0.02 cu mm.
So 0.20 cu mm contains N cells

Therefore 1 cu mm. contains N cells

0.02

Since the cells were diluted 200 times, 1 cu mm. of the undiluted blood will
therefore contain: N x 200 x 106 per litre
0.02

Thus by counting the cells in the 5 medium squares, and multiplying the Total by
106 you obtain the red cell count per Litre of undiluted blood.

9
 Each student should have his own red cell count, making at least two separate
determinations of it. He should also do two determinations on his partner’s blood.

The usually accepted value for the red cell counts in normal adults: 5 million/mm 3
for men, 4.5 million/mm3 for women (these values are probably too low). More
recent work puts these values at: 4.75 – 6.0 (mean 5.3) million/mm 3 for men, 4.4
– 5.4 (mean 4.8) million/mm3 for women.

Questions: 1. What does a (a) low or (b) high RBC count suggest?

2. How accurate are blood cells counts?

What are the inevitable sources of error?

B. ESTIMATION OF HAEMOGLOBIN

Haemoglobin is usually estimated colorimetrically either as oxy-haemoglobin or

as one of its conversion products much as acid haematin, alkaline haematin,
carboxyhaemoglobin, cyanomethaemoglobin. The coloured solutions obtained
may be matched with a standard visually or the determination of haemoglobin
carried out by a photoelectric colorimeter in which the transmitted light is
converted to electrical energy by a photoelectric cell and the result read on a
galvanometer. Photoelectric methods are much more accurate than visual
methods of matching.

The Sahli Method

This method is still used widely in medical clinics. It relies on the dark brown
colour of acid haematin which is obtained by mixing a blood sample with 0.1 N
HCl, the solution obtained is then diluted progressively with distilled water until it
matches visually with a dark brown glass standard which is supplied with each
Sahli haemoglobinometer.

Procedure
Wash the 0.02 ml pipette thoroughly with distilled water, alcohol, and ether, fill
the dilution tube to the mark 10 with fresh 0.1 N HCL. Prick the finger or lobe of
an ear and suck up 0.02 ml of blood, wipe off any blood on the outside, adjust the
volume exactly to the mark by tapping on the nail. Blow the blood gently into the
acid and suck up and down two or three times to mix thoroughly. Allow to stand
for exactly five minutes. Acid haematin is formed. Add distilled water drop by
drop from a pipette. After each addition stir with the glass rod. Continue to add
water till the tint in the diluting tube is just darker than that of the standard.
Compare the tubes against bright-diffused daylight and also while holding them
against a sheet of white paper. Take the reading of the upper level of fluid in the
dilution tube. Continue dilution until the test is just appreciably paler than the
standard. Take the average of this reading and the previous one as the correct
reading.

Calculation: The equivalent of 100% on the Sahli scale in grams Hb per 100 ml.
Varies with different makes of the instrument, and is engraved on the stand
carrying the tubes. If X=Hb for 100% reading, and Y=% reading obtained, Hb in
sample tested = X * Y/100.

10
C. DETERMINATION OF PACKED CELL VOLUME (P.C.V.)/HAEMATOCRIT

1. Fill the haematocrit tube with the pipette provided after obtaining blood from
a finger. The tube contains heparin to prevent the blood from clotting.
2. Spin the tubes (capillary tubes) in the micro-centrifuge for 10 minutes.
3. Read the height of the column of packed red cells using the reader provided
which gives the result as a percentage. This percentage value is known as the
PCV or haematocrit value.

HAEMATOLOGICAL INDICES

You are now in a position to calculate certain indices and standards which are useful in
the diagnosis of anaemia. The figures needed are:

Red blood cell count, the haemoglobin content, the packed cell volume and the diagnosis
of the red blood cell.

1. THE COLOR INDEX (CI) = Haemoglobin (% of normal)

R.B.C. count (% of normal)

Take the normal R.B.C. count as 5 million/cmm blood and normal haemoglobin
as 14.8g/100 ml blood.

Normal value of CI = 1.0; normal range 0.85 – 1.15

2. MEAN CORPUSCULAR HAEMOGLOBIN (MCH) = Hb X 10____

RBC (106/ul)

Haemoglobin in g/1000ml(dL) blood

R.B.C. count in million / cu mm

This value is expressed as Micro micrograms (uug) = picograms

Normal range: 26 – 34 pg

3. MEAN CORPUSCULAR VOLUME (MCV) = Hct X 10

RBC (106/ul)
R.B.C. count in million / cmm
This value is expressed as Cubic micron (c.u.)
Normal range = 78 – 94 c.u.

