Download Physiology Lab Question 1

Frédéric Zuhorn
Physiology
EXAMINATION OF BLOOD
Physiology Lab Question 1
Determine the bleeding time, clotting time and the prothrombin time (prothrombinindex).
Bleeding Time:
- time elapsed gives information about extrinsic pathway of blood coagulation and
platelet capacity
- puncture a finger, wipe off the first blood and measure time until bleeding stops
- normal bleeding time = 1-3 min
Clotting time:
- use venous blood, as blood from finger tip contains tissue fragments (extrinsic
pathway)
- measurement of intrinsic clotting activity (done before operations)
- 6 drops on a glassplate, dip in with a glass rod every 30 sec, measure time until it has
formed a clot
- normal clotting time = 5-8 min
Prothrombin time:
- anticoagulation of fresh blood with calcium ions, centrifugation to obtain plasma
- sample is heated up to 37 °C in a waterbath
- 0.1 ml of plasma and 0.2 ml of thromboplastin (for the conversion of prothrombin to
thrombin – which cleaves fibrinogen to fibrin) are mixed in a watch glass with a glass
rod and time is measured
- normal prothrombin time = 12-15 sec
- prothrombin index = normal time / measured time * 100
Physiology Lab Question 2
Determination of protein content of serum
-
Biuret reaction – photometric concentration measurement – presence of peptide bonds
5 ml biuret reagent to two test tubes
0.1 ml serum to one test tube and 0.1 phys. NaCl sol. to the other test tube (as a
standard solution)
incubate at room temperature for 30 min
measure extinction (E) at 555 nm
E of sample / E of standard * conc. of standard = conc. of sample
normal protein content = 60-80 g/L
Frédéric Zuhorn
Physiology
Physiology Lab Question 3
Describe the principle and the technique of the packed cell volume (haematocrit)
determination
-
percentage of RBC over total blood volume
anticoagulated blood is filled into capillary tubes (using capillary attraction)
tubes are closed with clay at one end
placed into centrifuge for about 5 min
values are measured with a special ruler, to read off the percentage
normal haematocrit value = 44 %
anemia = 30 % , polycythemia = 70 %
Physiology Lab Question 4
Determine the specific gravity of blood (according to Hammerschlag).
Determine the specific gravity of blood and blood plasma (according to Philips-Van
Slyke)
Hammerschlag:
- composed of 1 part of chloroform (spec. gravity = 1.45) and 2 parts of benzol (spec.
gravity = 0.85)
- add one droplet of blood:
o if floating on the surface, spec. gravity of solution is higher than of blood – add
benzol
o if floating in the middle, same spec. gravity
o if sinking to bottom, blood has a higher spec. gravity than solution – add
chloroform
- filter solution into a flask to get rid of the blood drop
- measure spec. gravity with floating ergometer
- normal spec. gravity of blood = 1.06 g/cm³
Philips-Van Slyke:
- coppersulfate solution with different concentration and known spec. gravity
- add one drop to each solution
- if floating in the middle, same spec. gravity
- normal spec. gravity of
o blood = 1.06 g/cm³
o plasma = 1.03 g/cm³
o RBC = 1.09 g/cm³
Frédéric Zuhorn
Physiology
Physiology Lab Question 5
Determination of osmotic resistance of RBCs
-
-
osmotic resistance = osmolarity of a solution that can be resisted by cells
o isotonic solution – 0.9 % NaCl (for frogs = 0.65%)
o hypotonic solution – less than 0.9 % NaCl
--- swelling of cells
o hypertonic solution – more than 0.9 % NaCl
--- shrinking of cells
take 8-10 test tubes with different NaCl concentrations
place a drop of blood in each tube, shake and centrifuge them
no hemolysis – RBCs can be seen on the bottom of the tubes with transparent fluid
above
if hemolysis happened (at lower NaCl conc.) – solution is more red and less RBCs are
seen on the bottom
normal values:
o start of hemolysis = 0.4 %
o complete hemolysis = 0.3 %
Physiology Lab Question 6
How do you determine the erythrocyte sedimentation rate of a blood sample?
-
-
blood is anticoagulated with sodium citrate
1 vol. of anticoagulant to 4 vol. of blood
thin glass tube is filled to mark zero and placed in a vertical position for one hour
normal sedimentation rate:
o male = 3-8 mm/h
o female = 6-11 mm/h
increased rate – pregnancy, inflamation, tumors, anemia
decreased rate – polycythemia, increased blood viscosity
Frédéric Zuhorn
Physiology
Physiology Lab Question 7
How do you determine the haemoglobin content of blood?
Spectroscopic examination of gas-compounds of haemoglobin.
Hemoglobinometer:
- drop of blood on cover glass with H-shaped groove
- mix with hemolyzing stick and put the other cover glass on top
- look through the meter, where you see a green field divided into two (one standard
colour and one changeable colour)
- change the colour until they are homogenous and read the value for the Hb-conc.
