PRACTICAL TASKS:

1. Electroneurogram (ENG) recording of mixed nerve (frog sciatic nerve)

ENG = summated biopotential registered from a peripheral nerve, i.e. the sum of action
potentials of the nerve fibres which it contains.
Procedure: ENG is recorded at the distal end of the sciatic nerve of an immobilised frog,
1-2ml of a curare (blocker of N-cholinergic receptors between Aa motor neuron and
skeletal muscle cell) like substance is injected into the dorsa lymph sac. Within 15 min,
proximal and distal sections of one of the sciatic nerves are dissected. For the proximal
portion, incision made parallel and adjacent to lateral rim of sacral bone, abdominal
cavity is opened, and frog placed on back, proximal portion reached using a curved glass
stick. String is threaded under it, the section is kept intact and hung on steel electrodes.
Distal part of nerve dissected above the knee joint (about 1cm) and another string
threaded under to position nerve on silver plated electrode
Recording: unipolar. The intact leg is placed on lead plate electrode and grounded to
deflect electrical surface charges and to reduce side effect of electrical stimulus on the
recording electrode. A minimum distance of 50mm must be kept between the
stimulating and recording electrodes. To avoid distortion from circuit currents, stand
supporting frog and electrodes is placed in faraday cage. Coaxial cables Connect
stimulation electrodes to output of stimulator recording electrodes to input of amplifier.
ENG recorded using rectangular impulses. Duration = 0.05ms, voltage = 0.8-1.0V and
frequency = 20Hz. ENG curve displayed on oscilloscope screen. Consists of 1,2 or 3 basic
waves depending on the distance between the stimulating and recording electrode and
nerve diameter. Before the first wave, ENG registers a minimal rectangular deflection
which is caused by the stimulus and labelled the stimulus artefact.
(see essay book for image you need to learn)
2. Measurement of conduction velocity of different nerve fibres united in the sciatic
nerve
ENG of sciatic nerve is registered as described previously. voltage is selected so all 3
waves appear on chart. Using the oscilloscope graticule, latent period (time between
stimulus artefact and peak of each wave) is calculated in ms at beam velocity of
0.25mm/ms. Distance between electrodes is measured in mm
3. Relatedness between stimulus intensity (I) and duration (t), and excitation (The
Horveg-Wess curve)
See essay 4
4. Clinically important reflexes
Conditions for reflexes -

• Comfortable environment
• patient’s attention must be diverted
• Perform symmetrical checks (to compare responses)
• Apply short brisk and equal in strength stimuli
• Maintaining intervals between stimuli
• Stimulus should be applied suddenly unexpectedly and painlessly
Two groups of reflexes
1. Exteroreceptive (Mucosal and cutaneous)
• Corneal reflex (mucosal) – closure of lids upon irritation of the cornea. Prior to
test, patient is instructed to look sideways and then the cornea is touched with
cotton wool
• Conjunctival reflex (mucosal) – closure of the upper eyelid when the conjunctiva
is touched with cotton wool
• Pharyngeal reflex (mucosal) – left and right half of posterior wall of pharynx are
brushed with a spatula, this elicits contraction of constrictor muscle, resulting in
urge to vomit
• Abdominal reflex (cutaneous) – subject in supine position with lower limbs
slightly bent at hip and knee joint, abdominal muscles are relaxed. Skin of
abdominal area is irritated with a pin/metal clip which elicits contraction of the
underlying muscles. Skin is stimulated under the rib cage, at naval level and in
the inguinal area
• Cremasteric reflex (cutaneous) – stimulation of skin on inner side of thigh causes
contraction of cremaster muscle and retraction of testis on same side
• Plantar reflex (cutaneous) – subject in supine position and skin on inner side of
sole, towards the big toe and along midline is stimulated with a pin. Response is
contraction (flexion) of toes. Extension of the toe instead of flexion (Babinski
response) is characteristic of babies up to one year
2. Proprioceptive (tendon and periosteal) reflexes
• Mandibular reflex (periosteal) – mouth of subject is open and masticatory
muscles are relaxed. Examiners index finger is placed on subject’s chin and a
tap with a reflex hammer on it causes contraction of masticatory muscles and
slight upward movement of lower jaw
• Styloradial reflex (periosteal) - subject in supine position. Forearms slightly