4. MEAN CORPUSCULAR HAEMOGLOBIN CONCENTRATION (MCHC)

(MCHC) = Hb g/1000ml x 100

Hct

This value is expressed as a g/dL. Normal range 32 – 38 g/dL.

11
D. BLOOD GROUPING AND CROSS-MATCHING

(i) Blood Grouping (typing)

On a white plate provided label A, B and D respectively. Place a drop of

anti-A serum on the side labeled A, a drop of Anti-B serum on the side
labeled B and a drop of anti-D serum on the side labeled D.

Make sure that the pipettes for the sera are kept separate. Add a drop or
two of the red cell sample to each drop of antiserum and mix by rocking
the white plate gently.

Agglutination is shown by the formation of dark red granules visible to the

naked eye. In the absence of agglutination the cells sediment, giving rise
to red streaks which are easily disturbed by movement of the plate.

Agglutination usually occurs within 10 minutes, but gentle rocking of the

glass slide hastens the process.

(a) From the results of your test: What is your ABO blood group: What
is your Rh blood group?
(b) If agglutination was not clearly visible to the naked eye, this means
that agglutination did not occur?

(ii) Cross-Matching
Place a drop of the fresh blood serum provided on a white plate provided.
Add a drop or two of your red cell suspension. Mix as before and observe
if any agglutination occurs.

(a) Can your blood be safely transfused into the donor of the serum which
you have just used? Explain.
(b) Can the blood of the donor of the serum which you have just used be
safely transfused into you?
(c) What is the importance of cross matching?
(d) Comment on the importance of the Rh group especially in obstetric
care.

12
Background information:
When blood from one person is mixed with that from another, the two blood samples
may either
(a) mix well and appear as one blood, when they are spoken of as Compatible or
(b) show clumping (agglutination of the red corpuscles into large particles visible to the
naked eye) and later undergoing haemolysis, when they are spoken of as incompatible
bloods.

Incompatibility is due to reaction between specific chemical bodies in the plasma, called
agglutinins and specific chemical bodies in the red corpuscles called agglutinogens, so
that the corpuscles agglutinate. The subject is of the utmost importance in connection
with blood transfusion.

Note: Agglutination is quite different from coagulation of blood.

There are two principal blood group systems; the Landsteiner or ABO system, and the
(Rh) factor system. The latter is important chiefly in obstetric practice.

The Landsteiner (ABO) system. A, B, AB and O.

Agglutination usually occurs within 10 minutes. Gentle agitation hastens the process. If
no clumping is seen wait for 1 hour before concluding that agglutination has not taken
place. To prevent evaporation cover the slides with some moist filter paper or with a
Petri dish. Sometimes rouleaux formation may cause difficulties; in such a case dilute
the red cell suspension further and repeat.

From your results, deduce your blood group. See table as given below.

TABLE
Slide A Slide B Group of Red cells
Anti A Serum Anti B Serum Under test
(Alpha-agglutinin) (Beta-agglutinin)

Agglutination Agglutination (I) AB

Agglutination No agglutination (II) A
No agglutination Agglutination (III) B
No agglutination No agglutination (IV) O

Rh Grouping
Requisites: 1. Slide D and 2. Anti-D Serum

Agglutination present…………………………………….. Rh + ve (Positive)

No Agglutination………………………………………… Rh – ve (Negative)

13
 4. BLOOD II

A. White Blood Cell Count

i. Total Leucocyte Count

Procedure: Make a 1 in 20 dilution by adding 20ul of well mixed blood into 0.38
ml of the diluents in a bijou bottle. Mix well for about two (2) minutes. Get a
clean improved Neubauer counting chamber gently breath over it and fix a cover
slip immediately on top of the ruled area of the improved Neubauer counting
chamber so that a series of concentrically arranged rings (Newton’s rings) are
seen.

Fill a clean dry counting chamber, with its cover glass already in position, without
delay. This is simply accomplished with the aid of a Pasteur pipette. Place the tip
of the pipette on the counting area of the haemocytometer at the edge of the cover
slip and release just enough fluid to fill the counting area completely. Care should
be taken that the counting chamber is filled in one action and that no fluid flows
into the surrounding moat.

