A STUDY ON SIZE AND SHAPE OF ERYTHROCYTES OF NORMAL AND MALARIAL BLOOD USING LASER DIFFRACTION TECHNIQUE by Amar Alansi

A STUDY ON SIZE AND SHAPE OF ERYTHROCYTES OF
NORMAL AND MALARIAL BLOOD USING LASER

DIFFRACTION TECHNIQUE
Amar Alansi
Osmaina University
Master Of Science
In
Physics
A STUDY ON SIZE AND SHAPE OF ERYTHROCYTES

OF NORMAL AND MALARIAL BLOOD
USING LASER DIFFRACTION TECHNIQUE
Supervisor:
Dr. Kaleem Jaleeli
Author:
Amar Alansi
May, 2015
Contents
Certificate
iii
Declaration
vi
Dedication
vii
Acknowledgements
viii
Abstract
ix
Preface
1 Introduction
1.1 Blood . . . . . . . . . . . . . . . . . . .
1.1.1 Properties of Blood . . . . . . .
1.2 Composition of Blood . . . . . . . . . .
1.3 Blood Cells . . . . . . . . . . . . . . . .
1.4 Plasma Proteins . . . . . . . . . . . . .
1.4.1 Plasma Proteins . . . . . . . . .
1.4.2 Properties of Plasma Proteins . .
1.4.3 Red Blood Cells . . . . . . . . .
1.4.4 Normal Size . . . . . . . . . . . .
1.4.5 Properties of red blood cells . . .
1.4.6 Function of Red Blood Cells . . .
1.4.7 Pathological Variations . . . . .
1.5 White Blood Cells . . . . . . . . . . . .
1.5.1 Classification . . . . . . . . . . .
1.5.2 Properties of White Blood Cells
1.5.3 Function of White Blood Cells .
1.6 Platelets . . . . . . . . . . . . . . . . . .
1.6.1 Size of Platelets . . . . . . . . . .
1.6.2 Shape of Platelets . . . . . . . .
1.6.3 Strucyure and Composition . . .
1.6.4 Properties of Platelets . . . . . .
1.6.5 Functions of Platelets . . . . . .
1.7 Malaria . . . . . . . . . . . . . . . . . .
1.7.1 Plasmodium vivax . . . . . . . .
1.7.2 Plasmodium malariae . . . . . .
1.7.3 Plasmodium ovale . . . . . . . .
1.7.4 Plasmodium falciparum . . . . .
1.8 Survey of Literature . . . . . . . . . . .
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1
1
1
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9
10
2 Theoretical aspects of diffraction

2.1 Diffraction and the Wave Theory of Light
2.2 Diffraction by a Circular Aperture . . . .
2.2.1 Resolvability . . . . . . . . . . . .
2.2.2 Babinet Principle . . . . . . . . . .
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13
13
14
15
16
3 Materials and Methods

3.1 Samples collection . . . . . . . . .
3.2 Preparation of a thin blood film on
3.3 Experimental set up . . . . . . . .
3.4 Procedure and Analysis . . . . . .
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19
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20
4 Results and Discussion

4.1 Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
23
44
. . . . .
the slide
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Appendix of Mathematica Programs
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47
Bibliography
55
List of Figures
57
List of Tables
59
ii
iv
Declaration
The Author declares that he carried out the project entitled A Study on Size and Shape
of Erythrocytes of normal and malarial blood using Laser Diffraction Technique
under the supervision of Dr. Kaleem Ahmed Jaleeli at Biophysics unit, Department of
Physics Nizam College (Autonomous), Osmania University, Hyderabad, India.
Amar Yahya Yahay Alansi
Dedicated to
my parents
my wife
my son
my brothers and my sisters
Amar alansi
Acknowledgements
The author solemnly offers his sincere gratitude to the almighty, the most beneficent and
merciful for bestowing the opportunity to complete this project. Peace and blessing of Allah
be upon his last Messenger MOHAMMED (Sallallah Alaihe Wasallam ), who guided us to
The right path.
The author declares that he carried out the project entitled A Study on Size and Shape
of Erythrocytes of normal and malarial blood using Laser Diffraction Technique under the
supervision of Dr. Kaleem Ahmed Jaleeli at Biophysics unit, Department of Physics Nizam
College, Osmania University, Hyderabad, India.
The author wishes to thank Dr.
Kaleem Ahmed Jaleeli for co-operation, constant
encouragement and guidance and useful discussion. Further the author thanks Prof.
Adeel Ahmed, former Head, Department of Physics, Nizam College Osmania University,
for useful discussion in completing the project. Special thanks to Dr. N.V. Prsad, Head
Department of physics, and Prof. T.L.N. Swamy, Principle, Nizam college for the interest
they have shown during the program.
The author would like to thank in particular his lovely classmates.
Amar Yahya Yahay Alansi
Abstract
The subtle changes in the physiology of erythrocytes at the cellular level are documented in
the present laser diffraction study. Using this technique one can differentiate the morphologies
of erythrocytes. The size and shape of blood cells are determined usually by microscope. This
method, besides being tedious, cannot be extended to a large number of cells and samples. In
view of this, a simple and quick method has been developed for determining the average size
and shape of blood cells by employing laser diffraction technique. Blood samples were collected
from normal healthy persons and patients suffering from malaria. The laser diffraction method
is very rapid and simple for assessing the average size of the cells. This could also be used
with advantage as a diagnostic tool for assessing the variation in the size of human RBC.
Preface
The blood serves as the principle transport medium of the body , carrying oxygen, nutrients messages
to tissues and waste product , synthesized metabolites and carbon dioxide to the organs of excretion
. In other words , Blood is described as a fluid connective tissue . The blood plays many important
roles in coordinating the individual cells into whole complex organisms. Human blood is characterized
by a number of physicochemical properties in disease analysis. With modern electronic instruments it is
possible to detect changes in the chemistry of blood cells that are responsible for severe disorders. The
change in the sublet interaction between erythrocytes and plasma may be termed as disease . this subtle
interaction perturbs the membrane physiology of erythrocytes because the proteins are very sensitive
detectors of environment and these are present in plenty on the erythrocyte membrane. Hence, the blood
is being studied extensively by the physiologists, biochemists and biomedical engineers , but it has not
drawn much attention of physicists. However , some reports are available on ultrasonic, dialectic and
dieelctrophoretic properties of human blood and its erythrocytes.
The application of concepts, principles and techniques of physics for the selection of problems in biology
at different levels of complexity to get an integral picture is drawing the attention of many researches.
The main aim of this project is to present the size of normal blood and malarial blood by using laser
diffraction technique.
This project contains four chapters namely
1. Introduction
2. Theoretical Aspects of diffraction
3. Materials, and methods
4. Results and Discussion
This project starts with the first chapter which presents a brief description of blood and its components,
functional properties of blood (erythrocytes, leukocytes, platelets) ,and malaria and its types .
The second chapter is concerned with materials and methods of the present investigation. A detailed
description of collection of blood, the procedure of preparation of sample and Theoretical aspects of
diffraction has been discussion.
The third chapter describes the theory of optical diffraction.
The forth Chapter reports results on size of normal human erythrocytes and malarial human erythrocytes
. A detailed discussion is mentioned.
Chapter 1
Introduction
1.1
Blood
Blood is a connective tissue in fluid form. It is considered as the fluid of life because it carries oxygen
from lungs to all parts of the body and carbon dioxide from all parts of the body to the lungs. It is known
as fluid of growth because it carries nutritive substances from the digestive system and hormones from
endocrine gland to all the tissues.
The blood is also called the fluid of health because it protects the body against the diseases and gets
rid of the waste products and unwanted substances by transporting them to the excretory organs like
kidneys.
1.1.1
Properties of Blood
Color
Blood is red in color. Arterial blood is scarlet red because it contains more oxygen and venous blood is
purple red because of more carbon dioxide.
Volume
Average volume of blood in a normal adult is 5 L. In a newborn baby, the volume is 450 ml. It increases
during growth and reaches 5 L at the time of puberty. In females, it is slightly less and is about 4.5 L.
It is about 8% of the body weight in a normal young healthy adult, weighing about 70 kg.
Reaction and pH
Blood is slightly alkaline and its pH in normal conditions is 7.4.
Specific gravity
Specific gravity of total blood : 1.052 to 1.061
Specific gravity blood cells : 1.092 to 1.101
Specific gravity of plasma : 1.022 to 1.026
Viscosity
Blood is five times more viscous than water. It is mainly due to red blood cells and plasma proteins.
1.2
Composition of Blood
Blood contains the blood cells which are called formed elements and the liquid portion known as plasma
Figure 1.1.
1
1.3. BLOOD CELLS
Figure 1.1: Composition of Blood
1.3
Blood Cells
Three types of cells are present in the blood:

Red blood cells or erythrocytes
White blood cells or leukocytes
Platelets or thrombocytes.
1.4
1.4.1
Plasma Proteins
Plasma Proteins
Plasma proteins are:

Serum albumin
Serum globulin
Fibrinogen
1.4.2
Properties of Plasma Proteins
Molecular Weight
Albumin : 69,000
Globulin : 1,56,000
Fibrinogen : 4,00,000
Specific Gravity
Specific gravity of the plasma proteins is 1.026.
Buffer Action
Acceptance of hydrogen ions is called buffer action. The plasma proteins have 1/6 of total buffering
action of the blood.
1.4.3
Red Blood Cells
Red blood cells (RBCs) are the non-nucleated formed elements in the blood. Red blood cells are also
known as erythrocytes (erythros = red). Red color of the red blood cell is due to the presence of the
coloring pigment called hemoglobin. RBCs play a vital role in transport of respiratory gases. RBCs are
larger in number compared to the other two blood cells, namely white blood cells and platelets.
2
CHAPTER 1. INTRODUCTION
1.4.4
Normal Size
Diameter : 7.2 (6.9 to 7.4 ).

Thickness : At the periphery it is thicker with 2.2 and at the center it is thinner with 1 Figure 1.2.
This difference in thickness is because of the biconcave shape.
Surface area : 120 sq .
Volume : 85 to 90 cu .
Figure 1.2: Shape and dimensions of a red blood cell
1.4.5
Properties of red blood cells
Rouleaux Formation
When blood is taken out of the blood vessel, the RBCs pile up one above another like the pile of coins.
This property of the RBCs is called rouleaux (pleural = rouleau) formation Figure 1.3. It is accelerated
by plasma proteins globulin and fbrinogen.
Figure 1.3: Rouleau formation
Specific Gravity
Specific gravity of RBC is 1.092 to 1.101.
Paked Cell Volume

Packed cell volume (PCV) is the proportion of blood occupied by RBCs expressed in percentage. It is
also called hematocrit value. It is 45% of the blood and the plasma volume is 55% .
1.4. PLASMA PROTEINS

Suspension Stability
During circulation, the RBCs remain suspended uniformly in the blood. This property of the RBCs is
called the suspension stability.
1.4.6
Function of Red Blood Cells
Major function of RBCs is the transport of respiratory gases. Following are the functions of RBCs:
Transport of Oxygen from the Lungs to the Tissues

Hemoglobin in RBC combines with oxygen to form oxyhemoglobin. About 97% of oxygen is transported
in blood in the form of oxyhemoglobin .
Transport of Carbon Dioxide from the Tissues to the Lungs
Hemoglobin combines with carbon dioxide and form carbhemoglobin. About 30% of carbon dioxide is
transported in this form.
RBCs contain a large amount of the carbonic anhydrase. This enzyme is necessary for the formation of
bicarbonate from water and carbon dioxide. Thus, it helps to transport carbon dioxide in the form of
bicarbonate from tissues to lungs. About 63% of carbon dioxide is transported in this form.
Buffering Action in Blood
Hemoglobin functions as a good buffer. By this action, it regulates the hydrogen ion concentration and
thereby plays a role in the maintenance of acidbase balance .
In Blood Group Determination
RBCs carry the blood group antigens like A antigen, B antigen and Rh factor. This helps in determination
of blood group and enables to prevent reactions due to incompatible blood transfusion .
1.4.7
Pathological Variations
Variations in size of red blood cells

Under physiological conditions, the size of RBCs in venous blood is slightly larger than those in arterial
blood. In pathological conditions, the variations in size of RBCs are:
Microcytes (smaller cells)
Macrocytes (larger cells)
Anisocytes (cells with different sizes)
MICROCYTES Microcytes are present in:
Iron-defciency anemia
Prolonged forced breathing
Increased osmotic pressure in blood
MACROCYTES Macrocytes are present in:
Megaloblastic anemia
Decreased osmotic pressure in blood
ANISOCYTES Anisocytes occurs in pernicious anemia.
Variations in Shape of Red Blood Cells
Shape of RBCs is altered in many conditions including different types of anemia.
1. Crenation: Shrinkage as in hypertonic conditions
2. Spherocytosis: Globular form as in hypotonic conditions
3. Elliptocytosis: Elliptical shape as in certain types of anemia
4. Sickle cell: Crescentic shape as in sickle cell anemia
5. Poikilocytosis: Unusual shapes due to deformed cell membrane. The shape will be of fask, hammer
or any other unusual shape
Variations in Structure of Red Blood Cells
PUNCTATE BASOPHILISM Striated appearance of RBCs by the presence of dots of basophilic
materials (porphyrin) is called punctate basophilism. It occurs in conditions like lead poisoning.
RING IN RED BLOOD CELLS Ring or twisted strands of basophilic material appear in the periphery
of the RBCs. This is also called the Goblet ring. This appears in the RBCs in certain types of anemia.
HOWELL-JOLLY BODIES In certain types of anemia, some nuclear fragments are present in the
ectoplasm of the RBCs. These nuclear fragments are called HowellJolly bodies.
1.5
White Blood Cells
White blood cells (WBCs) or leukocytes are the colorless and nucleated formed elements of blood (leuko
is derived from Greek word leukos = white). Alternate spelling for leukocytes is leucocytes Figure 1.4.
Figure 1.4: WBC

Compared to RBCs, the WBCs are larger in size and lesser in number. Yet functionally, these cells are
important like RBCs because of their role in defense mechanism of body and protect the body from
invading organisms by acting like soldiers.
1.5.1
Classification
Some of the WBCs have granules in the cytoplasm. Based on the presence or absence of granules in the
cytoplasm, the leukocytes are classifed into two groups:
Granulocytes which have granules
Agranulocytes which do not have granules
5
1.6. PLATELETS
Granulocytes
Depending upon the staining property of granules, the granulocytes are classifed into three types:
Neutrophils with granules taking both acidic and basic stains
Eosinophils with granules taking acidic stain
Basophils with granules taking basic stain
Agranulocytes
Agranulocytes have plain cytoplasm without granules. Agranulocytes are of two types:
Monocytes
Lymphocytes
1.5.2
Properties of White Blood Cells
Diapedesis
Diapedesis is the process by which the leukocytes squeeze through the narrow blood vessels.
Ameboid Movement
Neutrophils, monocytes and lymphocytes show amebic movement, characterized by protrusion of the
cytoplasm and change in the shape.
Chemotaxis
Chemotaxis is the attraction of WBCs towards the injured tissues by the chemical substances released
at the site of injury.
Phagocytosis
Neutrophils and monocytes engulf the foreign bodies by means of phagocytosis .
1.5.3
Function of White Blood Cells
Generally, WBCs play an important role in defense mechanism. These cells protect the body from
invading organisms or foreign bodies, either by destroying or inactivating them. However, in defense
mechanism, each type of WBCs acts in a different way.
1.6
Platelets
Platelets or thrombocytes are the formed elements of blood. Platelets are small colorless, non-nucleated
and moderately refractive bodies. These formed elements of blood are considered to be the fragments of
cytoplasm Figure 1.5.
1.6.1
Size of Platelets
Diameter : 2.5 (2 to 4 )
Volume : 7.5 cu (7 to 8 cu ).
1.6.2
Shape of Platelets
Normally, platelets are of several shapes, viz. spherical or rod-shaped and become oval or disk-shaped
when inactivated. Sometimes, the platelets have dumbbell shape, comma shape, cigar shape or any other
unusual shape. Inactivated platelets are without processes or flopodia and the activated platelets develop
processes or flopodia .
6
Figure 1.5: Platelets
1.6.3
Strucyure and Composition
Platelet is constituted by:

Cell membrane or surface membrane
Microtubules
Cytoplasm
1.6.4
Properties of Platelets
Platelets have three important properties (three As):

Adhesiveness
Aggregation
Agglutination
1.6.5
Functions of Platelets
Normally, platelets are inactive and execute their actions only when activated. Activated platelets
immediately release many substances. This process is known as platelet release reaction. Functions
of platelets are carried out by these substances.
Functions of platelets are:
Role in Blood Clotting
Platelets are responsible for the formation of intrinsic prothrombin activator. This substance is responsible
for the onset of blood clotting .
Role in Clot Retraction
In the blood clot, blood cells including platelets are entrapped in between the fbrin threads. Cytoplasm
of platelets contains the contractile proteins, namely actin, myosin and thrombosthenin, which are
responsible for clot retraction .
Role in Prevention of Blood Loss(Hemostasis)
Platelets accelerate the hemostasis by three ways:
Platelets secrete 5-HT, which causes the constriction of blood vessels
Due to the adhesive property, the platelets seal the damage in blood vessels like capillaries
By formation of temporary plug, the platelets seal the damage in blood vessels
7
1.7. MALARIA
Role in Repair of Ruptured Blood Vessel
Platelet-derived growth factor (PDGF) formed in cytoplasm of platelets is useful for the repair of the
endothelium and other structures of the ruptured blood vessels.
Role in Deffnse Mechanism
By the property of agglutination, platelets encircle the foreign bodies and destroy them.
1.7
Malaria
Malaria is one of the most successful parasites ever known to mankind. After thousands of years, it
remains the worlds most pervasive infection, affecting at least 91 different countries and some 300 million
people. The disease causes fever, shivering, joint pain, headache, and vomiting. In severe cases, patients
can have jaundice, kidney failure, and anemia, and can lapse into a coma.
It is ever-present in the tropics and countries in sub-Saharan Africa, which account for nearly 90 percent
of all malaria cases. The majority of the remaining cases are clustered in India, Brazil, Afghanistan, Sri
Lanka, Thailand, Indonesia, Vietnam, Cambodia, and China. Malaria causes 1 to 1.5 million deaths
each year, and in Africa, it accounts for 25 percent of all deaths of children under the age of five.
Plasmodium spp., which cause malaria, remain endemic throughout the world in tropical and subtropical
countries with an estimated 300 million to 500 million cases annually. Plasmodium vivax, Plasmodium
ovale, Plasmodium malariae, and Plasmodium falciparum are the etiologic agents of human malaria.
Along with schistosomiasis and amebiasis, malaria is a major cause of mortality in people in
underdeveloped countries. Between 1 and 2 million deaths worldwide are caused by malaria each year,
primarily due to infection with P. falciparum. Mortality in children who have malaria is also signifcantly
associated with infection by P. falciparum. In the United States, approximately 1000 cases are reported
annually, with P. falciparum being the etiologic agent in more than 50% of the cases. Most of these cases
are in travelers to endemic areas.
Plasmodium vivax has the widest geographic distribution and is the one most likely to be found in
temperate climates. P. vivax and P. falciparum cause the majority of infections. P. ovale is confned to
Africa; P. falciparum and P. malariae have similar distributions throughout Africa and tropical countries.
In general, infections caused by P. vivax, P. ovale, and P. malariae are less severe than those caused by
P. falciparum (12).
1.7.1
Plasmodium vivax
Plasmodium vivax has a tertian lifecycle patternthat is, it takes approximately 48 hours for the life cycle
to be completed. The invasion of a new group of RBCs begins on the third day. P. vivax usually invades
young RBCs (reticulocytes) and therefore is characterized by enlarged infected RBCs, often 1 12 to 2
times normal size. A fne pink stippling known as Schffners stippling (or dots) may be present in the
cell. The young trophozoite is characterized by its ameboid appearance; by maturity, it usually fills the
RBC, and golden brown malarial pigment is present. The mature schizont contains 12 to 24 merozoites,
with an average of 16. Gametocytes are rounded and fill the cell. Macrogametocytes are often difficult
to differentiate from mature trophozoites. Figure 1.6 shows several stages of P. vivax.
1.7.2
Plasmodium malariae
Plasmodium malariae usually invades older RBCs, perhaps accounting for the occasional darker
appearance of the invaded RBC. The life cycle is characterized as quartan, with reproduction occurring
every 72 hours and invasion of new RBCs every fourth day. The trophozoite is compact and may assume
a characteristic band appearance, in which it stretches across the diameter of the RBCs (Figure 1.7,A).
Notice the presence of dark, coarse, brown-black pigment in the band form. Occasionally, a few pink
cytoplasmic dots, called Ziemanns dots, may be seen.The mature schizont contains 6 to 12 merozoites
(Figure 1.7,B), with an average of 8. Merozoites may be arranged in a characteristic loose daisy petal
arrangement around the clumped pigment; however, they may also be randomly arranged.
1.7.3
Plasmodium ovale
Plasmodium ovale, the least commonly seen species, resembles P. vivax. In P. ovale infections, the RBC
is enlarged and may assume an oval shape with fimbriated or fringelike edges. Schffners dots are less
commonly seen than with P. vivax. The parasite remains compact, has golden-brown pigment, and has
8
Figure 1.6: A, Plasmodium vivax trophozoite. B, P. vivax mature schizont. C, P. vivax macrogametocyte.
D, P. vivax microgametocyte.
Figure 1.7: A, Plasmodium malariae band form trophozoite. B, P. malariae schizont

a range of 6 to 12 merozoites in the mature schizont. It also exhibits a tertian life cycle. Figure 1.8,A,
shows trophozoites of P. ovale. The Schffners stippling in Figure 1.8,B, has stained almost a bluish pink,
but the compact organism and fimbriated cell are characteristic.
Figure 1.8: A, Plasmodium ovale trophozoite. B, Schffners stippling of P. ovale clearly visible
1.7.4
Plasmodium falciparum
Although identifed as having a tertian life cycle, P. falciparum often demonstrates an asynchronous life
cycle with rupture of the RBCs taking place at irregular intervals ranging from 36 to 48 hours. The life
cycle stages seen in peripheral blood are usually limited to the ringform trophozoite and the gametocyte.
Other stages mature in the venules and capillaries of the major organs. P. falciparum invades RBCs of
any age and, for this reason, often exhibits the highest parasitemiareaching 50% in some cases. The ring
forms of P. falciparum (Figure 1.9,A) are more delicate than those of other species and often have two
9
1.8. SURVEY OF LITERATURE