bent and lie on the abdomen. Wrist positioned between pronation and
supination with 2nd and 3rd finger held by examiner. Tap with the reflex
hammer on the stylo-radial process elicits mild pronation and flexion at the
elbow and wrist joint
 • Biceps reflex (tendon) – subjects’ forearm is supported by examiners arm
and slightly bent at elbow. Tendon of biceps muscle tapped several times
resulting in contraction of muscle and flexion of forearm
• Triceps reflex (tendon) – subjects arm flexed at right angle while being
supported and relaxed. Spot 2cm above olecranon is tapped and causes
extension of arm
• Patellar (knee jerk) reflex (tendon) – examiner puts arm under knee for
support, legs raised at right angle, so muscles are relaxed. Patellar ligament is
tapped causing extension of the leg
• Achilles (ankle jerk) reflex (tendon) – patient in supine position. Leg is flexed
at hip and knee joints with one forelimb crossed over the other and hanging
free. Foot held by toes and flexed slightly to stretch Achilles tendon. Tap on
tendon elicits a jerk reflex towards plantar surface which is caused by
contraction of triceps surae muscle
Assessing these conditioned reflexes helps in assessment of the state of the nervous system
and assess the location and scope of neurological damage if one of these reflexes were to
be absent
5. EEG (of brain cortex) – a method for registering summated bioelectrical activity
EEG recording carried out in supine/sitting position in special chamber with metal plated
walls (faraday cage). Skeletal muscles as relaxed as possible to avoid adding muscle AP to
the recording. Electrodes are metal discs made of silver/platinum/gold attached to elastic
helmet.
Recording can be unipolar (one of the electrodes is indifferent and the other is active) or
bipolar (between two electrodes placed at points in the scalp, whose potentials BOTH
change as a result of brain activity (active electrodes))
Potential differences are very small so are boosted by an amplifier before recording
Recording: device includes nibs using ink or carbon paper to make a roll of paper which is
rotated by an electric motor at speeds of 15,30 and 60mm/s. potential differences are
registered on the EEG and represent waves of a given frequency and amplitude. In different
brain conditions, EEG consists of a series of waves with the same frequency and amplitude
(rhythms). Normal brain activity is characterised by alpha, beta theta and delta rhythms.
Alpha: 8-12Hz – in wakeful relaxation, eyes closed
Beta: 13-30Hz – awake and alert with eyes open
Delta: <4Hz – finding is normal in new-borns and young children during wakefulness and
recorded in stages 3 and 4 of slow wave sleep in adults (deep sleep). If present in adults
during wakefulness, it is a sign of pathology (brain swelling, intoxication)
Theta: 4-7Hz – normal in children under 10 and in adults prior to falling asleep and during
deep sleep.
Synchronisation = when eyes close, rhythm switches from beta to alpha
Desynchronization = when patient who has been relaxing with closed eyes is asked to open
them, rhythm changes from alpha to beta
6. Determination of blood types with test sera
Carried out with test sera containing known antibodies – sera from types A (B antibody), B(a
antibody) and O (A and B antibodies)
Three drops, one from each of the sera are placed on a slide and marked with a marker,
using the corners of a second slide, one for each drop of serum, blood is carried onto the
first slide and mixed with sera. Agglutination is checked after at least 5 minutes and appears
as small red dots or larger particles on a background of clear serum.
If agglutination occurs in both A and AB drops – blood type is A
If agglutination occurs in both B and AB drops – blood type is B
If agglutination occurs with all three drops – blood type is AB
If agglutination does not occur with any drop – blood type is 0
7. Drawing blood
Blood for routine lab tests is taken from a vein (venous blood) or by piercing the skin
(capillary blood) – venous used when larger amounts are needed. Veins used are those
closer to the skin and relatively fixed such as those in the cubital fossa, on the back of hand
etc. Blood from an artery is taken only when assessing arterial blood gases (O2 and CO2),
usually taken from the ear lobe after rubbing.
RULES: deal with anxiety (calm patient), ensure 12hour fasting period, ensure 24 hours with
no alcohol intake and ensure patient is at rest before and during procedure, patient must be
lying or sitting and motionless during procedure.
