Experiment: S 1

ANALYSIS OF BLOOD GASES

a) Determination of PO2, PCO2 and pHb) Calculation of Acid-Base Balance Parameters

I. Equipment

• Blood gases analysis equipment• Injector and anticoagulant substance

II. Purpose and General Information

The pressure that is applied by the gas to walls of chamber which it is put in ,is shown by ‘P’. This value can be expressed as mm Hg or torr. If it is assumed that there are more than one kind of gas in the chamber, the total pressure applied is the pressure that is applied by the every kind of gas, then they are put in the chamber by themselves (Daltons’ law). The pressure of each gas in the mixture is called as partial pressure. As it is known that the barometric pressure at sea level is 760 mm Hg. Dry air consists of %20,93 O2 and % 0.04 CO2 and % 79.03 N2. Oxygen’s partial pressure is shown as PO2 and carbondioxide’s partial pressure is shown as PCO2

In dry air, the values are:PO2 : 159,1 mm HgPCO2 : 0,3 mm HgPN2 : 600,6 mm Hg (including the gases found in small amounts)

The partial pressures of O2 and CO2 in the blood or any other body fluids can be measured easily by oxygen and carbodioxide electrodes. Lately,new equipments can measure the blood gases of tiny amounts of bood. These equipments can measure PO2, PCO2 and pH, normally in arterial blood, PO2 is 100 mm Hg, PCO2 is 40mm Hg. In arterial blood the normal pH may change between 7,36 and 7,44.

II.I. Oxygen Electrode: The partial pressure of oxygen in mixture can be measured with oxygen electrode when O2 molecules are in mixture with liquid or any other gases. Oxygen electrode is covered by a membrane that is permeable to oxygen. If it gets in contact with a sample, O2 molecules pass through the membrane to the electrolyte inside .At the end, the oxygen partial pressures inside comes to equilibruim at the same partial pressure of media. O2

molecules pass through the permeable membrane easily, but membrane is not permeable to the proteins and erythrocytes in the blood.

1

After some reactions betwen platinum cathode and silver-silver chlorure anode , an electrical current begins with flowing electrones and this current is increased by the amplifier. The increased current moves the scale needle. The movement of this needle is proportional with the flowing current which flows from the cathode to anode and O2 molecules arriving the cathode.

II.II. Carbondioxide Electrode: The CO2 electrode measures partial CO2 pressure (PCO2) which is formed by dissolved CO2 molecules in the blood or any kind of liquid, in mm Hg or torr units. The measurement of PCO2 is an indirect measurent. The CO2 electrode measures pH changes formed by CO2 molecules in the sodiumbicarbonate solution in electrode end. So, it is a pH electrode too. The teflon membrane on the CO2 electrode separates from sodiumbicarbonate solution then blood pattern. The teflon membrane permits CO2 molecules to pass, it prevents ion transfer. The teflon membrane is also electrical insulater, so inside of the CO2 electrode is completely insulated from the cuvette and water bath. When CO2 molecules dissolve into the aqueaus sodiumbicarbonate layer , CO2 reacts with the water. Carbonic acid forms and pH of the liquid decreases. When PCO2 increases tenfold, pH decreases one unit. On this occasion, pH is a lineer function of logarithm of PCO2. The pH of the thin layer is determined by measuring the potential differences between glass electrode and the calomel reference electrode. The lucite jacket surrounding the glass electrode is filled with electrolyte and a calomel reference electrode is in contact with this electrode. Only a thin layer of the electrolyte overlying the sensitive portion of the glass electrode comes into CO2 equilibrium with the sample. The diffusion in the edge side of the film can be neglected. The response time of the CO2 electrode is 0.5-3 minutes and this duration may change due to the different causes. Thickness of the teflon membrane is the most important factor.

2

Current Amplifier

PlatinumCathode

Silver-Silver ChlorureAnode

Scala

Membrane

ElectrolyteO2

Figure 1. Working of O2 electrode and amplifier.

II.III. pH Electrode: Measurement of pH in the blood and the other fluids is possible by reading a potential difference in a galvanic cell formed by the pH electrode.When electrodes put into the two sides of the capillary glass which is sensitive for H+ ions, the potential difference which is formed, is the value of pH. These electrodes are named pH electrode and pH reference electrode. Two buffer solutions known pH values are put into capillary tube. Calibration is done for each one. Then, blood sample is put into cleaned capillary tube and its pH is read.

III. Experimental Procedure

Equipments which are used for measuring blood gases,are perfect nowadays by the help of the technology.There are equipments which can make calibration itself and can be ready for measuring but, all these blood gas measuring have the similar systems.

3

Voltage Amplifier

Scala

GlassElectrode

Reference Electrode

ElectroliteMembrane

CO2 Sample

Figure 2. Working of CO2 electrode and amplifier.

Voltage Amplifier

Scala

Glass Electrode

Reference Electrode

Fluid of unknown pH

Figure 3.Working of pH electrode and amplifier.

III.I. Calibration Procedure: Before measurement, the calibration procedure must be done because the equipment needs to get ready for measuring. Different gas mixtures can be used for this aim.We can use this equation to reach PO2 of the gas whose % percent is known in the gas mixture:

PO2 (mm Hg) = O2 % × (barometric pressure - water steam pressure ) / 100

(water steam pressure at 37°C is 47mm Hg)

We can use this formula to get PCO2 of the gas if its % is known in the mixture. In every blood gas machines, PO2, PCO2 and pH electrodes look a capillary room. Capillary room is for blood samples whose volume is quite decreased, measurement. Capillary room and each three electrodes are in water bath. The temperature that measurements are done, is adjusted by changing the temperature of the water bath. Usually measurements are done at 37°C. Apply the calibration procedure given below for blood gas analysis.a) Water baths temperature is 37°C.b) For calibration of oxygen and carbondioxide electrodes, we use O2-CO2-N2 gas mixtures.By known concentration of the gas in the mixture, values of PO2 or PCO2 are found easily. In calibration of the oxygen electrode firstly, the zero gas is passed through the capillary room, in other words, in front of the electrodes. This gas is either pure nitrogen or pure carbondioxide. After a while when electrode gets in balance, oxygen electrode must show point zero. If not, by zero adjustment screw shown value is taken to zero. Then the gas, whose concentration is known and PO2 is calculated by the formula above, is passed through. After a while electrode has to show the known value. If it is not showing, it is adjusted by screw. In this manner, PO2 electrode is ready to measure.c) Calibration of the carbondioxide electrode is similar to calibration of the oxygen electrode. In other words; from capillary room that electrodes look to, two different gases which we know the CO2 concentrations are passed through. While each gas is passing from the capillary room, electrode is adjusted according to known value. In conclusion; if CO2 electrode knows two values of gas mixture, it can read unknown PCO2 value of blood.Notice: While PO2 electrode is being calibrated with a mixture of gas if this mixture has CO2, the first calibration of CO2 electrode can be possible, after the zero calibration. Because gas mixtures are passing in front of both PCO2 and PO2 electrodes.d) In calibration of pH electrode,two solutions of pH known are used. First one has a pH1=6.84 (at 37C) and the other is pH2=7.38 (at 37C). You see that the value of the buffers are very close to the arterial blood pH value. The first liquid is passed and setting of the electrode is done according to the pH value. After capillary room is washed and dried (usually air is passed from the capillary room to be dried), the second buffer solution is placed. Then the calibration is done due to the second buffer solution’s pH. So the equipment is now ready to be used.III.II. Experimental Procedure: There mustn't be any air bubble in the blood which will be taken (inner side of the syringe must be applied with heparin like a thin film).a) The blood sample is put to the capillary room where the three electrodes are in. During this procedure no air bubble must be in the capillary room. After any time, the blood gases and H+

iones come to a balance with the electrodes. Then PO2, pH and PCO2 values can be read.b) After these procedures, the blood must not be waited for a long time in the capillary room. That is to say, capillary room must be washed immediately. Vacum pump is runned. At the