Chemical method:
- venous blood is anticoagulated with EDTA
- prepare two test tubes:
o 5 ml drobkin reagent, 0.02 ml distilled water
o 5 ml drobkin reagent, 0.02 ml blood
- wait for 15 min
- measure absorbance (A) with a photometer at 540 nm
- Calculation: Hb (g/L) = A of sample / A of standard * concentration of standard
- Normal Values:
o Male = 140-180 g/L
o Female = 120-160 g/L
Spectroscopy:
- black tube containing two prisms, placed in front of test tubes
- Oxyhemoglobin: 2 absorbtion bands at 540 nm and 578 nm.
o Sodiumdithionate reduces Hb – two bands will fuse at 555 nm.
- Carbonmonoxidhemoglobin: 2 absorbtion bands at 535 nm and 572 nm.
o Cannot be reduced – bands do not fuse
- Methemoglobin (Fe3+ instead of Fe2+): 5 absorbtion bands
o Hb is oxidized by potassiumferricyanide
o binds oxygen covalently, adults have Met.Hb-convertase – babies don’t
o fatal if drinking water with too much nitrate is given to babies
Frédéric Zuhorn
Physiology
Physiology Lab Question 8
How do you determine the red blood cell count?
-
fill dilution pipette with blood until mark 1
fill the pipette with hayem solution until mark 101
hayem solution is a hypertonic solution, we use it so that RBCs don’t stick together
mix and discard the first few drops
fill both sides of the bürker chamber and place under microscope (magnif.: 40*)
count 20 small squares on each side and take the average
multiply the average count with 100 (for dilution) and 4000
normal RBC count:
o Male = 4.3-5.6 million per mm³
o Female = 3.9-5.3 million per mm³
Physiology Lab Question 9
How do you determine the white blood cell count?
-
fill dilution pipette with blood until mark 0.5
fill pipette with türk solution until mark 11.0
türk solution is a mixture of acetic acid and gentian violet, that hemolizes the RBCs
and stains the nuclei of WBCs blue
mix and discard the first few drops
fill both sides of the bürker chamber and count 25 squares on each side
take the average and multiply with 10 (for dilution), 10 (for height of chamber) and 25
(for the area)
eg.
90/25 = 3.6 3.6*10*10*25 = 9000 WBCs
normal WBC count = 4000-9000 cells per mm³
Increase – bacterial infection, leukemia
Decrease – viral infection, bone marrow damage
Frédéric Zuhorn
Physiology
Physiology Lab Question 10
Determine the diameter and volume of red blood cells.
Mean haemoglobin content (MCH) and haemoglobin concentration (MCHC) of
erythrocytes.
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-
use eyepiece micrometer and calibrate it according to bürker chamber
anticoagulated blood is diluted 10 times with 0.15 mol/L NaCl solution
one drop of diluted blood is placed on a slide and covered by a coverslip
measure diameters of 200 RBCs at the same magnification as used for calibration
plot a “Price-Jones Curve“
o normal – center is around 7 micrometer
o shift to the right – macrocytosis – sign of cancer
o shift to the left – microcytosis – Fe and Vitamin B12 deficiency
o flattened – unicytosis – bone marrow problems
normal RBC diameter = 7.0-7.5 micrometer
Volume of RBC (MCV):
Hct / RBCs per mm³ = MCV (femtoliter)
Mean Hb content (MCH): Hb (g/L) / RBCs per mm³ = MCH (picograms)
Mean Hb conc. (MCHC): Hb (g/L) / Hct = MCHC (g/L RBC)
normal values:
o MCV = 82-92 fl
o MCH = 28-36 pg
o MCHC = 310-360 g/L
Frédéric Zuhorn
Physiology
Physiology Lab Quetion 11
How do you make a blood film?
Describe the morphological and staining characteristics of the different white blood cell
types.
Give the differential leukocyte count.
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use two slides and put a drop of blood on one end of a slide, put the other slide in front
of the drop at an 45° angle and drag the blood to the other end
allow the blood film to dry in air
fixation of cells in conc. May-Grünwald solution for 3 min
staining with diluted May-Grünwald solution (1:1 with water) for 3 min
washing with water
staining with diluted Giemsa solution (1:15 with water) for 10 min
washing with water and drying in air
Leukocytes: 4500-8000 cells/microliter
o Neutrophils:
 multilobed nucleus
 no cytoplasm stained
 diameter = 10-12 micrometer
 50-70 %
o Eosinophils:
 bilobed nucleus
 granules in cytoplasm stained red
 diamenter = 13 micrometer
 2-4 %
o Basophils:
 blue granules of cytoplasm cover nucleus
 diameter = 8-14 micrometer
 0.5-1.0 %
o Monocytes:
 kidney shaped nucleus
 diameter = 12-15 micrometer
 2-8 %
o Lymphocytes:
 large dark nucleus
 blue cytoplasm
 diameter = 8-12 micrometer
 30-40 %
Frédéric Zuhorn
Physiology
Physiology Lab Question 12
How do you determine the reticulocyte count?