chromatin dots. Appliqu forms, parasites at the edge of the RBC, and multiple ring forms in a single RBC
are common. In this figure, the appliqu form is seen in the lower left and upper right of the photograph.
The mature trophozoite is small and compact and may have dark-brown pigment. The schizont has 8 to
36 merozoites, with an average of 20 to 24. Gametocytes have a characteristic banana or crescent shape
(Figure 1.9,B).
Figure 1.9: A, Plasmodium falciparum ring-form trophozoites. B, P. falciparum gametocyte
1.8
Survey of Literature
Rajendra Kumar et al (2003)(8) investigated the heat shock protein (Hsp-90) of Plasmodium Falciparum
and antimalarial activity of its inhibitor-galdanamycin and suggested that an active and essential Hsp-90
chaperone cycle exist in plasmodium and the favourable pharmacology of benzoquinone ansamycin
compound .
Warhurst et al (2003)(18) studied the relationship of physicochemical properties and structure of the
differential antiplasmodial activity of the cinchoma alkaloids and examined ionization constant, octomal
water distribution and haematin interaction for eight alkaloids to explain the influence of small structural
differences on activity.
Shiff (2002)(15), Najera (2002)(7), and Hyde (2002)(6) focused on finding a potent and reliable anti
parasitic drug that would inhibit plasmodium infection and growth and suggested that in nearly all
the malarial endepic populations , plasmodium has developed resistance against the hall mark drug
chloroquine and its derivatives.
Mohammed et al (2006)(1) studied various viscometric parameters , such as blood velocity, volume flow
rate and viscosity , of malarial blood , and suggested that the viscosity is high and velocity and flow rates
reduced.
Ramakrishna rao et al (2009)(13) studied size and shape of blood cells of cancer by using laser diffraction
technique ,and suggested that mean diameter of RBC of normal human is 7.12 and where as for cancer
cells is 9.35 .
Scanlon, Sanders (2007)(14) introduced some composition and properties of red blood cells and plasma
and white blood cells.
Omolade Okwa (2012)(11) described the malaria and its types. David Halliday, Robert Resnick, Jearl
Walker (2010)(4) described diffraction of light by a Circular Aperture and The Fresnel Bright Spot and
Rayleighs criterion.
Bruce Torrence, Torrence (2009)(16) reported introduces commands and procedures pertinent to the
linear algebra . The size and shape of Red blood cells (RBC) are of clinical importance not only to
characteristic different cells but also in differentiating abnormal from normal cells . For example the size
of red blood cells from one individual are not all equal but are distributed about mean value and hence
the average size has to be determined . Bernard oser (1954)(9) reported that in case pernicious anemia
, the mean size of RBC is greater than the normal and there is larger than normal variation among the
cells.
Hartell (1970)(5) mentioned that there exist to variation not only in size but also in the shape of yeast
cells during their cell cycle , leading to periodic fluctuation in density . Longhurst (1964)(10), Charles
meyer (1934)(3) and Calthrope (1952)(2) reported that average size of the cells can be measure by youngs
eriometer employed in the determination of the average size of lycopodium particles. This method based
on the babinets principle , given fraunhofer diffraction pattern on the retina of the observer .
Anwar Ali (1983)(17) developed a simple and rapid method for the determining the average size and
shape of biological cells by Appling laser diffraction , which is found to be free from practical definite of
the eriometer technique .
A Perusal of literature revals different methods and investigation for the control of malarial parasites but
10
not much alteration has been given to study the changes in RBC physiology of the blood drawn from the
patients suffering from malaria at the membrane level. Therefor an attempt has been made to study the
alteration in RBC physiology of malarial blood by employing Laser diffraction technique .
11
1.8. SURVEY OF LITERATURE
12
Chapter 2
Theoretical aspects of diffraction

2.1
Diffraction and the Wave Theory of Light
The light produces an interference pattern called a diffraction pattern. For example, when monochromatic
light from a distant source (or a laser) passes through a narrow slit and is then intercepted by a viewing
screen, the light produces on the screen a diffraction pattern like that in Figure 2.1. This pattern consists
of a broad and intense (very bright) central maximum plus a number of narrower and less intense maxima
(called secondary or side maxima) to both sides. In between the maxima are minima. Light flares into
those dark regions, but the light waves cancel out one another.
Figure 2.1: This diffraction pattern appeared on a viewing screen when light that had passed through a
narrow vertical slit reached the screen. Diffraction caused the light to flare out perpendicular to the long
sides of the slit. That flaring produced an interference pattern consisting of a broad central maximum
plus less intense and narrower secondary (or side) maxima, with minima between them
Such a pattern would be totally unexpected in geometrical optics: If light traveled in straight lines as
rays, then the slit would allow some of those rays through to form a sharp rendition of the slit on the
viewing screen instead of a pattern of bright and dark bands as seen in Figure 2.1.
Diffraction is not limited to situations when light passes through a narrow opening (such as a slit or
pinhole). It also occurs when light passes an edge, such as the edges of the razor blade whose diffraction
pattern is shown in Figure 2.2. Note the lines of maxima and minima that run approximately parallel
to the edges, at both the inside edges of the blade and the outside edges. As the light passes, say, the
vertical edge at the left, it flares left and right and undergoes interference, producing the pattern along
13
2.2. DIFFRACTION BY A CIRCULAR APERTURE

the left edge. The rightmost portion of that pattern actually lies behind the blade, within what would
be the blades shadow if geometrical optics prevailed.
Figure 2.2: The diffraction pattern produced by a razor blade in monochromatic light. Note the lines of
alternating maximum and minimum intensity
A common example of diffraction when one looks at a clear blue sky and see tiny specks and hairlike
structures floating in the view. These floaters, as they are called, are produced when light passes the
edges of tiny deposits in the vitreous humor, the transparent material filling most of the eyeball. What
is seen when a floater is in the field of vision is the diffraction pattern produced on the retina by one of
these deposits. If it is sight through a pinhole in a piece of cardboard so as to make the light entering the
eye approximately a plane wave, individual maxima and minima in the patterns can be distinguished.
Diffraction is a wave effect. That is, it occurs because light is a wave and it occurs with other types of
waves as well. For example, one might have probably seen diffraction in action at football games. When
a cheerleader near the playing field yells up at several thousand noisy fans, the yell can hardly be heard
because the sound waves diffract when they pass through the narrow opening of the cheerleaders mouth.
This flaring leaves little of the waves traveling toward the fans in front of the cheerleader. To offset the
diffraction, the cheerleader can yell through a megaphone. The sound waves then emerge from the much
wider opening at the end of the megaphone. The flaring is thus reduced, and much more of the sound
reaches the fans in front of the cheerleader.
2.2
Diffraction by a Circular Aperture
Diffraction is consider by a circular aperture - that is, a circular opening, such as a circular lens, through
which light can pass. Figure 2.3 shows the image formed by light from a laser that was directed onto
a circular aperture with a very small diameter. This image is not a point, as geometrical optics would
suggest, but a circular disk surrounded by several progressively fainter secondary rings. Comparison with
Figure 2.1 leaves little doubt that we are dealing with a diffraction phenomenon. Here, however, the
aperture is a circle of diameter rather than a rectangular slit.
The (complex) analysis of such patterns shows that the first minimum for the diffraction pattern of a
circular aperture of diameter d is located by
sin = 1.22
(2.1)
The angle here is the angle from the central axis to any point on that (circular) minimum. Compare
this with a sin =
sin =
(2.2)
which locates the first minimum for a long narrow slit of width a . The main difference is the factor 1.22,
which enters because of the circular shape of the aperture.
14
CHAPTER 2. THEORETICAL ASPECTS OF DIFFRACTION
Figure 2.3: The diffraction pattern of a circular aperture. Note the central maximum and the circular
secondary maxima. The figure has been overexposed to bring out these secondary maxima, which are
much less intense than the central maximum
2.2.1
Resolvability
The fact that lens images are diffraction patterns is important when one wishs to resolve (distinguish)
two distant point objects whose angular separation is small. Figure 2.4 shows, in three different cases,
the visual appearance and corresponding intensity pattern for two distant point objects (stars, say) with
small angular separation. In Figure 2.4a, the objects are not resolved because of diffraction; that is,
their diffraction patterns (mainly their central maxima) overlap so much that the two objects cannot be
distinguished from a single point object. In Figure 2.4b the objects are barely resolved, and in Figure 2.4c
they are fully resolved.
Figure 2.4: At the top, the images of two point sources (stars) formed by a converging lens. At the
bottom, representations of the image intensities. In (a) the angular separation of the sources is too small
for them to be distinguished, in (b) they can be marginally distinguished, and in (c) they are clearly
distinguished. Rayleighs criterion is satisfied in (b), with the central maximum of one diffraction pattern
coinciding with the first minimum of the other
In Figure 2.4b the angular separation of the two point sources is such that the central maximum of the
diffraction pattern of one source is centered on the first minimum of the diffraction pattern of the other, a
condition called Rayleighs criterion for resolvability. From (2.3), two objects that are barely resolvable.
by this criterion must have an angular separation R of
15
R = sin1 (1.22 )
a
Since the angles are small, we can replace sin R with R expressed in radians
(2.3)
(2.4)
a
Applying Rayleighs criterion for resolvability to human vision is only an approximation because
visual resolvability depends on many factors, such as the relative brightness of the sources and their
surroundings, turbulence in the air between the sources and the observer, and the functioning of the
observers visual system.
Experimental results show that the least angular separation that can actually be resolved by a person is
generally somewhat greater than the value given by (2.4). However, for calculations here, taking (2.4)
as being a precise criterion: If the angular separation between the sources is greater than R , we can
visually resolve the sources; if it is less, we cannot.
Rayleighs criterion can explain the arresting illusions of color in the style of painting known as pointillism
(Figure 2.5). In this style, a painting is made not with brush strokes in the usual sense but rather with a
myriad of small colored dots. One fascinating aspect of a pointillistie painting is that when one change
his distance from it, the colors shift in subtle, almost subconscious ways. This color shifting has to do
with whether he can resolve the colored dots. When one stands close enough to the painting, the angular
separations of adjacent dots are greater than R and thus the dots can be seen individually. Their
colors are the true colors of the paints used. However, when one stands far enough from the painting,
the angular separations are less than R and the dots cannot be seen individually. The resulting blend
of colors coming into the eye from any group of dots can then cause the brain to make up a color for
that group-a color that may not actually exist in the group. In this way, a pointillistic painter uses the
viewers visual system to create the colors of the art.
R = 1.22
Figure 2.5: The pointillistic painting The Seine at Herblay by Maximilien Luce consists of thousands of
colored dots. With the viewer very close to the canvas, the dots and their true colors are visible. At
normal viewing distances, the dots are irresolvable and thus blend
When anybody wishs to use a lens instead of our visual system to resolve objects of small angular
separation, it is desirable to make the diffraction pattern as small as possible.
According to (2.4), this can be done either by increasing the lens diameter or by using light of a shorter
wavelength. For this reason ultraviolet light is often used with microscopes because its wavelength is
shorter than a visible light wavelength.
2.2.2
Babinet Principle
Two pupil functions, A1 (x, y) and A2 (x, y) are complementary to each other if
16
CHAPTER 2. THEORETICAL ASPECTS OF DIFFRACTION
A1 (x, y) + A2 (x, y) = constant
(2.5)
If the amplitude is constant over the whole x, y plane, then it is say that two diffracting apertures are
complementary if the transparent parts in one are the opaque parts in the other, and vice versa. For
example, the complementary aperture of a circular aperture is the whole x, y plane, excluding only the
opaque disc of the same size as the circular aperture.
assuming that there are two arbitrary complementary apertures, giving diffraction pattern U1 (P ) and
U2 (P ). If U (P ) is the amplitude of the illumination (Figure 2.6) produced without any diffraction
apertures, the Babinet principle says that
U2 (P ) + U2 (P ) = U (P )
(2.6)
which can be very easily shown to be true.

