This document discusses various histological tools used to study tissues at the microscopic level. It describes light microscopes, which use visible light and magnification to examine thin tissue slices stained with histological dyes. Electron microscopes are also covered, using electron beams instead of light for higher resolution imaging of cell structures. Specific techniques covered include fluorescence microscopy using fluorescent dyes, polarizing microscopy examining birefringence, and transmission electron microscopy producing 2D images of cell organelles. The history and development of microscopy from early simple microscopes to modern compound and electron microscopes is summarized.

1. HISTOLOGICAL
TOOLS
PRESENTED BY:
PRABLEEN ARORA
MDS STUDENT

2. INTRODUCTION
 Histology is the study of microscopic anatomy
of cells and tissues of plants and animals.
 Performed by examining a thin slice of tissue
under a light or electron microscope enhanced
through the use of histological stains.
 Histotechniques constitute the backbone of
diagnostic pathology enabling the diagnosis of
diseased tissue possible for proper
management.
 Histopathology, the microscopic study of
diseased tissue, is an important tool in

3. MICROSCOPY
BIOPSY
TISSUE PROCESSING
STAINING
HISTOCHEMISTRY AND
CYTOCHEMISTRY
IMMUNOCYTOCHEMISTR
It includes :

4. MICROSCOPE
Microscope: It is the instrument that is used to see
objects not visible to naked eye.
Microscopy: It is the study of a fine structure of an
object under a microscope.
Microscope is the most commonly used piece of
apparatus in the laboratory. It produces magnified
images of minute objects.
The microscope magnifies as well as resolves the

5. Magnification power is the degree of image
enlargement.
Magnification power of the microscope =
Magnifying power of objective x Magnification power of
eyepiece.
Resolving power is the capacity of the opyical system
to produce separate image of objects very close to each
other.

6. HISTORY
In 1595, Zacharias Janssen built the first
microscope.
Magnified 3-9 times the original size.

7. The credit of invention of light microscope
goes to Anton van Leeuwenhoek (1632-1723)
who is now known as Father of microscopy.
He was the first to observe and describe microbes which
he referred to as ANIMALCULES under the microscope.
Robert Hooke made a copy of Leeuwenhoek's light
microscope and then improved upon his design.
The electron microscope was introduced in the 1930's
by Germans, Max Knoll and Ernst Ruska.

8. TYPES OF MICROSCOPE
(A) LIGHT MICROSCOPE
Microscopes utilizing visible light
1. Simple microscope
2. Compound microscope
•Dark field microscope
•Phase contrast microscope
•Polarizing microscope
•Interference microscope
Microscopes utilizing ultraviolet light
1. Fluorescence microscopy
(B) ELECTRON MICROSCOPE
Scanning electron microscope
Transmission electron microscope
Freeze fracture microscopy

9. SIMPLE MICROSCOPE:
•A simple microscope is a microscope that uses only one lens for
magnification.
•It is the original design of the light microscope.
•Even though it is now considered pre-historic, the use of a
single, convex lens for viewing is still found in simple
magnification devices, such as magnifying glass and the loupe.
Magnification power of hand
lens is from 20X to 200X.

10. COMPOUND MICROSCOPE
•The compound microscope consists essentially of two or more
double convex lenses fixed in the two extremities of a hollow
cylinder.
•The lower lens (nearest to the object) is called the objective; the
upper lens (nearest to the eye of the observer), the eyepiece.
•The cylinder is mounted upright on a screw device, which permits
it to be raised or lowered until the object is in focus, i.e., until a
clear image is formed.
PARTS: A) STAND
B) BODY
C) OPTICAL SYSTEM
D) LIGHT/ILLUMINATION SYSTEM

