Cell fractionation techniques are used to separate cell organelles and components based on size and density. Key techniques include homogenization to create a uniform cell mixture, and differential centrifugation which uses increasing speeds to separate components into pellets based on size. Other techniques discussed are staining, tissue culture, chromatography, spectrophotometry, electrophoresis, and microscopy which allows viewing and measuring cells.

1. CHAPTER : 01
CELL STRUCTURE AND FUNCTIONS

2. TECHNIQUES USED IN CELL
BIOLOGY
 In order to know the structure and functions of cells
and its orgenelles some of the techniques
are given below :

3. CELL FRACTIONATION
Definition:
The combination of various methods used to separate a cell
organelle and components based upon size and density.
It is very useful for electron microscopy of cell components.
The principle of cell fractionation consists of two steps :
1. Homogenization
2. Centrifugation

4. 1. HOMOGENIZATION
 The process in which different fractions of biological sample
becomes equal in composition.
OR
 It is the formation of a homogenous mass of cells.

5. PROCESS
 It involves the grinding of cells in a suitable medium with correct
pH, ionic composition and temperature. For example : In plants,
enzyme pectinase is added to digest middle lamella.
 This can be done in a blender.
 It gives rise to uniform mixture of cells which is then centrifuged.

6. 2. CENTRIFUGATION
 It is the process to separate substances on the basis of their
size and densities under the influence of centrifugal force.
 It is done by centrifuge machine.
PRINCIPLE :
 This machine can spin the tube at very high speed.
 Spinning the tube exerts centrifugal force on the contents.

7. TYPES OF CENTRIFUGATION
There are two types of centrifugation:
1.Density gradient centrifugation.
2.Differential centrifugation.

8. DENSITY GRADIENT CENTRIFUGATION.
 The cell components of different sizes and densities are separated
in different layers.
 The upper layers are less dense than lower layers.

9. DIFFERENTIAL CENTRIFUGATION.
The size and shape of particles determines how fast it settles.
PROCESS:
 A series of increasing speeds can be used. At each step, the
content which settles in the bottom of the tube are called pellet.
 The content which remains suspended above in the form of
liquid are called supernatant.
 After each speed, the supernatant can be drawn off and
centrifuge again.
 A series of pallets containing cell organelles of smaller and
smaller size can therefore obtained.

12. DIFFERENTIAL STAINING
Definition:
 Most biological structures are transparent. In order to
differentiate between these structures various colour dyes are
applied. Such techniques area called staining techniques.

13. SINGLE STAINING
 When only one stain is used it is called single staining.
e.g., Borax Carmine stain nucleus.
DOUBLE STAINING OF DIFFERENETIAL STAINING:
 When two stains are used the process is called double staining
or differential staining.
e.g., Haematoxylin stains nucleus and eosin that stains
cytoplasm.

14. MICRODISSECTION
 Variety of techniques where microscope is used to help in
dissection.
 It is done to remove tumors or granules from delicate tissues or
cell like brain, heart and nerve cells. In this technique the image is
seen on large TV screen or monitor while dissecting.

15. TISSUE CULTURE
 Growth of a cell or a tissue on chemically defined nutrient
medium under sterile conditions is called tissue culture.
 This technique can be used for both plants and animals.

16. PLANT TISSUE CULTURE
 Used for plant cloning i.e., production of genetically identical
plants ( clones ).

17. ANIMAL TISSUE CULTURE
 It is usually set up by growing individual cells to form a single
layer of cells over the surface of glass container.
 Animal tissue cultures are used to see any abnormality in the
cell.
e.g.,
Cancer
Chromosomal disorder

18. CHROMATOGRAPHY
 It is technique which is used to separate different chemical
compounds from a mixture.
 It is generally used for the separation of the mixture of
proteins, amino acids photosynthetic pigments.

19. TYPES OF CHROMATOGRAPHY
There are different types of chromatographic techniques.
1.Column Chromatography
2.Gas Chromatography
3.Ion Exchange Chromatography
4.Liquid Chromatography
5.Paper Chromatography
6.Thin-Layer Chromatography (TLC)….. Etc.,

20. PAPER CHROMATOGRAPHY
It is simple and most widely used technique.
PHASES OF PAPERCHROMATOGRAPHY:
1.Stationary Phase :
It is cellulose filter paper.
2.Mobile Phase:
• It is solvent in which sample mixture is dissolved.
• When the solvent travels over the paper, the mixture
sample begins to separate as a dot at different places on
paper according to their affinity.
• This paper is then called chromatogram.

