practicals

Practical 1) Electroneurogram (ENG) Recording of Mixed Nerve (Frog Sciatic Nerve)

• Immobilize the Frog

  • Inject 1–2 ml of curare into the dorsal lymph sac to prevent muscle movement.

• Dissect the Sciatic Nerve

  • Expose the nerve and separate it into proximal and distal sections.

• Prepare the Proximal Nerve for Recording

  • Place the frog on its back.

  • Use a curved glass rod to access the sciatic nerve.

  • Thread a string under the nerve and suspend it on steel electrodes.

• Prepare the Distal Nerve

  • Dissect the nerve above the knee joint.

  • Thread a string under the nerve and place it on silver-plated electrodes.

• Set Up the Recording Electrodes

  • Use a unipolar configuration:

    • Active electrode on the dissected nerve.

    • Indifferent electrode on the intact leg.

  • Ground both electrodes to reduce electrical interference.

• Minimize External Noise

  • Place the entire setup inside a Faraday cage to block external electrical signals.

• Connect Equipment

  • Use coaxial cables to connect:

    • Stimulating electrodes → stimulator output

    • Recording electrodes → amplifier input

• Stimulate the Nerve

  • Apply a rectangular impulse with:

    • Duration: 0.05 ms

    • Voltage: 0.8–1.0 V

    • Frequency: 20 Hz

• Observe and Record ENG

  • View the ENG waveform on an oscilloscope screen.

  • Expect 1 to 3 waves, depending on the distance between the stimulating and recording electrodes.

  • Look for a stimulus artifact—a small deflection before the first wave.

Practical 2) Measurement of Conduction Velocity of Different Nerve Fibers of the Sciatic Nerve

• ENG registered as previously described

• Set stimulus voltage high enough to produce all 3 waves on the oscilloscope.

• Use the oscilloscope grid to take measurements.

• Measure the latent period — the time from the stimulus artifact to the peak of each wave.

  • Use a beam speed of 0.25 ms/mm.

• Measure the distance between electrodes in millimeters (mm).

Practical 3) Relationship Between Stimulus Intensity and Duration (Horveg-Weiss Curve)

1. Shows the relationship between stimulus strength and duration produces curves showing intensity-duration relatedness for nerve fibers (A, B, C).

2. Rheobase is found where the curve flattens near the x-axis.

3. Chronaxie is the time at twice the rheobase.

4. Chronaxie values are marked on the y-axis and connected to the I/T curve with perpendicular lines.

5. Used to assess nerve excitability and monitor peripheral nerve disorders or treatments.

Practical 4) Clinically Important Reflexes

Conditions for Accurate Reflex Testing

• Room temperature: 20–22°C

• Patient relaxed and attention diverted

• Stimuli must be:

  • Short, brisk, and equal in strength

  • Sudden, painless, and given at intervals

• Always check both sides for comparison

Types of Reflexes 1. Exteroceptive Reflexes (from surface receptors)

a. Mucosal Reflexes:

• Corneal reflex: Eyelids close when cornea is touched

• Conjunctival reflex: Upper eyelid closes when conjunctiva is touched

• Pharyngeal reflex: Touching throat causes gag reflex

b. Cutaneous Reflexes:

• Abdominal reflex: Pinching skin causes abdominal muscle contraction

• Cremasteric reflex: Touching inner thigh causes testicle retraction

2. Proprioceptive Reflexes (from muscles/tendons)

a. Periosteal Reflexes:

• Mandibular reflex: Chin tap causes jaw movement

• Styloradial reflex: Tapping wrist bone causes elbow/wrist movement

b. Tendon Reflexes:

• Biceps reflex: Tap causes arm flexion

• Triceps reflex: Tap above elbow causes arm extension

• Patellar reflex (knee jerk): Tap below knee causes leg extension

Why Reflexes Matter

• Give objective signs about nervous system function

• Help detect location and severity of nerve damage

• Useful in diagnosing neurological diseases

Practical 5) EEG as a Method of Recording the Electrical Activity of the Brain

• Done with the person lying down or sitting inside a Faraday cage to block external electrical interference.

• Skeletal Muscles must be relaxed to avoid signals from muscle activity.

• Metal disk electrodes (silver, gold, or platinum) are attached to an elastic cap on the head.

• Bipolar recording: both electrodes detect brain activity.

• Unipolar recording: one active, one indifferent electrode.

• An amplifier boosts weak signals, which are sent through cables to the recording system.