4

same time, the poor water should send to enter to the capillary room. After the washing procedure is repeated 2-3 times, vacuum pump is runned 1-2 minutes without any water to dry the capillary room.III.III. Calculation of Acid-Base Parameters (By using Siggaard-Anderson Nomogram): After measurement of blood gases, acid -base balance parameters which are called as Act[HCO3], Std.[HCO3], Total-CO2 and Base Excess (B.E.) can be calculated with the known PO2, PCO2 and pH values by using a computer or a proper nomogram. Usually Siggaard-Anderson Alignment Nomogram is used. For these calculations, listed below procedures are applied.a) PCO2 and pH values are marked at the nomogram.b) A line, which is passing on these points, is drawned. From the points that the line cuts the bicarbonate scale and total carbondioxide scale, Act[HCO3] and total CO2 values are read.c) The point that the line cuts Hb=9mmol/L line, will give Base Excess (B.E.) (15gram Hb is nearly 9mmol), because of acceptance of the hemoglobin as 15 gram this line is used.d) If B.E. value and PCO2=40mm Hg point are united with a line, the point which this line intersects is Std.[HCO3].e) pH=pK+ log[HCO3]/PCO2 (Henderson-Hasselbach Equation). At this equation =0,0306 mmol/L mm Hg (at 37C) and pK=6,099 (pH=7,4 and at 37C) must be used. If PCO2 and pH values are put their places, Act.[HCO3] can also be found by calculation.

IV. Findings

PaO2: mm Hg (torr)

PaCO2: mm Hg (torr)

pHa:

Act [HCO3]: mEq/L

TCO2: mmol/L

Std.[HCO3]: mEq/L

B.E.: mEq/L

V. Discussion and Questions

1) Attention must be payed not to have any air bubble in the blood sample that taken by syringe. Why?2) The blood sample mustn't be stored for long periods except in water-ice mixture or outside of freezing compartment in ice box. Give reason ?3) Compare [HCO3] concentrations that are found by Henderson-Hasselbach equation and by nomogram. Are they different from each others?

5

Experiment: H 1

VISCOSITY MEASUREMENTS IN BODY FLUIDS

a) Determination of plasma and blood viscosity b) Determination of relation between viscosity and hematocrit

I. Equipment

• Harkness viscometer • Microhematocrit centrifuge• Microhematocrit tubes • Injector• Several liquid samples e.g. plasma, blood samples, distilled water, %0,9 NaCl solution, milimetric paper.

II. Purpose and General Information

Viscosity is defined mainly as the internal resistance of fluids and gases against flow.The difference of various fluids in viscosity is resulted by the particle content and structural characteristics.

The viscosity of fluids containing colloidal components, e.g. plasma, is determined by hydrophilic or hydrophobic characters of the colloidal components. Fluids containing hydrophilic colloids are more viscous than those containing hydrophobic colloids. The increase in viscosity of fluids containing hydrophilic colloids is more prominent than that containing hydrophobic colloids of raises the concentration in disperse phase concentration. (When hydrophilic disperse phase concentration increases, liquids viscosity increases more than the ratio of hydrophobic.) At Figure 1, viscosity diagram as disperse phase concentrations function can be seen.

Viscosities of body fluids such as blood, plasma, synovial vary with their contents of

6

Vis

cosi

ty

Hydrophilic

Hydrophobic

Figure-1 Viscosity diagram as disperse phase concentrations function

cells (erythrocytes, leukocyte etc.) and proteins (fibrinogen, albumin, globulin). These parameters are detectable by various laboratory methods (electrophoresis, viscometer, E.S.R.) and useful in clinical practice. The viscosity of pure water is 1mPa s (milipascal second) in the temperature of, 20oC, the whole blood viscosity is 3,0-4,0 m Pa s and plasma viscosity is 1,5 mPa s in the temperature of 37oC.

Erythrocytes are the main component affecting blood viscosity. The increase in hematocrit is reflected by the increase in blood viscosity (Figure 2). These changes are detected in large vessels. In vessels with diameter smaller than 100µm, however, the increase in viscosity per 1% increase in hematocrit is greater than in large vessels. In severe polycytemia, the increase in peripheral resistance increases the contractile force, which myocardium must produce. In the contrary in severe anemia, cardiac output increases despite decreased viscosity and peripheral resistance to provide 02 necessity hearts pumping volume in a minute increases. Thus no decrease in cardiac work ensues in anemia as expected.

The viscosity of fluid flowing in rigid pipes is calculated by this formula

=.r4.p.t / 8. v. l

In formula; =Viscosity(mpas),r = diameter (m), ∆p = pressure gradient(Pascal), t = time of passage(s), l=length of pipe(m). Note: The exercise uses kilograms, meters, and seconds, rather than grams, centimeters, and seconds. Viscosity can be measured in g-cm-s. with the resulting unit called the Poise, 10 Poise= 1 Pa s

Blood viscosity is inversely correlated with the deformability of erythrocytes. Although rise in plasma proteins is expected to increase blood viscosity, this effect may not be significant. In cases of increased erythrocyte rigidity e.g. hereditary spherocytosis, the blood viscosity rises.

The erythrocytes tend to flow in the center of the lumen vessels. Therefore, there are less erythrocytes in the branches originating from main vessels with a right angle of in 25%. This effect is called as plasma skimming and causes a decrease in viscosity in capillaries than in large vessels. The viscosity is higher in vitro than in vivo.

Figure 2. Effect of hematocrit difference on dependent viscosity.

Plasma proteins are the major determinant of plasma viscosity. These proteins are mainly fibrinogen that it has large and hydrophilic structure, albumin and globulins. Plasma

7

Rel

ativ

e bl

ood

visc

osity

Hematocrit%

Glass viscometer

The back extremity of the dog

protein levels are rise in inflammatory process and tissue damage. In acute inflammation, fibrinogen and -globulins levels increase. In chronic inflammation -globulins levels increase. These changes cause increase in plasma viscosity.

Figure 3- Harkness viscometer.

Glass parts(tubes and reservoirs) are assembled in a vertical plane. Capillaries and manometer parts are in water to be kept in a fixed temperature. Electronic timer with "start and stop" electrodes also assembled in the same vertical plane. A small vacuum pump to increase the Hg level and an order tap which commands the pump are the other parts of the tool (Figure 3).

III. Experimental Procedure

Important Notice: Don't touch any buttons or taps except the tool's timer reset button, vacuum pump and the tap connects with Hg reservoir. (If you have a problem, announce to the instructor.)III. I. Calibration of Viscosimetera) The tool is turned on by the “opening key”.b) 1 ml liquid is putted in the reference reservoir to determine its viscosity.c) The tap is turned on the left, which will secure the connection with the vacuum pump tomove Hg, level back to the start electrode. (If you have any problem on this step, announce it to the instructor.)d) When the timer is turned to the zero position, the tap is closed. The Hg, which slips out from the effect of vacuum, starts to flow back and it will drag the liquid on capillaries at this time.When start electrode contacts with Hg, the timer will start working, and when it reaches to thestop electrode, the timer will stop.e) According to the measure with distilled water, is calculated one of the liquid's relative viscosity (mPa s) by using formula given

n1=nw . t1 / tw

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Capillaries(2000.30mm)Timer electrode

Distilled water

Vacuum pump

Sample reservoir

Liquid sample(Plasma, blood)

Mercury

n1=viscosity of reference liquidnw=viscosity of water 0.693 mPa st1 =reference liquid's flowing timetw =water flowing time

Important Notice : Clean the reference reservoir and be careful, it has to be empty before all measurement.