How do you determine the platelet count?
Reticulocyte count:
- prepare a film of “brilliant cresyle blue staining“ on a slide and let it dry
- place a drop of blood (preferebly of a woman) in the middle and put a coverslip above
- leave it for 10 min incubation
- use immersion oil microscopy and count 500 RBCs and how many have blue stained
reticulum in the middle ( = reticulocytes)
- normal reticulocyte count = 5-15 per 1000 RBCs
Platelet count:
- mix 0.1 ml blood with 0.9 ml “Reer-Ecker solution“
- after centrifugation, the platelets are in the supernatent
- 1 drop of supernatent is placed in the bürker chambers
- count platelets of 40 rectangles and take the average
- multiply the average with 1000 (for volume) and 10 (for dilution)
- normal platelet count = 150000-300000 per mm³
Physiology Lab Question 13
How do you perform A, B, 0 and AB blood grouping?
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on RBCs are antigens coded by the specific genes one person has
have a white plate with three kinds of monoclonal antibodies (anti-B, anti-A and antiAB) and a control
mix with unknown blood and stir carefully
precipitating at anti-A and anti-AB, means it is Blood Group A
precipitating at anti-B and anti- AB, means it is Blood Group B
precipitating at all three, means it is Blood Group AB
Blood Group 0 does not react with any antigens
Physiology Lab Question 14
How do you perform Rh blood grouping?
-
6 Rh-antigens are expressed on the RBC surface, controlled by 6 genes in 3 allele
pairs (c,d,e,C,D,E)
Rh+ persons have D-antigen
Anti-D antibodies do not cause agglutination, unless reaction is done in a protein
containing medium or facilitated by anti-immunoglobin antisera
Place two drops of blood on each side of a slide
Put Anti-D on one drop and NaCl on the other
Place the slide in moisturising chamber, to prevent drying of blood and incubate for 30
min at 37°C
If agglutination occurs – Rh+
If no agglutination occurs – Rh-
Frédéric Zuhorn
Physiology
HEART AND CIRCULATION
Physiology Lab Question 15
Electric and Thermal Stimulation of the Heart. Demonstration of extra systole.
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anastatize a frog , decapitate it, destroy spinal cord, cut open the chest and cut the
pericardium
- attach the apex of the ventricle to the little serafin-clip, which is in connection to the
registration unit (transducer & computer)
Thermal Stimulation:
- use little cupper rods, that have been placed in hot or cold water, to stimulate the SA
Node
o HOT – frequency increases – due to increase of ion diffusion (Na+ influx)
o COLD – frequency decreases – due to decrease of ion diffusion (Na+ influx)
- place the rod at the ventricle
o HOT – larger contraction, same frequency – increase in calcium conc.
o COLD – smaller contraction, same frequency – decrease in calcium conc.
Electric Stimulation:
- with the biostimulator, you can control voltage, frequency and duration of stimulus (2
Hz frequency, 5 ms duration)
- stimulate right after a contraction, at the relative refractory period – thereby, you will
observe an extra systole, followed by a longer compensatory pause
- if you stimulate before a contraction, at the absolute refractory period, you will not
observe an extra systole
Frédéric Zuhorn
Physiology
Physiology Lab Question 16
Experiments on the excitatory and conductive system of the heart (ligatures of Stannius).
Demonstration of the “all or nothing“ law. Summation.
Stannius Ligation 1:
- thread between sinus venosus and the atrial border
- after performing the ligature, the heart stops beating for 15 min
- when the heart regains beating, it has two frequencies
o sinus venosus – normal (60 per min) – as it receives stimuli from the SA Node
o atria&ventricle – 40 per min – as it receives stimuli from the AV Node
StanniusLigation 2:
- thread between atria and ventricle
- first heart will stop, then regains beating – it will have three different frequencies
o sinus venosus – SA Node – 60 per min
o atria – AV Node – 40 per min
o ventricle – upper part of His bundle – 20 per min
The difference between the frequencies is because of the difference in relative membrane
potential of the SA Node, AV Node and His bundle
All or Nothing Law:
Once the heart stoped beating, we find its threshold. Then apply a stimulus higher than its
threshold – the whole heart will contract, regardless where the stimulus was given. Heart
muscles are connected by gap junction, so if one part is stimulated, the whole heart responds.
Summation:
Once the heart stoped beating and we know its threshold, we apply a stimulus at a voltage
just below threshold and increase the frequency.