• EEG records brain wave patterns based on frequency and amplitude.

Brain Waves

• Alpha waves: awake, relaxed, eyes closed

• Beta waves: awake, alert, eyes open

• Theta waves: drowsy or early sleep

• Delta waves: deep sleep

Practical 6) Drawing Blood

Blood Collection Summary

• Blood can be drawn from a vein, artery, or capillaries (skin puncture).

• Venous blood: taken from the cubital fossa, back of the hand, or foot for larger samples.

• Arterial blood: used for blood gas tests; drawn from brachial or femoral artery.

• Capillary (arterialized) blood: usually from the earlobe.

Blood Draw Procedure

1. Calm the patient and check for prep instructions (e.g., fasting, no alcohol or smoking).

2. Choose a suitable vein, clean the area with alcohol.

3. Apply a tourniquet briefly to locate the vein.

4. Fix the arm, insert the needle, then release pressure after puncture.

5. Let blood flow into tubes, then apply pressure and bandage the site.

Common Errors

• Prolonged tourniquet use

• Tapping the vein

• Pumping the fist

Finger Prick (for small samples)

• Clean the skin

• Wipe the first drop

• Collect the following drops

Practical 7) Determination of Blood Types with Test Sera

ABO Blood Typing (Simplified)

• Blood type is tested using anti-A, anti-B, and anti-AB sera.

• Place one drop of each serum on a marked slide.

• Add a small drop of blood (1/10th the serum amount) to each.

• Mix and wait 5 minutes.

• Look for agglutination (red clumps).

Results Interpretation

• Anti-A & Anti-AB: Type A

• Anti-B & Anti-AB: Type B

• All three: Type AB

• None: Type O

Practical 8) Chamber Method for RBC Count

• Blood is drawn and diluted in a mixing pipette, which has a capillary part with an ampoule containing a red pellet for mixing and recognition.

• Dilution is performed using Hayems solution, a colorless and isotonic fluid.

• A patient's finger is pierced, and blood is drawn into the capillary part of the pipette, up to the 0.5 mark.

• Hayems solution is added immediately after drawing blood, up to the 101 mark, to achieve a dilution ratio of 1:100.

• After vigorous stirring, the diluted blood is transferred to the chamber above the midsection of the Burker chamber and examined under a microscope at low magnification.

• Red blood cells (RBCs) are counted in 80 small squares of the chamber, excluding the first and last squares.

• RBC count is calculated using Burker's equation, taking into account:

  • number of counted RBCs

  • counted squares

  • blood dilution ratio

  • volume

  • conversion factor.

Practical 9) Chamber Method for WBC Count

• Blood is drawn and diluted in a mixing pipette, which has a capillary part with an ampoule containing a white pellet for mixing and recognition.

• Dilution is performed using Turk's solution, a violet-colored fluid.

• A patient's finger is pierced, and blood is drawn into the capillary part of the pipette, up to the 0.5 mark.

• Turk's solution is added immediately after drawing blood, up to the 11 mark, to achieve a dilution ratio of 1:10 (20 times dilution).

• After vigorous stirring, the diluted blood is transferred to the chamber above the midsection of the Burker chamber and examined under a microscope at low magnification.

• White blood cells (WBCs) are counted in 100 small squares of the chamber, including 6 big squares and 4 middle squares.

• WBC count is calculated using Burker's equation, considering:

  • number of counted WBCs

  • counted squares

  • blood dilution ratio

  • volume

  • conversion factor.

Practical 10: Examination of the Thyroid Gland (Pages 89-90)

Radioactive Uptake Test:

• Uses radioactive iodine (I-131) to check thyroid function.

• Given in the morning on an empty stomach.

• Gamma counter measures iodine absorption compared to a control.

• Absorption checked at 2, 4, and 24 hours after intake.

T3 Inhibition Test (Werners Test):

• Assesses pituitary control of iodine hormone production.

• 100 mcg of T3 given daily for 10 days.

• Measures thyroid’s I-131 uptake before and after T3.

• T3 lowers TSH, reducing iodine uptake by 50-100%.

• No reduction means pituitary control is lost (seen in hyperthyroidism).

Practical 11: Methods for Examining the Adrenal Glands (Pages 90-91)

The Thorn Test:

• based on how ACTH stimulates glucocorticoid secretion.

• Used to assess adrenal gland function.

• Rat given 20 IU ACTH by injection.

• Eosinophil count drops ~50% after 4 hours = positive test.

