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In 1832 "Robert Brown" discovered nucleus. It is the essential part of the cell.
 
A definite nucleus is absent in prokaryotic cell. Instead of it a DNA molecules is present. It is called by “Prokaryotic cell". Ex: Bacteria.
 
Definite nucleus is present. It is cov­ered by nuclear membrane.
 
The position of a nucleus in the cell is definite. It occupies more or less central position. Now and then it is pushed to die periphery.
 
Number:
 
  1. Anurfeate: Nucleus is absent Ex: RBC of mammal
  2. Mononucleate: It is very common. Usually the cell contains a single nucleus
  3. Binucleate: Two nuclei are present. Ex: cells of cartilage.
  4. Poly nucleate: Three to many nuclei are present. In animals poly nucleate cell is called "Syncytial cell". Ex: Syncytial cell of osteoblast.
.
Shape:
 
It is usually oval. But its shape is dependent on the cell type.
 
  1. Cylindrical: Ex: Columunar epithelium.
  2. Horse shoe shape: Ex: Paramecium meganucleus.
  3. Tri lobed nucleus: Ex: WBC of mammals.
  4. Branched nucleus: Ex: Silk spining cell of insect larvae.
  5. Irregular nucleus: Ex: Glandular cell of insect.
 
Size:
 
The size of the nucleus varies with cell type . It is directly proportional to the amount of cytoplasm. "Hertwig" gave nucleo plasmic index.
 
nucleas cell nucleoplasmic index 17
 
Structure:
 
The nucleus will show.
 
  1. Nuclear membrane.
  2. Nucleo-plasm or karyolymph.
  3. Chromatin network.
  4. Nucleolus.
 
Nuclear membrane:
 
It is called nuclear envelope. It is 100 to 150 A°. The membr< nes are unit membranes. The outer membrane bears ribosomes, It is cor, nuous with endoplasmic reticulum.
 
Nuclear pores:
 
Nuclear membrane is not continuous. Here and there pores are present. Each pore is 500 to 800 A° in diameter. Each pore is covered by annulus membrane The pore and annulus will be called "Pore Complex". Because of ithese pores nucleoplasm and cytoplasm will be continuous.
 
nucleus cell structure function9
 
Nucleoplasm:
 
Below the nuclear membrane a transparent semifluid is present. It is granular. In this a chromatin network is present. It contains proteins, ions, enzymes, and nuclic acids. It is believed that nucleoplasm will take part in spindle formation.
 
Chromatin network:
 
In the nucleoplasm chromatin net work is present. In the inter phase nucleus the chromosomes are fine threads, and in the form of a network.
 
Euchromatin & Heterochromatin:
 
In the interphase nucleus the chromatin net work shows dark stained regions. They are called heterochromatin regions, Light stained regions are called euchromatin.
 
In 1928 "Hertz" defined heterochromatin as condensed chromatin part. The hetero-chromatin is two types.
 
a) Facultative-hetero-chromatin:
 
This is a temporary state of inactivation of chromatin. In which one chromosome of the pair will becomes hetero chromatin. Ex : In mammals the second X chromosome in the somatic cells of female will become hetero-chromatin, it is called ban* body.
 
b) Constitutive hetero-chromatin:
 
It is present permanently. It is present in both chromosomes of the pair. It is common in nucleolar organising chromosomes.
 
Hetero-chromatic regions genitically inactive. Now a days it is believed that it contains polygens, for the synthesis of tRNA and 5S RNA.
 
Functions:
 
  1. It gives protection to the genome.
  2. Helps in the reflection of DNA.
  3. During synaptic pairing in meiosis it attracts the homologous chromosomes.
Nucleolus:
 
It was discovered by "Fontana". In almost all the cells in the inter-phase nucleus, nucleolus is present. The number may be one or two. In some cases hundreds of nuclei will be present. Ex. Amphibian oocyte. The size of the nucleolus is depended upon the activity of the cell. They may be smaller or big.
 
nucleolus nucleus cell13
 
The nucleous shows two parts, a) Amorphous part, b) Filamentous part.
 
a) Amphorophous part contains four parts
 
  1. Granular zone
  2. Fibrillar zone
  3. Protenaceious part,
  4. Nucleolar associated chromatid.
 
Nucleous contains RNA, proteins and enzymes. It is formed with the help of nucleolar organising chromosome.
 
Functions:
 
  1. It is helpful in biogenesis of ribosomes.
  2. It plays a major role in mitosis.
 
Constitution of Nucleus:
 
It contains nucleo proteins, enzymes and inorganic salts. Low molecular weight proteins like histones, protamines are present. High molecular proteins contain tryptophan, tyrosine are constituent particles, DNA, RNA nuclei acids will be present. Salts of sodium, magnesium, calcium etc., some phospholipids are also present.
 
Functions of Nucleus:
 
  1. Nucleus plays a major role in the general metabolism of the cell
  2. It is helpful in the synthesis of ribosomes.
  3. It is helpful in the synthesis of RNA.
  4. It controls the synthesis of proteins.
  5. It is the seat of heredity.
In the cytoplasm of the cell complex membrane bounded system is present. It was called cytoplasmic vacuolar system by "Sacz & De robertis" in 1975. This system contains endoplasmic reticule, nuclear envelop, and Golgi.
 
Endoplasmic Reticulum (E.R.) was first observed by "Porter, Claude, Fullam" in 1945.
 
Occurrence: In all eukaryotic cells E.R. is present. It is absent in R.B.C. of mammals and prokaryotes. In the embry­onic cells E.R. is small. It is undifferenti­ated.
 
Types of E.R.: E.R. occurs in two forms a) Smooth E.R. b) Rough E.R.
 
endoplasmic reticulu smooth er rough er 112
 
Smooth E.R.: The E.R. without ribosomes is called smooth E.R. This is present in cells which synthesis steroids. In intestine cell, liver cells, retinal cells it is present.
 
Rough E.R: The E.R. shows ribosomes on its outer surface. They take up protein synthesis. This reticulum is more in protein synthesing cells.
 
Morphology: E.R. is represented in three forms; a) Cisternae b) Vesicles c) Tubules.
 
a) Cisternae: These are long unbranched flat structures. They are arranged parallely. They show 40 to 50 mill microns in diameter. They are more in liver pancreas, brain cells, etc.
 
b) Vesicles: They are oval membrane bounded structures. They are 25 to 500 mill microns in diameter. They occur in liver and pancreatic cells.
 
c) Tubules: They are small and branched. They show 50 to 190 mill microns in diameter. They are arranged irregularly in cell. They are seen in almost all the cells.
 
Electron microscopic structure of E.R.: All the three forms of E.R. will show double unit membranes. Each membrane shows 60 A0 thickness. The unit membrane show fluid mosaic nature of plasma membrane. Usually E.R. mem­brane is in contact with plasma membrane, nuclear membrane and Golgi. In 1965 "Palade" observed secretory granules in the E.R.
 
Biogenesis of E.R.: The origin and development of E.R. is not clearly Known.
 
1) From nuclear membrane: The membrane of E.R. resembles nuclear membrane and plasma membrane. Hence we believe that E.R. might have come from them.
 
2) E.R. Developed 'Denovo': It developed as it is.
 
3) Multistep mechanism: Rough E.R. is developed in the following way. a) Annulated lamellae: It was reported by "Meculloch" in 1952. From the nuclear membrane small structures are liberated as buds. They unite to form annulate lamellae. These lamellae are united to form cisternae. Thus E.R. is formed.
 