PROCEDURE:
1. Choose vein
2. Choose section (must not be connected to a drip system)
3. Clean the spot with 70% alcohol,
4. Tighten the arm using a tourniquet,
5. Grasp the arm and pull skin in order to fixate the selected vein (don’t pump fist or pat the
vein),
6. After successful veni-puncture, relieve pressure on arm immediately
7. allow blood to flow freely into test tube
8. sequence of sampling should be - blood culture - Serum - With citrate for ESR, coagulation
- In tubes with heparin for testing of plasma, heavy metals… - EDTA-K3 tube –
haematological tests – test tube with glycolysis inhibitors - for glucose, lactate…
9. after necessary amount of blood is drawn, needle withdrawn, and puncture site is
compressed with sterile tampon and bandaged.
ERRORS:
- Prolonged compression – after two minutes local stasis will occur with acidosis and
haemoconcentration (particularly in patients with oedema), this will lead to higher
registered levels of albumin, Ca, RBC, WBC, haematocrit, platelets…
- Tapping vein – may cause haemolysis/activation of clotting factors
- ‘pumping fist’ – during the tourniquet compression may provoke local haemolysis
and acidosis
8. Chamber method for RBC count
Blood is drawn and diluted using a mixing pipette. Capillary to Ampulla volume ratio is
1:100, in the ampulla there is a red pellet. Ampulla starts at 0.5 and ends at 101. Ring finger
is pricked (ring finger used as its blood supply is isolated from the rest of the fingers
therefore if an infection occurs, it can be isolated), the first drop of blood is wiped off
(because first drop is mainly tissue fluid). Then, the capillary end of pipette held at
45degree angle is dipped into the second drop. Using a rubber tube blood is drawn up to or
slightly above 0.5 mark. Diluent (Hayems solution) is added up to the 101 mark (1 in 200
dilution). Diluted blood is transferred from capillary pipette to capillary space above mid-
section of Burker chamber. Counting RBC is done in smallest squares. (All 80 squares used
except last one). Burker’s rule states that all cells inside the big square are counted plus the
cells hitting 2 perpendicular lines out of 4 of that square, chosen at random. Burkers
Equation X = a/80 x 200 x 4000 x 106 (a= number of cells/ 80 which is number of squares,
200 is dilution factor, 4000 indicates how many times the volume is smaller than 1mm 3 and
106 is to recalculate according to a litre)
9.Chamber method for WBC count
Blood is drawn and diluted using a mixing pipette. Capillary to Ampulla volume ratio is 1:10.
Ampulla starts at 0.5 and ends at 11. In the ampulla there is a white pellet which facilitates
mixing and serves as a recognition mark. Turk’s solution used for dilution. Blood taken from
ring finger, first drop wiped off (see above for reasons why this is done). Capillary end of
pipette held at 45degree angle is dipped into the second drop. Blood drawn slowly up to 0.5
mark using rubber tube then Turk’s solution drawn up to 11 (1 in 20 dilution). After vigorous
stirring, diluted blood transferred to capillary space above mid-section of Burker’s chamber
and examined under microscope. WBC counted in mid-sized squares and in at least 100
squares which include ones in 6 of the big squares and 4 from the 7 th big one. Counting is
done according to Burker’s rule X= a/100 x 20 x 250 x 106 (a= number of cells/ 100 which is
number of squares, 20 is dilution factor, 250 indicates how many times the volume is
smaller than 1mm3 and 106 is to recalculate according to a litre)
10. Examination of the thyroid gland – radioiodine uptake and T3 inhibition (Werner’s)
tests
A) Radioiodine uptake test
Use of radioactive isotope of iodine (131J)
Determines the speed and scope of absorption of 131J by the thyroid gland as well as
uptake curve
PROCEDURE:
In morning, a known quantity of radioiodine (37 kBq 131J) is administrated on empty
stomach
A control dose which represents 100% of the administered dose is dissolved in a special
Using a gamma counter, activity of the isotope absorbed by the gland is measured against
that of the control 2, 4 and 24 hours after intake.
Absorption of 131J is registered in percentages
RESULTS
Normal = between 8-22% after 2 hours, 15-28% after 4 and 25-48% after 24.