12. STAND
Base or foot: It is usually shaped like a horse shoe. It imparts stability to the
microscope.
1. Body: It carries the body tube, stage, sub-stage and a mirror.
A. Body tube: It is attached to the limb. It is of 2 TYPES.
• External tube which carries at its lower end a revolving nosepiece having objective
lenses of different magnification.
• Internal tube carries at its upper end eyepieces.
B Stage: It is the large plate below the tube on which you have to place the slide
which you wish to see.
C. Sub-stage: It is placed below the stage. It carries the condenser.
MECHANICAL ADJUSTMENTS
1. Focusing mechanisms:
Screws for coarse adjustment: Used to bring the section into focus with a 10X objective.
It is controlled by a pair of large knobs.
Screws for fine adjustment : Required to focus when using higher magnification
objectives and oil immersion lenses. This is usually controlled by two smaller knobs on
each side of the body.

13. Optical system
CONDENSER
It brings the parallel light rays into focus on the plane of the
section or slide.
It is mounted on the sub-stage.
An iris diaphragm, situated below the condenser, can be used to
restrict the glare produced by too much light.
OBJECTIVE LENS
They are fitted to the nose piece.
It is near the object and produces an initial magnified image of the
object.
Their magnifying power is measured as the times of its diameter
they magnify. The four objective lens commonly used have
magnification of 4x, 10x ,40x, 100x with color coding as red,
yellow, light blue and white respectively for rapid identification.

14. Ocular or eye peice
Mononuclear - 1 eyepiece.
Binocular – 2 eyepiece
Their magnification can be 5x, 10x,15x
MIRROR
It is located below the condenser.
Its function is to direct the light rays from a light source to the condenser and
optical field of the microscope.
The mirror has two sides: one has a plane mirror (sunlight) and the other with a
concave mirror (artificial light). The plane mirror is used to direct the light to the
condenser to give a uniform illumination. In day light or when there is no
condenser in the microscope a concave mirror is used to direct the light to the
slide.
LIGHT SOURCE
Daylight: It is best to use reflected sunlight of a dull white background. It is not
sufficient for oil immersion lens and it is not available during evening or night.
Electric light: A built-in source of illumination of 60 watt electric lamp placed
18” away from the microscope is sufficient for most routine work. In the absence
of electricity, a battery lamp or an oil lamp can be utilized. Best however are

15. POLARISING MICROSCOPE
This method is used for demonstration of birefringence eg
amyloid, foreign body, hair etc.
 Birefringent objects rotate the light rays and therefore appear
bright in a dark background.
polariser
analyser

16. FLUORESCENT MICROSCOPE
This method is used for
demonstration of naturally occurring
fluorescent material and other non
fluorescent substances or micro-
organisms after staining them with
some fluorescent dyes
Principle:
Fluorescent substances are those
substances which absorb light of a
shorter wavelength and high energy
and emit light of longer wavelength
and less energy.
Many substances have the property
of emitting visible light when
irradiated by invisible rays, like
when ultraviolet light is focused
upon them, such a specimen glows
and can be observed by emitted
fluorescence. The phenomenon is
called fluorescence.

17. Uses
1. For identifying mycobacterium tuberculosis,
amyloid,lipids, elastic fibers.
2. It is used extensively in fluorescent antibody
techniques used in parasitology and bacteriology.
3. It is also used widely in histopathology of kidney; skin
etc. where immune/auto-immune basis of disease is
expected.
4. Widely used in cytogenetics.

18. Fluorescent antibody technique- USE IN PERIO
Principle:
Immunofluorescence is a technique which uses the
highly specific binding of an antibody to its antigen in
order to label specific proteins or other molecules within
the cell.
Method
1. Antibody to which a fluorescent dye is attached is
referred to as labeled antibodies.
2. Labeled antibodies are mixed with suspension of
bacteria and preparation is examined under
microscope.
3. Bacterial cells that have combined with the labeled
antibody will only be visible in the microscopic

19. ELECTRON MICROSCOPE
Most electron microscopes used to study biological material can
magnify objects up to 1 million times.
Although this does not make atoms visible, it does allow
researchers to distinguish individual molecules of biological
importance.
Drawback
Since no living specimen can survive under their high vacuum,
they cannot show the ever-changing movements that characterize
a living cell.
PRINCIPLE
By the substitution of an electron beam for light rays, a much
greater degree of resolution can be obtained, since at an
acceleration of 50,000 volts electrons have a wavelength of only
0.001nm; therefore a theoretical resolving power of 0.0005nm
could be attained, which would enable molecules to be seen.