Each stimulus results in a certain amount of ion transfer, but not enough to produce an action
potential. At a certain frequency (if high enough), we regain a regular systole
Frédéric Zuhorn
Physiology
Physiology Lab Question 17
Electrocardiogram. Recording of ECG.
-
SEE THIEME PAGE 196-199
ECG- registration of mass electric activity of the heart, conducted to the body surface
Leads:
o Bipolar Einthoven leads (Limb leads: 1, 2 & 3)
o Unipolar Goldberger leads (Augmented Limb leads)
o Unipolar Wilson leads (central terminal electrode)
Bipolar Einthoven Leads:
- two electrodes are connected and their potential differences are measured and
registered
- from these you can construct the electric axis of the heart (right arm is negativ, feet are
positiv)
o measure the high of the R-waves of the three limbs
o draw a triangle – from the mid point of each limb of the triangle, measure the
vectors so that the mid point of the vectors correspond to the midpoint of the
triangle limb
o draw perpendicular line at beginning and end of vectors towards middle of
triangle
o you can now draw an arrow in the middle where all three pairs of vectors meet
Unipolar Goldberger Leads:
- using the same electrodes as before, we combine two of them (so that they cancel
eachother out with their electric resistance) – this will give us an indirect electrode
with no electric charge
- pick the third electrode and register the electric potential between them
- this gives us multiple points of view on the frontal axis of the heart, by 30° changes
Unipolar Wilson Leads:
- all three electrodes are combined by a large electrical resistor
- by placing additional electrodes on the chest, we can see more of the frontal plane of
the heart
ECG Curves (P, Q, R, S, T, U)
- P
=
depolarisation of atria
- PQ
=
time before contraction
- QRS =
depolarisation of ventricles & repolarisation of atria
- ST
=
refractory period
- T
=
repolarisation of ventricles
- U
=
repolarisation of papillary muscls
Frédéric Zuhorn
Physiology
Physiology Lab Question 18
Heart Sounds (phonocardiogram)
-
„LUB-DUB“
Heart sounds are generated by and therefore corresponds to the different valves that
are closing
sounds correlate to ECG: 1st sound at QRS complex, 2nd sound at end of T-wave
aortic valve:
2nd intercostal space, right parasternal
pulmonic valve:
2nd intercostal space, left parasternal
mitral valve:
5th intercostal space, left midclavicular line
tricuspid valve:
4th intercostal space, right parasternal
Problems:
o Sternosis: scar tissue – shrinkage of valve – observed during diastole
o Regurgitation: cannot close properly – observed during systole
Physiology Lab Question 19
Effects of vagal and sympathetic stimulation on the heart. Goltz reflex.
- applying stimuli to nerves or administering chemotransmitters
- vagal stimulation – negative effect – acetylcholine
- sympathetic stimulation – positive effect – adrenaline
Procedure:
- put a small tube or stick into frog’s esophagus, to extend area – you will see vagus
nerve – isolate it!
- find treshold on one of the surrounding muscles and apply stimuli little above treshold
with frequency of 2 Hz and duration of 5 ms (bipolar electrode connected to
biostimulator) to vagus and sympathetic nerve
Results:
- CHRONOTROPIC effect heart rate (rate of impulse generation)
- DROMOTROPIC effect
velocity of impulse conduction
- BATHMOTROPIC effect iritability of impuls conductivity
- INOTROPIC effect
force of contraction
- TONOTROPIC effect
muscle tone during diastole (resting tone)
C
VAGAL
NERVE
D
B
SYMP.
NERVE
I
T
Goltz Reflex:
- do not destroy the frog’s spinal cord for this experiment
- place preperation on the back, disect to get the ventricle on the clip (transducercomputer)
- hit the belly of the frog – this may give large vagal stimulation, that may stop the heart
Frédéric Zuhorn
Physiology
Physiology Lab Question 20
Experiments on isolated frogs heart by means of Straub cannula.
Effects of various ions on isolated heart.
Examination of effects of acethylcholine and adrenaline.