B) T3 inhibition test (Werner’s test)
Demonstrates the existence of normal pituitary control over the production of iodine
containing hormones
PROCEDURE:
During a period of 10 days, 100 micrograms of T3 is administered daily
Uptake of 131J by the thyroid gland is measured before and after the radioiodine uptake
test
RESULTS:
Normal = exogenic T3 suppresses the secretion of TSH by the pituitary gland and absorption
of radioiodine is reduced by 50-100%
Hyperthyroidism = suppression does not occur, and absorption remains high which indicates
loss of pituitary control
11. Methods for examining adrenal glands – Thorn’s, dexamethasone suppression of
glucocorticoid secretion and Na salt inhibition of aldosterone secretion tests
A) Thorn test
Based on stimulatory effect of ACTH on synthesis of glucocorticoids which reduce number of
eosinophils in peripheral blood.
B) Dexamethasone suppression of glucocorticoid secretion
Demonstrates overall hypothalamo-pituitary regulation of adrenal glucocorticoid secretion
PROCEDURE:
Low dose – after measuring cortisone blood level, patient is given 0.5mg of dexamethasone
at 8am, 14.00pm, 24.00am, and 6am
2 hrs after last dose (at 8am), cortisol level measured again
RESULTS:
Normal = reduced on average by 70%
Hyperglucocortisism = no suppression, proves loss of pituitary control
High dose - test conducted with high daily dose of 8mg (2mg every 6 hours) for 3 days
(24mg total), level of plasma cortisol drops over 50% in cases of ACTH-dependent adrenal
hyperplasia.
C) Na salt inhibition of aldosterone secretion
Demonstrates the effect of Na plasma conc. on aldosterone secretion
PROCEDURE:
For 3 days patient put on diet rich in NaCl (11-14g/24h) with 20mg of deoxycorticosterone
acetate (DOCA) – a weak mineral corticoid daily.
Plasma renin activity and aldosterone level are checked before and after 3-day period
RESULTS:
Normal = external input and internal retention of Na inhibit aldosterone secretion up to 50%
of its initial level and reduce plasma renin activity and k+ excretion
Primary aldosteronism (increased aldosterone production usually due to tumour) = no
inhibition
12. Methods for examining the pancreas – oral glucose tolerance test
WHO recommends this test for the diagnosis of diabetes
PROCEDURE:
After 3 -5 days on a rich carb diet
In the morning (on an empty stomach), patient drinks 75g of glucose dissolved in 300ml
water and 5ml lemon juice, must be drunk within 5 min.
Plasma glucose levels are determined prior to the test and 4 times after it at 30min intervals
RESULTS:
NORMAL if after 1 hr = not above 11 mmol/l and after 2 hrs = not above 8 mmol/l
If between 8 -11 = Considered as lowered glucose tolerance and patient should undergo
constant follow-up
If above 11 mmol/l = Diabetes is diagnosed
13. Tests for early pregnancy – Galli-Mainini and immunological
A. The Galli-Mainini test
A bioassay for diagnosing early pregnancy, based on the stimulating effect of human
chorionic gonadotropin (HCG) on spermatogenesis in a male frog.
PROCEDURE:
- Male frog selected and check to see for spontaneous spermatorrhea (excessive
ejaculation), If not then investigation can proceed.
- 2-3ml of filtered urine (from female) is injected into the male dorsal lymph sac
- Secretion from the cloacae is tested for the presence of sperm cells (after 1, 2 and 3
hrs)
- If sperm is present – positive result (woman is pregnant)
B. Immunological tests
Based on the antigen/antibody reaction which can be visualised by adding an appropriate
indicator
Antigen = Glycoprotein molecule of HCG found in the plasma and urine of pregnant women.
Has 2 subunits (Alpha and Beta), beta determines the structural specificity of the hormone.
Antibody = Anti – HCG – antibodies are produced against the whole molecule (anti – alpha
Beta) or against the beta subunit (anti-beta).