20. ELECTRON MICROSCOPE
It is used to study ultrastructural details of the tissues and
cells.
Tissue is fixed in 4% glutaraldehyde at 4 degree celsius for
4 hrs.
Ultrathin microsections with thickness of 100 nm are cut with
diamond knives.
TYPES:
1) TRANSMISSION ELECTRON MICROSCOPY (TEM) :
helps to visualize cell’s cytoplasm and organelles.
2) SCANNING ELECTRON MICROSCOPY (SEM) : helps in
the study of cell surface.

21. TRANSMISSION ELECTRON MICROSCOPY (TEM)
TEM gives two dimensional image of the tissue.
TEM helps visualize cell’s cytoplasm and organelles.
Unfortunately the degree of correction that is currently feasible with TEM lenses permit a
resolution of 0.25nm, but this is still a thousand times greater than that possible with the
light microscope.
A further difficulty with TEM is that, since electrons have poor penetrating power, the
sections to be examined must be very thin, less than 50nm thick. This necessitates the
use of special hard embedding media (plastics) and special ultra microtomes to cut such
thin sections. Steel knives cannot be used to cut these sections; either glass or diamond
knives are used.
Principle and Method
In TEM, the electrons beam passes right through many parts of specimen.
The filament of electron microscope emits beam of electrons.
By means of magnetic coil, electrons are focused onto the object and electrons passing
through object are then received by another magnetic coil acting as objective lens which
gives a magnified image of the object.
These electrons are then received by third magnetic coil acting as ocular or projection
lens which further magnifies the primary image.
This final image can be visualized on a fluorescent screen or recorded on a
photographic plate.
As the electron cannot be seen so this image is formed on the fluorescent screen that
converts the energy of the electrons into light.

23. SCANNING ELECTRON MICROSCOPY (SEM)
In this 3-D image is produced. The image is produced on cathode ray oscillograph which
can also be amplified.
SEM helps in the study of cell surface.
SEM can also be used for fluorescent antibody techniques.
Resolution up to 50 nanometers can be achieved.
APPLICATION:
Secondary electrons are most valuable for showing morphology and topography of
samples and imaging.
Backscattered electrons are valuable for revealing chemical composition of samples.
Diffracted –backscattered electrons determine crystalline structure and orientation of
minerals in samples.
Photons are used for elemental analysis.

24. In SEM, accelerated electrons scans the
surface of specimen instead of passing
through it.
These accelerated electrons carries
significant amount of kinetic energy.
As the electron beam hits the specimen
energy is dissipated as variety of signals,
these signals include:
 Secondary electrons
Backscattered electrons
Diffracted-backscattered electrons
Photons
Heat
Detectors collect these signals and send to
a screen and final image is produced.
Electron micrographs are obtained by
photographing the image.

25. BIOPSY
It is the removal of tissue from living organism for the
purposes of microscopic examination and diagnosis.
PURPOSE:
Diagnosis of pathologic lesions.
Grading of tumors.
Diagnosis of metastatic lesions.
For evaluation of recurrence.
It is useful for therapeutic assessment of lesion by
differentiating between benign and malignant lesions.

26. To ensure obtaining a proper specimen for the pathologist,
following points must be considered:
1. Do not paint the surface of the area to be biopsied with
iodine or a highly coloured antiseptic.
2. If using infiltration anesthesia, do not inject LA solution
directly into the lesion. Instead, inject around the periphery
of the lesion.
3. Use a sharp scalpel to avoid tearing of the tissues.
4. Remove a border of normal tissue with the specimen, if at
all possible.
5. Use care not to mutilate the specimen when holding it with
the forceps.
6. Fix the tissue immediately upon removal in 10% formalin
or 70% alcohol. If the specimen is thin, place it upon a
piece of glazed paper and drop into fixative to prevent
curling of tissues.