Using the 2/3” objective count all the cells in 5 large squares.

Diluting Fluid:

A mixture of 1% glacial acid in distilled water, coloured with Gentian Violet is

used for white blood cells, this haemolyses the red blood cells and stains the
nuclei of the white cells so that they are easily recognized.

Calculation: No. of cells counted x dilution

Volume of fluid (measured in) mm3
6

= No. of cells counted x 20 x 10

0.1 x 5

= No. of cells counted x 10⁹/l

Questions:

1. Under what circumstances are WBC counts too high?

2. Under what circumstances are WBC counts too low?

3. What other haematologic information would you require to further evaluate

the significance of high white cell count?

14
 ii. DIFFERENTIAL WHITE BLOOD CELL (WBC) COUNT

You are provided with two blood slides. The first slide is from a normal smear
and the second one is from some ‘condition’ or abnormal smear.

Do a differential count on each as follows:-

First scan the slide to see that it is well stained. Using oil immersion objective,
count all the WBC you come across as you move the slide from left to right,
taking note of each type of white cell.

Avoid edges of the slide, when you reach the end of the slide move it slightly
forward to bring in another view and now count again as you move the slide from
right to left. Continue counting until you have counted a total of 100 WBC.

Questions:
1. Comment on your findings on the 2 slides.

2. What is the abnormality on the “abnormal condition” slide and what is the
significance of this finding?

15
B. ERYTHROCYTE SEDIMENTATION RATE (E.S.R)

Erythrocyte Sedimentation Rate is an empirical test whereby the rate of

sedimentation or the fall of red cells in their own anticoagulated plasma is
recorded. The rate of fall of red cells in whole blood depends on many factors.
The principle one being their power to form rouleaux i.e. they aggregate like a
pile of coins; this in turn is governed by the concentration of fibrinogen and
globulins in the plasma, the rate increasing in general with increasing
concentration of either or both of these proteins. In a wide variety of diseases
these proteins concentrations are altered and a rise in ESR results. Conversely
the ESR tends to return to normal as healing occurs. It is therefore, a useful
indicator of occult disease, and in differential diagnosis.

METHOD TO MEASURE ESR

The Disposable ESR tube is about 30 cm long and graduated over a 20 cm scale
in millimeters (mm) divisions, it has an internal diameter of 2.5 mm and when
filled would contain 0.7 ml of blood.

The Disposable ESR tube must GENTLY be inset in the vacutainer containing at
least 3 ml of anti coagulated venous blood until blood sample fills the ESR tube
up to the mark 0. Then allow it to stand vertically on ESR stand for ONE hour.

The distance red blood cells settle in ONE hour is the ESR, which is expressed as
“millimeters in the first hour”. One could also note the ESR at the end of the
second hour.

NORMAL ESR VALUES (WESTERGREN METHOD)

mm/first hour Men 0 - 10 mm

Women 0 - 20 mm

Precautions when deducing ESR results:

The test must be set up within 3 hours of blood collection otherwise the
sedimentation will be retarded:-

Blood containing the slightest trace of a clot must be discarded and there should
be no air bubbles in the ESR tube when filling it with blood; The ESR tube must
be placed in an absolutely vertical position and the test should be done at room
temperature since higher temperatures accelerate the sedimentation rate.

16
 9. RENAL FUNCTION TESTS

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 ADH from the posterior pituitary gland. The kidneys respond to the lower
levels of hormone by increasing the rate of production of urine. This has a 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 the meals.

Using Lab sticks measure each of the following from at least one sample of urine from
each of the tests below; pH, Glucose, Urea, Nitrite, Blood, Bilirubin, Urobilinogen and
Ketones.

1.(a) Subject No. I: will act as “Control” to establish the rate of urine secretion prior to
the test. On entering the laboratory he empties his bladder and this urine is
discarded. One hour afterwards he empties the bladder again and this urine
sample is preserved to measure its volume and specific gravity to establish the
rate of urine secretion.

(b) Test Subject No. II: Empties his bladder and discards this urine. Now he drinks
water, at the rate of 12mL/kg body weight. Bladder is emptied at twenty minute
intervals. Volume and specific gravity of each sample of urine is measured and
the results are expressed on a graph. How long does it take to excrete the quantity
that he drank i.e., restore the balance?