Applying this principle to the Fraunhofer diffraction patterns produced by a circular aperture and an
opaque disc, it can see that U (P ) is a very high and narrow peak at the origin, because all the light will
travel in a single direction if there is no diffracting aperture.
Thus, except for the point at the origin, the Fraunhofer diffraction patterns of complementary apertures
are identical in shape but with amplitudes of opposite sign.
Figure 2.6: Fraunhofer diffraction pattern for four different apertures

When the diffraction pattern is the equation for the first and second order minima can be written as
according to Rayleighs criterion
sin1 = 1.22
(2.7)
sin2 = 1.22
(2.8)
For small angles of diffraction , equations (2.7) and (2.8) may be approximated as
tan1 =
= 1.22
D1
d
17
(2.9)
tan2 =
= 1.22
D1
d
(2.10)
Where 1 and D1 are the angle of diffraction and the sample to screen distance respectively for the
first order minima, 2 and D2 are the corresponding quantities for the second order diffraction minima ,
the mean diameter of the cells was calls was calculated using the equations (2.9) and (2.10) taking into
account the wavelength of the laser light 6328 108 cm .
18
Chapter 3
Materials and Methods

This chapter describes the procedure of collection and preparation of blood samples for the
experimentation.
This experiment is based on Babinet principle, its theorem concerning diffraction that states that the
diffraction pattern from an opaque body is identical to that from a hole of the same size and shape except
for the overall forward beam intensity , that gives Fraunhofer diffraction pattern.
There are methods to determine of size of blood
Microscope
Eriometer
Diffraction of laser beam
3.1
Samples collection
Fresh samples of normal human blood and samples from the patients suffering from malaria (Plasmodium
vivax) were collected from fever hospital and apollo hospital Hyderabad.
Shape and size of blood was determined ,using the technique of laser diffraction in the biophysics
laboratory.
3.2
Preparation of a thin blood film on the slide
Clean the finger with cotton wool dampened with alcohol (Figure 3.1).
Figure 3.1: Clean the Finger

Using a sterile lancet and a quick rolling action, puncture the ball of the finger or toe (Figure 3.2).
Apply gentle pressure to the finger and collect a single small drop of blood about this size on the middle
of the slide. his is for the thin film (Figure 3.3).
Using another clean slide as a spreader and with the slide with the blood resting on a flat, firm surface,
touch the small drop of blood with the edge of the spreader, allowing the blood to run right along the edge.
Firmly push the spreader along the slide, keeping it at an angle of 45 . the edge of the spreader must
remain in even contact with the surface of the other slide while the blood is being spread (Figure 3.4).
19
3.3. EXPERIMENTAL SET UP
Figure 3.2: Puncture the Finger
Figure 3.3: Collect the Blood
Figure 3.4: Spread of the Blood on the Slide
3.3
Experimental set up
Figure 3.5 is the schematic experimental arrangement of the laser diffraction . A He-Ne laser (L) of power
2mw was employed for the diffraction purpose .
Figure 3.6 gives the experimental set up to obtain laser diffraction . The specimen slide B, was introduced
between the laser and the screen S , such that the smeared surface facing the screen .A well defined
diffraction pattern was obtained on the screen. Circular diffraction patterns were observed with malarial
cells and disc shaped RBC .
3.4
Procedure and Analysis
In this part of the experiment we will determine the size of blood d by passing a laser beam through
slide and examining the diraction pattern projected on a screen. The laser beam is parallel and
monochromatic. Experimental set up consist of He-Ne laser and slide with blood smear and screen .
here, both screen and slide surface are perpendicular to the incident beam. The distance D between the
20
CHAPTER 3. MATERIALS AND METHODS
Figure 3.5: Schematic Experimental Arrangement of Laser Diffraction
Figure 3.6: Experimental Arrangement of Laser Diffraction

slide and the screen was fixed. The laser beam is passing through the slide. A diffraction pattern is seen
in the screen, produced by RBC. Then the radius of first order diffraction was measured using meter
scale of least count 0.1cm.for distance between screen and slide to be D.
The radius r of the first order diffraction minima was measured for different values of the sample to
screen distance, D. A plot was drawn between radius of the diffraction minima r and the sample to
screen distance D, the slope of which gives the angle of diffraction . using the equation (2.9) to calculate
d for first order of diffraction.
21
3.4. PROCEDURE AND ANALYSIS
22
Chapter 4
Results and Discussion