22. SPECTROPHOTOMETRY
It is a technique which is used to determine the absorption of
different wavelength of light by a particular chemical compound or
a photosynthetic pigment.
SPECTROPHOTOMETER:
The instrument used in spectrophotometry is called
spectrophotometer.

23. ABSORPTION SPECTRUM
The amount of light absorbed at each wavelength is plotted in a
graph called the absorption spectrum.
USESOF SPECTROPHOTOMETRY:
1.To determine the wavelengths of light that take a part in
photosynthesis.
2.To determine the very minute quantity of a substance ( such
as DNA ) in a sample.

24. ELECTROPHORESIS
It is technique which is used to separate fragments of a charge
bearing polymer molecule according to their size, shape,
molecular weight and surface charge whether (+) or (-).
Such charge bearing polymer molecules are DNA, RNA, protein
etc.,

25. PROCESS
 This technique uses a gel medium for separation of fragments which is done under
the influence of an electric field.
 The gel is sandwiched between the glass or plastic plates to form a viscous slab.
 The two ends of the slab are suspended in two salt solutions that are connected
by electrodes to a power source.
 At one end of the slab, the samples are loaded.
 When voltage is applied to the apparatus, the molecules present in the gel migrate
through the electric field.
 The negative charged molecule will move towards the positive pole and the
molecules having the positive charge will move towards the negative pole.
 The velocity of movement of fragments is inversely proportional to the size.
 Smaller fragments move faster than larger.
 In this way, all the fragments are separated in the gel after some time.
 Later on, molecules can be pin pointed by staining the gel.

27. GEL USED IN ELECTROPHORESIS
1. Agarose Gel
2. Polyacrylamide Gel
Each type of gel is well suited to different types and sizes of the
analyte.
Polyacrylamide gels are usually used for proteins and have very
high resolving power for small fragments of DNA ( 5 - 500 bp )

28. RESOLUTION & MAGNIFICATION IN MICROSCOPY
RESOLUTION:
 DEFINITION:
The minimum capacity of the lens to differentiate between two adjacent points is called
resolution.
OR
The ability of optical instrument to separate or distinguish small or closely adjacent images.
RESOLUTION OF HUMAN NAKED EYE :
Resolution of human eye is 0.1 mm. This resolution can be increased by increasing
magnification.
0.1 mm
Less than 0.1 mm

29. The resolving power of light microscope is 250
nm.
The resolving power of electron microscope is 0.2
nm.

30. MAGNIFICATION
DEFINITION:
The capacity of an optical instrument to increase the size of an object than its
original size.
 The object that can not be seen by naked eye can also be observed by increasing
magnification.
 Different lenses have different magnification powers which are represented by letter
“X” that means the number of times than original size.
 Therefore a lens of 10 X magnification power can increase the size of an object of 1
μm to 10 μm.
MAGNIFICATION POWER OF DIFFERENT LENSES
OCULAR LENSES OBJECTIVE
ES
5 X 20 X
10 X 40 X
others also exist 100 X etc.,

31.  The magnification of light microscope is upto
4000 X.
 The magnification of electron microscope is
upto 2,000,000 X.

32. MICROSCOPY
 It is the technique used to view objects that cannot be seen by the naked eye.
 The range can be anything between mm and nm.
 Most animal cells and plant cell are between 10 μm and 30 μm.
 A common compound microscope consists of ocular lens and objective lens.
 The overall magnification power of such a microscope Is equal to the product of
magnification powers of both lenses.
HOWTODETERMINETHEMAGNIFICATIONPOWEROFMICROSCOPE:
Mag. Power = X value of ocular lens x X value of objective lens
e.g.,
Mag. Power of ocular lens = 10 X
Mag. Power of objective lens = 40 X
Magnification Power of Microscope = 10 x 40
= 400X

33. MICROMETRY
Micrometry is the measurement of the size of object under microscope.
OCULARMICROMETER:
 It is glass disc with 100 equal divisions with no absolute value.
 It is placed in the eye piece of microscope.
STAGEMICROMETER:
 Ocular micrometer is calibrated by using a stage micrometer.
 This is a glass slide with an exact scale like a miniature transparent ruler.
 By superimposing imaged of the ocular micrometer and stage micrometer scales, it is
calibrated so the size of any given object viewed under the microscope can be
estimated.