Functions:
 
  1. Mechanical support: E.R. will give mechanical support to cytoplasm. Hence it is called cytoskeleton.
  2. Intracellular transport: E.R. functions as circulatory system of the cell. It transport substance from one place to another place, in the cell it is called intracellular transport.
  3. Protein synthesis: ER. will provide surface for the attachment of ribosomes. These ribosomes will synthesise the proteins.
  4. Synthesis of lipoproteins: Smooth E.R. will synthesise lipids. In the Golgi complex the glycerides get associated with proteins produced by the E.R. Thus complex lipoproteins are formed.
  5. Detoxification: Smooth E.R. will detoxify internal, or external toxins. If toxins are more in the body more smooth E.R. will be produced.
  6. A.T.P. Synthesis: E.R. membranes are the sites of A.T.P, synthesis in the cell.
  7. Formation of other membranes: In the cell E.R. gives rise to.
    a) Cisternae of Golgi apparatus.
    b) Outer membrane of nucleus.
  8. Impulse conduction: In the muscle sarcoplasmic eticulum will release calcium which is responsible for the muscle action In 1926 "Porter" stated that E.R. membrane shows ionic grandients, and electric potential.
 
Interrelationship of different cell membranes:
 
  1. The outer membrane of the nucleus will give buds. They unite to form annulate lamellae.
  2. Annulate lamellae will give rough E.R.
  3. From smooth E.R. golgi will form.
  4. Vesicles will give plasma lemma.
  5. According to GERL system Golgi will give rise to primary lysosomes. Thus there is an interrelationship among the membrane systems.
In the animal and plant cells clus­ters of fat filled structures are present. They are called Golgi apparatus or complex. In 1898 Camillo Golgi' recognised it in the nerve cell of the owl. The Golgi complex of invertebrates is called dictyo­some.
 
Occurence: Golgi complex is seen in all eukaryotic cells. Golgi complex is not seen in mature sperm, red blood cell and prokaryotes.
 
Golgi complex occurs in two forms
 
a) Localized form: Golgi complex occurs singly and has a fixed position. (In between nucleus and secretory pore)
 
b) Diffused form: In the nerve and liver cells Golgi complex is scattered, in it each unit is called dictyosome.
 
Structure: Golgi body is seen in the form of three components.
 
1. Cisternae: These are tubular, flat, fluid filled sacs. They show 200 to 300A0 width. Each sac is covered by two membranes. In a dictyosome 3 to 7 cisterne are present. They are arranged one above the other. Their convex side is towards nucleus and their concave surface is towards plasma membrane. The convex side of the cisternae is called forming face. The concave surface is called maturing face. It shows big secreting vesicles. These secretory vesicles store secretory substances. They may develop into lysosomes.
 
Polarity of cisternae: The cisternae shows maturing face and forming face. Forming face is convex and towards nucleus. The smooth E.R. gives vesicles. They unite to form cisternae.
 
2. Golgi vesicles: On the forming face of golgi cisternae small vesicles are present. They are 400 A° width. They usually develop form E.R.
 
3. Secretory vesicles: On the maturing face of golgi cisternae secretory vesicles are present. They contain secretory products of golgi. They finally change into lysosomes.

Chemical Composition:
 
Golgi complex will be rich in chemical substances.
 
  1. Phospholipids: These substances have a composition which is in between the structure of phospholipids of endoplasmic reticulum and plasma membrane.
  2. Enzymes: ATP-ase, CPT-ase, transphorases, etc. enzymes are. present.
  3. Carbohydrates: Glucose, manose, galactose carbohydrates are seen.
 
Origin of Golgi Complex:
 
  1. From E R: Beams & Kessel' in 1968 proposed that Golgi system cisternae arise from Endoplasmic reticulum.
  2. Rough E.R. produces protein which will be transfer to smooth ER.
  3. Vesicles are developed from smooth E.R.
  4. By the fusion of vesicles cisternae are formed. From the cisternae secretory vesicles are formed on the maturing face. (These sides are called "G.E.R.L" -  Golgi Endoplasmic Reticulum Lysosome)
 
golgi apparatus 27

2) From nuclear membrane: In 1965 "Bouch" described the origin of golgi from the outer membrane of the nucleus. Vesicles are pinched off from the outer nuclear membrane and they are united to form cistemae.
 
3) By the division pre-existing dictyosome: During the cell division the number of dictyosomes will increase. In each daughter cell the number of dictyosomes will be equal to the dictyosome of parent cell.
 
4) Zones of exclusion: Each golgi complex is surrounded by a specific zone of cytoplasm. In this zone ribosomes and other organelles are absent. Such zone is called "Exclusive zone". E. R. present in this zone will show smooth surface.
 
Functions: Golgi complex is mainly connected with secretory function. In different types of cells different types of secretions are produced.
 
Golgi complex & secretion: The secretory mechanism will follow
 
  1. Proteins are produced by ribosome.
  2. They will be transmitted to smooth E.R.
  3. From there they are concentrated at Golgi complex.
  4. From the Golgi complex the secretory vesicles are formed.
 
They produce the secretion. The secretions are three types.
 
a) Holocrine secretion: The entire cell is filled with secretory product, it gives rise to secretions. Ex: Sebaceous gland.
b) Apocrine secretion: The secretory vesicles will come out of the plasma membrane. They take away small amount of cytoplasm. Ex: Apocrine sweat gland.
c) Merocrine secretion: Secretory vesicles will unit with P.M. and open to the exterior. Ex: Salivary gland.
 
S.No. ' Cell type -------- Golgi function
 
1. Pancreas ------- Digestive enzymes
2. Haepatic cells of liver ------ Secretion of lipids
3. Plasma cells of blood ------ Immunoglobulins
4. Thyroid gland ------ Thyroxin
 
2) Synthesis of glycoprotein: Most of the cells will produce glycopro­teins. The glycoprotein contains a protein part and carbohydrate part. They are important for secretions.
 
3) Storage of secretory products: Ribosome synthesize proteins, they are discharged into E.R. They are concentrated in the cistemae. They are stored in secretory vesicles. Thus the glycoproteins are stored in Golgi.
 
4) Formation of lysosomes: From the maturing face of cistemae granules are produced. They are united to form lysosomes.
 
5) Acrosome formation: "Burgos" stated that acrosome of the sperm is formed because of golgi spermatogensis golgi complex will develop into each vacuole a small proacrosomal granule is formed. If spherical body. Inside each vacuoleIt increases in size and they are many, they unite to form a single granule, acrosomal granule is formed It forms the acrosome. Acrosome is helps penetrate into ovum during fertilization.
Lysosome:
 
These are first ob­served in liver cells. They are 1.5 to 2 milii micron in size These are single mem­brane bounded structures. They were first called pericanalicular-dense bodies. "Chris­tian De Duve" called them lysosomes in 1955. They were named as lysosomes because they contain hydrolytic enzymes.
 
Occurence: They are present in all animal cells ,except mammalian R.B.C. They are more in liver cells spleen cells, kidney cells etc.. In bacteria they are absent.
 
Shape & size: They are round or spherical bodies. They are .4 to .8 milli microns in size.
 
Structure: Each lysosome is covered by a unit membrane. It encloses a dense matrix. It shows 2 regions, outer dense part and central less dense part.

Chemical Composition: Every lysosome will show hydrolytic enzyme. They are important in digestion of food such as 40 enzymes arc recognized in lysosome.
 
Polymorphism in lysosomes or kinds of lysosomes:
 
In the same cell at different times or in different cells 4 kinds of lysosome are reported.
 
a) Primary lysosome or storage granule:
 
  1. It is a newly formed lysosome.
  2. It is formed from golgi.
  3. It forms from G.E.R.L, which means 'Golgi associated with Endoplas­mic Reticulum will give Lysosome". This was stated by Dyson 1978.
  4. This is called original lysosome.
 
b) Phagosome or pinosome or digestive vacuole:
 
  1. A original lysosome units with a phagocytic or pinocync vesicle and forms a phagosome.
  2. In this phagosome the food is digested.
 
c) Autophagic vacuole or autolysosome:
 
When the organism is in a state of starvation the lysosome will start digesting the cell contents. Such lysosome is called autophagic vacuole.
 
d) Residual body:
 
After the process of digestion in phagosome or autophagic vacuole some materials are not digested. Such Iysosomes with undigested food is called residual body. This residual body will send the undigested matter through plasma membrane.
 