27. TYPES OF BIOPSY
EXCISIONAL
BIOPSY
INCISIONAL
BIOPSY
PUNCH
BIOPSY
CORE
NEEDLE
BIOPSY
EXFOLIATIVE
CYTOLOGY
BRUSH
BIOPSY
USH BIOPSY

28. EXCISIONAL BIOPSY
It is the total excision of a small
lesion for microscopic examination.
INDICATIONS :
a) Small lesion ( less than 1 cm).
b) 2-3mm normal tissue
surrounding the lesion is also
excised.
c) When the lesion is sessile or
pedunculated.
d) Tissues which are freely
movable and located above
the mucosa or just beneath the

29. INCISIONAL BIOPSY
It is performed by removing a wedge
shaped specimen of the pathological
tissue along with surrounding normal
zone.
Biopsy - deep and narrow biopsy rather
than a broad and shallow one, because
superficial changes may be different from
those deeper in the tissue.
INDICATIONS:
a) If the lesion is large (more than 1 cm)
and diffuse and extend deeply into the
surrounding tissue so that total
removal cannot be obtained easily
with LA.

30. PUNCH BIOPSY
It is rarely used in the oral cavity
as most of the oral lesions are
easily accessible.
In this technique, a sharpened
hollow tube is rotated until
underlying bone or muscle is
reached.
The tissue is then removed in the
same manner as excisional and
incisional biopsy.

31. BRUSH BIOPSY
Biopsy obtained by abrading the surface of a lesion with a
brush with stiff bristles to obtain cells and tissues for
microscopic examination.
Effective in obtaining tissue samples from inaccessible
areas.

32. CORE NEEDLE BIOPSY
Common needle biopsy procedures include fine-needle
aspiration and core needle biopsy. It may be used to take
tissue or fluid samples from muscles, bones, and other organs,
such as the liver or lungs

33. EXFOLIATIVE CYTOLOGY
It is the study of cells which exfoliate or abrade from the
surface.
It is not a substitute but an adjunct to surgical biopsy.
The cytologic smear is
reported in 5 classes:
I : NORMAL
II: ATYPICAL
III: INTERMEDIATE
IV: SUGGESTIVE OF
CANCER
V : POSITIVE FOR
CANCER

34. Oral exfoliative cytology is still controversial, particularly in premalignant
conditions
Normal oral squamous epithelium continuously sheds the most superficial cells
If the area is affected by malignant or other disease, the deeper cells lose their
cohesiveness and are exfoliated at the same time as the superficial cells.
Exfoliative cytology has not been a very diagnostic or useful screening method
for oral cancer because hyperkeratosis and keratin itself interfere with cell
obtainment and a greater proportion of diagnostic cells are below the surface

35. Class 1 (normal) indicates that only normal cells were observed
Class 2 (atypical) indicates presence of minor atypical but no
evidence of malignant change
Class 3 (intermediate) – this is an in between cytology that
separates cancer from non cancer diagnosis biopsy is
recommended.
Class 4 (suggestive of cancer) a few cells with malignant
characteristics or many cells with borderline characteristics
.biopsy is mandatory

36. TISSUE PROCESSING
FIXATION
DEHYDRATION
CLEARING
IMPREGNATION
EMBEDDING AND BLOCKING
SECTION CUTTING
ROUTINE STAINING
STEPS INCLUDE:

37. Tissue sample
Sample of a tissue is called tissue block which is
obtained by biopsy.
If a sample is taken from cadaver it is essential to
remove it promptly after death to avoid post
mortem degeneration.
Use Sharp instrument to avoid distortion of
microscopic features.
After obtaining sample it must be immersed in