-
usual preperation of frog and heart – then tye off the right aorta
make a cut into the left aorta and insert the cannula, until you reach the ventricle
as soon as the cannula is inside the ventricle, the ringer solution that is in the cannula
is pumping up and down according to the contrctions
attach apex to the clip (transducer and computer)
change ionic medium inside the heart
o excess Calcium – heart stops in systole
 as actin and myosin depends on itracellular calcium conc – so if there is
a high calcium conc, relaxation of muscle does not take place and
muscles contract until the whole heart stops
o excess Potassium – heart stops in diastole
 passive diffusion out of the cell is not possible, therefore, repolarisation
is impossible – heart stops
o acetylcholine decreases heart rate, adrenaline increases heart rate
 see Lab Question 19 – similar to vagal & sympathetic stimulation
Physiology Lab Question 21
Demonstration of neurotransmission on frog’s heart after Loewi
-
prepare two straub canulated hearts (one with intact isolated vagal nerve)
stimulate the vagal nerve electrically and add to its ringer solution “acetylcholine
esterase inhibitors“ (eg. eserin & physostigmin)
after constant vagal stimulation, the heart will stop
remove ringer solution from this preperation and place it to the second preperation
second preperation is not stimulated but will act in the same way
this proofs chemotransmitters, as chemicals released from preperation 1, that stoped
the heart, causes the preperation 2 to stop
CDBIT effects …
Frédéric Zuhorn
Physiology
Physiology Lab Question 22
Study of peripheral circulation in frog’s blood vessels
(examination of Frog’s tongue; experiment of Laewen and Trendelenburg)
-
-
frog is anastatized (turning effect must be absent)
take out the tongue and flatten it under the microscope to observe blood vessels
o a big vessel distibuting blood to smaller vessels = artery
o several small vessels distribute blood to one big = vein
o if walls are transparent and RBC strech one by one through a vessel = capillary
after injection of adrenalin, you observe shrinking/contration of vessels (esp. arteries)
Laewen & Trendelenburg experiment:
- cut frog in half and use the abdominal part for this experiment and the other for a heart
experiment
- open abdomen and remove all organs except kidneys
- tye off arteries of kidneys, the cloaca venous plexus and cystic arteries
- insert a small flexible cannula into the abdominal aorta and abdominal vein
- attach transfusion bottle to cannula – if enough hydrostatic pressure, you see drops
coming out of the abdominal vein
- count the drops per minute
- by changing the height of the transfusion bottle, more or less drops come out of the
abdominal vein
- when injecting adrenalin solution, you can observe a decrease in the number of drops,
due to vasoconstriction
- when injecting acethylcholine solution, you can observe the opposite, due to
vasodilation
Frédéric Zuhorn
Physiology
Physiology Lab Question 23
Examination of the arterial pulse. Index of physical condition.
-
normal pulse = 60-80 beats per minute
measure the pulse using three fingers at for example the temporal, carotid, axillary,
cubital, radial, femoral, popliteal, posterior tibial and dorsalis pedis ARTERIES
- check different pulse qualities:
o regularity – time interval between beats
o rate – number of beats per minute (tachicardia – fast; bradicardia – slow)
o amplitude – height of your finger lifted
o quickness – time for amplitude to reach min. and max.
o hardness – apply force with your finger and to block pulse wave
o width – how much the artery is filled with blood
- Hypertensive: fast rate, large amplitude, very hard
- Fainting: slow rate, small amplitude, soft and thin
Index of physical condition:
- physical fitness index (PFI), by measuring only pulse
- check how long it takes for a patient to get to normal pulse after physical exercise
o check pulse – climb on and off steps for 5 min – then measure pulse every
minute for three minutes
o PFI = 100 * exercise time (s) / 2 * sum of measured beats
o compare result with a chart according to age, sex, etc.
Physiology Lab Question 24
Methods of measuring blood pressure in human.
-
normal blood pressure = 120 / 80 mmHg
direct and indirect measurements
indirect: place cuff on left upper arm, fined cubital artery and place your stetoscope on
it, start to pump air until artery is blocked, then let out the air very slowly until you
hear the carotid artery beating – this gives you the first (systolic) blood pressure;
continue until beating disappears – this gives you the second (diastolic) blood pressure
Frédéric Zuhorn
Physiology
Physiology Lab Question 25
Direct measurement of blood pressure in animal experiment.
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-
-
-
-
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anastatize the cat and make following preperations:
o insert a cannula into the trachea
o canulate one carotid artery and one femoral or saphenous vein
o isolate vagal and sympathetic nerves on both sides and the sciatic nerve on
opposite leg of femoral vein cannula
record normal curves and waves:
o primary wave: gives systole and diastole
o secondary wave: due to respiration
o tertiary wave: not always found – only after blood has ben treated with phys.