In nerve and muscle cells residual bodies are more in number. They are called, "Lipofucine granules". (By the estimation of these granules the age can be decided)
The polymorphic tendency of lysosome is not real, it is connected with the digestive activity of the lysosome.

List of some hydrolytic enzymes seen in Lysosome:
 
Lysosome List
 
Bio genesis of Lysosomes:
 
The origin of lysosome is not clearly known. According to 'Dyson' 1978 the lysosomes arise from Golgi complex and endoplasmic reticulum. The protein granules produced by ribosome is stored in endoplasmic reticulum. They move in to smooth endoplasmic reticulum. From there they move into Golgi. There they are concentrated and modified as primary lysosomes.
 
Lysosome Formation18

Functions of Lysosomes:
 
Lysosome will perform the following functions.
 
1) Digestion of large extracellular particles on the out side.
 
Liposomal enzymes will be discharged out side of the cell and digest the material present out side the cell.
 
2) Digestion In cell or intracellular digestion:
 
Lysosomes will digest the food that enters into the cell.
 
3) Cellular digestion:
 
The lysosome can digest the entire cell. It is called autolysis. Because of which De Duve called them suicidal bags of the cells. Autolysis is very important to the organisms.
 
Eg: The degeneration of tadpole tail in the fife history of frog is a result of autolysis.
 
4) Autophagy:
 
When the cell is in starvation the lysosome of a cell, will start the digesting the cell contents. This is called autophagy.
 
5) Sperm penetration:
 
During fertilization the acrosome of the sperm will produce lysosomal enzymes. They are useful to dissolve the tissue present around the ovum.
 
6) Chromosome Breaks:
 
Lysosome shows acid DNA ase it will break the chromosome and cause the rearrangement.
DEFINITION OF MITOCHONDRIA:
 
Mitochondria are center for cellular respiration. It converts chemical energy into kinetic energy.
 
Information About Mitochondria
 
  1. In 1857 Kolliker observed mitochondria and called them as sarco-somes.
  2. Flemming called them as Fila.
  3. Altmann in 1890 called them as Bioplasts.
  4. Benda gave the name mito-chondria.
  5. Porter & Palade described their electron microscopic structure.
  6. Mitochondria are present in all eukaryotic cells.
 
Size: The length varies from 1.5 to 10 microns. The smallest mitochondrion is seen in yeast. It is 1 mill micron in length. The width is .5 to .7 microns (Oocyte of amphibian show 20 to 40 microns length mitochondria.)
 
Shape: They are filamentous or they may show rod, spherical, or thread like structures.
 
Number: In Micro monas only one mitochondrion is present. In a liver cell 1000 to 1600 mitochondria are present. The highest number of mitochondria are seen in the cell of flight muscles. However cells of green plants contain less number of mitochondria. In red blood corpuscles of mammals and other higher animals mitochondria are absent.
 
Structure Of Mitochondria: The mitochondria is covered by 2 layers. It shows outer membrane and inner membrane. In each mitochondrion 2 chambers are present.
 
  1. Outer chamber
  2. Inner chamber.
 
Outer membrane: It is a continuous membrane which covers and protects the mitochondrion. !t separates the mitochondrion from the cytosol. It is permeable.
 
Inner membrane: It is projected in wards as a cristae. The membrane shows two faces. The outer face "C" face (Cytosol face) and inner face is called "M" face (Matrix face). On this M-face a number of knob like structures are present.
 
Outer chamber: It is the space between outer and inner membranes. It is 60 to 80A°. It is filled with a fluid.
 
Inner chamber: It is called mitochondrial matrix. It is filled with jel like substance. It contains many enzyme systems. In 1963 "Nass" observed the presence of DNA molecule in the mitochondria.
 
Variations of cristae: Normally the cristae are perpendicular but various arrangements can be noticed.
 
Parallel cristae: In the nerve cells, and cells of striated muscles the mitochondria will show parallel cristae.
 
Concentric cristae: In the spermatogonia of man the mitochondria will show concentric cristae.
 
Haphazard arrangement: In choas-choas the mitochondria will show irregular arrangement of cristae
 
Spherical cristae: In the spermatocytes the cristae are spherical.
 
Mitochondria without cristae: Very rarely the inner membrane is smooth. It will not show cristae.
 
Reduced cristae: In the cells of opossum testis the aristae are reduced in the mitochondria.
 
Chemical composition: Mitochondria contain 73% of proteins, 25 to 30% of lipids, 5% of RNA and small amount of DNA. The enzyme complexes are more. The lipids contain 90% phospholipids, cholesterol, carotenoids etc.
 
Enzymes: In 1969 Lehninger gave the account of enzymes in mitochondria.
 
1) Enzymes of the outer membrane of mitochondria
a) Mono mine oxidase enzyme.
b) Fatty acid activating enzymes.
 
2) Enzymes of the outer chamber of mitochondria
1) Adenylate kinase.
2) Neuckocyte diphosphokinase.
 
3) Enzymes of the inner membrane
In the inner membrane electron transport enzymes are present. They are cytochromes, flavor proteins, dehydroginases etc.
a) ATP synthetase oxidase.
b) Carnitine fatty acid acyltransferase etc., enzymes are present. Enzymes of matrix : These enzymes systems bebng to krebs cycle and fatty acid cycle.
a) Fumarase.
b) Aconitase.
c) Citrate synthatase etc
 
Elementary particles: On the inner membrane of the mitochondrion stalked particles are present. They are called F, or elementary particles. They are equidistantly placed. Each particle shows a stalk and head piece. The stalk is 50A° in length. The head is 100A° in diameter. The distance between 2 particles is 100A°. The head piece contains enzymes of ATP-ase system and bring oxidative phosphorelation. Hence they are called oxysomes.
 
Respiratory chains: Respiratory chain contains a series of complex proteins and electron carriers. These electron carriers are represented by 4 complexes.
 
Complex I: It contains flavo-protein of NADH dehydroginase. It contains non-haemiron which combine with protein. It receives hydrogen from NADH.
 
Complex II: It contains flavo-protein of succinic dehydrogenase. It receives hydrogen from succinic dehydroginase.
 
Complex III: It contains Cytochrome b1, Cytochrome cr
 
Complex IV: It contains Cytochome at Cytochrome a3.
 
These complexes are connected by ubino-quinone. Cytochrome C will be present. Ubinoquinone connects complex 1,2 and 3. Cytochrome C will connect complex 384.
 
'Respiratory chain is proposed by 'Mochenan' and 'Green' and 'Baum' in 1970.
 
Biogenesis of mitochondria:
 
  1. Luck stated free existing mitochon­dria will elongate and divide and new mitochondria are formed.
  2.  Morrison stated mitochondria arise from either plasma membrane or endoplasmic reticulum.
  3. In the cytoplasm small particles may-be present they are called promitochondria they may give rise to mitochondria.
 
Mitochondrial D.N.A.: Mitochondria contain one or two molecules of D.N.A. Mitochondrial D.N.A. is circular. It is highly twisted double strand molecule. "Rabino- with" stated that mitochondrial D.N.A. contains more G and C content thatunclear D.N.A. Moleculer weight of mitochondrial D.N.A is 9 to 11 millions. This D.N.A. has the capacity of multiplication. D.N.A. polymerase enzyme is present. This D.N.A. will produce R.N.A. It is believed that it may take up the production of some proteins.
 
Origin of mitochondria
 
Mitochondria semi auto nous or prokaryotic origin:
 
In 1890 'Akmann' suggested that mitochondria and chloroplasts may be intracellular parasites of the cells which have entered the cytoplasm of eukaryotic cell. And they live as symbotonts. Hence Akmann called them as Bioplats.
 