38. FIXATION
Any tissue removed from the body starts decomposing immediately because
of loss of blood supply and oxygen, accumulation of products of metabolism
of action of autolytic enzymes and putrefaction by bacteria. This process of
decomposition is prevented by fixation.
Prevention of putrefaction and autolysis.
Hardens the tissue which helps in section cutting.
Makes cell insensitive to hypertonic or hypotonic solution.
EFFECTS:
1. FORMALIN
2. GLUTRALDEHYDE
3. PICRIC ACID
4. ALCOHOL
5. OSMIUM TETRAOXIDE
COMMONLY USED FIXATIVES:

39. DEHYDRATION
This is a process in which water from the tissues and cells is removed so that this
space so created is subsequently taken up by wax.
Dehydration is carried out by passing the tissues through a series of ascending grades
of alcohol (70%,80%,95% and absolute alcohol).
If ethyl alcohol is not available isopropryl alcohol or acetone can be used.
Purpose of dehydration is to remove excess fixative.
Done in two stages:
Stage 1 (Stage of gradual dehydration): Formalin fixed tissue block is passed through
increasing strength of alcohol to absolute alcohol.
Stage2 (clearing stage): Purpose is to remove dehydrating agent. This is the process
in which alcohol from the tissues and cells is removed and is replaced by a fluid in which
wax is soluble.It also makes the tissue transparent. Xylol is used for this purpose
because alcohol does not act as paraffin solvent so it is necessary to replace alcohol
with xylol which is miscible both with alcohol and paraffin. Alcohol dehydrated block is
passed through xylol until all alcohol has been replaced by xylol. Other clearing agents
are benzene and chloroform.

40. This is the process in which empty spaces in the tissues and
cells after removal of clearing agent are taken up molten by
paraffin wax. This harden the tissue which helps in section
cutting.
Impregnation is done in molten paraffin wax which has the
melting point ranging from 54-62 degree.
IMPREGNATION
Embedding is a process in which tissue sample are placed into
mould along with liquid embedding material (agar / gelatin /wax)
which is then hardened..
EMBEDDING AND BLOCKING

41. Purpose: to facilitate the sectioning of the tissue into slices
sufficiently thin to allow the passage of light through them (approx
4 to 10 micrometre in routine practice).
Most common medium used traditionally is paraffin.
Xylol permeated tissue block is passed through warm paraffin
which dissolve readily in xylol.
Melted wax fills in tissue spaces which hardens on cooling and
renders the block ready for sectioning.
 METALLIC MOULDS PLASTIC MOULDS

42. Select a mould having sufficient room for tissue and at least a 2 mm
surrounding margin of wax .
Lift tissue with warm forceps and place it at bottom of mould
Fill it with molten paraffin wax
Gently press & orient it so that surface to be sectioned is kept flat.
TECHNIQUE
When thin layer forms over surface, cool and submerge it under water.
After solidification, remove from mould.
Trimmed with razor blade in such a way that edges are parallel.
Ready to be sectioned on microtome.
Insert a label.

43. Technique of section cutting is MICROTOMY .
Equipment used for cutting section is MICROTOME.
Microtome is used to cut extremely thin slices of material known
as
SECTIONS
SECTION CUTTING
ROTARY : most commonly used
SLIDING
FREEZING
ROCKING
BASE-SLEDGE
TYPES OF MICROTOMES:
ROTARY MICROTOME

44. STAINING
There are innumerable tissue stains but commonly used
stain is HAEMATOXYLIN AND EOSIN .
HAEMATOXYLIN – natural dye obtained from log wood of
tree.
INACTIVE PRODUCT
ACTIVE INGREDIENT
(HAEMATEIN)
OXIDATION
NATURAL SUNNLIGHT
BY ADDING OXIDANTS
KMnO4, SODIUM IODATE

45. Dried slides placed vertically.
Sections are firstly deparaffinised ( removal of wax by
placing slide in xylene for 10-15 min .
Pass slide through descending grades of alcohol (
absolute alcohol , 95 % , 80 % , 60% ) & then to water (as
haematoxylin is water based dye)
Place slide in haematoxylin stain for 8-10 min.
Rinse in water.
.
TECHNIQUE

46. For differentiation (i.e. selective removal of excess dye from sect
ion)put in SCOTT’S TAP WATER containing NaHCO3 and MgS
O4.
Counter stain with 1 % aqueous sol. Of Eosin for 30 sec – 1 min.
Dip in tap water.
Before mounting , dehydration of section through ascending
grade of alcohol and finally cleared in xylene , 2-3 dips in each
solution.
Mount in DPX ( DEXTRENE POLYSTYRENE XYLENE ) /
CANADA BALSAM.