NaCl
Stimulation of Vagus nerve:
o Intensive stimulus will stop the heart – vagal escape
o After a while the heart will regain beating, but with a much lower frequency
o AV is now the pacemaker instead of SA (normally)
Injection into femoral vein:
o Acetylcholine – blood pressure decreases
o Low adrenaline conc. – stimulates β-adrenergic receptors – vasodilation –
decrease in blood pressure
o High adrenaline conc. – stimulates α-adrenergic receptors – vasoconstriction –
increase in blood pressure
Stimulation of sciatic nerve:
o Simulation of pain – stress – adrenaline is released in large dosage into the
blood – blood pressure increases up to 200 mmHg
Asphyxia simulation:
o Closing of trachea’s cannula - hypercapnia and hypoxia (increase in CO2 and
decrease in O2)
o Pressure response – intense chest movement
Frédéric Zuhorn
Physiology
Physiology Lab Question 26
Thoracic percussion and ausculation
Heart:
-
aortic valve:
pulmonic valve:
mitral valve:
tricuspid valve:
2nd intercostal space, right parasternal
2nd intercostal space, left parasternal
5th intercostal space, left midclavicular line
4th intercostal space, right parasternal
in addition to normal heart sound, we may have systolic or diastolic murmer caused b
stenosis and regurgitation
Lungs:
- ausculatory triangle: space between trapezius muscle, latissimus dorsi muscle and
lower medial margin of scapula
- inspiration: listen for normal “vesicular murmur“ (gental normal sound)
- expiration: no sounds in healthy individuals; however in allergic, asthmatic and
smoker patients, slight murmur may be observed
Trachea:
- normal bronchial murmur
- listen for additional sounds – blowing, rubbing, bubbling
Abdomen:
- 1-3 normal intestinal movements
- if these sounds are missing – life threatening – checked after delivery and surgery
Percussion:
- Dull: compact organs, no air, like heart and liver
- Resonating: organs filled with air, like lungs
- Box: large space filled with air, like stomach and colon
- Drumb: pathological – emphesema – rupture of alveolar walls – air filled space
Frédéric Zuhorn
Physiology
Physiology Lab Question 27
Spirometry. Volumes and capacities of the lungs. Vital capacity.
-
spirometer consist of a drum that is closed at one end and has an open bottom that is
placed in water
- tube with its end over the water level inside the drum, connects instrument with the
patient
- volume inspired or expired through the mouth piece is directly proportional to the
change in height of the drum (nose is closed with a noseclip!)
Volumes:
- Tidal volume (VT): patient at rest, calm breathing; 500 ml
- Inspiratory reserve volume (IRV): patient inhales as much as possible; 2500 ml
- Expiratory reserve volume (ERV): patient exhales as much as possible; 1000 ml
- Residual volume (RV): after max. exhaling, some air is still left in lungs; 1500 ml
- Minimal volume: air that stays in lungs even after death; 100ml
Capacities:
- Vital capacity:
VC = ERV + VT + IRV
- Inspiratory capacity:
IC = VT + IRV
- Functional residual capacity:
FRC = ERV + RV
- Total lung capacity:
TLC = all…
Physiology Lab Question 28
Determination of respiration pressure. Donders model.
- mercury manometer – transducer – computer
Maximal expiration pressure:
- patient blows out as much and as strong as possible into manometer
- normal exp. pressure = 40 mmHg
Maximal inspiration pressure:
- buccinator muscles must be relaxed
- normal insp. Pressure = 40 mmHg
Donder’s model:
- sealed container with buttom made of rubber and a balloon or rats lung placed into it
(both canulated, with canules open to the outside)
- rubber is analoque to the diaphragm – therefore demonstration of negative intrapleural
pressure
Respiratory muscles:
- Tidal Volume:
o Inspiration – diaphragm
o Expiration – no muscle activity
- Maximum Pressure:
o Inspiration – diaphragm & external intercostal muscles
o Expiration – abdominal muscles & internal intercostal muscles
Frédéric Zuhorn
Physiology
Physiology Lab Question 29
Effects of respiration on the filling of the heart
(Experiments of Müller and Valsalva)
Valsalva:
- after maximal inspiration, glottis is voluntarily shut, one tries to blow the air out
- pulse will weaken and disappears after a while, as intrathoracal pressure is very high –
this causes compression of the vena cava – therefor venous filling of the heart will
stop
Müller:
- after maximal expiration, glottis is volutarily shut, one tries to breath air in
- negative intrathoracal pressure will creat a vacuum – sucking effect of veins – large
amount of blood enters vena cava – overfilling and streching of the heart – pulse
disappears
Frédéric Zuhorn
Physiology
DIGESTION AND METABOLISM
Physiology Question 30
Examination of saliva
-
content of saliva is close to that of plasma, but also contains enzymes (amylase),
immunoglobins and lymphocytes
- daily excretion = 1.0-1.5 L
Examination:
- pH: (indicator paper) normal = pH 7
- Thiocyanite ions: found at high concentration in smokers
o Make a dilution of saliva (1:10) with distilled water
o Add ferrinitrate reagent – if saliva is positive, it will turn from yellow to
intense orange
- Enzyme activity:
o Mix 20 ml starch solution and 1 ml saliva
o Take 3 ml into another test tube and boil it
o Remainder is placed into water bath (40°C)