  1. In Bacteria & Mitochondria electron transport system is present in plasma membrane and in inner membrane respectively.
  2. Bacterial plasma membrane shows mesosomes, mitochondrial crystae can be compared with them.
  3. Both bacteria & Mitochondria will show circular D.N.A.
  4. In both bacteria and mitochondria ribosomes are reported.
  5. Both bacteria and mitochondria will produce AT.P. and R.N.A. Hence we consider the mitochondria might have originated from bacterial cell. In the cell mitochondria will function as semi-autonomous body.
 
Mitochondria Functions:
 
  1. A.T.P. Synthesis: It is the power house of the cell. It brings oxidation of food. Hence Kreb's cycle reactions, electron transport system enzymes are located in mitochondria. By the oxidation of food energy is liberated in the form of A.T.P. (Oxidative phosphorelation takes place.)
  2. Yolk formation: Mitochondria are responsible for the fcri soiydk in the developing ovum Granules are formed in the matrix They oeconie large masses Mitochondrion is converted into yolk storing body.
  3. Mitochondrian sperm formation: When spermatid become? <pe'm mitochondria will form a spiral around the axial filament. This is called Neben-kem. It forms the middle piece of the sperm.
  4. Origin of new system: It is believed that some of the ceil organelles may originate from mitochondria.
  5. Heat production: In the oxidation of food ATP is released. Only 45% of the total energy is trapped in the form of ATP. The remaining 35% of ATP will come out as heat. (In birds and mammals this heat is useful for the maintenance of body temperature.).
  6. ATP released during respiration (because of mitochondria ) will take part in many biosynthetic paths of the cell.
The cytoplasm of the animal cell is bounded by a thin limiting membrane called "Cell membrane".
 
  1. Nageli and Cramer called this membrane as cell membrane in 1855.
  2. In 1931 "Plowe" named this as plasmalemma.
  3. Modem cell biologists prefer to call it plasma membrane (According to De Robertis).
  4. Danielli & Davson in 1952 proposed molecular model of plasma membrane.
  5. Robertson proposed unit membrane concept in 1960.
 
1) Material used for study of plasma membrane:
 
  1. Human R.B.C. are kept in dilute solution (Hypotonic). Then it swells. It breakes and protoplasm will go out (Haemolysis). The remaining membrane is called Ghost. It is used for plasma membrane studies.
  2. Liver cell or the membrane surrounding the nerve fibre are used as a material for the study of plasma membrane.
 
2) Physical nature of plasma membrane:
 
Danielli described the nature of plasma membrane. Robertson by using electron microscope and X-ray diffraction methods proposed the structure of plasma membrane. The plasma membrane shows 3 layers. (Trilaminar).
 
  1. Outer layer is made by proteins....20 A°
  2. Middle layer is bimolecular lipid layer....35A°
  3. Inner layer is made by proteins ...20A°
 
3) Membrane model:
 
The membrane models are many. This model represents the molcular srganisation of the membrane.
 
  1. Danielli Davsons model: It shows thin lipid layer with protein absorbed on both the sides. The lipid contains polar heads pointing out sides and ion poiar parts run transversly. In 1954 Danielli modified this model and gave a new model.
  2. Robertson unit membrane concept: Robertson in 1959 described trilaminar structure of plasma membrane. It contains an outer and inner protein layers in between them a bimolecular lipid layer is present.
    1) The unit membrane is 75A° thick. 
    2) The Outer and inner protein layers are 20A° thick. 
    3) In between them the lipid layer will show 35A° units. 
    4) The polar hydrophyllic ends of the lipid layer will face the proteins. Where as the hydrophobic ends of the lipids are away from the proteins.
  3. Fluid Mosaic model: In 1972 "Singer" & "Nicholson" proposed this model.
  1. Cell membrane is a mosaic of lipids and proteins.
  2. Lipids are arranged in a bilayer way. It forms the structural frame work of plasma membrane.
  3. Protein molecules are arranged in 2 ways.

1) Extrinsic proteins: These proteins are located adjacent to the outer and inner surfaces of the lipid layer.
 
2) Intrinsic proteins: These proteins will penetrate into the lipid layers partially or totally. They are called integral or intrinsic proteins.
 
This fluid mosaic model is accepted universally. The differential distribu­tion of protein in the various regions of membrane is known as "Membrane asymmetry".
 
4) Chemical composition of plasma membrane:
 
Plasma membrane Shows proteins, lipids and small percent of carbo­hydrates.
 
a) Lipids: The lipids of the plasma membrane are phospholipid, lecithin cholesterol glycolipids. the distribution of phospholipids in the bilayer of plasma membrane is highly asymmetrical The phospholipid will show 2 ends.
 
  1. Hydrophobic end: It is water hating, non polar end.
  2. Hydrophilic end: It is water loving part. It is called polar end. It is present near the proteins.
 
Hydrophobic end contains fatty acids and Hydrophilic end contains a phosphate group.
 
b) Proteins: The peripheral proteins or extrinsic proteins are free from lipids. They are loosely attached to the membrane. The integral proteins or intrinsic proteins will penetrate into the lipid layer. These proteins will give mechanical strength to the plasma membrane. They regulate cell activities.
 
c) Carbohydrates: Very small percentage of carbohydrates are present. Hexoses, Hexosamine, Fructose etc., are present. They may be attached to lipids glycolipids; and they are attached to proteins... Glycoproteins. These glycolipids and glycoproteins are present on the external surface.
 
d) Enzymes: More than 30 enzymes are isolated from plasma membrane. They are phosphotase, ATP ase, RNA ase etc.
 
5) Pores in plasma membrane:
 
Plasma membrane shows some pores. The diameter is 35 Nm (nanometer). There are several models to describe the structure of pore.
 
  1. Structural pore: These are permanent openings.
  2. Dyanamic pores: They form during the intake of material.
  3. Protein channel pores: These are small channels of specific proteins through which some ions can diffuse.
  4. Modification of plasma membrane: To perform specialised functions plasma membrane will show some modifications.
  5. They are (a) Microvilli, (b) Inter digitations, (c) Desomosomes, tight junctions etc. 
    a) Microvilli: In certain parts the plasma membrane will show minute infloldings they are called microvilli. They show -1 mili micron diameter and 6 milli micron length. In some cases the villi are connected with endoplasmic reticulum. These microvilli are more in intestinal mucosa cells. The microvilli show cytoplasm with micro filaments. The microvilli will increase the absorptive surface of the intestinal epithelium. 
    b) Inter digitations: At some places the plasma membrane of adjacent cells will develop into finger like projections they are called inter digitations. 
    c) Desmosomes: The plasma membranes of adjacent cells become thicker in certain regions. On these thick areas fine, filaments are present. They are called tonbfilaments or tonofibrils. Such parts are called desmosomes. Desmosome is concerned with cell adhesion and maintenance of cell shape. 
    d) Terminal bar: It is a desmosome without tonofibrils. It is called terminal bar. 
    e) Zonula acculdens: These are special area of adjacent cells where the 2 plasma membranes fuse. These tight junctions are present below the apical boarder. It gives mechanical attachment between cells.
 
7) Properties of plasma membrane:
 
The substances which either enter the cell or leave the cell should pass through plasma membrane. The plasma membrane will' show permeability. It shows the following properties.
 
i) Osmosis: The diffusion of water molecular from low concentrated solution to high concentrated solution through plasma membrane is called osmosis.
 
ii) Passive transport: The movement of molecules from higher concentration to lower concentration without the expenditure of .energy, through plasma membrane is called passive transport.
 