(c) Test Subject No. III: After emptying his bladder and discarding urine, drinks
normal saline (0.9% NaCl) solution at the same rate as subject No. II. Bladder is
emptied at twenty minute intervals. Volume and specific gravity of each sample
is measured and the results are expressed on the same graph as Test No. II.
Comment on the response.

(d) Test Subject No. IV: After emptying his/her bladder and discarding urine, subject
drinks strong tea. Bladder is emptied at twenty minute intervals. Volume and
specific gravity of each sample is measured. Comment on the response.

17
Urine Dilution and Concentration Tests

In the normal kidney, the glomerular filtrate in passing the tubules becomes hypertonic
by reabsorption of water from distal segments (D.T. and C.D.)

Impairment of this part of nephron results in failure of concentrating power of the kidney.

The failing kidney excretes the low threshold substances (Urea, Uric acid, etc.) in lower
concentration than in normal urine. If fluids are withheld for sometime from a normal
person, he passes urine of Sp Gravity 1.030 or over. The impaired kidney is unable to
raise the S.G. much if at all, above that of protein free plasma (1.010)

The failing kidney is also unable to reduce specific gravity below that of the filtrate,
suggesting the impairment of the ability of the tubule to reabsorb the Na+ and Cl- from
isotonic fluid. Thus, when a large quantity of fluid is drunk, the specific gravity does not
fall to the low level (1.001-1.003) as in a health kidney, but remains around deproteinized
plasma (1.010). The fact that the urine is isotonic with protein-free plasma suggests that
the tubule cells have lost their ability of selective permeability and that substances are
returned to the blood in the peritubular capillaries by a process of diffusion. This is
usually a later manifestation of tubular damage than loss of concentrating power.

DILUTION TEST

Test Subject A: No precautions are taken before the test. On entering the laboratory, he
empties his bladder and discards the urine. Now he drinks water at the rate of 15m/kg
body weight. Separate half hourly specimens of urine are collected during the following
three hours. Volume and specific gravity of each sample is measured and results
expressed on a separate graph.

Interpretation: About one litre of fluid should be excreted within the three hour period,
the greater part during the first hour. The Specific Gravity of the largest specimen will
approach 1.002. When the renal function is impaired the elimination will be reduced and
the minimum Specific Gravity will be comparatively high, e.g. 1.010.

Recording of Specific Gravity: Check your hydrometer by floating it in distilled water.

Add or subtract any deviation from zero in subsequent determination of Specific Gravity
of urine.

Sp. Gravity of a Liquid = 1 + Reading of Hydrometer

1000

18
When the quantity of urine in any one specimen is insufficient to fill the hydrometer tube,
a measured volume of distilled water may be added to the urine. The determinations are
then made on the diluted urine. Allowance must be then made for added water in
subsequent calculations e.g. if urine is diluted to two volumes, multiply the hydrometer
by two. If urine is diluted to three volumes, multiply the last two digits of reading of
hydrometer by three. The normal Specific Gravity of urine varies from 1.001 to 1.025,
depending on the state of hydration and the time of the day. It may occasionally rise to
even 1.035.

The Specific Gravity is greatly influenced by temperature. Hence, a value of 1.020

which is reached when passed could change to 1.025 at room temperature. Hence, it is
best to make all examinations at current room temperature.

19
 10. EXPERIMENTS IN ENDOCRINOLOGY

A. INSULIN SHOCK IN MICE

Prepare three mice, weighing about 20g each, for the experiment by fasting them for 24
hours. Inject into each fasted mouse subcutaneously 0.25-0.5 units in 0.1 ml isotonic salt
solution and note the time of the injection. Immediately inject intraperitoneally into one
of the mice 1.0 ml of 10% glucose solution. Put each mouse under a separate beaker.
Observe the behaviour of the mice.

The dose of insulin should produce convulsion in about 30-60 min in mice which had not
been injected with glucose. Hypoglycemic shock will be indicated by unusual pose,
tacypnea, disturbance of coordination of movement (in addition, there will be
tachycardia, extrasystoles slight rise in blood pressure, irritability and hypoglycaemia).
The convulsions may last 20 seconds or more.

As soon as there are indications of convulsions inject intraperitoneally into one of the
mice 1ml of 10% glucose solution. Note the result. Record and explain the results seen
in the three mice.