III. II. Separation of Plasmaa) To obtain plasma sample 5 ml blood is centrifuged. b) After realization of all steps described above plasma viscosity measurement is done.

III.III. Measuring of Sample Liquida) 5 ml blood sample is taken,b) 1 ml blood is putted into four different tubes and 0.2 - 0.4 - 0.6 - 0.8 ml serum physiologic are added on them.c) Blood is taken from all tubes to the microhematocrit tubes and the Hct levels are read.d) The viscosity of blood sample are measured, which diluted with serum physiologic.e) The graphics relationship between viscosity and hematocrit are drawn.

IV. Findings

V. Discussion and Questions

1) Compare electrophoresis, E.S.R and viscometer in respect of their advantages and the disadvantages.2) Discuss the factors, which cause changes in liquid reference's relative viscosity.3) What's the connection between viscosity and Hct that you have found?

Experiment: T 1

9

MEASUREMENT OF BODY TEMPERATURE WITH THERMOCOUPLE DEVICE

a) Thermometryb) Regulation of Body Temperature

I. Equipment

• Electric universal thermometer (Type TE3)• Different type electrodes • Connection box that provides measurement from different electrodes at the same time.

II. Purpose and General Information

The measurement of body temperature is very important method in following a disease and its results. Mercury thermometers are used when the measurement of body temperature in a patient is not necessary to be measured continuously for example in clinics. In these days the measurement of body temperature with electronic devices is becoming widespread even they are not only used in researches they are started to be used in clinics too. For example the usage of these electronic thermometers is important to observe the skin temperature differences between various points of the body. The electronic thermometer that is sensitive to heat has two parts. The first part is called thermocouple, which is originated, from the combination of two different metals. The second part is called thermister, which is originated, from semi-conductor metal and its resistance changes with heat.In this experiment a thermometer will be used that depends on thermocouple principle. This thermometer’s working principle is as follows:Heating the combination point of two different metals that are combined to reach other causes potential difference in their free ends. This event is called ‘‘thermoelectric event’’. The voltage that is originated from these two metals depends on the type of the metals.

Figure 1. Heating the combination point of two different metals causes the origination of potential differences between free – ends.

The value of electromotor force(e.m.f.) for a unit of temperature difference depends on the distance of the chosen metal couple from each other at the order of electropositivity. Heating the combination point of these two metals causes the passage of the current from the preceding metal to the following one. This provides the conversion of heat energy to electric energy and this system of two different metals is called thermoelectric battery. The combination of one or more thermoelectric couple in the form of constituting e.m.f. is called

10

A B

Fe Cu

thermo battery. The increasing of thermoelectric e.m.f. with temperature provides a very good chance for measuring temperature.

Homoeothermic organisms can protect their body temperature, so their body temperature is always kept constant and independent from the external temperature. This is provided with the help of a control system of the body. Various regions temperature in the body is regulated with thermo receptors and internal thermo receptors that are located in skin and these are related and connected with the hypothalamus. If temperature of any region of the body shows a deviation from the ideal value the cooler and heater systems become activated. (For example: Contraction in the muscle structure that we say as shiving, changes in blood flow, heat production in brown fat tissue, activation of sweat glands.)

Figure 2. No current will pass from the galvanometer at the time that T1= T2 because of equality of e.m.f. s of the thermo battery at this time.

So in homoeothermic organisms thermo receptors are not only responsible for perceiving the external temperature they also take role in the control and regulation of the body temperature. They perceive the temperature of the regions that they are located and they are in connection with the hypothalamus. All these events in the body are called ’’thermoregulation’’

III. Experimental Procedure

III.I. Calibration:a) The button that is at right top of the equipment to control position.b) The light circle is adjusted that is seen on the screen to the red line on the scale with the adjustment (A) button at the left top of the equipment.c) The equipment is turned to ready position for measurement by taking the button at right top to measuring (M) position.

III.II. Observation of skin temperature differences in resting and after physical activity.a) The skin temperature is determined and recorded from at least five different points is chosen in the body in resting state.b) The experiment subject must make an exercise, which is related to this whole body at least five minutes.c) The skin temperature is determined and recorded at the points that measured in the resting state after the exercise.

11

A B

T1 T2

Cu

Fe

Cu

III.III. Regulation of temperature experiment.a) Temperature measurement from the left hand middle finger of the experiment subject with thermocouple is realized. (Middle finger and thermometer must be in contact with each other continuously in whole experiment and the measurement must be done continuously.)b) Experiment subject inserts his right hand to iced water for two minutes and than take it out of water.c) Measurements are continued from left hand middle finger for eight minutes and these measurements must be done in every minute for one time that must be started with inserting the hand to the iced water and must continue for eight minutes.d) A diagram is drawn that X axis substitutes for time (min) and Y axis substitutes for temperature (ºC )

IV. Findings

Before exercise After exercise Iced water Hot water

V. Discussion and Questions

1) Explain thermocouple principle.2) What are the factors effective on skin temperature?3) What is thermoregulation and how does it happen?4) What is importance of controlling the body temperature with classical mercury thermometer in clinics and what are the special cases that you need to control and follow the body temperature?5) In which cases the temperature control can be done in clinics and how?

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Experiment: R 4

DETERMINATON OF PLATEAU AND DEAD TIME WITH GEIGER-MULLER COUNTER SYSTEM

a) Appointment of plateau and optimum working voltageb) Appointment of dead timec) Inverse square law

I. Purpose and General Information

When an energetic charged particle passes through a gas, it ionizes some of its molecules. If the gas enclosed in a container between two charged electrodes, the negative ions (or electrons) are attracted to the positive electrode and the positive ions are attracted to the negative electrode. In order to collect the ions before they recombine with neutral molecules on their way towards the electrodes, a certain minimum voltage is required. If this voltage is raised, the ions are accelerated by the electric field between the electrodes, gaining increased velocity, until on collision with other atoms or molecules, they can ionize them in turn. This process continues, causing a greater number of ions to be collected by the electrodes than that of the original ions created by the direct hit of the charged particle; thus a greater current pulse is created.

There is a relationship between the size of the pulse and the applied voltage as shown at the figure below.

There are six main Geiger-Müller regions of operation. In region I (0-V0 Voltage) some ion pairs are collected on the electrode or there is a loss of ion pairs by recombination. The higher the voltage the less the recombination. In region II (V0-V1) the recombination loss

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is negligible, almost all ion pairs collects on anode, so this region is referred to as the saturation region or the ionization chamber region. In region III, there is a dependence of the collected charge on the initial ionization. This region is known as the proportional region. In region IV, the charge collected is independent of the ionization initiating it. It is known as the non-proportional region. In region V. there is not any dependence of the collected charge on the initial ionization, and known as the G-M region. In the region VI, there is a continuous disintegration.

I.I. Structure of Geiger-Müller Tube

The Geiger tube usually takes the form of a cylinder made of metal or glass coated inside with a conductive material (metal or graphite). A thin wire is stretched along the axis of the cylinder.

If the envelope is metal it may serve directly as the cathode. Stainless steal, nickel or other high work function materials make suitable cathode surfaces.

I.II. Characteristics of Geiger -Müller TubeAt very low voltages, the pulses will be so small as to escape detection. By gradually

increasing the voltage, a point is reached where the pulses begin to be detected. This is the threshold voltage”. From this point on, when voltage increased, the counting rate increases very quickly, until a point is reached where this rate remains constant. At this voltage the counting rate is almost independent of voltage. The voltage range, in which this condition prevails, is called the plateau”. In an ideal Geiger tube, this plateau is perfectly horizontal. In a practical tube there is usually a slight slope of a few percent. The range of the plateau is generally a few hundred volts. If the voltage is increased beyond the plateau, the count rate increases very quickly, until finally a state of continuous disintegration is reached in the gas, which usually causes damage to the tube. Obviously the tube should be operated within the plateau.Advantages of Geiger Müller:-A very large output pulse, independent of high voltage with no need for external amplification.-Simplicity and versality.