  1. Endocytosis: It is the process of by which large particles of food are engulfed. 1) Pinocytosis or cell drinking : Ingestion of liquid through plasma membrane is called pinocytosis.
  2. Micro pinocytosis is observed at submicroscopic level. When a liquid come nearer to plasma membrane, it forms a vesicles. It is called pinoc vesicle. After some time it is pinched off from the plasma membrane and becomes pinocytic vesicle in the cytoplasm
  3. Phagocytosis: The process of taking solid food or solid material through the cell membrane into the cell is called phagocytosis. Phagocytosis means to eat. It is first discovered by "Metknikoff
  4. Exocytosis: It is also called cell vomiting . The process of sending out products from the cell to the out side is called exocytosis or emeiocytosis.
  5. Active transport: If molecules or ions move against the concentrate gradient through plasma membrane, it is called active transport. For this energy is required. Now a days it is proved that carrier system is seen in the plasma membrane and it is responsible for active transport.
 
8) Origin of plasma membrane:
 
  1. It is believed that it develop de novo.
  2. It is formed by the assembly of lipids & proteins.
  3. It is believed that it develops from other membrane systems.
Cell is a fundamental, structural and functional unit of living organism. The science which deals with cells and their organelles is called cell-biology. The term cell was first used by Robert Hooke in 1665. He described the cell first as cella which means hollow space. Robert Hooke observed cells in the section of cork. In 1831 Robert Brown observed nucleus in plant cells. In 1858 Rudolf Virchow stated that new cells arise from pre-existing cells.
 
"Omnis cellulae cellula. Schilden, German Botanist in 1938 described cell theory with regard to plant cell. T. Schwann German Zoologist in 1939 described cell theory with regard to animal cells. Cell theory denotes that "Cell is the structural and functional unit of life."
 
SHAPE OF CELL:
 
The shape of the cells is variable. The cells of unicellular forms, leucocytes and-bacteria, exhibit a number of shapes and those of multicellular organisms exhibit still further variation. Their shapes may be rounded, cylindrical, irregular, triangular and tubular.
 
SIZE OF CELLS:
 
Size is extremely variable, measuring from one micron to 175 mm. The ostrich egg cell is 176 mm. in diameter, thus visible to the naked eye. The nerve cell found in mammals may reach a length of 3 or 3.5 feet. Smaller cells are those of the Pleuropneumonia like organisms.
 
Plasma Membrane:
 
A porous membrane surrounds the cytoplasm called plasma membrane. Electron microscopic studies reveal that the plasma membrane is composed of outer, inner protein layers and in between them double layered lipids are present Robertson called plasma membrane unit membrane.
 
The main function of plasma membrane is to regulate the entry and exist of substances.
 
Cytoplasm:
 
The part of protoplasm outside the nucleus is known as cytoplasm. It is distinguished as an outer non-granular thick ectoplasm and inner granular thin endoplasm. In the cytoplasm many organelles are present.
 
Cell Organells:
 
In the cytoplasm many cell organelles are present.
 
1. Centrosome:
 
It is the center of the cell discovered by van Benden in 1887. It is found near the nucleus and includes a specialised portion of cytoplasm, called centirosome. Its matrix is called as kinoplasm, in which two centrioles are embedded. Each centriole consists of nine fibrillar units and each fibrillar unit is found to contain three microtubules. The function of centrioles is to form the spindle at the time of cell division.
 
2. Endoplasmic Reticulum:
 
In the cytoplasm a network of tubules are present. It is called endoplasmic reticulum. This network of tubules will be two types.
 
i. Smooth endoplasmic reticulum:
 
On the surface of the tubules ribosomes are absent. Hence they are ce Sled smooth endoplasmic reticulum or agranular endoplasmic reticulum.
 
ii. Rough endoplasmic reticulum:
 
On the surface of the tubules ribosomes are present. It is rough endoplas­mic reticulum. This is called granular endoplasmic reticulum.
 
Endoplasmic reticulum will connect plasma-membrane nucleus and other organells.
 
Functions:
 
  1. It gives strength to the cell and forms cytoskeleton.
  2. Granular endoplasmic reticulum will produce proteins.
  3. Agranular endoplasmic reticulum will produce lipids.
  4. It forms the work bench for many biochemical reactions in the cell.
 
3. Ribosomes:
 
It is a small particle present in the cytoplasm. They will be attached to the cell organelles and they are also freely distributed in the cytoplasm.
 
In a Eukaryotic cell 80s ribosomes are present. This ribosome is made by 2 sub units. They are 40s, 60s sub units.
 
Ribosome is made by proteins and RNA. Ribosome shows 150 to 200A° diameter. Ribosome combine with m RNA and produce proteins. A group of ribosomes with m RNA is called polysome.
 
4. Golgi Complex:
 
They are described by Golgi. They are also called dictyosomes, Bpochondria, and idosomes. The complex shows three types of structures,
 
a. Cisternae:
 
These are flat sacs. They are arranged one above the other. They are 150 V in length, 60 A° in thickness.
 
b. Vacuoles:
 
These are oval in shape. They are big.
 
 
They are in the form of groups. All these structures totally called Golgi complex.
 
Functions:
 
They are more in secretory cells. Hence they are connected with secretory function. They store proteins and lipids. During cell division they produce cell, plate. During the formation of sperm they will form the acrosome of the sperm.
 
5. Mitochondria:
 
These are first described by Altamann as Bioplasts, in 1894. They were m i led as mitochondria by Benda in 1897.
 
They are filamentous or rod like structures. The mitochondria are covered by layers. Inner membrane is folded inside. Those folding's are called cristae. On these cristae oxysomes are present.
 
  1. In the central matrix of mitochondria respiratory enzymes are present. The take up Krebs cycle reactions.
  2. In the inner membrane of mitochondria electron transport enzymes are present.
  3. Mitochondria helps in the oxidation of the food material and liberates energy , Hence they are called power houses of cell.
  4. In the mitochondria a circular DNA is present. Hence mitochondria is also c. led semi autonomous body.
 
6. Lysosome:
 
These are described by De-Duve. Each lysosome is round in shape. It shows .4 to .8 microns in diameter. It is covered by lipoprotein layer. It contains hydrolytic enzymes. It is useful for intracellular digestion and autolysis of the cell.
 
Functions :
 
  1. Lysosome is helpful in the digestion of the food.
  2. At starvation lysosome will digest cell organelles.
  3. Lysosome can dissolve the cell. It is called suicide. Henc lysosomes are called suicidal bags of cells.
 
Inclusions of the Cytoplasm:
 
In the cytoplasm vacuoles and duetoplasmic bodies are pr-sent. In the young stages vacuoles are absent in the cytoplasm. When the cell is growing the .Cytoplasm vacuoles are formed. In a older cell big vacuole is present. It is filled with cell sap. A vacuole is covered by tonoplast. In the cell sap water, some excretory substances, some pigments, and other substances are present.
 
Duetoplasmic Bodies:
 
In the cytoplasm reserve food materials, excretory wastes a<\l secretory substances are stored. They are called duetoplasmic bodies.
 
Nucleus:
 
In a eukaryotic cell a definite nucleus is present. It is 5 to 25 microns in, size. It shows the following parts.
 
a) Nuclear Membrane:
 
Nucleus is covered by a nuclear membrane. It is made by 2 layers. In between the two layers perinuclear space is present. In the nuclear membrane small openings are present. Around each opening on the out side a small annulus is present.
 
Hammerling proved that nucleus is the seat of heredity through grafting experiments on Acetabularia.
 
b) Nucleoplasm:
 
Below the nuclear membrane nucleoplasm is present. In this glycoproteins, RNA and enzymes are present.
 
c) Chromatin Network:
 
In the nuclear spa many chromosomes are present. They are thin and filamentous. They are in the form of a network. On the chromosomes genes are present. They are units of heredity.
 
d) Nucleolus:
 
In the nucleoplasm one or two round structures are present They are called nucleoli. They contain proteins and RNA. They produce ribosomes.
 