48. Frozen sections
When rapid confirmation of nature or spread of
diseased tissue is required(during surgery) they
can be prepared in great haste by another procedure.
When a fresh tissue is rapidly frozen, the matter within the tissue
turns into ice and in this state the tissue is firm, the ice acting as
embedding medium. Therefore, sections are produced without the
use of dehydrating solution, clearing agent or wax embedding.
Everything present in the living tissue is still present in the frozen
sections but it is necessary to examine these sections promptly to
avoid fixation.

49. Merits
1. This is a quick diagnostic procedure (10minutes).
2. Every type of staining can be done.
3. There is minimal shrinkage of tissues as compared to paraffin
sections.
4. Lipids and enzymes which are lost in routine paraffin sections
can be demonstrated.
Demerits
1. It is difficult to cut serial sections.
2. It is not possible to maintain tissue blocks for future use.
3. Sections cut are thicker.
4. Structural details tend to be distorted due to lack of
embedding medium.

50. Preparation of ground sections of
teeth or bone
•Decalcification of bone and teeth often obscures the structures.
Teeth in particular are damaged because tooth enamel, being
about 96% mineral substance, is usually completely destroyed by
ordinary methods of decalcification.
•Undecalcified teeth and undecalcified bone may be studied by
making thin ground sections of the specimen.
•The equipment used for making ground sections includes a
laboratory lathe, a coarse and a fine abrasive lathe wheel, a
stream of water, a wooden block, adhesive tape, and a camel hair
brush.
•The finished ground section is then mounted on a microscope
slide.
Note: Teeth used for making ground sections should not be

51. HISTOCHEMISTRY AND
CYTOCHEMISTRY
Cytochemistry is a science of localizing chemical
components of cells and organelles on histological
sections by using various techniques.
Histochemistry has contributed greatly to the
understanding of structure-function relationships.
• Histochemistry
• Enzyme histochemistry
• Immunocytochemistry
• In situ hybridization
Histological technique
used for studying
chemistry of tissues
and cells

52. Application:
Histochemical methods depend on a reasonably straightforward staining reaction of a
dye for an organic molecule. For example:
1. Sudan black and oil red O stains are widely used for revealing the presence and
location of lipids within tissues.
2. The nucleic acids DNA and RNA are demonstrable histochemically (with the
Feulgen reaction and with toluidine blue and hematoxylin staining, respectively).
ENZYME HISTOCHEMISTRY: It is used by both the histologist and the pathologist to
localize enzymatic activity within tissue sections. E.g. acid and alkaline phosphatases,
esterases, and the dehydrogenases, oxidases, and ATPases of various metabolic
cycles.
The general principle of enzyme tissue chemistry involves incubation of the tissue slice
or section in a medium containing:
A substrate specific to the enzyme system under study.
A buffer chosen to maintain the ph optimum of the enzyme system.
Any cofactors required by the enzyme.