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The disadvantages of Geiger- Müller:-No disormination between types of radiation and the energy of radiation.-A limited counting rate, because of its high dead-time.

I.III. The Dead Time(Td) of Geiger -Müller TubeAfter an avalanche has occurred in the tube, there is a time duration called the dead-

time where the tube is not sensitive to the passage of new particles through it. The reason is that the electric field between the electrodes decreases because of the accumulation of positive ions in the tube. After the dead-time, when the positive ions have moved towards the cathode, the electric field again increases. So, new particles entering the tube are counted, but the current pulse obtained is small. The time interval between the dead time and the point where electric field reaches its primary value, is called “Recovery time”. If more than one particle has entered the tube within a time interval smaller than Td, then only one count is obtained. The dead-time in the Geiger tube is in the range of some hundreds of microseconds.

The dead-time may be measured by two methods:-By viewing the pulse on an oscilloscope and measuring the time difference between them-By counting the rate from two sources and wing the formula:

Td = (m1+m2-m1,2) / 2m1.m2

Where: m1 the counting rate from the first sourcem2 the counting rate from the second sourcem1,2 the counting rate from both sources together

Due to dead-time, the number of counts registered on the counter must be corrected. If Nc is the recorded counting rate, then the true counting rate NT is given by the formula:

NT= Nc / (1-Nc. Td)For example, if Nc=80counts/s and Td= 0,0005s, then:NT =80 / (1-(80)*(0,0005)) = 83 counts/s.

15

Thus the correction is only of 3,8 %. If the counting rate was 1000 per second, then the required correction would be 50 %. Thus we see that the Geiger-Müller tube is useful at low counting rates only.

II. Experimental Procedure

II.I. The plateau of Geiger-Müller tubePurpose: Determination of the plateau region and the choice of a working voltageRequired equipment : Geiger-Müller counter, radioactive source, stop-watch Working procedure:a) H.V. ADS is set to 0 volt.b) The G-M probe is connected to the DETECTOR INPUT socket.c) The instrument is switched ON.d) The radioactive source is attached to the round opening of the brass plate. The plate is inserted in a groove 2 cm away from the front of the Geiger tube.e) Pushed on the START switch and verify that the COUNT ON indicator lamp is illuminated.f) The H.V. ADS gradually increase is control until the threshold is reached.g) The counting time is fixed according to the desired accuracy and the count rate.h) The threshold voltage is started, the high voltage is increased in steps of 20 volts and the number of counts are measured at each voltage (for three times). All the measurements should be made during the same time interval chosen in step 7. Stop the measurement at the end of each interval and the number of counts are recorded.i) A graph of the number of counts are plotted as a function of the voltage level.j) From the graph is determined the plateau region. The slope is calculated.II.II. The dead-time (Td) of Geiger tube:Purpose: To measure the Td of the Geiger tube by two source method.Required equipment: Geiger Müller counter 2 gamma sources of equal intensity, stop-watchWorking procedures:a) The G-M counter is brought to operating condition.b) The first source is put as close as possible to the tube. The number of counts are measured until at least 40.000 count level is reached. The exact number is recorded and the time interval.c) The second source is added to the first one in front of the tube. The number of counts are obtained in a time interval longer than in the preceding step. Minimum error measurement will be obtained when this time is two times longer then in step 2.d) The first source is removed and repeated step 2 with the second source alone.e) The dead-time is calculated as explained before.Measurement of radioactivity require the subtraction of the background count rate from that of the sample plus background:II.III.a) The number of sample counts are measured for 2 minutes.b) The number of background counts are measured without radioactive sample for 2 minutes.c) The background counts are taken for 2 minutes and the number of sample counts per unittime.d) The absolute sample count rate is calculated from the equation below:e) Absolute sample count rate =(Sample plus background count rate) – (Background count rate /counting time).

III. Findings

16

Experiment: O 2

MEASUREMENTS BY THE MICROSCOPE

a) Measurements of the hair thicknessb) Measurements and comparison of human and frog erythrocyte diameter

I. Equipment

• Microscope • Ocular and objective micrometers• Dyes• Hair • Human and frog blood samples

II. Purpose and General Information

The optical equipment, which is used in order to observe too small specimen, is called microscope. As a principle of microscope consists of two approaching lenses. The biggest lens, which approaches to the object, is called objective, the lens, which is nearer to eye, is called ocular. The image of the object is reversed and magnified by the objective. This image is viewed at focal point. Observed at ocular the object is reversed, magnified and the image arrives to eye (figure 1)

The microscope, which will be used, is composed of two ocular. In one of them, measurement units are seen which are called ocular units. There are four different objectives related to the moveable table located at the top of the microscope tube. The part of microscope where objects are put on is called stage. In order to fix the specimen, slide stage position adjustments are used. These screws move it right-left or forward-backward to make them correspond. Above the stage there is a condenser and a mirror. At the right of microscope there are found three knobs. These knobs located from back: used to focus the light reflected from the mirror. Micro-knob and macro-knob. When measuring with the microscope, microscope tube approaches 1mm to specimen and elevating the objective only does the clearing of the view. Otherwise the specimen slide may be broken.

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ocularobjective

Figure 1.

Figure 2.

III. Experimental Procedure

III.I. Measuring hair measurement:a) A piece of hair is taken and put on the stage. Using the knobs the specimen is adjusted.b) Macro-knob the specimen is approached to the objective. Then looking from the oculars by using macro-knob the specimen’s view is cleared and gets larger. Using the micro-knob, you try to get the clearest view. Using the upper knob the glasses are slided in function to place the hair at the middle of glass ocular.c) The number of units covered (Figure 3) by hair thickness is calculated.

a’ =…. unitsd) Specimen slides are taken from stage. In their places objective micrometer is located. Above the objective micrometer it is fond 1mm scale at the center of the circle. As in B, we find a clear view. Both objective and ocular micrometer are compared and are calculated the amount of units occupied the objective micrometer into the ocular micrometer.

a”=…………units.

e) Thickness of hair is calculated as below:

18

Micron (µ)

Normal eyes Optic microscope Electron microscope

epith

el

eryt

hroc

yte

bact

eria

viru

s

prot

ein

mol

ecul

e

amin

o ac

id

atom

OBJECTIVES

OCULER

MICRO-KNOB

MACRO-KNOBMIRROR

a” (units)…..1mm a’ (units)…...x mmX= a’x 1mm/a”= …..mm

III.II. Finding out the diameter of erythrocytes (human and frog)a) Staining: A drop of blood is taken from the finger and dropped over the cover slip. Taking a second cover slip in order to have a homogenous preparation you apply it with an angle of 45 degrees. After dropping two drops of May Grunwald, you wait for two minutes and you distillate for one minute. Then after staining with GIEMSA you wait for seven minutes and wash it with tap water. Wiping the back of cover slip with alcohol, it’s ready to observe with microscope.b) Diameter Measurement: Before starting the experiment an appropriate objective has to be found (40 times). After the mirror is brought to the most shining position it is not moved anymore. The preparation is placed under the objective and a clear view is obtained. Then the amount of units occupied by the diameter of erythrocyte over ocular micrometer is calculated. This operation is reviewed 10 times. Found values are added and divided by 10, so you find the average diameter of an erythrocyte (a average=……) Erythrocyte preparations are taken and in their places objective micrometers are placed. Finding out the 1/100 mm scale of the objective micrometer you compare it with ocular micrometer (figure 3). Then you calculate how many ocular units are equal to 100 objective micrometer units. (a2=……).Erythrocyte diameter X= a average/ a2.By the same way, we can find the diameter of the frog erythrocyte.