Dexamethasone Suppression Test:

• tests the pituitary control of adrenal glucocorticoids.

• Patient takes 0.5 mg dexamethasone until 6 am next day.

• Cortisol measured before and 2 hours after last dose.

• Normal: cortisol drops by 70%.

• No drop = loss of pituitary control (hypercortisolism).

Na Salt Inhibition of Aldosterone:

• Tests how sodium affects aldosterone secretion.

• Patient on high-salt diet + 20 mg DOCA for 3 days.

• Renin and aldosterone measured before and after diet.

• Normal: aldosterone secretion drops by ~50%.

• No drop = primary aldosteronism (excess hormone secretion).

Practical 12: Methods for Examining the Pancreas (Page 91)

ORAL GLUCOSE TOLERANCE TESTS:

• Recommended by WHO for diagnosing diabetes

• Patient consumes 75g glucose dissolved in 300ml water and 5ml lemon juice

• Plasma glucose levels measured before and at 30-minute intervals after intake

• Normal blood sugar curve: ≤11mmol/L after 1 hour, ≤8mmol/L after 2 hours

• Levels of 8-11mmol/L indicate lowered glucose tolerance, requiring follow-up

• Blood sugar concentration >11mmol/L after 2 hours indicates diabetes mellitus

Practical 13: Pregnancy Tests (Pages 98-99)

Galli-Mainini Test:

• Uses hCG’s effect on frog sperm production to detect pregnancy.

• Male frog’s cloaca secretion checked for sperm.

• If no sperm, inject 2-3 ml of filtered morning urine into frog’s lymph sac.

• Check for sperm after 1, 2, and 3 hours.

• Presence of sperm = positive pregnancy test.

Immunological Pregnancy Tests:

• Detect hCG using antigen-antibody reactions.

1. Agglutination Test:

• Direct: hCG attached to particles, clump with pregnant urine.

• Inhibited: Anti-hCG antibodies clump unless urine contains hCG, which stops clumping.

2. Lateral Flow Assay (Dipstick):

• Dipstick placed in urine.

• hCG binds antibodies, moves up, forming colored test band if positive.

• Control band always shows to confirm test worked.

• Early morning urine preferred (highest hCG levels).

Practical 14) Measurement of lung volumes and capacities page 141

Lung Volumes and Capacities:

Static Volumes:

• Tidal Volume (TV): Air inhaled/exhaled at rest (~0.5 L)

• Inspiratory Reserve Volume (IRV): Max air inhaled after normal breath (2–3.3 L)

• Expiratory Reserve Volume (ERV): Max air exhaled after normal breath (0.7–1 L)

• Residual Volume (RV): Air left after max exhalation (1.1–1.2 L)

Static Capacities:

• Functional Residual Capacity (FRC): Air in lungs after normal exhalation

• Inspiratory Capacity (IC): Max air inhaled after normal exhalation

• Vital Capacity (VC): Max air exhaled after max inhalation (Men: 4.5–5 L, Women: 3–3.5 L)

Other measurments:

• Forced Expiratory Volume in 1 second (FEV1): Air forcefully exhaled in 1 sec (~80% of VC); reduced in asthma.

Measurement Procedure:

• Use flow spirometer; enter patient data (age, sex, height, weight).

• Patient breathes normally, then performs deep exhale and slow max inhale.

• Spirometer shows results vs. predicted values for assessment.

Practical 15) Measurement of oxygen consumption and carbon dioxide release page 15

• Prepare the patient by seating them on the veloergometer, attaching suction ECG electrodes, and placing the mask on their face.

• The testing is conducted in several stages:

  • Resting Stage: Measure the amount of inhaled oxygen (O2), exhaled carbon dioxide (CO2), and pulmonary ventilation at rest.

  • Loading Stages: Each stage lasts for 2 minutes and involves an increase in heart rate, breath rate, pulmonary ventilation, oxygen consumption, and CO2 release.

• The test continues with sequential loading stages until a specific criterion is met, determined by the investigator. The criteria may include ECG abnormalities, the subject's request, or reaching a plateau in oxygen consumption and CO2 release regardless of further increase in loading. • After termination of the test, the patient remains on the veloergometer for post-test procedures.

• Derived indices from the test include:

  • Oxygen consumption: Amount of oxygen used by the body in its oxidation processes per minute.

  • Oxygen consumption per kilogram of body weight: Oxygen consumption normalized for body weight.

  • Released CO2: Amount of carbon dioxide released by the body per minute.