o Take out 2 ml after 2, 5, 10 and 20 minutes and put in two test tubes (each 1 ml)
o Perform Fehling’s test for reducing sugars: the more sugars – the more red
o Perform Iodine test for starch: the more starch – the more blue
Physiology Question 31
Determination of gastric acid secretion (BAO, MAO, PAO)
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patient is not allowed to eat or drink for 12 hours
patient swollows a gastric tube with oliva at the end to be placedat the end of the
stomach (observed via x-ray)
the entire stomach’s content is sucked out through a syringe – start stopwatch
after 1 hour, suck out everything and place into beaker labeled 0-min-fraction
inject “pentagastrin“ (stimulates HCl production)
in 15 min intervals, suck out everything and place into beakers labelled 15-, 30-, 45and 60-min-fraction
measure volume of all beakers and take 10 ml sample of each for titration (using 0.1 N
NaOH solution)
BAO = basal acid output: given by 0-min-fraction and value in millimoles per hour
MAO = maximum acid output: sum of four stimulated fractions in millimoles per hour
PAO = peak acid output: sum of two highest values in millimoles per hour
normal values:
o BAO = 3 millimoles/hour
o MAO = 18 millimoles/hour
o PAO = 9 millimoles/hour
If too low – hypoacid gastric juice – sign of cancer
If too high – hyperacid gastric juice – can cause ulcer
Frédéric Zuhorn
Physiology
Physiology Lab Question 32
Determination of lactic acid and pepsin
Lactic acid – Uffelmann reaction:
- 1 drop ferrichloride, 1 drop phenol, 3 ml distilled water
- to this add gastric juice – if lactic acid is present, we get a intense yellow colour
- usually there is no lactic acid in gastric juice, as lactic acid is produced by bacteria. So
if for example in the case of cancer the stomach does not produce enough HCl, the
enviroment for bacteria gets better (lower pH) and lactic acid is produced
Pepsin – Mett tubes
- glass capillaries filled with egg white and boiled
- put these tubes in three tubes (HCl, pepsin and both)
- place it in a water bath (40°C) and leave it for 1 hour
- egg white will dissolve in the test tube with both HCl and pepsin (HCl activates pepsin)
Physiology Lab Question 33
Examination of the exocrine pancreas.
Amylase determination in the serum and urine.
Pancreatitis: Dangerous disease, characterised by high amylase activity in serum and urine.
In case of pancreas obstruction, the enzyme is active inside the pancreas,
leading to self digestion of internal organs – DEATH
Phadelson Test:
- starch tablets conjugated to a blue dye
- place a tablet into diluted specimen (important for urine, due to high enzyme activity:
1/5 and 1/10) and put this in hot water bath for 30 min – if there is no amylase activity,
we will have a sedimentation and a clear supernatent
- if there is amylase activity, we will have a blue supernatent that, after centrifugation,
can be measured for extinctions by photometry (at 620 nm and with a standard
dilution as blank!)
- normal extinction:
o Serum = 100-300 international units (IU)
o Urine = 200-2000 international units (IU)
- In case of higher values – dangerous damage of pancreas
Frédéric Zuhorn
Physiology
Physiology Lab Question 34
Examination of bile.
Determination of bile pigments.
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the patient need to swollow a tube, that is placed inside the duodenum (Vater Papilla)
x-ray controlled – once at correct position, balloon are air filled so that tube remains at
position and that we get an isolated area
we suck out all the fluid discard it and wash out the area with warm NaCl solution
three types of bile:
o A-Type: yellow bile, spontaneous coming from bile duct, bile + duodenal
secretion + pancreatic juice
o B-Type: after injecting “glanduitrin“ intravenously – stimulates gallbladder
contraction – secretion of dark concentrated bile (microbiological examination)
o C-Type: gallbladder is emptied – liver produces fresh bile
Detection for bile acid:
o 3 ml of diluted bile, some drops of saccharose and underlayered concentrated
sulphuric acid
o if bile acid is present, we can observe a thin red ring at the contact surfaces
Detection for bile pigments:
o Gmelin reaction: 3 ml of diluted bile + conc. nitric acid underlayered: if
present – coloured ring
o Rosenbach reaction: filter bile through filter paper, flatten paper and put one
drop of nitric acid on it: if present – coloured rings
o Rosin reaction: bile + 1 ml iodine solution (2%) layered on top: if present –
coloured ring
Physiology Lab Question 35
Examination of faeces
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general: amount, pH, smell, consistancy, color
microscopically: presence of eggs or parasites, lack of food digestion (eg. muscles)
detection of blood: esophageal bleeding, hemorroids, GI-tract bleeding
o Benzidine probe:
 Dissolve extract with “Eter“
 Add this to a test tube containing 2 ml benzidin solution and 2 ml
sulfuric acid solution
 If colour changes to dark green, blood is present
o Hematest:
 Put sample on filter paper and place 1 hematest tablet on it
 Pipette some distilled water on it
 If blue ring appears at filter paper, blood is present
Frédéric Zuhorn
Physiology
Physiology Lab Question 36
How do you perform a routine urinary screening?