Figure 3.

IV. Findings

19

OCULAR MICROMETER

OBJECTIVE MICROMETER

1) Measuring the thickness of the hair.The number of units occupied by the hair thickness………….a’ =………..The number of units occupied by the scale of objective microscope . a’ =………….Hair thickness …………. X=a’/a” =………………..2) Measuring the diameter of the erythrocytes.

The number of units occupied by the erythrocyte over ocular units.The number of units occupied by the objective micrometer scale over the ocular micrometer.

Human Froga average=………. b average=…………………..

a 2 =………………..

Diameters of the erythrocytesX1 = a average/a2 =………… X2 = b average/a2 =………….

Comparison of human and frog erythrocytesK= X2 / X1 =…………..

V. Discussion and Questions

1) What kind of microscopes is there?2) What’s the power of a microscope and what does it depend on?3) Explain the eye’s angle of view?

20

Experiment: S 2

DETERMINATION OF O2, CO2 AND N2 GAS CONCENTRATIONSIN EXPIRATION AND INSPIRATION GAS SAMPLES

a) Determination of respiratory quotientb) Determination of metabolism

I. Equipment

• Gallenkamp-Lloyd gas analysis apparatus • Paper with milimetric graphic• Polyethylene sample sacks • Gasmeter • Nose forceps• Chronometer

II. Purpose and General Information

Gallenkamp-Lloyd gas analysis apparatus detects the total volumes O2 and CO2 gases in expiration gases and in air within 0,02% error range. Working principle of apparatus depends on absorption of O2 and CO2 gases by certain solutions. The aim of the experiment is determination of any individual’s metabolism.

Figure 1.Gallenkamp-Lloyd Gas Analyzer

M: Five-way tap C1, C2: Semi saturated KOH and pirogallol reservoirs.K, P: CO2 and O2 absorption pipettes H: Mercury reservoir R: Connections for stabilityG: Inlet tubulure Ç: Outlet tubulure C.S: Glass cylinder B: Burette S: Back tap

21

Gallenkamp-Lloyd gas analyser is framed with wooden block. Each ‘U’ tubes placed on the left and right side of the item, has reservoirs. Between the ‘U’ tubes is placed a cylindrical graded tube(burette). The bottom edge opened of the burette is attached to reservoir full of mercury with solid plastic tube. The two peripheral reservoirs and central burette are connected to a five-way tap. Gallenkamp-Lloyd gas analyser is placed into a glass cylinder, which is full with water till tap level. Right reservoir is filled with 24 ml pirogallol and the left is filled with 24ml semi saturated KOH solution. Each of the solutions is covered with 12 ml liquid paraffin(the back reservoirs). And the mercury reservoir is covered with 30 ml pure, dry mercury.

The pirogallol absorbs O2 and the KOH absorbs CO2. But because of added sature KOH while preparing pirogallol, it has capability of absorbing CO2. So that initially we must make sample absorbed by KOH. If it is sure that there is no CO2 in the sample, we can directly start with pirogallol.

III. Experimental Procedure

After 25-30 measurements the solutions in apparatus have to change. Because the solution become saturated after using 25-30 use. After determination of O2% and CO2% values, N2 % is calculated by expelling the sum of O2% and CO2% from 100.

Make the item ready to use, it is initially calibrated with atmospheric air, for this procedure these steps are followed:a) To get rid of the air above the mercury while the main tap is connection with air, the mercury reservoir is elevated slowly.b) 10 ml air is taken inside by lowering the reservoir.c) The tap is connected to KOH.d) The level is regulated (according to reference line).e) The tap is connected to pirogallol.f) The level is regulated (according to reference line).g) The back valve is closed.h) Again the tap is connected to KOH, the level is regulated and the volume(V1)is read. i) The air sample is absorbed 3 times by elevating and depressing the mercury reservoir. j) The mercury level is read.k) The air sample is absorbed 3 times by elevating and depressing the mercury reservoir. l) The mercury level is read.m) Until the mercury level is stabilized, repeat the step “i”.n) The mercury level is read(V2)o)The tap is connected to pirogallol , this time don’t read mercury level.p) The air sample is absorbed 3 times by elevating and depressing the mercury reservoir. r) The mercury level is read.s) Until the mercury level is stabilized, step “p” is repeated. t) The mercury level is read.u) It is connected to KOH and absorbed 2 times. The level isn’t read.v) It is connected to pirogallol and absorbed 5 times, the level is regulated and read (V3 ).

22

Table 1.Nomogram to detect body surface (m2)(III), according to length(cm)(I), and weight (kg)(II).To put into practice put a ruler on weight and height values, read the body surface value (as m2) from the middle column.

23

Len

gth(

cm)

Bod

y su

rfac

e(m

2 )

Wei

ght(

kg)

Calculation:

First level for CO2: V1

Last level: V2 V1 – V2 = CO2 amount (ml).If the amount of CO2 is V1-V2 in V1; CO2 %amount;

100.(V1-V2)X=-----------------------=CO2% amount

V1

First level for O2 : V2

Last level : V3 V2 – V3= O2 amount (ml)

If the amount of O2 is V2 –V3 in V1; O2% amount;

100.(V2-V3)X= ----------------------= O2% amount

(V1)

For N2 : 100- (O2% +CO2% ) = N2% is found

All these procedures are done for calibration of the Gallenkamp-Lloyd gas analyser and the O2, CO2 and N2 percentage values of atmospheric air. After these, the following step is testing procedure of sample gas (unknown).

Table 2.Correction coefficients to convert standard condition of the sample gas volumes according to STPD ( 0oC, 760 mmHg and dry).

24

Gas temperature(0C)

Water steam

pressure(mm) Correction coefficients

III.I. Testing Procedure of Gas Sample a) The gas sample is collected and to KOH is connected. The level is regulated and the value

is read (V1=9,85ml).b) Gas is absorbed to KOH 5 times and then the level is read (9,85ml).c) Gas is absorbed to KOH 5 times and then the level is read.(V2=9,85 ml) (CO2 amount is zero)d) The tap is connected to pirogallol. The value is read (9,85ml).e) It is absorbed to pirogallol by the sample 10 times and the value is read (9,035ml).f) It is absorbed to pirogallol by the sample 10 times and the value is read (9,035ml).The value is read (9,02ml).g) The sample is connected to KOH and make it absorbed 2 times, but don’t read the value.h) The sample is connected to pirogallol and absorbed 10 times. The value is read (9,01ml).i) The sample is connected to pirogallol and absorbed 10 times. The value is read (9,01ml).j) By finding the same value is the end of procedure.

Calculation:

For CO2 V1= 9,85ml V2 = 9,85ml

V1 – V2 = No difference.CO2 amount is zero For O2 V2 =9,85ml

V3 = 9,01ml V2 – V3 = 0,84 ml 0.84ml O2 in 9,85ml X in 100ml-------------------------------------

100.0,84X=-----------------=8,27 O2% is obtained

9,85

For N2%: 100 – 8,27 = 91,83 is obtainedWe use the formula below to calculate the partial pressure of each gas by using their % value.

%value of gas (Barometric pressure- water vapor pressure)PO2=----------------------------------------------------------------------------

100

Example: O2% = 8,27Barometric pressure = 760 torrWater vapor pressure = 47 torr at 37 0CPO2 = 8,27 x (760-47) / 100 = 58,96 torr

25

R.Q. 0.68 0.70 0.72 0.74 0.76 0.78 0.80 0.82 0.84 0.86 0.88 0.90 0.92 0.94 0.96 0.98 1.00

Kal. 4.65 4.68 4.70 4.73 4.75 4.78 4.80 4.82 4.85 4.87 4.90 4.92 4.95 4.97 5.00 5.02 5.05

Table 3. Heat equivalences of 1liter O2 that fit to different RQ values.