  • Released CO2 per kilogram of body weight: Released CO2 normalized for body weight.

  • Heart rate: Number of heartbeats per minute.

  • Respiratory quotient: Ratio of CO2 production to O2 consumption, providing insights into the type of fuel being oxidized.

  • Oxygen pulse: Amount of oxygen uptake per heartbeat at rest. • These measurements help assess the individual's aerobic capacity, metabolic efficiency, and respiratory response to exercise.

Practical 16) Recording and analysis of the ECG. Page 172

ECG Leads and Placement:

• Bipolar leads:

  • Lead 1: Right arm (–), Left arm (+)

  • Lead 2: Right arm (–), Left leg (+)

  • Lead 3: Left arm (–), Left leg (+)

• Unipolar augmented limb leads:

  • aVR: Right arm + neutral point

  • aVL: Left arm + neutral point

  • aVF: Left leg + neutral point

• Unipolar chest leads:

  • V1 to V6 placed at specific chest points (4th to 5th intercostal spaces, various lines)

• Electrode placement:

  • Attach limb leads on volar forearms and medial lower legs

  • Use conducting gel or Ringer-soaked pads to reduce resistance

  • Color code electrodes for easy ID (e.g., red = right arm, yellow = left arm, green = left leg, black = right leg ground)

• Chest electrodes: Use suction cups with gel on marked chest spots

ECG Recording Settings:

• Calibrate stylus to deflect 10 mm for 1 mV signal

• Use paper speed of 25 or 50 mm/s

• Standard amplification settings

ECG Waveform Key Points:

• P wave: Atrial excitation; duration ≤ 0.08 s

• PQ segment: AV node delay; isoelectric line; ≤ 0.08 s

• PR interval: Time from atrial to ventricular excitation; ≤ 0.2 s

• QRS complex: Ventricular excitation; duration ≤ 0.08 s

• ST segment: Full ventricular excitation (plateau)

• T wave: Ventricular repolarization

Practical 17) Sphygmography (Page 195)

Sphygmography:

• Records arterial pulse graphically using a sensor.

• The sphygmogram shows the pulse wave of the arterial wall.

• Carotid sphygmogram: Graph of carotid artery pulsations.

Key parts of the sphygmogram:

• Anacrote: Rising slope of pulse

• Anacrotic peak: Highest point (systolic pressure)

• Catacrote: Sharp downward slope

• T wave: Late systolic wave

• I notch: Aortic valve closing

• D-dicrote: Small second rise (dicorotic notch)

Procedure:

• Patient lies down relaxed.

• Palpate carotid pulse.

• Attach sensor to carotid artery.

• Record pulse after a short breath pause.

Practical 18) Measuring the arterial blood pressure (Pages 205-207)

Blood Pressure Measurement Procedure:

• Use sphygmomanometer (cuff, pump, valve) and stethoscope.

• Perform in a quiet, comfortable room.

• Patient sits or lies with head raised 45°.

• Place cuff on upper arm, ~2.5 cm above elbow crease.

• Inflate cuff to about 30 mmHg above expected pressure (max 150 mmHg).

• Slowly release air while listening with stethoscope.

• Systolic pressure: First clear sound heard.

• Diastolic pressure: Sound disappears.

• Take 3 readings, 30 seconds apart, and average results.

Practical 19) Combined functional test of the cardiovascular system.

Exercise Protocol & Assessment (H.H Criteria)

Stage 1: 20 knee bends in 30 seconds (warm-up)
Stage 2: Sprint in place for 15 seconds (3 minutes after Stage 1)
Stage 3: Run in place for 3 minutes at 180 steps/min (4 minutes after Stage 2)

Monitoring:

• Heart rate & BP checked:

  • 3 min after Stage 1

  • 4 min after Stage 2

  • 5 min after Stage 3

• Pulse: Taken in first 10 sec of each minute

• BP: Taken after pulse

Response Types (H.H Criteria):

• Normotonic: HR & systolic ↑ (160–180 mmHg), diastolic stable; all return to normal within 3–4 min post-exercise.

• Hypertonic: Systolic >200–220 mmHg, diastolic normal; common in teens and athletes.

• Diastolic Type: Diastolic >95 mmHg, systolic rises with exercise intensity.

• Systolic-Diastolic Type: Systolic >220 mmHg & diastolic >110 mmHg; indicates hypertension.

• Hypotonic: Slight systolic rise, HR 180–190 bpm, delayed recovery; poor exercise adaptation.