Functions:
 
  1. It is the seat of life in the cell.
  2. It carries hereditary characters from one generation to another generation.
  3. It produces nucleic acids.
Feature Prokaryotic Cell Eukaryotic Cell
1. Nuclear envelope absent present
2. DNA single, circular and naked more than one and combined with proteins
3. Chromosome single multiple
4. Nucleolus absent present
5. Division amrtosis mitosis or meiosis
6. Ribosomes 70S(50S+30S scattered in cytoplasm 80S(60S+40S) Found attached to ER or free in cytoplasm.
7. Endo membranes absent present
8. Mitochondria absent present
9. Chloroplasts absent present in plant cells, absent in animal cell
10. Lysosomes absent present
11. Peroxisomes absent present
12. Cytoskeleton absent present
13. Cell wall non-cellulose cellulose only in plants cells
14. Respiratory enzymes located in plasma membrane enclosed in mitochondria.
The cells which lack true nuclear membrane are called prokaryotic cells. Blue green algae, bacteria are the examples for prokaryotic cells.
 
Prokaryotic cells lack chloroplasts mitochondria, Golgi complex and endoplasmic reticulum.
 
Prokaryotic cells are simple and primitive cells.
 
  1. In blue green algae cell shows a cell wall, enclosing protoplasm. In the protoplasm the peripheral coloured part is the chromateplasm and the central colorless part is the centroplasm. In the centroplasm there is DNA as the genetic material.
  2. Bacterial cell consists of a rigid cell enclosing protoplasm. The rigid cell is surrounded by slime layer or capsule. The protoplasm shows a peripheral plasma membrane which is often producing coiled mesosomes to carry respira­tion. The central part of the cell shows a long coiled thread like DNA. Protoplasm contains many ribosomes, fat bodies, volutin granules etc. The cells also show pili and flagella.
Cell Biology (Gr., kytos-hollow vessel or cell, logos-to discourse) is a biological science which deals with the study of cells. The cell itself can be regarded as the vital unit of organisms.
 
'Aristotle' and 'Paracelsus' concluded that "all animals and plants, how­ever, complicated are constituted by few elements which are repeated in each one of them."
 
The beginning of cell biology dates back to the 15th century, when 'Da Vinci in 1485 has stressed upon the use of lenses in viewing small objects. In 1658, 'Jan Swammerdam' gave the first description of the cell in his account of the red blood cells of the frog. The cytology came in its actual existence with the discovery of cell in 1665, by "Robert Hooke", while examining a thin slice of cork under his crude compound microscope, Hooke observed its honey-combed structure. He gave them the name "cells" (cellulae -little room).
 
Malphighi studied a variety of animal tissues microscopically and there­fore, he is generally considered as the father of 'microscopic anatomy '.
 
'A.V. Leeuwen Hoek (1632-1723) discovered the animalcules, infusoria (Protozoa), bacteria, etc., and made microscopical observations on protozoa, ants, aphids, spermatozoa, red blood cells etc.
 
The cell theory was proposed by two German biologists 'M.J. Schleiden' (1804-1881), and Theodor Schwann' (1810-1882) independently in 1838 and 1839, respectively. The cell theory holds that the animals and plants have same pattern of organization and construction. The bodies of both animals and plants are composed of cells and that each cell can act independently. In words of 'Schwann' and 'Schleiden' cell is "functional biological unit".
 
"Rudolf Virchow" in 1885 stated, "where a cell exists there must have been a pre-existing cell, just as the animal arises only from an animal and the plants only from a plant".
 
"Purkinje" in 1840 coined the term protoplasm. The protoplasm was first of all observed by "Corti" (1772) and the French Zoologist 'Dujardin' (1835) called it sarcode.
 
"Huxley" in 1868 referred to protoplasm as the "physical basis of life".
 
The protoplasm theory states that all living matter of animals and plants is protoplasm. The part of the protoplasm which occurs between the plasma membrane and nucleus is named as cytoplasm.
 
20th century brought many modern micro techniques. New histo chemical and cyto chemical methods have been developed to detect various molecular components of the cell. Different biochemical events of the cell could be known by autoradiography. Methods of tissue culturing have made possible the study of living cells.
 
Year Names of contributor Cytological contribution
1824 R.J.H. Dutrochet Showed that all animals and plants composed of cells.
1826 Turpin Reported the occurrence of cell division.
1831 R. Brown Described the nucleus
1835 Felix Dujardin Described protoplasm as ("Sarcode")
1838 M.J.Schleiden Proposed "Cell theory"
1839 T.Schwann Applied "Cell theory" to animals.
1840 J.E. Purkinje Named the cell contents as Protoplasm.
1855 R.Virchow Stated that all cells arise from pre-existing cell.
1857 Kollieker Discovered mitochondrian
1863 Waldeyer Chromosomes of cell
1871 F. Miescher discovered nucleo-protein and nucleic acid.
     
1882 Strasburger described mitosis in plant cells
1887 E.Van Benden discovered centrioles.
1888 T.Boweri described the centrioles.
1888 Waldeyer Introduced the term chromosome.
1902 W.S.Sutton Proposed "the chromosome theory" heredity.
1905 J.B.Farmer along with J.E.Moore. Coined the term meiosis 
1943 A.Claude Isolated cell components like ribosomes, mitochondria and nuclei
1952 C. Du Duve Identified hysosomes.
1953 J.D.Watson and F.H.C. Crick Proposed the double helixmodel for the DNA molecule.
     
1959 S.Ochoa Synthesis of polyribonuclotide in vitro.
1959 A.Kornberg Synthesis of polydeoxiri-
1968 M.W.Nirenberg and H.G.Khorana. Triplet genetic code.
1968 R.H.HoIley discovery of base sequence of RNA

This method is optional for estimation of hemoglobin and this method is recommended by International Committee for Standardization in hemotology. This is because in this method all type of hemoglobin are transformed to cyanmethemoglobin (except sulfhemoglobin), and a firm and trustworthy standard is available.

Principle

When Blood is mixed with a solution of potassium cynide,  potassium ferricyanide and Drabkin’s solution, the erythrocytes are lysed by producing evenly disturbed hemoglobin solution. Potassium ferricyanide transforms hemoglobin to methemoglobin, and methemoglobin combines with potassium cyanide to produce hemiglobincyanide (cyanmethemoglobin). This way all types of hemoglobin present in blood are entirely transformed to a single compound cyanmethemoglobin. When the reaction is entire, absorbance of the solution is deliberate in a spectrophotometer at 540 nanometer. Hemoglobincyanide has a wide absorbance peak at this wavelength. The absorbance is compared with that of the standard hemiglobincyanide solution by using a formula to obtain the amount of hemoglobin.

Equipment

  1. Spectrophotometer or  photoelectric colorimeter
  2. Pipette 5 ml
  3. Sahli’s pipette

Reagents

  1. Drabkin’s Solution
  2. Cyanmethemoglobin standard solution with known hemoglobin value

Specimen

Blood obtained from skin puncture or EDTA-anticoagulated venous blood.

Procedure

  1. Take 5 ml of Drabkin’s solution in a test tube and add 20 μl of blood. This way, we will get the dilution of 1:25. Now mix the mixture and allow to stand for atleast 5 minutes. This time is adequate for transformation of hemoglobin to hemiglobincyanide.
  2. Pour the test sample to a cuvette and read the absorbance of the test sample in a spectrophotometer at 540 nanometer or in a photoelectric colorimeter using a yellow-green filter. Also read the absorbance of the standard solution. Absorbance must be read against Drabkin’s solution.
  3. From the formula given below, the hemoglobin value is derived.

Hemoglobin in gm/dl = [Absorbance of test sample ÷ Absorbance of standard] x concentration of standard x [Dilution factor ÷ 100]

Preparation of table and graph

Result can be obtained quickly, if the table of graph is prepared which correspond absorbance with hemoglobin concentration. This is markedly acceptable when huge number of samples are daily processed on the same instrument.