III.II. Determination of Respiratory Quotient (RQ) Collect expiratory gas in a 100 lt volume Douglas bag, which is made of polyethylene and

is shouldered on the subject. For this, apply the tip of expiration - inspiration ventile to the subject’s mouth and close the nose with forceps. Connect the expiration tip of the ventile to the Douglas bag. Let the inspiration tip out being in contact with the atmospheric air. By the way the subject inspires the atmospheric air and let us collect expired air into bag during the experiment. Do this procedure for certain period. At the end of the experiment, collect atmospheric air in one of the bags and expiration gas in Douglas bag to the other bag. Measure the air volume that collected in Douglas bag by transferring it through a gas meter. This measured volume gives us the gas volume that saturated with water vapor, in body temperature, conditional pressure (B.T.P.S: Body Temperature Pressure Dry). Converting this measure into standard conditions (S.T.P.D: Standard Temperature Pressure Dry) 0oC, 760 torr and dry gas volume is required. For this multiply with appropriate correcting coefficient (Table 1) or calculate by using the formula below.

Vo = Vt (P – b) 273 / 760TVt = air volume by the measurement done in the normal conditions.P = conditional barometric pressure b = conditional water vapor pressureT = absolute temperaturet = conditional temperatureTo determine the concentrations of O2, CO2 and N2 in atmospheric air and expiration air, the concentrations O2, CO2 and N2 in sample sacks is determineted by using Gallenkamp-Lloyd gas analyzer. Some of the inspired O2 combines with hidrogen forming H2O, while some are converted to CO2. Besides that certain amount of the taken O2 is used in other ways in the organism.As a matter of fact as all of the consumed O2 doesn’t appear in the form of CO2 in the expired air, there will form a change in the percentage of the N2. Whereas the percentage of the N2 gas must be equal. To help keeping this equality, there is used N2 correcting formula.

N 2 correcting formula:Exp. N2% value

Insp. O2 %value x-------------------------Insp. N2% value

The new insp. O2 %value is considered the basis for the other calculations.

The spent O2 : New O2% value-exp O2% value STPD-----------------------------------------x Vo volume -----------=O2 ml

100 (For one minute)

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The excreted CO2: exp. CO2 %value-insp. CO2 %value STPD-------------------------------------------x Vo volume --------= CO2 ml

100 (For one minute)

brought out CO2

RQ (Respiration Quotient) =---------------------------spent O2

The caloric or heat equivalent that we gained by the combustion of 1 litter O 2 is found from table-2 according to RQ value.This value is multiplied with the consumed O2 quantity during experiment and by the way we found the person’s energy consumption or metabolism value. We find the subject’s body surface by calculating his / her length and weight (Table 3) in order to translate the metabolism value into standard units. The found metabolism value is divided by this value and the result is considered by the way, as ( Cal/m2/ h) that is in hours, calorie for unit body surface (m2).The metabolism value found by this way is a metabolism measurement based on open system’s principle.There’s an example of this method:

Time: 10 minute (time to collect air volume) Subject: 60kg, 165cm and 1,64m2 body surfaceTotal volume: V1=70 lt(B.T.P.S.) Temperature: t= 20oC B.B.= 751,5 torr S.B.B.20oC-------------------17.5 torr 751,5-17,5 =734 torr

STPD

= 62,95 lt(10′) = 6,295 lt(1′) = 6,295 . 1000 = 6295 ml

%İnsp. air %Exp. air N2 correction

CO2 = 0,04 3,50

O2 = 20,93 16,90 20,93 . 21,09 O2 value

N2 = 79,03 79,60

O2 consumption: . 6295 = 264 ml (for one minute)

CO2 expired:

. 6295 = 218 ml (for one minute)

CO2 218

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RQ = ----------= -----------------=0,82 O2 264

the equivalence calorie is 4,825 cal according to R.Q=0,82

4,825. = 1,27 cal (for one minute) 60.1, 27 = 76,2 cal (for 60 minute)

(during experiment) = 1,27. 10 = 12,7 cal

The metabolism value; = 76,2/ 1,64 = 46,4 cal/m 2 /h

III.III. The Applying Method of Orsat Gas AnalyserThere are two Gallenkamp Gas Analysers in our laboratory. One of them is also Gallenkamp-Orsat Gas Analyser (Figure 2)a) The absorption tubes are filled with appropriate absorbant solutionsb) The water reservoir is filled with waterc) If you like, can be use color liquid with an organic dye the instead of waterd) The water reservoir is elevated to the top of the analyzere) The tree way tape at end of manifold is opened and let the the liquid in reservoir fills into graded burette.f) The thre way tape is turned to vertical position and the air in the connection tube is removed by using the blow-ball.g) The three way tape is adjusted to appropriate position. The reservoir is lowered down to the lowest position of graded burette so that sample gase is taken inside the burette.h) The water is added or extracted to the water reservoir until fluid in the burette is same amount with the liquid in the bottle.i) All absorption solutions are applied absorption procedure by holding the water reservoir up and down. This operation must be started with CO2.

j) The tape that connect the burette to the absorption tube which contains KOH is turned on and level of the water reservoir is elevated.The water reservoir is depressed down again and the gas sample is pushed into burette.k) While depressing the water reservoir, KOH is reached to the marked level in the KOH tube. This procedure must be kept on until the absorption procedure is completed.l) The value is read on graded burette. The procedure is repeated for O2 and record the value.

IV. Findings

V. Discussion and Questions

1) What are the normal values of gases in inspiration and expiration air in resting state?2) What are the alterations of expiration gas components during exercises? What are the minimum and maximum value of these gas components?3) What is the temperature of water bath of Gallenkamp-Lloyd gas analyzer? What is its important?4) What is the calorie requirement of normal person and its alteration during exercises?5) Write if there is a relation between calorie consumption and nutrition? Explain

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Figure 2.Gallenkamp-Orsat Gas Analyser

29

water reservoir

Graded burrette

Blow-Ball

U tubes

Collector

Absorption tubes

Mercury reservoir

Burning tub

Burrettecuvette

Adjusment box

Experiment: H 4

DETERMINATION OF SURFACE TENSION CONSTANT IN BODY

FLUIDS [APPLICATION OF TATE LAW]

I. Equipment

• Pipette• Watch glass• Sensitive scales• Blood• Serum• Distilled water

II. Purpose and General Information

When a fluid containing pipette with (r) radius is held vertically, the force balancing the drop at end of the pipette is F=2 π r α, α (dyn/cm) is the surface tension constant of the fluid. The surface tension force acts on the unit surface vertically on the surface is called “surface tension constant”. This constant depends on type of fluid, cleanness of surface of fluid, and temperature. The blood plasma, that surface tension constant will be measured composed of 90% water and 10% organic and inorganic compounds. The blood plasma proteins are divided into three groups as albumin, globulin and fibrinogen.

Weight of a drop of the fluid, which surface tension constant is known is F 1 while unknown is F2. Because of same pipette is used (r) radius does not change.

F1=2 π r α1 F2=2 π r α2 sF1/ F2= α1 /α2

α2 = F2 / F1 .α1 (dyn/cm)

III. Experimental Procedure

[Attention: in all weighing double weighing method must be carried out. According to this method, the fluid is weighed first in right scale then in the left scale.The results are divided after addition, namely arithmetical mean is taken.]

a) The mass of the watch glass in balance by double weighing method is found carefully, after washing with distilled water and drying it.