Immunological agglutination tests:
Based on agglutination, made visible by attaching HCG or antibodies to inert particles (latex)
or to RBC
Direct agglutination method = latex particles coated with anti-HCG antibodies, then urine
from a pregnant woman (containing HCG) is added and an antigen/antibody complex forms,
resulting in agglutination
Inhibited agglutination method = requires an antiserum containing anti-HCG antibodies, a
urine sample and suspension of inert particles or sheep RBC coated with HCG
Serum with anti-HCG antibodies + urine with HCG + inert particles coated with HCG = no
agglutination (positive result)
Serum with anti-HCG antibodies + urine with no HCG + inert particles coated with HCG =
agglutination (negative result)
Lateral flow immune-chromatographic assay method = Dipstick is put into urine sample
containing HCG, hormone molecule binds to the antibodies on the particles. They move
upwards and upon reaching the test line form a complex of the HCG. If HCG is present, the
test band is coloured. The control line (mouse anti-Ig antibodies) will always be coloured
regardless of positive or negative test

14. Measurement of lung values and capacities

Lung Volumes
Residual volume = The volume of air remaining in the lungs after max expiration (approx.
1.1-1.2 l)
Tidal volume = The volume of air inspired or expired with each normal breath (approx. 0.5l)
Inspiratory reserve volume = Volume of air that can be inspired above a tidal volume
(approx. 2-3l)
Expiratory reserve volume = Volume of air that can be expired after the expiration of tidal
volume (approx. 0.7 – 1l)
Lung Capacity
Total lung capacity = Amount of volume of air after max inspiration (approx. 6l in male, 4.2l
females (Cannot be measured by a spirometer)
Vital capacity = The amount of gas that can be exhaled after max inspiration (TV+IRV+ERV) -
approx. 4.5l men, 3.5l females
Inspiratory capacity = Tidal volume + IRV
Functional residual capacity = ERV + RV, volume of air remaining in the lungs after a tidal
volume is expired (cannot be measured by a spirometer)
Forced expiratory volume in 1 sec = volume of air that is expired in the 1st second of a
forced maximal expiration, normally 80% of the vital capacity. Expressed as FEV1/FVC ratio =
0.8
15. Measurement of oxygen consumption and carbon dioxide release
PROCEDURE:
- Get patient ready
- Seat them on the veloergometer
- Attach the suction ECG electrodes
- Put mask on
- Test is carried out in several stages
- First stage measures amount of inhaled O2 and exhaled CO2 and pulmonary
ventilation at rest (approx. 40s of normal breathing without loading measured
- Next is the loading steps (each step has duration of 2min)
- At each step there is an increase in heart rate, breathing frequency, pulmonary
ventilation, oxygen consumption, CO2 release
 - The test is terminated at 1. Reaching the loading stage determined by the
investigator, 2. Registration of ECG abnormalities 3. At the subjects request or 4. At
reaching a plateau of oxygen consumption and carbon dioxide release
- After termination, patients remains on veloergometer
- Indices which are derived = Oxygen consumption, oxygen consumption per Kg of
body weight, released CO2, released CO2 per Kg of body weight, heart rate,
respiratory quotient and oxygen pulse – amount of oxygen uptake per heartbeat at
rest
16. Recording and analysis of the ECG
Method for recording summated bioelectrical activity of a beating heart.
ECG leads can be bipolar (both electrodes detect bioelectrical activity) or unipolar (one
active electrode detects activity and one passive electrode doesn’t)
Standard bipolar leads:
1st lead – right arm (R-negative pole), left arm (L-positive pole)
2nd lead – right arm (R-negative pole) and left leg (F-positive pole)
3rd lead – left arm (L-negative pole) and left leg (F-positive pole)
Augmented limb leads (unipolar)
Avr (augmented vector right) – positioned on right wrist
Avl (augmented vector left) – positioned on left wrist
Avf (augmented vector foot) – positioned on left foot
Chest leads (unipolar)
V1 – on 4th intercostal space to right of sternum
V2 – on 4th intercostal space to left of sternum
V3 – between points for V2 and V4
V4 – 5th intercostal space, 2cm medial to midclavicular line
V5 – on level of V4 on anterior axillary line
V6 – on level of V4 on midaxillary line
PROCEDURE:
1. Leads of ECG placed on palmar surface of two forearms and medial side of lower legs
on pads soaked in ringer solution or directly on skin rubbed with conducting gel.