53. IMMUNOCHEMISTRY
•It is the application of immunologic techniques to the cellular pathology.
•It is a powerful technique for the detection of tissue and cellular components,
particularly protein molecules.
•It involves coupling, and subsequently visualizing, a labeled antibody to the component
molecule of interest within a tissue section or cell population under study.
•PRINCIPLE
•To detect the status and localisation of particular antigen in the cells (membrane,
cytoplasm or nucleus) by use of specific antibodies which are then visualised by
chromogen. This helps in confirming a specific infection.
•TECHNIQUES
1. Peroxidase –antiperoxidase (PAP) in which PAP reagent is linked to primary
antibody by a bridging antibody.
2. Avidin-biotin conjugate (ABC) immunoenzymatic technique in which biotinylated
secondary antibody serves to link the primary antibody to large performed complex of
avidin, biotin, peroxidase

54. The coupling site within the tissue is revealed microscopically by
tagging the antibody either:
1. With a colored molecule visible in the light microscope.
2. With a molecule of specific shape or electron opacity that can
be recognized under the electron microscope.
3. GOLD/PROTEIN A labeling
4. FLUORESCENT labeling
APPLICATION
1. Tumors of uncertain histogenesis
2. Prognostic marker in cancer.
3. prediction of response to therapy.
4. Infections

55. HYBRIDIZATION TECHNIQUE
Working of cell is imp to understand and this requires that technique that permit
analysis of molecules involved in process of information flow from DNA to
protein.
Differrnt types of hybridization:
Blotting is the technique in which nucleic acids or proteins are immobilized onto
a solid support, generally nylon or nitrocellulose membranes. There are
different blotting procedures depending on the type of molecule being
transferred.
When DNA fragments are transferred the procedure is called a Southern blot.
With Northern blotting, RNA molecules are transferred.
With Western blotting, protein molecules are transferred.

56. SOUTHERN ANALYSIS –characterizes and quantifies the presence of DNA of
a specific gene in the presence of all other genes in a eukaryotic organisms.
NORTHERN ANALYSIS –Identifies and quantifies specific messenger RNA
transcripts in the presence of all RNA transcripts expressed with in a single cell
type
WESTERN ANALYSIS- detects a single protein species from among all other
proteins expressed in a single cell or tissue. It is based on high affinity and
specificity between antibodies and antigens

57. BASICS OF HYBRIDIZATION
PROCEDURE
•All blotting procedures begin with a standard
process called gel electrophoresis.
•During this step, DNA, RNA, or proteins are
loaded on to an agarose or polyacylamide gel
(that functions like a molecular sieve) and are
then run through an electric field.
•wo types of gels are commonly used to
separate molecules according to size and/or
charge: agarose gels are used to separate DNA
and RNA, and polyacrylamide gel
electrophoresis (PAGE) is used to separate
proteins.
•Shorter molecules move faster and migrate
further than longer ones because the shorter
molecules migrate more easily through the
pores of the gel.

58. •Transfer is initiated when the gel is retrieved
from the electrophoresis apparatus and the
nylon / nitrocellulose membrane is laid on top
of the gel.
•The objective now is to transfer the bands of
molecules found in the gel to the membrane.
The molecules are immobilized (fixed) on the
membrane.
•Short fragments of DNA that have a
complementary nucleotide sequence to the
molecule being analyzed are normally used as
probes in Southern and Northern blots.
• Proteins/antigens that react with the
proteins/antibodies being analyzed are used

59. Conclusion
•Microscope plays a very important role in viewing
organisms and structures which are not visible to naked
eye.
•A basic understanding of working and principles of
various types of microscopes enable us to have a
complete knowledge of the any subject at molecular
level.
•Moreover other special techniques act as an adjunct. As
the science is advancing, one has to keep himself
updated with the latest techniques and armamentarium

60. REFERENCES
HISTOLOGY by Ronald A. Bergman, Adel K. Afifi, Paul M. Heidger JR
Medical laboratory technology methods and interpretations by Ramnik
Sood
Textbook of histology and A practical guide , 2nd edition, by J.P.
Gunasegaran
Pathology practical book, 2nd edition, by Harsh Mohan.
Basic Histology (9th Edition) Lange
Shafer’s Oral Pathology (5th Edition)
Immunihistochemistry (Eldem Sadikoglou)
Molecular Hybridization Technique Of Nucleic Acid: ISSN 1843-6099
Oral Histology (Antonio Nancy 6th Edition)