The forth chapter concerns with the result of the present investigation on size of erythrocytes of normal
persons and also of patients suffering from malaria. In this chapter, results are presented for ten blood
samples each of normal and malarial blood.
Tables 4.1-4.10 present data on radius (r) of first order diffraction ring and corresponding distance (D)
for the erythrocytes of blood collected from 10 healthy donors. These parameters are required for the
calculation of size of erythrocytes. Figures 4.1 - 4.10 show the plots drawn between D on x axis and
r on y - axis for 20 samples. Plots are straight lines passing through origin and the slopes of which are
used to calculate average size of erythrocytes. The parameters are concerned with first order diffraction.
Similarly, Tables 4.11 - 4.20 present data on D and r for the erythrocytes of blood of 10 patients suffering
from Malaria. Figures 4.11 - 4.20 show the plots drawn between D on x axis and r on y - axis for 20
samples. Here also plots are straight lines passing through origin, The slope of which gives the angle
diffraction.
Table 4.21 reveals data on average size of erythrocytes of normal and malarial blood for 10 samples each.
It is evident from Table 4.21 that size of malarial erythrocytes is more that of normal. In the present
study, the size of normal erythrocytes is in the range of 7.70 - 8.16 m, while it is in the range of 9.46 10.04 mum for malarial erythrocytes. The same is represented by bar graphs to have at a glance picture
for the comparison purpose (Figure 4.21).
Figure 4.22 shows micrographs of normal and malarial erythrocytes taken from light microscope of
L. C. 10 m interfacing with computer. Significantly more size in the case of malarial erythrocytes in
comparison with the normal is evident. In the care of malaria still the erythrocytes are circular in shape.
No morphological abnormalities can be seen in malarial erythrocytes. Figure 4.23 presents diffraction
patterns of both normal and malarial, when Red laser light is used. The diffraction patterns are circular
rings, because of the fact that human erythrocytes are of biconcave disc type. First and second order
diffraction can be seen. Malarial erythrocytes of relatively larger size give diffraction rings with smaller
radius in comparison with normal erythrocytes. This aspect is confirmed with the formula obtained for
the size of the erythrocytes (ie circular aperture).
23
Table 4.1: Data on the distance r of first order of diffraction as a function of distance D between the
slide and the screen for sample 1 (Normal sample)
Figure 4.1: The radius r of first order of diffraction ring as a function of distance D between the slide
and the screen for sample 1 (Normal sample)
24
CHAPTER 4. RESULTS AND DISCUSSION
25
26
27
28
29
30
31
32
33
slide and the screen for sample 11 (Malarial sample)
and the screen for sample 11 (Malarial sample)
34
35
36
37
38
39
40
41
42
43
4.1. CONCLUSIONS
Table 4.21: Size (d) of normal and malarial erythrocytes
Figure 4.21: A comparison of Normal and Malarial erythrocytes

The parasite exists on the surface of RBC as a result the size gets affected. The size of human erythrocytes
of malaria patients is more, when compared with the normal (Table 4.21), as the parasite enters into the
blood. The blood gets affected so also the erythrocytes (Figure 4.22). The diffraction pattern of normal
and malarial bloods as seen in the Figure 4.23.
The study reveals that the size of the malaria is more in comparison with the normal blood.
The mean diameter of RBC of normal human obtained by this method is 7.83 m and where as for
malaria cells is 9.75m (Figure 4.21). In the case of malaria , the size obtained is drastically increased
due to changes taken place on the RBC cell membrane, because of high metabolic activity in the case
of malaria and this leads to the changes in the cell morphology and size. The width of the diffraction
pattern is a function of the variation in the size of the particles. similarly the sharpness of the minima
depends upon the consistency in the cellular size and shape. All human blood cells are of uniform shape
and hence the diffraction pattern produced by these cells are very sharp and clear. In the case of malarial
blood cells the variation in the shape is considerably more and hence the sharpness of the pattern.
4.1
Conclusions
1. Laser diffraction method is very rapid and simple for assessing the average size of the cells.
2. At a glance picture of diffraction pattern gives an insight into the diffraction of light by small
particles.
44
Figure 4.22: Micrograph of RBCs (A) normal B) malaria
Figure 4.23: Diffractograms of human erythrocytes (A) normal (B) malaria

3. The method is not only simple but also elegant.
4. It can be readily handled and can be easily demonstrated to a large gathering at a time especially
to moderately sophisticated health science students which would provide them to look at physical
optics as a set of relevant phenomena.
5. This could also be used with advantage as a diagnostic tool for assessing if there is more than
normal variation in the size distribution of human RBC from the width of the diffraction ring.
6. It is inexpensive, besides being accurate.
7. Size of malarial is significantly large when compared with normal erythrocytes.
8. There is variation in size of malarial erythrocytes to some extent within the same blood.
45
4.1. CONCLUSIONS
46
Appendix of Mathematica Programs

In this appendix the commands present some Mathematica programs aimed at the numerical and
graphical investigation of discrete dynamical systems.
Sample 1
datan1={{5,0.45},{6,0.55},{7,0.7},{8,0.8},{9,0.95},{10,1},{11,1.1},{12,1.25},{13,1.35},{14,1.4},{15,1.45
}};
Text@Grid[Prepend[datan1,{D(cm),r(cm)}],FrameAll,Dividers{Center,{False,True}},Spacings
2,FrameStyleRed]
gp=ListPlot[datan1,PlotStyle{Black},FrameTrue,FrameLabel{D(cm),r(cm)},PlotRange{
{0,20},{0,1.5}},AxesOrigin{0,0},LabelStyleDirective[Black,10]];
be=Fit[datan1,{0,x},x]
gbe=Plot[be,{x,0,15},PlotStyle{Blue},FrameTrue,FrameLabel{D(cm),r(cm)},PlotRange{
Show[gp,gbe,EpilogText[Style[r=0.100 D, Black,10],{5,1}]]
Sample 2
datan2={{5,0.45},{6,0.55},{7,0.65},{8,0.75},{9,0.9},{10,1},{11,1.1},{12,1.3},{13,1.35},{14,1.4},{15,1.45
}};
2,FrameStyleRed]
Sample 3
datan3={{5,0.45},{6,0.55},{7,0.65},{8,0.7},{9,0.85},{10,1},{11,1.1},{12,1.3},{13,1.35},{14,1.4},{15,1.45
}};
2,FrameStyleRed]
47
4.1. CONCLUSIONS
Sample 4
datan4={{5,0.5},{6,0.55},{7,0.65},{8,0.75},{9,0.9},{10,1},{11,1.2},{12,1.25},{13,1.3},{14,1.4},{15,1.45}
};
2,FrameStyleRed]
Sample 5
datan5={{5,0.6},{6,0.65},{7,0.7},{8,0.75},{9,0.9},{10,1},{11,1.1},{12,1.2},{13,1.35},{14,1.4},{15,1.45}}
;
2,FrameStyleRed]
Sample 6
datan6={{5,0.45},{6,0.5},{7,0.7},{8,0.75},{9,0.85},{10,0.9},{11,1.15},{12,1.25},{13,1.35},{14,1.4},{15,1
.45}};
2,FrameStyleRed]
48