2. Placement facilitated by different coloured leads. Red = right hand, yellow = left
hand, green = left foot and black = right foot (used for grounding)
3. For chest electrodes, electrodes placed on specific locations on the chest and rubbed
with conducting gel
 4. Recording made using standard amplification. Deflection of 10mm*mV^-1. The
paper moves out of the machine at speeds of 25 or 50mm/s. At these speeds each
mm mark between two vertical lines equals 0.04s or 0.02s respectively
INTERPRETATION
(include diagram in essay 35 of PQRST wave and what each peak represents)
17. Sphygmography
A method for graphic registration of the arterial pulse
Sphygmogram = graphic recording of pulsations of the arterial wall. A carotid Sphygmogram
is a graphic image of the pulsations of the carotid artery.
(See essay book for image displaying Sphygmography of carotid artery)
ELEMENTS:
Anacrote – steep ascending limb of the trace
Anacrotic peak (P) – corresponds to the highest value of systolic pressure in the vascular
system
Catacrote – sharply descending portion of trace
T wave – late systolic wave
I notch – closure of the aortic valve
Dicrote (D) – dicrotic increase
PROCEDURE:
1. ECG + PCG – allows simultaneous recording of two or more Sphygmograms from
central and peripheral arteries
2. Mechanical pulsations of arterial wall transformed into electrical signals which are
amplified and recorded by a sphygmograph
3. Consists of transformer (sensor), amplifier and recording device
4. Patient lies supine, facing upwards and slightly to the left with neck muscles as
relaxed as possible
5. Carotid pulse palpated in region of anterior triangle of neck
6. Applying moderate pressure, sensor is fixed with a band or by hand and registrations
begin.
7. Recording made during brief breathing pause (approx. 10 secs)
8. Speed of paper is set at 50 mm/s
18. Measuring arterial blood pressure
PROCEDURE
1. A sphygmomanometer (consisting of manometer connected to inflatable cuff with a
pumping bulb and air valve) and a stethoscope are used.
 2. Must take place in quiet room with ambient temp
3. Subject must be sitting or lying down with head raised above shoulders at 450 angle
4. The arm (free of any tight clothing) must be abducted, slightly bent at elbow and
relaxed
5. Cuff placed about 2.5cm above the antecubital fossa
6. Cuff is pumped, and pumping should not exceed more than 30mmHg above systolic
pressure
7. Air valve is opened slowly
8. Compression decreases, and auscultation is performed
9. Systolic pressure determined by first, distinct but slightly weak sound
10. Diastolic pressure determined by disappearance of the sound
11. Carried out 3 times with at least 30 second intervals
19. Combined functional test of the cardiovascular system
Use = To asses functional condition of the cardiovascular system by monitoring heart rate
and blood pressure Immediately after 3 different physical exercises.
PROCEDURE:

Test has 3 stages

1. 20 knee bends for 30 seconds (warm up)
2. Sprint in place for 15 seconds (must be done 3 minutes after stage 1)
3. Run in place for 3 minutes (done 4 minutes after stage 2)
HR and BP are measured – 3 minutes after stage 1, 4 minutes after stage 2 and 5 minutes
after stage 3
Pulse is taken – in first 10 seconds of each minute, BP is taken after this is done
INTERPRETATION
Normotonic if - Heart rate increases, systolic pressure rises to 160 -180 mmHg, diastolic
pressure fluctuates around initial value but all return to normal 3 min after stage 2 and 4
min after stage 3
Hypertonic (of systolic type) if - Sharp rise of systolic pressure (above 200-220 mmHg),
normal diastolic values.
Occurs in extreme excitability of sympathetic division of autonomic division
Seen in puberty and healthy sportsmen
Hypertonic (of diastolic type) if - Substantial rise of diastolic pressure (Above 95 mmHg),
systolic pressure changes are adequate to loading levels
A sign of extreme fatigue
Hypertonic (of systolic-diastolic type) if – Systolic pressure rises above 220 mmHg, diastolic
rises above 110 -110 mmHg
A Sign of a disorder (high blood pressure)
Hypotonic if - Systolic pressure rises slightly, heart rate increases to 180 – 190 bpm,
recovery is delayed, diastolic pressure fluctuates around initial values
A Sign of poor adaptation to loading