M=..........g.

b) After cleaning the pipette with distilled water, draw a little amount of distilled 10 drops of water are put on the glass of watch. Then the weight of 10 drops of water and the glass are

30

measured by double weighing method. The weight of the glass is subtracted from the total weight. M1 =…....….g M1` = M1- M =..........g

This is the weight of 10 drops distilled water. Then this value is divided to 10 [M1¨ = M1` /10=.........g] and the weight of 1-drop distilled water is found. c) Watch glass is cleaned and dried again. A little amount of blood (serum) , which the surface

tension constant will be found is drawn. Ten drops is dropped by pipette on the clean watch

glass. The mass of watch glass with 10 drops of blood (serum) is measured by double

weighing method.

M2 =............g M2`= M2 –M =................g

This is the weight of 10 drops blood plasma and this value is divided to 10 [ M2¨= M2`/10=..........g] and the weight of 1-drop blood plasma is found.

mass unitMass of watch glass M ..........gMass of 10 drops distilled water M1` ..........gMass of 1 drop distilled water M1¨ ..........gMass of 10 drops blood plasma M2` ..........gMass of 1 drop blood plasma M2¨ ..........g

d) Weight of 1 drop distilled water is F1= M1`.g and weight of 1 drop blood (serum) is F2= M2¨.g .The rate of weight is equal to the rate of mass because of any changes in gravity haste [g= 980 cm/sn²] (F1/F2= M1¨ / M2¨)So using weight instead of mass occurs no difference in base relation. The comparison liquid that is used in this experiment is distilled water and its surface tension constant is:

α=73 dyn/cm.α2 = F2 / F1 .α1 = M2¨ / M1¨.73

IV. Findings

α2=.............. dyn/cm

V. Discussion and Questions

1) Write the surface tension constant’s dimension equation and unit in SI unit system.2) Define the Tate Rule and write mathematical relation.3) Explain these concepts: capillarity “wetting” and “nonwetting” liquids.4) Define adhesion and cohesion forces. Compare the two forces in liquid with examples.5) What are the factors that reduce the surface tension constant? How do capillary active and capillary inactive affect the surface tension constant?6) Explain the importance of the surface tension for living organism with examples.

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Experiment: H 5

DETERMINATION OF INTRA-ERYTHROCYTE FLUID VOLUME

a) Measurement of Hctb) Erythrocyte count

I. Equipment

• 0.33% N perchloric acid• 6-7 mL venous blood• Microhematocrit tube• Injector• Hct ruler• Centrifugal machine• Electromagnetic mixer

II. Purpose and General Information

The body fluid that is very important for main physiological functions that divided into two compartment as EXTRACELLULAR and INTRACELLULAR compartments.

Approximately 25 liters of 40 liters of whole body water is stored in 75 billion cells and called intracellular fluids. All fluids that is out of these cells are named as extracellular fluids. The extracellular fluid compartment is composed of blood plasma which is in a closed system (in heart and blood vessels). In a 70 kg adult, the amount of extracellular fluid is almost 15 liters.

II.I. Blood VolumeBlood contains both extracellular fluid (plasma fluid) and intracellular fluid (intra

erythrocyte fluid). Because of the circulation of blood in a closed system, properties of blood and blood volume is very important.

Healthy adult’s person blood volume is 5000 mL. 3000 mL of this amount is plasma and 2000 mL is erythrocytes. These values are subject to change by age, sex, weight and other factors. Hematocrit(Hct) is the percentage of various components (erythrocyte) assembled at the lower part of tube after centrifuge. Real hematocrit value is about 40% for men, 36% for women. Measured hematocrit values are 3-8% greater than the real value as a result of the plasma at this proportion between erythrocytes.

Blood is a complex fluid formed as a suspension of various cellular components, has unique rheological (fluidity) feature. It is possible to examine rheological features of blood depending on blood viscosity. Main factors of the blood viscosity are: 1-Erythrocyte Deformability 2- Hematocrit 3- Plasma Viscosity 4- Thrombocyte Aggregation 5- Erythrocyte Aggregation.

When the factors which affect blood viscosity are examined, it can be seen that erythrocytes are effective. Therefore, shape, structure, size and deformability of erythrocytes affect the blood viscosity. These features may change in different circumstances. But a normal

32

erythrocyte must reshape into a biconcave disk shape. Biconcave disk shape of erythrocyte is the most convenient shape to complete its life, gas transport and flow.

Changes in the shape (or volume) of the erythrocytes, affect the erythrocyte function in a negative way. Changes in the blood volume effects the transport function because of the changes in the erythrocyte’s shape also it effects the rheology in a negative way because of the increase in viscosity. It is determinated that especially in hypertensive person intra erythrocyte fluid volume has increased.

If intra erythrocyte fluid volume increases in healthy and young body, it can be said that there is a hypertension possibility at middle ages for that person. This is important for protective medicine.

III. Experimental Procedurea) 0.5 mL blood sample is put into a tube and 2.5 mL 0.33% N HClO4 (perchloric acid) is added. The solution is mixed in the Vorteks Mixer.b) 2 microhematocrit tubes are filled with and mixed, centrifuged and Hct value is read(X:denatured total blood hematocrit)c) 0.5 mL serum sample is put into tube and added 2.5 mL 0.33% N HClO4. Mixed until it is homogenised.d) 2 microhematocrit tubes are filled with and mixed, centrifuged and Hct value is read. (Y:denatured serum protein hematocrit)e) 1 microhematocrit tube is filled with blood sample and centrifuged. The observed value is the Hct of total blood.f) The erythrocytes of the same blood sample is counted in the Neu-bauer counting cabin near the experiment mechanism.MCV = Hct x 10 / Erythrocyte number (mm3/million)Write the value in the formula and calculate intracellular fluid volume (intra erythrocyte fluid volume).

IEFV = MCV - [ (6X-6Y)x(MCV) / 100 ]IEFV : Intra Erythrocyte Fluid VolumeMCV : Mean Corpuscular VolumeX : Denatured Total Blood HematocritY : Denatured Serum Protein Hematocrit

IV. Findings

MCV:………. IEFV:……….

V. Discussion and Questions

1) What are the compartments of the body fluids?2) In which circumstances the hematocrit value increases or decreases? What does this change mean?3) What is the difference between the measured Hct value and the real Hct value? What is the reason?4) What kind of situations change the concentration of the intracellular fluid of the erythrocytes?5) What are the blood proteins? What are their functions?6) What is the difference between serum and plasma?7) What are the plasma proteins?

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Experiment: R 2

THE ABSORPTION OF NUCLEAR RADIATIONS

a) The Absorption of Gamma Radiation In Various Tissuesb) Finding Half Value Thickness For Various Materials’ Densities

I. Equipment

• Scintillation or Geiger-Müller counter• Radioactive source Iodine─131 (I131), Eγ = 364 kev , T1/2 = 8,05 day

Cessium─137 (Cs137) Eγ = 662 kev , T1/ 2 = 30 yıl.• Several absorbative samples• Non-living material: several plaques in different thicknesses such as Cu , Zn , Pb (1-2 mm)• Living originated materials: plasma, blood (in tube), tissue samples cut in several thicknesses (liver brain tissues)• Semi-logaritmic graphic paper

II. Purpose and General InformationGamma rays emitted from a radioactive material are absorbed by media and their

severity is reduced as a result of interaction of media`s atoms reciprocally (photoelectric effect, compton effect, pair production ) .

II.I. The absorbtion by photoelectric effectWhole energy of photon that occurs in this effect is transferred to a bounded electron

and this electron leaves the atom with a kinetic energy of E=hf (h=Planck’s constant, f (frequency)= c / λ(vave length) This electron can be thrown away from absorber or if the absorber is thin enough, electron is absorbed again because of the shortness of reaching distance in solid. In low photon energy, photoelectric event under 500kev has a big role for lead in absorption.