For the preparation of a calibration graph, adulterate cyanmethemoglobin standards are commercially available. As another option, standard cyanmethemoglobin solution is diluted serially with Drabkin’s solution. Concentration of hemoglobin (horizontal axis) in each dilution is arranged against the absorbance (vertical axis) on a linear graph paper. A straight line connecting the points and passing through the origin is obtained. A table can be prepared relating absorbance to concentration of hemoglobin from the help of this graph.

Notes:

  1. The hemiglobincyanide solution is stable so that delay in getting the reading of absorbance does not influence the result.
  2. High TLC (total leukocyte cunt) (> 25,000/μl), abnormal plasma proteins (e.g. in Waldenström’s macroglobulinemia, multiple myeloma) or lipemic blood (hypertriglyceridemia), can cause the error in results.

Principle

When blood is mixed with an acid solution, the hemoglobin converts into the brown-colored acid hematin. The acid hematin is then diluted with distilled water till the color of the acid hematin matches that of the brown glass standard. The hemoglobin is estimated by reading the value directly from the scale.

Equipments

  1. Sahli’s hemoglobinometer: This equipment consist of a comparator with a brown glass standard and  Sahli’s graduated hemoglobin tube which is marked in percent and gram.
  2. Hemoglobin pipette or Sahli’s pipette (marked at 0.02 ml or 20 μl).
  3. Stirrer (a small glass rod).
  4. Dropping pipette (dropper).

Reagents

  1. N/10 hydrochloric acid
  2. Distilled water

Specimen

Blood is obtained directly by skin puncture or EDTA-anticoagulated venous blood.

Method

  1. The N/10 hydrochloric acid is place into Sahli’s graduated tube up to mark 2 grams.
  2. With the help of Sahli’s pipette, take blood sample exactly up to 20 μl mark. Blood adhering to the outer part of the pipette is wiped away with the help of tissue paper (absorbent paper) or cotton (gauze piece).
  3. Add blood sample to the N/10 hydrochloric acid solution which is placed into Sahli’s graduated tube, mix the mixture with the help of a glass stirrer, and allow the tube to stand for 10 minutes.
  4. Add distilled water drop by drop into the mixture placed in Sahli’s graduated tube, till the color of the solution matches that of the brown glass standard.
  5. Take the reading of the lower meniscus from the Sahli’s graduated tube in grams.

Note:

  1. All the Sahli’s graduated tube is marked in both percent and grams figures, this is because (a) different manufacturers of hemoglobinometers have different values as 100%, so that blood sample will yield different results on different instruments and (b) no single hemoglobin is can be evaluate as 100% since it is different according to the sex and age of the individual and altitude.
  2. Disadvantages of Sahli’s method:
    • It is impossible to match the color perfectly of the mixture into the Sahli’s graduated tube with the brown glass standard.
    • Minimum 1 hour is required for the maximum color development of acid hematin because 95% color of acid hematin is attained at the end of 10 minutes.
    • Sulfhemoglobin, methemoglobin and carboxyhemoglobin cannot be converted into acid hematin. Fetal hemoglobin is also not converted to acid hematin and therefore this technique is not appropriate for in small infants.
    • The acid hematin solution is not firm and stable, and the color development is slow.
    • Lights may affect the visual comparison of color.
    • Color of the brown glass standard dims with time.
    • Personal error in matching the color of the mixture in Sahli’s graduated tube with the brown glass standard is 10%.

HEMOGLOBIN

Hemoglobin is composed of heme (iron + protoporphyrin) and globin polypeptide chains. It is present in the red blood cells of all vertebrates except Channichthyidae (the family of fish: white-blooded fish also called crocodile fish found in southern South America and the Southern Ocean around Antarctica). It carries oxygen from the lungs to the tissues and carbon dioxide from tissues to the lungs.

In humans, hemoglobin is not homogeneous and normally different variants and derivatives exist. Normal hemoglobin variants are fetal hemoglobin (Hb F), adult hemoglobin (Hb A), Hb A2 and embryonic hemoglobins (Gower I, Gower II and Portland). They differ from each other on the basis of the structure and the type of polypeptide chains.

INDICATION FOR HEMOGLOBIN ESTIMATION

  1. Screening for polycythemia: Polycythemia is a disease state in which the hemoglobin level and hematocrit (HCT) or packed cell volume (PCV) value is elevated. It may be primary, secondary or relative.
  2. To determine presence and severity of anemia: Anemia is a disease state in which the hemoglobin concentration or oxygen-carrying capacity of blood is low. Clinical signs and symptoms (conjunctival vessels, polar of skin, mucosal membranes) are unreliable for the diagnosis of anemia. Anemia is best determined by estimation of hemoglobin and hematocrit (HCT) or packed cell volume (PCV).
  3. To assess response to specific therapy in anemia.
  4. Estimation of red cell indices (along with hematocrit (HCT) or packed cell volume (PCV) and red cell count) i.e. mean cell volume (MCV), mean cell hemoglobin (MCH) and mean cell hemoglobin concentration (MCHC).
  5. Selection of blood donors in the blood bank.

METHOD FOR ESTIMATION OF HEMOGLOBIN

There are different methods for estimation of hemoglobin. These are:

(1) Colorimetric methods: In these methods, the color comparison is made between the standard and the test sample, either visually or by colorimetric methods.

(2) Gasometric method: In this method, oxygen-carrying capacity of red blood cells (RBCs) is measured in a Van Slyke apparatus. The amount of hemoglobin is then derived from the formula that 1 gram of hemoglobin carries 1.34 ml of oxygen. However, this method measures only physiologically active hemoglobin, which can carry oxygen. It does not measure methemoglobin, sulfhemoglobin, and carboxyhemoglobin. Also, this method is expensive and time-consuming, and the result is about 2% less than other methods.

(3) Chemical method: In this method, iron-content of hemoglobin is first evaluated. The value of hemoglobin is then derived indirectly from the formula that 100 grams of hemoglobin contain 374 mg of iron. This method is tiresome and time-consuming.

(4) Specific gravity method: In this method, an approximate value of hemoglobin is estimated from the specific gravity of blood as determined from copper sulfate technique. This method is simple and rapid. This method is useful and most common in mass screening like the selection of blood donors. See procedure.

Tallqvist Hemoglobin Chart

Tallqvist hemoglobin chart consists of a series of lithographed colors said to correspond to hemoglobin values ranging from 10% to 100%. In this method, a drop of blood obtained by finger puncture is placed on a piece of absorbent paper. The color produced is matched against the color on the chart and the corresponding reading is taken. The room of error is 20-50%. Although this method is very cheap and simple.

Red Cell Indices

  • 19 Jul 2016
Red Cell Indices

Red cell indices are mean cell volume (MCV), mean cell hemoglobin (MCH), and mean cell hemoglobin concentration (MCHC). They are also called as “absolute values”. They are derived from the values of hemoglobin, packed cell volume (PCV or hematocrit), and red cell count. Red cell indices are accurately measured by automated hematology analyzers. Recently, a new parameter called red cell distribution width (RDW) has been introduced.
 
USES OF RED CELL INDICES
 
(1) Morphological classification of anemia: Based on values of red cell indices, anemia is classified into three main types: normocytic normochromic, microcytic hypo-chromic, and macrocytic normo-chromic. Calculation of red cell indices is especially helpful in mild or moderate anemia when red cell changes are subtle and often difficult to appreciate on stained blood smear.
(2) Differentiation of iron deficiency anemia from thalassemia trait: In iron deficiency, MCV, MCH, and MCHC are low, while in thalassemia trait, MCV and MCH are low and MCHC is normal.
 
MEAN CELL VOLUME
 
MCV is a measure of average size of the red cells. It is measured directly by automated instruments from the measurement of volume of each red cell. With semiautomated instruments and by manual method, it is obtained by dividing PCV by red cell count.
 
MCV =                PCV in%                  x 10
            RBC count in million/cmm
 
     MCV is expressed in femtoliters or fl (10⁻¹⁵ of a liter). It corresponds with red cell diameter on blood smear. Normal MCV is 80-100 fl.
 