B. IMMUNOLOGICAL TESTS FOR EARLY DIAGNOSIS OF PREGNANCY

Rapid tests for early diagnosis of pregnancy aim at detecting chorionic zonadotrophin in
urine. This can be done by using an animal for bioassay (female mice in the Aschhein-
Zondeh test and male or female toad in the Hogben test). The animal bioassay techniques
are, nevertheless, relatively cumbersome and slow (at least 12 hours) as compared to the
immunological techniques about 4 minutes). A number of pharmaceutical firms now
have special diagnostic kits which are easy and fast and convenient to use. The
procedure presented below is the one used in “Searle” Pregnancy Latex Test which will
be used in this practical.

“Immunological methods for the detection of human chorionic nadotrophin (HCG)

in urine are now well established as presumptive tests for pregnancy. The HCG is
detected by an immunological reaction between this hormone and its antibodies.
Flocculation occurs when a suspension of latex particles, on which HCG has been
absorbed, is mixed with the corresponding HCG anti-Serum. This flocculation will not
take place when the anti-serum is mixed with urine containing free HCG, as the
antibodies in the antiserum will have been neutralized. This principle provides a rapid
test for the presumptive diagnosis of pregnancy.”

20
 11. CNS I: SPINAL AND CRANIAL REFLEXES

The reflexes described in this section are those usually tested in the routine examination
of patients. Changes in reflex activity give valuable information concerning the nature
and location of diseases involving the nervous system.

If a normal response is obtained it indicates that the reflexes pathway-receptor, afferent

nerve, central intergrating areas, efferent nerve, effector (e.g. muscle) – is intact. There is
no absolute standard as to what is the normal degree of briskness in a reflex. It is,
therefore, important to become familiar with the range and the extent to which a normal
reflex may be influenced by other simultaneous events in the central nervous system (for
instance, reinforcement).

Reflexes may be normal, diminished, absent, changed in character, or asymmetrical i.e.

different on the two sides of the body.

When demonstrating reflexes in man, now in the laboratory and later clinically,
remember that usually your subject or patient is conscious and that reflex activity is
modified by the activity of the higher levels of the nervous system. Treat your subject
kindly, make him/her comfortable both physically, mentally and distract his attention
from the part under investigation. Get into the habit of giving simple and precise
instructions in non-technical language. Don’t say “Focus on infinity” say: “Stare hard at
the mark on the ceiling over there”.

Stretch Reflexes (Tendon Jerks)

Knee Jerk
The best position for the subject is sitting on the bench or a stool that is too high for him,
so that the legs hang free and the knees are flexed at a right angle. The subject must relax
fully, and should have his attention diverted from the examination. With the tendon
hammer, tap the patellar tendon (not patella or the tibia) sharply and cleanly just below
the patella. If the operator’s other hand is placed on the front of the subject’s thigh on the
quadriceps muscle, a weak contraction may be felt when there is no visible movement at
the knee joint.

Reinforcement
Ask the subject to hook his fingers together, close his eyes, and make a vigorous effect as
though to pull his hand apart. While he is doing so, test the knee jerk again. It will
probably be increased, and effect that is especially noticeable if the response was weak
previously. This is due partly to the distraction of the subject’s attention and partly to a
general increase of muscle tone which accompanies any muscular activity, even though it
is in another part of the body.

21
Ankle Jerk
The subject kneels on a chair so that both legs are supported but the feet hang free. Tap
the Achilles tendon smartly and note the extension of the foot, at the same time feeling
the contraction of the gastrocnemius muscle. Repeat with reinforcement. Now dorsiflex
the ankle by pressing on the sole of the foot so that the muscles attached to the tendo-
achillis are stretched. Repeat the ankle jerk with and without reinforcement. Note the
differences in response.

Biceps Jerk
The operator grasps the subject’s left arm with his own left hand and places the fingers
round the back of the subject’s elbow. The subject’s forearm lies relaxed, along that of
the operator, whose thumb is placed firmly over the biceps tendon. The operator strikes
his own thumb and feels the tendon tighten as the biceps contracts. The contractions may
be visible, but there may not be visible movement in the forearm.

Cutaneous or superficial reflexes

Abdominal reflexes
When the skin of the abdominal wall is lightly stroked or scratched, the underlying
muscles, innervated from the spinal segment corresponding to the sensory innervations of
the stimulated area, respond by a short-lived contraction.