General: routine “sticks”, daily amount (normal 1 – 1.5 L), colour, smell, foam, pH (normal
pH 4.5-7.8)
Test tube tests: poteins, glucose, ketone bodies, blood, billirubin, specific gravity
Microscopic: urine sediment (cells like RBCs, WBCs and epithelial cells, parasites, chrystals)
Physiology Lab Question 37
How do you determine the specific gravity of urine?
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fill urine sample in a cylinder
put urometer into the sample and read of the scale
normal spec. gravity = 1.02 g/cm²
diluting probe: patient drinks 2 liters tea in ten minutes
o measure spec. gravity – must be around 1.002 – if not the kidney cannot dilute
the urine properly
concentrating probe: salty meal but no drinks
o measure spec. gravity – must be around 1.04 – if not the kidney cannot
concentrate the urine
Physiology Lab Question 38
Examination of protein, glucose and ketone bodies in the urine
Protein:
- usually not present in urine
- take a sample and add sulfosalicilic acid – precipitation – if positive
- if present we must check the “daily protein loss“ – biuret reaction (photometry)
Glucose:
- usually not present in urine
- Fehling’s and Nylander tests based on reducing properties
- Fehling’s test: mix Fehling 1 and 2 with urine sample and heat it up – if glucose is
present, colour changes from blue to red
- Nylander test: bismuth + urine heated – if glucose is present, colour changes from
colourless to black
Ketone bodies:
- usually not present in urine
- we use Legal probe, based on sodium-nitroprusside solution
- 5 ml urine + few drops of NaOH (to have a basic medium) + 10 drops of legal probe –
if ketone bodies are present, colour changes to violet
Frédéric Zuhorn
Physiology
Physiology Lab Question 39
Determination of haemoglobin, bilirubin and urobilinogen in the urine
Haemoglobin:
- must not be found in the urine
- Benzidine probe:
 Dissolve urine with “Eter“
 Add this to a test tube containing 2 ml benzidin solution and 2 ml
sulfuric acid solution
 If colour changes to dark green, blood is present
Bilirubin:
- present in small amount
- Rosenbach reaction: drops of urine on filter paper, conc. nitric acid drob – coloured
rings if positiv
Urobilinogen:
- normally found in urine (colourless & water soluble)
- Ehrlich reagent:
o Add it to urine sample
 no change at room temp. – colour change to red when boiled – normal
 no change at room temp. – no change when boiled – lack
 colour change at room temp. – too high
- lack of urobilinogen can be caused by bile stones or bacteria in the GI-tract
- high amount of urobilinogen can be caused by internal wounds, hepatitis, etc.
Physiology Lab Question 40
How do you examine urine sediment?
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put urine sample into centrifuge
discard the supernatant
shake pallet and place some on a slide, put coverslip over it and lie it under the
microscope
look for:
o cells
 usually not found
 RBCs, leukocytes, cancer cells, uroepithel cells
o crystals:
 usually not found
 caused by uric acid or calcium oxalate kidney stones
o cylinders:
 usually not found
 caused by kidney problems
Frédéric Zuhorn
Physiology
Physiology Lab Question 41
Determination of basal metabolic rate.
Measurement of oxygen consumption in rat.
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indirect method to measure the energy (E) cosumption involves the measurement of
oxygen consumption and from this the calculation ofenergy consumption
respiratory quotient (RQ) = carbon dioxide formed / oxygen used
for a certain RQ diet, a certain amount of E is released by usage of 1 liter of oxygen
o this is known as – “Heat-Energy-Equivalent of oxygen“
in an average diet:
o RQ = 0.82
o Heat-E-Equivalent = 20 kj / liter of oxygen
we use a spirometer, that is full of oxygen and soda lime, to absord carbon dioxide
expired
the constant usage of oxygen by the patient and th time is measured
Calculation example:
o 1.5 L oxygen used in 6 min – therefore – 15 L oxygen used in 1 hour
o oxygen consumed * Heat-E-equ. = 15 * 20 = 300 kj/h
o compare this with the body surface area (using the anogram given)
o Basal metabolic rate (BMR) = 300 / 1.8 = 200 kj/h/m²
o BMR = Oxygen consumed (per hour) * Heat-Energy-Equ. / body surface
following criteria have to be considered:
o patient is awake, resting, laying down
o no previous physical exercise
o avoid stress and emotions
o neutral temperature (28°C for naked person)
o patient did not est or drink for 12 hours
o no drugs
normal BMR values:
o males = 180 kj/h/m²
o female = 150 kj/h/m²
Rat experiment:
- place the rat into a closed container with oxygen and soda lime (to absord carbon
dioxide)
- immerse the container into a water bath (37°C)
- oxygen used by the rat, creates a vacuum inside the container, that is equalised by
NaOH solution (measured with a biuret)
- after measuring the time and the consumed oxygen (equivalent with NaOH sol. used)
we can calculate the BMR as before (using rat anogram)