Causes of Increased MCV
 
• Megaloblastic anemia
• Non-megaloblastic macrocytosis: Chronic alcoholism, liver disease, hypothyroidism, normal pregnancy, reticulocytosis
• Newborns.
 
Causes of Low MCV
 
• Microcytic hypochromic anemia
 
MCV is normal in normocytic normochromic anemia (acute blood loss, hemolysis, aplastic anemia).
     In the presence of large number of abnormal red cells like sickle cells, and in dimorphic anemia (e.g. mixed normocytic and microcytic), MCV may be normal (since it is an average value) and thus unreliable for morphological classification.
     Mentzer index is derived by dividing MCV with red cell count. Ratio of less than 13 is seen in thalassemia while ratio is more than 13 in iron deficiency anemia.
 
MEAN CELL HEMOGLOBIN (MCH)
 
MCH is the average amount of hemoglobin in a single red cell. It is obtained by dividing hemoglobin value by red cell count.
 
MCH =    Hemoglobin in grams/dl     x 10
           RBC count in millions/cmm
 
MCH is expressed in picograms or pg (10⁻¹² gram). Reference range is 27-32 pg.
     MCH is decreased in microcytic hypochromic anemia, and increased in macrocytic anemia and in newborns.
 
MEAN CELL HEMOGLOBIN CONCENTRATION (MCHC)
 
MCHC is obtained by dividing hemoglobin value by PCV and expressed in grams/dl or grams/liter. It refers to concentration of hemoglobin in 1 dl or 1 liter of packed red cells.
 
MCHC = Hemoglobin in grams/dl  x 100
                        PCV in %
 
Reference range is 30-35 grams/dl. MCHC is raised in hereditary spherocytosis, and is decreased in hypochromic anemia. MCHC corresponds with degree of hemoglobinization of red cells on a blood smear. If MCHC is normal, red cell is normochromic, and if low, red cell is hypochromic.
 
Red Cell Distribution Width (RDW)
 
Some automated analyzers measure red cell distribution width or RDW. It is a measure of degree of variation in red cell size (anisocytosis) in a blood sample. It is helpful in differential diagnosis of some anemias. Amongst microcytic anemias, RDW is low in ß thalassemia trait, high in iron deficiency anemia, and normal in anemia of chronic disease. Normal RDW is 9.0 to 14.5.
 
REFERENCE RANGES
 
• Mean cell volume: 80-100 fl
• Mean cell hemoglobin: 27-32 pg
• Mean cell hemoglobin concentra-tion: 30-35 g/dl
• Red cell distribution width: 9.0-14.5
 
REFERENCES
 
1. Henry JB. Clinical diagnosis and management by laboratory methods (20th Ed). Philadelphia: WB Saunders Company, 2001.
2. Wallach J. Interpretation of Diagnostic Tests (7th Ed). Philadelphia: Lippincott Williams and Wilkins, 2000¹⁵

Reticulocytes

Reticulocytes are young or juvenile red cells released from the bone marrow into the bloodstream and that contain remnants of ribonucleic acid (RNA) and ribosomes but no nucleus. After staining with a supravital dye such as new methylene blue, RNA appears as blue precipitating granules or filaments within the red cells. Following supravital staining, any nonnucleated red cell containing 2 or more granules of bluestained material is considered as a reticulocyte (The College of American Pathology). Supravital staining refers to staining of cells in a living state before they are killed by fixation or drying or with passage of time. Reticulocyte count is performed by manual method.

PRINCIPLE

A few drops of blood (collected in EDTA) are incubated with new methylene blue solution which stains granules of RNA in red cells. A thin smear is prepared on a glass slide from the mixture and reticulocytes are counted under the microscope. Number of reticulocytes is expressed as a percentage of red cells.

REAGENT

New methylene blue solution is prepared as follows:

  • New methylene blue: 1.0 gm
  • Sodium citrate: 0.6 gm
  • Sodium chloride: 0.7 gm
  • Distilled water: 100 ml

Reagent should be kept stored in a refrigerator at 2-6°C and filtered before use.
Suitable alternatives to new methylene blue are brilliant cresyl blue and azure B.

SPECIMEN

Capillary blood or EDTA anticoagulated venous blood can be used.

METHOD

(1) Take 2-3 drops of filtered new methylene blue solution in a 12 × 75 mm test tube.

(2) Add equal amount of blood and mix well.

(3) Keep the mixture at room temperature or at 37°C for 15 minutes.

(4) After gentle mixing, place a small drop from the mixture on a glass slide, prepare a thin smear, and allow to dry in the air.

(5) Examine under the microscope using oil-immersion objective. Mature red cells stain pale green blue. Reticulocytes show deep blue precipitates of fine granules and filaments in the form of a network (reticulum). Most immature reticulocytes show a large amount of precipitated material in the form of a mass, while the most mature reticulocytes show only a few granules or strands. Any nonnucleated red cell is considered as a reticulocyte if it contains 2 or more blue-stained particles of ribosomal RNA.

(6) Count 1000 red cells and note the number of red cells that are reticulocytes. Counting error is minimized if size of the microscopic field is reduced. This is achieved by using a Miller ocular disk inserted in the eyepiece; it divides the field into two squares (one nine times larger in size than the other). Reticulocytes are counted in both the squares and the red cells are counted in the smaller square.

REPORTING THE RESULT

(1) Reticulocyte percentage: The most common method of reporting is reticulocyte percentage which is calculated from the following formula:

Reticulocyte% =  NR   x 100
                           NRBC

Where NR is the Number of reticulocyte counted and NRBC is number of red blood cell counted.

Reference range is 0.5%-2.5% in adults and children. Reticulocyte count is higher in newborns.

(2) Absolute reticulocyte count = Reticulocyte percentage × Red cell count
Normal: 50,000 to 85,000/cmm

(3) Corrected reticulocyte count (Reticulocyte index)

                    = Reticulocyte % x PCV of Patient
                                                 Normal PCV

Corrected reticulocyte count > 2% indicates reticulocyte release appropriate for the degree of anemia. If < 2%, reticulocyte release is inappropriate.

(4) Reticulocyte maturation production index

  =         Corrected reticulocyte count
         Estimated maturation time in days

REFERENCE RANGES

  • Reticulocyte percentage: 0.5 2.5%
  • Absolute reticulocyte count: 50,000-85,000/cmm

Reticulocyte

  • 24 May 2016
Reticulocytes are young or juvenile red cells released from the bone marrow into the bloodstream and that contain remnants of ribonucleic acid (RNA) and ribosomes but no nucleus. After staining with a supravital dye such as new methylene blue, RNA appears as blue precipitating granules or filaments within the red cells. Following supravital staining, any nonnucleated red cell containing 2 or more granules of bluestained material is considered as a reticulocyte (The College of American Pathology). Supravital staining refers to staining of cells in a living state before they are killed by fixation or drying or with passage of time. Reticulocyte count is performed by manual method.
 
USES
  • As one of the baseline studies in anemia with no obvious cause
  • To diagnose anemia due to ineffective erythropoiesis (premature destruction of red cell precursors in bone marrow seen in megaloblastic anemia and thalassemia) or due to decreased production of red cells: In hypoplastic anemia or in ineffective erythropoiesis, reticulocyte count is low as compared to the degree of anemia. Increased erythropoiesis (e.g. in hemolytic anemia, blood loss, or specific treatment of nutritional anemia) is associated with increased reticulocyte count. Thus reticulocyte count is used to differentiate hypoproliferative anemia from hyperproliferative anemia.
  • To assess response to specific therapy in iron deficiency and megaloblastic anemias.
  • To assess response to erythropoietin therapy in anemia of chronic renal failure.
  • To follow the course of bone marrow transplantation for engraftment
  • To assess recovery from myelosuppressive therapy
  • To assess anemia in neonate
 
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