Plantar reflexes
With the subject lying down, hold the foot firmly with one hand, and with the other
scratch the sole with a hard, blunt object such as a key. Scratch firmly along the outer
edge of the sole of the foot, beginning at the heel and ending near the base of the little
toe. The normal response is the plantar flexion of the great toe, with plantar flexion and
bunching together of the other toes. There is often plantar flexion of the whole foot and
attempted withdrawal.

In upper motor neuron lesions, the plantar response is changed in character. There is
dorsiflexion of the great toe and fanning out of the other toes. This is known as the
Babinski response: there is probably no more important single physical sign in clinical
neurology. Although the Babinski response is characteristic of an upper motor neuron
lesions, the response is present in a normal child in the first year of life when the
pyramidal fibres are still being myelinated, and is also present in some adults during deep
sleep.

A plantar response of any kind may be difficult or impossible to elicit in a subject,

because of going barefoot all his life, has very thickened and cornified skin on the soles
of his feet.

What segments of the cord mediate the above spinal stretch reflexes and superficial
reflexes.

22
Corneal and Conjunctival reflexes

These are superficial reflexes, quantitatively similar to the cutaneous reflexes. Very
gently touch periphery of the cornea and the bulbar conjunctiva with a fine wisp of clean
cotton wool. Describe the responses. What is the nervous pathway?

23
 12. CNS II: SPECIAL SENSES

1. Taste:
The subject extends his tongue from the mouth and dries it with a blotting paper.
The observer using small pieces of blotting paper on cotton buds paints the
following solutions in term of:- the tip of the tongue, the sides, the center and the
back of the tongue.

Solutions:-

A. 5% Cane Sugar
B. Saturated Quinine/fragile solution
C. 1% Sodium Chloride
D. Chili Powder/Solution

2. Smell:

I. The subject closes his eyes, pinches his nose and opens his mouth. The observer
places a piece of …………………………… on the subjects tongue. This piece is
in turn replaced by a piece of ……………………..the substance may be rolled
with the tongue but not chewed.

Repeat the exercise with the nose open.

II. The subject, with eyes closed, sniffs at each substance provided and attempts to
identify the odour.

3. Hearing:

I. The subject closes one ear with a finger, the observer brings up a watch behind his
head and notices the distance at which it is first head. Repeat with the other ear.
II. Hold the vibrating tuning fork with its base firmly on mastoid process. When the
sound is just audible, take it off the mastoid process. When the sound is just
audible, take it off the mastoid process and hold the prongs near the outer ear. It
should be heard now, if it is not heard, repeat the test the other way round.
III. Rest the base of vibrating tuning fork on the forehead (in the midline) or the top
of the head. Describe your sensations.
IV. If in the proceeding test the sound was heard equally in both ears, repeat the
exercise but block one ear with your finger. Describe your sensations.
V. The subject closes his/her eyes. The observer makes clicking sounds/noise with
forceps or anything else form various positions around the subject’s head. The
subject tries to localize the sound source.

Block one auditory meatus with wet cotton wool or tip of the subject’s finger.
Repeat the test. Compare the responses.

24
4. Vision:

I. Let the subject fix his gaze on an object held at arm’s length.
Observe the diameter of the pupil bring the object slowly towards the face and
observe the diameter of the pupil and the position of the eyeballs.
II. Hold and open book in front of the eyes. Bring it nearer until the print can no
longer be seen clearly. Measure the distance.
III. Place the subject at 6 meters from Snellen’s chart and let him/her use one eye at a
time. Indicate the letter on the chart with a pointer and the subject reads them off.
Note the number corresponding to the last row that can be read correctly. The
result is written as 6 over this number.
IV. Visual Fields Assessment will be demonstrated to you using the spot test used in
the clinic and the confrontation method.

5. COLOUR BLINDNESS TEST:

The subject is spun rapidly clockwise in a rotating chair ten times and then
suddenly stopped. Observe the following:
1. The eye movements. (Do you see nystagmus?) In which direction is the fast
phase? By convection the direction of nystagmus is the direction of the fast
phase.
2. The subject is asked to point at an object held three feet in front of him first with
his eyes open and then with eyes closed. What do you observe?
3. The subject is asked to walk on a straight line. Does he/she tend to fall, and if so
in what direction? Stand by the subject ready to give support. Explain the
observations.

25