Sample 7
datan7={{5,0.4},{6,0.55},{7,0.7},{8,0.8},{9,0.85},{10,0.9},{11,1.1},{12,1.2},{13,1.25},{14,1.3},{15,1.35
}};
2,FrameStyleRed]
Sample 8
datan8={{5,0.45},{6,0.55},{7,0.7},{8,0.85},{9,0.95},{10,1},{11,1.1},{12,1.25},{13,1.3},{14,1.35},{15,1.4
}};
2,FrameStyleRed]
Sample 9
datan9={{5,0.55},{6,0.6},{7,0.65},{8,0.85},{9,0.9},{10,1},{11,1.1},{12,1.2},{13,1.3},{14,1.35},{15,1.45}
};
2,FrameStyleRed]
49
4.1. CONCLUSIONS
Sample 10
datan10={{5,0.5},{6,0.65},{7,0.7},{8,0.85},{9,0.9},{10,1},{11,1.1},{12,1.2},{13,1.3},{14,1.35},{15,1.4}}
;
Text@Grid[Prepend[datan10,{D(cm),r(cm)}],FrameAll,Dividers{Center,{False,True}},Spacing
s2,FrameStyleRed]
gp=ListPlot[datan10,PlotStyle{Black},FrameTrue,FrameLabel{D(cm),r(cm)},PlotRange
{{0,20},{0,1.5}},AxesOrigin{0,0},LabelStyleDirective[Black,10]];
Sample 11
datam1={{5,0.35},{6,0.4},{7,0.5},{8,0.55},{9,0.6},{11,0.7},{12,0.8},{13,0.9},{14,1.1},{15,1.2}};
Text@Grid[Prepend[datam1,{D(cm),r(cm)}],FrameAll,Dividers{Center,{False,True}},Spacing
s2,FrameStyleRed]
gp=ListPlot[datam1,PlotStyle{Black},FrameTrue,FrameLabel{D(cm),r(cm)},PlotRange{
be=Fit[datam1,{0,x},x]
Sample 12
datam2={{5,0.4},{6,0.45},{7,0.5},{8,0.65},{9,0.7},{10,0.75},{11,0.8},{12,0.95},{13,1.1},{14,1.2},{15,1.3
}};
s2,FrameStyleRed]
Sample 13
datam3={{5,0.35},{6,0.4},{7,0.5},{8,0.6},{9,0.65},{10,0.8},{11,0.9},{12,1},{13,1.1},{14,1.2},{15,1.3}};
s2,FrameStyleRed]
50

Sample 14
datam4={{5,0.4},{6,0.45},{7,0.5},{8,0.65},{9,0.7},{11,0.8},{12,0.95},{13,1},{14,1.1},{15,1.2}};
s2,FrameStyleRed]
Sample 15
datam5={{5,0.35},{6,0.45},{7,0.55},{8,0.65},{9,0.75},{10,0.7},{11,0.8},{12,1},{13,1.1},{14,1.2}};
s2,FrameStyleRed]
Sample 16
datam6={{5,0.4},{6,0.45},{7,0.55},{8,0.6},{9,0.75},{10,0.8},{11,0.9},{12,0.95},{13,1},{14,1.1},{15,1.3}
};
s2,FrameStyleRed]
51
4.1. CONCLUSIONS
Sample 17
datam7={{5,0.4},{6,0.45},{7,0.5},{8,0.55},{9,0.65},{10,0.7},{11,0.9},{12,1},{13,1.1},{14,1.2},{15,1.3}};
s2,FrameStyleRed]
Sample 18
datam8={{5,0.35},{6,0.5},{7,0.55},{8,0.6},{9,0.65},{10,0.8},{11,0.9},{12,1},{13,1.1},{14,1.2}};
s2,FrameStyleRed]
Sample 19
datam9={{5,0.35},{6,0.4},{7,0.55},{8,0.6},{9,0.7},{10,0.8},{11,0.85},{12,0.9},{13,1.1},{14,1.2},{15,1.3}
};
s2,FrameStyleRed]
Sample 20
datam10={{5,0.3},{6,0.4},{7,0.5},{8,0.6},{9,0.7},{10,0.8},{11,0.85},{12,0.9},{13,1},{14,1.1},{15,1.2}};
52

Text@Grid[Prepend[datam10,{D(cm),r(cm)}],FrameAll,Dividers{Center,{False,True}},Spacin
gs2,FrameStyleRed]
gp=ListPlot[datam10,PlotStyle{Black},FrameTrue,FrameLabel{D(cm),r(cm)},PlotRange
{{0,20},{0,1.5}},AxesOrigin{0,0},LabelStyleDirective[Black,10]];
Table of size for all samples

dataa={{1,10.73,7.96},{2,9.53,7.73},{3,9.46,7.79},{4,9.93,7.73},{5,9.61,7.7},{6,9.6,7.83},{7,9.54,8.16},{8
,9.48,7.8},{9,9.55,7.79},{10,10.04,7.82}};
Text@Grid[Prepend[dataa,{sample,sizeofmalarialblood,sizeofnormalblood}],FrameAll,Dividers
{Center,{False,True}},Spacings4,FrameStyleRed]
Bar graph
Needs[BarCharts]
BarChart[{7.83,9.75}, BarLabels{normal blood, malarial blood},AxesLabelsize]
53
4.1. CONCLUSIONS
54
Bibliography
[1] Mohammed Gulam Ahamad, Kaleem Ahmed Jaleeli, and Adeel Ahmad. J.Pure & Appl. Phys.,
18(4), 2006.
[2] J. Calthrope. Advanced experiments in practical optics. William Heinemann Ltd.,London, 1952.
[3] Charles and F Mayer. The diffraction of light x-ray and material particles. University of Chicago
Press, Chicago, Illinois, 1934.
[4] David Halliday, Robert Resnick, and Jearl Walker. Fundamentals of Physics. Wiley, 10th edition,
2010.
[5] Leland H. Hartwell. Periodic density fluctuation during the yeast cell cycle and the selection of
synchronous cultures. Journal of Bacteiology, 104(3):12801285, December 1970.
[6] John E. Hyde. Mechanisms of resistance of plasmodium falciparum to antimalarial drugs. Microbes
and Infection, 4(2):165174, February 2002.
[7] Njera JA. Malaria control: achievements, problems and strategies. Parassitologia, 43(12):189, Jun
2001.
[8] Rajinder Kumar, Alla Musiyenko, and Sailen Barik. The heat shock protein 90 of plasmodium
falciparum and antimalarial activity of its inhibitor, geldanamycin. Malaria Journal, 2(30),
September 2003.
[9] Bernard Levussove and Oser. Hawks Physiological Chemistry. McGraw-Hill, 14th edition, 1965.
[10] R.S. Longhurst. Geometrical and Physical Optics. Longman, 3th edition, June 1974.
[11] Omolade O. Okwa. Malaria Parasites. InTech, 1st edition, March 2012.
[12] World Health Organization. Basic malaria microscopy,Part I. Learners guide. WHO press, 2nd
edition, 2010.
[13] Ramakrishna Rao, Kaleem Ahmed Jaleeli, Bellubbi BS, and Adeel Ahmad. J. Pure & Appl. Phys.,
21(2):201203, 2009.
[14] Valerie C. Scanlon and Tina Sanders. Essentials of Anatomy and Physiology. F. A. Davis Company,
5th edition, 2007.
[15] Clive Shiff. Integrated approach to malaria control. Clinical Microbiology Reviews, 15(2):278293,
April 2002.
[16] Bruce F. Torrence and Eve A. Torrence. The Students Introduction to MATHEMATICA. Cambridge
University Press, 2nd edition, 2009.
[17] Anwar Ali A K W. ultrasonic and dielectrophoresis studies in biological media. PhD thesis, Osmania
university, Hyderabad, india, 1983.
[18] David C Warhurst, John C Craig, Ipemida S Adagu1, David J Meyer1, and Sylvia Y Lee. The
relationship of physico-chemical properties and structure to the differential antiplasmodial activity
of the cinchona alkaloids. Malaria Journal, 2(26), September 2003.
55
BIBLIOGRAPHY
56
List of Figures
1.1
1.2
1.3
1.4
1.5
1.6
1.7
1.8
1.9
2.1
Composition of Blood . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Shape and dimensions of a red blood cell . . . . . . . . . . . . . . . . . . . . . . . .
Rouleau formation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
WBC . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Platelets . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
A, Plasmodium vivax trophozoite.
B, P. vivax mature schizont.
C, P.
macrogametocyte. D, P. vivax microgametocyte. . . . . . . . . . . . . . . . . . . .
A, Plasmodium malariae band form trophozoite. B, P. malariae schizont . . . . . .
A, Plasmodium ovale trophozoite. B, Schffners stippling of P. ovale clearly visible .
A, Plasmodium falciparum ring-form trophozoites. B, P. falciparum gametocyte .
. . . .
. . . .
. . . .
. . . .
. . . .
vivax
. . . .
. . . .
. . . .
. . . .
2.6
This diffraction pattern appeared on a viewing screen when light that had passed through
a narrow vertical slit reached the screen. Diffraction caused the light to flare out
perpendicular to the long sides of the slit. That flaring produced an interference pattern
consisting of a broad central maximum plus less intense and narrower secondary (or side)
maxima, with minima between them . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
The diffraction pattern produced by a razor blade in monochromatic light. Note the lines
of alternating maximum and minimum intensity . . . . . . . . . . . . . . . . . . . . . . .
The diffraction pattern of a circular aperture. Note the central maximum and the circular
secondary maxima. The figure has been overexposed to bring out these secondary maxima,
which are much less intense than the central maximum . . . . . . . . . . . . . . . . . . . .
At the top, the images of two point sources (stars) formed by a converging lens. At
the bottom, representations of the image intensities. In (a) the angular separation of
the sources is too small for them to be distinguished, in (b) they can be marginally
distinguished, and in (c) they are clearly distinguished. Rayleighs criterion is satisfied in
(b), with the central maximum of one diffraction pattern coinciding with the first minimum
of the other . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
The pointillistic painting The Seine at Herblay by Maximilien Luce consists of thousands
of colored dots. With the viewer very close to the canvas, the dots and their true colors
are visible. At normal viewing distances, the dots are irresolvable and thus blend . . . . .
Fraunhofer diffraction pattern for four different apertures . . . . . . . . . . . . . . . . . .
3.1
3.2
3.3
3.4
3.5
3.6
Clean the Finger . . . . . . . . . . . . . . . . . . . . . . .