E=hf=W+(1/2)mv2

photon Electron Scattered electron

The emission of electrons by a substance being irradiated with photon is known as the

Photoelectic Effect.

‾ ‾

34

II.II. The absorption by compton effectCompton effect is a main factor in absorption of photon coming from radiation waves

when radiation energy increases. In compton effect coming photon diffuses with atomic electron. This way electron leaves atom. Photon deviates with an angle compared to the first direction and since it gives some of its energy to the electron., its energy decreases. This process is considered as a diffusion or an electron with a photon because photon energy coming from source is greater than the binding energy of atomic electron. As a result of diffusion of each electron in atom by itself, the coefficient of compton absorption per atom is proportional to atomic number Z.

II. III. The absorption by pair productionWhen the energy is great enough, the photoelectric effect and compton effect can be

replaced by pair producion. In pair production in the “couloumb” area of the nucleus, a ray that has enough energy is lost and an electron and a positron occur. The total energy of this couple is equal to energy of photon's energy.

For lead the probablity of double formation is greater than compton event in photon energies greater than 1.02 MeV. In double formation because of atomic number Z and the demand of energy, absorption coefficient is important for heavy elements that has high energy. Attenuation Of Gamma Radiations

The photon energy severity that passes through a material between photon waves and the detector that measures the rays and by this way the photon energy that is absorbed in material is defined in exponential law (BEER) below :

(I) I = Io . e─ µ. x

Io = the photon severity that is measured in the absence of absorber on the bunch way.I = the photon severity that passes through the absorber material in x thickness.x = absorber thickness (cm)µ = the coefficient of total linear absorption ( cm -1) (The fractional decrease in the bunch severity for unit absorber thickness )

‾Target electron

E=hf

Incident photon

Scattered photon

E′= hf′

Scattered electron

Loss in photon energy =gain in electron energyhf ▬ hf′ = KEIf the electron energy after the collision is KE, then, from the law of conservation of energy..

‾

35

The absorber thickness that halves the photon severity ''Half Value Thickness'' (HVT). It is found as I = Io /2 in the first absorption equation.

HVT = X = 0.693 / µ (cm)

To be protected against biological effects of radiation, radiation severity should be decreased to a certain value. If the photon’s energy and severity is known , the thickness of obstruction can be calculated for the protection from these rays by finding half value thickness from counting thickness graphic which belongs to that radionuclide.

Generally for protection, decreasing the radiation severity ten times is enough. To calculate this ;

X1/10 = Tenth Value Thickness (TVT) = 2,3 / µ (cm) equation is used.

The ratio of the linear absorption coefficient and the density of absorber gives mass absorption coefficient.

µm = µ / ρ (cm2 / gr)

Accoding to this , the absorption equation is written as below :

I = Io . e- (µ / ρ) x. ρx

Given in the equation p.x multiplication is called the unit area mass or surface density. Its unit is gr/cm2.

EXAMPLE: How many times will the lead plate with a thickness of 1cm decrease the gamma beam of I - 131 radionuclide with 360keV energy? (Density of lead : ρ = 11.3 gr/cm3 ; µm = 0.25 gr / cm2 )

Linear Absorption Coefficientµ = µm . ρ= 11,3 x 0,25 = 2.8 cm -1

Half Value Thickness (HVT) = 0.693 / µ = 0.693 / 2.8 = 0.25 cm

1 cm lead with half value thickness unit 1 / 0.25 = 4 Half Value Thickness

Beam Severity Decreasing Factor = (1/2) 4 = 1/16

Thus lead in 1 cm thickness will decrease the severity 16 times.The effective linear absorption coefficient of materials which are composed of light

elements (muscle, adipose and bone tissue or water ) is proportional to their density in the gamma beam energy which is important for compton event probability (nearly 100 keV ─ 200 keV). In other words µ/ρ = µm is appoximately constant. Namely, gamma beam severity with certain energy is constant at a certain ratio. Namely several material weights which are needed to decrease gamma beam severity with a certain energy in a certain ratio are same. But the volume and so the thickness for higher dense material is lesser than lower dense material.

The value of absorption coefficient is relative to the material's environment and energy of radiation. For an element higher energy has a lower absorption coefficient.I - 131 (364 keV ) ; for Pb µ = 2.761 cm-1

Co - 60 (1.17 ─ 1.3 MeV) ; for Pb µ = 0.6447 cm-1

36

As a practice of second formula, if the change of logarithm of decreasing ( log I / Io ) ratio of beam severity is examined according to absorbent’s surface density (graphical), matters with same surface density ( in same thickness gr/cm2) are observed to do almost the same absorption, whatever their kinds are .The important thing is the mass that hits the surface of the absorber plate in 1cm thickness.

For this reason it is more appropiate to use 1cm3 mass of material instead of using material’s (absorbent) thickness. For example; the absorption of gamma beams with certain energy of 30.5 gr/cm2 lead and aluminium is the same. But 30.5 gr/cm2 is lead's thickness and 11.3 is aluminium's.

Important notice : In this experiment Avoid contamination of yourself and your environment. If any contamination occurs accidentally notify the experiment member / lecturer.

III . Experimental ProcedureIII.I. Calibration of scintilation countera) Put detector to suitable entrance of analyser ( scintillation) form counter system.b) Place radioactive source on detector axis direction.c) Adjust the counter system to the optimum working voltage.

ATTENTION : Before making the power button of the device on ''0''(zero) ''on '' or ''off'' position, absolutely adjust the ''H.V adj'' button to zero position. make this process decreasing the voltage arrow slowly.d) If scintillation counter is being used, adjust the ''Df /Int '' button to Int position and adjust ''baseline'' button to %10-20 of source energy.e) Measure ''background ''counting and source countings in determined time interval. (To obtain net counting, subtract ''BG'' back ground value from each of the countings).f) Make new countings by placing different absorbed plaques between detector and source and note if countings change or not.g) Make countings by changing source to different directions. Note the differences between countings.

III.II. Experimental ProcedureLet a, b, c, d, e alternatives as the same as the experimental procedure given at III.I.f) Fix a constant point by regulating the geometry at the radiation source as absorbed materials can enter this interval.g) Localizing absorber tablets between the source and the detector one by one note each of the obtained countings.III.III. Determination of HVT by explanation on graphic.a) Repeat this process with different absorbed materials.b) Draw alterations of countings (or counting ratios like I / Io) as absorbent (or area densities ) thickness function severally on semilogaritmic graphic paper.

Note: Io = is the net counting value when no absorbent material is present in the interval.

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III.IV. Calculation of the real count.Substances a, b, c, d are the same ones as experimental procedure given at III. I.e) Note the back ground counting.f) Calculate the law of ''reverse squares'' by taking countings from different distances and calculate the real counting.(N)g) Find the distance that reduces the intensity of the radiation that is taken from the radioactive source to 10%.

IV. Findings

V. Discussion and Questions V.I.a) What kind of correlation between the distance change of source to detector and counting sensitivity?b) How do the absorber sustances affect the counts?c) What is the effect of differerent geometry and absorbers to the counts?V.II. Calculate the following by using the chart :a) The effective linear absorption coefficient of each absorber (µ)b) Half Value Thickness (HVT) (cm) and surface density (gr/cm2)c) Compare HVT values of tissues and metals.V.III.a) Prove the law of ''reverse squares'' by taking countings from different distances. Nd = N / d2 N = real counting (on the surface of the detector) Nd = the counting taken in a distance d. d = distance between the source and the detector.b) What is the practical importance of the 10th (tenth) Value Thickness?V.IV.a) Discuss the tissue factors that play a role in absorption of a radiation of a certain energy.b) Discuss the importance of absorption and scatter of radiation in Radiology Nuclear Medicine ( syntigraphy imaging ) and Radiotherapy.

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