Puncture the Finger . . . . . . . . . . . . . . . . . . . . .
Collect the Blood . . . . . . . . . . . . . . . . . . . . . . .
Spread of the Blood on the Slide . . . . . . . . . . . . . .
Schematic Experimental Arrangement of Laser Diffraction
Experimental Arrangement of Laser Diffraction . . . . . .
4.1
The radius r of first order of diffraction ring as a function of

and the screen for sample 1 (Normal sample) . . . . . . . .
2.2
2.3
2.4
2.5
4.2
4.3
4.4
4.5
57
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distance
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distance
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distance
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distance
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distance
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D between the slide

. . . . . . . . . . . .
D between the slide
. . . . . . . . . . . .
D between the slide
. . . . . . . . . . . .
D between the slide
. . . . . . . . . . . .
D between the slide
. . . . . . . . . . . .
2
3
3
5
7
9
9
9
10
13
14
15
15
16
17
19
20
20
20
21
21
24
25
26
27
28
LIST OF FIGURES
4.6
4.7
4.8
4.9
4.10
4.11
4.12
4.13
4.14
4.15
4.16
4.17
4.18
4.19
4.20
4.21
4.22
4.23
The radius r of first order of diffraction ring as a function of distance

and the screen for sample 6 (Normal sample) . . . . . . . . . . . . .
and the screen for sample 11 (Malarial sample) . . . . . . . . . . . .
A comparison of Normal and Malarial erythrocytes . . . . . . . . . .
Micrograph of RBCs (A) normal B) malaria . . . . . . . . . . . . . .
Diffractograms of human erythrocytes (A) normal (B) malaria . . .
58
D between the slide

. . . . . . . . . . . .
D between the slide
. . . . . . . . . . . .
D between the slide
. . . . . . . . . . . .
D between the slide
. . . . . . . . . . . .
D between the slide
. . . . . . . . . . . .
D between the slide
. . . . . . . . . . . .
D between the slide
. . . . . . . . . . . .
D between the slide
. . . . . . . . . . . .
D between the slide
. . . . . . . . . . . .
D between the slide
. . . . . . . . . . . .
D between the slide
. . . . . . . . . . . .
D between the slide
. . . . . . . . . . . .
D between the slide
. . . . . . . . . . . .
D between the slide
. . . . . . . . . . . .
D between the slide
. . . . . . . . . . . .
. . . . . . . . . . . .
. . . . . . . . . . . .
. . . . . . . . . . . .
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
45
List of Tables
4.1
4.2
4.3
4.4
4.5
4.6
4.7
4.8
4.9
4.10
4.11
4.12
4.13
4.14
4.15
4.16
4.17
4.18
4.19
4.20
4.21
Data on the distance r of first order of diffraction as a function of distance D between the
slide and the screen for sample 1 (Normal sample) . . . . . . . . . . . . . . . . . . . . . .
slide and the screen for sample 11 (Malarial sample) . . . . . . . . . . . . . . . . . . . . .
Size (d) of normal and malarial erythrocytes . . . . . . . . . . . . . . . . . . . . . . . . . .
59
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44

A STUDY ON SIZE AND SHAPE OF ERYTHROCYTES OF NORMAL AND MALARIAL BLOOD USING LASER DIFFRACTION TECHNIQUE by Amar Alansi

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A STUDY ON SIZE AND SHAPE OF ERYTHROCYTES OF NORMAL AND MALARIAL BLOOD USING LASER DIFFRACTION TECHNIQUE by Amar Alansi

Hochgeladen von

Copyright:

Verfügbare Formate

A STUDY ON SIZE AND SHAPE OF ERYTHROCYTES OF

NORMAL AND MALARIAL BLOOD USING LASER

A STUDY ON SIZE AND SHAPE OF ERYTHROCYTES

2 Theoretical aspects of diffraction

3 Materials and Methods

4 Results and Discussion

Appendix of Mathematica Programs

Amar Yahya Yahay Alansi

my brothers and my sisters

Amar Yahya Yahay Alansi

1.3. BLOOD CELLS

Figure 1.1: Composition of Blood

Three types of cells are present in the blood:

Plasma proteins are:

Properties of Plasma Proteins

Red Blood Cells

Diameter : 7.2 (6.9 to 7.4 ).

Figure 1.2: Shape and dimensions of a red blood cell

Properties of red blood cells

Figure 1.3: Rouleau formation

Paked Cell Volume

1.4. PLASMA PROTEINS

Function of Red Blood Cells

Transport of Oxygen from the Lungs to the Tissues

Variations in size of red blood cells

White Blood Cells

Figure 1.4: WBC

Properties of White Blood Cells

Function of White Blood Cells

Figure 1.5: Platelets

Strucyure and Composition

Platelet is constituted by:

Platelets have three important properties (three As):

Figure 1.7: A, Plasmodium malariae band form trophozoite. B, P. malariae schizont

1.8. SURVEY OF LITERATURE

Figure 1.9: A, Plasmodium falciparum ring-form trophozoites. B, P. falciparum gametocyte

1.8. SURVEY OF LITERATURE

Theoretical aspects of diffraction

Diffraction and the Wave Theory of Light

2.2. DIFFRACTION BY A CIRCULAR APERTURE

Diffraction by a Circular Aperture

CHAPTER 2. THEORETICAL ASPECTS OF DIFFRACTION

2.2. DIFFRACTION BY A CIRCULAR APERTURE

CHAPTER 2. THEORETICAL ASPECTS OF DIFFRACTION

A1 (x, y) + A2 (x, y) = constant

which can be very easily shown to be true.

Figure 2.6: Fraunhofer diffraction pattern for four different apertures

2.2. DIFFRACTION BY A CIRCULAR APERTURE

Materials and Methods

Preparation of a thin blood film on the slide

Figure 3.1: Clean the Finger

3.3. EXPERIMENTAL SET UP

Figure 3.2: Puncture the Finger

Figure 3.3: Collect the Blood

Figure 3.4: Spread of the Blood on the Slide

Procedure and Analysis

CHAPTER 3. MATERIALS AND METHODS

Figure 3.5: Schematic Experimental Arrangement of Laser Diffraction

Figure 3.6: Experimental Arrangement of Laser Diffraction

3.4. PROCEDURE AND ANALYSIS

Results and Discussion

CHAPTER 4. RESULTS AND DISCUSSION

CHAPTER 4. RESULTS AND DISCUSSION

CHAPTER 4. RESULTS AND DISCUSSION